Single Chain Research Articles

Dual blockade of IL-17A and IL-36 pathways via a bispecific antibody exhibits enhanced anti-inflammatory potency

Antibody drugs targeting single inflammatory cytokines have revolutionized the treatment of immune-mediated inflammatory diseases. To investigate whether dual targeting interleukin-17 (IL-17) and IL-36 enhances anti-inflammatory activity, bispecific Ab HB0043 was generated by linking the single chain fragment variables (scFvs) from humanized anti-IL-36R antibody (HB0034) to the C-terminus of the heavy chain of anti-IL-17A IgG1 (HB0017) Fc using a flexible peptide linker. HB0043 largely maintained the binding affinities and biological activities of the two parent monoclonal antibodies (mAbs) in vitro. IL-17 and IL-36 cooperated to amplify the expression of pro-inflammatory and pro-fibrotic genes in normal human dermal fibroblasts (NHDF). However, HB0043 more effectively blocked IL-6 and IL-8 production in NHDF stimulated by IL-17A and IL-36 compared to two monoclonal antibodies. In a mouse model of Oxazolone (OXA)-induced atopic dermatitis and Imiquimod (IMQ)-induced skin inflammation, administration of both anti-IL17A mAb HB0017 and anti-mouse IL-36R surrogate antibody HB0034SA showed improved effectiveness in alleviating skin thickening and inflammation based on histological assessment. Further, in cynomolgus monkeys, HB0043 showed no enhanced target-related toxicity compared with the two parental mAbs in vivo and with a moderate increase in production of anti-drug antibodies. Together, dual blockade of IL-17A and IL-36R in the form of a bispecific antibody may have advantages in blocking the overlapping and non-overlapping functions of these two cytokines in skin inflammation that could not optimally be curtailed with single mAbs. In conclusion, as monotherapy may reach therapeutic celling for certain difficult-to-treat inflammatory and fibrotic diseases, dual targeting could potentially pave a way to combat these diseases.

Abstract 4130641: The Role of Oxidized Phospholipids in Triggering Trained Immunity and Accelerating Atherosclerotic Plaque Inflammation during Cholesterol Cycling

Introduction: Oxidized phospholipids (OxPL) derived from LDL play a crucial role in the development and progression of atherosclerosis by promoting chronic inflammation and formation of foam cells. The natural antibody E06, binding to OxPL, diminishes macrophage uptake of OxLDL in vitro and decreases atherosclerosis progression in vivo. In our prior work using a model of plasma cholesterol cycling (high-low-high), resulting in atherosclerotic plaque Progression (P)-Regression (PR)-re-Progression (PRP), respectively, we observed accelerated plaque inflammation in the PRP group relative to mice with non-cycling high cholesterol. Hypothesis: OxPL promotes plaque inflammation in the cholesterol cycling model via trained immunity and that the E06 antibody will mitigate this process. Methods: We utilized transgenic "E06 mice" that expressed a single-chain variable fragment of E06 (E06-scFv) using the Apoe promoter on the Ldlr−/− background. Both E06 mice and Ldlr-/- mice were subjected to high cholesterol diets (HCD) and categorized into three groups in the cholesterol cycling model: Baseline Progression (P), PR and PRP. Plaque regression was induced by ApoB-ASO injection, which significantly lowered cholesterol levels by inhibiting LDL production. Results: As before, Ldlr-/- mice in the PRP group exhibited accelerated plaque inflammation post-regression shown by CD68 staining on aortic roots. Notably, this acceleration was significantly inhibited in the E06 mice, highlighting the protective effects of E06 against plaque re-progression. Additionally, flow cytometry of peripheral blood exhibited reduced total monocytes and Ly6Chi-monocytes (thought to become inflammatory macrophages in plaques), alongside increased Ly6Clo-monocytes (thought to become inflammation resolving macrophages in plaques) among E06 mice in the PRP group compared to Ldlr-/- mice. Ex-vivo stimulation of bone marrow cells with the inflammatory stimulus LPS demonstrated higher IL-6 levels in supernatant in Ldlr-/- mice versus E06 mice in the PRP group, suggesting OXPL-induced trained immunity in bone marrow precursors of monocytes and macrophages is involved. Conclusion: The acceleration of plaque inflammation in cholesterol cycling model was significantly inhibited in the E06 mice. These results provide insight into the increased risk of cardiovascular disease in individuals with intermittent adherence to statins who undergo cholesterol cycling.

EXTH-05. STEM CELL-BASED THERAPEUTIC STRATEGY FOR LEPTOMENINGEAL METASTASIS OF SOLID CANCERS

Abstract Leptomeningeal dissemination (LMD), known as carcinomatous meningitis, presents an extremely poor prognosis with an average life expectancy of 4-6 weeks. Although whole brain/spinal cord irradiation and intrathecal injection of anticancer drugs are sometimes performed, their effects are limited, so new therapeutic strategies for LMD are urgently needed. Based on the following greatest advantage of stem cell (SC) based therapeutic strategy for LMD, we tested its efficacy in LMD of various solid cancers; LMD is a condition in which cancer cells spread within a huge single space without separation, and the SCs with the characteristic of cancer tropism and chemotaxis can reach cancer cells easily; Repetitive intrathecal injection of therapeutic agents for patients is an invasive and risky procedure, but SCs can continuously secrete/release therapeutic agents into the cerebrospinal fluid after a few injections and increase local concentration of therapeutic substances. We have shown that treatment using engineered SCs may be effective treatment option against LMD from breast cancer, lung cancer, and malignant melanoma. Specifically, we reported that engineered allogeneic SCs which simultaneously targeting epidermal growth factor receptor (EGFR) and death receptor (DR) has efficacy for LMD of triple negative breast cancer and non-small cell lung cancer. Also, we reported that SCs releasing oncolytic herpes virus, granulocyte-macrophage colony-stimulating factor (GM-CSF), and single-chain variable region fragments (scFv) against programmed death receptor-1 (PD-1) has efficacy in LMD of malignant melanoma. Overall, we believe that SC therapy has the potential to become a new therapeutic option for LMD in the future.

In Situ Tumor-Infiltrating Lymphocyte Therapy by Local Delivery of an mRNA Encoding Membrane-Anchored Anti-CD3 Single-Chain Variable Fragment

<i>In Situ</i> Tumor-Infiltrating Lymphocyte Therapy by Local Delivery of an mRNA Encoding Membrane-Anchored Anti-CD3 Single-Chain Variable Fragment

Exponential Wiener index of some silicate networks

Any graph that depicts a particular molecular structure can be given a topological graph index, also known as a molecular descriptor. This index can be used to examine numerical data and discover more about specific physical properties of molecules. Silicates are hard crystals that can be sliced into little pieces. Therefore, silicon play a vital role in many real time applications. It is the authentic material to make microchips which run in PDA, PC and other game tools. In the current study, we have determined the new topological index for a few silicate structures, including cyclic, single-chain, and Pyro silicates by grouping the vertices of the corresponding molecular graphs based on their distance sums. This index is known as the exponential Wiener index. It is expanded to include their line graphs as well. The numerical values of exponential Wiener index and multiplicative exponential Wiener index for the molecular graphs of cyclic silicates, single chain silicates and their line graphs with n vertices where n = 3, 4, 5 are explored.

Development and Optimization of a Redox Enzyme-Based Fluorescence Biosensor for the Identification of MsrB1 Inhibitors

MsrB1 is a thiol-dependent enzyme that reduces protein methionine-R-sulfoxide and regulates inflammatory response in macrophages. Therefore, MsrB1 could be a promising therapeutic target for the control of inflammation. To identify MsrB1 inhibitors, we construct a redox protein-based fluorescence biosensor composed of MsrB1, a circularly permutated fluorescent protein, and the thioredoxin1 in a single polypeptide chain. This protein-based biosensor, named RIYsense, efficiently measures protein methionine sulfoxide reduction by ratiometric fluorescence increase. We used it for high-throughput screening of potential MsrB1 inhibitors among 6868 compounds. A total of 192 compounds were selected based on their ability to reduce relative fluorescence intensity by more than 50% compared to the control. Then, we used molecular docking simulations of the compound on MsrB1, affinity assays, and MsrB1 activity measurement to identify compounds with reliable and strong inhibitory effects. Two compounds were selected as MsrB1 inhibitors: 4-[5-(4-ethylphenyl)-3-(4-hydroxyphenyl)-3,4-dihydropyrazol-2-yl]benzenesulfonamide and 6-chloro-10-(4-ethylphenyl)pyrimido[4,5-b]quinoline-2,4-dione. They are heterocyclic, polyaromatic compounds with a substituted phenyl moiety interacting with the MsrB1 active site, as revealed by docking simulation. These compounds were found to decrease the expression of anti-inflammatory cytokines such as IL-10 and IL-1rn, leading to auricular skin swelling and increased thickness in an ear edema model, effectively mimicking the effects observed in MsrB1 knockout mice. In summary, using a novel redox protein-based fluorescence biosensor, we identified potential MsrB1 inhibitors that can regulate the inflammatory response, particularly by influencing the expression of anti-inflammatory cytokines. These compounds are promising tools for understanding MsrB1’s role during inflammation and eventually controlling inflammation in therapeutic approaches.

Characterization of anti-EBNA-1 antibodies and exploration of their molecular mimicry potential in an EBV-infected Sjögren's syndrome patient

There is a potential link between autoimmune diseases and Epstein-Barr virus (EBV) infections, with EBV playing a substantial role in the onset of Sjögren's syndrome (SjS). Some EBV proteins could mimic host self-antigens post-infection, leading to molecular mimicry. This similarity may cause the immune system to attack its tissues mistakenly. Among the various proteins associated with EBV, nuclear antigen 1 (EBNA-1) is essential for the latent replication of infected cells and is prevalent in all EBV-related diseases. In the study, single-chain variable fragment (scFv) antibodies targeting EBNA-1 were isolated using phage display technology from a primary SjS patient who also had a chronic active EBV infection. The specific clones were enriched after panning, and the binding activity of selected scFvs targeting EBNA-1 was confirmed. Sequence analysis indicated that the scFvs exhibiting positive signals could be grouped into five clones, all of which used homologous heavy chain V regions derived from germline Vh4-39, and two types of light chain V regions stemming from germline Vλ1-44 and Vλ3-15. These scFvs were found to exhibit a high degree of somatic mutations, likely indicative of antigen selection. Of the scFvs, P1-3 demonstrated the strongest binding affinity to EBNA-1, exhibiting a determined value of 7.3 x 10-8 M, and showed cross-reactivity to the SjS associated La/SSB self-antigen. The experimental results combined with AlphaFold 3 predictions revealed a potential epitope for scFv P1-3 binding to EBNA-1. Additionally, scFv P1-3 could also cross-bind to the modeled structure of La/SSB. We inferred a possible structural correlation between EBNA-1 and La/SSB involving an X2AX6PG epitope motif. This research contributes to our understanding of the structural basis of the interactions between antibodies and EBNA-1, shedding light on the VH and VL gene usage of anti-EBNA-1 antibodies in EBV-infected SjS patients and the potential origins of autoantibodies.

Recombinant Antibody Fragments for Neurological Disorders: An Update.

Recombinant antibody fragments are promising alternatives to full-length immunoglobulins, creating big opportunities for the pharmaceutical industry. Nowadays, antibody fragments such as antigen-binding fragments (Fab), single-chain fragment variable (scFv), single-domain antibodies (sdAbs), and bispecific antibodies (bsAbs) are being evaluated as diagnostics or therapeutics in preclinical models and in clinical trials. Immunotherapy approaches, including passive transfer of protective antibodies, have shown therapeutic efficacy in several animal models of Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal dementia (FTD), Huntington's disease (HD), transmissible spongiform encephalopathies (TSEs) and multiple sclerosis (MS). There are various antibodies approved by the Food and Drug Administration (FDA) for treating multiple sclerosis and two amyloid beta-specific humanized antibodies, Aducanumab and Lecanemab, for AD. Our previous review summarized data on recombinant antibodies evaluated in pre-clinical models for immunotherapy of neurodegenerative diseases. Here, we explore recent studies in this fascinating research field, give an update on new preventive and therapeutic applications of recombinant antibody fragments for neurological disorders and discuss the potential of antibody fragments for developing novel approaches for crossing the blood-brain barrier (BBB) and targeting cells and molecules of interest in the brain.

Redefining copy number variation and single-nucleotide polymorphism counting via novel concepts based on recent PCR enhancements.

Human genes have numerous copy number variations (CNVs) and single-nucleotide polymorphisms (SNPs) that control most of the body’s core functions. On average, 12–16% of human genes have CNVs, and a single gene can have a few hundred to several thousand SNPs. Numerous genome-wide association studies (GWAS) have shown that CNVs and SNPs can coexist in certain genomic regions, amplifying their effects on gene expression and regulation and disease susceptibility. Researchers initially categorized CNVs and SNPs into two types: homozygous and heterozygous. However, copy numbers were soon found to have a much wider range, underscoring their significance in certain diseases and microbial interactions. Because of the significant impact of CNVs and SNPs, research groups worldwide have eagerly sought effective methods for detecting both simultaneously. Despite yielding some minor results, these simultaneous counting methods have failed to meet expectations, leaving researchers to measure CNVs and SNPs separately. To overcome these limitations, we developed a novel approach by combining primers designed using the STexS method with matching probes used in the STexS II method. This method successfully detected both CNVs and SNPs in CYP2A6 and CYP2A7 using a single quantitative polymerase chain reaction. Once properly adjusted based on the three core principles, this new method markedly improved the time, cost-effectiveness, and overall accuracy of determining an individual’s genetic status. Further testing of 100 human genomic DNA samples enabled calculations of the overall frequency of the [T] and [G] alleles of the CYP2A6 -48T>G SNP within an East Asian population yielded results that were highly congruent with those in a National Institutes of Health (NIH) database. This novel method will redefine genetic profiling and provide a means to successfully predict genetic characteristics and enhance personalized medicine by pinpointing appropriate individualized treatments.

Highly selective split intein method for efficient separation and purification of recombinant therapeutic proteins from mammalian cell culture fluid

Biologics and vaccines have been successfully developed over the last few decades to treat many diseases. Each of these drugs must be highly purified for clinical use. Monoclonal antibodies (mAbs), the dominant therapeutic modality on the market, can be easily purified using the standard Protein A affinity platform. However, no generally applicable affinity platforms are available for the manufacture of other therapeutic proteins for clinical use. Thus, multicolumn chromatography processes for widely being used for product purification. These processes demand significant optimization to meet desired product quality attributes, where each step also decreases final yields. In this work, we demonstrate the novel self-removing iCapTagTM affinity tag, which provides a new platform for capturing, concentrating, and purifying recombinant proteins. Importantly, this system provides a tagless target protein, which is suitable for research and clinical use, where the only requirement for tag removal is a small change in buffer pH. No additional proteins, reagents or cofactors are required. We also present case studies demonstrating the use of iCapTagTM for highly efficient purification of untagged interferon alpha 2b, the ML39 single chain variable fragment (scFv), and the receptor binding domain (RBD) of SARS-CoV-2 spike protein. These proteins were expressed and secreted by Expi293 cells with the self-removing tag fused to their N-terminus. We were able to obtain highly pure (> 99%) tagless protein in a single purification step with high clearance of host cell DNA, tagged precursor, higher and lower molecular weight impurities. Based on these preliminary results, we propose the iCapTagTM as a universal capture platform for diverse classes of recombinant therapeutic proteins.

New prospects for 89Zr-immuno-PET in brain applications – Alpha-synucleinopathies

BackgroundRecently, 89Zr-immuno-PET imaging of therapeutic antibodies, actively transported over the blood-brain-barrier via transferrin-mediated transcytosis, was demonstrated using the chelator DFO*. In these studies, aducanumab targeting amyloid-beta was fused with a transferrin binding unit: a single-chain Fab fragment derived from 8D3 (scFab8D3). Alpha-synuclein is a hallmark protein of several neurodegenerative diseases such as Parkinson's Disease, Lewy-Body-Dementia, and Multiple System Atrophy. 89Zr-immuno-PET imaging of alpha-synuclein can be a valuable tool for image-guided drug development and assessment of target engagement. The goal of this study was to compare two currently used constructs of 8D3 for targeting potential, namely a single moiety of scFab8D3 fused to the alpha-synuclein antibody HLu-3 (HLu-3-scFab8D3) versus HLu-3 fused with two 8D3 single-chain variable fragments (HLu-3-(scFv8D3)2), by 89Zr-immuno-PET in an alpha-synuclein pre-formed fibril (PFF) deposition model. HLu-3 and the HIV-targeting B12-scFab8D3 were used as controls. The best-performing compound was further investigated in an animal model with predominantly intraneural target aggregation. MethodsAntibodies were conjugated with DFO* using DFO*-NCS and subsequently radiolabeled with 89Zr. Assessment of binding affinity was done by alpha-synuclein ELISA and with FACS analysis using mTfR1 expressing CHO-S cells. Radioimmunoconjugates were first evaluated in an extracellular alpha-synuclein deposition model established by intracranial injection of non-sonicated PFFs into the left striatum of C57Bl/6 WT mice, whereas saline was injected into the contralateral side as control. PET imaging was performed 1, 3, and 7 days post-injection, followed by ex vivo biodistribution, autoradiography and immunofluorescence analysis. Based on the results from these studies, the better-performing antibody candidate was tested similarly in an alpha-synuclein seeding model. The seeding model has intraneural alpha-synuclein aggregation and was established by intracranial injection of sonicated PFFs into both striata of F28tg mice, which overexpress human wild-type alpha-synuclein. Untreated F28tg and C57Bl/6 WT mice served as controls. ResultsThe radioconjugate was produced in sufficient radiochemical yields and purity. There was no impairment of binding affinity towards alpha-synuclein, and acceptable binding with negligible losses to mTfR1. PET imaging with [89Zr]Zr-HLu-3-scFab8D3 and [89Zr]Zr-HLu-3-(scFv8D3)2 in the deposition model showed uptake at the site of alpha-synuclein deposits. However, uptake was variable between mice. Reliable PET quantification was hampered due to the small deposition volume (~2 μL). Immunofluorescence revealed specific alpha-synuclein target engagement of both constructs with PFF deposits in the striatum, in contrast to the [89Zr]Zr-B12-scFab8D3 control. Unexpectedly, ex vivo autoradiography showed uptake in some controls ([89Zr]Zr-B12-scFab8D3 in the contralateral striatum without PFFs), potentially related to astrocyte activation at the injection sides. Ex vivo and PET brain uptake was higher for [89Zr]Zr-HLu-3-scFab8D3 when compared to [89Zr]Zr-HLu-3-(scFv8D3)2 and was therefore selected for further testing in the alpha-synuclein seeding model. No significant difference in in vivo and ex vivo brain uptake of [89Zr]Zr-HLu-3-scFab8D3 between PFF-injected F28tg, F28tg and C57Bl/6 mice was observed. Furthermore, ex vivo immunofluorescence and autoradiography showed no specific alpha-synuclein target engagement. ConclusionsSuccessful target engagement of [89Zr]Zr-HLu-3-scFab8D3 and [89Zr]Zr-HLu-3-(scFv8D3)2 with alpha-synuclein was shown in a PFF deposition model. PET imaging showed variable results, and in vivo detection of the depositions was possible in some cases. Due to the better performance in the deposition model, [89Zr]Zr-HLu-3-scFab8D3 was further investigated in an alpha-synuclein seeding model with intraneural Lewy-body pathology, showing no difference between the control groups and PFF-seeded mice. Furthermore, immunostaining of seeded F28tg mice manifested sufficient intraneural alpha-synuclein pathology but no corresponding antibody accumulation. These results underscore the ongoing challenge of imaging intraneural inclusions via immuno-PET.

How Does Supply Network Structure Influence Firms’ Financial Performance During Disruption?

In a post pandemic world, supply chain mapping has become a key concern for almost any supply chain operation. A single focal supply chain network might have hundreds of suppliers including those that supply both goods and services. These increase exponentially as one traces the network downstream. Using a pair of networks from the auto industry (Ford and General Motors) as a basis for the analysis, we apply a data mining technique to filter for significant supply chain relationships in each of these networks over the period spanning 2018–2022. We then examine the structural metrics for each of these networks and compare these measures with applicable financial measures to elucidate how supply chain network structure affects financial performance. Our regression analyses reveal that supplier network structures significantly affect customers’ market value. Specifically, Tobin’s Q is negatively related to the number of suppliers they source from and the importance of the customer (e.g., Ford) to its suppliers, but it is positively associated with the tightness of supplier customer connection and the density of the network.

Shotgun Sequencing of 512‐mer Copolyester Allows Random Access to Stored Information

AbstractDigital information encoded in polymers has been exclusively decoded by mass spectrometry. However, the size limit of analytes in mass spectrometry restricts the storage capacity per chain. In addition, sequential decoding hinders random access to the bits of interest without full‐chain sequencing. Here we report the shotgun sequencing of a 512‐mer sequence‐defined polymer whose molecular weight (57.3 kDa) far exceeds the analytical limit of mass spectrometry. A 4‐bit fragmentation code was implemented at aperiodic positions during the synthetic encoding of 512‐bit information without affecting storage capacity per chain. Upon activating the fragmentation code, the polymer chain splits into 18 oligomers, which could be individually decoded by tandem‐mass sequencing. These sequences were computationally reconstructed into a full sequence using an error‐detection method. The proposed sequencing method eliminates the storage limit of a single polymer chain and allows random access to the bits of interest without full‐chain sequencing.

Structural insights into the toxicity of type II ribosome inactivating proteins (RIPs): a molecular dynamics study

Ribosome Inactivating Proteins (RIPs) act by irreversibly depurinating the 28S rRNA ricin-sarcin loop (SRL) of the eukaryotic ribosome resulting in protein synthesis inhibition. In general, they consist of two variants: Type I which is single chained (∼30 kDa), and Type II, a more toxic variant which is a Type I N-glycosidase chain covalently linked to a lectin chain. These proteins are believed to play a pivotal role in defence mechanisms. Intriguingly, non-toxic variants of such toxic proteins do exist in nature. To explore their mode of action, in the present study we have selected three toxic (Ricin, Ebulin and HmRIP) as well as two non-toxic (BGSL and SGSL) RIPs and performed molecular docking and molecular dynamic simulations with the SRL loop. This study throws light on the structural stability and plasticity of the toxic and non-toxic RIP complexes. Furthermore, analysis of the active site cavity volume and binding free energy calculations reveal that the SRL, particularly the specific adenine (A4605), is relatively unstable in the case of non-toxic RIPs which is also supported by the free binding energy calculations, and the pocket size analysis indicates the abnormal increase in active site cavity volume of non-toxic RIPs with time. This first-of-its-kind comprehensive study of toxic and non-toxic RIPs gives insights about the mode of action and the dynamic nature of their interaction with the SRL loop. These observations will be helpful in the development of toxoids against RIPs and also in designing novel therapeutic approaches against human diseases.

EuDockScore: Euclidean graph neural networks for scoring protein-protein interfaces.

Protein-protein interactions are essential for a variety of biological phenomena including mediating bio-chemical reactions, cell signaling, and the immune response. Proteins seek to form interfaces which reduce overall system energy. Although determination of single polypeptide chain protein structures has been revolutionized by deep learning techniques, complex prediction has still not been perfected. Additionally, experimentally determining structures is incredibly resource and time expensive. An alternative is the technique of computational docking, which takes the solved individual structures of proteins to produce candidate interfaces (decoys). Decoys are then scored using a mathematical function that assess the quality of the system, know as a scoring functions. Beyond docking, scoring functions are a critical component of assessing structures produced by many protein generative models. Scoring models are also used as a final filtering in many generative deep learning models including those that generate antibody binders, and those which perform docking. In this work we present improved scoring functions for protein-protein interactions which utilizes cutting-edge euclidean graph neural network architectures, to assess protein-protein interfaces. These euclidean docking score models are known as EuDockScore, and EuDockScore-Ab with the latter being antibody-antigen dock specific. Finally, we provided EuDockScore-AFM a model trained on antibody-antigen outputs from AlphaFold-Multimer which proves useful in re-ranking large numbers of AlphaFold-Multimer outputs. The code for these models is available at https://gitlab.com/mcfeemat/eudockscore.

Self-Assembly and Wound Healing Activity of Biomimetic Cycloalkane-Based Lipopeptides.

The self-assembly of lipopeptide (peptide amphiphile) molecules bearing single linear lipid chains has been widely studied, as has their diverse range of bioactivities. Here, we introduce lipopeptides bearing one or two cycloalkane chains (cycloheptadecyl or cyclododecyl) conjugated to the collagen-stimulating pentapeptide KTTKS used in Matrixyl formulations. The self-assembly of all four molecules is probed using fluorescence probe measurements to detect the critical aggregation concentration (CAC), and cryogenic-TEM and small-angle X-ray scattering (SAXS) to image the nanostructure. The peptide conformation is studied using circular dichroism (CD) and FTIR spectroscopies. All the cycloalkane lipopeptides show excellent compatibility with dermal fibroblasts. The compounds bearing one or two cyclododecyl chains (denoted as DKT and DDKT, respectively) show wound healing in diabetic rats, the improvement being markedly enhanced for DDKT. Interestingly, the revival of hair follicles and blood vessels in the dermis were observed, which are the critical markers of effective wound repair. Analysis of H&E-stained tissue images (from a rat model) shows that the rat groups treated with DDKT and DKT displayed a significantly increased amount of regenerated hair follicles, indicating a faster healing process for DDKT compared to the control group. Collagen deposition was also enhanced, especially for DDKT, and by day 20, the DDKT-treated groups had developed a dense collagen network accompanied by a regenerated epidermis. At the same time, the number of blood vessels in DDKT-treated diabetic wounds was significantly higher than in control groups and neovascularization was substantially enhanced, as assayed using α-SMA (a marker for vascular smooth muscle cells) and CD31 (a marker specific to vascular endothelial cells). These results suggest that the lead lipopeptide DDKT exhibits a remarkable pro-vascularization capability and shows great promise for future application as a wound-healing biomaterial.

Targeting overexpressed antigens in glioblastoma via CAR T cells with computationally designed high-affinity protein binders.

Chimeric antigen receptor (CAR) T cells targeting receptors on tumour cells have had limited success in patients with glioblastoma. Here we report the development and therapeutic performance of CAR constructs leveraging protein binders computationally designed de novo to have high affinity for the epidermal growth factor receptor (EGFR) or the tumour-associated antigen CD276, which are overexpressed in glioblastoma. With respect to T cells with a CAR using an antibody-derived single-chain variable fragment as antigen-binding domain, the designed binders on CAR T cells promoted the proliferation of the cells, the secretion of cytotoxic cytokines and their resistance to cell exhaustion, and improved antitumour performance in vitro and in vivo. Moreover, CARs with the binders exhibited higher surface expression and greater resistance to degradation, as indicated by bulk and single-cell transcriptional profiling of the cells. The de novo design of binding domains for specific tumour antigens may potentiate the antitumour efficacy of CAR T cell therapies for other solid cancers.

Semisynthetic Phage Display Library Construction: Design and Synthesis of Diversified Single-Chain Variable Fragments and Generation of Primary Libraries.

Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the first of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Libraries. The three-step method involves (1) primary library (PL) construction, (2) filtered library construction, and (3) secondary library construction. The first step, described here, entails design, synthesis, and cloning of four PLs. These PLs are designed with specific properties amenable to therapeutic antibody development using one universal variable heavy (VH) scaffold and four distinct variable light (VL) scaffolds. The scaffolds are diversified in positions that bind both protein and peptide targets identified in antibody-antigen complexes of known structure using the amino acid frequencies found in those positions in known human antibody sequences, avoiding residues that may lead to developability liabilities. The diversified scaffolds are combined with 90 synthetic neutral HCDR3 sequences designed with developable human diversity genes (IGHD) and joining heavy genes (IGHJ) in germline configuration, and assembled as single-chain variable fragments (scFvs) in a VL-linker-VH orientation. The four designed PLs are synthesized using trinucleotide phosphoramidites (TRIMs) and cloned independently into a phagemid vector for M13 pIII display. Quality control of the cloning of the four PLs is also described, which involves sequencing scFvs in each library.

Semisynthetic Phage Display Library Construction: Generation of Filtered Libraries.

Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the second of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Gold Plus Libraries. The three-step method involves (1) primary library (PL) construction, (2) filtered library (FL) construction, and (3) secondary library construction. The second step, described here, involves display of the PLs as single-chain variable fragment (scFv) fusions to protein pIII of the M13 phage, as well as heat shock treatment and subsequent selection of well-folded and thermostable scFvs via protein L binding, whereas unstable and defective scFvs are removed by washing steps and centrifugation. The quality of the filtration process is assessed by sequencing clones chosen at random from the FLs. These libraries, enriched with thermostable antibodies, are then ready to be used for the third and final step of the process: generation of secondary libraries.

Semisynthetic Phage Display Library Construction: Generation of Single-Chain Variable Fragment Secondary Libraries.

Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the third and final step of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Libraries. The three-step method involves (1) primary library construction, (2) filtered library (FL) construction, and (3) secondary library (SL) construction. In the third step, described here, the nucleotide sequences encoding the single-chain variable fragments (scFvs) of FLs are amplified by PCR and combined with the heavy- chain CDR3 region (HCDR3) and joining fragments (H3J) obtained from a pool of donors to maximize diversity ("natural H3J fragments"). These natural H3J fragments are amplified with a set of primers designed to capture >95% of the natural H3J repertoire. The resultant fragments replace the neutral H3J fragments of the FLs, resulting in the final semisynthetic secondary libraries. The quality of these libraries is assessed by sequencing clones chosen at random from the libraries, typically 96 clones. These libraries are then ready to be used for phage selections on targets of interest, providing a robust antibody discovery platform.