By providing broad resistance to environmental biocides, transporters from the small multidrug resistance (SMR) family drive the spread of multidrug resistance cassettes among bacterial populations. ...A fundamental understanding of substrate selectivity by SMR transporters is needed to identify the types of selective pressures that contribute to this process. Using solid-supported membrane electrophysiology, we find that promiscuous transport of hydrophobic substituted cations is a general feature of SMR transporters. To understand the molecular basis for promiscuity, we solved X-ray crystal structures of a SMR transporter Gdx-Clo in complex with substrates to a maximum resolution of 2.3 Å. These structures confirm the family's extremely rare dual topology architecture and reveal a cleft between two helices that provides accommodation in the membrane for the hydrophobic substituents of transported drug-like cations.
Phosphorylation of the MLKL pseudokinase by the RIPK3 kinase leads to MLKL oligomerization, translocation to, and permeabilization of, the plasma membrane to induce necroptotic cell death. The ...precise choreography of MLKL activation remains incompletely understood. Here, we report Monobodies, synthetic binding proteins, that bind the pseudokinase domain of MLKL within human cells and their crystal structures in complex with the human MLKL pseudokinase domain. While Monobody-32 constitutively binds the MLKL hinge region, Monobody-27 binds MLKL via an epitope that overlaps the RIPK3 binding site and is only exposed after phosphorylated MLKL disengages from RIPK3 following necroptotic stimulation. The crystal structures identified two distinct conformations of the MLKL pseudokinase domain, supporting the idea that a conformational transition accompanies MLKL disengagement from RIPK3. These studies provide further evidence that MLKL undergoes a large conformational change upon activation, and identify MLKL disengagement from RIPK3 as a key regulatory step in the necroptosis pathway.
The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage ...Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) “killer” domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the “necrosome” and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation tomembranes where activatedMLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobodybinding site centered on the α4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the α4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediatemembrane translocation, distinct from known phospholipid binding sites.
RAS guanosine triphosphatases (GTPases) are mutated in nearly 20% of human tumors, making them an attractive therapeutic target. Following our discovery that nucleotide-free RAS (apo RAS) regulates ...cell signaling, we selectively target this state as an approach to inhibit RAS function. Here, we describe the R15 monobody that exclusively binds the apo state of all three RAS isoforms in vitro, regardless of the mutation status, and captures RAS in the apo state in cells. R15 inhibits the signaling and transforming activity of a subset of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., fast exchange mutants). Intracellular expression of R15 reduces the tumor-forming capacity of cancer cell lines driven by select RAS mutants and KRAS(G12D)-mutant patient-derived xenografts (PDXs). Thus, our approach establishes an opportunity to selectively inhibit a subset of RAS mutants by targeting the apo state with drug-like molecules.
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•Development of a high-affinity monobody, R15, selective for apo RAS•R15 inhibits RAS mutants with elevated spontaneous nucleotide release rates•>50% of oncogenic RAS mutants may be susceptible to inhibitors binding to apo RAS•Targeting apo RAS represents a viable approach for inhibiting RAS-driven tumors
Khan et al. develop a high-affinity monobody to nucleotide-free RAS that, when expressed intracellularly, inhibits oncogenic RAS-mediated signaling and tumorigenesis. This study reveals the feasibility of targeting the nucleotide-free state to inhibit tumors driven by oncogenic RAS mutants that possess elevated nucleotide exchange activity.
Abstract
Background: Targeting and engineering γδ T cells has emerged as an orthogonal therapeutic approach in oncology with capacity to modulate both innate and adaptive immunity. Solid tumors (e.g. ...pancreatic ductal adenocarcinoma (PDAC), melanoma, glioblastoma, ovarian-, breast- cancer etc.), can be enriched for γδ1T cells, where they mediate a robust, multi-faceted immunosuppression. We verified the phenotypic and functional features, and the immunosuppressive potential of cancer patient γδ1T cells and have developed a therapeutic antibody (mAb), LYT-210, for selective depletion of these immune evasion culprits. LYT-210 is highly specific, fully human, species cross-reactive IgG1 antibody, aimed for IND enabling studies and clinical development.
Methods: γδ1T cells were profiled and studied from blood of healthy and cancer donors, and from surgically excised primary or metastatic tumors. A proprietary, synthetic Fab-phage library was used to generate LYT-210, a fully human IgG1 mAb. Functional assays (ADCC/ADCP) confirmed an expected and desired, Fcγ-dependent LYT-210 mode of action. We used healthy and patient PBMCs to assess the dynamics, dose dependence and specificity of γδ1 T cell killing with LYT-210. Patient tumor organoids were used for ex vivo efficacy studies. Purified proteins and engineered cell lines served to determine specificity and affinity of LYT-210. Retrogenix protein array (>5500 cell surface and secreted proteins) was used to identify possible off-target binding of LYT-210.
Results: Anti-δ1antibody was used to treat patient tumor organoids (n = 29) and showed potent intra-tumoral T cell activation across tumor types (e.g. colorectal and liver cancer). Additionally, LYT-210 exerted potent ADCP and ADCC on: i) stable cell lines expressing γδ1TCRs and ii) γδ1T cells from patient PBMCs and did so by sparing αβ T cells and γδ2 T cells. Furthermore, LYT-210 induced ADCP and ADCC were Fcγ receptor dependent, confirming LYT-210-specific and effector cell dependent mode of action. LYT-210 induced γδ1 T cell-specific death rapidly, i.e. within 30 minutes of treatment with an EC50 of 26 pM and with the effect sustained over 70 hours of culture. Cell surface proteomics microarray analysis showed very weak cross reaction of LYT-210 with CEACAM5 (a known human cancer cell surface antigen), but in confirmatory assays had minimal binding to CEACAM5 expressed onto HEK293 cells (~200 nM) and no binding at all to purified CEACAM5. The Retrogenix array and additional assays therefore confirm the profound specificity of LYT-210 to γδ1T cells.
Conclusions: We have developed a novel, therapeutic immuno-oncology strategy specifically targeting imunosuppressive γδ1cells. Our data support that this therapeutic approach may have the potential to be transformative for the treatment of cancers where γδ1T cells drive a pro-tumorigenic, immunosuppressive environment.
Citation Format: Tatyana Panchenko, Wei Wang, Eric Denbaum, Akiko Koide, Takamitsu Hattori, Aleksandra Filipovic, George Miller, Shohei Koide. Efficacy and characterization of a specific and potent monoclonal antibody, LYT-210, against immunosuppressive gd1 T cell in cancer 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 1827.
Abstract
RAS is the most frequently mutated human oncogene and about 20% of all human cancers harbor mutations in one of three RAS oncogenes (K, N and H-RAS) with pancreatic cancers harboring RAS ...mutations in >90% of tumors. Therapeutically targeting RAS has been highly challenging and other than allele specific KRAS (G12C) inhibitors, no direct RAS inhibitor has been approved for clinical trial. However, KRAS (G12C) mutations are less abundant in some cancers like pancreatic cancer, thus there remains an unmet need for inhibitors that target the more common RAS mutants. This demand devising novel strategies to identify new approaches to inhibit RAS. Previously, we developed the H/KRAS-specific monobody called NS1 that allosterically inhibit RAS-mediated signaling by targeting the α4-α5 dimerization interface. When used as a genetically encoded intracellular reagent, NS1 inhibited the growth of H/KRAS-mutant human tumor lines both in vitro and in xenograft tumor models. However, these tumor models lack a functional immune system. Here, we evaluated the potency of targeting KRAS dimerization interface with NS1 in an immune-competent murine model of pancreatic cancer. Although human and murine KRAS differ by a single amino acid Asp (D) to Glu (E) at position 132 in the NS1 binding region of the allosteric lobe, NS1 bound similarly to human and murine KRAS both in vitro and in vivo. Furthermore, NS1 expression inhibited ERK-MAPK activation, proliferation and anchorage-independent growth of engineered KPC cells derived from pancreatic tumor in the KPC genetic mouse model. Finally, when these engineered KPC cells were injected orthotopically in immune-competent C57/B6 mice, we observed a dramatic decrease in tumor growth kinetics and tumor burden in NS1 expressing cohorts. Similarly, NS1 expression resulted in decreased ERK-MAPK activation and increased apoptosis. NS1 expressing tumors were characterized by increased infiltration of helper CD4+ T cells and macrophage. Thus, inhibition of mutant KRAS by NS1 results in an enhanced anti-tumor immune response in a syngeneic model for pancreatic cancer. These results establish that targeting α4-α5 dimerization interface of RAS can be a viable therapeutic option for targeting KRAS-mutant pancreatic cancer.
Citation Format: Imran Khan, Julia Lefler, Catherine Marelia, Mariyam Zuberi, Eric Denbaum, Akiko Koide, Cynthia Timmers, Michael Ostrowski, Shohei Koide, John O’Bryan. Targeting the KRAS dimerization interface enhances T-cell mediated anti-tumor response in vivo abstract. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-032.
Abstract
Background: Targeting and engineering γδ T cells has recently emerged as an orthogonal therapeutic approach in oncology with capacity to effectively modulate both innate and adaptive immune ...properties. We and others have shown that in solid tumors such as pancreatic ductal adenocarcinoma (PDA), melanoma, glioblastoma, breast cancer etc., γδ1 T cells express immunosuppression-related molecules and possess a pro-tumorigenic capacity. Previously, we have shown that intra-tumoral γδ T cells from patients bearing PDA, colorectal cancer (CRC) and hepatocellular carcinoma (HCC) potently inhibit patients’ αβ T cells, rendering them immunosuppressed. We hypothesized that anti-δ1 monoclonal antibody would be a potent and effective, novel therapeutic. In order to harness the therapeutic potential of γδ1 T cell blockade we have developed a set of highly specific, fully human anti-δ1 T cell receptor (TCR) antibodies.
Methods: We have sequenced δ chains from 19 cancer patients with primary PDA, primary CRC and gastric cancer (GC) to identify tumor-specific δ1. A proprietary synthetic, human antibody library was screened via phage display to identify high-affinity antibodies. Surface plasmon resonance and bead-based assays were used to measure binding affinity. We used a panel of ex-vivo experiments to assess the immunosuppressive features of γδ T cells. Antibody efficacy was assayed using patient-derived organotypic tumor spheroids (PDOTS) which recapitulate complex tumor architecture. PDOTS of n = 20 patients (PDA, CRC and liver metastases, HCC) were treated with the antibodies and resulting immune profiles analyzed by flow cytometry.
Results: Because the δ1 chains in patients showed diverse CDR3 sequences, we used a selection strategy to identify antibodies that bind diverse δ1 TCRs. Our first-in-class anti-δ1 antibodies have low nanomolar affinity to human δ1 TCRs and show no binding to δ2 TCRs. Furthermore, we chose the lead clinical candidate that showed no preference for the γ chains of the TCR, as patients may harbor a diverse set of γδ heterodimers. Importantly, we show that our lead anti-δ1 antibody achieves reproducible and robust efficacy in the PDOTS system as shown by the up-regulation of pro-inflammatory T cell markers (IFNγ, TNF-α, CD44). Ongoing experiments in γδ knockout mice will investigate the efficacy of combination of γδ1 T cell targeting and existing checkpoint inhibitors (anti- PD1, PDL1, and CTLA4).
Conclusion: We have defined a novel therapeutic immuno-oncology strategy and translated it to develop and characterize a lead clinical candidate anti-δ1 monoclonal antibody. Overall, our results demonstrate that we have an
efficacious, novel immunotherapy that has the potential to be transformative for the treatment of cancers where γδ1 T cells drive a pro-tumorigenic, immunosuppressive environment.
Citation Format: Tatyana Panchenko, Wei Wang, Eric Denbaum, Takamitsu Hattori, Akiko Koide, Aleksandra Filipovic, George Miller, Shohei Koide. Development and testing of the first in class immunotherapy targeting immuno-suppressive δ1 containing γδ T cells for the treatment of pancreatic ductal adenocarcinoma and other solid tumors 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 2382.
Abstract
RAS GTPases are important mediators of oncogenesis with nearly 20% of human tumors harboring mutant RAS proteins. However, pharmacological inhibition of RAS has proven challenging. We have ...employed Monobody technology to discover novel vulnerabilities in RAS that can be exploited to inhibit RAS signaling and tumorigenesis. Monobodies are single-domain synthetic binding proteins that achieve levels of affinity and selectivity similar to antibodies. In contrast to antibodies, Monobodies are fully functional in the reducing environment of the cytoplasm and thus are particularly suitable as genetically encoded “tool biologics”. We previously developed the NS1 Monobody that inhibited RAS by targeting the α4-α5 allosteric lobe to prevent RAS self-association and nanoclustering, and NS1 has become a widely used tool in the RAS research community. Following on this success, we sought to identify additional vulnerabilities in RAS. Based on our discovery that nucleotide-free RAS (apoRAS) inhibits PIK3C2B function, we assessed the feasibility of selectively targeting this state of RAS as an approach to inhibit RAS. Here, we have developed several Monobodies and extensively characterized one of them, R15. Although NS1 was agnostic to the nucleotide state of RAS, R15 bound exclusively to the apo state of all three RAS isoforms but did not interact with nucleotide-loaded RAS. When expressed in cells, R15 inhibited the signaling and transforming activity of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., “fast exchange mutants”), such as G13D and Q61L, but was ineffective against slow exchange mutants such as G12V. Surprisingly, R15 also bound and inhibited RAS(G12D), the most common oncogenic KRAS mutation in pancreatic cancer. Biochemical studies demonstrated that R15 captured RAS from cell lysates in the nucleotide-free state, suggesting that R15 traps apoRAS and prevents nucleotide reloading. Disruption of SOS binding by mutation of D69N further enhanced R15 binding to these fast exchange RAS mutants. Finally, inducible expression of R15 selectively inhibited the tumor forming capacity of tumor lines and patient derived xenografts driven by fast exchange RAS mutants, including RAS(G12D), but not RAS(G12V). Thus, in contrast to conventional wisdom, our approach has established a new opportunity to selectively inhibit the tumorigenic activity of certain RAS mutants by targeting the apo state of RAS with drug-like molecules.
Citation Format: Imran Khan, Akiko Koide, Mariyam Zuberi, Eric Denbaum, Gayatari Ketavar, Kai-Wen Teng, Matthew Rhett, Russell Sepncer-Smith, Ernest R. Camp, Shohei Koide, John O’Bryan. Inhibition of RAS signaling and tumorigenesis through novel targeting novel vulnerabilities abstract. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-064.
RAS is the most frequently mutated oncogene in human cancer with nearly ~20% of cancer patients possessing mutations in one of three RAS genes (K, N or HRAS). However, KRAS is mutated in nearly 90% ...of pancreatic ductal carcinomas (PDAC). Although pharmacological inhibition of RAS has been challenging, KRAS(G12C)-specific inhibitors have recently entered the clinic. While KRAS(G12C) is frequently expressed in lung cancers, it is rare in PDAC. Thus, more broadly efficacious RAS inhibitors are needed for treating KRAS mutant-driven cancers such as PDAC. A RAS-specific tool biologic, NS1 Monobody, inhibits HRAS- and KRAS-mediated signalling and oncogenic transformation both in vitro and in vivo by targeting the α4-α5 allosteric site of RAS and blocking RAS self-association. Here, we evaluated the efficacy of targeting the α4-α5 interface of KRAS as an approach to inhibit PDAC development using an immunocompetent orthotopic mouse model. Chemically regulated NS1 expression inhibited ERK and AKT activation in KRAS(G12D) mutant KPC PDAC cells and reduced the formation and progression of pancreatic tumours. NS1-expressing tumours were characterized by increased infiltration of CD4 + T helper cells. These results suggest that targeting the #x3B1;4-#x3B1;5 allosteric site of KRAS may represent a viable therapeutic approach for inhibiting KRAS-mutant pancreatic tumours.
Abstract
RAS GTPases are important mediators of oncogenesis with nearly 30% of human tumors harboring mutant RAS proteins. However, pharmacological inhibition of RAS has proven challenging. We have ...employed Monobody technology to discover novel vulnerabilities in RAS that can be exploited to inhibit RAS signaling and tumorigenesis. Monobodies are single-domain synthetic binding proteins that achieve levels of affinity and selectivity similar to antibodies. In contrast to antibodies, Monobodies are fully functional in the reducing environment of the cytoplasm and thus are particularly suitable as genetically encoded “tool biologics”. We previously developed the NS1 Monobody that inhibited RAS by targeting the α4-α5 allosteric lobe to prevent RAS self-association and nanoclustering, and NS1 has become a widely used tool in the RAS research community. Following on this success, we sought to identify additional vulnerabilities in RAS. Based on our discovery that nucleotide-free RAS (apoRAS) inhibits PIK3C2B function, we assessed the feasibility of selectively targeting this state of RAS as an approach to inhibit oncogenic RAS function. Here, we have developed several Monobodies and extensively characterized one of them, R15. Although NS1 was agnostic to the nucleotide state of RAS, R15 bound exclusively to the apo state of all three RAS isoforms but did not interact with nucleotide-loaded RAS. When expressed in cells, R15 selectively inhibited the signaling and transforming activity of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., “fast exchange mutants”), such as G13D and Q61L. Surprisingly, R15 bound and inhibited RAS(G12D) mutants which are not reported to be fast exchange. Biochemical studies demonstrated that RAS captured with R15 from cell lysates was indeed nucleotide free, suggesting that R15 traps apoRAS and prevents nucleotide reloading. Finally, intracellularly expressed R15 selectively inhibited the tumor forming capacity of human cell lines driven by fast exchange RAS mutants but not RAS(G12V) in mouse xenografts. Thus, in contrast to conventional wisdom, our approach has established a new opportunity to selectively inhibit certain RAS mutants by targeting the apo state of RAS with drug-like molecules.
Citation Format: Imran Kahn, Akiko Koide, Mariyam Zuberi, Gayatri Ketavarapu, Eric Denbaum, Kai Wen Teng, J. Matthew Rhett, Russell Spencer-Smith, G. Aaron Hobbs, Earnest Ramsay Camp, Shohei Koide, John P. O'Bryan. Inhibition of RAS signaling and tumorigenesis through targeting novel vulnerabilities abstract. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B023.
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