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Antibody-directed therapies for hematological malignancies
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Antibody-directed therapies for hematological malignancies Michael L. Linenberger, David G. Maloney and Irwin D. Bernstein Malignant hematopoietic cells express lineage-restricted antigens that serve as suitable targets for antibody-directed therapy. Although several highly specific, potent and relatively nontoxic, engineered antibodies, immunoglobulin fragments and antibody conjugates have been developed, only three have gained approval for clinical use. Of these, a chimeric mouse–human anti-CD20 antibody has yielded the most impressive clinical results. Encouraging data with the other approved antibodies, and with agents in clinical trials, suggest that rational antibody design will generate effective products for several different hematological malignancies. Despite these advances, significant challenges remain in the identification of optimal cellular targets, antibody forms and treatment schedules for therapeutic applications.
Michael L. Linenberger* David G. Maloney Clinical Research Division, Fred Hutchinson Cancer Research Center, and Dept of Medicine, University of Washington, Seattle, WA, USA. *e-mail: [email protected] Irwin D. Bernstein Clinical Research Division, Fred Hutchinson Cancer Research Center, and Dept of Pediatrics, University of Washington, Seattle, WA, USA.
Since the seminal description of murine hybridoma methodology in 1975 [1], monoclonal antibodies (mAbs) have been developed as diagnostic tools and therapeutic agents for hematological malignancies [2–5]. Initially, rodent mAbs were used to identify useful tumor target antigens, characterize their function and define important biological variables, such as surface density, internalization kinetics and shedding. Subsequent in vivo studies with unmodified antibodies demonstrated the critical roles of immunoglobulin (Ig) structure in eliciting the desirable effect of host anti-tumor activation and the undesirable effect of host antiglobulin response. More recently, genetic engineering has enabled the development of chimeric or humanized antibodies, Ig fragments and multivalent antibodies, in order to avoid immunogenicity and augment anti-tumor potency. It has also been possible to link antibodies and Ig fragments with toxins, radionuclides, enzymes, chemotherapeutic agents, cytokines and drug-containing liposomes for therapeutic purposes. This review will summarize the molecular and biochemical advances leading to the rational design of antibody-based treatment approaches for hematological malignancies. Examples of relevant current strategies will be discussed to illustrate the key elements of antibody construction, target selection, chemical modification, therapeutic applications and mechanisms of resistance. For more-comprehensive summaries of individual antibody therapies for specific disorders the reader is referred to excellent recent reviews [3–5]. Antibody design
Recombinant technologies have facilitated the development of hybrid, truncated and conjugated antibodies [2]. These reagents often avoid the limitations of unmodified rodent mAbs, such as: http://tmm.trends.com
(1) immunogenicity (formation of antiglobulin antibodies); (2) suboptimal avidity to the antigen target; (3) inefficiency at activating human complement; (4) limited potential to activate antibodydependent cell-mediated cytotoxicity (ADCC) mechanisms; and (5) unfavorable pharmacokinetics. Chimeric antibodies, which consist of monoclonal antigen-binding (Fab) or variable (Fv) domains combined with human constant (Fc) regions, are less immunogenic and incorporate Fc isotypes that optimally activate host effector mechanisms (usually of the IgG1 or IgG3 subclasses). Similar advantages derive from humanized antibodies, which contain the three antigen-binding loops within the complementarity-determining regions (CDRs) of the rodent mAb heavy (H)- and light (L)-chain V domains grafted onto the framework regions of the human V and C domains (Fig. 1). Recombinant antibody fragments, including Fv fragments (noncovalently associated H- and L-chain V domains) and single-chain Fv (scFv) fragments (H- and L-chain V domains covalently linked by short polypeptide linkers) (Fig. 1), are used as vehicles to deliver toxins and drug conjugates. The small size (roughly 50 kDa for scFv fragments) facilitates tumor tissue penetration with more-favorable biodistribution and plasma clearance than full-length antibodies. However, because of their more-rapid clearance, very high doses are required, thereby detracting from their clinical utility. A variety of more-novel antibody forms have been developed to improve their usefulness as cytocidal and/or imaging agents. These include multivalent antibodies (consisting of two or more different antigen-binding arms), bispecific mAbs (consisting of two complete antibodies, against different targets, joined by a chemical crosslinker) and bispecific F(ab)′2 fragments. Diabodies, which are linked recombinant scFv fragments (Fig. 1), might be either bivalent and monospecific (i.e. both Fv arms recognize the same antigen) or bispecific (i.e. each Fv arm recognizes a different antigen). Although these constructs should optimize specificity and avoid the problem of antiglobulin formation, they have not yet been shown to be superior to conventional therapies in early clinical trials. Target selection
Clonal malignant cells of lymphoid and myeloid origin commonly express lineage-restricted surface
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Fig. 1. Evolution of monoclonal antibodies (mAbs). Initially, mAbs were derived from rodent hybridoma cell lines that were isolated and propagated in tissue culture. Subsequently, recombinant technologies allowed the selection, modification and largescale expression of rodent sequences encoding the antigen-binding regions. Chimeric or humanized antibodies, which incorporate human constant domains either with the rodent variable (V) regions or with the three antigen-binding loops of the complementaritydetermining regions (CDRs), respectively, were developed to reduce immunogenicity and optimize host effector mechanisms. Heavy and light chain V-region fragments (Fv) and singlechain fragments (scFv; which consist of the two chains joined by a polypeptide linker) were developed as smaller delivery vehicles with more-favorable biodistribution than fulllength antibodies. Although these fragments lack Fc regions, and therefore cannot activate host effector functions, they can be conjugated to toxins, drugs or radionuclides. They might also be combined to form diabodies, which can be either monospecific or bispecific (i.e. each Fv arm recognizes a different antigen). Fab domains indicate monoclonal antigen-binding fragment. Abbreviations: Amp, ampicillin; Ig, immunoglobulin.
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Rodent hybridoma
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differentiation antigens, signaling proteins, growth factor receptors and, in the case of B-cell malignancies, Ig molecules. These antigens are similar to those expressed by the non-malignant precursors from which they were derived and are potential antigens for antibody-directed therapy. However, among these, the V-region epitopes of the surface idiotype Ig represent the only true tumor-specific targets [6]. To date, the majority of therapeutic reagents have been generated against tumor-associated antigens. These antigens might be either preferentially expressed or overexpressed by the tumor cells and/or they might play a more critical function in the malignant cells than in the normal cell counterparts. Because these antigens are also expressed by at least some normal myeloid or lymphoid cells, the success of the antibody treatment relies on the ability of the normal cells to either tolerate ‘collateral damage’ or to be reconstituted by antigen-negative precursors. The ideal surface antigen is found at high density on the tumor cells (i.e. >104–105 binding sites per cell) with limited expression on normal counterparts and with little or no circulating cell-free antigen. The antigenic epitope should have minimal variations or heterogeneity of expression among tumor cell subclones. Target molecules that serve critical host–cell functions are desirable http://tmm.trends.com
because immunological ‘escape’, due to loss of expression, is less likely to occur. In some cases, such as with anti-idiotype antibodies, antibody binding, and perhaps surface hypercrosslinking, activates signaling pathways that facilitate cell death mechanisms [7]. Non-internalized surface molecules
Antigen–antibody complexes that are slowly or minimally internalized by the target cell are optimal for approaches that rely on extracellular mechanisms of cytotoxicity. These include: enzymelinked antibodies that activate a prodrug in the tumor microenvironment; bispecific or multivalent antibodies that crosslink the tumor cell to local immune effector cells; full-length antibodies that activate ADCC and/or complement-dependent cytotoxicity (CDC) through the Fc domain; and certain radioimmunoconjugates that would be rapidly degraded if internalized (Fig. 2). Clinically relevant examples of cell-surface molecules with low rates of antibody-mediated internalization or modulation include the cluster of differentiation (CD) antigens CD20, CD45 and CD52. CD20, a 35 kDa membrane phosphoprotein that functions as a Ca2+ channel and is involved in normal B-cell activation and proliferation, is expressed on
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(a) Radioimmunoconjugates
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(d) Antibody–drug conjugates
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Fig. 2. Cytotoxic mechanisms of antibody-based treatments. Noninternalizing antigen targets are ideal for most radioimmunoconjugates and full-length antibodies that rely on Fc-mediated activation of host effector functions. (a) Radioimmunoconjugates containing iodine-131(131I ) emit β-radiation over many cell diameters, and therefore can kill inaccessible or antigen-negative cells at a distance. (b) Fc domains extending from antibody-coated tumor cells might activate antibody-dependent cell-mediated cytotoxicity (ADCC), through Fc receptors on natural killer (NK) cells or macrophages. Alternatively, complement-dependent cytotoxicity (CDC) might be initiated by the binding of complement protein C1 to Fc domains. In addition, some noninternalizing antibodies might activate receptor-mediated signaling pathways that inhibit growth or induce cell death. Internalizing antigens are required for intracellular delivery of toxins or antitumor drugs. (c) Plant and bacterial immunotoxins traffic through endosomes or the Golgi and endoplasmic reticulum. Free toxin is then released to the cytoplasm where it inhibits protein synthesis and ultimately causes cell death. (d) Antibody–drug conjugates also undergo dissociation after internalization. As an example, gemtuzumab ozogamicin, which consists of calicheamicin conjugated to an anti-CD33 antibody by a stable bifunctional linker, undergoes hydrolysis in the acidic environment of the lysosome. After free calicheamicin is released, it translocates to the nucleus where it cleaves double-stranded DNA and induces cell death.
the majority of B-cell low-grade and aggressive non-Hodgkin’s lymphomas (NHLs). A chimeric mouse–human anti-CD20 antibody, rituximab (Rituxan, Genentech, Inc.), was approved by the Food and Drug Administration (FDA) in 1997 for treatment of relapsed and refractory follicular NHL [8]. http://tmm.trends.com
Rituximab inhibits proliferation and induces cytotoxicity by activating CDC, ADCC and, to a limited extent, apoptosis [9,10]. Since approval, this agent has been used to treat several different B-lymphoid malignancies that express variable amounts of surface CD20. Particularly noteworthy is the efficacy of rituximab against post-transplant B-cell NHL induced by Epstein–Barr virus (EBV) [11]. Other murine anti-CD20 mAbs have been conjugated with iodine-131 (131I) or yttritum-90 (90Y) (both β-emitting radionuclides) to deliver tumoricidal doses of radiation to sites of residual lymphoma [12,13]. Similarly, radioimmunoconjugates against CD45, a noninternalizing pan-hematopoietic antigen, have been used to augment high-dose chemotherapy and/or radiotherapy before hematopoietic stem cell transplantation for acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and myelodysplasia [14]. CD52, a 12 amino acid glycosylphosphatidylinositol (GPI)-linked glycoprotein that might function as a cytoadhesion molecule, is expressed by some B- and T-cell malignancies, as well as by normal lymphocytes and monocytes. Engagement of CD52 by a humanized anti-CD52 antibody (Campath-1H) appears to trigger CDC and ADCC in vivo, and induces apoptosis in some malignant lymphoid cell types [15]. The anti-CD52
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antibody alemtuzumab (Campath, Berlex, Inc.) was approved by the FDA in May 2001 for treatment of chemotherapy-resistant B-cell chronic lymphocytic leukemia (B-CLL). Internalized surface molecules
Several therapeutic antibodies recognize cell-surface antigens that are internalized after binding. Internalization is required for antibody conjugates that are designed to deliver toxins, chemotherapy drugs or, less commonly, radionuclides to the cytoplasm. The intracellular pathways followed by these antibodies and conjugates have not been fully characterized in most cases. In general, the antigen–antibody complexes coalesce on the surface, undergo endocytosis, and ultimately translocate into lysosomes. Proteolytic processing occurs during trafficking in endosomes, lysosomes or, in some cases, the Golgi and endoplasmic reticulum (ER). Importantly, the antigen–antibody complexes are dissociated and the conjugate moieties are released, allowing them to travel to the cytoplasm or nucleus to induce a cytopathic effect [2,3,16,17] (Fig. 2). Rates and efficiencies of internalization vary, depending on the antigen and the phenotypic features of the host cell. However, the cytotoxic effectiveness of toxin- or chemotherapy-conjugated antibodies often relies more on efficient intracellular processing and the intrinsic potency of the conjugate rather than on the rate of internalization [18]. Gemtuzumab ozogamicin, a recombinant humanized anti-CD33 antibody linked to a derivative of the anti-tumor antibiotic calicheamicin (Mylotarg, Wyeth-Ayerst Laboratories), is the only antibody–drug conjugate approved by the FDA. It is indicated for the treatment of relapsed AML in elderly patients [19]. This agent rapidly saturates CD33 sites, an internalizing antigen commonly found on AML cells, and is quickly endocytosed. After internalization, calicheamicin is released from the lysosomes. It subsequently enters the nucleus where it cleaves double-stranded DNA and induces cell death (Fig. 2). Radioimmunoconjugates
Most malignant hematopoietic cells are susceptible to the cytotoxic effects of radiation, and radionuclides have therefore been extensively utilized in antibody conjugates [2,3,5]. These agents are constructed by covalently binding the radioisotope directly to the antibody or by crosslinking through a chelating molecule or chemical linker. The linker design and methodology are carefully chosen to optimize radiolabeling efficiency, stability, biodistribution, pharmacokinetics and/or intracellular retention (for internalizing antibodies). The cytotoxic efficacy of a radioimmunoconjugate depends on the kinetics of antibody localization and retention of the radionuclide. These variables relate, in turn, to: (1) the size and vascularity of the tumor; (2) the http://tmm.trends.com
density, heterogeneity and modulation of the antigen; and (3) the size, specificity and stability of the radioimmunoconjugate. The path length, half-life and linear energy transfer of the radioisotope are also important. β-emitting isotopes such as copper-67 (67Cu), 131I and 90Y have longer path lengths (0.6, 0.8 and 5.3 mm, respectively) and longer half-lifes (2.5, 8.1 and 2.5 days, respectively) than the α-emitting isotopes bismuth-212 and astatine-211 (path lengths of 0.06 mm and half-lifes of 1 and 7 h, respectively). Conversely, the particulate energy of α-emitters are 2.5–10-fold greater than the β-emitters. Because of the long path length, β-emissions could theoretically kill neighboring tumor cells that are antigennegative or inaccessible to antibody (Fig. 2) [20]. 131I and 67Cu, unlike 90Y, also emit γ-radiation, which facilitates imaging and dosimetry determinations [3]. 90Y or 67Cu are the isotopes of choice with internalized antibodies because they are retained and maintain activity much longer than 131I, which is rapidly dehalogenated within lysosomes [21,22]. As a consequence of their availability, stability and ease of manipulation, 131I and 90Y have been utilized in most constructs. Several clinical trials have studied polyclonal, monoclonal or humanized antibodies conjugated with 131I or 90Y [3,5,22]. In all cases, dose-limiting toxicity to normal tissues remains the major safety concern. The most successful approach to date involves the use of 131I-labeled anti-CD20 antibodies for the treatment of relapsed B-cell NHL. Anti-CD20 radioimmunoconjugates have been used alone [23] or with additional myeloablative chemotherapy [12], with or without subsequent hematopoietic stem cell transplantation. The efficacy in relapsed patients appears to be at least equivalent to, if not superior to, conventional therapies [12,23]. After demonstrating efficacy in relapsed patients, 131I-conjugated anti-CD20 is now being studied as part of initial therapy for previously untreated patients and for patients that relapse after treatment with rituximab. Similar encouraging preliminary results have been reported with 90Y-conjugated anti-CD20 [13]. In other clinical studies of B-cell NHL, radiolabeled antibodies against CD21, CD22, CD37, HLA-DR or idiotypic Ig have been tested [3]. 131I-labeled anti-CD45 has shown promise as an adjunctive therapy in stem cell transplant approaches for acute leukemia [14]. 131I- and 90Y-conjugated polyclonal antiferritin antibodies were found to be active against relapsed NHL [3]; however, this approach was not superior to available conventional salvage treatment options. Radioimmunoconjugates with anti-CD25 or anti-CD5 have been used to treat T-cell malignancies [3], whereas radiolabeled anti-CD33 has been investigated in AML [5,24]. Although some clinical effects have been documented, the utility of these
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agents remain undefined. To date, only a few α-emitting radioimmunoconjugates have been developed for hematological malignancies and only one has been tested for safety in patients [24]. Immunotoxins
Immunotoxins consist of complete antibodies or scFv fragments fused to plant- or bacteria-derived catalytic proteins [17]. The toxins currently in use are exceedingly potent, potentially requiring only one molecule to induce cell death. Because of this potency and native host-cell binding ability, they can only be safely used in modified forms with targeted approaches. Immunotoxins are generated by either chemically linking the modified toxin to the antibody (using disulfide bond chemistry or unique linker construction) or by splicing the Fv and toxin genes into a vector and expressing the recombinant product in bacteria. After internalization, the toxin is released from the antibody during trafficking through the endosome or Golgi and ER (lysosomal acidity degrades the toxin). Intracytoplasmic toxin then interferes with protein synthesis, ultimately causing apoptotic cell death (Fig. 2). Plant phytotoxins, including ricin, saporin, gelonin, bryodin and pokeweed antiviral protein, are ribosomeinactivating proteins. They have been combined with antibodies against a variety of internalizing antigens expressed by malignant myeloid and lymphoid cells [2,3,17]. Recombinant constructs have been developed to incorporate modifications that optimize intracellular routing, reduce immunogenicity and eliminate the binding activity of the native toxin. To date, the majority of phytotoxin-containing immunotoxins have been tested only in preclinical models. In small Phase I clinical trials, blocked ricin or deglycosylated ricin A-chains conjugated with antiCD7, anti-CD19 or anti-CD22 antibodies have shown some activity against relapsed/aggressive T- or B-cell NHLs [3,17]. However, liver toxicity, vascular leak syndrome and other adverse effects have prevented the use of these agents at higher doses. In addition, patients frequently formed antibodies against the immunotoxin. In comparison, a recent small study demonstrated that combinations of anti-CD3 and antiCD7 ricin A-chain immunotoxins were tolerable and effective in ameliorating graft-versus-host disease, with significant reductions in circulating T cells [25].These combined agents might, therefore, be useful against T-cell malignancies. The bacterial toxins diphtheria toxin and Pseudomonas exotoxin A (PEA) have also been used to target hematological malignancies [17,26,27]. These agents inhibit protein synthesis by ADP ribosylation activity, which inactivates elongation factor-2. A recombinant anti-CD25 Fv fragment fused with truncated PEA has demonstrated clinical activity in a small number of advanced T- and B-cell malignancies [26]. Of note, systemic side-effects and hepatotoxicity were mild and only 1 out of 35 patients http://tmm.trends.com
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developed antibodies, thus allowing administration of multiple doses. Similarly, multiple doses of a PEA-fused anti-CD22 Fv fragment were generally well tolerated in patients with chemotherapyresistant hairy cell leukemia [27]. More importantly, 11 out of 16 patients in this dose-escalation trial achieved a complete response. Antibody–drug conjugates
Conventional chemotherapy agents have been linked to antibodies as a means to localize drug to the site of the tumor. Immunoconjugates containing methotrexate, 5-fluorouracil, cytarabine, chlorambucil, melphalan, doxorubicin, idarubicin or bleomycin have been tested in in vitro and experimental animal models [2,16]. Unfortunately, early chemical conjugation processes often compromised drug potency and antibody avidity. Subsequently, branched linkers were developed to minimize steric hinderance between the drug and antibody, and to increase the number of drug molecules carried by each antibody [28]. An alternative approach has been to join drug-containing liposomes to antibodies. In murine tumor xenotransplant models, such immunoliposomes exhibit improved pharmacokinetics, enhanced delivery of drug to the tumor, and greater cytotoxicity, compared with liposomal drug alone or drug given with unmodified antibodies [29]. More recently, potent anticancer drugs with unacceptable toxicity when administered systemically have been linked to antibodies against internalizing tumor antigens. For example, the tyrosine kinase inhibitor genistein has been combined with an anti-CD19 antibody. This agent induces cell death by inhibiting tyrosine kinasemediated anti-apoptotic signals derived from the membrane complex CD19–LYN. In a small clinical trial, this immunoconjugate was well tolerated and three responses were seen among patients with treatment-refractory B-cell NHLs and leukemias; however, anti-mouse antibodies also developed in a third of patients [30]. N-acetyl-gamma calicheamicin is another compound with prohibitive systemic toxicity that can be safely administered as a component of the antiCD33 conjugate, gemtuzumab ozogamicin [19]. Calicheamicins induce cell death at sub-picomolar concentrations by cleaving double-stranded DNA at sequence-specific sites [31]. Gemtuzumab ozogamicin utilizes a stable bifunctional linker that prevents dissociation of calicheamicin except within the acidic lysosomal environment. Normal myelomonocytic progenitors and monocytes express CD33, whereas pluripotent hematopoietic stem cells are CD33−. Thus, treatment with gemtuzumab ozogamicin causes transient marrow suppression but does not ablate hematopoiesis. Two doses of gemtuzumab ozogamicin, given 14 days apart, induced a clinical remission in 30% of 142 adult patients with relapsed AML [19]. Infusion-related symptoms and transient
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liver function abnormalities constituted the major non-hematological side-effects. Formation of anticonjugate or anti-mouse antibodies was rare. This agent is currently being studied as part of combined therapy for AML and as monotherapy for other myeloid malignancies (e.g. high-risk myelodysplasia). The safety and efficacy of gemtuzumab ozogamicin, the first antibody chemotherapy conjugate to be approved by the FDA, renewed hope that similar approaches using other novel drug conjugates will also be beneficial. Potential mechanisms of resistance to antibody-based therapies
Malignant hematopoietic cells might escape the cytotoxic effects of antibody-directed therapies through several mechanisms. High interstitial pressure within large tumor masses limits the delivery and/or diffusion of complete antibody molecules or conjugates. Alternatively, antiglobulin or anticonjugate immune responses alter the biodistribution and pharmacokinetics of the agents during subsequent treatments. To overcome these roadblocks, engineered Ig fragments and humanized antibodies have been developed. With respect to the tumor cell itself, decreased antigen density, increased surface modulation and altered internalization kinetics could limit or prevent activation of antibody-induced cytotoxic mechanisms. If complement-dependent killing is important, it might also be antagonized by expression of membrane-bound complement regulators [10,32]. In rare cases, antigennegative tumor subclones have been shown to arise (or be selected) following treatment [33,34]. Antibody–drug conjugates might be affected by multidrug resistance (MDR) mechanisms that are active against conventional chemotherapy agents. For example, AML cell lines that overexpress the drug transporter P-glycoprotein (Pgp, the product of the MDR1 gene) are usually resistant to gemtuzumab ozogamacin, and Pgp antagonists reverse this resistance [35]. Furthermore, functional Pgp expression on blast cells is associated with inferior clinical responses to gemtuzumab ozogamicin in patients with relapsed AML [36]. Theoretically, other drug transporters and non-transporter resistance mechanisms, such as those mediating DNA repair and apoptosis, could compromise the effectiveness of drug- or toxin-containing immunoconjugates. Future directions
Several questions still need to be addressed to optimize antibody-directed therapies. First, can antibodies and immunoconjugates be better designed? Fully human mAbs, generated from transgenic mice that express the human H- and L-chain gene repertoire [37], could prove superior to chimeric and humanized antibodies in avoiding antiglobulin responses. To date, transgenic-mouse-derived human antibodies against the epidermal growth factor http://tmm.trends.com
receptor (EGFr) have shown high potency against EGFr-expressing solid tumor xenografts in athymic mice [38]; however, human studies have not yet been published. Phage- and ribosome-display technologies are facilitating the selection of antibody V regions with optimal binding affinities and pharmacokinetics [39]. These high-throughput methods could also help identify novel tumor-associated antigens by using a subtractive panning approach to select antibodies that react against tumor cells but do not react against the normal, nonmalignant cell counterpart [40]. Recombinant techniques allow the creation of site-specific mutations in variable gene sequences to ‘reshape’ the antigen-binding domains. In immunotoxin construction, genetic modifications of relevant sequences might further reduce immunogenicity and non-tumor toxicity. Second, can tumor-specific cytotoxicity be enhanced? Multispecific antibodies have been developed to improve the localization of immune effector cells or cytotoxic agents. In preclinical models, antitumor responses have been observed with bispecific antibodies that contain arms that activate T cells (anti-CD3 or anti-CD28), natural killer cells (anti-CD16) or myeloid effector cells (anti-CD64 or antibodies against other Fc receptors). Bispecific anti-CD16/CD30 and antiCD3/CD19 antibodies have been tested in small Phase I/II clinical trials; safety and some responses have so far been demonstrated [41,42]. Antibodydirected enzyme prodrug therapy is an alternative strategy that delivers an enzyme to the tumor microenvironment where it activates a systemically administered chemotherapy prodrug [2,43]. Novel toxins, such as the amphibian ribonuclease onconase [44], and high-energy α-emitting radioisotopes [24] offer theoretical advantages over the toxins and radionuclides currently in use. Adjunctive therapies, such as cytokines and lymphokines, have been used to augment host ADCC. Monensin and nigericin can potentiate the cytotoxicity of ricin-containing immunotoxins [45]. Finally, approaches currently under evaluation for solid tumors, such as antibodies against tumor microvascular endothelial cell antigens [46] or antibodies that block metabolically active receptors [47], could serve as useful models for the treatment of hematological malignancies. Third, are antibody-based treatments most effective as monotherapy or in combination with other modalities? Recently completed studies of rituximab plus conventional chemotherapy demonstrated superior responses compared with those achieved with either treatment alone [48–50]. Radiolabeled anti-CD20 antibodies are a valuable component of combination pretransplant conditioning therapy for NHL [12]. Ongoing trials are similarly addressing the efficacy and safety of gemtuzumab ozogamacin in combination with standard anti-leukemic agents and in stem cell
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transplant regimens. As a further extension of this principle, combinations of antibodies with other unmodified antibodies or with immunotoxins appear to be more effective in in vitro and murine tumor models than the single agents [51–53]. The pace of discovery and clinical applications for antibody-directed therapies are rapidly accelerating. This flourishing activity, often References 1 Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497 2 Farah, R.A. et al. (1998) The development of monoclonal antibodies for the therapy of cancer. Crit. Rev. Eukaryotic Gene Expr. 8, 321–356 3 Maloney, D.G. and Press, O.W. (1998) Newer treatments for non-Hodgkin’s lymphoma: monoclonal antibodies. Oncology 12, 63–76 4 Vose, J.M. (2001) Immunotherapy for nonHodgkin’s lymphoma. Oncology 15, 141–147 5 Ruffner, K.L. and Matthews, D.C. (2000) Current uses of monoclonal antibodies in the treatment of acute leukemia. Semin. Oncol. 27, 531–539 6 Brown, S.L. et al. (1989) Antiidiotype antibody therapy of B-cell lymphoma. Semin. Oncol. 16, 199–210 7 Vuist, W.M. et al. (1994) Lymphoma regression induced by monoclonal anti-idiotypic antibodies correlates with their ability to induce Ig signal transduction and is not prevented by tumor expression of high levels of bcl-2 protein. Blood 83, 899–906 8 Grillo-Lopez, A.J. et al. (1999) Overview of the clinical development of rituximab: first monoclonal antibody approved for the treatment of lymphoma. Semin. Oncol. 26, 66–73 9 Shan, D. et al. (2000) Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells. Cancer Immunol. Immunother. 48, 673–683 10 Golay, J. et al. (2000) Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis. Blood 95, 3900–3908 11 Milpied, N. et al. (2000) Humanized anti-CD20 monoclonal antibody (Rituximab) in post transplant B-lymphoproliferative disorder: a retrospective analysis on 32 patients. Ann. Oncol. 11, Suppl. 1, 113–116 12 Press, O.W. et al. (2000) A phase I/II trial of iodine-131-tositumomab (anti-CD20), etoposide, cyclophosphamide, and autologous stem cell transplantation for relapsed B-cell lymphomas. Blood 96, 2934–2942 13 Witzig, T.E. et al. (1999) Phase I/II trial of IDEC-Y2B8 radioimmunotherapy for treatment of relapsed or refractory CD20(+) B-cell non-Hodgkin’s lymphoma. J. Clin. Oncol. 17, 3793–3803 14 Matthews, D.C. et al. (1999) Phase I study of (131)I-anti-CD45 antibody plus cyclophosphamide and total body irradiation for advanced acute leukemia and myelodysplastic syndrome. Blood 94, 1237–1247 15 Flynn, J.M. and Byrd, J.C. (2000) Campath-1H monoclonal antibody therapy. Curr. Opin. Oncol. 12, 574–581 16 Trail, P.A. and Bianchi, A.B. (1999) Monoclonal antibody drug conjugates in the treatment of cancer. Curr. Opin. Immunol. 11, 584–588
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conducted by partners from industry and academia, has been fueled by several recent clinical successes. Antibody-based approaches currently offer a versatile, potent and, in many cases, less-toxic alternative to conventional treatments. Although effective as single agents, the greatest benefit of antibodies and immunoconjugates might be as components of combination or adjuvant therapies.
17 Kreitman, R.J. (1999) Immunotoxins in cancer therapy. Curr. Opin. Immunol. 11, 570–578 18 van Horssen, P.J. et al. (1995) Cytotoxic potency of CD22-ricin A depends on intracellular routing rather than on the number of internalized molecules. Scand. J. Immunol. 41, 563–569 19 Sievers, E.L. et al. (2001) Efficacy and safety of gemtuzumab ozogamicin in patients with CD33-positive acute myeloid leukemia in first relapse. J. Clin. Oncol. 19, 3244–3254 20 Nourigat, C. et al. (1990) Treatment of lymphoma with radiolabeled antibody: elimination of tumor cells lacking target antigen. J. Natl Cancer. Inst. 82, 47–50 21 Press, O.W. et al. (1996) Comparative metabolism and retention of iodine-125, yttrium-90, and indium-111 radioimmunoconjugates by cancer cells. Cancer Res. 56, 2123–2129 22 DeNardo, S.J. et al. (1999) A new era for radiolabeled antibodies in cancer? Curr. Opin. Immunol. 11, 563–569 23 Kaminski, M.S. et al. (2000) Radioimmunotherapy with iodine (131)I tositumomab for relapsed or refractory B-cell non-Hodgkin lymphoma: updated results and long-term follow-up of the University of Michigan experience. Blood 96, 1259–1266 24 Sgouros, G. et al. (1999) Pharmacokinetics and dosimetry of an alpha-particle emitter labeled antibody: 213Bi-HuM195 (anti-CD33) in patients with leukemia. J. Nucl. Med. 40, 1935–1946 25 van Oosterhout, Y.V. et al. (2000) A combination of anti-CD3 and anti-CD7 ricin A-immunotoxins for the in vivo treatment of acute graft versus host disease. Blood 95, 3693–3701 26 Kreitman, R.J. et al. (2000) Phase I trial of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) in patients with hematologic malignancies. J. Clin. Oncol. 18, 1622–1636 27 Kreitman, R.J. et al. (2001) Efficacy of the anti-CD22 recombinant immunotoxin BL22 in chemotherapy-resistant hairy-cell leukemia. New Engl. J. Med. 345, 241–247 28 King, H.D. et al. (1999) Monoclonal antibody conjugates of doxorubicin prepared with branched linkers: A novel method for increasing the potency of doxorubicin immunoconjugates. Bioconjugate Chem. 10, 279–288 29 Tseng, Y.L. et al. (1999) Sterically stabilized anti-idiotype immunoliposomes improve the therapeutic efficacy of doxorubicin in a murine B-cell lymphoma model. Int. J. Cancer 80, 723–730 30 Uckun, F.M. et al. (1999) Treatment of therapy-refractory B-lineage acute lymphoblastic leukemia with an apoptosis-inducing CD19-directed tyrosine kinase inhibitor. Clin. Cancer Res. 5, 3906–3913 31 Zein, N. et al. (1988) Calicheamicin gamma 1I: an antitumor antibiotic that cleaves doublestranded DNA site specifically. Science 240, 1198–1201
32 Maio, M. et al. (1998) Structure, distribution, and functional role of protectin (CD59) in complementsusceptibility and in immunotherapy of human malignancies. Int. J. Oncol. 13, 305–318 33 Davis, T.A. et al. (1999) Therapy of B-cell lymphoma with anti-CD20 antibodies can result in the loss of CD20 antigen expression. Clin. Cancer Res. 5, 611–615 34 Meeker, T. et al. (1985) Emergence of idiotype variants during treatment of B-cell lymphoma with anti-idiotype antibodies. New Engl. J. Med. 312, 1658–1665 35 Naito, K. et al. (2000) Calicheamicin-conjugated humanized anti-CD33 monoclonal antibody (gemtuzumab zogamicin, CMA-676) shows cytocidal effect on CD33-positive leukemia cell lines, but is inactive on P-glycoprotein-expressing sublines. Leukemia 14, 1436–1443 36 Linenberger, M.L. et al. (2001) Multidrugresistance phenotype and clinical responses to gemtuzumab ozogamicin. Blood 98, 988–994 37 Green, L.L. et al. (1994) Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs. Nat. Genet. 7, 13–21 38 Yang, X.D. et al. (2001) Development of ABX-EGF, a fully human anti-EGF receptor monoclonal antibody for cancer therapy. Crit. Rev. Oncol. Hematol. 38, 17–23 39 Dall’Acqua, W. and Carter, P. (1998) Antibody engineering. Curr. Opin. Struct. Biol. 8, 443–450 40 Ridgeway, J.B. et al. (1999) Identification of a human anti-CD55 single-chain Fv by subtractive panning of a phage library using tumor and nontumor cell lines. Cancer Res. 59, 2718–2723 41 Hartmann, F. et al. (2001) Anti-CD16/CD30 bispecific antibody treatment for Hodgkin’s disease: role of infusion schedule and costimulation with cytokines. Clin. Cancer Res. 7, 1873–1881 42 Manzke, O. et al. (2001) Locoregional treatment of low-grade B-cell lymphoma with CD3×CD19 bispecific antibodies and CD28 costimulation: I. clinical phase I evaluation. Int. J. Cancer 91, 508–515 43 Haisma, H.J. et al. (1998) Construction and characterization of a fusion protein of single-chain anti-CD20 antibody and human β-glucuronidase for antibody-directed enzyme prodrug therapy. Blood 92, 184–190 44 Newton, D.L. et al. (2001) Potent and specific antitumor effects of an anti-CD22-targeted cytotoxic ribonuclease: potential for the treatment of non-Hodgkin lymphoma. Blood 97, 528–535 45 van Horssen, P.J. et al. (1999) Influence of cytotoxicity enhancers in combination with human serum on the activity of CD22recombinant ricin A against B cell lines, chronic and acute lymphocytic leukemia cells. Leukemia 13, 241–249
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46 Ran, S. et al. (1998) Infarction of solid Hodgkin’s tumors in mice by antibody-directed targeting of tissue factor to tumor vasculature. Cancer Res. 58, 4646–4653 47 Fan, Z. and Mendelsohn, J. (1998) Therapeutic application of anti-growth factor receptor antibodies. Curr. Opin. Oncol. 10, 67–73 48 Czuczman, M.S. et al. (1999) Treatment of patients with low-grade B-cell lymphoma with the combination of chimeric anti-CD20 monoclonal antibody and CHOP chemotherapy. J. Clin. Oncol. 17, 268–276
49 Vose, J.M. et al. (2001) Phase II study of rituximab in combination with CHOP chemotherapy in patients with previously untreated, aggressive nonHodgkin’s lymphoma. J. Clin. Oncol. 19, 389–397 50 Coiffier, B. et al. (2000) Mabthera (rituximab) plus CHOP is superior to CHOP alone in elderly patients with diffuse large B-cell lymphoma (DLCL): interim results of a randomized GELA trial. Blood 96, 223a 51 Benoit, N.E. and Wade, W.F. (1996) Increased inhibition of proliferation of human B cell lymphomas following ligation of CD40, and either
CD19, CD20, CD95 or surface immunoglobulin. Immunopharmacology 35, 129–139 52 Flavell, D.J. et al. (1997) Systemic therapy with 3BIT, a triple combination cocktail of anti-CD19, -CD22, and -CD38-saporin immunotoxins, is curative of human B-cell lymphoma in severe combined immunodeficient mice. Cancer Res. 57, 4824–4829 53 Herrera, L. et al. (2000) Immunotoxins against CD19 and CD22 are effective in killing precursorB acute lymphoblastic leukemia cells in vitro. Leukemia 14, 853–858
Molecular basis of resistance to azole antifungals Antonella Lupetti, Romano Danesi, Mario Campa, Mario Del Tacca and Steven Kelly The increased incidence of invasive mycoses and the emerging problem of antifungal drug resistance has prompted investigations of the underlying molecular mechanisms, particularly for the azole compounds central to current therapy. The target site for the azoles is the ERG11 gene product, the α-demethylase, which is part of the ergosterol cytochrome P450 lanosterol 14α biosynthetic pathway. The resulting ergosterol depletion renders fungal cells vulnerable to further membrane damage. Development of azole resistance in fungi may occur through increased levels of the cellular target, upregulation of genes controlling drug efflux, alterations in sterol synthesis and decreased affinity of azoles for the cellular target. Here, we review the adaptative changes in fungi, in particular Candida albicans, in response to inhibitors of ergosterol biosynthesis. The molecular mechanisms of azole resistance might help in devising more effective antifungal therapies. Antonella Lupetti Mario Campa Dept of Experimental Pathology, Medical Biotechnologies, Infectious Diseases and Epidemiology, University of Pisa, 35–39, Via S. Zeno, 56127 Pisa, Italy. Romano Danesi* Mario Del Tacca Division of Pharmacology and Chemotherapy, Dept of Oncology, Transplants and Advanced Technologies in Medicine, University of Pisa, 55, Via Roma, 56126 Pisa, Italy. *e-mail: [email protected] Steven Kelly Wolfson Laboratory of P450 Biodiversity, Institute of Biological Sciences, Edward Llwyd Building, The University of Wales, Aberystwyth, Ceredigion, UK SY23 3DA.
Mucosal and invasive opportunistic fungal infections have increased during the past two decades as a consequence of the rising number of immunocompromised hosts, such as HIV-infected individuals, transplant recipients and patients given immunosuppressive therapy or broad-spectrum antibiotics. Besides the most commonly isolated Candida and Aspergillus species, new emerging opportunistic fungi include Mucor, Fusarium, Rhizomucor and Absidia species. In addition, the spectrum of infective fungi includes Saccharomyces cerevisiae. Another factor that contributes to the severity of opportunistic infections is the development of resistance to antifungal agents. Indeed, molecular alterations often result in the development of drugresistant Candida albicans and other fungi from an initially susceptible population [1]. The aim of this paper is to review the molecular determinants of antifungal resistance, the understanding of which could provide new perspectives for improved treatment of invasive fungal infections confronting high-risk patients. Current treatment of systemic mycoses is mainly based on the use of polyenes http://tmm.trends.com
(e.g. amphotericin B) and azoles, such as triazoles (e.g. itraconazole and fluconazole) (Fig. 1). Ergosterol, the major component of fungal membrane, is the target of polyene antibiotics and the ergosterol biosynthesis pathway is the target of azole derivatives. Ergosterol contributes to a variety of cellular functions, including fluidity and integrity of the membrane and the proper function of membrane-bound enzymes such as proteins associated with nutrient transport and chitin synthesis. Ergosterol is also a major component of secretory vesicles in S. cerevisiae, and has an important role in mitochondrial respiration; indeed, mutants defective in ergosterol biosynthesis and yeasts treated with azole compounds are induced to a respiratory-deficient ‘petite’ status at a high frequency [2]. This review focuses on the various mechanisms of azole resistance in C. albicans. Polyenes
Polyenes target ergosterol in fungal membranes. They are fungicidal agents used in short treatment regimens due to associated toxicity, which may account for the low incidence of resistance encountered. Consistent with this mode of action, amphotericin B-resistant Candida strains often have a marked decrease in ergosterol content compared with amphotericin B-susceptible control isolates [1]. Resistance in clinical isolates of Cryptococcus neoformans was also associated with a mutation preventing ergosterol biosynthesis at the C8-isomerization step [3]. Resistance may also be associated with altered phospholipids or increased catalase activity with decreasing susceptibility to oxidative damage [4]. Other drugs
Flucytosine, which inhibits cellular DNA and RNA synthesis, is mainly used in combination therapy as
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Antibody-directed therapies for hematological malignancies Michael L. Linenberger, David G. Maloney and Irwin D. Bernstein Malignant hematopoietic cells express lineage-restricted antigens that serve as suitable targets for antibody-directed therapy. Although several highly specific, potent and relatively nontoxic, engineered antibodies, immunoglobulin fragments and antibody conjugates have been developed, only three have gained approval for clinical use. Of these, a chimeric mouse–human anti-CD20 antibody has yielded the most impressive clinical results. Encouraging data with the other approved antibodies, and with agents in clinical trials, suggest that rational antibody design will generate effective products for several different hematological malignancies. Despite these advances, significant challenges remain in the identification of optimal cellular targets, antibody forms and treatment schedules for therapeutic applications.
Michael L. Linenberger* David G. Maloney Clinical Research Division, Fred Hutchinson Cancer Research Center, and Dept of Medicine, University of Washington, Seattle, WA, USA. *e-mail: [email protected] Irwin D. Bernstein Clinical Research Division, Fred Hutchinson Cancer Research Center, and Dept of Pediatrics, University of Washington, Seattle, WA, USA.
Since the seminal description of murine hybridoma methodology in 1975 [1], monoclonal antibodies (mAbs) have been developed as diagnostic tools and therapeutic agents for hematological malignancies [2–5]. Initially, rodent mAbs were used to identify useful tumor target antigens, characterize their function and define important biological variables, such as surface density, internalization kinetics and shedding. Subsequent in vivo studies with unmodified antibodies demonstrated the critical roles of immunoglobulin (Ig) structure in eliciting the desirable effect of host anti-tumor activation and the undesirable effect of host antiglobulin response. More recently, genetic engineering has enabled the development of chimeric or humanized antibodies, Ig fragments and multivalent antibodies, in order to avoid immunogenicity and augment anti-tumor potency. It has also been possible to link antibodies and Ig fragments with toxins, radionuclides, enzymes, chemotherapeutic agents, cytokines and drug-containing liposomes for therapeutic purposes. This review will summarize the molecular and biochemical advances leading to the rational design of antibody-based treatment approaches for hematological malignancies. Examples of relevant current strategies will be discussed to illustrate the key elements of antibody construction, target selection, chemical modification, therapeutic applications and mechanisms of resistance. For more-comprehensive summaries of individual antibody therapies for specific disorders the reader is referred to excellent recent reviews [3–5]. Antibody design
Recombinant technologies have facilitated the development of hybrid, truncated and conjugated antibodies [2]. These reagents often avoid the limitations of unmodified rodent mAbs, such as: http://tmm.trends.com
(1) immunogenicity (formation of antiglobulin antibodies); (2) suboptimal avidity to the antigen target; (3) inefficiency at activating human complement; (4) limited potential to activate antibodydependent cell-mediated cytotoxicity (ADCC) mechanisms; and (5) unfavorable pharmacokinetics. Chimeric antibodies, which consist of monoclonal antigen-binding (Fab) or variable (Fv) domains combined with human constant (Fc) regions, are less immunogenic and incorporate Fc isotypes that optimally activate host effector mechanisms (usually of the IgG1 or IgG3 subclasses). Similar advantages derive from humanized antibodies, which contain the three antigen-binding loops within the complementarity-determining regions (CDRs) of the rodent mAb heavy (H)- and light (L)-chain V domains grafted onto the framework regions of the human V and C domains (Fig. 1). Recombinant antibody fragments, including Fv fragments (noncovalently associated H- and L-chain V domains) and single-chain Fv (scFv) fragments (H- and L-chain V domains covalently linked by short polypeptide linkers) (Fig. 1), are used as vehicles to deliver toxins and drug conjugates. The small size (roughly 50 kDa for scFv fragments) facilitates tumor tissue penetration with more-favorable biodistribution and plasma clearance than full-length antibodies. However, because of their more-rapid clearance, very high doses are required, thereby detracting from their clinical utility. A variety of more-novel antibody forms have been developed to improve their usefulness as cytocidal and/or imaging agents. These include multivalent antibodies (consisting of two or more different antigen-binding arms), bispecific mAbs (consisting of two complete antibodies, against different targets, joined by a chemical crosslinker) and bispecific F(ab)′2 fragments. Diabodies, which are linked recombinant scFv fragments (Fig. 1), might be either bivalent and monospecific (i.e. both Fv arms recognize the same antigen) or bispecific (i.e. each Fv arm recognizes a different antigen). Although these constructs should optimize specificity and avoid the problem of antiglobulin formation, they have not yet been shown to be superior to conventional therapies in early clinical trials. Target selection
Clonal malignant cells of lymphoid and myeloid origin commonly express lineage-restricted surface
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Fig. 1. Evolution of monoclonal antibodies (mAbs). Initially, mAbs were derived from rodent hybridoma cell lines that were isolated and propagated in tissue culture. Subsequently, recombinant technologies allowed the selection, modification and largescale expression of rodent sequences encoding the antigen-binding regions. Chimeric or humanized antibodies, which incorporate human constant domains either with the rodent variable (V) regions or with the three antigen-binding loops of the complementaritydetermining regions (CDRs), respectively, were developed to reduce immunogenicity and optimize host effector mechanisms. Heavy and light chain V-region fragments (Fv) and singlechain fragments (scFv; which consist of the two chains joined by a polypeptide linker) were developed as smaller delivery vehicles with more-favorable biodistribution than fulllength antibodies. Although these fragments lack Fc regions, and therefore cannot activate host effector functions, they can be conjugated to toxins, drugs or radionuclides. They might also be combined to form diabodies, which can be either monospecific or bispecific (i.e. each Fv arm recognizes a different antigen). Fab domains indicate monoclonal antigen-binding fragment. Abbreviations: Amp, ampicillin; Ig, immunoglobulin.
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Ig variable sequences CDRs
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differentiation antigens, signaling proteins, growth factor receptors and, in the case of B-cell malignancies, Ig molecules. These antigens are similar to those expressed by the non-malignant precursors from which they were derived and are potential antigens for antibody-directed therapy. However, among these, the V-region epitopes of the surface idiotype Ig represent the only true tumor-specific targets [6]. To date, the majority of therapeutic reagents have been generated against tumor-associated antigens. These antigens might be either preferentially expressed or overexpressed by the tumor cells and/or they might play a more critical function in the malignant cells than in the normal cell counterparts. Because these antigens are also expressed by at least some normal myeloid or lymphoid cells, the success of the antibody treatment relies on the ability of the normal cells to either tolerate ‘collateral damage’ or to be reconstituted by antigen-negative precursors. The ideal surface antigen is found at high density on the tumor cells (i.e. >104–105 binding sites per cell) with limited expression on normal counterparts and with little or no circulating cell-free antigen. The antigenic epitope should have minimal variations or heterogeneity of expression among tumor cell subclones. Target molecules that serve critical host–cell functions are desirable http://tmm.trends.com
because immunological ‘escape’, due to loss of expression, is less likely to occur. In some cases, such as with anti-idiotype antibodies, antibody binding, and perhaps surface hypercrosslinking, activates signaling pathways that facilitate cell death mechanisms [7]. Non-internalized surface molecules
Antigen–antibody complexes that are slowly or minimally internalized by the target cell are optimal for approaches that rely on extracellular mechanisms of cytotoxicity. These include: enzymelinked antibodies that activate a prodrug in the tumor microenvironment; bispecific or multivalent antibodies that crosslink the tumor cell to local immune effector cells; full-length antibodies that activate ADCC and/or complement-dependent cytotoxicity (CDC) through the Fc domain; and certain radioimmunoconjugates that would be rapidly degraded if internalized (Fig. 2). Clinically relevant examples of cell-surface molecules with low rates of antibody-mediated internalization or modulation include the cluster of differentiation (CD) antigens CD20, CD45 and CD52. CD20, a 35 kDa membrane phosphoprotein that functions as a Ca2+ channel and is involved in normal B-cell activation and proliferation, is expressed on
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(a) Radioimmunoconjugates
(c) Immunotoxins
(b) Chimeric antibodies
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Fig. 2. Cytotoxic mechanisms of antibody-based treatments. Noninternalizing antigen targets are ideal for most radioimmunoconjugates and full-length antibodies that rely on Fc-mediated activation of host effector functions. (a) Radioimmunoconjugates containing iodine-131(131I ) emit β-radiation over many cell diameters, and therefore can kill inaccessible or antigen-negative cells at a distance. (b) Fc domains extending from antibody-coated tumor cells might activate antibody-dependent cell-mediated cytotoxicity (ADCC), through Fc receptors on natural killer (NK) cells or macrophages. Alternatively, complement-dependent cytotoxicity (CDC) might be initiated by the binding of complement protein C1 to Fc domains. In addition, some noninternalizing antibodies might activate receptor-mediated signaling pathways that inhibit growth or induce cell death. Internalizing antigens are required for intracellular delivery of toxins or antitumor drugs. (c) Plant and bacterial immunotoxins traffic through endosomes or the Golgi and endoplasmic reticulum. Free toxin is then released to the cytoplasm where it inhibits protein synthesis and ultimately causes cell death. (d) Antibody–drug conjugates also undergo dissociation after internalization. As an example, gemtuzumab ozogamicin, which consists of calicheamicin conjugated to an anti-CD33 antibody by a stable bifunctional linker, undergoes hydrolysis in the acidic environment of the lysosome. After free calicheamicin is released, it translocates to the nucleus where it cleaves double-stranded DNA and induces cell death.
the majority of B-cell low-grade and aggressive non-Hodgkin’s lymphomas (NHLs). A chimeric mouse–human anti-CD20 antibody, rituximab (Rituxan, Genentech, Inc.), was approved by the Food and Drug Administration (FDA) in 1997 for treatment of relapsed and refractory follicular NHL [8]. http://tmm.trends.com
Rituximab inhibits proliferation and induces cytotoxicity by activating CDC, ADCC and, to a limited extent, apoptosis [9,10]. Since approval, this agent has been used to treat several different B-lymphoid malignancies that express variable amounts of surface CD20. Particularly noteworthy is the efficacy of rituximab against post-transplant B-cell NHL induced by Epstein–Barr virus (EBV) [11]. Other murine anti-CD20 mAbs have been conjugated with iodine-131 (131I) or yttritum-90 (90Y) (both β-emitting radionuclides) to deliver tumoricidal doses of radiation to sites of residual lymphoma [12,13]. Similarly, radioimmunoconjugates against CD45, a noninternalizing pan-hematopoietic antigen, have been used to augment high-dose chemotherapy and/or radiotherapy before hematopoietic stem cell transplantation for acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and myelodysplasia [14]. CD52, a 12 amino acid glycosylphosphatidylinositol (GPI)-linked glycoprotein that might function as a cytoadhesion molecule, is expressed by some B- and T-cell malignancies, as well as by normal lymphocytes and monocytes. Engagement of CD52 by a humanized anti-CD52 antibody (Campath-1H) appears to trigger CDC and ADCC in vivo, and induces apoptosis in some malignant lymphoid cell types [15]. The anti-CD52
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antibody alemtuzumab (Campath, Berlex, Inc.) was approved by the FDA in May 2001 for treatment of chemotherapy-resistant B-cell chronic lymphocytic leukemia (B-CLL). Internalized surface molecules
Several therapeutic antibodies recognize cell-surface antigens that are internalized after binding. Internalization is required for antibody conjugates that are designed to deliver toxins, chemotherapy drugs or, less commonly, radionuclides to the cytoplasm. The intracellular pathways followed by these antibodies and conjugates have not been fully characterized in most cases. In general, the antigen–antibody complexes coalesce on the surface, undergo endocytosis, and ultimately translocate into lysosomes. Proteolytic processing occurs during trafficking in endosomes, lysosomes or, in some cases, the Golgi and endoplasmic reticulum (ER). Importantly, the antigen–antibody complexes are dissociated and the conjugate moieties are released, allowing them to travel to the cytoplasm or nucleus to induce a cytopathic effect [2,3,16,17] (Fig. 2). Rates and efficiencies of internalization vary, depending on the antigen and the phenotypic features of the host cell. However, the cytotoxic effectiveness of toxin- or chemotherapy-conjugated antibodies often relies more on efficient intracellular processing and the intrinsic potency of the conjugate rather than on the rate of internalization [18]. Gemtuzumab ozogamicin, a recombinant humanized anti-CD33 antibody linked to a derivative of the anti-tumor antibiotic calicheamicin (Mylotarg, Wyeth-Ayerst Laboratories), is the only antibody–drug conjugate approved by the FDA. It is indicated for the treatment of relapsed AML in elderly patients [19]. This agent rapidly saturates CD33 sites, an internalizing antigen commonly found on AML cells, and is quickly endocytosed. After internalization, calicheamicin is released from the lysosomes. It subsequently enters the nucleus where it cleaves double-stranded DNA and induces cell death (Fig. 2). Radioimmunoconjugates
Most malignant hematopoietic cells are susceptible to the cytotoxic effects of radiation, and radionuclides have therefore been extensively utilized in antibody conjugates [2,3,5]. These agents are constructed by covalently binding the radioisotope directly to the antibody or by crosslinking through a chelating molecule or chemical linker. The linker design and methodology are carefully chosen to optimize radiolabeling efficiency, stability, biodistribution, pharmacokinetics and/or intracellular retention (for internalizing antibodies). The cytotoxic efficacy of a radioimmunoconjugate depends on the kinetics of antibody localization and retention of the radionuclide. These variables relate, in turn, to: (1) the size and vascularity of the tumor; (2) the http://tmm.trends.com
density, heterogeneity and modulation of the antigen; and (3) the size, specificity and stability of the radioimmunoconjugate. The path length, half-life and linear energy transfer of the radioisotope are also important. β-emitting isotopes such as copper-67 (67Cu), 131I and 90Y have longer path lengths (0.6, 0.8 and 5.3 mm, respectively) and longer half-lifes (2.5, 8.1 and 2.5 days, respectively) than the α-emitting isotopes bismuth-212 and astatine-211 (path lengths of 0.06 mm and half-lifes of 1 and 7 h, respectively). Conversely, the particulate energy of α-emitters are 2.5–10-fold greater than the β-emitters. Because of the long path length, β-emissions could theoretically kill neighboring tumor cells that are antigennegative or inaccessible to antibody (Fig. 2) [20]. 131I and 67Cu, unlike 90Y, also emit γ-radiation, which facilitates imaging and dosimetry determinations [3]. 90Y or 67Cu are the isotopes of choice with internalized antibodies because they are retained and maintain activity much longer than 131I, which is rapidly dehalogenated within lysosomes [21,22]. As a consequence of their availability, stability and ease of manipulation, 131I and 90Y have been utilized in most constructs. Several clinical trials have studied polyclonal, monoclonal or humanized antibodies conjugated with 131I or 90Y [3,5,22]. In all cases, dose-limiting toxicity to normal tissues remains the major safety concern. The most successful approach to date involves the use of 131I-labeled anti-CD20 antibodies for the treatment of relapsed B-cell NHL. Anti-CD20 radioimmunoconjugates have been used alone [23] or with additional myeloablative chemotherapy [12], with or without subsequent hematopoietic stem cell transplantation. The efficacy in relapsed patients appears to be at least equivalent to, if not superior to, conventional therapies [12,23]. After demonstrating efficacy in relapsed patients, 131I-conjugated anti-CD20 is now being studied as part of initial therapy for previously untreated patients and for patients that relapse after treatment with rituximab. Similar encouraging preliminary results have been reported with 90Y-conjugated anti-CD20 [13]. In other clinical studies of B-cell NHL, radiolabeled antibodies against CD21, CD22, CD37, HLA-DR or idiotypic Ig have been tested [3]. 131I-labeled anti-CD45 has shown promise as an adjunctive therapy in stem cell transplant approaches for acute leukemia [14]. 131I- and 90Y-conjugated polyclonal antiferritin antibodies were found to be active against relapsed NHL [3]; however, this approach was not superior to available conventional salvage treatment options. Radioimmunoconjugates with anti-CD25 or anti-CD5 have been used to treat T-cell malignancies [3], whereas radiolabeled anti-CD33 has been investigated in AML [5,24]. Although some clinical effects have been documented, the utility of these
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agents remain undefined. To date, only a few α-emitting radioimmunoconjugates have been developed for hematological malignancies and only one has been tested for safety in patients [24]. Immunotoxins
Immunotoxins consist of complete antibodies or scFv fragments fused to plant- or bacteria-derived catalytic proteins [17]. The toxins currently in use are exceedingly potent, potentially requiring only one molecule to induce cell death. Because of this potency and native host-cell binding ability, they can only be safely used in modified forms with targeted approaches. Immunotoxins are generated by either chemically linking the modified toxin to the antibody (using disulfide bond chemistry or unique linker construction) or by splicing the Fv and toxin genes into a vector and expressing the recombinant product in bacteria. After internalization, the toxin is released from the antibody during trafficking through the endosome or Golgi and ER (lysosomal acidity degrades the toxin). Intracytoplasmic toxin then interferes with protein synthesis, ultimately causing apoptotic cell death (Fig. 2). Plant phytotoxins, including ricin, saporin, gelonin, bryodin and pokeweed antiviral protein, are ribosomeinactivating proteins. They have been combined with antibodies against a variety of internalizing antigens expressed by malignant myeloid and lymphoid cells [2,3,17]. Recombinant constructs have been developed to incorporate modifications that optimize intracellular routing, reduce immunogenicity and eliminate the binding activity of the native toxin. To date, the majority of phytotoxin-containing immunotoxins have been tested only in preclinical models. In small Phase I clinical trials, blocked ricin or deglycosylated ricin A-chains conjugated with antiCD7, anti-CD19 or anti-CD22 antibodies have shown some activity against relapsed/aggressive T- or B-cell NHLs [3,17]. However, liver toxicity, vascular leak syndrome and other adverse effects have prevented the use of these agents at higher doses. In addition, patients frequently formed antibodies against the immunotoxin. In comparison, a recent small study demonstrated that combinations of anti-CD3 and antiCD7 ricin A-chain immunotoxins were tolerable and effective in ameliorating graft-versus-host disease, with significant reductions in circulating T cells [25].These combined agents might, therefore, be useful against T-cell malignancies. The bacterial toxins diphtheria toxin and Pseudomonas exotoxin A (PEA) have also been used to target hematological malignancies [17,26,27]. These agents inhibit protein synthesis by ADP ribosylation activity, which inactivates elongation factor-2. A recombinant anti-CD25 Fv fragment fused with truncated PEA has demonstrated clinical activity in a small number of advanced T- and B-cell malignancies [26]. Of note, systemic side-effects and hepatotoxicity were mild and only 1 out of 35 patients http://tmm.trends.com
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developed antibodies, thus allowing administration of multiple doses. Similarly, multiple doses of a PEA-fused anti-CD22 Fv fragment were generally well tolerated in patients with chemotherapyresistant hairy cell leukemia [27]. More importantly, 11 out of 16 patients in this dose-escalation trial achieved a complete response. Antibody–drug conjugates
Conventional chemotherapy agents have been linked to antibodies as a means to localize drug to the site of the tumor. Immunoconjugates containing methotrexate, 5-fluorouracil, cytarabine, chlorambucil, melphalan, doxorubicin, idarubicin or bleomycin have been tested in in vitro and experimental animal models [2,16]. Unfortunately, early chemical conjugation processes often compromised drug potency and antibody avidity. Subsequently, branched linkers were developed to minimize steric hinderance between the drug and antibody, and to increase the number of drug molecules carried by each antibody [28]. An alternative approach has been to join drug-containing liposomes to antibodies. In murine tumor xenotransplant models, such immunoliposomes exhibit improved pharmacokinetics, enhanced delivery of drug to the tumor, and greater cytotoxicity, compared with liposomal drug alone or drug given with unmodified antibodies [29]. More recently, potent anticancer drugs with unacceptable toxicity when administered systemically have been linked to antibodies against internalizing tumor antigens. For example, the tyrosine kinase inhibitor genistein has been combined with an anti-CD19 antibody. This agent induces cell death by inhibiting tyrosine kinasemediated anti-apoptotic signals derived from the membrane complex CD19–LYN. In a small clinical trial, this immunoconjugate was well tolerated and three responses were seen among patients with treatment-refractory B-cell NHLs and leukemias; however, anti-mouse antibodies also developed in a third of patients [30]. N-acetyl-gamma calicheamicin is another compound with prohibitive systemic toxicity that can be safely administered as a component of the antiCD33 conjugate, gemtuzumab ozogamicin [19]. Calicheamicins induce cell death at sub-picomolar concentrations by cleaving double-stranded DNA at sequence-specific sites [31]. Gemtuzumab ozogamicin utilizes a stable bifunctional linker that prevents dissociation of calicheamicin except within the acidic lysosomal environment. Normal myelomonocytic progenitors and monocytes express CD33, whereas pluripotent hematopoietic stem cells are CD33−. Thus, treatment with gemtuzumab ozogamicin causes transient marrow suppression but does not ablate hematopoiesis. Two doses of gemtuzumab ozogamicin, given 14 days apart, induced a clinical remission in 30% of 142 adult patients with relapsed AML [19]. Infusion-related symptoms and transient
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liver function abnormalities constituted the major non-hematological side-effects. Formation of anticonjugate or anti-mouse antibodies was rare. This agent is currently being studied as part of combined therapy for AML and as monotherapy for other myeloid malignancies (e.g. high-risk myelodysplasia). The safety and efficacy of gemtuzumab ozogamicin, the first antibody chemotherapy conjugate to be approved by the FDA, renewed hope that similar approaches using other novel drug conjugates will also be beneficial. Potential mechanisms of resistance to antibody-based therapies
Malignant hematopoietic cells might escape the cytotoxic effects of antibody-directed therapies through several mechanisms. High interstitial pressure within large tumor masses limits the delivery and/or diffusion of complete antibody molecules or conjugates. Alternatively, antiglobulin or anticonjugate immune responses alter the biodistribution and pharmacokinetics of the agents during subsequent treatments. To overcome these roadblocks, engineered Ig fragments and humanized antibodies have been developed. With respect to the tumor cell itself, decreased antigen density, increased surface modulation and altered internalization kinetics could limit or prevent activation of antibody-induced cytotoxic mechanisms. If complement-dependent killing is important, it might also be antagonized by expression of membrane-bound complement regulators [10,32]. In rare cases, antigennegative tumor subclones have been shown to arise (or be selected) following treatment [33,34]. Antibody–drug conjugates might be affected by multidrug resistance (MDR) mechanisms that are active against conventional chemotherapy agents. For example, AML cell lines that overexpress the drug transporter P-glycoprotein (Pgp, the product of the MDR1 gene) are usually resistant to gemtuzumab ozogamacin, and Pgp antagonists reverse this resistance [35]. Furthermore, functional Pgp expression on blast cells is associated with inferior clinical responses to gemtuzumab ozogamicin in patients with relapsed AML [36]. Theoretically, other drug transporters and non-transporter resistance mechanisms, such as those mediating DNA repair and apoptosis, could compromise the effectiveness of drug- or toxin-containing immunoconjugates. Future directions
Several questions still need to be addressed to optimize antibody-directed therapies. First, can antibodies and immunoconjugates be better designed? Fully human mAbs, generated from transgenic mice that express the human H- and L-chain gene repertoire [37], could prove superior to chimeric and humanized antibodies in avoiding antiglobulin responses. To date, transgenic-mouse-derived human antibodies against the epidermal growth factor http://tmm.trends.com
receptor (EGFr) have shown high potency against EGFr-expressing solid tumor xenografts in athymic mice [38]; however, human studies have not yet been published. Phage- and ribosome-display technologies are facilitating the selection of antibody V regions with optimal binding affinities and pharmacokinetics [39]. These high-throughput methods could also help identify novel tumor-associated antigens by using a subtractive panning approach to select antibodies that react against tumor cells but do not react against the normal, nonmalignant cell counterpart [40]. Recombinant techniques allow the creation of site-specific mutations in variable gene sequences to ‘reshape’ the antigen-binding domains. In immunotoxin construction, genetic modifications of relevant sequences might further reduce immunogenicity and non-tumor toxicity. Second, can tumor-specific cytotoxicity be enhanced? Multispecific antibodies have been developed to improve the localization of immune effector cells or cytotoxic agents. In preclinical models, antitumor responses have been observed with bispecific antibodies that contain arms that activate T cells (anti-CD3 or anti-CD28), natural killer cells (anti-CD16) or myeloid effector cells (anti-CD64 or antibodies against other Fc receptors). Bispecific anti-CD16/CD30 and antiCD3/CD19 antibodies have been tested in small Phase I/II clinical trials; safety and some responses have so far been demonstrated [41,42]. Antibodydirected enzyme prodrug therapy is an alternative strategy that delivers an enzyme to the tumor microenvironment where it activates a systemically administered chemotherapy prodrug [2,43]. Novel toxins, such as the amphibian ribonuclease onconase [44], and high-energy α-emitting radioisotopes [24] offer theoretical advantages over the toxins and radionuclides currently in use. Adjunctive therapies, such as cytokines and lymphokines, have been used to augment host ADCC. Monensin and nigericin can potentiate the cytotoxicity of ricin-containing immunotoxins [45]. Finally, approaches currently under evaluation for solid tumors, such as antibodies against tumor microvascular endothelial cell antigens [46] or antibodies that block metabolically active receptors [47], could serve as useful models for the treatment of hematological malignancies. Third, are antibody-based treatments most effective as monotherapy or in combination with other modalities? Recently completed studies of rituximab plus conventional chemotherapy demonstrated superior responses compared with those achieved with either treatment alone [48–50]. Radiolabeled anti-CD20 antibodies are a valuable component of combination pretransplant conditioning therapy for NHL [12]. Ongoing trials are similarly addressing the efficacy and safety of gemtuzumab ozogamacin in combination with standard anti-leukemic agents and in stem cell
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transplant regimens. As a further extension of this principle, combinations of antibodies with other unmodified antibodies or with immunotoxins appear to be more effective in in vitro and murine tumor models than the single agents [51–53]. The pace of discovery and clinical applications for antibody-directed therapies are rapidly accelerating. This flourishing activity, often References 1 Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497 2 Farah, R.A. et al. (1998) The development of monoclonal antibodies for the therapy of cancer. Crit. Rev. Eukaryotic Gene Expr. 8, 321–356 3 Maloney, D.G. and Press, O.W. (1998) Newer treatments for non-Hodgkin’s lymphoma: monoclonal antibodies. Oncology 12, 63–76 4 Vose, J.M. (2001) Immunotherapy for nonHodgkin’s lymphoma. Oncology 15, 141–147 5 Ruffner, K.L. and Matthews, D.C. (2000) Current uses of monoclonal antibodies in the treatment of acute leukemia. Semin. Oncol. 27, 531–539 6 Brown, S.L. et al. (1989) Antiidiotype antibody therapy of B-cell lymphoma. Semin. Oncol. 16, 199–210 7 Vuist, W.M. et al. (1994) Lymphoma regression induced by monoclonal anti-idiotypic antibodies correlates with their ability to induce Ig signal transduction and is not prevented by tumor expression of high levels of bcl-2 protein. Blood 83, 899–906 8 Grillo-Lopez, A.J. et al. (1999) Overview of the clinical development of rituximab: first monoclonal antibody approved for the treatment of lymphoma. Semin. Oncol. 26, 66–73 9 Shan, D. et al. (2000) Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells. Cancer Immunol. Immunother. 48, 673–683 10 Golay, J. et al. (2000) Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis. Blood 95, 3900–3908 11 Milpied, N. et al. (2000) Humanized anti-CD20 monoclonal antibody (Rituximab) in post transplant B-lymphoproliferative disorder: a retrospective analysis on 32 patients. Ann. Oncol. 11, Suppl. 1, 113–116 12 Press, O.W. et al. (2000) A phase I/II trial of iodine-131-tositumomab (anti-CD20), etoposide, cyclophosphamide, and autologous stem cell transplantation for relapsed B-cell lymphomas. Blood 96, 2934–2942 13 Witzig, T.E. et al. (1999) Phase I/II trial of IDEC-Y2B8 radioimmunotherapy for treatment of relapsed or refractory CD20(+) B-cell non-Hodgkin’s lymphoma. J. Clin. Oncol. 17, 3793–3803 14 Matthews, D.C. et al. (1999) Phase I study of (131)I-anti-CD45 antibody plus cyclophosphamide and total body irradiation for advanced acute leukemia and myelodysplastic syndrome. Blood 94, 1237–1247 15 Flynn, J.M. and Byrd, J.C. (2000) Campath-1H monoclonal antibody therapy. Curr. Opin. Oncol. 12, 574–581 16 Trail, P.A. and Bianchi, A.B. (1999) Monoclonal antibody drug conjugates in the treatment of cancer. Curr. Opin. Immunol. 11, 584–588
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conducted by partners from industry and academia, has been fueled by several recent clinical successes. Antibody-based approaches currently offer a versatile, potent and, in many cases, less-toxic alternative to conventional treatments. Although effective as single agents, the greatest benefit of antibodies and immunoconjugates might be as components of combination or adjuvant therapies.
17 Kreitman, R.J. (1999) Immunotoxins in cancer therapy. Curr. Opin. Immunol. 11, 570–578 18 van Horssen, P.J. et al. (1995) Cytotoxic potency of CD22-ricin A depends on intracellular routing rather than on the number of internalized molecules. Scand. J. Immunol. 41, 563–569 19 Sievers, E.L. et al. (2001) Efficacy and safety of gemtuzumab ozogamicin in patients with CD33-positive acute myeloid leukemia in first relapse. J. Clin. Oncol. 19, 3244–3254 20 Nourigat, C. et al. (1990) Treatment of lymphoma with radiolabeled antibody: elimination of tumor cells lacking target antigen. J. Natl Cancer. Inst. 82, 47–50 21 Press, O.W. et al. (1996) Comparative metabolism and retention of iodine-125, yttrium-90, and indium-111 radioimmunoconjugates by cancer cells. Cancer Res. 56, 2123–2129 22 DeNardo, S.J. et al. (1999) A new era for radiolabeled antibodies in cancer? Curr. Opin. Immunol. 11, 563–569 23 Kaminski, M.S. et al. (2000) Radioimmunotherapy with iodine (131)I tositumomab for relapsed or refractory B-cell non-Hodgkin lymphoma: updated results and long-term follow-up of the University of Michigan experience. Blood 96, 1259–1266 24 Sgouros, G. et al. (1999) Pharmacokinetics and dosimetry of an alpha-particle emitter labeled antibody: 213Bi-HuM195 (anti-CD33) in patients with leukemia. J. Nucl. Med. 40, 1935–1946 25 van Oosterhout, Y.V. et al. (2000) A combination of anti-CD3 and anti-CD7 ricin A-immunotoxins for the in vivo treatment of acute graft versus host disease. Blood 95, 3693–3701 26 Kreitman, R.J. et al. (2000) Phase I trial of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) in patients with hematologic malignancies. J. Clin. Oncol. 18, 1622–1636 27 Kreitman, R.J. et al. (2001) Efficacy of the anti-CD22 recombinant immunotoxin BL22 in chemotherapy-resistant hairy-cell leukemia. New Engl. J. Med. 345, 241–247 28 King, H.D. et al. (1999) Monoclonal antibody conjugates of doxorubicin prepared with branched linkers: A novel method for increasing the potency of doxorubicin immunoconjugates. Bioconjugate Chem. 10, 279–288 29 Tseng, Y.L. et al. (1999) Sterically stabilized anti-idiotype immunoliposomes improve the therapeutic efficacy of doxorubicin in a murine B-cell lymphoma model. Int. J. Cancer 80, 723–730 30 Uckun, F.M. et al. (1999) Treatment of therapy-refractory B-lineage acute lymphoblastic leukemia with an apoptosis-inducing CD19-directed tyrosine kinase inhibitor. Clin. Cancer Res. 5, 3906–3913 31 Zein, N. et al. (1988) Calicheamicin gamma 1I: an antitumor antibiotic that cleaves doublestranded DNA site specifically. Science 240, 1198–1201
32 Maio, M. et al. (1998) Structure, distribution, and functional role of protectin (CD59) in complementsusceptibility and in immunotherapy of human malignancies. Int. J. Oncol. 13, 305–318 33 Davis, T.A. et al. (1999) Therapy of B-cell lymphoma with anti-CD20 antibodies can result in the loss of CD20 antigen expression. Clin. Cancer Res. 5, 611–615 34 Meeker, T. et al. (1985) Emergence of idiotype variants during treatment of B-cell lymphoma with anti-idiotype antibodies. New Engl. J. Med. 312, 1658–1665 35 Naito, K. et al. (2000) Calicheamicin-conjugated humanized anti-CD33 monoclonal antibody (gemtuzumab zogamicin, CMA-676) shows cytocidal effect on CD33-positive leukemia cell lines, but is inactive on P-glycoprotein-expressing sublines. Leukemia 14, 1436–1443 36 Linenberger, M.L. et al. (2001) Multidrugresistance phenotype and clinical responses to gemtuzumab ozogamicin. Blood 98, 988–994 37 Green, L.L. et al. (1994) Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs. Nat. Genet. 7, 13–21 38 Yang, X.D. et al. (2001) Development of ABX-EGF, a fully human anti-EGF receptor monoclonal antibody for cancer therapy. Crit. Rev. Oncol. Hematol. 38, 17–23 39 Dall’Acqua, W. and Carter, P. (1998) Antibody engineering. Curr. Opin. Struct. Biol. 8, 443–450 40 Ridgeway, J.B. et al. (1999) Identification of a human anti-CD55 single-chain Fv by subtractive panning of a phage library using tumor and nontumor cell lines. Cancer Res. 59, 2718–2723 41 Hartmann, F. et al. (2001) Anti-CD16/CD30 bispecific antibody treatment for Hodgkin’s disease: role of infusion schedule and costimulation with cytokines. Clin. Cancer Res. 7, 1873–1881 42 Manzke, O. et al. (2001) Locoregional treatment of low-grade B-cell lymphoma with CD3×CD19 bispecific antibodies and CD28 costimulation: I. clinical phase I evaluation. Int. J. Cancer 91, 508–515 43 Haisma, H.J. et al. (1998) Construction and characterization of a fusion protein of single-chain anti-CD20 antibody and human β-glucuronidase for antibody-directed enzyme prodrug therapy. Blood 92, 184–190 44 Newton, D.L. et al. (2001) Potent and specific antitumor effects of an anti-CD22-targeted cytotoxic ribonuclease: potential for the treatment of non-Hodgkin lymphoma. Blood 97, 528–535 45 van Horssen, P.J. et al. (1999) Influence of cytotoxicity enhancers in combination with human serum on the activity of CD22recombinant ricin A against B cell lines, chronic and acute lymphocytic leukemia cells. Leukemia 13, 241–249
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46 Ran, S. et al. (1998) Infarction of solid Hodgkin’s tumors in mice by antibody-directed targeting of tissue factor to tumor vasculature. Cancer Res. 58, 4646–4653 47 Fan, Z. and Mendelsohn, J. (1998) Therapeutic application of anti-growth factor receptor antibodies. Curr. Opin. Oncol. 10, 67–73 48 Czuczman, M.S. et al. (1999) Treatment of patients with low-grade B-cell lymphoma with the combination of chimeric anti-CD20 monoclonal antibody and CHOP chemotherapy. J. Clin. Oncol. 17, 268–276
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Molecular basis of resistance to azole antifungals Antonella Lupetti, Romano Danesi, Mario Campa, Mario Del Tacca and Steven Kelly The increased incidence of invasive mycoses and the emerging problem of antifungal drug resistance has prompted investigations of the underlying molecular mechanisms, particularly for the azole compounds central to current therapy. The target site for the azoles is the ERG11 gene product, the α-demethylase, which is part of the ergosterol cytochrome P450 lanosterol 14α biosynthetic pathway. The resulting ergosterol depletion renders fungal cells vulnerable to further membrane damage. Development of azole resistance in fungi may occur through increased levels of the cellular target, upregulation of genes controlling drug efflux, alterations in sterol synthesis and decreased affinity of azoles for the cellular target. Here, we review the adaptative changes in fungi, in particular Candida albicans, in response to inhibitors of ergosterol biosynthesis. The molecular mechanisms of azole resistance might help in devising more effective antifungal therapies. Antonella Lupetti Mario Campa Dept of Experimental Pathology, Medical Biotechnologies, Infectious Diseases and Epidemiology, University of Pisa, 35–39, Via S. Zeno, 56127 Pisa, Italy. Romano Danesi* Mario Del Tacca Division of Pharmacology and Chemotherapy, Dept of Oncology, Transplants and Advanced Technologies in Medicine, University of Pisa, 55, Via Roma, 56126 Pisa, Italy. *e-mail: [email protected] Steven Kelly Wolfson Laboratory of P450 Biodiversity, Institute of Biological Sciences, Edward Llwyd Building, The University of Wales, Aberystwyth, Ceredigion, UK SY23 3DA.
Mucosal and invasive opportunistic fungal infections have increased during the past two decades as a consequence of the rising number of immunocompromised hosts, such as HIV-infected individuals, transplant recipients and patients given immunosuppressive therapy or broad-spectrum antibiotics. Besides the most commonly isolated Candida and Aspergillus species, new emerging opportunistic fungi include Mucor, Fusarium, Rhizomucor and Absidia species. In addition, the spectrum of infective fungi includes Saccharomyces cerevisiae. Another factor that contributes to the severity of opportunistic infections is the development of resistance to antifungal agents. Indeed, molecular alterations often result in the development of drugresistant Candida albicans and other fungi from an initially susceptible population [1]. The aim of this paper is to review the molecular determinants of antifungal resistance, the understanding of which could provide new perspectives for improved treatment of invasive fungal infections confronting high-risk patients. Current treatment of systemic mycoses is mainly based on the use of polyenes http://tmm.trends.com
(e.g. amphotericin B) and azoles, such as triazoles (e.g. itraconazole and fluconazole) (Fig. 1). Ergosterol, the major component of fungal membrane, is the target of polyene antibiotics and the ergosterol biosynthesis pathway is the target of azole derivatives. Ergosterol contributes to a variety of cellular functions, including fluidity and integrity of the membrane and the proper function of membrane-bound enzymes such as proteins associated with nutrient transport and chitin synthesis. Ergosterol is also a major component of secretory vesicles in S. cerevisiae, and has an important role in mitochondrial respiration; indeed, mutants defective in ergosterol biosynthesis and yeasts treated with azole compounds are induced to a respiratory-deficient ‘petite’ status at a high frequency [2]. This review focuses on the various mechanisms of azole resistance in C. albicans. Polyenes
Polyenes target ergosterol in fungal membranes. They are fungicidal agents used in short treatment regimens due to associated toxicity, which may account for the low incidence of resistance encountered. Consistent with this mode of action, amphotericin B-resistant Candida strains often have a marked decrease in ergosterol content compared with amphotericin B-susceptible control isolates [1]. Resistance in clinical isolates of Cryptococcus neoformans was also associated with a mutation preventing ergosterol biosynthesis at the C8-isomerization step [3]. Resistance may also be associated with altered phospholipids or increased catalase activity with decreasing susceptibility to oxidative damage [4]. Other drugs
Flucytosine, which inhibits cellular DNA and RNA synthesis, is mainly used in combination therapy as
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