Beruflich Dokumente
Kultur Dokumente
ISSN 058229879 ACTA BIOCHIMICA et BIOPHYSICA SIN ICA 2003 , 35 (6) : 503 - 510 CN 3121300/ Q
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
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 ,
designated as p TR I [ Fig. 1 ( A) ] . In order to increase concent ration of 1. 5 mg/ L . Bot h t hese two kinds of
t he solubility of TR I , t he t rispecific f ragment was cells were incubated for 30 min at 37 ° C , 5 % CO2 ,
cloned to plasmid p TRFA [ 18 ] by using primer ‘ TR I washed twice , and t hen resuspended in RPM I21640
up ’and ‘ TR I down ’ ( Table 1 ) ( LA Taq DNA ( p H 7. 2 ) independently. The concent ration of
polymerase ) . The orientation was confirmed by activated J urkat cells and S K2OV23 cells were
sequencing and t his plasmid was named as psTR I adjusted to 2 ×109 and 4 ×108 cells/ L , respectively. To
[ Fig. 1 (B) ] . demonstrate cross2linking , equal volumes of F ITC
1. 3 Conf irmation of the expression of trispecif ic labeled activated J unkat cells and TR ITC labeled S K2
antibody OV23 cells[ 21 ] were mixed at a ratio of 5∶
1 . sTR I lyses
E. coli BL21 Star ( D E3 ) ( Invit rogen Co . , supernatant ( 60 mg/ L total protein ) were added to
U SA) was t ransformed wit h p TR I or psTR I , t he t he cells , mixed , and incubated at 37 ° C for 30 min ,
cells were cult ured to A 600 ≈ 0. 4 at 37 ° C in 1 L LB and t hen subjected to FACS analysis. Analysis were
media containing 50 mg/ L kanamycin. The cult ure per2formed using CellQuest g software ( Becton ,
was t hen induced at 30 ° C for 4 h wit h 0. 4 mmol/ L Dickinsin) .
isopropyl2β2D2t hiogalactopyranoside ( IP T G ) . The 1. 4. 3 I n vit ro cytotox icity assay Human
cells were t hen harvested by cent rif ugation at 4000 r/ perip heral blood leukocyte cells ( PBL ) of healt hy
min for 10 min at 4 ° C. The pellet s were resuspended donors were isolated f rom perip heral blood by met hod
in 100 mL p hosp hate2buffered saline ( PBS ) , and of Ficoll density gradient cent rif ugation , monocyte/
t hen lysed by sonication , and t hen cent rif uged at 10 macrop hage f raction was depleted by glass adherence
000 r/ min for 30 min to get t he supernatant . The met hod for 2 h at 37 ° C , 5 % CO2 . S K2OV23 target
proteins were f ractionated by 10 % SDS2PA GE and cells were plated in 962well flat2bottom plate and
t hen blotted to nit rocellulose membrane. Because t he incubated wit h different dilutions of supernatant
sc TsAb recombinant proteins , TR I and sTR I for containing sTR I overnight to prepare cell monolayer
p TR I and psTR I respectively , contains t he c2myc tag bound wit h antibody. Freshly isolated effector cells
for detection , Western blotting was performed using ( PBL ) were added to t he monolayer of t umor cells at
mouse anti2c2myc antibody ( 9 E10 ) ( SantaCruz , appropriate ratios. Plates were incubated at 37 ° C for
U SA) and horseradish peroxidase ( HRP) 2conjugated 72 h. Ot her procedures were carried out as
goat anti2mouse Ig G antibody ( J ackson , U SA ) as described [ 22 ] .
described [ 19 ] . 1. 4. 4 M orphologic analysis 2 ×102 S K2OV23
1. 4 Functional characterization of trispecif ic target cells were incubated in 62well flat2bottom plate
antibody overnight. Effector cells ( PBL s ) were pret reated
1. 4. 1 T he anti gen bi n di ng assay The antigen wit h sTR I overnight , washed two times , and t hen
binding activities of sTR I to J urkat cell membrane added into t he S K2OV23 plate wit h E/ T ratio of 20 :
antigen , S K2OV23 cell membrane antigen , and 1. The cells were co2incubated wit h appropriate sTR I
rhCD28/ Fc antigen were st udied by enzyme2linked supernatant for 2 h , 4 h and 10 h , respectively. The
immunosorbant assay ( EL ISA ) as described morp hology of t he cells was investigated.
before [ 19 ] . Briefly , EL ISA was performed wit h t he
antigen immobilized on 962well plates. The sTR I
2 Results
protein bound wit h antigen was detected by mouse
2. 1 Construction of trispecif ic antibody scTsAb
anti2c2myc tag 9 E10 antibody , followed by HRP2
To express sc TsAb t hat was composed of partial
conjugated goat anti2mouse antibody , incubation wit h
anti2CD28 ( AF336117 ) , anti2CD3 and anti2OC
peroxidase subst rate , and finally measurement of
antibodies , it s expression vectors p TR I and psTR I
absorbance at 490 nm. The supernatant of expression
product of empty vector was set as negative cont rol. were const ructed as shown in Fig. 1 ( A , B ) . The
1. 4. 2 B ri dge FA CS ( f l uorescence acti v ated cell universally acknowledged flexible glycine2rich
sorti ng ) assay Bridge FACS analysis was
( )
sequence G4 S 3 was inserted between each V H and
performed as described[ 20 ] . Briefly , J urkat cells was VL to rest rict int ra2chain pairing. In order to provide
preactivated wit h 80 ku/ L IL22 ( Instit ute of sufficient flexibility to allow each molecule to bind
Hematology & Hospital of Blood Diseases , China ) , t heir respective receptor simultaneously , interlinker2
50 μg/ L CD3 monoclonal antibody for t hree days , Fc ( NST YRVVSVL TVL HQDWL N GKE YKC K )
and t hen washed wit h RPM I21640 for two times. and interlinker2HSA ( FQNALL V R YT KKV PQ2
J urkat cells were resuspended in RPM I21640 ( p H VSTP TPV EVS) were alsoint roduced. c2myc tag was
6. 8) and mixed wit h F ITC ( fluorescein added for immunodetection. In psTR I [ Fig. 1 ( B ) ] ,
isot hiocyanate , Sigma) at a final concent ration of 0. 5 t he sc TsAb was co2expressed wit h FkpA under t he
mg/ L . S K2OV23 were resuspended in RPM I21640 cont rol of t he same T 7 promoter and l ac operator.
( p H 7. 2) and mixed wit h TR ITC at a final 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 order to obtain large amount s of soluble sc TsAb ,
( anti2OCV H ) 2 ( anti2OCVL ) 2interlinker2 ( anti2 FkpA2based co2expression vector psTR I was
CD3VL ) 2 ( anti2CD3V H ) 2interlinker2anti2CD28V H ) const ructed. The expression profiles and Western
[ Fig. 1 (B) ] . blotting result s suggested t he chaperone molecule
2. 2 Conf irmation of the expression of trispecifc FkpA could improve t he solubility of t he sc TsAb
antibody 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
Fig. 2 SDS2PAGE ( left) and Western blotting ( right) of sTR I had relatively high binding affinities wit h
pTRI and psTRI
J urkat membrane CD3 antigen , CD28 antigen , and
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
S K2OV23 membrane antigen , respectively , while t he
SDS2PA GE and Western blotting is : 1 and 2 , supernatant ( S) and binding activities of cont rol were relatively low ( Fig.
pellet ( P) of E. coli cells harboring p TMF ; 3 and 4 , supernatant and 3) . The result s implied t he ot her ingredient s in t he
pellet of E. coli cells harboring p TRI ; 5 , molecular weight marker (in supernatant had little effect on t he binding of sTR I to
Western , prestained marker) ; 6 and 7 , supernatant and pellet of E.
antigens. Besides , t he antibody dilution ratio among
coli cells harboring psTRI ; 8 and 9 , supernatant and pellet of E. coli
cells harboring p TRFA vector. 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.
The expression result was detected by SDS2 FACS analysis demonst rated t hat sTR I could
PA GE and Western blotting ( Fig. 2 ) . sc TsAb could p hysically cross2link J urkat cells and ovarian
be observed wit h t he expected molecular weight of 84 carcinoma S K2OV23 cells ( Fig. 4 ) . CD3 + and
kD. TR I protein accounted for about 4 % of t he total
CD28 + J urkat cells were prelabled wit h F ITC , and
bacteria protein and 37. 67 % was in supernatant . In
S K2OV23 cells wit h TR ITC , respectively. In a t umor cells , whereas in t he cont rol experiment wit h
representative experiment , only in t he presence of antibody f ree or vector , t he expression supernatant
sTR I , double2positive colored cell population could not . The percentage of S K2OV23 t hat bound to
increased greatly in t he FACS analysis [ top right J urkat cells in t he presence of sTR I was
quadrant in Fig. 4 ( A ) right ] , which implied t hat calculated wit h CellQuest software to be 92. 14 %
sc TsAb could mediate J urkat cells ’ binding wit h
compared wit h 41. 43 % , 49. 82 % when antibody t umor cells effectively , a series of cont rols have been
f ree and vector supernatant were used [ Fig. 4 (B) ] . set : BSCD28 [ anti2OC × anti2CD3 bispecific
2. 3. 2 T he cytotoxcit y assay of s T R I To f urt her antibody ( 2. 1 mg/ L ) [ 16 ] + anti2CD28 antibody ( 40
examine t he utility of t his new t rispecific antibody for μg/ L ) ] , BS [ anti2OC2CD3 bispecific antibody ( 2. 1
application in t umor t herapy , M T T based mg/ L ) ] , CD3 ( 2. 8 mg/ L ) [ Fig. 5 (B) ] . The
cytotoxicity assay was performed to evaluate t he induced lysate supernatant of E. coli cells carrying
ability of effector T cells to dest roy S K2OV23 t umor p TRFA ( about 12 mg/ L total bacterial protein) was
cells in t he presence of sc TsAb. Different f rom t he used as negative cont rol. The supernatant of sTR I
BsAb M T T cytotoxicity assay , t he PBL s used in ( 12 mg/ L total bacterial protein ) had almost t he
cytotoxicity assay had not been pre2t reated wit h low same cytotoxicity behavior as positive cont rol
dose of IL22 or anti2CD3 monoclonal antibody. The BSCD28 , which is higher t han BsAb and CD3 as
cytotoxicity result s [ Fig. 5 ( A ) ] showed t hat t he expected. In Fig. 5 ( C) , t he supernatant of sTR I ( 60
sc TsAb has an effective cytotoxicity to t umor cells. mg/ L total bacterial protein ) lysed t he t umor cell
sTR I f unctioned best at t he concent ration of about 12 more efficiently t han OCCD3CD28 [ anti2OC scFv ( 4
mg/ L total bacterial protein and wit h t he increase of mg/ L ) , anti2CD3 monoclonal antibody ( 20 μg/ L ) +
dilution ratio , t he killing ratio decreased gradually. anti2CD28 monoclonal antibody ( 40 μg/ L ) ] , OCCD3
So t his concent ration was used in f urt her [ anti2OC scFv ( 4 mg/ L ) , anti2CD3 monoclonal
experiment s. To confirm t he cytotoxicity against 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 sc TsAb decreased. This finding suggested t hat wit h
t umor cells. Cytotoxicity experiment revealed a higher affinity to t umor cells , t he sc TsAb had more
correlationship between an increase in binding ability time to adhere to t umor cells and created more
for S K2OV23 and t he ability of t he sc TsAb to chances for t he T cells to bind and kill t umor cells.
argument lysis of S K2OV23 cells [ Fig. 3 ( B ) , Fig. 5 Most of t he t umor cells were dest royed wit hin
( A ) ] . Wit h t he decrease of t he binding ability to 24 h after exposure to effective cells in t he presence of
ovarian carcinoma , t he cytotoxicity mediated by sc TsAb. At E / T ratio of 25∶1 , after coincubation wit h
sTR I for 2 h , S K2OV23 cells were adhered wit h CD28 by agonistic antibody has been shown to
effector T cells forming t he wreat h ; when prevent A ICD of native T cells during primary
coincubated for 4 h , T cells started to lyse S K2OV23 stimulation [ 26 ] .
cells , t he profile of S K2OV23 was blurred ; after 10 h We describe t he antit umor properties of a new
coincubation , most of t he t umor cells collapsed [ Fig. class of t rispecific antibody. To our knowledge , it is
5 ( D ) ] . Finally , t he S K2OV23 cells vanished in t he t he first report t hat a single2chain t rispecific antibody
medium , leaving t he PBL s alone. can activate t he two signals on T cells simultaneously.
An anti2OC , anti2CD3 ε chain and anti2CD28
3 Discussion composed sc TsAb was const ructed wit h t he intent of
T2cell based anti2t umor immunot herapy requires recruiting T cells to t he ovarian t umor cells , where
f ull and efficient T2cell activation [ 23 ] . It is generally t hey were activated to dest roy t he ovarian carcinoma
accepted t hat T2cell activation requires two distinct cells [ Fig. 5 ( D ) ] . The EL ISA result and FACS
signals. Signal t hrough t he T2cell receptor alone can bridge analysis showed t he sc TsAb could recognize
lead to anergy or activation2induced cell deat h and bind specifically wit h ovarian carcinoma member
( A ICD) [ 24 ] . Costimulation wit h CD28 decreases t he antigen , CD3 complex and CD28 molecule on T2
probability of lymp hocytes apoptosis and prolongs t he lymp hocytes. In t his const ruction , anti2CD3 ε chain
lifespan of lymp hocytes i n vit ro [ 25 ] . Ligation of 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 509
stimulation of T cells as an antigen ( signal 1 ) , V H In summary , t he multif unctional sc TsAb has
domain of anti2CD28 antibody simulates t he B7 many advantages. ( 1 ) It can bind to cytotoxicity T
molecule expressed on antigen processing cell ( APC) , cells and t umor cells simultaneously and t hen active T
interact wit h CD28 on T cells ( signal 2 ) . The cell to kill t umor cells in a highly t umor specific
sc TsAb set up a bridge between ovarian carcinoma manner. ( 2 ) It can initiate and sustain high level of
cells and T cells and created a microenvironment for cytotoxicity reactivity t hrough CD3 and CD28 “two
t he T cell activation and T cell killing effect ( Fig. 6) . signal”t riggering. ( 3 ) It has optimal intermediate
The cytotoxicity mediated by sTR I revealed t hat t he molecule size ( about 84 kD) which is small enough to
sc TsAb f unctions as scBsAb plus CD28 monoclonal penet rate t umors and large enough to sustain in
antibody [ Fig. 5 ( B ) ] and was better t han anti2OC circulation for an appropriate period of time. ( 4) It is
scFv plus anti2CD3 , anti2CD28 monoclonal antibody genetically engineered to minimize t he human anti2
[ Fig. 5 ( C) ] . antibody ( mouse Ig ) ( HAMA ) reaction which has
To ensure properly and f unctionally refolding of hindered t he clinical application of many mouse
each antibody wit hin sc TsAb , partial sequences of monoclonal antibody [ 30 ] and easy of manufact uring. It
HSA and Fc were int roduced as interlinkers. EL ISA would set up a new approach on t he way to successf ul
and FACS analysis demonst rated t hat each antibody elimination of disseminated t umor cells.
wit hin sc TsAb refolded correctly. Poznansky et
[ 27 ]
al . have demonst rated t hat t he stability of HSA References
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510 ACTA B IOCHIM ICA et B IOPH YSICA SIN ICA Vol. 35 , No. 6
一种新型的重组单链三特异抗体的构建与表达