In vitro evolution of an antibody fragment population to?nd high-af?nity hapten binders
Helena Persson1,Henrik Wallmark1,Anne Ljungars2, Johan Hallborn2and Mats Ohlin1,3
1Department of Immunotechnology,Lund University,BMC D13SE-22184, Lund and2BioInvent International AB,So¨lvegatan41SE-22370,Lund, Sweden
3To whom correspondence should be addressed.
E-mail:mats.ohlin@immun.lth.se
Recently,we constructed a focused antibody library tai-lored to interact with haptens.High functionality of this library was demonstrated,as speci?c binders could be retrieved to a range of different haptens.In the current study we have developed a mutagenesis and selection strategy in order to further?ne-tune the hapten binding properties of these antibody fragments.Testosterone was chosen as model antigen for the investigation.A popu-lation,rather than a single clone,originating from this focused library and enriched for testosterone binders, was subjected to random mutagenesis and different phage display selection strategies of various stringencies. These included consecutively lowering the antigen con-centration and having,or not having,soluble hapten present during the phage capture and elution steps.The different selection procedures resulted in a considerable increase in apparent af?nities for several of the selected populations,from which the highest af?nity antibody iso-lated had a K D of2nM,corresponding to an 200-fold af?nity improvement compared with the best clone of the starting population.Importantly,the polyclonal nature of the starting material allowed for the identi?cation of novel unrelated variants that differed in?ne-speci?city, demonstrating that this approach is valuable for explor-ing different parts of structure space.
Keywords:af?nity maturation/antibody library/hapten/phage display/testosterone
Introduction
Besides their importance in the adaptive immune response, antibodies have emerged as an invaluable tool in applications ranging from basic research to disease diagnostics and therapy.The hybridoma technology(Ko¨hler and Milstein, 1975),which relies on animal immunizations,is still the most common way of obtaining antibodies,in particular for diagnostic applications.However,within the past two decades,since the cloning of the?rst antibody repertoires (Huse et al.,1989;Ward et al.,1989)and with the successful production of antibody fragments in bacteria(Skerra and Plu¨ckthun,1988)combined with the functionally display of these on phage(McCafferty et al.,1990;Kang et al.,1991), antibody library technology has gradually become the method of choice for the development of speci?c binders. Over the years numerous antibody libraries have been reported(So¨derlind et al.,2001;Hoogenboom,2002).Various strategies,using natural as well as synthetic diversity,have been utilized,often with the goal to create very large libraries with diversity high enough to provide binding speci?city against any antigen.Although in many cases successful,it has,despite the size of these so called universal or single-pot libraries,been dif?cult to isolate high-af?nity antibodies to some particularly troublesome antigens.In speci?c appli-cations,libraries tailored to interact with a certain antigen or group of antigens,so called focused libraries,have therefore been proposed as an attractive alternative(Collis et al.,2003; Almagro,2004;Almagro et al.,2006;Kehoe et al.,2006; Sidhu and Fellouse,2006).Recently,we successfully con-structed such a focused library with features biased to recog-nize haptens(Persson et al.,2006).
The main principle behind the design of the focused hapten library was based on the fact that the topography of the binding site correlates with the size of its antigen (Webster et al.,1994;MacCallum et al.,1996).As small-molecule antigens almost exclusively interact with cavity-shaped paratopes,a hapten-speci?c antibody,displaying a pronounced binding pocket,was used as structural backbone for library construction.By keeping framework and canoni-cal structure determining residues constant,we reasoned that a large portion of the library would maintain the cavity topo-graphy as seen in the wild-type https://www.doczj.com/doc/2417942271.html,ing site-directed mutagenesis,diversity was mainly restricted to positions and residue types that are commonly involved in hapten contacts (MacCallum et al.,1996;Almagro,2004),resulting in vari-ation of mainly centrally located cavity-lining residues.To possibly allow for the formation of cavities of variable depth (Collis et al.,2003),some variation in loop length was also introduced.Despite the relatively small size of this so-called cavity library(5?107),we were able to identify highly speci?c antibodies to a range of different haptens. Importantly,we could show that the cavity library was a superior source of hapten-binders compared with a consider-ably larger general-purpose repertoire(Persson et al.,2006). In order to be generally applicable,a library has to be able to provide binders of high speci?city as well as of high af?-nity.However,as no particular efforts were made to select for binding strength in the initial study(Persson et al.,2006), the af?nity of the obtained hapten-binders were relatively modest(K D?1026–1027M).Therefore,in the present investigation we set out to further explore the evolution potential of the cavity library to deliver hapten-binding anti-bodies with af?nities required for practical applications. Methods
Construction of libraries
The starting material for library construction was based on a sub-population of the previously described cavity library (Persson et al.,2006).This library had been selected twice on testosterone-biotin(testosterone3-(5-(6-(5-(biotin) pentanamido)hexanamido)pentylcarbamoyl)propanoate) coupled to avidin-coated magnetic beads(Persson
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et al .,2006).Plasmid DNA was isolated from this population,from here on designated CT (C for cavity library and T for testosterone),and submitted to one or two rounds of random mutagenesis by error-prone PCR using an excess of guanine and/or thymine (Fig.1).In the ?rst round,the reaction mix (100m l)contained 10mM Tris–HCl (pH 8.3),50mM KCl,9mM MgCl 2,0.75mM MnCl 2,0.001%gelatin (w/v),0.75mM dNTP and an additional 3.2mM dGTP or dTTP,5U AmpliTaq DNA polymerase (Applied Biosystems,Foster City,CA,USA),0.4m M each of the vector-speci?c primers GGCCGATTCATTAATGCAGC and GCCTTTAGCGTCAG ACTGTAG and as template 10ng of the puri?ed vector was used.The DNA was ampli?ed for 26cycles (948C 1min,568C 1min,728C 2min).The two different PCR products,resulting from either an excess in dGTP (denoted CTep1)or dTTP,were pooled and used as a template in the second PCR.The same conditions as for the ?rst round were used,however,only an excess of dGTP was used this time,creat-ing the library CTep2.The mutated gene fragments CTep1and CTep2were gel puri?ed and digested with S?I and Not I (New England Biolabs,Beverly,MA,USA)followed by lig-ation into the corresponding sites of a modi?ed version of the pFab5c.His phagemid (Engberg et al .,1995),shortened to encode only the ?nal C-terminal domain of protein III.The libraries were subsequently transformed into electrocom-petent Top10F’Escherichia coli (Invitrogen,Carlsbad,CA,USA)and random clones sequenced from both libraries.Mutational rates were determined by calculating the percen-tage of mutated residues at the nucleotide or amino acid level,excluding those positions that were targeted for diver-si?cation in the cavity library [38H ,40H ,55H ,57H ,59H ,66H ,107H -109H ,113H ,40L ,105-117L ;the subscript relates to its position in the heavy (H)or light chain (L)](Persson et al .,2006).Throughout this study,residue numbering follows the IMGT nomenclature (Lefranc et al .,2003),inserting gaps at the top of the loops.The three loops of the heavy and light chain are denoted complementarity determining region (CDR)H1-3and CDRL1-3,respectively.
Phage selections
Phage stocks of the two libraries and CT were prepared by VCSM13helper phage infection (Stratagene,La Jolla,CA,USA)as described (Cicortas Gunnarsson et al .,2004).An overview of the selection procedure is illustrated in Fig.1and Table I.Brie?y,three selection rounds were performed,using gradually lower concentration of testosterone-biotin (Sigma-Aldrich Inc.,St Louis,MO,USA)(Fig.2).Also between the rounds,the coating of the paramagnetic beads was alternated between streptavidin (Dynal A/S,Oslo,Norway)and avidin (Spherotech Inc.,Libertyville,IL,USA)to reduce the risk of obtaining carrier-speci?c binders.In the ?rst round,the three different libraries (CT,CTep1and CTep2)were handled separately.The libraries were pre-incubated with testosterone-biotin diluted in selection buffer [0.2%gelatin,0.05%(v/v)Tween 20in phosphate-buffer saline (PBS)]for 2h on rotation at room temperature.Magnetic beads,washed twice in selection buffer,were added and the mixture was allowed to incubate for another 30min.After ?ve washes in selection buffer,followed by two washes in PBS,binding phages were eluted by the addition of trypsin (Invitrogen,Pasley,UK),which cleaves at a site located between the displayed single chain antibody fragment (scFv)and protein III.The cleavage reaction was subsequently inhib-ited by the addition of aprotinin (Invitrogen).Eluted phages were propagated and titers determined as previously described (Cicortas Gunnarsson et al .,2004).Equal amounts of phages from the three selections were pooled and used for the second round of selection,which was carried out essentially as described for the ?rst round but with the following changes;the amount of panning antigen was decreased (from 5?1028to 1?1028M)and the phage capture step was varied,by excluding or including soluble testosterone (1026M,Sigma-Aldrich)as competitor at the time of addition of the magnetic beads.The two resulting populations (C2L and C2H )were treated separately in the third selection round.In addition to varying the antigen concentrations and conditions during the capturing step,the elution method was also altered,yield-ing in total eight different selection strategies (Table I).The strategies were named with three letters;A,C and E,coding for antigen concentration,capture condition and elution method,respectively.The letters were indexed with high (H)or low (L)to denote the selection pressure for each variable.A H for the lower antigen concentration (2?1029M),A L for the higher (1028M),C H if soluble testosterone (1026M)
was
Fig.1.A ?owchart outlining the experimental steps of the study.The starting material of the af?nity maturation process (library CT)was a phage-selected sub-population of the previously described cavity library (Persson et al .,2006).Library CT,which was highly enriched for testosterone binders,was submitted to one or two rounds of random mutagenesis by error-prone PCR,resulting in the two libraries CTep1and CTep2.Different phage display selection strategies were subsequently used on these libraries to ?nd antibody fragments of increased af?nity (for further details on selection parameters see Table I).After a ?rst round of selection,in which the three starting libraries (CT,CTep1and CTep2)had been handled separately,the libraries were pooled and used for a second round of selection.By varying the conditions during the phage capture step,two populations (C2L and C2H )were obtained.These were treated separately in the third selection round,giving in total 8different selection strategies.These were named with three letters;A,C and E,coding for Antigen concentration,Capture condition and Elution method,respectively,and furthermore indexed with H (high)or L (low)to denote selection pressure for each variable.All 16populations depicted were analyzed by competitive phage ELISA and individual scFv obtained from the 10populations of selection rounds two and three were also assayed in soluble format (for further details see Table II).
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present during the capturing step and C L if it was not,E L if the phages were eluted with trypsin or E H if eluted with soluble testosterone (1025M,2h).In all three selection rounds an excess of magnetic beads were used to allow all antigen-bound phages to be captured and to minimize the risk of selecting multivalently displaying phages that otherwise,due to avidity effects,most likely would have a selection advantage.
Phage ELISA and sequencing of selected populations
The unselected libraries (CT,CTep1and CTep2)as well as the different populations from the three selection rounds were analyzed for speci?city using phage ELISA.The popu-lations were tested on testosterone-biotin and FITC-biotin (5(6)-(biotinamidohexanoylamido)pentylthioureidyl?uores-cein)(Sigma-Aldrich),both bound to the plate via streptavi-din,and on bovine serum albumin.Antigen-bound phages were detected by a horseradish peroxidase (HRP)-conjugated anti-M13antibody (GE Healthcare,Uppsala,Sweden)using o -phenylenediamine as chromogen.In addition,six clones,randomly picked from each of the eight strategies of the third selection round,were similarly analyzed for speci?city and subsequently sent for automated DNA sequencing (MWG Biotech,Ebersberg,Germany).
To estimate the binding af?nity for soluble testosterone,the different populations were further analyzed in a competi-tive phage ELISA format.Testosterone was used to compete with testosterone-biotin immobilized on streptavidin as described (Persson et al .,2006).This allowed for the deter-mination of IC 50values,i.e.the concentration of competing hapten that inhibited 50%of phages binding to the immobi-lized antigen.IC 50values were similarly determined for estradiol (Sigma-Aldrich),?uorescein (Sigma-Aldrich)and testosterone-biotin.Before the addition of the phage solution containing the inhibiting antigen (1–20000ng/ml),care was taken to saturate the biotin-binding sites of streptavidin by adding an excess of testosterone-biotin,a crucial step when inhibiting with biotinylated substances as in the case of testosterone-biotin.For comparison studies,two testosterone-speci?c clones (TC-5and TC-20,GenBank accession numbers DQ250208and DQ250223,respectively),which previously had been isolated from the cavity library (Persson et al .,2006),were also
analyzed.
Fig.2.Molecular structures of testosterone-biotin (A),the derivative used for the phage display selections and the subsequent ELISA and BIAcore assays,and testosterone (B).
T a b l e I .O v e r v i e w o f t h e s e l e c t i o n s t r a t e g i e s
S e l e c t i o n 1
S e l e c t i o n 2
S e l e c t i o n 3
I n p u t A n t i g e n
C a t c h i n g E l u t i o n
O u t p u t
I n p u t
A n t i g e n C a t c h i n g
E l u t i o n
O u t p u t
I n p u t A n t i g e n
C a t c h i n g E l u t i o n O u t p u t
C T
5*1028M T B
S A T r y p s i n C T *
C T *C T e p 1*C T e p 2*p o o l e d t o g e t h e r
1028M T B
A v i d i n
T r y p s i n
C 2L
C 2L
2*1029M T B S A T r y p s i n A H C L E L
C 2L
2*1029M T B S A 1025M T A H C L E H
C T e p 1
5*1028M T B
S A
T r y p s i n C T e p 1*
C 2L
1028M T B S A T r y p s i n A L C L E L
C 2L
1028M T B S A 1025M T A L C L E H
C T *C T e p 1*C T e p 2*p o o l e d t o g e t h e r 1028M T B
A v i d i n t1026M T
T r y p s i n
C 2H
C 2H
2*1029M T B S A t1026M T T r y p s i n A H C H E L
C 2H
2*1029M T B S A t1026M T 1025M T A H C H E H
C T e p 25*1028M T B S A
T r y p s i n C T e p 2*
C 2H
1028M T B S A t1026M T T r y p s i n A L C H E L
C 2H
1028M T B
S A t1026M T 1025M T
A L C H E H T
B ,t e s t o s t e r o n e -b i o t i n .S A ,s t r e p t a v i d i n .T ,t e s t o s t e r o n e .
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High-throughput ELISA screening
For the subsequent analysis steps,an automated ELISA screening procedure was performed using integrated robotic workstations as reviewed by Hallborn and Carlsson(2002). For this purpose a change to a compatible vector,that allows the production of soluble scFv,was needed.Phagemide DNA from the ten populations of selection rounds two and three as well as from the control clones TC-5and TC-20was isolated and digested with the restriction enzymes S?I and Avr II(New England Biolabs).The fragment encoding the scFv was gel-puri?ed and subsequently inserted into a vector that provides the secreted scFv with three FLAG-epitopes and a hexahistidine tag at their C-termini(BioInvent International AB,Lund,Sweden).The constructs were trans-formed into chemically competent Top10E.coli using stan-dard heat chock treatment.
A total of1152clones from each strategy were picked, transferred to384-well plates containing media supplemented with isopropyl thiogalactoside and scFv were produced over-night at378C with vigorous shaking.After sedimentation of the bacteria,the supernatants containing the scFv were screened for binding to biotinylated testosterone in a primary and a secondary assay.Brie?y,the produced scFv were cap-tured by anti-FLAG antibodies(M2,Sigma-Aldrich)that had been adsorbed on the surface of microtiter plates.By limiting the anti-FLAG antibody,differences in scFv production levels should minimally affect the assay,making any observed differences in signal dependent on binding ability. Following a washing-step,testosterone-biotin was added (40nM).After1h of incubation,unbound antigen was removed and HRP-labeled streptavidin(Dako Cytomation, Glostrup,Denmark)added.After a last washing-step,lumi-nescent substrate(Pierce supersignal,Pierce,Rockford,IL, USA)was added and the plates were read in a Tecan Ultra instrument(Tecan AG,Ma¨nnedorf,Switzerland).To get a value of the binding properties of the scFv,a threshold was set using the clones TC-5and TC-20with known af?nity (Persson et al.,2006).FITC-biotin was used as a negative reference to assess the speci?city of the interaction.The48 clones from each strategy that gave the highest speci?c signals in the primary assay were selected for a secondary analysis.The basic set-up of both screenings was the same; however,the secondary assay was much more stringent, using lower testosterone-biotin concentration(10nM)and an increased number of washes.From the secondary screen-ing twelve clones were picked for sequencing from each selection strategy,the?ve clones that gave the highest signal and seven clones picked at random.Of the96sequenced clones,nine unique clones were identi?ed(designated TM-1–9).
Competitive immunoassays of soluble scFv
To investigate the binding af?nity of the nine unique clones for testosterone and testosterone-biotin,competitive ELISA on crude expression supernatants was performed,essentially as described for the phage-displayed scFv but with the following changes:scFv fragments were detected using the HRP-labeled anti-FLAG M2antibody(Sigma-Aldrich)and the reaction developed using luminescent substrate.The binding to estradiol and?uorescein was also accessed.The concentration range used for the inhibiting antigens were0.05–20000ng/ml.
The ELISA IC50values were subsequently veri?ed in a competitive BIAcore assay using a BIAcore3000instrument (BIAcore AB,Uppsala,Sweden).Streptavidin was covalently coupled to a CM5sensor chip using amine coupling accord-ing to the manufacturer’s instructions and an excess of testosterone-biotin,enough to cover all available biotin-binding sites,was added.A second?ow cell was treated with the same chemical procedure but without the addition of antigen and used as reference.A?xed concentration of scFv (TM-2,TM-5,TM-7,TC-5and TC-20),puri?ed from the periplasmic space using af?nity chromatography on Ni-NTA agarose(Qiagen,Hilden,Germany),was mixed with a range of concentrations(0–20000ng/ml)of the inhibiting antigens (testosterone,testosterone-biotin,estradiol and?uorescein), allowed to reach equilibrium and subsequently injected onto the chip at a?ow rate of20m l/min.All experiments were performed at258C and the chip was regenerated in20mM HCl,150mM NaCl.Response curves were generated by sub-tracting the signal obtained from the control?ow cell and IC50values determined.Of note,none of the analyzed scFv showed any signs of reactivity with streptavidin.
Results
Library construction and analysis
The aim of the study was to develop a mutagenesis and selection system(Fig.1)to?nd high-af?nity binders for haptens,in this study exempli?ed by testosterone.The CT library,a sub-population of the previously described cavity library(Persson et al.,2006),was chosen as our initial source of diversity.The cavity library had been designed to include binders of high frequency and functionality against a broad range of different haptens.This library had been sub-jected to two rounds of phage selections on testosterone, yielding the CT library.Analysis of individual members of this pool by ELISA showed that$90%of these were testos-terone speci?c.Also,sequencing revealed a high diversity among those residues targeted for modi?cation(Persson et al.,2006),providing a polyclonal nature of the starting material used for evolution.
Additional diversity was introduced randomly by error-prone PCR(Fig.1).In this way the two libraries CTep1and CTep2were created,each having an estimated size of3–4?107clones of which89%had an insert and80%were in-frame(data not shown).If excluding those positions tar-geted in the design of the cavity library,the random nucleo-tide substitution frequencies of CTep1and CTep2were0.77 and0.87%,respectively,giving an average of3.8(range1–6)and3.9(range2–7)amino acid substitutions per scFv (data not shown).This procedure resulted in$6–8times higher mutation frequencies compared with the CT library (0.12%).Importantly,the extensive diversity found in the cavity-lining residues of the CT library was maintained in both error-prone evolved libraries.
Binding characteristics of the selected populations
We used different selection strategies of various stringencies to select clones with increased testosterone binding activity. Differences were related to the amount of antigen
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(testosterone-biotin)used,whether or not soluble antigen (testosterone)was present during the phage capturing step and choice of elution method.The different approaches are summarized in Table I.
In order to follow the contributions of the different selec-tion parameters,the initial libraries(CT,CTep1and CTep2) and the various pools obtained after each round of selection were analyzed for binding speci?city using phage ELISA. All populations were highly speci?c for testosterone-biotin (data not shown),suggesting that the selection procedures preserved the original speci?city of the starting material. None of the populations showed reactivity to?uorescein,the speci?city of clone FITC8,which was used as diversity-carrying scaffold when constructing the cavity library,nor to estradiol,a steroid highly similar to testosterone.
A competitive ELISA analysis to assess the apparent af?-nities to testosterone-biotin and to soluble testosterone was also performed(Table II).In the?rst selection round,a rela-tively low selection pressure was applied,allowing the removal of non-functional clones formed by the mutagenesis process and also allowing the ampli?cation of rare and poorly expressed scFv.Consequently,this round did not alter the obtained IC50values.In the two following rounds the selection pressure was increased considerably.As one might expect,there was a correlation between binding af?nity for the biotinylated target antigen and the number of selections, with the best result obtained for strategy A H C H E L(IC50?4?1028M)of the third round(Table II).Thus,resulting in at least a500-fold increase in apparent af?nity for testosterone-biotin compared with the starting material.The most prominent increase in binding strength was obtained by decreasing the antigen concentration.The presence of soluble testosterone(C H)during the catching step also seemed to promote lower IC50values;however,no obvious positive effect of the elution method could be observed. Whereas the af?nity for testosterone-biotin showed a peak after the third selection round,it is apparent that the binding to the soluble hapten did not follow the same trend.An initial decrease in IC50values was seen when going from the ?rst to the second selection round.This was especially appar-ent for population C2L.The subsequent selection of these clones,including higher stringency with respect to both antigen concentration and elution method,did not signi?-cantly change the af?nity of the bulk population for testoster-one.In contrast,as illustrated by the differences in IC50 values of the two populations C2H and C2L(33-fold),the presence of soluble testosterone during the phage-catching phase had a major deleterious effect on the binding to free testosterone of the recovered population.This effect was further observed in selection round three,where half of the populations no longer had a detectable testosterone reactivity, suggesting that the addition of soluble testosterone comple-tely drained the populations of testosterone binders. Nevertheless,C2L of the second round of selection and four of the eight populations of the third round showed an $100-fold increase in apparent af?nity for the native antigen compared with the starting populations.
Screening of individual clones
A total of1152clones were picked from each of the total10 strategies from selection round two and three and their scFv were produced in soluble form.In a high-throughput ELISA the clones were assessed for binding to testosterone-biotin.A primary screening assay identi?ed numerous clones that gave signals10–50-fold higher than the control clones TC-5and TC-20,which had previously been isolated from the CT
T able II.Characteristics of the initial libraries and the different selected populations
Population Competitive phage ELISA on
populations of binders High-throughput ELISA screening of individual
soluble scFv
Number of
unique scFv c
Number of scFv
binding to the
soluble hapten d
IC50(TB,nM)IC50(T,nM)Primary screening a(%)Secondary screening b(%)
Unselected CT.2000050000--
CTep1.2000030000––
CTep2.2000070000––
Selection1CT*.2000050000––
CTep1*.2000070000––
CTep2*.2000040000––
Selection2C2L100030040
C2H10001000016
Selection3A H C L E L50060033923/121/3
A H C L E H20060018943/121/3
A L C L E L70040056315/123/5
A L C L E H70040028465/123/5
A H C H E L40.7000033981/120/1
A H C H E H200.70000441002/120/2
A L C H E L2007000041923/121/3
A L C H E H200.7000033903/120/3
TB,testosterone-biotin.
T,testosterone.
a Percentage of clones having a signal greater than5000.1152clones from each strategy were analyzed in this assay.In this assay,the control antibody fragments TC-5and TC-20,which had previously been isolated from the CT library,had signals in the range of500to700.
b Percentage of clones having a signal greater than10000.48clones from each strategy were analyzed in this assay.TC-5and TC-20did not result in any detectable signal in this assay.
c The number of unique scFv was determine
d by sequencing of positiv
e scFv as de?ned by the secondary ELISA screening.
d Th
e number o
f scFv bindin
g the soluble hapten was assessed by competitive ELISA.
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population,prior to the error-prone evolution and the strin-gent selections (Table II).Forty-eight of the clones display-ing the highest signal from each strategy were subjected to a secondary screening assay of higher stringency,as evidenced by the fact that neither of the two control scFv showed any detectable signal in this assay.In both the primary and sec-ondary screening assays,the frequency of clones displaying high signals were considerably lower for the populations of the second selection round compared with the third.
Based on the result of the secondary screening,twelve clones from each of the strategies in selection round three were sequenced.Out of 96sequenced scFv,nine different variants were found (denoted TM-1–9,Fig.3).Notably,one of the sequences,TM-7,was found in all strategies and rep-resented 75%of all sequenced clones.In contrast,some of the sequence variants were only found in one or a few of the populations,thus suggesting that the selection strategies were different enough to tap different parts of the available sequence space.It can also be seen that higher stringency selections,with respect to antigen concentration and catching conditions,had an adverse effect on the diversity of the selected population (Table II).
The nine unique scFv were further analyzed in a competi-tive ELISA to assess the binding af?nity to testosterone and testosterone-biotin.As shown in Table III,?ve of the nine clones (TM-1–5)had similar binding af?nities to testosterone and testosterone-biotin,whereas four of the scFv (TM-6–9)only recognized the biotinylated form of the antigen.These data were later con?rmed by competitive BIAcore analysis (Table III).The best clone,TM-4,had an IC 50value of 2?1029M for soluble testosterone (Fig.4),corresponding to a 200-fold increase in apparent af?nity compared with TC-5.
Sequence and structure of selected scFv
Based on sequence similarity,the nine isolated scFv could be grouped into two groups (Fig.3).Interestingly,this division correlates with the observed reactivity pattern (Table III).Whereas the ?ve clones that bound soluble tes-tosterone had a CDRL3length of 11,the remaining four scFv had a nine amino acids long CDRL3loop.This ?ne-speci?city could further be coupled to highly conserved resi-dues within each of the two groups,in particular residues 40H ,55H and 107L .The preferred combination of these CDRL3lengths and these three residues was also evident among the 48random sequenced clones picked from the 8strategies of selection round three,before the high-throughput ELISA analyses (data not shown).
The random mutagenesis process introduced in total 32amino acid changes among the nine sequence variants.As shown in Fig.3,each clone has between 1and 8amino
acid
Fig.3.Alignment of sequence variants (TM-1-9)identi?ed after three cycles of phage display and 2rounds of high-throughput ELISA screening.The wild-type sequence is a ?uorescein-speci?c scFv whose gene served as diversity carrying scaffold.Mutated regions include those targeted by site-directed mutagenesis,introduced in a previous study (Persson et al .,2006),and those randomly introduced in the current study through an error-prone PCR process.Our previous design strategy (Persson et al .,2006)also allowed for the observed length variations in CDRH2and CDRL3.The scFv TC-5and TC-20that were isolated directly from the CT library (Persson et al .,2006)before the error-prone evolution,are also included in the ?gure.Residues identical with the wild-type sequence are shown with dots while absent residues are indicated with gaps.Residues of the heavy and light chains are numbered according to IMGT (Lefranc et al .,2003)and the linker region ((G 4-S)3)from 1to 15.Amino acids are abbreviated according to their one letter https://www.doczj.com/doc/2417942271.html,plete gene sequences are available in GenBank,accession numbers EU095633-41.
T able III.Binding characteristics of the nine isolated scFv (TM-1-9)as determined by competitive BIAcore and ELISA measurements scFv
ELISA (IC 50)BIAcore (IC 50)TB (nM)
T (nM)TB (nM)T (nM)TM-15030ND ND TM-220305030TM-340200ND ND TM-442ND ND TM-550207050TM-640.70000ND ND TM-720.7000030.70000
TM-870.70000ND ND TM-940.70000
ND ND TC-5ND ND 500400TC-20
ND
ND
600
700
For comparisons,scFv TC-5and TC-20,previously isolated from the CT library (Persson et al .,2006),are also included.TB,testosterone-biotin.T,testosterone.
ND,not determined.
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changes (average 3.6)distributed over the variable domain of the heavy chain (V H ),the light chain (V L )and the linker.This number is similar to the level of diversi?cation found in CTep1and CTep2,but considerable higher than the number of substitutions found in the unmutated population (CT)or among the randomly picked clones after three rounds of selection,which had an average of 0.26and 1.4amino acid substitutions per scFv,respectively.No hot spots of mutations could be observed,as the locations of the substi-tutions were all unique.Also,none of the positions targeted for diversi?cation in the cavity library were changed into residues not intended by the design.Some accumulation of mutations was observed in the CDRs (amino acid frequency change: 1.7%)compared with framework regions (1.3%).Also,an increased mutational frequency was observed in the linker region (3.7%).
Discussion
Anti-hapten antibodies are often of lower af?nity than those of high molecular weight antigens like proteins (Chappey et al .,1994).Being small and hydrophobic,haptens generally contain few functional groups that can provide speci?c inter-action with the antibody-binding site.Furthermore,owing to their non-immunogenic nature,haptens need to be covalently attached to a larger carrier molecule,such as a protein,in order to be used as an immunogen.Consequently,the anti-bodies,whether raised in vivo or in vitro ,typically show higher af?nities for the immunogen than for the free hapten (Franek,1987;Charlton et al .,2001;Moghaddam et al .,2001).However,there are not any inherent restrictions in the structure of antibodies that prevent them from binding small molecules strongly and several recombinant antibody libraries have been reported to contain antibodies against soluble haptens (Grif?ths et al .,1994;Vaughan et al .,1996;So
¨derlind et al .,2000).In a study by Boder et al .(2000),the af?nity of a ?uorescein-speci?c antibody was evolved to a K D of 48fM,to our knowledge the highest monovalent ligand-binding so far reported for an antibody.Nevertheless,
haptens remain challenging targets that often require opti-mized mutagenesis and selection strategies in order to ?nd high-af?nity binders (Charlton et al .,2001;Moghaddam et al .,2001;Sheedy et al .,2007).
The aim of the current study was to explore the potential of a focused antibody library for the development of high-af?nity hapten binders.Based on known structure–function relation-ships of antibodies,a focused library,designated the cavity library,had previously been designed to have an improved rec-ognition for hapten targets (Persson et al .,2006).Highly speci?c binders were retrieved to a range of different haptens and importantly these could not only bind the hapten bound to its carrier but also the free,soluble hapten.It appeared that by focusing the diversity solely to those residues that commonly interact with the hapten,unwanted interactions (e.g.with the linker or the carrier)that skew the selection process could be minimized (Persson et al .,2006).The selection pressure was in the previous study kept relatively low and as a consequence the retrieved binders were of moderate af?nity (106–107M 21).However,the high diversity of the obtained binders suggested that the standardized selection procedure did not utilize the full capacity of the library and that there was considerable room for improvement.It is also likely that the introduction of additional diversity would help to increase the af?nity even further.Several studies have shown a lack of correspondence between the residues in contact with the antigen and those modi?ed in af?nity maturation processes (Tomlinson et al .,1996;Daugherty et al .,2000;Ramirez-Benitez and Almagro,2001).Mutations of buried or peripherally located residues have been shown,through long-range effects,to provide ?ne-tuning of the antigen-binding site and an increase in af?nity.Therefore,to increase the likelihood of isolating high-af?nity binders,additional diversity was introduced to include more peripheral positions of the paratope that were not included in the original diversity design.Furthermore,by comparing the
antibody repertoires of na?
¨ve and secondary responses in vivo it has been demonstrated that not all clones are suitable for effective maturation.Several studies have described dominat-ing clones in the initial repertoire that are absent or rarely found in secondary responses.It has therefore been suggested that some clones will,due to the VDJ recombination,be allo-cated to a part of structural space that make them non-competitive in the subsequent hypermutation process (Alzari et al .,1990;Furukawa et al .,1999;George and Gray,1999).However,the molecular mechanisms governing the maturation pathway of antibodies are still poorly understood and thus dif?-cult to predict.Rather than utilizing a single clone as starting material for diversi?cation,which is standard procedure when performing af?nity maturation experiments,we therefore based the new library on an entire pool of clones that had been enriched by phage display for variants speci?c for testosterone,the hapten chosen as model antigen for this investigation.
The various selection procedures resulted in a signi?cant improvement of the obtained IC 50values for several of the selected populations,an improvement that was mainly achieved by the reduction in antigen concentration between each round.By using a high concentration of competing soluble antigen during the period when antigen-bound phages are caught onto the magnetic beads,the recovery of scFv with fast off-rates,including those that are displayed multivalently,should ef?ciently be prevented (Hawkins et al .,1992).Although having some positive effect on the binding
af?nity
Fig.4.Dose-dependent binding of scFv TM-4to testosterone (squares)and testosterone-biotin (circles)as determined by competitive ELISA.The mean binding signal,average of duplicates,at each antigen concentration was divided by the antibody binding response obtained in the absence of inhibiting antigen to give percentage binding.The IC 50values,being equal to the antigen concentration at which the binding response is half-maximum (50%),are 2and 4nM for testosterone and testosterone-biotin,respectively,thus making this antibody fragment suitable for measuring testosterone within the clinically relevant range (0.7–70nM)(Fitzgerald and Herold,1996).
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to testosterone-biotin,this procedure adversely affected the binding to free testosterone.It appears that this process selected for variants that had matured by making contacts with the biotinylated conjugate compared with the naked mol-ecule,an evolution facilitated by the small size of the antigen, suggesting that this step was too stringent with respect to the kinetics of the population.The use of soluble antigen to com-petitively elute phages rather than using non-speci?c elution methods have in several studies been utilized to enhance the selection of binders with higher af?nity for the free hapten (Sheedy et al.,2007).However,in the current study this approach had no detectable effect on the quality of the selected populations as assessed by binding to soluble testos-terone.Despite the fact that the presence of soluble testoster-one during the catching or elution steps did not have the intended effect,our diversi?cation and selection strategy still allowed us to successfully evolve several of the populations to at least a100-fold increase in mean af?nity for the biotiny-lated as well as for the unmodi?ed hapten.
Importantly,the best clone showed an$200-fold af?nity improvement for soluble testosterone compared with the best clone obtained from the original population,corresponding to an af?nity useful for analyses of clinically relevant levels of testosterone(Fitzgerald and Herold,1996).The nine isolated clones could,based on sequence similarity,be clustered into two distinct groups.The most obvious feature differing between these were CDRL3length(9or11residues)and type of residue found in40H,55H and107L.Interestingly,there was a striking correlation between these groups and their?ne speci-?city.Only antibody fragments harboring the longer CDRL3 loop could bind soluble testosterone,whereas clones of the other group were highly linker-dependent.Among the total34 sequenced clones of the starting material(CT,CTep1and CTep2),75%contained the longer CDRL3version(data not shown).In contrast,none of these randomly picked sequences were found to have a CDRL3of length9,suggesting that there was a substantial selection advantage for clones having this loop length.These results clearly imply that some of the selec-tion parameters as well as the subsequent screening process preferentially‘selected’clones that recognized the conjugated form of the hapten over antibodies that speci?cally recognized the free hapten.It is therefore likely that the introduction of a method early in the screening program that also assesses for recognition of the free hapten should substantially improve the chances of yielding even better soluble hapten-binders.
The observed change in antibody repertoire is not surpris-ing since an increased selection pressure put higher demands on the antigen–antibody interaction and accordingly new ways have to be found to increase the af?nity.As outlined above,repertoire shifts have frequently been observed during immune responses such as those to hapten conjugates(Berek and Milstein,1987)and to some extent also to protein anti-gens(Newman et al.,1992;Kalinke et al.,1996).Related to these?ndings,Brown et al.(2000)noted that antibodies of the late phase of an immune response were better suited for interacting with the hapten in context of the carrier in com-parison to clones of the initial response.This was structurally explained by a widening of the original deep pocket,result-ing in an elongated shallow groove that more ef?ciently accommodated parts of the carrier or the linker in addition to the hapten itself.Whether a similar structural change resulted in the observed speci?city change of clones TM-6-9is conceivable but unclear.The polyclonal starting population allowed for the observed repertoire shift.Although it did not, for the purpose of this study,provide any additional ben-e?cial binding characteristics,the results demonstrate, together with a recent study by Groves et al.(2006),the potential of using polyclonal populations to more ef?ciently exploit structural space.
Whereas a clear connection existed between the obtained reactivity pattern and residues targeted in the original design, no such correlation could be attributed to any of the ran-domly introduced mutations.No consensus mutations were found among these,and at a?rst glance they could easily be disregarded as neutral mutations accumulated during the ran-domization process.However,the considerably higher mutation frequency found among the nine clones isolated after the high-throughput screening assay compared with the number of mutations found after the three rounds of phage selections indicates the importance of these additional substi-tutions in creating novel binders of higher af?nity.The lack of consensus mutations suggests that there are numerous paths to improved?tness.Although,some accumulation of mutations was seen in the CDRs,none of the substituted amino acids were among those proposed to line the antigen-binding cavity(Persson et al.,2006),supporting the notion that af?nity maturation of antibodies can effectively be achieved through peripheral or‘second-sphere’changes (Tomlinson et al.,1996;Ramirez-Benitez and Almagro, 2001).An increased mutational frequency was found in the scFv linker,which might possibly in?uence the stability of the scFv or the orientation of its two domains and thereby the af?nity of the antigen–antibody interaction.
In conclusion,we have demonstrated that the process of in vitro af?nity maturation can successfully be applied on a population of molecules in order to?ne-tune the binding activity and speci?city of hapten-binders originating from the cavity library.Similar to the natural system,we started out with an anti-body repertoire having diversity mainly focused to the central parts of the paratope.An initial enrichment allowed for the retrieval of a population of binders having an effective imprint of the ligand in their binding pockets.In analogy with the somatic hypermutation process,additional diversity was intro-duced to include more peripherally located regions.This allowed adjustments of the initially obtained imprints,resulting in the retrieval of antibody fragments with signi?cantly improved binding af?nities for the hapten target of investigation. Acknowledgements
We gratefully acknowledge?nancial support from BioInvent International AB and the Swedish Research Council.
Funding
BioInvent International AB;the Swedish Research Council (grant number2006-5446).
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Received April1,2008;revised April1,2008;
accepted April10,2008
Edited by Carl Borrebaeck
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1.细胞周期非特异性药物（CCNSC）和细胞周期特异性药物（CCSC ）的区别 CCSC是周期特异性药物，特异性地杀伤处于特定时相的肿瘤细胞，需等肿瘤细胞处于对应时相才有效，故应慢滴。 2.化疗药物给药剂量按体表面积计算（文生氏公式） 体表面积（m2）=0.0061×身高（cm）+0.128×体重（kg）-0.1529 工作中计算体表面积：以身高1.6m，体重60公斤，体表面积为1.6m2，做调整 3.肿瘤病人治疗后，无复发，生存率≥5年，算治愈 4.联合化疗方案药物成原则 a.两种以上作用机制不同的药物组成 b.周期非特异性药物和不同时相的周期特异性药物配合 c.各药的毒性不相重复 d.一般3～4个药物最好,临床上一般2-3个药合用，4个药合用一般用于复发的肿瘤患者或者难治性的血液病患者， 5.化疗药物的器官毒性 ADR（多柔比星，阿霉素）：心脏毒性 BLM（博来霉素）：肺纤维化 DDP（顺铂）：肾毒性 L-OHP（奥沙利铂）、VCR（长春新碱）、PTX（紫杉醇）：神经毒性 BCNU（卡莫司汀）：肝毒性 6.化疗分为 a.诱导化疗:又称新辅助化疗，实施局部治疗方法（如手术或放疗）前所做的全身化疗，目的是使肿块缩小、及早杀灭看不见的转移细胞，以利于后续的手术、放疗等治疗。对于早期和晚期肿瘤患者不采用新辅助化疗的方法。 b.辅助化疗:手术治疗和放疗的后，杀灭手术无法清除的微小病灶，减少复发，提高生存率。
c.姑息化疗:对于手术后复发、转移或就诊时不能切除的肿瘤病人，目的并不是彻底地消灭肿瘤，而在于能够平稳地控制肿瘤的进展，缓解患者的痛苦，延长其生命。这时的化疗称作“姑息化疗”。 7.常用细胞周期特异性药物 S 期特异性药物： 抗叶酸类：甲氨蝶呤（MTX ，胸腺嘧啶、嘌呤）、培美曲塞（PEM ，胸腺 嘧啶、嘌呤，）、雷替曲塞（胸腺嘧啶）、六甲蜜胺（嘧啶） 抗嘧啶类：氟尿嘧啶（5-FU ）、卡培他滨（CAPE ，希罗达，5-FU 前体药）、 替加氟（FT207，5-FU 衍生物）、替吉奥（替加氟+吉美嘧啶 +奥替拉西钾）、优福定（替加氟+尿嘧啶）、卡莫氟（HCFU ， 5-FU 衍生物）、 阿糖胞苷（Ara-c ）、吉西他滨(GEM ，作用机制同Ara-c)、 安西他滨（Ara-c 衍生物） 抗嘌呤类：6-巯嘌呤（6-MP ）、硫唑嘌呤（体内转化6-巯嘌呤起作用）、 氟达拉滨（Ara-A ，阿糖腺苷类似物，抗病毒类）、硫鸟嘌呤 （6-TG ）、喷他司丁（DCF ，新的抗代谢药，本品是从链霉素 菌中分离得的抗生素） 干扰嘌呤和嘧啶合成 ：羟基脲(HU) 拓扑异构酶I ：喜树碱类，羟喜树碱（HCPT ），伊立替康（CPT-11）、 拓扑替康 拓扑异构酶II ：依托泊苷（VP-16）、替尼泊苷（VM-26） M 期特异性药物：长春碱类：长春碱（VLB ）、长春新碱（VCR ）、长春瑞滨（NVB ）、 长春地辛（VDS ） 紫杉类：紫杉醇（PTX ）、多西他赛（DOC ） G 1期特异性药物：L-ASP （L-门冬酰胺酶），肾上腺皮质类固醇 G 2期特异性药物：博来霉素（BLM ，国外）、平阳霉素 （PYM ，国内，与BLM 成 分相近） 影响蛋白质功能与合成的药 门冬酰胺酶、培门冬酶、高三尖杉酯碱 抗代谢类 拓扑异构酶抑制剂
单克隆抗体在肿瘤治疗中的应用 抗体分子是生物学及医学领域中用途最为广泛的蛋白质分子。利用传统的免疫方法或通过细胞工程和基因工程技术制备的抗肿瘤特异性抗原、肿瘤相关抗原、独特型决定簇、某些细胞因子受体、激素及一些癌基因产物的多克隆抗体、单克隆抗体或基因工程抗体等使肿瘤的被动免疫治疗发生了改观。人们可以用单抗单独应用于肿瘤治疗,也可以以单抗为特异性载体而将与其偶联的放射性核素、抗癌药物、毒素、酶和其他类型生物制剂“携运”至肿瘤部位,发挥相应的抗瘤效应,这种免疫偶联物亦称为“生物导弹”。 人们最初期望用类似于抗感染的被动免疫方法来治疗肿瘤,即用特异性的同种或异种抗血清或患同类肿瘤“痊愈”病人的血清注射给肿瘤病人。由于人类肿瘤细胞抗源性、肿瘤细胞异质性等诸多理论上的问题未能解决,因而要获取特异性强且效价高的抗肿瘤抗血清很不现实。直到20世纪70年代中期B 淋巴细胞杂交瘤技术的建立,人类在这领域的研究才向前迈进一大步。B淋巴细胞与鼠的骨髓瘤细胞融合,在选择性培养基的条件下,筛选出杂交瘤细胞,筛选出的杂交瘤细胞继承了其亲代细胞的性质,既可分泌抗体,又能无限传代。由特异抗原致敏的某个B细胞克隆所产生的抗体即为单克隆抗体。这种由杂交瘤技术制备的单抗是杂交瘤细胞所分泌的抗体,其质地均一,纯度高,效价高,且能重复大量生产。由于单克隆抗体特异性高,能在多种抗原中识别特异性抗原决定簇,已帮助人类鉴定出多种肿瘤相关抗原,但某种肿瘤是否存在特异性抗原至今未获普遍认同。 目前认为单抗的作用机制有阻断作用、信号传导作用以及靶向作用等三种作用机制:1１阻断作用 现用于临床的大部分未偶联单抗主要用于自身免疫和免疫抑制,是通过阻断和调节作用完成的。几乎在所有的单抗应用中,通常都是通过阻断免疫系统的一种重要的胞浆或受体-配体相互作用而实现的。另一种相类似的阻断活性可能存在于单抗的抗病毒感染中,通过阻断和抵消病原体的进入和扩散表现出对机体的防御功能,短期给予单抗后可取得长期疗效。2１信号传导作用许多抗癌单抗是通过恢复效应因子,直接启动信号机制而获得细胞毒效应的。在抗-Id的临床试验中,B细胞受体(BCR)与抗体的交联导致正常细胞和肿瘤细胞的生长受抑制和凋亡。对trastuaumab而言,单抗结合可诱导一系列在肿瘤生长控制中起作用的信号传递,该抗原是生长因子受体家族的一个成员,能提供重要的有丝分裂信号,其单抗似乎能阻断与促进肿瘤生长有关的重要的配体-受体相互作用。3１靶向作用单抗靶向肿瘤细胞的首要目的是产生肿瘤特异性反应物,然后由免疫系统中的活化因子将其消灭,如早期抗-Id单抗在淋巴瘤中的应用。研究表明:利用单抗与化学药物、放射性核素以及毒素形成的偶联物具有对肿瘤细胞的选择性杀伤作用,同时具有更高的疗效,并且对耐药性肿瘤细胞也有杀伤作用。这些研究结果为应用于肿瘤治疗的可行性提供了重要依据。单克隆抗体用于抗肿瘤治疗有2种基本的方式,一是单抗的单独应用,二是
抗肿瘤药物的分类和临床应用 抗肿瘤药物的分类和临床应用 1.根据药物的化学结构和来源分：烷化剂、抗代谢药物、抗肿瘤抗生素、抗肿瘤植物药、激素和杂类。 2.根据抗肿瘤作用的生化机制分：干扰核酸生物合成的药物、直接影响DNA结构与功能的药物、干扰转录过程和阻止RNA合成的药物、干扰蛋白质合成与功能的芗、影响激素平衡的药物和其他。 3.根据药物作用的周期或时相特异性分：细胞周期非特异性药物和细胞周期（时相）特异性药物。 恶性肿瘤是危害人类健康的最危险的疾病之一，肿瘤的治疗强调综合治疗的原则，化疗是其中的一个重要手段。近年来抗肿瘤药物的研究取得了飞速发展，出现了一些新型的抗肿瘤药物，作用于肿瘤发生和转移的不同环节和新靶点。按照抗肿瘤药物的传统分类和研究进展，将抗肿瘤药物分为细胞毒药物；影响激素平衡的药物；其他抗肿瘤药物，包括生物反应调节剂和新型分子靶向药物等；抗肿瘤辅助用药。 一、细胞毒药物 1．破坏DNA结构和功能的药物 氮芥烷化剂类的代表药物，高度活泼，在中性或弱碱条件下迅速与多种有机物质的亲核基团结合，作用强但缺乏选择性。进入血中后水解或与细胞的某些成分结合，在血中停留的时间只有几分钟，作用短暂而迅速。G1期及M期细胞对氮芥的作用最敏感，大剂量时对各周期的细胞和非增殖细胞均有杀伤作用。主要用于恶性淋巴瘤及癌性胸膜、心包及腹腔积液。目前已很少用于其他肿瘤。不良反应包括消化道反应、骨髓抑制脱发、注射于血管外可引起溃疡。 环磷酰胺周期非特异性药，作用机制与氮芥相同。在体外无活性，主要通过肝p450酶水解成醛磷酰胺再形成磷酰胺氮芥发挥作用。抗瘤谱广，对白血病和实体瘤都有效。环磷酰胺口服后易被吸收，约1小时后血浆浓度达最高峰，在体内t1/2 4—6小时，约50%由肾脏排出，对泌尿道有毒性。大部分不能透过血脑屏障。环磷酰胺临床广泛应用，对恶性淋巴瘤、白血病、多发性骨髓瘤均有效，
新型铂类抗肿瘤药物发展概况 作者：韩学亮 201100260102 摘要 奈达铂、奥沙利铂等新型铂类抗肿瘤药物在临床上广泛应用,其具有抗癌谱广、活性强、不良反应低、与顺铂无交叉耐药性等特点。此外,尚处于临床研究中的亲脂性铂配合物、多核铂配合物等新型铂类化合物,也显示出提高疗效、降低不良反应的优势。该文对铂类抗肿瘤药物的发展历程、作用机制和一些具体药物进行了综述。 关键字 铂类抗肿瘤药物、卡铂、顺铂、奥沙利铂、作用机制 引言 铂族金属包括铂、铑、铱、锇、钌6个元素，它们具有许多独特和卓越的理化性质，一直在高新技术方面发挥着重要的作用。铂配合物，在肿瘤的治疗方面以其显著的疗效，被广泛应用于临床，并成为许多肿瘤联合用药方案中的重要组成部分。 铂类药物的研究最早兴起于40年前，1967年美国密执安州立大学教授Roserlberg和Camp首次发现顺铂具有抗癌症活性。这一发现迅速引起了肿瘤界的广泛兴趣，之后铂类抗肿瘤药物的研究成为热点，随着一个个有历史意义的试验结果的公布，肿瘤化疗疗效有了大幅的飞跃。1995年WHO对上百种抗肿瘤药物进行排名，顺铂在疗效及市场等方面的综合评价位于第二位。有统计数据表明，我国所有的化疗方案中的有70％～80％以铂为主或有铂类药物参加配伍。同时，铂类金属药物也是当前抗肿瘤药物最为活跃的研究开发领域之一，新的铂类抗肿瘤药奥沙利铂、乐铂等已相继推出。铂类药物的抗肿瘤作用机制也有了进一步了解。目前第三代铂类抗肿瘤药沙铂和4-NDDP等已进入临床试验［1］。 自 20世纪 60年代发现顺铂具有抗肿瘤活性以来 ,铂类金属抗肿瘤药物的应用和研究得到了迅速的发展。迄今为止 ,人们大约已合成了数千种铂类化合物 ,但作为抗肿瘤药物投入临床的仅 30种左右 ,其中 20 多种因抗肿瘤活性弱或毒
1 单克隆抗体药物----科普知识 单克隆抗体药物 2009-10-19 15:47 1986年，美国FDA批准了第一个单克隆抗体药物上市，距今已经整整20年了。截止到现在，全世界共有21个治疗用抗体药物被批准上市，实现300亿美元的销售额，在国际，也在国内形成了抗体药物开发热潮。巨大的市场前景和现存的技术问题及壁垒并存的现实不可避免地引发抗体药物新一轮技术革命。而其结果又将毫无疑问地改变抗体药物的市场格局。抗体药物的研究开发能否真能成为中国生物技术药物开启国际市场大门的新钥匙？什么是我们首选的切入点，又如何形成我们自己的特色和竞争优势？回顾国际抗体药物20年风雨飘摇的发展经历，总结其中的经验教训无疑会给我们一些有益的启示。 1986年，美国FDA批准上市了第一个抗体药物Orthoclone，用于治疗移植物抗宿主病（GVHD），翻开了生物医药历史崭新的一页。时隔8年，美国才批准了第二个抗体药物上市。进入21世纪，抗体药物研发上市的速度明显加快。20年后的今天，全球共批准上市21个抗体药物。进入临床验证的数量也直线上升，从上个世纪80年代的70个，到90年代新增140个，以及2000年至2005年6月又增加的130个，预计2010年将再增 240个【1】，显示抗体药物研究异常活跃。目前共有150余个抗体药物正在临床评估中，其中16个已进入III期临床【2】。 抗体药物研发进展迅速及竞争激烈的主要原因是1）抗体药物具有高度特异性，是靶向治疗的基础，在这一方面远优于小分子药物；2）虽然在世界范围内20年仅仅批准上市了21个抗体药物，事实上其淘汰率仍明显低于小分子药物，临床转化率以及批准成功率都较高，以治疗癌症的抗体药物为例，其批准成功率为29％；3）抗体药物即使在专利保护到期后，由于其生产条件的复杂性，也很难受到通用名药价格的威胁；4）更为重要的是已上市的抗体药物具有很高的市场回报率，大大刺激了投资热情。目前，上市抗体药物中已盈利的有8个，其中4个已经成为年销售额10亿美元以上的“重磅弹”，5个销售总额超过30亿美元【3】。预计2005-2010年抗体药物销售的平均增长率为20％，年销售额将超过300亿美元，市场潜力巨大。 但具有讽刺意味的是，从药物经济学的角度看，抗体药物并非很好的药物候选者。首先，单克隆抗体是大分子糖蛋白，结构复杂、不利储存、不能口服、进入体内5-7天才能到达靶位置。其次，抗体药物研发费用较高，达10－18亿美元，高于小分子药物平均研发费的9亿美元。第三，目前抗体药物的单剂用量大，药物的质量标准高，生产成本高昂，导致价格昂贵，以致被喻为“经济负作用”。以治疗肿瘤的抗体药物Avastin为例，单个病人年度费用高达5万美元【4】。然而，正在形成的巨大市场是抗体药物研发的不竭驱动力。 我国在单克隆抗体技术起步非常早，80年代曾出现过研究热潮，但产业化成就还微不足道。目前，受国际抗体研发进展的影响，又出现了新一轮的“抗体热”，北京、上海、广州等纷纷成立了由研究机构、企业和投资商的联合抗体研发中心和公司。面对国际抗体药物竞争的新态势，我国抗体药物产业是否遇到了真正的机遇？我们选择的切入点是什么，又如何形成自己的特色和竞争优势？抗体药物的研究开发能否成为中国生物技术药物开启国际市场大门的新钥匙？回顾国际抗体药物20年风雨飘摇的发展经历，总结其中的经验教训无疑会给我们一些有益的启示，这是本文的主要目的。 一、上市抗体药物的发展现状 从第一个抗体药物上市至今20年内，全球共批准了21个抗体药物，其中美国18个（包括9个被欧盟
新型铂类抗肿瘤药物发展概况 作者：韩学亮201100260102 摘要 奈达铂、奥沙利铂等新型铂类抗肿瘤药物在临床上广泛应用,其具有抗癌谱广、活性强、不良反应低、与顺铂无交叉耐药性等特点。此外,尚处于临床研究中的亲脂性铂配合物、多核铂配合物等新型铂类化合物,也显示出提高疗效、降低不良反应的优势。该文对铂类抗肿瘤药物的发展历程、作用机制和一些具体药物进行了综述。 关键字 铂类抗肿瘤药物、卡铂、顺铂、奥沙利铂、作用机制 引言 铂族金属包括铂、铑、铱、锇、钌6个元素，它们具有许多独特和卓越的理化性质，一直在高新技术方面发挥着重要的作用。铂配合物，在肿瘤的治疗方面以其显著的疗效，被广泛应用于临床，并成为许多肿瘤联合用药方案中的重要组成部分。 铂类药物的研究最早兴起于40年前，1967年美国密执安州立大学教授Roserlberg和Camp首次发现顺铂具有抗癌症活性。这一发现迅速引起了肿瘤界的广泛兴趣，之后铂类抗肿瘤药物的研究成为热点，随着一个个有历史意义的试验结果的公布，肿瘤化疗疗效有了大幅的飞跃。1995年WHO对上百种抗肿瘤药物进行排名，顺铂在疗效及市场等方面的综合评价位于第二位。有统计数据表明，我国所有的化疗方案中的有70％～80％以铂为主或有铂类药物参加配伍。同时，铂类金属药物也是当前抗肿瘤药物最为活跃的研究开发领域之一，新的铂
类抗肿瘤药奥沙利铂、乐铂等已相继推出。铂类药物的抗肿瘤作用机制也有了进一步了解。目前第三代铂类抗肿瘤药沙铂和4-NDDP等已进入临床试验［1］。 自20世纪60年代发现顺铂具有抗肿瘤活性以来,铂类金属抗肿瘤药物的应用和研究得到了迅速的发展。迄今为止,人们大约已合成了数千种铂类化合物,但作为抗肿瘤药物投入临床的仅30种左右,其中20 多种因抗肿瘤活性弱或毒性过强而被淘汰［2］。 1 铂类抗肿瘤药物发展历程 在过去的30余年里，人们对几千个新的铂类化合物进行了筛选，其中有几十个化合物进入了临床研究，目前已上市的铂类抗肿瘤药物有4个品种，其发展大致经历了3个阶段。 1.1 第1代铂类抗肿瘤药物 顺铂是顺二氯二氨合铂(Ⅱ)的简称，缩写为DDP或CDDP(Ⅱ）。1979年首次在美国上市，是第一个上市的铂类抗肿瘤药物，目前已被收录入中、美、英等国的药典。顺铂对睾丸癌和卵巢癌的疗效明显，也可以用来治疗膀胱、颈部、头部、食管的肿瘤以及小细胞肺癌，还可与博来霉素、阿霉素及5-氟尿嘧啶配伍进行联合用药。顺铂抗癌谱广、作用强、活性高，易与其他抗肿瘤药配伍，因其交叉耐药性少而有利于联合用药，但其严重的毒副反应（包括肾毒性、胃肠道毒性、耳毒性及神经毒性）及低溶解性、耐药性限制了临床的大剂量和长期使用。 1995年WHO对上百种治癌药物进行排名，顺铂的综合评价(疗效、市场等)名列前茅，列第2位。另据统计，在我国以顺铂为主或有顺铂参与配伍的化疗方案占所有化疗方案的70%～80%。顺铂仍是目前应用最广泛的药物之一，含铂类化疗方案是晚期非小细胞肺癌的首选方案，亦是小细胞肺癌的主要组方之一。顺铂是头颈癌单药有效率最高的药物之一；顺铂加五氟尿嘧啶（5-FU）是头颈癌化疗的标准方案之一，联合紫杉醇、吉西他滨亦是非常有前景的头颈癌化疗方案。顺铂及卡铂一直是治疗睾丸癌（尤其是非精原细胞性）、卵巢癌的主要治疗药物。顺铂与其他化疗药物联合是侵袭性膀胱癌、骨肉瘤、食管癌、胃癌等的标准化疗方案。 早期的顺铂合成是以K2PtCl4为原料与氨水反应得到，但因重现性差且含有大量的杂质被放弃。目前顺铂合成(收率为80%)以K2PtCl4为起始原料，通过下面三个步骤来实现: (1)加入KI ,转化成K2 PtI4，与氨水反应，制备出相应cis-[Pt(II)I2(NH3)2]中间体。(2)中间体与AgNO3 (或Ag2SO4/Ag2O)反应，过滤分离AgI得到cis-[Pt(NH3)2(H2O)2 ]2 +母液。(3)母液与KCl反应,制得顺铂［3］。
单克隆抗体制备与应用 姓名：王志豪学号：10073485 班级：工优070 关键词：单克隆抗体，人抗体，杂交瘤细胞 摘要：1975年德国学者Kohler和英国学者Milstein成功地将骨髓瘤细胞和产生抗体的B淋巴细胞融合为杂交瘤细胞,其分泌的抗体是由识别一种抗原决定簇 的细胞克隆所产生的均一性抗体,称之为单克隆抗体。从鼠源单抗之后,单抗历经了鼠源性抗体、嵌合抗体、人源化抗体、人源性抗体4个发展阶段。随着分子生物学和细胞生物学的发展,单抗理论几乎应用到生物学研究的每一个区域。 1975 年, Kohler 和Milstein 创立了杂交瘤技术制备单克隆 抗体,此后单克隆抗体迅速广泛地应用于生物学和医学的各个领域。单克隆抗体可用于分析抗原的细微结构及检验抗原抗体未知的结构 关系;生产出针对复杂生物混合物中的特定分子的抗体,可用于分离、分析及纯化该特定分子抗原;其试剂可用于临床诊断和治疗,或用于 以单抗为弹头的“生物导弹”药物等。但单克隆抗体技术自问世以来,在临床治疗方面进展缓慢,主要原因是目前单克隆抗体大多是鼠源性的,而鼠源单抗应用于人体治疗时存在诸多问题:鼠源单抗在人体中 常不能有效激活补体和Fc 受体相关的效应系统;被人体免疫系统所 识别,产生人抗鼠抗体(HAMA) 反应;且在人体循环系统中很快被清除。因此,在保持对特异抗原表位的高亲和力的基础上人源化和全人化的改造,减少异源抗体的免疫原性成为单抗研究的重点。此外,传统杂交瘤技术还存在制备周期较长,成本较高,杂交瘤细胞不稳定抗性会丢
失等缺陷。近年来,随着分子生物学技术的发展,出现了嵌合单克隆抗体和由转基因小鼠、噬菌体展示技术、核糖体展示技术及共价展示技术所制备的单克隆抗体。这些技术可有效解决传统杂交瘤技术所存在的问题,为单克隆抗体的应用提供更广阔的空间。 1994 年, 美国Cell Genesys 公司和Genpharm公司宣布转基因小鼠作为生产全人抗体的载体问世。这项技术是将人抗体基因微位点转入小鼠体内,产生能分泌人抗体的转基因小鼠。其前提是人的抗体基因片段在小鼠体内进行重排并表达,并且这些片段能与小鼠细胞的信号机制相互作用,即在抗原刺激后,这些片段可被选择、表达并活化B 细胞分泌人抗体。这些转基因小鼠的不足之处在于转移基因片段较小,仅30kb 左右,因此这种抗体库在面对抗原多样性时,其抗体应答显得单薄而不足。此后,Green 等人利用基因打靶技术将编码人抗体轻重链的基因片段大约18Mb 的DNA 全部转到自身抗体基因位点已被灭活的小鼠基因组中,再经过繁育筛选,建立了稳定的转基因小鼠品系。这样得到的转基因小鼠对特异的抗原能产生高亲和力的人抗体。用传统的杂交瘤技术,将表达特异抗体的转基因小鼠B 细胞和骨髓瘤细胞融合,获得杂交瘤细胞系,产生人源抗体。利用转基因小鼠技术已获得了一系列抗IL8 、TNFα以及EGFR 的人单克隆抗体,这些细胞因子在肿瘤或其他疾病中起着重要的作用,因此其单克隆抗体作为导向剂具有重要的临床治疗意义。目前生产的单抗大多是鼠源性的，但其在临床应用方面还存在着很大的弊端，主要是鼠源单抗与NK 等免疫细胞表面Fc 段受体亲和力弱,产生的抗体依赖性细胞介导的细胞毒
单克隆抗体在肿瘤治疗中的应用 【摘要】单克隆抗体在一段相当短的时间内成为治疗癌症的主流方法。它们的第一个用途是作为致癌受体酪氨酸激酶受体拮抗剂，但今天单克隆抗体已成为长期寻求的有效化疗药物靶向递送的载体并作为操纵抗癌免疫反应的功能的强大的工具。在临床上有更加可喜的成果，未来将有可能看到持续增长治疗性抗体和它们的衍生物的发展。 由于单克隆抗体药物专一性强、疗效显著，为抗肿瘤治疗开辟了一条新的途径，因此成为近年来研究的热点药物之一。单克隆抗体抗体是由B 淋巴细胞转化而来的浆细胞分泌的，每个B淋巴细胞株只能产生一种它专有的、针对一种特异性抗原决定簇的抗体。这种从一株单一细胞系产生的抗体就叫单克隆抗体，简称单抗。这些抗体具有相同的结构和特性。抗体与特异性表达的肿瘤细胞表面蛋白质结合，从而阻碍蛋白质的表达，起到抗肿瘤作用。抗体还可使B 淋巴细胞产生免疫反应，诱导癌细胞凋亡。早期单抗为鼠源性单抗，易被人体免疫系统识别，应用受到限制。后来采用基因工程的方法生产人源或人鼠嵌合型单抗，广泛应用于临床。 单抗药物治疗主要是利用其靶向性来干预肿瘤发生发展过程中的各个通路，或是激活宿主对肿瘤的免疫等。随着生物医学的不断发展，一定会出现具有更高靶向性的单抗药物。但是，单抗药物还存在一些尚未解决的问题，最突出的问题是如何降低单抗的免疫原性，单抗的异源性所引起的抗体反应，不但降低了单抗的效价，而且会给患者带来严重的后果。因此，对异源性单抗进行改造以及人源性单抗的研制成为单抗研究的重要方向 1.EGEG疗法 表皮生长因子受体EGFR是一种细胞表面蛋白，与多种癌症密切相关，也是癌症治疗的主要靶标。基因编码信息被翻译为特定蛋白，不过，许多蛋白必须经由翻译后程序激活，比如自身磷酸化。蛋白激活影响着许多重要的细胞过程，包括细胞增殖、分化和迁移。若EGFR 出现故障使这些过程脱离控制，就会导致癌症。然而，尽管EGFR与癌症有着密切关联，人们对EGFR的激活机制还并不完全了解。 受体酪氨酸激酶是一个细胞表面受体大家族，EGFR也是其中一员。EGFR有一个细胞外的配体结合域，和一个细胞内的激酶区域。EGFR激活是其配体EGF结合到配体结合域，诱导受体二聚化，随后二聚体的两个激酶区域相互磷酸化。因为在相对较低的浓度下，即使没有EGF诱导的二聚化，单独的激酶域在溶液中也能自激活。二聚化是指两个同样的分子聚合形成单个化合物。研究发现，除了配体EGF结合以外，EGFR激活还需要EGFR跨膜螺旋和
1、不属于烷化剂类的抗肿瘤药物的结构类型是 A.氮芥类 B.乙撑亚胺类 C.亚硝基脲类 D.甲磺酸酯类 E.硝基咪唑类 参考答案：E 试题难度：本题共被作答83次，正确率59% ，易错项为E,A 。 参考解析：本题考查烷化剂的结构类型。E为合成抗茵药，其余均为烷化剂类。故本题答案应选E。 2、烷化剂环磷酰胺的结构类型是 A.氮芥类 B.乙撑亚胺类 C.甲磺酸酯类 D.多元卤醇类 E.亚硝基脲类 参考答案：A 试题难度：本题共被作答113次，正确率82% ，易错项为A,B 。 参考解析：本题考查环磷酰胺的结构类型及结构特征。塞替派为乙撑亚胺类，白消安为甲磺酸酯类，卡莫司汀为亚硝基脲类。故本题答案应选A。 3、属于嘌呤类抗代谢的抗肿瘤药物是 A.米托蒽醌 B.紫杉醇 C.巯嘌呤 D.卡铂
E.甲氨蝶呤 参考答案：C 试题难度：本题共被作答145次，正确率72% ，易错项为C,E 。 参考解析：本题考查药物的结构类型及特征。米托蒽醌为醌类，紫杉醇为环烯二萜类，甲氨蝶呤为叶酸类，卡铂为金属配合物类。故本题答案应选C。 4、具有以下结构的化合物，与哪个药物性质及作用机制相同 A.顺铂 B.卡莫司汀 C.氟尿嘧啶 D.多柔比星 E.紫杉醇 参考答案：A 试题难度：本题共被作答288次，正确率81% ，易错项为A,C 。 参考解析：本题考查金属配合物类抗肿瘤药的化学结构。该化学结构为卡铂，属于顺铂的第二代，故药物性质及作用机理与顺铂相同。故本题答案应选A。 5、甲氨蝶呤中毒时可使用亚叶酸钙进行解救，其目的是提供 A.二氢叶酸 B.叶酸 C.四氢叶酸 D.谷氨酸 E.蝶呤酸 参考答案：C
单克隆抗体的制备 摘要:单克隆抗体技术是现代生命科学研究的重要工具,在基因和蛋白质的结构和功能研究方面有着不可或缺的作用。近年来,随着分子生物学技术的发展,出现了嵌合单克隆抗体和由转基因小鼠、噬菌体展示技术、核糖体展示技术及共价展示技术所产生的单克隆抗体。这些技术将有效解决单克隆抗体的鼠源性等问题。下面主要讲述制备单抗的实验过程。 关键词：抗体，单克隆，肿瘤，细胞融合，淋巴细胞 现代生物技术制药工业始于1971年，现已创造出35个重要治疗药物，全球大约有2500多家公司，主要产品有重组蛋白质药品、重组疫苗和诊断、治疗用的单克隆机体三大类。我国自80年代在采用现代生物技术改造传统生物技术制药产业方面已取得初步成果。但我国生物技术诊断试剂、酶工程、动植物细胞工程医药产品、现代生物技术支撑技术、后处理技术和制剂技术等方面与国外还存在差距。 1．国外现代生物技术产业发展的现状 自1971年Cetus公司成立至今，现代生物技术制药工业已走完了二十五年的路程，创造出35个重要的治疗药物，目前已在治疗癌症、多发性硬化症、贫血、发育不良，糖尿病、肝炎、心力衰竭、血友病、囊性纤维变性和一些罕见的遗传性疾病中取得良好效果。在医药工业中，传统生物技术（包括近代生物技术）已为人类提供了许多重要药品，在保障人类生命健康和推动社会进步中发挥了巨大作用；现代生物技术以其特有的高新技术又为人类提供了传统生物技术难以获得的极微量的珍贵药品。由于这一系列现代生物技术新型药物的出现，使过去无法治疗的疑难疾病得到了治疗。同时，应用现代生物技术DNA重组，细胞融合以及细胞大规模培养等现代生物技术发展和提高传统生物技术的生产水平，为抗生素、氨基酸、维生素以及基体激素等药品的生产，构建了高产新菌株，创造新工艺，提高生产能力，降低生产成本，促进生产发展。
单抗类抗肿瘤药物概述 单抗类抗肿瘤药物单抗类抗肿瘤药物作用机制为当机 体受抗原刺激时，抗原分子上的许多决定簇分别激活各个具有不同基因的B 淋巴细胞。 被激活的B 细胞分裂增殖形成效应B 细胞（浆细胞）和记忆B 细胞，大量的浆细胞克隆合成和分泌大量的抗体分子分布到血液、体液中。如果能选出一个制造一种专一抗体的浆细胞进行培养，就可得到由单细胞经分裂增殖而形成细胞群，即单克隆。 单克隆细胞将合成针对一种抗原决定簇的抗体，称为单克隆抗体。单克隆抗体以其高特异性、有效性和低毒性，可以准确地攻击靶分子, 且毒副作用较低,已成为一类重要的抗肿 瘤药物。单克隆抗体抗肿瘤机制包括：免疫介导的效应功能，包括抗体依赖性细胞介导的细胞毒性反应(ADCC)和补体依 赖性细胞毒性反应(CDC)。单抗与肿瘤细胞靶抗原特异性结合后，其Fc段可以与NK细胞、巨噬细胞和中性粒细胞等 效应免疫细胞表面的Fc受体(FcR)结合，激活细胞内信号，发挥效应功能。NK细胞通过释放细胞毒性颗粒(穿孔素和颗粒酶)导致靶细胞的凋亡；释放细胞因子和趋化因子抑制细胞增殖及血管生成。 巨噬细胞可以吞噬肿瘤细胞，有释放蛋白酶、活性氧和细胞
因子等加强ADCC作用。此外，一些偶联抗体通过连接细胞毒化合物或放射性物质来杀伤肿瘤细胞，如TDM1（trastuzumab emtansine）、Zevalin等。1997-2013年FDA 和CFDA批准的抗肿瘤单抗类药物列表如图15。图15：1997-2013年FDA和CFDA批准的抗肿瘤单抗类药物（点开大图观看更清晰?）截至目前，全球上市的单克隆抗体共51个，其中鼠源单克隆4个、嵌合抗体7个、人源化单克隆抗体23个、全人单克隆抗体17个。单抗药物中，抗肿瘤药物占了一半左右。截至目前，中国上市的抗肿瘤单抗类药物共有7个，其中进口4个，国产3个，国内自主研发的第一个单克隆抗体类抗肿瘤药物为百泰药业治疗鼻咽癌的药物尼妥珠单抗（泰欣生）2008年4月被正式批准联合放疗治疗EGFR表达阳性的Ⅲ/Ⅳ期鼻咽癌（比埃克替尼早了3年），这是全球第一个以EGFR为靶点的人源化单抗药物。2015年，中国国内单抗药物销售额约为72亿元人民币，其中肿瘤药占了80%，约为57亿元，同比约占全球抗药市场的1.13%。对比小分子靶向药物，2014年国内22重点城市样本医院靶向小分子抗肿瘤药物市场为13.21亿元，根据2015年样本医院全年靶向小分子药物购入金额为14.92亿元，占全球市场的1.34%。 从全球市场上看，2015年靶向抗肿瘤药物TOP10中有6个是单抗，前3名全是单抗，且销售额差距明显，前3名2015
6 种铂类化疗药对比 一代：顺铂（DDP） 顺铂是多种实体瘤的一线用药，可作为放疗增敏剂；疗效可观，抗癌谱广（肺癌、卵巢癌、骨肉瘤、神经母细胞瘤、宫颈癌及泌尿系肿瘤等）。 部分不良反应： 1.消化道反应：常见，如食欲减退、恶心、呕吐、腹泻等，急性反应一般用药1～2小时后发生，可持续1周以上；用药时应给予强效止吐药。 2.肾毒性：也是剂量限制毒性，重复用药可加剧肾毒性；表现为用药后 1～2 周出现肾小管损伤、血尿、血尿素氮或肌酐升高等；每日＞90 mg/m2 需警惕，除了水化暂无与其他预防手段；DDP肾小管的损伤在一般剂量下多为可逆性的；但剂量过大或用药过频，可导致药物在体内的蓄积，使肾小管损伤为不可逆的，产生肾功能衰竭，甚至死亡。 3.神经毒性：与用药总量有关，表现为头晕、耳鸣、耳聋、不可逆的高频听力丧失，少数人表现为球后神经炎、感觉异常、味觉丧失。与氨基糖甙类抗生素、两性霉素B、头孢噻吩等合用，可产生致命性肾衰，并致耳聋。 4.骨髓抑制：较轻，主要表现为白细胞减少，血小板减少相对较轻；剂量≤100mg/m2 ，发生率10-20%，剂量≥120 mg/m2 则40％，亦与联合化疗中其它抗癌药出现重叠反应，白细胞＜ 3.5x109/L、血小板＜75x109/L 需谨慎。部分注意事项： 1.既往肾病史、中耳炎史者慎用； 2.若“WBC低于3500/mm3或PLT低于8万/mm3、用药后持续性严重呕吐、 早期肾毒性表现”时应停用； 3.密切监测血常规及肝肾功能； 4.避光。 二代：卡铂（CBP） 第二代铂类抗肿瘤药；广谱（细胞肺癌、卵巢癌、睾丸肿瘤、头颈部鳞癌等，也可用于非小细胞肺癌、膀胱癌、子宫颈癌、胸膜间皮瘤、黑色素瘤、子宫内膜癌等）。 男性肌酐清除率 = [（140 - 年龄）×体重× 1.23]/ 血清肌酐 女性肌酐清除率 = 男性肌酐清除率× 0.85 卡铂剂量（mg）= 所设定的 AUC（mg/mL/min）×[肌酐清除率（mL/min）+25] 部分不良反应： 1.在体内存留时间比顺铂短，因而消化道毒性低于顺铂、肾毒性轻微且不常见、耳毒性和神经毒性罕见。 2.骨髓抑制比顺铂强，有剂量限制性；血小板减少严重，最低点出现在 2～ 3 周，恢复在治疗后第4周。 3.治疗前不需水化，但同时应用氨基糖甙类会增加肾毒性和耳毒性。 4.卡铂无明显肾毒性，但治疗前肾功能状况却能显著影响卡铂所致血小板减少的程度，治疗前肾小球滤过率（GFR）低下者血小板减少较普遍。 部分注意事项： 1.与顺铂有不完全交叉耐药，既往顺铂无效者，改用卡铂仍有可能取得疗效。
骨细胞相关因子在骨重建中的作用 骨细胞是一种动态的、具有复杂功能的细胞，也是骨组织中含量最丰富、分布最广泛的细胞。近几年研究发现，骨细胞在骨重建中的调节作用越来越明显，其分泌的骨硬化蛋白、RANKL及OPG是调节骨形成和骨吸收的重要调控因子。骨细胞特异性地分泌的骨硬化蛋白对骨形成具有特殊的抑制效果，主要机制是结合LRP5/LRP6，从而阻止经典Wnt信号通路。而骨硬化蛋白的单克隆抗体则通过拮抗其作用而保证Wnt信号通路的正常传导，引起骨形成、骨密度和骨强度增加。骨细胞同样会分泌RANKL及OPG，两者在生理和病理条件下直接或间接调节破骨细胞分化和功能，调控骨重吸收。该文就这一领域近年研究现状和发展方向作一综述。 骨组织总是在连续不断地进行骨重建。两种类型细胞参与到骨重建，包括来源于血液系统的破骨细胞和来源于骨髓基质干细胞的成骨细胞和骨细胞。成骨细胞和破骨细胞在骨组织内只是短暂的存在，且数量少，位置不定。骨细胞则是骨组织中含量最丰富的细胞，在骨组织中形成遍布矿化骨基质的三维细胞网络。近几年研究发现，骨细胞在骨重建中的调节作用越来越明显，其分泌的骨硬化蛋白、RANKL及OPG是调节骨形成和骨吸收的重要调控因子。 1 骨细胞及其功能 骨细胞起源于成骨细胞，成熟成骨细胞的5%～20%包埋在自身分泌的基质中，并分化为骨细胞。成骨细胞的寿命是数周，破骨细胞仅为数天，而骨细胞的平均半寿期大约为25年[1]。这使骨细胞成为骨组织中数量最多的细胞，约占骨组织中细胞总数的90%以上。骨细胞目前被认为是骨组织中主要的应力感受器，其通过骨基质和骨陷窝-小管网络系统高度的连通性，能感知来自流体的各种力[2]。 骨细胞表达成骨细胞的大多数基因，包括成骨细胞特异性的转录因子和蛋白，尽管表达水平可能不尽相同。骨细胞中碱性磷酸酶和Ⅰ型胶原的表达较低，而骨钙蛋白表达较高。角膜蛋白在成骨细胞的表达显著高于骨细胞[3]。在骨组织矿化及磷酸代谢相关基因表达上骨细胞比成骨细胞富含更多基因，如PHEX、DMP1、MEPE、FGF23[4]。骨细胞既分泌磷蛋白质（如Dmp1），也参与骨基质的矿物质沉积[5]。骨细胞还参与体内磷酸盐平衡，作为磷元素和纤维原细胞因子（FGF）的主要供应者[5]。骨细胞也表达某些影响骨形成的分子微粒，包括Dkk1和SOST，但Dkk1也存在于成骨细胞，而SOST仅在骨细胞表达[4]。SOST 基因编码的骨硬化蛋白对骨形成蛋白（BMP）家族的成员蛋白具有很强的拮抗作用。此外，骨硬化蛋白和Dkk1与LRP5和LPR6结合，阻止Wnt信号通路的激活。此类研究表明骨细胞中少量表达的基因可能作为骨组织重建过程中的分子调解者。 2 骨细胞调节骨形成和骨吸收的分子机制
单克隆抗体药物治疗肿瘤的研究现状与展望近几年单抗药物已广泛应用于临床肿瘤治疗，源于其对肿瘤相关靶点显示特异性结合，对肿瘤细胞的选择性杀伤作用。抗肿瘤单抗药物研究的发展趋势是继续寻求新的分子靶点、抗体人源化及偶联物分子的小型化。 单抗药物能用于治疗肿瘤、病毒性感染、心血管以及其它病患，尤其在治疗肿瘤方面，已显示出良好的效果。抗肿瘤单抗药物一般包括两类，一是抗肿瘤单抗；二是抗肿瘤单抗偶联物，或称免疫偶联物。免疫偶联物分子由单抗与“弹头”药物两部分组成。单抗所针对的靶标通常为肿瘤细胞表面的肿瘤相关抗原或特定的受体。 用作“弹头”的物质主要有三类，及放射性核素、药物和毒素；其与单抗连接分别构成放射性免疫偶联物、化学免疫偶联物和免疫毒素。 单抗药物的研究进展 单抗药物对肿瘤细胞的选择性杀伤作用：单抗与药物偶联物或与毒素偶联物对肿瘤靶细胞显示选择性杀伤作用，对表达有关抗原的肿瘤细胞作用强，对抗原性无关细胞的作用弱或无作用。单抗药物偶联物对肿瘤细胞的杀伤活性比无关抗体偶联物的活性强；药物与单抗偶联后对肿瘤细胞的活性比游离药物强。 单抗药物具有更高的疗效：偶联物与相应的游离药物比较，一般具有更高的疗效或显示较低的毒性。曾与单抗进行偶联并在裸鼠进行疗效观察的抗癌药物有
阿霉素、柔红霉素、平阳霉素等长春碱类衍生物。 单抗药物对肿瘤相关靶点的特异性作用：特定受体或特定的基因表达蛋白可能作为单抗药物的靶点。 单抗药物对抗药性肿瘤细胞的杀伤作用：单抗偶联物对于抗药性肿瘤细胞仍显示较强的杀伤活性。例如长期服用氨甲喋呤而出现抗药性的成骨肉瘤细胞，单抗氨甲喋呤偶联物仍显示较强的杀伤作用。 存在的问题与解决途径 单抗药物的临床研究结果已为其应用于治疗肿瘤展现出良好的前景，但仍存在涉及免疫学和药理学两方面的问题。免疫学方面的问题主要是人抗鼠抗体（HAMA）反应。药理学方面的问题主要是到达肿瘤的药量不足，由于单抗药物是异体蛋白，会被网状内皮系统摄取，有相当数量将积聚于肝、脾和骨髓。单抗药物是大分子物质，通过毛细血管内皮层以及穿透肿瘤细胞外间隙均受到限制，解决途径主要包括： 降低单抗药物的免疫原性：使用人源化单抗仍不能解决“弹头”（毒素）的免疫原性问题，使用化疗药物为“弹头”则可避免此部分的抗体反应。 提高单抗药物在肿瘤组织的浓度：单抗药物在体内的运送过程与肿瘤局部的供血状况有关，动物实验表明，血管丰富、血流量较大的肿瘤，用单抗药物治疗的效果也比较好。 加强单抗药物对肿瘤细胞的杀伤作用：单抗能特异性结合到相关的肿瘤靶细胞，但通常难以杀伤靶细胞。单抗治疗剂一般须调动体内的免疫系统，包括细胞免疫和体液免疫效以达到治疗肿瘤的效果。如上所述，单抗药物实际到达肿瘤细胞的数量有限，为取得良好的结果，单抗药物需要高效化，仅有微量的偶联物到达靶部位即可杀伤肿瘤细胞。 进一步提高药物作用的选择性：将单抗偶联的活化酶与前体药物联合使用，在靶肿瘤进行酶性活化。前体药物是指该药无治疗活性或仅有较低活性，需在体内经过代谢转化为活性型才显示药物活性。其优越性是可能降低毒性和延长药物体内作用的时间。
抗肿瘤药分类 1 烷化剂抗肿瘤药 环磷酰胺Cyclophosphamide 塞替派Thiotepa 司莫司汀Semustine 盐酸氮芥Chlormethine Hydrochloride 白消安（马利兰）Busulfan 苯丁酸氮芥Chlorambucil 氮甲Formylmerphalan 卡莫司汀Carmustine 六甲蜜胺Altretamine 洛莫司汀Lomustine 苯丙氨酸氮芥DL-Phenylalanine Mustard 硝卡芥Nitrocaphane 异环磷酰胺Ifosfamide 二溴甘露醇Mitobronitol 2 抗代谢类抗肿瘤药 阿糖胞苷Cytarabine 氟尿嘧啶Fluorouracil 甲氨蝶呤Methotrexate 羟基脲Hydroxycarbamide 替加氟Tegafur 甲异靛Meisoindigotin 巯嘌呤Mercaptopurine 3抗生素类抗肿瘤药 放线菌素D(更生霉素) Dactinomycin 丝裂霉素Mitomycin 盐酸阿霉素Doxorubicin Hydrochloride 盐酸平阳霉素Bleomycin A5 Hydrochloride 盐酸表柔比星Epirubicin Hydrochloride 盐酸吡柔比星Pirarubicin Hydrochloride 盐酸柔红霉素Daunorubicin Hydrochloride 4天然来源抗肿瘤药 高三尖杉酯碱Homoharringtonine 硫酸长春新碱(醛基长春碱) Vincristine Sulfate 羟喜树碱Hydroxycamptothecin 依托泊苷Etoposide 硫酸长春地辛Vindesine Sulfate 硫酸长春碱Vinblastine Sulfate 重酒石酸长春瑞宾Vinorelbine Bitartrate 紫杉醇Paclitaxel 长春质碱转移因子长春瑞宾碱多烯紫杉醇莪术油 人参多糖秋水仙碱9-氨基喜树碱7-乙基喜树碱榄香烯 5激素类抗肿瘤药 氨鲁米特Aminoglutethimide 他莫昔芬Tamoxifen 氟他胺Flutamide 戈那瑞林Gonadorelin 醋酸亮丙瑞林Leuprorelin Acetate 来曲唑Lelrozol 6其他 卡铂Carboplatin 盐酸丙卡巴肼(甲基巴肼) Procarbazine Hydrochloride 安吖啶Amsacrine 枸橼酸达卡巴嗪Dacarbazine Citrate 门冬酰胺酶(L-门冬酰胺酶) Asparaginase 顺铂Cisplatin 盐酸米托蒽醌Mitoxantrone Hydrochloride 烷化剂抗肿瘤药的部分药物简介 (1) 环磷酰胺是一种直接作用于肿瘤细胞的药物，对增殖细胞群的各期均有杀伤作用。进入人体后肝脏或肿瘤组织内存在的过量磷酰胺酶或磷酸酶水解，释放出氮芥基而杀伤肿瘤细胞抑制其生长的作用。异环磷酰胺在体外无抗癌活性，进入体内被肝脏或肿瘤内存在的磷酰胺酶或磷酸酶水解，变为活化作用型的磷酰胺氮芥而起作用。其作用机制为与DNA发生交叉联结，抑制DNA的合成，也可干扰RNA的功能，属细胞周期非特异性药物。本品抗瘤谱广，对多种肿瘤有抑制作用。 (2) 硝卡介Nitrocaphane化学名称为：2-[双(β-氯乙基)胺甲基]-5-硝基苯丙氨酸，为细胞周期非特异性
铂类抗肿瘤药的简介 摘要：本文综述了铂类抗肿瘤药的分类、发展和各类铂类抗肿瘤药的简介、药理作用、临床应用及不良反应。 关键词：铂类、抗肿瘤药、分类、简介 癌症，是威胁人类健康的第二大杀手，全球每年有上百万人死于癌症。因此，加强抗肿瘤药物研究，降低癌症死亡率，是医药工作者亟待解决的难题。目前临床上治疗癌症主要以化疗为主，而铂类药物作为临床上不可或缺的一线化疗药物，在研究与临床应用中取得迅猛发展。铂类药物的抗癌活性是由美国学者于1965年发现的，之后第一代铂类药物顺铂、锡铂、顺氯氨铂，第二代铂类药物奈达铂、依铂、卡铂、卡波铂、碳铂，第三代铂类药物洛铂、奥沙利铂、诺贝铂、赛特铂等相继用于临床治疗（1）。 自从第一代铂类药物顺铂用于临床，铂类药物沿着两个较宽方向发展，一是改善顺铂的毒副作用；二是克服其在瘤体内的耐药性。自70年代开始，人们从数千种铂化合物中仅筛选出10余种进行了临床试验。至1986 年卡铂上市, 成为第二代铂类药物，目前顺铂与卡铂在世界范围内被接受。随着近几年对铂类药物的作用机理、构效关系、以及临床治疗的深入研究内外的深入研究, 发现以二氨基环己烷为载铂配体的一类铂配合物，不仅改善了顺铂及卡铂的毒副作用，而且扩大了它们的活性谱，对许多耐顺铂或卡铂的细胞株或瘤株具有活性（2）。这种新型的铂类药物被称为第三代抗癌药，其代表药物有洛铂、奥沙利铂等。
1995年，世界卫生组织对世界上近百种抗癌药物进行评价，顺铂的疗效、市场等综合评价得分名列前茅，排在第2位。另据统计，在我国医院抗癌化疗治疗方案中，以顺铂为主或有顺铂参加配伍的方案占所有化疗方案的70%~ 80%（3）。通过这些数据表明，铂类药物在化疗治疗中占有举足轻重的地位。下面本文就对一些临床上常用的各类铂类抗肿瘤药物做一下简单的介绍。 1.第一代铂类抗癌药 1.1．顺铂 顺铂的化学名为顺式二氨基二氯络铂，又名顺氯氨铂，英文缩写为DDP。1967年，美国密执安州立大学教授Rosenberg等人发现，其顺式异构体有抗癌作用，而反式异构体无此作用。1969年，顺铂开始应用于临床（4）。 在血液中氯离子浓度相对高(100mM )的环境中,顺铂以完整分子形态和血浆蛋白相结合。在胞内氯离子浓度相对较低(4~20mM)的环境中发生水化，其中的双氯配体全部或其中一个解离，这样顺铂才能和靶标DNA相结合。它作用于鸟嘌呤的N7位，部分通过共价键与腺嘌呤结合，当结合上第2个DNA键时形成一个闭合的环状复合物，扭转了DNA 分子构象，从而阻止复制和转录，最终致细胞凋亡。