DT389-YP7, a Recombinant Immunotoxin against Glypican-3 That Inhibits Hepatocellular Cancer Cells: An In Vitro Study

Hepatocellular carcinoma (HCC) is one of the high-metastatic types of cancer, and metastasis occurs in one-third of patients with HCC. To maintain the effectiveness of drug compounds on cancer cells and minimize their side effects on normal cells, it is important to use new approaches for overcoming malignancies. Immunotoxins (ITs), an example of such a new approach, are protein-structured compounds consisting of toxic and binding moieties which can specifically bind to cancer cells and efficiently induce cell death. Here, we design and scrutinize a novel immunotoxin against an oncofetal marker on HCC cells. We applied a truncated diphtheria toxin (DT389) without binding domain as a toxin moiety to be fused with a humanized YP7 scFv against a high-expressed Glypican-3 (GPC3) antigen on the surface of HCC cells. Cytotoxic effects of this IT were investigated on HepG2 (GPC3+) and SkBr3 (GPC3−) cell lines as positive- and negative-expressed GPC3 antigens. The dissociation constant (Kd) was calculated 11.39 nM and 18.02 nM for IT and YP7 scfv, respectively, whereas only IT showed toxic effects on the HepG2 cell line, and decreased cell viability (IC50 = 848.2 ng/mL). Changing morphology (up to 85%), cell cycle arrest at G2 phase (up to 13%), increasing intracellular reactive oxygen species (ROSs) (up to 50%), inducing apoptosis (up to 38% for apoptosis and 23% for necrosis), and an almost complete inhibition of cell movement were other effects of immunotoxin treatment on HepG2 cells, not on SkBr3 cell line. These promising results reveal that this new recombinant immunotoxin can be considered as an option as an HCC inhibitor. However, more extensive studies are needed to accomplish this concept.

A Single-Domain Antibody-Based Anti-PSMA Recombinant Immunotoxin Exhibits Specificity and Efficacy for Prostate Cancer Therapy

Prostate cancer (PCa) is the second most common cancer in men, causing more than 300,000 deaths every year worldwide. Due to their superior cell-killing ability and the relative simplicity of their preparation, immunotoxin molecules have great potential in the clinical treatment of cancer, and several such molecules have been approved for clinical application. In this study, we adopted a relatively simple strategy based on a single-domain antibody (sdAb) and an improved Pseudomonas exotoxin A (PE) toxin (PE24X7) to prepare a safer immunotoxin against prostate-specific membrane antigen (PSMA) for PCa treatment. The designed anti-PSMA immunotoxin, JVM-PE24X7, was conveniently prepared in its soluble form in an Escherichia coli (E. coli) system, avoiding the complex renaturation process needed for immunotoxin preparation by the conventional strategy. The product was very stable and showed a very strong ability to bind the PSMA receptor. Cytotoxicity assays showed that this molecule at a very low concentration could kill PSMA-positive PCa cells, with an EC50 value (concentration at which the cell viability decreased by 50%) of 15.3 pM against PSMA-positive LNCaP cells. Moreover, this molecule showed very good killing selectivity between PSMA-positive and PSMA-negative cells, with a selection ratio of more than 300-fold. Animal studies showed that this molecule at a very low dosage (5 × 0.5 mg/kg once every three days) completely inhibited the growth of PCa tumors, and the maximum tolerable dose (MTD) was more than 15 mg/kg, indicating its very potent tumor-treatment ability and a wide therapeutic window. Use of the new PE toxin, PE24X7, as the effector moiety significantly reduced off-target toxicity and improved the therapeutic window of the immunotoxin. The above results demonstrate that the designed anti-PSMA immunotoxin, JVM-PE24X7, has good application value for the treatment of PCa.

Phase I study of mesothelin-targeted immunotoxin LMB-100 in combination with tofacitinib in patients with advanced pancreatobiliary cancer.

3051 Background: LMB-100 recombinant immunotoxin consists of a mesothelin-binding Fab for targeting a modified Pseudomonas exotoxin A payload to tumors. Previous clinical trials demonstrated that almost all patients formed anti-drug-antibodies (ADAs) to LMB-100 that made administration beyond cycle 2 ineffective. Tofacitinib is an oral JAK inhibitor that prevented formation of ADAs against a closely related immunotoxin in pre-clinical studies. The primary objective of the dose escalation cohort was assessment of safety and tolerability of LMB-100 given with tofacitinib to patients with mesothelin-expressing solid tumors. The primary objective of the expansion cohort was to determine whether co-administration of tofacitinib delays formation of neutralizing LMB-100 ADAs. Methods: Patients (n = 13) with pancreatic adenocarcinoma and other mesothelin-expressing solid tumors (n = 3; cholangiocarcinoma, appendix, cystadenocarcinoma) were treated for up to 3 cycles with LMB-100 as a 30-minute infusion on days 4, 6, and 8 at two dose levels (100 and 140 mcg/kg) and co-treated with oral tofacitinib for the first 10 days of the cycle (10 mg BID). Results: Dose level 1 of LMB-100 was started at 100 mcg/kg one dose level below the single agent MTD. Dose escalation to 140 mcg/kg (dose level 2) resulted in DLTs in 2 of the 3 patients treated: grade 3 cardiac toxicity and grade 4 hyponatremia, both attributed to capillary leak syndrome. Ultimately, 7 patients were treated at dose level 1 without DLTs and 100 mcg/kg was chosen as the LMB-100 dose for the expansion cohort. The last of 6 patients treated in the expansion cohort developed grade 4 pericardial effusion leading to early closure of the study for toxicity. No objective responses were seen. Of the 8 patients who received two cycles of treatment at MTD, 4 met prespecified criteria for ADA prevention, and 2 patients who went on to receive cycle 3 had detectable LMB-100 plasma drug levels after administration. Conclusions: LMB-100 was unable to be co-administered safely with tofacitinib. ADA formation was prevented in 2 patients through 3 cycles, a rare occurrence. Clinical trial information: NCT04034238.

Phase 1 trial of anti-CD22 recombinant immunotoxin moxetumomab pasudotox combined with rituximab for relapsed/refractory hairy cell leukemia.

7036 Background: Anti-CD22 recombinant immunotoxin moxetumomab pasudotox (Moxe) is FDA-approved for hairy cell leukemia (HCL) patients who have received at least two prior systemic therapies including a purine nucleoside analog. In phase 3 testing the complete remission (CR) rate was 41%, and response was higher in patients with lower tumor burden and lower titers of antidrug antibodies (ADA). Phase 1 testing indicated that most CRs were without minimal residual disease (MRD) and eradication of MRD was associated with prolonged CR duration. Monoclonal antibody (Mab) rituximab binds to CD20 on HCL cells and induces apoptosis or immune-mediated killing, but as a single-agent achieved only 13% CRs in relapsed HCL requiring therapy. In a phase 1 trial to determine safety, rituximab was combined with Moxe, with the goal to help reduce tumor burden and to prevent or delay ADA by killing normal B-cells. Methods: To allow rituximab sufficient time to accomplish both goals, it was infused 3 days before day 1 of cycle 1 at 375 mg/m2, and Moxe was given by 30-minute infusion on days 1, 3 and 5. On repeat cycles of Moxe days 1, 3 and 5, rituximab was given on day 1. Cycles were generally spaced 4 weeks apart. Moxe was begun at a lower dose, 30 rather than the 40 mcg/kg dose used in phase 3 in case the rituximab would increase its toxicity. Bone marrow aspirate flow cytometry, which can detect 0.002% HCL cells, was the most sensitive test used for MRD detection, much more sensitive than BRAF V600E digital droplet PCR (ddPCR) or bone marrow biopsy immunohistochemistry (IHC). Patients could receive 4 cycles past MRD-free CR, but not more than 8 cycles. Results: Three patients received Moxe at 30 mcg/Kg/dose and 6 received 40 mcg/Kg/dose, all without dose limiting toxicity (DLT). There was no evidence of hemolytic uremic syndrome or capillary leak syndrome. To prevent intravascular hypovolemia due to expected third spacing, patients were encouraged to drink one cup per hour of water or other fluid from days 1 to 8 and take dexamethasone 4 mg orally if headache or nausea prevented good oral hydration. Of the 9 patients, 7 (78%) achieved CR after 2 (n = 6) or 3 (n = 1) cycles, and achieved MRD-free CR after 2 (n = 3), 4 (n = 3) or 6 (n = 1) cycles. No patients became infected with COVID-19. Conclusions: This phase 1 trial met its primary endpoint of determining whether rituximab could be safely combined with Moxe and will enroll 4 additional patients to further access clinical activity. Further testing will determine whether addition of a CD20 Mab to Moxe significantly improves clinical outcome compared to Moxe alone, particularly long-term MRD-free CR rate. Clinical trial information: NCT03805932.

Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells

Background KRAS oncogenes harboring codon G12 and G13 substitutions are considered gatekeeper mutations which drive oncogenesis in many cancers. To date, there are still no target-specific vaccines or drugs available against this genotype, thus reinforcing the need towards the development of targeted therapies such as immunotoxins. Methods This study aims to develop a recombinant anti-mKRAS scFv-fused mutant Hydra actinoporin-like-toxin-1 (mHALT-1) immunotoxin that is capable of recognizing and eradicating codon-12 mutated k-ras antigen abnormal cells. One G13D peptide mimotope (164-D) and one G12V peptide mimotope (68-V) were designed to elicit antigen specific IgG titres against mutated K-ras antigens in immunised Balb/c mice. The RNA was extracted from splenocytes following ELISA confirmation on post-immunized mice sera and was reverse transcribed into cDNA. The scFv combinatorial library was constructed from cDNA repertoire of variable regions of heavy chain (VH) and light chain (VL) fusions connected by a flexible glycine-serine linker, using splicing by overlap extension PCR (SOE-PCR). Anti-mKRAS G12V and G13D scFvs were cloned in pCANTAB5E phagemid and superinfected with helper phage. After few rounds of bio-panning, a specific mKRAS G12V and G13D scFv antibody against G12V and G13D control mimotope was identified and confirmed using ELISA without any cross-reactivity with other mimotopes or controls. Subsequently, the anti-mKRAS scFv was fused to mHALT-1 using SOE-PCR and cloned in pET22b vector. Expressed recombinant immunotoxins were analyzed for their effects on cell proliferation by the MTT assay and targeted specificity by cell-based ELISA on KRAS-positive and KRAS-negative cancer cells. Results The VH and VL genes from spleen RNA of mice immunized with 164-D and 68-V were amplified and randomly linked together, using SOE-PCR producing band sizes about 750 bp. Anti-mKRAS G12V and G13D scFvs were constructed in phagemid pCANTAB5E vectors with a library containing 3.4 × 106 and 2.9 × 106 individual clones, respectively. After three rounds of bio-panning, the anti-mKRAS G12V-34 scFv antibody against G12V control mimotope was identified and confirmed without any cross-reactivity with other controls using ELISA. Anti-mKRAS G12V-34 scFv fragment was fused to mHALT-1 toxin and cloned in pET22b vector with expression as inclusion bodies in E. coli BL21(DE3) (molecular weight of ~46.8 kDa). After successful solubilization and refolding, the mHALT-1-scFv immunotoxin exhibited cytotoxic effects on SW-480 colorectal cancer cells with IC50 of 25.39 μg/mL, with minimal cytotoxicity effect on NHDF cells. Discussion These results suggested that the development of such immunotoxins is potentially useful as an immunotherapeutic application against KRAS-positive malignancies.

Phase I study of mesothelin-targeted immunotoxin LMB-100 in combination with tofacitinib in persons with pancreatobiliary cancer or other mesothelin expressing solid tumors.

TPS452 Background: LMB-100 recombinant immunotoxin consists of a mesothelin-binding Fab for targeting with a modified Pseudomonas exotoxin A payload. Formation of anti-drug antibodies (ADAs) is thought to contribute significantly to limited clinical efficacy of LMB-100 seen in prior clinical trials. Most patients develop clinically meaningful ADAs after 1-2 cycles of LMB-100, resulting in rapid neutralization of LMB-100 during subsequent cycles and undetectable plasma drug levels. Tofacitinib is an oral Janus Kinase-1 and -3 (JAK) inhibitor approved by the FDA for the treatment of rheumatoid arthritis and ulcerative colitis. Pre-clinical studies have shown that tofacitinib can prevent the formation of ADAs against recombinant immunotoxin (Onda et al. Journal of Immunology 2014), and that co-administration of tofacitinib with LMB-100 increases immunotoxin serum half- life in mice and anti-tumor efficacy (Simon et al. JCI Insight 2019). We hypothesize that co-administration of tofacitinib with LMB-100 will prevent or delay the formation of high titer ADAs to LMB-100, such that 2 effective cycles of immunotoxin can be administered to patients. Methods: This phase I clinical trial consists of a dose escalation phase using 3+3 design to determine the maximum tolerated dose (MTD) of LMB-100 that can be administered with tofacitinib in participants (n = 18 max) with mesothelin-expressing solid tumors, followed by a dose expansion phase at the MTD for participants (n = 15) with pancreatic adenocarcinoma or extrahepatic cholangiocarcinoma. The primary objective of the expansion phase is to determine whether co-administration of tofacitinib delays formation of neutralizing anti-LMB-100 ADAs for cycle 2 of treatment as measured by LMB-100 plasma drug levels. A positive outcome will be reached if percent of participants achieving threshold LMB-100 drug levels during cycle 2 increases from 50% to 80% (83.6% probability if >10 of 15 evaluable participants meet this milestone). Plasma drug levels during cycle 3 will also be analyzed as a secondary endpoint. Key inclusion criteria include adults with histologically confirmed previously treated solid tumor malignancies. Participants will receive tofacitinib 10 mg twice daily on days 1-10 and LMB-100 at 65, 100, or 140 mcg/kg on days 4, 6 and 8 of a 21-day cycle. The dose escalation phase has been completed and enrollment onto dose expansion phase is ongoing. Clinical trial information: NCT04034238.