A murine–human chimeric IgG antibody against vascular endothelial growth factor receptor 2 inhibits angiogenesis in vitro

Vascular endothelial growth factor receptor 2 (VEGFR2) has been reported to play an important role in angiogenesis and tumorigenesis. A murine anti-VEGFR2 mAb (A8H1) has been established in a previous study. To reduce the incompatibility of the murine mAb for human use, the chimeric anti-VEGFR2-IgG was developed by genetic recombination of the variable regions of the A8H1 antibody and the constant regions of human IgG, and was expressed in Sp2/0 cells transfected with the two recombinant vectors containing the heavy chain and the light chain regions. After screening, clone 2F12 was selected and was found to stably secrete the murine–human chimeric anti-VEGFR2-IgG (coded 2F12). This chimeric IgG maintained the specificity and the affinity of the parental murine antibody against VEGFR2, and effectively identified VEGFR2 expressed on the surface of HUVECs and BEL-7402 cells. Furthermore, the 2F12 antibody demonstrated inhibition of angiogenesis in vitro, such as proliferation, migration, invasion and tube formation of HUVECs. This murine–human chimeric IgG may be considered for further development as an anti-angiogenesis and anti-tumor agent.     

Antitumour activity of a chimeric antibody against the leucocyte antigen CD48

Preclinical studies with the murine anti-CD48 antibody, mHuLym3 (IgG2a) have shown it to be a potentially useful therapeutic reagent in the treatment of human leukaemia and lymphoma. For clinical use, humanised antibodies can have a number of advantages over their original murine version, including mediation of higher effector cell function with human cells, longer serum half-life and lower immunogenicity. In this study, we have produced a mouse/human chimeric HuLym3 antibody (cHuLym3) where the murine antibody constant regions have been replaced with human constant regions. We report the production and preclinical characterisation of the antibody, cHuLym3, with potent in vitro and in vivo antitumour activity. The genes encoding the variable heavy and light chains were amplified by the polymerase chain reaction, sequenced and cloned into eukaryotic expression vectors containing the human light- and heavy-chain constant regions (κ and IgG1). The chimeric and murine HuLym3 antibodies had similar cell-binding specificity and affinity. In the human Raji cell severe combined immunodeficient mouse model the i.v. injection of cHuLym3 and mHuLym3 produced similar antitumour responses. Doses of cHuLym3 and mHuLym3 (100 μg) on days 1, 2 and 4 after i.v. Raji cell injection produced a 40% longer time to hind-leg paralysis than when a control antibody was used. cHuLym3 had more potent activity than mHuLym3 in antibody-dependent cellular cytotoxicity (ADCC) assays in vitro, with human peripheral blood mononuclear cells as effectors. Up to 60% specific cell lysis was observed with cHuLym3 in ADCC assays. These properties suggest that anti-CD48 antibodies may be useful in the treatment of a number of diseases including lymphoid leukaemias and lymphoma. Received: 5 May 1999 / Accepted: 12 August 1999     

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.     

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。     

Human monocytes and U937 cells bear two distinct Fc receptors for IgG

Several convergent lines of evidence have led us to propose that human monocytes and the related cell line U937 possess a second class of IgG Fc receptor (FcR) in addition to the 72-Kd high affinity FcR previously described. IgG affinity purification from detergent lysates of surface radiolabeled U937 cells has yielded both a 40-Kd IgG-binding membrane protein (p40) and the 72-Kd FcR protein. By the same procedure, only the p40 was isolated from the erythroblast cell line K562 and from the B cell lines, Daudi and Raji. Serologic cross-reactivity between the 40-Kd FcR on U937 and Daudi cells was demonstrated using a goat anti-FcR antiserum. A murine (m) monoclonal antibody, raised against the FcR of K562 cells, precipitated the 40-Kd FcR from lysates of U937 and K562 cells but not from Daudi or Raji cells. This antibody, referred to as anti-p40 (IV.3), selectively inhibited the binding of murine IgG1-coated erythrocytes to U937 cells, whereas monomeric human IgG selectively inhibited binding of human anti-Rh(D)-coated erythrocytes to U937 cells. Both Daudi and U937 cells mediated mIgG1 anti-T3 (Leu-4)-induced stimulation of T lymphocytes. In contrast, mIgG2a anti-T3 (OKT3)-induced stimulation was supported effectively by U937 cells but only modestly by Daudi cells. Intact IgG or Fab fragments of anti-p40 (IV.3) blocked mIgG1 anti-T3 (Leu-4) stimulation but not mIgG2a anti-T3 (OKT3) stimulation of T cells; monomeric human IgG blocked only OKT3-induced stimulation. The simplest interpretation of these results is that human monocytes and U937 cells bear two classes of IgG FcR, one of 72 Kd and the other, as described above, of 40 Kd. We propose that the 72-Kd FcR mediates rosette formation with red cells coated by human anti-Rh IgG as well as T cell stimulation by mIgG2a anti-T3 (OKT3) and that the 40-Kd FcR mediates rosette formation with erythrocytes bearing mIgG1 as well as T cell stimulation by mIgG1 anti-T3 (Leu-4). Furthermore, we suggest that these two FcR are the human homologues of the murine macrophage FcRI (binding mIgG2a) and FcRII (binding mIgG2b/1).     

Chimeric and humanized antibodies with specificity for the CD33 antigen.

L and H chain cDNAs of M195, a murine mAb that binds to the CD33 Ag on normal and leukemic myeloid cells, were cloned. The cDNAs were used in the construction of mouse/human IgG1 and IgG3 chimeric antibodies. In addition, humanized antibodies were constructed which combined the complementarity-determining regions of the M195 antibody with human framework and constant regions. The human framework was chosen to maximize homology with the M195 V domain sequence. Moreover, a computer model of M195 was used to identify several framework amino acids that are likely to interact with the complementarity-determining regions, and these residues were also retained in the humanized antibodies. Unexpectedly, the humanized IgG1 and IgG3 M195 antibodies, which have reshaped V regions, have higher apparent binding affinity for the CD33 Ag than the chimeric or mouse antibodies.     

Construction and characterization of the chimeric antibody 8C11 to the hepatitis E virus

Homodimers of the truncated hepatitis E virus (HEV) capsid proteins, E2 and p239, were conformed to model the dominant antigenic determinants of HEV. Using E2 as an immunogen, two neutralizing monoclonal antibodies (mAbs), namely 8C11 and 8H3, were produced. We constructed a mouse-human chimeric antibody derived from 8C11 and its expression in Chinese hamster ovary (CHO) cells. cDNAs encoding variable regions of heavy and light chains were isolated from hybridoma cells and inserted into mammalian expression vectors containing cDNA of human gamma-1 and kappa constant regions, respectively. The vectors were then cotransfected into CHO cells, and a stable cell line was established. Results from indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showed that the chimeric antibody was assembled correctly to the native IgG molecule and could be secreted from the cells. Similar to the original mAb, the expressed chimeric antibody displayed HEV antigen-binding activity and an enhancement effect on 8H3 binding to HEV antigen. The chimeric antibody could specifically inhibit the binding of p239 to HepG2 cells and compete with HEV IgG in positive serum by antibody-competitive ELISA. The chimeric antibody is expected to be less immunogenic in human and more suitable for antibody therapy of hepatitis E.     

Synthesis and functional characterization of a recombinant monoclonal antibody directed against the alpha-chain of the human interleukin-2 receptor.

We have determined the sequence of the light and heavy chains of mAb 3G-10 (IgG1), a monoclonal antibody competing with interleukin 2 (IL2) for binding to the human IL2 receptor Tac protein. The antibody-encoding genes were chimerized by introducing splice donor and part of the intron sequences into the cDNA and subsequently linking it to the constant parts of the human IgG1 gene. The chimeric mAb was produced in mouse myeloma cells and purified. Murine and chimeric mAbs showed similar properties with respect to inhibition of T-cell proliferation. In contrast to its murine counterpart, the chimeric mAb exhibited Ab-dependent cellular cytotoxicity and, when combined with an Ab recognizing a different epitope on the IL2 receptor Tac protein, was able to activate human complement. The chimerized mAb might therefore have improved therapeutic efficacy.     

Enhancement of antibody-dependent cytotoxicity with a chimeric anti-GD2 antibody

A mouse/human chimeric antibody (ch14.18) was developed that reacts with the disialoganglioside GD2 on the surface of tumor cells of neuroectodermal origin. ch14.18 has the constant regions of a human IgG1 antibody and was expressed in a murine hybridoma. This antibody was produced in tissue culture at concentrations up to 180 mg/liter of spent culture fluid. ch14.18 was characterized and compared to 14.G2a, a murine mAb against GD2 of IgG2a isotype derived from the same parental hybridoma as ch14.18. Scatchard plot analysis of data from saturation binding studies on M21 melanoma cells showed identical binding for ch14.18 and 14.G2a. Indirect immunofluorescence revealed the same staining pattern for ch14.18 and 14.G2a on different melanoma cell lines. Both antibodies were equally capable of targeting M21 xenografts in athymic nude mice. ch14.18- and 14.G2a-activated human C-mediated cytolysis of melanoma cell; however, ch14.18-mediated antibody-dependent cytotoxicity of human effector cells against melanoma cells 50- to 100-fold more efficiently than 14.G2a.     

Humanization of a mouse monoclonal antibody by CDR-grafting: the importance of framework residues on loop conformation.

A mouse monoclonal antibody (mAb 425) with therapeutic potential was 'humanized' in two ways. Firstly the mouse variable regions from mAb 425 were spliced onto human constant regions to create a chimeric 425 antibody. Secondly, the mouse complementarity-determining regions (CDRs) from mAb 425 were grafted into human variable regions, which were then joined to human constant regions, to create a reshaped human 425 antibody. Using a molecular model of the mouse mAb 425 variable regions, framework residues (FRs) that might be critical for antigen-binding were identified. To test the importance of these residues, nine versions of the reshaped human 425 heavy chain variable (VH) regions and two versions of the reshaped human 425 light chain variable (VL) regions were designed and constructed. The recombinant DNAs coding for the chimeric and reshaped human light and heavy chains were co-expressed transiently in COS cells. In antigen-binding assays and competition-binding assays, the reshaped human antibodies were compared with mouse 425 antibody and to chimeric 425 antibody. The different versions of 425-reshaped human antibody showed a wide range of avidities for antigen, indicating that substitutions at certain positions in the human FRs significantly influenced binding to antigen. Why certain individual FR residues influence antigen-binding is discussed. One version of reshaped human 425 antibody bound to antigen with an avidity approaching that of the mouse 425 antibody.     

Stability of murine, chimeric and humanized antibodies against pre-S2 surface antigen of hepatitis B virus

We have constructed a humanized antibody with specificity for the pre-S2 surface antigen of hepatitis B virus (HBV) by grafting the complementarity determining regions (CDRs) of parental murine monoclonal antibody (mAb) into human anti-Sm antibody framework regions. The humanized antibody has a substitution at position 94 in a framework region of the heavy chain variable region, and exhibits the same antigen binding affinity as the parental murine monoclonal and chimeric antibodies. In order to assess the stability of these antibodies, thermal inactivation of the parental, chimeric and humanized antibodies was analyzed. Fifty percent inactivation of the chimeric and humanized antibodies was observed at 63.7 degrees C and 68.7 degrees C, respectively, compared to 55.0 degrees C for murine antibody. The humanized antibody also exhibited increased stability against denaturant. Guanidine-induced unfolding monitored by the changes in fluorescence intensity at 360 nm showed that midpoints of the transition of the chimeric and humanized antibodies were 2.47 M and 2.56 M, respectively, whereas that of the murine antibody was 1.36 M.     

Characterization of a human T cell-specific chimeric antibody (CD7) with human constant and mouse variable regions

A chimeric human-mouse anti-T lymphocyte mAb (CHT2; SDZ 214-380) has been constructed by cloning the variable region exons of both the L and H chains from the murine hybridoma RFT2 which have CD7 specificity and reactivity with a 40-kDa Ag. The variable L chain exon was joined to the human C kappa, and the variable H chain exon was joined to the human IgG1 region exon encoding the human allotype nGlm(z), nGlm(a). The gene constructs were introduced by electroporation into SP2/0, a non-Ig-producing murine myeloma. The identical tissue reactivity of the newly made CHT2 and the original murine RFT2 mAb (CD7) was confirmed by blocking experiments as well as by immunohistology and flow cytometry. Because this new mAb may have clinical use, the CD7 Ag expression of T lineage cells has also been quantitated in double and triple immunofluorescence assays in combinations with mAb to restricted forms of leukocyte common Ag that designate unprimed (CD45R+) and primed T lymphocyte populations (UCHL1+). CHT2 shows very strong reactivity with large thymic blast cells and cortical thymocytes from which T-ALL originates. Strong staining is seen on CD45R+ unprimed "virgin" T lymphocytes, whereas the expression on UCHL1+ primed "memory" cell types is weaker unless these cells are reactivated by mitogens or Ag. Thus CHT2 may spare a substantial population of resting memory T cells which is relevant to its potential therapeutic use. In addition the chimeric antibody had a greater in vitro antibody dependent cytotoxicity and a prolonged half-life (4.2 to 5.0 days) in Rhesus monkeys.     

从抗TNF抗体的CDR肽库中获得拮抗TNF的短肽

为设计来自抗体的短肽 ,以抗肿瘤坏死因子 (TNF)嵌合抗体 (cA2 )CDRs为模板 ,在其两侧各加 3个随机氨基酸残基 ( X3 CDR X3 ) ,构建了 6个以CDR为基础的肽库 .经过 3轮亲和选择 ,挑取单克隆 ,进一步经ELISA检测TNF阳性噬菌体克隆 ,分离得到 7个ELISA阳性较好的噬菌体肽克隆 ,分别命名为CDR2L1、CDR2L2、CDR2L3、CDR1L1、CDR2H1、CDR3H1、CDR3H 2 .应用MTT方法 ,检测 7个克隆对TNF生物学活性的拮抗作用 .结果显示 :来自CDR2L ,CDR3H肽库中的CDR2L2、CDR2L3,CDR3H2噬菌体肽具有明显的拮抗TNF诱导L92 9细胞的细胞毒作用 ,其中以CDR2L2噬菌体肽的拮抗活性最强 .而来源于CDR1L ,CDR2H肽库的CDR1L1和CDR2H1噬菌体肽和来自CDR2L ,CDR3H肽库中的CDR2L1和CDR3H1噬菌体肽没有明显的拮抗TNF作用 .研究结果初步表明 :从cA2抗体CDR肽库中筛选得到的噬菌体CDR模拟肽具有亲本抗体相似的结合活性和生物学效应 ,从而为开发已知抗体 (特别是治疗用抗体 )CDR为基础的肽药物创建一个技术平台奠定基础     

Expression of a functional chimeric Ig-MHC class II protein.

We have generated a chimeric protein molecule composed of the alpha- and beta-chains of the MHC class II I-E molecule fused to antibody V regions derived from anti-human CD4 mAb MT310. Expression vectors were constructed containing the functional, rearranged gene segments coding for the V region domains of the antibody H and L chains in place of the first domains of the complete structural genes of the I-E alpha- and beta-chains, respectively. Cells transfected with both hybrid genes expressed a stable protein product on the cell surface. The chimeric molecule exhibited the idiotype of the antibody MT310 as shown by binding to the anti-idiotypic mAb 20-46. A protein of the anticipated molecular mass was immunoprecipitated with anti-mouse IgG antiserum. Furthermore, human soluble CD4 did bind to the transfected cell line, demonstrating that the chimeric protein possessed the binding capacity of the original mAb. Thus, the hybrid molecule retained: 1) the properties of a MHC class II protein with regard to correct chain assembly and transport to the cell surface; as well as 2) the Ag binding capacity of the antibody genes used. The generation of hybrid MHC class II molecules with highly specific, non-MHC-restricted binding capacities will be useful for studying MHC class II-mediated effector functions such as selection of the T cell repertoire in thymus of transgenic mice.     

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.     

Monoclonal antibodies to meningococcal factor H binding protein with overlapping epitopes and discordant functional activity

Background

Meningococcal factor H binding protein (fHbp) is a promising vaccine candidate. Anti-fHbp antibodies can bind to meningococci and elicit complement-mediated bactericidal activity directly. The antibodies also can block binding of the human complement down-regulator, factor H (fH). Without bound fH, the organism would be expected to have increased susceptibility to bacteriolysis. Here we describe bactericidal activity of two anti-fHbp mAbs with overlapping epitopes in relation to their different effects on fH binding and bactericidal activity.

Methods and Principal Findings

Both mAbs recognized prevalent fHbp sequence variants in variant group 1. Using yeast display and site-specific mutagenesis, binding of one of the mAbs (JAR 1, IgG3) to fHbp was eliminated by a single amino acid substitution, R204A, and was decreased by K143A but not by R204H or D142A. The JAR 1 epitope overlapped that of previously described mAb (mAb502, IgG2a) whose binding to fHbp was eliminated by R204A or R204H substitutions, and was decreased by D142A but not by K143A. Although JAR 1 and mAb502 appeared to have overlapping epitopes, only JAR 1 inhibited binding of fH to fHbp and had human complement-mediated bactericidal activity. mAb502 enhanced fH binding and lacked human complement-mediated bactericidal activity. To control for confounding effects of different mouse IgG subclasses on complement activation, we created chimeric mAbs in which the mouse mAb502 or JAR 1 paratopes were paired with human IgG1 constant regions. While both chimeric mAbs showed similar binding to fHbp, only JAR 1, which inhibited fH binding, had human complement-mediated bactericidal activity.

Conclusions

The lack of human complement-mediated bactericidal activity by anti-fHbp mAb502 appeared to result from an inability to inhibit binding of fH. These results underscore the importance of inhibition of fH binding for anti-fHbp mAb bactericidal activity.     

"Superhumanized" antibodies: reduction of immunogenic potential by complementarity-determining region grafting with human germline sequences: application to an anti-CD28

Humanized Abs are created by combining, at the genetic level, the complementarity-determining regions of a murine mAb with the framework sequences of a human Ab variable domain. This leads to a functional Ab with reduced immunogenic side effects in human therapy. In this study, we report a new approach to humanizing murine mAbs that may reduce immunogenicity even further. This method is applied to humanize the murine anti-human CD28 Ab, 9.3. The canonical structures of the hypervariable loops of murine 9.3 were matched to human genomic V gene sequences whose hypervariable loops had identical or similar canonical structures. Framework sequences for those human V genes were then used, unmodified, with the 9.3 complementarity-determining regions to construct a humanized version of 9.3. The humanized 9.3 and a chimeric 9.3 control were expressed in Escherichia coli as Fab. The humanized Fab showed a moderate loss in avidity in a direct binding ELISA with immobilized CD28-Ig fusion protein (CD28-Ig). Humanized 9.3 blocked ligation of CD28-Ig to cells expressing the CD28 receptor CD80. Lastly, the humanized 9.3 showed biological activity as an immunosuppressant by inhibiting a MLR.     

Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing Fc gamma RI and/or Fc gamma RII receptors.

Aglycosylated human IgG1 and IgG3 monoclonal anti-D (Rh) and human IgG1 and IgG3 chimaeric anti-5-iodo-4-hydroxy-3-nitrophenacetyl (anti-NIP) monoclonal antibodies produced in the presence of tunicamycin have been compared with the native glycosylated proteins with respect to recognition by human Fc gamma RI and/or Fc gamma RII receptors on U937, Daudi or K562 cells. Human red cells sensitized with glycosylated IgG3 form rosettes via Fc gamma RI with 60% of U937 cells. Inhibition of rosette formation required greater than 35-fold concentrated more aglycosylated than glycosylated human monoclonal anti-D (Rh) antibody. Unlabelled polyclonal human IgG and glycosylated monoclonal IgG1 and anti-D (Rh) antibody inhibited the binding of 125I-labelled monomeric human IgG binding by U937 Fc gamma RI at concentrations greater than 50-fold lower than the aglycosylated monoclonal IgG1 anti-D (Rh) (K50 approximately 3 x 10(-9) M and approximately 6 x 10(-7) M respectively). Similar results were obtained using glycosylated and aglycosylated monoclonal human IgG1 or IgG3 chimaeric anti-NIP antibody-sensitized red cells rosetting with Fc gamma RI-/Fc gamma RII+ Daudi and K562 cells. Rosette formation could be inhibited by the glycosylated form (at greater than 10(-6) M) but not by the aglycosylated form. Haemagglutination analysis using a panel of murine monoclonal antibodies specific for epitopes located on C gamma 2, C gamma 3 or C gamma 2/C gamma 3 interface regions did not demonstrate differences in Fc conformation between the glycosylated or aglycosylated human monoclonal antibodies. These data suggest that the Fc gamma RI and Fc gamma RII sites on human IgG are highly conformation-dependent and that the carbohydrate moiety serves to stabilize the Fc structure rather than interacting directly with Fc receptors.     

Construction, expression, and biologic activity of murine/human chimeric antibodies with specificity for the human alpha/beta T cell receptor.

Murine/human chimeric antibodies with specificity for the human TCR-alpha/beta have been produced by genetic engineering. The L and H chain V region exons encoding the murine mAb BMA 031 were isolated and inserted into mammalian expression vectors containing the human kappa and gamma 1 or gamma 4 C region exons. The chimeric genes were transfected into murine Sp2/O hybridoma cells by electroporation and transfectomas secreting chimeric antibody were isolated. Secretion levels ranged from 1 to 7 pg/cell/24 h. The chimeric antibodies bound specifically to T cells and competed effectively with the parental murine mAb for binding to these sites. The ability to promote antibody-dependent cell-mediated cytolysis was significantly enhanced in the chimeric antibodies as compared with murine BMA 031. C-dependent cytolysis, however, was not detectable with any of the antibodies. Chimeric BMA 031 is a clinically relevant, genetically engineered antibody with potential uses in transplantation, graft-vs-host disease, autoimmune diseases and other T cell-related disorders.