A proximity biotinylation-based approach to identify protein-E3 ligase interactions induced by PROTACs and molecular glues

Proteolysis-targeting chimaeras (PROTACs) as well as molecular glues such as immunomodulatory drugs (IMiDs) and indisulam are drugs that induce interactions between substrate proteins and an E3 ubiquitin ligases for targeted protein degradation. Here, we develop a workflow based on proximity-dependent biotinylation by AirID to identify drug-induced neo-substrates of the E3 ligase cereblon (CRBN). Using AirID-CRBN, we detect IMiD-dependent biotinylation of CRBN neo-substrates in vitro and identify biotinylated peptides of well-known neo-substrates by mass spectrometry with high specificity and selectivity. Additional analyses reveal ZMYM2 and ZMYM2-FGFR1 fusion protein—responsible for the 8p11 syndrome involved in acute myeloid leukaemia—as CRBN neo-substrates. Furthermore, AirID-DCAF15 and AirID-CRBN biotinylate neo-substrates targeted by indisulam and PROTACs, respectively, suggesting that this approach has the potential to serve as a general strategy for characterizing drug-inducible protein–protein interactions in cells.

MARKETING RESEARCH OF THE MEDICINE MARKET FOR THE TREATMENT OF BREAST CANCER IN UKRAINE

the article presents the results of the study of the marketing market of medicines for the treatment of breast cancer in Ukraine during 2019-2020. Medicine treatment schemes based on a clinical protocol were analyzed. On December 1, 2019, 184 drugs were registered for the treatment of breast cancer in Ukraine, and on December 1, 2020 - 194. In 2020, the largest share among of the studied drugs belongs to the group of antineoplastic and immunomodulatory drugs - 82.0 %, and the group of drugs that affect the musculoskeletal system - 18.0 %. On December 1, 2020, drugs were represented by 43 manufacturers, of which 18.6 % were Ukrainian and 81.4 % were foreign. Among Ukrainian companies, the primacy belongs to Pharmex Group LLC (4.1 %), Teva Ukraine LLC (3.1 %) and Farmak JSC (2.6 %). The largest share of foreign suppliers occupies Great Britain - 26.3 %, Switzerland and India by 11.9 %. At that time, in 5 pharmacotherapeutic groups (L01AA01, L01BA01, L01CA04, L01XC07, M05BA03) is not represented by any drug Ukrainian manufacturer. Analysis of the assortment of drugs for the treatment of breast cancer by dosage form in 2020 showed that the main share (35.6 %) is represented by concentrate for solution for infusion, 21.6 % - tablets and 20.6 % - lyophilisate for solution for infusion. Other dosage forms form a total of 22.2 % of the total assortment of drugs, studied in the Ukrainian market.

Discovery and Early Clinical Development of Selective Immunoproteasome Inhibitors

Inhibitors of the proteolytic activity of the 20S proteasome have transformed the treatment of multiple B-cell malignancies. These agents have also been employed with success in the treatment of patients with autoimmune diseases and immune-mediated disorders. However, new agents are needed to fully unlock the potential of proteasome inhibitors as immunomodulatory drugs. The discovery that selective inhibitors of the immunoproteasome possess broad anti-inflammatory activity in preclinical models has led to the progression of multiple compounds to clinical trials. This review focuses on the anti-inflammatory potential of immunoproteasome inhibition and the early development of KZR-616, the first selective inhibitor of the immunoproteasome to reach clinical testing.

Development of PRPK Directed Phthalimides

Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide (Pom) bind to cereblon (CRBN) and trigger proteasomal degradation of neo-substrates IKZF1/3 leading to multiple myeloma (MM) cell apoptosis. Pomalidomide (Pom) also binds albeit weakly to p53-related protein kinase (PRPK, aka TP53RK), an understudied kinase reported to be associated with poor prognosis in MM patients. Here, we developed a series of IMiDs based on Pom and conducted a structure-activity relationship (SAR) study to identify a potent and selective PRPK binder. Structural analysis showed that IMiDs bind PRPK in a fundamentally different way from CRBN, and suggested specific derivatization to improve affinity. We employed a structure-guided strategy to develop compound TXM-02-118, which exhibited nanomolar affinityfor PRPK in binding assays, and showed high selectivity for PRPK over CRBN. Overall, the work represents an initial effort to develop tool compounds for studying PRPK. Moreover, it illustrates how a single class of molecules can use different recognition elements to bind diverse targets using fundamentally different binding poses. This has broad implications for chemical probe and lead compound selectivity profiling, and argues for more wide-spread use of global proteomics or similar methodologies.

Immunomodulatory drug discovery from herbal medicines: Insights from organ-specific activity and xenobiotic defenses

Traditional herbal medicines, which emphasize a holistic, patient-centric view of disease treatment, provide an exciting starting point for discovery of new immunomodulatory drugs. Progress on identification of herbal molecules with proven single agent activity has been slow, in part because of insufficient consideration of pharmacology fundamentals. Many molecules derived from medicinal plants exhibit low oral bioavailability and rapid clearance, leading to low systemic exposure. Recent research suggests that such molecules can act locally in the gut or liver to activate xenobiotic defense pathways that trigger beneficial systemic effects on the immune system. We discuss this hypothesis in the context of four plant-derived molecules with immunomodulatory activity: indigo, polysaccharides, colchicine, and ginsenosides. We end by proposing research strategies for identification of novel immunomodulatory drugs from herbal medicine sources that are informed by the possibility of local action in the gut or liver, leading to generation of systemic immune mediators.

A Case of Intestinal Tuberculosis Mimicking Crohn’s Disease: A Clinical and Diagnostic Dilemma

This case highlights the importance of differentiating between Crohn’s disease and intestinal tuberculosis. The rates of misdiagnosis of Crohn’s disease and intestinal tuberculosis range from 50% to 70% because of their non-specific and clinically similar manifestations.If intestinal tuberculosis is misdiagnosed as Crohn’s disease, use of immunomodulatory drugs commonly used for Crohn’s disease can increase the risk of disseminated tuberculosis. Here we present a case highlighting the clinical similarity between these two distinct medical conditions and suggest how a similar scenario can be approached, which can help to differentiate between the two otherwise very similar conditions.

The Combination of Anti-Tigit and Lenalidomide Promotes Synergistic Myeloma-Specific Immunity after ASCT

Multiple myeloma is a largely incurable bone marrow (BM) resident plasma cell malignancy that is increasing in incidence. Autologous stem cell transplantation (ASCT) is the current standard consolidation therapy and a subset of patients achieve durable progression free survival that is suggestive of long-term immune control. Utilizing novel preclinical models, we have provided definitive evidence that this is largely mediated by T cell-dependent myeloma-specific immunity. In both patients and preclinical models, myeloma progression is associated with T cell dysfunction and expression of multiple inhibitory receptors suggesting a loss of immunosurveillance. In mice, we have demonstrated potent anti-myeloma efficacy of TIGIT blockade in both ASCT and non-transplant settings. Here we utilized identical TIGIT Abs that do or do not Fc bind to demonstrate that immunological efficacy after ASCT was absolutely dependent on ADCC (median survival was unreached (>110 days) in Fc-binding vs 73 days in Fc-dead and 71 days in control Ig (cIg)-treated mice). Since TIGIT inhibition does not protect against myeloma relapse in all mice, it is apparent that combinational approaches are required to target non-responders. Therefore, we hypothesized that TIGIT blockade could be combined with immunomodulatory drugs (IMiDs) to provide synergistic anti-myeloma activity after ASCT. To that end, we utilized CRBN transgenic mice to investigate the efficacy of TIGIT blockade in combination with lenalidomide, the standard of care IMiD used in maintenance therapy after clinical ASCT. Briefly, B6 Vk*MYC myeloma-bearing (MM-bearing) mice were lethally irradiated and transplanted with B6 bone marrow (BM) and a suboptimal dose of T cells followed by anti-TIGIT or control Ig (100 mg twice weekly) for 5 weeks with lenalidomide (50 mg/kg daily gavage) or control diluent from D+14 for 3 weeks (Figure 1A). The combination of anti-TIGIT and lenalidomide provided synergistic anti-myeloma efficacy evidenced by prolonged median survival (109 days in combination vs < 60 days in monotherapy/control-treated mice, p<0.01; Figure B). Myeloma M bands were also suppressed in the combination treated mice relative to monotherapy or cIg-treated mice (p<0.01; Figure 1). Analysis of BM CD8 T cells 6 weeks after ASCT demonstrated that combination therapy significantly decreased terminal exhaustion (TOX + TIM3 + CD101 + PD-1 + DNAM-1 ─) with an average of only 20% of CD8 T cells with an exhausted phenotype in the combination group compared to greater than 50% exhausted CD8 T cells in monotherapy or cIg-treated mice (p<0.05; Figure 1C-D). The combination also increased the frequency of central memory and tissue-resident memory subsets (CD49b +CD69 +; p<0.05; Figure 1C-D), and increased IFNγ production from activated (PD-1 +; p<0.05; Figure 1E) cells compared to monotherapy or control Ig-treated mice. Importantly, these phenotypic changes were specific to the BM tumor microenvironment as we observed no effect of combination or monotherapy treatment on CD8 or CD4 T cells in peripheral blood. In sum, these data provide a strong rationale for combining TIGIT inhibition with immunomodulatory drugs to prevent the progression of myeloma. Figure 1 Figure 1. Disclosures Driessens: iTeos Therapeutics: Current Employment, Current equity holder in publicly-traded company. Holmberg: Up-To-Date: Patents & Royalties; Bristol Myers Squibb: Research Funding; Janssen: Research Funding; Merck: Research Funding; Millennium-Takeda: Research Funding; Sanofi: Research Funding; Seattle Genetics: Research Funding. Hill: NeoLeukin Therapeutics: Consultancy; Compass Therapeutics: Research Funding; NapaJen Pharma: Consultancy; Generon Corporation: Consultancy; Roche: Research Funding; iTeos Therapeutics: Consultancy, Research Funding; Syndax Pharmaceuticals: Research Funding; Applied Molecular Transport: Research Funding.

Real-World Assessment of Prior Treatment Patterns in Patients Receiving Belantamab Mafodotin Using a Longitudinal Pharmacy and Medical Open Source Claims Database

Introduction: Belantamab mafodotin (belamaf) is a first-in-class B-cell maturation antigen-targeting antibody-drug conjugate, with a manageable adverse event profile, that has shown durable efficacy in a broad range of patients with relapsed or refractory multiple myeloma (RRMM) (Lonial et al., Lancet Oncol. 2020). As belamaf is an approved therapy, it is important to understand how it is being integrated into the management of patients with RRMM. The aim of this analysis was to examine baseline demographic and treatment patterns of these patients prior to belamaf in a real-world clinical practice. Methods: This descriptive, retrospective cohort study used IQVIA's longitudinal pharmacy and medical open source claims database, a nationally representative real-world database. The study covered the period from January 2001 to April 2021. Patient inclusion into the study required belamaf treatment initiation and ≥18 years of age at initiation. The first claim date for belamaf during the index period (August 2020 to April 2021) was the index date (ID). Key measures included patient demographics and prior MM treatments. Patient demographics were identified at the ID. Prior MM treatments were assessed during the prior treatment period, which extended from the database start date (January 2001) until the ID. The definition of a line of therapy (LOT) used in this analysis was consistent with published guidance (Rajkumar SV, et al. Blood, 2015;126:921-922). Treatments of interest were identified within the first 28 days of the first observed claim for MM treatment. A new LOT was identified if a new treatment was added after the initial 28 days after the start of the LOT, ≥1 treatment was discontinued (defined by a coverage gap >60 days), or the same LOT was restarted at a later date if ≥1 regimen was administered in between. Different therapeutic classes (e.g. PIs, immunomodulatory drugs, anti-CD38 antibodies) could be used in each LOT either singly or in combinations. Addition/deletion of corticosteriods did not constitute a new LOT. Results: 304 patients with a claim for belamaf were identified in the database and included in the analysis; median age was 70 years and 50.3% were female. Median (range) time from the earliest observed claim for MM treatment during the prior treatment period to the ID was 1,812 (104─6,676) days. Median (range) time from the earliest observed diagnosis for MM during the prior treatment period to the ID was 2,123 (22-7,186) days (~5.8 years). The median number of MM therapeutic classes, overall, received during the prior treatment period was 5; 82.9% of patients with MM received ≥4 classes of MM therapy. Prior therapy for MM was observed for 303 patients; the therapeutic classes of those MM treatments are shown in the Table. The median number of observed LOTs received by patients in the prior treatment period was 5 and 73.0% had ≥4 LOTs. LOT for belamaf may be over/under estimated as reporting on some of the MM medications was embargoed by manufacturers and/or specialty pharmacies for certain periods of time, leading to some incompleteness of data. Embargoed products included lenalidomide, pomalidomide, selinexor, thalidomide, ixazomib, and bortezomib. Although data capture for these MM treatments may not be complete in open source claims, some of the claims data for these medications were included in this analysis, i.e., medical claims for infused medications and pharmacy claims from switch suppliers for oral medications. Conclusions: The real-world evidence analyzed in this study is in line with the baseline characteristics reported for the DREAMM-1 and DREAMM-2 trials (Lonial et al., Lancet Oncol. 2020, Trudel et al., Blood Cancer J., 2019). Patients receiving belamaf have triple-class refractory myeloma with multiple LOTs generally aligned with the current indication. The most frequent prior treatments included proteasome inhibitors, anti-CD38 monoclonal antibodies, immunomodulatory drugs, and corticosteroids. Corticosteroid use is high as it is required to be prescribed in combination with other drugs according to their indications. This study identified patients in the US receiving belamaf and provides early insights into the patient demographic and clinical characteristics as well as number and range of treatments prior to treatment with belamaf. Funding: GSK (Study 214283). Figure 1 Figure 1. Disclosures Boytsov: GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company. Stockl: IQVIA: Current Employment; GlaxoSmithKline: Research Funding. Chen: GlaxoSmithKline: Research Funding; IQVIA: Current Employment. Wang: GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company. Wang: IQVIA: Current Employment; GlaxoSmithKline: Research Funding. Cao: IQVIA: Current Employment. Doherty: GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company. Paka: GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company.

611 Exploiting T cells as vehicles of liposomal SHP2i to enhance adoptive cell therapy

BackgroundAdoptive T cell therapy (ACT) is often accompanied by supporting immunomodulatory drugs to protect T cells from the suppressive tumor microenvironment (TME) [1]. However, systemic administration of these immunomodulators can cause serious side effects and fail to distribute optimally to exert sufficient lymphocyte stimulation within the tumor and lymphoid compartments. Loading T cells with adjuvant drugs or cytokines prior to cell transfer provides a solution to this issue, showing the potential to use T cells as vehicles to carry immunomodulatory molecules to target sites [2]. SHP2 is an important hub connecting several intracellular oncogenic signaling pathways including PD-1/PD-L1, representing a notable target for cancer immunotherapy. SHP2 inhibition has been shown to elicited tumor regression by improving CD8+ T cells activity [3]. Herein we present a lipid nanoparticle system encapsulating an SHP2 inhibitor (SHP2i) that allows high T cell loading capacity and enhances their therapeutic activity.MethodsRemote-loading gradients were used to achieve high encapsulation efficiency of SHP2i into the lipid nanoparticle platform. Mouse cytotoxic T cells were loaded with SHP2i, and loading efficiency and release rates from the T cells were evaluated in vitro. Flow cytometry was used to assess T cell viability, proliferation, and phenotype. In vivo biodistribution of loaded T cells was evaluated by labeling lipid nanoparticles with gadolinium and T cells with Cell-trace-marker, which were measured with ICP-MS and Flow respectively. The therapeutic anti-tumor efficacy of the loaded T cells was demonstrated on EG.7-OVA tumor-bearing mice.ResultsThe developed formulation allowed high T cell loading efficiency of SHPi and extended-release over 5 days. Loading T cells with lipid formulated SHP2i did not compromise cell viability and proliferation and resulted in T cells retaining a central memory phenotype than unloaded counterparts. Adoptively transferred T cells loaded with lipid nanoparticles showed the same distribution and proliferation behavior as the unloaded T cells in vivo, accumulating into tumor tissue three days post cell infusion. Loaded OT.I T cells significantly improved tumor growth inhibition and overall survival than OT.I T cells alone, with 5 out of 6 mice completely tumor-free, resulting in durable long-term responders.ConclusionsLoading T cells with liposomal SHP2i before ACT allowed specific and controlled delivery of immunomodulatory drugs by T cells. The loaded T cells showed improved anti-tumor efficacy. The developed lipid formulation allows the loading of a variety of immunomodulatory drugs to T cells, which serve both as a drug delivery vehicle and enhance the tumor efficacy of the transferred cells.ReferencesWaldman AD, Fritz JM, Lenardo MJ. A guide to cancer immunotherapy: from T cell basic science to clinical practice. Nat. Rev. Immunol. 2020. p. 651–68.Combes F, Meyer E, Sanders NN. Immune cells as tumor drug delivery vehicles. J Control Release. Elsevier; 2020;327:70–87.Yuan X, Bu H, Zhou J, Yang CY, Zhang H. Recent Advances of SHP2 Inhibitors in Cancer Therapy: Current Development and Clinical Application. J Med Chem. 2020;63:11368–96.Ethics ApprovalThe study has been approved by the Danish Animal Experiments Inspectorate with the permit number 2020-15-0201-00482. The participants gave informed consent before taking part.