生物化学与生物物理学报

ISSN 058229879 ACTA BIOCHIMICA et BIOPHYSICA SIN ICA 2003 , 35 (6) : 503 - 510 CN 3121300/ Q

A Ne w Model of Trispecif ic Antibody Resulting the Cytotoxicity

Directed against Tumor Cells

SON G Li2Ping , CHEN G J u2Long , WAN G Xiang2Bin , ZHAN G Zhong ,

FAN G Min , ZHOU Zhi2 Yong , HUAN G Hua2Liang 3
( I nstit ute of Genetics and Develop mental Biology , t he Chi nese A cademy of Sciences , Beiji ng 100101 , Chi na )

Abstract 　　Bispecific antibodies (BsAb) wit h specificity to bot h t umor cells and CD3 molecule were
believed to be promising immunological tools for t he t herapy wit h minimal residual diseases by
activating cytotoxic T cells. However , wit hout costimulatory molecule CD28 , t he activated T cells
tended to apoptosis. In order to kill t umor cells more efficiently , a recombinant multif unctional single2
chain t rispecific antibody ( sc TsAb) , which contains anti2ovarian carcinoma ( OC) scFv , anti2CD3 scFv
and V H domain of anti2CD28 antibody , was const ructed and expressed in E. coli BL21 Star st rain.
The sc TsAb showed st rong binding avidities to membrane antigen of S K2OV23 cell , CD3 molecule on
J urkat cell , and recombinant CD28 antigen. It was f urt her demonst rated t hat t his sc TsAb could
activate perip heral blood T cells to elicit st rong cytotoxicity against S K2OV23 cells. This new type of
recombinant scFv antibody set up a new technological platform for T cells based immunot herapy
against cancer , especially wit h t he failure on M HC antigen presentation or absence of costimulating
signal.

Key words 　　t rispecific antibody ( TsAb) ; scFv ; ovarian carcinoma ( OC) ; CD3 ; CD28

　　According to t he immune surveillance T cells demanded a“second ”costimulatory signal in

hypot hesis , t umor antigens could be t he target s for
t umor immune dest ruction. However , down
regulation of anti2t umor immune response in t umor
patient s often leads to t umor immune escape [ 1 ] .
Considerable effort s had been made to induce efficient
immune reaction. One promising st rategy was
bispecific antibodies ( BsAb ) based approaches to
retarget effective cells to mediate cytotoxicity against
t umor cells[ 2 ,3 ] .
Two st rategies had been int roduced so far :
firstly , FcγR2bearing effective cells such as
macrop hages and N K cells were recruited toward
t umor cells using anti2FcγR × anti2 TAA ( t umor
associated antigen) antibodies[ 4 ] , secondly , anti2CD3
× anti2 TAA antibodies att racted effective T cells to
kill t umor cells in coordination wit h costimulating
signals[ 5 ] . The latter st rategy was more promising
because it aimed at t he activation of T lymp hocytes
t hat were highly specific effector in mediating
cytotoxicity. However , according to t he 22signal
model of T cell activation , t he effective activation of
Received : February 17 , 2003 　　Accepted : March 24 , 2003
This work was supported by a grant from t he National High Tech2
nology Research and Development Program of China ( 863 Program)
(No . 8632102209204201)
3 Corresponding aut hor : Tel , 86210264857285 ; Fax , 862102
64857285 ; e2mail , HL huang @genetics. ac. cn
addition to t riggering TCR2CD3 complex. Anti2CD3
antibody signals via CD3 simulated p hysiological
antigen2 TCR/ CD3 complex by M HC concomitant
antigen. Costimulatory signals , such as CD28/ B7 ,
CD154/ CD40 , CD2/ CD58 , and L FA21/ ICAM21 [ 6 ] ,
ensured t he optimal T cell activation. The best2
characterized second signal molecule was CD28. Anti2
CD28 antibody resembled CD28 ligands B721 and B72
2 , which could interact wit h CD28 to promote T2cell
survival and initiate T2cell clonal expansion and
differentiation [ 7 ] . By now , almost all t he t umor
immunot herapy wit h CD28 costimulation employed
two forms , eit her anti2 TAA × anti2CD3 bispecific
antibody in combination wit h anti2 TAA × anti2CD28
bispecific antibody [ 8 ,9 ] , or anti2 TAA × anti2CD3
bispecific antibody and anti2CD28 antibody
conjugate [ 10 ]
.
The develop ment of BsAb had undergone
chemical conjugation period , somatic f usion period
and genetic engineering period [ 11 ] . Trispecific
antibody ( TsAb ) had almost undergone t he same
route. Using t he sulf hydryl2specific cross2linker o2
p henylenedi2 maleimide ( o2PDM ) , a chemical
conjugated TsAb [ F ( ab’
)3] had been
[ 12 ,13 ]
developed . However , t he high cost , low
efficiency , laborious work and by2product s in
producing clinical TsAbs hindered t heir develop ment .
 504 　　　ACTA B IOCHIM ICA et B IOPH YSICA SIN ICA Vol. 35 , No. 6 　　　　　

Genetic engineering approaches offered a new cell lines ( human leukemia T cell lymp hoblast ) were
potential means. Recombinant disulfide stabilized kept by our laboratory. Anti2OC scFv , anti2OC ×
Fab2 ( scFv) 2 TsAbs were built by C2terminal f usion of anti2CD3 bispecific antibodies were prepared by
scFv molecules to bot h of t he VL2CL (L ) and V H2CH1 renat uring and purifying t he inclusion body expressed
( Fd) chains in Fab chains , which could be expressed in E. coli [. 16 ]
in mammalian cells[ 14 ,15 ] . 1. 2 　Construction of trispecif ic antibody
In t he present st udy , a recombinant scFv2based The backbone of sc TsAb was obtained via
TsAb was const ructed which combined activating and splicing by overlapping extention ( SO E ) 2PCR
costimulating f unctions wit hin t he single chain met hod wit h six oligonucletides P1 , P2 , P3 , RE1 ,
molecule. In t he sc TsAb , anti2OC antigen scFv , RE2 , RE3 ( Table 1) . The backbone of sc TsAb was
anti2CD3 ε chain scFv and V H domain of anti2CD28
antibody were linked by two specifically designed Table 1 　Primer sequences
interlinkers. The two interlinkers were HSA and Fc 　Name Sequence
2CCCAAGCTTATGAAATACCTATTGCCTACGGC 23′
f ragment s which were supposed to prolong t he half2 　 P1 5′
P2 2 GCCCAGGTGAAACTGCCGTGCCGTCCATGTA 2
5′
life of sc TsAb i n vi vo and perhaps append it s 　
CTCACACCACTGACGGTCTGCCGACCAAATTG2
complement2dependent cytotoxicity ( CDC ) and GAAGGTGGTGGTGGTTC23′
antibody2dependent cellular cytotoxicity ( ADCC ) 2CTGCTGGTTCGTTACACCAAGAAAGTACCCCA 2
5′
　 P3
f unctions. The sc TsAb could promote t he formation AGTGTCAACTCCAACTCCTGTAGAGGTCTCAGG2
of conjugate of t he ovarian carcinoma cell and CD3/ TGGTGGTGGTTCTCAT23′
TCR complex , juxtaposing CD28 molecule on t he 　 RE1 2CCGGAATTCCATATGAGAACCACCACCACC 23′
5′
surface of T cell. Cytolytic T cells t hat could lyse 　 RE2 2TTCTTGGTGTAACGAACCAGCAGCGCATTCTG2
5′
GAAAGAACCACCACCACCGGATCC CTCGAGAG2
t umor cells in M T T assay were generated when AACCACCACCACCTTCC23′
perip heral blood leukocytes were activated in t he 2 GGCACGGCAGTTTCACCTGGGCCATGGCTGG2
5′
　 RE3
presence of t umor cells and sc TsAb. These result s TTGGGCAGCGAGTAATAACAATCCAGCGGCTG 2
demonst rated t hat sc TsAb could delivery signal 1 and CCGTAGGCAATAGGTATT 23′
signal 2 in T cells and efficiently boost t umor lysis 　 TRI up 2CATCACCATCACCATCACCCGTGCCGTCCATG 2
5′
TACTCAC23′
activity of T cells. It put forward a new way for
2TTACGGGCAAGGTGGACAAGT23′
5′
CD32based
　 TRI down
powerf ul immunot herapy wit hout
simultaneous administ ration of ot her costimulatory
molecules. cloned into Hi n d III and Eco R I sites of p UC19 to
yield p U HM1 [ Fig. 1 ( A ) ] . The anti2OC scFv ×
1 　Materials and Met hods anti2CD3 scFv bispecific antibody coding f ragment ,
1. 1 　Antibodies and cell l ines cloned f rom plasmid pAL M2Fc/ BsAb [ 16 ] wit h X ho I
Anti2CD3 monoclonal antibody was purchased and B am HI double digestion , was inserted to
f rom Wuhan Instit ute of Biological Product s p U HM1 to get p U HM2. Finally t he f ragment
( Wuhan , China ) . RhCD28/ Fc Chimera , containing BsAb and backbone of t he sc TsAb was
recombinant human CD28 ext racellular domain f used shifted f rom p U HM2 to p TM F2CD28 ( containing V H
to human Ig G1 Fc were product s of R &D System domain of anti2CD28 antibody ) [ 17 ] by N de I and
Inc. ( U SA ) . Protein prestained markers were Hi n d III digestion , Finally t he expression vector of
purchased f rom N EB Biolabs. S K2OV23 and J urkat t rispecific single chain antibody was const ructed ,

Fig. 1 　Construction , structure and expression of scTsAb

(A) Intermediate plasmid for construction p TRI. pU HM1 , pAL M2Fc/ BsAb , p TMF2CD28 pU HM2 , p TRI. (B) Structure of psTRI.
 J un. , 2003 SON G Li2Ping et al . : A New Model of TsAb wit h Cytotoxicity against Tumor Cells
designated as p TR I [ Fig. 1 ( A) ] . In order to increase
t he solubility of TR I , t he t rispecific f ragment was
cloned to plasmid p TRFA [ 18 ] by using primer ‘ TR I
up ’and ‘ TR I down ’ ( Table 1 ) ( LA Taq DNA
polymerase ) . The orientation was confirmed by
sequencing and t his plasmid was named as psTR I
[ Fig. 1 (B) ] .
1. 3 　Conf irmation of the expression of trispecif ic
antibody
E. coli BL21 Star ( D E3 ) ( Invit rogen Co . ,
U SA) was t ransformed wit h p TR I or psTR I , t he
cells were cult ured to A 600 ≈ 0. 4 at 37 ° C in 1 L LB
media containing 50 mg/ L kanamycin. The cult ure
was t hen induced at 30 ° C for 4 h wit h 0. 4 mmol/ L
isopropyl2β2D2t hiogalactopyranoside ( IP T G ) . The
cells were t hen harvested by cent rif ugation at 4000 r/
min for 10 min at 4 ° C. The pellet s were resuspended
in 100 mL p hosp hate2buffered saline ( PBS ) , and
t hen lysed by sonication , and t hen cent rif uged at 10
000 r/ min for 30 min to get t he supernatant . The
proteins were f ractionated by 10 % SDS2PA GE and
t hen blotted to nit rocellulose membrane. Because t he
sc TsAb recombinant proteins , TR I and sTR I for
p TR I and psTR I respectively , contains t he c2myc tag
for detection , Western blotting was performed using
mouse anti2c2myc antibody ( 9 E10 ) ( SantaCruz ,
U SA) and horseradish peroxidase ( HRP) 2conjugated
goat anti2mouse Ig G antibody ( J ackson , U SA ) as
described [ 19 ] .
1. 4 　 Functional characterization of trispecif ic
antibody
1. 4. 1 　 T he anti gen bi n di ng assay 　　The antigen
binding activities of sTR I to J urkat cell membrane
antigen , S K2OV23 cell membrane antigen , and
rhCD28/ Fc antigen were st udied by enzyme2linked
immunosorbant assay ( EL ISA ) as described
before [ 19 ] . Briefly , EL ISA was performed wit h t he
antigen immobilized on 962well plates. The sTR I
protein bound wit h antigen was detected by mouse
anti2c2myc tag 9 E10 antibody , followed by HRP2
conjugated goat anti2mouse antibody , incubation wit h
peroxidase subst rate , and finally measurement of
absorbance at 490 nm. The supernatant of expression
product of empty vector was set as negative cont rol.
1. 4. 2 　B ri dge FA CS ( f l uorescence acti v ated cell
sorti ng ) assay 　 　 Bridge FACS analysis was
performed as described[ 20 ] . Briefly , J urkat cells was
preactivated wit h 80 ku/ L IL22 ( Instit ute of
Hematology & Hospital of Blood Diseases , China ) ,
50 μg/ L CD3 monoclonal antibody for t hree days ,
and t hen washed wit h RPM I21640 for two times.
J urkat cells were resuspended in RPM I21640 ( p H
6. 8) and mixed wit h F ITC ( fluorescein
isot hiocyanate , Sigma) at a final concent ration of 0. 5
mg/ L . S K2OV23 were resuspended in RPM I21640
( p H 7. 2) and mixed wit h TR ITC at a final
505
concent ration of 1. 5 mg/ L . Bot h t hese two kinds of
cells were incubated for 30 min at 37 ° C , 5 % CO2 ,
washed twice , and t hen resuspended in RPM I21640
( p H 7. 2 ) independently. The concent ration of
activated J urkat cells and S K2OV23 cells were
adjusted to 2 ×109 and 4 ×108 cells/ L , respectively. To
demonstrate cross2linking , equal volumes of F ITC
labeled activated J unkat cells and TR ITC labeled S K2
OV23 cells[ 21 ] were mixed at a ratio of 5∶
1 . sTR I lyses
supernatant ( 60 mg/ L total protein ) were added to
t he cells , mixed , and incubated at 37 ° C for 30 min ,
and t hen subjected to FACS analysis. Analysis were
per2formed using CellQuest g software ( Becton ,
Dickinsin) .
1. 4. 3 　 I n vit ro cytotox icity assay 　 　 Human
perip heral blood leukocyte cells ( PBL ) of healt hy
donors were isolated f rom perip heral blood by met hod
of Ficoll density gradient cent rif ugation , monocyte/
macrop hage f raction was depleted by glass adherence
met hod for 2 h at 37 ° C , 5 % CO2 . S K2OV23 target
cells were plated in 962well flat2bottom plate and
incubated wit h different dilutions of supernatant
containing sTR I overnight to prepare cell monolayer
bound wit h antibody. Freshly isolated effector cells
( PBL ) were added to t he monolayer of t umor cells at
appropriate ratios. Plates were incubated at 37 ° C for
72 h. Ot her procedures were carried out as
described [ 22 ] .
1. 4. 4 　M orphologic analysis 　　2 ×102 S K2OV23
target cells were incubated in 62well flat2bottom plate
overnight. Effector cells ( PBL s ) were pret reated
wit h sTR I overnight , washed two times , and t hen
added into t he S K2OV23 plate wit h E/ T ratio of 20 :
1. The cells were co2incubated wit h appropriate sTR I
supernatant for 2 h , 4 h and 10 h , respectively. The
morp hology of t he cells was investigated.
2 　Results
2. 1 　Construction of trispecif ic antibody scTsAb
To express sc TsAb t hat was composed of partial
anti2CD28 ( AF336117 ) , anti2CD3 and anti2OC
antibodies , it s expression vectors p TR I and psTR I
were const ructed as shown in Fig. 1 ( A , B ) . The
universally acknowledged flexible glycine2rich
( )
sequence G4 S 3 was inserted between each V H and
VL to rest rict int ra2chain pairing. In order to provide
sufficient flexibility to allow each molecule to bind
t heir respective receptor simultaneously , interlinker2
Fc ( NST YRVVSVL TVL HQDWL N GKE YKC K )
and interlinker2HSA ( FQNALL V R YT KKV PQ2
VSTP TPV EVS) were alsoint roduced. c2myc tag was
added for immunodetection. In psTR I [ Fig. 1 ( B ) ] ,
t he sc TsAb was co2expressed wit h FkpA under t he
cont rol of t he same T 7 promoter and l ac operator.
To avoid wrong assembling of each antibody in sTR I ,
 506 　　　ACTA B IOCHIM ICA et B IOPH YSICA SIN ICA Vol. 35 , No. 6 　　　　　

t he orientation of sTR I was specifically designed as
( anti2OCV H ) 2 ( anti2OCVL ) 2interlinker2 ( anti2
CD3VL ) 2 ( anti2CD3V H ) 2interlinker2anti2CD28V H )
[ Fig. 1 (B) ] .
2. 2 　Conf irmation of the expression of trispecifc
antibody
Fig. 2 　SDS2PAGE ( left) and Western blotting ( right) of
pTRI and psTRI
The molecular weight was marked at right . The arrows indicated t he
band of desired TRI and psTRI ( about 84 kD) . The loading order in
SDS2PA GE and Western blotting is : 1 and 2 , supernatant ( S) and
pellet ( P) of E. coli cells harboring p TMF ; 3 and 4 , supernatant and
pellet of E. coli cells harboring p TRI ; 5 , molecular weight marker (in
Western , prestained marker) ; 6 and 7 , supernatant and pellet of E.
coli cells harboring psTRI ; 8 and 9 , supernatant and pellet of E. coli
cells harboring p TRFA vector.
　 　 The expression result was detected by SDS2
PA GE and Western blotting ( Fig. 2 ) . sc TsAb could
be observed wit h t he expected molecular weight of 84
kD. TR I protein accounted for about 4 % of t he total
bacteria protein and 37. 67 % was in supernatant . In
order to obtain large amount s of soluble sc TsAb ,
FkpA2based co2expression vector psTR I was
const ructed. The expression profiles and Western
blotting result s suggested t he chaperone molecule
FkpA could improve t he solubility of t he sc TsAb
greatly ( Fig. 2 ) . The sTR I protein accounted for
about 9. 81 % of t he total bacteria protein and t he
soluble part of sTR I reached it s 62. 63 %. Due to it s
high solubility , sTR I was used in f urt her
experiment s.
2. 3 　Functional characterization of trispecif ic
antibody
2. 3. 1 　B i n di n g p roperties of s T R I 　　To confirm
t he antigen binding properties of each component in
sc TsAb , EL ISA was performed as mentioned in
materials and met hod. The result s demonst rated t hat
sTR I had relatively high binding affinities wit h
J urkat membrane CD3 antigen , CD28 antigen , and
S K2OV23 membrane antigen , respectively , while t he
binding activities of cont rol were relatively low ( Fig.
3) . The result s implied t he ot her ingredient s in t he
supernatant had little effect on t he binding of sTR I to
antigens. Besides , t he antibody dilution ratio among
1∶5 to 1 ∶
80 ( about 50 - 1. 25 mg/ L ) was optimal in
EL ISA assays and t he cytotoxicity assay described
below got t he similar result s.
　　FACS analysis demonst rated t hat sTR I could
p hysically cross2link J urkat cells and ovarian
carcinoma S K2OV23 cells ( Fig. 4 ) . CD3 + and
CD28 + J urkat cells were prelabled wit h F ITC , and

Fig. 3 　EL ISA result of trispecif ic antibody sTRI

(A) sTRI2anti2CD28 EL ISA assay. 100 μL of CD28 (1 mg/ L) antigen (rhCD28/ FcChimera) was coated on EL ISA plate overnight , incubated wit h
different dilution of supernatant of sTRI and control , respectively. sTRI CD28 stands for lysed supernatant of induced E. coli cells harboring psTRI ;
Control CD28 , stands for supernatant of induced E. coli cells harboring plasmid p TRFA. (B) sTRI2anti2CD3 and sTRI2anti2SK2OV23 EL ISA assay.
J urkat and SK2OV23 cell membrane antigens were prepared by sonication for 80 s and t hen centrifugated for 10 min at 12 000 r/ min. 100 μL of J urkat
and SK2OV23 membrane antigen (100 mg/ L) were coated on EL ISA plate overnight at 4 ° C. Control SKOV , SK2OV23 cell membrane antigen was
coated and t he expression product of p TRFA was used as t he first antibody ; sTRI SKOV , SK2OV23 cell member antigen was coated and t he expression
product of psTRI was used as t he first antibody ; Control J urk , J urkat cell membrane antigen was coated and t he expression product of p TRFA was used
as t he first antibody ; sTRI J urk , J urkat cell membrane antigen was coated and t he expression product of psTRI was used as t he first antibody. The
original lysed supernatant concentration was 250 mg/ L .
 J un. , 2003 SON G Li2Ping et al . : A New Model of TsAb wit h Cytotoxicity against Tumor Cells 507

S K2OV23 cells wit h TR ITC , respectively. In a
representative experiment , only in t he presence of
sTR I , double2positive colored cell population
increased greatly in t he FACS analysis [ top right
quadrant in Fig. 4 ( A ) right ] , which implied t hat
sc TsAb could mediate J urkat cells ’ binding wit h
t umor cells , whereas in t he cont rol experiment wit h
antibody f ree or vector , t he expression supernatant
could not . The percentage of S K2OV23 t hat bound to
J urkat cells in t he presence of sTR I was
calculated wit h CellQuest software to be 92. 14 %

Fig. 4 　Bridge FACS analysis

Bridge FACS analysis of FITC2labled J urkat cells and TRITC2labled SK2OV23 cells in t he presence of sTRI lysed supernatant (12 mg/ L) . (A) The
cells in t he upright quadrant represented t he cross2linking of t he two different types of cells. (B) SK2OV23 cells t hat acquired FITC fluorescence were
a result of binding to T cells. The marker region ( M1) stands for SK2OV23 cells t hat linked wit h FITC labeled T cells. FL1 represented FITC ; FL2 ,
represented TRITC ; Auto , t he supernatant of vector expression production was set as negative control.

compared wit h 41. 43 % , 49. 82 % when antibody
f ree and vector supernatant were used [ Fig. 4 (B) ] .
2. 3. 2 　 T he cytotoxcit y assay of s T R I 　　To f urt her
examine t he utility of t his new t rispecific antibody for
application in t umor t herapy , M T T based
cytotoxicity assay was performed to evaluate t he
ability of effector T cells to dest roy S K2OV23 t umor
cells in t he presence of sc TsAb. Different f rom t he
BsAb M T T cytotoxicity assay , t he PBL s used in
cytotoxicity assay had not been pre2t reated wit h low
dose of IL22 or anti2CD3 monoclonal antibody. The
cytotoxicity result s [ Fig. 5 ( A ) ] showed t hat t he
sc TsAb has an effective cytotoxicity to t umor cells.
sTR I f unctioned best at t he concent ration of about 12
mg/ L total bacterial protein and wit h t he increase of
dilution ratio , t he killing ratio decreased gradually.
So t his concent ration was used in f urt her
experiment s. To confirm t he cytotoxicity against
t umor cells effectively , a series of cont rols have been
set : BSCD28 [ anti2OC × anti2CD3 bispecific
antibody ( 2. 1 mg/ L ) [ 16 ] + anti2CD28 antibody ( 40
μg/ L ) ] , BS [ anti2OC2CD3 bispecific antibody ( 2. 1
mg/ L ) ] , CD3 ( 2. 8 mg/ L ) [ Fig. 5 (B) ] . The
induced lysate supernatant of E. coli cells carrying
p TRFA ( about 12 mg/ L total bacterial protein) was
used as negative cont rol. The supernatant of sTR I
( 12 mg/ L total bacterial protein ) had almost t he
same cytotoxicity behavior as positive cont rol
BSCD28 , which is higher t han BsAb and CD3 as
expected. In Fig. 5 ( C) , t he supernatant of sTR I ( 60
mg/ L total bacterial protein ) lysed t he t umor cell
more efficiently t han OCCD3CD28 [ anti2OC scFv ( 4
mg/ L ) , anti2CD3 monoclonal antibody ( 20 μg/ L ) +
anti2CD28 monoclonal antibody ( 40 μg/ L ) ] , OCCD3
[ anti2OC scFv ( 4 mg/ L ) , anti2CD3 monoclonal
antibody ( 2. 8 mg/ L ) ] . The result s showed t he
 508 　　　ACTA B IOCHIM ICA et B IOPH YSICA SIN ICA Vol. 35 , No. 6 　　　　　

sc TsAb has st rong ability to redirect T cells to kill
t umor cells. Cytotoxicity experiment revealed a
correlationship between an increase in binding ability
for S K2OV23 and t he ability of t he sc TsAb to
argument lysis of S K2OV23 cells [ Fig. 3 ( B ) , Fig. 5
( A ) ] . Wit h t he decrease of t he binding ability to
ovarian carcinoma , t he cytotoxicity mediated by
sc TsAb decreased. This finding suggested t hat wit h
higher affinity to t umor cells , t he sc TsAb had more
time to adhere to t umor cells and created more
chances for t he T cells to bind and kill t umor cells.
　　Most of t he t umor cells were dest royed wit hin
24 h after exposure to effective cells in t he presence of
sc TsAb. At E / T ratio of 25∶1 , after coincubation wit h

Fig. 5 　sTRI2mediated cytotoxicity in vit ro

(A) Different concentration of sTRI2mediated cell deat h of SK2OV23 cells by activated human PBL as determined by M TT assay. Original , 12 mg/ L
total bacterial protein ; Dilution10 , 120 mg/ L total bacterial protein ; Dilution100 , 1. 2 mg/ L total bacterial protein ; Dilution1000 , 0. 12 mg/ L total
bacterial protein. (B) and ( C) , sTRI2mediated cytotoxicity wit h different control. sTRI , stands for anti2CD3 ×anti2CD28 ×anti2OC trispecific
antibody ; Vector control , t he lysed supernatant of E. coli cells carrying plasmid p TRFA ;BSCD28 , anti2OC × anti2CD3 bispecific antibody + anti2
CD28 antibody ; BS , anti2OC ×anti2CD3 bispecific antibody ; CD3 , anti2CD3 antibody ; OCCD3CD28 , anti2OC scFv + anti2CD3 + anti2CD28
monoclonal antibody ; OCCD3 , anti2OC scFv + anti2CD3 monoclonal antibody. (D) Morphology change of SK2OV23 and J urkat cells after treated
wit h sTRI for 2 h , 4 h , and 10 h (40 ×) . The lateral arrows indicated t he PBL s , t he vertical arrows indicated t he SK2OV23 cells.

sTR I for 2 h , S K2OV23 cells were adhered wit h
effector T cells forming t he wreat h ; when
coincubated for 4 h , T cells started to lyse S K2OV23
cells , t he profile of S K2OV23 was blurred ; after 10 h
coincubation , most of t he t umor cells collapsed [ Fig.
5 ( D ) ] . Finally , t he S K2OV23 cells vanished in t he
medium , leaving t he PBL s alone.
3 　Discussion
T2cell based anti2t umor immunot herapy requires
f ull and efficient T2cell activation [ 23 ] . It is generally
accepted t hat T2cell activation requires two distinct
signals. Signal t hrough t he T2cell receptor alone can
lead to anergy or activation2induced cell deat h
( A ICD) [ 24 ] . Costimulation wit h CD28 decreases t he
probability of lymp hocytes apoptosis and prolongs t he
lifespan of lymp hocytes i n vit ro [ 25 ] . Ligation of
CD28 by agonistic antibody has been shown to
prevent A ICD of native T cells during primary
stimulation [ 26 ] .
We describe t he antit umor properties of a new
class of t rispecific antibody. To our knowledge , it is
t he first report t hat a single2chain t rispecific antibody
can activate t he two signals on T cells simultaneously.
An anti2OC , anti2CD3 ε chain and anti2CD28
composed sc TsAb was const ructed wit h t he intent of
recruiting T cells to t he ovarian t umor cells , where
t hey were activated to dest roy t he ovarian carcinoma
cells [ Fig. 5 ( D ) ] . The EL ISA result and FACS
bridge analysis showed t he sc TsAb could recognize
and bind specifically wit h ovarian carcinoma member
antigen , CD3 complex and CD28 molecule on T2
lymp hocytes. In t his const ruction , anti2CD3 ε chain
scFv act s wit h TCR2CD3 complex , mimics t he
 J un. , 2003 SON G Li2Ping et al . : A New Model of TsAb wit h Cytotoxicity against Tumor Cells
stimulation of T cells as an antigen ( signal 1 ) , V H
domain of anti2CD28 antibody simulates t he B7
molecule expressed on antigen processing cell ( APC) ,
interact wit h CD28 on T cells ( signal 2 ) . The
sc TsAb set up a bridge between ovarian carcinoma
cells and T cells and created a microenvironment for
t he T cell activation and T cell killing effect ( Fig. 6) .
The cytotoxicity mediated by sTR I revealed t hat t he
sc TsAb f unctions as scBsAb plus CD28 monoclonal
antibody [ Fig. 5 ( B ) ] and was better t han anti2OC
scFv plus anti2CD3 , anti2CD28 monoclonal antibody
[ Fig. 5 ( C) ] .
　　To ensure properly and f unctionally refolding of
each antibody wit hin sc TsAb , partial sequences of
HSA and Fc were int roduced as interlinkers. EL ISA
and FACS analysis demonst rated t hat each antibody
wit hin sc TsAb refolded correctly. Poznansky et
[ 27 ]
al . have demonst rated t hat t he stability of HSA
conjugated protein in animal body increased 20 - 40
times. In t he animal test of recombinant single chain
BsAb in our lab , t he half2life of t he BsAb containing
HSA linker elonged to t hree times compared wit h t he
BsAb wit hout t he HSA ( unpublished data ) . To
enhance t he solubility of t he sc TsAb expression in E.
coli , FkpA , a heat shock periplasmic peptidyl2prolyl
cis/ t rans isomerase ( PPIase) was int roduced , which
could suppress t he formation of inclusion
[ 28 ,29 ]
bodies . The result s proved t hat it could
facilitate t he soluble expression of sc TsAb greatly
when it was co2expressed wit h target protein ( Fig.
2) .
Fig. 6 　Cartoon model for the role of recombinant scTsAb
(A) In t he native condition , wit h t he costimulation of CD28 , t he TCR
expressed on CTL cell recognized t he antigen peptide2MHC on tumor
cell and elicited a signal cascade to active T cell to attack tumor cell.
However t his pat hway might be disrupted by t he deficiency of tumor
antigen expression , MHC I molecule , B7 co2stimulatory signal and
antigen presentation ability of patients. (B) In t he modified model , TRI
contained anti2tumor arm to bind to tumor cells , CD3 and CD28 arms to
‘pull’and active T cells to attack tumor cells. At t he same time , t he
HSA fragment on TRI molecule could prolong t he half2life2time of sTRI
in sera and t he Fc fragment on t he TCR might trigger t he FcγR + cell to
kill tumor cells too .
509
　　In summary , t he multif unctional sc TsAb has
many advantages. ( 1 ) It can bind to cytotoxicity T
cells and t umor cells simultaneously and t hen active T
cell to kill t umor cells in a highly t umor specific
manner. ( 2 ) It can initiate and sustain high level of
cytotoxicity reactivity t hrough CD3 and CD28 “two
signal”t riggering. ( 3 ) It has optimal intermediate
molecule size ( about 84 kD) which is small enough to
penet rate t umors and large enough to sustain in
circulation for an appropriate period of time. ( 4) It is
genetically engineered to minimize t he human anti2
antibody ( mouse Ig ) ( HAMA ) reaction which has
hindered t he clinical application of many mouse
monoclonal antibody [ 30 ] and easy of manufact uring. It
would set up a new approach on t he way to successf ul
elimination of disseminated t umor cells.
References
1 　GilBoa E. How tumors escape immune destruction and what we can
do about it . Cancer I m m unol I m m unot her , 1999 , 48 (7) : 382 -
385
2 　Zhu Z , Lewis GD , Carter P. Engineering high affinity humanized
anti2p185 HER2/ anti2CD3 bispecific F (ab’ ) 2 for efficient lysis of
p185 HER2 overexpressing tumor cells. I nt J Cancer , 1995 , 62
(3) : 319 - 324
3 　Kroesen BJ , ter Haar A , Spakman H , Willemse P , Sleijfer D T , de
Vries EG , Mulder N H et al . Local antitumor treat ment in
carcinoma patients wit h bispecific2monoclonal2antibody2redirected T
cells. Cancer I m m unol I m m unot her , 1993 , 37 (6) : 400 - 407
4 　Valerius T , Stockmeyer B , van Spriel AB , Graziano RF , van den
Herik2Oudijk I , Repp R , Deo YM et al . FcalphaRI ( CD89) as a
novel trigger molecule for bispecific antibody t herapy. B lood ,
1997 , 90 (11) : 4485 - 4492
5 　Segal DM , Weiner GJ , Weiner L M. Introduction : Bispecific
antibodies. J I m m unol Met hods , 2001 , 248 (122) : 1 - 6
6 　Salomon B , Bluestone J A. Complexities of CD28/ B7 : CTLA24
costimulatory pat hways in autoimmunity and transplantation.
A nnu Rev I m m unol , 2001 , 19 : 225 - 252
7 　Sharpe AH , Freeman GJ . The B72CD28 superfamily. N at Rev
I m m unol , 2002 , 2 (2) : 116 - 126
8 　Holliger P , Manzke O , Span M , Hawkins R , Fleischmann B , liu
Q H , Wolf J et al . Carcinoembryonic antigen ( CEA) 2specific T2
cell activation in colon carcinoma induced by anti2CD3 × anti2CEA
bispecific diabodies and B7 × anti2CEA bispecific fusion proteins.
Cancer Res , 1999 , 59 (12) : 2909 - 2916
9 　Grosse2Hovest L , Brandl M , Dohlsten M , Kalland T , Wilmanns
W , J ung G. Tumor2growt h inhibition wit h bispecific antibody
fragments in a syngeneic mouse melanoma model : The role of
targeted T2cell co2stimulation via CD28. I nt J Cancer , 1999 , 80
(1) : 138 - 144
10 　Daniel PT , Kroidl A , Kopp J , Sturm I , Moldenhauer G , Dorken
B , Pezzutto A. Immunot herapy of B2cell lymphoma wit h CD3x19
bispecific antibodies : Costimulation via CD28 prevents “ veto ”
apoptosis of antibody2targeted cytotoxic T cells. B lood , 1998 , 92
(12) : 4750 - 4757
11 　Kriangkum J , Xu B , Nagata L P , Fulton RE , Suresh MR.
Bispecific and bifunctional single chain recombinant antibodies.
Biomol Eng , 2001 , 18 : 31 - 40
12 　Tutt A , Stevenson GT , Glennie MJ . Trispecific F ( ab ’ ) 3
derivatives t hat use cooperative signaling via t he TCR/ CD3
complex and CD2 to activate and redirect resting cytotoxic T cells.
J I m m unol , 1991 , 147 (1) : 60 - 69
13 　Wong WM , Vakis SA , Ayre KR , Ellwood CN , Howell WM ,
Tutt AL , Cawley MI et al . Rheumatoid art hritis T cells produce
 510 　　　ACTA B IOCHIM ICA et B IOPH YSICA SIN ICA
Th1 cytokines in response to stimulation wit h a novel trispecific
antibody directed against CD2 , CD3 , and CD28. Scand J
R heu m atol , 2000 , 29 (5) : 282 - 287
14 　Schoonjans R , Willems A , Schoonooghe S , Leoen J , Grooten J ,
Mertens N. A new model for intermediate molecular weight
recombinant bispecific and trispecific antibodies by efficient
heterodimerization of single chain variable domains t hrough fusion
to a Fab2chain. Biomol Eng , 2001 , 17 (6) : 193 - 202
15 　Schoonjans R , Willems A , Schoonooghe S , Fiers W , Grooten J ,
Mertens N. Fab chains as an efficient heterodimerization scaffold
for t he production of recombinant bispecific and trispecific antibody
derivatives. J I m m unol , 2000 , 165 (12) : 7050 - 7057
16 　Fang M , Zhao R , Li H , Jiang X , Yin CC , Lin Q , Huang HL .
Construction and expression of an anti2human ovarian carcinoma ×
anti2human CD3 bispecific single2chain antibody and its refolding
studies. Hi gh Technology L etters , 2002 , 12 (143) : 47 - 50
( 引自 : 高技术通讯)
17 　Cheng JL , Wang XB , Zhang Z , Liu J , Yao XS , Huang HL . A
met hod for construction reshaping single2domain antibody. A cta
Genetics S i nica , 2002 , 29 (3) : 189 - 195
( 引自 : 遗传学报)
18 　Zhang Z , Song L P , Fang M , Wang F , He D , Zhao R , Huang HL
et al . Overexpression of bacterial peptidyl2prolylisomerase f kpA
markedly improves soluble and functional production of engineering
antibodies in Escherichia coli . Biotechniques , 2003 , Submited
19 　Zhang Z , Li ZH , Wang F , Fang M , Yin CC , Zhou ZY , Lin Q et
al . Overexpression of DsbC and Dsb G markedly improves soluble
and functional expression of single2chain Fv antibodies in
Escherichia coli . Protei n Ex pr Purif , 2002 , 26 : 218 - 228
20 　Kriangkum J , Xu B , Gervais C , Paquette D , Jacobs FA , Martin
L , Suresh MR. Development and characterization of a bispecific
single2chain antibody directed against T cells and ovarian
carcinoma. Hybri dom a , 2000 , 19 (1) : 33 - 41
21 　Kreutz FT , Xu DZ , Suresh MR. A new met hod to generate
quadromas by electrofusion and FACS sorting. Hybri dom a , 1998 ,
一种新型的重组单链三特异抗体的构建与表达
宋利萍 　程巨龙 　王祥斌 　张 　众 　方 　敏 　周志勇 　黄华　 3
( 中国科学院遗传与发育生物学研究所 , 北京 100101 )
摘要 　　双特异抗体由于其缺乏共刺激信号 , 激活的 T 细胞往往会导致凋亡 。为了更有效地杀伤肿瘤细胞 , 构建了抗卵巢
癌单链 ×抗 CD3 单链 ×抗 CD28 单域重组三特异抗体的表达载体 。利用大肠杆菌中的分子伴侣 FkpA 与三特异抗体共表达 ,
提高了三特异抗体的在 BL21 Star 菌中的可溶性原核表达 。EL ISA 结果显示 , 可溶性的重组单链三特异抗体 ( sTRI) 与 SK2
OV23 细胞的膜抗原 , J urkat 细胞上的 CD3 分子以及重组 CD28 抗原均有较强的结合活性 。桥连试验证明该抗体能同时与
SK2OV23 细胞和 J urkat 细胞结合 。体外杀伤试验表明该抗体能激活外周血 T 细胞来杀伤肿瘤细胞 。形态学观察也进一步说
明该抗体具有良好的结合活性以及体外杀伤活性 。这种新型的重组单链三特异抗体为基于 T 细胞的癌症免疫治疗建立了一
个新的技术平台 。
关键词 　　三特异抗体 ; 重组单链抗体 ; 卵巢癌 ; CD28 ; CD3
收稿日期 : 2003202217 　　接受日期 : 2003203224
国家高技术研究发展计划 (863 计划) 资助项目 (No . 8632102209204201)
3 联系人 : Tel , 010264857285 ; Fax , 010264857285 ; e2mail , HL huang @genetics. ac. cn
Vol. 35 , No. 6 　　　　　
17 : 267 - 273
22 　Heo DS , Park J G , Hata K , Day R , Herberman RB , Whiteside
TL . Evaluation of tetrazolium2based semiautomatic colorimetric
assay for measurement of human antitumor cytotoxicity. Cancer
Res , 1990 , 50 (12) : 3681 - 3690
23 　Chambers CA. The expanding world of co2stimulation : The two2
signal model revisited. T rends I m m unol , 2001 , 22 (4) : 217 -
223
24 　Daniel PT , Kroidl A , Cayeux S , Bargou R , Blankenstein T ,
Dorken B. Costimulatory signals t hrough B7. 1/ CD28 prevent T
cell apoptosis during target cell lysis. J I m m unol , 1997 , 159 (8) :
3808 - 3815
25 　Levine BL , Bernstein WB , Connors M , Craighead N , Lindsten T ,
Thompson CB , J une CH. Effects of CD28 costimulation on
long2term proliferation of CD4 + T cells in t he absence of exogenous
feeder cells. J I m m unol , 1997 , 159 (12) : 5921 - 5930
26 　Boise L H , Minn AJ , Noel PJ , J une CH , Accavitti MA , Lindsten
T , Thompson CB. CD28 costimulation can promote T cell survival
by enhancing t he expression of Bcl2XL . I m m unity , 1995 , 3 (1) :
87 - 98
27 　Poznansky MJ , Halford J , Taylor D. Growt h hormone2albumin
conjugates. Reduced renal toxicity and altered plasma clearance.
FEB S . L ett , 1988 , 239 (1) : 18 - 22
28 　Arie J P , Sassoon N , Betton J M. Chaperone function of FkpA , a
heat shock prolyl isomerase , in t he periplasm of Escherichia coli .
Mol Microbiol , 2001 , 39 (1) : 199 - 210
29 　Bot hmann H , Pluckt hun A. The periplasmic Escherichia coli
peptidylprolyl cis , t rans2isomerase FkpA. I. Increased functional
expression of antibody fragments wit h and wit hout cis2prolines. J
Biol Chem , 2000 , 275 (22) : 17100 - 17105
30 　Yang G , Ran YL , Sun L X , Liu J , Yu L , Yang ZH. High
expression in CHO cells and activity of an anti2P185 erbB2 mouse/
human chimeric antibody. A cta Biochi m Biophys S i n , 2001 , 33
(1) : 87 - 92
( 引自 : 生物化学与生物物理学报)