Biology subjects

Krackhardt, A. M.

Publications and source records attributed to Krackhardt, A. M..

4 recordsLinked to original sources

bioRxiv2025

Single-domain antibodies targeting the pan-T-cell markers CD2 and CD7 as universal immunotherapy T-cell tracers

RationaleDespite significant advancements in immunotherapeutic approaches, such as immune checkpoint modulation and CAR-T cell therapy, reliable and universally applicable surrogate markers to monitor and evaluate the wide variety of therapeutic responses are still missing. This gap of information can lead to delayed assessment of responders versus non-responders and to misinterpretation, potentially resulting in premature or erroneous treatment decisions. Thus, a universal surrogate marker may be of preeminent value in monitoring immune responses, guiding immunotherapies, and ultimately improving patient outcomes. To address this challenge, we developed two immunoPET tracers based on single-domain antibodies (sdAbs) that target the pan T-cell markers CD2 and CD7 to monitor and visualize T cells distribution in vivo. MethodsThis study aimed to establish a comprehensive approach for clinical translation by assessing the feasibility and key characteristics of CD2- and CD7-sdAb for safe, effective clinical application. Binding specificity was confirmed through antigen transduction and knockout in tumor and T-cell leukemia cell lines. In vitro, cytokine secretion and T-cell cytotoxicity were evaluated, while a xenogeneic myeloid sarcoma mouse model was used to analyze T-cell cytotoxicity and visualize TCR-transduced CD8+ T cells at the tumor site using PET/MR imaging. ResultsThis study demonstrates the high specificity, affinity, and thermal stability of CD2- and CD7-sdAb, enabling efficient radiolabeling without impairing T-cell functionality. Specifically, binding to human CD8 T cells did not alter cytokine secretion or cytotoxicity in vitro. Furthermore, in vivo, the cytotoxic ability of transferred TCR-transduced human CD8+ T cells was not compromised upon i.v. application of CD2- and CD7-sdAb. Ultimately, intravenously injected 68Ga-NOTA-CD2-sdAb and 68Ga-NOTA-CD7-sdAb were respectively able to clearly depict and visualize previously administered TCR-transduced CD8+ T cells at the tumor site by PET/MRI. ConclusionsIn this study we have developed two pan T-cell tracers that are able to visualize human T cells and clearly differentiate between responding- and non-responding tumors while not impacting T-cell functionality in vitro and in vivo. The results provide a base for clinical use of these tracers as pan T-cell tracers applicable for any form of immunotherapy, making them a crucial tool in guiding immunotherapeutic decision-making.

immunology↗

bioRxiv2022

High-resolution profiling of neoantigen-specific T cell receptor activation signatures links moderate stimulation patterns to resilience and sustained tumor control

Neoantigen-specific T cell receptors (neoTCRs) promise a safe, highly personalized therapeutic approach in anti-tumor immunotherapy. Substantial progress has been made regarding their identification whereas detailed functional assessment of single TCR characteristics impacting therapeutic efficacy is lacking. We previously identified and functionally characterized neoTCRs specific for neoepitopes derived from KIF2C and SYTL4 demonstrating differences in functional avidity in a patient with metastatic melanoma. In this work, we now combined single-cell TCR- and RNA-sequencing using stimulated peripheral blood derived CD8+ T cells of this patient and thereby identified two new neoTCRs recognizing the previously identified mutated epitope KIF2CP13L. Analyzing patient-derived neoTCR expressing T cells, we detected distinct activation patterns as a measure for substantial heterogeneity within oligoclonal T cell responses towards neoantigens upon specific ex vivo-restimulation. Moreover, neoTCR-transgenic T cells from healthy donors were employed for detailed in vitro and in vivo fine-characterization focusing on TCR-intrinsic functional patterns. Most importantly, in a xenogeneic mouse model experimentally simulating rechallenge of tumor infiltrating lymphocytes (TILs) after adoptive T cell transfer, we found that T cells expressing neoTCRs with a moderate activation profile provide a stable and more sustained anti-tumor response upon repeated in vivo tumor challenge as compared to neoTCRs with a stronger, burst-like reactivity. These insights have significant implications for engineering TCR-transgenic T cells for therapeutic purposes. One Sentence SummaryCombining TCR specificity linked single-cell transcriptomics with in vitro and in vivo characterization of transgenic T cells helps to decipher functional potential and persistence of neoantigen-specific T cell receptors (TCRs) for TCR-transgenic T cell-based adoptive cellular anti-tumor immunotherapy.

immunology↗

bioRxiv2022

Proteogenomic analysis reveals RNA as an important source for tumor-agnostic neoantigen identification correlating with T-cell infiltration

Systemic pan-tumor analyses may reveal the significance of common features implicated in cancer immunogenicity and patient survival. Here, we provide a comprehensive multi-omics data set for 32 patients across 25 tumor types by combining proteogenomics with phenotypic and functional analyses. By using an optimized computational approach, we discovered a large number of novel tumor-specific and tumor-associated antigens including shared common target candidates. To create a pipeline for the identification of neoantigens in our cohort, we combined deep DNA and RNA sequencing with MS- based immunopeptidomics of tumor specimens, followed by the assessment of their immunogenicity. In fact, we could detect a broad variety of non-wild type HLA-binding peptides in the majority of patients and confirmed the immunogenicity of 24 neoantigens. Most interestingly, the majority of total and immunogenic neoantigens originated from variants identified in the RNA dataset, illustrating the importance of RNA as a still understudied source of cancer antigens. Moreover, the amount of these mainly RNA-based immunogenic neoantigens correlated positively with overall CD8+ tumor-infiltrating T cells. This study therefore underlines the importance of RNA-centered variant detection for the identification of shared biomarkers and potentially relevant neoantigen candidates. Statement of significanceThe significance of this study lies not only in the potential of our optimized proteogenomic workflow for the discovery of neoantigens (in particular RNA-derived neoantigens) for clinical application, but sheds light on the entity-agnostic prevalence of HLA class I peptide presentation of RNA processing events to be used for tumor targeting.

cancer biology↗

bioRxiv2021

Spatial and temporal plasticity of neoantigen-specific T-cell responses bases on characteristics associated to antigen and TCR

Neoantigens derived from somatic mutations have been demonstrated to correlate with therapeutic responses mediated by treatment with immune checkpoint inhibitors. Neoantigens are therefore highly attractive targets for the development of personalized medicine approaches although their quality and associated immune responses is not yet well understood. In a case study of metastatic malignant melanoma, we performed an in-depth characterization of neoantigens and respective T-cell responses in the context of immunotherapy with Ipilimumab. Three neoantigens identified either by immunopeptidomics or in silico prediction were investigated using binding affinity analyses and structural simulations. We isolated seven T-cell receptors (TCRs) from the patient immune repertoire recognizing these antigens. TCRs were compared in-vitro and in-vivo with multi-parametric analyses. Identified immunogenic peptides showed similar binding affinities to the human leukocyte antigen (HLA) complex and comparable differences to their wildtype counterparts in molecular dynamic simulations. Nevertheless, isolated TCRs differed substantially in functionality and frequency. In fact, TCRs with comparably lower functional avidity and higher potential for cross-reactivity provided at least equal anti-tumor immune responses in vivo. Of note, these TCRs showed a reduced activation pattern upon primary in vitro stimulation. Exploration of the TCR-{beta} repertoire in blood and in different tumor-related tissues over three years, offered insights on the high frequency and particular long-term persistence of low-avidity TCRs. These data indicate that qualitative differences of neoantigen-specific TCRs and their impact on function and longevity need to be considered for neoantigen targeting by adoptive T-cell therapy using TCR-transgenic T cells. Statement of translational relevanceImmunotherapy has demonstrated high efficacy in diverse malignancies. Neoantigens derived from mutations provide promising targets for safe and highly tumor-specific therapeutic approaches. Yet, single determinants of an effective and enduring T-cell mediated tumor rejection are still not well understood. We analyzed in detail seven neoantigen-specific T-cell receptors (TCRs) derived from a melanoma patient targeting three different altered peptide ligands identified by mass spectrometry and prediction analyses. Functional characterization of these TCRs revealed potent anti-tumor reactivity of all TCRs. Of special interest, TCRs with comparably lower affinity demonstrated effective in vivo activity as well as dominant spatial and temporal distribution in blood and tissue. Functional differences of TCR may require further T-cell and/or TCR engineering and should be considered for future clinical trial designs.

immunology↗