Ronit Mazor

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.

Removing T-cell epitopes with computational protein design.

Significance Proteins represent the fastest-growing class of pharmaceuticals for a diverse range of clinical applications. Computational protein design has the potential to create a novel class of therapeutics with tunable biophysical properties. However, the immune system reacts to T-cell epitope sequences in non-human proteins, leading to neutralization and elimination by the immune system. Here, we combine machine learning with structure-based protein design to identify and redesign T-cell epitopes without disrupting function of the target protein. We test the method experimentally, removing T-cell epitopes from GFP and Pseudomonas exotoxin A while maintaining function. Immune responses can make protein therapeutics ineffective or even dangerous. We describe a general computational protein design method for reducing immunogenicity by eliminating known and predicted T-cell epitopes and maximizing the content of human peptide sequences without disrupting protein structure and function. We show that the method recapitulates previous experimental results on immunogenicity reduction, and we use it to disrupt T-cell epitopes in GFP and Pseudomonas exotoxin A without disrupting function.

Increased Levels of Numerical Chromosome Aberrations after In Vitro Exposure of Human Peripheral Blood Lymphocytes to Radiofrequency Electromagnetic Fields for 72 Hours

Abstract Mazor, R., Korenstein-Ilan, A., Barbul, A., Eshet, Y., Shahadi, A., Jerby, E. and Korenstein, R. Increased Levels of Numerical Chromosome Aberrations after In Vitro Exposure of Human Peripheral Blood Lymphocytes to Radiofrequency Electromagnetic Fields for 72 Hours. Radiat. Res. 169, 28–37 (2008). We investigated the effects of 72 h in vitro exposure of 10 human lymphocyte samples to radiofrequency electromagnetic fields (800 MHz, continuous wave) on genomic instability. The lymphyocytes were exposed in a specially designed waveguide resonator at specific absorption rates (SARs) of 2.9 and 4.1 W/kg in a temperature range of 36–37°C. The induced aneuploidy of chromosomes 1, 10, 11 and 17 was determined by interphase FISH using semi-automated image analysis. We observed increased levels of aneuploidy depending on the chromosome studied as well as on the level of exposure. In chromosomes 1 and 10, there was increased aneuploidy at the higher SAR, while for chromosomes 11 and 17, the increases were observed only for the lower SAR. Multisomy (chromosomal gains) appeared to be the primary contributor to the increased aneuploidy. The effect of temperature on the level of aneuploidy was examined over the range of 33.5–40°C for 72 h with no statistically significant difference in the level of aneuploidy compared to 37°C. These findings suggest the possible existence of an athermal effect of RF radiation that causes increased levels of aneuploidy. These results contribute to the assessment of potential health risks after continuous chronic exposure to RF radiation at SARs close to the current levels set by ICNIRP guidelines.

Elimination of murine and human T-cell epitopes in recombinant immunotoxin eliminates neutralizing and anti-drug antibodies in vivo.

Antibodies against the toxin portion of recombinant immunotoxins (RIT) reduce their efficacy and pose a potential safety risk. To overcome this problem we mutated the very immunogenic immunotoxin SS1P to produce LMB-T20, a de-immunized RIT that has the eight human T-cell epitopes in SS1P modified or removed. To determine the effect of T-cell epitope removal in vivo we mapped the T-cell epitopes in immune-competent BALB/c mice and found that these mice recognize two epitopes. One corresponds to the human immunodominant T-cell epitope and the other to a human subdominant epitope; both were eliminated in LMB-T20. We found that mice immunized with LMB-T20 did not have T-cell activation and did not develop anti-drug antibodies (ADA), whereas mice immunized with SS1P, showed T-cell activation, and developed ADA detected by both ELISA and drug neutralizing assays. The ability of the mice treated with LMB-T20 to respond to other antigens was not compromised. We conclude that elimination of T-cell epitopes is sufficient to prevent formation of antibodies to an immunogenic foreign protein.

Dual B- and T-cell de-immunization of recombinant immunotoxin targeting mesothelin with high cytotoxic activity

Recombinant immunotoxins (RITs) are genetically engineered proteins being developed to treat cancer. They are composed of an Fv that targets a cancer antigen and a portion of a protein toxin. Their clinical success is limited by their immunogenicity. Our goal is to produce a new RIT that targets mesothelin and is non-immunogenic by combining mutations that decrease B- and T-cell epitopes. Starting with an immunotoxin that has B-cell epitopes suppressed, we added mutations step-wise that suppress T-cell epitopes. The final protein (LMB-T14) has greatly reduced antigenicity as assessed by binding to human anti-sera and a greatly decreased ability to activate helper T-cells evaluated in a T-cell activation assay. It is very cytotoxic to mesothelioma cells from patients, and to cancer cell lines. LMB-T14 produces complete remissions of a mesothelin expressing cancer (A431/H9) xenograft. The approach used here can be used to de-immunize other therapeutic foreign proteins.

Tolerogenic nanoparticles restore the antitumor activity of recombinant immunotoxins by mitigating immunogenicity.

Significance Protein-based drugs are very active in treating cancer, but their efficacy is limited by the formation of neutralizing antidrug antibodies (ADAs). Recombinant immunotoxins are proteins that are very effective in patients with leukemia, in whom immunity is suppressed, but induce ADAs, which compromise their activity, in patients with intact immunity. Here we used an immunomodulator that is encapsulated in a nanoparticle delivery system (SVP-R) to induce specific immune tolerance to immunotoxins in mice. SVP-R induces immune tolerance, prevents ADA formation, and prevents the drug neutralization and clearance that results in restoration of its antitumor activity. Importantly, the combination is also efficacious in mice with preexisting antibodies, indicating that this approach can benefit patients who often have such antibodies. Protein-based drugs are very active in treating cancer, but their efficacy can be limited by the formation of neutralizing antidrug antibodies (ADAs). Recombinant immunotoxins are proteins that are very effective in patients with leukemia, where immunity is suppressed, but induce ADAs, which compromise their activity, in patients with intact immunity. Here we induced a specific, durable, and transferable immune tolerance to recombinant immunotoxins by combining them with nanoparticles containing rapamycin (SVP-R). SVP-R mitigated the formation of inhibitory ADAs in naïve and sensitized mice, resulting in restoration of antitumor activity. The immune tolerance is mediated by colocalization of the SVP-R and immunotoxin to dendritic cells and macrophages in the spleen and is abrogated by depletion of regulatory T cells. Tolerance induced by SVPs was not blocked by checkpoint inhibitors or costimulatory agonist monoclonal antibodies that by themselves enhance ADA formation.

Role of HLA-DP in the Presentation of Epitopes from the Truncated Bacterial PE38 Immunotoxin

Identification of helper T-cell epitopes is important in many fields of medicine. We previously used an experimental approach to identify T-cell epitopes in PE38, a truncated bacterial toxin used in immunotoxins. Here, we evaluated the ability of antibodies to DR, DP, or DQ to block T-cell responses to PE38 epitopes in 36 PBMC samples. We predicted the binding affinities of peptides to DR, DP, and DQ alleles using computational tools and analyzed their ability to predict the T-cell epitopes. We found that HLA-DR is responsible for 65% of the responses, DP 24%, and DQ 4%. One epitope that is presented in 20% of the samples (10/50) is entirely DP restricted and was not predicted to bind to DR or DP reference alleles using binding algorithms. We conclude that DP has an important role in helper T-cell response to PE38.

Abstract 68: Methotrexate prevents primary immune responses against recombinant immunotoxin in murine models

Recombinant immunotoxins (rITs) are composed of a tumor antigen-targeting antibody fragment fused to a portion of Pseudomonas exotoxin A. rITs have been effective in clinical trials for patients with hematologic malignancies. The CD22 targeting rIT Moxetumomab Pasudotox has achieved overall response rates of 86% and complete remission rates of 46% in patients with relapsed-refractory Hairy Cell Leukemia. However, the therapeutic efficacy of rITs against solid tumors is limited by their immunogenicity in immune-competent patients. In clinical trials to treat mesothelioma patients with SS1P, a rIT targeting mesothelin, 90% of patients developed neutralizing antibodies against SS1P after one cycle of treatment. When immunosuppressive chemotherapy and SS1P were combined, more cycles of rIT could be given and several patients with advanced chemo-refractory mesothelioma had striking tumor regressions. This implicates high therapeutic potential for rITs against solid tumors once immunogenicity is surmounted. Methotrexate (MTX) is a folate antagonist which interferes with purine biosynthesis, and is used to treat osteosarcomas and other cancers. MTX also interferes with T cell responses and is used to treat autoimmune diseases. Based on its immunosuppressive properties, Joly et al. demonstrated that low-dose MTX prevented the formation of ADAs against recombinant human alglucosidase alfa in mice in an antigen-specific manner. We hypothesized that MTX would similarly prevent the formation of ADAs against rITs in an antigen-specific manner. To test our hypotheses, mice were treated with the mesothelin-targeting rIT RG7787 with or without MTX given 0, 24, and 48 hours after RG7787 treatment. Serum was collected and anti-RG7787 ADAs were measured by direct ELISA. We found that six doses of RG7787 combined with low dose MTX (1 mg/kg) inhibited the formation of ADAs against RG7787. This inhibition was sustained through six challenges with RG7787 without additional MTX. Further, we found that immunization with RG7787 plus MTX induced RG7787-specific tolerance, and had no effect on the ADA response against a second protein, ovalbumin. We conclude that combination of MTX and RG7787 is effective at preventing primary immune responses in a durable, antigen-specific manner. We propose to combine this agent in immune-competent cancer patients receiving rIT therapy to prevent rIT immunogenicity. Citation Format: Emily M. King, Ronit Mazor, Ira Pastan. Methotrexate prevents primary immune responses against recombinant immunotoxin in murine models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 68. doi:10.1158/1538-7445.AM2017-68

Abstract 72: Induction of immune tolerance to recombinant immunotoxin LMB-100 using synthetic vaccine particles encapsulating rapamycin

Recombinant Immunotoxins (RITs) are genetically engineered proteins designed for cancer therapy. LMB-100 is a second generation RIT that is composed of a humanized Fab targeting mesothelin and a de-immunized fragment of Pseudomonas Exotoxin A. Due to the bacterial origin of the toxin, LMB-100 is immunogenic, although less immunogenic than first generation immunotoxins. Almost all patients treated with LMB-100 made anti-drug antibodies (ADAs) after two or more cycles of treatment that neutralized the RIT and greatly lowered efficacy. Kishimoto et al. demonstrated that Synthetic Vaccine Particles containing Rapamycin (SVP-R) inhibited the formation of ADAs, when administered with a foreign protein such as KLH or Pegsiticase. The SVP-R are taken up by macrophages and dendritic cells and increase the number of regulatory T cells in treated mice. Here we evaluated the efficacy of combination therapy of LMB-100 and SVP-R to eradicate mesothelin expressing tumors while preventing ADA formation. To evaluate if SVP-R can prevent ADAs against LMB-100, we immunized immune-competent mice with a combination of SVP-R and RIT and measured ADAs titers by ELISA and by a functional neutralization assay. We found that the treatment reduced ADA titers by more than 99%. To determine if the mice were tolerized to LMB-100, we treated the mice with two doses of SVP-R and six doses of LMB-100 to induce tolerance; and followed by nine challenges of LMB-100 given over 6 weeks. We found that the ADAs were reduced by 98%, indicating development of immunological tolerance to LMB-100. To show that the tolerance is transferable, we tolerized the mice with six doses of SVP-R and LMB-100 and adoptively transferred their splenocytes to naive recipient mice. The recipient mice were challenged with six doses of LMB-100 and ADA titers were measured. We found that splenocytes from tolerized mice induced a 66% decrease in ADA formation in the recipient mice indicating that the tolerance was mediated by cells of the immune system. To demonstrate that immune suppression is useful in treating tumors, we implanted mouse breast cancer cells expressing human mesothelin into Balb/c mice with a normal immune system and treated them with LMB-100. LMB-100 produced tumor regressions when given before ADAs developed, but was inactive in mice with pre-existing ADAs. However, when mice with preexisting ADAs were treated with LMB-100 and SVP-R, anti-tumor activity was restored and ADAs suppressed (P≤0.0001). SVP-R are being evaluated in humans to prevent ADA to Pegsiticase, an enzyme for refractory gout treatment. Our data indicates that combining SVP-R with LMB-100 should be useful in treating cancer by allowing more treatment cycles and better efficacy. This approach can be used to increase the efficacy of other immunogenic agents such as CAR-T cells, antibody drug conjugates and viral gene therapy vectors Citation Format: Ronit Mazor, Emily King, Takashi Kei Kishimoto, Ira Pastan. Induction of immune tolerance to recombinant immunotoxin LMB-100 using synthetic vaccine particles encapsulating rapamycin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 72. doi:10.1158/1538-7445.AM2017-72