Effects of humanization and gene shuffling on immunogenicity and antigen binding of anti-TAG-72 single-chain Fvs.

One major constraint in the clinical application of murine monoclonal antibodies (MAbs) is the development of a human antimurine antibody response. The immunogenicity of MAbs can be minimized by replacing nonhuman regions with corresponding human sequences. The studies reported in our article were undertaken to analyze the immunoreactivity and the immunogenicity of the CC49 single-chain antibody fragments (scFvs): (i) an scFv construct comprised of mouse CC49 VL and VH (m/m scFv), (ii) a light chain shuffled scFv with human VL (Hum4 VL) and mouse CC49 VH (h/m scFv), and (iii) a humanized scFv assembled from Hum4 VL and CC49 VH complementary determining regions (CDRs) grafted onto a VH framework of MAb 21/28' CL (h/CDR scFv). The CC49 scFvs competed for an antigen binding site with CC49 IgG in a similar fashion in a competition radioimmunoassay and were able to inhibit the binding of CC49 IgG to the antigen completely. The immunogenicity of CC49 scFvs was tested using sera with antiidiotypic antibodies to MAb CC49 obtained from patients treated by CC49 IgG in clinical trials. All tested sera exhibited the highest reactivity to the m/m scFv. However, the sera demonstrated differential reactivities to h/CDR scFv and h/m scFv. Replacement of the mouse chain in h/m scFv and h/CDR scFv decreased or completely averted serum reactivity. Our studies compared for the first time the antigen binding and immunogenicity of different scFv constructs containing the mouse, CDR grafted or human variable chains. These results indicate that the humanized CC49 scFv is potentially an important agent for imaging and therapeutic applications with TAG-72-positive tumors.     

全人源抗乳腺癌噬菌体单链抗体库的构建

目的：利用噬菌体展示技术构建全人源性抗乳腺癌单链抗体库。方法：从临床获取未化疗乳腺癌病人外周血样30份,分离出单个核细胞（PBMC）,提取总RNA,用RT-PCR技术逆转录获得cDNA,并扩增出全套人抗体重链（VH）和轻链（VL）基因,经重叠延伸PCR（SOE-PCR）,在体外将两者连接成单链抗体（scFv）基因片段,将该片段用Sfi I和Not I酶切后克隆至pCantab5E噬菌体载体,电转化TG1感受态菌,收集培养后平板上的菌落,即构建初级噬菌体单链抗体库。结果：得到长度约为360bp和340bp的VH和VL,拼接后得到的scFv长度约为750bp;经PCR初步鉴定插入率约为80%,BstN 1多样性酶切检验,酶切图谱呈多样性。经测序验证,最终获得库容约为2.4×106pfu/ml初级单链抗体库。结论：本研究获得了全人源抗乳腺癌噬菌体单链抗体库,为下一步筛选抗人乳腺癌细胞特异性单链抗体奠定了基础。     

Construction and selection of human anti-idiotypic antibody single chain variable fragments or CDR3 fragments of nasopharyngeal carcinoma

Peripheral blood mononuclear cells (PBMCs) of patients with NPC were immunized in vitro by anti-NPC monoclonal antibody FC2 and transformed by Epstein-Barr virus (EBV). Detection showed that of 10 NPC patients, 8 patients' B cells immunized by FC2 and transformed by EBV produced anti-idiotypic antibodies to NPC. Five types of VH genes and 7 types of VL genes were obtained by RT-PCR amplification and then connected with (Gly4Ser)3 linker to form 14 types of scFv genes. ScFv genes digested with Sfi I were cloned into vector fUSE5 and transformed into E. coli MC 1061. Phage anti-idiotypic antibody library with 1.5 x 10(8) clones was obtained. After four rounds of panning, 270 phage clones were selected randomly and 91 FC2-positive clones were obtained by Sandwich ELISA, the positive ratio was 33.7%. 5 clones (D83, E92, G22, I50, I54), which might display beta type Ab2 scFv, were selected by binding inhibition test. These 5 phage anti-idiotypic antibodies were further analyzed by DNA sequencing. The VDJ regions of G22, I50, I54 belonged to VH4-39-D4-11-JH3-linker-V1-19-JL2, VH4-4-D4-11-JH6 and VH4-31-D4-11-JH6, respectively. E92 had the same VDJ regions with G22; D83 had the same VDJ regions with 150. So, a strategy for preparing and selecting beta type Ab2 scFv or CDR by means of immunization in vitro, EBV transformation and phage display technique is feasible, which paves a way for preparing cancer vaccine using beta type Ab2 scFv.     

全人源抗乳腺癌噬菌体单链抗体库的构建

目的:利用噬菌体展示技术构建全人源性抗乳腺癌单链抗体库。方法:从临床获取未化疗乳腺癌病人外周血样30份,分离出单个核细胞(PBMC),提取总RNA,用RT-PCR技术逆转录获得cDNA,并扩增出全套人抗体重链(VH)和轻链(VL)基因,经重叠延伸PCR(SOE-PCR),在体外将两者连接成单链抗体(scFv)基因片段,将该片段用Sfi I和Not I酶切后克隆至pCantab5E噬菌体载体,电转化TG1感受态菌,收集培养后平板上的菌落,即构建初级噬菌体单链抗体库。结果:得到长度约为360bp和340bp的VH和VL,拼接后得到的scFv长度约为750bp;经PCR初步鉴定插入率约为80%,BstN 1多样性酶切检验,酶切图谱呈多样性。经测序验证,最终获得库容约为2.4×106pfu/ml初级单链抗体库。结论:本研究获得了全人源抗乳腺癌噬菌体单链抗体库,为下一步筛选抗人乳腺癌细胞特异性单链抗体奠定了基础。     

Optimizing the generation of recombinant single-chain antibodies against placental alkaline phosphatase

Recombinant technologies to engineer ordinary hybridoma monoclonal antibodies (MAbs) to single-chain fragment variable (scFv) may cause loss of antibody affinity, increased tendency to aggregate, increased temperature sensitivity, and low yield of active protein. In the present investigation, the well-characterized MAb H7 against placental alkaline phosphatase (PLAP), used as a model antibody, was engineered to improve solubility and stability of scFv with retained high affinity. The original procedure to generate single-chain antibodies with a 10-amino acid linker between VH and VL yielded an almost insoluble product. By site-directed mutagenesis, four selective sequence substitutions were made in the VL fragment and one in the VH fragment to improve solubility. The importance of the linker length was investigated, and a 25/30 amino acid linker was found to improve solubility. In order to further increase the stability of the single-chain antibody, an additional covalent -S-S- bond was introduced between amino acid 100 in the VL fragment and amino acid 44 in the VH region, to make a single-chain disulphide stabilized variable fragment (scdsFv). Altogether five different antibody constructs were produced and compared in terms of solubility, stability, affinity, and production properties. Immunospecificity was tested by enzyme-linked immunosorbent assay (ELISA) against the target antigen, temperature sensitivity by exposing the purified scFv to higher temperatures. All the new constructs retained almost equal activity and high affinity for their target antigen, placental alkaline phosphatase (PLAP), compared to the intact MAb H7, up to +42 degrees C as evaluated by ELISA. The overall affinity K(A) > 10(9) (M(1)) of the new antibodies could be maintained in the same order of magnitude as the original one (H7), when evaluated by Biacore technology. The best final single-chain antibody was obtained by performing the specific site-directed mutations and introducing a linker of 30 amino acids, but not by additional stabilizing disulphide bonds. The yield of the final antibody was improved approximately 10-fold by the modifications. This antibody could easily be expressed in a bacterial system using the PET-32a TrxA vector and the Escherichia coli strain BL21 Origami B (DE3). Purified antibody, which could be kept at concentrations up to 0.8 mg/mL, was obtained, which is sufficient for clinical testing of therapeutic applications.     

鼻咽癌人源抗独特型单链抗体的制备及筛选

背景与目的:抗独特型抗体作为肿瘤抗原替代物可用于肿瘤治疗,这已在临床试验中得到证实。但由于目前所使用的抗独特型抗体多为鼠源性,用于人体可产生人抗鼠抗体反应,从而影响疗效。本实验拟构建噬菌体人源抗独特型抗体库,并从中筛选出能模拟鼻咽癌相关抗原的β型抗独特型单链抗体scFv(Ab2βscFv),以解决鼠源性抗独特型抗体用于临床所产生的人抗鼠抗体反应。方法:体外致敏并用EB病毒(Epstein-Barrvirus,EBV)转化鼻咽癌患者的外周血单个核细胞(peripheralbloodmononuclearcell,PBMC),用RT-PCR分别扩增VH和VL基因并连接成scFv基因,将scFv基因与载体fUSE5连接后,转化大肠杆菌MC1061,构建噬菌体呈现型scFv库。在用单抗FC2对文库进行4轮筛选后,用SandwichELISA和结合抑制法从中筛选出β型Ab2scFv。结果:用单抗FC2体外致敏并经EBV转化的10例鼻咽癌患者的PBMC中,8例有鼻咽癌抗独特型抗体产生。经PCR分别扩增出5种VH(γ、μ)和7种VL(κ、λ)基因,经连接组成14种scFv基因。在与载体连接后,导入大肠杆菌MC1061,得到库容为1.5×108的初级噬菌体抗独特型抗体库。经富集筛选后,从中随机挑取270个克隆进行ELISA筛选,得到91个Ab2scFv单克隆,阳性率为33.7%。再用结合抑制法从中初步筛选出5个可能为β型的Ab2scFv。结论:联     

肿瘤干细胞表面标志物CD133单链抗体基因的分离与鉴定

 【摘要】　目的　分离、构建和表达抗人CD133单链抗体（scFv CD133）,测定其生物学活性。方法　用抗体工程技术从抗人CD133单克隆抗体（mAb）杂交瘤细胞中分离抗体可变区基因（VL和VH）,测定DNA序列并确定抗体互补决定区（CDR）;将scFv CD133基因克隆至pET32a中,转化Origami菌株,IPTG诱导,Ni2+-NTA His树脂纯化单链抗体,梯度硫氰酸盐洗脱法和酶联免疫吸附（ELISA）法检测其亲和性和特异性。结果　scFv CD133 基因VL和VH长度分别为339 bp和342 bp,各编码113和114个氨基酸,其中VL隶属于小鼠Igκ轻链,VH隶属于小鼠Ig重链I亚类。scFv CD133经SDS-PAGE和Western blot分析证明相对分子质量为27×103,体外实验具有一定的亲和性和特异性。结论　获得scFv CD133基因,为CD+133肿瘤干细胞的靶向治疗奠定了基础。     

Human antibody derivatives against the fibroblast activation protein for tumor stroma targeting of carcinomas

The fibroblast activation protein (FAP) is selectively expressed on activated fibroblasts of the tumor stroma on more than 90% of lung, breast and colon carcinomas. The high prevalence and abundance of FAP(+) stroma make it a promising target for in vivo diagnosis and therapy of a variety of carcinomas. We describe the humanization of the murine FAP-specific MAb, F19, which has already been clinically used for in vivo diagnostic purposes. Using phage display technology and human V-repertoires, VL and VH regions of F19 were replaced by analogous human V-regions while retaining the original HCDR3 sequence in order to maintain F19 epitope specificity. The resulting human single-chain fragments of immunoglobulin variable regions (scFv 34, scFv 18) showed affinities of 6 nM on cell membrane-bound FAP. scFv 34 was expressed as a bivalent minibody (Mb 34). The antigen-binding characteristics of Mb 34 were comparable to the parental and a complementarity-determining region (CDR)-grafted version of F19. This was revealed by binding competition studies, FACS analyses and immunohistochemistry on various tumor samples including breast, colon and lung carcinomas. Importantly, compared with the CDR-grafted humanized scFv version of F19, the V-regions of the selected human scFv 34 showed sequence identity with the parental antibody (Ab) only over the short, 15-amino acid long HCDR3. Thus, a largely reduced xenoantigenic potential is expected. These human Ab derivatives are suitable to develop novel therapeutic concepts with broad applicability for a wide variety of histological carcinomas based on tumor stroma targeting.     

结肠癌单抗MC5的噬菌体呈现型单链可变区片段的制备

背景与目的：MC5是一种特异性良好的针对人结肠癌的鼠源性单克隆抗体,而将鼠源性抗体小型化可使其用于在体研究时引起人抗鼠抗体反应的可能性大大降低。本研究的目的是制备MC5的噬菌体呈现型单链可变区片段（ScFv)。方法：从分泌MC5的杂交瘤细胞分离mRNA,RT-PCR分别扩增抗体的重,轻链可变区DNA（VH和VL DNA）,两者经linker DNA连接形成ScFvDNA,将ScFvDNA与噬粒载体pCANTAB5E的连接产物转化于大肠杆菌TG1,经M13KO7辅助噬菌体感染后,获得重组噬菌体抗体ScFv,以高表达MC5结合抗原的细胞株SW480对重组噬菌体抗体ScFv进行两轮筛选后,随机挑取克隆经ELISA筛选呈现MC5 ScFv的噬菌体单克隆,经竞争ELISA对阳性克隆结合抗原的能力进行鉴定。结果：VH,VL和ScFvDNA分别约为340bp,320bp和750bp,在随机筛检的25个克隆中得到10个呈现MC5ScFv的噬菌体单克隆,其中结合抗原能力强的克隆有3个,结论：用噬菌体呈现技术成功地制备了单抗MC5的ScFv,为拓展该抗体的应用范围奠定了基础。     

Generation and characterization of a recombinant/chimeric B72.3 (human gamma 1)

We report here the generation and characterization of a recombinant/chimeric construct of murine gamma 1 monoclonal antibody (MAb) B72.3, containing the murine variable region and a human gamma 1 constant region [designated cB72.3(gamma i)]. cB72.3(gamma 1) was generated by first isolating functionally rearranged VH and VL genes of B72.3 from partial genomic libraries in phage vectors. Construction of mouse-human chimeric heavy and light chain genes was performed by inserting restriction fragments carrying VL and VH regions of B72.3 into unique sites of expression vectors which contains sequences encoding constant regions of human kappa and gamma 1, respectively. The expression constructs were subsequently electroporated into SP2/0 cells. The transfected SP2/0 murine cell line has been shown to synthesize cB72.3(gamma 1) at a level of 10-20 micrograms/ml. Reciprocal competition radioimmunoassays demonstrated that cB72.3(gamma 1), a previously described cB72.3(gamma 4), and native B72.3 (designated nB72.3) competed similarly. A rat anti-idiotype MAb made against nB72.3 was shown to bind equally well to cB72.3(gamma 1) and to the nB72.3. Immunochemical studies of the nB72.3, cB72.3(gamma 4), and cB72.3(gamma 1) revealed slight differences in size among the three MAb forms on sodium dodecyl sulfate gels and revealed a higher isoelectric point for the cB72.3(gamma 1). Antibody-dependent cell-mediated cytotoxicity experiments using human lymphokine-activated killer effector cells indicated better tumor cell killing by the cB72.3(gamma 1) than the nB72.3 or cB72.3(gamma 4). Dual label studies of coinjected cB72.3(gamma 1) and nB72.3 revealed that both MAbs could efficiently localize human tumor xenografts in athymic mice. Pharmacokinetic studies, analyzing the blood clearance of cB72.3(gamma 1), cB72.3(gamma 4), and nB72.3 in mice, showed that the nB72.3 beta phase of clearance was slower than that of other MAb forms. However, when the pharmacokinetic patterns of these three MAbs forms were analyzed in monkeys, the cB72.3(gamma 1) and the nB72.3 showed similar clearance curves, while the cB72.3(gamma 4) showed a much slower plasma clearance. In view of the binding properties of nB72.3 and its ability to localize a range of carcinomas in clinical trials, the studies reported here demonstrate that the cB72.3(gamma 1) may serve as a potentially useful diagnostic and/or therapeutic reagent.     

Anti-idiotypic antibody facilitates scFv chimeric immune receptor gene transduction and clonal expansion of human lymphocytes for tumor therapy

Chimeric immune receptors (CIR) transduced into lymphocytes link target recognition by single chain antibody Fv (scFv) to activation through CD28/TCRzeta signaling. As surrogate antigens, anti-idiotypic antibodies may facilitate gene-transduction and clonal expansion of human lymphocytes for in vivo tumor therapy. The murine monoclonal antibody (MAb) 8H9 reacts with a novel antigen widely expressed on solid tumors. A CIR consisting of human CD8-leader sequence, 8H9-scFv, CD28 (transmembrane and cytoplasmic domains), and TCR-zeta chain was constructed, ligated into the pMSCVneo vector, and used to transfect the packaging line GP + envAM12 bearing an amphotropic envelope. Rat anti-idiotypic MAb 2E9 (IgG2a) was used to clone retroviral producer line as well as to expand gene-modified primary human lymphocytes. Sequential enrichments using either affinity chromatography or cell sorting using anti-idiotypic MAb 2E9 significantly improved the percentage of producer clones positive for surface 8H9-scFv and the efficiency of their supernatant in transducing the indicator cell line K562. By 3 weeks of in vitro culture, >95% of transduced primary human lymphocytes were CIR-positive. Upon periodic stimulation with 2E9, these lymphocytes underwent >10(6)-fold expansion by 6 months in culture. They mediated antigen-specific non-MHC restricted cytokine release and tumor cytotoxicity, and inhibited human xenograft engraftment in SCID mice. Anti-idiotypic antibody may provide a useful tool for optimizing gene transduction of CIR fusion constructs into primary human lymphocytes and their continual expansion in vitro.     

Production and characterization of a murine/human chimeric anti-idiotype antibody that mimics ganglioside.

The VL and VH from a murine anti-idiotypic antibody that mimics ganglioside have been cloned, sequenced, and expressed as a chimeric mouse/human IgG1 antibody. The chimeric antibody retained a binding specificity indistinguishable from the original murine antibody. The VH was a member of Vgam 3.8 family. The sequences are discussed in terms of ways in which proteins may mimic ganglioside epitopes.     

Selection of human single chain Fv antibody fragments binding and inhibiting Helicobacter pylori urease.

Single chain Fv antibody fragments (scFv) binding to purified Helicobacter pylori urease were selected from a nonimmune human antibody repertoire displayed on filamentous phage. After three rounds of screening on solid phase urease, 44 clones were found to bind the enzyme and four distinct scFv were identified by sequencing their heavy and light chain variable region genes (V(H) and V(L)). Two of the selected human scFv (scFv B4 and scFv D9) inhibited the activity of H. pylori urease with inhibitory constants (K(i)) of 7 and 2 microM, respectively. Their affinity (K(d)) for H. pylori urease as determined by surface plasmon resonance ranged from 17 to 42 nM. Both scFv were able to bind to urease present on the surface of living H. pylori organisms as demonstrated by flow cytometry analysis. The binding sites of scFv B4 and D9 were mapped by the use of two random hexapeptide libraries (X6 and CX6C) displayed on filamentous bacteriophage. The selected peptide sequences were shown to inhibit scFv binding to H. pylori urease and thus could be used in a vaccination strategy as epitopes mimicking (mimotopes) the region of urease recognized by these human scFv antibody fragments.     

Efficient in vivo targeting of malignant melanoma by single-chain Fv antibody fragments

Single-chain antibody fragments (scFvs) have superior pharmacokinetic and biodistribution characteristics compared with larger antibody fragments when used for radioimaging of human tumours. An scFv specific for malignant melanoma (B4 scFv) was tested in a mouse xenograft model for human melanoma and compared with the monoclonal antibody (MAb) LHM2. LHM2 is specific for the high molecular weight proteoglycan melanoma-associated antigen (HMW-MAA), while B4 scFv binds a novel melanoma-specific antigen. The B4 scFv was cleared rapidly from the circulation (t(1/2)alpha = 7 min) compared with LHM2 MAb (t(1/2)alpha = 37 min). However, the B4 scFv had an unusually long t(1/2)beta (437 min compared with 384 min for LHM2 MAb). Biodistribution studies showed that B4 accumulated specifically in the tumour grafts. Although non-specific accumulation in the liver, lung, spleen and blood was lower than with LHM2, non-specific accumulation of B4 in the kidney was higher. Despite this, the overall targeting efficiency in this study, as determined by tumour:normal tissue ratios, showed that B4 was superior to whole antibody. Immunoscintigraphy images showed good correlation with the biodistribution data for B4 and LHM2. This study represents the first use of an anti-melanoma scFv in vivo.     

A single-chain antibody fragment against human thyroglobulin: construction and evaluation of immunoreactivity

Smaller recombinant antibody fragments are at the forefront of in vivo diagnosis and therapy. These units possess better distribution and faster clearance than larger molecules. Among these, single chain antibody fragments (scFv) are emerging as credible alternatives. These proteins are shown to have same specificities and affinities for their antigens as the parental monoclonal antibody (MAb). We have attempted to produce scFv against human thyroglobulin (H-Tg) using anti-Tg secreting hybridoma cells and PCR-based cloning approach. Hybridoma secreting anti-Tg MAb B10IV was established. cDNA was prepared from hybridoma cells. The V(H) and V(L) genes were amplified and cloned. The gene sequences were submitted to Genebank database (accession nos. AJ508533 and AM072962, respectively.) V(L) and V(H) genes were then linked together with a linker peptide and successfully cloned in pET28a and expressed as His-tag fusion protein in expression host BL21 (DE3). The scFv protein from IPTG-induced cells was purified under native conditions by immobilized metal affinity chromatography on a Ni-NTA agarose column. The yield expressed in Escherichia coli was approximately 8 mg/L. ScFv could be labeled with (125)I and its immunoreactivity evaluated in radioassays. Although scFv demonstrated specific binding to H-Tg, the immunoreactivity was low (10.3%) compared to the parental MAb B10IV, which showed immunoreactivity of 37.27%. Inhibition radioassays exhibited that scFv and MAb interact with the same epitope on the target antigen, indicating its specificity.     

Characterization of a novel monoclonal antibody with restricted specificity to the free beta 2 integrin alpha M CD11b subunit

Leukocyte cell surface expression and function of beta2 integrins require the intracellular association of alpha subunits, CD11a, b, c, d, respectively, with the common CD18 beta2 subunit. We have raised and characterized a murine MAb -- ME-MDF -- directed against the low affinity form of the human integrin alphaM subunit CD11b A-domain. MAb ME-MDF is an IgG2a that has a kDa of 2,45461 +/- 0.12 x 10(-9) M. MAb ME-MDF recognizes both the low and high affinity forms of the CD11b A-domain. Flow cytometry showed that ME-MDF does not recognize the heterodimeric CD11b/CD18 molecule at the surface of polymorphonuclear cells and the human monoblast cell line U937. Western blot analysis of U937 cell line cell surface proteins demonstrated that ME-MDF reacts specifically with the CD11b subunit but does not react with the heterodimeric CD11b/CD18 complex, a feature that differentiates it from other CD11b A-dom-specific MAbs. These observations suggest that ME-MDF recognizes an epitope that is involved in the association of the two subunits and hence is not accessible within the heterodimeric form of the CD11b/CD18 molecule. These data show that the CD11b A-dom engages not only the MIDAS but also the ME-MDF-specific epitope to associate with the CD18 subunit. We have also constructed, and expressed in the yeast Pichia pastoris, the corresponding recombinant scFv form of MAb ME-MDF and characterized the CDRs. MAb ME-MDF is characterized by short VH and VL CDR3. MAb ME-MDF and/or its recombinant scFv form would be very useful to study the structural basis of the association between the alpha and beta2 integrin subunits and to investigate the possibility of modulating CR3 cell surface expression by preventing subunit association.     

Establishment and characterization of Epstein-Barr virus gp350-expressing transfected human lymphoid (Raji) cell clones.

Gp350, a late Epstein-Barr-virus (EBV) glycoprotein expressed on both the envelope of viral particles and EBV-producing cells, is also the candidate for the development of an anti-EBV subunit vaccine. This glycoprotein is thought to play an important role in anti-EBV immunity. However, studies on the role of this viral antigen in cellular cytotoxicity and other immune functions have been hampered by the lack of a suitable model expressing gp350. We describe here a study in which we successfully transfected a gp350-negative cell line resistant to natural-killer(NK)-cell activity (i.e., Raji) with a recombinant plasmid (pZIP-MA) containing the EBV-gp350 and the neomycin resistance gene. Three clones with a stable and strong expression of gp350 on their surface membrane, as demonstrated using a gp350-specific (i.e., 2LI0) monoclonal antibody (MAb) were isolated, characterized and used as targets in an antibody-dependent cellular cytotoxicity (ADCC) assay. However, gp350 expression on 2 of the 3 isolated clones was not recognized by an anti-gp350 MAb (72AI) which is specific to a unique gp350 epitope with a dual function (i.e., involved in both EBV binding to its target cell receptors and in inducing virus-neutralizing antibody). We have also found that gp350 expression on our 3 selected clones does not affect EBV-receptor (CR2) density. Our model of gp350-expressing, NK-cell-activity-resistant targets revealed very useful in determining that gp350 serves as a target antigen for EBV-specific ADCC. These gp350-expressing cell clones appear to represent a valuable tool for diagnostic purposes (i.e., for detecting and titrating gp350 antibodies in patients with EBV-associated diseases). Our approach should also prove useful for studying the expression of other cell-surface-associated viral and tumor antigens and their role in specific cellular immunity and immunosurveillance.     

Human anti-CD30 recombinant antibodies by guided phage antibody selection using cell panning

In various clinical studies, Hodgkin's patients have been treated with anti-CD30 immunotherapeutic agents and have shown promising responses. One of the problems that appeared from these studies is the development of an immune response against the nonhuman therapeutics, which limits repeated administration and reduces efficacy. We have set out to make a recombinant, human anti-CD30 single-chain variable fragment (scFv) antibody, which may serve as a targeting moiety with reduced immunogenicity and more rapid tumour penetration in similar clinical applications. Rather than selecting a naive phage antibody library on recombinant CD30 antigen, we used guided selection of a murine antibody in combination with panning on the CD30-positive cell line L540. The murine monoclonal antibody Ki-4 was chosen as starting antibody, because it inhibits the shedding of the extracellular part of the CD30 antigen. This makes the antibody better suited for CD30-targeting than most other anti-CD30 antibodies. We have previously isolated the murine Ki-4 scFv by selecting a mini-library of hybridoma-derived phage scFv-antibodies via panning on L540 cells. Here, we report that phage display technology was successfully used to obtain a human Ki-4 scFv version by guided selection. The murine variable heavy (VH) and light (VL) chain genes of the Ki-4 scFv were sequentially replaced by human V gene repertoires, while retaining only the major determinant for epitope-specificity: the heavy-chain complementarity determining region 3 (CDR3) of murine Ki-4. After two rounds of chain shuffling and selection by panning on L540 cells, a fully human anti-CD30 scFv was selected. It competes with the parental monoclonal antibody Ki-4 for binding to CD30, inhibits the shedding of the extracellular part of the CD30 receptor from L540 cells and is thus a promising candidate for the generation of anti-CD30 immunotherapeutics.     

Development and characterization of a syngeneic monoclonal antibody to a rat mammary tumor metastasis-associated mucin-like cell-surface antigen (gp580)

A rat hybridoma producing IgM monoclonal antibody (MAb) GP21:56 was generated with specificity for a high-molecular-weight, mucin-like glycoprotein (gp580) present on highly metastatic 13762NF rat mammary adenocarcinoma cells. The hybridoma was made by fusing rat Y3 Ag1.2.3 myeloma cells with spleen cells from a rat immunized i.d. with purified gp580. The gp580 appeared to be of low immunogenicity in syngeneic F344 rats because a total of 27 fusions were required to produce one hybridoma with specificity for this glycoprotein. Immunoblotting of purified gp580 after electrophoresis in 1% agarose and antibody-binding assays using purified gp580 linked to microtiter plates confirmed that MAb GP21:56 bound specifically to gp580. Other MAbs made against breast mucins were negative for gp580 reactivity. Enzyme-linked immunoabsorbent assays (ELISA) and radiolabelled antibody binding assays demonstrated that MAb GP21:56 bound to 13762NF adenocarcinoma cell lines and clones in relation to their spontaneous metastatic potentials; significantly more MAb GP21:56 bound to highly metastatic MTLn3 cells than to low metastatic MTC cells, and MAb GP21:56 showed little reactivity towards the majority of other cell lines tested, whether of rodent or of human origin. Kinetic binding studies indicated that MAb GP21:56 does not have a high affinity for gp580 but, once bound, it shows high avidity for this sialogalactoprotein. Localization studies using frozen tissue sections of 13762NF tumors indicated that MAb GP21:56 reacts with tumor cells grown in vivo in an analogous manner to in vitro cultured cells. Using immunoperoxidase techniques, less than 50% of the highly metastatic MTLn3 tumor cells were stained, whereas approximately 20% of the intermediate metastatic MTF7 and MTLn2 cells and less than 10% of low metastatic MTC and MTPa cells were stained with MAb GP21:56. The cell-to-cell reactivity was heterogeneous and mainly associated with the tumor-cell surface and extracellular matrix.     

Single-chain variable fragment antibody against human aspartyl/asparaginyl beta-hydroxylase expressed in recombinant Escherichia coli

The human aspartyl beta-hydroxylase (HAAH) is a highly conserved enzyme that hydroxylates epidermal growth factor-like domains in transformation-associated proteins. Previous studies showed that the gene of HAAH was overexpressed in many human malignancies. In the present study, the HAAH-specific single-chain variable fragment (ScFv) antibody was produced in recombinant Escherichia coli. The variable regions of the genes of the heavy chain (VH) and light chain (VL) cloned from the hybridoma cells G3/F11 were connected with a flexible linker using an overlap extension polymerase chain reaction. Nucleotide sequence analysis revealed that the anti-HAAH VH was a member of the VH V gene family and the VL gene belonged to the Vκ gene family VI subgroup. Extensive efforts to express the functional ScFv antibody in E. coli have been made by using two different prokaryotic expression vectors-pHEN1 and pET-16b-to compare the expression level and solubility of the antibody. The recombinant pHEN1/E1-anti-HAAH vector could express soluble ScFv, although the yield was only 7.8% of the total cellular protein. However, the pET-16b/E2-anti-HAAH vector produced the ScFv as inclusion bodies inside the host cytoplasm, although the expression level of the antibody was quite high (28.5% of the total cellular protein). Soluble ScFv antibody produced by pHEN1/E1-anti-HAAH was characterized for its antigen-binding characteristics. Its antigen affinity as antibody was measured by indirect enzyme linked immunosorbent assay analysis and proved to have high binding activity to the antigen HAAH.