Site-specific attachment to recombinant antibodies via introduced surface cysteine residues

Many diagnostic and therapeutic applications of monoclonal antibodies require the covalent linking of effector or reporter molecules to the immunoglobulin polypeptides. Existing methods generally involve the non-selective modification of amino acid side chains, producing one or more randomly distributed attachment sites. This results in heterogeneous labelling of the antibody molecules and often to a decrease in antigen-binding due to the modification of residues close to the antigen-binding site. We report a novel strategy for site-specifically labelling antibodies through surface cysteine residues. Examination of molecular structures was used to identify amino acids of the CH1 domain of the IgG heavy chain that were accessible to solvent but not to larger molecules. Site-directed mutagenesis was used to substitute cysteine residues at these positions in the heavy chain of a mouse/human chimaeric version of the tumour-binding monoclonal antibody, B72.3. Expression of the modified antibody genes in mammalian cells yielded correctly assembled proteins that had thiol groups in pre-determined positions and showed no loss of antigen-binding activity. One of the mutants was used to demonstrate the site-specific attachment of a radio-iodinated ligand to the chimaeric B72.3 antibody.     

Crystallization and preliminary X-ray diffraction study of a chimaeric Fab' fragment of antibody binding tumour cells

Crystals have been obtained of a chimaeric Fab' fragment that binds to a tumour-associated mucin-like glycoprotein TAG72. The Fab' fragment comprises the variable heavy and light-chain domains of a murine monoclonal antibody, B72.3, coupled to human gamma 4 and kappa constant regions. The crystals are orthorhombic and belong to the space group P2(1)2(1)2(1), with unit cell dimensions a = 67.9 A, b = 94.2 A and c = 208.8 A. Diffraction to 2.6 A resolution was observed using synchrotron radiation. Despite the acute radiation sensitivity of the crystals a full native data set has been collected using the Weissenberg camera at the Photon Factory synchrotron. These data will be used for molecular replacement calculations in an attempt to elucidate the structure of this chimaeric Fab' fragment.     

Comparative biodistribution and antibody-dependent cellular cytotoxicity of native and heavy chain chimeric antibody.

We have recently chimerized the heavy chain of the pan-carcinoma monoclonal antibody (mAb) B72.3. Studies were undertaken to compare the IgG1 chimeric antibody, B72.3-1-3 with native murine B72.3 (nB72.3). Using fluorescence-activated cell sorting analysis, B72.3-1-3 demonstrated specific binding to fresh LS174T tumor cells. Biodistribution of 131I B72.3-1-3 was similar to 131I nB72.3 in nude mice bearing LS174T xenografts. Peak radiolocalization indices were noted on day 6 for B72.3-1-3 and day 8 for nB72.3. Both antibodies were capable of imaging LS174T tumors by radioimmunoscintigraphy. Antibody-dependent cellular cytotoxicity of LS174T by human peripheral blood lymphocytes was tested in 8h 51Cr release assays. With either no antibody or nB72.3, lymphocytes were not capable of killing LS174T cells. However, B72.3-1-3 at a concentration of 5 and 50 micrograms/ml mediated significant lysis of tumor cells by human lymphocytes. These results suggest that chimeric antibodies retain their binding properties to tumor cells and display biodistribution patterns similar to their unmodified counterparts. Such modifications may reduce the deleterious human antimouse antibody response to murine mAbs as well as augment antibody-dependent cellular cytotoxicity of tumor cells by human effectors.     

Autoradiographic analysis of monoclonal antibody distribution in human colon and breast tumor xenografts

Summary The targeting of monoclonal antibodies to human tumor xenografts in nude mice was investigated by analysis of the cellular distribution of two radioiodinated monoclonal antibodies, B6.2 and B72.3, which recognize different tumor-associated antigens. The time course of distribution of each antibody within Clouser human mammary carcinoma (B6.2 positive, B72.3 negative) and LS174T human colorectal carcinoma (B6.2 positive, B72.3 positive) following i. v. injections was compared using autoradiographic techniques, which were also used to determine the pattern of binding after in vitro incubation with radiolabeled antibody. Both in vivo and in vitro localization of ¹²⁵I-B72.3 in LS174T were characterized by the binding of antibody to antigen-rich mucin globules. In contrast, in vivo localization of B6.2 was restricted to groups of cells in well vascularized regions. Thus, the in vivo accumulations of B6.2 and B72.3 although quantitatively similar showed very different spatial distributions within LS174T tumors. The in vivo binding of B6.2 in Clouser tumors was restricted to small clusters of cells scattered fairly evenly throughout the tumor. There was no evidence for the presence of such antigen-rich foci after in vitro incubation of tumor sections with B6.2 suggesting that heterogeneity of regional uptake may be due to differences in antibody delivery. This type of information may provide a rational basis for the selection of appropriate therapeutic isotopes for radioimmunotherapy studies using these and other tumor models.     

Class II genes of miniature swine. IV. Characterization and expression of two allelic class II DQB cDNA clones

Two cDNA clones coding for allelic miniature swine MHC class II Ag DQB chains have been isolated, characterized, and shown to be expressed after transfection into mouse fibroblasts. The two alleles differ at the nucleotide level by an overwhelming proportion of replacement substitutions, suggesting the influence of selection for polymorphism. Most of the resulting predicted amino acid replacements are in regions commonly polymorphic in mouse Ab and human DQB sequences, corresponding to the predicted Ag recognition site. Nucleotide and amino acid sequence comparisons to homologous mouse and human sequences show more similarity between swine and man than between either swine and mouse or man and mouse. This tendency is most pronounced when comparing the 3' untranslated regions. However, an examination of unique cross-species sharing of amino acid residues suggests a closer relationship between both man and miniature swine and man and mouse than between miniature swine and mouse. The simplest explanation we can envision for these findings is that the mouse DQB gene homologue (Ab) has been subject to a higher substitution rate than either swine or human DQB genes. An additional cytoplasmic exon expressed in mouse Ab gene products and in putative human DQB2 gene products is lacking in both swine and human DQB cDNA clones. Its absence suggests either that the expression of this exon in mouse Ab genes was activated after mammalian speciation or that the expression of this exon was independently inactivated in swine DQB and human DQB1 genes. Alternatively, the mouse Ab gene may be derived from the same primordial gene as human DQB2, whereas the pig DQB gene may be derived from the same primordial gene as the human DQB1 gene.     

A genetically engineered murine/human chimeric antibody retains specificity for human tumor-associated antigen

Chimeric immunoglobulin genes were constructed by fusing murine variable region exons to human constant region exons. The ultimate goal was to produce an antibody capable of escaping surveillance by the human immune system while retaining the tumor specificity of a murine monoclonal. The murine variable regions were isolated from the functionally expressed kappa and gamma 1 immunoglobulin genes of the murine hybridoma cell line B6.2, the secreted monoclonal antibody of which reacts with a surface antigen from human breast, lung, and colon carcinomas. The kappa and gamma 1 chain fusion genes were co-introduced into non-antibody producing murine myeloma cells by electroporation. Transfectants that produced murine/human chimeric antibody were obtained at high frequency as indicated by immunoblots probed with an antisera specific for human immunoglobulin. Enzyme-linked immunoabsorbent assay analysis demonstrated that this chimeric antibody was secreted from the myeloma cells and retained the ability to bind selectively to membrane prepared from human tumor cells. The chimeric immunoglobulin was also shown by indirect fluorescence microscopy to bind to intact human carcinoma cells with specificity expected of B6.2. The ability of chimeric antibody to recognize human tumor-associated antigen makes feasible a novel approach to cancer immunotherapy.     

抗人前列腺干细胞抗原单克隆抗体可变区基因的5＇RACE扩增及序列分析

目的：克隆并分析抗人前列腺干细胞抗原单克隆抗体轻链和重链的可变区基因。方法：从分泌抗人前列腺干细胞抗原单克隆抗体的杂交瘤细胞株中提取总RNA,根据小鼠IgG恒定区序列设计特异性引物,通过5’RACE法扩增其轻链和重链的可变区基因,克隆入pMD18-T载体,测序并分析其可变区序列。结果：3株抗人前列腺干细胞抗原单克隆抗体的重链可变区基因序列全长均为423bp,编码141个氨基酸残基;轻链可变区基因序列全长均为393bp,编码131个氨基酸残基;在GenBank中对氨基酸序列进行比对分析,均符合小鼠IgG可变区基因的特征;根据Kabat法则对3株抗体轻链和重链可变区氨基酸序列进行分析,确定了3个抗原互补决定区、4个框架区和前导肽。结论：通过5＇RACE法得到了3株抗人前列腺干细胞抗原单克隆抗体轻链与重链可变区基因,为进一步研究抗体三维结构、人源化改造奠定了基础。     

Characterization of neutralizing mouse‐human chimeric and shuffling antibodies against botulinum neurotoxin A

Mouse‐human chimeric monoclonal antibodies that could neutralize botulinum neurotoxins were developed and an attempt was made to establish mouse hybridoma cell clones that produced monoclonal antibodies that neutralized botulinum neurotoxin serotype A (BoNT/A). Four clones (2–4, 2–5, 9–4 and B1) were selected for chimerization on the basis of their neutralizing activity against BoNT/A and the cDNA of the variable regions of their heavy (V_H) and light chains (V_L) were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO‐DG44 cells were transfected with these plasmids and mouse‐human chimeric antibodies (AC24, AC25, AC94 and ACB1) purified to examine their binding and neutralizing activities. Each chimeric antibody exhibited almost the same capability as each parent mouse mAb to bind and neutralize activities against BoNT/A. From the chimeric antibodies against BoNT/A, shuffling chimeric antibodies designed with replacement of their V_H or V_L domains were constructed. A shuffling antibody (AC2494) that derived its V_H and V_L domains from chimeric antibodies AC24 and AC94, respectively, showed much higher neutralizing activity than did other shuffling antibodies and parent counterparts. This result indicates that it is possible to build high‐potency neutralizing chimeric antibodies by selecting and shuffling V_H and V_L domains from a variety of repertoires. A shuffling chimeric antibody might be the best candidate for replacing horse antitoxin for inducing passive immunotherapy against botulism.     

Technetium labeling of monoclonal antibodies with functionalized BATOs. 1. TcCl(DMG)3PITC

BATO (boronic acid adduct of technetium dioximes) complexes, TcCl(dioxime)3BR, were prepared in which the boron substituent (R) was the protein-reactive m-phenyl isothiocyanate (PITC). The 99TcCl(dioxime)3PITC complexes [dioxime = dimethylglyoxime (DMG) or cyclohexanedione dioxime (CDO)] were prepared from 99Tc(dioxime)3(mu-OH)SnCl3 and characterized. The X-ray crystal structure of 99TcCl(DMG)3PITC was determined. The 99mTc complexes were prepared from 99mTcO4- in a process using a freeze-dried kit, either in a one-step procedure or via 99mTcCl(dioxime)3. Initial labeling studies with 99mTcCl(dioxime)3PITC were performed on glycine and polylysine and, subsequently, on mouse IgG and the B72.3 monoclonal antibody. Covalent attachment of 99mTcCl(DMG)3PITC to B72.3 was demonstrated by SDS-PAGE electrophoresis. B72.3 labeled with 99mTcCl(DMG)3PITC displayed high binding to a TAG 72 affinity column and had a distribution in normal mice similar to that reported for iodine-labeled B72.3.     

TP-PCR法构建抗人CD28嵌合抗体双启动子昆虫杆状病毒重组转移载体

为构建能同时表达抗人CD28嵌合重链和嵌合轻链基因的双启动子杆状病毒转移载体,利用PCR法扩增出抗人CD28鼠源性单抗的可变区基因和人IgG1的恒定区基因,再采用一种不需要限制性核酸内切酶和连接酶的新方法——三引物PCR（TP-PCR）法将两者拼接后分别插入杆状病毒转移载体pAcUW3的ph和p10启动子后,并通过酶切、电泳、PCR扩增和测序对重组体进行了鉴定。研究结果表明,TP-PCR法快速、方便、准确,无需设计外源的DNA序列,就能构建完好的融合表达基因。该转移载体的构建成功为嵌合抗体基因在昆虫细胞中的表达奠定了基础。     

Genomic organization of adrenergic and serotonin receptors in the mouse: linkage mapping of sequence-related genes provides a method for examining mammalian chromosome evolution.

Five sequence-related genes encoding four adrenergic receptors and a serotonin receptor were localized to specific regions of four mouse chromosomes with respect to 11 other genetic markers. Linkage was established by the analysis of the haplotypes of 114 interspecific backcross mice. Adra2r (alpha 2-C10) and Adrb1r (beta 1) receptors mapped to the distal region of mouse chromosome 19. These genes were separated by 2.6 +/- 1.5 cM in a segment of mouse chromosome 19 that has a similar organization of these genes on the long arm of human chromosome 10. The Adra1r (alpha 1B), Adrb2r (beta 2), and Htra1 (5HT1A) genes mapped to proximal mouse chromosome 11, proximal mouse chromosome 18, and distal mouse chromosome 13, respectively. The organization of genes linked to these loci on regions of the three mouse chromosomes is consistent with the organization of homologous human genes on human chromosome 5. These findings further define the relationship of linkage groups conserved during the evolution of the mouse and human genomes. We have identified a region that may have been translocated during evolution and suggest that the human genomic organization of adrenergic receptors more closely resembles that of a putative primordial ancestor.     

Construction of chimaeric genes for mapping a surface-exposed epitope on the pilus of non-typable Haemophilus influenzae strain M37

A murine monoclonal antibody (mAb), designated 3H12, reacts with a surface-exposed conformational epitope on the pilus of non-typable Haemophilus influenzae strain M37. This antibody does not recognize the related pilus from H. influenzae type b, strain MinnA. Although mAb 3H12 does not recognize strain M37 pilin on Western blots, mAb 3H12 recognizes the recombinant M37 pilin protein expressed by Escherichia coli. In order to map the epitope recognized by mAb 3H12, we constructed a series of chimaeric genes. The chimaeric genes were expressed in E. coli and the chimaeric proteins characterized with respect to their reactivity with mAb 3H12. Residues between 37 and 100 of the M37 pilin protein are essential for the expression of the mAb 3H12 epitope. Residues in the carboxyl half of the M37 protein enhance the reactivity of mAb 3H12 when expressed in the presence of residues 37-100. Construction of chimaeric genes may provide a general methodology for mapping of conformational epitopes expressed by one of a related pair of proteins.     

The sulfatase gene family: cross-species PCR cloning using the MOPAC technique.

Several human sulfatase cDNAs have recently been cloned, revealing highly conserved domains of protein similarity. We have used this information for the isolation of sulfatase genes in different species using the polymerase chain reaction (PCR). Degenerate oligonucleotide primers corresponding to these regions of identity among human arylsulfatases A, B, and steroid sulfatase (ARSA, ARSB, and STS) were designed. The primers were used in the PCR amplification of reverse transcribed RNA (RT-PCR) from multiple tissues in human and mouse. Amplification products were obtained from mouse liver and from human liver, lymphoblasts, kidney, intestine, heart, muscle, and brain cDNA samples. Each of the PCR products was subcloned into a plasmid vector, and several subclones were characterized by colony hybridization and DNA sequencing. All the previously identified human ARSA, ARSB, and STS were found among our clones, indicating the power of the technique. Sequence analysis of two mouse clones showed high degrees of homology with the human ARSA and ARSB sequences, respectively, and likely represent the murine homologues of these enzymes. These are the first sulfatase genes isolated in the mouse. A murine equivalent for STS could not be identified, suggesting its strong diversity from the human homologue.     

Characterization of a mouse/human chimeric monoclonal antibody (17-1A) to a colon cancer tumor-associated antigen

Mouse monoclonal antibody 17-1A is specific for an antigen expressed on cells of human gastrointestinal malignancies and has been used in radioimmune imaging and therapy trials for patients with colon and pancreatic cancer. The cell line SG3/5 was generated by transfection of a nonproducing mouse myeloma line (SP2/0) with a chimeric gene construct composed of variable regions from the mouse 17-1A immunoglobulin (gamma 2a, kappa) and constant regions of human k and gamma 3 immunoglobulin genes. The secreted immunoglobulin was bound by mouse monoclonal antibodies to human IgG(Fc) and IgG3 but not by staphylococcal protein A. Gel filtration HPLC profiles of purified chimeric antibody were similar to normal human IgG3 but quite different from native 17-1A and normal human IgG1, 2, and 4. Native and chimeric 17-1A had similar patterns of reactivity with colon cancer, other adenocarcinoma, and leukemic cell lines. Competitive inhibition documented that native and chimeric 17-1A had identical capacities to inhibit radiolabeled native 17-1A binding to colon cancer cell lines. Thus, the chimeric 17-1A exhibits molecular characteristics of normal human IgG3 but retains the specificity and binding affinity of the native 17-1A murine monoclonal antibody. The native and chimeric 17-1A mediated similar modest degrees of human lymphocyte and monocyte ADCC in a 4-hr 51Cr release assay, and both failed to mediate complement lysis of colon carcinoma cell lines in the presence of human complement. This human/mouse chimeric monoclonal antibody may be a good candidate for use in clinical trials because it retains the tumor antigen specificity and human effector cell recognition of the native 17-1A, would presumably have a fivefold to 10-fold longer circulating half-life in man, and should be considerably less immunogenic as compared with native murine immunoglobulins.     

Identification of novel simple sequence length polymorphisms (SSLPs) in mouse by interspersed repetitive element (IRE)-PCR.

Interspersed repetitive element (IRE)-PCR is a useful method for identification of novel human or mouse sequence tagged sites (STSs) from contigs of genomic clones. We describe the use of IRE-PCR with mouse B1 repetitive element primers to generate novel, PCR amplifiable, simple sequence length polymorphisms (SSLPs) from yeast artificial chromosome (YAC) clones containing regions of mouse chromosomes 13 and 14. Forty-two IRE-PCR products were cloned and sequenced from eight YACs. Of these, 29 clones contained multiple simple sequence repeat units. PCR analysis with primers derived from unique sequences flanking the simple sequence repeat units in seven clones showed all to be polymorphic between various mouse strains. This novel approach to SSLP identification represents an efficient method for saturating a genomic interval with polymorphic genetic markers that may expedite the positional cloning of genes for traits and diseases.     

Conjugation of metallothionein to a murine monoclonal antibody

A method of conjugation of the metal-binding protein, metallothionein, to an anticarcinoma murine monoclonal antibody, B72.3, and its F(ab')2 fragment has been developed utilizing the heterobifunctional crosslinking reagent, succinimidyl 4-(N-maleimidomethyl)-cyclohexane 1-carboxylate. This crosslinking reagent is first reacted with the free amines on the immunoglobulin. After removal of unreacted crosslinker, conjugation is affected through a sulfhydryl group on metallothionein. Under the conditions employed all immunoglobulin aggregates contained metallothionein. The degree of undesired aggregation is directly proportional to the number of metallothioneins attached to the immunoglobulin. This aggregation can be controlled by the amount of crosslinker and metallothionein presented to the immunoglobulin. The immunoglobulin conjugate retains full immunoreactivity and can be readily purified from the unreacted metallothionein and high molecular weight aggregates. The metallothionein-B72.3 conjugate functions as an efficient and stable chelator of radiometals. Thus metallothionein-monoclonal antibody conjugates have potential utility in cancer diagnosis and therapy.     

Characterization and humanization of a monoclonal antibody that neutralizes human leukocyte interferon: a candidate therapeutic for IDDM and SLE

We have developed a panel of murine monoclonal antibodies that recognize human interferon alpha. One of these mononclonal antibodies binds and neutralizes, with high affinity, all of seven tested recombinant human interferon alphas. This mononclonal antibody also neutralizes the interferon activity present in two independent pools of interferon alphas prepared following stimulation of human peripheral blood leukocytes. The complementary determining regions from this murine mononclonal antibody were transferred to a human IgG2 heavy chain and to a human kappa1 light chain. In addition, six (heavy chain) and two (light chain) amino acids were transferred from the framework regions. This generated a humanized mononclonal antibody that retained the specificity of the mouse parent. The humanized anti-interferon alpha antibody is a candidate therapeutic for those diseases, such as insulin-dependent diabetes, systemic lupus erythematosis, psoriasis and Crohn's disease, which are all characterized by pathological expression of interferon alpha.