Small-molecule Immunomodulators Research Articles

Abstract B66: Deep TLR PrimedTM T cells induce potent antitumor activity without systemic toxicity

Abstract TLR7 agonists augment immune responses in the tumor microenvironment (TME), primarily through antigen presenting cell (APC) engagement, enhancement of antigen presentation and T cell costimulation. However, multiple TLR agonists have displayed considerable toxicities upon systemic administration. To circumvent this problem, we developed a T cell-mediated delivery system of TLR7 agonists that can target the TME and lymphoid organs to maximize efficacy while avoiding systemic toxicities. Torque has developed the Deep PrimedTM T-cell therapy technology to surface-tether immunomodulators to multitargeted T cells (MTCs) specific for tumor-associated antigens (TAAs) before adoptive transfer. These Deep PrimedTM T cells target multiple tumor antigens and direct the stimulatory activity of cytokines or small-molecule immunomodulators within the tumors but not in circulation. To create Deep PrimedTM MTCs loaded with TLR7 agonists, we screened several liposomal formulations containing two different TLR7 agonists. To determine the best liposome formulations, TLR7 agonists and liposomal formulations were screened for optimal T-cell loading and viability as well as drug release by HPLC both before and after cell freeze and thaw. Liposome formulations with desired characteristics were then tethered to antigen-specific murine TCR-transgenic CD8 T cells to generate Deep TLR Primed T cells. Following ACT into immunocompetent syngeneic tumor-bearing animals, various Deep Primed T-cell products were evaluated for immune cell activation and efficacy by survival and tumor growth inhibition. Multiple liposomal formulations of TLR7 agonist delivered drug onto MTCs with minimal effect on viability and proliferative capacity. However, drug release over time varied considerably with liposome formulation. Additionally, the TLR7 agonist affected drug release after cell freeze and thaw. In vivo, both the TLR7 agonist and liposomal formulation contributed to the efficacy of the Deep TLR Primed T cells in immunocompetent syngeneic tumor-bearing mice. Optimal liposome liposomal formulations tethered to cells inhibited tumor growth significantly more than ACT of T cells alone or combined with systemically delivered TLR agonists. In conclusion, we found select liposomal formulations successfully delivered TLR7 agonist onto MTCs with low toxicity and exhibited controlled drug release. Furthermore, once the agonist was loaded onto MTCs it was not significantly released after cell freeze and thaw. This formulation stability and extended release will be critical for product safety and efficacy. In vivo, Deep TLR Primed T cells displayed superior efficacy and similar safety compared to T cells alone. Deep TLR PrimedTM tumor antigen-specific autologous T cells have the potential to treat a wide variety of tumors and their distant metastases, to turn a cold tumor hot, enabling a new immunotherapy treatment. Citation Format: Nathan Westcott, Austin Boesch, Vasily Rybakin, Ji Young Hwang, Kira Jørgensen, Rasmus Lassen, Martin Kraemer, Martin Bak, Gael Veiga, Jonas Bruun, Carlos Tassa, Harrison Rodts, Manny Sequeira, Karsten Sauer, Thomas Andresen. Deep TLR PrimedTM T cells induce potent antitumor activity without systemic toxicity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B66.

Small-molecule PD-L1 inhibitor BMS1166 abrogates the function of PD-L1 by blocking its ER export

ABSTRACT Therapeutic monoclonal antibodies against the PD-L1/PD-1 (programmed death ligand-1/programmed cell death protein-1) axis have achieved great successes in cancer treatments, but the development of small-molecule immunomodulators of the pathway has lagged far behind. We established a cellular coculture assay with two stable transfectant cell lines, a PD-L1-expressing tumor cell line PC9/PD-L1 and a PD-1-expressing T cell line Jurkat/PD-1. Western blotting analyses were used to monitor the PD-L1/PD-1 interaction and signaling. We analyzed PD-L1 glycosylation by lectin binding assay and glycosidase digestion, and examined subcellular localization of PD-L1 by immunocytochemical staining. Luciferase assay and real-time PCR were used to evaluate T cell activation in the coculture experiments. We found that coculturing of the PC9/PD-L1 cells with the Jurkat/PD-1 cells induced a lysosomal degradation of PD-1. A small-molecule PD-L1 inhibitor BMS1166 developed by Bristol-Myers Squibb inhibited the coculture-induced PD-1 degradation through a unique mechanism. BMS1166 specifically affected PD-L1 glycosylation and prevented transporting of the under-glycosylated form of PD-L1 from endoplasmic reticulum (ER) to Golgi, leading to accumulation of PD-L1 in ER. In doing so, BMS1166 blocked PD-L1/PD-1 signaling. Coculturing PD-L1-expressing cells with PD-1-expressing cells induced degradation of PD-1, which could be used as a readout to identify inhibitors of PD-L1/PD-1 signaling. The small-molecule PD-L1 inhibitor BMS1166 abolished the glycosylation and maturation of PD-L1 by blocking its exporting from ER to Golgi. Our study discovered a new strategy to identify inhibitors of the PD-L1/PD-1 signaling pathway and to develop new drugs for the treatment of cancer.

Potent Lymphatic Translocation and Spatial Control Over Innate Immune Activation by Polymer-Lipid Amphiphile Conjugates of Small-Molecule TLR7/8 Agonists.

Uncontrolled systemic inflammatory immune triggering has hampered the clinical translation of several classes of small-molecule immunomodulators, such as imidazoquinoline TLR7/8 agonists for vaccine design and cancer immunotherapy. By taking advantage of the inherent serum-protein-binding property of lipid motifs and their tendency to accumulate in lymphoid tissue, we designed amphiphilic lipid-polymer conjugates that suppress systemic inflammation but provoke potent lymph-node immune activation. This work provides a rational basis for the design of lipid-polymer amphiphiles for optimized lymphoid targeting.

Selective Toll-like receptor 7 agonists with novel chromeno[3,4-d]imidazol-4(1H)-one and 2-(trifluoromethyl)quinoline/ quinazoline-4-amine scaffolds

Toll-like receptors (TLRs) are promising targets for treatment of viral infections, autoimmune diseases, and cancers. Here, two new series of selective small-molecule TLR7 agonists with novel scaffolds and good selectivity over TLR8 are described, some with potencies in the low micromolar range. 8-Hydroxy-1-isobutylchromeno[3,4-d]imidazol-4(1H)-one (26) from the first series was designed and synthesized on the basis of previously described TLR7 antagonist 2, and is shown to be a selective TLR7 agonist (EC50, 1.8 μM). The second series was based on 2-(trifluoromethyl)quinolin-4-amine and 2-(trifluoromethyl)quinazolin-4-amine scaffolds, which were defined according to our in-house ligand-based virtual screening protocol. Further synthesis of a focused library of analogs, biological evaluation, and docking studies provided systematic exploration of the structure−activity relationships, which indicate that a secondary or tertiary amine with smaller flexible alkyl substituents up to three carbon atoms in length, or bulkier rigid aliphatic rings is required at position 4 on 2-(trifluoromethyl)quinoline/quinazoline scaffold for potent TLR7 agonist activity. The influence of selected TLR7 agonists on cytokine production is also reported showing that N-cyclopropyl-2-(trifluoromethyl)quinazolin-4-amine (46) is able to induce increased levels of IL-6 and IL-8. These data demonstrate successful in-silico definition of novel TLR7 versus TLR8-selective compounds as promising chemical probes for further development of potent small-molecule immunomodulators.

HTiP: High-Throughput Immunomodulator Phenotypic Screening Platform to Reveal IAP Antagonists as Anti-cancer Immune Enhancers

Protein- and cell-based immunotherapeutic agents have revolutionized cancer treatment. However, small-molecule immunomodulators with favorable pharmacological properties for reaching intracellular targets remain to be developed. To explore the vast chemical space, a robust method that recapitulates the complex cancer-immune microenvironment in a high-throughput format is essential. To address this critical gap, we developed a high-throughput immunomodulator phenotypic screening platform, HTiP, which integrates the immune and cancer cell co-culture system with imaging- and biochemical-based multiplexed readouts. Using the HTiP platform, we have demonstrated its capability in modeling an oncogenic KRAS mutation-driven immunosuppressive phenotype. From a bioactive chemical library, multiple structurally distinct compounds were identified, all of which target the same class of proteins, inhibitor of apoptosis protein (IAP). IAP has demonstrated roles in cancer immunity. Identification of IAP antagonists as potent anti-tumor immune enhancers provides strong validating evidence for the use of the HTiP platform to discover small-molecule immunomodulators.

Lymph-Node-Targeted Immune Activation by Engineered Block Copolymer Amphiphiles-TLR7/8 Agonist Conjugates.

Small molecule immuno-modulators such as agonists of Toll-like receptors (TLRs) are attractive compounds to stimulate innate immune cells toward potent antiviral and antitumor responses. However, small molecules rapidly enter the systemic circulation and cause "wasted inflammation". Hence, synthetic strategies to confine their radius of action to lymphoid tissue are of great relevance, to both enhance their efficacy and concomitantly limit toxicity. Here, we demonstrate that covalent conjugation of a small molecule TLR7/8 agonist immunomodulatory to a micelle-forming amphiphilic block copolymer greatly alters the pharmacokinetic profile, resulting in highly efficient lymphatic delivery. Moreover, we designed amphiphilic block copolymers in such a way to form thermodynamically stable micelles through π-π stacking between aromatic moieties, and we engineered the block copolymers to undergo an irreversible amphiphilic to hydrophilic transition in response to the acidic endosomal pH.

PO-427 Establishment of KRAS (G12D)/Trp53 null/Pdx1-cre (KPC) mouse homograft tumour models to facilitate preclinical efficacy evaluation of combinatory immunotherapies

IntroductionPancreatic ductal adenocarcinoma (PDAC) has a poor 5 year survival rate of 6% and remains one of the most difficult to treat cancer types, with very few standard of care options. Fueled by the clinical success of immune checkpoint inhibitors in other solid tumours, several clinical trials have been conducted in PDAC patients. Unfortunately, these attempts have achieved limited clinical benefit as single agent treatments. Now investigators are eager to test combinatory regimens of chemotherapies, small molecule immunomodulators, and immune checkpoint antibodies. Therefore, highly relevant preclinical models are much needed for proof of principle efficacy evaluation studies.KPC models, first described by Tuveson et al;Cancer Res 20166:242–47) such as LSL-KrasG12D/+, LSL-Trp53R172H/+, Pdx1-Cre, and later established as a variety of derivatives with Trp53 heterozygous or homozygous knockouts, recapitulate human PDAC tumours in many aspects including: morphological small ductal tumours with enriched stromal contents, key features of the PDAC immune microenvironment representing robust inflammatory reactions, e.g. high levels of B cells and macrophages, and exclusion of effector T cells. Most importantly, no response to a series of chemotherapies and immune checkpoint inhibitors, including PD-1 and CTLA-4 antibodies, is observed. These features make KPC models an ideal platform to test combinatory therapies for PDAC within a hostile tumour microenvironment.Material and methodsWe have generated a transplantable KPC tumour model by passaging the primary tumour subcutaneously in C57BL/6 mice. These KPC homografts (KPC MuPrime™) retain morphological similarity to human PDAC.Results and discussionsImmunoprofiling of both subcutaneously and orthotopically grafted tumours demonstrated highly enriched CD45 +lymphocytes, particularly B cells and macrophages, in the tumour. The tumour responded poorly to gemcitabine as well as other CTX and targeted treatments, in line with data from the parental line. Orthotopically engrafted tumours led to lethality of the host with a median survival time of 25 days.ConclusionWe are now testing a variety of combinatory therapies including chemotherapies, immunomodulators, and immune checkpoint antibodies with this model.

Abstract 4709: Cell-specific gene program-based small-molecule immunomodulators targeting solid-tumor microenvironments

Abstract Cancer immunotherapy includes promising strategies based on immune checkpoint blockade (e.g., anti-PD-1/PD-L1, anti-CTLA-4). A limitation of such therapies for solid tumors stems from other immune-suppressive mechanisms mediated by myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages. We hypothesize that molecules that target specific suppressive immune cells in the tumor microenvironment can reprogram the pro-tumor microenvironment towards antitumor immunity. Using our proteome-scale target/antitarget network-based lead optimization method (CANDESIGN), we designed and synthesized cell-specific nontoxic chemical libraries with modular functions for anticancer potency and immunomodulation. We used machine learning iteratively on experimental data to identify cell-specific target/anti-target networks (gene programs) as well as designed and synthesized compound libraries targeting cell-specific programs for desired cancer and immune cell function ex vivo. Specifically, we altered suppressive function of MDSCs by targeting upregulated genes in activated monocytic MDSCs in the tumor microenvironment compared to cells in spleen. We synthesized a potent nontoxic compound that specifically modulates activated MDSC function and corresponding changes in CD4+ and CD8+ T-cell activity in a mouse bladder cancer model. We observed a significant reduction in tumor mass following treatment by oral gavage. Interestingly, the bladder cancer cells used in our mouse model (as well as human and dog bladder cancer cells) are insensitive to our lead compound in vitro, suggesting in vivo antitumor activity via immunomodulation. Our lead decreased frequency of monocytic and granulocytic MDSCs in the ascites and the tumor. Moreover, a decreased frequency of MDSC expressing suppressive functional markers (e.g., Arg1, iNOS, PD-L1) and increased IFNγ+CD8+ T cells were observed in the tumor microenvironment after compound treatment. Finally, we performed a small clinical trial on pet dogs with bladder tumors treated with our compound tablets for 4-5 weeks that resulted in ~50% reduction in tumor volume. We conclude that designing cell-specific compounds perturbing the tumor microenvironment to combat immune suppression gives a selective advantage to the immune system to combat solid tumors via single and combination drug/cell therapies. Citation Format: Erin Kischuk, Joydeb Majumder, Jonathan A. Fine, Travis C. Lantz, Deepika Dhawan, Deborah W. Knapp, Timothy L. Ratliff, Gaurav Chopra. Cell-specific gene program-based small-molecule immunomodulators targeting solid-tumor microenvironments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4709.

Retinoic Acid Receptor-Related Orphan Receptor γt (RORγt) Agonists as Potential Small Molecule Therapeutics for Cancer Immunotherapy.

The recent success of PD-1/PD-L1 antibodies for advanced cancer treatment has led to the conclusion that activating the immune system can be employed to fight cancer. These results also encourage the development of small molecule immunomodulators for cancer immunotherapy. RORγt is a key transcription factor mediating Th17 cell differentiation and IL-17 production, which is able to activate CD8+ T cells and elicit antitumor efficacy. Since RORγt agonists have been shown to increase basal activity of RORγt and promote Th17 cell differentiation, development of RORγt agonists could provide a unique approach to cancer immunotherapy. In this review, we summarize RORγt sterol and synthetic agonists, analyze the common ground of their mode of actions, and discuss the potential role of RORγt agonists as small molecule therapeutics for cancer immunotherapy.

Controlling the pro-inflammatory function of 6-sulfo LacNAc (slan) dendritic cells with dimethylfumarate

BackroundThe fumaric acid ester (FAE) dimethylfumarate (DMF) is a small molecule immunomodulator successfully used for the treatment of psoriasis and multiple sclerosis (MS). DMF is thought to inhibit pathogenic immune responses with Th17/Th1T cells, and IL-23/IL-12 producing dendritic cells (DCs). 6-sulfo LacNAc expressing dendritic cells (slanDCs) are a human pro-inflammatory cell type found frequently among the infiltrating leukocytes in skin lesions of psoriasis and brain lesions of MS. ObjectiveTo explore the influence of DMF on functional properties and cell signaling pathways of slanDCs. MethodsIn the context of slanDCs we studied the role of DMF in modulating cell migration, phenotypic maturation, cytokine production, cell signaling and T cell stimulation. ResultsInitially, we observed the reduction of slanDCs numbers in psoriasis skin lesions of FAE treated patients. Studying whether DMF controls the migratory capacity of slanDCs to chemotactic factors expressed in psoriasis we observed an inhibition of the CX3CL1 and C5a depedent cell migration. DMF also attenuated the rapid spontaneous phenotypic maturation of slanDCs, as judged by a reduced CD80, CD86, CD83 and HLA-DR expression. In addition, we observed a DMF-dependent decrease of IL-23, IL-12, TNF-α and IL-10 secretion, and noticed a reduced capacity to stimulate Th17/Th1 responses. DMF targeted in slanDCs different intracellular cell signaling pathways including NFκB, STAT1 and HO-1. ConclusionsWith this study we identify a frequent pro-inflammatory cell type found in psoriasis and MS as a relevant target for the therapeutic immunomodulatory effects of DMF.

Enhancing Adoptive Cell Therapy of Cancer through Targeted Delivery of Small-Molecule Immunomodulators to Internalizing or Noninternalizing Receptors.

Adoptive cell therapy (ACT) has achieved striking efficacy in B-cell leukemias, but less success treating other cancers, in part due to the rapid loss of ACT T-cell effector function in vivo due to immunosuppression in solid tumors. Transforming growth factor-β (TGF-β) signaling is an important mechanism of immune suppression in the tumor microenvironment, but systemic inhibition of TGF-β is toxic. Here we evaluated the potential of targeting a small molecule inhibitor of TGF-β to ACT T-cells using PEGylated immunoliposomes. Liposomes were prepared that released TGF-β inhibitor over ∼3 days in vitro. We compared the impact of targeting these drug-loaded vesicles to T-cells via an internalizing receptor (CD90) or noninternalizing receptor (CD45). When lymphocytes were preloaded with immunoliposomes in vitro prior to adoptive therapy, vesicles targeted to both CD45 and CD90 promoted enhanced T-cell expression of granzymes relative to free systemic drug administration, but only targeting to CD45 enhanced accumulation of granzyme-expressing T-cells in tumors, which correlated with the greatest enhancement of T-cell antitumor activity. By contrast, when administered i.v. to target T-cells in vivo, only targeting of a CD90 isoform expressed exclusively by the donor T-cells led to greater tumor regression over equivalent doses of free systemic drug. These results suggest that in vivo, targeting of receptors uniquely expressed by donor T-cells is of paramount importance for maximal efficacy. This immunoliposome strategy should be broadly applicable to target exogenous or endogenous T-cells and defines parameters to optimize delivery of supporting (or suppressive) drugs to these important immune effectors.

Small molecule agonists of IRF3 activation function as influenza vaccine adjuvants by modulating the humoral and cellular anti-viral immune response

Abstract We have identified a panel of small molecule immunomodulators that activate IRF3 and induce innate immune signaling to drive an antigen-specific protective immune response against viral infections. Our lead adjuvant candidate, KIN1148, binds to retinoic acid inducible gene-I (RIG-I) and induces RIG-I signaling to drive IRF3 activation. Studies using the H1N1 influenza virus challenge model demonstrate that immunization with monovalent influenza split vaccine (SV) and KIN1148 is dose sparing and protects mice against a lethal H1N1 A/California/04/2009 challenge. The SV-H1N1/KIN1148 adjuvant system induces functional antibodies neutralizing viral infectivity and inhibiting influenza hemagglutinin-mediated blood agglutination. SV-H1N1/KIN1148 prime/boost immunizations mediate a strong influenza-specific Th2 response and enhance the production of the immunoregulatory cytokine IL-10 by lung and draining lymph-node-derived T cells from challenged mice. In addition, prime immunization with SV-H1N1/KIN1148 alone provides protection against a 10xLD50 H1N1 challenge by reducing the viral load in the lungs of infected mice. Passive transfer experiments suggest that the protection after prime immunization is at least partially mediated by a cellular immune component. In summary, the KIN1148 split vaccine adjuvant system controls influenza virus infection by boosting the production of functional antibodies, reducing viral load, and inducing a Th2-type immune response. The induction of Th2 and immunoregulatory cytokines by KIN1148-adjuvanted SV might be beneficial to ameliorate the immunopathogenesis of an immune response to highly pathogenic avian influenza virus in infected individuals.

Small molecule drugs with immunomodulatory effects in cancer

Immunotherapy shows promise for positively changing the landscape of the management of many advanced solid tumors, including gastrointestinal (GI) malignancies. Many of these developments have been focused on vaccine-based, monoclonal antibody therapies and more recently, checkpoint inhibitors, although many small molecule inhibitors can function as immunomodulators. Small molecule compounds have several advantages over conventional immunotherapeutic agents including: ease of production and the potential for oral administration. There is a potential niche for small molecule immunomodulators to enhance the efficacy of existing immunotherapeutic and cytotoxic agents. This article focuses on two categories of small molecule compounds with immunomodulatory effects: IDO and MEK inhibitors. Indoleamine -2, 3- dioxygenase (IDO) is known for its effects in tumor immunity. IDO inhibitors are generally well-tolerated and have the potential to enhance anti-tumor responses when combined with checkpoint inhibitors. MEK inhibitors affect signal transduction of the RAS-RAF-MEK pathway and numerous MEK inhibitors are currently being investigated in solid tumors. Small molecule immunomodulators are currently being investigated for their potential role in augmenting the effects of conventional immunotherapeutic agents although further research is required to identify those patients most likely to respond to combination therapy.

Nanoparticulate STING agonists are potent lymph node-targeted vaccine adjuvants.

Cyclic dinucleotides (CDNs) are agonists of stimulator of IFN genes (STING) and have potential as vaccine adjuvants. However, cyclic di-GMP (cdGMP) injected s.c. shows minimal uptake into lymphatics/draining lymph nodes (dLNs) and instead is rapidly distributed to the bloodstream, leading to systemic inflammation. Here, we encapsulated cdGMP within PEGylated lipid nanoparticles (NP-cdGMP) to redirect this adjuvant to dLNs. Compared with unformulated CDNs, encapsulation blocked systemic dissemination and markedly enhanced dLN accumulation in murine models. Delivery of NP-cdGMP increased CD8+ T cell responses primed by peptide vaccines and enhanced therapeutic antitumor immunity. A combination of a poorly immunogenic liposomal HIV gp41 peptide antigen and NP-cdGMP robustly induced type I IFN in dLNs, induced a greater expansion of vaccine-specific CD4+ T cells, and greatly increased germinal center B cell differentiation in dLNs compared with a combination of liposomal HIV gp41 and soluble CDN. Further, NP-cdGMP promoted durable antibody titers that were substantially higher than those promoted by the well-studied TLR agonist monophosphoryl lipid A and comparable to a much larger dose of unformulated cdGMP, without the systemic toxicity of the latter. These results demonstrate that nanoparticulate delivery safely targets CDNs to the dLNs and enhances the efficacy of this adjuvant. Moreover, this approach can be broadly applied to other small-molecule immunomodulators of interest for vaccines and immunotherapy.

Preclinical assessment of a novel small molecule inhibitor of tryptophan 2,3-dioxygenase 2 (TDO2)

Meeting abstracts iTeos leverages the science of LICR to target metabolism of the tumor microenvironment and develops small molecule immunomodulators. TDO2 is a heme-containing enzyme highly expressed in the liver, in specific brain regions and liver/brain tumors. It catalyses the first and rate-

Inhibition of B Cell Differentiation to the Plasmablast and Plasma Cell Lineage by CC-220, a Potent Modulator of the CRBN E3 Ubiquitin Ligase Complex

AbstractAbstract 3279 Background:CC-220 is an immunomodulatory compound which binds to the CUL4 family E3 ubiquitin ligase complex protein cereblon (CRBN) with high affinity and modulates the ubiquitination of substrate proteins. To explore the effects of CRBN targeting on the differentiation of B cells to the plasmablast and plasma cell lineages, an in vitro model of primary human B cell differentiation was developed based on the methods of Jourdan et al. (Jourdan M, et al. Blood. 2009). Methods:CD19+ peripheral blood human B cells from normal donors, or total PBMC for patients with systemic lupus erythematosus (SLE), were cultured in the presence of interleukin (IL)-2, IL-10, IL-15, TLR9 agonist, and CD40L for 4 days, followed by IL-2, IL-6, IL-10, and IL-15 for another 3 days. Cells were counted, viability assessed, and expression of CD20, CD38, CD44, and CD83 were measured by flow cytometry. Plasmablast lineage factors IRF-4, BLIMP-1, XBP-1, and IgJ, and germinal center markers PAX-5 and BCL-6 were measured by qRT-PCR. Intracellular protein expression was measured by laser scanning cytometry. Secreted immunoglobulins IgG and IgM were measured by ELISA. Results:In normal B cell cultures, CC-220 reduced the percentage of total viable cells in these cultures after 4 days to 69.6% (P≤0.05) or 35.8% of control (P≤0.001) at 20 nM or 200 nM CC-220, respectively. CC-220 decreased the percentage of viable CD20-CD38+ plasmablasts on day 7 from 30.4% in control cultures in a dose-dependent manner to 27.3%, 2.1%, and 0.4% at 2 nM, 20 nM, and 200 nM CC-220, respectively. On Day 7, qRT-PCR analysis showed that CC-220 (20 nM) reduced expression of the plasmablast lineage factors IRF-4, BLIMP-1, XBP-1, and IgJ gene expression to 20.5%, 14.3%, 15.1%, and 31.5% of control, respectively (P ≤0.001). By intracellular flow cytometry, CC-220 (20 nM) significantly decreased IRF-4 (P<0.5), BLIMP-1 (P< 0.05), and XBP-1 (P<0.05) protein expression at Day 4, but significantly increased BCL-6 (P<0.05) protein expression on Day 7. By laser scanning cytometry on Day 7, CC-220 (20 nM) reduced CD38+ cell intracellular protein expression of IRF-4 (P≤0.001), and BLIMP-1 (P≤0.001), and increased BCL-6 expression (P≤0.05) (n=3). CC-220 inhibited secreted IgG production with an IC50=1.8 nM (n=3).In PBMC from SLE patients, CC-220 (20 nM) had similar effects as in normal B cells, reducing BLIMP-1, XBP-1, and IgJ gene expression to 52.8%, 49.2%, and 13.6% of control, respectively (P≤0.001, n=3). CC-220 (20 nM) significantly reduced CD38+ plasmablast intracellular protein expression of BLIMP-1 (P≤0.01) and IRF-4 (P ≤0.001), and increased BCL-6 (P≤0.05) (n=3). CC-220 inhibited secreted IgM and IgG production by SLE patient PBMC with IC50s of 0.9 nM and 3.2 nM, respectively (n=3). Conclusions:These results demonstrate that targeting of the E3 ubiquitin ligase complex substrate co-receptor CRBN with the small molecule immunomodulator compound CC-220 results in potent inhibition of B cell differentiation to the plasmablast lineage, as shown by a reduction in the percentage of viable CD38+ cells, a decrease in BLIMP-1, XBP-1, IRF4, and IgJ gene and protein expression, and inhibition of secreted immunoglobulin production. These data implicate the CUL4-CRBN complex in the differentiation of B cells to the plasma cell lineage. CC-220 is currently entering clinical development for the treatment of immune diseases associated with autoantibody production. Disclosures:Schafer:Celgene: Employment, Equity Ownership. Capone:Celgene Corp: Employment, Equity Ownership. Wu:Celgene Corp: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership.

Small Molecule Immunomodulatory Drugs

As the molecular pathobiology of immunologically based diseases, such as rheumatoid arthritis, has become clearer, pharmaceutical researchers have responded with highly efficacious and selective biological compounds. In contrast to older, nonspecific small-molecule therapeutics, the exquisite species sensitivity of monoclonal antibodies has introduced new challenges to preclinical safety studies. Repeated exposure of animals to biopharmaceutical compounds tends to be restricted in the species in which these compounds have pharmacological action, and it tends to stimulate antidrug immune responses with acceleration of clearance, thereby limiting the duration of repeat-dose studies and potentially resulting in hypersensitivity reactions. Thus, the safety testing of biopharmaceutical compounds has necessitated the use of relatively short-term studies in rodents, whereas nonhuman primates have become the primary tool for large-animal, repeat-dose studies. However, as the number of highly targeted and efficacious small-molecule immunomodulators rapidly increases, these molecules will be developed in a manner similar to that of other small molecules with regard to safety assessment. Because such approaches inherently push drug levels to achieve maximally tolerated doses, the pharmacologic specificity of these new small-molecule drugs may be lost as they affect additional receptors and pathways. Therefore, toxicologic pathologists must refamiliarize themselves with the consequences of profound immunosuppression in species other than nonhuman primates. The interrelationships of cytokine signaling and receptor biology are complex, highly integrated, and at times paradoxical, and the loss of specificity at high doses may result in unforeseen consequences caused by the impact on complex down-stream pathways that culminate in exaggerated and adverse responses. The species specificity of such responses may not be inherently familiar or anticipated.

Catalytic Enantioselective Electrophilic Aminations of Acyclic α-Alkyl β-Carbonyl Nucleophilies.

Highly enantioselective aminations of acyclic α-alkyl β-keto thioesters and trifluoroethyl α-methyl α-cyanoacetate (12) with as low as 0.05 mol % of a bifunctional cinchona alkaloid catalyst were established. This ability to afford highly enantioselectivity for the amination of α-alkyl β-carbonyl compounds renders the 6'-OH cinchona alkaloid-catalyzed amination applicable for the enantioselective synthesis of acyclic chiral compounds bearing N-substituted quaternary stereocenters. The synthetic application of this reaction is illustrated in a concise asymmetric synthesis of α-methylserine, a key intermediate previously utilized in the total synthesis of a small molecule immunomodulator, conagenin.

Toxicity of targeted therapies in elderly patients

Targeted therapies have revolutionized the treatment of cancer in recent years. However, treating elderly patients with these new biological therapies remains a challenge. Bevacizumab, a monoclonal antibody directed toward VEGF, appears to increase cardiovascular events in patients who are aged 65 years and over and increases the toxicity in these patients with lung cancer when added to chemotherapy. Owing to the less significant data available concerning the toxicity of trastuzumab and cetuximab, one cannot consider their use to be too toxic for the elderly patient. Rituximab has revolutionized the treatment of lymphomas with no apparent toxic effects in the elderly population. Small-molecule tyrosine kinase inhibitors and immunomodulators – thalidomide and lenalidomide – are emergent drugs that also appear to be efficient and safe for use in these patients. Few data are available concerning the toxicity of biological drugs in the elderly population, hence their use must be evaluated in each single case, always taking into consideration the risk–benefit ratio for the individual patient.

New targets for lymphoma treatment

The rapid development in understanding the biology oflymphoid cells has led to the identification of multiplepathways and targets for therapy in recent years. These rangefrom cell surface interactions involving the B-cell receptor(BCR), through signalling pathways such as that mediated viathe mammalian target of rapamycin (mTOR) and NF-jB,modulation of gene expression by histone acetylation, controlof protein degradation by the proteosome or heat shockproteins, and regulation of apoptosis by both surface deathreceptors and intracellular proteins. In addition, a variety ofstrategies for driving immune responses to lymphoma havebeen investigated, including the use of small moleculeimmunomodulators, and agonistic antibodies which targetimmune cells rather than the malignant cells themselves.Finally, the idiotype remains an attractive target for B-celllymphoma, being wholly patient specific, and vaccinationstrategies aimed at raising humoral and cellular responses are atan advanced stage of clinical study. The development of allthese agents remains at an experimental stage, with none havingas yet delivered clear advantages in terms of overall survival inphase III trials, although some are getting close. In many casesnovel combinations are in clinical testing, with eitherconventional cytotoxics, monoclonal antibodies or several newagents together. A deeper understanding of their mechanismsof action should allow the rational development of suchcombinations in the near future.