Natural killer (NK) cells are innate lymphoid cells with antitumor functions. Using an N-ethyl-N-nitrosourea (ENU)-induced mutagenesis screen in mice, we identified a strain with an NK cell ...deficiency caused by a hypomorphic mutation in the Bcl2 (B cell lymphoma 2) gene. Analysis of these mice and the conditional deletion of Bcl2 in NK cells revealed a nonredundant intrinsic requirement for BCL2 in NK cell survival. In these mice, NK cells in cycle were protected against apoptosis, and NK cell counts were restored in inflammatory conditions, suggesting a redundant role for BCL2 in proliferating NK cells. Consistent with this, cycling NK cells expressed higher MCL1 (myeloid cell leukemia 1) levels in both control and BCL2-null mice. Finally, we showed that deletion of BIM restored survival in BCL2-deficient but not MCL1-deficient NK cells. Overall, these data demonstrate an essential role for the binding of BCL2 to BIM in the survival of noncycling NK cells. They also favor a model in which MCL1 is the dominant survival protein in proliferating NK cells.
Abstract
Early attempts at using TNF superfamily members for anticancer therapies, TNF and FAS, led to serious systemic toxicities. However, the discovery of tumor necrosis factor-related ...apoptosis-inducing ligand (TRAIL) introduced an agonist capable of killing tumor cells via apoptosis without the side effects observed with TNF and FAS agonists. First generation TRAIL agonists included a recombinant version of human TRAIL (dulanermin), as well as multiple DR4 and DR5 agonist antibodies. Despite some isolated responders, the initial clinical results were poor. The first-generation TRAIL agonists were limited by poor pharmacokinetics (the half-life for dulanermin was between 30 and 60 minutes) or by poor agonist activity and the need for Fc-mediated cross linking. Here we present the development and evaluation of two second generation TRAIL agonists, MM-201a and MM-201b. Both versions are composed of an IgG1 Fc fused to a single chain TRAIL trimer (Fc-scTRAIL). Mutations within the TRAIL domains, selected from a random mutagenesis library, were introduced to improve stability, expression, and DR5 binding. MM-201a has 5 mutations in each monomeric unit (R130G/N228S/I247V/Y213W/S215D) and MM-201b has 3 mutations in each monomer (R130G/N228S/I247V). In a panel of 27 colorectal carcinoma and sarcoma cell lines, both versions of MM-201 were observed to be significantly more active than all comparators, including the TRAIL cytokine and both DR4 and DR5 antibodies. MM-201a had a level of activity similar to ABBV-621, a single chain TRAIL fused to the N-terminus of an IgG1 Fc that is currently the subject of a Phase 1 trial. However, MM-201b was significantly more active than both MM-201a and ABBV-621, with up to 11-fold lower IC50 across a panel of 12 CRC cell lines. MM-201b treatment reduced cell viability to less than 20% in 10 out 12 colorectal cancer cell lines and in 8 of these cell lines, this was achieved at concentrations less than 1 nM. MM-201 also induced complete cell death at 1 nM or less in 3 of 8 synovial sarcoma and chondrosarcoma cell lines tested. For example, MM-201b reduced the viability of the SW-982 synovial sarcoma cell line to 17% at a dose of 1.5 pM, which is nearly twice the reduction in viability from the same dose of ABBV-621. We next evaluated both versions of MM-201 in multiple colorectal cancer and sarcoma patient-derived xenograft (PDX) models. In the Ewing’s sarcoma PDX model TM01617, MM-201b treatment resulted in 90% tumor growth inhibition. In the same model, treatment with 10 mg/kg docetaxel resulted in 73% growth inhibition; however, in combination with MM-201a, the same dose resulted in a 100% complete response rate. Similar results were observed in the SK-UT1 uterine sarcoma xenograft. Based on this evidence, we believe that MM-201b is best in class and, when combined with an appropriate patient selection strategy, has significant potential for the treatment of sarcomas and colorectal cancer in patients.
Citation Format: Andrew J. Sawyer, Sara Ghassemifar, Christina Wong, Jennifer Richards, Stephanie Grabow, James Suchy, Alexander Koshkaryev, Maja Razlog, Eric Tam, Daryl C. Drummond. Engineering and preclinical activity of MM-201, a best-in-class TRAIL receptor agonist 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 2491.
Studies of gene‐targeted mice identified the roles of the different pro‐survival BCL‐2 proteins during embryogenesis. However, little is known about the role(s) of these proteins in adults in ...response to cytotoxic stresses, such as treatment with anti‐cancer agents. We investigated the role of BCL‐XL in adult mice using a strategy where prior bone marrow transplantation allowed for loss of BCL‐XL exclusively in non‐hematopoietic tissues to prevent anemia caused by BCL‐XL deficiency in erythroid cells. Unexpectedly, the combination of total body γ‐irradiation (TBI) and genetic loss of Bcl‐x caused secondary anemia resulting from chronic renal failure due to apoptosis of renal tubular epithelium with secondary obstructive nephropathy. These findings identify a critical protective role of BCL‐XL in the adult kidney and inform on the use of BCL‐XL inhibitors in combination with DNA damage‐inducing drugs for cancer therapy. Encouragingly, the combination of DNA damage‐inducing anti‐cancer therapy plus a BCL‐XL inhibitor could be tolerated in mice, at least when applied sequentially.
SYNOPSIS
The pro‐survival BCL‐2 family member BCL‐XL is critical for the survival of renal proximal tubular epithelial cells during radiation therapy in cancer patients. Genetic loss of BCL‐XL, but not its pharmacological inhibition, causes fatal kidney damage and secondary anemia in adult mice after total body irradiation.
Inducible loss of BCL‐XL in all cells in adult mice causes primary anemia due to apoptosis of erythroid and megakaryocytic cell populations.
γ‐radiation and BCL‐XL deficiency in all non‐hematopoietic cells cause secondary anemia resulting from kidney damage.
γ‐radiation or DNA damage‐inducing drugs in combination with specific BCL‐XL inhibitor A1331852 is tolerated in mice.
Genetic loss of anti‐apoptotic BCL‐XL causes fatal kidney damage and secondary anemia in adult mice subjected to total body irradiation.
Abstract
TNFR2 is an emerging therapeutic target in immuno-oncology. We have previously shown that an agonistic mouse TNFR2 antibody was able to activate CD8+ T cells in vitro and has anti-tumor ...activity in multiple syngeneic mouse tumor models. This activity required CD8+ T cell and NK cell response and involves Fc γ receptor engagement. Based on this compelling evidence, we have developed MM-401, a human antibody targeting the TNFR2 receptor. MM-401 was humanized from a mouse hybridoma antibody by CDR grafting and includes additional mutations for improved affinity and biophysical properties. Consequently, MM-401 binds with low nanomolar affinity to a region in human TNFR2 that corresponds to the mouse TNFR2 epitope of our mouse surrogate antibody. Although the antibody competes with TNF α in binding the receptor, MM-401 has agonistic activity and induces TNFR2 signaling as observed using a NFκB reporter cell assay. Upon incubation of MM-401 with CD4+ and CD8+ T cells from healthy human blood, we observed upregulation of activation markers and cytokine production comparable to utomilumab (anti-4-1BB), MEDI6469 (anti-OX40), and TRX518 (anti-GITR). We also observed that MM-401 promotes antibody-dependent cellular cytotoxicity (ADCC) in an NK cell-mediated in vitro assay and a reduction in the number regulatory T (Treg) cells in ovarian cancer ascites samples. These data suggest that MM-401 could also promote anti-tumor immunity by mediating ADCC, as well as by direct co-stimulation of T cell responses. Currently, we are evaluating MM-401 in patient derived xenograft (PDX) models in humanized mice generated using NSG-SGM3 mice with cord blood CD34+ hematopoietic stem cells. These results justify the continued development of MM-401 as a modulator of anti-tumor immunity for the treatment of cancer.
Citation Format: James F. Sampson, Vinodh B. Kurella, Violette Paragas, Sandeep Kumar, James E. Lulo, James A. Qiu, Maja Razlog, Ross B. Fulton, Adam J. Camblin, Jennifer M. Richards, Christina S. Wong, Alexander Koshkaryev, James J. Suchy, Stephanie Grabow, Marco Muda, Andreas Raue, Daryl C. Drummond, Eric M. Tam. A novel human TNFR2 antibody (MM-401) modulates T cell responses in anti-cancer immunity 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 555.
Abstract
TNFR2 has been implicated as a novel target for cancer immunotherapy. While TNFR2 has been linked to enhanced suppressive activity of regulatory T cells (Tregs) in auto-immune models, the ...effect of TNFR2-targeted therapy in cancer remains unclear. Here we present a novel monoclonal anti-TNFR2 antibody that provides T cell co-stimulation and yields robust anti-tumor activity in in vitro and in vivo models. In syngeneic murine tumor models, treatment with a murine surrogate anti-TNFR2 antibody results in robust anti-tumor activity both alone and in combination with checkpoint inhibitor antibodies targeting PD-1 and PDL-1. Complete responders exhibited immunological memory months after initial tumor clearance. Furthermore, significant anti-tumor activity was observed in anti-TNFR2-treated mice even in a model that proved resistant to PD1-targeted antibody treatment. Depletion studies suggest that CD8+ T and NK cells are required for activity, while TNFR2 knockout models suggest that TNFR2 expression on cancer cells is not required for activity. Using an antibody with a mutant-Fc, we show that activity is dependent on FcγR binding. Studies in FcγR knockout mice, complemented by studies using different antibody-Fc variants, confirm that enhanced agonism via FcγR binding is the dominant mechanism of action. Contrary to antibodies targeting other TNF superfamily receptors, treatment does not lead to strong depletion of TNFR2-expressing cell types such as Tregs. Consistent with its proposed mechanism, long-term dosing of the anti-TNFR2 antibody did not cause toxicity in two inbred mouse strains when compared to an anti-CTLA4 antibody, which caused weight loss, splenomegaly and elevated inflammatory cytokines in serum. Following anti-TNFR2 treatment, we observed a broad reversal of immunosuppression in the tumor characterized by downregulation of suppressive markers. A human anti-TNFR2 antibody (MM-401) with low nanomolar affinity and binding to the same epitope as the murine surrogate antibody has been developed. MM-401 is being developed as a potential novel treatment option for cancer patients.
Citation Format: Jennifer Richards, Christina Wong, Alexander Koshkaryev, Ross Fulton, Adam Camblin, James Sampson, Lia Luus, James Suchy, Stephanie Grabow, Vinodh Kurella, Sandeep Kumar, James Lulo, James Qiu, Yang Jiao, Lihui Xu, Violette Paragas, Maja Razlog, Marco Muda, Eric Tam, Andreas Raue, Daryl Drummond. MM-401, a novel anti-TNFR2 antibody that induces T cell co-stimulation, robust anti-tumor activity and immune memory 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 4846.
Impaired apoptosis is a cancer hallmark, and some types of lymphomas and other cancers harbor mutations that directly affect key cell death regulators, such as Bcl-2 family members. However, because ...the majority of tumors seem to lack such mutations, we are examining the hypothesis that tumorigenesis can be sustained at least initially by the normal expression of specific endogenous pro-survival Bcl-2 family members. We previously demonstrated that the lymphomagenesis in Εμ-myc transgenic mice, which constitutively overexpress the c-Myc oncoprotein in B-lymphoid cells and develop pre-B and B-cell lymphomas, does not require endogenous Bcl-2. In striking contrast, we report here that loss in these mice of its close relative Bcl-xL attenuated the pre-neoplastic expansion of pro-B and pre-B cells otherwise driven by c-Myc overexpression, sensitized these cells to apoptosis and ablated lymphoma formation. Remarkably, even loss of a single bcl-x allele delayed the lymphomagenesis. These findings identify Bcl-xL as a prerequisite for the emergence of c-Myc–driven pre-B/B lymphoma and suggest that BH3 mimetic drugs may provide a prophylactic strategy for c-Myc–driven tumors.
Abstract
TNFR2, an emerging therapeutic target in immuno-oncology, is upregulated upon T cell activation and is expressed by tumor-infiltrating effector and regulatory T (Treg) cells. We generated a ...novel monoclonal antibody, Y9, specific to murine TNFR2 and investigated its mechanism of action. In vitro, Y9 stimulation of purified T cells increased proliferation and effector function, indicating that Y9 acts as an agonist and can provide co-stimulation.
In vivo, Y9 treatment of mice with established tumors resulted in complete tumor clearance across a variety of models. The activity of Y9 on immune cells was confirmed by its decreased activity in mice depleted of NK or CD8+ T cells. Unlike the proposed Treg depletion mechanism of other therapeutic antibodies, depletion of Treg cells is not the primary mechanism of action of Y9 treatment. Instead, decreased TNFR2 and other co-inhibitory receptor surface expression was observed following treatment. Y9 activity depended on FcγR binding, which facilitated enhanced agonist activity.
Based on this evidence, we developed MM-401, a human antibody targeting TNFR2. MM-401 has agonistic activity; upon incubation of MM-401 with human CD4+ and CD8+ T cells, we observed upregulation of activation markers and cytokine production comparable to MEDI6469 (anti-OX40). We also observed that MM-401 promotes antibody-dependent cellular cytotoxicity (ADCC) in an NK cell-mediated in vitro assay and a reduction in the number of Treg cells in human ovarian cancer ascites. These data suggest that MM-401 could also promote anti-tumor immunity by mediating ADCC, as well as by direct co-stimulation of T cell responses, and justifies the continued development of MM-401 as a novel cancer immunotherapy.
Abstract
Somatic gain of function mutations in nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor or loss of function mutations in Kelch-like ECH Associated Protein 1 (KEAP1) E3 ...ligase, which regulates NRF2 protein levels, are frequently identified in many solid cancers such as non-small cell lung cancer (NSCLC), esophageal squamous cell carcinoma (ESCC), and head and neck squamous cell carcinoma (HNSCC). These genomic alterations drive the activation of cytoprotective NRF2 transcriptional programs. In addition to mutational activation, NRF2 pathway activation has been documented with high frequency in patients without mutations in the pathway. Since tumor cells with hyperactivated NRF2 have been demonstrated to be dependent on this pathway for survival, targeting NRF2 represents an attractive therapeutic approach in tumors with aberrant NRF2 activation. Here, we report the identification of VVD-065, a first-in-class NRF2 inhibitor that acts via an unprecedented mechanism of action. Covalent modification of sensor cysteines on KEAP1 during electrophilic or oxidative stress is known to inhibit KEAP1 mediated NRF2 degradation. In complete contrast, covalent modification of a KEAP1 sensor cysteine by VVD-065 dramatically enhances NRF2 degradation. At low nM concentrations, VVD-065 covalently targets KEAP1 E3 ligase, and allosterically increases the affinity between KEAP1 and CUL3, thereby promoting the formation of active KEAP1-CUL3 E3 ligase complexes and increasing the rate of NRF2 degradation. VVD-065 profoundly reduces NRF2 protein levels in WT KEAP1/NRF2 settings, as well as various KEAP1 and NRF2 mutant settings. Consequently, VVD-065 substantially inhibits NRF2-dependent gene expression and proliferation of NRF2-dependent cell lines. In vivo, oral administration of VVD-065 results in robust degradation of NRF2 and decreased expression of NRF2 target genes. Tumor growth inhibition studies with cell-line derived, and patient derived NSCLC/ESCC xenograft (CDX/PDX) models revealed robust dose-dependent tumor growth inhibition and tumor regression in the absence of any overt toxicity. Since constitutive NRF2 activation often contributes to chemotherapeutic resistance, we also conducted TGI studies with 100+ PDX models to assess combination opportunities with chemotherapeutic agents. A marked combination response was achieved with cisplatin and nab-paclitaxel in various solid tumor types such as NSCLC, ESCC, HNSCC, and uterine cancers. Combination with chemotherapy was safe and well-tolerated in these mouse studies. In summary, we have identified VVD-065, a potent and selective KEAP1 activator that promotes the degradation of NRF2. Degradation of NRF2 led to robust monotherapy response in NRF2-activated cancers and also chemo-sensitized chemo-refractory tumors. A related molecule, acting through the same mechanism of action has now entered into Phase I clinical trials.
Citation Format: Nil Roy, Tine Wyseure, I-Chung Lo, Jordon Inloes, Aaron Snead, Steffen Bernard, Justine Metzger, Jonathan Pollock, Stephanie Grabow, Christie Eissler, Melaminah Williams, Sarah Jacinto, Gabe Simon, Todd Kinsella, David Weinstein, Matt Patricelli. Discovery of VVD-065, a first-in-class allosteric molecular glue of the Keap1-Cul3 E3-ligase complex for the treatment of NRF2-activated cancers abstract. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR011.
Summary
Apoptosis is required to maintain tissue homeostasis in multicellular organisms. Platelets, the anucleate cells that are essential for blood clotting, are a prime example. Their brief life ...span in the circulation is regulated by the intrinsic apoptosis pathway. Pro‐survival BCL‐XL (also termed BCL2L1) is essential for platelet viability. It functions to restrain the pro‐apoptotic BCL‐2 family members BAK (also termed BAK1) and BAX, the essential mediators of intrinsic apoptosis. Genetic deletion or pharmacological inhibition of BCL‐XL results in thrombocytopenia. Conversely, deletion of BAK in platelets doubles their circulating life span. However, what triggers platelet apoptosis in vivo remains unclear. The pro‐apoptotic BH3‐only proteins are essential for initiating apoptosis in nucleated cells, and there is some evidence to suggest they also play a role in platelet biology. We investigated whether PUMA (also termed BBC3), a potent BH3‐only protein that can inhibit all pro‐survival BCL‐2 family members as well as directly activate BAX, regulates the death of platelets. Surprisingly, loss of PUMA had no impact on the loss of platelets caused by loss of BCL‐XL. It therefore remains to be established whether other BH3‐only proteins play a critical role in induction of apoptosis in platelets or whether their death is controlled solely by the interactions between BCL‐XL with BAK and BAX.
Due to the myriad interactions between prosurvival and proapoptotic members of the Bcl-2 family of proteins, establishing the mechanisms that regulate the intrinsic apoptotic pathway has proven ...challenging. Mechanistic insights have primarily been gleaned from in vitro studies because genetic approaches in mammals that produce unambiguous data are difficult to design. Here we describe a mutation in mouse and human Bak that specifically disrupts its interaction with the prosurvival protein Bcl-x sub(L). Substitution of Glu75 in mBak (hBAK Q77) for leucine does not affect the three-dimensional structure of Bak or killing activity but reduces its affinity for Bcl-x sub(L) via loss of a single hydrogen bond. Using this mutant, we investigated the requirement for physical restraint of Bak by Bcl-x sub(L) in apoptotic regulation. In vitro, BakQ75L cells were significantly more sensitive to various apoptotic stimuli. In vivo, loss of Bcl-x sub(L) binding to Bak led to significant defects in T-cell and blood platelet survival. Thus, we provide the first definitive in vivo evidence that prosurvival proteins maintain cellular viability by interacting with and inhibiting Bak.
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