Immunotoxin Therapy of Hematologic Malignancies

Arthur E. Frankel, David M. Neville, Thomas A. Bugge, Robert J. Kreitman, and Stephen H. Leppla

Patients with chemotherapy relapsed or refractory he-matologic malignancies may be effectively treated withallogeneic or autologous stem cell transplants. How-ever, many patients cannot be transplanted due to age,comorbidities, or lack of suitable donors. Further, afraction of patients relapse post-transplant. Novel ther-apeutic agents that can kill multidrug-resistant malig-nant stem cells and are not myelosuppressive areneeded. One class of such agents is immunotoxins.Immunotoxins consist of cell-selective ligands co-valently linked to peptide toxins. The ligand deliversthe molecule to specific cell surface receptors on ma-lignant cells. The toxin triggers cell death either byreaching the cytosol and catalytically inactivating vitalcell processes or by modifying the tumor cell surfacemembrane. We have synthesized immunotoxins fortherapy of chemoresistant hematologic diseases. Inthis review, we will detail the synthesis of a number ofthese drugs and describe their preclinical and clinicalactivity. Several of these agents have shown dramaticantitumor effects in patients with hematologic neo-plasms, and one immunotoxin has been approved foruse by the US Food and Drug Administration (FDA).Over the next several decades, a growing number ofthese agents should reach the clinic.Semin Oncol 30:545-557. © 2003 Elsevier Inc. All rightsreserved.

PATIENTS WITH CHEMOTHERAPY re-lapsed or refractory hematologic malignancies

may be effectively treated with allogeneic or au-tologous stem cell transplants.1 However, manypatients cannot be transplanted due to age, comor-bidities, or lack of suitable donors. Further, a frac-tion of patients relapse post-transplant. Noveltherapeutic agents that can kill multidrug-resistantmalignant stem cells and are not myelosuppressiveare needed. One class of such agents is immuno-toxins. Immunotoxins consist of cell-selective li-gands covalently linked to peptide toxins. Theligand delivers the molecule to specific cell surfacereceptors on malignant cells. The toxin triggerscell death either by reaching the cytosol and cat-alytically inactivating vital cell processes or bymodifying the tumor cell surface membrane. Wehave synthesized immunotoxins for therapy ofchemoresistant hematologic diseases. In this re-view, we will detail the synthesis of a number ofthese drugs and describe their preclinical and clin-ical activity. Several of these agents have showndramatic antitumor effects in patients with hema-tologic neoplasms, and one immunotoxin has beenapproved for use by the US Food and Drug Ad-

ministration (FDA). Over the next several de-cades, a growing number of these agents shouldreach the clinic.

SYNTHESIS OF IMMUNOTOXINS

Monoclonal antibodies, antibody fragments,and cytokines reactive with cell surface antigensand receptors with restricted expression to humanhematopoietic cells have been used in immuno-toxin synthesis. The cell surface targets have in-cluded the cluster designation differentiation an-tigens: CD2, CD3, CD4, CD5, CD6, CD7, CD8,CD10, CD19, CD22, CD24, CD25, CD26, CD29,CD30, CD33, CD37, CD38, CD45, CD45RO,CD52, CD54, CD56, CD64, CD72, CD80, CD86,and CD122. Additional cell surface targets includethe cytokine receptors: interleukin-2 receptor (IL-2R), granulocyte-macrophage colony-stimulatingfactor receptor (GM-CSFR), interleukin-3 recep-tor (IL-3R), interleukin-4 receptor (IL-4R), inter-leukin-6 receptor (IL-6R), interleukin-7 receptor(IL-7R), interleukin-9 receptor (IL-9R), interleu-kin-13 receptor (IL-13R), granulocyte colony-stimulating factor receptor (G-CSFR), urokinasereceptor (uPAR), transferrin receptor (TfR), andinterleukin-15 receptor (IL-15R). Finally, there is aheterogeneous group of hematopoietic receptors,including CCR5, human leukocyte antigen (HLA)-DR, immunoglobulin (Ig) idiotype, IgMFc, theCLL2m chronic lymphocytic leukemia antigen,the ML30 heat shock protein, the kappa-myelomaantigen (KMA) lymphoblastoid antigen, the M5acute myeloid leukemia (AML) antigen, theHAE9 and HAE3 erythroblast antigens, the SN7B-cell antigen, the 8A and 62B1 myeloma anti-gens, neurokinin-1, and the NDA4, IORT6, andSOKT1 T-cell antigens. Not all ligand-toxins

From the Wake Forest University School of Medicine, Winston-Salem, NC; National Institute of Mental Health, Bethesda, MD;National Institute of Dental and Craniofacial Research, Bethesda,MD; National Cancer Institute, Bethesda, MD; National Instituteof Allergy and Infectious Diseases, Bethesda, MD.

Address reprint requests Arthur E. Frankel, MD, Hanes 406,Wake Forest University of School, Medical School of Medicine,Medical Center Blvd, Winston-Salem, NC 27157.

© 2003 Elsevier Inc. All rights reserved.0093-7754/03/3004-0008$30.00/0doi:10.1016/S0093-7754(03)00241-0

545Seminars in Oncology, Vol 30, No 4 (August), 2003: pp 545-557

Table 1. In Vitro Potency of Immunotoxins for Hematologic Malignancies

Agent Receptor Normal Tissue IC50 (pmol/L) Reference

Directed at CD AntigensOKT11-saporin CD2 T cells 0.5 2A-dmDT390-bisFv(G4S) CD3 T cells 0.2 3TEC-T4-saporin CD4 T cells 1,000 4H65-rGeloninD247C CD5 T cells, rare B cells 41 5Anti–CD6-blocked ricin CD6 Thymocytes 4 8BA1e-deglycosylated ricin A CD7 T cells 5 9WT82-ricin A CD8 T cells �10,000 10SN5-ricin A CD10 Granulocytes, fibroblasts 2,000 11My7/Fab�-GAMIg-ricin A CD13 Granulocytes, monocytes �10,000 201G10/Fab�-GAMIg-ricin A CD15 Granulocytes, monocytes 5,000 20BU12-saporin CD19 B cells, dendritic cells 35 12RTB4(Fv)100THW100A-PE38 CD22 B cells 16 13SWAII-ricin A CD24 B cells, granulocytes 70 25Anti–Tac(Fv)-PE38 CD25 T cells, B cells, macrophages 2 141F7-blocked ricin CD26 T cells �1,000 154B4-blocked ricin CD29 Lymphocytes, endothelium 500 16T25(dsFv)-PE38 CD30 T cells, B cells 160 17HuM195-rgelonin CD33 Monocytes, granulocytes 50 18MB-1/anti-saporin/saporin CD37 T cells, B cells, myeloid �30,000 19OKT10-saporin CD38 Lymphocytes, macrophages 38 21G28-5sFv-bryodin 1 CD40 B cells, dendritic cells 100 22BMAC1-ricin A CD45 Leukocytes 3,000 23UCHL1-ricin A CD45RO Leukocytes 1,000 24Campath-1-saporin CD52 Leukocytes 200 26UV3-deglycosylated ricin A CD54 Granulocytes, monocytes 40 27N901-blocked ricin CD56 NK cells, neural tissues 50 28H22-deglycosylated ricin A CD64 Monocytes, macrophages 4,000 29J3-109-PAP CD72 B cells 100 30M24-saporin CD80 B cells, macrophages 0.3 311G10-saporin CD86 Monocytes, dendritic cells 3 31Mik-beta1(Fv)-PE40 CD122 Leukocytes 100 32

Directed at Cytokine ReceptorsDAB389IL2 IL-2R Activated T, B cells 4 33DT388IL3 IL-3R Mast cells, myeloid stem 15 34DAB389IL4 IL-4R Hepatocytes, endothelium 50 35IL6-PE664Glu IL-6R Hepatocytes 30 36DAB389IL7 IL-7R Thymocytes, B cells, T cells 100 37rhIL9-ETA� IL-9R Thymocytes, mast cells 6,000 38IL13-PE38QQR IL-13R Skin, lung, heart, liver cells 0.8 39DAB389IL15 IL-15R Leukocytes, skin, lung, liver 7,000 40GCSF-PE40 G-CSFR Granulocytes 100 41DT388GMCSF GM-CSFR Granulocytes, macrophages 3 42DTAT uPAR Endothelium 20 43

Directed at Miscellaneous AntigensRANTES-PE38 CCR5 T cells �5,000 44DAB389SP Neurokinin-1 Neurons, endothelium 18 452G5-ricin A HLA-DR Bcells, T cell, dendritic �10,000 4638-13-ricin A Ceramide TH Pk1/Pk2 cells 200 47CLL2m-ricin A CLL2m antigen ND 23 48HAE3-ricin A Glycophorin RBC 3,100 49HAE9-ricin A Erythroid antigen Erythroblasts 320 49TEC-saporin IgM B cells 100 50IOR-T6-HT Thymocyte antigen Thymocytes 5,000 518A-saporin 8A myeloma antigen B cells 60 5262B1-saporin 62Bmyeloma antigen B cells 1 52

546 FRANKEL ET AL

have shown potent selective toxicity. In somecases, the ligand-receptor is expressed at very lowlevels on the malignant cells or is poorly internal-ized (CD4, CD8, CD10, CD13, CD15, CD26,CD37, CD45, glycophorin, HLA-DR, IL-9R, andIL-15R). In other cases, receptor is present oncritical normal tissues (TfR, IL-4R and IL-6R).Table 1 shows a list of the immunotoxins synthe-sized for therapy of human hematologic cancers,important normal tissues with the targeted recep-tor when present, and a representative concentra-tion of the agent reducing malignant cell viabilityby 50% (IC50). In addition to receptor specificity,other ligand variables in immunotoxin potencyinclude the affinity, distance of the antigen/recep-tor epitope from the cell membrane, and size andconformation of the ligand.6,7

All the toxins used in immunotoxin synthesisfor treatment of hematologic tumors have beencytotoxins. Cytotoxins can directly induce celldeath. There are three classes of peptide cytotox-ins, and each has been used. Class I toxins areintracellular enzymes; class II proteins bind to cellsurfaces and trigger intracellular signal pathways;and class III cytotoxins are pore-forming peptidesthat cause leaks in the plasma membrane. Most ofthe immunotoxins used for hematologic malignan-cies have been made with class I cytotoxins.

The exceptions that have not used class I cyto-toxins include the class II recombinant cytotoxinconjugate, anti-Tac(Fab)-PLC, and the class III

cytotoxin immunoconjugates: IOR-T6-HT, ScFv-mel-FLAG, and OKT1-PT . The class II immuno-toxin consists of the Fab fragment of the anti-CD25 monoclonal antibody (anti-Tac) fused tophospholipase C.57 This conjugate reacts with theIL-2R �-subunit (CD25) on activated or malig-nant T and B cells. The molecule deliversphospholipase C to the surface of the malignantlymphocyte, which in turn hydrolyzes phosphati-dylcholine triggering abnormal cell signaling anddeath. The conjugate had an IC50 of 20 pmol/L.The class III cytotoxin conjugates—IOR-T6-HT,ScFv-mel-FLAG, OKT1-PT, and T3-3A1-CTX—have been studied in vitro. IOR-T6-HT binds to aT-cell differentiation antigen on malignant T cellsand the hemolytic toxin creates pores in the mem-brane.51 ScFv-mel-FLAG binds the KMA antigenon human myeloma cells where the associatedinsect class III toxin, mellitin amphipathic pep-tide, punches holes in the myeloma cell mem-brane.58 OKT1-PT consists of the anti-CD5 anti-body OKT1 coupled to the plant Pyrularia thioninmembrane active peptide.59 Again, delivery of themolecule to T-cell surfaces is followed by mem-brane pore formation and leakage. Finally, T3-3A1-CTX consists of an anti–T-cell monoclonalantibody coupled to the cobra venom cationic 63amino acid membrane lytic peptide toxin.60 Un-fortunately, there is no information on the in vivosafety of any of the class II and class III cytotoxinconjugates. Since all of these toxins may react

Table 1. In Vitro Potency of Immunotoxins for Hematologic Malignancies (Contd.)

Agent Receptor Normal Tissue IC50 (pmol/L) Reference

Directed at Miscellaneous Antigens (Cont’d)SN7-RTA SN7 B cell antigen B cells 2 53LAM3-saporin M5b-AML antigen Monocytes, platelets 70 54NDA4-gelonin NDA4 T-cell antigen T cells, B cells 200 55ML30-saporin HSP65 Virus infected cells 1,000 56

NOTE. Cell lines, toxin used, and duration of incubation vary, which makes comparisons of different ligands difficult. Normal tissue reactivityaffected by toxin. In the case of blocked ricin, residual galactosyl binding causes endothelial reactivity and toxicity and may contribute to tumorcell killing.

Abbreviations: rgelonin, recombinant gelonin; PE, Pseudomonas exotoxin; PE38, 38-kd fragment of PE; PE40, 40-kd fragment of PE; PAP,pokeweed antiviral protein; DT, diphtheria toxin; DAB389, DT amino acids residues 1-389; DT388, DT amino acid residues 1-388; PE664Glu,PE-modified with 4 lysines changed to glutamic acids to reduce normal tissue binding; ETA; mutant PE with reduced normal tissue binding;PE38QQR, modified 38-kd PE fragment; uPAR, urokinase-plasminogen-activator receptor; G-CSF, granulocyte colony-stimulating factor;GM-CSF, granulocyte-macrophage colony-stimulating factor; DTAT, diphtheria toxin fragment fused to the N-terminal fragment of urokinase;TH, trihexoside; RBC, red blood cells, Pk1/Pk2, rare Pk blood group allotypes; HT, hemolytic toxin; HSP65, heat shock protein 65 kd Mr; ND,not determined.

IMMUNOTOXIN THERAPY OF HEMATOLOGIC MALIGNANCIES 547

with cell surface membranes of blood cells andendothelium, it will be important to determine thein vivo safety of these immunotoxins.

The class I toxin conjugates offer additionalsafety, since they only damage cells after ligand-receptor internalization. Most of the employedclass I toxins inactivate cytosolic protein synthesiseither by modifying elongation factor 2 (diphthe-ria toxin [DT} and Pseudomonas exotoxin [PE]) orby degrading ribosomal RNA (ricin, abrin, vis-cumin, gelonin, saporin) or total RNA (ribonucle-ase, angiogenin,). Bax triggers mitochondrial-me-diated apoptosis. Some of the type I cytotoxinshave normal tissue binding sites, which must beremoved or modified prior to conjugation. Thesetoxins include ricin, abrin, viscumin, DT, and PE.A large group of cytotoxins do not have normaltissue binding sites and must be linked to cellsurface reactive ligands to intoxicate cells. Thesetoxins include gelonin, saporin, pokeweed antivi-ral protein (PAP), bryodin, bouganin, momordin,

dianthin, momorcochin, trichokirin, luffin, restric-tocin, mitogillin, �-sarcin, Onconase, pancreaticribonuclease, Bax, eosinophil-derived neurotoxin,and angiogenin. Examples of the class I toxins usedin hematologic cancer immunotoxins are shown inTable 2. Which toxins provide the greatest spec-ificity and potency? Complete inactivation of thenormal tissue binding sites is necessary. Partialinactivation, as was observed with the blockedricin conjugates, yields a poor therapeutic index.Direct comparisons of immunotoxins targeting thesame antigen/receptor on nonmyeloid cells withdifferent toxins in which the normal tissue bindingsites have been eliminated have been made withimmunotoxins reactive with CD2, CD3, CD5,CD7, CD19, CD22, CD25, CD30, IL-2R, GM-CSFR, IL-4R, IL-6R, uPAR, IL-13R, and TfR.The anti-TfR immunoconjugates include the mostmembers for comparisons among toxins (Table 3).The best cytotoxins for conjugate synthesis in-clude the bacterial holotoxins—DT and PE—in

Table 2. Peptide Class I Cytotoxins Used in Hematologic Neoplasm Immunotoxins

Conjugate Toxin Receptor IC50 (pmol/L) Reference

A-dmDT390-bisFv(G4S) DT CD3 0.2 3RTB4(Fv)100THW100A-PE38 PE CD22 16 133A1e-deglycosylated ricin A Ricin CD7 5 9Anti-CD25-MLA Viscumin CD25 400 61M24-bouganin Bouganin CD80 10 31BU12-saporin Saporin CD19 35 12H65-rGeloninD247C Gelonin CD5 41 5G28-5sFv-bryodin 1 Bryodin CD40 100 22J3-109-PAP PAP CD72 100 308A-momordin Momordin 8A myeloma antigen 200 62BerH2-rdianthin 30 Dianthin 30 CD30 100 63UCHT1/anti-Igdianthin 32 Dianthin 32 CD3 10 64UCHT1/anti-Igmomorcochin Momorcochin CD3 68 64UCHT1/anti-Igtrichokirin Trichokirin CD3 32 64HB21-luffin Luffin TfR 50 79RSFv Restrictocin TfR 40 72H65-MS-mitogillin Mitogillin CD5 150 65HB21-�-sarcin �-sarcin TfR 30 80LL2-onconase Onconase CD22 20 66T101-ribonuclease A RibonucleaseA CD5 120,000 67IL2-Bax Bax IL2R 140,000 68hpRNase1-IL2 hpRNase 1 IL2R 20,000 69EDNsFv EDN TfR 500 70AngFBsFvL2 Angiogenin TfR 5,000 71Tf-ribonuclcase Ribonuclease TfR 100,000 73

Abbreviations: rdianthin, recombinant dianthin; hpRNase, human pancreatic ribonuclease; EDN, eosinophil-derived neurotoxin; TfR, trans-ferrin receptor; Tf, transferrin.

548 FRANKEL ET AL

which the binding domains have been selectivelymodified with preservation of the translocationdomains. These conjugates show similar potencyin non-myeloid cells. The immunotoxins withplant ribosome-inactivating proteins—ricin Achain, viscumin A chain, gelonin, saporin,pokeweed antiviral protein, bryodin, bouganin,momordin, dianthin, luffin, momorcochin, and tri-chokirin—have intermediate potency. The mech-anism by which they reach the cytosol remainsunknown. Immunotoxins with the fungal nucle-ases—restrictocin, mitogillin, and �-sarcin—alsohave intermediate affinity, The amphibian, bo-vine, and human nucleases—onconase, ribonucle-ase, eosinophil-derived neurotoxin, angiogeninand the human pro-apoptotic protein Bax—havethe least potency. Immunotoxins for myeloid leu-kemias are a special case. Because of the rapidtrafficking of endosomes to lysosomes and the re-duced routing to the Golgi and endoplasmic retic-ulum in myeloid cells, DT conjugates work best.74

DT can escape to the cytosol from early endo-somes to the cytosol, while PE and ricin toxin onlyreach the cytosol after passage through the Golgiand endoplasmic reticulum. Another factor influ-encing efficacy is immunogenicity. Patients withantitoxin antibodies clear immunotoxins rapidlyfrom the bloodstream and have a marked reducedarea under the curve (AUC). Since most peopleare immunized with DT, there is a significant

pretreatment antibody titer in the blood of manypatients and an amnestic response occurs in addi-tional patients treated with DT conjugates. Ofintermediate immunogenicity are the toxins thatare foreign antigens to which people have notbeen previously exposed. These toxin conjugatesevoke antibodies after a lag of 3 to 4 weeks. Theseinclude most of the cytotoxins with the exceptionof human proteins—Bax, ribonuclease, eosinophilneurotoxin, and angiogenin. Since these toxinshave the least potency, selection of the cytotoxinfor a particular application involves compromisesbetween immunogenicity, potency, and specificity.Pharmacologic problems due to the toxin havebeen observed in vivo. Vascular leak syndromefrom vascular injury may be due to selective re-ceptors for peptide toxins on endothelial cells.Modification of the endothelial binding residueson toxins may reduce this toxicity and has beentested on ricin A chain conjugates.75 Some of thecytotoxin conjugates are small, less than 50 kd Mr.Such conjugates may be rapidly cleared by thekidney glomeruli leading to a short half-life, poorAUC, and renal tubular injury. One approach toovercome this problem is to enlarge the size of theconjugate so that glomerular filtration is reduced.This has been successfully accomplished with sev-eral anti-CD3 recombinant DT fusion proteinsleading to less renal toxicity and improvedAUC.3,76 Another approach to reduce the nontar-get tissue toxicities of immunotoxins is to addanother tissue-specific requirement for cell intox-ication. Since several bacterial toxins requirefurin-mediated cleavage prior to translocation ofthe catalytic domain to the cytosol, replacing thefurin sequences with sequences specific to tumor-selective proteases would provide additional spec-ificity. This has been accomplished for anthraxprotective antigen in combination with a fusionmolecule of the N-terminal fragment of lethalfactor with the translocation and catalytic do-mains of PE (FP59).77,78 The resulting toxins showtumor-selective cytotoxicity and safety in animals.Similar modifications should be possible with DTand PE conjugates. Also important in immuno-toxin design is the choice of linker between theligand and toxin.

The covalent attachment of the toxin and li-gand has to meet several requirements. It must bestable in the bloodstream and interstitial fluid. Itshould not impair ligand affinity to its receptor,and it must not impede toxin translocation or

Table 3. Comparison of the Potency of Anti-transferrin Receptor Immunoconjugates

Immunotoxin ToxinIC50

(pmol/L) Reference

DT388anti-TfR(Fv) DT 3 85Anti-TfR(Fv)PE38 PE 6 85454A12-ricin A Ricin 60 845E9-gelonin Gelonin 50 83HB21-bryodin II Bryodin 50 82HB21-luffin Luffin 50 79B2/25-saporin Saporin 50 81HB21-�-sarcin �-sarcin 30 80RSFv Restrictocin 40 72Anti-TfR(Fv)-EDN EDN 500 70Anti-TfR(Fv)AngL2 Angiogenin 5,000 71Anti-TfR(Fv)-pancRNase PancRNase 100,000 73

Abbreviations: DT, diphtheria toxin; PE, Pseudomonas exo-toxin; TfR, transferrin receptor; EDN, eosinophil-derived neu-rotoxin; pancRNase, pancreatic ribonuclease; Ang, angiogenin

IMMUNOTOXIN THERAPY OF HEMATOLOGIC MALIGNANCIES 549

enzymatic activity. The conjugation of ligand andtoxin has employed both chemical conjugationand genetic fusion. The former has been accom-plished with bifunctional reagents such as thethiolating compounds—3-(2-pyridyldithio)propri-onic acid N-hydroxysuccinimide ester (SPDP) or3-maleimidobenzoic acid N-hydroxysuccinimideester (MBS). Such chemical conjugates bind to avariety of lysines on the ligand and/or toxin yield-ing a heterogeneous production with at least mildto moderate effects on ligand and toxin func-tions.2,4-8,10-12,15,16,18-21,23-31,46-56,59-65,79-84,86,87 Moresuccessful immunotoxin design has employed ge-netic engineering where an amide bond with orwithout a linker peptide connects the ligand andtoxin. Such fusions have been more successful ispreserving receptor affinity and toxin domainfunctions.3,4,9,13,17,22,32-45,57,58,66-74,76-78,85

CLINICAL RESULTS WITHIMMUNOTOXINS

Among the several hundred hematologic cancerimmunotoxins discussed above, about two dozenhave been tested in phase I-III clinical trials. Ex-citingly, a number of these compounds haveshown significant antileukemia and antilymphoma

activity, and one (DAB389IL2 or ONTAK, LigandPharmaceuticals, San Diego, CA) has been ap-proved by the US FDA for therapy of cutaneousT-cell lymphoma. Table 4 lists the published stud-ies of immunotoxins in hematologic malignancypatients. The table lists the compounds, the dis-eases for which they were tested, their responserates, and their toxicities. Direct comparisons aredifficult due to the different conjugate construc-tions, the different patient populations, and thedifferent routes, doses, and schedules of drug usedin the various trials. Nevertheless, we will addressthe efficacy, pharmacokinetics, immune responses,and toxicities for each agent and try to presentcommon principles.

Efficacy

Immunotoxins with the highest response rate inclinical studies have been active in tissue culturein the picomolar range and have been fully recom-binant molecules.

BL22 consists of a disulfide-stabilized anti-CD22sFv fused to PE38. When given at doses of 3 to 50�g/kg intravenously over 30 minutes every otherday for three doses to patients with purine analog-resistant hairy cell leukemia (HCL), there were 13

Table 4. Results of Clinical Studies of Hematologic Neoplasm Immunotoxins

Immunotoxin Disease Response Rate Toxicities References

RFB4(dsFv)-PE38 (BL22) Hairy cell leukemia 13/16 HUS 88Anti–Tac(Fv)-PE38 (LMB-2) Lymphoma 8/35 Transaminasemia 89DAB389IL2 (ONTAK) CTCL 33/91 VLS, transaminasemia 90, 93B43-PAP B-cell ALL 5/17 VLS 95IgRFB4-dg. ricin A Lymphomas 11/45 VLS 96-98FabRFB4-dg ricin A Lymphomas 5/14 VLS 101BerH2-saporin Hodgkin’s disease 3/4 None 102DAB486IL2 Lymphomas 12/109 Transaminasemia, HUS, renal insufficiency 103IgG-HD37-dg ricin A Lymphomas 5/54 VLS, HUS, acrocyanosis 104, 1053A1e-dg ricin A T-cell leukemias 2/11 VLS 106DT388GMCSF AML 4/37 Transaminasemia, liver failure 100Anti–B4-blocked ricin Lymphomas 8/75 VLS, transaminasemia, akinetic mutism 113-115T101-ricin A CLL 0/5 None 107H65-ricin A CLL, CTCL 2/10 CLL; 4/14 CTCL VLS 108, 109SPV-T3a-dg ricin A/WT-1-dg

ricin A GVHD 2/4 None 99Anti–Tac-PE ATL 0/4 Liver failure 110IgRFT5-dg ricin A Hodgkin’s disease 2/15 VLS 111K1-4-dg ricin A Hodgkin’s disease 1/15 VLS 112

Abbreviations: HUS, hemolytic-uremic syndrome; VLS, vascular leak syndrome; dg, deglycosylated; PAP, pokeweed antiviral protein; ALL, acutelymphoblastic leukemia; AML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia; CTCL, cutaneous T cell lymphoma; GVHD,graft-versus-host disease; PE, Pseudomonas exotoxin; ATL, adult T-cell leukemia; DT, diphtheria toxin; DAB, diphtheria toxin A and B chain.

550 FRANKEL ET AL

of responses among 16 patients, including 11 com-plete remissions.88 The three nonresponders eitherreceived a low dose or had pre-existing toxin-neutralizing antibodies. The remissions were dura-ble with only three of 11 responders relapsing after10 to 23 months. Re-treatment again producedcomplete remissions. Thus, BL22 immunotoxinappears to be the current best salvage treatmentfor relapsed HCL patients.

LMB2 is composed of an anti-CD25 sFv fused toPE38. LMB2 has been administered to 35 patientswith lymphomas at doses of 2 to 63 �g/kg intra-venously over 30 minutes four times per day for 3days.89 A complete remission was observed inHCL, which was ongoing at 20 months. Therewere seven partial remissions distributed as fol-lows: three patients with HCL, one with cutane-ous T-cell lymphoma (CTCL), one with chroniclymphocytic leukemia (CLL), one with Hodgkin’sdisease (HD), and one with adult T-cell leukemia(ATL). Responders received at least 60 �g/kg totaldose of LMB2 per cycle. The duration of remis-sions have not been determined but exceeds 20and 6 months for two patients and 1 month for theremaining patients. Responding patients hadclearance of circulating malignant cells, improve-ment in skin lesions, and regression of lymphnodes and splenomegaly.

ONTAK is a fusion protein composed of thecatalytic and translocation domains of DT fused tohuman IL-2. Among 71 CTCL patients with stageIB to IVA disease refractory to other therapies andtreated with 9 or 18 �g/kg/d ONTAK for 5 daysevery 3 weeks, there were 21 remissions, includingseven complete remissions.90 The median durationof remission was 7 months. When combined withdexamethasone 8 mg/d, responses were seen in 12of 20 CTCL patients.91 ONTAK also produced apartial remission in a patient with peripheral T-cell lymphoma (PTCL).92 In the phase I study,ONTAK produced a durable complete remission(ongoing after 5 years) in a patient with trans-plant-refractory large cell lymphoma.93 Two pa-tients with low-grade lymphoma obtained partialremissions. Based on the above results, in 1999 theFDA made ONTAK the first approved immuno-toxin.94

B43-PAP consists of an anti-CD19 monoclonalantibody chemically conjugated to PAP. Among17 children with relapsed B-cell acute lymphocyticleukemia (ALL), there were four complete remis-

sions and one partial remission to five daily intra-venous infusions of 0.5 to 1,250 �g/kg B43-PAP.95

IgRFB4-dgA is composed of an anti-CD22 an-tibody coupled to deglycosylated ricin A chain.When given either by a 192-hour continuous in-fusion or by four infusions over 4 hours each tochemoresistant B-cell non-Hodgkin’s lymphoma(NHL) patients and one post-kidney transplantlymphoma, there were 11 remissions among 54treated patients.96-98 These included two completeremissions lasting 2 and 32� months and ninepartial remissions lasting 2, 2, 3, 3, 3, 5, 6, 8, and14 months.

A combination of anti-CD3 (SPV-T3a-dgA)and anti-CD7 (WT1-dgA) immunotoxins wasgiven as three or four infusions of 2 or 4 mg/m2 ofa 1:1 combination of the two conjugates to foursteroid-refractory patients with graft-versus-hostdisease (GVHD) and two nearly complete remis-sions were observed.99 One patient had a resolu-tion of grade 4 skin GVHD. The other patient hada resolution of garde 2 intestinal GVHD.

DT388GMCSF consists of the catalytic andtranslocation domains of DT fused to human GM-CSF. A 1- to 5-�g/kg quantity was administeredintravenously over 15 minutes daily for 5 days to37 patients with relapsed or refractory AML.100

One patient had a complete remission with recov-ery of normal hematopoeisis lasting 1 year. After ayear, she had falling blood counts and increasedmarrow blasts. She was again treated and showedclearance of marrow blasts on day 12, but devel-oped sepsis and died on day 21. Three other pa-tients had partial remissions lasting months withclearance of marrow blasts on day 30, but did notachieve recovery of normal hematopoiesis. In onecase, the patient received a cord blood transplantafter achievement of blast cytoreduction.

Responses have been observed with other im-munotoxins as noted in Table 4, including Fab-�RFB4-dg ricin A, BerH2-saporin, DAB486IL2,IgG-HD37-dg ricin A, anti–B4-blocked ricin,H65-ricin A, IgRFT5-dg ricin A, and Ki-4-dg ricinA, but these molecules have not undergone furtherdevelopment.

Pharmacokinetics and Tissue Distribution

Larger molecules have longer half-lives in thecirculation and poorer tissue penetration. Thehalf-life of monoclonal antibody conjugates withRTA, ricin, PAP, and PE ranged from 9 to 24hours.95,98,102,106 The molecular weights were all

IMMUNOTOXIN THERAPY OF HEMATOLOGIC MALIGNANCIES 551

around 200,000 Mr. There was likely little clear-ance of these molecules via renal glomeruli, andpenetration into extravascular sites such as nodes,marrow, or skin nodules was poor.107,115 In con-trast, the smaller recombinant immunotoxins(60,000 to 70,000 Mr) have had shorter half-livesof 1 to 5 hours88,89,93,100,103 and should have betterpenetration into extravascular sites of disease.However, there are no reports to date regardingthe saturation of extravascular tumor deposits inclinical trials with recombinant immunotoxins.The shorter circulation times and increased vas-cular permeability of the smaller recombinant im-munotoxins may contribute to their greater anti-tumor efficacy and reduced endothelial toxicity(see below).

Immune Responses

Because most individuals in the United Statesare immunized with diphtheria toxoid as children,close of half of adults have anti-DT antibody titerspretreatment with DT conjugates.116 Further, be-tween 10% and 20% of patients have had previousPseudomonas infections (often subclinical) yield-ing pretreatment anti-PE antibody titers.117 Pa-tients exposed to castor oil may show anti-ricinantibodies. These circulating antibodies reducethe half-life and AUC for immunotoxins and, insome cases, neutralize cell cytotoxicity.90,100,118

Even if patients lack pretreatment immunity, afteradministration of the foreign protein, most pa-tients develop anti-immunotoxin antibodies. Theexceptions include CLL patients and some pa-tients with severe immunosuppression.

Toxicities

There are two general classes of side effects dueto immunotoxins. In some instances, the targetedtoxin receptor/antigen is present on normal tis-sues. This can lead to significant toxicities.DT388GMCSF reacts with normal liver macro-phages—Kupffer cells. Subsequent cytokine re-lease triggers dose-limiting hepatocyte damage.100

Toxicities that are independent of ligand havebeen observed in almost every immunotoxin clin-ical study. The similarity of the toxicities observedwith different immunotoxin trials suggests that thelesions are due to the toxin moieties. The fiveclasses of side effects are (1) transient constitu-tional symptoms with fever, chills, nausea, vomit-ing, myalgias, arthralgias, asthenia, and hypoten-sion; (2) transient hepatotoxicity with elevated

transaminases and, rarely, other liver enzymes; (3)transient vascular leak syndrome (VLS) consistingof edema, hypoalbuminemia, weight gain, and,rarely, dyspnea and aphasia; (4) assorted syn-dromes including rhabdomyolysis, hemolytic ure-mic syndrome, and renal insufficiency with pro-teinuria and renal tubular acidosis; and (5) allergicreactions. The toxicities may be due to binding ofthe toxin moieties to normal tissues. Reaction ofimmunotoxins with macrophages, lymphocytes, orendothelium may lead to cytokine release withconsequent constitutional symptoms and elevatedcirculating cytokines. Elevations of IL-6 andTNF-� have been reported in some but not allcases.88,96,98,100 Where studied, corticosteroids orinfliximab � rofecoxib appeared to dampen oreliminate this side effect.88,91 The hepatotoxicitymay be directly due to immunotoxin binding af-fected by the pI of the ligand in the conjugate orsecondary release of cytokine from macrophagesmay damage the liver.100,119,120 In most cases, im-pairments of hepatic function such as VII produc-tion or bilirubin clearance were not observed andthe liver injury was reversible. VLS is a syndromeobserved with IL-2, monoclonal antibodies, somechemotherapy drugs such as taxotere, and manyimmunotoxins. The onset of the syndrome is usu-ally 4 to 6 days after initiating therapy and it lasts4 to 10 days. “Leaky” capillaries throughout thebody produce low serum albumin, edema, weightgain, dyspnea, aphasia, rhabdomyolysis, and pro-teinuria. The abnormal vascular endothelium maybe intoxicated by the immunotoxin directly ormay be “activated” by elevated circulating cyto-kines. Evidence from tissue culture and animalmodels has been reported for a direct binding siteof toxins on vascular endothelial cells and for anindirect toxic effect of immunotoxin-triggered in-flammatory cytokines on endothelial cells.121,122

There do not appear to be effective methods ofprophylaxis, although paradoxically active hydra-tion appears to reduce the incidence and severityof VLS. Further, smaller recombinant immunotox-ins appear to have a lower incidence, possibly dueto their shorter circulating half-life. Excluding pa-tients with prior radiotherapy may also lower theincidence or severity of the VLS syndrome.86 Therare cases of hemolytic-uremic syndrome (HUS)and rhabdomyolysis may be patient-specific typesof vascular injury or due to ligand (particularlyanti-CD22 antibodies). Allergic or anaphylactoidreactions occur very rarely, are IgE-mediated, and

552 FRANKEL ET AL

are treated similar to allergic reactions to otherforeign proteins such as L-asparaginase. Fluids, car-diac monitoring, oxygen, corticosteroids, diphen-hydramine, and, rarely, epinephrine can be given.Patients with anaphylactoid reactions should notbe re-treated with the same immunotoxin.

CURRENT IMMUNOTOXIN CLINICALTRIALS IN HEMATOLOGIC

MALIGNANCIES

Current and near future applications of immu-notoxins in hematologic malignancies are focusedon expanding the use of some of the active, estab-lished agents, and testing novel compounds. Thesestudies and the sites for the studies are listed inTable 5.

Further protocols with DAB389IL2 (ONTAK)include testing in CD25� CTCL, B-cell NHL,PTCL,92 GVHD,123 and CLL.124 Combinationstudies with dexamethasone, bexarotene (an RXR-agonist), or tributyrin are being tested in CTCL,NHL, and CLL.91 Bexarotene and butyrates up-regulate IL-2R subunits on CTCL and CLL cellsand sensitize the malignant cells to killing byDAB389IL2.125,126 RFB4(Fv)-PE38 (BL22) is beingtested in HCL, CD22� HCL B-cell NHL, and

CD22� B-cell ALL. Anti-Tac(Fv)-PE38 (LMB-2)is being tested in patients with CLL and CTCL.DT388GMCSF is being tested in adults with re-lapsed AML in the absence of corticosteroids.SPV-T3a-dgA plus WT1-dgA is continuing phaseII evaluation for therapy of steroid-refractoryGVHD.

A number of novel immunotoxins have startedor will soon start clinical trials. HuM195-recom-binant gelonin has been given to 20 AML patientsto date and dose escalation is proceeding.DT388IL3 has been aseptically vialed, is undergo-ing monkey toxicology testing, and will enter clin-ical trials in the spring. BU12-saporin is beinggiven to children and adults with B-cell ALL andNHL, respectively. OKT10-saporin is being testedin patients with advanced multiple myeloma.A-dmDT390-bisFv is undergoing production andwill enter phase I study in patients with refractoryaplastic anemia and T-cell malignancies.

These studies should help define the role forimmunotoxins in the care for patients with hema-tologic malignancies. The last two decades havesuggested potential clinical applications for thesechimeric proteins. The next few years should seeimproved success of these agents as improvements

Table 5. Ongoing and Planned Immunotoxin Studies in Patients With Hematologic Neoplasms

Agent Target Receptor Disease Contact (telephone)

DAB389IL2 (ONTAK) IL-2R CD25� CTCL Foss (617-636-5558)NHL Dang (713-792-2860)GVHD Ho (617-632-5938)MDS Rosmarin (401-793-4648)

�bexarotene (Targretin) CTCL, NHL, CLL Foss (617-636-5558)�bexarotene (Targretin) CLL Frankel (336-716-3313)

RFB4dsFv-PE38(BL22) CD22 HCL Kreitman (301-402-5633)CLL, NHL Kreitman (301-402-5633)ALL Wayne (301-435-2257)

Anti-Tac(Fv)-PE38 (LMB2) CD25 CLL Kreitman (301-402-5633)CTCL Kreitman (301-402-5633)

DT388GMCSF GM-CSFR AML Sweetenham (303-315-5608)DT388IL3 IL-3R AML Rizzieri (919-684-8111)A-dmDT390bisFv CD3� AA, T-NHL Frankel (336-716-3313)HuM195-rgelonin CD33 AML Talpaz (713-792-3525)BU12-saporin CD19 B-cell ALL Flavell (44-1703796947)OKT10-saporin CD38 Myeloma Flavell (44-1703796947)SPV-T3a-dgA/WT1-dgA CD3/CD7 GVHD De Witte (31-2-4361476)

Abbreviations: AA, aplastic anemia; NHL, non-Hodgkin’s lymphoma; HD, Hodgkin’s disease; GVHD, graft-versus-host disease; ALL, acutelymphoblastic leukemia; AML, acute myeloid leukemia; HCL, hairy cell leukemia; MDS, myelodysplastic syndrome; CTCL, cutaneous T-celllymphoma.

IMMUNOTOXIN THERAPY OF HEMATOLOGIC MALIGNANCIES 553

are made in protein design to reduce normal tissuetoxicities and the new molecules are matched withthe molecular profile of particular patients to en-hance response rates.

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