Generation of a mouse monoclonal antibody against chicken interleukin-6

A monoclonal antibody (MAb) specific for chicken interleukin-6 (chIL-6) was generated by using Balb/c mice immunized with recombinant chIL-6 (rchIL-6). On Western blot analysis, the MAb, designated E3, reacted with rchIL-6 but not with recombinant murine IL-6 (rmIL-6). The MAb E3 also reacted with supernatant of the chicken macrophage-like cell line HD11 stimulated with lipopolysaccaride. The rchIL-6-induced phosphorylation of STAT3 in a chicken hybridoma cell line was inhibited by addition of MAb E3 in a dose-dependent manner. These results indicate that the MAb E3 specific for chIL-6 is useful for detection and functional analysis of chIL-6.     

Photodynamic killing of human squamous cell carcinoma cells using a monoclonal antibody-photosensitizer conjugate

We have developed procedures in which the photosensitizer benzoporphyrin derivative monoacid ring A (BPD) can be covalently linked to carrier molecules of modified polyvinyl alcohol (PVA) to produce water-soluble PVA-BPD conjugates with a molecular mass in the range of 30 kd. These carriers can subsequently be covalently linked to monoclonal antibodies (MoAbs) using heterobifunctional linking agents. We describe here such a conjugate in which the MoAb (5E8) has specificity for a glycoprotein detected on human squamous cell carcinomas of the lung. We provide evidence that the conjugates produced were covalently linked and retained both their photosensitizing and antigen-binding activities. We show further that the MoAb-PVA-BPD conjugate, in the presence of 10% fetal calf serum, exhibited highly enhanced phototoxic killing of the target cell line (A549) over that exhibited by free BPD or a control MoAb-PVA-BPD conjugate. These results demonstrate, therefore, both the selectivity and specificity of this MoAb conjugate.     

Targeting T cells against brain tumors with a bispecific ligand-antibody conjugate

High-affinity receptors expressed on the surface of some tumors can be exploited by chemically conjugating the ligand for the receptor and an antibody against immune effector cells, thus redirecting their cytolytic potential against the tumor. Ovarian carcinomas and some brain tumors express the high-affinity folate receptor (FR). In this report, a transgenic mouse model that generates endogenously arising choroid plexus tumors was used to show that folate/anti- T-cell receptor antibody conjugates can direct infiltration of T cells into solid brain tumor masses. An engineered single-chain Fv form of the anti-T-cell receptor antibody KJ16 was conjugated with folate, to produce a bispecific agent that was substantially smaller than most previously characterized bispecific antibodies. Folate conjugation to the antibody increased T-cell infiltration into the tumors by 10- to 20-fold, and significantly prolonged survival of the mice. Int. J. Cancer 76:761–766, 1998.© 1998 Wiley-Liss, Inc.     

A monoclonal antibody against human FXYD6

Previous research has shown that FXYD6 (FXYD domain-containing ion transport regulator 6) is highly increased in bile duct tumor. However, the biological function of FXYD6 is unclear. We aim to prepare and identify a monoclonal antibody against FXYD6, which will be used in diagnostics and as a tool in understanding the role of FXYD6 in pathogenesis of hepatobiliary cancer. In this study, hybridoma cell fusion technology is used for production of FXYD6 monoclonal antibody. BALB/c mice are immunized with FXYD6 synthetic peptide fragment. Hybridoma clones are screened using indirect enzyme-linked immunosorbent assay (ELISA). FXYD6 monoclonal antibody is produced by ascites revulsion. Protein A affinity chromatography is used for the purification of FXYD6 monoclonal antibody. Titer and specificity of monoclonal antibody are assessed by ELISA. Expression of FXYD6 in pancreatic cancer is detected by immunohistochemistry. As a result, one stable hybridoma cell clone producing FXYD6 monoclonal antibody has been established. 2.86?mg monoclonal antibody against FXYD6 with high specificity is prepared with titer of 1:5400. Immunohistochemistry shows that FXYD6 positive staining occurs in the cell membrane of pancreatic cancer, which results in an advantage in investigating the tissue distribution and biological function of FXYD6.     

Selective cytotoxicity against human tumour cells by a vindesine-monoclonal antibody conjugate.

The anti-mitotic drug vindesine was coupled chemically to a monoclonal antibody raised originally against the human osteogenic sarcoma cell line, 791T. The cytotoxicity of the conjugate in vitro was tested, in comparison with free vindesine, against sarcoma 791T and other antigenically cross-reactive osteogenic sarcoma-cell lines, and also against tumour cell lines which have no detectable reaction with the monoclonal antibody. Continuous exposure of cultured 791T cells indicated that the vindesine was partially inactivated following conjugation since the conjugate was less toxic than the free drug. However, antibody-binding activity was essentially preserved following conjugation. Despite diminished drug activity in the conjugate, assays designed to mimic antibody binding to tumour in which target cells were treated with conjugate and washed before culture, showed selective cytotoxicity for osteogenic sarcoma lines with little or no effect on non-cross reactive control cells. In comparison, free vindesine was toxic equally for all cell lines and free antibody was non-toxic. These studies indicate that conjugation of a cytotoxic agent to a monoclonal antibody can confer on that agent selectivity for a particular target cell type which is recognised by the antibody.     

Cell-specific cytotoxicity expressed by a conjugate of ricin and murine monoclonal antibody directed against thy 1.1 antigen

The isolation and characterization are described of a conjugate between ricin, and a thy 1.1-specific monoclonal IgG2a murine antibody synthesized using N-succinimidyl-3-(2-pyridyldithio)propionate. The conjugate selectively inhibited protein synthesis in Thy 1.1-positive (AKR SL3) mouse leukemia cells compared to Thy 1.2-positive (AKR/Cu SL1) cells.     

A bispecific monoclonal antibody against methotrexate and a human tumour associated antigen augments cytotoxicity of methotrexate-carrier conjugate

A bispecific monoclonal antibody, reactive with methotrexate (MTX) and a tumour associated antigen (gp72) has been produced by fusing spleen cells from MTX immunised mice with 791T/36/3 (anti-gp72) hybridoma. The hybrid antibody was purified from anti-MTX and anti-gp72 antibodies present in the hybridoma culture supernatant by combinations of affinity chromatography on a MTX-agarose immunoabsorbent and stepwise acid elution from Sepharose-Protein A. A particular feature of the present antibody is that it reacts with conjugated MTX; this would allow in vivo targeting of conjugates, increasing many fold the number of molecules of drug carried or localising to pre-targeted antibody. Dual binding between tumour cell surface antigen and MTX was demonstrated by the ability of the hybrid antibody to bridge between tumour cells and MTX as MTX-HSA conjugate, reaction here being detected by flow cytofluorimetry. Purified hybrid antibody specifically enhanced the in vitro cytotoxicity of MTX-HSA for gp72 positive tumour cells.     

Cytotoxicity against human tumor cells mediated by the conjugate of anti-epidermal growth factor receptor monoclonal antibody to recombinant ricin A chain

We have produced monoclonal antibodies against the epidermal growth factor (EGF) receptor which bind to the receptor with high affinity, compete with EGF for binding, block EGF-induced tyrosine kinase activity, and activate internalization and down-regulation of the receptor. These antibodies are cytostatic against cultured A431 cells at concentrations of 5-20 nM. In addition, they prevent the growth of A431 tumor xenografts in athymic mice. In the present experiments, we have attempted to improve the antitumor activity of monoclonal antibody 528 IgG2a against the EGF receptor by linking it to recombinant ricin A chain (rRA). The immunoconjugate (528 IgG-rRA) showed a potent cytotoxic effect on A431 cells in vitro. At a concentration of 10 pM, it inhibited the proliferation of cultured A431 cells by 50% and also inhibited protein synthesis in these cells by 50%. Proliferation was prevented and cell death occurred at 528 IgG-rRA concentrations of 60 pM or greater. Recombinant free ricin A chain was far less toxic. The cytotoxic effect of the immunoconjugate was neutralized by 528 IgG at concentrations 100-fold higher than 528 IgG-rRA. When the cytotoxic effect of 528 IgG-rRA was compared among several human cell lines expressing different numbers of EGF receptors, the capacity to inhibit both proliferation and protein synthesis generally correlated with the number of EGF receptors on the plasma membranes of these cells. Since 528 IgG-rRA is a very potent immunotoxin against A431 cells in culture, we designed experiments to test its in vivo antitumor activity against A431 xenografts in athymic mice. To measure the clearance of 528 IgG-rRA, 50 micrograms of immunotoxin were injected i.p. into athymic mice, blood was collected from the animals at regular intervals, and the level of immunotoxin in the serum was assayed by protein synthesis inhibition in cultured A431 cells. The blood level of active immunoconjugate reached a maximum 6 h after i.p. injection. The half-life of the absorption phase was 2.2 h, the half-life for elimination was 9.2 h, and blood levels which could be potentially cytotoxic were maintained for 48-72 h. We investigated a number of immunotoxin treatment schedules, including every other day for 4 days, based on these data. The results demonstrate that, while 528 IgG-rRA has higher in vivo antitumor activity than 528 IgG against A431 cell xenografts, this is accompanied by toxicity against the murine host.     

Targeted killing of squamous carcinoma cells by a monoclonal antibody-peplomycin conjugate which recognizes the EGF receptor.

We determined in vitro the antitumor activity of a conjugate prepared by binding a monoclonal antibody (B4G7), which recognizes the human epidermal growth factor (EGF) receptor, with peplomycin (PEP), which is an antitumor agent effective against squamous cell carcinoma. This B4G7-PEP conjugate was prepared by coupling of B4G7 and carboxymethylpeplomycin active ester. The conjugate killed A431 cells of squamous cell carcinoma which overexpress EGF receptors at lower concentrations than PEP alone. On the basis of its IC50, the conjugate was six times more potent than PEP alone. A simple mixture of B4G7 and PEP was as effective as PEP alone in cytotoxicity. The addition of ten times the amounts of B4G7 to this conjugate decreased its cytotoxicity. When other squamous cell carcinoma cell lines with different levels of EGF receptors (NA, Ca9-22, TE-1, TE-8) were treated with the conjugate, cells were killed dose-dependently and the cytotoxicity was dependent on the number of EGF receptors. When each squamous cell carcinoma cell line was treated with a control conjugate prepared by combining PEP with mouse IgG instead of B4G7, no cytotoxicity was observed. These results indicate that B4G7-PEP will be a useful weapon in multidisciplinary treatment which utilizes the EGF receptor, as these receptors are detected in a higher incidence in squamous cell carcinoma.     

Tumor-associated antigen defined by a monoclonal antibody against neuraminidase-treated human cancer cells

A monoclonal antibody, MH-A6, was produced by immunization with a human gastric cancer cell line, MKN 74, treated with neuraminidase. The antigen defined by the monoclonal antibody was detected on various tumor tissues and a limited number of normal tissues in immunoperoxidase assay, and the expression of MH-A6 antigen was not influenced by neuraminidase treatment except for some cases of tumor tissues. Interestingly, neuraminidase treatment enhanced binding of the antibody on some adenocarcinomas, but diminished binding of the antibody on squamous cell carcinomas. Both treatment of the immunizing tissues with trypsin and periodic acid diminished binding of the antibody. In isolation of MH-A6 antigen from MKN 74 cells by the monoclonal antibody coupled-affinity column, the epitope exists on molecules with molecular weights of 30,000 and 72,000, and with an acidic pH range in two-dimensional electrophoresis. CEA and CA 19-9 activities were not detected in purified MH-A6 antigen by solid-phase radioimmunoassay, and the reactivity of the MH-A6 antibody with CEA and CA 19-9 was not detected in enzyme-linked immunosorbent assay. Hemagglutination observed between erythrocytes (Lewisa, Lewisb, or NE-treated) and anti-Lewisa, anti-Lewisb sera, or anti-T-agglutinin (peanut lectin), respectively, was not inhibited by MH-A6 antigen. The results suggest that MH-A6 antigen is a tumor-associated antigen, probably glycoprotein, and different from CEA, CA 19-9, Lewisa, Lewisb, and Thomsen-Friedreich (T) antigen.     

Elimination of B-lymphoma cells from human bone marrow: model experiments using monodisperse magnetic particles coated with primary monoclonal antibodies

Conditions for removing B-lymphoma cells from human bone marrow using "immunobeads" (IBs) were investigated. The IBs were prepared by coupling monoclonal antibodies directly to a new type of monodisperse magnetic polymer particles (M 450). Two monoclonal immunoglobulin M antibodies, AB-1 (CD 19), a B-cell-specific antibody, and AB-4, an HLA-DR-specific antibody, were used. The IBs were incubated with Rael Burkitt lymphoma cells admixed to fresh, mononuclear human bone marrow cells. After incubation for 30 min at 4 degrees C, the IBs were removed using cobalt samarium magnets. The number of remaining clonogenic tumor cells was assayed by the Courtenay and Mills soft agar procedure, and the clonogenic capacity of the bone marrow progenitor cells was measured by granulocyte-monocyte and granulocyte-erythroid-monocyte-megakaryocyte assays. With a ratio of tumor cells to normal bone marrow cells of 0.1 or 0.01 and a ratio of immunobeads to tumor cells in excess of 75, a tumor cell depletion of more than 3 logs was achieved with the AB-4 IBs and slightly less with the AB-1 beads. After two consecutive cycles of purification with the AB-4 beads, no colonies were found, corresponding to more than 6 logs of purification. In the case of the AB-1 beads, 4 to 5 logs of purification were achieved. The concomitant reduction in clonogenic bone marrow progenitor cells was only 30 to 40%. Flow cytometric studies showed that the tumor cell population contained appreciable proportions of cells binding only small amounts of the antibodies used. The results indicate that the IB procedure is highly efficient and capable of removing tumor cells expressing low levels of antigen. Compared to other purging methods in use the procedure described seems to offer several advantages with respect to efficacy, speed, and simplicity. By the use of a panel of suitable antibodies the new immunobead procedure may be potentially useful in autologous bone marrow transplantation of B-lymphomas and non-T-leukemias with poor prognosis.     

Reactivity of a new Pan-B monoclonal antibody (MA6) against human leukaemias

The reactivity of a new Pan-B monoclonal antibody, MA6, against 69 cases of ALL, three plasmacytoma, 10 AML and five CML was evaluated. The antibody reacted positively against five of the 11 cases of U-ALL, 15 of the 26 C-ALL and all the cases of Pre B-ALL (one), B-ALL (one), B-CLL (12) and HCL (two). The MA6 did not react against the 16 cases of T-ALL, three plasmacytoma, 10 AML and five CML. The antibody has a broader spectrum of B-cell reactivity than CD9 and CD20 but is similar to the Pan-B antibody, CD19. MA6 appears to react against some of the very immature B cells and is therefore potentially useful, in conjunction with other antibodies such as CD9 and CD19, to confirm the B-lineage of some cases of U-ALL. The difference in the spectrum of reactivity against B-cell malignancies between MA6 and the other CDw40 antibody, G28-5, confirms their difference in antigenic specificity.     

Cure of SCID mice bearing human B-lymphoma xenografts by an anti-CD74 antibody-anthracycline drug conjugate.

PURPOSE: The purpose of this research was to test the therapeutic efficacy of an anthracycline-antibody conjugate for the treatment of human B-cell lymphoma in a preclinical animal model. EXPERIMENTAL DESIGN: Doxorubicin (dox) conjugates of the murine and humanized versions of the anti B-cell antibody LL1, targeting CD74, were prepared, along with a nonspecific control dox-antibody conjugate, targeting carcinoembryonic antigen. Antibody conjugates carried approximately 8-10 drug molecules attached site-specifically at thiols of reduced interchain disulfide bonds. Conjugates were tested, initially in vitro, and then for therapeutic efficacy in a systemic model, using a lethal i.v. dose of Raji cells in SCID mice. RESULTS: Dox-LL1 conjugates were shown to be stable and 3-fold more effective in vitro against the human B-cell Burkitt's lymphoma line, Raji, compared with the nonspecific control conjugate that did not target CD74 or B cells. When SCID mice were given an i.v. dose of 2.5 million Raji cells, they would die of disseminated disease within 15-25 days postinjection. A single dose of dox-LL1 conjugate, 117-350 micro g, given 5 days to 14 (advanced disease) days after injection of the Raji cells resulted in cure of most animals out to 180 days after injection of the cells, whereas animals in treatment control groups were not cured. The dose of dox-LL1 found useful in this work corresponds with a significantly lower drug dose than reported previously with other drug-antibody conjugates CONCLUSION: CD74 appears to be a uniquely useful target antigen for delivery of drugs, effecting cures of animals with single, low doses of conjugate.     

SC-1, a functional human monoclonal antibody against autologous stomach carcinoma cells

Human monoclonal antibodies were isolated from stomach carcinoma patients by the fusion of spleen and lymph node lymphocytes with the heteromyeloma line SPM4-0. Initial screening was carried out on autologous primary tumor cell cultures in an adhesion assay in order to select surface-reactive functional antibodies. This was followed by live cell immunoperoxidase staining assays. The human monoclonal antibody SC-1 was found to selectively react with cultured cells isolated from autologous and allogeneic stomach carcinoma patients, and with cryostat sections of the primary tumors. More extensive screening revealed that the antibody showed no reactivity with a wide range of tumor tissues and normal cells. Some reactivity was present on fetal tissues. SC-1 inhibits movement of the autologous tumor cells and identifies a protein with a molecular weight of 50,000. This study demonstrates that, with the use of selective screening assays on primary tumor material, it is possible to isolate antibodies which not only result from an immune response in the patient but also interfere with intercellular processes of tumor cells.     

Sensitive detection of human IgG in ELISA using a monoclonal anti-IgG-peroxidase conjugate

Enzyme-antibody (Ab) conjugates specific for IgG are widely used in indirect immunological assays and have been until recently routinely prepared with polyclonal IgG-specific animal Abs. The use of monoclonal Abs (MAbs) could permit a better standardization of the IgG-specific conjugate reagents but is expected to result in lower reactivity due to the recognition of a single epitope by the MAbs. In this work, we have characterized a monoclonal anti-human IgG-peroxidase (HRP) reagent and compared its reactivity with commercial reagents. The murine C5-1 anti-human IgG MAb was selected for conjugation because of its high affinity (K(a) = 1.9 x 10(10)M), pan-IgG reactivity and absence of cross-reactivity with various structures including animal IgGs. The specific activity and binding kinetics of the C5-1:HRP conjugate were similar to the ones of two polyclonal anti-IgG:HRP conjugates when tested with immobilized human IgG. The C5-1:HRP conjugate could detect low amounts of human IgG much more effectively than two commercial monoclonal conjugates although it was slightly less effective than a polyclonal conjugate. However, the C5-1 conjugate yielded reduced background reactivity compared to the polyclonal conjugate, resulting in similar signal-to-noise ratios. These results indicate that the C5-1:HRP conjugate could be a suitable substitute for anti-human IgG conjugates prepared from animal antisera.     

Chk1-dependent slowing of S-phase progression protects DT40 B-lymphoma cells against killing by the nucleoside analogue 5-fluorouracil

Chk1 plays a crucial role in the DNA damage and replication checkpoints in vertebrates and may therefore be an important determinant of tumour cell responses to genotoxic anticancer drugs. To evaluate this concept we compared the effects of the nucleoside analogue 5-fluorouracil (5FU) on cell cycle progression and clonogenic survival in DT40 B-lymphoma cells with an isogenic mutant derivative in which Chk1 function was ablated by gene targeting. We show that 5FU activates Chk1 in wild-type DT40 cells and that 5FU-treated cells accumulate in the S phase of the cell cycle due to slowing of the overall rate of DNA replication. In marked contrast, Chk1-deficient DT40 cells fail to slow DNA replication upon initial exposure to 5FU, despite equivalent inhibition of the target enzyme thymidylate synthase, and instead accumulate progressively in the G1 phase of the following cell cycle. This G1 accumulation cannot be reversed rapidly by exogenous thymidine or removal of 5FU, and is associated with increased incorporation of 5FU into genomic DNA and severely diminished clonogenic survival. Taken together, these results demonstrate that a Chk1-dependent replication checkpoint which slows S phase progression can protect tumour cells against the cytotoxic effects of 5FU.     

抗肝癌干细胞功能性单克隆抗体的研制

摘 要? 目的：研制抗肝癌干细胞的功能性单克隆抗体,为肝癌干细胞的靶向治疗提供候选抗体药物。方法：从人肝癌组织中分离人肝癌干细胞样细胞（human liver cancer stem-like cells, hLCSLCs）,免疫BALB/c裸鼠,采用脾细胞融合法制备大容量单抗库。应用细胞免疫荧光、无血清成球培养、裸鼠皮下成瘤等方法筛选、鉴定特异识别肝癌干细胞的单克隆抗体。流式细胞仪分选hLCSLCs侧群细胞（hLCSLCs side population cells, hLCSLCs-SP）,无血清悬浮培养法和CCK-8法检测杂交瘤单抗对hLCSLC-SP自我更新和增殖能力的影响。结果：细胞融合后获得2 964株杂交瘤克隆,在能与hLCSLCs反应的237株克隆中,有116株单抗能与hLCSLCs的细胞膜结合,其中的33株杂交瘤单抗只与hLCSLC-SP反应（阳性率为2%~5%）、不与非hLCSLC-SP反应。该33株单抗中有6株能与CD133阳性细胞有不同比例的共染,并且与无血清悬浮培养的成球细胞呈阳性反应(阳性率为3%~26%),明显高于hLCSLCs-SP。裸鼠皮下接种1×104个15D2单抗阳性的hLCSLCs,成瘤率为100%。功能性筛选实验发现,6株单抗中的4株能显著抑制hLCSLC-SP的增殖和成球生长,其抑制率分别为24%~42%和13%~50%。结论：采用自建的大容量单克隆抗体库技术,筛选获得了4株特异性识别hLCSLC-SP的功能性单抗,为肝癌干细胞的抗体靶向治疗奠定了基础。     

Establishment of a monoclonal antibody against glycosylated CD271 specific for cancer cells in immunohistochemistry

Various proteins are highly expressed in cancer (e.g., epidermal growth factor receptor); however, the majority are also expressed in normal cells, although they may differ in expression intensity. Recently, we reported that CD271 (nerve growth factor receptor), a glycosylated protein, increases malignant behavior of cancer, particularly stemlike phenotypes in squamous cell carcinoma (SCC). CD271 is expressed in SCC and in normal epithelial basal cells. Glycosylation alterations generally occur in cancer cells; therefore, we attempted to establish a cancer‐specific anti‐glycosylated CD271 antibody. We purified recombinant glycosylated CD271 protein, immunized mice with the protein, and screened hybridomas using an ELISA assay with cancer cell lines. We established a clone G4B1 against CD271 which is glycosylated with O‐glycan and sialic acid. The G4B1 antibody reacted with the CD271 protein expressed in esophageal cancer, but not in normal esophageal basal cells. This specificity was confirmed in hypopharyngeal and cervical cancers. G4B1 antibody recognized the fetal esophageal epithelium and Barrett''s esophagus, which possess stem cell–like characteristics. In conclusion, G4B1 antibody could be useful for precise identification of dysplasia and cancer cells in SCC.     

抗胰腺癌干细胞单克隆抗体的筛选和鉴定

目的：从抗胰腺癌干细胞单抗库中筛选、鉴定识别胰腺癌干细胞的功能性单抗,为胰腺癌干细胞靶向治疗提供候选抗体药物。方法：无血清悬浮培养及PKH26染色确定胰腺癌HPAC细胞系中肿瘤干细胞的存在。流式细胞术检测HPAC的干细胞标志物CD133在球体细胞中的阳性比例,检测20株杂交瘤单抗在HPAC亲本和球体细胞中的阳性表达。双色荧光标记流式细胞术检测CD133和单抗在HPAC亲本和球体细胞中的共表达比例;无血清悬浮培养法观察单抗15E9对HPAC成球细胞自我更新的影响。CCK一8法检测单抗15E9对HPAC细胞增殖和耐药的影响。结果：HPAC细胞能在无血清培养基中存活、增殖并形成细胞球,成球率为4．8％±0．6％。HPAC球体细胞中CD133’细胞的比例较亲本细胞提高至11．6倍。20株候选杂交瘤单抗中有3株单抗能识别HPAC球体细胞中CD133’细胞,其中单抗15E9共染比例为3．5％,并能显著抑制HPAC细胞的成球,抑制率达到30．4％。单抗15E9联合吉西他滨能显著抑制HPAC球体细胞的增殖,联合组和对照组Ic50分别为30．8nmol／L和58．1nmol／L。结论：本研究成功筛选出1株杂交瘤单抗可以识别胰腺癌干细胞,并且可识别CD133＋的胰腺癌干细胞;体外功能显示该抗体具有抑制HPAC干细胞的自我更新能力,抗体干预后显著降低HPAC耐药能力,可能是潜在的胰腺癌干细胞的靶向治疗抗体药物。     

采用肝癌球体细胞筛选鉴定识别肝癌干细胞的单抗

目的 采用肝癌球体细胞筛选鉴定识别肝癌干细胞的单抗,为靶向肿瘤干细胞治疗肝癌提供候选治疗单抗。方法 采用无血清悬浮培养的方法富集培养肝癌干细胞。通过细胞免疫荧光、顺铂耐药实验、Real-time PCR、裸鼠皮下成瘤实验等方法筛选、鉴定抗肝癌干细胞单抗。采用免疫组织化学的方法鉴定单抗识别的抗原在肝癌组织中的表达情况。质谱鉴定抗原。结果 MHCC97L细胞能在无血清悬浮培养条件下形成细胞球,并能被PKH26染料标记。流式细胞检测发现MHCC97L球体细胞中CD90表达比例较亲本细胞提高了3.4倍。无血清成球抑制实验获得6株显著抑制MHCC97L细胞在无血清成球的单抗,抑制率分别为54.67%,50.33%,45.73%,42.26%,39.11%,37.63%。细胞免疫荧光结果显示单抗28C10和CD90在MHCC97L细胞中能共定位。Real-Time PCR检测结果显示MHCC97L 28C10⁺细胞Sox-2和Oct-4表达显著高于MHCC97L 28C10^-细胞。流式细胞检测,在MHCC97L及其sphere细胞中28C10⁺细胞比例分别为7.98%和10.7%,28C10⁺细胞比例提高了1.34倍。流式细胞术分选获得的28C10⁺细胞的体外成球能力、侵袭能力显著高于28C10^-细胞。CCK-8法检测结果显示28C10⁺细胞较28C10^-细胞显示出对化疗药物顺铂的耐药,IC₅₀分别为1.96 μg/mL和1.16 μg/mL。裸鼠致瘤实验结果显示,28C10⁺细胞裸鼠皮下接种2×10⁴ cells/只,2个月可形成肿瘤,成瘤率为40%。另外1只未形成肿瘤的裸鼠形成了肺部转移灶(1/5)。免疫组化检测显示单抗28C10的靶抗原阳性率为72.0%(77/107),而在癌旁组织中低表达,差异有统计学意义。质谱结果显示28C10识别的抗原为HSP90α。结论 采用MHCC97L球体细胞模型成功鉴定一株特异识别肝癌干细胞的单抗,为靶向肝癌干细胞的抗体治疗奠定基础。