Richard Beers

An immunotoxin with greatly reduced immunogenicity by identification and removal of B cell epitopes

Recombinant immunotoxins are hybrid proteins composed of an Fv that binds to a tumor antigen fused to a bacterial or plant toxin. Immunotoxin BL22 targets CD22 positive malignancies and is composed of an anti-CD22 Fv fused to a 38-kDa fragment of Pseudomonas exotoxin A (PE38). BL22 has produced many complete remissions in drug-resistant Hairy cell leukemia, where many treatment cycles can be given, because neutralizing antibodies do not form. In marked contrast, only minor responses have been observed in trials with immunotoxins targeting solid tumors, because only a single treatment cycle can be given before antibodies develop. To allow more treatment cycles and increase efficacy, we have produced a less immunogenic immunotoxin by identifying and eliminating most of the B cell epitopes on PE38. This was accomplished by mutation of specific large hydrophilic amino acids (Arg, Gln, Glu, Lys) to Ala, Ser, or Gly. The new immunotoxin (HA22–8X) is significantly less immunogenic in three strains of mice, yet retains full cytotoxic and anti-tumor activities. Elimination of B-cell epitopes is a promising approach to the production of less immunogenic proteins for therapeutic purposes.

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.

INCREASED BINDING AFFINITY ENHANCES TARGETING OF GLIOMA XENOGRAFTS BY EGFRVIII-SPECIFIC scFv

Combinatorial variation of CDR3 of VH and VL, followed by phage display, was used to select affinity mutants of the parental anti‐epidermal growth factor receptor‐vIII (EGFRvIII) scFv MR1. One mutant, MR1‐1(scFv), had increased specific binding affinity for EGFRvIII. It was produced and radiolabeled, and its biodistribution was evaluated in human glioma‐bearing athymic mice. MR1‐1 targeted the same EGFRvIII epitope as MR1 with an approximately 15‐fold higher affinity (Kd = 1.5 × 10−9 M) measured by surface resonance analysis. Labeling with 131I or 125I was performed, and the immunoreactive fraction of the labeled MR1‐1(scFv) was 50% to 55%. After incubation at 37°C for 4 days, the binding affinity was maintained at 60% of initial levels. The specificity of MR1‐1 for EGFRvIII was demonstrated in vitro by flow cytometry and incubation of FITC‐labeled scFv with the EGFRvIII‐expressing U87MG.ΔEGFR cell line or with the EGFRvIII‐negative U87MG cell line in the presence or absence of competing unlabeled MR1‐1(scFv). We also investigated the internalization and processing of MR1‐1 compared with MR1; MR1‐1 exhibited levels of both cell surface retention and internalization up to 5 times higher than those by MR1. In biodistribution studies performed in athymic mice bearing s.c. U87MG.ΔEGFR tumor xenografts, animals received paired‐label intratumoral infusions of 131I‐labeled MR1‐1(scFv) and 125I‐labeled MR1(scFv). Our results showed an up to 244% ± 77% increase in tumor uptake for MR1‐1 compared with that for MR1. The improved tumor retention of MR1‐1(scFv) combined with its rapid clearance from normal tissues also resulted in sustained higher tumor:normal organ ratios. These results suggest that the improved affinity of MR1‐1 can significantly impact in vivo glioma‐specific targeting and immunotherapy. Int. J. Cancer 88:962–969, 2000.

Recombinant immunotoxin against B-cell malignancies with no immunogenicity in mice by removal of B-cell epitopes

Many nonhuman proteins have useful pharmacological activities, but are infrequently effective in humans because of their high immunogenicity. A recombinant immunotoxin (HA22, CAT8015, moxetumomab pasudotox) composed of an anti-CD22 antibody variable fragment fused to PE38, a 38-kDa portion of Pseudomonas exotoxin A, has produced many complete remissions in drug-resistant hairy-cell leukemia when several cycles of the agent can be given, but has much less activity when antibodies develop. We have pursued a strategy to deimmunize recombinant immunotoxins by identifying and removing B-cell epitopes. We previously reported that we could eliminate most B-cell epitopes using a combination of point mutations and deletions. Here we show the location and amino acid composition of all of the B-cell epitopes in the remaining 25-kDa portion of Pseudomonas exotoxin. Using this information, we eliminated these epitopes to produce an immunotoxin (HA22-LR-8M) that is fully cytotoxic against malignant B-cell lines, has high cytotoxic activity against cells directly isolated from patients with chronic lymphocytic leukemia, and has excellent antitumor activity in mice. HA22-LR-8M does not induce antibody formation in mice when given repeatedly by intravenous injection and does not induce a secondary antibody response when given to mice previously exposed to HA22. HA22-LR-8M also has greatly reduced antigenicity when exposed to sera from patients who have produced antibodies to HA22. The properties of HA22-LR-8M make it an excellent candidate for further clinical development.

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.

New Monoclonal Antibodies to Mesothelin Useful for Immunohistochemistry, Fluorescence-Activated Cell Sorting, Western Blotting, and ELISA

Purpose: Mesothelin is a cell surface protein that is highly expressed in some malignant tumors, and is a promising target for immunotherapy. Recent data suggests that mesothelin is an adhesive protein and may have a role in the metastases of ovarian cancer. Although a few monoclonal antibodies (MAb) to mesothelin have been produced, they have limitations for the study of expression of native mesothelin because of their low affinity or reactivity only with denatured mesothelin protein. We have produced novel MAbs to mesothelin to help study mesothelin function and to develop improved diagnosis and immunotherapy of mesothelin-expressing tumors. Experimental Design: Mesothelin-deficient mice were immunized with plasmid cDNA encoding mesothelin, and boosted with a mesothelin-rabbit IgG Fc fusion protein prior to cell fusion. Hybridomas were screened by an ELISA using plates coated with mesothelin-Fc protein. Results: Seventeen hybridomas producing anti-mesothelin antibodies were established and shown to react with two epitopes on mesothelin. One group reacts with the same epitope as the low affinity antibody K1 that was originally used to identify mesothelin. The other is a new group that reacts with a new epitope. One antibody from each group was chosen for further study and shown to react strongly on ELISA, on immunohistochemistry, and by fluorescence-activated cell sorting on living cells. Conclusion: Our two newly established MAbs, MN and MB, have different and useful properties compared with current antibodies used for the detection of mesothelin by immunohistochemistry, fluorescence-activated cell sorting, ELISA, and Western blotting.

Identification and elimination of an immunodominant T-cell epitope in recombinant immunotoxins based on Pseudomonas exotoxin A

Recombinant immunotoxins (RITs) are chimeric proteins that are being developed for cancer treatment. We have produced RITs that contain PE38, a portion of the bacterial protein Pseudomonas exotoxin A. Because the toxin is bacterial, it often induces neutralizing antibodies, which limit the number of treatment cycles and the effectiveness of the therapy. Because T cells are essential for antibody responses to proteins, we adopted an assay to map the CD4+ T-cell epitopes in PE38. We incubated peripheral blood mononuclear cells with an immunotoxin to stimulate T-cell expansion, followed by exposure to overlapping peptide fragments of PE38 and an IL-2 ELISpot assay to measure responses. Our observation of T-cell responses in 50 of 50 individuals correlates with the frequency of antibody formation in patients with normal immune systems. We found a single, highly immunodominant epitope in 46% (23/50) of the donors. The immunodominant epitope is DRB1-restricted and was observed in subjects with different HLA alleles, indicating promiscuity. We identified two amino acids that, when deleted or mutated to alanine, eliminated the immunodominant epitope, and we used this information to construct mutant RITs that are highly cytotoxic and do not stimulate T-cell responses in many donors.

Recombinant immunotoxin for cancer treatment with low immunogenicity by identification and silencing of human T-cell epitopes.

Significance Recombinant immunotoxins have produced complete remissions in leukemia patients where many doses can be given but are less active in patients with solid tumors because their immune system makes antidrug antibodies, which inactivate the immunotoxin. To suppress the immune response, we have identified and largely silenced the T-cell epitopes responsible for the immune response. A redesigned immunotoxin with T-cell epitope mutations is highly cytotoxic to cell lines and to cells isolated from cancer patients and produces complete remissions in mice with human cancer xenografts. The approach described can be applied to deimmunize other therapeutically useful foreign proteins. Nonhuman proteins have valuable therapeutic properties, but their efficacy is limited by neutralizing antibodies. Recombinant immunotoxins (RITs) are potent anticancer agents that have produced many complete remissions in leukemia, but immunogenicity limits the number of doses that can be given to patients with normal immune systems. Using human cells, we identified eight helper T-cell epitopes in PE38, a portion of the bacterial protein Pseudomonas exotoxin A which consists of the toxin moiety of the RIT, and used this information to make LMB-T18 in which three epitopes were deleted and five others diminished by point mutations in key residues. LMB-T18 has high cytotoxic and antitumor activity and is very resistant to thermal denaturation. The new immunotoxin has a 93% decrease in T-cell epitopes and should have improved efficacy in patients because more treatment cycles can be given. Furthermore, the deimmunized toxin can be used to make RITs targeting other antigens, and the approach we describe can be used to deimmunize other therapeutically useful nonhuman proteins.

A Recombinant Immunotoxin against the Tumor-Associated Antigen Mesothelin Reengineered for High Activity, Low Off-Target Toxicity, and Reduced Antigenicity

SS1P is a recombinant immunotoxin (RIT) engineered for the targeted elimination of malignant cells that express the tumor-associated antigen mesothelin. It is composed of an antimesothelin antibody variable fragment (Fv) linked to a cytotoxic fragment of Pseudomonas exotoxin A (PE) that includes domains II and III of native PE. The clinical use of SS1P is limited by its propensity to induce neutralizing antibodies and to cause a dose-limiting capillary leak syndrome (CLS) in patients. In this article, we describe a reengineered SS1P with improved properties that overcome these deficits. The redesign of SS1P consists of (i) removing the bulk of PE domain II (residues 251–273 and 284–394 of native PE), leaving only an 11-residue furin cleavage site, (ii) adding a Gly–Gly–Ser peptide linker after the furin cleavage site, and (iii) replacing eight highly solvent-exposed residues in the catalytic domain of PE. The new molecule, SS1-LR/GGS/8M, has cytotoxic activity comparable with SS1P on several mesothelin-expressing cell lines and remarkably improved activity on primary cells from patients with mesothelioma. In a mouse xenograft tumor model, high doses of SS1-LR/GGS/8M elicit antitumor activity superior to the activity of SS1P at its maximum-tolerated dose. In addition, SS1-LR/GGS/8M has greatly decreased ability to cause CLS in a rat model and reduced antigenicity or reactivity with antibodies to the sera of patients previously treated with SS1P. Mol Cancer Ther; 12(1); 48–57. ©2012 AACR.

A recombinant immunotoxin targeting CD22 with low immunogenicity, low nonspecific toxicity, and high antitumor activity in mice.

Recombinant immunotoxins (RITs) are genetically engineered proteins designed to kill cancer cells. The RIT HA22 contains the Fv portion of an anti-CD22 antibody fused to a 38 kDa fragment of Pseudomonas exotoxin A (PE38). As PE38 is a bacterial protein, patients frequently produce antibodies that neutralize its activity, preventing retreatment. We have earlier shown in mice that PE38 contains 7 major B-cell epitopes located in domains II and III of the protein. Here we present a new mutant RIT, HA22-LR-6X, in which we removed most B-cell epitopes by deleting domain II and mutating 6 residues in domain III. HA22-LR-6X is cytotoxic to several lymphoma cell lines, has very low nonspecific toxicity, and retains potent antitumor activity in mice with CA46 lymphomas. To assess its immunogenicity, we immunized 3 MHC-divergent strains of mice with 5 μg doses of HA22-LR-6X, and found that HA22-LR-6X elicited significantly lower antibody responses than HA22 or other mutant RITs with fewer epitopes removed. Furthermore, large (50 μg) doses of HA22-LR-6X induced markedly lower antibody responses than 5 μg of HA22, indicating that high doses can be administered with low immunogenicity. Our experiments show that we have correctly identified and removed B-cell epitopes from PE38, producing a highly active immunotoxin with low immunogenicity and low animal toxicity. Future studies will determine if these properties carry over to humans with cancer.