Anti-CD37 Alpha-Amanitin Conjugated Antibodies As Therapeutic Weapons for Richter's Syndrome

Abstract

The leukocyte surface antigen CD37 (TSPAN26), a member of the tetraspanin superfamily, is widely expressed on most malignant B cells, making it an actionable target for treatment of patients with chronic lymphocytic leukemia (CLL) and other B-cell non-Hodgkin lymphoma (NHL) indications. Accordingly, αCD37 antibodies have shown promising results in phase 1/2 clinical trials for CLL and NHL. Richter's syndrome (RS) is the transformation of CLL into an aggressive and rapidly fatal lymphoma, typically a diffuse large B cell lymphoma (DLBCL). RS is a challenging disease since very few effective treatment options exist for these patients and the available regimens, mainly based on R-CHOP scheme, show limited efficacy. We recently established patient-derived xenograft (PDX) models from RS patients and have shown that they can be used to test the efficacy of novel drugs and drug combinations 1,2. All available RS-PDX models were characterized by high-levels of CD37 expression, when assessed by RNA sequencing, reverse-transcriptase-polymerase chain reaction (RT-PCR), flow cytometry (FACS), western blot (WB) and immunohistochemistry (IHC). More precisely, two models (RS1316 and IP867/17) showed slightly higher CD37 levels compared to the others (RS9737 and RS1050). These models were used to test three different αCD37-ATACs®, ADCs which comprise amanitin-derivatives as payload. Amanitin (the main poison in the green deathcap mushroom) belongs to the well-known amatoxin family. Amanitin is taken up by OATP1B3 transporter, solely expressed on hepatocytes. Upon mushroom intoxication, it can lead to severe liver toxicity by inhibiting the RNA polymerase II. Upon conjugation to target-specific antibodies, the maximal tolerated dose is significantly increased by reducing the non-specific liver uptake. By binding to its antigen, ATACs deliver amanitin only into target-positive cancer cells while target negative cells show no off-target toxicity. Consistent with CD37 expression on the cell surface, ex-vivo treatment of freshly purified cells from RS-PDX tumor masses to αCD37-ATACs® resulted in increased apoptosis after 72 hours of treatment, with only minor differences among the 3 ATACs® and the models used. Since alpha-amanitin is a deadly toxin known to target human RNA polymerase II and, at high doses, also RNA polymerase III, we checked messenger RNA levels in basal conditions and after CD37-ATAC® treatment by looking at different housekeeping genes, and confirmed a reduction in global mRNA levels. αCD37-ATAC® efficacy was then assessed in vivo in systemic RS-PDX models where RS cells are intra-venously (i.v.) injected in the tail vein and cells distribute to different tissues (blood, spleen and bone marrow), resembling the human disease. Cells from RS1316, RS1050 and RS9737 models were injected into the tail vein and left to engraft 14 days, before mice were randomly assigned to vehicle or ATAC® groups. A single i.v. treatment for each αCD37-ATAC® was administered, testing two different doses for each compound, and mice were then monitored for survival. Overall, the single administration of all three ATACs® caused highly significant disease regression. In the RS1316 model, independently of the dose or tested ATAC®s, all treated mice, except one, were alive and disease-free until the end of the experiment, 140 days post cells injection, while survival of vehicle-treated mice was 65 days. FACS analysis to trace neoplastic cells in parenchymatous organs and bone marrow confirmed the absence of neoplastic cells. In the other 2 models, RS9737 and RS1050, even though ATAC® treatment did not result in complete disease eradication, a single administration of αCD37-ATAC® resulted in a dramatically increased survival (approximately 35-60 days, depending on the model and ATAC® used). Finally, CD37 expression was confirmed by RNA sequencing on a cohort of 15 primary RS samples, even though with variable levels. Compared to CLL cells, RS samples showed CD37 expression levels comparable to those reported for DLBCL cells. Overall, these data indicate CD37 as a potential target to treat RS patients with selective targeting αCD37-ATACs®. ATACs® is a registered trade mark of Heidelberg Pharma Research GmbH, Germany References Vaisitti T et al. Blood. 2021;137(24):3365-3377. Iannello A, et al. Blood. 2021;137(24):3378-3389. Disclosures Orlik: Heidelberg Pharma: Current Employment. Kulke: Heidelberg Pharma: Current Employment. Pahl: Heidelberg Pharma: Current Employment. Deaglio: Heidelberg Pharma: Research Funding; Astra Zeneca: Research Funding.

CD20 expression regulates CD37 levels in B-cell lymphoma – implications for immunotherapies

“…Overall, our study adds new knowledge to the current state of the art on the regulation of CD37, which in light of recent findings 11 , 12 , 18 , 26 , 31 , 32 constitutes an attractive therapeutic target for immunotherapies. The results of our study once again prove that CD37 is highly variable in DLBCL patients.…”

“… 26 , 31 On the other hand, using ADCs based on the preclinically explored ADC – naratuximab emtansine we show that CD20 loss facilitates CD37 endocytosis and it may translate to increased efficacy of anti-CD37 ADCs that have recently been presented as a highly efficient strategy in B-cell malignancies in vitro and in murine models. 32 The efficacy of ADCs relies on the endocytosis of the target antigen-ADC complex and the release of the cytotoxic payload to the cytoplasm. It has already been reported that rituximab synergizes with naratuximab emtansine by increasing the endocytosis of CD37.…”

CD20 expression regulates CD37 levels in B-cell lymphoma – implications for immunotherapies

“…All in all, our study adds new knowledge on the current state of the art on the regulation of CD37, which in the light of the recent findings (11,12,18,24,29,30) constitutes an attractive therapeutic target for immunotherapies. The results of our study once again prove that CD37 is highly variable in DLBCL patients.…”

“…On the other hand, we hypothesize that CD20 loss facilitates CD37 endocytosis and it may translate to increased efficacy of anti-CD37 ADCs that have recently been presented as a highly efficient strategy in B-cell malignancies in vitro and in murine models (30). The efficacy of ADCs relies on the endocytosis of the target antigen-ADC complex and the release of the cytotoxic payload to the cytoplasm.…”

Novel Approaches for the Treatment of Patients with Richter’s Syndrome

“…Treatment of RS cells with these compounds, both ex vivo and in vivo, induced apoptosis and significantly prolonged survival of treated mice, after a single-dose administration of these ADCs, making CD37 an interesting target for RS patients (Fig. 1A) [59].…”