Intracellular expression of a single-chain antibody directed against type IV collagenase inhibits the growth of lung cancer xenografts in nude mice

It was documented that type IV collagenase with two subtypes of 72 ku/MMP-2 and 92 ku/MMP-9 plays an important role in tumor invasion and metastasis. The endoplasmic reticulum (ER)- retained, single chain Fv antibody fragment (scFv) was used to inhibit the function of type IV collagenase. For expression in mammalian cells, the assembled scFv M97 gene with ER retention signal encoding 6 additional amino acids (SEKDEL) was reamplified by PCR. The amplified fragments were cloned into the pcDNA3.1 vector. The resulting plasmid was sequenced and then introduced into PG cells, a highly metastatic human lung cancer cell line, by lipofectAMINE method. The result of intrabody gene therapy showed that type IV collegenase expression was down regulated significantly as measured by ELISA. The biological behavior of PG cell, such as the ability of in vitro invasion through Matrigel, colony formation on soft agar, was also inhibited by scFv M97 transfection. Animal experiments in a xenograft model of human lung cancer showed that scFv M97 transfection significantly prolonged the survival time of nude mice. The results indicate that intracellular antibody technology represents a novel and efficient way to abrogate selectively the activity of type IV collagenase.     

Intracellular expression of a single-chain antibody directed against type IV collagenase inhibits the growth of lung cancer xenografts in nude mice

It was documented that type IV collagenase with two subtypes of 72 ku/MMP-2 and 92 ku/MMP-9 plays an important role in tumor invasion and metastasis. The endoplasmic reticulum (ER)- retained, single chain Fv antibody fragment (scFv) was used to inhibit the function of type IV collagenase. For expression in mammalian cells, the assembled scFv M97 gene with ER retention signal encoding 6 additional amino acids (SEKDEL) was reamplified by PCR. The amplified fragments were cloned into the pcDNA3.1 vector. The resulting plasmid was sequenced and then introduced into PG cells, a highly metastatic human lung cancer cell line, by lipofectAMINE method. The result of intrabody gene therapy showed that type IV collegenase expression was down regulated significantly as measured by ELISA. The biological behavior of PG cell, such as the ability of in vitro invasion through Matrigel, colony formation on soft agar, was also inhibited by scFv M97 transfection. Animal experiments in a xenograft model of human lung cancer     

一种新型肿瘤血管抗体Fab的基因克隆与表达

鼠单克隆抗体AA98是我室研制的一株新型抗肿瘤血管抗体,体内实验证明它可以抑制血管生成,抑制肿瘤生长。从分泌抗体AA98的杂交瘤细胞中提取总RNA,采用逆转录PCR(RT-PCR)分别扩增重链Fd及轻链κ,并进行序列测定。将Fd和κ链依次与噬菌粒pComb3H连接,构建pComb3H-AA98Fab表达载体。在大肠杆菌中表达了AA98Fab。免疫印迹表明该Fab片段识别分子量为100kD的蛋白,具有原抗体AA98的抗原特异性。AA98Fab是研究抗体AA98作用机理的工具,也为制备重组免疫毒素,尝试肿瘤血管靶向治疗实验打下了基础。     

Laminin increases the release of type IV collagenase from malignant cells

We have studied the effect of laminin on type IV collagenolytic activity elaborated by malignant cells in culture. Laminin (at concentrations of 4-8 micrograms/ml) added to serum-free culture supernatants of subconfluent A2058 human melanoma cells significantly increased the release of the type IV collagenolytic activity (200-300%). The induction of type IV collagenase was more pronounced (580%) using a fragment of laminin which binds to the cell surface laminin receptor. A monoclonal antibody against the human laminin receptor blocked the effect of laminin on type IV collagenase, suggesting that occupation of the laminin receptor may be necessary for the effect. Increase in the type IV collagenolytic activity mediated by laminin was also demonstrated in two other malignant cell lines, HT fibrosarcoma (168%) and mouse melanoma (B16-F10) (271%). The increase in type IV collagenase was found to be specific for laminin because another cell-binding matrix protein, fibronectin, did not have any effect, and epidermal growth factor and transferrin actually decreased the type IV collagenase in human melanoma culture medium (epidermal growth factor, 50% at 20 ng/ml; and transferrin, 20% at 10 micrograms/ml). These studies suggest that tumor cell binding to laminin, which comprises the first step of basement membrane invasion, will induce the second step, namely the collagenolytic dissolution of the basement membrane.     

Activation of 72-kDa Type IV Collagenase/Gelatinase by Normal Fibroblasts in Collagen Lattices Is Mediated by Integrin Receptors but Is Not Related to Lattice Contraction

The matrix metalloproteinase 72-kDa type IV collagenase (also known as gelatinase A) is thought to be involved in both normal connective tissue remodeling and invasive pathological processes. Like other matrix metalloproteinases, 72-kDa type IV collagenase is secreted by fibroblast monolayers as an inactive proenzyme, but is unique among this enzyme family in that it is not activated by serine proteinases such as plasmin. However, when fibroblasts are cultured in a collagen lattice, a situation thought to better approximate in vivo conditions, we have invariably found much of the secreted 72-kDa type IV collagenase in its enzymatically active 62-kDa form. Although collagen lattice contraction appeared to be required for the activation of 72-kDa type IV collagenase, we have found that the process of contraction can be dissociated from proenzyme activation. Both cytochalasin D and α-methylmannoside completely blocked lattice contraction, but not proenzyme activation. Furthermore, the monoclonal antibody M-13, which is directed against the β₁ integrin chain, blocked collagen lattice contraction but not 72-kDa type IV procollagenase activation. At concentrations significantly higher than required to block lattice contraction or cell adhesion to collagen, M-13 was able to inhibit proenzyme activation. A second monoclonal antibody to the β₁ integrin, P5D2, had little effect on collagen lattice contraction at low concentrations, but could significantly inhibit the activation of 72-kDa type IV procollagenase. Antibodies to the integrin α₂ chain also inhibited proenzyme activation. These data show that the activation of 72-kDa type IV collagenase proenzyme, like collagen lattice contraction, is mediated by β₁ integrin receptors, possibly α₂β₁. Although both anti-β₁ antibodies used are directed to the same site on the integrin chain, the fact that each antibody preferentially blocks a different event, either lattice contraction or activation of 72-kDa type IV collagenase, suggests the existence of branch points in the receptor-mediated signal transduction pathway.     

Use of the polymerase chain reaction to clone and sequence a cDNA encoding the bovine alpha 3 chain of type IV collagen

A novel type IV collagen, alpha 3(IV), has previously been isolated from a collagenase digest of bovine and human glomerular and lens basement membranes. The cloning and sequencing of a cDNA encoding the alpha 3(IV) chain is described here. Using the polymerase chain reaction, with primers derived from the known 27-residue bovine alpha 3(IV) amino acid sequence, a 68-base pair bovine genomic fragment (KEM68) which encodes the known peptide sequence, was synthesized. KEM68 was then used to screen a bovine lens cDNA library and a 1.5-kilobase partial cDNA clone obtained, encoding 471 residues of the bovine alpha 3(IV) chain: 238 residues from the triple helical collagenous domain and all 233 residues of the noncollagenous domain. The collagenous repeat sequence has three interruptions, coinciding with those in the alpha 1(IV) chain. The noncollagenous domain has 12 cysteine residues in identical positions to those of other type IV collagens and 71, 61, and 70% overall similarity with the human alpha 1(IV), alpha 2(IV), and alpha 5(IV) chains. The noncollagenous domain of alpha 3(IV) is of particular interest as it appears to be the component of glomerular basement membrane that reacts maximally with the Goodpasture antibody. Furthermore, such antigenicity is absent from collagenase digests of the glomerular basement membrane of some patients with Alport syndrome. The alpha 3(IV) cDNA clone described here now permits study of the molecular pathology of COL4A3 in Alport syndrome.     

抗GPC3单链抗体的制备及其在肝细胞癌靶向检测中的应用

磷脂酰肌醇蛋白聚糖3 (Glypican-3,GPC3) 是Glypican家族的关键成员,它在肝细胞癌 (HCC) 的发展、血管生成和转移中起着重要作用。大多数HCC过表达GPC3,而在正常的成人肝脏和良性肝脏病变中几乎检测不到GPC3,因此其可以作为HCC高度特异性的诊断标志和理想的治疗靶标。文中将前期筛选得到的靶向GPC3的单克隆抗体1B11轻重链基因通过柔性肽 (Linker) 连接,经原核表达纯化,成功获得抗GPC3单链抗体1B11-scFv。通过蛋白印迹法鉴定验证了单链抗体的表达和装配正确;流式细胞术检测其与GPC3阳性细胞株具有与全长抗体相当的结合活性;将单链抗体与近红外荧光探针Cy5.5-NHS偶联,发现其在荷瘤裸鼠体内可精准靶向肿瘤部位,提示其具有实现探针引导下的多方位肝癌精准手术导航的潜力。     

Modulation of plasminogen activation and type IV collagenase activity by a synthetic peptide derived from the laminin A chain.

Laminin is a large multidomain glycoprotein with diverse biological activities which include stimulation of neurite outgrowth, enhancement of tumor metastasis, and promotion of cell growth, adhesion, and differentiation. A 19 amino acid synthetic peptide derived from the E8 fragment of the laminin A chain (Cys-Ser-Arg-Ala-Arg-Lys-Gln-Ala-Ala-Ser-Ile-Lys-Val-Ala-Val-Ser-Ala-Asp -Arg- NH2) was identified which promotes metastasis and stimulates collagenase IV activity in the culture medium of B16 melanoma cells (Kanemoto et al., 1990). We report that this peptide, here designated LamA2091-2108, is also a potent stimulator of tissue plasminogen activator (t-PA)-catalyzed plasminogen activation, resulting in a 22-fold increase in the kcat/Km of the activation reaction. The activity of purified type I and type IV collagenase was inhibited by LamA2091-2108 with IC50 values of 3 and 43 microM, respectively. These data support an alternative mechanism for the appearance of collagenase activity in the culture media of melanoma cells, namely, that the peptide stimulates plasminogen activation, subsequently generating collagenase activity.     

Antitumor effects of the molecule-downsized immunoconjugate composed of lidamycin and Fab′fragment of monoclonal antibody directed against type IV collagenase

Type IV collagenase plays an important role in tumor invasion and metastasis through cleaving type IV collagen in the basement membrane and extracellular matrix. In this study a molecule-downsized immunoconjugate (Fab'-LDM) was constructed by linking lidamycin (LDM), a highly potent antitumor antibiotic, to the Fab' fragment of a monoclonal antibody directed against type IV collagenase and its antitumor effect was investigated. As assayed in 10% SDS-PAGE gel, the molecular weight of Fab'-LDM conjugate was 65 kD with a 1 : 1 molecular ratio of Fab' and LDM. The Fab'-LDM conjugate maintained most part of the immunoreactivity of Fab' fragment to both type IV collagense and mouse hepatoma 22 cells by ELISA. By MTT assay, Fab'-LDM conjugate showed more potent cytotoxicity to hepatoma 22 cells than that of LDM. Administered intravenously, Fab'-LDM conjugate proved to be more effective against the growth of subcutaneously transplanted hepatoma 22 in mice than free LDM in two experiment settings. In Experiment     

Partial purification and characterization of a neutral protease which cleaves type IV collagen

A neutral protease has been extracted from the media of cultured metastatic tumor cells and purified approximately 1000 times after sequential ammonium sulfate fractionization, concanavalin A column chromatography, and molecular sieve chromatography. The protease has an apparent molecular weight of 70 000--80 000, is inactive at acid pH, requires trypsin activation, and is inhibited by ethylene-diaminetetraacetic acid but not by phenylmethanesulfonyl fluoride, N-ethylmaleimide, or soybean trypsin inhibitor. The enzyme produces specific cleavage products for both chains of pro type IV collagen isolated without pepsinization and apparently cleaves at one point in a major pepsin-extracted chain of placenta type IV collagen. The partially purified enzyme fails to significantly degrade other collagens or fibronectin under digestion conditions in which specific reaction products are produced for type IV collagen. The existence of this enzyme is significant since previously described animal collagenases fail to degrade type IV collagen. Such a type IV specific collagenase could play a role in tumor invasion and may be secreted by other cells such as endothelial cells, epithelial cells, and immune cells.     

Mn-SOD与抗癌胚抗原单链抗体基因的融合及高效表达

将Ｍｎ－ＳＯＤ与抗癌胚抗原（ＣＥＡ）单链抗体基因（Ｓｃ－Ｆｖ ｇｅｎｅ）融合,重组到含Ｔ７启动子的表达载体ｐＥＴ－２２ｂ（＋）中,构建表达质粒ｐＥＴＭｎ－ＳＯＤ－ＳｃＦｖ,并转化大肠杆菌ＢＬ２１（ＤＥ３）,进行高效表达,表达物占菌体可溶性总蛋白的２４％。ＳＤＳ－ＰＡＧＥ和蛋白质和迹图谱显示表达物分子量为４５ｋＤ与融合基因编码蛋白质的理论值相符。该蛋白质在大肠杆菌中为泌型表达有利于纯化。ＲＩＡ测定表     

一株针对人EGFR的单链抗体克隆与哺乳细胞表达

目的:制备特异性抗人表皮生长因子受体(EGFR)的单链抗体(sc Fv),鉴定其生物学活性,为进一步研究基于单链抗体的免疫治疗奠定基础。方法:从分泌抗人EGFR单克隆抗体的杂交瘤细胞系提取总RNA,利用5'RACE技术扩增轻链和重链可变区(VL、VH)基因,构建具有VL、VH基因的单链抗体基因,并将构建的单链抗体基因克隆到真核细胞表达载体pc DNA3.1中进行表达和鉴定。ELISA鉴定单链抗体对抗原的特异性;Fortebio检测抗原抗体间的亲和力,流式细胞术检测单链抗体结合肺癌细胞系天然EGFR的功能活性。结果:获得唯一的轻重链可变区序列VL、VH,成功构建EGFR-sc Fv,特异性与天然EGFR蛋白结合,亲和力达3.22×10-9mol/L。结论:成功构建了抗人EGFR单链抗体,为肺癌免疫导向治疗研究奠定了基础。     

抗IV型胶原酶单链抗体在毕赤酵母中分泌表达*

利用毕赤酵母系统表达抗 IV型胶原酶人单链抗体。首先把目的基因克隆到毕赤酵母表达载体上 ,电击转化受体菌。在甲醇诱导下表达单链抗体。 SDS- PAGE和免疫印迹显示毕赤酵母分泌表达人单链抗体 ,表达量约 2 0 mg/ L酵母培养物。该表达系统与大肠杆菌相比 ,简化了表达产物的分离纯化程序。     

Antitumor effects of the molecule-downsized immunoconjugate composed of lidamycin and Fab’ fragment of monoclonal antibody directed against type IV collagenase

Type IV collagenase plays an important role in tumor invasion and metastasis through cleaving type IV collagen in the basement membrane and extracellular matrix. In this study a molecule-downsized immunoconjugate (Fab’-LDM) was constructed by linking lidamycin (LDM), a highly potent antitumor antibiotic, to the Fab’ fragment of a monoclonal antibody directed against type IV collagenase and its antitumor effect was investigated. As assayed in 10% SDS-PAGE gel, the molecular weight of Fab’-LDM conjugate was 65 kD with a 1: 1 molecular ratio of Fab’ and LDM. The Fab’-LDM conjugate maintained most part of the immunoreactivity of Fab’ fragment to both type IV collagense and mouse hepatoma 22 cells by ELISA. By MTT assay, Fab’-LDM conjugate showed more potent cytotoxicity to hepatoma 22 cells than that of LDM. Administered intravenously, Fab’-LDM conjugate proved to be more effective against the growth of subcutaneously transplanted hepatoma 22 in mice than free LDM in two experiment settings. In Experiment I, the drugs were given intravenously on day 1 and day 8. Fab’-LDM at the doses of 0.025 mg/kg, 0.05 mg/kg and 0.1 mg/kg inhibited tumor growth by 76.7%, 93.3% and 94.8%, while free LDM at 0.05 mg/kg inhibited tumor growth by 76.1%, respectively. In experiment II, the drugs were given intravenously on day 4 and day 11, Fab’-LDM at the doses of 0.025 mg/kg and 0.05 mg/kg inhibited tumor growth by 74.2%, 80.9%, while free LDM at 0.05 mg/kg inhibited tumor growth by 60.5%, respectively. In terms of survival time, Fab’-LDM was more effective than free LDM. The results suggest that the molecule-downsized immunoconjugate directed against type IV collagenase is of high efficacy in experimental cancer therapy.     

H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen

H-ras-transformed human bronchial epithelial cells (TBE-1) secrete a single major extracellular matrix metalloprotease which is not found in the normal parental cells. The enzyme is secreted in a latent form of 72 kDa, which can be activated to catalyze the cleavage of the basement membrane macromolecule type IV collagen. The substrates in their order of preference are: gelatin, type IV collagen, type V collagen, fibronectin, and type VII collagen; but the enzyme does not cleave the interstitial collagens or laminin. This protease is identical to gelatinase isolated from normal human skin explants, normal human skin fibroblasts, and SV40-transformed human lung fibroblasts. Based on its ability to initiate the degradation of type IV collagen in a pepsin-resistant portion of the molecule, it will be referred to as type IV collagenase. This enzyme is most likely the human analog of type IV collagenase detected in several rodent tumors, which has the same molecular mass and has been linked to their metastatic potential. Type IV collagenase consists of three domains. Two of them, the amino-terminal domain and the carboxyl-terminal domain, are homologous to interstitial collagenase and human and rat stromelysin. The middle domain, of 175 residues, is organized into three 58-residue head-to-tail repeats which are homologous to the type II motif of the collagen-binding domain of fibronectin. Type IV collagenase represents the third member of a newly recognized gene family coding for secreted extracellular matrix metalloproteases, which includes interstitial fibroblast collagenase and stromelysin.     

具有谷胱甘肽过氧化物酶活性的含硒单链抗体酶制备

 利用RT PCR从分泌有谷胱甘肽结合部位的单克隆抗体杂交瘤细胞株 2F3中 ,扩增出单抗重链可变区和轻链可变区基因 .经DNA测序后 ,用Linker(Gly4 Ser1) 3 构建成单链抗体 (scFv)表达载体pTMF scFv ,将重组质粒pTMF scFv转化到大肠杆菌BL2 1(DE3) ,实现了单链抗体的高效表达 .表达的单链抗体占菌体总蛋白 2 5%～ 30 % .该重组蛋白以包涵体形式存在 ,分子量为 30kD .经过金属螯合亲和层析纯化、复性和凝胶过滤纯化 ,得到电泳均一的单链抗体 .再经化学诱变 ,得到含硒单链抗体酶 ,其谷胱甘肽过氧化物酶活性为 330 0U μmol.采用荧光滴定法测定了单链抗体对谷胱甘肽的结合常数     

Enzyme-antibody histochemistry. A method for detection of collagens collectively

Different types of distinct molecular forms of collagen are components of the extracellular matrix in most tissues. The common types can usually be detected by immunohistochemical methods but others may escape detection for lack of specific antisera. However, all these collagens are substrates for the collagenase of Clostridium histolyticum. In this report we describe a method that allows the visualization of collagens, collectively, in a tissue preparation. The method is based on the affinity between clostridial collagenase and collagen on one hand, and collagenase and its antibody on the other. Under the conditions of low temperature used in the procedure, collagenase binds to collagen, but digestion does not occur. Subsequent reaction of the bound collagenase with the specific collagenase antibody is followed by reaction with a tagged anti-IgG reagent. This allows the visualization of the enzyme-substrate complex. The procedure is illustrated in sections of the heart and the aorta, as well as in the isolated cardiomyocytes and the collagen distribution is verified using collagens type I and IV specific antibodies. In all instances the collagenase staining pattern includes all structural features seen individually with the type specific anticollagen antibodies.     

Monoclonal antibodies to type IV collagenase recognize a protein with limited sequence homology to interstitial collagenase and stromelysin

Type IV collagenase is a metalloproteinase associated with metastatic tumor cells. It specifically cleaves the triple helical basement membrane (type IV) collagen molecule at a single site. Monoclonal antibodies which block the activity of the human type IV collagenase were developed and used to purify this antigen. The purified type IV collagenase was partially sequenced following cyanogen bromide and trypsin cleavage. The amino acid sequence of the human type IV collagenase fragments revealed a region homologous to the human interstitial collagenase and stromelysin. However, several sequences in type IV collagenase were identified which are distinct from the latter. Polyclonal antibodies were raised against a synthetic peptide derived from such a sequence. Following affinity purification, the antibodies recognized the denatured human type IV collagenase in Western immunoblotting. These data indicate that type IV collagenase is a distinct member of a general family of metalloproteinases.     

A new recombinant single chain trispecific antibody recruits T lymphocytes to kill CEA (carcinoma embryonic antigen) positive tumor cells in vitro efficiently

Anti-tumor associated antigen (TAA).CD3.CD28 trispecific antibody(TsAb) is able to provide two signals for fully and continuously activation of T lymphocytes and recruit them around tumor cells, presenting an attractive concept in tumor immunotherapy. Here, a new single chain trispecific antibody (scTsAb), named CEA-scTsAb, was constructed by fusion of anti-CEA (Carcinoma Embryonic Antigen) single chain antibody (scFv), anti-CD3 scFv and anti-CD28 VH, spaced by polypeptide interlinkers taken from the fragment of constant region (FC) of human IgG and human serum albumin (HSA). It was expressed in Escherichia coli at low temperature (30 degrees C) with up to 50% of the antibody being present in soluble form. After one step of DEAE anion chromatography, the soluble product was sufficiently pure for further in vitro activity assays. First, it was proved that CEA-scTsAb could recognize three antigens (CEA, CD28 and Jurkat cell membrane antigen) specifically and could distinguish antigen positive cells from antigen negative cells in vitro. Then fresh PBMC (peripheral blood mononuclear cells), without being pre-treated by co-stimulatory reagents, such as IL-2 or CD28 mAb, were used as effector cells to test their ability to mediate tumor specific cytolysis of CEA-positive tumor cells, SW1116. It was found by photomicrography that T lymphocytes were attracted to SW1116 cells in the presence of CEA-scTsAb, which resulted in effective cytolysis of tumor cells. As shown by MTT assay, the efficacy of tumor specific cytolysis mediated by CEA-scTsAb related to both the quantity and activation of PBMC. At an effector cells/target cells ratio (E/T) of 5, it was proved by dual-color FACS with propidium iodide (PI) and FITC-annexin V that both necrosis and apoptosis of tumor cells were causes of tumor specific cytolysis. In summary, a new single chain trispecific (CEA x CD3 x CD28) antibody was constructed and characterized carefully in this paper and was found to possess functions: (i) to activate T lymphocytes independently of additional co-stimulatory signal, (ii) to attract activated T lymphocytes around CEA-positive tumor cells, (iii) to attack CEA-positive tumor cells with recruited T lymphocytes. Because it recognizes a widely distributed tumor antigen (CEA), with moderate molecular weight (about 75 kDa) and a simple production procedure, and is able to mediate a high level of tumor specific cytolysis without any additional co-stimulating reagents, CEA-scTsAb is very promising for the task of immunotherapy in future.     

Purification and characterization of human 72-kDa gelatinase (type IV collagenase). Use of immunolocalisation to demonstrate the non-coordinate regulation of the 72-kDa and 95-kDa gelatinases by human fibroblasts.

Human gingival fibroblast gelatinase (type IV collagenase) has been purified to homogeneity using a combination of ion exchange chromatography, gel filtration and affinity chromatography. The purified proenzyme electrophoresed under reducing conditions as a single band of 72 kDa which could be activated to a species of 65 kDa. Gelatinase was activated by organomercurials by a process apparently initiated by a conformational change and involving self-cleavage. It was not activated by trypsin or plasmin unlike the other family members, collagenase and stromelysin. Gelatinase otherwise exhibited properties typical of the metalloproteinases: it was inhibited by metal chelating agents and by the specific inhibitor TIMP (tissue inhibitor of metalloproteinases). Its major substrate was shown to be denatured collagen although it was also able to degrade native type IV and V collagens. A polyclonal antibody was raised in a sheep using the purified enzyme as antigen. The antiserum recognised and specifically inhibited the 72-kDa gelatinase but not a 95-kDa gelatinase from pig leukocytes. It was used in immunolocalisation studies on human fibroblasts to investigate the regulation of the production of the two Mr forms of gelatinase. These studies clearly demonstrate that human fibroblasts constitutively synthesize and secrete 72-kDa gelatinase but that 95-kDa gelatinase was inducible by agents such as cytokines. The significance of these results in relation to the likely in vivo r?le of gelatinases is discussed.