TCR-based Immunotherapy Research Articles

Strengths and limitations of web servers for the modeling of TCRpMHC complexes

Cellular immunity relies on the ability of a T-cell receptor (TCR) to recognize a peptide (p) presented by a class I major histocompatibility complex (MHC) receptor on the surface of a cell. The TCR-peptide-MHC (TCRpMHC) interaction is a crucial step in activating T-cells, and the structural characteristics of these molecules play a significant role in determining the specificity and affinity of this interaction. Hence, obtaining 3D structures of TCRpMHC complexes offers valuable insights into various aspects of cellular immunity and can facilitate the development of T-cell-based immunotherapies. Here, we aimed to compare three popular web servers for modeling the structures of TCRpMHC complexes, namely ImmuneScape (IS), TCRpMHCmodels, and TCRmodel2, to examine their strengths and limitations. Each method employs a different modeling strategy, including docking, homology modeling, and deep learning. The accuracy of each method was evaluated by reproducing the 3D structures of a dataset of 87 TCRpMHC complexes with experimentally determined crystal structures available on the Protein Data Bank (PDB). All selected structures were limited to human MHC alleles, presenting a diverse set of peptide ligands. A detailed analysis of produced models was conducted using multiple metrics, including Root Mean Square Deviation (RMSD) and standardized assessments from CAPRI and DockQ. Special attention was given to the complementarity-determining region (CDR) loops of the TCRs and to the peptide ligands, which define most of the unique features and specificity of a given TCRpMHC interaction. Our study provides an optimistic view of the current state-of-the-art for TCRpMHC modeling but highlights some remaining challenges that must be addressed in order to support the future application of these tools for TCR engineering and computer-aided design of TCR-based immunotherapies.

A Computational Strategy for the Rapid Identification and Ranking of Patient-Specific T Cell Receptors Bound to Neoantigens.

T cell receptor (TCR) recognition of a peptide-major histocompatibility complex (pMHC) is crucial for adaptive immune response. The identification of therapeutically relevant TCR-pMHC protein pairs is a bottleneck in the implementation of TCR-based immunotherapies. The ability to computationally design TCRs to target a specific pMHC requires automated integration of next-generation sequencing, protein-protein structure prediction, molecular dynamics, and TCR ranking. A pipeline to evaluate patient-specific, sequence-based TCRs to a target pMHC is presented. Using the three most frequently expressed TCRs from 16 colorectal cancer patients, the protein-protein structure of the TCRs to the target CEA peptide-MHC is predicted using Modeller and ColabFold. TCR-pMHC structures are compared using automated equilibration and successive analysis. ColabFold generated configurations require an ≈2.5× reduction in equilibration time of TCR-pMHC structures compared to Modeller. The structural differences between Modeller and ColabFold are demonstrated by root mean square deviation (≈0.20nm) between clusters of equilibrated configurations, which impact the number of hydrogen bonds and Lennard-Jones contacts between the TCR and pMHC. TCR ranking criteria that may prioritize TCRs for evaluation of in vitro immunogenicity are identified, and this ranking is validated by comparing to state-of-the-art machine learning-based methods trained to predict the probability of TCR-pMHC binding.

Abstract 3598: A new strategy for T cell therapy: T cells secreting TCR anti-CD3 bispecific T-cell engager

Abstract TCR-engineered T (TCR-T) cells and TCR-anti-CD3 bispecific T-cell engagers (TCEs) are potent TCR-based therapeutic agents with distinct advantages and limitations in tumor treatment. TCR-T cells offer durable persistence within patients but necessitate personalized manufacturing and lack the capacity to harness bystander T cells. Conversely, TCEs are readily available as "off-the-shelf" products and can recruit bystander T cells, yet they exhibit a shorter lifespan. In our study, we sought to merge the merits of both approaches by engineering T cells to secrete a TCR-anti-CD3 TCE specific for alpha fetoprotein (AFP), a tumor-associated antigen abundantly expressed in hepatocellular carcinoma (HCC). We initially identified a TCR with specificity for the AFP158-166 peptide bound to HLA-A*02:01 and enhanced its affinity to picomolar via phage display. To facilitate efficient secretion by T cells, we adapted the high-affinity TCR to a single-chain format (scTCR) and fused it with a CD3-specific single-chain antibody fragment (scAFP-TCE). Our findings demonstrated that scAFP-TCE effectively redirected bystander T cells to engage in the lysis of HCC cells. Moreover, scAFP-TCE could be secreted by T cells transduced with lentiviral particles encoding the TCE gene. These transduced T cells exhibited potent antitumor activity both independently and by enlisting bystander T cells. This innovative T cell strategy, combining the bystander-recruiting ability of fusion proteins with the durable persistence seen in T cell therapy after a single infusion, presents a promising alternative to conventional TCR-based therapeutic agents. We anticipate that this novel approach may hold substantial potential for enhancing HCC treatment and expanding the scope of TCR-based immunotherapies. Citation Format: Hanli Sun, Jiao Huang, Kai Zhan, Wanli Wu, Xianqing Tang, Min Liu, Shanshan Guo, Hongjun Zheng, Yingjie Huang, Shi Zhong. A new strategy for T cell therapy: T cells secreting TCR anti-CD3 bispecific T-cell engager [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 3598.

Enhanced T cell receptor specificity through framework engineering.

Development of T cell receptors (TCRs) as immunotherapeutics is hindered by inherent TCR cross-reactivity. Engineering more specific TCRs has proven challenging, as unlike antibodies, improving TCR affinity does not usually improve specificity. Although various protein design approaches have been explored to surmount this, mutations in TCR binding interfaces risk broadening specificity or introducing new reactivities. Here we explored if TCR specificity could alternatively be tuned through framework mutations distant from the interface. Studying the 868 TCR specific for the HIV SL9 epitope presented by HLA-A2, we used deep mutational scanning to identify a framework mutation above the mobile CDR3β loop. This glycine to proline mutation had no discernable impact on binding affinity or functional avidity towards the SL9 epitope but weakened recognition of SL9 escape variants and led to fewer responses in a SL9-derived positional scanning library. In contrast, an interfacial mutation near the tip of CDR3α that also did not impact affinity or functional avidity towards SL9 weakened specificity. Simulations indicated that the specificity-enhancing mutation functions by reducing the range of loop motions, limiting the ability of the TCR to adjust to different ligands. Although our results are likely to be TCR dependent, using framework engineering to control TCR loop motions may be a viable strategy for improving the specificity of TCR-based immunotherapies.

CD22 TCR-Engineered T Cells Exert Anti-Leukemia Cytotoxicity without Causing Inflammatory Responses

Chimeric antigen receptor (CAR) T cells are remarkably effective, especially against B-cell malignancies. However, CAR-T cell therapy is also associated with cytokine-driven inflammatory toxicities such as cytokine release syndrome. Similar cytokine-related toxicity can occur after bispecific T-cell engager (BiTE) administration. It is unknown whether these inflammatory responses are specifically related to T-cell-mediated therapies that are activated through artificial CAR or BiTE signals. Cytokine-driven inflammatory toxicities were not commonly encountered prior to the wide-spread use of CAR-T cells in the clinic–neither from the adoptive transfer of ex vivo-expanded autologous polyclonal antigen-specific T cells, tumor-infiltrating lymphocytes, nor TCR-engineered T cells (TCR-T cells). However, it is challenging to compare these diverse cell products, utilized in different clinical contexts and against different antigens. Preclinical comparisons of CARs and TCRs consistently showed lower sensitivity of CARs for antigen as compared to that of TCRs, at least partly due to inefficient downstream signaling of the former (Salter AI et al. Science Signaling. 2021; Gudipati V et al. Nat Immunol. 2020; Harris DT et al. J Immunol. 2018, Oren R et al. J Immunol. 2014). Nonetheless, it is not known whether these differences in signaling and T cell effector functions affect the preponderance of eliciting inflammatory responses. Here, we discovered a TCR targeting the CD22 antigen expressed by various B-cell malignancies in order to evaluate whether anti-leukemia/lymphoma T-cell cytotoxicity could be achieved without causing inflammatory responses by using TCR-activated T cells rather than CAR-activated T cells for the same antigen, CD22. A TCR specific to HLA-A*02:01-restricted epitope of human CD22 was isolated from an HLA-A2 negative healthy donor T cells through allogeneic in vitro stimulations. TCR alpha/beta-paired single cell sequencing was performed, and the sequence of the most enriched TCR clonotype was reconstructed in a retroviral expression vector. Primary human T cells transduced with this CD22 TCR recognized malignant B-cell lines only when they expressed both HLA-A*02:01 and CD22, confirming the intended specificity of the TCR. The TCR did not cross-react with other human proteins. CD22 TCR-T cells were then compared with CD22 CAR-T cells that are successful in the treatment of patients with CD22+ leukemia/lymphoma but associated with dose-limiting inflammatory toxicities (Shah NN et al. J Clin Oncol. 2020, Frank MJ et al. Blood. 2021). CD22 TCR-T cells mediated efficient in vitro cytotoxicity against CD22-dim lymphoma cells whereas CD22 CAR-T cells exerted suboptimal cytotoxicity. Importantly, TCR-T cells (1e7-3e7 cells/mouse) also cleared leukemia in xenograft models without inducing systemic proinflammatory cytokine elevation (Figure). In contrast, CD22 CAR-T cells, given at the dose necessary to control in vivo leukemia growth (5e6 cells/mouse), led to significantly increased levels of circulating proinflammatory cytokines such as IFNγ (Figure), TNFα, GM-CSF, MIP-1α and MIP-1β. Systemic cytokine elevation after CAR-T cell infusion exacerbated when NSG xenograft mice were pre-infused (24 hours prior to adoptive T cell transfer) with PBMC containing myeloid cell populations that were autologous to T cells. TCR-T cell therapy was not associated with proinflammatory cytokine elevation even in the presence of myeloid cell populations. Consistent with these in vivo observations, in vitro cytokine production by CAR-T cells were higher compared to TCR-T cells in response to CD22+ leukemia cells. Moreover, T cells activated through the CD22 TCR and CD22 CAR by the identical leukemia cell line demonstrated differential transcriptional signatures; CAR-T cells exhibited a disproportionate and significant upregulation of inflammatory pathways compared to TCR-T cells while effector response pathways were either equivalent or enriched in TCR-T cells. Together, these results highlight the opportunity for TCR-T cells to mediate anti-leukemia responses with a potentially safer toxicity profile compared to CAR-T cells. Our work provides the basis for incorporating TCR-based immunotherapy into the treatment paradigm for patients with relapsed/refractory B-cell malignancies, improving the safety of adoptive cell therapy.

T-Cell Receptor-Engineered T Cells Targeting FLT3-D835 Mutation-Derived Neoantigens in Acute Myeloid Leukemia

The adoptive transfer of T-cell receptor (TCR)-engineered T cells has demonstrated a remarkable anti-leukemic potential in mice models and human research for acute myeloid leukemia (AML). Neoantigens arising from somatic mutations are ideal targets for immunotherapy due to their high immunogenicity and harmless to normal cells. Therefore, neoantigen-specific TCR-based immunotherapy may have great prospects for AML. Activating mutations in FMS-like tyrosine kinase 3 ( FLT3) represent the most frequent genomic alterations of AML, including internal tandem duplication (ITD) in the juxtamembrane domain and point mutations within the tyrosine kinase domain, mostly at the activation loop residue D835. A number of FLT3 inhibitors have been clinically employed for FLT3-mutated AML. However, a durable response is limited by resistance-conferring secondary kinase domain mutations, notably point mutations at D835. Therefore, there is a critical need for novel and potent treatments to eliminate AML cells harboring FLT3-D835 mutations. Here, we show the construction of TCR-engineered T cells targeting FLT3-D835 mutants, presented in the context of HLA-A *02:01. We first identified neoepitopes derived from D835Y/H/V/F/I with strong affinities to HLA-A *02:01 using NetMHCpan 4.1. These neoepitopes could form the stable complex with HLA-A *02:01 on the cell surface. Furthermore, CD8 + T cells specific for D835H were detected in the peripheral blood of AML patients carrying D835H mutant at different stages of the illness, most of which were memory T cells. Moreover, one neoepitope (Neo-D835H) induced IFN-γ secretion when co-cultured with peripheral blood mononuclear cells (PBMCs) isolated from AML patients with D835H mutant. Neo-D835H-specific T cells were effectively generated through the rapid stimulation of CD8 + T cells isolated from an HLA-A *02:01-positive healthy donor with Neo-D835H-loaded autologous dendritic cells. These T cells were recognized by Neo-D835H-HLA-A2-Tetramer (0.37%) and consequently isolated by flow cytometry to perform TCRα and TCRβ sequencing. Both the TCRA and TCRB clonotypes showed dominant sequences that accounted for more than 50% of the total, suggesting that T cells with these dominant TCRA and TCRB-paired sequences were likely Neo-D835H-specific. We thus next generated TCR-engineered T cells with these TCRA- and TCRB-paired sequences for further functional analysis. These TCR-engineered T cells recognized Neo-D835H and showed cytotoxic activity against Neo-D835H-pulsed T2 cells in an antigen dose-dependent manner, while showed no responses to the T2 cells pulsed with the corresponding wild-type peptide. Taken together, we present novel neoantigens with promise as immunotherapy targets for AML and other hematologic malignancies with FLT3-D835 mutations. Future studies will evaluate the efficacy of Neo-D835H-specific TCR-engineered T-cell-based immunotherapy in vivo in patient-derived xenograft murine model and TCR-based therapeutic approaches targeting other FLT3-TKD mutations.

Abstract 895: Tumor-wide neoantigen-specific T-cells infiltrating mutant IDH1 low-grade gliomas and persisting in peripheral blood allow for personalized TCR-based immunotherapies

Abstract BACKGROUND: The low mutational burden and immunologically “cold” microenvironment of mutant IDH1 low-grade gliomas (LGG) are considerable challenges facing immunotherapy against these tumor types. However, we hypothesize that LGG-targeting T-cells may exist at low frequency and with limited regional infiltration within the tumor. Multi-region tumor sampling coupled with high-throughput T-cell receptor (TCR) profiling across the LGG landscape detected neoantigen-specific T-cells that persisted in peripheral blood. TCR-engineered T-cells transduced with these TCRs demonstrated neoantigen-specific immunogenicity. METHODS: Maximal-anatomical sampling of at least 10 distinct tumor regions were collected at the initial resection for three WHO Grade II diffuse astrocytoma patients for exome-based prediction of clonally and subclonally expressed neoantigens, RNAseq analysis of regional immune cell composition, and TCR beta deep sequencing. We used these predictions to generate a barcoded library of patient-specific peptide-HLA multimers loaded with predicted neoepitopes. With this library, neoantigen-specific CD8+ T-cells were captured and isolated from patient peripheral blood. Single cell TCR sequencing allowed us to identify the neoantigen-reactive TCR clonotypes which were transduced subsequently into Jurkat76 cell lines for functional validation. RESULTS: We screened patient-derived peripheral blood drawn two years after initial resection in 3 mutant IDH1 LGG patients and detected a total of 20 TCR clonotypes recognizing neoepitopes derived from truncal, tumor-wide mutations in CNTNAP1 (n=8), TP53 (n=3), and MRPL46 (n=2) as well as subclonal mutations in PRMT5 (n=1) and ZDHHC5 (n=6). Multi-sampling RNAseq analysis indicated varying degrees of interpatient and intratumoral immune infiltration as well as distally located populations of neoantigen-reactive T-cells within the tumor, suggesting widespread migration of neoantigen-specific T-cells across the glioma landscape. We proceeded with TCR functional analysis for one patient (P375) with 5 detected TCR clonotypes recognizing neoantigens derived from mutations in PRMT5, MRPL46, and TP53. Jurkat76 cells transduced with the mutant-PRMT5-specific TCR demonstrated a neoantigen-specific immune response when co-cultured with mutant-PRMT5 pulsed-antigen presenting cells expressing HLA-A*0201 (T2 cells). CONCLUSION: Our study demonstrates the existence and persistence of neoantigen-targeting T-cells within the blood and tumor of mutant IDH1 LGG patients. We identified a TCR clonotype that successfully recognizes and induces an immune response against mutant-PRMT5. These findings suggest a feasible methodology to develop personalized T-cell-based immunotherapies for patients with mutant IDH1 LGGs. Citation Format: Darwin W. Kwok, Michael Y. Zhang, Cliff Wang, Nicholas Stevers, Tyler Borrman, Zheng Pan, Benjamin Yuen, Songming Peng, Diana Nguyen, Michael Martin, Chibo Hong, Stephanie Hilz, Joanna Phillips, Anny Shai, Nancy Ann Oberheim Bush, Shawn Hervey-Jumper, Michael McDermott, Stefanie Mandl, Hideho Okada, Joseph Costello. Tumor-wide neoantigen-specific T-cells infiltrating mutant IDH1 low-grade gliomas and persisting in peripheral blood allow for personalized TCR-based immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 895.

Abstract IA011: Identifying tumor-restricted target antigens for adoptive cellular immunotherapy to treat Ewing Sarcoma using multi-omic discovery platforms

Abstract Background: In the last four decades, no significant improvement in survival rates for children with Ewing Sarcoma (EWS) has been made. Less than 30% of patients with metastatic disease are long- term survivors and standard therapy incorporates dose-intensive cytotoxic chemotherapy risking lifelong late effects. Engineered T cells (TCRs/CARs) can mediate impressive clinical anti-tumor activity and would represent a promising treatment option for patients. Development of potent and safe adoptive cellular immunotherapies (ACT) relies on antigents that are absent from any normal, vital tissue in the body to avoid on-target/off-tumor recognition, which conventional methods often fail to identify. Therefore, novel pipelines for rational target antigen discovery are critical for advancing ACT for the treatment of the EWS. Methods: To identify tumor antigens that meet the requirement for ACT targets, we performed differential gene expression analysis of compiled transcriptomes from EWS tumors (n=120) and normal tissues (n=42). Bioinformatic cell-surface annotation identified top differentially expressed targets best suited for CAR T cells (Heitzeneder et. al, JNCI 2019). However, more than 70% of the human proteome represent intracellular molecules, a target-pool accessible to TCRs through presentation of peptides via MHC. To empirically determine the immunopeptidome, we profiled EWS cells by immunoprecipitation with a pan-MHC-I antibody, followed by liquid chromatography and tandem mass spectrometry (LC-MS/MS). To identify peptide-MHC antigens best suited for TCRs, this data was incorporated with the differential tumor-to-normal transcriptome. Results: We identified 584 genes as overexpressed in EWS(LogFC>1, P<.01) compared to normal tissue. A total of 24 genes showed high abundance in EWS (AVElog2[TPM+1]>5) and low normal tissue expression (AVElog2[TPM+1]<2). Of those, 11 were predicted to be cell surface-associated and possess the potential to serve as CAR T-cell targets. This identified pregnancy-associated plasma protein-A (PAPP-A), a placental antigen expressed at the maternal-fetal interface (Heitzeneder et al, JNCI 2019). Immunopeptidome analysis of HLA-A*0201+EW8 cells showed that peptides of ~30% of the top 24 genes were presented by MHC-I. No neoantigenic peptides caused by common mutations in EWS or the EWS-Fli1 breakpoint region were found. Instead, immunopeptidome analysis identified oncofetal and cancer-testis antigens for TCR-based treatment approaches. Conclusions: Truly tumor-specific antigens are rare in pediatric solid tumors, yet, they often manifest stalled fetal developmental programs and continue to express placental, oncofetal and cancer testis antigens. Those represent an attractive pool of target antigens for the development of ACT to treat EWS, which is otherwise characterized by lofe immunogenicity. Comprehensive analysis of the surfaceome and immunopeptidome has proven an efficient method to identify such target antigens and facilitates the preclinical development of CAR-T cell and TCR-based immunotherapies. Citation Format: Sabine Heitzeneder. Identifying tumor-restricted target antigens for adoptive cellular immunotherapy to treat Ewing Sarcoma using multi-omic discovery platforms [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA011.

A Costimulatory CAR Improves TCR-based Cancer Immunotherapy

T-cell receptors (TCR) recognize intracellular and extracellular cancer antigens, allowing T cells to target many tumor antigens. To sustain proliferation and persistence, T cells require not only signaling through the TCR (signal 1), but also costimulatory (signal 2) and cytokine (signal 3) signaling. Because most cancer cells lack costimulatory molecules, TCR engagement at the tumor site results in incomplete T-cell activation and transient antitumor effects. To overcome this lack of signal 2, we genetically modified tumor-specific T cells with a costimulatory chimeric antigen receptor (CoCAR). Like classical CARs, CoCARs combine the antigen-binding domain of an antibody with costimulatory endodomains to trigger T-cell proliferation, but CoCARs lack the cytotoxic CD3ζ chain to avoid toxicity to normal tissues. We first tested a CD19-targeting CoCAR in combination with an HLA-A*02:01-restricted, survivin-specific transgenic TCR (sTCR) in serial cocultures with leukemia cells coexpressing the cognate peptide-HLA complex (signal 1) and CD19 (signal 2). The CoCAR enabled sTCR+ T cells to kill tumors over a median of four additional tumor challenges. CoCAR activity depended on CD19 but was maintained in tumors with heterogeneous CD19 expression. In a murine tumor model, sTCR+CoCAR+ T cells improved tumor control and prolonged survival compared with sTCR+ T cells. We further evaluated the CoCAR in Epstein-Barr virus-specific T cells (EBVST). CoCAR-expressing EBVSTs expanded more rapidly than nontransduced EBVSTs and delayed tumor progression in an EBV+ murine lymphoma model. Overall, we demonstrated that the CoCAR can increase the activity of T cells expressing both native and transgenic TCRs and enhance antitumor responses.

The Promise of Personalized TCR-Based Cellular Immunotherapy for Cancer Patients.

Mutation-derived neoantigens are now established as attractive targets for cancer immunotherapy. The field of adoptive T cell transfer (ACT) therapy was significantly reshaped by tumor neoantigens and is now moving towards the genetic engineering of T cells with neoantigen-specific T cell receptors (TCRs). Yet, the identification of neoantigen-reactive TCRs remains challenging and the process needs to be adapted to clinical timelines. In addition, the state of recipient T cells for TCR transduction is critical and can affect TCR-ACT efficacy. Here we provide an overview of the main strategies for TCR-engineering, describe the selection and expansion of optimal carrier cells for TCR-ACT and discuss the next-generation methods for rapid identification of relevant TCR candidates for gene transfer therapy.

Cooperative ectodomain interaction among TCRαβ, CD3δɛ and CD3γɛ

Abstract The T-cell receptor (TCR) complex comprises of the ligand-binding subunit TCRαβ, and the signaling subunits CD3δɛ, CD3γɛ and CD3ζζ, with the Cα/Cβ in proximity to both CD3δɛ and CD3γɛ extracellularly. Direct measurements of the ectodomain interactions had not been successful, although they are believed to be important for TCR stability and functionality. Mechanical force has been shown to modulate TCR–ligand interactions. The TCR mechanosensor hypothesis predicts that force-encoded information may transmit from pMHC to CD3 via TCR-CD3 interaction. Evaluating ectodomain interactions among TCRαβ, CD3δɛ and CD3γɛ can help elucidate the TCR triggering mechanism and further guide the design of TCR-based immunotherapy. Using two mechanical based assays, we were able to measure the weak two-dimensional (2D) affinities among ectodomains of human TCRαβ (2B4-LC13), and human CD3δɛ or CD3γɛ and showed catch bond formation where lifetimes of TCRαβ–CD3δɛ and TCRαβ–CD3γɛ bonds are prolonged by forces <15 pN. Remarkably, CD3δɛ and CD3γɛ bind TCRαβ cooperatively, forming more bonds that last longer when both CD3s interact with TCRαβ as a whole than the sum of either CD3 interacting with TCRαβ individually. Interestingly, these measurements are comparable to 2D affinity and force-dependent bond lifetime of the 2B4 TCR interaction with its cognate ligand K5:I-Ek, supporting their relevance to TCR function. Using molecular dynamics simulations based on a published Cryo-EM structure, we identified the formation of long-lasting CD3δɛ–TCRαβ–CD3γɛ trimolecular bonds as the structural mechanisms of the cooperativity. Our work helps explain TCR function under force and suggests strategies for engineering of TCR for immunotherapy applications.

Targeting KRAS mutations with HLA class II-restricted TCRs for the treatment of solid tumors

ABSTRACT T-cell receptor (TCR) redirected T cells are considered as the next generation of care for the treatment of numerous solid tumors. KRAS mutations are driver neoantigens that are expressed in over 25% of all cancers and are thus regarded as ideal targets for Adoptive Cell Therapy (ACT). We have isolated four KRAS-specific TCRs from a long-term surviving pancreatic cancer patient vaccinated with a mix of mutated KRAS peptides. The sequence of these TCRs could be identified and expressed in primary cells. We demonstrated stable expression of all TCRs as well as target-specific functionality when expressing T cells were co-incubated with target cells presenting KRAS peptides. In addition, these TCRs were all partially co-receptor independent since they were functional in both CD4 and CD8 T cells, thus indicating high affinity. Interestingly, we observed that certain TCRs were able to recognize several KRAS mutations in complex with their cognate Human leukocyte antigen (HLA), suggesting that, here, the point mutations were less important for the HLA binding and TCR recognition, whereas others were single-mutation restricted. Finally, we demonstrated that these peptides were indeed processed and presented, since HLA-matched antigen presenting cells exogenously loaded with KRAS proteins were recognized by TCR-transduced T cells. Taken together, our data demonstrate that KRAS mutations are immunogenic for CD4 T cells and are interesting targets for TCR-based cancer immunotherapy.

PRAME mRNA Expression in AML/MDS and HLA Genotype Analysis: Impact on Population Coverage and Design of TCR-Based Immunotherapies

The cancer-testis antigen PRAME (PReferentially expressed Antigen in MElanoma) is expressed at high levels in several oncological indications, including leukemias and solid tumors, as confirmed by mRNA or protein expression studies. Its low to absent expression on normal tissues allows suitable discrimination of tumors for targeted immunotherapy. As an intracellular protein, PRAME-derived peptides can be presented by several HLA class I allotypes, including HLA-A*02:01, and specific T cell recognition has been reported for different PRAME peptide-HLA (pHLA) ligands. Thus, developing a small set of TCRs covering different HLAs could increase the number of PRAME-positive patients eligible for treatment with T cell receptor (TCR)-based immunotherapy approaches. We determined PRAME expression in a group of Caucasian patients with acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (MDS). In addition, we analyzed the HLA-A genotype distribution in a set of healthy blood donors to determine how many and which different TCRs would be needed to optimally cover a Caucasian population in Germany. For the analysis of PRAME expression, bone marrow (BM) and peripheral blood (PB) samples from 165 patients (AML=133, MDS=32), obtained from multiple clinical sites across Germany, were studied. Ninety-six samples were from newly diagnosed patients (58%) while 68 samples were from relapsed/refractory patients (41%), irrespective of percentage of leukemic blasts. Disease status was not known for one patient. PRAME mRNA expression was measured by quantitative reverse transcriptase polymerase chain reaction (RT-qPCR). A threshold for PRAME-negativity was derived by measuring average PRAME mRNA expression in 22 healthy individuals. Detection of 99 or more copies of PRAME, as measured in 12.5 ng RNA, was considered positive. Of the 165 AML/MDS patients, 79 (48%; AML= 69, MDS=10) were positive for PRAME mRNA. Among relapsed/refractory patients, 52% (35/68) were classified as PRAME-positive, versus 45% (43/96) of first-diagnosis AML/MDS patients. NGS high-resolution HLA-A genotyping showed that 55% of all patients had an HLA-A*02:01 allotype, which is higher than the approximately 45% expected for a random Caucasian population and accounted for by the fact that some patients were included with known HLA-A2 status due to prior assessment for transplant eligibility. In combination, 43 (26%) AML/MDS patients were positive for PRAME above the threshold level as well as for HLA-A*02:01, thereby being potential candidates for treatment with PRAME-specific, HLA-A*02:01-restricted TCR immunotherapy. In a group of 141 healthy local blood donors, NGS high-resolution HLA-A genotyping showed an expected distribution of HLA-A*02:01, HLA-A*01:01, HLA-A*03:01, HLA-A*24:02 and HLA-A*11:01 of 43%, 26%, 25%, 19% and 8% respectively, with some donors heterozygous for two of these particular allotypes. To determine the smallest number of TCRs needed to address the largest number of patients, we assessed how the non-overlapping distribution for each potential HLA-A allotype would contribute to patient coverage. Starting with the 43% of patients who would be covered by an HLA-A*02:01-restricted TCR immunotherapy product, additional coverage of 21%, 14%, 9%, and 1% of patients could be achieved with TCR products using HLA-A*01:01-, HLA-A*03:01-, HLA-A*24:02- and HLA-A*11:01-restricted TCRs, respectively. In conclusion, approximately half of AML/MDS patients are PRAME-positive and could be addressed with PRAME-specific TCR immunotherapy approaches. An HLA-A*02:01-restricted TCR addresses the most patients in a population in Germany at 43%, while coverage could be increased to 87% with addition of three TCRs restricted by the next most common HLA-A allotypes, contingent upon suitable TCR discovery. Disclosures Addo: Medigene Immunotherapies GmbH: Current Employment. Davari:Medigene Immunotherapies: Current Employment. Raffegerst:Medigene Immunotherapies GmbH: Current Employment, Current equity holder in publicly-traded company. Tippmer:Medigene Immunotherapies: Current Employment. Schendel:Medigene AG: Current Employment, Current equity holder in publicly-traded company. Pinkernell:Medigene Immunotherapies GmbH: Current Employment, Current equity holder in publicly-traded company.

Abstract 3231: The chimeric co-stimulatory receptor PD1-41BB enhances the function of T cell receptor (TCR)-modified T cells targeting solid tumors

Abstract Introduction: The use of cellular immunotherapies has led to impressive complete and durable clinical responses in patients with certain types of hematological cancers. However, positive clinical results in solid tumor indications are still rare and many patients are in urgent need of alternative treatment options for several different indications. It has become clear that expression of inhibitory immune checkpoint molecules as well as harsh metabolic conditions in the tumor microenvironment (TME) are responsible for lack of activity of T cell immunotherapies in several settings, especially solid tumors. Here additional strategies are necessary to efficiently employ cellular immunotherapies. With the aim to further enhance the clinical efficacy of TCR-based immunotherapies under immunosuppressive conditions found in tumors, we analyzed the ability of PD1-41BB, a chimeric co-stimulatory receptor, to reverse the natural inhibitory PD-1/PD-L1 interaction into a supporting co-stimulatory signal in TCR-modified T cells encountering tumor cells. Methods: We evaluated the ability of the chimeric co-stimulatory receptor PD1-41BB to improve activity of TCR-modified T cells using 2-dimensional or 3-dimensional in vitro assays that model different immunosuppressive conditions found in tumors. Results: We demonstrate that chronic stimulation as well as several immunosuppressive factors of the TME, such as tumor cell expression of inhibitory immune checkpoint molecules or glucose restriction, impede the ability of TCR-transduced T cells to produce inflammatory cytokines and to efficiently lyse tumor cells. By using a chimeric co-stimulatory receptor consisting of the extracellular part of PD-1 and the co-stimulatory domain of 4-1BB we reversed the naturally occurring inhibitory PD-1/PD-L1 interaction to provide a co-stimulatory signal for improved T cell activity under immunosuppressive conditions or chronic stimulation. Addition of the chimeric co-stimulatory receptor PD1-41BB to TCR-modified T cells led to enhanced release of Interferon-γ, increased tumor cell killing, T cell proliferation and persistence in these T cell-tumor cell models. Conclusions: These preclinical studies support our approach to enhance the clinical efficacy of TCR-T therapies in PD-L1-positive malignancies by reversing naturally occurring inhibitory signals enabling counteraction of checkpoint-mediated dysfunction and metabolic insufficiency. The chimeric co-stimulatory PD1-41BB receptor has the potential to further enhance the clinical efficacy of TCR-modified T cells in patients with PD-L1-positive malignancies. Further preclinical in vitro and in vivo studies are ongoing to investigate the safety and efficacy of PD1-41BB in combination with multiple TCR candidates to explore its feasibility for the treatment of various cancers. Citation Format: Nadja Sailer, Melanie Salvermoser, Maria Gerget, Sarah Thome, Angelika J. Fischbeck, Svenja Ruehland, Luis F. Olguín-Contreras, Maja Buerdek, Christian Ellinger, Elfriede Noessner, Dolores J. Schendel, Patrik Kehler. The chimeric co-stimulatory receptor PD1-41BB enhances the function of T cell receptor (TCR)-modified T cells targeting solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3231.

T cell receptor-based cancer immunotherapy: Emerging efficacy and pathways of resistance.

Adoptive cell transfer (ACT) using chimeric antigen receptor (CAR)‐modified T cells can induce durable remissions in patients with refractory B‐lymphoid cancers. By contrast, results applying CAR‐modified T cells to solid malignancies have been comparatively modest. Alternative strategies to redirect T cell specificity and cytolytic function are therefore necessary if ACT is to serve a greater role in human cancer treatments. T cell receptors (TCRs) are antigen recognition structures physiologically expressed by all T cells that have complementary, and in some cases superior, properties to CARs. Unlike CARs, TCRs confer recognition to epitopes derived from proteins residing within any subcellular compartment, including the membrane, cytoplasm and nucleus. This enables TCRs to detect a broad universe of targets, such as neoantigens, cancer germline antigens, and viral oncoproteins. Moreover, because TCRs have evolved to efficiently detect and amplify antigenic signals, these receptors respond to epitope densities many fold smaller than required for CAR‐signaling. Herein, we summarize recent clinical data demonstrating that TCR‐based immunotherapies can mediate regression of solid malignancies, including immune‐checkpoint inhibitor refractory cancers. These trials simultaneously highlight emerging mechanisms of TCR resistance. We conclude by discussing how TCR‐based immunotherapies can achieve broader dissemination through innovations in cell manufacturing and non‐viral genome integration techniques.

Key Features Relevant to Select Antigens and TCR From the MHC-Mismatched Repertoire to Treat Cancer.

Adoptive transfer of T cells transgenic for tumor-reactive T-cell receptors (TCR) is an attractive immunotherapeutic approach. However, clinical translation is so far limited due to challenges in the identification of suitable target antigens as well as TCRs that are concurrent safe and efficient. Definition of key characteristics relevant for effective and specific tumor rejection is essential to improve current TCR-based adoptive T-cell immunotherapies. We here characterized in-depth two TCRs derived from the human leukocyte antigen (HLA)-mismatched allogeneic repertoire targeting two different myeloperoxidase (MPO)-derived peptides presented by the same HLA-restriction element side by side comprising state of the art biochemical and cellular in vitro, in vivo, and in silico experiments. In vitro experiments reveal comparable functional avidities, off-rates, and cytotoxic activities for both TCRs. However, we observed differences especially with respect to cytokine secretion and cross-reactivity as well as in vivo activity. Biochemical and in silico analyses demonstrate different binding qualities of MPO-peptides to the HLA-complex determining TCR qualities. We conclude from our biochemical and in silico analyses of peptide-HLA-binding that rigid and high-affinity binding of peptides is one of the most important factors for isolation of TCRs with high specificity and tumor rejection capacity from the MHC-mismatched repertoire. Based on our results, we developed a workflow for selection of such TCRs with high potency and safety profile suitable for clinical translation.

Induction of neoantigen-reactive T cells from healthy donors.

The identification of immunogenic neoantigens and their cognate T cells represents the most crucial and rate-limiting steps in the development of personalized cancer immunotherapies that are based on vaccination or on infusion of T cell receptor (TCR)-engineered T cells. Recent advances in deep-sequencing technologies and in silico prediction algorithms have allowed rapid identification of candidate neoepitopes. However, large-scale validation of putative neoepitopes and the isolation of reactive T cells are challenging because of the limited availablity of patient material and the low frequencies of neoepitope-specific T cells. Here we describe a standardized protocol for the induction of neoepitope-reactive T cells from healthy donor T cell repertoires, unaffected by the potentially immunosuppressive environment of the tumor-bearing host. Monocyte-derived dendritic cells (DCs) transfected with mRNA encoding candidate neoepitopes are used to prime autologous naive CD8+ T cells. Antigen-specific T cells that recognize endogenously processed and presented epitopes are detected using peptide-MHC (pMHC) multimers. Single multimer-positive T cells are sorted for the identification of TCR sequences, after an optional step that includes clonal expansion and functional characterization. The time required to identify neoepitope-specific T cells is 15 d, with an additional 2-4 weeks required for clonal expansion and downstream functional characterization. Identified neoepitopes and corresponding TCRs provide candidates for use in vaccination and TCR-based cancer immunotherapies, and datasets generated by this technology should be useful for improving algorithms to predict immunogenic neoantigens.

NK cells specifically TCR-dressed to kill cancer cells.

BackgroundAdoptive T-cell transfer of therapeutic TCR holds great promise to specifically kill cancer cells, but relies on modifying the patient's own T cells ex vivo before injection. The manufacturing of T cells in a tailor-made setting is a long and expensive process which could be resolved by the use of universal cells. Currently, only the Natural Killer (NK) cell line NK-92 is FDA approved for universal use. In order to expand their recognition ability, they were equipped with Chimeric Antigen Receptors (CARs). However, unlike CARs, T-cell receptors (TCRs) can recognize all cellular proteins, which expand NK-92 recognition to the whole proteome.MethodsWe herein genetically engineered NK-92 to express the CD3 signaling complex, and showed that it rendered them able to express a functional TCR. Functional assays and in vivo efficacy were used to validate these cells.FindingsThis is the first demonstration that a non-T cell can exploit TCRs. This TCR-redirected cell line, termed TCR-NK-92, mimicked primary T cells phenotypically, metabolically and functionally, but retained its NK cell effector functions. Our results demonstrate a unique manner to indefinitely produce TCR-redirected lymphocytes at lower cost and with similar therapeutic efficacy as redirected T cells.InterpretationThese results suggest that an NK cell line could be the basis for an off-the-shelf TCR-based cancer immunotherapy solution.FundThis work was supported by the Research Council of Norway (#254817), South-Eastern Norway Regional Health Authority (#14/00500-79), by OUS-Radiumhospitalet (Gene Therapy program) and the department of Oncology at the University of Lausanne.

Enhanced interaction between SEC2 mutant and TCR Vβ induces MHC II–independent activation of T cells via PKCθ/NF-κB and IL-2R/STAT5 signaling pathways

SEC2, a major histocompatibility complex class II (MHC II)-dependent T-cell mitogen, binds MHC II and T-cell receptor (TCR) Vβs to induce effective co-stimulating signals for clonal T-cell expansion. We previously characterized a SEC2 mutant with increased recognition of TCR Vβs, ST-4, which could intensify NF-κB signaling transduction, leading to IL-2 production and T-cell activation. In this study, we found that in contrast to SEC2, ST-4 could induce murine CD4+ T-cell proliferation in a Vβ8.2- and Vβ8.3-specific manner in the absence of MHC II+ antigen-presenting cells (APCs). Furthermore, although IL-2 secretion in response to either SEC2 or ST-4 stimulation was accompanied by up-regulation of protein kinase Cθ (PKCθ), inhibitor of κB (IκB), α and β IκB kinase (IKKα/β), IκBα, and NF-κB in mouse splenocytes, only ST-4 could activate CD4+ T cells in the absence of MHC II+ APCs through the PKCθ/NF-κB signaling pathway. The PKCθ inhibitor AEB071 significantly suppressed SEC2/ST-4-induced T-cell proliferation, CD69 and CD25 expression, and IL-2 secretion with or without MHC II+ APCs. Further, SEC2/ST-4-induced changes in PKCθ/NF-κB signaling were significantly relieved by AEB071 in a dose-dependent manner. Using Lck siRNA, we found that Lck controlled SEC2/ST-4-induced phosphorylation of PKCθ. We also demonstrated that the IL-2R/STAT5 pathway is essential for SEC2/ST-4-induced T-cell activation. Collectively, our data demonstrate that an enhanced ST-4-TCR interaction can compensate for lack of MHC II and stimulate MHC II-free CD4+ T-cell proliferation via PKCθ/NF-κB and IL-2R/STAT5 signaling pathways. Compared with SEC2, intensified PKCθ/NF-κB and IL-2R/STAT5 signals induced by ST-4 lead to enhanced T-cell activation. The results of this study will facilitate better understanding of TCR-based immunotherapies for cancer.

A Potent Tumor-Reactive p53-Specific Single-Chain TCR without On- or Off-Target Autoimmunity InVivo.

Genetic engineering of Tcells with a Tcell receptor (TCR) targeting tumor antigen is a promising strategy for cancer immunotherapy. Inefficient expression of the introduced TCR due to TCR mispairing may limit the efficacy and adversely affect the safety of TCR gene therapy. Here, we evaluated the safety andtherapeutic efficiency of an optimized single-chain TCR (scTCR) specific for an HLA-A2.1-restricted (non-mutated) p53(264-272) peptide in adoptive Tcell transfer (ACT) models using our unique transgenic mice expressing human p53 and HLA-A2.1 that closely mimic the human setting. Specifically, we showed that adoptive transfer of optimized scTCR-redirected Tcells does not induce on-target and off-target autoimmunity. Furthermore, ACT resulted in full tumor protection and led to a long-lived effective, antigen-specific memory Tcell response in syngeneic and xenograft models. Taken together, the study demonstrated that our scTCR specific for the broadly expressed tumor-associated antigen p53(264-272) can eradicate p53+A2.1+ tumor cells without inducing off-target or self-directed toxicities in mouse models of ACT. These data strongly support the improved safety and therapeutic efficacy of high-affinity p53scTCR for TCR-based immunotherapy of p53-associated malignancies.