Single-chain Variable Fragment Antibody Research Articles

Anti-CAMP1 IgG promotes macrophage phagocytosis of Cutibacterium acnes Type II

Among 5 types of the Christie-Atkins-Munch-Petersen factor (CAMP) of Cutibacterium acnes, CAMP1 is highly expressed in phylotype II as well as IB, and thought to be a virulence factor of opportunistic but fatal blood, soft tissue, and implant-related infections. The target of a human single-chain variable antibody fragment (scFv), recently isolated from a phage display library, has been identified as CAMP1 of phylotype II, using immunoprecipitation followed by mass spectrometry, phage display peptide biopanning, 3D-modelling, and ELISA. The IgG1 format of the antibody could enhance phagocytosis of C. acnes DMST 14916 by THP-1 human monocytes. Our results suggest that the antibody-dependent phagocytosis process is mediated by the caveolae membrane system and involves the induction of IL-1β. This is the first report on the study of a human antibody against CAMP1 of C. acnes phylotype II, of which a potential use as therapeutic antibody against virulence C. acnes infection is postulated.

Combining Cellular Immunization and Phage Display Screening Results in Novel, FcγRI-Specific Antibodies.

Antibodies that specifically bind to individual human fragment crystallizable γ receptors (FcγRs) are of interest as research tools in studying immune cell functions, as well as components in bispecific antibodies for immune cell engagement in cancer therapy. Monoclonal antibodies for human low-affinity FcγRs have been successfully generated by hybridoma technology and are widely used in pre-clinical research. However, the generation of monoclonal antibodies by hybridoma technology that specifically bind to the high-affinity receptor FcγRI is challenging. Monomeric mouse IgG2a, IgG2b, and IgG3 bind human FcγRI with high affinity via the Fc part, leading to an Fc-mediated rather than a fragment for antigen binding (Fab)-mediated selection of monoclonal antibodies. Blocking the Fc-binding site of FcγRI with an excess of human IgG or Fc during screening decreases the risk of Fc-mediated interactions but can also block the potential epitopes of new antibody candidates. Therefore, we replaced hybridoma technology with phage display of a single-chain fragment variable (scFv) antibody library that was generated from mice immunized with FcγRI-positive cells and screened it with a cellular panning approach assisted by next-generation sequencing (NGS). Seven new FcγRI-specific antibody sequences were selected with this methodology, which were produced as Fc-silent antibodies showing FcγRI-restricted specificity.

Adding value to banana farming: Antibody production in post-harvest leaves

Banana, a globally popular fruit, is widely cultivated in tropical and sub-tropical regions. After fruit harvest, remaining banana plant materials are low-value byproducts, mostly composted or used as fibre or for food packaging. As an aim to potentially increase farmer income, this study explored underutilised banana biomass as a novel plant tissue for production of a high-value product. Protein scFvTG130 used in this study, is an anti-toxoplasma single chain variable fragment antibody that can be used in diagnostics and neutralising the Toxoplasma gondii pathogen. Using detached banana leaves, we investigated the factors influencing the efficacy of a transient expression system using reporter genes and recombinant protein, scFvTG130. Transient expression was optimal at 2 days after detached banana leaves were vacuum infiltrated at 0.08MPa vacuum pressure for a duration of 3min with 0.01% (v/v) Tween20 using Agrobacterium strain GV3101 harbouring disarmed virus-based vector pIR-GFPscFvTG130. The highest concentration of anti-toxoplasma scFvTG130 antibody obtained using detached banana leaves was 22.8µg/g fresh leaf tissue. This first study using detached banana leaf tissue for the transient expression of a recombinant protein, successfully demonstrated anti-toxoplasma scFvTG130 antibody expression, supporting the potential application for other related proteins using an underutilised detached banana leaf tissue.

Preparation of Bispecific IgY-scFvs Inhibition Adherences of Enterotoxigenic Escherichia coli (K88 and F18) to Porcine IPEC-J2 Cell.

Enterotoxigenic Escherichia coli (ETEC) strains are significant contributors to postweaning diarrhea in piglets. Of the ETEC causing diarrhea, K88 and F18 accounted for 92.7%. Despite the prevalence of ETEC K88 and F18, there is currently no effective vaccine available due to the diversity of these strains. This study presents an innovative approach by isolating chicken-derived single-chain variable fragment antibodies (scFvs) specific to K88 and F18 fimbrial antigens from chickens immunized against these ETEC virulence factors. These scFvs effectively inhibited adhesion of K88 and F18 to porcine intestinal epithelial cells (IPEC-J2), with the inhibitory effect demonstrating a dose-dependent increase. Furthermore, a bispecific scFv was designed and expressed in Pichia pastoris. This engineered construct displayed remarkable potency; at a concentration of 25.08 μg, it significantly reduced the adhesion rate of ETEC strains to IPEC-J2 cells by 72.10% and 69.11% when challenged with either K88 or F18 alone. Even in the presence of both antigens, the adhesion rate was notably decreased by 57.92%. By targeting and impeding the initial adhesion step of ETEC pathogenesis, this antibody-based intervention holds promise as a potential alternative to antibiotics, thereby mitigating the risks associated with antibiotic resistance and residual drug contamination in livestock production. Overall, this study lays the groundwork for the development of innovative treatments against ETEC infections in piglets.

Abstract 1327: Affinity tuning anti-CD123 chimeric antigen receptor natural killer cell therapy to treat acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is a highly prevalent blood and bone marrow cancer characterized by the uncontrolled growth of abnormal myeloblasts. Current treatments for AML often result in systemic toxicities for patients, many of whom will still experience relapse. There is thus an unmet need to develop safer and more effective therapies targeting AML. Chimeric antigen receptor (CAR) T cells, which are engineered to express a cancer-targeting extracellular antibody single-chain variable fragment (scFv) linked to an intracellular T cell-activating domain, have shown promise in treating B cell leukemias, but their success has been limited in myeloid leukemias. This is due in part to the lack of AML-specific target antigens. Moreover, impaired T cell function, a hallmark of AML, is also problematic when relying on autologous T cell activation upon reinfusion. We overcame the limitations of CAR T therapy in AML by developing CAR-expressing natural killer (NK) cells targeting CD123. While present on both healthy and AML cells, CD123 is expressed at >10-fold higher levels on malignant cells. Furthermore, use of NK allows use of fully functional allogeneic cells from healthy donors, since NK cells do not induce graft-versus-host-disease. To optimize the tumor cell selectivity of CAR NK cells, we employed the yeast display directed evolution platform to isolate anti-CD123 scFvs with varying affinities and are identifying and characterizing clones that maximize the interaction with CD123high AML cells over healthy CD123low cells, as determined by binding, cytotoxicity, cytokine secretion, and intracellular signaling studies. Collectively, our efforts introduce a new paradigm for engineered cell therapeutics with significant promise for AML treatment. Citation Format: Monika Kizerwetter, Huilin Yang, Ruyan Rahnama, Challice Bonifant, Jamie Spangler. Affinity tuning anti-CD123 chimeric antigen receptor natural killer cell therapy to treat acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1327.

Construction of a Human Immune Library from Gallbladder Cancer Patients for the Single-Chain Fragment Variable (scFv) Antibody Selection against Claudin 18.2 via Phage Display.

Gallbladder cancer (GBC) is a very aggressive malignant neoplasm of the biliary tract with a poor prognosis. There are no specific therapies for the treatment of GBC or early diagnosis tools; for this reason, the development of strategies and technologies that facilitate or allow an early diagnosis of GBC continues to be decisive. Phage display is a robust technique used for the production of monoclonal antibodies (mAbs) involving (1) the generation of gene libraries, (2) the screening and selection of isoforms related to an immobilized antigen, and (3) the in vitro maturation of the affinity of the antibody for the antigen. This research aimed to construct a human immune library from PBMCs of GBC patients and the isolation of scFv-phage clones with specificity against the larger extracellular loop belonging to claudin 18.2, which is an important biomarker overexpressed in GBC as well as gastric cancer. The immune-library-denominated GALLBLA1 was constructed from seven GBC patients and has a diversity of 6.12 × 1010pfu mL-1. After three rounds of panning, we were able to identify clones with specificity against claudin 18.2. GALLBLA1 can contribute to the selection, isolation, and recombinant production of new human mAbs candidates for the treatment of gastrointestinal cancers.

Assessment of Grapefruit Expressing Anti-NodT Antibody for Huanglongbing Resistance

Growers rely on broad-spectrum agrochemicals to manage one of the most economically important fruit tree diseases, huanglongbing (HLB, citrus greening disease), presumptively caused by the gram-negative bacterium ‘ Candidatus Liberibacter asiaticus’ ( CLas). Although genetic resistance would be an attractive alternative to chemical management, this option is not yet available for HLB. Here, we tested whether a single chain variable fragment (scFv) antibody targeting the predicted CLas outer membrane transporter NodT can inhibit CLas growth or HLB disease development when expressed in ‘Duncan’ grapefruit ( Citrus × paradisi). CLas NodT is similar to TolC and could be involved in Type I secretion and virulence. The scFv antibody was expressed in grapefruit trees as a C-terminal translational fusion to the Flowering Locus T protein of Poncirus trifoliata (FT-scFv) to promote expression in the phloem. Wild-type and FT-scFv-expressing transgenic lines were challenged with CLas using three inoculation approaches: psyllid-mediated inoculation, vegetative graft inoculation, and natural exposure in grove-like conditions. With the first two approaches, HLB symptom expression and CLas bacterial titers in FT-scFv grapefruit lines were not significantly different from wild-type controls. In the grove trial, one FT-scFv line exhibited a slight, but significant, reduction in canopy chlorosis compared to wild-type controls. Wild-type and FT-scFv lines were similar in all other metrics. We conclude that the expression of anti-NodT FT-scFv antibody in grapefruit did not decrease HLB susceptibility. Although our approach was unsuccessful, we hope that documenting our results will be useful for those seeking to develop HLB-resistant citrus germplasm in the future. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Preparation and recognition mechanism study of an scFv targeting chloramphenicol for a hybridization chain reaction-CRISPR/Cas12a amplified fluoroimmunoassay

Recombinant antibody-based immunoassays have emerged as crucial techniques for detecting antibiotic residues in food samples. Developing a stable recombinant antibody production system and enhancing detection sensitivity are crucial for their biosensing applications. Here, we bioengineered a single-chain fragment variable (scFv) antibody to target chloramphenicol (CAP) using both Bacillus subtilis and HEK 293 systems, with the HEK 293-derived scFv demonstrating superior sensitivity. Computational chemistry analyses indicated that ASP-99 and ASN-102 residues in the scFv play key roles in antibody recognition, and the hydroxyl group near the benzene ring of the target molecule is critical for in antibody binding. Furthermore, we enhanced the scFv's biosensing sensitivity using an HCR-CRISPR/Cas12a amplification strategy in a streptavidin-based immunoassay. In the dual-step amplification process, detection limits for CAP in the HCR and HCR-CRISPR/Cas12a stages were significantly reduced to 55.23 pg/mL and 3.31 pg/mL, respectively. These findings introduce an effective method for developing CAP-specific scFv antibodies and also propose a multi-amplification strategy to increase immunoassay sensitivity. Additionally, theoretical studies also offer valuable guidance in CAP hapten design and genetic engineering for antibody modification.

Production and characterization of single-chain variable fragment antibodies targeting the breast cancer tumor marker nectin-4.

Nectin-4 is a novel biomarker overexpressed in various types of cancer, including breast cancer, in which it has been associated with poor prognosis. Current literature suggests that nectin-4 has a role in cancer progression and may have prognostic and therapeutic implications. The present study aims to produce nectin-4-specific single-chain variable fragment (scFv) antibodies and evaluate their applications in breast cancer cell lines and clinical specimens. We generated recombinant nectin-4 ectodomain fragments as immunogens to immunize chickens and the chickens' immunoglobulin genes were amplified for construction of anti-nectin-4 scFv libraries using phage display. The binding capacities of the selected clones were evaluated with the recombinant nectin-4 fragments, breast cancer cell lines, and paraffin-embedded tissue sections using various laboratory approaches. The binding affinity and in silico docking profile were also characterized. We have selected two clones (S21 and L4) from the libraries with superior binding capacity. S21 yielded higher signals when used as the primry antibody for western blot analysis and flow cytometry, whereas clone L4 generated cleaner and stronger signals in immunofluorescence and immunohistochemistry staining. In addition, both scFvs could diminish attachment-free cell aggregation of nectin-4-positive breast cancer cells. As results from ELISA indicated that L4 bound more efficiently to fixed nectin-4 ectodomain, molecular docking analysis was further performed and demonstrated that L4 possesses multiple polar contacts with nectin-4 and diversity in interacting residues. Overall, the nectin-4-specific scFvs could recognize nectin-4 expressed by breast cancer cells and have the merit of being further explored for potential diagnostic and therapeutic applications.

Towards detection of structurally-diverse glycated epitopes in native proteins: Single-chain antibody directed to non-A1c epitope in human haemoglobin

Over 500 million people worldwide are affected by diabetes mellitus, a chronic disease that leads to high blood glucose levels and causes severe side effects. The predominant biological marker for diagnosis of diabetes is glycated haemoglobin (GHb). In human blood the predominant reducing sugar, glucose, irreversibly conjugates onto accessible amine groups within Hb. Most methods for diagnosis and monitoring of diabetes selectively detect N-terminal glycation at Val-1 on the β-globin chain, but not glycation at other sites. Detection of other glycated epitopes of GHb has the potential to provide new information on the extent, duration and timing of elevated glucose, facilitating personalised diagnosis and intelligent diabetic control. In this work, a new anti-GHb Fab antibody (Fab-1) specific for haemoglobin A1c (HbA1c) with nanomolar affinity was discovered via epitope-directed immunisation and phage display. A single chain variable fragment (scFv) antibody derived from Fab-1 retained affinity and specificity for HbA1c, and affinity was enhanced tenfold upon addition of an enhanced green fluorescent protein tag. Both the scFv and Fab-1 recognised an epitope within HbA1c that was distinct from β-Val-1, and our data suggest that this epitope may include glycation at Lys-66 in the β-globin chain. To our knowledge, this is the first report of an scFv/Fab anti-glycated epitope antibody that recognises a non-A1c epitope in GHb, and confirms that fructosamine attached to different, discrete glycation sites within the same protein can be resolved from one another by immunoassay.

Alpha-Synuclein Oligomers Driven T1-T2 Switchable Nanoprobes for Early and Accurate Diagnosis of Parkinson's Disease.

The alpha-synuclein (α-syn) oligomers hold a central role in the pathology of Parkinson's disease (PD). Achieving accurate detection of α-syn oligomers in vivo presents a promising avenue for early and accurate diagnosis of PD. Magnetic resonance imaging (MRI), with non-invasion and exceptional tissue penetration, offers a potent tool for visualizing α-syn oligomers in vivo. Nonetheless, ensuring diagnostic specificity remains a formidable challenge. Herein, a novel MRI probe (ASOSN) is introduced, which encompasses highly sensitive antiferromagnetic nanoparticles functionalized with single-chain fragment variable antibodies, endowing it with the capacity for discerning recognition and binding to α-syn oligomers and triggering a switchable T1-T2 MRI signal. Significantly, ASOSN possesses the unique capability to accurately discriminate α-syn oligomers from neuroinflammation in vivo. Moreover, ASOSN facilitates the non-invasive and precise visualizing of endogenous α-syn oligomers in living systems. This innovative design heralds the development of a non-invasive visualization strategy for α-syn oligomers, marking a pivotal advancement for early and accurate diagnosis of PD.

BG4 antibody can recognize telomeric G-quadruplexes harboring destabilizing base modifications and lesions.

BG4 is a single-chain variable fragment antibody shown to bind various G-quadruplex (GQ) topologies with high affinity and specificity, and to detect GQ in cells, including GQ structures formed within telomeric TTAGGG repeats. Here, we used ELISA and single-molecule pull-down (SiMPull) detection to test how various lengths and GQ destabilizing base modifications in telomeric DNA constructs alter BG4 binding. We observed high-affinity BG4 binding to telomeric GQ independent of telomere length, although three telomeric repeat constructs that cannot form stable intramolecular GQ showed reduced affinity. A single guanine substitution with 8-aza-7-deaza-G, T, A, or C reduced affinity to varying degrees depending on the location and base type, whereas two G substitutions in the telomeric construct dramatically reduced or abolished binding. Substitution with damaged bases 8-oxoguanine and O6-methylguanine failed to prevent BG4 binding although affinity was reduced depending on lesion location. SiMPull combined with FRET revealed that BG4 binding promotes folding of telomeric GQ harboring a G to T substitution or 8-oxoguanine. Atomic force microscopy revealed that BG4 binds telomeric GQ with a 1:1 stoichiometry. Collectively, our data suggest that BG4 can recognize partially folded telomeric GQ structures and promote telomeric GQ stability.

Isolation and characterization of a novel single-chain variable fragment (scFv) against Lymphocyte function-associated antigen-1 (LFA-1) using phage display method.

Lymphocyte function-associated antigene-1 (LFA-1) is a well-described integrin found on lymphocytes and other leukocytes, which is known to be overexpressed in leukemias and lymphomas. This receptor plays a significant role in immune responses such as T-cell activation, leukocyte cell-cell interactions, and trafficking of leukocyte populations. Subsequently, binders of LFA-1 emerge as potential candidates for cancer and autoimmune therapy. This study used the phage display technique to construct and characterize a high-affinity single-chain fragment variable (scFv) antibody against LFA-1. After expression, purification, dialysis, and concentration of the recombinant LFA-1 protein, four female BALB/c mice were immunized, splenocyte's mRNA was extracted, and cDNA was synthesized. A scFv library was constructed by linking the amplified VH/Vκ fragments through a 72-bp linker using SOEing PCR. Next, the scFv gene fragments were cloned into the pComb-3XSS phagemid vector; thus, the phage library was developed. The selection process involved three rounds of phage-bio-panning, polyclonal, and monoclonal phage ELISA. AF17 was chosen and characterized among the positive clones through SDS-PAGE, Western blotting, indirect ELISA, and in-silico analyses. The results of the study showed the successful construction of a high-affinity scFv library against LFA-1. The accuracy of the AF17 production and its ability to bind to the LFA-1 were confirmed through SDS-PAGE, Western blot, and ELISA. This study highlights the potential application of the high-affinity AF17 against LFA-1 for targeting T lymphocytes for therapeutic purposes.

Chimeric single-chain variable fragment-human immunoglobulin G crystallizable fragment antibody against GD2 for neuroblastoma targeted immunotherapy

Aim: The present study aims to generate chimeric mouse single-chain variable fragment (scFv) and immunoglobulin G1 (IgG1) crystallizable fragment (Fc) antibody against disialoganglioside (GD2) for the treatment of neuroblastoma (NB). The generated scFv-IgG Fc antibody, lacking first constant domain of heavy chain (CH1), is of a smaller size than the natural antibody and has anti-tumor activity. Methods: Vector for scFv-IgG Fc antibody was constructed and scFv-IgG Fc antibody was expressed in human embryonic kidney 293T (HEK293T) cell line. Purification of scFv-IgG Fc antibody from the culture supernatant of transfected HEK293T cells was performed by Protein G affinity chromatography. The structure and binding activity of scFv-IgG Fc antibody were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting (WB), and immunofluorescence techniques. Anti-tumor activities by antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) were determined. Results: Using plasmid fusion-human IgG1-Fc2 tag vector (pFUSE-hIgG1-Fc2), a plasmid vector encoding chimeric mouse scFv and hIgG1 Fc antibody against GD2 was successfully constructed. This vector was transfected into human HEK293T cells to produce scFv-IgG Fc antibody. The transfected HEK293T cells could produce chimeric scFv-IgG Fc antibody against GD2, which lacks the IgG heavy chain CH1 domain but carries CH2 and CH3 domains. The chimeric antibodies could be purified from the culture supernatant of the transfected HEK293T culture in the presence of zeocin drug. The produced GD2 scFv-IgG Fc antibodies, which are smaller in size than the intact antibody, could trigger the killing of GD2 expressed NB cell line SH-SY5Y by ADCC and ADCP mechanisms. Conclusions: The results indicate that chimeric scFv-hIgG Fc antibody, lacking heavy chain CH1 domain, could mediate antibody induced anti-tumor activities. The small size of this type of chimeric antibody may be employed as anti-GD2 antibody for NB therapy.

Canine distemper virus (CDV)-neutralizing activities of an anti-CDV canine-derived single-chain variable antibody fragment 4-15 (scFv 4-15) screened by phage display technology

Canine distemper virus (CDV) is a highly contagious pathogen that causes severe diarrhea, fever and vomiting in domestic dogs, posing a serious threat to the dog breeding industry. Currently, there are no effective therapeutic agents for emergency treatment despite the availability of vaccines against CDV infection. Single-chain fragment variable (scFv) antibody has been demonstrated to effectively inhibit virus infections, suggesting a potential candidate as a therapeutic agent for canine distemper. In this study, a phage-displayed scFv library was constructed from the peripheral blood lymphocytes of dog immunized intramuscularly with live-attenuated CDV vaccine, and was subjected to four rounds of pannings against CDV. Subsequent indirect enzyme-linked immunosorbent assay screening revealed high-affinity scFv antibodies specific to CDV, and indirect immunofluorescence assay screening revealed CDV-neutralizing activity of scFv antibodies. Our results showed that a scFv antibody 4–15 (scFv 4–15) with high-affinity binding to CDV and neutralizing activity against CDV was obtained, which displayed effective therapeutic potential in vivo for dogs challenged with a lethal dose of CDV. Conclusively, the scFv 4–15 with high-affinity binding and neutralizing activity to CDV that was obtained by phage display technology provides a promising candidate for the therapeutic agents against CDV infection.

Abstract B134: Targeting Philadelphia chromosome-like acute lymphoblastic leukemia with a novel CRLF2 antibody fragment-drug conjugate

Abstract Introduction: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk hematological malignancy with poor outcome, and new treatments are urgently required. We address this challenge by targeting cytokine receptor-like factor 2 (CRLF2), which is overexpressed in ~50% of Ph-like ALL cases conferring chemoresistance and survival of Ph-like ALL cells through JAK/STAT signaling. CRLF2 is an ideal target due to its role in leukemogenesis, limiting the likelihood of relapse associated with antigen loss, and its minimal expression in healthy tissue. Here, we developed a novel CRLF2-targeting antibody fragment-drug conjugate (CRLF2-FDC) and evaluated its efficacy in preclinical models. Methods: A single-chain anti-CRLF2 variable monoclonal antibody fragment (scFv-Fc) was engineered by conventional hybridoma technology, PCR and expression in HEK293T cells. The scFv-Fc was conjugated to the anti-microtubule drug DM1 via a non-cleavable linker. The selective association and internalization of the CRLF2-FDC was determined in vitrousing Ph-like ALL cell lines and their CRLF2-knockdown counterparts, and ex vivo using patient-derived xenograft (PDX) cells. The in vivo efficacy of the CRLF2-FDC (equivalent to 10 mg/kg scFv-Fc, 0.4 mg/kg DM1, once weekly x 4 weeks via intravenous injection) was tested against pediatric ALL PDX models in immune-deficient (NSG) mice, in comparison to scFv-Fc or DM1 alone. The efficacy of the CRLF2-FDC was further evaluated following two weeks of standard-of-care induction chemotherapy [vincristine (0.15 mg/kg, once weekly), dexamethasone (5 mg/kg) and L-asparaginase (1,000 U/kg) both 5 days on 2 days off (VXL)]. Treatment efficiency was calculated by subtracting the median event-free survival (EFS) of the control and treated groups (T-C) and stringent objective response criteria (progressive disease, PD; partial response, PR; complete response, CR; maintained CR, MCR) and leukemia infiltration in major organs at day 28 post treatment initiation. Results: CRLF2-dependent association (P≤0.01) and internalization of the CRLF2-FDC was confirmed both in vitro and in vivo in Ph-like ALL cells. While the scFv-Fc alone or free DM1 elicited PDs in a Ph-like ALL PDX in vivo (T-C 6 and 1.6 days, respectively, P=0.0013 for both), the CRLF2-FDC achieved a PR (T-C 32 days, P=0.0088). Besides targeted drug delivery, the CRLF2-FDC also demonstrated a significant antagonistic impact on STAT5 signaling in vivo 8 h after administration, suggesting a two-pronged therapeutic approach. Notably, the CRLF2-FDC achieved an MCR when administered after VXL (T-C 53.9 days, P <0.0001) compared to a CR (T-C 25.2 days, P <0.0001) by VXL alone. Furthermore, the CRLF2-FDC significantly decreased leukemic burden in major organs at 28 days post treatment initiation. Conclusions: This study describes the selective potency and promising preclinical activity of a novel CRLF2-FDC for the treatment of Ph-like ALL when administered either as a single agent or following standard-of-care induction therapy. Citation Format: Richard B Lock, Sara M.A. Mohamed, Peter Schofield, Karl-Heinz Friedrich, Daniel Christ, Maria Kavallaris, Narges Bayat. Targeting Philadelphia chromosome-like acute lymphoblastic leukemia with a novel CRLF2 antibody fragment-drug conjugate [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B134.

Human Single Domain Antibody-Based CAR-T Cells Targeting BAFF-R Demonstrate Promising Preclinical Activity in B Cell Malignancies

Background: B-cell activating factor-receptor (BAFF-R) is a potential B-cell specific target, highly expressed in B cell malignancies and regulates B cell proliferation and survival. Single-chain antibody-based CAR-T cells targeting BAFF-R (PMB101) has demonstrated antitumor effects against human B cell malignancies and can overcome CD19 antigen loss (Qin et al. Sci Transl Med. 2019;11(511)). However, CARs built on antigen-specific single chain antibody variable fragment (scFv) may have some immunological risks. Recently, single variable domain of heavy chain antibodies are becoming an alternative to scFv to construct CARs. To improve the function of BAFF-R CAR-T cells, we designed CARs with only a fully human heavy-chain variable domain. Methods: A fully human antibody phage display library (IMARS, IASO Biotherapeutics) was used to select anti-BAFF-R clones using optimal protein/cell alternate panning. The enzyme-linked immunosorbent assay (ELISA) and flow cytometry were applied to evaluate the specificity of clones. The screened clones were grafted into a second-generation CAR with CD8α hinge, 4-1BB costimulatory and CD3ζ intracellular signaling domains to generate CAR-T cells. PMB101 and CAR-T cells constructed with VAY736 were used as positive controls. Non-transduced T cells from the same donor were used as a negative control. Results: Four candidate clones including clone 1, 5, 77 and 80 were finally screened by in vitro functional evaluation, among which clone 5, 77 and 80 were variable domain heavy chain antibodies. The expression levels of the degranulation marker CD107a on CAR-T cells constructed with the four candidate clones were significantly upregulated after coculture with BAFF-R positive tumor cells, while BAFF-R negative tumor cells could not effectively activate CAR-T cells (data not shown). The four clones exhibited similar cytotoxicity in CAR-T cells compared with the PMB101 and VAY736 CAR-T cells using the luciferase-based cytotoxicity assay (data not shown). Xenografts were established in NPG mice following intravenous injection of 1 x 10 6 jeko-1 cells expressing the firefly luciferase gene on day - 5, and a single dose of 4 x 10 6 CAR T cells were infused IV on day 0. Compared to the other groups, no progressive tumor growth was discovered in mice treated with clone 5 CAR-T cells until day 49. Clone 5 CAR-T cells showed excellent in vivo antitumor activity to PMB101 and conferred long-term survival (data not shown). Conclusion: We developed a novel BAFF-R CAR-T cell product structured on single-domain antibody. This product demonstrated promising preclinical activity, and it may provide an alternative choice for patients with B cell malignancies.

Novel sandwich immunoassay detects a shrimp AHPND-causing binary PirABVp toxin produced by Vibrio parahaemolyticus.

The binary PirA/PirB toxin expressed by Vibrio parahaemolyticus (PirABVp) is a virulent complex that causes acute hepatopancreatic necrosis disease (AHPND) in shrimps, affecting the global shrimp farming industry. AHPND is currently diagnosed by detecting pirA and pirB genes by PCR; however, several V. parahaemolyticus strains do not produce the two toxins as proteins. Thus, an immunoassay using antibodies may be the most effective tool for detecting toxin molecules. In this study, we report a sandwich ELISA-based immunoassay for the detection of PirABVp. We utilized a single-chain variable fragment (scFv) antibody library to select scFvs against the PirA or PirB subunits. Phage display panning rounds were conducted to screen and identify scFv antibodies directed against each recombinant toxin subunit. Selected scFvs were converted into IgGs to develop a sandwich immunoassay to detect recombinant and bacterial PirABVp. Antibodies produced as IgG forms showed sub-nanomolar to nanomolar affinities (KD), and a pair of anti-PirA antibody as a capture and anti-PirB antibody as a detector showed a limit of detection of 201.7 ng/mL for recombinant PirABVp. The developed immunoassay detected PirABVp in the protein lysates of AHPND-causing V. parahaemolyticus (VpAHPND) and showed a significant detectability in moribund or dead shrimp infected with a VpAHPND virulent strain compared to that in non-infected shrimp. These results indicate that the developed immunoassay is a reliable method for diagnosing AHPND by detecting PirABVp at the protein level and could be further utilized to accurately determine the virulence of extant or newly identified VpAHPND in the global shrimp culture industry.

Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach.

Single-chain fragment variable (scFv) antibodies were previously constructed of variable light and heavy chains joined by a (Gly4-Ser) 3 linker. The linker was created using molecular modeling software as a loop structure. Here, we introduce a protocol forin silico analysis of a complete scFv antibody that interacts with the epidermal growth factor receptor (EGFR). The homology modeling, with Pyrx of protein-protein docking and molecular dynamic simulation of the interacting scFv antibody and EGFR First, the authors used a protein structure modeling program and Python for homology modeling, and the antibody scFv structure was modeled for homology. The investigators downloaded Pyrx software as a platform in the docking study. The Molecular dynamic simulation was run using modeling software. Results show that when the MD simulation was subjected to energy minimization, the protein model had the lowest binding energy (-5.4 kcal/M). In addition, the MD simulation in this study showed that the docked EGFR-scFv antibody was stable for 20-75 ns when the movement of the structure increased sharply to 7.2 Å. In conclusion, in silicoanalysiswas performed, and the molecular docking and molecular dynamics simulations of the scFv antibody proved the effectiveness of the designed immune-therapeutic drug scFv as a specific drug therapy for EGFR.

Development of an Anti-HER2 Single-Chain Variable Antibody Fragment Construct for High-Yield Soluble Expression in Escherichia coli and One-Step Chromatographic Purification.

Although single-chain variable fragment (scFv) is recognized as a highly versatile scaffold of recombinant antibody fragment molecules, its overexpression in Escherichia coli often leads to the formation of inclusion bodies. To address this issue, we devised and tested four different constructs, named v21, v22, v23 and v24, for producing anti-human epidermal growth factor receptor 2 (HER2) scFv. Among them, the v24 construct obtained from N-terminal fusion of maltose-binding protein (MBP) and subsequent tobacco etch virus protease (TEV) was identified as the most efficient construct for the production of anti-HER2 scFv. Aided by an MBP tag, high-yield soluble expression was ensured and soluble scFv was liberated in cells via autonomous proteolytic cleavage by endogenously expressed TEV. The isolated scFv containing a C-terminal hexahistidine tag was purified through a one-step purification via nickel-affinity chromatography. The purified scFv exhibited a strong (nanomolar Kd) affinity to HER2 both in vitro and in cells. Structural and functional stabilities of the scFv during storage for more than one month were also assured. Given the great utility of anti-HER2 scFv as a basic platform for developing therapeutic and diagnostic agents for cancers, the v24 construct and methods presented in this study are expected to provide a better manufacturing system for producing anti-HER2 scFv with various industrial applications.