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.     

Designing immunotoxins for cancer therapy

Immunotoxins are the rapeutic agents with a high degree of specificity and unique mechanism of action. An immunotoxin is achimeric protein consisting of a targeting moiety linked to a toxin. The targeting moiety selectively binds to a tumor cell and targets it for death via the attached toxin. Generally, immunotoxins are specifically potent against cancer cells in vitro and in animal models of human malignancies. However, immunotoxins can be limited clinically by immunogenicity, toxicity, and instability. In this review, weofferwaysto overcome these limitations to create “ideal immunotoxins” for cancer therapy. These include producing single chain targeting/toxin fusion proteins of fully human origin that are extracellularly stable but once internalized, can be cleaved by intracellular proteases to free the toxin and facilitate its translocation to the cytosol.     

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     

Immunotoxins in cancer therapy.

Immunotoxins are composed of a protein toxin connected to a binding ligand such as an antibody or growth factor. These molecules bind to surface antigens (which internalize) and kill cells by catalytic inhibition of protein synthesis within the cell cytosol. Immunotoxins have recently been tested clinically in hematologic malignancies and solid tumors and have demonstrated potent clinical efficacy in patients with malignant diseases that are refractory to surgery, radiation therapy and chemotherapy - the traditional modalities of cancer treatment. This therapy is thus evolving into a separate modality of cancer treatment, capable of rationally targeting cells on the basis of surface markers. Efforts are underway to obviate impediments to clinical efficacy, including immunogenicity and toxicity to normal tissues. Immunotoxins are now being developed to new antigens for the treatment of cancer.     

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: 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.     

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.     

Are immunoconjugates useful for therapy with autoimmune diseases?

Immunoconjugates or immunotoxins (ITs) are targeting molecules which consist of a monoclonal antibody together with a toxin-thereby they can selectively kill target cells in a highly efficient manner. The use of ITs as a drug targeting approach is one of the most attractive research fields for tumor therapy; however, the study of ITs for the treatment of autoimmune diseases has been given little attention until recently. It has been shown that ITs could help alleviate the symptoms of myasthenia gravis and rheumatoid arthritis in animal models. In the last 3 yr ITs have been used in clinical trials (phase I and phase II) for the treatment of various autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, insulin-dependent diabetes mellitus and psoriasis. This article reviews the main progress on the application of ITs for the therapies of autoimmune diseases. The preliminary results suggest the future may hold some promise, but side effects, in addition to there being no convincing efficacy, remain unresolved.     

Immunotoxins: New therapeutic reagents for autoimmunity,cancer, and AIDS

Immunotoxins consist of cell-reactive ligands coupled to toxins or their toxic subunits. The ligands are usually antibodies, hormones, or growth factors and the toxins are of bacterial or plant origin.In vitro studies using A chain-containing immunotoxins specifically to kill tumor cells were successful and led to further experimentsin vivo. Such studies, carried out over the past 5 years in both animals and humans, have demonstrated that the efficacy of immunotoxinsin vivo is often poor, due to problems involving instability of the conjugate, inferior potency, inaccessibility of tumor cells, nonspecific binding to cells other than the target cells, and survival of antigen-negative mutants. In addition, immune responses against both the ligand and the A chain are usually elicited, precluding repeated therapy. During the past several years, there have been attempts to solve these problems and develop more effective immunotoxins.     

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。     

抗T细胞免疫毒素的T细胞清除效率及其对造血细胞的影响

抗T细胞免疫毒素主要用于同种异体骨髓移植中清除T细胞,减轻移植物抗宿主反应(GVHD),提高移植成功率,也可用于白血病自体骨髓移植中清除残留白血病细胞,减少移植后白血病复发的可能性。本实验结果表明抗T细胞免疫毒素在10~(-8)M浓度对靶细胞的杀伤达3.0log以上,即可清除99.99%的靶细胞,并可有效地抑制周血T细胞转化功能和骨髓T细胞分泌IL—2的功能,同时对骨髓粒单系和红系造血细胞的增殖无明显的抑制作用。     

Therapeutic intervention with inhibitors of co-stimulatory pathways in autoimmune disease

Many humanized antibodies and fusion proteins targeting T-cell co-stimulatory molecules are now in late-stage clinical development (phase II, phase III) or have recently completed phase III clinical trials. Both Amevive, an LFA-3-Ig fusion protein targeting CD2, and Xanelim, a humanized anti-CD11a antibody, have shown efficacy in pivotal phase III trials in patients with plaque psoriasis. These new medicines are poised to enter clinical use in 2002.     

Selective killing of T cells by immunotoxins directed at distinct Vβ epitopes of the T cell receptor

The potency and specificity of anti-T cell receptor (TcR)-directed immunotoxins were studied in two T cell leukemia lines, HPB-ALL and Jurkat, and in primary T cells. Immunoconjugates were synthesized using anti-CD3? or distinct anti-V_β antibodies cross-linked to CRM9, a binding site-mutant of diphtheria toxin. All TcR-expressing cells display the CD3 complex on the plasma membrane. HPB-ALL cells express the V_β5 gene product in the β subunit of the TcR, while Jurkat cells express V_β8. V_β expression in primary T cells isolated from buffy coats is heterogeneous. Primary T cell populations expressing specific V_β epitopes in the TcR were generated by plating CD3⁺ T cells on V_β-specific antibody-coated flasks or by positive immunomagnetic selection. Immunotoxins directed against the invariant CD3? epitope target and kill all T cells. Immunoconjugates targeted at distinct anti-V_β epitopes are specific for cells that express the corresponding gene product in the TcR. The results demonstrate the ability of antiTcR-based immunotoxins selectively to kill T cells with defined V_β epitopes. These reagents may be clinically useful in disorders mediated by autoreactive T cell populations exhibiting V_β restriction and in the treatment of clonal TcR-expressing lymphomas.     

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。     

Non-Hodgkin’s Lymphoma

Non-Hodgkin's lymphomas (NHL) represent a heterogenous group of diseases including low, intermediate and high grade histological subtypes. Most entities are sensitive to chemotherapy and radiotherapy. However, most relapsed patients are incurable with conventional treatment. The major reason for unsatisfactory long-term results in NHL is tumour cells that persist after standard treatment. New sensitive techniques have been developed to detect occult lymphoma cells. These cells might be eradicated by new immunotherapeutic agents with different modes of action, such as cytokines or antibody-based agents. In NHL, most experience has been accumulated with interferon-alpha, which seems to be effective against minimal residual disease (MRD). The experience with interleukin-2 and interleukin-3 is less convincing. Monoclonal antibodies have been used in their native form, or conjugated with radioisotopes or toxins to selectively destroy lymphoma cells. Such immunotoxins and radioisotope-coupled antibodies have shown promising results in early clinical trials, and are now being evaluated in patients with smaller tumour burdens.     

Pseudomonas exotoxin A: from virulence factor to anti-cancer agent

The pathogenic bacterium Pseudomonas aeruginosa has the ability to cause severe acute and chronic infections in humans. Pseudomonas exotoxin A (PE) is the most toxic virulence factor of this bacterium. It has ADP-ribosylation activity and decisively affects the protein synthesis of the host cells. The cytotoxic pathways of PE have been elucidated, and it could be shown that PE uses several molecular strategies developed under evolutionary pressure for effective killing. Interestingly, a medical benefit from this molecule has also been ascertained in recent years and several PE-based immunotoxins have been constructed and tested in preclinical and clinical trials against different cancers. In these molecules, the enzymatic active domain of PE is specifically targeted to tumor-related antigens. This review describes the current knowledge about the cytotoxic pathways of PE. Additionally, it summarizes preclinical and clinical trials of PE-based immunotoxins and furthermore discusses current problems and answers with these agents.     

A comparison of anti-lymphocyte immunotoxins containing different ribosome-inactivating proteins and antibodies.

Immunotoxins were prepared with several single-chain ribosome-inactivating proteins (RIPs type 1) and with the A-chain of ricin linked to the F(ab')2 fragment of sheep anti-mouse IgG. The cytotoxic activity of these conjugates was tested on human lymphocytes pretreated with an anti-CD3 murine MoAb. The immunotoxins inhibited DNA synthesis in phytohaemagglutinin (PHA)-stimulated lymphocytes with IC50S (concentrations causing 50% inhibition) ranging from 8.9 x 10(-13) to 5.7 x 10(-11) M (immunotoxins containing dianthin 32, saporin, pokeweed antiviral protein from seeds (PAP-S), bryodin, momordin, momorcochin, and trichokirin), 1 x 10(-8) M (immunotoxin containing gelonin) and 5 x 10(-9) M (immunotoxin containing ricin A-chain). The immunotoxin containing saporin linked to the anti-mouse IgG F(ab')2 fragment was also highly toxic to human lymphocytes pretreated with anti-CD2, -CD3, -CD5 and -CD45 MoAbs, with IC50S less than or equal to 10(-11) M. Immunotoxins were prepared also with saporin linked to MoAbs against various CD antigens. The immunotoxin prepared with the anti-CD3 antibody had the highest specific cytotoxicity to human lymphocytes.     

Sensitivity of target cells to immunotoxins: possible role of cell-surface antigens.

In this paper, we describe the activity of conjugates constituted of monoclonal antibodies chemically coupled to ricin and the ricin A-chain-like toxin gelonin. Several antibody-toxin conjugates (immunotoxins) were assayed against a Thy 1.2+ and Lyt 2.2+ T-cell line. Anti-Thy 1.2-gelonin and anti-Lyt 2.2-ricin immunotoxins (IT) had a good cytotoxic activity against target cells, whereas anti-Lyt 2.2-gelonin showed weak or no cytotoxicity. An anti-Lyt 2.2-gelonin IT displayed detectable cytotoxicity only when cells were pretreated with ammonium chloride. IT binding activity was very similar for anti-Thy 1.2 and anti-Lyt 2.2 IT, and surface antigen density was of the same order of magnitude for Thy 1.2 and Lyt 2.2 antigens. Our results suggest that intrinsic properties of the surface target antigen are able to influence the IT cytotoxic efficacy.     

Immunoablation of cells expressing the NG2 chondroitin sulphate proteoglycan

Expression of the transmembrane NG2 chondroitin sulphate proteoglycan (CSPG) defines a distinct population of NG2‐glia. NG2‐glia serve as a regenerative pool of oligodendrocyte progenitor cells in the adult central nervous system (CNS), which is important for demyelinating diseases such as multiple sclerosis, and are a major component of the glial scar that inhibits axon regeneration after CNS injury. In addition, NG2‐glia form unique neuron–glial synapses with unresolved functions. However, to date it has proven difficult to study the importance of NG2‐glia in any of these functions using conventional transgenic NG2 ‘knockout’ mice. To overcome this, we aimed to determine whether NG2‐glia can be targeted using an immunotoxin approach. We demonstrate that incubation in primary anti‐NG2 antibody in combination with secondary saporin‐conjugated antibody selectively kills NG2‐expressing cells in vitro. In addition, we provide evidence that the same protocol induces the loss of NG2‐glia without affecting astrocyte or neuronal numbers in cerebellar brain slices from postnatal mice. This study shows that targeting the NG2 CSPG with immunotoxins is an effective and selective means for killing NG2‐glia, which has important implications for studying the functions of these enigmatic cells both in the normal CNS, and in demyelination and degeneration.     

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.