Computational Analysis of T-Cell Receptor Repertoire Workflow: From T-Cell Isolation to Bioinformatics Analysis.

The T-cell receptor (TCR) is the key molecule involved in the adaptive immune response. It is generated by the V(D)J recombination, responsible of the enormous diversity of the TCR repertoire, a crucial feature determining the individual capability to response to antigens and to build immunological memory. A pivotal role in the recognition of antigen is played by the hypervariable complementarity-determining region 3 (CDR3) of the V-beta chain of TCR. Investigating the CDR3 supports the understanding of the adaptive immune system dynamics in physiological processes, such as immune aging, and in disease, especially autoimmune disorders in which T cells are main actors. High-throughput sequencing (HTS) paved the way for a great progress in the investigation of TCR repertoire, enhancing the read depth in the process of library generation of sequencing and the number of samples that can be analyzed simultaneously. Therefore, the leverage of big datasets stressed the need to develop computational approach, by bioinformatics, to unravel the characteristics of the TCR repertoire.

Insights into cytomegalovirus-associated T cell receptors in recipients following allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Cytomegalovirus (CMV) reactivation is a serious problem in recipients of allogeneic hematopoietic stem cell transplantation. Long-term latency depends on specific T cell immune reconstitution, which identifies various pathogens by T cell receptors (TCRs). However, the mechanisms underlying the selection of CMV-specific TCRs in recipients after transplantation remain unclear. METHODS: Using high-throughput sequencing and bioinformatics analysis, the T cell immune repertoire of seven CMV reactivated recipients (CRRs) were analyzed and compared to those of seven CMV non-activated recipients (CNRs) at an early stage after transplant. RESULTS: The counts of unique complementarity-determining region 3 (CDR3) were significantly higher in CNRs than in CRRs. The CDR3 clones in the CNRs exhibit higher homogeneity compared to the CRRs. With regard to T cell receptor ß-chain variable region (TRBV) and joint region (TRBJ) genotypes, significant differences were observed in the frequencies of TRBV6, BV23, and BV7-8 between the two groups. In addition to TRBV29-1/BJ1-2, TRBV2/BJ2-2, and TRBV12-4/BJ1-5, 11 V-J combinations had significantly different expression levels between CRRs and CNRs. CONCLUSIONS: The differences in TCR diversity, TRBV segments, and TRBV-BJ combinations observed between CNRs and CRRs might be associated with post-transplant CMV reactivation and could serve as a foundation for further research.

Development and experimental validation of computational methods for human antibody affinity enhancement.

High affinity is crucial for the efficacy and specificity of antibody. Due to involving high-throughput screens, biological experiments for antibody affinity maturation are time-consuming and have a low success rate. Precise computational-assisted antibody design promises to accelerate this process, but there is still a lack of effective computational methods capable of pinpointing beneficial mutations within the complementarity-determining region (CDR) of antibodies. Moreover, random mutations often lead to challenges in antibody expression and immunogenicity. In this study, to enhance the affinity of a human antibody against avian influenza virus, a CDR library was constructed and evolutionary information was acquired through sequence alignment to restrict the mutation positions and types. Concurrently, a statistical potential methodology was developed based on amino acid interactions between antibodies and antigens to calculate potential affinity-enhanced antibodies, which were further subjected to molecular dynamics simulations. Subsequently, experimental validation confirmed that a point mutation enhancing 2.5-fold affinity was obtained from 10 designs, resulting in the antibody affinity of 2 nM. A predictive model for antibody-antigen interactions based on the binding interface was also developed, achieving an Area Under the Curve (AUC) of 0.83 and a precision of 0.89 on the test set. Lastly, a novel approach involving combinations of affinity-enhancing mutations and an iterative mutation optimization scheme similar to the Monte Carlo method were proposed. This study presents computational methods that rapidly and accurately enhance antibody affinity, addressing issues related to antibody expression and immunogenicity.

ABodyBuilder3: improved and scalable antibody structure predictions.

SUMMARY: In this article, we introduce ABodyBuilder3, an improved and scalable antibody structure prediction model based on ABodyBuilder2. We achieve a new state-of-the-art accuracy in the modelling of CDR loops by leveraging language model embeddings, and show how predicted structures can be further improved through careful relaxation strategies. Finally, we incorporate a predicted Local Distance Difference Test into the model output to allow for a more accurate estimation of uncertainties. AVAILABILITY AND IMPLEMENTATION: The software package is available at https://github.com/Exscientia/ABodyBuilder3 with model weights and data at https://zenodo.org/records/11354577.

CDR L3 Loop Rearrangement Switches Multispecific SPE-7 IgE Antibody From Hapten to Protein Binding.

The monoclonal IgE antibody SPE-7 was originally raised against a 2,4-dinitrophenyl (DNP) target. Through its ability to adopt multiple conformations, the antibody is capable of binding to a diverse range of small haptens and large proteins. The present study examines a dataset of experimentally determined crystal structures of the SPE-7 antibody to gain insight into the mechanisms that contribute to its multispecificity. With the emergence of more and more therapeutic antibodies against a huge repertoire of different targets, our research could be of great interest for future drug development. We are able to discriminate between the different paratope-binding states in the conformational ensembles obtained by enhanced sampling molecular dynamics simulations, and to calculate their transition timescales and state probabilities. Furthermore, we describe the key residues responsible for discriminating between the different binding capacities and identify a tryptophan in a central position of the CDR L3 loop as the residue of greatest interest. The overall dynamics of the paratope appear to be mainly influenced by the CDR L3 and CDR L1 loops.

Direct recognition of an intact foreign protein by an αß T cell receptor.

αß T cell receptors (αßTCRs) co-recognise antigens when bound to Major Histocompatibility Complex (MHC) or MHC class I-like molecules. Additionally, some αßTCRs can bind non-MHC molecules, but how much intact antigen reactivities are achieved remains unknown. Here, we identify an αß T cell clone that directly recognises the intact foreign protein, R-phycoerythrin (PE), a multimeric (αß)6γ protein complex. This direct αßTCR-PE interaction occurs in an MHC-independent manner, yet triggers T cell activation and bound PE with an affinity comparable to αßTCR-peptide-MHC interactions. The crystal structure reveals how six αßTCR molecules simultaneously engage the PE hexamer, mediated by the complementarity-determining regions (CDRs) of the αßTCR. Here, the αßTCR mainly binds to two α-helices of the globin fold in the PE α-subunit, which is analogous to the antigen-binding platform of the MHC molecule. Using retrogenic mice expressing this TCR, we show that it supports intrathymic T cell development, maturation, and exit into the periphery as mature CD4/CD8 double negative (DN) T cells with TCR-mediated functional capacity. Accordingly, we show how an αßTCR can recognise an intact foreign protein in an antibody-like manner.

Estimates of Sequences with Ultralong and Short CDR3s in the Bovine IgM B Cell Receptor Repertoire Using the Long-read Oxford Nanopore MinION Platform.

Cattle produce Abs with an H chain ultralong CDR3 (40-70 aa). These Abs have been shown to have features such as broad neutralization of viruses and are investigated as human therapeutics. A common issue in sequencing the bovine BCR repertoire is the sequence length required to capture variable (V) and isotype gene information. This study aimed to assess the use of Oxford Nanopore Technologies' MinION platform to perform IgM BCR repertoire sequencing to assess variation in the percentage of ultralong CDR3s among dairy cattle. Blood was collected from nine Holstein heifers. B cells were isolated using magnetic bead-based separation, RNA was extracted, and IgM+ transcripts were amplified using PCR and sequenced using a MinION R10.4 flow cell. The distribution of CDR3 lengths was trimodal, and the percentage of ultralong CDR3s ranged among animals from 2.32 to 20.13% in DNA sequences and 1.56% to 17.02% in productive protein sequences. V segment usage varied significantly among heifers. Segment IGHV1-7, associated with ultralong CDR3s, was used in 5.8-24.2% of sequences; usage was positively correlated with ultralong CDR3 production (r = 0.99, p < 0.01). To our knowledge, this is the first study to sequence the bovine BCR repertoire using Oxford Nanopore Technologies and demonstrates the potential for cost-efficient long-read repertoire sequencing in cattle without assembly. Findings from this study support literature describing the distribution of length and percentage of ultralong CDR3s. Future studies will investigate changes in the bovine BCR repertoire associated with age, antigenic exposure, and genetics.

Rapid affinity optimization of an anti-TREM2 clinical lead antibody by cross-lineage immune repertoire mining.

We describe a process for rapid antibody affinity optimization by repertoire mining to identify clones across B cell clonal lineages based on convergent immune responses where antigen-specific clones with the same heavy (VH) and light chain germline segment pairs, or parallel lineages, bind a single epitope on the antigen. We use this convergence framework to mine unique and distinct VH lineages from rat anti-triggering receptor on myeloid cells 2 (TREM2) antibody repertoire datasets with high diversity in the third complementarity-determining loop region (CDR H3) to further affinity-optimize a high-affinity agonistic anti-TREM2 antibody while retaining critical functional properties. Structural analyses confirm a nearly identical binding mode of anti-TREM2 variants with subtle but significant structural differences in the binding interface. Parallel lineage repertoire mining is uniquely tailored to rationally explore the large CDR H3 sequence space in antibody repertoires and can be easily and generally applied to antibodies discovered in vivo.

Invariant VD and DJ Motifs Define a Novel Class of Human Antibodies and TCRs Prototyped by antigen receptors of Dual-Expresser Lymphocytes.

INTRODUCTION: Dual-expressing lymphocytes (DEs) are unique immune cells that express both B cell receptors (BCRs, surface antibody) and T cell receptors (TCRs). In type 1 diabetes, DE antibodies are predominated by one antibody (x-mAb), an IgM monoclonal antibody with a germline-encoded CDR3 that recognizes self-reactive TCRs. We explored if x-mAb and its interacting TCRs have distinct structural features. METHODS: Using bioinformatics, we compared x-mAb and its most common interacting TCRαß to billions of antigen receptor sequences to determine if they were unique or randomly generated. RESULTS: X-mAb represents a unique class of human antibodies with a conserved CDR3 sequence (CARx1-4DTAMVYYFYDW), consisting of a fixed DJH motif (DTAMVYYFDYW) paired with various VH genes. A public TCRß clonotype (CASSPGTEAFF) associated with x-mAb on DEs features two invariant segments, VßD (CASSPGT) and DJß (PGTEAFF), key to two large families of public TCRß clonotypes-CASSPGT-Jßx and CASSPGT-Jßx-formed by recombining the VßD motif with Jß genes and the DJß motif with Vß genes. B cells also use CASSPGT as a VHD motif for public IGH clonotypes (CASSPGT-Jßx). DISCUSSION: DEs, unlike conventional T and B cells, use invariant motifs to create public antibodies and TCRs, a trait previously seen only in cartilaginous fish.

The T-cell repertoire of Spanish patients with COVID-19 as a strategy to link T-cell characteristics to the severity of the disease.

BACKGROUND: The architecture and dynamics of T cell populations are critical in orchestrating the immune response to SARS-CoV-2. In our study, we used T Cell Receptor sequencing (TCRseq) to investigate TCR repertoires in 173 post-infection COVID-19 patients. METHODS: The cohort included 98 mild and 75 severe cases with a median age of 53. We amplified and sequenced the TCR ß chain Complementary Determining Region 3 (CDR3b) and performed bioinformatic analyses to assess repertoire diversity, clonality, and V/J allelic usage between age, sex and severity groups. CDR3b amino acid sequence inference was performed by clustering structural motifs and filtering validated reactive CDR3b to COVID-19. RESULTS: Our results revealed a pronounced decrease in diversity and an increase in clonal expansion in the TCR repertoires of severe COVID-19 patients younger than 55 years old. These results reflect the observed trends in patients older than 55 years old (both mild and severe). In addition, we identified a significant reduction in the usage of key V alleles (TRBV14, TRBV19, TRBV15 and TRBV6-4) associated with disease severity. Notably, severe patients under 55 years old had allelic patterns that resemble those over 55 years old, accompanied by a skewed frequency of COVID-19-related motifs. CONCLUSIONS: Present results suggest that severe patients younger than 55 may have a compromised TCR repertoire contributing to a worse disease outcome.

Characteristics of T-Cell Receptor Repertoire for Differential Response to Methotrexate Treatment for Rheumatoid Arthritis.

BACKGROUND: Methotrexate (MTX) serves as the initial treatment for rheumatoid arthritis (RA). However, a substantial proportion of RA patients, estimated between 30% and 50%, do not respond positively to MTX. While the T-cell receptor (TCR) is crucial for the immune response during RA, its role in differentiating MTX responsiveness has not been thoroughly investigated. METHODS: This study used next-generation sequencing to analyze the TCR ß-chain complementary determining region sequences in peripheral blood mononuclear cells obtained from RA patients before MTX treatment. This study aimed to compare the characteristics of the TCR repertoire between the MTX responder and non-responder groups. RESULTS: The study identified a significant difference in the TRBV6-6 gene (p = .003) concerning MTX treatment response. Additionally, a significant difference was found in the number of "3" nucleotide deletions at the 5'Jdels site (p = .023) in the VDJ rearrangement. CONCLUSION: These findings suggest distinct TCR repertoire characteristics between MTX responder and non-responder groups among RA patients. This discovery offers new insights into understanding the variable responses of RA patients to MTX therapy.

Antibody-SGM, a Score-Based Generative Model for Antibody Heavy-Chain Design.

Traditional computational methods for antibody design involved random mutagenesis followed by energy function assessment for candidate selection. Recently, diffusion models have garnered considerable attention as cutting-edge generative models, lauded for their remarkable performance. However, these methods often focus solely on the backbone or sequence, resulting in the incomplete depiction of the overall structure and necessitating additional techniques to predict the missing component. This study presents Antibody-SGM, an innovative joint structure-sequence diffusion model that addresses the limitations of existing protein backbone generation models. Unlike previous models, Antibody-SGM successfully integrates sequence-specific attributes and functional properties into the generation process. Our methodology generates full-atom native-like antibody heavy chains by refining the generation to create valid pairs of sequences and structures, starting with random sequences and structural properties. The versatility of our method is demonstrated through various applications, including the design of full-atom antibodies, antigen-specific CDR design, antibody heavy chains optimization, validation with Alphafold3, and the identification of crucial antibody sequences and structural features. Antibody-SGM also optimizes protein function through active inpainting learning, allowing simultaneous sequence and structure optimization. These improvements demonstrate the promise of our strategy for protein engineering and significantly increase the power of protein design models.

Development of novel humanized VHH synthetic libraries based on physicochemical analyses.

Due to the high affinity and specificity of antibodies toward antigens, various antibody-based applications have been developed. Recently, variable antigen-binding domains of heavy-chain antibodies (VHH) have become an attractive alternative to conventional fragment antibodies due to their unique molecular characteristics. As an antibody-generating strategy, synthetic VHH libraries (including humanized VHH libraries) have been developed using distinct strategies to constrain the diversity of amino acid sequences. In this study, we designed and constructed several novel synthetic humanized VHH libraries based on biophysical analyses conducted using the complementarity determining region-grafting method and comprehensive sequence analyses of VHHs deposited in the protein data bank. We obtained VHHs from the libraries, and hit clones exhibited considerable thermal stability. We also found that VHHs from distinct libraries tended to have different epitopes. Based on our results, we propose a strategy for generating humanized VHHs with distinct epitopes toward various antigens by utilizing our library combinations.

Computational detection of antigen-specific B cell receptors following immunization.

B cell receptors (BCRs) play a crucial role in recognizing and fighting foreign antigens. High-throughput sequencing enables in-depth sampling of the BCRs repertoire after immunization. However, only a minor fraction of BCRs actively participate in any given infection. To what extent can we accurately identify antigen-specific sequences directly from BCRs repertoires? We present a computational method grounded on sequence similarity, aimed at identifying statistically significant responsive BCRs. This method leverages well-known characteristics of affinity maturation and expected diversity. We validate its effectiveness using longitudinally sampled human immune repertoire data following influenza vaccination and SARS-CoV-2 infections. We show that different lineages converge to the same responding Complementarity Determining Region 3, demonstrating convergent selection within an individual. The outcomes of this method hold promise for application in vaccine development, personalized medicine, and antibody-derived therapeutics.

Similar usage of T-cell receptor ß-chain between tumor and adjacent normal tissue in hepatocellular carcinoma.

BACKGROUND: In this study, we comprehensively profiled the T-cell receptor (TCR) repertoire of the tumor and adjacent normal tissue in patients with HBV-associated hepatocellular carcinoma (HCC) and determined the baseline characteristics and clinical significance of TCR. METHODS: High-throughput sequencing was used to determine the profile of complementarity-determining region 3 (CDR3) of the TCR-ß chain variable (TRBV) in the tumor and normal tissue samples of 14 HCC patients. At the same time, TRBV diversity and differences in expression between tumor and normal tissues were investigated. The cumulative frequency of top 100 CDR3 (CF100), clonality, and Shannon entropy as indices to evaluate diversity, RESULTS: The diversity of TRBV CDR3 showed no significant difference between tumor and normal tissues. Of the 58 V gene segments in TRBV, TRBV16 and TRBV7-6 had a significantly higher frequency in the tumor group than in the normal group (p < 0.05). The frequency of 14 J gene segments showed no significant difference between tumor and normal tissues. In contrast, the frequency of 22 TRBVx/BJx combinations was significantly higher in the tumor than in the normal tissue. In addition, the length and type of TRBV CDR3 were similar in tumor and normal tissues, and a Gaussian distribution was observed in both groups. CONCLUSION: This study provided a large amount of information about the TCR lineage in HBV-associated HCC, laying the foundation for further research. In addition, the fact that the immune repertoire (TRBV CDR3) hardly differs between tumor and adjacent normal tissue provides a new clue for exploring the mechanism of the liver as an organ with immune privileges.

Dysfunctional tumor-infiltrating Vδ1 + T lymphocytes in microsatellite-stable colorectal cancer.

Although γδ T cells are known to participate in immune dysregulation in solid tumors, their relevance to human microsatellite-stable (MSS) colorectal cancer (CRC) is still undefined. Here, using integrated gene expression analysis and T cell receptor sequencing, we characterized γδ T cells in MSS CRC, with a focus on Vδ1 + T cells. We identified Vδ1+ T cells with shared motifs in the third complementarity-determining region of the δ-chain, reflective of antigen recognition. Changes in gene and protein expression levels suggested a dysfunctional effector state of Vδ1+ T cells in MSS CRC, distinct from Vδ1+ T cells in microsatellite-instable (MSI). Interaction analysis highlighted an immunosuppressive role of fibroblasts in the dysregulation of Vδ1+ T cells in MSS CRC via the TIGIT-NECTIN2 axis. Blocking this pathway with a TIGIT antibody partially restored cytotoxicity of the dysfunctional Vδ1 phenotype. These results define an operative pathway in γδ T cells in MSS CRC.

Chemical complementarity of tumor resident, T-cell receptor CDR3s and renalase-1 correlates with increased melanoma survival.

Overexpression of the secretory protein renalase-1 negatively impacts the survival of melanoma and pancreatic cancer patients, while inhibition of renalase-1 signaling drives tumor rejection by promoting T-cell activation. Thus, we investigated the chemical complementarity between melanoma-resident, T-cell receptor (TCR) complementarity-determining region 3 (CDR3) amino acid sequences (AAs) and the renalase-1 protein. Increasing complementarity of TCR CDR3s to renalase-1 AAs, as assessed by a chemical complementarity scoring algorithm, was associated with improved overall survival (OS) in melanoma patients. The expression levels of several immune signature genes were significantly, positively correlated with increasing TCR CDR3-renalase-1 complementarity scores. Additionally, the survival association observed with high complementarity of TCR CDR3s to renalase-1 AAs was more robust in cases with low renalase-1 gene expression levels. Mapping of TCR CDR3-renalase-1 in silico interaction sites identified major epitope candidates including RP220, the signaling module of the renalase-1 protein, consistent with the fact that a monoclonal antibody to RP220 is a potent inhibitor of melanoma growth. These findings indicate that renalase-1 is a potential antigen for TCR recognition in melanoma and could be considered as a target for immunotherapy.

De novo generation of SARS-CoV-2 antibody CDRH3 with a pre-trained generative large language model.

Artificial Intelligence (AI) techniques have made great advances in assisting antibody design. However, antibody design still heavily relies on isolating antigen-specific antibodies from serum, which is a resource-intensive and time-consuming process. To address this issue, we propose a Pre-trained Antibody generative large Language Model (PALM-H3) for the de novo generation of artificial antibodies heavy chain complementarity-determining region 3 (CDRH3) with desired antigen-binding specificity, reducing the reliance on natural antibodies. We also build a high-precision model antigen-antibody binder (A2binder) that pairs antigen epitope sequences with antibody sequences to predict binding specificity and affinity. PALM-H3-generated antibodies exhibit binding ability to SARS-CoV-2 antigens, including the emerging XBB variant, as confirmed through in-silico analysis and in-vitro assays. The in-vitro assays validate that PALM-H3-generated antibodies achieve high binding affinity and potent neutralization capability against spike proteins of SARS-CoV-2 wild-type, Alpha, Delta, and the emerging XBB variant. Meanwhile, A2binder demonstrates exceptional predictive performance on binding specificity for various epitopes and variants. Furthermore, by incorporating the attention mechanism inherent in the Roformer architecture into the PALM-H3 model, we improve its interpretability, providing crucial insights into the fundamental principles of antibody design.

Worse Wilms' Tumor Outcomes Associated With Chemical Complementarity for Multiple T-Cell Receptor CDR3-CMV Epitope Pairs.

BACKGROUND/AIM: Wilms' tumors are pediatric renal tumors that generally have a good prognosis and outcomes. Viral illnesses have been linked to development of neoplasms and should be considered as a factor that could modulate overall survival. MATERIALS AND METHODS: We considered recently developed adaptive immune receptor, genomics and bioinformatics approaches to assess the potential impact of cytomegalovirus (CMV) infections in Wilms' tumor. RESULTS: T-cell receptor (TCR) complementarity determining region-3 (CDR3) amino acid sequences from Wilms' tumor specimens represented by the Therapeutically Applicable Research to Generate Effective Treatments dataset were compared with known anti-CMV TCR CDR3s, indicating that cases representing the anti-CMV TCR CDR3s had worse outcomes. Then, a chemical complementarity scoring approach for the Wilms' tumor, TCR CDR3s and a series of CMV antigens further indicated that cases representing a higher chemical complementarity to the CMV antigens had worse outcomes. CONCLUSION: Overall, we present a potentially novel method to assess CMV infections and identify patients who could benefit from therapies that address such infections.

Generation of rabbit single-chain variable fragments with different physicochemical and biological properties by complementary determining region-grafting technology.

In this study, we have demonstrated a complementary-determining region (CDR) grafting technology for the generation of rabbit scFvs with different antigen recognition and physicochemical properties. The antigen-binding affinity of the CDR-grafted anti-CRP scFv, C1R/B1R (V1), which was generated by the CDR/framework region (CDR/FR) definition based on the traditional numbering rule, was insufficient when compared to that of the original clone, C1R, suggesting that the amino acid residues outside the original CDRs might significantly contribute to antigen recognition in rabbit scFvs. We redefined new CDRs and FRs to maintain antigen-binding affinities through the extension of multiple amino acid residues for CDRH1 and CDRH2, based on the amino acid sequence alignments of rabbit scFvs isolated from phage libraries. The new version successfully maintained the antigen binding affinity. CDR-grafted scFvs possessing a common CDR sequence and different FR sequences were successfully generated based on this new CDR/FR definition, and their physicochemical properties were further investigated. The antigen-binding activities of rabbit scFvs on Maxisorp varied between the tested clones in sandwich ELISA, supporting the idea that the combination of CDR with different FRs might change the physicochemical properties of scFvs on a solid material. The CDR-grafted scFvs possessing a frame sequence of anti-CRP scFv C2R maintained the ability to bind to protein L and were successfully purified. Expression titers showed improved solubility by diminishing the amount of insoluble scFvs. Thus, the method developed in this study is promising for generating alternatives with strict antigen binding recognition and different physicochemical properties.