Immunotoxins and cancer therapy

In the past decade,an increased amount of clinicallyloriented research involving immunotoxins has been published.Immunotoxins are a group of artificially-made cytotoxic molecules targeting cancer cells.These moleculescomposed of a targeting moiety,such as a ligand or an antibody,linked to toxin moiety,which is a toxin with eithertruncated or deleted cell-binding domain that prevents it from binding to normal cells.Immunotoxins can bedivided into two categories:chemically conjugated immunotoxins and recombinant ones.The immunotoxins of thefirst category have shown limited efficacy in clinical trials in patients with hematologic malignancies and solidtumors.Within the last few years,single-chain immunotoxins provide enhanced therapeutic efficacy overconjugated forms and result in improved antitumor activity.In this review,we briefly illustrate the design of theimmunotoxins and their applications in clinical trials.Cellular & Molecular Immunology.2005;2(2):106-112.     

Immunotoxins in cancer therapy: Review and update

Immunotoxins are a novel class of cancer therapeutics that contains a cytotoxic agent fused to a targeting moiety. Various toxic agents from different sources are used in immunotoxin development, including bacterial, plant and human origin cytotoxic elements. Although bacterial and plant-derived toxins are highly toxic and commonly used in immunotoxins, their immunogenicity for human restricted their application in cancer therapy. Here, we discuss the advantages and limitations of bacterial toxins such as Pseudomonas and Diphtheria toxins, plant toxins such as ricin and gelonin, and some endogenous protein of human origin such as RNases and Granzymes. This article will also review different generations of immunotoxins with special focus on immunotoxins which are under clinical trials or approved for clinical use. Finally, current deimmunization strategies for development of new less-immunogenic recombinant immunotoxins will be discussed.

Abbreviations: mAbs: Monoclonal antibodies; EF2: elongation factor 2; ITs: Immunotoxins; DT: Diphtheria toxin; PE: Pseudomonas exotoxin; dgA: de-glycosylated A-chain of ricin; rGel: recombinant de-glycosylated form of gelonin; NKC: natural killer cells; HTR: human transferrin receptor; EGF: epidermal growth factor; GM-CSF: granulocyte-macrophage colony-stimulating factor; DAB389: truncated Diphtheria toxin; B-CCL: B-cell chronic lymphocytic leukemia; RCC: renal cell carcinoma; GVHD: Graft-versus-host disease; EGFR: epidermal growth factor receptor; AML: acute myeloid leukemia; Fab: fragment antigen-binding; dsFv: disulfide-stabilized fragment variable; scFv: single-chain fragment variable; B-ALL: B-lineage Acute Lymphoblastic Leukemia; Fv: fragment variable; HCL: hairy cell leukemia; IL-2R: Interleukin-2 receptor; CR: complete response; CLL: chronic lymphocytic leukemia; ATL: adult T-cell leukemia; DARPins: designed Ankyrin repeat proteins; pmol: picomolar; HAMA: human-anti mouse antibody     

Recombinant immunotoxins for treating cancer

Recombinant immunotoxins are antibody-toxin chimeric molecules that kill cancer cells via binding to a surface antigen, internalization and delivery of the toxin moiety to the cell cytosol. In the cytosol, toxins catalytically inhibit a critical cell function and cause cell death. The antibody portion of the chimera targets antigens that are expressed preferentially on the surface of cancer cells. Truncated versions of either diphtheria toxin (DT) or Pseudomonas exotoxin (PE) can be used to construct fusions with cDNAs encoding antibody fragments or cell-binding ligands. Recombinant immunotoxins are routinely produced in E. coli and purified using standard chromatographic methods. Before they can be evaluated for anticancer activity in humans, recombinant immunotoxins undergo extensive preclinical testing. Immunotoxins must demonstrate cell-killing activity in tissue culture, antitumor activity in an animal model and have favorable pharmacokinetic and toxicity profiles. Candidate molecules with favorable characteristics are then evaluated in clinical trials. Here we report on the initial evaluation of BL22, a recombinant immunotoxin targeted to CD22 expressed on the surface of B-cell malignancies.     

Recombinant Immunotoxins Containing Truncated Bacterial Toxins for the Treatment of Hematologic Malignancies

Immunotoxins are molecules that contain a protein toxin and a ligand that is either an antibody or a growth factor. The ligand binds to a target cell antigen, and the target cell internalizes the immunotoxin, allowing the toxin to migrate to the cytoplasm where it can kill the cell. In the case of recombinant immunotoxins, the ligand and toxin are encoded in DNA that is then expressed in bacteria, and the purified immunotoxin contains the ligand and toxin fused together. Among the most active recombinant immunotoxins clinically tested are those that are targeted to hematologic malignancies. One agent, containing human interleukin-2 and truncated diphtheria toxin (denileukin diftitox), has been approved for use in cutaneous T-cell lymphoma, and has shown activity in other hematologic malignancies, including leukemias and lymphomas. Diphtheria toxin has also been targeted by other ligands, including granulocyte-macrophage colony-stimulating factor and interleukin-3, to target myelogenous leukemia cells. Single-chain antibodies containing variable heavy and light antibody domains have been fused to truncated Pseudomonas exotoxin to target lymphomas and lymphocytic leukemias. Recombinant immunotoxins anti-Tac(Fv)-PE38 (LMB-2), targeting CD25, and RFB4(dsFv)-PE38 (BL22, CAT-3888), targeting CD22, have each been tested in patients. Major responses have been observed after failure of standard chemotherapy. The most successful application of recombinant immunotoxins today is in hairy cell leukemia, where BL22 has induced complete remissions in most patients who were previously treated with optimal chemotherapy.     

Immunotoxins and cytokine toxin fusion proteins

Paul Ehrlich first suggested the simple and elegant concept of creating specific cell toxins or 'magic bullets' through the fusion of cell specific antibodies and toxins. In practice it has proven difficult to create safe and effective 'magic bullets'. In the past several years, several immunotoxins have been applied to clinical testing. These immunotoxins have been created by the biochemical coupling of cell or lineage specific monoclonal antibodies to plant toxins or fragments thereof. These immunotoxins have been used to treat bone marrow transplant recipients and patients with autoimmune disorders. In recent years, another strategy has also been pursued to create hybrid toxins. Rather than use antibodies as the targeting moiety, cytokines have been used to target a select population of cells bearing a high copy number of receptors for the specific cytokine. Rather than biochemically couple a cytokine to the toxin, the cytokine and toxin are fused by a peptide bond established via genetic engineering. A prototype IL-2 diphtheria toxin-related fusion protein is now being tested in the clinic for treatment of hematopoietic malignancies and autoimmune disorders.     

Modeling Recombinant Immunotoxin Efficacies in Solid Tumors

Effectiveness of cancer therapy is improved by the use of recombinant immunotoxins (RITs) that target membrane proteins unique to malignant tumor cells. Although RIT antitumor activity in vivo can always be improved with larger doses, clinical restriction on the dose toleration makes it critical to explore how RIT antitumor activity can be maximized without resorting to dose elevation. In this work, a mathematical model was developed to explore functional correlations between the properties of several recombinant immunotoxins and their antitumor efficacies in vivo. Simulations were compared with experimental data of human tumor xenografts grown on nude mice to assess parameters critical to optimal antitumor activity. We dissected out or held constant as many parameters of the model as possible to investigate the effect of the remaining parameters on the behavior of the system as a whole. Empirical correlations between immunotoxin binding affinity and the target binding site density were obtained for several recombinant immunotoxins targeting either human A431 carcinoma or CD46 Burkitt’s lymphoma. Simulations reinforced the idea of binding site barrier for drug diffusion and suggested that optimal antitumor activity was achieved when the binding affinity is logarithmically dependent on the target binding site density.     

核糖核酸酶特殊生物学功能及药理作用研究新进展

核糖核酸酶作为一种模型蛋白被普遍用于分子生物学研究。近年来的一系列研究表明，核糖核酸酶及其结构类似物具有许多重要的生物学功能，如控制肿瘤血管生成，杀灭肿瘤细胞及抑制病毒（包括HIV－1病毒）的复制等，引起人们的广泛关注，核糖核酸酶家族大多具有裂解RNA的活性，因此可与抗体连接构成具有显著的靶向性及人源化的免疫毒素，被用来杀伤癌细胞及其它异常增殖细胞。我们将对有关核糖核酸酶家族生物学功能的新发现及研究进展作一介绍。     

rRTA:DS27免疫毒素的制备及胞内运输研究

目的：探讨免疫毒素ｒＲＴＡ：ＤＳ２７的内化行为。方法：用原核系统来获得重组蓖麻毒素Ａ链（ｒＲＴＡ）;用胶体金双标记法进行Ｍｏｌｔ４细胞中免疫毒素的导向研究。结果：ＣＭ－Ｓｅｐｈｏｒｏｓｅ一步纯化到电泳纯的ｒＲＴＡ,电镜观察到ｒＲＴＡ：ＤＳ２７遵循特异的胞内运输途径。结论：上述结果对于深入了解免疫毒素的作用机理,并进而改善其临床应用具有重要作用。     

Human kappa light chain targeted Pseudomonas exotoxin A--identifying human antibodies and Fab fragments with favorable characteristics for antibody-drug conjugate development

Antibody-drug conjugates (ADC) represent promising agents for targeted cancer therapy. To allow rational selection of human antibodies with favorable characteristics for ADC development a screening tool was designed obviating the need of preparing individual covalently linked conjugates. Therefore, α-kappa-ETA' was designed as a fusion protein consisting of a human kappa light chain binding antibody fragment and a truncated version of Pseudomonas exotoxin A. α-kappa-ETA' specifically bound to human kappa light chains of human or human-mouse chimeric antibodies and Fab fragments. Antibody-redirected α-kappa-ETA' specifically inhibited proliferation of antigen-expressing cell lines at low toxin and antibody concentrations. Selected antibodies that efficiently delivered α-kappa-ETA' in the novel assay system were used to generate scFv-based covalently linked immunotoxins. These molecules efficiently triggered apoptosis of target cells, indicating that antibodies identified in our assay system can be converted to functional immunoconjugates. Finally, a panel of human epidermal growth factor receptor (EGFR) antibodies was screened--demonstrating favorable characteristics with antibody 2F8. These data suggest that antibodies with potential for Pseudomonas exotoxin A-based ADC development can be identified using the novel α-kappa-ETA' conjugate.     

Targeting TARP, a novel breast and prostate tumor-associated antigen, with T cell receptor-like human recombinant antibodies

MHC class I molecules are important components of immune surveillance. There are no available methods to directly visualize and determine the quantity and distribution of MHC/peptide complexes on individual cells or to detect such complexes on antigen-presenting cells in tissues. MHC-restricted recombinant antibodies with the same specificity of T cell receptors (TCR) may become a valuable tool to address these questions. They may also serve as valuable targeting molecules that mimic the specificity of cytotoxic T cells. We isolated by phage display a panel of human recombinant Fab antibodies with peptide-specific, MHC-restricted TCR-like reactivity directed toward HLA-A2-restricted T cell epitopes derived from a novel antigen termed TCRgamma alternative reading frame protein (TARP) which is expressed on prostate and breast cancer cells. We have characterized one of these recombinant antibodies and demonstrated its capacity to directly detect specific HLA-A2/TARP T cell epitopes on antigen-presenting cells that have complexes formed by naturally occurring active intracellular processing of the antigen, as well as on the surface of tumor cells. Moreover, by genetic fusion we armed the TCR-like antibody with a potent toxin and demonstrated that it can serve as a targeting moiety killing tumor cells in a peptide-specific, MHC-restricted manner similar to cytotoxic T lymphocytes.     

Monoclonal antibodies to tissue-specific cell surface antigens. I. Characterization of an antibody to a prostate tissue antigen

Monoclonal antibodies were raised to PC-3 human prostate adenocarcinoma cells, and one hybridoma, designated F77-129, was extensively purified and used to characterize a PC-3 antigen. The F77-129 antibody also showed serological reactivity with the Du-145 prostate cancer line and with three of four breast carcinoma lines tested; it showed variable binding to a colon carcinoma line. Several other lines tested, including melanomas, fibrosarcomas, and leukemias, were completely negative. Immunoperoxidase staining of frozen surgical specimens showed binding to both normal and malignant prostate and breast tissue. Injection of radioiodinated F77-129 into tumor-bearing nude mice showed specific in vivo targeting to prostatic cancer implants. The antigen also showed surface modulation by bound antibody, suggesting possible clinical utility of this antibody in delivering immunotoxins to tumors.     

Influence of relative binding affinity on efficacy in a panel of anti-CD3 scFv immunotoxins.

The in vitro cell killing potency of an immunotoxin reflects the aggregate of several independent biochemical properties. These include antigen binding affinity; internalization rate, intracellular processing and intrinsic toxin domain potency. This study examines the influence of antigen binding affinity on potency in various immunotoxin fusion proteins where target antigen binding is mediated by single chain antibody variable region fragments (scFv). Firstly, the relationship between affinity and potency was examined in a panel of four scFv immunotoxins generated from different anti-CD3 monoclonal antibodies fused to the 38 kDa fragment of Pseudomonas aeruginosa exotoxin A (PE38). Of these four scFv-PE38 immunotoxins, the one derived from the anti-CD3 monoclonal antibody UCHT1 has highest cell killing potency. Analysis of these four scFv-PE38 immunotoxins indicated a correlation between antigen binding affinity and immunotoxin potency in the cell killing assay with the exception of the scFvPE38 immunotoxin derived from the antibody BC3. However this scFv appeared to suffer a greater drop in affinity ( approximately 100x), relative to the parent Mab than did the other three scFvs used in this study (2-10x). Secondly, the scFv(UCHT1)-PE38 immunotoxin was then compared with a further panel of scFv(UCHT1)-derived immunotoxins including a divalent PE38 version and both monovalent and divalent Corynebacterium diphtheriae toxin (DT389) fusion proteins. When the scFv-UCHT1 domain was amino-terminally positioned relative to the toxin, as in the scFv(UCHT1)-PE38, an approximately 10-fold higher antigen-binding affinity was observed than with the C-terminal fusion, used in the DT389-scFv(UCHT1) molecule. Despite this lower antigen-binding activity, the DT389-scFv immunotoxin had a 60-fold higher potency in the T-cell-killing assay. Thirdly, a divalent form of the DT389-scFv construct, containing tandem scFv domains, had a 10-fold higher binding activity, which was exactly reflected in a 10-fold increase in potency. Therefore, when comparing immunotoxins in which scFvs from different antibodies are fused to the same toxin domain (DT or PE) a broad correlation appears to exist between binding affinity and immunotoxin potency. However, no correlation between affinity and potency appears to exist when different toxin domains are combined with the same scFv antibody domain.     

Construction,expression, and characterization of an anti-tumor immunotoxin containing the human anti-c-Met single-chain antibody and PE38KDEL

Recombinant immunotoxins consisting of small antibody fragments fused to cytotoxic moieties are being evaluated for use in prospective antibody-targeted cancer therapies. A receptor tyrosine kinase known as c-Met is overexpressed in a vast range of human malignancies, making it an ideal target for antibody-mediated delivery of numerous cytotoxic agents. A single Fab molecule capable of binding to human c-Met with high affinity and specificity was previously identified using antibody phage-display technology. In order to develop a molecule to increase both the cytotoxicity and anti-tumor activity of the anti-c-Met molecule, a recombinant immunotoxin anti-c-Met/PE38KDEL was constructed and expressed by fusing the human anti-c-Met single-chain variable fragment (ScFv) with a modified Pseudomonas exotoxin A (PE38KDEL). Purified anti-c-Met/PE38KDEL was demonstrated to specifically bind to cells of c-Met-positive human hepatoma cell lines, causing a proliferation defect by inducing caspase-3/8-mediated apoptosis, as observed by in vitro assays. Furthermore, anti-c-Met/PE38KDEL administration was shown to inhibit the growth of hepatocellular carcinoma xenografts in vivo through suppression of Ki-67 expression and enhancement of tumor cell apoptosis rates. Cumulatively, the current findings demonstrate the successful construction of a recombinant immunotoxin capable of accurately targeting c-Met-positive human hepatoma cell lines both in vitro and in vivo, providing a novel compound with potential for applications as an alternative therapy for c-Met-positive cancer management.     

Human monoclonal antibodies with a protective activity against tetanus toxin

The antibodies and their protective activity in response to tetanus toxoid in man were studied by producing human antitetanus monoclonal antibodies after transformation of peripheral blood lymphocytes with Epstein Barr virus. Two human monoclonal IgG that reacted with the heavy chain of the toxin were obtained. One of them binds the COOH-terminal moiety and the other the NH2-terminal moiety. Only the NH2-terminal specific monoclonal antibody neutralized toxin in mouse, but in doses approximately 100-fold higher than those of a polyclonal antiserum. However, the association of these 2 antibodies was protective with doses lower than necessary for the monoclonal antibodies alone. To replace the polyclonal antibodies used, a good protection could be achieved by mixed human monoclonal antibodies against different epitopes of tetanus toxin.     

Treatment of prostate carcinoma with (galectin-3)-targeted HPMA copolymer-(G3-C12)-5-Fluorouracil conjugates

Galectin-3 (Gal-3), over-expressed on a variety of human tumor cells, is a potential binding site for targeted metastatic prostate cancer therapy. The aim of this study was to develop a G3-C12-mediated drug delivery system based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers targeting to Gal-3-expressed human PC-3 prostate carcinoma cells. 5-Fluorouracil (5-Fu), an anti-tumor agent, was selected as a model drug. G3-C12, a binding peptide, which specifically binds to the carbohydrate-recognition domain (CRD) of Gal-3, was attached to HPMA copolymers as a targeting moiety. Compared with non-targeted conjugates (P-Fu), Gal-3-targeted HPMA copolymer-(G3-C12)-5-Fu conjugates (P-(G3-C12)-Fu) displayed a superior intracellular internalization followed by enhanced cytotoxicity and apoptosis-induction. Subsequently, the in vitro migration study on PC-3 cells indicated that P-(G3-C12)-Fu was able to efficiently inhibit the cell migration ability after wounding. On PC-3 tumor-bearing mice model, G3-C12-modified copolymers showed a higher tumor accumulation coupled with a faster clearance from blood circulation than non-modified ones. Finally, Gal-3-targeted conjugates significantly improved the anti-tumor activity of 5-Fu in nude mice bearing PC-3 tumor xenografts. Consequently, G3-C12 would be a promising targeting moiety for cell-specific prostate cancer therapy in future.     

Antigen-specific targeting and elimination of EBV-transformed B cells by allergen toxins

BACKGROUND: With the exception of antigen-specific immunotherapy, current treatments for atopic diseases provide only symptomatic relief. Because of the increasing incidence of such diseases, the development of novel strategies and concepts for the treatment of allergies is urgently needed. OBJECTIVE: Here we present a new approach for the treatment of atopic diseases. The strategy is comparable to the application of immunotoxins in cancer therapy, in which a cytotoxic peptide is coupled to a cancer cell-specific antibody fragment or ligand. In the case of so-called allergen toxins (ATs), the target cell-specific moiety is an allergen or allergen-derived fragment, which should be bound only by allergen-reactive cells. After receptor-mediated internalization, allergen-specific cells are killed, and the allergic pathogenesis is interrupted. METHODS: Proof of the AT principle was shown by using a human ex vivo system in which EBV was used to transform human B cells specific for the timothy grass pollen allergen Phl p 5b. The AT is composed of the major B-cell and T-cell epitopes of the Phl p 5b (P5) allergen fused to a truncated form of the highly toxic Pseudomonas aeruginosa exotoxin A (ETA'). RESULTS: Allergen-specific and nonspecific B cells were challenged with P5-ETA', but only the Phl p 5b-reactive B cells showed selective binding and cytotoxicity. CONCLUSION: This approach represents an initial step toward a novel therapeutic strategy in the treatment of atopic diseases.     

Recombinant immunotoxins for the treatment of Hodgkin's disease (Review)

In recent years, substantial experience has been accumulated with tumor-specific immunotherapeutics which seem to be effective against minimal residual disease. The coupling of toxins to monoclonal antibodies has indicated promising results in early clinical trials. Recombinant DNA technology makes it possible to genetically fuse coding regions of V genes or cytokines to modified toxin domains. These recombinant immunotoxins can easily be manipulated to increase the cytotoxic potency or affinity. Binding single-chain variable fragments (scFv) expressed as chimeric fusion proteins on the surface of filamentous bacteriophages were selected on Hodgkin-derived cell lines. This technique was also used to create a new humanized anti-CD30 scFv which exhibits similar binding to the CD30 antigen when compared to its murine predecessor. ScFvs were then inserted into a new bacterial expression vector and thus fused to a deletion mutant of Pseudomonas exotoxin. Anti-CD25(scFv)-ETA' and anti-CD30(scFv)-ETA' were isolated from E. coli periplasm and purified by metal chelate affinity and size exclusion chromatography. All immunotoxins produced showed specific cytotoxicity against Hodgkin lymphoma cell lines as documented by competitive assays. In addition, these constructs were highly efficient in the treatment of disseminated human Hodgkin's disease in SCID mice. These in vivo data indicate a possible clinical impact for patients with relapsed CD25- and/or CD30-positive lymphoma.     

Histone modification enhances the effectiveness of IL-13 receptor targeted immunotoxin in murine models of human pancreatic cancer

Background  

Interleukin-13 Receptor α2 (IL-13Rα2) is a tumor-associated antigen and target for cancer therapy. Since IL-13Rα2 is heterogeneously overexpressed in a variety of human cancers, it would be highly desirable to uniformly upregulate IL-13Rα2 expression in tumors for optimal targeting.     

High specific cytotoxicity of antibody-toxin hybrid molecules (immunotoxins) for target cells

In an attempt to develop highly efficient antibody—drug conjugates for passive immunotherapy of cancer the A-chain of the potent toxin, ricin, was coupled to antibodies in order to render them specifically cytotoxic for target cells without the participation of complement. The antibody—toxin conjugates (immunotoxins) showed no loss of antibody or A-chain activity. In vitro, highly purified A-chain was about 5000 times less toxic on HeLa cells than whole ricin. Unconjugated A-chain made no difference between TNP-HeLa and HeLa cells but when coupled to anti-DPN antibodies it became about 500 times more cytotoxic to TNP-HeLa cells than to HeLa cells. In vivo A-chain (LD₅₀: 20 mg/kg) was about 3000 times less toxic than whole ricin and treatment with immunotoxin significantly inhibited tumor take and tumor growth of TNP-HeLa cells in nude mice.     

Immunotoxins: the power and the glory.

Immunotoxins are hybrid proteins in which the potent cytocidal action of a toxin is harnessed for the selective destruction of target cells by attachment to a specific monoclonal antibody (mAb) or growth factor. This brief article describes the latest advances in the molecular and cellular biology, pharmacology and clinical evaluation of immunotoxins, as discussed at a recent meeting.