A3--a novel colon and pancreatic cancer reactive antibody from a primate phage library selected using intact tumour cells

The identification of novel tumour-associated antigens (TAAs) is pivotal for progression in the fields of tumour immunotherapy and diagnosis. In the present study, we have developed, based on flow cytometric evaluation and use of a mini-library composed of specific antibody clones linked to different antibiotic resistance markers, methods for positive and subtractive selection of phage antibodies employing intact cells as the antigen source. An scFv phage library (2.7 x 10(7)) was constructed from a primate (Macaca fascicularis) immunised with pooled human colon carcinomas. This library was selected for 3 rounds by binding to Colo 205 colon adenocarcinoma cells and proteolytic elution followed by phage amplification. Several antibodies reactive with colon carcinomas and with restricted reactivity to a few epithelial normal tissues were identified by immunohistochemistry. One clone, A3 scFv, recognised an epitope that was homogeneously expressed in 11/11 of colon and 4/4 pancreatic carcinomas studied and in normal tissue restricted to subtypes of epithelia in the gastrointestinal tract. The A3 scFv had an apparent overall affinity approximately 100-fold higher than an A3 Fab, suggesting binding of scFv homodimers. The cell surface density of the A3 epitope, calculated on the basis of Fab binding, was exceptionally high, approaching 3 million per cell. We also demonstrate efficient T-cell-mediated killing of colon cancer cells coated with A3 scFv fused to the low MHC class II binding superantigen mutant SEA(D227A). The identified A3 molecule thus represents a TAA with properties that suggest its use for immunotherapy of colon and pancreatic cancer.     

Isolation and characterisation of a human anti-idiotypic scFv used as a surrogate tumour antigen to elicit an anti-HER-2/neu humoral response in mice

HER-2/neu is a tumour antigen that is overexpressed in human breast tumours. Among the vaccine strategies developed to overcome immune tolerance to self-proteins, vaccination with anti-idiotypic (anti-Id) antibodies has been described as a promising approach for treatment of several malignant diseases. To develop an active immunotherapy for cancer patients positive for HER-2/neu, we investigated immunisation with human anti-Id single-chain fragments (scFv) mimicking the conformation of HER-2/neu protein to induce a humoral response in mice. We selected by phage display two human anti-Id scFv (Ab2beta) directed against trastuzumab F(ab')2 fragments (Ab1), a humanised anti-HER-2/neu monoclonal antibody. Using competitive ELISA and Biacore biosensor analysis, we showed that anti-Id scFv 40 and scFv 69 could inhibit HER-2/neu binding to trastuzumab. Following vaccination of BALB/c mice with the soluble or phage-displayed scFv, Ab3 polyclonal antibodies, and among them Ab1' antibodies able to bind HER-2/neu, were detected in the sera of the immunised mice. These results demonstrate that the human anti-Id scFv could act as a surrogate antigen for HER-2/neu. The present study strongly suggests that the novel 30 kDa human mini-antibody could be used as an anti-idiotype-based vaccine formulation to induce an effective humoral response in patients bearing HER-2/neu-positive tumours.     

Isolation and characterization of the human monoclonal antibodies C10 in single-chain fragment variable (scFv) format to glucose oxidase from Aspergillus niger

Despite biotechnological and clinical applications very few monoclonal antibodies (MAbs) directed to the enzyme glucose oxidase, have been produced so far because of the heavy side effects of the immunization schedule for conventional MAb preparation. In contrast, the phage display method allows for the selection of monoclonal human antibody fragments against any antigens, including toxic proteins. Furthermore, cDNA sequences encoding selected antibody fragments are readily identified, facilitating various molecular targeting approaches. In order to obtain such human fragments recognizing glucose oxidase, we used the large synthetic ETH-2 library based on the principle of protein design. Phage displaying glucose oxidase reactive scFvs were obtained after three rounds of selection on glucose oxidase-coated immunotubes and subsequent amplification in TG1 E. coli cells. Eventually, one high reactive scFv clone was selected and further examined. The anti-glucose oxidase scFv C10 was found suitable for Western blot; Biacore analysis showed that the binding affinity of the glucose oxidase-reactive scFv is almost equal that of MAbs prepared with conventional hybridoma technology. Finally, the cDNA sequence of this human scFv may be exploited to generate bispecific antibodies to target in the tumor environment-specific toxic enzymatic reaction.     

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.     

Isolation of human tumor-specific antibodies by selection of an antibody phage library on melanoma cells.

A human single-chain Fv (scFv) library as fusion to phage was constructed from donors with a high titer of autoantibodies. The library was subjected to three rounds of positive selection on human melanoma cells and negative selection on human peripheral blood mononuclear cells. Two scFv clones, B3 and B4, were isolated that bound melanoma cells in cell ELISA and fluorescence-activated cell sorting. The scFvs were characterized further by immunohistochemistry on a large number of normal human tissues. No cross-reactivity with normal tissues was observed. On the other hand, the target antigens were expressed in sections from several different melanoma patients and in some breast cancer and basal cell carcinoma sections. The unusually high tumor specificity of the B3 and B4 antigens makes them attractive targets for the specific therapy of melanoma. The selection strategy used should be generally applicable to the identification of novel cell surface antigens by antibody phage display.     

Evaluation of monoclonal antibodies for the development of breast cancer immunotoxins

Eighty-five antibodies recognizing breast cancer-selective antigens were conjugated to ricin toxin A-chain using a disulfide linkage. The cytotoxicities of the resulting immunotoxins were determined on breast cancer cells and normal human fibroblasts. Twenty-four antibodies formed immunotoxins that were toxic to at least one breast cancer cell line at concentrations of 10 nM or less but were nontoxic to human fibroblast lines used as negative controls. Some of the breast tumor-selective immunotoxins were as toxic as a conjugate between monoclonal anti-transferrin receptor and ricin toxin A-chain (50% inhibition of cellular protein synthesis at approximately 0.1 nM). Another set of four immunotoxins were indiscriminately toxic to human breast tumor cell lines, two human fibroblast cell lines, and a human lymphoblastoid line. Several of the antibodies the toxin conjugates of which specifically killed breast cancer cell lines may be useful in cancer therapy, since they show a wide range of binding to individual breast tumors and cell lines and a limited range of binding to normal tissue types.     

Monoclonal anti-idiotype antibody 6G6.C4 fused to GM-CSF is capable of breaking tolerance to carcinoembryonic antigen (CEA) in CEA-transgenic mice

Internal image anti-idiotypic antibodies are capable of mimicking tumor-associated antigens and thus may serve as surrogate for vaccination strategies in cancer patients. The monoclonal antibody (mAb) 6G6.C4 mimics an epitope specific for the human carcinoembryonic antigen (CEA) and generates a CEA-specific response (Ab3) in various experimental animals. In humans, however, 6G6.C4 only yields a very limited humoral anti-CEA reaction presumably due to tolerance against the CEA autoantigen. In this study, we investigated the CEA-specific Ab3 response in mice transgenic for the human CEA and tested whether the antigen tolerance could be overcome by fusing a recombinant single-chain variable fragment of 6G6.C4 (scFv6G6.C4) to the murine granulocyte macrophage colony-stimulating factor (GM-CSF).Like mAb 6G6.C4, the fusion protein (scFv6G6.C4/GM-CSF) retained binding to the CEA-specific idiotype mAb T84.66. Also, scFv6G6.C4/GM-CSF was biologically active as measured by proliferation of the GM-CSF-dependent murine FDC-P1 cells in vitro. After immunization with the scFv6G6.C4/GM-CSF fusion protein, CEA-transgenic animals showed significantly enhanced Ab3 antibody responses to scFv6G6.C4 (P=0.005) and to CEA (P=0.012) compared with the scFV6G6.C4 alone. Sera from mice immunized with the fusion protein specifically recognized CEA in Western blot analyses with no cross-reaction to CEA-related antigens. Finally, the Ab3 antisera detected single CEA-expressing tumor cells in suspension as shown by flow cytometry. Taken together, these data show in a model antigenically related to the human system that vaccination with scFv6G6.C4/GM-CSF improves vaccination against an endogenous tumor-associated antigen resulting in a highly specific humoral Ab3 response in vivo that is capable of bind single circulating CEA-positive tumor cells.     

Monoclonal antibodies applied to primary human breast carcinoma: Relationship to menopausal status,lymph node status,and steroid hormone receptor content

Summary Monoclonal antibodies were raised against purified human milk fat globule membranes. The binding of three of these (Mam-3, HMFG 1, and HMFG 2) to fixed histological sections of 100 primary breast carcinomas has been investigated. Tissue specificity was investigated by testing the binding of the antibodies to carcinomas from other organs and to benign proliferative breast lesions.In breast tissue, antigens were found only in the epithelial cells, while the stromal component, myoepithelium, and any inflammatory cells present were negative. The reaction was characterized by considerable heterogeneity, both with regard to the percent of cells positive and the intensity of the reaction. No relationship was observed between binding and histological type of breast carcinoma. However, in ductal infiltrating carcinomas, a tendency towards a greater proportion of tumors that bound the antibodies was observed among the highly differentiated compared to the low differentiated carcinomas.Data on estrogen and progesterone receptors were available in 53 and 22 of the 100 carcinomas, respectively. There were no apparent relationships between the presence of surface antigens and the menopausal status, lymph node status, or estrogen receptor status, but the tumors lacking progesterone receptor apparently lacked the antigens as well.The presence of surface antigens probably defines a differentiation in primary breast carcinoma. Whether this differentiation is of any prognostic significance remains to be established.     

Relationship of pancreatic cancer apomucin to mammary and intestinal apomucins

Pancreatic cancer mucins have several carbohydrate antigens that are potentially useful in the detection of pancreatic cancers, but little is known about the core polypeptides of pancreatic cancer mucins. In this study, purified mucin from SW1990 pancreatic cancer xenografts was deglycosylated by treatment with hydrogen fluoride to give pancreatic cancer apomucin. Consistent with near-complete removal of carbohydrate, the apomucin had 10- to 70-fold decreased binding of lectins and, unlike the native mucin, served as an acceptor for polypeptidyl N-acetylgalactosaminyl transferase. Antibodies prepared against the apomucin did not bind to native mucin, and antibodies that bound to native mucin did not bind to apomucin. On the basis of cross-reaction with deglycosylated colon cancer mucin and intestinal mucin repeat peptide, apomucins from SW1990 pancreatic cancer xenografts contain the intestinal mucin repeat peptide. On the basis of binding of breast cancer-reactive monoclonal antibodies 139H2, DF3, and HMFG-2, apomucins from SW1990 pancreatic cancer xenografts also have the mammary mucin repeat peptide. Using complementary DNA probes specific for intestinal mucin and breast mucin sequences, both types of apomucin mRNA were detected in nude mouse xenografts of SW1990 cells. In immunohistochemical staining, antibody against deglycosylated SW1990 mucin stained normal breast and pancreas but not normal colon. Some pancreatic and mammary cancers and most colonic cancers, however, were stained by antibodies against both intestinal apomucin and mammary apomucin. We conclude that pancreatic cancers can produce mucins with the intestinal repeat peptide as well as those with mammary repeat peptide sequences.     

Human monoclonal antibodies derived from lymph nodes of a patient with breast carcinoma react with MuMTV polypeptides

Nine human hybridoma cell lines were established from a fusion of axillary lymph node lymphocytes of a patient with breast ductal carcinoma with a human lymphoblastoid cell line. The human hybridoma nature of the fusion products was confirmed by chromosomal analysis and HLA typing. The hybridomas are stable over a year of growth, and can be frozen, thawed and regrown. The carcinoma cells of the patient harbor mouse mammary tumor virus (MuMTV) cross-reacting antigens. The patient's serum and the purified monoclonal antibodies reacted with MuMTV polypeptides. Radioimmunoprecipitation studies using labeled MuMTV confirmed the binding of the patient's serum to the viral proteins. None of the control immunoglobulins reacted with the virus. No binding of the hybridoma immunoglobulins was observed with two other retroviruses (avian myeloblastosis virus and simian sarcoma virus). The ligand binding characteristics of the monoclonal antibodies suggest binding to epitopes on the various structural virus polypeptides. These monoclonal antibodies may serve as a probe to analyze the MuMTV-human breast carcinoma relationship.     

A cell surface-displayed anti-c-myc single-chain antibody: new perspectives for the genetic improvement of cellular tumor vaccines

We have shown recently that cell surface-bound, single-chain Fv antibodies (scFv) are a powerful tool for the improvement of cellular tumor vaccines. To simplify this approach and to develop a general tool for the generation and improvement of cellular tumor vaccines, we chose an scFv against a peptide from the human proto-oncogene c-myc that could anchor any c-myc-tagged protein to the cell surface. The retroviral vector p50-Mx-neo (pMESV) was used to express scFv on the surface of the human melanoma line SkMel63. The cell-bound anti-c-myc scFv bound specifically to a soluble purified anti-CD28 scFv carrying a c-myc peptide-tag at its C terminus. Proof of principle was determined by incubating human peripheral blood lymphocytes with a mixture of (a) anti-c-myc-transfected SkMel63 cells binding the anti-CD28 scFv and (b) SkMel63 cells transfected with an anti-CD3 scFv. A clear synergistic effect on T-cell activation was observed that was comparable with that obtained in previous studies using SkMel63 cells transfected with the gene for the anti-CD28 scFv. As the cell surface-displayed anti-c-myc scFv can bind any c-myc-tagged protein of interest, this technique facilitates the genetic engineering of cellular vaccines for the therapy of virtually all human neoplasias.     

抗CEA和CA19-9的单链抗体在HEK293细胞中的融合表达及亲和力鉴定

 目的 在HEK293细胞中高效表达抗CEA scFv和抗CA19-9 scFv两种单链抗体的Fc融合蛋白（anti-CEA scFv-Fc和anti-CA19-9 scFv-Fc），并测定两种单链抗体亲和力。方法 从GenBank中获取两种scFv基因序列；经优化后合成，并与pET32a（+）构成原核表达载体anti-CEA scFv/pET32a（+）和anti-CA19-9 scFv/pET32a（+），在E.coli BL21（DE3）中进行IPTG诱导表达；两种scFv经PCR扩增技术删除末端终止序列，利用重叠区基因扩增法在anti-CEA scFv上游加入人IL-2信号肽，分别与含人IgG Fc段基因的pTT5载体（Fc/pTT5）构成真核表达载体（anti-CEA scFv（-t）-Fc/pTT5、IL2-anti-CEA scFv（-t）-Fc/pTT5和anti-CA19-9 scFv（-t）-Fc/pTT5）；使用脂质体转染法，将3个重组质粒转入HEK293细胞中进行表达；产物经rProtein-A FF亲和层析纯化，竞争性细胞ELISA和Western blot检测。结果 免疫印迹显示，3个scFv-Fc融合蛋白均在HEK293细胞中表达；竞争结合实验表明，anti-CEA scFv-Fc和anti-CA19-9 scFv-Fc的抗体浓度分别为8.2 nmol/L和6.0 nmol/L。结论 anti-CEA scFv和anti-CA19-9 scFv能在HEK293细胞内表达；虽然表达量较低，但纯化后表达产物能够识别肿瘤细胞表面抗原CEA和CA19-9。     

Specific monoclonal antibodies reacting with human breast cancer cells

Spleen cells from Balb/c mice immunized with human breast cancer cells (MCF-7) were fused with murine myeloma SP2/0 cells. Screening of the monoclonal antibodies produced was carried out on glutaraldehyde fixed cells coated on microtiterplates. An initial evaluation of the specificity was obtained by comparing the binding of the monoclonal antibodies to MCF-7 cells with the binding to human peripheral blood lymphocytes. Eight monoclonal antibodies reacting with different epitopes on the MCF-7 cells were obtained. On the basis of their clonal origin, isotype and reaction pattern towards the MCF-7 cells these monoclonal antibodies were subdivided into two classes. Both groups of antibodies reacted with fixed and unfixed MCF-7 cells. The cellular distribution of the antigens recognized by the monoclonal antibodies was determined. To check for specificity a panel of different cells (of human and animal origin) was evaluated by immunocytochemical techniques.     

Targeting immune effector molecules to human tumor cells through genetic delivery of 5T4-specific scFv fusion proteins

Although several clinical trials have shown beneficial effects by targeting tumor-associated antigens (TAAs) with monoclonal antibodies, a number of issues, including poor penetration of the tumor mass and human antimouse antibody responses, remain. The use of recombinant single-chain Fv (scFv) fragments has the potential to address these and other issues while allowing the addition of different effector functions. To develop therapeutic strategies that recruit both humoral and cellular arms of the immune response, we have constructed chimeric proteins linking either the human IgG1 Fc domain or the extracellular domain of murine B7.1 to a scFv specific for the oncofetal glycoprotein, 5T4. This TAA is expressed by a wide variety of carcinomas and is associated with metastasis and poorer clinical outcome. We have engineered retroviral constructs that produce fusion proteins able to interact simultaneously with both 5T4-positive cells and with the receptor/ligands of the immune effector moieties. Genetic delivery through a murine leukemia virus vector to 5T4-positive tumor cells results in the secreted scFv fusion protein binding to the cell surface. Furthermore, the scFv-HIgG1 fusion protein is able to direct lysis of 5T4-expressing human tumor cell lines through antibody-dependent cell cytotoxicity, indicating its potential as a gene therapy for human cancers.     

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.     

Anti-idiotypic antibody as the surrogate antigen for cloning scFv and its fusion proteins

Single-chain variable fragment (ScFv) is a versatile building block for novel targeting constructs. However, a reliable screening and binding assay is often the limiting step for antigens that are difficult to clone or purify. Anti-idiotypic antibodies may be useful as surrogate antigens for cloning scFv and their fusion proteins. 8H9 is a murine IgG(1) monoclonal antibody (MAb) specific for a novel antigen expressed on the cell surface of a wide spectrum of human solid tumors, but not in normal tissues. Rat anti-8H9-idiotypic hybridomas (clones 2E9, 1E12, and 1F11) were produced by somatic cell fusion between rat lymphocytes and mouse SP2/0 myeloma. In direct binding assays enzyme-linked immunosorbant assay--(ELISA)--they were specific for the 8H9 idiotope. Using 2E9 as the surrogate antigen, 8H9-scFv was cloned from hybridoma cDNA by phage display. 8H9scFv was then fused to human-gamma1-CH2-CH3 cDNA for transduction into CHO and NSO cells. High expressors of mouse scFv-human Fc chimeric antibody were selected. The secreted homodimer reacted specifically with antigen-positive tumor cells by ELISA and by flow cytometry, inhibitable by the anti-idiotypic antibody. The reduced size resulted in a shorter half-life in vivo, while achieving comparable tumor to nontumor ratio as the native antibody 8H9. However, its in vitro activity in antibody-dependent cell-mediated cytotoxicity was modest.     

High affinity restricts the localization and tumor penetration of single-chain fv antibody molecules.

Antitumor monoclonal antibodies must bind to tumor antigens with high affinity to achieve durable tumor retention. This has spurred efforts to generate high affinity antibodies for use in cancer therapy. However, it has been hypothesized that very high affinity interactions between antibodies and tumor antigens may impair efficient tumor penetration of the monoclonal antibodies and thus diminish effective in vivo targeting (K. Fujimori et al., J. Nucl. Med., 31: 1191-1198, 1990). Here we show that intrinsic affinity properties regulate the quantitative delivery of antitumor single-chain Fv (scFv) molecules to solid tumors and the penetration of scFv from the vasculature into tumor masses. In biodistribution studies examining a series of radioiodinated scFv mutants with affinities ranging from 10(-7)-10(-11) M, quantitative tumor retention did not significantly increase with enhancements in affinity beyond 10(-9) M. Similar distribution patterns were observed when the scFv were evaluated in the absence of renal clearance in anephric mice, indicating that the rapid renal clearance of the scFv was not responsible for these observations. IHC and IF evaluations of tumor sections after the i.v. administration of scFv affinity mutants revealed that the lowest affinity molecule exhibited diffuse tumor staining whereas the highest affinity scFv was primarily retained in the perivascular regions of the tumor. These results indicate that antibody-based molecules with extremely high affinity have impaired tumor penetration properties that must be considered in the design of antibody-based cancer therapies.     

Generation and immunohistological characterization of human monoclonal antibodies to mammary carcinoma cells

The purpose of this study was to identify human antibodies generated against autologous breast tumor cells by the host's immune response. Accordingly, lymphocytes from lymph nodes of seven different patients with metastatic breast carcinomas were immortalized by fusing them with a nonsecreting variant of murine myeloma cells. The screening for binding of antibodies to tumor cells was performed by indirect immunoperoxidase staining of paraffin-embedded tissue sections of the autologous tumor. The selected hybrid cells, after being cloned three times, were stable for the secretion of immunoglobulins for over 2 years. A total of 81 human immunoglobulin-producing clones was obtained from an initial 595 wells with hybrid growth. Nine of these clones produced immunoglobulin M, none of which showed detectable binding to tissue antigens in breast. Seventy-two clones produced immunoglobulin G monoclonal antibodies, and 15 of these showed preferential binding to breast carcinoma cells. Three of these immunoglobulin G monoclonal antibodies were subjected to detailed immunohistological evaluations. Using these antibodies at concentrations ranging from 10 to 100 ng/tissue section, the morphologically normal mammary epithelial cells could be discriminated from their malignant counterparts. The antibodies showed diffuse staining of cytoplasmic components in the malignant counterparts. Under these conditions, lymphocytes, erythrocytes, and stromal cells in breast tissues were unstained. The antibodies showed variable reactivity with malignant epithelial cells of colon and stomach, and with normal epithelial cells lining the renal tubules and sebaceous glands in skin. Antigenic heterogeneity of malignant mammary epithelial cells was revealed. The antibodies may have value in the characterization of tumor-associated antigens responsible for inducing autologous immune responses.