Abstract: Despite recent advances, treatment of leukemia is often not curative. New insights indicate that this may be attributable to a small population of therapy-resistant malignant cells with self-renewal capacity and the ability to generate large numbers of more differentiated leukemia cells. These leukemia-initiating cells are commonly referred to as Leukemia Stem Cells (LSCs). LSCs are regarded as the root of leukemia origin and leukemia recurrence after seemingly successful therapy. Not surprisingly therefore, contemporary leukemia research has focused on ways to specifically eliminate LSCs, leading to the identification of several promising anti-LSC strategies. Firstly, LSCs may be eliminated by antibody- or ligand-based cell surface delivery of therapeutics such as naked antibodies, immunotoxins, and immunocytokines. This approach exploits LSC-associated surface antigens, such as CD33, CD44, CD96, CD123 and CLL-1 for LSC-selective therapy and aims to spare normal hematopoietic stem cells. A second strategy aims to disrupt the interactions between LSCs and their highly specialized niche. These interactions appear to be pivotal for maintenance of the stem cell-like characteristics of LSCs. A third strategy centers on the selective modulation of aberrantly activated signaling pathways central to LSC biology. A fourth strategy, dubbed 'epigenetic reprogramming', aims to selectively reverse epigenetic alterations that are implicated in ontogeny and maintenance of LSCs. In this review, we will discuss the rationale for these LSCs-targeted strategies and highlight recent advances that may ultimately help pave the way towards selective LSCs-elimination.
Abstract: Gemtuzumab ozogamicin (GO, Mylotarg) is a targeted therapeutic agent in which an anti-CD33 antibody is chemically coupled to a highly cytotoxic calicheamicin derivative through a hydrolysable linker. GO has improved the treatment outcome for a subgroup of acute myeloid leukemia (AML) patients, but its use is associated with severe myelosuppression and hepatotoxicity. Here, we report on a novel anti-leukemia agent, designated scFvCD33:sTRAIL, in which an anti-CD33 single chain fragment of variable regions (scFv) antibody fragment is genetically linked to soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). Normal CD33-positive monocytes were fully resistant to prolonged treatment with scFvCD33:sTRAIL, whereas treatment with GO resulted in substantial cytotoxicity. The activity of scFvCD33:sTRAIL towards AML cells was up to 30-fold higher than GO. The CD33-restricted anti-leukemia activity of scFvCD33:sTRAIL remained stable during prolonged storage at 37 degrees C, whereas GO showed a rapid increase in CD33-independent cytotoxicity. Moreover, scFvCD33:sTRAIL showed potent anti-leukemia activity towards CD33+ CML cells when treatment was combined with the Bcr-Abl tyrosine kinase inhibitor, Gleevec. Importantly, ex vivo treatment of patient-derived CD33+ AML tumor cells with scFvCD33:sTRAIL resulted in potent apoptosis induction that was enhanced by valproic acid, mitoxantrone and 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG). Taken together, scFvCD33:sTRAIL is superior to GO in terms of tumor selectivity, activity and stability, warranting its further development for the treatment of CD33-positive leukemias.
Abstract: Antibody-based therapeutic approaches are yielding more and more of the promise they have held since the conception of the 'magic bullet' theory by Paul Ehrlich. The beneficial effect of antibody-based therapies is directly related to antibody-dependent functions, such as neutralization and antibody-dependent cellular cytotoxicity, but in many cases also relies on the delivery of toxic compounds to cancerous cells. However, the clinical utility of toxic antibody conjugates can be significantly hampered by side effects. Ideal effector compounds are inactive 'en route', but gain full activity once the antibody conjugate has bound to cancerous cells. Of significant potential in this respect are the pro-apoptotic ligands Tumor Necrosis Factor (TNF), fibroblast-associated cell-surface ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL). TNF ligands are normally present as homotrimeric transmembrane proteins, but can also be processed into a soluble trimeric form. Compared to their corresponding transmembrane counterpart, soluble TNF, FasL and TRAIL have a strongly reduced capacity to activate TNF receptor 2, Fas and TRAIL receptor 2. However, all sequence information required for full activation of these receptors is latently retained in these soluble ligands and can be unmasked by oligomerization or cell surface immobilization. The latter provides a clear rationale for the use of these ligands as effectors in antibody-based therapy. The antibody-targeted ligand will be in a relatively inactive soluble form while en route. However, once bound to the targeted cancer cell the soluble TNF ligand fusion proteins will be converted into fully active membrane ligand-like molecules. Here we will, after briefly detailing the biology of TNF, TRAIL and FasL, focus on the promises and pitfalls of targeted TNF ligand fusion proteins in achieving a 'magic bullet' with maximum cancer selective activity and minimal side effects.
Abstract: OBJECTIVE: Cell membrane antigens like the gastrin-releasing peptide receptor (GRPR), the prostate stem cell antigen (PSCA), and the prostate-specific membrane antigen (PSMA), expressed in prostate cancer, are attractive targets for new therapeutic and diagnostic applications. Therefore, we investigated in this study whether these antigens are expressed in metastasized prostate cancer. METHODS: Formalin-fixed, paraffin-embedded specimens of 15 patients with uni- or bilateral lymph node metastases of prostate cancer (totaling 21 cases) and 17 patient-cases of bone metastases were processed for immunohistochemistry with anti-GRPR, anti-PSCA, and anti-PSMA antibodies. A pathologist blinded to clinical and pathological data scored the immunoreactivity for these antibodies on a four-point scale (0 = no staining; 1+ = weak staining; 2+ = moderate staining; 3+ = strong staining) and documented the distribution pattern. RESULTS: GRPR staining in lymph node metastases was seen in 85.7% of cases (18 of 21 cases), PSCA in 95.2% (20/21), and PSMA in 100% (21/21). GRPR in bone metastases was seen in 52.9% of cases (9/17), PSCA in 94.1% (16/17), and PSMA in 100% (17/17). CONCLUSION: We have shown for the first time that GRPR is expressed in the vast majority of lymph node metastases and in 52.9% of bone metastases of prostate cancer. PSCA and PSMA are both highly expressed in lymph node and bone metastases. Although PSCA and PSMA are mostly expressed in prostate cancer metastases, GRPR offers an interesting alternative target as it can be targeted relatively easy with peptide-based (radio)pharmaceuticals.
Abstract: Previously, we have shown that epidermal growth factor receptor (EGFR)-selective delivery of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL), by genetic fusion to antibody fragment scFv425, enhances the tumor-selective pro-apoptotic activity of sTRAIL. Insight into the respective contribution of the agonistic receptors TRAIL-R1 and TRAIL-R2 to TRAIL-induced apoptosis may provide a rational approach to further optimize TRAIL-based therapy. Recently, this issue has been investigated using sTRAIL mutants designed to selectively bind to either receptor. However, the relative contribution of the respective TRAIL receptors, in particular TRAIL-R1, in TRAIL signaling is still unresolved. Here, we fused scFv425 to designed sTRAIL mutant sTRAILmR1-5, reported to selectively activate TRAIL-R1, and investigated the therapeutic apoptotic activity of this novel fusion protein. EGFR-specific binding of scFv425:sTRAILmR1-5 potently induced apoptosis, which was superior to the apoptotic activity of scFv425:sTRAIL-wt and a nontargeted MOCK-scFv:sTRAILmR1-5. During cotreatment with cisplatin or the histone deacetylase inhibitor valproic acid, scFv425:sTRAILmR1-5 retained its superior pro-apoptotic activity compared to scFv425:sTRAIL-wt. However, in catching-type Enzyme-Linked ImmunoSorbent Assays with TRAIL-R1:Fc and TRAIL-R2:Fc, scFv425:sTRAILmR1-5 was found to not only bind to TRAIL-R1 but also to TRAIL-R2. Binding to TRAIL-R2 also had functional consequences because the apoptotic activity of scFv425:sTRAILmR1-5 was strongly inhibited by a TRAIL-R2 blocking monoclonal antibody. Moreover, scFv425:sTRAILmR1-5 retained apoptotic activity upon selective knockdown of TRAIL-R1 using small inhibitory RNA. Collectively, these data indicate that both agonistic TRAIL receptors are functionally involved in TRAIL signaling by scFv425:sTRAILmR1-5 in solid tumor cells. Moreover, the superior target cell-restricted apoptotic activity of scFv425:sTRAILmR1-5 indicates its therapeutic potential for EGFR-positive solid tumors.
Abstract: Ischemia-reperfusion injury is a leading cause of acute renal failure and a major determinant in the outcome of kidney transplantation. Here we explored systemic gene therapy with a modified adenovirus expressing Interleukin (IL)-13, a cytokine with strong anti-inflammatory and cytoprotective properties. When ischemia was induced we found that the IL-13 receptor is expressed in both the normal and experimental kidneys. Prior to the induction of ischemia, rats received adenovirus-IL-13, control adenovirus or saline. IL-13 plasma levels increased more than 50-fold in adenovirus-IL-13 treated animals, confirming successful IL-13 gene delivery. Histological analysis showed decreased tubular epithelial cell damage with adenovirus-IL-13 therapy, accompanied by reduced kidney injury molecule-1 expression. Interstitial infiltration by neutrophils and macrophages was reduced by half as was interstitial fibrosis and expression of alpha-smooth muscle actin. IL-13 treatment significantly diminished the expression of E-selectin, IL-8, MIP-2, TNF-alpha and MCP-1 mRNA. These results suggest that the use of systemic IL-13 gene therapy may be useful in reducing renal tubulointerstitial damage and inflammation caused by ischemia-reperfusion.
Abstract: The gastrointestinal tract is a complex niche and the main port of entry of many pathogens that trigger a wide range of diseases like inflammatory bowel disease (IBD) and colon cancer. Antibodies are effective for treating such diseases, but a system capable of local delivery at the site of the pathology, thus avoiding systemic side effects, is not yet available. Here we report a novel recombinant scFvSIgA1 protein produced by Lactococcus lactis, anchored to the bacterial membrane, which retains its full immuno-recognizing potential. This scFv fragment employed was specific for a colon cancer epitope, epithelial glycoprotein protein-2 (EGP-2). Accordingly L. lactis expressing this chimeric protein was capable of binding cells expressing this epitope. Expression of specific antibodies on bacteria may allow local delivery of anticancer agents produced by such bacteria in conjunction with the antibody and provides a new avenue in the quest for targeted drug delivery.
Abstract: The clinical efficacy of the CD20-specific chimeric monoclonal antibody rituximab is significantly hampered by intrinsic or acquired resistance to therapy. Rituximab activates antibody-dependent cellular cytotoxicity/complement-dependent cytotoxicity-dependent lysis but also induces apoptosis by cross-linking of its target antigen CD20. Recent reports indicate that this apoptotic activity of rituximab can be synergized by cotreatment with Fas agonists. Here, we report on a strategy designed to exploit and optimize the synergy between rituximab and Fas signaling by genetically fusing a rituximab-derived antibody fragment to soluble Fas ligand (sFasL). The resultant fusion protein, designated scFvRit:sFasL, potently induced CD20-restricted apoptosis in a panel of malignant B-cell lines (10 of 11) and primary patient-derived malignant B cells (two of two non-Hodgkin lymphoma and five of six B cell chronic lymphocytic leukemia). ScFvRit:sFasL efficiently activated CD20 and Fas apoptotic signaling, resulting in a far superior proapoptotic activity compared with cotreatment with rituximab and Fas agonists. ScFvRit:sFasL lacked activity toward normal human B cells and also lacked systemic toxicity in nude mice with no elevation of aspartate aminotransferase and alanine aminotransferase levels or liver caspase-3 activity. In conclusion, scFvRit:sFasL efficiently activates CD20 and Fas-apoptotic signaling and may be useful for the elimination of malignant B cells.
Abstract: EpCAM is a well-established pancarcinoma-associated target antigen that has been used in a variety of therapeutic approaches. Of particular appeal are those strategies that aim to retarget and locally activate immune effector mechanisms involving apoptosis. Cancer cells typically employ various strategies to evade recognition and elimination by immune effector cells, including low or absent expression of MHCI molecules and active elimination of tumor infiltrating immune cells. In addition, cancer cells show an increased resistance towards endogenous pro-apoptotic stimuli due to aberrancies in their apoptotic machinery. However, compelling evidence indicates that cancer cells are often reliant on these molecular aberrations for continued cell survival. This pivotal role of immune evasion and apoptosis resistance has fueled the quest for therapeutic strategies that can selectively retarget and reactivate immune effector cells or molecules, whereby the balance of cellular fate of cancer cells is selectively tipped towards apoptosis. Here we review and discuss the perspectives for EpCAM-targeted apoptosis induction in cancer by EpCAM-selective bispecific antibodies and TRAIL fusion proteins.
Abstract: Prostate cancer is one of the most common causes of cancer in men. Evaluating the different stages of prostate cancer with conventional imaging techniques still proves difficult. Nuclear imaging might provide a technique that is able to evaluate prostate cancer, but clinical application has been limited due to lack of accuracy of current radiopharmaceuticals. The development of radiopharmaceuticals that can be targeted to specific antigens, overexpressed in prostate cancer, but sparse in normal tissue, is crucial. Peptides are of particular interest because of their favourable characteristics, leading to increased attention for nuclear imaging of the gastrin-releasing-peptide-receptor (GRPR) with radiolabelled bombesin-like peptides. Several derivatives of bombesin and its truncated form have been prepared for imaging with single photon emission computed tomography (SPECT) or positron emission tomography (PET), thereby delivering potent candidates for further clinical evaluation. This article provides an overview of the development and preclinical evaluation of radiolabelled bombesin analogues for in vivo targeting of GRPR in prostate cancer. The effect of the radionuclide, chelator, spacer and unnatural amino acids on affinity, metabolic stability and image quality are discussed, as well as agonistic or antagonistic properties. Potent candidates are proposed based on these selection criteria: (I) high affinity for GRPR, with rapid and specific tumour uptake (II) high hydrophilicity resulting in the preferred renal-urinary mode of excretion and low hepatobiliary excretion, (III) high stability, but relatively rapid clearance from blood. Also, a summary is made of clinical studies that report on the detection of prostate cancer with GRPR targeted radiopharmaceuticals.
Abstract: Previously, we demonstrated potent tumor cell-selective pro-apoptotic activity of scFv425:sTRAIL, a recombinant fusion protein comprised of EGFR-directed antibody fragment (scFv425) genetically fused to human soluble TNF-related apoptosis-inducing ligand (sTRAIL). Here, we report on the promising therapeutic systemic tumoricidal activity of scFv425:sTRAIL when produced by the replication-deficient adenovirus Ad-scFv425:sTRAIL. In vitro treatment of EGFR-positive tumor cells with Ad-scFv425:sTRAIL resulted in the potent induction of apoptosis of not only infected tumor cells, but importantly also of up to 60% of noninfected EGFR-positive tumor cells. A single intraocular injection of Ad-scFv425:sTRAIL in tumor-free nu/nu mice resulted in predominant liver infection and concomitant high blood plasma levels of scFv425:sTRAIL. These mice showed no sign of Ad-scFv425:sTRAIL-related liver toxicity. Identical treatment of mice with established intraperitoneal renal cell carcinoma xenografts resulted in rapid and massive tumor load reduction and subsequent long-term survival. Taken together, adenoviral-mediated in vivo production of scFv425:sTRAIL may be exploitable for systemic treatment of EGFR-positive cancer.
Abstract: Ab binding to CD20 has been shown to induce apoptosis in B cells. In this study, we demonstrate that rituximab sensitizes lymphoma B cells to Fas-induced apoptosis in a caspase-8-dependent manner. To elucidate the mechanism by which Rituximab affects Fas-mediated cell death, we investigated rituximab-induced signaling and apoptosis pathways. Rituximab-induced apoptosis involved the death receptor pathway and proceeded in a caspase-8-dependent manner. Ectopic overexpression of FLIP (the physiological inhibitor of the death receptor pathway) or application of zIETD-fmk (specific inhibitor of caspase-8, the initiator-caspase of the death receptor pathway) both specifically reduced rituximab-induced apoptosis in Ramos B cells. Blocking the death receptor ligands Fas ligand or TRAIL, using neutralizing Abs, did not inhibit apoptosis, implying that a direct death receptor/ligand interaction is not involved in CD20-mediated cell death. Instead, we hypothesized that rituximab-induced apoptosis involves membrane clustering of Fas molecules that leads to formation of the death-inducing signaling complex (DISC) and downstream activation of the death receptor pathway. Indeed, Fas coimmune precipitation experiments showed that, upon CD20-cross-linking, Fas-associated death domain protein (FADD) and caspase-8 were recruited into the DISC. Additionally, rituximab induced CD20 and Fas translocation to raft-like domains on the cell surface. Further analysis revealed that, upon stimulation with rituximab, Fas, caspase-8, and FADD were found in sucrose-gradient raft fractions together with CD20. In conclusion, in this study, we present evidence for the involvement of the death receptor pathway in rituximab-induced apoptosis of Ramos B cells with concomitant sensitization of these cells to Fas-mediated apoptosis via Fas multimerization and recruitment of caspase-8 and FADD to the DISC.
Abstract: Gemtuzumab ozogamicin (GO) is a calicheamicin-conjugated antibody directed against CD33, an antigen highly expressed on acute myeloid leukemic (AML) cells. CD33-specific binding triggers internalization of GO and subsequent hydrolytic release of calicheamicin. Calicheamicin then translocates to the nucleus, intercalates in the DNA structure and subsequently induces double-strand DNA breaks. GO is part of clinical practice for AML, but is frequently associated with severe side effects. Therefore, combination of GO with other therapeutics is warranted to reduce toxicity, while maximizing therapeutic selectivity. We hypothesized that the histone deacetylase inhibitor valproic acid (VPA) sensitizes AML cells to GO. VPA-induced histone hyperacetylation opens the chromatin structure, whereby the DNA intercalation of calicheamicin should be augmented. We found that clinically relevant concentrations of VPA potently augmented the tumoricidal activity of GO towards AML cell lines and primary AML blasts. Moreover, VPA treatment indeed augmented the DNA intercalation of calicheamicin and enhanced DNA degradation. Importantly, synergy was restricted to CD33-positive AML cells and did not require caspase activation. In conclusion, the synergistic proapoptotic activity of cotreatment of AML cells with VPA and GO indicates the potential value of this strategy for AML.
Abstract: BACKGROUND: Kidney transplantation is possible by virtue of systemic immunosuppression, which is in turn accompanied by serious side effects. The search for novel therapeutic agents and strategies is ongoing. Here we investigate the effects of adenovirus-mediated gene therapy with interleukin (IL)-13, which is a cytokine with strong immunomodulatory properties, on acute renal allograft injury. In addition, we compare the effects of local (intrarenal) and systemic (intramuscular) IL-13 gene therapy in kidney transplantation. METHODS: The experiments were performed in a rat Fisher to Lewis acute rejection model of kidney transplantation. An adenovirus-IL-13 or adenovirus-luciferase was injected either into the donor kidney before transplantation (local treatment) or into the hind leg muscle of recipient rats (systemic treatment). A group with no treatment served as control. No additional immunosuppressive therapy was applied. The rats were sacrificed after 8 days and inflammatory markers and renal pre-fibrosis were assessed. RESULTS: Efficient gene transfer was confirmed by ELISA, immunohistochemistry and real-time PCR. IL-13 gene therapy diminished graft infiltration with macrophages and cytotoxic T cells and limited up-regulation of mRNA levels of the adhesion molecule E-selectin and pro-inflammatory cytokines TNF-alpha and IFN-gamma. Moreover, reduced renal interstitial pre-fibrosis was found in the rats receiving IL-13 gene therapy. The effects of local and systemic therapy were similar. CONCLUSIONS: This study demonstrates that IL-13 gene therapy in the graft significantly attenuates acute renal allograft damage, suggesting local therapy with IL-13 as a strategy to reduce the need for systemic immunosuppressive medication and thereby its side effects.
Abstract: Agonistic anti-Fas antibodies and multimeric recombinant Fas ligand (FasL) preparations show high tumoricidal activity against leukemic cells, but are unsuitable for clinical application due to unacceptable systemic toxicity. Consequently, new antileukemia strategies based on Fas activation have to meet the criterion of strictly localized action at the tumor-cell surface. Recent insight into the FasL/Fas system has revealed that soluble homotrimeric FasL (sFasL) is in fact nontoxic to normal cells, but also lacks tumoricidal activity. We report on a novel fusion protein, designated scFvCD7:sFasL, that is designed to have leukemia-restricted activity. ScFvCD7:sFasL consists of sFasL genetically linked to a high-affinity single-chain fragment of variable regions (scFv) antibody fragment specific for the T-cell leukemia-associated antigen CD7. Soluble homotrimeric scFvCD7:sFasL is inactive and acquires tumoricidal activity only after specific binding to tumor cell-surface-expressed CD7. Treatment of T-cell acute lymphoblastic leukemia (T-ALL) cell lines and patient-derived T-ALL, peripheral T-cell lymphoma (PTCL), and CD7-positive acute myeloid leukemia (AML) cells with homotrimeric scFvCD7:sFasL revealed potent CD7-restricted induction of apoptosis that was augmented by conventional drugs, farnesyl transferase inhibitor L-744832, and the proteasome inhibitor bortezomib (Velcade; Millenium, Cambridge, MA). Importantly, identical treatment did not affect normal human peripheral-blood lymphocytes (PBLs) and endothelial cells, with only moderate apoptosis in interleukin-2 (IL-2)/CD3-activated T cells. CD7-restricted activation of Fas in T-cell leukemic cells by scFvCD7:sFasL revitalizes interest in the applicability of Fas signaling in leukemia therapy.
Abstract: Apoptosis is a ubiquitous, highly coordinated process of self-destruction. Conventional cancer therapeutic agents can cause, to a certain degree, apoptosis of cancer cells. However, they do so in an indiscriminate manner leading to unacceptable side effects. Strategies of selectively activating apoptosis of cancer cells are described.
Abstract: OBJECT: Patients with astrocytic tumors in the central nervous system (CNS) have low survival rates despite surgery and radiotherapy. Innovative therapies and strategies must be developed to prolong survival of these patients. The alginate microencapsulation method, used to continuously release a certain cytotoxic agent in the vicinity of the tumor, is such a novel therapeutic strategy. The biological functionality of the apoptosis inducing scFv425:sTRAIL protein, which was released through the microencapsulation method, was studied in vitro. Analysis of the intracerebral biocompatibility of alginate capsules was performed by implantation of empty alginate capsules in the brain of mice. METHOD: Chinese Hamster Ovary cells (CHO-K1) were recombinantly engineered to produce the single chain anti-EGFR-sTRAIL protein (scFv425:sTRAIL). The CHO-K1 producer cells were encapsulated in an alginate capsule with a semi-permeable membrane through which the scFv425:sTRAIL protein could be released. RESULTS: In vitro studies show maintained biological functionality of the released scFv425:sTRAIL protein. There was no immunological tissue response detectable after intracerebral implantation of the alginate capsules in mice brains. CONCLUSION: Biological functionality of the produced scFv425:sTRAIL protein is maintained and intracerebral biocompatibility of the capsules is warranted. Alginate encapsulation of CHO-K1--scFv425:sTRAIL--producer cells and subsequently their intracerebral implantation is technically feasible. This study justifies further in vivo experiments.
Abstract: Important breakthroughs in cancer therapy include clinical application of antibodies, such as Rituximab, and small inhibitory molecules, such as Iressa and Velcade. In addition, recent reports have indicated the therapeutic potential of physiological pro-apoptotic proteins such as TRAIL and galectin-1. Although unrelated at first glance, each strategy relies on the deliberate and selective induction of apoptosis in malignant cells. Importantly, therapy-resistance in cancer is frequently associated with de-regulation in the mechanisms that control apoptosis. However, cancer cells are often reliant on these molecular aberrations for survival. Therefore, selective induction of apoptosis in cancer cells but not normal cells seems feasible. Here, we review recent progress and prospects of selected novel anti-cancer approaches that specifically target and sensitize cancer cells to apoptosis.
Abstract: Epidermal growth factor receptor (EGFR) signaling inhibition by monoclonal antibodies and EGFR-specific tyrosine kinase inhibitors has shown clinical efficacy in cancer by restoring susceptibility of tumor cells to therapeutic apoptosis induction. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent with tumor-selective apoptotic activity. Here we present a novel approach that combines EGFR-signaling inhibition with target cell-restricted apoptosis induction using a TRAIL fusion protein with engineered specificity for EGFR. This fusion protein, scFv425:sTRAIL, comprises the EGFR-blocking antibody fragment scFv425 genetically fused to soluble TRAIL (sTRAIL). Treatment with scFv425:sTRAIL resulted in the specific accretion to the cell surface of EGFR-positive cells only. EGFR-specific binding rapidly induced a dephosphorylation of EGFR and down-stream mitogenic signaling, which was accompanied by cFLIP(L) down-regulation and Bad dephosphorylation. EGFR-specific binding converted soluble scFv425:sTRAIL into a membrane-bound form of TRAIL that cross-linked agonistic TRAIL receptors in a paracrine manner, resulting in potent apoptosis induction in a series of EGFR-positive tumor cell lines. Co-treatment of EGFR-positive tumor cells with the EGFR-tyrosine kinase inhibitor Iressa resulted in a potent synergistic pro-apoptotic effect, caused by the specific down-regulation of c-FLIP. Furthermore, in mixed culture experiments binding (L)of scFv425:sTRAIL to EGFR-positive target cells conveyed a potent apoptotic effect toward EGFR-negative bystander tumor cells. The favorable characteristics of scFv425:sTRAIL, alone and in combination with Iressa, as well as its potent anti-tumor bystander activity indicate its potential value for treatment of EGFR-expressing cancers.
Abstract: Current treatment of human T-cell leukemia and lymphoma is predominantly limited to conventional cytotoxic therapy and is associated with limited therapeutic response and significant morbidity. Therefore, more potent and leukemia-specific therapies with favorable toxicity profiles are urgently needed. Here, we report on the construction of a novel therapeutic fusion protein, scFvCD7:sTRAIL, designed to induce target antigen-restricted apoptosis in human T-cell tumors. ScFvCD7:sTRAIL consists of the death-inducing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) genetically linked to an scFv antibody fragment specific for the T-cell surface antigen CD7. Treatment with scFvCD7:sTRAIL induced potent CD7-restricted apoptosis in a series of malignant T-cell lines, whereas normal resting leukocytes, activated T cells, and vascular endothelial cells (human umbilical vein endothelial cells) showed no detectable apoptosis. The apoptosis-inducing activity of scFvCD7:sTRAIL was stronger than that of the immunotoxin scFvCD7:ETA. In mixed culture experiments with CD7-positive and CD7-negative tumor cells, scFvCD7:sTRAIL induced very potent bystander apoptosis of CD7-negative tumor cells. In vitro treatment of blood cells freshly derived from T-acute lymphoblastic leukemia patients resulted in marked apoptosis of the malignant T cells that was strongly augmented by vincristin. In conclusion, scFvCD7:sTRAIL is a novel recombinant protein causing restricted apoptosis in human leukemic T cells with low toxicity for normal human blood and endothelial cells.
Abstract: The apparent tumor selective apoptosis-inducing activity of recombinant soluble TNF-related apoptosis-inducing ligand (TRAIL) has aroused much interest for use in clinical application. However, to exploit fully its therapeutic potential, the characteristics of both the TRAIL receptor system and soluble TRAIL (sTRAIL) should be taken into account: first, the widespread expression of the various TRAIL receptors throughout the human body; second, the differential binding affinities and crosslinking requirements of the agonistic receptors TRAIL-R1 and TRAIL-R2; and third, the solution behavior of particular sTRAIL preparations. Therefore, we constructed a novel TRAIL fusion protein, designated scFvC54:sTRAIL, comprising the human scFv antibody fragment C54 genetically linked to the N-terminus of human sTRAIL. The scFvC54:sTRAIL fusion protein was designed to induce apoptosis by crosslinking of agonistic TRAIL receptors only after specific binding of scFvC54:sTRAIL to the abundantly expressed carcinoma-associated cell surface antigen EGP2 (alias EpCAM). Target antigen-restricted apoptosis induction was demonstrated for various EGP2-positive tumor cells and could be inhibited by an EGP2 competing antibody. Target antigen binding converted soluble scFvC54:sTRAIL into a membrane-bound form of TRAIL that was capable of signaling apoptosis not only through TRAIL-R1 but also through TRAIL-R2. Size-exclusion fast protein liquid chromatography (FPLC) indicated that scFvC54:sTRAIL was produced as stable and homogeneous trimers in the absence of detectable TRAIL aggregates. The favorable characteristics of the scFvC54:sTRAIL fusion protein potentially reduce the amount of sTRAIL required for antitumor activity and may be of value for the treatment of various human carcinomas.
Abstract: Previously, we reported on the target cell-restricted fratricide apoptotic activity of scFvC54:sTRAIL, a fusion protein comprising human-soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) genetically linked to the antibody fragment scFvC54 specific for the cell surface target antigen EGP2. In the present study, we report that the selective binding of scFvC54:sTRAIL to EGP2-positive target cells conveys an exceptionally potent pro-apoptotic effect toward neighboring tumor cells that are devoid of EGP2 expression (bystander cells). The anti-tumor bystander activity of scFvC54:sTRAIL was detectable at target-to-bystander cell ratios as low as 1:100. Treatment in the presence of EGP2-blocking or TRAIL-neutralizing antibody strongly inhibited apoptosis in both target and bystander tumor cells. In the absence of target cells, bystander cell apoptosis induction was abrogated. The bystander apoptosis activity of scFvC54:sTRAIL did not require internalization, enzymatic conversion, diffusion, or communication (gap junctional intracellular communication) between target and bystander cells. Furthermore, scFvC54:sTRAIL showed no detectable signs of innocent bystander activity toward freshly isolated blood cells. Further development of this new principle is warranted for approaches where cancer cells can escape from antibody-based therapy due to partial loss of target antigen expression.
Abstract: EGP-2, also known as Ep-CAM, is expressed at high levels on the surface of most carcinomas and is therefore considered an attractive target for anticancer strategies. To explore the mechanisms regulating the expression of EGP-2, sequences 3.4 kb upstream of the transcription start site were isolated and assayed for their ability to control the expression of the EGP-2 cDNA, the green fluorescent protein, the luciferase reporter gene and the thymidine kinase and cytosine deaminase suicide genes. Expression of these chimeric constructs as assessed in a range of different cell lines was restricted to cell lines expressing EGP-2. In addition, only cells expressing EGP-2 were sensitive for gancyclovir after being transiently transfected with EGP-2 promoter-driven thymidine kinase. Deletion analyses defined 687 bp upstream as the basic proximal promoter region, which could confer epithelial-specific expression to the GFP reporter gene in vitro. As these EGP-2 sequences can confer promoter activity to reporter and suicide genes in an EGP-2 restricted manner, they may be useful for gene therapy of EGP-2 expressing carcinomas.
Abstract: The effectiveness of cellular immunotherapy of solid tumors is often hampered by the lack of specific infiltration of immune effector cells into the tumor mass. Therefore, we studied the potential of tumor antigen-specific antibodies to elicit tumor-specific myeloid cell activation, to induce or enhance tumor infiltration by immune cells. To this end, we developed an in vitro model system using the human myeloid cell line MonoMac-6. Incubation of IFN-gamma-primed MonoMac-6 cells with serum-opsonized zymosan or EGP-2-directed, mouse IgG2a-opsonized, EGP-2-positive tumor cells resulted in the production of ROS and TNF-alpha and induced E-selectin and ICAM-1 expression on HUVECs. FcR-mediated MonoMac-6 cell activation was strictly dependent on the activation of MonoMac-6 cells with IFN-gamma. In addition, no myeloid cell activation was observed in the presence of human serum or using tumor antigen-specific mouse antibody subclasses other than IgG2a, suggesting the crucial involvement of CD64 (FcgammaR1) in the effects observed. However, serum-inhibited myeloid cell activation was completely restored employing a 2-step targeting approach in which tumor cell opsonization with mouse anti-EGP-2 antibodies was followed by incubation with human antimouse Ig antibodies. Moreover, using this 2-step approach, not only anti-EGP-2-directed mouse IgG2a but also mouse IgG1 antibodies effectively induced tumor-specific myeloid cell activation. In conclusion, we describe a method to induce efficient and tumor-specific activation of myeloid cells based on the sequential use of mouse tumor antigen-specific and human antimouse Ig antibodies. Targeted myeloid cell activation may provide a means to aid in the induction of a tumor-directed immune response and as such, the method described here could be of clinical significance.
Abstract: Bi-specific antibodies (BsAbs) combine immune cell activation with tumor cell recognition as a result of which tumor cells are killed by pre-defined effector cells. In this review a brief introduction to monoclonal antibodies will precede a more in-depth presentation of the current status of BsAb therapy for cancer. Target molecules and effector mechanisms aimed at tumor cells or aimed at tumor vasculature, and the application of recombinant DNA technology in the construction of antibodies, will be discussed. The lessons learned from the last decade will be discussed in consideration of the potential future development of BsAbs for cancer therapy.
Abstract: To overcome dose limiting toxicities and to increase efficacy of immunotherapy of cancer, a number of strategies are under development for selectively redirecting effector cells/molecules towards tumor cells. Many of these strategies exploit the specificity of tumor associated antigen recognition by monoclonal antibodies. Using either hybridoma fusion, chemical derivatization or molecular biology technology, antibodies with dual specificity can be constructed. These so called biospecific antibodies (BsAbs) have been used to redirect the cytolytic activity of a variety of immune effector cells such as cytotoxic T lymphocytes, natural killer cells, neutrophils and monocytes/macrophages to tumor cells. Local administration of BsAbs, either alone or in combination with autologous effector cells, is highly effective in eradicating tumor cells. In contrast, systemic application of BsAb at present is only suitable for adjuvant treatment of minimal residual disease due to poor tumor cell accessibility. As an alternative, angiogenesis related determinants on tumor blood vessels can be exploited for the selective delivery of effector cells/molecules apart from being used to inhibit angiogenesis. Important advantages of this strategy is that the endothelial cell associated target epitope(s) are easy accessible. The dependence of tumor growth on the tumor's blood supply also renders tumor endothelial cells an attractive target for therapy. Although still in its infancy, attacking the tumor's blood supply for example by delivering coagulation factors or toxins, or by BsAb directed immunotherapies holds great promise for antineoplastic therapy.
Abstract: Bispecific antibody (BsMAb) BIS-1 has been developed to redirect the cytolytic activity of cytotoxic T lymphocytes (CTL) to epithelial glycoprotein-2 (EGP-2) expressing tumour cells. Intravenous administration of BIS-1 F(ab')2 to carcinoma patients in a phase I/II clinical trial, caused immunomodulation as demonstrated by a rapid lymphopenia prior to a rise in plasma tumour necrosis factor-alpha and interferon-gamma levels. Yet, no lymphocyte accumulation in the tumour tissue and no anti-tumour effect could be observed. These data suggest a BsMAb-induced lymphocyte adhesion to blood vessel walls and/or generalized redistribution of the lymphocytes into tissues. In this study, we describe the effects of BIS-1 F(ab')2 binding to peripheral blood mononuclear cells (PBMC) on their capacity to interact with resting endothelial cells in vitro. Resting and pre-activated PBMC exhibited a significant increase in adhesive interaction with endothelial cells when preincubated with BIS-1 F(ab')2, followed by an increase in transendothelial migration (tem). Binding of BIS-1 F(ab')2 to PBMC affected the expression of a number of adhesion molecules involved in lymphocyte adhesion/migration. Furthermore, PBMC preincubated with BIS-1 F(ab')2 induced the expression of endothelial cell adhesion molecules E-selectin, VCAM-1 and ICAM-1 during adhesion/tem. These phenomena were related to the CD3 recognizing antibody fragment of the BsMAb and dependent on lymphocyte-endothelial cell contact. Possibly, in patients, the BIS-1 F(ab')2 infusion induced lymphopenia is a result of generalized activation of endothelial cells, leading to the formation of a temporary sink for lymphocytes. This process may distract the lymphocytes from homing to the tumour cells, and hence prevent the occurrence of BIS-1 F(ab')2 - CTL-mediated tumour cell lysis.
Abstract: A versatile expression vector is described for the rapid construction and evaluation of bispecific scFvs and scFv-based fusion proteins. An important feature of this vector is the presence of two multiple cloning sites (MCS) separated by an in frame linker sequence. The first MCS was specifically designed to contain unique SfiI and NotI restriction enzyme sites that can be used for directional and in frame insertion of scFvs (or potentially any molecule) selected from established phage-display systems. Using this new vector, a functional bs-(scFv)(2) (2C11-MOC31) was constructed for retargeted T-cell cytotoxicity towards EGP2 positive tumor cells. The vector was also used for grafting of a number of promising biological effector principles onto scFv MOC31, including the prodrug converting enzyme cytosine deaminase, the anti-angiogenic factor angiostatin, and the thrombogenic molecule tissue factor. We aimed at producing biologically active fusion proteins by directing them through the endoplasmic reticulum-based protein folding machinery of eukaryotic cells (COS-7) using a kappa light chain leader, thereby taking advantage of the associated quality control mechanisms that allow only fully folded and processed fusion proteins to be secreted into the medium. Supernatants derived from fusion protein transfected COS-7 cells, which were transiently transfected at low transfection rates, were directly assayed for the biological and/or targeting activity of the excreted fusion proteins without any prior purification steps. This procedure might help to identify those fusion proteins that have favourable characteristics like stability and biological activity in the presence of serum and at low protein concentrations. Targeted delivery of all effector principles was subsequently assessed in an in vitro model system. The method we devised is both rapid and versatile and can be useful to construct and identify series of new chimeric proteins with enhanced therapeutic potential in human cancer therapy.
Abstract: The UBE1L gene isolated from the chromosome 3p21 region has an extremely reduced level of mRNA in lung cancer. Sequence analysis showed a 45% homology to the human ubiquitin-activating enzyme E1 at the amino acid level. To further characterize the protein product, we generated UBE1L protein-specific antibodies. Immunoblot analysis revealed a full-length gene product of approximately 112 kDa. Assessment of the level and distribution pattern of the UBE1L protein in normal and tumor tissue using the generated antibodies showed that the UBE1L protein was present in normal lung cells and non-lung cancer cell lines, but was undetectable in all 14 human lung cancer cell lines analyzed. This difference in expression of the UBE1L protein between normal lung tissue and lung tumor-derived cell lines suggests a possible involvement of an E1-like protein in the origin and/or progression of lung tumors.
Abstract: A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab). The purified huMab had an affinity of 5 nM and effectively mediated tumor cell killing in in vitro and in vivo assays. These experiments show that nonimmunized phage antibody display libraries can be used to obtain high-affinity, functional, and clinically applicable huMabs directed against a tumor-associated antigen.
Abstract: We describe the construction of a recombinant bispecific antibody fragment in the diabody format with specificity for both the well-established human pancarcinoma associated target antigen EGP2 (epithelial glycoprotein 2, also known as the CO17-1A antigen or KSA) and the CD3epsilon chain of human TCR/CD3 complex. The murine anti-EGP2 (MOC31) single chain variable fragment (scFv) and the humanized anti-CD3 (Ucht1v9) scFv were cast into a diabody format (designated Dia5v9) using a short 5 amino acid Gly-Ser linker between immunoglobulin heavy-chain and light-chain variable domains. Purification of the poly-histidine tagged Dia5v9 was achieved from extracts of protease deficient Escherichia coli by IMAC chromatography. The Dia5v9 diabody showed strong binding to both EGP2 and CD3 in transfected cells. The in vitro efficacy of Dia5v9 in mediating tumor cell lysis by interleukin-2 activated human T cells appeared to be similar to that of the hybrid-hybridoma-derived BsF(ab')2 Bis1 (anti-EGP2/anti-CD3) in a standard 4-hr 51Cr-release assay. This small and partially humanized recombinant bispecific antibody fragment may be valuable for T-cell-based immunotherapeutical treatment protocols, retargeting activated peripheral blood T lymphocytes to lyse various human carcinomas in vivo.
Abstract: In this report, we describe a new and simple method for flow cytometric quantitation of lymphocyte numbers in lymphocyte-endothelial adhesion/ transendothelial migration assays. The method exploits fluorescent flow cytometer alignment beads as a counting reference. Known amounts of beads are added to samples with unknown lymphocyte numbers. Lymphocytes and a preset number of fluorescent beads are simultaneously analyzed. The total number of cells present in the sample can be subsequently calculated from the fixed ratio of added to analyzed fluorescent beads. Using this fluorescent-beads-based flow cytometric cell counting of lymphocyte numbers in adhesion/migration assays, labeling of cells and other time-consuming calibration procedures are not required and analysis time is short. Furthermore, we demonstrate that this cell counting method can be combined with concurrent single- or double-label fluorescence flow cytometric phenotyping of adherent and migrated lymphocytes. The method was applied to the in vitro study of the effects of lymphocyte activation status and binding of bispecific antibody (directed against CD3 x tumor cell-associated antigen) on lymphocyte adhesion and transendothelial migration.
Abstract: The tumour-associated antigen epithelial glycoprotein-2 (EGP-2) is a promising target for detection and treatment of a variety of human carcinomas. Antibodies to this antigen have been successfully used in patients for imaging of small-cell lung cancer and for adjuvant treatment of minimal residual disease of colon cancer. We describe here the isolation and complete characterization of high-affinity single-chain variable fragments (scFv) to the EGP-2 antigen. First, the binding kinetics of four murine whole antibodies directed to EGP-2 (17-1A, 323/A3, MOC-31 and MOC-161) were determined using surface plasmon resonance (SPR). The MOC-31 antibody has the lowest apparent off-rate, followed by MOC-161 and 323/A3. The V-genes of the two MOC hybridomas were cloned as scFv in a phage display vector and antigen-binding phage were selected by panning on recombinant antigen. The scFvs compete with the original hybridoma antibodies for binding to antigen and specifically bind to human carcinomas in immunohistochemistry. MOC-31 scFv has an off-rate which is better than those of the bivalent 17-1A and 323/A3 whole antibodies, providing it with an essential characteristic for tumour retention in vivo. The availability of these high-affinity anti-EGP-2 antibody fragments and of their encoding V-genes creates a variety of possibilities for their future use as tumour-targeting vehicles.
Abstract: Immunotherapy is a powerful anti-cancer treatment modality. However, despite numerous encouraging results obtained in pre-clinical studies, a definite breakthrough towards an established clinical treatment modality has as yet not occurred. Antibodies against tumor antigens have been shown to localise at the site of the tumor, but inadequate triggering of immune effector mechanisms have thwarted clinical efficacy thus far. Cellular immunotherapy has been hampered by limitations such as lack of specificity, down-regulation of major histocompatibility complex (MHC)-expression or Fas ligand up-regulation on tumor cells. This review focuses on the use of bispecific antibodies (BsAbs) for immunotherapy of cancer. Using BsAbs, it is possible to take advantage of the highly specific binding characteristics of antibodies and combine these with the powerful effector functions of cytotoxic immune effector cells. BsAbs share two different, monoclonal antibody-derived, antigen-recognizing moieties within one molecule. By dual binding, BsAbs reactive with a trigger molecule on an immune effector cell on the one hand and a surface antigen on a tumor target cell on the other are thus able to functionally focus the lytic activity of the immune effector cell towards the target cell. Over the last few years, the concept of BsAb-mediated tumor cell killing has been studied extensively both in preclinical models and in a number of phase I clinical trials. Promising pre-clinical results have been reported using tumor models in which diverse immune effector cell populations have been used. Despite this pre-clinical in vivo efficacy, the first clinical trials indicate that we are still not in a position to successfully treat human malignancies. This review discusses the production of BsAbs, the choice of trigger molecules in combination with potential effector cells and the preclinical models that have led to the current use of BsAbs in experimental clinical trials. It has become clear that appropriate immune cell activation and establishing a favourable effector-to-target cell ratio will have direct impact on the efficacy of the therapeutic approaches using BsAbs. New directions are discussed, i.e. finding appropriate dosage schemes by which immune effector cells become redirected without inducing hyporesponsiveness, defining possibilities for combining different immune effector cell populations and creating an in situ tumor environment that allows maximal tumoricidal activity
Abstract: The presence of tumour cells in the circulation may predict disease recurrence and metastasis. To improve on existing methods of cytological or immunocytological detection, we have developed a sensitive and quantitative technique for the detection of carcinoma cells in blood, using the reverse transcriptase polymerase chain reaction (RT-PCR) identifying transcripts of the pancarcinoma-associated tumour marker EGP-2 (KSA or 17-1A antigen). The amount of EGP2 mRNA was quantified using an internal recombinant competitor RNA standard with known concentration and which is both reversely transcribed and co-amplified in the same reaction, allowing for a reliable assessment of the initial amount of EGP2 mRNA in the sample. Calibration studies, seeding blood with MCF-7 breast carcinoma cells, showed that the assay can detect ten tumour cells among 1.0 x 10(6) leucocytes. The PCR assay revealed that normal bone marrow expresses low levels of EGP2 mRNA, although immunocytochemistry with the anti-EGP2 MAb MOC31 could not identify any positively stained cell. Analyses using this RT-PCR assay may prove to have applications to the assessment of circulating tumour cells in clinical samples.
Abstract: The bispecific monoclonal antibody (bsAb) BIS-1 combines a monoclonal-antibody(mAb)-defined specificity for the CD3 complex, as present on all T lymphocytes, with a mAb-defined specificity for the pancarcinoma/epithelium associated glycoprotein EGP-2. In vitro studies indicate that BIS-1 can direct T lymphocytes to kill EGP-2-positive tumour target cells. T cell pre-activation is necessary for this activity and can be obtained either via incubation of isolated peripheral blood mononuclear cells with CD3 mAb, followed by short culturing in recombinant interleukin-2-containing medium, or via costimulation with CD5- and CD28-based bsAb. Clinical application of BIS-1 was started in a pilot study in which carcinoma patients suffering from malignant ascites or intrapleural effusion were treated. In this study, ex vivo activated autologous lymphocytes were applied locally, i.e. intraperitoneally or intrapleurally, in the presence of BIS-1. Local inflammation and antitumour activity were observed, whereas no or only minor systemic toxicity was seen in these patients. Intravenous administration of BIS-1 F(ab')2 in combination with subcutaneously given recombinant interleukin-2 (i.v. bsAb/rIL-2 treatment) induced transient but considerable toxicity including peripheral vasoconstriction, dyspnoea and fever with a maximal tolerated dose of 5-8 micrograms/kg. High plasma concentrations of the inflammatory cytokines tumor necrosis factor alpha and interferon gamma were observed at this dose. Whereas bsAb-dictated antitumour activity could be demonstrated to be present in blood samples of these patients in an in vitro assay, no clear clinical responses were observed. In a rat model it was found that i.v. bsAb/rIL-2 treatment of EGP-2-positive tumours was effective when a low systemic tumour burden was present, suggesting that systemic bsAb/rIL-2 treatment might be effective in situations of minimal residual disease.
Abstract: In this report we describe the role of apoptosis in the process of tumour cell killing by bispecific monoclonal antibody (BsMAb)-redirected cytolytic T cells. The BsMAb used, BIS-1, has dual specificity for the CD3 complex on T cells and the pancarcinoma-associated 38 kDa transmembrane antigen EGP-2. BIS-1 allows activated T cells to specifically recognise and kill EGP-2-positive but not EGP-2-negative target cells. An assay was developed to quantify apoptosis in cells by separation of 3H-thymidine-labelled low-molecular, i.e. fragmented, from high-molecular, i.e. non-fragmented DNA. The presence of low molecular weight DNA was measured both within the target cells and in the cell-free supernatant. After exposure to BIS-1-redirected, -activated T cells, apoptosis was observed in EGP-2-positive target cells but not in EGP-2-negative target cells. Also no DNA fragmentation proved to be induced in the activated effector cells during assay. The degree of EGP-2-positive target DNA fragmentation depended on the concentration of BsMAb, the E/T ratio and the incubation time. Using a low E/T ratio (1/1), DNA fragmentation in and 51Cr release from target cells showed similar characteristics and kinetics. At higher E/T ratio (20/1), the 51Cr release from the target cells increased to a greater extent than the percentage fragmented target cell DNA. Inhibitors of DNA fragmentation added to the cytotoxicity assay inhibited not only DNA fragmentation, but also the release of chromium-51 from the target cells, suggesting that apoptosis and cell lysis are closely related in BsMAb-mediated cell killing.
Abstract: Effectiveness of bispecific-monoclonal-antibody (BsMAb)-mediated cellular anti-tumour activity was evaluated in vitro and in vivo in relation to the additional need for T-cell activation in a new immunocompetent rat tumour model. L37 tumour cells, derived from a squamous-cell carcinoma of the lung of Wag/Rij rats, were transfected with the cDNA coding for the human 38-kDa transmembrane pan-carcinoma-associated antigen EGP-2. Intravenous inoculation of EGP-2-positive L37 cells resulted in a rapid outgrowth of EGP-2-positive tumour nodules in the lungs. A BsMAb BIS-19, recognizing EGP-2 on the transfected tumour cells and the T-cell receptor of the rat, was made and allowed specific lysis of EGP-2-transfected L37 tumour cells by activated rat T lymphocytes in vitro. In vivo T-cell activation, assessed by up-regulation of IL-2-receptor expression, could be induced by daily injection of rat rIL-2. Intravenous treatment of tumour-bearing EGP-2-positive L37 tumour with BIS-19 together with rat rIL-2 resulted in almost complete disappearance of established tumour. In contrast, animals treated with BIS-19 alone, IL-2 alone or a combination of anti-EGP-2, anti-TcR and IL-2 showed much less or no tumour reduction. These results show effectiveness of systemic treatment with BsMAbs to induce anti-tumour activity in established tumours. Immune activation prior to or during treatment with BsMAbs, as achieved with IL-2, appears to be a prerequisite for successful treatment.
Abstract: Western blot analysis proved that all cluster-2 MAbs recognize identical or overlapping disulfide-bond-dependent epitopes, indicating the presence of a disulfide-bond-stabilized EGP-2 domain carrying highly immunodominant non-linear epitopes. The apparent immunodominance of this domain makes it difficult to generate and select antibodies against other potentially useful EGP-2 epitopes. Using PCR, we have generated mutant EGP-2 cDNA (delta EGP-2) from which the coding sequences for a putative immunodominant 6-kDa intra-chain loop structure has been removed. delta EGP-2 transfected COS-7 cells reacted with MM104, an antibody detecting a linear epitope on EGP-2, but were not recognized by any cluster-2 MAb. To generate new anti-EGP-2 antibodies we constructed another mutant EGP-2 protein (delta EGP-2) from which additional domains, irrelevant for antibody generation (signal peptide, trans-membrane and cytoplasmic domains), were removed. delta EGP-2 was introduced in a prokaryotic expression system that adds a polyhistidine affinity tag to the delta EGP-2 N-terminus, making possible one-step purification by immobilized metal-ion-affinity chromatography (IMAC). Western blot analysis showed that sera derived from mice immunized with purified delta EGP-2 had high-titer antibodies to reduced EGP-2 samples. We conclude that the availability of prokaryotic and eukaryotic EGP-2-expression constructs might facilitate the selection of new anti-EGP-2 MAbs otherwise difficult to obtain.
Abstract: The cluster-2 antigen, also called EGP-2, is a 38-kDa transmembrane glycoprotein with a distribution that is largely confined to human epithelial cells and their derived carcinomas. Monoclonal antibodies (MAbs) directed against EGP-2 have been extensively studied as anti-tumor agents, yet the function of the antigen is not known. In the present study we used a biotinylated recombinant soluble derivative of the EGP-2 (sEGPbio) as a probe to detect a possible EGP-2 ligand, using various carcinoma cell lines as a substrate. The recombinant soluble EGP-2 was expressed in the Autographa californica nuclear polyhedrosis virus (baculovirus) expression system. The sEGP-2, to which we engineered a poly-histidine affinity tag, was purified from infected Spodoptera frugiperda insect cells using immobilized metal-ion-affinity chromatography (IMAC). In Western blot analysis the sEGPbio probe bound to a 30-kDa protein band in 2 out of 5 of the assessed carcinoma cell lines, suggesting that this band may be an EGP-2 ligand. Interestingly, binding only occurred when, prior to SDS-PAGE, cell lysates had been subjected to a reducing agent (2-mercapto-ethanol). The physiological significance of this phenomenon and nature of the detected 30-kDa protein band remains to be determined.
Abstract: Expression of the neural cell adhesion molecule (NCAM) on small-cell lung carcinoma (SCLC) cell lines and tumour tissue has been investigated. Cell lines were found to express highly sialylated NCAM. Neuraminidase treatment revealed the presence of the 140- and 120-kDa isoforms with differential expression of a 95-kDa protein. Similar data were obtained with SCLC tumour tissues. These results were corroborated by Northern blotting where mRNA of 6.7 and 5.5 kb coding for the 140- and 120-kDa isoforms, respectively, were identified. In a few tumours, a weaker band of 7.4-kb mRNA coding for the 180-kDa NCAM was also identified. This result could not be confirmed biochemically due to shortage of material. Finally, a 5-kb transcript was identified in all SCLC samples examined. The NCAM isoform coded by this mRNA remains unknown. Using the polymerase chain reaction (PCR), we have demonstrated the presence of the VASE mini-exon in some isoforms of SCLC NCAM. The VASE mini-exon sequence in human SCLC differs from the published murine sequence by only one base change. This substitution does not result in altered amino-acid sequence.
