Specific Linker Research Articles

Combinatorial Targeting of Multiple Shared Antigens By Adapter-CAR-T Cells (aCAR-Ts) Allows Target Cell Discrimination and Specific Lysis Based on Differential Expression Profiles

▪Despite tremendous clinical success of chimeric antigen receptor (CAR) expressing T cell (CAR-T) therapies, targeting B-phenotypic antigens in ALL, CLL, NHL or multiple myeloma, there are still major limitations for broader clinical application. CAR-Ts are capable to generate a specific immune response against defined surface-expressed antigens leading to sustained depletion of target antigen expressing tissues e.g. B cells. While B cell function can be substituted by repetitive IgG infusions, prolonged depletion of vitally essential tissues is not compatible with life. In AML for instance, most promising target antigens are expressed along myeloid lineage differentiation, limiting the therapeutic applicability. Therefore, CAR-Ts targeting essential shared antigens mustallow tight regulation of CAR-T function and/or be able to differentiate between cancerous and healthy tissue.To address these issues, we have developed the adapter CAR-T cell (aCAR-T) system. By splitting antigen recognition and CAR-T activation, introducing adapter molecules (AMs), the system allows precise quantitative (on-/off-switch) as well as qualitative (change and combine target antigens) regulation of CAR-T function. aCAR-Ts are based on the unique properties of a novel scFv targeting a “neo”-epitope-like structure consisting of the endogenous vitamin biotin in the context of a specific linker, referred to as linker-label-epitope (LLE). LLEs can be easily conjugatedtonovel or preexisting AM formats like monoclonal antibodies (mAbs) or mAb fragments in a GMP-compliant manner. We were able to demonstrate that aCAR-Ts allow simultaneous targeting of various antigens (“OR”-gate),preventing antigen evasion by selection of antigen or epitope-loss variants.In the present study we intended to investigate whether aCAR-Ts are capable to identify and differentiate target cells due toversatileantigenexpression profiles (“AND”-gate). In theory, AMs against different target antigens can be assembled on the surface of a target cell,leading to aCAR-T activation independent of the targeted antigens (surface painting),by binding to the presented LLE-tags. Therefore, combinatorial AMs treatment might allow to translate complex and multiple antigen-dependent targetcellidentification into an aCAR-T activation.To test this hypothesis, we have generated LLE-AMs against ALL/NHL-(CD10, CD19, CD20, CD22, CD37, CD138, ROR1) and AML-(CD32, CD33, CD38, CD123, CD135, CD305, CLL1) associated antigens. Individual threshold concentrations for aCAR-T activation by different AMs, targeting the model cell lines Nalm6 (ALL), JeKo1 (NHL), HL-60,U973andMolm13 (all AML), have been analyzed. Cutoffswerefound to be between 10 and 100 pg/ml, dependent on target expression and target cell line. Importantly, combinations of 2, 3 or 5 AMs, targeting different antigens expressed on thesame target cell, cause target-cell lysis at concentrations below the activation threshold for single AMs(exemplified for HL-60 in Figure A). Our results clearly demonstrate an additive effect in combining different AMs to hurdle the activation threshold.Moreover, in a JeKo1 CD19 and/or CD20 knock out (KO) antigen-loss model, combinations of AMs targeting CD19, CD20 and ROR1 can differentiate between wild type and antigen -1 (CD19 or CD20 KO) or antigen -2 (CD19 and CD20 KO) variants in mediating target-cell lysis, even though at least one target antigen is expressed. Finally, we found that combinations of CD10, CD19, CD22 and CD138 sufficiently eliminate Nalm-6 BCP-ALL cells, while sparing healthy B cells in co-culture experiments. Similar results were obtained in co-culture experiments of HL-60 AML cells with monocytes, neutrophils as well asCD34-enriched hematopoietic progenitor cells, applying combinations of CD32, CD33, CD38, CD123, CD305 and CLL1. Co-culturing experiments using autologous blasts, monocytes, neutrophils and aCAR-Ts are ongoing.Together, our results indicate that aCAR-Ts in combination with selected AM combinations might have the ability to identify and specifically eliminate cancer cells based on complex antigen expression profiles. This would have major implications for clinical translation, enabling combinatorial therapy, essential to avoid antigen evasion, and the possibility to spare vitally essential tissue from elimination. [Display omitted] DisclosuresSeitz:Miltenyi Biotec: Patents & Royalties, Research Funding. Mittelstaet:Miltenyi Biotec: Employment, Patents & Royalties. Lock:Miltenyi Biotec: Employment. Kaiser:Miltenyi Biotec: Employment, Patents & Royalties. Handgretinger:Miltenyi Biotec: Patents & Royalties: Co-patent holder of TcR alpha/beta depletion technologies, Research Funding. Lang:Miltenyi Biotec: Patents & Royalties, Research Funding. Schlegel:Miltenyi Biotec: Patents & Royalties, Research Funding.

Adapter Chimeric Antigen Receptor (aCAR)-Engineered NK-92 Cells: An Off-the-Shelf Cellular Therapeutic for Universal Tumor Targeting

▪Chimeric antigen receptor (CAR) expressing T cells (CAR-Ts) have demonstrated tremendous clinical success, especially when targeted against the B-phenotypic antigens CD19 and CD22 in ALL, CLL as well as NHL. Despite the recent success, production of CAR-Ts still requires an extensive and time consuming manufacturing process. Moreover, CAR-Ts have to be prepared individually for each patient. Especially in heavily pretreated and rapidly progressing patients, which often lack sufficient numbers of healthy T cells for CAR-T production, alternatives are a significant clinical need. NK cells might represent a promising alternative effector cell source. The continuously expandable and well established NK cell line NK-92 can provide a safe and consistent way to produce NK effector cells in a GMP-compliant and cost-effective way. Irradiated NK-92 CARs as an “off-the-shelf on-demand” cell therapeutic are currently tested in pre-clinical and early-phase clinical trials. Furthermore, NK-92 can be redirected by CARs to mediate direct antigen specific lysis.We have recently developed a universal adapter CAR (aCAR) system. By splitting antigen recognition and CAR-immune cell activation, introducing adapter molecules (AMs), the system allows precise quantitative (on-/off-switch) as well as qualitative (change and combination of target antigens) regulation of immune cell function. aCARs are based on the unique properties of a novel scFv targeting a “neo”-epitope-like structure consisting of the endogenous vitamin biotin in the context of a specific linker, referred to as linker-label-epitope (LLE). LLEs can be easily conjugated on novel or preexisting AM formats like monoclonal antibodies (mAbs) or mAb fragments in a GMP-compliant manner.In the present study, we intended to combine the universal and flexible targeting as well as controllability of the aCAR with the “off-the-shelf” properties of NK-92 cells. NK-92 was obtained from ATCC and transduced with aCARs containing either CD28 or 4-1BB co-stimulatory plus CD3-ζ signaling domains. Importantly, only CD28 containing aCARs sufficiently mediated specific target cell lysis in the presence of biotinylated antibodies (LLE-AMs). Using single cell sorting, aCAR NK-92 clones with the highest CAR expression were selected and demonstrated significantly improved target cell lysis, in a LLE-AMs dependent manner. Both, LLE-AMs against CD19 and CD20, were capable of inducing significant NK-92-mediated lysis against the NHL cell lines Raji, Daudi and JeKo-1. Cytotoxicity experiments using aCAR NK-92 cells and primary lymphoma cells are ongoing. Specificity of the LLE-AM directed effector cell elimination was further proven using a JeKo-1 CD19 and/or CD20 knock out (KO) antigen-loss model. aCAR NK-92 mediated antigen specific lysis only in the presence of the target antigen and the specific LLE-AM. Moreover, combinations of anti-CD19 and anti-CD20 LLE-AMs are capable of avoiding antigen evasion. To test the universal applicability of aCAR NK-92, specific target cell lysis against a variety of different tumor entities was demonstrated using LLE-conjugated therapeutic antibodies. Importantly, irradiation of effector cells, as required in all active clinical trials using NK-92, prior to testing had no observable effect on target cell lysis. Finally, the investigation of potential on-target off-tumor reactivity against healthy B cells showed no cytotoxic effects of aCAR NK-92 cells in combination with LLE-AMs against CD19 or CD20.In conclusion, we have generated an NK cell line, aCAR NK-92, whose effector function can be tightly regulated and redirected against one or multiple antigens allowing tunable and universal targeting. Moreover, aCAR NK-92 cells can be manufactured as an “off-the-shelf on-demand” standardized product improving the practicality of NK CAR therapy combined with the possibility of tailoring a specific LLE-AM platform for patient-individualized treatment. DisclosuresSeitz:Miltenyi Biotec: Patents & Royalties, Research Funding. Mittelstaet:Miltenyi Biotec: Employment, Patents & Royalties. Kaiser:Miltenyi Biotec: Employment, Patents & Royalties. Schlegel:Miltenyi Biotec: Patents & Royalties, Research Funding. Handgretinger:Miltenyi Biotec: Patents & Royalties: Co-patent holder of TcR alpha/beta depletion technologies, Research Funding.

Bile Acid Oligomers and Their Combination with Antibiotics To Combat Bacterial Infections

The ever-growing risk of bacterial resistance is a critical concern. Among the various antimicrobial resistant bacterial strains, methicillin and vancomycin resistant Staphylococcus aureus are among the most dreadful, causing serious complications. On the basis of the hypothesis that microbes have reduced ability to develop resistance against membrane targeting antibiotics, bile acid oligomers having unique facially amphiphilic topologies were designed and synthesized. The oligomers with specific linkers exhibited potent and selective antibacterial activity against Gram-positive bacteria. The lead compounds also improved the efficacy of a range of known antibiotics belonging to different classes when tested in combination. The active dimers were found to be effective against antibiotic-resistant clinical isolates of S. aureus, including multidrug resistant isolates. A significant inhibitory activity against S. aureus biofilm, a highly drug-resistant bacterial phenotype often unresponsive to antibiotic therapy, was also noticed. No adverse effects were observed by these dimers in a cell viability assay against HEK293 cells.

Flexible Peptide Linkers Enhance the Antimicrobial Activity of Surface-Immobilized Bacteriolytic Enzymes.

Chemical linkers are frequently used in enzyme immobilization to improve enzyme flexibility and activity, whereas peptide linkers, although ubiquitous in protein engineering, are much less explored in enzyme immobilization. Here, we report peptide-linker-assisted noncovalent immobilization of the bacteriolytic enzyme lysostaphin (Lst) to generate anti- Staphylococcus aureus surfaces. Lst was immobilized through affinity tags onto a silica surface (glass slides) and nickel nitrilotriacetic acid (NiNTA) agarose beads via silica-binding peptides (SiBPs) or a hexahistidine tag (His-tag) fused at the C-terminus of Lst, respectively. By inserting specific peptide linkers upstream of the SiBP or His-tag, the immobilized enzymes killed >99.5% of S. aureus ATCC 6538 cells (108 CFU/mL) within 3 h in buffer and could be reused multiple times without significant loss of activity. In contrast, immobilized Lst without a peptide linker was less active/stable. Molecular modeling of Lst-linker-affinity tag constructs illustrated that the presence of the peptide linkers enhanced the molecular flexibility of the proximal Lst binding domain, which interacts with the bacterial substrate, and such increased flexibility correlated with increased antimicrobial activity. We further show that Lst immobilized onto NiNTA beads retained the ability to kill ∼99% of a 108 CFU/mL microbial challenge even in the presence of 1% of a commercial anionic surfactant, C12-14 alcohol EO 3:1 sodium sulfate, when the Lst construct contained a decapeptide linker containing glycine, serine, and alanine residues. This linker-assisted immobilization strategy could be extended to an unrelated lytic enzyme, the endolysin PlyPH, to target Bacillus anthracis Sterne cells either in buffer or in the presence of anionic surfactants. Our approach, therefore, provides a facile route to the use of antimicrobial enzymes on surfaces.

JNK regulates muscle remodeling via myostatin/SMAD inhibition

Skeletal muscle has a remarkable plasticity to adapt and remodel in response to environmental cues, such as physical exercise. Endurance exercise stimulates improvements in muscle oxidative capacity, while resistance exercise induces muscle growth. Here we show that the c-Jun N-terminal kinase (JNK) is a molecular switch that when active, stimulates muscle fibers to grow, resulting in increased muscle mass. Conversely, when muscle JNK activation is suppressed, an alternative remodeling program is initiated, resulting in smaller, more oxidative muscle fibers, and enhanced aerobic fitness. When muscle is exposed to mechanical stress, JNK initiates muscle growth via phosphorylation of the transcription factor, SMAD2, at specific linker region residues leading to inhibition of the growth suppressor, myostatin. In human skeletal muscle, this JNK/SMAD signaling axis is activated by resistance exercise, but not endurance exercise. We conclude that JNK acts as a key mediator of muscle remodeling during exercise via regulation of myostatin/SMAD signaling.

Chromatin remodeling in Drosophila preblastodermic embryo extract

Chromatin is known to undergo extensive remodeling during nuclear reprogramming. However, the factors and mechanisms involved in this remodeling are still poorly understood and current experimental approaches to study it are not best suited for molecular and genetic analyses. Here we report on the use of Drosophila preblastodermic embryo extracts (DREX) in chromatin remodeling experiments. Our results show that incubation of somatic nuclei in DREX induces changes in chromatin organization similar to those associated with nuclear reprogramming, such as rapid binding of the germline specific linker histone dBigH1 variant to somatic chromatin, heterochromatin reorganization, changes in the epigenetic state of chromatin, and nuclear lamin disassembly. These results raise the possibility of using the powerful tools of Drosophila genetics for the analysis of chromatin changes associated with this essential process.

Abstract LB-001: Development and evaluation of T-Zap: a novel antibody-drug conjugate for the treatment of Her2 positive breast cancer

Abstract Background: Breast cancer is the most common type of cancer worldwide and HER2-overexpressing types are especially associated with poor prognosis. Antibody-drug conjugates (ADCs) have been developed as a targeted approach to deliver toxins to cancer cells specifically with the ADC trastuzumab-emtansine (T-DM1) already in clinical use. A novel ADC T-Zap has been developed by linking the antibody trastuzumab to saporin - a ribosome inactivating protein. This study aims to evaluate the novel ADC T-Zap for the treatment of HER2 positive breast cancer. Methods: The plant toxin saporin was linked to the monoclonal antibody tratuzumab via a streptavidin-biotin bridge. Binding kinetics were assessed using flow cytometry, and MTS cell viability assays were performed to compare efficacy to T-DM1. Toxicity towards immune cells was investigated by MTS assays using immune cells. Results: Binding to target cells of T-Zap was confirmed. Comparison of T-Zap efficacy in breast cancer cell lines with and without resistance against trastuzumab showed a trend for higher efficacy of cell killing by T-Zap in trastuzumab resistant cells compared to T-DM1. Toxicity assays revealed no impact of T-Zap on cell viability in immune cells. Conclusions: Cell viability caused by T-Zap was more efficient compared to T-DM1 in the trastuzumab resistant cell line HCC1945. This strengthens the hyposthesis that T-Zap might have potential as new ADC overcoming trastuzumab resistance. Future work will include the development of a specific linker to link trastuzumab to saporin with the aim to develop an ADC that could prove a novel therapeutic agent for the treatment of HER2 positive breast cancer. Citation Format: Ricarda M. Hoffmann, Silvia Crescioli, David E. Thurston, Sophia N. Karagiannis. Development and evaluation of T-Zap: a novel antibody-drug conjugate for the treatment of Her2 positive breast cancer [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 LB-001.

Correction to "Divinylsulfonamides as Specific Linkers for Stapling Disulfide bonds in Peptides".

Correction to "Divinylsulfonamides as Specific Linkers for Stapling Disulfide bonds in Peptides".

Chiral Molecule-Enhanced Extinction Ratios of Quantum Dots Coupled to Random Plasmonic Structures.

Devices based on self-assembled hybrid colloidal quantum dots (CQDs) coupled with specific organic linker molecules are a promising way to simply realize room-temperature, spectrally tunable light detectors. Nevertheless, this type of devices usually has low quantum efficiency. Plasmonics has been shown as an efficient tool in guiding and confining light at nanoscale dimensions. As plasmonic modes exhibit highly confined fields, they locally increase light-matter interactions and consequently enhance the performance of CQD-based photodetectors. Recent publications presented experimental results of large extinction enhancement from a monolayer of CQDs coupled to random gold nanoislands using a monolayer of organic alkyl linkers. We report here that a twofold larger extinction enhancement in the visible spectrum is observed when a monolayer of helical chiral molecules connects the CQDs to the gold structure instead of a monolayer of achiral linkers. We also show that this effect provides insight into the chirality of the molecules within the monolayer. In future work, we plan to evaluate the potential of these results to be used in the construction of a more efficient and sensitive photon detector based on surface QDs, as well as to supply a simple way to map the chirality of a single chiral monolayer.

Abstract B106: Antibody-drug conjugates targeted at human aspartyl (asparaginyl) β-hydroxylase

Abstract Antibody-drug conjugates (ADCs) consist of an antibody that is specific for a disease antigen linked to one or more drug molecules. The goal is to utilize the antibody as a means of specific delivery of the drug to diseased cells. Theoretically this should decrease systemic exposure to the drug and allow for use of a lower dose. In cancer, the approach employs an antibody targeted at a tumor-associated antigen and a cytotoxic or cytostatic drug. To date there have been three ADCs approved by the US FDA for cancer treatment, and many more are now in the development stage. The efficacy of any ADC is dependent on three factors: the antibody, the attached drug, and the linker used to connect them. While each of these factors is of import, with the growth of interest in ADCs for cancer therapy, the use of specific drugs and linkers has become more routine. Thus, the specificities of the antibody and target antigen are now perhaps the primary obstacles towards the development of novel ADCs. Human aspartyl (asparaginyl) β-hydroxylase (HAAH) is an embryonic/developmental protein, which is downregulated in normal cells after birth but overexpressed on the surface of many malignant cells. It has been demonstrated to be sufficient to induce cancer cell proliferation, motility, and invasiveness. The enzyme hydroxylates important residues in the NOTCH protein altering signal transduction pathways that lead to increased growth and metastatic potential. Increased levels of HAAH have been detected by immunohistochemistry in a diverse array of solid and hematologic cancers (n > 20), including liver, bile duct, brain, breast, colon, prostate, ovary, pancreas, and lung cancers as well as various leukemias. HAAH is not found in measurable quantities in normal tissue (n > 500), including normal adjacent tissue within cancer biopsy specimens or in benign proliferative disorders. We have developed a fully human antibody against HAAH, PAN-622,that displays exquisite specificity for cancer. Here we explore PAN-622 drug conjugates for ultimate use in treating both hematologic and solid tumors. PAN-622 was conjugated to three different drugs: a maytansinoid (DM1), monomethyl auristatin E (MMAE), and duocarmycin (DUO). DM1 was conjugated via a noncleavable thio-ether linker while MMAE and DUO were conjugated using valine-citruline containing linkers that are cleavable by cathepsin B in the endosomal compartment. Conjugation of drugs to the PAN-622 antibody had little effect on the affinity of the antibody for HAAH. Binding affinities were determined by immunoassay on fixed cancer cells (H460, human lung cancer line) and were ~0.1, 0.2, and 0.5nM for PAN-622-DM1, PAN-622-MMAE, and PAN-622-DUO, respectively. The affinity of PAN-622 for HAAH as expressed on live cells has previously been shown to be ~1nM. Efficacies of the three ADCs for killing of H460 cells were determined using an MTS assay. The PAN-622-DM1 had an EC50 of ~15nM. The EC50 for PAN-622-MMAE was ~60nM, and that for PAN-622-DUO was ~300nM. Importantly, both unconjugated PAN-622 and a nonrelevant antibody conjugated to MMAE did not display any killing of the H460 cell line. Efficacy was also measured on a representative hematologic cancer line, MOLM-14 (acute myelogenous leukemia) where EC50s were in the 20-50nM range for all three ADCs. This work serves as a proof of concept, laying the groundwork for further development of HAAH-targeted ADCs. Citation Format: Kanam Malhotra, Susan Walker, Chen Fang, Karamveer Birthare, Michael S. Lebowitz, Steven Fuller, Muctarr Sesay, Hossein A. Ghanbari. Antibody-drug conjugates targeted at human aspartyl (asparaginyl) β-hydroxylase [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B106.

Growth-modulating agents for the synthesis of Al-MOF-type materials based on assembled 1D structural subdomains.

Novel aluminium MOF-type materials structured by 1D subdomains, such as organic-inorganic nanoribbons, were synthesized by modifying the conditions of solvothermal synthesis and the nature of the solvents in the presence of aryl monocarboxylate linkers with long alkyl chains, which acted as growth-modulating agents. Specifically, three different families of materials were prepared with various morphological characteristics: (i) isoreticular MIL-53(Al)-type materials, (ii) mesoscopic metalorganic structures and (iii) lamellar aluminium MOFs. The length of the alkyl chain in the aryl linker and the hydrophobic/hydrophilic nature of the solvothermal synthesis media determined the structuration level that was achieved. The derived Al-MOFs are active and stable catalysts for the synthesis of fine chemicals. This was illustrated by the efficient synthesis of 2,3-dihydro-2,2,4-trimethyl-1H-1,5-benzodiazepine.

Divinylsulfonamides as Specific Linkers for Stapling Disulfide Bonds in Peptides.

A new class of N-phenyl-divinylsulfonamides which can be easily prepared have been successfully developed and utilized as efficient linkers in the field of disulfide bond modification. Functional divinylsulfonamides provide opportunities for the specific introduction of various functionalities, including affinity probes, fluorescent tags, and drugs, into peptides.

Exploring metal organic frameworks for energy storage in batteries and supercapacitors

High energy density batteries and high power density supercapacitors have attracted much attention because they are crucial to the power supply of future portable electronic devices, electric automobiles, unmanned aerial vehicles, etc. The electrode materials are key components for batteries and supercapacitors, which influence the practical energy and power density. Metal-organic frameworks possessing unique morphology, high specific surface area, functional linkers, and metal sites are excellent electrode materials for electrochemical energy storage devices. Herein, we review and comment on recent progress in metal-organic framework-based lithium-ion batteries, sodium-ion batteries, lithium-air batteries, lithium-sulfur/selenium batteries, and supercapacitors. Future perspectives and directions of metal-organic framework-based electrochemical energy storage devices are put forward on the basis of theoretical knowledge from the reported literature and our experimental experience.

Smad2/3 Linker Phosphorylation Is a Possible Marker of Pancreatic Stem/Progenitor Cells in the Regenerative Phase of Acute Pancreatitis.

The aims of this study are to characterize cell proliferation and differentiation during regeneration after pancreatitis and pancreatic buds during development to evaluate the role of Smad2/3, phosphorylated at the specific linker threonine residues (pSmad2/3L-Thr) in positive cells. Male C57BL/6 mice received hourly intraperitoneal injections of cerulein and were analyzed after induced pancreatitis. Pancreatitis-affected tissue sections and pancreatic buds were immunostained for pSmad2/3L-Thr, with other markers thought to be stem/progenitor markers of the pancreas. pSmad2/3L-Thr immunostaining-positive cells increased as the pancreatitis progressed. The expression of pSmad2/3L-Thr was seen in acinar cells and ductlike tubular complexes. These results suggest that pSmad2/3L-Thr is expressed during acinar-ductal metaplasia. Immunohistochemical colocalization of pSmad2/3L-Thr with Ki67 was never observed. pSmad2/3L-Thr-positive cells may remain in an undifferentiated state. During the pancreatic development process, pSmad2/3L-Thr was expressed as other markers. pSmad2/3L-Thr develops in duct structure of the undifferentiated cell population in the last part of viviparity that acinar structure is formed clearly. pSmad2/3L-Thr expression occurs during acinar-ductal metaplasia after pancreatitis and may represent the contribution of stem cells and/or progenitor cells to the differentiation of the pancreas.

From R&D to Clinical Supplies

In the development of new antibody drug conjugates (ADCs), the activities performed by discovery groups typically focus on the rapid and comprehensive screening of many conjugates to find the ones with the desired efficacy and safety profiles. These conjugates are typically prepared in a combinatorial approach whereby various monoclonal antibodies (mAbs) for a specific target, linkers, and payloads are combined. These efforts usually rely on efficient screening methodologies and high-throughput tools, such as solid phase conjugation and purification arrays. The development of robust and consistent processes suitable to produce the selected candidate for clinical trials is typically not a priority for discovery. Many of the ADCs in the clinic today are based on one of two conjugation technologies and associated linker-payloads (LP). The traditional cysteine conjugation technology utilizes an auristatin as a payload with a cleavable or noncleavable linker. The other technology is based on conjugating a mayt...

Rigidity and flexibility in the tetrasaccharide linker of proteoglycans from atomic-resolution molecular simulation.

Proteoglycans (PGs) are covalent conjugates between protein and carbohydrate (glycosaminoglycans). Certain classes of glycosaminoglycans such as chondroitin sulfate/dermatan sulfate and heparan sulfate utilize a specific tetrasaccharide linker for attachment to the protein component: GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser. Toward understanding the conformational preferences of this linker, the present work used all-atom explicit-solvent molecular dynamics (MD) simulations combined with Adaptive Biasing Force (ABF) sampling to determine high-resolution, high-precision conformational free energy maps ΔG(φ, ψ) for each glycosidic linkage between constituent disaccharides, including the variant where GlcA is substituted with IdoA. These linkages are characterized by single, predominant (> 97% occupancy), and broad (45° × 60° for ΔG(φ, ψ) < 1 kcal/mol) free-energy minima, while the Xyl-Ser linkage has two such minima similar in free-energy, and additional flexibility from the Ser sidechain dihedral. Conformational analysis of microsecond-scale standard MD on the complete tetrasaccharide-O-Ser conjugate is consistent with ABF data, suggesting (φ, ψ) probabilities are independent of the linker context, and that the tetrasaccharide acts as a relatively rigid unit whereas significant conformational heterogeneity exists with respect to rotation about bonds connecting Xyl to Ser. © 2017 Wiley Periodicals, Inc.

Functional and Evolutionary Analyses Identify Proteolysis as a General Mechanism for NLRP1 Inflammasome Activation.

Inflammasomes are cytosolic multi-protein complexes that initiate immune responses to infection by recruiting and activating the Caspase-1 protease. Human NLRP1 was the first protein shown to form an inflammasome, but its physiological mechanism of activation remains unknown. Recently, specific variants of mouse and rat NLRP1 were found to be activated upon N-terminal cleavage by the anthrax lethal factor protease. However, agonists for other NLRP1 variants, including human NLRP1, are not known, and it remains unclear if they are also activated by proteolysis. Here we demonstrate that two mouse NLRP1 paralogs (NLRP1AB6 and NLRP1BB6) are also activated by N-terminal proteolytic cleavage. We also demonstrate that proteolysis within a specific N-terminal linker region is sufficient to activate human NLRP1. Evolutionary analysis of primate NLRP1 shows the linker/cleavage region has evolved under positive selection, indicative of pathogen-induced selective pressure. Collectively, these results identify proteolysis as a general mechanism of NLRP1 inflammasome activation that appears to be contributing to the rapid evolution of NLRP1 in rodents and primates.

Synthesis and Protein Engineering Applications of Cyclotides

Cyclotides are a group of plant-derived peptides with a head-to-tail cyclized backbone that is stabilized by three knotted disulfide bonds. Their exceptional stability and tolerance for residue substitutions have led to interest in their application as drug design scaffolds. To date, chemical synthesis has been the dominant methodology for producing cyclotides and their analogues. Native chemical ligation is the most common strategy to generate the cyclic backbone and has been highly successful at producing a wide range of cyclotides for studies of structure–activity relationships. Both this and other chemical approaches require a specific linker at the C-terminus and typically involve a non-directed folding (disulfide oxidation) regimen, which can sometimes be a limiting factor in final yields. Following the recent discovery of enzymes involved in peptide cyclization in planta, site-specific and highly efficient enzymatic ligations have been used for synthetic cyclotide backbone cyclization. In this review, chemical synthesis strategies and approaches involving cyclization via enzymes for the production of cyclotides are described.

Abstract B066: The hexavalent CD40 agonist APG1233 is a superior inducer of immune cell activation compared to trimeric formats or agonistic anti-CD40 antibodies

Abstract Introduction: The co-stimulatory receptor CD40 is strongly expressed on B cells, monocytes and antigen-presenting cells (APC). By promoting their maturation, activation and survival, CD40 signaling greatly contributes to anti-tumor responses of the immune system. The HERA-Technology developed by Apogenix is a powerful engineering platform for the production of modular fusion proteins targeting the TNF-receptor superfamily. Structurally, these novel ligands comprise a single-chain arrangement of three TNFSF protomers forming one TNFSF-receptor binding domain (RBD) with specific linkers and the Fc-part of human IgG as dimerization scaffold. The result is a stable and defined hexavalent receptor agonist with high clustering capacity for the cognate receptor. Here we compared the efficacy of different CD40 agonist formats, including the novel hexavalent scCD40L-RBD-Fc (APG1233), and the functional consequences of differential receptor clustering. In addition, the in vivo pharmacokinetics of the fully human APG1233 is compared with a chimeric mouse/human surrogate molecule APG1274. Materials and Methods: Immune cells were isolated from healthy-donor blood samples and profiled by multicolor flow cytometry (MC-FC). Subsequently, immune cells were cultured in growth media containing various forms of CD40 agonists. Upregulation of activation markers on B cells and monocytes (e.g. CD69, CD86, HLA-DR) and T cell-induced killing of tumor cells in direct co-culture was assessed by MC-FC and employing a real-time cell analysis system (xCELLigence), respectively. Secretion of cytokines in response to CD40 ligation and the pharmacokinetic properties of the fully human APG1233 and the chimeric murine/human APG1274 were determined by ELISA. Results: In vivo stability of APG1233 was demonstrated in a single dose mouse PK study revealing a terminal half-life of 84 hours. The human CD40L, however, does not bind murine CD40. Therefore, we used APG1274, a chimeric protein comprising the murine CD40L fused to human IgG and binding to mouse CD40, to determine serum pharmacokinetics. As a result of functional binding of APG1274 to murine CD40, the surrogate molecule is eliminated much quicker (t1/2 of 4 hours) demonstrating the specificity of both compounds. In vitro only the hexavalent APG1233 displayed activity in terms of stimulation of B cells, monocytes and PBMCs. In contrast, neither trimeric CD40L nor an agonistic antibody against CD40 were able to upregulate expression of activation markers. Similarly, the secretion of proinflammatory cytokines such as IL-12, CD95L and IFNγ by PBMCs was only stimulated after exposure to APG1233 and not in the presence of other CD40 agonists. In functional co-culture assays, after exposure to APG1233, in vitro generated M2-macrophages underwent conversion and acquired M1-type surface markers which strongly enhanced proliferation of naïve CD4+ T cells. Induction by M2-macrophages in this setup was much lower. Consistent with these data, only the hexavalent CD40 agonist APG1233 efficiently increased direct cytotoxic activity of immune cells against tumor cells measured by a real-time cell analysis assay. Conclusion: The CD40 agonist APG1233 is a member of a novel class of hexavalent TNFRSF agonists which binds its target with high specificity, exhibits excellent in vivo stability and superior biological activity over other agonistic formats (e.g. agonistic antibodies). Citation Format: Christian Merz, Jaromir Sykora, Meinolf Thiemann, David M. Richards, Thamara Beyer, Stefanie Kühn, Harald Fricke, Christian Gieffers, Oliver Hill. The hexavalent CD40 agonist APG1233 is a superior inducer of immune cell activation compared to trimeric formats or agonistic anti-CD40 antibodies [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B066.

Creating a polybenzene network.

Microporous Compounds Metal-organic framework (MOF) compounds are microporous materials in which inorganic centers are linked with organic ligands. Some network topologies, such as the diamond lattice, are formed by many MOFs, whereas others require specific linker positioning. One of the most