Gene therapy strategies hold great promise for complementing or even replacing our current methods of treating cancer. A critical component for successful cancer gene therapy is cancer-specific transgene expression in order to spare normal cells. This goal should be approachable by cell-targeted gene delivery and/or by imposing stringent transcriptional regulation. In theory, tightly restricted transgene expression is feasible because the expression of most genes is naturally under strict control. To this end, many investigators have begun to exploit knowledge about gene regulation to achieve cancer-specific gene expression. For example, a cancer-specific chimeric transcription factor resulting from a chromosomal translocation has been harnessed to selectively activate a toxin gene. More generally, targeted expression of therapeutic genes has been demonstrated using regulatory sequences from (i) genes that are ectopically expressed in cancers, (ii) viral genes expressed in virus-associated cancers, and (iii) tissue-specific genes expressed in cancers and their tissues of origin. The latter approach has been applied to models of melanoma, B- and T-lymphoid malignancies, osteosarcoma, prostate cancer, glioma, lung cancer, and various other carcinomas. Regulatory sequences from genes expressed in tumor vasculature and from cell cycle-regulated genes are also candidates for therapeutic transcriptional targeting. In addition, much progress has been made in developing systems for regulating transgene transcription using low molecular weight drugs, especially tetracyclines, facilitating precise temporal control of expression. Finally, pharmacologic principles must be considered in controlling the expression of therapeutic genes. This review summarizes progress in the above areas and discusses prospects for transcriptional targeting of transgene expression in relation to cancer therapy.
Gene transfer vectors based on adeno-associated virus (AAV) show great promise for human gene therapy. However, the efficiency of AAV-mediated gene delivery to different cell types can vary greatly, The ability to mediate targeted and specific delivery of therapeutics remains one of the most important hurdles in effectively treating human disease. Targeted AAV vectors are being developed with the goal of overcoming this hurdle, Currently, AAV vector targeting has been achieved via transcriptional regulation and with bispecific antibodies or specific ligands that are capable of mediating novel interactions between the AAV vector and specific cell surface receptors, This review presents an overview of current and potential methods for developing targeted AAV vectors. Future studies will determine if the efficacy of therapeutic AAV vectors is enhanced using these modifications.
Single-chain antibody (scFv) dimers (also known as diabodies) provide a facile route for production of engineered fragments with favorable tumor targeting and biodistribution properties in vivo, A scFv was constructed from the variable regions of the anti-carcinoembryonic antigen (CEA) monoclonal antibody T84.66 using a peptide linker of only eight amino acid residues in order to prevent formation of scFv monomers and produce dimers (diabodies) exclusively, The anti-CEA diabody was secreted at high level from E. coli and was purified by affinity chromatography, The T84.66/GS8 diabody bound antigen bivalently and retained a high apparent affinity for CEA (K-A = 8.19 x 10(10) M-1) as determined by surface plasmon resonance. Targeting properties of I-123-labeled T84.66 diabody were evaluated in vivo using athymic mice bearing LS174T human colorectal carcinoma xenografts. I-123-labeled anti-CPA diabodies exhibited rapid targeting of CPA-positive xenografts, reaching a maximum uptake level of 13.68 +/- 1.49% injected dose per gram 2 h after administration. Rapid blood clearance resulted in high tumor/blood uptake ratios; for example, 9.37 at 6 h and 48.69 at 24 h. Using a gamma camera equipped with a pinhole collimator, LS174T xenografts of masses 0.12 to 0.48 gm were readily imaged 6 h after injection, Performance of the anti-CPA diabody (bivalent, 55 kDa) was compared with previously described cognate fragments of T84.66, namely the scFv (28 kDa, monovalent) and minibody (scFv-C(H)3 dimer; bivalent, 80 kDa) by calculation of imaging figures of merit, Results show that diabodies labeled with short-lived radionuclides such as I-123 Or F-18 Should provide useful rapid clinical imaging agents.
The advantages of antibody- and T cell-based immunotherapy approaches can be combined by grafting cells with chimeric receptor genes encoding single-chain polypeptides that are able to recognize tumor antigens in an MHC-independent manner, and to transduce activating signals Into the cell. Upon encountering specific antigens, T cells harboring chimeric receptors are able to undergo specific stimulation and kill tumor cells in model systems both in vitro and In vivo. Although initial studies have focused on T-cell immunotherapy protocols, recent data suggest an important role for the manipulation of myeloid and NK cells in the treatment of cancer. Therefore, stem cell gene therapy approaches may have attractions providing longterm generation of targeted T and non-T cells. Although further scientific progress is required regarding the selection of the ideal effector cell(s), the definition of the optimal chR gene format and the development of improved gene-delivery systems, clinical trials, recently initiated in patients with advanced cancer, should help us to determine the real efficacy of these approaches.
We describe the engineering of a bispecific single-chain diabody secreted from mammalian producer cells for recruitment of enzyme to tumor cells. A bispecific diabody (Db-CEAGal) directed against carcinoembryonic antigen (CEA) and E. coli beta-galactosidase (Gal) was converted to a single-chain diabody (scDb-CEAGal) by covalently joining the VHA-VLB and the VHB-VLA fragments with a 15 amino acids flexible linker. Bacterially expressed scDb-CEAGal functionally recruited beta-galactosidase to plastic-bound CEA as well as the CEA-expressing colon carcinoma cell line LoVo. Compared to dimeric diabody CEAGal, the single-chain version showed an increased stability in serum at 37 degrees C, Active scDb-CEAGal was secreted from stably transfected human cells (HEK 293), specifically recruited beta-galactosidase to co-cultivated LoVo cells and mediated tumor cell killing through conversion of the prodrug daunomycin-beta-D-galactopyranoside to the toxic drug daunomycin, Single-chain diabodies secreted from mammalian producer cells may be useful for gene-directed antibody therapy by combining antibody-mediated effector recruitment with a gene-therapeutic approach.
A long-standing goal in gene therapy for cancer is a systemic delivery system that selectively targets tumor cells including metastases. We optimized a folate containing cationic liposome system for the systemic delivery of wtp53 to squamous cell carcinoma of the head and neck. The folate ligand, which serves to target the complier to tumor cells, increased the transfection efficiency by facilitating transient gene transfection. This system was demonstrated to be exceedingly tumor-selective in that normal tissues, including the highly proliferative gut and bone marrow, were not transfected. The systemic delivery by this method of wild-type p53 to established mouse xenografts markedly sensitized these human tumors to radiotherapy. This combination of systemic p53 gene therapy and conventional radiotherapy resulted in complete tumor regression and inhibition of their recurrence long-term, Similar results were also demonstrated with another model system, prostate cancer cell line DU145, This addition of a molecular component could provide an improved therapeutic approach for cancers of the head and neck and other forms of cancer as well.
Vectors derived from the autonomous parvovirus MVM(p) [Minute Virus of Mice (prototype strain)] target the expression of transgenes to transformed eel Is and are therefore of interest for cancer gene therapy. Their production is however difficult and stocks are contaminated by wild-type MVM that is generated through recombination between cotransfected vector and helper DNA sequences, Integration of helper sequences into a packaging cell line has allowed to decrease recombination and to generate vector stocks with no detectable helper virus. However, serial infection of this packaging cell line did not allow one to efficiently amplify recMVM, presumably because the initial titres were too low. We have now generated a new packaging cell line that produces ten-fold higher stocks after transfection and that allows us to reach recMVM titres of up to 10(7) infectious units/ml after three to four rounds of infection with no concentration procedure, Although undetectable in the initial stocks obtained by transfection,wild-type MVM appeared during serial infections, This rapid and simple method should be easily scaled up, and will allow the production and purification of vectors suitable for in vivo testing of the therapeutic efficiency of recMVM vectors.
We have applied a bispecific antibody (BsAb) approach to gene-modified T cells so as to both redirect T cell cytoxicity toward tumor and augment expression of an introduced transgene. Although activated T cells (ATC) can be expanded ex vivo to large numbers for clinical adoptive transfer, the cells are polyclonal and generally not tumor antigen-specific. Previously, we demonstrated that ATC modified with a retrovirus encoding the proinflammatory cytokine interleukin-1 beta (IL-1 beta) downregulate IL1-beta expression during culture expansion concomitant with a decline in T cell activation functions, including endogenous cytokine expression. To circumvent the lack of tumor specificity of ATC, as well as their reduced activity and transgene expression, we have utilized a bispecific antibody (BsAb) prepared by chemical heteroconjugation of the murine OKT3 monoclonal antibody (mAb) that binds the T cell CD3 receptor and the T84.66 mAb that is specific for carcinoembryonic antigen (CEA), a known tu mor-associated antigen (TAA). Expanded ATC populations (day 14) were preincubated, or armed, with the BsAb prior to exposure to tumor cells. ATC cytotoxicity against CEA(+) LS174T tumor cells was augmented six-fold by arming with the BsAb (5-500 ng BsAb/10(6)ATC), Furthermore, the secretion of endogenous cytokines was enhanced, indicative of BsAb-mediated reactivation. ATC that were gene modified with a IL-1 beta retroviral vector and armed with the BsAb showed increased cytotoxicity and endogenous cytokine expression in the presence of LS174T tu mor, as well as IL-1 beta expression that was augmented two to five-fold. Our results support a novel adoptive immunotherapy strategy for cancer by applying gene-modified ATC armed with an anti-CD3/anti-TAA BsAb. This approach could serve not only to retarget and reactivate ATC effector functions against tumor but also provide a means of directed delivery of additional, immunostimulatory molecules to the tumor site.
The characterization of immunogenic epitopes in murine V-regions is vital to the understanding of molecular mechanisms responsible for patient antibody response to genetically engineered molecules. We have prepared a purified light and heavy chains of mouse and mouse/human chimeric CC49 in an attempt to characterize the human immune response of this reagent. Intact antibody was reduced with Tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP . HC1) and purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoreils (SDS-PAGE) using BIO-RAD's Model 491 Prep Cell. All final preparations were analyzed by Western blot, SDS-PAGE and were used in competitive inhibition assays to characterize Immunogenicity, The final products proved to be stable for several months. The light and heavy chains of mouse and chimeric CC49 inhibited human anti-mouse antibody (HAMA) response in human serum equally in most cases but did not demonstrate an additive effect. All 15 patients, Infused with mCC49, had an immune response to mCC49 by day 14 with 13/15 patients having Immune response to the V-region of mCC49. V-region immunogenicity varied from patient to patient and during the course of response in each individual patient. The light and heavy chain of mouse and chimeric CC49 inhibited equally. The epitope specificity study of the human V-region immune response indicated that almost all of V-region immuno-genicity resides in the CDR epitopes.
A variety of strategies have been developed to accomplish gene therapy for cancer. One of these approaches is mutation compensation, whereby gene transfer is used for abrogating the function of dysregulated dominant oncogenes or for restoration of the function of deficient tumor suppressor genes. Bax, a member of the Bcl-2 family that can act as a tumor suppressor, potently induces apoptosis by caspase-dependent and independent mechanisms. We were able to generate a recombinant adenoviral vector encoding bax by using the inducible Cre-loxP system. Bax expression was tightly induced specifically by Cre recombinase, therefore allowing viral production. Furthermore, expression of Bax resulted in apoptotic cell death in human ovarian cancer cells. In contrast, Bax-mediated cell death was not observed in normal human peritoneal mesothelial cells. These results indicate that production and delivery of Bax via recombinant adenovirus vector is feasible, and that its preferential killing effect in human ovarian cancer cells might allow its use for gene therapy of ovarian cancer.
Modification of the tissue tropism and specificity of viral gene therapy vectors remains a major technical hurdle, despite the development of a wide variety of technical strategies for engineering the viral proteins. In this review we compare the advantages of the different approaches in use for modifying viral tropism. A new approach, called DNA shuffling or molecular breeding (by analogy to classical breeding), involves the homologous recombination of related DNA sequences obtained from the natural diversity of viral strains. This approach has proven to be very useful in many non-viral systems as well as in one viral system. DNA shuffling is unique in that it permutates natural mutations, which nature has already selected for function, leading to libraries of unusually high quality from which a wide variety of modified properties can be obtained.
Renal cell carcinomas (RCC) are highly resistent to conventional chemotherapy. The monoclonal antibody 138H11 was developed against human gamma-glutamyltransferase as a marker for RCC. This antibody strongly reacts with over 98% of both clear cell and papillary RCC, which together account for over 85% of the malignant renal tumours, Extracorporeal perfusion of human kidneys revealed a specific tumor uptake. The cytotoxic agent calicheamicin theta (Cam theta) belongs to the chemical group of enediynes which are DNA cleaving molecules that show very potent anti-cancer activity, To target the cytotoxic agent in RCC, we produced chemoimmunoconjugates of mAb 138H11 and Cam theta with a biocleavable linker and then tested this conjugate on the RCC cell line Caki-2. This novel conjugate was active and released the drug from the mAb under the assay conditions. Loss of toxicity through conjugation was tolerable. The 138H11-Cam theta conjugates were up to four logs more toxic for RCC than conventional cytostatic drugs.
A study is made of the efficacy of CD45-targeted immunoliposomes entrapping c-myb antisense phosphorothioate oligonucleotides to increase survival in a scid mouse model of human leukemia. The encapsulation efficiency of oligonucleotides in backbone liposomes was optimized using a dehydration-rehydration procedure. Pharmacokinetic parameters indicate that the t(1/2) of free oligonucleotide (0.14 +/- 0.02 h) was increased 63-fold when the oligonucleotide was encapsulated in small liposomes, whereas clearance decreased 50-fold accordingly. Multivalent liposomes for targeting were prepared by covalently coupled streptavidin on the liposome surface. The ability of streptavidin-liposomes to bind biotinylated antibodies was confirmed using biotin-peroxidase as tracer and size exclusion chromatography as it allows differentiation of the proportion of enzymatic activity in the liposome fraction versus the free enzyme. The in vivo efficacy of CD45-targeted liposomes was evaluated on scid mice transplanted with K562 human leukemia cells, Three weeks after transplant, mice were treated with HEPES (N-[2-Hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid]), free antisense oligonucleotide or liposomes encapsulating sense or antisense oligonucleotides, In order to improve the pharmacokinetic properties of free and encapsulated oligonucleotides, administration was performed by continuous infusion employing mini-osmotic pumps, We observed that CD45-targeted liposomes entrapping antisense oligonucleotides greatly increased survival, which was > 60% six months after tumor transplant, However, free antisense or encapsulated sense oligonucleotide in CD45-targeted liposome did little to increase survival with respect to the animals treated with HEPES, These results suggest that targeted immunoliposomes can contribute to the success of antisense therapeutic strategies in leukemia.
High dose recombinant human interleukin 2 (rhIL-2) therapy has been used in the treatment of established tumors in both animal models and patients with advanced melanoma or renal carcinoma. However, because high dose rhIL-2 therapy causes severe systemic toxicity in normal tissues, its clinical use has been limited. Therefore, targeting interleukin-2 (IL-2) to the tumor site should improve its anti-tumor-immune response and decrease its systemic toxicity. In this study, we describe the preparation and characterization of a recombinant humanized single-chain Fv (sFv) antibody/IL-2 fusion protein. This recombinant fusion protein consists of humanized variable heavy (VH) and light (V-L) domains of monoclonal antibody (mAb) 520C9 directed against the human HER-2/neu(c-erbB2) proto-oncogene product p185 and human IL-2. The fusion protein was stably expressed in baby hamster kidney cells and shown to retain the immunostimulatory activities of IL-2 as measured by IL-2-dependent cell proliferation and cytotoxicity assays. In addition to its IL-2 activity, this fusion protein also possesses binding specificity against the HER-2/neu(c-erbB2) proto-oncogene product, p185, as determined by enzyme linked immunosorbent assay (ELISA) using SKOV 3ip1 cells. Taken together, these findings suggest that this recombinant humanized sFv antibody/IL-2 fusion protein may provide an effective means of targeting therapeutic doses of IL-2 to p185 positive tumors without increasing systemic toxicity or immunogenicity.
By proteolytic cleavage, bovine RNaseA can be dissected into S peptide and S protein, from which the functional RNase can be reconstituted. Here, the cloning of homologous fragments of human placental RNase, huS peptide and huS protein, is described. Further, plasmids based on the pOPE vector family were constructed to express fusion proteins of huS peptide and huS protein with two different scFv (single chain) antibody fragments. The fusion proteins were produced by secretion to the periplasmic space of E. coli. Interspecies complementation was demonstrated by combining an scFv fusion protein containing huS peptide or a synthetic huS peptide with bovine S protein. In the reverse setup, RNase activity was reconstituted by combining an scFv fusion protein containing huS protein with synthetic bovine S peptide or synthetic human S peptide. RNase activity was also reconstituted by combining two different scFv antibodies carrying the two fragments of human pancreatic RNase enriched from E. coli periplasmic extracts by immobilised metal affinity chromatography. The results demonstrate the formation of a bispecific antibody conjugate with RNase activity. We have already shown that the human pancreatic RNase can efficiently lyse tumour cells in a recombinant immunotoxin setup.(1) The principle described in this study therefore not only provides a new small linker domain of human origin for the construction of bispecific antibodies, but might be employed to construct 'binary immunotoxins' with increased affinity, specificity and tumour uptake.
MOv18 binds an epitope on membrane folate receptors, highly expressed on ovarian carcinoma cells. Previous extensive clinical studies with c-MOv18 demonstrated preferential localization in tumour tissue of ovarian cancer patients. To study the safety and logistics of a treatment with I-131-labelled c-MOv18, 2 patients were injected intravenously (i.v.) with 740 MBq I-131-c-MOv18 IgG. Toxicity was evaluated according to WHO toxicity scales. Blood sampling was performed for 12 weeks post injection. Imaging was performed within Ih and frequently thereafter. Dose rates were obtained twice a day at various distances from patients with a portable dose rate measure. According to Dutch governmental regulations, the dose rate at 1 m from the patient should be below 20 mu Sv/h (400 MBq) upon discharge. Quantitative activity analysis of several organs was performed with the region of interest technique. Absorbed doses were calculated using MIRDOSE 3. For red marrow dose calculations, an activity ratio between red marrow and blood of 0.3 was used. Administration of I-131-c-MOv18 was uneventful. Mean isolation time was 4 1/2 d. No significant changes in haematological, biochemical, or urine profile were observed for more than 12 weeks. Blood kinetics were as reported earlier. Dosimetric analyses showed a mean absorbed dose of 39, 92, 115, and 51 cGy for whole body, liver, spleen, and red marrow, respectively. In conclusion, i.v. administration of I-131-c-MOv18 IgG was safe. No toxicity or psychological side effects were observed. The efficiency of dose rate measurements with a portable device adequately predicted the day of discharge from the hospital.
Reliable methods of determining antibody avidities are essential for comparing different antibodies (Abs) and for evaluating novel Ab constructs. Although it is accepted that true affinity applies only to monovalent binding, the concept of 'functional affinity' has been widely applied to multivalent binding interactions. We herein summarize the data indicating that 'functional affinities' are invalid. In many cases, binding of IgG Abs to multivalent antigens such as the cell surface is essentially irreversible, which is a consequence of bivalent binding and which implies that use of 'functional affinity', which means 'functional equilibrium association constant', cannot be valid. Alternative methods of evaluating Ab avidity are proposed. Widely used methods to measure either the total number of binding sites per cell or the immunoreactive fraction of Ab are also unreliable, primarily because of their dependence on extrapolation.
A recombinant adenovirus, AdE1A-tk, was constructed with a complete El A transcription unit and a therapeutic transgene (herpes simplex virus thymidine kinase). Both genomic and protein analysis of cells infected with AdE1A-tk demonstrated the presence of the expected coding sequences, and products of those sequences in the absence of any functional E1B. Evidence presented here demonstrated that the E1A-encoding sequences enhanced the expression of the CMV-directed HSVTK and inhibited growth of carcinoma cell lines even in the absence of ganciclovir. Importantly, the E1A also supported limited and preferential replication of AdE1A-tk in a lung cancer cell line compared to primary, non-transformed human respiratory epithelial cells. It is concluded that both the El A and therapeutic transgene within this adenoviral prototype confer antineoplastic effects, and hence are designated 'multimodality adenoviruses'.
