Immunotoxins are cytolytic fusion proteins developed for cancer therapy, composed of an antibody fragment that binds to a cancer cell and a protein toxin fragment that kills the cell.
exotoxin A (PE) ...is a potent toxin that is used for the killing moiety in many immunotoxins. Moxetumomab Pasudotox (Lumoxiti) contains an anti-CD22 Fv and a 38 kDa portion of PE. Lumoxiti was discovered in the Laboratory of Molecular Biology at the U.S. National Cancer Institute and co-developed with Medimmune/AstraZeneca to treat hairy cell leukemia. In 2018 Lumoxiti was approved by the US Food and Drug Administration for the treatment of drug-resistant Hairy Cell Leukemia. Due to the bacterial origin of the killing moiety, immunotoxins containing PE are highly immunogenic in patients with normal immune systems, but less immunogenic in patients with hematologic malignancies, whose immune systems are often compromised. LMB-100 is a de-immunized variant of the toxin with a humanized antibody that targets mesothelin and a PE toxin that was rationally designed for diminished reactivity with antibodies and B cell receptors. It is now being evaluated in clinical trials for the treatment of mesothelioma and pancreatic cancer and is showing somewhat diminished immunogenicity compared to its un modified parental counterpart. Here we review the immunogenicity of the original and de-immunized PE immunotoxins in mice and patients, the development of anti-drug antibodies (ADAs), their impact on drug availability and their effect on clinical efficacy. Efforts to mitigate the immunogenicity of immunotoxins and its impact on immunogenicity will be described including rational design to identify, remove, or suppress B cell or T cell epitopes, and combination of immunotoxins with immune modulating drugs.
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 fragment of a bacterial toxin that ...kills tumor cells. Because the toxin is a foreign protein, it is immunogenic. The clinical success of RITs in patients with a normal immune system is limited by their immunogenicity. In this review, we discuss our progress in therapeutic protein deimmunization and the balancing act between immunogenicity and therapeutic potency. One approach is to prevent the activation of B cells by mapping and elimination of B-cell epitopes. A second approach is to prevent helper T-cell activation by interfering with major histocompatibility complex II presentation or T-cell recognition. Immunizing mice with RITs that were deimmunized by elimination of the murine B- or T-cell epitopes showed that both approaches are effective. Another approach to control immunogenicity is to modify the host immune system. Nanoparticles containing synthetic vaccine particles encapsulating rapamycin can induce immune tolerance and prevent anti-drug antibody formation. This treatment restores RIT anti-tumor activity that is otherwise neutralized because of immunogenicity.
During gene therapy trials, immune responses against adeno-associated virus (AAV) vectors are monitored by antibody assays that detect the humoral and T-cell mediated cellular responses to AAV ...vectors. T cell assays commonly utilize the collection of patients' peripheral blood mononuclear cells (PBMCs) and stimulation with AAV-derived overlapping peptides. We recently described that spontaneous deamidation coincides with T cell epitopes in AAV capsids and that spontaneous deamidation may enhance or decrease immunogenicity in some individuals. This raised the concern for false negative results of antibody detection and PBMC immune monitoring assays because these assays use wild-type (WT) AAV or WT peptides for T cell re-stimulation and these peptides may not re-activate T cells that were stimulated with deamidated AAV capsid. To investigate this concern, we modeled the scenario by expanding T cells with deamidated peptides and evaluated the cross-reactivity of expanded T cells to WT peptides. In the majority of samples, cells that were expanded with deamidated peptides and restimulated with WT peptide had significantly lowered IL-2 and IFN-γ production. Spiking the four deamidated peptides to the WT peptide pool used for re-stimulation, restored the signal and corrected the performance of the assay. We also evaluated the impact of deamidation on anti AAV binding antibodies and did not observe a major impact on seroprevalence detection of AAV9. These data indicate that a high level of deamidation in AAV therapy may result in underestimation or even failure to detect immune responses against WT peptides during cellular immune monitoring.
Tac (CD25) is expressed on multiple hematologic malignancies and is a target for cancer therapies. LMB-2 is an extremely active anti-Tac recombinant immunotoxin composed of an Fv that binds to Tac ...and a 38-kDa fragment of
exotoxin A (PE38). Although LMB-2 has shown high cytotoxicity toward Tac-expressing cancer cells in clinical trials, its efficacy was hampered by the formation of anti-drug antibodies against the immunogenic bacterial toxin and by dose-limiting off-target toxicity. To reduce toxin immunogenicity and nonspecific toxicity, we introduced six point mutations into domain III that were previously shown to reduce T-cell immunogenicity and deleted domain II from the toxin, leaving only the 11aa furin cleavage site, which is required for cytotoxic activity. Although this strategy has been successfully implemented for mesothelin and CD22-targeting immunotoxins, we found that removal of domain II significantly lowered the cytotoxic activity of anti-Tac immunotoxins. To restore cytotoxic activity in the absence of PE domain II, we implemented a combined rational design and screening approach to isolate highly active domain II-deleted toxin variants. The domain II-deleted variant with the highest activity contained an engineered disulfide-bridged furin cleavage site designed to mimic its native conformation within domain II. We found that this approach restored 5-fold of the cytotoxic activity and dramatically improved the MTD. Both of these improvements led to significantly increased antitumor efficacy
We conclude that the next-generation anti-Tac immunotoxin is an improved candidate for targeting Tac-expressing malignancies.
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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.
Recombinant immunotoxins (RITs) are chimeric proteins being developed for cancer treatment. They are composed of an Ab fragment that targets a cancer Ag and a cytotoxic portion of
exotoxin A. They ...are effective for patients with hematologic malignancies with defective immunity, but their efficacy against solid tumors is limited by anti-drug Ab (ADA) responses in immune-competent patients. Pre-existing Abs or immune memory owing to previous toxin exposure represent additional hurdles because they induce rapid and strong ADA responses. Here, we evaluated the efficacy of methotrexate (MTX) to prevent ADA formation against the mesothelin-targeting RIT LMB-100 in naive mice and in mice with pre-existing Abs. We found that low-dose MTX combined with LMB-100 completely suppressed the formation of ADAs in a dose- and frequency-dependent manner. Suppression of the immune response restored blood levels of LMB-100 and prevented its neutralization. Furthermore, combination of MTX with LMB-100 did not compromise the immune response against a second Ag given after stopping MTX, indicating specific immune tolerance. Adoptive transfer of splenocytes suppressed Ab responses to LMB-100 in recipient mice, indicating a durable immune tolerance. We conclude that combination of MTX and LMB-100 is effective at preventing immune responses in a durable, Ag-specific manner. We propose combining low-dose MTX in immune-competent cancer patients receiving RIT therapy to prevent immunogenicity. This approach could be applied to other immunogenic therapeutic agents and to proteins for which there is pre-existing immunity.
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.
SS1P is an anti-mesothelin immunotoxin composed of a targeting antibody fragment genetically fused to a truncated fragment of Pseudomonas exotoxin A. Delayed responses reported in mesothelioma ...patients receiving SS1P suggest that anti-tumor immunity is induced. The goal of this study is to evaluate if SS1P therapy renders mesothelioma tumors more sensitive to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) immune checkpoint blockade. We evaluated the ability of SS1P to induce adenosine triphosphate (ATP) secretion and calreticulin expression on the surface of AE17M mouse mesothelioma cells. Both properties are associated with immunogenic cell death. Furthermore, we treated these tumors with intra-tumoral SS1P and systemic CTLA-4. We found that SS1P increased the release of ATP from AE17M cells in a dose and time-dependent manner. In addition, SS1P induced calreticulin expression on the surface of AE17M cells. These results suggest that SS1P promotes immunogenic cell death and could sensitize tumors to anti-CTLA-4 based therapy. In mouse studies, we found that the combination of anti-CTLA-4 with intra-tumoral SS1P induced complete regressions in most mice and provided a statistically significant survival benefit compared to monotherapy. The surviving mice were protected from tumor re-challenge, indicating the development of anti-tumor immunity. These findings support the use of intra-tumoral SS1P in combination with anti-CTLA-4.
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 ...SSIP to produce LMB-T20, a de-immunized RIT that has the eight human T-cell epitopes in SSIP 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 SSIP, 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.
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.
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