David J. FitzGerald

Immunotoxin therapy of cancer

Rationally designed anticancer agents that target cell-surface antigens or receptors represent a promising approach for treating cancer patients. However, antibodies that bind these targets are often, by themselves, non-cytotoxic. By attaching potent toxins we can dramatically improve the clinical utility of some anti-tumour antibodies. Here we describe the construction and clinical utility of several recombinant immunotoxins; each of which is composed of antibody Fv fragments fused to powerful bacterial toxins. Results from clinical trials indicate that recombinant immunotoxins and similar agents that are designed to combine antibody selectivity with toxin cell-killing potency will be useful additions to cancer therapy.

Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia.

Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3 constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL.

Adenovirus-induced release of epidermal growth factor and pseudomonas toxin into the cytosol of KB cells during receptor-mediated endocytosis

Abstract Using electron microscopy, we find that adenovirus and epidermal growth factor attached to colloidal gold (EGF-Au) enter KB cells together via coated pits and receptosomes. At 5 min, adenovirus begins to appear free in the cytosol and promotes the release of EGF-Au into the cytosol. Pseudomonas toxin (PE) also is internalized via coated pits and receptosomes. Adenovirus enhances the toxicity of PE 100-fold, and enhances a conjugate of PE and epidermal growth factor (PE-EGF), which enters cells via the EGF receptor, 10,000-fold. These results suggest that adenovirus disrupts receptosomes, enhancing delivery of material contained in them into the cytosol.

A protease-resistant immunotoxin against CD22 with greatly increased activity against CLL and diminished animal toxicity

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.

Characterization of the B Cell Epitopes Associated with a Truncated Form of Pseudomonas Exotoxin (PE38) Used to Make Immunotoxins for the Treatment of Cancer Patients

Recombinant immunotoxins composed of an Ab Fv fragment joined to a truncated portion of Pseudomonas exotoxin A (termed PE38) have been evaluated in clinical trials for the treatment of various human cancers. Immunotoxin therapy is very effective in hairy cell leukemia and also has activity in other hemological malignancies; however, a neutralizing Ab response to PE38 in patients with solid tumors prevents repeated treatments to maximize the benefit. In this study, we analyze the murine Ab response as a model to study the B cell epitopes associated with PE38. Sixty distinct mAbs to PE38 were characterized. Mutual competitive binding of the mAbs indicated the presence of 7 major epitope groups and 13 subgroups. The competition pattern indicated that the epitopes are discrete and could not be reproduced using a computer simulation program that created epitopes out of random surface residues on PE38. Using sera from immunotoxin-treated patients, the formation of human Abs to each of the topographical epitopes was demonstrated. One epitope subgroup, E1a, was identified as the principal neutralizing epitope. The location of each epitope on PE38 was determined by preparing 41 mutants of PE38 in which bulky surface residues were mutated to either alanine or glycine. All 7 major epitope groups and 9 of 13 epitope subgroups were identified by 14 different mutants and these retained high cytotoxic activity. Our results indicate that a relatively small number of discrete immunogenic sites are associated with PE38, most of which can be eliminated by point mutations.

Complete regression of human B-cell lymphoma xenografts in mice treated with recombinant anti-CD22 immunotoxin RFB4(dsFv)-PE38 at doses tolerated by cynomolgus monkeys.

RFB4(dsFv)‐PE38 is a recombinant immunotoxin in which the variable light domain (VL) is disulfide bonded via cysteine residues to the variable heavy domain (VH), which in turn is fused to PE38, a mutant form of Pseudomonas exotoxin A. RFB4 binds to CD22, which is a differentiation antigen expressed on the majority of B‐cell leukemias and lymphomas. To examine the potential efficacy of RFB4(dsFv)‐PE38 when administered at a dose schedule appropriate for phase I testing, mice bearing CA46 human CD22+ Burkitts lymphoma xenografts were treated on alternate days i.v. for 3 doses (QOD ×3). Complete regressions were observed in 80% and 100% of mice treated with 200 and 275 μg/kg QOD ×3, respectively. The higher dose was 27% of the LD50 and 34% of the LD10 in mice. Because RFB4(dsFv)‐PE38 is stable at 37°C, it could also be given by continuous infusion using pumps placed in the peritoneal cavity; complete regressions also resulted from this mode of administration. To study toxicology, a pilot toxicology study of RFB4(dsFv)‐PE38 was undertaken in cynomolgus monkeys, which like humans but unlike mice have CD22, which binds RFB4. Doses of 100 and 500 μg/kg i.v. QOD ×3 were well tolerated, indicating that a dose that cured tumors in mice was tolerated by primates. Based on these preclinical results, RFB4(dsFv)‐PE38 is being developed for the treatment of patients with CD22‐positive leukemias and lymphomas. Int. J. Cancer 81:148–155, 1999.

Differential Cellular Internalization of Anti-CD19 and -CD22 Immunotoxins Results in Different Cytotoxic Activity

B-cell malignancies routinely express surface antigens CD19 and CD22. Immunotoxins against both antigens have been evaluated, and the immunotoxins targeting CD22 are more active. To understand this disparity in cytotoxicity and guide the screening of therapeutic targets, we compared two immunotoxins, FMC63(Fv)-PE38-targeting CD19 and RFB4(Fv)-PE38 (BL22)-targeting CD22. Six lymphoma cell lines have 4- to 9-fold more binding sites per cell for CD19 than for CD22, but BL22 is 4- to 140-fold more active than FMC63(Fv)-PE38, although they have a similar cell binding affinity (Kd, approximately 7 nmol/L). In 1 hour, large amounts of BL22 are internalized (2- to 3-fold more than the number of CD22 molecules on the cell surface), whereas only 5.2% to 16.6% of surface-bound FMC63(Fv)-PE38 is internalized. The intracellular reservoir of CD22 decreases greatly after immunotoxin internalization, indicating that it contributes to the uptake of BL22. Treatment of cells with cycloheximide does not reduce the internalization of BL22. Both internalized immunotoxins are located in the same vesicles. Our results show that the rapid internalization of large amounts of BL22 bound to CD22 makes CD22 a better therapeutic target than CD19 for immunotoxins and probably for other immunoconjugates that act inside cells.

Recombinant immunotoxins for treating cancer

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

The morphologic pathway of binding and internalization of epidermal growth factor in cultured cells: Studies on A431, KB, and 3T3 cells, using multiple methods of labelling

We have prepared several electron and light microscopic labels of epidermal growth factor (EGF) to analyse the morphologic features of its binding and internalization by cultured cells. These include a ferritin conjugate of EGF, a covalent conjugate of EGF and horseradish peroxidase (EGF-HRP), a colloidal gold marker system using EGF-HRP as a primary antigen, and a covalent complex of EGF with rhodamine-labelled lactalbumin. All of the light and electron microscopic labels showed similar patterns of binding. EGF initially bound to diffusely distributed cell surface receptors at 4 degrees C. The EGF-receptor complexes clustered into clathrin-coated pits on the cell surface only when the temperature was raised to 37 degrees C. In KB and Swiss 3T3 cells, this was followed by rapid internationalization into receptosomes, compartmentalization into the Golgi system, clustering in the clathrin-coated regions of the Golgi, and finally delivery into lysosomes from the Golgi. This general pathway was seen in Swiss 3T3 cells which have a low number of EGF receptors, KB cells which have a moderate number of receptors and A431 cells that have a high number of receptors. However, the ruffling activity induced in A431 cells by EGF produced some internalization through macropinosomes, making the pathway of entry more difficult to evaluate. Double label experiments showed that EGF is internalized together with alpha 2-macroglobulin and adenovirus particles. These data clarify the route of entry of EGF in different cell types using multiple labels, and shows that it enters cells through the same coated pit entry pathway as most other ligands previously examined.

Immunotoxins for leukemia.

Unconjugated monoclonal antibodies that target hematopoietic differentiation antigens have been developed to treat hematologic malignancies. Although some of these have activity against chronic lymphocytic leukemia and hairy cell leukemia, in general, monoclonal antibodies have limited efficacy as single agents in the treatment of leukemia. To increase their potency, the binding domains of monoclonal antibodies can be attached to protein toxins. Such compounds, termed immunotoxins, are delivered to the interior of leukemia cells based on antibody specificity for cell surface target antigens. Recombinant immunotoxins have been shown to be highly cytotoxic to leukemic blasts in vitro, in xenograft model systems, and in early-phase clinical trials in humans. These agents will likely play an increasing role in the treatment of leukemia.