Abstract The major hurdles in the treatment of solid tumors with chimeric antigen receptor (CAR) T cell therapies are antigen heterogeneity and the immunosuppressive tumor microenvironment. To address these challenges, we developed a universal and switchable CAR T cell therapy platform (zCART) that utilizes cotinine, a pharmacologically inert hapten, as a molecular bridge between anti-cotinine CAR T cells and tumor cells via cotinine-conjugated affibodies. First, we generated two affibody switches targeting distinct tumor-associated antigens and one affibody switch targeting an immune-oncology molecule. In vitro studies demonstrated that all three cotinine-conjugated affibodies, when combined with anti-cotinine CAR T cells, induced potent, dose-dependent cytotoxicity against target-expressing tumor cells. The combination of distinct affibody switches resulted in enhanced anti-tumor activity. These results highlight that the cotinine-based, switchable CAR T cell therapy platform enables flexible, multi-target control of CAR T cell activity and effective elimination of heterogeneous solid tumors. By decoupling antigen recognition from CAR T cell activation, the zCART platform offers a safe, versatile, and next-generation approach to overcoming tumor antigen variability and the immunosuppressive microenvironment, positioning it as a promising strategy for solid-tumor immunotherapy. Citation Format: Ki Hyun Kim, Soohwan Kim, Eun-Hoe Lee, Soo-Youn Lim, Tack-Jin Yoo, Sung Min Kim, E-Young Kim, Ji-Hun Park, Hyun-Jong Lee, Seong Yeol Kim, Min Yoon, Youngha Lee, In-Sik Hwang, Yoon Lee, Jong-Hoon Kim, Jong-Seo Lee, Junho Chung. A universal and switchable CAR T cell therapy platform (zCART) targeting tumor antigens and the immunosuppressive microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1533.

Gut microbiota and humoral immunity have been suggested as key players in the pathogenesis of Sjögren disease (SjD), but their mechanisms remain unclear. In this study, we transferred the gut microbiota of SjD-like autoimmune dry eye disease model mice to B6 mice, then characterized the resulting gut microbiome composition, clinical ocular phenotype, and B cell receptor (BCR) repertoire. Notable changes were observed in the gut microbiome of NOD-FMT mice, accompanied by SjD-like clinical features, including elevated corneal fluorescein staining scores, reduced tear production, increased IL-6 mRNA levels, and decreased MUC5AC mRNA levels. Additionally, stereotypic B cell receptor (BCR) clonotypes were shared at significantly higher frequencies in NOD-FMT mice than in controls. The majority of B cell clones encoding these stereotypic clonotypes developed and expanded locally in the lacrimal gland, and some also achieved systemic presence. These results uncover a gut–ocular immune axis in which microbiota transfer induces stereotyped, systemically disseminating BCR clonotypes that contribute to the immunopathogenesis of autoimmune dry eye disease.

This study aimed to optimize Zirconium-89 (89Zr) labeling strategies for in vivo tracking of chimeric antigen receptor (CAR)-T cell migration using positron emission tomography (PET). The performances of the [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine (DFO) and [89Zr]Zr-oxine methods were compared to identify the most suitable chelator and activity concentration for cell labeling. CD19 CAR-Jurkat T cells were labeled with each [89Zr]Zr complex at different activity concentrations following optimization of the chelator amount and incubation time, and were then evaluated for cell viability, proliferation, and function over seven days. The refined synthesis procedures achieved radiochemical purities greater than 95%. Labeling efficiencies were 84.2 ± 5.4% for [89Zr]Zr-DFO and 43.7 ± 1.2% for [89Zr]Zr-oxine. [89Zr]Zr-DFO-labeled cells demonstrated high viability (94.2 ± 1.1%) and maintained 82% of baseline proliferation capacity over seven days, whereas [89Zr]Zr-oxine-labeled cells showed reduced viability (77.3 ± 7.8%) and a markedly impaired proliferation capacity, retaining only 7% of baseline. These results indicate that [89Zr]Zr-DFO provided superior labeling efficiency and cell preservation compared with [89Zr]Zr-oxine, supporting its potential use for PET-based tracking of CAR-T cells in cell therapy applications.

Abstract While AI offers transformative potential for therapeutic antibody design, the lack of ground-truth data fundamentally constrains our ability to model the epistatic topology of fitness landscapes. Here, we establish a high-throughput workflow to characterize tens of thousands of antibody variants per week with gold-standard biophysical precision. By combinatorially assembling functional variants from deep mutational scanning, we charted antibody fitness landscapes comprising over 17,000 data points, which revealed an extremely rugged, non-navigable epistatic topology. Yet, navigating at this unprecedented scale enabled the discovery of rare peak clusters exhibiting simultaneous enhancements in affinity and productivity. Strikingly, ProteinMPNN predicted the CDR-dependent productivity landscape with remarkable accuracy, suggesting that sequence-structure compatibility within CDRs gates cellular productivity. This insight enabled a structure-guided rescue strategy combining AlphaFold3 and ProteinMPNN, which successfully restored the cellular productivity of high-affinity, low-productivity clones via single amino acid substitutions. Two elite variants drawn directly from peak clusters further demonstrated 20- to 100-fold in vivo efficacy gains in a murine psoriasis model. Our findings establish CDR structural fitness as a fundamental determinant of antibody cellular productivity and validate landscape-scale navigation as a powerful framework for therapeutic antibody optimization.

Background/Objectives: Calsequestrin (CSQ) is a calcium-binding protein that is highly soluble and can serve as a solubility-enhancing fusion tag in recombinant protein expression. Its unique property of calcium-induced precipitation followed by EDTA-mediated resolubilization enables efficient purification. However, the broader application of CSQ-tagged proteins in research have been hampered by the lack of reliable anti-CSQ detection reagents. This study aimed to develop single-domain antibodies (sdAbs) against CSQ for use in diverse immunoassays and cell-based analyses. Methods: Single-domain antibodies were selected from phage-displayed chicken VH libraries generated from CSQ-immunized chickens. After biopanning, CSQ-specific VH sdAb clones were isolated and expressed as VH–human kappa light chain constant region (VH-Cκ) fusion proteins in E. coli. The PE06 clone was chosen for further characterization and conjugated to horseradish peroxidase (HRP) and Alexa Fluor 647 for assay applications. Results: PE06 VH-Cκ fusion protein demonstrated specific binding to CSQ-tagged proteins and enabled reliable detection in enzyme-linked immunosorbent assay (ELISA), immunoblotting, and flow cytometry. These results validated its utility as a chemically defined detection reagent for CSQ fusion proteins expressed in E. coli. Conclusions: This study establishes a CSQ-specific chicken VH sdAb as a versatile detection tool for CSQ-tagged proteins. The approach expands the utility of CSQ as a protein fusion tag and enables the development of recombinant antibodies fused with CSQ, such as scFv-CSQ constructs, for broad application in research and assay systems.

Bispecific T cell engagers (bispecific TCEs) are engineered antibodies that redirect T cells to mediate tumor cell killing by simultaneously binding to CD3 on T cells and tumor-associated antigens. As of July 2025, ten bispecific TCEs are clinically available. The CD3-binding antibodies in these bispecific TCEs can be classified into 6 groups based on the amino acid sequence similarity across their 6 complementarity-determining regions (CDRs). Specifically, antibodies were assigned to the same family if their six CDRs-HCDR1-3 and LCDR1-3-exhibited ≥80% pairwise sequence identity upon multiple sequence alignment. Family 1, derived from OKT3-a mouse hybridoma generated by immunizing BALB/c mice with human T cells-includes only blinatumomab; Family 2, derived from SP34-a rhesus monkey (Macaca mulatta) derived hybridoma specific for human T cells-comprises 5 antibodies; and Family 6, derived from UCHT1-a mouse hybridoma generated by immunizing mice with human T cells-contains only tebentafusp. The origin of the remaining 3 antibodies has not been disclosed and they possess unique CD3-binding sequences. We classified them into their own distinct families (Families 3, 4, and 5). Interestingly, mosunetuzumab (Family 4) showed remarkably lower incidence of adverse events such as cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and infection compared to other bispecific TCEs even though its affinity for CD3ε was not significantly different. The epitopes of 4 antibodies in Family 2, teclistamab, talquetamab, glofitamab, and tarlatamab were previously defined to be located at the N-terminal region of CD3ε via hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis. In our in silico epitope prediction analysis, the N-terminal region was included in the epitope region of all bispecific TCEs regardless of their family. Blinatumomab (Family 1) and tebentafusp (Family 6) did not bind to the CD3ε homolog of the cynomolgus monkey, whereas the other 8 bispecific TCEs did. This lack of cross-reactivity poses clear disadvantages in their preclinical development, particularly for toxicity and safety evaluation in nonhuman primate models.

Antibody production against an antigen of interest is highly efficient in chickens, and the use of chicken antibody libraries in phage display can result in high-affinity single-chain variable fragments (scFvs) for multiple applications. After library preparation from an animal immunized with the antigen of interest, the next step involves the identification of antigen binders. Here, we describe a process for the screening of a phage display chicken library using a technique called bio-panning. It consists of several rounds of binding scFv-displaying phage to antigens, followed by washing, elution, and reamplification. We also describe the steps for assessing clone pools obtained after bio-panning via an ELISA-based procedure known as "phage ELISA" to identify single clones. Last, we provide the steps for using high-throughput sequencing to analyze the pool of selected clones.

Phage-displayed antibody libraries can be constructed using any species that is easily immunized. The pComb3XSS phagemid vector is commonly used for library cloning and phage display. This phagemid encodes the origin of replication of the filamentous bacteriophage f1 but lacks all the genes required for replication and assembly of phage particles. The replication and the assembly of phage from these phagemids thus requires a "helper" phage that provides the genes essential for those steps during library production and bio-panning. One of those helper phages is VCSM13. In this protocol, we describe the preparation of VCSM13 helper phage. Users should prepare VCSM13 helper phage for library reamplification and for bio-panning.

Phage-displayed antibody fragment libraries can be constructed using essentially any species that is easily immunized, as long as the immunoglobulin variable region gene sequences are known. This protocol describes the procedures for the generation of a phage-displayed chicken single-chain variable fragment (scFv) library after immunization with a target antigen. Briefly, the rearranged heavy chain variable region (V H ) genes and the λ light chain variable region (V λ ) genes are amplified separately and are linked through two separate PCR steps to give the final scFv genes. The genes are then cloned into pComb3XSS to generate the phage display chicken scFv library, which can then be used for test and final library ligations.

Effective isolation of specific antibodies from immunological repertoires requires the generation of a diverse library against a specific antigen of interest, as well as efficient selection procedures, such as bio-panning and phage ELISA. Key to this is the generation of a good immune response in the host, followed by preparation of high-quality RNA and cDNA from which a library can be constructed by the amplification and cloning of immunoglobulin heavy and light chain genes. The first step in the construction of such an “immune library” is a successful course of immunization. Detection of a strong serum antibody titer will theoretically then result in a pool of extracted RNA that is enriched for transcripts of genes encoding the antibody of interest. Chicken antibodies have been widely used for research and diagnostic purposes, largely because of both their cross-reactivity to epitopes shared by humans, mice, primates, and other mammals, and their simple characteristics, with chickens featuring single functional copies of V H / J H and V λ / J λ gene pairs. In chickens, antibodies against an antigen of interest can be detected in the serum as soon as 5–7 d after immunization. Once the antibody titer reaches an appropriate level in the serum, the spleen, bursa of Fabricius, and bone marrow are then harvested, and antibody libraries can be prepared from extracted RNA. Here, we describe a protocol for chicken immunization with an antigen of interest, followed by RNA extraction from the relevant tissues and cDNA synthesis, which users can use for antibody library construction.

Chicken antibodies have been widely used for research and diagnostic purposes. Chicken antibodies are often cross-reactive to epitopes shared by humans, nonhuman primates, and other mammals, and can be tested in many mouse disease models, which provides an advantage for their preclinical study and evaluation. In addition, the variable region of chicken antibodies has unique structural characteristics, including noncanonical cysteine residues in the heavy chain complementarity-determining region (CDR)3 and a long heavy chain CDR3, which together with a short light chain CDR enable the formation of unconventional antibody paratopes. As chickens have single functional copies of the V H and J H genes, and the somatic gene conversion process usually involves D H genes, all functional VDJ gene fragments can be obtained from the B-cell repertoire using a single PCR primer set, without any primer bias. As for the light chain, chickens only have a V λ light chain, composed of a single V λ and J λ gene pair. Therefore, the chicken light chain repertoire can also be accurately amplified using a single primer set. This unbiased reconstitution of the chicken B-cell repertoire provides a great advantage not only in the construction of phage display libraries but also for the in silico selection of antigen binders from a virtual B-cell receptor repertoire. Here, we introduce the use of chicken antibodies in research, diagnostic, and therapeutic fields. In addition, the chromosomal organization of chicken immunoglobulin genes and its diversification mechanisms for shaping the antibody repertoire are also discussed.

B-cell maturation antigen (BCMA; TNFRSF17) has rapidly evolved from a plasma cell survival receptor within the BAFF/APRIL network to a central therapeutic hub in multiple myeloma (MM). In this review, we first outline the gene organization, expression pattern, and ligand biology of BCMA in the context of its sister receptors BAFF-R and TACI, emphasizing shared structural motifs-such as the conserved helix-loop-helix hairpin and DxL motif-that govern APRIL/BAFF recognition. We next detail how BCMA-proximal signaling through TRAF adaptors integrates canonical and noncanonical NF-κB, MAPK, and PI3K-AKT pathways to sustain long-lived plasma cells and drive myeloma progression, and how γ-secretase-mediated shedding generates soluble BCMA (sBCMA), which function as both a dynamic disease biomarker and an antigen sink for BCMA-directed agents. We then summarize the clinical development and distinguishing features of currently approved BCMA-targeted modalities-CAR T-cell therapies (ide-cel, cilta-cel), bispecific T-cell engagers (teclistamab, elranatamab, linvoseltamab), and the antibody-drug conjugate (belantamab mafodotin)-highlighting their efficacy, toxicity profiles, and practical positioning in relapsed/refractory MM. Finally, we review emerging resistance mechanisms, including γ-secretase-driven sBCMA elevation, ligand-rich APRIL/BAFF niches, and therapy-induced TNFRSF17 lesions, ranging from biallelic deletions to epitope-altering missense mutations and in-frame deletions within the BCMA extracellular domain. These insights inform rational strategies such as γ-secretase inhibition, dual-target CAR T-cells and bispecific T-cell engagers.

Chimeric antigen receptor T (CAR-T) cells show remarkable efficacy for some hematological malignancies. However, CAR targets that are expressed at high level and selective to tumors are scarce. Several strategies have been proposed to tackle the on-target off-tumor toxicity of CAR-T cells that arise from suboptimal selectivity, but these are complicated, with many involving dual gene expression for specificity. In this study, we show that switchable CAR-T cells with a tumor targeting adaptor can mitigate on-target off-tumor toxicity against a low selectivity tumor antigen that cannot be targeted by conventional CAR-T cells, such as CD40. Our system is composed of anti-cotinine murine CAR-T cells and cotinine-labeled anti-CD40 single chain variable fragments (scFv), with which we show selective tumor killing while sparing CD40-expressing normal cells including macrophages in a mouse model of lymphoma. Simple replacement of the tumor-targeting adaptor with a suicidal drug-conjugated tag may further enhance safety by enabling permanent in vivo depletion of the switchable CAR-T cells when necessary. In summary, our switchable CAR system can control CAR-T cell toxicity while maintaining therapeutic efficacy, thereby expanding the range of CAR targets. The therapeutic success of CAR-T cells depends on the availability of selective and high-density targets, which limits their applicability due to on-target off-tumor toxicity. Here authors overcome this limitation in a mouse model of immune therapy in which an adaptor is an epitope-tagged single chain variable fragment targeting the tumour antigen, and the CAR-T cells are targeting the epitope, thus enabling a precise dose-switch.

The immune escape of Omicron variants significantly subsides by the third dose of an mRNA vaccine. However, it is unclear how Omicron variant-neutralizing antibodies develop under repeated vaccination. We analyzed blood samples from 41 BNT162b2 vaccinees following the course of three injections and analyzed their B-cell receptor (BCR) repertoires at six time points in total. The concomitant reactivity to both ancestral and Omicron receptor-binding domain (RBD) was achieved by a limited number of BCR clonotypes depending on the accumulation of somatic hypermutation (SHM) after the third dose. Our findings suggest that SHM accumulation in the BCR space to broaden its specificity for unseen antigens is a counter protective mechanism against virus variant immune escape.

Abstract Chimeric antigen receptor (CAR) T cells targeting CD19 or the B cell maturation antigen (BCMA) have demonstrated impressive initial clinical responses. However, on follow-up assessment, a notable fraction of the patients with significant initial clinical response eventually experienced relapse. CAR T cell therapies targeting solid tumors have shown an even lower rate of clinical response as well as shorter remission periods. A prior study has shown that re-injection of the anti-HER2 CAR T cells was effective in a rhabdomyosarcoma relapse patient (Cancer Res 79, LB147 (2019)), but the repeated production of CAR T cells is costly, thus raising questions about its feasibility in practice. Previously, we developed a cotinine-based CAR T cell system (zCART) comprised of a tumor antigen-specific affibody conjugated with cotinine and an anti-cotinine scFv CAR, and demonstrated the anti-tumor activity of an anti-HER2 zCART treatment in both in vitro and in vivo settings. In this study, we tested the efficacy of anti-HER2 zCART treatment in vivo using an intraperitoneal (IP) tumor model with a single infusion of zCART cells. The anti-HER2 switch was injected daily for 7 days. When the HER2 positive tumor relapsed, the mice were re-treated with the same dose of anti-HER2 switch treatment for an additional 7 days. We observed that re-injection of only the anti-HER2 switch and not combined with zCART cells effectively and potently inhibited relapse of HER2 positive tumors. Furthermore, the efficacy of the anti-HER2 switch re-injection treatment was maintained in one additional relapse. In summary, the zCART system has potential to treat cancer relapse following CAR T cell therapy without the need for an additional production round of CAR T cells. Citation Format: Ki Hyun Kim, Soohwan Kim, Eun-Hoe Lee, Soo-Yoon Lim, Sung Min Kim, E-Young Kim, Hyun-Jong Lee, Seong Yeol Kim, Min Yoon, Young-Ha Lee, In-Sik Hwang, Yoon Lee, Jong-Hoon Kim, Jong-Seo Lee, Junho Chung. Potent inhibition of tumor relapse after anti-HER2 affibody tagged with cotinine (switch) plus anti-cotinine switchable CAR T cell therapy by repeated re-injection of the switch [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 6314.

In this Journal Club, Chae and Chung discuss a study characterizing the differentiation and maturation of both tumour-resident and circulating B cells in patients with melanoma.