Figure S1 shows BsAb 4 and BsAb 5 concentration over time in each mouse for half-life determination.

Figure S2 shows mesothelin expression on A431-Luc cells by flow cytometry.

The mechanisms leading to the formation of sclerotic lesions in focal segmental glomerulosclerosis (FSGS) remain incompletely understood; however, podocyte detachment and loss are considered key pathogenic events. Ubiquitin-specific protease 40 (USP40) is a deubiquitylating enzyme expressed in podocytes. In the present study, we investigated the role of USP40 in podocytes, focusing on its impact on the adhesion molecule integrin β1, which is essential for anchoring podocytes to the glomerular basement membrane. When USP40 knockout mice were subjected to an experimental FSGS model, they exhibited significantly more severe proteinuria and glomerulosclerosis than control mice, along with a marked reduction in podocyte number and integrin β1 expression. Consistently, knockdown of USP40 in cultured podocytes resulted in decreased integrin β1 expression and impaired adhesive properties compared with sham-treated cells. In HEK293 cells transfected with ubiquitin constructs, USP40 suppressed integrin β1 monoubiquitylation. In a separate internalization assay, USP40 prevented the clathrin-mediated endocytosis of integrin β1. In USP40 knockout mice, clathrin-coated vesicles colocalizing with integrin β1 were more frequently observed in podocyte foot processes than in control mice. Together, these findings suggest that USP40 functions as a deubiquitylating enzyme that stabilizes integrin β1 at the podocyte plasma membrane by preventing its endocytosis. We therefore propose that the USP40-integrin β1 axis represents a potential therapeutic target for FSGS.

Figure S3 shows that PBMCs and bispecific antibodies alone are insufficient to kill OVCAR8-Luc cancer cells.

Abstract Mesothelin (MSLN) is a cell surface protein that is overexpressed in various cancers, including mesothelioma, pancreatic, and ovarian cancer. Its expression in normal tissue is limited to the mesothelial cells lining the pleura, peritoneum and pericardium; make it an attractive target for antibody-based therapeutics. Many efforts have been dedicated towards the development of these antibody-based approaches but proteases in the tumor microenvironment promote the cleavage of MSLN from cancer cells. High concentrations of shed MSLN in the tumor microenvironment bind to the antibody inhibiting its activity and preventing the death of cancer cells. To address the presence of shed MSLN, an antibody called 15B6 was designed. It binds the membrane-proximal, protease sensitive region of MSLN that is not shed in the tumor microenvironment. This study investigates the ability of 15B6-targeted bispecific antibodies to eliminate MSLN expressing cancers. We designed humanized and murine versions of a CD3x15B6 bispecific antibody, which binds the CD3 on T cells and the MSLN (15B6) epitope on cancer cells serving as a bridge to promote the activation of T cells to kill MSLN positive cancer cells. These 15B6-based antibodies were compared to SS1-based antibodies that target the distal N-terminal domain of MSLN that is shed by proteases. In-vitro, the SS1 and 15B6 antibodies hold similar cytotoxic activity, but when cocultured with MSLN 296-591, a recombinant protein mimicking shed MSLN, the SS1 antibodies activity is inhibited. In-vivo, in a human mesothelioma model in immunodeficient mice, tumor shrinkage and growth inhibition were observed when treated with the 15B6-based antibody but not those treated with the SS1-based antibody. In an immunocompetent mouse model, the complete regression of colon and breast tumors was observed when treated with the 15B6-based antibody compared to the SS1 antibody and control samples. Transcriptional analysis revealed that 15B6-treated mice had higher levels of activation of both innate and adaptive immune cells, along with significant upregulation of cytokine and STAT5 signaling pathways. Through in-vitro and in-vivo studies, we have demonstrated that this 15B6-targeted antibody binds to the protease-sensitive region, is highly active against MSLN-expressing cancer cell lines in vitro, is not inhibited by shed MSLN, and promotes upregulation of immune cell populations and signaling pathways that support robust anti-tumor activity. Citation Format: Eber Antonio Guzman-Cruz, Masanori Onda, Xiufen Liu, Tara O’Shea, Qi Zhou, Wenlong Liu, Jing Bian, Chin-Hsien Tai, Ira Pastan, Mitchell Ho, . A T-cell engager antibody targeting the non-shed site of mesothelin in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2633.

Table S1 shows the amino acid sequences for the BsAbs investigated in the article.

Supplementary Method 1 explains the preparation of BsAb 5.

The tumor-associated antigen mesothelin is highly expressed in many human cancers, while its expression in normal tissues is limited. Its interaction with the cancer antigen 125 promotes heterotypic cell adhesion and tumor metastasis. Mesothelin-targeted immunotherapies are being intensively investigated, which is aided by growing structural knowledge of the protein and its interactions with antibodies. Recent studies have produced a complete atomic model showing mesothelin as a compact, right-handed, conformationally flexible solenoid composed of nine layers of helices, with glycans attached at all three predicted N-glycosylation sites. Structural analyses reveal that most therapeutic antibodies target the rigid and immunogenic N-terminal domain, while a few bind to middle domain or C-terminal linear tail, revealing correlation between immunogenicity and structural stability. Crystallographic studies have also extended to the interactions between mesothelin and CA-125. These structural advances offer insights into the potential function of mesothelin and guidance for further development of therapeutic antibodies.

Background:Mesothelin (MSLN) is a surface antigen highly expressed in several solid tumors, including mesothelioma, ovarian, and pancreatic cancers. However, therapeutic efficacy of MSLN-targeted agents is often compromised by shed MSLN (SM), which acts as a soluble decoy and accumulates in tumor microenvironments, reducing antibody engagement at the tumor surface. Methods:To overcome this barrier, we generated antibodies targeting the membrane-proximal, non-shed region of MSLN using a peptide encompassing major cleavage sites for rabbit immunization. From 200 B-cell clones, 14 antibodies specific to the juxtamembrane region of MSLN were identified. The lead candidate, RO4, underwent detailed characterization and humanization to improve clinical applicability. Results:Humanized RO4 (hRO4) exhibited enhanced binding affinity to MSLN and specifically recognized tumor-associated, non-shed epitopes. Structural analysis confirmed precise epitope engagement near the cleavage site. hRO4 effectively inhibited mesothelin shedding in vitro and enabled potent tumor eradication when expressed in chimeric antigen receptor (CAR) T cells in NOD scid gamma mouse models. Conclusions:Targeting a non-shed epitope of MSLN with hRO4 overcomes a critical limitation of conventional MSLN-directed therapies. By avoiding decoy interference and enhancing tumor-specific targeting, hRO4-based therapeutics offer promising clinical potential for improving outcomes in MSLN-positive cancers.

Canine lymphoma is one of the most common and aggressive hematopoietic tumors in dogs. Despite recent advances in veterinary cancer treatments, the lack of specificity, side effects, and resistance to conventional chemotherapies has opened an urgent need to develop more targeted and safe therapeutics to address this unmet need in dogs. Thus, in the present study, we aimed to generate a new class of therapeutics based on a recombinant single-domain antibody (sdAb) immunotoxin derived from the PE38 Pseudomonas aeruginosa exotoxin A. For this purpose, we fused the PE38 toxin with the specific C5 sdAb antibody, previously developed by our group for canine B-cell lymphoma. This resulted in a stable and highly specific C5-PE38 immunotoxin against canine B-cell lymphoma. The C5-PE38 immunotoxin revealed a potent cytotoxic activity (EC50 = 9.50 ± 0.04 μg/mL) against CLBL-1 canine B-cell lymphoma cells, while promoting inhibition of protein synthesis and, consequently, cell death. Importantly, in vivo results in a CLBL-1 xenograft mouse model demonstrated specific targeted tumor uptake and strong tumor growth inhibition in C5-PE38 treated mice compared with control vehicle-treated mice. The results obtained provide new data validating immunotoxins and recombinant sdAb-PE38 based scaffolds as a novel and promising anti-cancer therapy for the treatment of dog-related tumors, while contributing to comparative oncology.

Direct intratumoral injection of therapeutic drugs can minimize total dose and adverse effects compared to systemic administration. Leakage from the injection site may cause variable drug distribution and efficacy, and off-target toxicity. This study aimed to evaluate the co-injection of an iodinated contrast agent with an imageable surrogate drug (fluorescent albumin) to estimate spatial drug distribution. Fluorescent albumin and iodixanol were injected (1, 2, or 4 mL at 1 mL/min) into ex vivo bovine liver. Distribution of iodine on CT was compared to fluorescent albumin on fluorescence microscopy, including comparison of distribution volume in liver as a function of injected volume. Physical properties (hydrodynamic diameter, zeta potential) of both iodixanol and fluorescent albumin were measured. In comparison to fluorescent albumin, iodixanol is smaller (2.7 ± 0.4 nm vs. 17.0 ± 1.7 nm) and more neutrally charged (2.3 ± 0.5 mV vs. − 17.7 ± 1.4 mV). The distribution volume of iodixanol in tissue is approximately 7 times greater than that of fluorescent albumin. However, the correlation of distribution volumes of both agents is R2 = 0.89. The variance in distribution volume in tissue of iodixanol and fluorescent albumin increased with injection volume. The distributions of contrast and surrogate drug were correlated; however, differing physicochemical properties caused differences in their distribution. Although drug and contrast may not colocalize, contrast may serve as an imageable surrogate to inform preclinical development and clinical applications. This study suggests that dividing an interstitial injection into multifocal small-volume injections results in better localization of the injected drug at the target. NCT04840615.

e19015 Background: Anti-CD22 recombinant immunotoxin moxetumomab pasudotox (Moxe) was FDA-approved for relapsed/refractory hairy cell leukemia (HCL) but is now unavailable due to vial expiration until a company resumes development. The phase 3 complete remission (CR) rate was 41%, higher in patients with lower anti-drug antibodies (ADA). Minimal residual disease (MRD) eradication led to longer CR duration. HCL and the poorer prognosis variant HCLv strongly express CD22 and CD20. A clinical trial was done to determine if Rituximab could decrease immunogenicity by killing normal B-cells in HCL patients receiving Moxe and hasten MRD-free CR by reducing HCL tumor burden. Methods: To permit Rituximab enough time to reduce ADA and tumor burden, it was administered 3 days before cycle 1 day 1 at 375 mg/m2, and Moxe was given by 30-minute infusion days 1, 3 and 5. On subsequent 28-day cycles, patients received Moxe days 1, 3 and 5, and Rituximab prior to Moxe on day 1. Patients received up to 4 cycles past MRD-free CR, or up to 8 total cycles if MRD-free CR was achieved after cycle 4. The ADA assay determined percent neutralization by serum of the cytotoxicity of Moxe on CD22+ Raji cells. Results: After 13 patients received Moxe-Rituximab (MoxeR) without dose-limiting toxicity (DLT), meeting the phase 1 endpoint, 5 additional patients received Moxe with the biosimilar Ruxience (MoxeR). The first 3 patients received Moxe at 30 mcg/Kg/dose and subsequent patients 40 mcg/Kg/dose. All 18 were evaluable for toxicity and response. Although no DLT, one patient had transient grade 3 hemolytic uremic syndrome during cycle 3 without significant symptoms or need for therapy. Of the 18 patients, 15 (83%) responded, 14 (78%) achieved CR and 13 (72%) MRD-free CR by blood and bone marrow (BM) aspirate flow cytometry and BM biopsy immunohistochemistry. MRD-free CRs included one with HCLv. Even though Moxe vial expiration prevented enrollment of the 26 planned patients needed for a 1-sided p-value <0.025 compared to Moxe alone where 30 (47%) of 64 phase 1-3 patients achieved MRD-free CR, the 53% relative improvement with MoxeR achieved a 1-sided p-value of 0.05. Compared to 29 (48%) of 61 evaluable phase 1-3 patients who received Moxe alone with high ADA levels, 4 (29%) of 18 patients had high ADA levels to MoxeR (p=0.048). At 14-51 (median 35.3) months of follow-up, all but 2 of 13 MRD-free CRs are continuing, with relapse-free survival 14-45 (median 34.5) months. Conclusions: Despite enrolling slightly fewer patients than planned, MoxeR was safe and more effective than Moxe alone at achieving MRD-free CR, probably due to lower immunogenicity and faster reduction of HCL/HCLv tumor burden. Since non-Hodgkin’s lymphoma (NHL) cells from patients are sensitive to Moxe like HCL, MoxeR could be tested after NHL treatment to convert MRD+ to MRD-free CRs. Clinical trial information: NCT03805932 .

Mesothelin (MSLN) is a cell-surface protein that is expressed in many cancers, which makes it a popular target for Ab-based cancer therapy. However, MSLN is shed from cancer cells at high levels via proteases that cleave at its membrane-proximal C-terminal region. Shed MSLN accumulates in patients' fluids and tumors and can block Ab-based MSLN-targeting drugs from killing cancer cells. A previously established mAb, 15B6, binds MSLN at its protease-sensitive C-terminal region and does not bind shed MSLN. Moreover, 15B6 variable fragment (Fv)-derived chimeric antigen receptor T cells are not inhibited by shed MSLN and kill tumors in mice more effectively than mAb SS1 Fv-derived chimeric antigen receptor T cells, which bind an epitope retained in shed MSLN. In this study, we have established 15B6 Fv-derived MSLN × CD3 bispecific antibodies (BsAb) that target MSLN-expressing cancers. We identified our lead candidate BsAb 5 after screening multiple 15B6-derived BsAb formats in vitro for cytotoxic activity. BsAb 5 activates T cells to kill various cancer cell lines in a MSLN-specific manner. MSLN 296-591 His, a recombinant protein mimicking shed MSLN, does not inhibit 15B6-derived BsAb 5 but completely inhibits humanized SS1-derived BsAb 7. Furthermore, BsAb 5 inhibits and delays tumor growth and is not inhibited by MSLN 296-585 His in mice. Our findings indicate that by targeting the protease-sensitive region of MSLN, BsAb 5 has high MSLN-specific anticancer activity that is not inhibited by shed MSLN. BsAb 5 may be a promising immunotherapy candidate for MSLN-expressing cancers.

Despite many clinical trials, CAR-T cells are not yet approved for human solid tumor therapy. One popular target is mesothelin (MSLN) which is highly expressed on the surface of about 30% of cancers including mesothelioma and cancers of the ovary, pancreas, and lung. MSLN is shed by proteases that cleave near the C terminus, leaving a short peptide attached to the cell. Most anti-MSLN antibodies bind to shed MSLN, which can prevent their binding to target cells. To overcome this limitation, we developed an antibody (15B6) that binds next to the membrane at the protease-sensitive region, does not bind to shed MSLN, and makes CAR-T cells that have much higher anti-tumor activity than a CAR-T that binds to shed MSLN. We have now humanized the Fv (h15B6), so the CAR-T can be used to treat patients and show that h15B6 CAR-T produces complete regressions in a hard-to-treat pancreatic cancer patient derived xenograft model, whereas CAR-T targeting a shed epitope (SS1) have no anti-tumor activity. In these pancreatic cancers, the h15B6 CAR-T replicates and replaces the cancer cells, whereas there are no CAR-T cells in the tumors receiving SS1 CAR-T. To determine the mechanism accounting for high activity, we used an OVCAR-8 intraperitoneal model to show that poorly active SS1-CAR-T cells are bound to shed MSLN, whereas highly active h15B6 CAR-T do not contain bound MSLN enabling them to bind to and kill cancer cells.

BackgroundLMB-100 is a mesothelin (MSLN)-targeting recombinant immunotoxin (iTox) carrying a Pseudomonas exotoxin A payload that has shown promise against solid tumors, however, efficacy is limited by the development of neutralizing anti-drug antibodies (ADAs). Tofacitinib is an oral Janus Kinase (JAK) inhibitor that prevented ADA formation against iTox in preclinical studies.MethodsA phase 1 trial testing LMB-100 and tofacitinib in patients with MSLN-expressing cancers (pancreatic adenocarcinoma, n=13; cholangiocarcinoma, n=1; appendiceal carcinoma, n=1; cystadenocarcinoma, n=1) was performed to assess safety and to determine if tofacitinib impacted ADA formation. Participants 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 µg/kg) while oral tofacitinib was administered for the first 10 days of the cycle (10 mg BID). Peripheral blood was collected for analysis of ADA levels, serum cytokines and circulating immune subsets.ResultsThe study was closed early due to occurrence of drug-induced pericarditis in 2 patients. Pericarditis with the combination was not reproducible in a transgenic murine model containing human MSLN. Two of 4 patients receiving all 3 cycles of treatment maintained effective LMB-100 levels, an unusual occurrence. Sustained increases in systemic IL-10 and TNF-α were seen, a phenomenon not observed in prior LMB-100 studies. A decrease in activated T cell subsets and an increase in circulating immunosuppressive myeloid populations occurred. No radiologic decreases in tumor volume were observed.DiscussionFurther testing of tofacitinib to prevent ADA formation is recommended in applicable non-malignant disease settings.Clinical trial registrationhttps://www.clinicaltrials.gov/study/NCT04034238.

PDF file - 93K, Summary of the results of SS1P toxicity after IR knock down or addition of inhibitors in KB31 cells.

Supplementary Table from Development of Highly Effective Anti-Mesothelin hYP218 Chimeric Antigen Receptor T Cells With Increased Tumor Infiltration and Persistence for Treating Solid Tumors