Intermediate Affinity Research Articles

Enhanced Drug Delivery to Solid Tumors via Drug-Loaded Nanocarriers: An Image-Based Computational Framework.

ObjectiveNano-sized drug delivery systems (NSDDSs) offer a promising therapeutic technology with sufficient biocompatibility, stability, and drug-loading rates towards efficient drug delivery to solid tumors. We aim to apply a multi-scale computational model for evaluating drug delivery to predict treatment efficacy.MethodologyThree strategies for drug delivery, namely conventional chemotherapy (one-stage), as well as chemotherapy through two- and three-stage NSDDSs, were simulated and compared. A geometric model of the tumor and the capillary network was obtained by processing a real image. Subsequently, equations related to intravascular and interstitial flows as well as drug transport in tissue were solved by considering real conditions as well as details such as drug binding to cells and cellular uptake. Finally, the role of periodic treatments was investigated considering tumor recurrence between treatments. The impact of different parameters, nanoparticle (NP) size, binding affinity of drug, and the kinetics of release rate, were additionally investigated to determine their therapeutic efficacy.ResultsUsing NPs considerably increases the fraction of killed cells (FKCs) inside the tumor compared to conventional chemotherapy. Tumoral FKCs for two-stage DDS with smaller NP size (20nm) is higher than that of larger NPs (100nm), in all investigate release rates. Slower and continuous release of the chemotherapeutic agents from NPs have better treatment outcomes in comparison with faster release rate. In three-stage DDS, for intermediate and higher binding affinities, it is desirable for the secondary particle to be released at a faster rate, and the drug with slower rate. In lower binding affinities, high release rates have better performance. Results also demonstrate that after 5 treatments with three-stage DDS, 99.6% of tumor cells (TCs) are killed, while two-stage DDS and conventional chemotherapy kill 95.6% and 88.5% of tumor cells in the same period, respectively.ConclusionThe presented framework has the potential to enable decision making for new drugs via computational modeling of treatment responses and has the potential to aid oncologists with personalized treatment plans towards more optimal treatment outcomes.

Tripodal scaffolds with three appended imidazole thiones for Cu(I) chelation and protection from Cu-mediated oxidative stress

Imidazole thiones appear as interesting building blocks for Cu(I) chelation and protection against Cu-mediated oxidative stress. Therefore, a series of tripodal molecules derived from nitrilotriacetic acid appended with three imidazole thiones belonging either to histamine-like or histidine-like moieties were synthesized. These tripods demonstrate intermediate affinity between that previously measured for tripodal analogues bearing three thiol moieties such as cysteine and those grafted with three thioethers, like methionines, consistently with the thione group in the imidazole thione moiety existing as a tautomer between a thiol and a thione. The two non-alkylated tripods derived from thioimidazole, TH and TH* demonstrated three orders of magnitude larger affinity for Cu(I) (logKpH 7.4 = 14.3) than their analogues derived from N,N′-dialkylated thioimidazole TMe and TEt (logKpH 7.4 = 11–11.6). Their efficiency to inhibit Cu-mediated oxidative stress is demonstrated by several assays involving ascorbate consumption or biomolecule damages and correlates with their ability to chelate Cu(I), related to their conditional complexation constants at pH 7.4. The two non-alkylated tripods derived from thioimidazole, TH and TH* are significantly more powerful in reducing Cu-mediated oxidative stress than their analogues derived from N,N′-dialkylated thioimidazole TMe and TEt.

Zero-Valent Iron Nanoparticles Remediate Nickel-Contaminated Aqueous Solutions and Biosolids-Amended Agricultural Soil.

Nickel (Ni+2) accumulation in wastewater treatment sludge poses a potential environmental risk with biosolids-land application. An incubation experiment was conducted to evaluate the effect of nanoparticles of zero-valent iron (nZVI) on Ni+2 sorption in biosolids-treated agricultural soils. Two application rates of biosolids (0, 5%, w/w) and four treatment levels (0, 1, 5, and 10 g/kg) of nZVI were examined, either separately or interactively. The results of this study showed significant differences in Ni+2 sorption capacity between different nZVI treatments. The initial Ni+2 concentration in biosolids-amended soil significantly affected Ni sorption in the soil treated with nZVI. The “H-shape” of sorption isotherm in nZVI-treated soil reflects strong interaction between the Ni concentration and the nZVI treatment, while the C-shape of sorption isotherm in biosolids-amended soil without the nZVI treatment indicates intermediate affinity for Ni+2 sorption. Nickel retention in soil was increased with the increase of nZVI levels. The removal efficiency of Ni+2 by nZVI from solution was increased with the increase of pH from 5 to 11 and reached a maximum of 99.56% at pH 11 and nZVI treatment of 10 g/kg. The Ni+2 desorption rate decreased from 92 to 7, 4, and 1% with increasing nZVI treatment levels from 0 to 1, 5, and 10 g/kg, respectively, with a soil Ni+2 concentration of 50 mg/L. The maximum adsorption capacity (𝑞max) of 10 g/kg nZVI-treated soil was 333.3 mg/g, which was much higher than those from the other treatments of 0 (5 mg/g), 1 (25 mg/g), and 5 g/kg (125 mg/g). The underlying mechanism for Ni+2 immobilization using nZVI in an aquatic environment is controlled by a sorption process, reduction of metal ion to zero-valent metal, as well as (co)precipitation. Moreover, increasing the nZVI treatment level in biosolids-amended soil significantly decreased bioavailable Ni+2 concentrations in the soil.

Anti-Tumor Functions of Prelatent Antithrombin on Glioblastoma Multiforme Cells.

Antithrombin, the main physiological inhibitor of the coagulation cascade, exerts anti-tumor effects on glioblastoma multiforme cells. Antithrombin has different conformations: native, heparin-activated, prelatent, latent, and cleaved. The prelatent form has an intermediate affinity between latent and native antithrombin, although it is the most antiangiogenic form. Herein, we investigate the effect of this conformation on the tumorigenic processes of glioblastoma multiforme cells. Antithrombin forms were purified by chromatography. Chromogenic/fluorogenic assays were carried out to evaluate enteropeptidase and hepsin inhibition, two serine proteases involved in these processes. Wound healing, Matrigel invasion and BrdU incorporation assays were performed to study migration, invasion and proliferation. E-cadherin, Vimentin, VEGFA, pAKT, STAT3, pSTAT3, and pERK1/2 expression was assessed by Western blot and/or qRT-PCR. Prelatent antithrombin inhibited both enteropeptidase and hepsin, although it was less efficient than the native conformation. Exposure to prelatent antithrombin significantly reduced migration and invasion but not proliferation of U-87 MG, being the conformation most efficient on migration. Prelatent antithrombin down-regulated VEGFA, pSTAT3, and pERK1/2 expression in U-87 MG cells. Our work elucidates that prelatent antithrombin has surprisingly versatile anti-tumor properties in U-87 MG glioblastoma multiforme cells. This associates with resistance pathway activation, the decreased expression of tumorigenic proteins, and increased angiogenesis, postulating the existence of a new, formerly unknown receptor with potential therapeutic implications.

Comparison of adsorption behaviors of selected endocrine-disrupting compounds in soil.

Bisphenol-A (BPA), 17α-ethinylestradiol (EE2), and 4-nonylphenol (4NP) are endocrine-disrupting chemicals (EDCs) that are useful models for studying the potential fate and transport of EDCs in soil and water environments. Two alluvial soils with contrasting physicochemical properties were used as adsorbents for this study. The Zook soil material had more organic matter and clay than the sandy loam Hanlon soil material. Batch equilibrium experiments were performed to generate adsorption isotherms, to determine the adsorption parameters, and to assess desorption hysteresis. Adsorption of BPA to both soils followed an L-type isotherm, and 4NP adsorbed to both Hanlon and Zook soils exhibited S-shape isotherms. EE2 adsorbed to the Zook soil also followed an S-shaped isotherm, but EE2 adsorbed to the Hanlon soil showed an H-type isotherm. Overall, the Sips model fit the data well, with standard errors of prediction generally ≤6%. The adsorption affinity (KLF ) values were highest for 4NP, and BPA had the lowest hysteresis indices. The data suggest that BPA was most likely adsorbed by soil organic matter via hydrogen bonding involving its two phenolic groups. In contrast, isotherm shape, model affinity indices, lack of desorption, and molecular-scale characteristics led us to infer that 4NP was adsorbed largely by the retention of molecular clusters, perhaps in clay nanopores. Finally, the adsorption of EE2 exhibited different isotherm shapes for the two soils as well as intermediate affinity and desorption indices, suggesting that EE2 molecules could be retained both by soil organic matter and by clay.

CD122-SELECTIVE IL-2 COMPLEXES TREAT OVARIAN CARCINOMAS, INDUCE TREG FRAGILITY AND PROMOTE T CELL STEM CELLS

Abstract Regulatory T cells (Treg) engage IL-2 by high affinity CD25, and anti-tumor effector T cells (Teff) use intermediate affinity CD122. We studied IL-2 complexes (IL-2c) that selectively activate CD122+ Teff over CD25+ Tregs. We found IL-2c but not aPD-L1 potently inhibits ID8agg, an aggressive mouse ovarian cancer (OC) model. IL-2c decreased ascites Treg functional markers (CD25, GranzymeB) while upregulating them on Teffs. IL-2c inhibited Treg suppressive function in ascites but not TDLN. Ascites Tregs after IL-2c showed a fragile phenotype (increased PD-1, T-bet, and IFNg with maintained FoxP3) known to contribute to better immunotherapy response. These data suggest IL-2c inhibits Treg in the ascites through inducing fragile Tregs. Neither CD8+ T cells nor gd T cells is required for IL-2c induced Treg fragility. Studies testing other populations are ongoing. CD8+TCF-1+T cell stem cells (TCSC) improve immune checkpoint blockade in many cancer types. In ID8agg tumors, IL-2c induces CD8+TCF-1+ TCSC with tumor inhibitory capacity when transferred into Rag KO mice. tSNE analysis revealed IL-2c-induced TCSC are CXCR5+PD-1− express CCR2 CXCR3 and produce TNFa, while aPD-L1 induced TCSC are CXCR5+ and PD-1+, consistent with previous reports. These data suggest IL-2c induces beneficial CD8+TCSC distinct from aPD-L1. Although aPD-L1 is not efficacious alone in ID8agg, it significantly promoted IL-2c efficacy. Mice treated with IL-2c+αPD-L1 are resistant to tumor re-challenge even 6 weeks after final dose, suggesting durable immune memory. In sum, IL-2c is a novel OC immunotherapy that targets distinct immune pathways simultaneously, including inhibiting Tregs, boosting TCSCs, and inducing immune memory when combined with aPD-L1.

Lysine modification of human serum albumin and its effect on protein conformation and nalidixic acid binding

In order to investigate the involvement of lysine residues of human serum albumin (HSA) in nalidixic acid (NA) binding, various modified preparations of HSA such as 44% carbamylated (C44), 83% carbamylated (C83) and 85% acetylated (A85) were made by treating the HSA solution with a different molar excess of potassium cyanate and acetic anhydride. The extent of modification, charge homogeneity and conformational changes of these derivatives were checked by TNBSA reaction method, polyacrylamide gel electrophoresis (PAGE) and gel filtration using Sephacryl S-200 HR column, respectively. Binding of NA to HSA and its derivatives was examined using fluorescence quenching titration method to determine the binding constant. The emergence of a single band in PAGE and single symmetrical peak in gel filtration results confirmed the charge and size homogeneity of these derivatives. Hydrodynamic properties such as Stokes radius and frictional ratio, as obtained from the analytical gel filtration results suggested molecular expansion in C83 and A85 HSAs while C44 HSA retained the native conformation. Addition of NA to both native and modified HSA derivatives quenched the fluorescence intensity of the protein at 344 ​nm to a different extent. Whereas the values of the Stern-Volmer constant (KSV) and bimolecular quenching rate constant (kq) suggested, NA-HSA complex formation, binding constant (Ka) value suggested an intermediate binding affinity between NA and HSA. Furthermore, the decrease in the Ka value with the extent of modification was indicative of the involvement of lysine residues in NA-HSA interaction.

Suramin Targets the Conserved Ligand-Binding Pocket of Human Raf1 Kinase Inhibitory Protein.

Suramin was initially used to treat African sleeping sickness and has been clinically tested to treat human cancers and HIV infection in the recent years. However, the therapeutic index is low with numerous clinical side-effects, attributed to its diverse interactions with multiple biological macromolecules. Here, we report a novel binding target of suramin, human Raf1 kinase inhibitory protein (hRKIP), which is an important regulatory protein involved in the Ras/Raf1/MEK/ERK (MAPK) signal pathway. Biolayer interference technology showed that suramin had an intermediate affinity for binding hRKIP with a dissociation constant of 23.8 µM. Both nuclear magnetic resonance technology and molecular docking analysis revealed that suramin bound to the conserved ligand-binding pocket of hRKIP, and that residues K113, W173, and Y181 play crucial roles in hRKIP binding suramin. Furthermore, suramin treatment at 160 µM could profoundly increase the ERK phosphorylation level by around 3 times. Our results indicate that suramin binds to hRKIP and prevents hRKIP from binding with hRaf1, thus promoting the MAPK pathway. This work is beneficial to both mechanistically understanding the side-effects of suramin and efficiently improving the clinical applications of suramin.

Discovery and Characterization of an ALFA-Tag-Specific Affinity Resin Optimized for Protein Purification at Low Temperatures in Physiological Buffer.

Epitope tags are widely employed as tools to detect, purify and manipulate proteins in various experimental systems. We recently introduced the ALFA-tag together with two ALFA-specific single-domain antibodies (sdAbs), NbALFA and NbALFAPE, featuring high or intermediate affinity, respectively. Together, the ALFA system can be employed for a broad range of applications in microscopy, cell biology and biochemistry requiring either extraordinarily stable binding or mild competitive elution at room temperature. In order to further enhance the versatility of the ALFA system, we, here, aimed at developing an sdAb optimized for efficient elution at low temperatures. To achieve this, we followed a stringent selection scheme tailored to the specific application. We found candidates combining a fast capture of ALFA-tagged proteins with an efficient competitive elution at 4 °C in physiological buffer. Importantly, by employing a structure-guided semisynthetic library based on well-characterized NbALFA variants, the high specificity and consistent binding of proteins harboring ALFA-tags at either terminus could be maintained. ALFA SelectorCE, a resin presenting the cold-elutable NbALFACE, is an ideal tool for the one-step purification of sensitive protein complexes or temperature-labile enzymes. We believe that the general approach followed during the selection and screening can be transferred to other challenging sdAb discovery projects.

CD4 Inhibits Helper T Cell Activation at Lower Affinity Threshold for Full-Length T Cell Receptors Than Single Chain Signaling Constructs

CD4+ T cells are crucial for effective repression and elimination of cancer cells. Despite a paucity of CD4+ T cell receptor (TCR) clinical studies, CD4+ T cells are primed to become important therapeutics as they help circumvent tumor antigen escape and guide multifactorial immune responses. However, because CD8+ T cells directly kill tumor cells, most research has focused on the attributes of CD8+ TCRs. Less is known about how TCR affinity and CD4 expression affect CD4+ T cell activation in full length TCR (flTCR) and TCR single chain signaling (TCR-SCS) formats. Here, we generated an affinity panel of TCRs from CD4+ T cells and expressed them in flTCR and three TCR-SCS formats modeled after chimeric antigen receptors (CARs) to understand the contributions of TCR-pMHCII affinity, TCR format, and coreceptor CD4 interactions on CD4+ T cell activation. Strikingly, the coreceptor CD4 inhibited intermediate and high affinity TCR-construct activation by Lck-dependent and -independent mechanisms. These inhibition mechanisms had unique affinity thresholds dependent on the TCR format. Intracellular construct formats affected the tetramer staining for each TCR as well as IL-2 production. IL-2 production was promoted by increased TCR-pMHCII affinity and the flTCR format. Thus, CD4+ T cell therapy development should consider TCR affinity, CD4 expression, and construct format.

High resolution structure of human apolipoprotein (a) kringle IV type 2: beyond the lysine binding site

Lipoprotein (a) [Lp(a)] is characterized by an LDL-like composition in terms of lipids and apoB100, and by one copy of a unique glycoprotein, apo(a). The apo(a) structure is mainly based on the repetition of tandem kringle domains with high homology to plasminogen kringles 4 and 5. Among them, kringle IV type 2 (KIV-2) is present in a highly variable number of genetically encoded repeats, whose length is inversely related to Lp(a) plasma concentration and cardiovascular risk. Despite it being the major component of apo(a), the actual function of KIV-2 is still unclear. Here, we describe the first high-resolution crystallographic structure of this domain. It shows a general fold very similar to other KIV domains with high and intermediate affinity for the lysine analog, ε-aminocaproic acid. Interestingly, KIV-2 presents a lysine binding site (LBS) with a unique shape and charge distribution. KIV-2 affinity for predicted small molecule binders was found to be negligible in surface plasmon resonance experiments; and with the LBS being nonfunctional, we propose to rename it "pseudo-LBS". Further investigation of the protein by computational small-molecule docking allowed us to identify a possible heparin-binding site away from the LBS, which was confirmed by specific reverse charge mutations abolishing heparin binding. This study opens new possibilities to define the pathogenesis of Lp(a)-related diseases and to facilitate the design of specific therapeutic drugs.

Relationship of 2D Affinity to T Cell Functional Outcomes.

T cells are critical for a functioning adaptive immune response and a strong correlation exists between T cell responses and T cell receptor (TCR): peptide-loaded MHC (pMHC) binding. Studies that utilize pMHC tetramer, multimers, and assays of three-dimensional (3D) affinity have provided advancements in our understanding of T cell responses across different diseases. However, these technologies focus on higher affinity and avidity T cells while missing the lower affinity responders. Lower affinity TCRs in expanded polyclonal populations almost always constitute a significant proportion of the response with cells mediating different effector functions associated with variation in the proportion of high and low affinity T cells. Since lower affinity T cells expand and are functional, a fully inclusive view of T cell responses is required to accurately interpret the role of affinity for adaptive T cell immunity. For example, low affinity T cells are capable of inducing autoimmune disease and T cells with an intermediate affinity have been shown to exhibit an optimal anti-tumor response. Here, we focus on how affinity of the TCR may relate to T cell phenotype and provide examples where 2D affinity influences functional outcomes.

Sustained IL-2R signaling of limited duration by high-dose mIL-2/mCD25 fusion protein amplifies tumor-reactive CD8+ T cells to enhance antitumor immunity.

High-dose IL-2 induces cancer regression but its therapeutic use is limited due to high toxicities resulting from its broad cell targeting. In one strategy to overcome this limitation, IL-2 has been modified to selectively target the intermediate affinity IL-2R that broadly activates memory-phenotypic CD8+ T and NK cells, while minimizing Treg-associated tolerance. In this study, we modeled an alternative strategy to amplify tumor antigen-specific TCR transgenic CD8+ T cells through limited application of a long-acting IL-2 fusion protein, mIL-2/mCD25, which selectively targets the high-affinity IL-2R. Here, mice were vaccinated with a tumor antigen and high-dose mIL-2/mCD25 was applied to coincide with the induction of the high affinity IL-2R on tumor-specific T cells. A single high dose of mIL-2/mCD25, but not an equivalent amount of IL-2, amplified the frequency and function of tumor-reactive CD8+ T effector (Teff) and memory cells. These mIL-2/mCD25-dependent effects relied on distinctive requirements for TLR signals during priming of CD8+ tumor-specific T cells. The mIL-2/mCD25-amplified tumor-reactive effector and memory T cells supported long-lasting antitumor responses to B16-F10 melanoma. This regimen only transiently increased Tregs, yielding a favorable Teff-Treg ratio within the tumor microenvironment. Notably, mIL-2/mCD25 did not increase non-tumor-specific Teff or NK cells within tumors, further substantiating the specificity of mIL-2/mCD25 for tumor antigen-activated T cells. Thus, the selectivity and persistence of mIL-2/mCD25 in conjunction with a tumor vaccine supports antitumor immunity through a mechanism that is distinct from recombinant IL-2 or IL-2-based biologics that target the intermediate affinity IL-2R.

MYELOID DERIVED SUPPRESSOR CELLS (MDSC) IN KIDNEY RECIPIENTS: IDENTIFICATION OF CD25+EMDSC, AND AUGMENTATION OF MONOCYTIC-MDSC EARLY AFTER TRANSPLANTATION IN PATIENTS LATER DEVELOPING CANCER

Background: MDSC increase in cancer and associate with poor prognosis, however their role in transplantation (Tx) is unknown. We aimed to study the MDSC effects on the evolution of kidney transplant recipients (KTR). Methods: A cohort of229 KTR was prospectively studied at pre- and day 7 and 14, month 1, 3, 6, and 12 post-Tx.Myeloid cells CD11b+CD33+CD14+CD15-HLA-DR-/low(M-MDSC), CD33+CD11b+CD14-HLA-DRlow/-(early stage MDSC, eMDSC) and CD11b+CD33+CD14+CD15-HLA-DR+(monocytes) were enumerated, and their suppressive capacity was tested in cocultures with autologous lymphocytes. Anti-HLA antibodies, total antioxidant capacity (TAC), arginase-1 (Arg-1), matrix metalloproteinase 9 (MMP9) and suppressive cytokines were measured in serum and supernatants. In some experiments, IL2 receptor (IL2R) expression of chains α (CD25), β (CD122) and γ (CD132) and STAT5 phosphorylation (pSTAT5) after incubation with IL2 were tested. The induction of myeloid cell death by basiliximab (Bx, anti-CD25) after adding complement (C´) was also analyzed. Results: Before Tx, HLA-DR-/low and HLA-DR+ myeloid subsets were similar among KTR and healthy volunteers. After Tx, CD11b+CD33+CD14+CD15-HLA-DR-/lowcells expanded and remained high for the first year. In vitro, these cells inhibited CD4 and CD8 T lymphocytes proliferation and IFNγ, TNFα and GM-CSF synthesis, thus confirming them as M-MDSC. The expansion of M-MDSC was not related with post-Tx events excepting for the development of cancer. Patients with M-MDSC>45.2% and >179.2 cell/µl at post-Tx day 14 had a significantly lower cancer-free survival in the 2 years after Tx (p<0.05), and showed a higher risk for cancer than patients with low M-MDSC (p=0.029 and 0.031, respectively). Multivariate Cox regression showed that M-MDSC>179.2 cell/µl was a risk factor for cancer in KTR independent of age, sex and immunosuppression (HR=6.98, p=0.02). Circulating M-MDSCs high counts correlated with high Arg-1 (p<0.01) and IL-10 (p=0.01), and low TAC (p=0.03) in serum. M-MDSC were lower in patients with enhanced alloimmune response as represented by anti-HLA sensitization. eMDSC levels also increased after Tx, excepting in patients who received Bx. CD25+eMDSC counts and the CD25per cell expression (MFI CD25) increased after Tx, but were not detected in patients who received Bx. eMDSC were found to express all three IL2R chains (high affinity IL2R), while M-MDSC and monocytes only expressed β and γ (intermediate affinity IL2R). After incubation of eMDSC with IL2, pSTAT5 was detected. Preincubation with Bx blocked both detection of CD25 and pSTAT5, but did not result in cell death after adding C´. Conclusions: Early post-transplant mobilisation of M-MDSC predicts cancer and adds risk as an independent factor in KTR. M-MDSC may favor an immunosuppressive environment that promotes the tumoral development. A CD25+eMDSC subset has been identified which is under study. ISCIII project 45/2013. CAM project B2017/BMD3731. AECC project IDEAS206PAZ.

Concentration-dependent splicing is enabled by Rbfox motifs of intermediate affinity.

The Rbfox family of splicing factors regulate alternative splicing during animal development and in disease, impacting thousands of exons in the maturing brain, heart, and muscle. Rbfox proteins have long been known to bind to the RNA sequence GCAUG with high affinity, but just half of Rbfox binding sites contain a GCAUG motif in vivo. We incubated recombinant RBFOX2 with over 60,000 mouse and human transcriptomic sequences to reveal substantial binding to several moderate-affinity, non-GCAYG sites at a physiologically relevant range of RBFOX concentrations. We find that many of these “secondary motifs” bind Rbfox robustly in cells and that several together can exert regulation comparable to GCAUG in a trichromatic splicing reporter assay. Furthermore, secondary motifs regulate RNA splicing in neuronal development and in neuronal subtypes where cellular Rbfox concentrations are highest, enabling a second wave of splicing changes as Rbfox levels increase.

Abstract 2202: The combination of a mouse ortholog of ALKS 4230, a selective agonist of the intermediate affinity IL-2 receptor, and the angiogenesis inhibitor lucitanib enhances antitumor activity

Abstract ALKS 4230, an engineered cytokine designed for selective binding to the intermediate affinity interleukin-2 receptor (IL-2R), is being evaluated as a monotherapy and in combination with pembrolizumab in patients with advanced solid tumors. The selectivity of ALKS 4230 is achieved through stable fusion of circularly permuted IL-2 to IL-2Rα, with the goal of driving antitumor responses via selective activation of CD8+ T cells and NK cells, while avoiding activation of regulatory T cells (Tregs), which express the high affinity IL-2R. High levels of vascular endothelial growth factor (VEGF) are associated with an immunosuppressive tumor microenvironment and diminished response to high-dose IL-2. Lucitanib is an anti-angiogenic, multi-tyrosine kinase inhibitor targeting VEGFR1-3, PDGFRα/β, and FGFR1-3. We evaluated the antitumor efficacy and mechanism of the combination of RDB 1462, the mouse ortholog of ALKS 4230, and lucitanib in the MC38 mouse syngeneic tumor model. C57BL/6 mice were subcutaneously implanted with MC38 cells and treated with RDB 1462 (1.5 mg/kg and 3 mg/kg once every 3 days for 3 weeks), lucitanib (10 mg/kg daily for 28 days), or the combination of both molecules. After 11 days of treatment, lucitanib monotherapy resulted in 100% tumor growth inhibition (TGI), while 1.5 mg/kg and 3 mg/kg RDB 1462 monotherapy resulted in 25% and 51% TGI, respectively. Lucitanib monotherapy extended survival, with a mean survival time (MST) of 39 days compared to 15 days for vehicle, with no complete tumor regressions. MSTs were 18-22 days for RDB 1462 monotherapy groups, 60 days for the lower dose combination arm, and &amp;gt;60 days for the higher dose combination. Durable complete tumor regressions were observed in 1/20 mice treated with either dose of RDB 1462 monotherapy and 5/10 mice treated with the lower dose of RDB 1462 in combination with lucitanib. 10/10 mice treated with the higher dose of RDB 1462 in combination with lucitanib exhibited complete responses on Day 60. Flow cytometric analyses revealed that RDB 1462 treatment induced significant peripheral CD8+ T cell expansion compared to vehicle (p&amp;lt;0.05) without an increase in Foxp3+ Tregs. This effect was enhanced by the combination of RDB 1462 with lucitanib. The combination led to a significant (p&amp;lt;0.05) increase in intratumoral CD8+ T cells and dendritic cells (CD11c+F4/80−), together with a reduction in tumor-associated macrophages (CD11b+F4/80+). The combination treatment elicited a distinct gene expression profile in tumors by RNA-Seq, merging the immunostimulatory effects of RDB 1462 with the anti-angiogenic effects of lucitanib. The combination of an intermediate affinity IL-2R-selective cytokine and an angiogenesis inhibitor resulted in durable dose-dependent antitumor efficacy in the MC38 mouse tumor model. Citation Format: Jared E. Lopes, Rachel L. Dusek, Liliane Robillard, Minh Nguyen, Bruce Roth, Bryan Vought, Arthur Liberzon, Raymond J. Winquist, Heather C. Losey. The combination of a mouse ortholog of ALKS 4230, a selective agonist of the intermediate affinity IL-2 receptor, and the angiogenesis inhibitor lucitanib enhances antitumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2202.

Abstract 3639: Fusion-associated neoantigen burden and predicted immunogenicity of CDK12 biallelic loss-of-function tumors vary substantially across cancer types

Abstract Background: Biallelic inactivation of CDK12 is associated with a distinct genomic signature of focal tandem duplications (FTDs). Gene fusions resulting from CDK12-associated FTDs increase neoantigen load, raising interest in CDK12 as a biomarker of response to immune checkpoint inhibitors (ICIs). Although FTDs have been detected at higher rates in CDK12-altered tumors compared to CDK12 WT tumors for multiple cancer types, fusion-associated neoantigen burden has only been evaluated in metastatic castration-resistant prostate cancer. Methods: By retrospective review of molecular profiles, we identified tumors with CDK12 biallelic loss-of-function (LOF) in a primary cohort of over 9000 patient tumors, representing 39 cancer types, and assessed fusions detected by whole transcriptome sequencing for immune epitopes. Biomarker and fusion results for a second cohort of over 13,000 patient tumors were reviewed for validation of the initial findings. Results: Tumors with CDK12 biallelic LOF (bLOF) (n=29) had significantly higher mean fusion rates (11.7 fusions/tumor) than CDK12 monoallelic LOF (mLOF) and WT tumors (4.1 and 2.6, respectively). Analysis of fusion topologies and breakpoints indicated a predominance of duplication events for CDK12 bLOF tumors and a bimodal distribution of breakpoint distances (modes at ~250-500 kb and ~1.0-2.5 Mb), consistent with prior reports. Fusion rates correlated with the rate of predicted neoantigens with high, intermediate, and low affinity for MHC class I. Fusion-associated neoantigen burden was significantly higher in CDK12 bLOF tumors (145.0 high affinity peptides/tumor) than mLOF (49.7) and WT (26.6) tumors. Among CDK12-altered tumors, fusion rates were significantly higher in prostate (16.4 fusions/tumor, n=11) and ovarian (19.7, n=6) than other cancer types (3.4 overall, n=12), potentially reflecting cancer type-specific roles for CDK12. Co-occurrence of mismatch repair deficiency (dMMR)/high microsatellite instability (MSI-High) with CDK12 bLOF (n=5), most often in colorectal tumors (n=4), correlated with a lower fusion rate (0.2 fusions/tumor); recurrent CDK12 frameshift mutations in these cases (G1461fs, T1463fs, and Q1291fs) coincide with poly-nucleotide tracts, suggesting CDK12 mutations are a secondary effect. In a separate cohort of over 13,000 patient tumors, additional CDK12 bLOF tumors (n=47) were identified, showing a similar distribution and association with high fusion rate (11.0 fusions/tumor). Conclusion: Fusion rates and predicted neoantigen load varied significantly between CDK12 biallelic tumors across cancer types, highlighting the value of biomarkers with a quantitative immunogenic/phenotypic readout. We propose that fusion rates are linked to CDK12 alterations and may serve as useful biomarker to enhance our ability to identify responders of ICI therapy. Citation Format: Andrew Elliott, Phillip Stafford, Jian Zhang, Qing Zhang, Jeff Swensen, Daniel Martin, Joanne Xiu, Zoran Gatalica, Daniel Vaena, Elisabeth Heath, W. Michael Korn. Fusion-associated neoantigen burden and predicted immunogenicity of CDK12 biallelic loss-of-function tumors vary substantially across cancer types [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3639.

Abstract 6050: Transient and selective depletion of MDSCs and Tregs as an effective immunotherapy against triple-negative breast cancer

Abstract Background: Checkpoint inhibitors (CPI) have revolutionized the field of cancer immunotherapy. However, CPIs have shown only modest efficacy in clinical trials for TNBC which may be attributed to the presence of immunosuppressive cells both myeloid-derived suppressor (MDSCs) and T regulatory (Treg) cells in the tumor microenvironment (TME). We hypothesized that transient depletion of Tregs and MDSCs along with the administration of checkpoint inhibitors would markedly reduce immunosuppressive signals to the effector arm of the immune system and thereby lead to a more robust anti-tumor response causing tumor regression and a better disease outcome. To target Treg/MDSCs cells, we have developed two diphtheria-toxin based fusion protein toxins and are currently evaluating the activity of these molecules both as monotherapies and dual therapies in combination with CPIs. Methods: To target MDSCs and TNBC tumor cells, we genetically constructed, expressed and purified a diphtheria toxin-based fusion protein, s-DAB389mIL-4 that targets the IL-4 receptor (IL-4Rα) on these cell populations. This fusion protein toxin was tested in vitro against murine TNBC 4T1 cells and in vivo in a syngeneic murine adenocarcinoma model both as a monotherapy and in combination with s-DABIL-2(V6A) which targets Tregs cells through high and intermediate affinity IL-2 receptors. Results: We found that s-DAB389mIL-4 is highly potent against 4T1 cells in vitro (IC50 ranging from 10 pM to ~3 nM), and adversely affects 4T1 cell survival, proliferation, invasion, and migratory abilities. Recombinant IL-4 and anti-IL-4 antibody block the cytotoxic action of s-DAB389mIL-4 confirming that the fusion protein's activity is mediated through the IL-4Rα. s-DAB389mIL-4 treatment in a 4T1 cell-based murine adenocarcinoma model resulted in a significant reduction of (a) tumor volume and weight, (b) MDSCs, (c) splenomegaly, (d) 4T1 metastasis to the lung and, (d) increased infiltration of IFNϒ+ effector T-cells. Sequential combination therapy with both s-DABIL-4 and s-DABIL-2(V6A) resulted in (1) reduction in both tumor volume and weight, (2) transient depletion of activated Tregs, and (3) increased infiltration of T-effector cells in the TME. NanoString analysis of RNA isolated from tumor tissues treated with these two fusion protein toxins show enrichment of gene signatures which enhance the infiltration of anti-tumor immune cells in the TME and concomitant inhibition of processes which encourage tumor growth and metastasis. Conclusions: These findings suggest that s-DABmIL-4 both as a monotherapy and in combination with s-DABIL-2(V6A) results in transient reduction of both MDSCs and Tregs. These observations suggest that these novel fusion protein toxins may serve as an effective immunotherapeutic regimen for the treatment of TNBC. Based upon these results, we are currently testing these fusion protein toxins in combination with checkpoint blockade therapy. Citation Format: Sadiya Parveen, Sumit Siddharth, Laurene Cheung, John R. Murphy, Dipali Sharma, William R. Bishai. Transient and selective depletion of MDSCs and Tregs as an effective immunotherapy against triple-negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6050.

Nimotuzumab for Patients With Inoperable Cancer of the Head and Neck.

EGFR activation induces cell proliferation, neoformation of blood vessels, survival, and metastasis of the cancer cells. Nimotuzumab is an engineered, intermediate affinity anti-EGFR antibody, that apart from other drugs in its class, is very safe and does not cause hypomagnesemia or grade 3–4 cutaneous rash. The antibody inhibits cell proliferation and angiogenesis, activates natural killer cells, stimulates dendritic cell maturation, and induces cytotoxic T cells. Nimotuzumab restores MHC-I expression on tumor cells, hindering one of the EGFR immune-escape ways. The antibody has been extensively studied in 7 clinical trials, concurrently with irradiation or irradiation plus chemotherapy in subjects with inoperable head and neck tumors. Nimotuzumab was safe and efficacious in unfit patients receiving irradiation alone and in subjects treated with cisplatin and radiotherapy. In patients with locally advanced squamous cell carcinomas of the head and neck, nimotuzumab in combination with low dose cisplatin and radiotherapy was superior to cisplatin and radiotherapy in progression free survival, disease free survival, and locoregional tumor control.

Molecular requirements for B cell receptor engagement and activation

Abstract B cells are a central component of the adaptive branch of the immune system, responsible for humoral immunity through production of antibodies. Antigen engagement of the B cell receptor (BCR) is the first step in B cell activation; yet despite its recognized importance, the exact mechanism of BCR activation remains controversial. A central point of contention has been whether monovalent antigen is capable of activating the BCR or not. To address this question, we leveraged the B1-8hi knock-in BCR model, in which λ light-chain positive B cells display intermediate and high affinity binding to the haptens NP ((4-hydroxy-3-nitrophenyl)acetyl) and NIP ((4-hydroxy-3-iodo-5-nitrophenyl)acetyl), respectively, and combined this with a recently developed nucleic acid based scaffolding technology (Holliday junctions [HJs]) to prepare defined mono-, di- and trivalent displays of NP and NIP. Using confocal imaging, flow and imaging cytometry, as well as calcium-flux analyses, we find that HJs carrying 2 or 3 copies of either NP or NIP can bind and activate B1-8hi B cells across a wide range of concentrations, whereas HJs carrying a single NP require high concentrations for any measurable activity. Most interestingly, HJs carrying a single NIP are able to bind B1-8hi B cells at low concentrations, approaching equimolar stoichiometry, but despite measurable BCR engagement do not elicit activation. Our results may reconcile previous contradictory reports regarding monovalent antigen activation, by providing a framework for understanding complex interaction effects of antigen affinity, valency and concentration.