VL Domains Research Articles

A Simple Analysis of the Second (Extra) Disulfide Bridge of VHHs.

Camelids produce a special type of antibody, known as VHHs, that has lost the VL domain, providing a more optimised VH domain. This particular highly stable antibody domain has interesting properties for biotechnological development. Ordinarily, those molecules possess only one disulphide bridge, but surprisingly, at least a quarter of these VHHs have a second one. Curiously, this does not seem to be essential for the stability and the function of this domain. In an attempt to characterise precisely the role and impact of this disulphide bridge at the molecular level, several in silico mutants of a VHH were created to disrupt this second disulphide bridge and those systems were submitted to molecular dynamics simulation. The loss of the second disulphide bridge leads to an increase in the flexibility of CDR1 and CDR3 and an unexpected rigidification of CDR2. Local conformational analysis shows local differences in the observed protein conformations. However, in fact, there is no exploration of new conformations but a change in the equilibrium between the different observed conformations. This explains why the interaction of VHHs is not really affected by the presence or absence of this second bridge, but their rigidity is slightly reduced. Therefore, the additional disulphide bridge does not seem to be an essential part of VHHs function.

Molecular basis for antibody recognition of multiple drug–peptide/MHC complexes

The HapImmuneTM platform exploits covalent inhibitors as haptens for creating major histocompatibility complex (MHC)-presented tumor-specific neoantigens by design, combining targeted therapies with immunotherapy for the treatment of drug-resistant cancers. A HapImmune antibody, R023, recognizes multiple sotorasib-conjugated KRAS(G12C) peptides presented by different human leukocyte antigens (HLAs). This high specificity to sotorasib, coupled with broad HLA-binding capability, enables such antibodies, when reformatted as T cell engagers, to potently and selectively kill sotorasib-resistant KRAS(G12C) cancer cells expressing different HLAs upon sotorasib treatment. The loosening of HLA restriction could increase the patient population that can benefit from this therapeutic approach. To understand the molecular basis for its unconventional binding capability, we used single-particle cryogenic electron microscopy to determine the structures of R023 bound to multiple sotorasib-peptide conjugates presented by different HLAs. R023 forms a pocket for sotorasib between the VH and VL domains, binds HLAs in an unconventional, angled way, with VL making most contacts with them, and makes few contacts with the peptide moieties. This binding mode enables the antibody to accommodate different hapten-peptide conjugates and to adjust its conformation to different HLAs presenting hapten-peptides. Deep mutational scanning validated the structures and revealed distinct levels of mutation tolerance by sotorasib- and HLA-binding residues. Together, our structural information and sequence landscape analysis reveal key features for achieving MHC-restricted recognition of multiple hapten-peptide antigens, which will inform the development of next-generation therapeutic antibodies.

A Conditionally Activated Cytosol-Penetrating Antibody for TME-Dependent Intracellular Cargo Delivery.

Currently, therapeutic and diagnostic applications of antibodies are primarily limited to cell surface-exposed and extracellular proteins. However, research has been conducted on cell-penetrating peptides (CPP), as well as cytosol-penetrating antibodies, to overcome these limitations. In this context, a heparin sulfate proteoglycan (HSPG)-binding antibody was serendipitously discovered, which eventually localizes to the cytosol of target cells. Functional characterization revealed that the tested antibody has beneficial cytosol-penetrating capabilities and can deliver cargo proteins (up to 70 kDa) to the cytosol. To achieve tumor-specific cell targeting and cargo delivery through conditional activation of the cell-penetrating antibody in the tumor microenvironment, a single-chain Fc fragment (scFv) and a VL domain were isolated as masking units. Several in vitro assays demonstrated that fusing the masking protein with a cleavable linker to the cell penetration antibody results in the inactivation of antibody cell binding and internalization. Removal of the mask via MMP-9 protease cleavage, a protease that is frequently overexpressed in the tumor microenvironment (TME), led to complete regeneration of binding and cytosol-penetrating capabilities. Masked and conditionally activated cytosol-penetrating antibodies have the potential to serve as a modular platform for delivering protein cargoes addressing intracellular targets in tumor cells.

The dispensability of VH-VL pairing and the indispensability of VL domain integrity in the IgG1 secretion process.

Introduction: The CH1 domain of IgG antibodies controls assembly and secretion, mediated by the molecular chaperone BiP via the endoplasmic reticulum protein quality control (ERQC) mechanism. However, it is not clear whether the variable domains are necessary for this process. Methods: Here, we generated IgG1 antibodies in which the V domain (VH and/or VL) was either removed or replaced, and then assessed expression, assembly, and secretion in HEK293 cells. Results: All Ig variants formed a covalent linkage between the Cγ1 and Cκ, were successfully secreted in an assembled form. Replacement of the cognate Vκ with a non-secretory pseudo Vκ (ψVκ) hindered secretion of individual or assembled secretion of neither heavy chains (HCs) nor light chains (LCs). The ψLC (ψVκ-Cκ) exhibited a less folded structure compared to the wild type (wt) LC, as evidenced by enhanced stable binding to the molecular chaperone BiP and susceptibility to proteolytic degradation. Molecular dynamics simulation demonstrated dramatic alterations in overall structure of ψFab (Fd-ψLC) from wt Fab. Discussion: These findings suggest that V domains do not initiate HC:LC assembly and secretion; instead, the critical factor governing IgG assembly and secretion is the CH-CL pairing. Additionally, the structural integrity of the VL domain is crucial for IgG secretion. These data offer valuable insight into the design of bioactive molecules based on an IgG backbone.

Humanized single-domain antibody targeting HER2 enhances function of chimeric antigen receptor T cells.

Chimeric antigen receptors (CARs) can redirect T cells against antigen-expressing tumors, and each component plays an important role in the function and anti-tumor efficacy. It has been reported that using human sequences or a low affinity of CAR single-chain variable fragments (scFvs) in the CAR binding domains is a potential way to enhance the function of CAR-T cells. However, it remains largely unknown how a lower affinity of CARs using humanized scFvs affects the function of CAR-T cells until recently. We used different humanized anti-HER2 antibodies as the extracellular domain of CARs and further constructed a series of the CAR-T cells with different affinity. We have observed that moderately reducing the affinity of CARs (light chain variable domain (VL)-based CAR-T) could maintain the anti-tumor efficacy, and improved the safety of CAR therapy both in vitro and in vivo compared with high-affinity CAR-T cells. Moreover, T cells expressing the VL domain only antibody exhibited long-lasting tumor elimination capability after multiple challenges in vitro, longer persistence and lower cytokine levels in vivo. Our findings provide an alternative option for CAR-T optimization with the potential to widen the use of CAR T cells.

Multispecific Hybrid T Cell Receptors for Sensitive Targeting of Cancer

CAR T cells represent a promising therapy for a growing list of tumors. Despite high initial response rates, 50-60% of patients relapse within 1 year after CAR T cell infusion. Heterogeneity of target antigen expression can lead to outgrowth of tumor cells with low or negative antigen levels. Conventional CARs require high antigen levels for efficient T cell activation and tumor elimination. By contrast, T cell receptors (TCRs) are 10-100 times more sensitive than CARs. To achieve greater antigen sensitivity, we and others designed synthetic hybrid T cell receptors by fusing antibody-based recognition domains to TCRa and TCRb constant chains. Here we extend the initial monospecific design to incorporate two antigen recognition domains. We evaluated 2 monospecific hybrid receptors, one with VH and VL domains fused to TCRa and TCRb constant chains respectively (Split TCR/CAR) and a second where the entire scFv (VL-linker-VH) is fused only to the TCRa chain (Full TCR/CAR) leaving TCRb available for a second scFv. We used based editing to knock out endogenous TCRa and b chains to avoid mispairing. Using CD19 as a model target we found that both TCR/CAR designs lacked tonic signaling, induced TCR-like changes in Ca+ flux, more efficiently phosphorylated LAT, and exhibited superior recognition of Nalm-6 clones expressing a range of CD19 levels compared to conventional CD19 28z or 4-1BBz CARs. Interestingly, the Full TCR/CAR outperformed the Split format in these assays. Using soluble bilayer and TIRF microscopy, we demonstrated that unlike CARs, TCR/CAR T cells recapitulate a classical TCR-like immunological synapse. I n vivo studies using NSG mice engrafted with CD19+ Raji cells demonstrated potent anti-tumor efficacy, and the Full format outperformed the Split TCR/CAR. Capitalizing on the superiority of the Full TCR/CAR format, we next designed bispecific hybrid receptors composed of two scFvs of different specificities fused to the TCRa and TCRb chains. Multiple Myeloma is particularly suitable for bispecific targeting as relapse following CAR T cell therapy is common and multiple target antigens have been described. We designed bispecific Full TCR/CARs specific for pairs of myeloma antigens (BCMA/SLAMF7 and BCMA/GPRC5D) and expressed them in primary T cells. We used base editing to knock out SLAMF-7 which can be expressed in T cells and could cause fratricide. Bispecific TCR/CAR receptors conferred sensitive T cell recognition of target cells expressing BCMA and SLAMF-7 as measured by cytokine production, proliferation, and cytotoxicity. T cells equipped with these novel bispecific receptors maintained function when exposed to L363 cells BCMA ko or SLAMF-7 ko demonstrating that both antigen binding domains can trigger T cell functions against target cells. In vivo studies using NSG mice implanted with a mixture composed of 33% of Nalm-6 BCMA+, Nalm-6 SLAMF-7+ and Nalm-6 BCMA+SLAMF-7+ cells to mimic target antigen expression heterogeneity and antigen loss showed superior anti-tumor efficacy for bispecific TCR/CAR cells compared to conventional BCMA and SLAMF-7 BBz CARs. Our hybrid bispecific TCR/CAR also demonstrated superior anti-tumor efficacy compared to a previously described, and systematically optimized bispecific CAR format (Zah et al., 2020). Bispecific hybrid T cell receptors embedded with natural TCR signaling machinery represent a promising therapeutic option to address antigen downmodulation/loss and antigen heterogeneity for treatment of cancer.

Nanobodies counteract the toxicity of an amyloidogenic light chain by stabilizing a partially open dimeric conformation

Light chain amyloidosis (AL) is a systemic disease where fibrillar deposition of misfolded immunoglobulin light chains (LCs) severely affects organ function and results in poor prognosis for patients, especially when heart involvement is severe. Particularly relevant in this context is the cardiotoxicity exerted by still uncharacterized soluble LC species. Here, with the final goal of identifying alternative therapeutic strategies to tackle AL amyloidosis, we produced five llama-derived nanobodies (Nbs) specific against H3, a well-characterized amyloidogenic and cardiotoxic LC from an AL patient with severe cardiac involvement. We found that Nbs are specific and potent agents capable of abolishing H3 soluble toxicity in C. elegans in vivo model. Structural characterization of H3-Nb complexes revealed that the protective effect of Nbs is related to their ability to bind to the H3 VL domain and stabilise an unexpected partially open LC dimer in which the two VL domains no longer interact with each other. Thus, while identifying potent inhibitors of LC soluble toxicity, we also describe the first non-native structure of an amyloidogenic LC that may represent a crucial step in toxicity and aggregation mechanisms.

Sequence tolerance of immunoglobulin variable domain framework regions to noncanonical intradomain disulfide linkages

Most immunoglobulin (Ig) domains bear only a single highly conserved canonical intradomain, inter-β-sheet disulfide linkage formed between Cys23-Cys104, and incorporation of rare noncanonical disulfide linkages at other locations can enhance Ig domain stability. Here, we exhaustively surveyed the sequence tolerance of Ig variable (V) domain framework regions (FRs) to noncanonical disulfide linkages. Starting from a destabilized VH domain lacking a Cys23-Cys104 disulfide linkage, we generated and screened phage-displayed libraries of engineered VHs, bearing all possible pairwise combinations of Cys residues in neighboring β-strands of the Ig fold FRs. This approach identified seven novel Cys pairs in VH FRs (Cys4-Cys25, Cys4-Cys118, Cys5-Cys120, Cys6-Cys119, Cys22-Cys88, Cys24-Cys86, and Cys45-Cys100; the international ImMunoGeneTics information system numbering), whose presence rescued domain folding and stability. Introduction of a subset of these noncanonical disulfide linkages (three intra-β-sheet: Cys4-Cys25, Cys22-Cys88, and Cys24-Cys86, and one inter-β-sheet: Cys6-Cys119) into a diverse panel of VH, VL, and VHH domains enhanced their thermostability and protease resistance without significantly impacting expression, solubility, or binding to cognate antigens. None of the noncanonical disulfide linkages identified were present in the natural human VH repertoire. These data reveal an unexpected permissiveness of Ig V domains to noncanonical disulfide linkages at diverse locations in FRs, absent in the human repertoire, whose presence is compatible with antigen recognition and improves domain stability. Our work represents the most complete assessment to date of the role of engineered noncanonical disulfide bonding within FRs in Ig V domain structure and function.

In silico analysis of the different variable domain oriented single-chain variable fragment antibody-antigen complexes

Single-chain variable fragment (scFv) antibodies hold great potential as diagnostic tools and therapeutic agents, especially for tumor cells. Since these applications require their production with improved properties, the design strategy of scFvs is crucial for their active, soluble, and high yield expression with high affinity towards their antigens. The order of VL and VH domains is one of the important parameters that affect the expression and binding affinity properties of scFvs. In addition, the optimum order of VL and VH domains could change for each scFv. In the present study, we used computer simulation tools to evaluate the effect of variable domain orientation on structure, stability, interacting residues of scFvs, and binding free energies of scFv-antigen complexes. We selected anti-HER2 scFv, which is specific for human epidermal growth receptor 2 (HER2) overexpressed in breast cancer, and anti-IL-1β scFv against IL-1β which is an important inflammatory biomarker, as model scFvs. Molecular dynamics simulations of the scFv-antigen complexes for 100 ns resulted in stability and compactness for both scFv constructs. Interaction and binding free energies calculated by the Molecular Mechanics–Poisson–Boltzmann Surface Area (MM-PBSA) approach suggested that the relative binding energies of anti-HER2 scFv-VLVH and anti-HER2 scFv-VHVL constructs had similar binding affinity towards HER2, while a relatively more negative binding free energy obtained between anti-IL-1β scFv-VHVL and IL-1β pointed to a higher binding affinity. The in silico approach and the results obtained here could be applied as a guide for future experimental interaction studies for highly specific scFvs used as biotechnological tools. Communicated by Ramaswamy H. Sarma

Fully synthetic platform to rapidly generate tetravalent bispecific nanobody–based immunoglobulins

Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human VH3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor-binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the VH and VL domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules.

Genetic code expansion in E. coli enables production of a functional ‘ready-to-click’ T cell receptor-specific scFv

Antibody-based cancer therapies have been evolving at a rapid pace in the pharmaceutical market. Bispecific antibody-drug conjugates that engage immune cells to target and kill cancer cells with precision have inspired the development of immunotherapy. Miniaturized antibody fragments such as diabodies, nanobodies, or single-chain variable fragments (scFvs) hold great promise as antibody-drug conjugates as they specifically target tumor tissue and can penetrate it. Here, we optimized the soluble periplasmic expression of the scFv OKT3 comprising the variable VH and VL domains of the mouse anti-human CD3 antibody muromonab-CD3 (trade name Orthoclone OKT3) in E. coli. By an expansion of the genetic code, we site-specifically incorporated the reactive non-canonical amino acid Nε-((2-azidoethoxy)carbonyl)-L-lysine (AzK) into scFv OKT3 using an orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair. To confirm the AzK incorporation and to demonstrate the accessibility of the reactive azide group, we conjugated a fluorophore to scFv OKT3 AzK variants by copper-free strain-promoted alkyne-azide cycloaddition (‘click chemistry’). The scFv OKT3 wild type and the AzK variants bound T cells at nanomolar concentrations. In this study, a ‘ready-to-click’ scFv OKT3 was successfully developed for future applications, e.g. as controlled anti-T cell antibody-drug conjugate or bispecific T cell engager and for imaging immune T cell migration in cancers.

A constant domain mutation in a patient-derived antibody light chain reveals principles of AL amyloidosis

Light chain (AL) amyloidosis is a debilitating disease in which mutant antibody light chains (LC), secreted by aberrant plasma cell clones, misfold and form insoluble fibrils, which can be deposited in various organs. In the majority of cases, the fibrillar deposits consist of LC variable domains (VL) containing destabilizing mutations compared to their germline counterparts. This is also true for the patient LC FOR005. However, this pathogenic LC sequence contains an additional mutation in the constant domain (CL). The mechanistic impact of CL mutations is not yet understood in the context of AL amyloidosis. Our analysis reveals that the FOR005 CL mutation influences the amyloid pathway in specific ways: (1) folding and stability of the patient CL domain are strongly impaired; (2) the mutation disrupts the LC dimer interface and weakens dimerization; (3) the CL mutation promotes proteolytic cleavage of the LC monomers resulting in an isolated, amyloidogenic VL domain while dimeric LCs are not cleaved. The enhanced proteolysis rates and the inability of full-length LCs to form amyloid fibrils even in the presence of a destabilized CL domain support a model for AL amyloidosis in which the CL domain plays a protective role and in which proteolytic cleavage precedes amyloid formation.

AbYpap: Improvements to the Prediction of Antibody V H/V L Packing Using Gradient Boosted Regression.

The Fv region of the antibody (comprising VH and VL domains) is the area responsible for target binding and thus the antibody's specificity. The orientation, or packing, of these two domains relative to each other influences the topography of the Fv region, and therefore can influence the antibody's binding affinity. We present abYpap, an improved method for predicting the packing angle between the VH and VL domains. With the large data set now available, we were able to expand greatly the number of features that could be used compared with our previous work. The machine-learning model was tuned for improved performance using 37 selected residues (previously 13) and also by including the lengths of the most variable 'complementarity determining regions' (CDR-L1, CDR-L2, and CDR-H3). Our method shows large improvements from the previous version, and also against other modelling approaches, when predicting the packing angle. Supplementary information: Supplementary data are available at Protein Engineering Design and Selection online.

Construction of scFv Antibodies against the Outer Loops of the Microsporidium Nosema bombycis ATP/ADP-Transporters and Selection of the Fragment Efficiently Inhibiting Parasite Growth

Traditional sanitation practices remain the main strategy for controlling Bombyx mori infections caused by microsporidia Nosema bombycis. This actualizes the development of new approaches to increase the silkworm resistance to this parasite. Here, we constructed a mouse scFv library against the outer loops of N. bombycis ATP/ADP carriers and selected nine scFv fragments to the transporter, highly expressed in the early stages of the parasite intracellular growth. Expression of selected scFv genes in Sf9 cells, their infection with different ratios of microsporidia spores per insect cell, qPCR analysis of N. bombycis PTP2 and Spodoptera frugiperda COXI transcripts in 100 infected cultures made it possible to select the scFv fragment most effectively inhibiting the parasite growth. Western blot analysis of 42 infected cultures with Abs against the parasite β-tubulin confirmed its inhibitory efficiency. Since the VL part of this scFv fragment was identified as a human IgG domain retained from the pSEX81 phagemid during library construction, its VH sequence should be a key antigen-recognizing determinant. Along with the further selection of new recombinant Abs, this suggests the searching for its natural mouse VL domain or "camelization" of the VH fragment by introducing cysteine and hydrophilic residues, as well as the randomization of its CDRs.

Clip Formation in the Complementarity Determining Region of Bevacizumab Lowers Monomer Stability and Affinity for Both FcRn and FcγR: A Comprehensive Characterization of the Clipped Variant Including its Higher Order Structure

The presence of monoclonal antibody (mAb) fragments in pharmaceutical mAb products is a critical quality attribute and should be controlled for safety. Several mAb fragments derived from clip formation in the complementarity determining regions (CDRs), as well as from cleavage in the hinge region, have been reported. However, the properties of CDR-clipped variants are not fully understood because of difficulties in separating them from intact mAbs under non-denaturing conditions due to similarities in size. We have established a method for separating CDR-clipped variants under non-denaturing conditions using an appropriate size exclusion chromatography column.1 In this report, we provide a comprehensive characterization of a CDR-clipped variant from bevacizumab. The variant exhibited a lower pI, a higher tendency to form dimers, and a lower affinity for both neonatal Fc receptor (FcRn) and Fcγ receptor (FcγR). The effects of clip formation in CDR H3 on the higher order structure were analyzed by hydrogen/deuterium exchange mass spectrometry, and the observed changes in the structures of the VH, CH2, and VL domains were in agreement with the lowered affinity for antigen, FcRn, and FcγR. These findings suggest that clip formation in the CDR may affect the efficacy, safety, and pharmacokinetics of pharmaceutical mAbs.

New Anti-Flavivirus Fusion Loop Human Antibodies with Zika Virus-Neutralizing Potential.

Zika virus infections exhibit recurrent outbreaks and can be responsible for disease complications such as congenital Zika virus syndrome. Effective therapeutic interventions are still a challenge. Antibodies can provide significant protection, although the antibody response may fail due to antibody-dependent enhancement reactions. The choice of the target antigen is a crucial part of the process to generate effective neutralizing antibodies. Human anti-Zika virus antibodies were selected by phage display technology. The antibodies were selected against a mimetic peptide based on the fusion loop region in the protein E of Zika virus, which is highly conserved among different flaviviruses. Four rounds of selection were performed using the synthetic peptide in two strategies: the first was using the acidic elution of bound phages, and the second was by applying a competing procedure. After panning, the selected VH and VL domains were determined by combining NGS and bioinformatic approaches. Three different human monoclonal antibodies were expressed as scFvs and further characterized. All showed a binding capacity to Zika (ZIKV) and showed cross-recognition with yellow fever (YFV) and dengue (DENV) viruses. Two of these antibodies, AZ1p and AZ6m, could neutralize the ZIKV infection in vitro. Due to the conservation of the fusion loop region, these new antibodies can potentially be used in therapeutic intervention against Zika virus and other flavivirus illnesses.

More than 370-Fold Increase in Antibody Affinity to Estradiol-17β by Exploring Substitutions in the VH-CDR3.

Antibodies that specifically target biomarkers are essential in clinical diagnosis. Genetic engineering has assisted in designing novel antibodies that offer greater antigen-binding affinities, thus providing more sensitive immunoassays. We have succeeded in generating a single-chain Fv fragment (scFv) targeted estradiol-17β (E2) with more than 370-fold improved affinity, based on a strategy focusing the complementarity-determining region 3 in the VH domain (VH-CDR3). Systematic exploration of amino acid substitutions therein, using a clonal array profiling, revealed a cluster of four substitutions, containing H99P and a serial substitution E100eN-I100fA-L100gQ that lead to a 90-fold increase in E2-binding affinity. This substitution quartet in the VH-CDR3, combined with the substitution cluster I29V/L36M/S77G in the VL domain, resulted in a scFv fragment with a further increase in the affinity (Ka, 3.2 × 1010 M-1). This enabled a highly sensitive enzyme-linked immunosorbent assay capable of detecting up to 0.78 pg/assay. The current study has, thus, focused on the significance of reevaluating the potential of mutagenesis targeting the VH-CDR3, and encouraging the production and use of engineered antibodies that enable enhanced sensitivities as next-generation diagnostic tools.

Abstract LB213: Potent antitumor activity of a FGFR4 CAR-T in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma originating from skeletal muscle in children and adolescent young adults. Despite multi-modal aggressive therapies, relapsed, refractory or metastatic rhabdomyosarcoma remains a lethal disease with no significant improvement in outcome over decades of clinical trials. Therefore novel therapies are needed. FGFR4 is a developmentally regulated cell surface receptor tyrosine kinase that is overexpressed in RMS when compared with normal tissues, and mutationally activated in about 7.5% of RMS. Recently we showed that PAX3-FOXO1 establishes a super-enhancer in the FGFR4 genomic locus driving its high expression in fusion positive RMS. CAR T-cell therapy is effective in treating refractory and relapsed B-cell leukemia and lymphoma, with three CARs targeting CD19 approved by the FDA. Multiple CART trials are currently underway for solid tumors. Since FGFR4 is a cell surface protein, we hypothesized that FGFR4 will provide a rational target for immunotherapy in RMS. We confirmed by immunohistochemistry staining, western analysis, and Meso Scale Discovery that FGFR4 protein is highly differentially expressed in RMS samples. We developed a murine anti-FGFR4 antibody, 3A11, by immunizing mouse with FGFR4-IG fusion protein. 3A11 showed high affinity and specificity of binding to FGFR4. We then developed a second-generation CAR using the VL and VH domain of 3A11 antibody and found that the scFvFc retained its specificity and high affinity at nanomolar range. Human T cells transduced with 3A11 CAR construct were found to be highly potent at inducing IFN-γ, TNF-α, IL-2 and cytotoxicity when the FGFR4-CART was co-cultured with RMS cells, but not with RMS cells with FGFR4 knocked out or FGFR4 negative cells. 3A11 CART incubated with human primary cells obtained from liver, kidney, heart, and pancreas, did not elicit a cytokine response, indicating a low potential for “on-target off-tumor” toxicity. In vivo testing also found that 3A11 CART eliminated RMS cells in both murine xenograft metastatic and localized subcutaneous models. Therefore we have developed a CART targeting FGFR4 that shows high potency for treating RMS. A phase 1 FGFR4-CART clinical trial is planned for children and adolescent young adults with relapsed/refractory rhabdomyosarcoma. Citation Format: Adam Tai Chi Cheuk, Meijie Tian, Nityashree Shivaprasad, Steven Highfill, David Milewski, G Tom Brown, Peter Azorsa, Dina Schneider, Berkley Gryder, Jun S Wei, Young Kwok Song, Hsien-Chao Chou, Jerry Wu, Joon-Yong Chung, Brian Belyea, Corinne Linardic, Stephen M Hewitt, Boro Dropulic, Rimas Orentas, Javed Khan. Potent antitumor activity of a FGFR4 CAR-T in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB213.

Fibrillary Glomerulonephritis and Monoclonal Gammopathy: Potential Diagnostic Challenges.

Fibrillary glomerulonephritis (FGN) is a rare glomerular disease featured by the randomly arranged 12- to 24-nm fibrils under electron microscopy (EM). Up to 10% of FGN patients have monoclonal gammopathy. However, distinguishing between FGN as monoclonal gammopathy of renal significance (MGRS) and FGN from other causes with incidental monoclonal gammopathy of undetermined significance (MGUS) can be challenging, as the current way of demonstrating monoclonality is flawed due to (1) the suboptimal sensitivity of kappa staining by immunofluorescence in frozen tissue (IF-F) as compared to pronase-digested paraffin sections (IF-P), causing incorrect labeling of light chain restriction; (2) the unavailability of immunoglobulin G (IgG) subtyping in some centers; and (3) the unavailability of tests demonstrating the monoclonality of highly variable VH or VL domains in immunoglobulin structures in clinical use. The discovery of DnaJ homolog subfamily B member 9 (DNAJB9) allows diagnosis for FGN with less reliance on EM, and the summary of recent studies revealed that genuine MGRS is extremely rare among FGN. Further research integrating IF-P, IgG subtyping, VH or VL domain monoclonality confirmation, and DNAJB9 as diagnostic modalities, with corresponding clinical data including treatment response and prognosis, is required for a better understanding of this subject.

Generation and Characterization of an Anti-diclazuril Monoclonal Antibody and Development of a Diagnostic Enzyme-Linked Immunosorbent Assay for Poultry.

An anti-diclazuril monoclonal antibody (mAb) was developed for use in enzyme-linked immunosorbent assay (ELISA)-based detection of diclazuril with high sensitivity and specificity, which can be used to measure anti-coccidial drug residues. The anti-diclazuril mAb had a half-maximal inhibitory concentration of 0.449–0.517 ng/mL. The mAb cross-reactivity with toltrazuril, toltrazuril 18 sulfone, clozaril, monesin, madurmycin, and salinomycin was very minimal (< 0.1%). The detection limit of the ELISA using this mAb was 0.10 ng/mL and the sensitivity was 0.05 ng/mL. A standard curve generated in the range of 0.05–16.2 ng/mL had a linear correlation coefficient value of ≥ 0.99. The average recoveries of diclazuril from chicken and duck samples ranged from 85.0 to 102.5%.Intra- and inter-assay coefficients of variation ranged from 5.9 to 8.5% and 9.2 to 12.6%, respectively. Using the International Immunogenetics Information System®, the VH domain of the mAb was found to be encoded by an IGHV3 family gene and had the following complementarity determining region (CDR) sequences: GFTFSRY (CDR1), SRGGS (CDR2), and GDDNYAFAY (CDR3). The VL domain was encoded by an IGKV1 family gene and had the following CDR sequences: KSSQSLLNSRTRKNYLA (CDR1), WASTRES (CDR2), and KQSYNLHT (CDR3). This study provides a method to generate anti-diclazuril mAbs and determine their variable region sequences. The diagnostic ELISA developed using this mAb may drive additional studies on the monitoring and detection of food and veterinary drug residues.