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1.
J Clin Oncol ; 27(18): 2983-90, 2009 Jun 20.
Article in English | MEDLINE | ID: mdl-19414673

ABSTRACT

PURPOSE: To conduct a phase II trial in chemoresistant hairy cell leukemia (HCL) with BL22, a recombinant anti-CD22 immunotoxin which showed phase I activity in HCL. PATIENTS AND METHODS: Eligible patients had relapsed/refractory HCL and needed treatment based on blood counts. Patients were stratified into three groups: response to cladribine less than 1 year, those with a response lasting 1 to 4 years, or no response and uncontrolled infection. Patients received BL22 40 microg/kg every other day for three doses on cycle 1. Those achieving hematologic remission (HR), defined as neutrophils > or = 1,500/mm(3), hemoglobin > or = 11 g/dL, and platelets > or = 100,000/mm(3), were observed. Patients without HR were re-treated at 30 microg/kg every other day for three doses every 4 weeks beginning at least 8 weeks after cycle 1. RESULTS: Thirty-six patients were enrolled including 26, nine, and one in groups 1 to 3. The response after one cycle (CR, 25%; PR, 25%) improved when 56% were re-treated (CR, 47%; PR, 25%). CR rate was similar in groups 1 and 2 (P = .7). Twenty-two with baseline spleen height lower than 200 mm had higher CR (64% v 21%; P = .019) and OR rates (95% v 36%; P = .0002) compared to 14 with spleens either absent or higher than 200 mm. The only serious toxicity was reversible grade 3 hemolytic uremic syndrome, not requiring plasmapheresis, in two patients (6%). High neutralizing antibodies were observed in four patients (11%) and prevented re-treatment. CONCLUSION: BL22 activity in HCL is confirmed. Best responses to BL22 after cladribine failure are achieved before the patients develop massive splenomegaly or undergo splenectomy.


Subject(s)
Antibodies/therapeutic use , Enterotoxins/therapeutic use , Leukemia, Hairy Cell/drug therapy , Adult , Aged , Antibodies/administration & dosage , Antibodies/toxicity , Antineoplastic Agents/therapeutic use , Cladribine/therapeutic use , Drug Resistance , Enterotoxins/administration & dosage , Enterotoxins/pharmacokinetics , Enterotoxins/toxicity , Female , Humans , Male , Middle Aged , Prospective Studies , Spleen , Treatment Outcome
2.
Blood ; 113(16): 3792-800, 2009 Apr 16.
Article in English | MEDLINE | ID: mdl-18988862

ABSTRACT

Immunotoxins based on Pseudomonas exotoxin A (PE) are promising anticancer agents that combine a variable fragment (Fv) from an antibody to a tumor-associated antigen with a 38-kDa fragment of PE (PE38). The intoxication pathway of PE immunotoxins involves receptor-mediated internalization and trafficking through endosomes/lysosomes, during which the immunotoxin undergoes important proteolytic processing steps but must otherwise remain intact for eventual transport to the cytosol. We have investigated the proteolytic susceptibility of PE38 immunotoxins to lysosomal proteases and found that cleavage clusters within a limited segment of PE38. We subsequently generated mutants containing deletions in this region using HA22, an anti-CD22 Fv-PE38 immunotoxin currently undergoing clinical trials for B-cell malignancies. One mutant, HA22-LR, lacks all identified cleavage sites, is resistant to lysosomal degradation, and retains excellent biologic activity. HA22-LR killed chronic lymphocytic leukemia cells more potently and uniformly than HA22, suggesting that lysosomal protease digestion may limit immunotoxin efficacy unless the susceptible domain is eliminated. Remarkably, mice tolerated doses of HA22-LR at least 10-fold higher than lethal doses of HA22, and these higher doses exhibited markedly enhanced antitumor activity. We conclude that HA22-LR advances the therapeutic efficacy of HA22 by using an approach that may be applicable to other PE-based immunotoxins.


Subject(s)
ADP Ribose Transferases/pharmacology , Antibodies, Monoclonal/pharmacology , Bacterial Toxins/pharmacology , Exotoxins/pharmacology , Immunoglobulin Variable Region/pharmacology , Immunotoxins/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Sialic Acid Binding Ig-like Lectin 2 , Virulence Factors/pharmacology , Xenograft Model Antitumor Assays , ADP Ribose Transferases/adverse effects , ADP Ribose Transferases/genetics , ADP Ribose Transferases/pharmacokinetics , Animals , Antibodies, Monoclonal/adverse effects , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/pharmacokinetics , Bacterial Toxins/adverse effects , Bacterial Toxins/genetics , Bacterial Toxins/pharmacokinetics , Clinical Trials as Topic , Endosomes/metabolism , Exotoxins/adverse effects , Exotoxins/genetics , Exotoxins/pharmacokinetics , Female , Humans , Immunoglobulin Variable Region/adverse effects , Immunoglobulin Variable Region/genetics , Immunotoxins/adverse effects , Immunotoxins/genetics , Immunotoxins/pharmacokinetics , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Lysosomes/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Mutation , Virulence Factors/adverse effects , Virulence Factors/genetics , Virulence Factors/pharmacokinetics , Pseudomonas aeruginosa Exotoxin A
3.
Blood ; 112(6): 2272-7, 2008 Sep 15.
Article in English | MEDLINE | ID: mdl-18596230

ABSTRACT

CD22 is an important immunotherapeutic target on B-cell malignancies, particularly hairy cell leukemia (HCL), but its soluble extracellular domain, sCD22, has not yet been reported in the blood. By immunoaffinity and enzyme-linked immunosorbent assay techniques using anti-CD22 monoclonal antibodies, we identified the 100-kDa extracellular domain of CD22 and an 80-kDa processed form in serum of patients with HCL. The median sCD22 level measured by enzyme-linked immunosorbent assay was 18 ng/mL for 93 patients with HCL. sCD22 levels varied from 2.1 to 163 ng/mL and were higher (P < .001) than 23 normal donors (median, 0.6 ng/mL). More than 95% of normal donors had sCD22 levels less than 1.9 ng/mL. sCD22 levels were proportional to concentrations of circulating HCL cells (P = .002), and HCL spleen size (P < .001). sCD22 levels normalized with complete but not partial response to treatment. sCD22 levels up to 300 ng/mL had less than a 2-fold effect on the cytotoxicity of the anti-CD22 recombinant immunotoxin BL22. sCD22 levels may be useful to follow in patients with HCL and may be more specific than sCD25 in patients with CD22(+)/CD25(-) disease. Trials are listed on www.cancer.gov as NCT00002765, NCT00021983, NCT00074048, NCT00085085, NCT00337311, and NCT00462189.


Subject(s)
Leukemia, Hairy Cell/diagnosis , Sialic Acid Binding Ig-like Lectin 2/blood , Biomarkers, Tumor/blood , Case-Control Studies , Diagnostic Techniques and Procedures , Enzyme-Linked Immunosorbent Assay , Humans , Immunotoxins/toxicity , Leukemia, Hairy Cell/drug therapy , Molecular Weight , Solubility , Tumor Burden
4.
Leuk Res ; 32(9): 1400-6, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18295331

ABSTRACT

PRAME has been proposed as a useful marker for solid tumors and acute B-cell malignancies. Several studies demonstrate expression in CLL. To further examine its B-cell tumor distribution, we studied PRAME in both CLL and hairy cell leukemia (HCL). While by conventional PCR only 8% of 37 HCL and 27% of 22 CLL patients were positive, nearly all patients and normal donors expressed PRAME by real-time quantitative (TaqMan) PCR. We conclude that HCL and CLL differ in PRAME overexpression, and that basal normal expression of PRAME may limit its usefulness for following patients with minimal residual CLL or HCL.


Subject(s)
Antigens, Neoplasm/genetics , Biomarkers, Tumor/genetics , Gene Expression Regulation, Leukemic , Leukemia, Hairy Cell/genetics , Antigens, Neoplasm/metabolism , Biomarkers, Tumor/metabolism , Case-Control Studies , Flow Cytometry , Humans , Leukemia, Hairy Cell/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Lymphoma, Follicular/genetics , Lymphoma, Follicular/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction
5.
Clin Cancer Res ; 12(9): 2804-11, 2006 May 01.
Article in English | MEDLINE | ID: mdl-16675574

ABSTRACT

Cladribine induces long-term complete remission in hairy cell leukemia (HCL) patients but does not clear minimal residual disease (MRD) according to high-sensitivity PCR assays. To quantify MRD in patients after anti-CD22 recombinant immunotoxin BL22 and other agents, we used a relative quantitative PCR (RQ-PCR) assay using a primer and probe, both patient specific for the immunoglobulin heavy chain rearrangement. Using this method, we were able to detect one Bonna 12 HCL cell in either 10(6) Jurkat cells or in 10(6) normal mononuclear cells. We studied 84 samples from 10 patients, taken before or after treatment with BL22 and other agents. Patient-specific RQ-PCR was much more sensitive than flow cytometry, which in turn was (as recently reported) more sensitive than PCR using consensus primers. RQ-PCR was positive in 62 of 62 (100%) flow-positive samples in 10 patients and in 20 of 22 (91%) flow-negative samples in six patients. The relative level of MRD as quantified by RQ-PCR correlated with disease status and remission. Thus, patient-specific RQ-PCR is the most sensitive test for MRD in HCL patients and could be used to determine maximal response in patients obtaining multiple cycles of nonmyelotoxic biological treatment for this disease.


Subject(s)
Leukemia, Hairy Cell/blood , Neoplasm, Residual/diagnosis , Polymerase Chain Reaction/methods , Aged , Antigens, CD/genetics , Base Sequence , Cloning, Molecular , DNA Primers , Humans , Immunoglobulin Heavy Chains/genetics , Leukemia, Hairy Cell/genetics , Leukemia, Hairy Cell/immunology , Male , Middle Aged , Molecular Sequence Data
6.
Clin Cancer Res ; 8(4): 995-1002, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11948105

ABSTRACT

Recombinant immunotoxins are fusion proteins composed of the Fv domains of antibodies fused to bacterial or plant toxins that are being developed for the targeted therapy of cancer. RFB4 (Fv)-Pseudomonas exotoxin 38 (PE38) is an immunotoxin that targets CD22 expressed on B cells and B-cell malignancies. A disulfide-stabilized form of RFB4 (Fv)-PE38 is being evaluated in a Phase I clinical trial. The aim of the present study was to improve the activity of RFB4 (Fv)-PE38 to more effectively treat patients with leukemias and lymphomas. To increase the affinity of RFB4 (Fv), we used the techniques of phage display and hot spot mutagenesis. We identified mutational hot spot sequences in heavy chain complementary determining region 3 (V(H) CDR3) and randomized these in a phage display library. Mutant phages were panned on CD22-positive Daudi cells. A variety of mutant Fvs were obtained, and the corresponding immunotoxins were prepared. Several mutant immunotoxins with increased binding affinity and cytotoxic activity were obtained. The most active immunotoxin contained amino acid residues Thr-His-Trp (THW) in place of Ser-Ser-Tyr (SSY) at positions 100, 100A, and 100B of the Fv and had an affinity improved from 85 nM to 6 nM. The THW mutant had a 5- to 10-fold increase in activity on various CD22-positive cell lines and was up to 50 times more cytotoxic to cells from patients with chronic lymphocytic leukemia and hairy-cell leukemia.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antigens, CD/immunology , Antigens, Differentiation, B-Lymphocyte/immunology , Cell Adhesion Molecules , Immunotoxins/pharmacology , Lectins , Leukemia/drug therapy , Amino Acid Sequence , Antibodies, Monoclonal/genetics , Antibody Affinity , Bacteriophages/genetics , Base Sequence , Cell Line , Gene Expression , Gene Library , Humans , Immunoglobulin Fragments/genetics , Immunoglobulin Fragments/pharmacology , Immunotoxins/genetics , Immunotoxins/immunology , Inhibitory Concentration 50 , Leukemia/pathology , Mutation , Plasmids/genetics , Protein Biosynthesis , Proteins/drug effects , Sialic Acid Binding Ig-like Lectin 2 , Tumor Cells, Cultured/drug effects
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