Anetumab Ravtansine Research Articles

Randomized trial of anetumab ravtansine and pembrolizumab compared to pembrolizumab for mesothelioma

PurposeThe mesothelin-targeting antibody-drug conjugate anetumab ravtansine was evaluated in combination with the programmed cell death-1 (PD-1) inhibitor pembrolizumab based on the common expression of mesothelin and reports of activity in mesothelioma. Patients and methodsA phase 1 safety run-in of the combination of anetumab ravtansine (6.5 mg/kg iv q3weeks) and pembrolizumab (200 mg, IV q3weeks) was conducted, followed by a phase 2 randomization to the combination or pembrolizumab alone at medical centers across the United States and Canada in the National Cancer Institute’s Experimental Therapeutics Clinical Trials Network. Patients with pleural mesothelioma that expressed mesothelin and had previously received platinum-based therapy were eligible. ResultsIn phase 1 (n = 12) only one dose limiting toxicity was observed and the rules for dose reduction were not met. In phase 2, there was no difference in the confirmed response rates between the combination group (n = 18, 2 partial responses [PR], 11 %) and the pembrolizumab group (n = 17, 1 PR, 6 %; z = -0.5523, p = 0.29116). The median PFS was 12.2 months (95 % CI 5.1–not evaluable [NE]) for the combination, and 3.9 months for pembrolizumab (95 % CI 2.1-NE)(HR=0.55, p = 0.20). Patients with high baseline levels of soluble mesothelin who received anetumab ravtansine had a median PFS of 5 months. ConclusionsThe numeric difference in PFS between treatment groups was not statistically significant, likely related to a smaller than planned sample size. High levels of soluble mesothelin should potentially be considered to select against the use of mesothelin-targeting therapies in development that are neutralized by soluble mesothelin.

Soluble mesothelin neutralizes mesothelin antibody-based therapies.

2565 Background: Mesothelin (MSLN) is overexpressed in mesothelioma and other solid tumors making it the focus of multiple targeted therapies, including antibody drug conjugates (ADCs), T cell receptor (TCR) fusion constructs, and chimeric antigen receptor T-cell (CAR-T) products. However, the clinical efficacy of these treatments remains limited. We hypothesize that soluble MSLN (sMSLN) in the plasma binds to MSLN-targeted therapies before reaching the tumor. Our study aimed to evaluate the effects of sMSLN on a MSLN-targeting antibody, anetumab, and explore methods to reduce sMSLN. Methods: Exploratory analysis in NCT03126630: sMSLN testing was performed on blood samples from participants enrolled in NCT03126630 at the Molecular Targets Core Lab, National Cancer Institute. The median was used to categorize high and low levels of sMSLN. Survival was estimated with the Kaplan-Meier method and compared between groups with the log rank test. Anetumab immunoprecipitation: Anetumab was covalently coupled to Dynabeads at 5 µg per mg of beads to immunoprecipitate MSLN from two plasma samples. Assays were performed in duplicate. Therapeutics plasma exchange (TPE):Whole blood samples were collected before and after one plasma volume of TPE with albumin as the replacement fluid in patients undergoing routine TPE for various medical conditions, such as autoimmune diseases and hyperviscosity syndromes. Plasma levels of sMSLN were measured with an ELISA assay in matched pre- and post-TPE plasma samples. Results: We obtained sMSLN levels from 40 patients enrolled in NCT03126630. For patients treated with both anetumab ravtansine and pembrolizumab, median progression free survival (PFS) was shorter in the high sMSLN group (5 months) vs the low sMSLN group (12 months). For patients treated with pembrolizumab alone, PFS was similar for patients with high and low sMSLN (4 vs 3.4 months). Anetumab significantly reduced the concentration of sMSLN in plasma samples as detected by MSLN ELISA (p<0.05), demonstrating that sMSLN can bind to and sequester anetumab. Next, we evaluated TPE as a mechanism to reduce sMSLN. We obtained pre- and post-TPE plasma samples from 15 patients undergoing routine TPE for various medical conditions. TPE consistently reduced sMSLN (p<0.05) with an average decrease of 43.6% or 15.4 ng/mL. Conclusions: We found that high sMSLN levels are associated with shorter PFS for anetumab ravtansine, but not pembrolizumab. Additionally, anetumab binds to sMSLN in the plasma, which suggests that sMSLN can sequester anti-MSLN antibodies and may limit the efficacy of MSLN-targeted therapies. High levels of sMSLN could potentially be used as a biomarker to select which patients should not receive MSLN-targeting therapies. Furthermore, our results indicate that sMSLN reduction is feasible with TPE. Decreasing sMSLN could restore the activity of MSLN-targeted therapies.

OA02.04 Phase 1/2 Randomized Trial of Anetumab Ravtansine and Pembrolizumab Compared to Pembrolizumab for Pleural Mesothelioma - NCT03126630

OA02.04 Phase 1/2 Randomized Trial of Anetumab Ravtansine and Pembrolizumab Compared to Pembrolizumab for Pleural Mesothelioma - NCT03126630

Safety and activity of anti-mesothelin antibody–drug conjugate anetumab ravtansine in combination with pegylated-liposomal doxorubicin in platinum-resistant ovarian cancer: multicenter, phase Ib dose escalation and expansion study

ObjectivesAnetumab ravtansine is an antibody-drug conjugate consisting of a fully human anti-mesothelin monoclonal antibody conjugated to cytotoxic maytansinoid tubulin inhibitor DM4. Mesothelin is highly expressed in ovarian cancer. This phase...

Phase Ib study of anetumab ravtansive in combination with immunotherapy or immunotherapy plus chemotherapy in mesothelin-enriched advanced pancreatic adenocarcinoma: NCI10208.

4136 Background: Mesothelin (MSLN) is overexpressed in 80-85% of pancreatic adenocarcinomas (PDAC). Anetumab ravtansine (AR) is a fully human anti-MSLN immunoglobulin G1 antibody conjugated to the anti-tubulin maytansinoid DM4. Through NCI-ETCTN, the North American Star Consortium conducted a phase I study to evaluate the safety/tolerability of AR in various combinations in patients (pts) with PDAC. Here, we report preliminary results of the escalation part. Methods: Pts with advanced PDAC after at least one line of treatment were included. AR was combined with nivolumab (ARM1), nivolumab/ipilimumab (ARM 2), or nivolumab plus gemcitabine (Gem) (ARM3), using an integrated biomarker analysis. Two dose levels (DL) of AR were evaluated, DL1=5.5mg/kg and DL2=6.5mg/kg (established RP2D). Key eligibility criterion was MSLN expression in >5% of tumor cells by immunohistochemistry. Pts with prior anti-PD1/anti-CTLA4 treatment were excluded but treatment with prior Gem was allowed. Mandatory blood and paired tumor samples were collected for investigation of the immune microenvironment, genomic/transcriptomic changes and for an in-depth description of AR pharmacokinetics. Results: Data cut-off date was 22/01/2022. A total of n=33 pts were enrolled, n=11 (ARM 1), n=13 (ARM 2) and n=9 (ARM3). Median age of pts was 66 (40-83), 33% of PS=0 and 66% of PS=1. Twenty-six pts (79%) had previously been exposed to Gem. Median number of prior lines of treatment was 3 (1-7). Twenty-eight patients were evaluable for DLT. Grade (G)3/4 TRAEs: 0% in ARM1DL1, 5.3% in ARM1DL2, 0% in ARM2DL1, 16.9% in ARM2DL2, 8.6% in ARM3DL1 and 19.5% in ARM3DL2. There were 2 dose-limiting toxicities in ARM2DL2, one G3 upper gastrointestinal haemorrhage, possibly related to AR, and one G3 thrombocytopenia and G3 anaemia, definitely related to AR. Ocular toxicity events were G1/2 blurred vision in 5/33 (15%) and G1 xerophthalmia in 1/33 (3%), related to both AR and anti-PD1; G2 keratitis in 1/33 (3%), related to AR only. Only G1 peripheral neuropathy was observed in 4/33 (12%) pts. Efficacy data is presented in the table. In ARM3, the range of tumor measurement (ΤΜ) change was ΔTM=–16.8% to +16.2% and 3/8 (36%) pts with SD had previously been exposed to Gem. Conclusions: Based on the observed disease control rate and acceptable toleratbility, ARM3 (both DL1 and DL2) will be tested in the expansion part. A further 20 patients will be recruited for dose confirmation and comprehensive biomarker evaluation. Pharmacokinetic/pharmacodynamic analysis is under way. Clinical trial information: NCT03816358. [Table: see text]

A randomized phase II study of bevacizumab and weekly anetumab ravtansine or weekly paclitaxel in platinum-resistant or refractory ovarian cancer NCI trial#10150.

5514 Background: Mesothelin and its binder, antigen-CA125, are highly expressed in high grade serous and endometrioid ovarian cancers (HGOC) and, can inhibit paclitaxel-induced cell death. Anetumab ravtansine (AR) is a fully-human antibody directed at the mesothelin antigen, conjugated to a tubulin polymerization inhibitor. We assessed the safety and activity of the combination AR/bevacizumab (ARB) versus weekly paclitaxel/bevacizumab (PB) in patients (pts) with platinum resistant HGOC. Methods: An initial run-in phase I assessed the safety of ARB. After determination of the recommended phase 2 dose (RP2D), a multicenter 1:1 randomized phase 2 trial was designed to evaluate the progression free survival (PFS) in pts with platinum resistant/refractory HGOC. Pts were stratified by platinum resistant or refractory and prior bevacizumab (bev). Eligibility required measurable disease and mesothelin tested positive centrally on archival tissue by IHC. No limitation on the number of prior lines of therapy. A futility analysis was planned at 35 PFS events. The control arm was weekly intravenous paclitaxel 80mg/m2 with bev 10mg/kg every 2 weeks. A CT was performed every 8 weeks for RECIST1.1 assessment. The toxicities were reported according to CTCAE version 5.1. NCT03587311. Results: 7 pts were enrolled in the run-in phase 1 and the RP2D determined as bev (10mg/kg) biweekly with AR (2.2mg/kg) weekly on a 28 day-cycle. In the phase 2, 57 pts were enrolled, 28 pts in the ARB and 29 pts in the control group. The positivity rate for mesothelin screening was 88%. Pts were heavily pre-treated, median prior lines of 3 (range (1-9) with 24 pts received prior bev (42%) and 13 pts were platinum refractory (7 in ARB and 6 in PB). At the time of 35 PFS events, one CR and 4 PR were observed (ORR = 18%) in the ARB arm, versus no CR and 16 PR in the weekly PB (ORR = 55%). The estimated median PFS was 5.3 (95% CI: 3.7-7.4) months for ARB and 9.6 (95% CI: 7.4-17.4) months for PB (HR = 1.7(95% CI: 0.9-3.4)). The median number of cycles were 4 (1, 29) and 8 (1, 19) respectively. The most common treatment-related AEs in the ARB arm were mostly grade 1/2 increase AST (71%) and ALT (64%), thrombocytopenia (61%), fatigue (57%), and peripheral neuropathy (46%). In the PB arm, the most common treatment-related AE were anemia (66%), neutropenia (59%), epistaxis (48%), fatigue (45%) and peripheral neuropathy (45%). Conclusions: At the time of futility analysis, weekly PB had better outcome than weekly ARB leading to the study termination. Molecular and blood analyses are on-going to assess potential biomarkers of response. This study highlights the importance of randomization in assessment of novel therapies and potential for re-challenge with bevacizumab. These data show that weekly PB is an effective regimen and can be considered as the control arm in platinum resistant HGOC. Clinical trial information: NCT03587311.

Exploiting mesothelin in thymic carcinoma as a drug delivery target for anetumab ravtansine.

Thymic epithelial tumours (TETs) are rare tumours comprised of thymomas and thymic carcinoma. Novel therapies are needed, especially in thymic carcinoma where the 5-year survival rate hovers at 30%. Mesothelin (MSLN), a surface glycoprotein that is cleaved to produce mature MSLN (mMSLN) and megakaryocyte potentiating factor (MPF), is expressed in limited tissues. However, its expression is present in various cancers, including thymic carcinoma, where it is expressed in 79% of cases. We utilised flow cytometry, in vitro cytotoxicity assays, and an in vivo xenograft model in order to demonstrate the ability of the MSLN targeting antibody-drug conjugate (ADC) anetumab ravtansine (ARav) in inhibiting the growth of thymic carcinoma. Thymoma and thymic carcinoma cell lines express MSLN, and anetumab, the antibody moiety of ARav, was capable of binding MSLN expressing thymic carcinoma cells and internalising. ARav was effective at inhibiting the growth of thymic carcinoma cells stably transfected with mMSLN in vitro. In vivo, 15 mg/kg ARav inhibited T1889 xenograft tumour growth, while combining 7.5 mg/kg ARav with 4 mg/kg cisplatin yielded an additive effect on inhibiting tumour growth. These data demonstrate that anetumab ravtansine inhibits the growth of MSLN positive thymic carcinoma cells in vitro and in vivo.

Corneal Epitheliopathy Associated With Antibody-Drug Conjugates

This report highlights corneal epithelial changes in 3 patients receiving different antibody-drug conjugate (ADC) therapies that inhibit tubulin assembly: anetumab ravtansine for malignant pleural mesothelioma (Figure A), ado-trastuzumab emtansine for human epidermal growth factor receptor–positive ductal carcinoma of the breast (Figure B), and belantamab mafodotin for refractory myeloma (Figures C and D). The ADCs are a novel approach for treating cancer by using targeted monoclonal antibodies to direct cytotoxic payloads to malignant cells while reducing systemic toxicity.

Abstract CT105: Preliminary safety and efficacy of gavocabtagene autoleucel (gavo-cel, TC-210), a T cell receptor fusion construct (TRuC™), in patients with treatment refractory mesothelin overexpressing solid tumors

Abstract Background: Mesothelin overexpression is present in over 80,000 patients/year with either malignant mesothelioma (MPM), ovarian cancer, cholangiocarcinoma, or non-small cell lung cancer (NSCLC) in the U.S. alone. We are testing a novel genetically engineered anti-mesothelin TRuC™ T cell therapy called gavo-cel (TC-210) in a Phase 1 study in treatment refractory patients with any of the four aforementioned cancers (NCT03907852). Methods: Gavo-cel was engineered by transducing T cells with a lentiviral vector encoding for an anti-mesothelin llama-derived single domain antibody fused to the CD3epsilon subunit using a flexible glycine serine sequence. Upon translation, gavo-cel TRuCs integrate and reprogram intact TCR complexes to recognize tumor surface mesothelin in an HLA-independent manner. Eligibility criteria required demonstration of mesothelin overexpression on tumors (≥50% of tumor cells expressing 2+ or 3+ staining by IHC) at a central laboratory. Gavo-cel dose escalation proceeded following a modified 3+3 schema. TC-210 expansion and serum cytokine levels were serially measured. Results: Seven patients (6 MPM, 1 ovarian) received a single gavo-cel intravenous infusion at the initial dose of 5x107/m2 either alone (dose level [DL] 0, n=1) or following lymphodepletion (LD) (DL1, n=6) with fludarabine (30mg/m2/day x4) and cyclophosphamide (600mg/m2/day x3). The median age was 69 years (range, 36-84) and the median number of prior therapies was 5 (range, 2-9). Four patients had received ≥4 lines of therapy, including immune checkpoint inhibitors (n=5), the anti-mesothelin ADC anetumab ravtansine (n=1), and anti-mesothelin mRNA CAR-T (n=1). Four patients required bridging therapy. No on-target/off-tumor toxicities or neurotoxicity events of any grade were observed. Grade (gr) ≥3 treatment emergent AEs occurred in 2 (29%) patients with MPM treated at DL1: 1 with gr3 CRS, and 1 with gr3 CRS, gr3 pneumonitis and gr5 fungal sepsis (unrelated to gavo-cel). CRS and pneumonitis events resolved with tocilizumab and corticosteroids. Grade 3 pneumonitis was considered a DLT, resulting in DL1 cohort expansion to 6 patients. At data cut-off date (November 8, 2020), 6 patients have been followed for >3 months, including 4 followed through month 6. The disease control rate was 100%. All patients experienced target lesion regression by CT: median 61% (range, 5-75%). Three (43%) patients (2 with mesothelioma and 1 with ovarian cancer) had a partial response by RECIST v1.1 criteria, including 1 achieving a complete metabolic response. After a median follow-up of 6.5 months, the median OS at 6 months was (86%). The addition of LD increased TC-210 peak expansion and persistence, with cells being detected up to 30 days post infusion in peripheral blood. Conclusion: Intravenous systemic administration of gavo-cel was generally safe and resulted in three of seven patients having objective partial response. Dose escalation is ongoing at 1x108/m2. Updated clinical and translational data will be presented. Citation Format: David S. Hong, Melissa Johnson, Janos L. Tanyi, Lauren MacMullen, Robert Tighe, Lizzy Jalbert, Viera P. Muzithras, Kevin Zikaras, Alfonso Quintas Cardama, Raffit Hassan. Preliminary safety and efficacy of gavocabtagene autoleucel (gavo-cel, TC-210), a T cell receptor fusion construct (TRuC™), in patients with treatment refractory mesothelin overexpressing solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT105.

Mesothelin Is a Novel Disease Marker and Potential Therapeutic Target in Pediatric Acute Myeloid Leukemia

Acute myeloid leukemia (AML) remains a significant therapeutic challenge in children with only 50% cure rate with highly intensive cytotoxic chemotherapy. Further intensification is limited by acute and late effects, including severe infectious and cardiac toxicities; therefore, alternative therapeutic strategies are needed. As part of the NCI/COG TARGET AML Initiative, using transcriptome sequencing (RNA Seq) in 635 children and 178 adult AML, we demonstrated that mesothelin (MSLN) was highly-overexpressed in over one-third of pediatric AML. This transcript is not expressed in normal hematopoiesis, making it an ideal target for therapeutic intervention. MSLN is a cell-surface protein normally found in pleura and peritoneum and overexpressed in a variety of solid tumors, such as mesothelioma. The protein product of the MSLN gene exists in 4 forms in the body, including cell surface MSLN, soluble MSLN, and soluble megakaryocyte potentiating factor (MPF), which is the N-terminus of the MSLN pro-protein. Elevated serum soluble MSLN is an established diagnostic test for mesothelioma, with an FDA-approved ELISA-based assay (Mesomark™).Three different AML RNA Seq data sets totaling 813 cases, including TARGET validation cohort (n= 475), TARGET discovery phase (n= 160), and TCGA AML (n= 178), were interrogated for expression of MSLN, with normal marrow cases as a control. MSLN transcript expression at >5 transcripts per million (TPM) was observed in 268 patients (range 0-1225 TPM) with observed prevalence of 48% in TARGET discovery set, 33% in TARGET validation and 15% in adult TCGA set with a collective prevalence of 33% in the entire cohort. MSLN expression was enriched in patients with t(8;21), inv(16), and KMT2A fusions and rare in those with common AML-associated mutations (FLT3- ITD, NPM1, CEBPa mutations). MSLN was not expressed in 20 normal marrow samples (<1 TPM in all cases).We conducted orthogonal validation of the transcriptome data by evaluating diagnostic specimens from an independent cohort of 323 newly diagnosed pediatric AML patients for the presence of MSLN using quantitative RT-PCR and ELISA. RNA extracted from leukemic blasts or plasma obtained from diagnostic specimens were tested for MSLN expression by quantitative RT-PCR or sandwich ELISA, respectively. Optimized q-RT-PCR identified 36% of patients with MSLN expression. Similarly, sandwich ELISA (similar to Mesomark™) confirmed expression of soluble MSLN in 34% of patients tested, with strong concordance between the two tests (p <0.0001). We have also optimized diagnostic multi-dimensional flow cytometry (MDF) and MPF ELISA for evaluation of MSLN.Mesothelin is a cell surface protein highly expressed in AML blasts but absent in other hematopoietic cells, making it an attractive target for immunotherapy. We sought to test the principle of MSLN targeting in leukemia with antibody-drug conjugate (ADC) anetumab ravtansine (AR; BAY94-9343), comprised of an anti-MSLN antibody conjugated to tubulin inhibitor DM4. MSLN -overexpressing cells were created by lentivirally-transducing MSLN into K562 cells (K562+ MSLN). K562+ MSLN exhibited exquisite sensitivity to AR in vitro with an IC50 of 1.4 nM, compared to 360 nM in the non-transduced K562 and 250-260 nM in isotype control conditions (Fig. 1). In vitro cytotoxicity of AR in two primary pediatric MSLN + AML samples also demonstrated MSLN -dependent killing. Additionally, mice bearing K562+ MSLN xenografts were treated in vivo with AR, which demonstrated significant MSLN -dependent efficacy, resulting in median survival of 87 days compared to 32-41 days in control conditions, including chemotherapy with daunorubicin and cytarabine (Fig. 2).MSLN is a cell-surface protein uniquely overexpressed in one-third of pediatric AML, as well as a subset of adult AML. We have shown that MSLN can be detected through several sensitive diagnostic mechanisms including MDF, q-RT-PCR and serum soluble MSLN, making it an optimal marker for disease monitoring. The leukemic expression of MSLN and absence from normal hematopoietic tissue make it an ideal target for immunotherapeutic targeting, and our findings demonstrate that MSLN can be exploited for therapeutic benefit using ADCs. Further studies in MSLN + AML on optimal detection and immunotherapeutic strategies are needed, as MSLN shows great promise for enhanced disease monitoring and leukemia-specific therapeutic targeting. [Display omitted] DisclosuresLoken:Hematologics Inc: Employment, Equity Ownership. Pardo:Hematologics Inc: Employment.

Mesothelin is a novel cell surface disease marker and potential therapeutic target in acute myeloid leukemia

AbstractIn an effort to identify acute myeloid leukemia (AML)-restricted targets for therapeutic development in AML, we analyzed the transcriptomes of 2051 children and young adults with AML and compared the expression profile with normal marrow specimens. This analysis identified a large cohort of AML-restricted genes with high expression in AML, but low to no expression in normal hematopoiesis. Mesothelin (MSLN), a known therapeutic target in solid tumors, was shown to be highly overexpressed in 36% of the AML cohort (range, 5-1077.6 transcripts per million [TPM]) and virtually absent in normal marrow (range, 0.1-10.7 TPM). We verified MSLN transcript expression by quantitative reverse transcription polymerase chain reaction, confirmed cell surface protein expression on leukemic blasts by multidimensional flow cytometry, and demonstrated that MSLN expression was associated with promoter hypomethylation. MSLN was highly expressed in patients with KMT2A rearrangements (P < .001), core-binding factor fusions [inv(16)/t(16;16), P < .001; t(8;21), P < .001], and extramedullary disease (P = .001). We also demonstrated the presence of soluble MSLN in diagnostic serum specimens using an MSLN-directed enzyme-linked immunosorbent assay. In vitro and in vivo preclinical efficacy of the MSLN-directed antibody-drug conjugates (ADCs) anetumab ravtansine and anti-MSLN–DGN462 were evaluated in MSLN+ leukemia cell lines in vitro and in vivo, as well as in patient-derived xenografts. Treatment with ADCs resulted in potent target-dependent cytotoxicity in MSLN+ AML. In this study, we demonstrate that MSLN is expressed in a significant proportion of patients with AML and holds significant promise as a diagnostic and therapeutic target in AML, and that MSLN-directed therapeutic strategies, including ADCs, warrant further clinical investigation.

Favorable therapeutic response after anti-Mesothelin antibody\u2013drug conjugate treatment requires high expression of Mesothelin in tumor cells

PurposeThe cell surface glycoprotein Mesothelin is overexpressed in ovarian, fallopian tube, endometrial, cervical and primary peritoneal cancer and, therefore, might become a particular interesting tumor target in gynecologic oncology. However, even in malignant tumors of the same entity the level of Mesothelin expression varies between individuals, hence it can be expected that the response to Mesothelin-targeting therapies will be variable as well. In this study we explored the therapeutic potency of a novel anti-Mesothelin antibody–drug conjugate (Anetumab ravtansine) as a function of Mesothelin expression in the targeted tumor cells.MethodsAnti-tumor activity studies were performed in human uterine xenograft tumor models that express Mesothelin at high, moderate or low levels. The antibody–drug conjugate (ADC) was applied in varying doses ranging from 2 to 15 mg/kg at variable intervals in tumor bearing SCID/beige mice and the impact on tumor growth was monitored.ResultsThe therapeutic response to the anti-Mesothelin ADC correlated closely with the level of Mesothelin expression in tumor cells. Within the applied dose levels complete tumor regression was achieved only in tumors which expressed Mesothelin at particularly high levels (Hela cell tumors). The application of high anti-Mesothelin ADC doses less frequently was more efficious than giving lower doses at a higher frequency.ConclusionThe studies confirm the great therapeutic potential of Anetumab ravtansine. However, a favorable treatment outcome requires strong Mesothelin expression in tumor cells. Future clinical trials may benefit from a more rigorous selection of appropriate patients based on the level of Mesothelin expression in their tumor tissue. If, in addition, it is possible to better control side effects by introducing protective measures and by doing so to increase the maximum tolerated dose, Anetumab ravtansine has the potency to become a valuable therapeutic tool, especially in the field of gynecological oncology.

Abstract 6318: Acquired resistance in a malignant pleural mesothelioma preclinical model after treatment with Anetumab ravtansine

Abstract Mesothelin (MSLN) is a cell surface glycoprotein with high expression in various cancers and only limited expression in normal tissues. It is expressed in the majority of malignant pleural mesothelioma (MPM), a rare but very aggressive disease. Thus, several anti-MSLN-directed therapies including antibody-drug conjugates (ADCs), immunotoxins, targeted thorium-227 conjugates (TTCs), and CAR-T are currently being explored in MPM and further cancer indications. We developed an anti-mesothelin antibody-drug conjugate, anetumab ravtansine (BAY 94-9343, ARav) in which the anti-MSLN antibody is conjugated via an SPDB linker to the microtubule disrupting payload DM4, a derivative of maytansine. In order to explore potential mechanisms of acquired resistance to treatment with ARav in MPM, the cell line-derived MSLN-positive tumor model NCI-H226 was selected. We had previously shown that this model expresses high levels of MSLN and that ARav dosed for two cycles with 11.2 mg/kg Q3Dx3 inhibited tumor growth by 94% as compared to the vehicle control and achieved a 63% response rate (partial regression in 5 out of 8 mice). NCI-H226 xenograft tumors were transplanted subcutaneously onto mice and treated with 10 mg/kg ARav Q3Dx3 for 3 cycles. A tumor on one mouse that initially responded and afterwards started to regrow despite ARav treatment was excised and tumor tissue was transplanted onto 10 new mice (first in vivo passage). These mice were treated with ARav at 10 mg/kg (Q3Dx3) in the 1rst and 20 mg/kg in the 2nd cycle (n=5; Q3Dx3) or vehicle (n=5). From this approach, 2 tumors (A1, A4) with progressive disease under ARav treatment were obtained. These tumors were each transplanted onto 6 mice (second in vivo passage) which were treated with 20 mg/kg ARav (Q3Dx3) or vehicle. After implantation onto a new host, tumors were not sensitized to ARav treatment indicating that fundamental changes in the tumor cells had happened. To analyze potential molecular changes, tumors from ARav treated and vehicle treated mice were collected and FFPE tumors were analyzed by immunohistochemistry for MSLN and P-gp (MDR1) expression. In addition, MSLN mRNA levels were analyzed by qRT-PCR. In the NCI-H226 tumors serially passaged and with increased growth in the presence of ARav, MSLN protein was reduced to background level when compared to the MSLN Level in freshly established NCI-H226 xenograft tumors. The decrease of MSLN expression was also observed on the RNA level. The experimental data from serial passaging of the NCI-H226 cell line-derived MPM in vivo model with reduced response to ARav show that loss of the MSLN antigen occurs on the mRNA and protein level. Reduction of MSLN expression may decrease binding and internalization of ARav and may thus be a mechanism for acquired resistance in MSLN-expressing tumor models. Citation Format: Christoph Schatz, Sabine Zitzmann-Kolbe, Reimo Tetzner, Marlen Keil, Jens Hoffmann, Dominik Mumberg, Anette Sommer. Acquired resistance in a malignant pleural mesothelioma preclinical model after treatment with Anetumab ravtansine [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6318.

Abstract 1776: Exploring anetumab ravtansine in a preclinical model of thymic carcinoma

Abstract Thymic epithelial tumors (TETs) are a group of rare tumors (0.13 per 100,000 persons), comprised of thymomas and thymic carcinoma, arising from the epithelial cells of the thymus. The biology of these tumors is not well understood, and there are no approved targeted therapies against TETs. One promising drug target is mesothelin (MSLN), which is a surface glycoprotein that is cleaved to produce two proteins: mature MSLN (mMSLN) and megakaryocyte potentiating factor (MPF). In normal physiology, expression of MSLN is limited to mesothelial cells lining the pleura, pericardium, and peritoneum. Its expression is also present in various cancers, including thymic carcinoma, where it is expressed in 79% of cases. In this study, we investigate the ability of the MSLN targeting, fully human monoclonal antibody-drug conjugate (ADC) anetumab ravtansine (ARav) to inhibit growth of thymic carcinoma in in-vitro and in-vivo models. We utilized immunoblotting and RT-qPCR to characterize MSLN expression in human thymoma and thymic carcinoma derived cell lines. Binding of anetumab, the antibody moiety in ARav, to thymoma and thymic carcinoma cell lines was assessed by flow cytometry. In-vitro, ARav's effect on cell proliferation was tested against MP57 cells (thymic carcinoma), stably transfected with MSLN, utilizing CellTiter-Glo. ARav's activity in-vivo as a monotherapy, and as a combination therapy with cisplatin, was tested against MSLN expressing T1889 xenograft tumors in athymic nude mice and compared to vehicle, isotype control ADC, and standard-of-care (SoC) cisplatin. Using the anetumab antibody, thymoma (T1682) and thymic carcinoma (T1889) cell lines were shown to express MSLN on the cell surface. In-vitro, ARav was effective at inhibiting growth of MP57 cells stably transfected with mMSLN. In-vivo, 15 mg/kg ARav inhibited T1889 xenograft tumor growth more efficaciously than vehicle, the isotype control ADC (all given every 2 weeks), and 4 mg/kg cisplatin (given every week). Combining ARav at a dose of 7.5 mg/kg with 4 mg/kg cisplatin yielded an additive effect on inhibiting tumor growth. MSLN is expressed in thymoma and thymic carcinoma derived cell lines. Anetumab ravtansine inhibits growth of MSLN positive thymic carcinoma in our preclinical model; when combined with SoC cisplatin, ARav confers enhanced tumor growth inhibition. Citation Format: Vincent Chen, Anette Sommer, Christoph Schatz, Sabine Zitzmann-Kolbe, In-Kyu Kim, Giuseppe Giaccone. Exploring anetumab ravtansine in a preclinical model of thymic carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1776.

Abstract 1808: Loss of BAP1/BRCA1 counteracts spindle assembly checkpoint activation by the anti-mesothelin antibody drug-conjugate anetumab ravtansine

Abstract Mesothelioma is a rapidly lethal cancer with limited treatment options. Spindle poisons have demonstrated clinical usefulness in mesothelioma, however, predictive biomarkers are lacking. Anetumab ravtansine (ARav) is an antibody drug-conjugate (ADC) comprising a mesothelin targeting antibody coupled to the payload, DM4, a microtubule destabilizer. We have recently identified BRCA1 as a direct regulator of the spindle assembly checkpoint (SAC), which is activated by spindle poisons and mediates mitotic arrest. BAP1 is one of the most frequently inactivated tumor suppressors in mesothelioma and it interacts with and regulates expression of BRCA1. We therefore hypothesized that BAP1 inactivation may confer resistance to ARav, highlighting a potential opportunity to refine personalization with this ADC. Three primary cell lines were derived from mesotheliomas resected during pleurectomy decortication expressed mesothelin (MSLN) and further characterized via whole exome sequencing. Two of the three primary cell lines have BAP1 mutation (R417 frameshift (fs); E631fs) and show reduced BRCA1/MAD2 protein expression. MSLN expression was found to be independent of BAP1 inactivation status or BRCA1 expression level. BAP1 mutant primary cell lines exhibited resistance to ARav by overriding the mitotic arrest with continued cell cycle progression, indicating a defective SAC whereas the cell line with wt BAP1 underwent mitotic arrest followed by cell death. Increase in mitotic index was also observed in primary cell line with wt BAP1 following ARav treatment without any significant change in BAP1 mutant cell lines. Resistance to ARav in BAP1 deleted cell line NCI-H226 was reversed by stable expression of wildtype BAP1 in NCI-H226 indicating critical regulation of SAC by BAP1. Vinorelbine resistant cell line RVR with a defective SAC also exhibited resistance to ARav compared to the parental REN with no change in MSLN expression suggesting that an active SAC is required for prolonged mitotic arrest and subsequent cell death. Wildtype BAP1 and MSLN is critical for efficacy of ARav. Inactivation of BAP1 correlates with loss of BRCA1 expression and dysregulation of the SAC. Citation Format: Anita Singh, Jin-li Luo, Sara Busacca, Alan Dawson, Krishna Kalyan Kolluri, Samuel Janes, Andrew Fry, Anette Sommer, Dean Fennell. Loss of BAP1/BRCA1 counteracts spindle assembly checkpoint activation by the anti-mesothelin antibody drug-conjugate anetumab ravtansine [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1808.

First-in-Human, Multicenter, Phase I Dose-Escalation and Expansion Study of Anti-Mesothelin Antibody–Drug Conjugate Anetumab Ravtansine in Advanced or Metastatic Solid Tumors

PURPOSEThis phase I study, which to our knowledge is the first-in-human study of this kind, investigates the safety, tolerability, pharmacokinetics, and clinical activity of anetumab ravtansine, an antibody–drug conjugate of anti-mesothelin antibody linked to maytansinoid DM4, in patients with advanced, metastatic, or recurrent solid tumors known to express the tumor-differentiation antigen mesothelin.PATIENTS AND METHODSThis phase I, open-label, multicenter, dose-escalation and dose-expansion study of anetumab ravtansine enrolled 148 adult patients with multiple solid tumor types. Ten dose-escalation cohorts of patients with advanced or metastatic solid tumors (0.15-7.5 mg/kg) received anetumab ravtansine once every 3 weeks, and 6 expansion cohorts of patients with advanced, recurrent ovarian cancer or malignant mesothelioma received anetumab ravtansine at the maximum tolerated dose once every 3 weeks, 1.8 mg/kg once per week, and 2.2 mg/kg once per week.RESULTSForty-five patients were enrolled across the 10 dose-escalation cohorts. The maximum tolerated dose of anetumab ravtansine was 6.5 mg/kg once every 3 weeks or 2.2 mg/kg once per week. Thirty-two patients were enrolled in the 6.5 mg/kg once-every-3-weeks, 35 in the 1.8 mg/kg once-per-week, and 36 in the 2.2 mg/kg once-per-week expansion cohorts. The most common drug-related adverse events were fatigue, nausea, diarrhea, anorexia, vomiting, peripheral sensory neuropathy, and keratitis/keratopathy. There were no drug-related deaths. Anetumab ravtansine pharmacokinetics were dose proportional; the average half-life was 5.5 days. Among 148 patients with mesothelioma or ovarian, pancreatic, non–small-cell lung, and breast cancers, 1 had a complete response, 11 had partial responses, and 66 had stable disease. High levels of tumor mesothelin expression were detected in patients with clinical activity.CONCLUSIONAnetumab ravtansine exhibited a manageable safety and favorable pharmacokinetic profile with encouraging preliminary antitumor activity in heavily pretreated patients with mesothelin-expressing solid tumors. The results allowed for the determination of recommended doses, schedules, and patient populations for anetumab ravtansine in phase II studies.

Mesothelin expression in esophageal adenocarcinoma and squamous cell carcinoma and its possible impact on future treatment strategies.

Background:Mesothelin is expressed at very low levels by normal mesothelial cells but is overexpressed in several human cancers. This makes mesothelin a promising target for immunotherapy. Limited data exist about mesothelin expression in esophageal carcinoma. In a current clinical trial, the highly potent anti-mesothelin antibody anetumab ravtansine is used in patients with mesothelin-positive tumors. Response rates are correlated with mesothelin expression (using the Ventana antibody) in tumor cells. No data are available on expression levels using the Ventana antibody. Most data have been generated using the Novocastra antibody. As patients are selected for clinical trials based on antibody staining of tumor samples, a comparison of these two available antibodies is crucial.Methods:We analyzed 481 esophageal carcinomas [373 esophageal adenocarcinomas (EACs), 108 esophageal squamous cell carcinomas (ESCCs)] using two different monoclonal antibodies (Novocastra and Ventana) for mesothelin expression (low–mid and high-level expression, as used in one clinical trial). We also checked for the correlation of these results with clinical and molecular data.Results:We revealed different staining results for both antibodies in EACs: Ventana: 53.6% (low expression: 25.3%; high expression: 28.3%) and Novocastra: 35.7% (low expression: 21.2%; high expression 14.5%). In ESCC we found comparable staining results: Ventana: 13.3% (low expression: 9.5%; high expression: 3.8%) and Novocastra: 13% (low expression: 11.1%; high expression: 1.9%). ARID1a-deficient EAC patients demonstrated significantly higher rates of mesothelin-positive tumors than ARID1a intact EAC patients. No correlations were found with other molecular alterations (TP53 mutation, ERBB2 amplification) or survival rates.Conclusion:To the best of our knowledge, this is the largest study analyzing the importance of mesothelin expression in esophageal carcinoma. This study revealed a significant number of mesothelin-positive esophageal carcinomas, especially adenocarcinomas. New therapeutic targets are urgently required to improve the outcome of patients with locally advanced or metastasized esophageal carcinoma. The inhibition of mesothelin can be a new attractive target.

In Vivo Evaluation of Mesothelin As a Therapeutic Target in Pediatric Acute Myeloid Leukemia

Pediatric acute myeloid leukemia (AML) continues to have a cure rate of only 50% despite the use of highly intensive cytotoxic chemotherapy. Transcriptome sequencing of several AML samples by the NCI/COG TARGET AML Initiative identified mesothelin (MSLN) to be highly overexpressed in about one-third of pediatric AML (Tarlock et al., Blood, 128:2873, 2016). Because MSLN is not expressed in normal bone marrow samples (Fan et al., Blood, 130:3792, 2017) and only to a low level in other human organs and tissues, MSLN is an attractive therapeutic target for pediatric AML (Kaeding et al., Blood, 130:2641, 2017). The anti-MSLN antibody-drug conjugate (ADC) anetumab ravtansine (BAY 94-9343) generated by conjugating MSLN-antibody with tubulin inhibitor DM4 (Meso-ADC), and isotype control antibody conjugated with the same drug (Iso-ADC) were used to evaluate the efficacy of MSLN targeting in vivo. MSLN-overexpressing K562 (K562-MSLN) CML cells and MV4;11 (MV4;11-MSLN) AML cells were generated by lentiviral transduction of MSLN cDNA. Cell line-derived xenografts (CDX) were created by injecting the MSLN-transduced or parental (MSLN-) cells into NSG-SGM3 mice via the tail vein. Mice were randomly assigned to treatment groups when the median percentage of human cells in mouse peripheral blood was greater than 0.5%. K562-MSLN CDX mice treated with Meso-ADC (5 mg/Kg Q3dx3, i.v.) survived a median of 46 days longer than those treated with Iso-ADC (P=0.0011) and significantly longer than comparison groups, including K562-MSLN CDX mice treated with daunorubicin and Ara-C (DA, P=0.0008) or untreated (P=0.0018) (Fig. 1A). Median survival of K562 CDX mice treated with Meso-ADC, Iso-ADC, or untreated was similar (Fig. 1B). MV4;11-MSLN CDX mice treated with Meso-ADC exhibited complete remission and remained disease-free at 1 year post cell injection, with AML cell burden remaining &amp;lt;0.1% throughout the study period (Fig. 1C). In contrast, MV;11-MSLN CDX mice treated with Iso-ADC or untreated succumbed to disease at 72 and 38 days, respectively. Taken together, these results indicate that Meso-ADC was efficacious in reducing leukemia burden, and this effect required MSLN expression in target cells. We have generated a panel of patient-derived xenograft (PDX) lines by transplanting and serially propagating primary pediatric AML samples into NSG-SGM3 mice. The efficacy of Meso-ADC was also evaluated in a systemic PDX model using a MSLN+ PDX line (NTPL-146). NTPL-146 PDX mice treated with Meso-ADC (5 mg/Kg, Q3dx3 -x2 cycles) survived a median of 50 days longer than those treated with Iso-ADC (P=0.0018, Fig. 1D, arrows indicate time when each treatment cycle was initiated). In an independent experiment with NTPL-146 PDX mice, a survival benefit of Meso-ADC treatment over no treatment was observed after 1 cycle of Meso-ADC treatment (5 mg/Kg, Q3dx3, P=0.0019, Fig. 1E). Additionally, a combination therapy strategy with daunorubicin and Ara-C followed by Meso-ADC (DA -&amp;gt; Meso-ADC) resulted in improved median survival over Meso-ADC (P=0.0027) or DA treatment alone (P=0.0018) (Fig. 1E). The disseminated MSLN+ leukemia mouse models described herein support MSLN-targeted antibody-drug conjugate as a potential treatment strategy in MSLN+ AML. Furthermore, we provide the first in vivo demonstration of synergy between MSLN-targeted therapy and conventional chemotherapy in MSLN+ AML, warranting additional investigation to validate and optimize novel strategies for combination therapy. Figure 1 Disclosures Kaeding: Celgene: Employment. Schatz:Bayer AG: Employment. Sommer:Bayer AG: Employment, Equity Ownership.

Abstract 4816: Anetumab ravtansine has monotherapy efficacy in mesothelin positive patient-derived NSCLC tumor models and in a syngeneic tumor model in immunocompetent mice

Abstract Chemotherapy and immune checkpoint inhibitors (ICIs) are approved for treatment of non-small-cell lung cancer (NSCLC). However, there still remains a high medical need in NSCLC, eg. in patients non-responsive to ICIs or progressed after treatment with ICIs. Mesothelin (MSLN) is expressed in ~60% of lung adenocarcinomas. Here, we describe the mesothelin targeting antibody drug conjugate anetumab ravtansine (ARav) with the maytansinoid payload (DM4) as a novel treatment option for NSCLC. In the NSCLC cell line-derived xenograft model NCI-H322, ARav dosed at 2.5 mg/kg or 10 mg/kg, Q3Dx3, i.v., showed significant antitumor activity and was superior to cisplatin (dosed 3 mg/kg, Q3Dx12, ip). In addition, ARav monotherapy also showed antitumor activity in MSLN-positive NSCLC patient-derived xenograft (PDX) tumor models ST1243 and ST1684 at 15 mg/kg, Q2W. Maytansinoids and maytansinoid-based ADCs have been described to induce immunogenic cell death and immune response in vitro and in vivo, respectively. To explore the effects of ARav alone or in combination with anti PD-L1 Ab on tumor growth and the immune system, the MC38 C57BL/6 mouse colon cancer cell line was stably transfected with human mesothelin (MC38-hMSLN). MC38-hMSLN cells were transplanted s.c. in immunocompetent C57BL/6 mice to evaluate the anti-tumor activity of ARav. MC38-hMSLN had a high hMSLN expression level as shown by IHC. A dose of 10 mg/kg of ARav (Q3Dx3, i.v.) was highly efficacious with 11/12 animals showing complete regression of the tumor. Tumor free survivors (TFS) re-challenged 80 days after treatment all rejected MC38-hMSLN (11/11 animals), indicating the development of an immune memory response. The specificity of the immune response was further confirmed in an independently conducted experiment, where previously ARav treated MC38-hMSLN TFS mice were re-challenged with B16-F10 melanoma cells, which grew. Next, a lower dose of ARav (3 mg/kg, Q3Dx3, i.v.) was combined with an anti PD-L1 antibody in the MC38-hMSLN tumor bearing mice. The combination led to increased frequency of TFS compared to each monotherapy (12/12 TFS in the combination versus 7/12 for the anti PD-L1 and 2/12 for ARav). Further studies are currently ongoing to optimize the combination dosing and schedule as well as to characterize the immune cells involved in the response. In summary, the data supports the development of ARav in NSCLC and further exploration of ARav in combination with immune checkpoint inhibitors in MSLN-positive cancer indications. Anetumab ravtansine clinical activity is currently assessed at phase I studies in ovarian cancer in combination with pegylated liposomal doxorubicin (Phase 1b, NCT02751918), in multiple indications including NSCLC (Phase Ib, NCT03102320), and in combination with the anti PD-L1 antibody atezolizumab in NSCLC (Phase I /II, NCT03455556). Citation Format: Anette Sommer, Pascale Lejeune, Sabine Hoff, Annette O. Walter, Sandra Berndt, Lars Roese, Andreas Schlicker, Michael J. Wick, Cem Elbi, Dominik Mumberg, Christoph Schatz. Anetumab ravtansine has monotherapy efficacy in mesothelin positive patient-derived NSCLC tumor models and in a syngeneic tumor model in immunocompetent mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4816.

A Clinical Study of Anetumab Ravtansine in Adults With Solid Tumors Who Have Been Treated in Previous Bayer-sponsored Anetumab Ravtansine Studies

A Clinical Study of Anetumab Ravtansine in Adults With Solid Tumors Who Have Been Treated in Previous Bayer-sponsored Anetumab Ravtansine Studies