Báo cáo khoa học: Application of the Fc fusion format to generate tag-free bi-specific diabodies doc

Keywords bi-specific diabody; Fc fusion format; preparation method; small therapeutic antibody; tag-free protein Correspondence I.. The BsAb was purified by protein A affinity chromatograp

bi-specific diabodies

Ryutaro Asano1, Keiko Ikoma1, Hiroko Kawaguchi1, Yuna Ishiyama1, Takeshi Nakanishi1,

Mitsuo Umetsu1, Hiroki Hayashi2, Yu Katayose2, Michiaki Unno2, Toshio Kudo3and Izumi Kumagai1

1 Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan

2 Division of Gastroenterological Surgery, Department of Surgery, Graduate School of Medicine, Tohoku University, Sendai, Japan

3 Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan

Introduction

Bi-specific antibodies (BsAbs) are attractive formats

for recombinant antibodies that can bind to two

differ-ent epitopes on antigens This bi-specificity can be used

in cancer immunotherapy by cross-linking tumor cells

to immune cells such as cytotoxic T cells, natural killer

cells and macrophages This linkage accelerates the destruction of the tumor cells by immune cells, so that the dose of therapeutic antibodies can be reduced from that required in the case of mono-specific anti-bodies [1,2]

Keywords

bi-specific diabody; Fc fusion format;

preparation method; small therapeutic

antibody; tag-free protein

Correspondence

I Kumagai, Aoba 6-6-11-606, Aramaki,

Aoba-ku, Sendai 980-8579, Japan

Fax: +81 22 795 6164

Tel: +81 22 795 7274

E-mail: kmiz@kuma.che.tohoku.ac.jp

(Received 1 May 2009, revised 9

November 2009, accepted 17 November

2009)

doi:10.1111/j.1742-4658.2009.07499.x

We previously reported the use of a humanized bi-specific diabody that targets epidermal growth factor receptor and CD3 (hEx3-Db) for cancer immunotherapy Bacterial expression can be used to express small recombi-nant antibodies on a large scale; however, their overexpression often results

in the formation of insoluble aggregates, and in most cases artificial affinity peptide tags need to be fused to the antibodies for purification by affinity chromatography Here, we propose a novel method for preparing refined, functional, tag-free bi-specific diabodies from IgG-like bi-specific antibodies (BsAbs) in a mammalian expression system We created an IgG-like BsAb

in which bi-specific diabodies were fused to the human Fc region via a designed human rhinovirus 3C (HRV3C) protease recognition site The BsAb was purified by protein A affinity chromatography, and the refined tag-free hEx3-Db was efficiently produced from the Fc fusion format by protease digestion The tag-free hEx3-Db from the Fc fusion format showed a greater inhibition of cancer growth than affinity-tagged hEx3-Db prepared directly from Chinese hamster ovary cells We also applied our novel method to another small recombinant antibody fragment, hEx3 sin-gle-chain diabody (hEx3-scDb), and demonstrated the versatility and advantages of our proposed method compared with papain digestion of hEx3-scDb This approach may be used for industrial-scale production of functional tag-free small therapeutic antibodies

Abbreviations

BsAbs, bi-specific antibodies; CHO, Chinese hamster ovary; Db, diabody; EGFR, epidermal growth factor receptor; hEx3-Db, humanized bi-specific diabody that targets epidermal growth factor receptor and CD3; hEx3-scDb, hEx3 single-chain diabody; HRV3C, human rhinovirus 3C; MTS, 3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt; scDb, single-chain diabody; scFv, single chain Fv; T-LAK, lymphokine-activated killer cells with the T-cell phenotype; tanDb, tandem single-chain diabody;

taFv, tandem scFv.

Conventionally, BsAbs are produced by chemical

conjugation or somatic fusion of two hybridomas,

form-ing a quadroma that can produce bi-specific IgG

mole-cules [1,3] Clinical studies of these BsAbs have been

performed, and some impressive local anti-tumor

responses have been reported; however, these trials have

also been limited by the occurrence of human

anti-mouse antibody and⁄ or Fc-mediated side-effects such as

the induction of a cytokine storm [4,5] Furthermore,

these methods cannot be utilized for large-scale

produc-tion, and a quadroma cannot control the heterogeneity

of the antibodies produced; for instance, ten possible

variants of antibodies can be generated when two heavy

and two light chains are randomly associated

There-fore, steady production of homogeneous BsAbs requires

the use of a host-vector system

Advances in antibody engineering techniques and

host-vector expression systems have facilitated the

gen-eration of recombinant BsAbs with improved

proper-ties A variety of recombinant BsAbs have been

developed from two antibody fragments such as

single-chain Fv fragments (scFv; 25 kDa) [6,7], and diabodies

(Db; 55 kDa) [8] that recognize different antigens The

most common BsAb formats that have been produced

from these fragments are tandem scFv (taFv) [9],

tan-dem single-chain diabodies (tantan-dem scDb, tanDb) [10]

and mini-bodies (dimeric scDb–CH3 fusion protein)

[11] Compared with classic BsAbs prepared by

chemi-cal conjugation or production of a quadroma, small

antibody molecules, such as diabodies, are of a

suit-able size for rapid tissue penetration, high target

reten-tion and rapid clearance [12,13] Their smaller size also

enables expression of BsAbs in bacteria, and as the

structure is composed only of antibody variable

regions, this eliminates the Fc-mediated side-effects of

BsAbs Although the rapid blood clearance and

monovalency of bi-specific diabodies, scDbs and taFv

(all approximately 55 kDa) may limit their therapeutic

application, engineering the length and amino acid

composition of the middle linker in scDb, for example,

may enable them to assemble into multimers, such as

tanDb (114 kDa), with higher molecular weight and

bivalency for each target antigen [14,15]

Small bi-specific antibody fragments prepared in

bacteria are often expressed as insoluble aggregates in

the cytoplasmic or periplasmic space [10,16–18], and

require fusion of artificial affinity peptide tags, such as

a polyhistidine tag, hemagglutinin tag or FLAG tag,

at the N- or C-terminus of the BsAbs to allow

com-plete removal of the vast amount of host-derived

proteins by affinity chromatography [16,19] The

requirement for such tags raises concerns about

immu-nogenicity We have previously reported significant

anti-tumor activity in vitro and in vivo for a humanized bi-specific diabody targeting epidermal growth factor receptor (EGFR) and CD3 (hEx3-Db) [20] However, even though the yield of hEx3-Db was over 10 mgÆL)1 culture, it was also expressed as insoluble aggregates, and fusion of an affinity tag was necessary for purifica-tion before the re-folding process

We have also reported the construction of a mam-malian expression system for affinity-tagged bi-specific diabodies and their Fc fusion formats [21] Here, we developed a novel method for the production of highly purified tag-free diabodies using the mammalian expression system Diagrams of the various gene con-structs are shown in Fig 1 The tag-free hEx3-Db alone was expressed sufficiently to be purified by ion-exchange chromatography Expression of the hEx3 diabodies fused to the human Fc region via a designed protease recognition site enabled high-efficiency purifi-cation by protein A affinity chromatography and increased the yield of tag-free hEx3-Db We also used our method to produce tag-free small BsAbs to hEx3-scDb For hEx3-scDb, use of the designed protease recognition site had advantages over papain digestion, which caused unwanted degradation Both tag-free hEx3-Db and hEx3-scDb prepared by restriction pro-tease digestion from the Fc fusion format showed a greater inhibition of cancer growth in vitro than previ-ously produced affinity-tagged diabodies directly pre-pared from the supernatant of Chinese hamster ovary (CHO) transfectants [21] Thus, this approach appears

to improve both the yield and efficacy of the bi-specific antibody fragments

Results

Preparation of tag-free bi-specific diabodies Tag-free hEx3-Db was directly secreted from mamma-lian cells and purified by cation-exchange chromatog-raphy as described in Experimental procedures Purified hEx3-Db was applied to a gel filtration col-umn for further analysis and purification (Fig 2A) The first small peak, second large peak and the shoul-der of the major peak seen in the chromatograph were identified as the multimeric, dimeric and monomeric structures of tag-free hEx3-Db, respectively Equivalent amounts of hOHh5L (humanized OKT3 VH -linker - humanized 528 VL) and h5HhOL (humanized

528 VH - linker - humanized OKT3 VL) were con-firmed in the dimeric fraction by SDS–PAGE analysis (Fig 2B) Thus, purified tag-free hEx3-Dbs were obtained without affinity chromatography at a final yield of approximately 1 mgÆL)1culture

To prepare the high-quality, tag-free bi-specific

dia-bodies, we fused the hEx3-Db to the human IgG1 Fc

region We inserted a recognition site for HRV3C

pro-tease between the diabody fragments and the Fc

por-tion of hEx3-Fc A schematic illustrapor-tion of the

preparation of tag-free hEx3-Db from its Fc fusion

format is shown in Fig 3A The expressed IgG-like

BsAbs were purified by protein A affinity

chromatog-raphy and digested using glutathione S-transferase

(GST)-fused HRV3C protease The treated solution

was loaded onto a glutathione-immobilized column

and then a protein A column to remove added

prote-ase and digested Fc SDS–PAGE analysis of each

puri-fication step showed the successful preparation of

tag-free hEx3-Db from its Fc fusion format (Fig 3B) Gel

filtration chromatography showed that tag-free

hEx3-Db predominantly formed dimers, with a small

amount of multimeric forms (Fig 4A) The

homogene-ity of tag-free hEx3-Db in the eluted fraction was also

confirmed by SDS–PAGE (Fig 4B) The final yield of

tag-free hEx3-Db from the Fc fusion format was

approximately 5 mgÆL)1 culture, i.e five times that of the secreted tag-free hEx3-Db Thus, secretion of BsAbs as the Fc fusion format increased the amount

of prepared tag-free diabodies due to the high produc-tivity (approximately 10 mgÆL)1) and the efficient puri-fication using protein A

Mass spectrometry of tag-free bi-specific diabodies

We previously reported that the strong inter-domain interaction between cognate VH and VL domains of hEx3-Db leads to the spontaneous formation of func-tional heterodimers [22] In the present study, the molecular weight of the monomorphous heterodimer

of the tag-free hEx3-Db prepared from the Fc fusion format was confirmed by MALDI-TOF mass spec-trometry (Fig 4C) The mass spectrum for the diabod-ies prepared from the Fc fusion format had two peaks, one at m⁄ z 26 424 and another at m ⁄ z 25 970, which correspond to the calculated molecular weights of

h5H

pcDNA-h5HhOL-3C-Fc

Tag-free hEx3-Db

hEx3-Db-3C-Fc(tool for tag-free hEx3-Db)

hEx3-scDb-3C-Fc(tool for tag-free hEx3-scDb)

pcDNA-hEx3-scDb-3C-Fc

CMV promoter Kozak sequence Leader peptide

Peptide linker (GGGGS) Peptide linker [(GGGGS)4]

HRV3C protease recognition site (LEVLFQGP) Hinge

Neo Neomycin resistance Hyg Hygromycin resistance

hOL

Fig 1 Schematic illustration of the BsAb

gene constructs in pCDNA3.1 The V H and

V L regions of humanized 528 Fv are

desig-nated h5H and h5L, and those of humanized

OKT3 Fv are designated hOH and hOL,

respectively The positions of important

restriction enzyme sites used and the key

components are shown.

hOHh5L digested from the Fc fusion (26 442) and

h5HhOL without the peptide tag (25 991), respectively

These results indicate that Db–3C–Fc fusion proteins

can serve as a tool for preparing tag-free diabodies

with high yield and purity

Binding affinity of tag-free bi-specific diabodies

and its effect on growth inhibition

The binding affinity of tag-free hEx3-Dbs for

CD3-positive lymphokine-activated killer cells with the

T-cell phenotype (T-LAK cells) and EGFR-positive

TFK-1 cells was measured by flow cytometry using

polyclonal antibody to hEx3-Db Tag-free hEx3-Dbs interacted with each targeted antigen (Fig 5A), and the binding profiles were comparable with those previ-ously reported for affinity-tagged hEx3-Db [20,22] These results indicate that the diabody prepared by HRV3C protease digestion from the Fc fusion format retained sufficient binding activity and bi-specificity

To evaluate the inhibition of cancer growth by tag-free hEx3-Db, an MTS assay was performed for

TFK-1 cells by using T-LAK cells at an effector⁄ target ratio

of 5 : 1 Tag-free hEx3-Db prepared from the Fc fusion format inhibited cancer cell growth more effec-tively than did affinity-tagged hEx3-Db (Fig 5B) Imperceptible differences in purity and local structural perturbations that are dependent on the preparation method might affect these activities

67 kDa

A

B

25 kDa

43 kDa

47.5–

5 mAU

32.5–

Tag-free hOHh5L

25– Tag-free h5HhOL

16.5–

150 200 250 300

Elution volume (mL)

Fig 2 (A) Gel filtration of tag-free hEx3-Db The elution volume is

shown on the x axis, and the molecular mass (kDa) is shown

above The eluted fractions containing the bi-specific diabody are

indicated by the two-headed arrow (B) SDS–PAGE analysis under

reducing conditions of the eluted fraction Molecular size markers

are shown on the left.

HRV3C protease site

A

B

Tag-free hEx3-Db hEx3-Db-3C-Fc

1

175 –

83 –

62 –

–

Tag-free hOHh5L

16.5 –

Fig 3 (A) Schematic illustration of the hEx3-Db–3C–Fc fusion pro-tein The HRV3C protease cleavage site used for preparation of tag-free hEx3-Db is indicated (B) Reducing SDS–PAGE of each purification step for preparation of tag-free hEx3-Db from hEx3-Db– 3C–Fc Lane 1, protein A chromatography-purified hEx3-Db–3C–Fc; lane 2, after HRV3C protease digestion; lane 3, after removal of HRV3C protease by glutathione Sepharose 4B chromatography; lane 4, purified tag-free hEx3-Db after removal of the Fc region by protein A chromatography.

Application of method to tag-free bi-specific

sin-gle-chain diabody

To demonstrate the utility of this novel method, we

applied it to the preparation of tag-free hEx3-scDb,

which is a single-chain form of hEx3-Db (Fig 6A) An

HRV3C protease recognition site was inserted between

hEx3-scDb and the Fc portion, and the recognition

sequence for papain was conserved Papain is a

cyste-ine protease that is generally used in the preparation

of Fab fragments from IgG, because the recognition

site for papain naturally exists around the hinge region

of intact antibody

When we digested hEx3-scDb–3C–Fc with HRV3C

protease, hEx3-scDb was separated from the Fc

por-tion with no degradapor-tion Similar to the tag-free

hEx3-Db, the Fc portion was completely removed by protein

A affinity chromatography (Fig 6B) To confirm the

benefit of the design of the HRV3C protease digestion

site, we also followed the time course of papain

digestion of hEx3-scDb–3C–Fc (Fig 6C) Although

tag-free hEx3-scDb was successfully prepared by

papain digestion, especially with an incubation time

of 1 h, two unexpected bands corresponding to

hOHh5L and h5HhOL caused by a break in the

mid-dle linker from scDb also appeared This digestion

proceeded as the incubation time increased, and

further degradation of h5HhOL was observed after

incubation for 10 h

The binding affinity of tag-free hEx3-scDb prepared from the Fc fusion format for both targeted cells was confirmed by flow cytometry (Fig 7A), and its enhanced cytotoxicity was compared with affinity-tagged hEx3-scDb [21] in the MTS assay with the use

of T-LAK cells as effector cells (Fig 7B) These results strongly support the utility and general applicability of our method for the preparation of homogeneous tag-free small recombinant antibodies

Discussion Recombinant BsAbs have several advantages over clas-sic BsAbs prepared by chemical cross-linkage or fusion

of two hybridoma clones [16,23–25] The IgG-like BsAbs containing human Fc regions are highly effec-tive recombinant antibodies [25–27] because of the antibody-dependent cellular cytotoxicity effect By comparison, small bi-specific diabodies without Fc have the advantages of rapid tissue penetration, high target retention and a distance between the two anti-gen-binding sites of the diabodies that is large enough

to bring two cells together for recruitment of immune cells [1,2,28]

Large-scale production of bi-specific diabodies in bacterial expression systems would be expected because

of their small size; however, the yield is typically only

a few mg per L in most cases [10,16,17] We previously proposed an in vitro refolding system to prepare

250

Fig 4 (A) Gel filtration of purified hEx3-Db after removal of HRV3C protease and the Fc fragment The elution volume is shown on the x axis, and the molecular mass (kDa) is shown above The eluted fractions containing the bi-specific diabody are indicated by the two-headed arrow (B) SDS–PAGE analysis under reducing conditions of the eluted fractions Molecular size markers are shown on the left (C) MALDI-TOF mass spectra of the tag-free hEx3-Db prepared from hEx3-Db–3C–Fc.

functional bi-specific diabodies from the insoluble

frac-tion, but solubilizing the expressed proteins from

insol-uble fraction required purification from the vast

amount of host-derived proteins, which forced us to

utilize an artificial tag [20,22,29] The immunogenicity

of the artificial peptide tag has not been determined,

and preparation of tag-free formats from insoluble

fractions may be difficult to achieve [16] For these reasons, a new preparation method for bi-specific dia-bodies was needed that required minimal artificial amino acid sequences and produced high yields

In the present study, we successfully purified tag-free hEx3-Db from the supernatant of transfected CHO

T-LAK

A

B

TFK-1

a b

Fluorescent intensity

100 E:T = 5 *

*

50

Affinity-tagged hEx3-Db Tag-free hEx3-Db from Fc fusion 0

0 1 10 100 1000

0 1 10 100 1000

(T LAK)

Concentration of BsAb (fmol·mL –1 )

100

101 102 103 104 100

101 102 103 104

Fig 5 (A) Flow cytometry analysis of tag-free hEx3-Db prepared

from hEx3-Db–3C–Fc Cells were incubated with NaCl ⁄ P i as a

nega-tive control (a) and with either OKT3 parental IgG (for T-LAK cells)

or 528 IgG (for TFK-1 cells), followed by staining with fluorescein

isothiocyanate-conjugated anti-mouse IgG as a positive control (b).

The shaded areas correspond to the fluorescence intensity

distribu-tions of the cells incubated with hEx3-Db Each mixture was

stained with rabbit anti-hEx3-Db serum followed by fluorescein

isothiocyanate-conjugated anti-rabbit IgG (B) Growth inhibition of

EGFR-positive TFK-1 cells by tag-free and affinity-tagged hEx3

diabodies Each bi-specific diabody and T-LAK cells (effectors, E)

were added to TFK-1 cells (T) at a ratio of 5 : 1 The tag-free

hEx3-Db inhibited growth significantly better (*P < 0.005) than the

affinity-tagged hEx3-Db did [21] Data are mean values ± SD and

are representative of at least three independent experiments with

similar results.

HRV3C protease site

A

B

C

Tag-free hEx3-scDb hEx3-scDb-3C-Fc

Tag-free hEx3-scDb hEx3-scDb-3C-Fc

1

hEx3-scDb-3C-Fc (monomer) Tag-free hEx3-scDb

Fc

1 2

175-hEx3-scDb-3C-Fc

Tag-free hOHh5L Tag-free h5HhOL

25 -16.5

-1 h 5 h 10 h

2 3 4

3 1 2 3 1 2 3

Fig 6 (A) Schematic illustration of the hEx3-scDb–3C–Fc fusion protein The HRV3C protease cleavage site used for preparation of tag-free hEx3-scDb is indicated (B) Reducing SDS–PAGE of each purification step for preparation of tag-free scDb from scDb–3C–Fc Lane 1, protein A chromatography-purified hEx3-scDb–3C–Fc; lane 2, after HRV3C protease digestion; lane 3, after removal of HRV3C protease by glutathione Sepharose 4B chroma-tography; lane 4, purified tag-free hEx3-scDb after removal of the

Fc region by protein A chromatography (C) Reducing SDS–PAGE

of hEx3-scDb–3C–Fc incubated with papain for 1, 5 and 10 h Lane

1, digested hEx3-scDb–3C–Fc; lane 2, flowthrough from protein

A chromatography; lane 3, eluted protein from protein A chroma-tography.

cells using cation-exchange and gel filtration

chroma-tography (Fig 2) However, the final yield of this

secreted tag-free hEx3-Db was approximately 1 mgÆL)1

culture We thus developed a novel method using

IgG-like BsAb and a restriction protease with high

specific-ity The fusion of Fc to diabodies resulted in high

productivity and enabled affinity purification using

protein A The homogeneous dimer structure and molecular weight of the tag-free hEx3-Db prepared from the Fc fusion format (hEx3-Db–3C–Fc) were confirmed by gel filtration and mass spectrometry, and the yield was approximately five times that of the directly secreted tag-free hEx3-Db (Figs 3 and 4) The specific binding affinity and bi-specificity of the tag-free hEx3-Db for T-LAK and TFK-1 cells were observed by flow cytometry (Fig 5A) Interestingly, the result of the MTS assay showed that growth inhi-bition by tag-free hEx3-Db from the Fc fusion was more intense than that by affinity-tagged hEx3-Db (Fig 5B) Although it is unclear why the tag-free dia-bodies prepared from the Fc fusion format had such high activity, imperceptible differences in purity and local structural perturbations that are dependent on the preparation method might have affected the activ-ity of the diabodies The reasons for this difference in activity are now under investigation Furthermore, we were able to reproduce our results with tag-free hEx3-scDb, which indicates the utility and applicability of our method for the preparation of tag-free small recombinant antibodies (Figs 6 and 7) The single-chain format has additional advantages: scDbs can be produced from a single expression vector and are expected to have improved stability in vivo because the two chains in the diabody are connected to each other via a linker [14,30]

In general, papain and pepsin have been used in the preparation of antibody fragments from IgG-like anti-bodies, and successful preparation of scFv from scFv–

Fc has also been reported [31] However, for hEx3 sin-gle-chain diabodies fused with Fc, papain digestion led

to undesired degradation (Fig 6C) Thus, the advanta-ges of using the designed protease recognition site were confirmed, especially in recombinant antibodies that included a number of artificial sequences

To date, several different small BsAb formats have been proposed to increase efficacy and availability, such as scDbs [30], taFv [9,32] and mini-bodies [11] Further, dimeric scDbs known as tanDbs, with biva-lency for each target antigen, can be produced by engi-neering the length and amino acid composition of middle linker of scDb [15] Here, we selected diabodies and scDb monomers with a 20-amino-acid middle lin-ker [(GGGGS)4] as small BsAbs, because they are one

of the simplest construction formats [20,22] Use of our preparation method for other BsAbs formats is currently in progress

We previously reported for BsAbs with affinity pep-tide tags that hEx3-scDb has comparable function to that of hEx3-Db in vitro [22] In this work, we have shown that tag-free formats behave quantitatively

T-LAK

A

B

TFK-1

a b

Fluorescent intensity

100 E:T = 5 *

*

50

Affinity-tagged hEx3-scDb Tag-free hEx3-scDb from Fc fusion 0

0 1 10 100 1000

0

(T-LAK)

Concentration of BsAb (fmol·mL –1 )

100

101 102 103 104 100

101 102 103 104

Fig 7 (A) Flow cytometric analysis of purified tag-free hEx3-scDb.

Cells were incubated with NaCl ⁄ P i as a negative control (a) and

with either OKT3 parental IgG (for T-LAK cells) or 528 IgG (for

TFK-1 cells), followed by staining with fluorescein

isothiocyanate-conju-gated anti-mouse IgG as a positive control (b) The shaded areas

correspond to the fluorescence intensity distributions of the cells

incubated with hEx3-Db Each mixture was stained with rabbit

anti-hEx3-Db serum followed by fluorescein

isothiocyanate-conjugated anti-rabbit IgG (B) Growth inhibition of EGFR-positive

TFK-1 cells by tag-free and affinity-tagged hEx3 single-chain

diabod-ies Each bi-specific diabody and T-LAK cells (effectors, E) were

added to TFK-1 cells (T) at a ratio of 5 : 1 The tag-free hEx3-scDb

inhibited growth significantly better (*P < 0.005) than the

affinity-tagged hEx3-scDb did [21] Data are mean values ± SD and are

representative of at least three independent experiments with

similar results.

similarly in in vitro cell growth inhibition studies

(Figs 5B and 7B) Therefore, regardless of the presence

or absence of an affinity tag, the activity of hEx3-Db

is comparable to that of hEx3-scDb Several reports

have demonstrated a higher stability of scDb than

other formats such as Db and taFv [14,33–35]

Although hEx3-Db and hEx3-scDb showed similar

activities in vitro, there is a possibility the hEx3-scDb

may exhibit a higher activity in vivo because of higher

stability Stability tests under physiological conditions

between hEx3-Db and hEx3-scDb are currently in

pro-gress

Issues such as rapid blood clearance and the

rela-tively low affinity caused by low molecular weight and

monovalent binding may limit the therapeutic

applica-tion of bi-specific diabodies [14] In such cases,

conver-sion into more effective formats such as tanDb may be

required The approach described here is also expected

to be applicable for convenient preparation of such

antibody fragments

In conclusion, we prepared tag-free bi-specific

diabodies in a mammalian expression system and

devel-oped a novel method using IgG-like antibodies and

protease digestion to prepare highly purified, tag-free

bi-specific diabodies Our method may allow

industrial-scale production of functional tag-free small biological

agents such as small recombinant antibodies

Experimental procedures

Preparation of secreted Ex3 diabodies

In accordance with the convention used in a previous

report, we describe the two hetero scFvs of hEx3-Db as

h5HhOL and hOHh5L [20] The gene constructs (Fig 1)

were inserted into pcDNA3.1⁄ Neo or pcDNA3.1 ⁄ Hygro

mammalian expression vectors (both from Invitrogen,

Groningen, Netherlands) The leader peptide sequences for

protein secretion were derived from the mouse OKT3 IgG

[36] The methods for expression and purification of the

affinity-tagged hEx3-Db and hEx3-scDb have been

described previously [21] For production of tag-free

hEx3-Db, CHO cells were co-transfected with pcDNA-h5HhOL

()) and pcDNA-hOHh5L()) (Fig 1), and cell clones

expressing tag-free hEx3-Db were established in the

pres-ence of neomycin (G418) and hygromycin as described

pre-viously [21] CHO clones that stably expressed tag-free

hEx3-Db were selected by screening for a growth inhibition

effect of each individual clone The established CHO clone

was cultured as previously described [27] The secreted

tag-free hEx3-Db was purified from pooled supernatants using

a 5 mL HiTrap SP XL column (GE Healthcare Bio-Science

Corp., Piscataway, NJ, USA) with a 5–250 mm gradient of

NaCl in 50 mm phosphate solution (pH 6.0)

Preparation of tag-free hEx3 diabodies from the

Fc fusion format

To construct the expression vector for preparing tag-free diabodies by using IgG-like BsAbs, we connected the hEx3 diabodies and the human IgG1 Fc region via a recognition site (LEVLFQGP) for human rhinovirus 3C (HRV3C) pro-tease CHO cells were co-transfected with equal amounts of the pcDNA-h5HhOL-3C-Fc and pcDNA-hOHh5L()) vec-tors (Fig 1), and grown in presence of neomycin (G418) and hygromycin as described previously [21] A CHO clone that stably expressed the hEx3-Db–3C–Fc fusion protein was selected in a manner similar to that for tag-free

hEx3-Db For tag-free hEx3-scDb, CHO cells were transfected with the pcDNA-hEx3-scDb–3C–Fc vector, and selection for a stably expressed clone was performed in the presence

of 500 lgÆmL)1of G418 (Nacalai Tesque, Kyoto, Japan) IgG-like BsAbs of hEx3–3C–Fc and hEx3-scDb–3C–Fc were first purified by affinity chromatography on a protein

A column (GE Healthcare) and then digested by HRV3C protease fused to GST (PreScission protease; GE Health-care) according to the protocol described by the manufac-turer The protease was removed using a glutathione Sepharose 4B column (GE Healthcare), and the flow-through was re-loaded onto the protein A column to remove the digested Fc and undigested hEx3-scDb–3C–Fc fusion protein The presence of the BsAbs in each stage of purification were confirmed by SDS–PAGE under reducing conditions

To illustrate the applicability of this novel method, papain digestion of hEx3-scDb–3C–Fc was performed by use of an ImmunoPure Fab preparation kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) The influence of papain digestion was confirmed by SDS–PAGE analysis under reducing conditions at 1, 5 and 10 h after digestion

Gel filtration chromatography

Gel filtration analysis with a Hiload Superdex 200 pg col-umn (26⁄ 60; GE Healthcare) was used to evaluate the structure of the bi-specific diabodies The column was equil-ibrated using NaCl⁄ Pi, and then 5 mL of purified recombi-nant antibodies was applied to the column at a flow rate of 2.5 mLÆmin)1

Mass spectrometry

Mass spectra were measured using a REFLEX III MALDI-TOF mass spectrometer (Bruker Daltonics Inc., Billerica, MA, USA) equipped with a nitrogen laser (337 nm) Sinapic acid was applied as a matrix, and was dissolved to saturation in water:acetonitrile (2 : 1 v⁄ v) con-taining 0.067% trifluoroacetic acid Sample solutions from each stage were mixed with the sinapic acid-saturated solution in a 1 : 1 v⁄ v ratio, and then 1 lL of the mixed

solution was loaded onto the sample target After

co-crys-tallization on the target, the crystals were washed twice

with 2 lL of water containing 0.1% trifluoroacetic acid to

remove residual salts Analysis was performed in positive

and linear modes with an accelerating voltage of 27 kV,

and 200 scans were averaged The spectra obtained were

calibrated externally using the [M + H+] ions from

two protein standards: cytochrome c from horse heart

(m⁄ z 12 360.08) and bovine trypsin (m ⁄ z 23 311.53) [37]

Preparation of T-LAK cells

Peripheral blood mononuclear cells were isolated by

den-sity-gradient centrifugation of heparin-containing blood

from healthy volunteers To induce proliferation of T-LAK

cells, peripheral blood mononuclear cells were cultured for

48 h at a density of 1· 106cells per mL in medium

supple-mented with 100 IUÆmL)1 of recombinant human IL-2

(kindly supplied by Shionogi Pharmaceutical Co., Osaka,

Japan) in a culture flask (A⁄ S Nunc, Roskilde, Denmark)

that had been pre-coated with OKT3 monoclonal antibody

(10 lgÆmL)1) Proliferated cells were then transferred to

another flask, and expanded for 2–3 weeks in a culture

medium containing 100 IUÆmL)1 IL-2, as reported

previ-ously [38]

Flow cytometric analyses

Test cells (1· 106

) were incubated on ice with 200 pmol of BsAb for 30 min After washing with NaCl⁄ Pi containing

0.1% NaN3, they were exposed for 30 min on ice to rabbit

anti-hEx3-Db serum (kindly supplied by

Immuno-Biologi-cal Laboratories Co Ltd, Gunma, Japan) as the second

antibody, and fluorescein isothiocyanate-conjugated

anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA,

USA) as the third antibody The stained cells were analyzed

by flow cytometry (FACSCalibur, Becton Dickinson, San

Jose, CA, USA) [20]

In vitro growth inhibition assay

In vitrogrowth inhibition of TFK-1 (human bile duct

carci-noma) was assayed using a

3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium

inner salt (MTS) assay kit (CellTiter 96 aqueous

non-radio-active cell proliferation assay; Promega, Madison, WI,

USA) as reported previously [39]

Acknowledgements

This work was supported by Grants-in-Aid for

Scien-tific Research from the Ministry of Education, Science,

Sports, and Culture of Japan (to R.A and I.K.) and

by grants from the New Energy and Industrial

Technology Development Organization of Japan Additional support was provided through the Program for Promotion of Fundamental Studies in Health Sci-ences of the National Institute of Biomedical Innova-tion

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