Supplementary Table 1 from Differential Cellular Internalization of Anti-CD19 and -CD22 Immunotoxins Results in Different Cytotoxic Activity

Table S1. Cytotoxic activity in vitro in five cell lines; Table S2. In vivo toxicity of RITs; Fig S1. Cytotoxic activity in patients cells curves

PDF file - 231K, Human antigenicity of SS1-LR/GGS/8M

Significance Mesothelin (MSLN) is a cell-surface protein that is a popular target for antibody-based therapies. We have identified shed MSLN as a major obstacle to successful antibody therapies and prepared a monoclonal antibody that inhibits shedding and makes very active CAR T cells whose activity is not blocked by shed MSLN and merits further preclinical development.

Moxetumomab pasudotox is a fusion protein of a CD22-targeting antibody and Pseudomonas exotoxin. Minutes of exposure to Moxetumomab achieves similar cell killing than hours of exposure to a novel deimmunized variant against some acute lymphoblastic leukemia (ALL). Because blood levels fall quickly, Moxetumomab is more than 1000-fold more active than the deimmunized variant in vivo. We aimed to identify which part of Moxetumomab increases in vivo efficacy and generated five immunotoxins, tested time-dependent activity, and determined the efficacy in a KOPN-8 xenograft model. Full domain II shortened the time cells had to be exposed to die to only a few minutes for some ALL; deimmunized domain III consistently extended the time. Against KOPN-8, full domain II accelerated time to arrest protein synthesis by three-fold and tripled PARP-cleavage. In vivo efficacy was increased by more than 10-fold by domain II and increasing size, and therefore half-life enhanced efficacy two- to four-fold. In summary, in vivo efficacy is determined by the time cells have to be exposed to immunotoxin to die and serum half-life. Thus, domain II is most critical for activity against some ALL treated with bolus doses; however, immunotoxins lacking all but the furin-cleavage site of domain II may be advantageous when treating continuously.

AbstractSS1P is a recombinant immunotoxin (RIT) that targets mesothelin. It consists of an antimesothelin Fv fused to a portion of Pseudomonas exotoxin A. In clinical studies, it has produced dramatic responses in patients with advanced mesothelioma, when combined with immunosuppressive therapy so that several treatment cycles could be given. Otherwise its activity is limited by its immunogenicity. In this work, we describe the development and characterization of LMB-T20, a highly potent RIT targeted at mesothelin-expressing cancers with low immunogenicity due to removal of its eight T-cell epitopes. LMB-T20 was more active than SS1P when tested on four different mesothelin-expressing cell lines as well as on cells obtained from patients with mesothelioma. It also has potent antitumor activity in mice, and has reduced immunogenicity as measured by cytokine secretion assays. In conclusion, LMB-T20 is a favorable candidate for evaluation in clinical trials due to its reduced immunogenicity and excellent activity. Mol Cancer Ther; 14(12); 2789–96. ©2015 AACR.

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.

Abstract 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.

Moxetumomab pasudotox and SS1P are immunotoxins that have been developed in our lab to treat cancer. Moxetumomab pasudotox, which can be given for many treatment cycles, targets CD22 and has produced many complete remissions in drug-resistant hairy cell leukemia. However, SS1P, which targets mesothelin, could only be given for one cycle before ADA (anti drug antibodies) developed and neutralized the protein. We hypothesize that SS1P could be more effective if it were less immunogenic and more treatment cycles could be given. To de-immunize the immunotoxin we sought to identify and remove T cell epitopes. To identify the epitopes we stimulated PBMCs from 50 normal donors with whole immunotoxin for 14 days to expand the T cells and then stimulated the T cells with peptides (15 mers) derived from the toxin, followed by ELISpot to identify the peptides that stimulated the T cells. We found that all 50 donors gave a positive response, which correlates with the frequency of ADAs in patients with normal immune systems. We used alanine-scanning mutagenesis to determine which amino acids in each peptide are important for T cell stimulation. SS1P contains domains II and III of PE. In 46% of the donors we found a single highly immunodominant and promiscuous epitope that is located in domain II. We previously showed we could eliminate almost all of domain II while retaining excellent cytotoxic activity. These data indicate that immunogenicity should be eliminated in 34% of normal individuals donors and diminished in another 42% by deletion of domain II. In domain III we identified 5 peptides that stimulated T cell responses in different donors and also identified alanine mutations within each peptide that eliminated or greatly lowered T cell stimulation in most donors. We are using this information to make immunotoxins with mutations in domain III that should have a further decrease in immunogenicity. We have shown that among many individuals with different HLA alleles there is a dominant T cell epitope in domain II of PE38 and 5 subdominant epitopes in domain III. We plan to use this information to make new immunotoxins in which the large majority of T cell epitopes are silenced resulting in diminished immunogenicity of the immunotoxin. We plan to combine these T cell mutations with B cell mutations previously described to make immunotoxins with very low immunogenicity allowing more treatment cycles to be given and better anti-tumor activity achieved. Citation Format: Ronit Mazor, Aaron N. Vassall, Jaime A. Eberle, Richard Beers, Xiaobo Hu, Itai Benhar, Ira Pastan. Identification and elimination of T-cell epitopes in recombinant immunotoxins used to treat cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4334. doi:10.1158/1538-7445.AM2013-4334

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.

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.

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 microg 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 microg) doses of HA22-LR-6X induced markedly lower antibody responses than 5 microg 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.

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.

Abstract 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, ∼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. [Cancer Res 2008;68(15):6300–5]

Recombinant immunotoxins exhibit targeting and cytotoxic functions needed for cell-specific destruction. However, antitumor efficacy, safety, and pharmacokinetics of these therapeutics might be improved by further macromolecular engineering. SS1P is a recombinant anti-mesothelin immunotoxin in clinical trials in patients with mesothelin-expressing tumors. We have modified this immunotoxin using several PEGylation strategies employing releasable linkages between the protein and the PEG polymers, and observed superior performance of these bioconjugates when compared to similar PEG derivatives bearing permanent linkages to the polymers. PEGylated derivatives displayed markedly diminished cytotoxicity on cultured mesothelin-overexpressing A431-K5 cells; however, the releasable PEGylated immunotoxins exhibited increased antitumor activity in A431-K5 xenografts in mice, with a diminished animal toxicity. Most significantly, complete tumor regressions were achievable with single dose administration of the bioconjugates but not the native immunotoxin. Pharmacokinetic analysis of the releasable PEGylated derivatives in mice demonstrated an over 80-fold expansion of the area under the curve exposure of bioactive protein when compared to native immunotoxin. A correlation in degree of derivatization, release kinetics, and polymer size with potency was observed in vivo, whereas in vitro cytotoxicity was not predictive of efficacy in animal models. The potent antitumor efficacy of the releasable PEGylated mesothelin-targeted immunotoxins was not exhibited by similar untargeted PEG immunotoxins in this model. Since the bioconjugates can also exhibit the attributes of passive targeting via enhanced permeability and retention, this is the first demonstration of a pivotal role of active targeting for immunotoxin bioconjugate efficacy.