Soluble T-cell Receptor Research Articles

ABBV-184: A novel survivin-specific TCR/CD3 bispecific T cell engager is active against both solid tumor and hematological malignancies.

CD3 bispecific T cell engagers (TCE), comprised of a tumor targeting domain linked to a CD3 binding domain, function by bridging target-positive tumors and CD3-expressing effector T cells enabling redirected T cell-mediated killing of tumor cells. Although the majority of CD3 bispecific molecules in clinical development incorporate tumor-targeting antibody-based binding domains, many tumor-associated antigens derive from intracellular proteins and are not accessible to targeting via antibody. Intracellular proteins processed into short peptide fragments and presented on the cell surface by major histocompatibility complex proteins (MHC) are recognized by T cell receptors (TCR) on the surface of T cells. Here we describe the generation and preclinical evaluation of ABBV-184, a novel TCR/anti-CD3 bispecific composed of a highly selective soluble TCR that binds a peptide derived from the oncogene survivin (BIRC5) bound to the Class I MHC allele human leukocyte antigen (HLA)-A*02:01 expressed on tumor cells, linked to a specific binder to the CD3 receptor on T cells. ABBV-184 drives an optimal distance between T cell and target cell thereby enabling sensitive recognition of low-density peptide/MHC targets. Consistent with the expression profile of survivin across a broad range of both hematological and solid tumors, treatment of AML and NSCLC cell lines with ABBV-184 results in T cell activation, proliferation, and potent redirected cytotoxicity of HLA-A2 positive target cell lines, both in vitro and in vivo, including patient-derived AML samples. These results indicate that ABBV-184 is an attractive clinical candidate for the treatment of patients with AML and NSCLC.

Redirecting Polyclonal T Cells against Cancer with Soluble T-Cell Receptors.

Cancer cells accumulate genetic mutations in coding proteins that may be presented by HLA as neoantigenic peptides (peptide HLA, pHLA). T cells scan for neoantigenic pHLA by the T-cell receptor (TCR):CD3 complex. This complex has the dual function of binding pHLA, by the TCR, and triggering T-cell activation by CD3. Checkpoint therapy activates exhausted T cells to kill cancer cells and generally work best against tumors with high neoantigen burden and in patients with neoantigenic-reactive T cells. TCR T-cell engagers (TCE) are a novel class of immunotherapy that bypasses these two requirements by redirecting polyclonal T cells, regardless of their native specificity, to kill a cancer cell independent of neoantigen burden. This is accomplished through deconstructing the membrane-bound TCR:CD3 complex into a soluble bispecific protein comprised of a targeting domain (TCR) and activating domain (usually anti-CD3 single-chain variable fragment). The pool of targets for TCR TCE is larger than for antibody therapeutics and includes >90% of human intra- or extracellular proteins. Most tumor-associated antigens for solid tumors are intracellular and accessible only by a TCR therapeutic. Tebentafusp, a TCR TCE directed to a peptide derived from the gp100 melanoma protein presented by HLA*A02:01, demonstrated a survival benefit in metastatic uveal melanoma (mUM). This survival benefit highlights the promise of TCR TCEs because mUM is a solid tumor with a very low neoantigen burden and has poor response to checkpoints and chemotherapy. Other TCR TCE programs are now in clinical studies for a broader range of tumors.

832P Treatment sequence with tebentafusp (tebe) and anti-PD1/ipilimumab (PD1+IPI) in HLA-A2*02:01 patients (pts) with metastatic uveal melanoma (mUM)

mUM has poor prognosis. Unlike cutaneous melanoma, PD1+IPI shows modest efficacy. In phase 2 trials median progression-free survival (mPFS) ranges between 3-5.5 mo. Tebe, a soluble T-cell receptor and CD3-directed bispecific fusion protein, resulted in prolonged overall survival (OS) (21.7 mo) in a phase 3 study, but median duration of response (mDOR) was short and PFS benefit modest. The optimal sequence of tebe and PD1+IPI is unknown.

Abstract 2891: Enhanced anti-tumor responses with a novel dual pMHC T-cell engager bispecific antibody

Abstract Intracellular tumor antigens presented as peptides on MHC (pMHC) class I molecules are attractive targets for more tumor-selective immunotherapeutic approaches with promising data already emerging from clinical trials. pMHCs have been targeted by TCR-engineered T cells or soluble recombinant T-cell receptors (TCRs) fused to an anti-CD3 fragment. Naturally occurring cancer-reactive TCRs have weak affinity and require substantial affinity enhancements for their cognate pMHC. However, the outcome of this process is difficult to predict and bears the risk for off-target cross reactivities in normal tissues, which may lead to severe adverse events in the clinic. Here, we describe a highly potent dual pMHC T-cell engager (TCE) antibody format with high specificity towards tumor-specific pMHCs utilizing an HLA-A*02:01 restricted MAGE-A4 epitope to validate the concept. A series of monovalent and bivalent antibody constructs composed of anti-MAGE-A4 binding arms, ranging in affinities from 30 nM to 100 pM, were fused to an anti-CD3 Fab fragment with lower affinity compared to that commonly used for TCR-fusions. The different antibody constructs were evaluated for selective killing of MAGE-A4/HLA-A*02 positive human U2OS osteosarcoma and A375 melanoma cancer cells versus a panel of different MAGE-A4-negative/HLA-A*02-positive human cell lines. Bivalent bispecific antibody variants mediated a 7-fold greater degree of cancer cell killing and similarly increased T cell activation compared to their monovalent bispecific counterparts. EC50 values ranged as low as single digit picomolar, while the overall cross reactivity against MAGE-A4-negative/HLA-A*02-positive cells was not substantially affected. These results prove that dual targeting of pMHCs on cancer cells provides selective and efficient T cell-mediated target cell killing and T cell activation, even at very low levels of pMHC on the cell surface, highlighting the pivotal roles played by the affinity of the individual arms, valency, and epitope densities. We further analyzed the dual pMHC TCE for potential off-target effects by recognition of similar and physiologically relevant non-MAGE-A4 peptides. T2 cells pulsed with similar peptides and co-cultured with PBMC effector cells showed no significant T cell activation or IFNg release in the presence of the dual pMHC TCE in comparison to MAGE-A4 peptide-pulsed T2 cells. Finally, we compared the dual pMHC TCE against a recombinant TCR fused to an anti-CD3 scFv, a construct that is currently in clinical development. Interestingly, the dual pMHC TCE resulted in a 3-fold more potent cancer cell killing while having significantly lower effect on cytokine production. In conclusion, dual pMHC targeting with TCE antibodies is an attractive alternative to soluble affinity-enhanced TCR-based cancer immunotherapies as they facilitate potent tumor targeting without the need for extensive affinity enhancements. Citation Format: Stephanie Jungmichel, Fabian Scheifele, Anna Sobieraj, Severin Wendelspiess, Thomas Schleier, Philip Knobel, Camilla Winnewisser, Melissa Vrohlings, Philipp Richle, Hannes Merten, Jacqueline Blunschi, Christian Leisner, Leonardo Borras. Enhanced anti-tumor responses with a novel dual pMHC T-cell engager bispecific antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2891.

A Leucine Zipper Dimerization Strategy to Generate Soluble T Cell Receptors Using the Escherichia coli Expression System.

T cell-mediated adaptive immunity plays a key role in immunological surveillance and host control of infectious diseases. A better understanding of T cell receptor (TCR) recognition of pathogen-derived epitopes or cancer-associated neoantigens is the basis for developing T cell-based vaccines and immunotherapies. Studies on the interaction between soluble TCR α:β heterodimers and peptide-bound major histocompatibility complexes (pMHCs) inform underlying mechanisms driving TCR recognition, but not every isolated TCR can be prepared in soluble form for structural and functional studies using conventional methods. Here, taking a challenging HIV-specific TCR as a model, we designed a general leucine zipper (LZ) dimerization strategy for soluble TCR preparation using the Escherichia coli expression system. We report details of TCR construction, inclusion body expression and purification, and protein refolding and purification. Measurements of binding affinity between the TCR and its specific pMHC using surface plasmon resonance (SPR) verify its activity. We conclude that this is a feasible approach to produce challenging TCRs in soluble form, needed for studies related to T cell recognition.

Uveal Melanoma Metastasis.

Simple SummarySurvival after diagnosis of metastatic uveal melanoma has not significantly improved over decades, most patients die within a year from diagnosis. Uveal melanoma is clearly distinct from cutaneous melanoma and the therapies developed for the latter do not work for the former. This is apparently due to three major aspects of UM: (i) the mutations that drive UM tumorigenesis activate two oncogenic signaling pathways and one of the two cannot yet be targeted by therapy; (ii) UM has relatively few tumor specific neo-antigens that could be presented to the immune system and therefore evades immune control; and (iii) UM shows an infiltrate of inflammatory cells that promote tumor progression. However, in the future, there might be drugs to target both oncogenic pathways that are activated in UM, new immune therapy approaches might circumvent the paucity of neo-antigens and liver directed, local therapy might improve response to cytostatic therapy. Hopefully, there will be therapies that can repeat the success obtained with targeted and immune therapy for cutaneous melanoma.Uveal melanoma (UM) is characterized by relatively few, highly incident molecular alterations and their association with metastatic risk is deeply understood. Nevertheless, this knowledge has so far not led to innovative therapies for the successful treatment of UM metastases or for adjuvant therapy, leaving survival after diagnosis of metastatic UM almost unaltered in decades. The driver mutations of UM, mainly in the G-protein genes GNAQ and GNA11, activate the MAP-kinase pathway as well as the YAP/TAZ pathway. At present, there are no drugs that target the latter and this likely explains the failure of mitogen activated kinase kinase inhibitors. Immune checkpoint blockers, despite the game changing effect in cutaneous melanoma (CM), show only limited effects in UM probably because of the low mutational burden of 0.5 per megabase and the unavailability of antibodies targeting the main immune checkpoint active in UM. The highly pro-tumorigenic microenvironment of UM also contributes to therapy resistance. However, T-cell redirection by a soluble T-cell receptor that is fused to an anti-CD3 single-chain variable fragment, local, liver specific therapy, new immune checkpoint blockers, and YAP/TAZ specific drugs give new hope to repeating the success of innovative therapy obtained for CM.

Soluble Expression of Fc-Fused T Cell Receptors Allows Yielding Novel Bispecific T Cell Engagers.

The specific recognition of T cell receptors (TCR) and peptides presented by human leukocyte antigens (pHLAs) is the core step for T cell triggering to execute anti-tumor activity. However, TCR assembly and soluble expression are challenging, which precludes the broad use of TCR in tumor therapy. Herein, we used heterodimeric Fc to assist in the correct assembly of TCRs to achieve the stable and soluble expression of several TCRs in mammalian cells, and the soluble TCRs enable us to yield novel bispecific T cell engagers (TCR/aCD3) through pairing them with an anti-CD3 antibody. The NY-ESO-1/LAGE-1 targeted TCR/aCD3 (NY-TCR/aCD3) that we generated can redirect naïve T cells to specific lysis antigen-positive tumor cells, but the potency of the NY-TCR/aCD3 was disappointing. Furthermore, we found that the activation of T cells by NY-TCR/aCD3 was mild and unabiding, and the activity of NY-TCR/aCD3 could be significantly improved when we replaced naïve T cells with pre-activated T cells. Therefore, we employed the robust T cell activation ability of staphylococcal enterotoxin C2 (SEC2) to optimize the activity of NY-TCR/aCD3. Moreover, we found that the secretions of SEC2-activated T cells can promote HLA-I expression and thus increase target levels, which may further contribute to improving the activity of NY-TCR/aCD3. Our study described novel strategies for soluble TCR expression, and the optimization of the generation and potency of TCR/aCD3 provided a representative for us to fully exploit TCRs for the precision targeting of cancers.

Novel Antivirals in Clinical Development for Chronic Hepatitis B Infection.

Globally, chronic hepatitis B (CHB) infection is one of the leading causes of liver failure, decompensated cirrhosis, and hepatocellular carcinoma. Existing antiviral therapy can suppress viral replication but not fully eradicate the virus nor the risk of liver-related complications. Novel treatments targeting alternative steps of the viral cycle or to intensify/restore the host’s immunity are being developed. We discuss novel drugs that have already entered clinical phases of development. Agents that interfere with specific steps of HBV replication include RNA interference, core protein allosteric modulation, and inhibition of viral entry or viral protein excretion (NAPs and STOPS). Agents that target the host’s immunity include toll-like receptor agonists, therapeutic vaccines, immune checkpoint modulators, soluble T-cell receptors, and monoclonal antibodies. Most have demonstrated favorable results in suppression of viral proteins and genomic materials (i.e., HBV DNA and/or pre-genomic RNA), and/or evidence on host-immunity restoration including cytokine responses and T-cell activation. Given the abundant clinical experience and real-world safety data with the currently existing therapy, any novel agent for CHB should be accompanied by convincing safety data. Combination therapy of nucleos(t)ide analogue, a novel virus-directing agent, and/or an immunomodulatory agent will be the likely approach to optimize the chance of a functional cure in CHB.

Phase 1 first-in-human study of ABBV-184 monotherapy in adult patients with previously treated acute myeloid leukemia or non-small cell lung cancer.

TPS2674 Background: Survivin, a member of the inhibitor of apoptosis protein family, is an attractive therapeutic target in cancer, due to its broad expression in solid tumors and hematologic malignancies but limited expression in normal tissues. Elevated survivin expression is associated with an increased invasive phenotype and worse clinical outcomes. ABBV-184 is a first-in-class T-cell receptor (TCR)/anti-cluster of differentiation 3 (CD3) bispecific molecule. It is composed of a soluble TCR that binds to a survivin-derived peptide bound to the class I MHC allele HLA-A2:01 expressed on tumor cells and to the CD3 receptor on T cells. Preclinical data have demonstrated that treatment with ABBV-184 results in T-cell activation, proliferation, and redirected cytotoxicity of HLA-A2:01–positive target cell lines. This first-in-human trial evaluates ABBV-184 monotherapy in patients with previously treated acute myeloid leukemia (AML) or non-small cell lung cancer (NSCLC). Methods: Patients (≥18 years, Eastern Cooperative Oncology Group performance status ≤2, HLA-A2:01 restricted genotype) with relapsed or refractory AML or NSCLC are currently enrolling in this phase 1 multicenter, open-label trial (NCT04272203), which includes parallel dose-escalation and dose-expansion phases for both diseases. Primary objectives are to determine the recommended phase 2 dose (RP2D) of ABBV-184 (dose escalation) and to assess its preliminary efficacy (dose expansion). Secondary objectives include safety, tolerability, pharmacokinetics (PK), and immunogenicity assessments (dose escalation and dose expansion) and duration of response (dose expansion). Patients will receive intravenous infusion of ABBV-184 once weekly. Dose escalation of ABBV-184 is guided by a Bayesian optimal interval design and the RP2D will be determined on the basis of clinical safety, PK, and pharmacodynamic data. For patients with AML, disease assessment is performed according to modified European LeukemiaNet-International Working Group criteria. For patients with NSCLC, response will be assessed using Response Evaluation Criteria In Solid Tumors (RECIST) v1.1 and immune RECIST. Treatment can continue until disease progression or intolerable toxicity. Biomarker assessments will include longitudinal profiling of peripheral blood immune cells and cytokines, analysis of HLA-A2 and survivin levels on AML bone marrow blasts and NSCLC tumor biopsies, and retrospective correlations of biomarker data with antitumor activity. Enrollment initiated in Sep 2020, with 7 patients enrolled as of Jan 2021. Clinical trial information: NCT04272203.

Engineering soluble T-cell receptors for therapy.

Immunotherapy approaches that target peptide–human leukocyte antigen (pHLA) complexes are becoming highly attractive because of their potential to access virtually all foreign and cellular proteins. For this reason, there has been considerable interest in the development of the natural ligand for pHLA, the T‐cell receptor (TCR), as a soluble drug to target disease‐associated pHLA presented at the cell surface. However, native TCR stability is suboptimal for soluble drug development, and natural TCRs generally have weak affinities for pHLAs, limiting their potential to reach efficacious receptor occupancy levels as soluble drugs. To overcome these limitations and make full use of the TCR as a soluble drug platform, several protein engineering solutions have been applied to TCRs to enhance both their stability and affinity, with a focus on retaining target specificity and selectivity. Here, we review these advances and look to the future for the next generation of soluble TCR‐based therapies that can target monomorphic HLA‐like proteins presenting both peptide and nonpeptide antigens.

A phase I/II first-in-human study of a novel anti-MAGE-A4 TCR/anti-CD3 bispecific (IMC-C103C) as monotherapy and in combination with atezolizumab in HLA-A*02:01-positive patients with MAGE-A4-positive advanced solid tumors (IMC-C103C-101).

TPS3165 Background: ImmTAC bispecifics are unique T cell receptor (TCR)/anti-CD3 bispecific molecules that are designed to redirect polyclonal T cells against intracellular antigens, in contrast to antibody-based therapies, which are limited to extracellular antigens. ImmTAC molecules recognize a specific peptide presented on defined Class I HLA molecules via an affinity enhanced, engineered, soluble TCR. Through the addition of an anti-CD3 scFv effector domain fused to the TCR targeting domain, ImmTAC molecules redirect T cell activity against cancer cells, regardless of the specificity of the T cell. IMC-C103C is an ImmTAC being investigated against MAGE-A4, which is among the most commonly expressed cancer testis antigens in solid malignancies, but with minimal to absent expression on normal tissues and/or hematopoietic cells. The most advanced ImmTAC in development, tebentafusp (IMCgp100), directed against melanocyte-associated lineage antigen gp100, has shown monotherapy activity in uveal melanoma and PD-1 refractory advanced cutaneous and uveal melanoma. Tebentafusp is being further evaluated in combination with durvalumab and tremelimumab. Methods: IMC-C103C-101 is a multi-center, open-label, Phase 1/2 first-in-human study of IMC-C103C as monotherapy and combination with atezolizumab in HLA-A*02:01-positive patients with MAGE-A4-positive advanced cancers. The study includes IMC-C103C monotherapy (Q1W) dose escalation, followed by expansion into indication specific arms to test for efficacy in defined patient cohorts. Concurrently, combinations with atezolizumab are planned. Primary objectives of dose escalation are to identify the MTD/RP2D, and characterize safety/tolerability. Secondary objectives include an assessment of efficacy (best overall response by RECIST v1.1), PK, PD, and ADA. IMC-C103C monotherapy dose escalation is in progress. Clinical trial information: NCT03973333 .

Abstract A24: Antigen-targeted soluble bispecific T-cell receptor (ImmTACTM) molecules in immunotherapy

Abstract Cancer immunotherapies function to harness the body’s own immune system to eradicate cancer cells. Critical to this process is the T cell, capable of directing potent and antigen-specific immune responses. T-cell receptor (TCR) antigens are short intracellularly processed peptides presented on the cell surface. As the majority of the proteome is processed and presented on the surface of cells and hence can be targeted by a TCR, TCR-based therapies offer distinct advantages over antibody-based therapies that are limited to targeting only secreted or cell surface proteins. In this study, we describe our approach to address the limitations and utilize the natural advantages of the human TCR to target a diverse range of tumor indications. Immune mobilizing monoclonal TCRs Against Cancer (ImmTAC) molecules are a novel class of bispecific biologic formed from an affinity-engineered, soluble, targeted TCR fused to a T cell redirecting anti-CD3 scFv. We describe our process to engineer high-affinity ImmTAC molecules towards novel cancer antigens that have been identified and validated in house. Additionally, we discuss the challenges and opportunities in the preclinical testing of an exquisitely human-specific biologic that led to the development of an entirely in vitro preclinical package that is proven to support the clinical development of ImmTAC molecules to clinical development. In summary, we present a novel approach to cancer immunotherapy, detailing the engineering, optimization, preclinical testing and clinical development of ImmTAC molecules, a new bispecific biologic. Note: This abstract was not presented at the conference. Citation Format: Annelise Vuidepot. Antigen-targeted soluble bispecific T-cell receptor (ImmTACTM) molecules in immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A24.

Abstract A92: Assessing the T-cell exhaustion status of TCR discovery outputs

Abstract Identification of antigen-specific T cell receptors (TCRs) is an essential component of the ImmTAC (Immune mobilizing monoclonal TCRs Against Cancer) platform at Immunocore. ImmTAC molecules are formed of a soluble TCR fused to an anti-CD3 effector function that engages, redirects and activates T cells to kill cancerous cells. TCRs recognize peptide antigens presented on the surface of the cancer cell by human leukocyte antigen (HLA). Thus, a fundamental step in our pipeline is to isolate T-cell receptors specific for validated cancer antigens for downstream development. T cells are isolated from whole blood of healthy donors to identify antigen-specific T cell clones using an in vitro model that consists of several stimulation steps. Activated CD8+ T cells are sorted into single cells for subsequent analysis and characterization. Despite the relative success of this approach, often analysis of T-cell clones is limited by low or absent mRNA expression for either or both TCR alpha (TRAV) and beta (TRBV) chains. We predict that TCR downregulation may result from overstimulation of T cells, leading to exhaustion. Here, we aimed to establish an in vitro model of T-cell exhaustion that would enable us to better understand the molecular mechanisms behind this process and thus facilitate method development. We induced extreme conditions of stimulation using CD3/CD28 beads and recombinant human IL-2 (rhIL-2) for 2-3 weeks. Whole-transcriptome analysis of the resulting activated T cells was compared to rested cells using a 5′ single-cell RNA-seq method from 10X Genomics (California, USA). Our preliminary analysis indicates that the activated T cells express several checkpoint inhibitors at higher levels. Our findings will be further investigated by performing full-length RNA sequencing of previously isolated single T-cell clones with low TRAV and/or TRBV detection rates. By identifying a molecular signature that represents the phenotype of exhausted T cells, we can potentially assess the state of T cells in TCR discovery outputs. Most importantly, determination of the expression levels of inhibitory receptors that are responsible for the decrease or loss of TCR expression is expected to enhance our understanding of the potential impact of T-cell exhaustion. Citation Format: Paraskevi Mallini, Filipa Bravo-Lopes, Karolina Lech, Sunir Malla, Maria Busz, Nathaniel Davies, Jacob Hurst, Luke Williams, Sterenn Davis, Michelle Teng. Assessing the T-cell exhaustion status of TCR discovery outputs [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A92.

Stabilization of soluble high-affinity T-cell receptor with denovo disulfide bonds.

Soluble T‐cell receptors (TCRs) have recently gained visibility as target‐recognition units of anticancer immunotherapeutic agents. Here, we improved the thermal stability of the well‐expressed high‐affinity A6 TCR by introducing pairs of cysteines in the invariable parts of the α‐ and β‐chain. A mutant with a novel intradomain disulfide bond in each chain also tested superior to the wild‐type in the accelerated stability assay. Binding of the mutant to the soluble cognate peptide (cp)–MHC and to the peptide‐loaded T2 cell line was equal to the wild‐type A6 TCR. The same stabilization motif worked efficiently in TCRs with different specificities, such as DMF5 and 1G4. Altogether, the biophysical properties of the soluble TCR molecule could be improved, without affecting its expression level and antigen‐binding specificity.

TCR-ligand interactions are required for murine epidermal Vγ3Vδ1 T cell development

Abstract Dendritic epidermal T cells (DETC), which bear the Vγ3Vδ1 T cell receptor (TCR), are the exclusive T cell population resident in the murine epidermis where they act as sentinels for neighboring keratinocytes. γδ T cells differentiate from lymphoid precursors largely within the thymic microenvironment in a highly organized manner. Cellular selection processes are also believed to be involved and indeed several lines of evidence suggest that proper development and homing of the Vγ3Vδ1+ subset does require positive selection events in the fetal thymus. To evaluate the role of TCR interactions with cognate ligand in Vγ3Vδ1 T cell development and maturation, we used a soluble DETC TCR tetramer to characterize ligand expression in the fetal thymus and skin. We show that temporal expression of ligand in the thymus closely mimics the window of DETC intrathymic development. Ligand expression is independent of Skint1, a molecule shown to be important for thymic maturation of DETC and their subsequent residence in the epidermis. Furthermore, using the soluble DETC TCR tetramer as a ligand blocking reagent, we show that in fetal thymic reaggregation cultures, TCR-ligand interactions are essential for DETC development and maturation. Current experiments are investigating the nature of the ligand-expressing thymic epithelial cell population. Understanding TCR-ligand interactions involved in DETC development may provide important insight into identifying the enigmatic DETC antigen.

Expression and Purification of Soluble, Glycosylated T‐cell Receptors in Chinese Hamster Ovary Cells

T‐cell receptors (TCRs) are glycoproteins that are expressed on the surface of T cells, which are a major component of the adaptive immune response. The glycans on the constant domain of this protein are involved in interactions with other proteins, such as galectin‐3, which mediates the immune response1. Additionally, altering the glycosylation of this protein has been associated with diseases such as Multiple Sclerosis1. Herein, we aim to express a soluble, glycosylated TCR in Chinese hamster ovary (CHO) suspension cells and purify it with the goal of characterizing the effects of glycosylation on TCR structure and function. To date, a soluble, unglycosylated TCR was expressed once, in 1990,2 but has not been replicated since. This project aims to express a glycosylated, soluble TCR with more current technology, which should result in more efficient expression.This project began with the design of a solubilized glycoprotein. We selected a starting sequence that has known binding affinity to ovalbumin and proceeded with modifications for optimized expression. The transmembrane sequence was removed after the last cysteine and replaced with a glycosylphosphatidylinositol (GPI) ‐ anchored sequence. This targets the protein to the endoplasmic reticulum (ER) and Golgi apparatus to ensure glycosylation. Following expression and glycosylation, the TCR should be localized to the surface of the cell, where the GPI sequence can be enzymatically cleaved using phosphatidylinositol‐phospholipase C (PI‐PLC), resulting in a soluble released protein. The α and β chains of the heterodimer were combined into a single plasmid using a 2A peptide. The use of 2A peptides instead of a more traditional technique, such as internal ribosome entry site (IRES), results in more efficient folding due to expression of stoichiometric ratios of both chains of the protein, as compared to a higher concentration of a single chain.The protein is transfected in CHO‐S cells and the expressed protein is harvested after 24 hours. The protein of interest is purified by affinity chromatography using a nickel column, followed by analysis with SDS‐PAGE. Imaging of the gels after surface cleavage with PI‐PLC and cell lysis confirmed that the protein was not localized to the plasma membrane as it was not recovered after enzymatic cleavage. However, the protein is purified from the inside of the cell after cell lysis. To determine the cellular localization of the protein we are using immunofluorescence using fluorescently labeled antibodies to target the His tag on the protein, as well as the ER, Golgi, and plasma membrane. Currently we are addressing the incorrect localization and analyzing methods to redirect the protein to the cell surface.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract B001: Generation of ImmTACTM molecules: Engineering high-affinity soluble T-cell receptors for the treatment of cancer

Abstract Immunotherapeutic strategies are centered on harnessing the human immune system to recognise and destroy cancer cells. At Immunocore, we have developed bi-specific Immune-mobilising monoclonal TCRs Against Cancer (ImmTAC) molecules comprising a soluble TCR fused to an anti-CD3 effector function that redirect T-cells to destroy cancer cells. The TCR-targeting domain overcomes a key limitation of traditional antibody-based therapies by targeting short peptide fragments derived from intracellularly processed proteins presented on the cell surface by human leukocyte antigens (HLAs), offering up to nine-fold more potential targets than are accessible to antibodies. To mediate efficient T-cell-mediated tumor clearance, ImmTAC molecules are engineered to overcome the weak affinities of natural TCRs imposed by thymic selection through a complex multistep engineering process described herein.T-cell clones recognising in-house validated cancer-specific peptides are isolated and affinity-enhanced up to a million fold by introducing mutations to the complementarity determining regions, resulting in picomolar affinity TCRs capable of recognizing exceptionally low numbers of target on the cancer cell surface. The TCR is fused to an anti-CD3 scFv to generate an ImmTAC molecule and is made soluble through the inclusion of a non-native disulphide bond. The efficacy and specificity of the ImmTAC molecule is scrutinized using a range of cellular and molecular assays. The potential application of the ImmTAC platform is exemplified by the expanding portfolio of ImmTAC molecules targeting diverse disease indications. Our lead candidate, IMCgp100, recognizes the melanoma-associated gp100 peptide and is in pivotal trials for the treatment of patients with metastatic uveal melanoma. Citation Format: Izabela Bombik, Alessio Bortoluzzi, Nicole Mai, Andrew Preston, Annelise Vuidepot, Bent K. Jakobsen, Nathaniel Liddy. Generation of ImmTACTM molecules: Engineering high-affinity soluble T-cell receptors for the treatment of cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B001.

PO-418 ImmTAC® molecules: cancer treatment through soluble, high affinity T cell receptors

Introduction Tumour associated antigen (TAA) peptides presented on cancer cells in complex with human leukocyte antigen (HLA) molecules provide a marker for targeting by cytotoxic T cells. The ability of T cells to identify aberrant peptides presented on malignant cells is achieved through a membrane-anchored a/b heterodimer called the T cell receptor (TCR). Whilst patients frequently mount anti-tumour T cell responses, often these are insufficient to clear the tumour. This can be attributed to the low level expression of tumour peptides on cancer cells and to the low binding affinity of TCRs targeting cancer antigens. To overcome the target recognition limitations of the naturally occurring T cell pool, we have developed a new class of soluble bi-specific molecules called ImmTAC (immune mobilising monoclonal TCRs against cancer) which combine a tumour targeting domain, an affinity-engineered TCR and a T cell-activating effector function, an anti-CD3 scFv. Material and methods T cell clones are isolated from human blood, which harbour wild-type TCRs recognising peptides derived from TAAs. The TCR α- and β- chains are sequenced and a stabilising non-native inter-chain disulphide bond is introduced. The TCR chains are expressed as inclusion bodies in Escherichia coli and are refolded in vitro to produce soluble and stable protein. The binding affinity is determined by surface plasmon resonance (SPR) and is typically in the range ~1–400 mM with half-lives of 1–10 s. For effective tumour targeting, the TCR is engineered using phage display to enhance affinity for cognate antigen by a million-fold or greater. To generate an ImmTAC molecule, the soluble TCR is fused to a humanised anti-CD3 scFv effector function and potency and specificity is assessed in vitro against HLA-relevant antigen positive cancer cell lines and cultured primary cells isolated from normal human tissues. Results and discussions Our lead clinical candidate, IMCgp100, targets the HLA-A*02/gp100280–288 epitope expressed at the surface of melanoma cells. Using directed molecular evolution and phage display targeting the complementarity-determining regions (CDRs) we have produced a bispecific reagent with picomolar binding affinity and a binding half-life of several hours. IMCgp100 has entered pivotal monotherapy trials for the treatment of patients with metastatic uveal melanoma. Conclusion The ImmTAC technology platform is a new class of bi-specific biologic based on T cell receptors. IMCgp100 is the first ImmTAC in clinical trials to treat advanced melanoma.

Soluble T-cell receptor design influences functional yield in an E. coli chaperone-assisted expression system.

There is a quest for production of soluble protein of high quality for the study of T-cell receptors (TCRs), but expression often results in low yields of functional molecules. In this study, we used an E. coli chaperone-assisted periplasmic production system and compared expression of 4 different soluble TCR formats: single-chain TCR (scTCR), two different disulfide-linked TCR (dsTCR) formats, and chimeric Fab (cFab). A stabilized version of scTCR was also included. Additionally, we evaluated the influence of host (XL1-Blue or RosettaBlueTM) and the effect of IPTG induction on expression profiles. A celiac disease patient-derived TCR with specificity for gluten was used, and we achieved detectable expression for all formats and variants. We found that expression in RosettaBlueTM without IPTG induction resulted in the highest periplasmic yields. Moreover, after large-scale expression and protein purification, only the scTCR format was obtained in high yields. Importantly, stability engineering of the scTCR was a prerequisite for obtaining reliable biophysical characterization of the TCR-pMHC interaction. The scTCR format is readily compatible with high-throughput screening approaches that may enable both development of reagents allowing for defined peptide MHC (pMHC) characterization and discovery of potential novel therapeutic leads.

Subtle changes at the variable domain interface of the T-cell receptor can strongly increase affinity

Most affinity-maturation campaigns for antibodies and T-cell receptors (TCRs) operate on the residues at the binding site, located within the loops known as complementarity-determining regions (CDRs). Accordingly, mutations in contact residues, or so-called "second shell" residues, that increase affinity are typically identified by directed evolution involving combinatorial libraries. To determine the impact of residues located at a distance from the binding site, here we used single-codon libraries of both CDR and non-CDR residues to generate a deep mutational scan of a human TCR against the cancer antigen MART-1·HLA-A2. Non-CDR residues included those at the interface of the TCR variable domains (Vα and Vβ) and surface-exposed framework residues. Mutational analyses showed that both Vα/Vβ interface and CDR residues were important in maintaining binding to MART-1·HLA-A2, probably due to either structural requirements for proper Vα/Vβ association or direct contact with the ligand. More surprisingly, many Vα/Vβ interface substitutions yielded improved binding to MART-1·HLA-A2. To further explore this finding, we constructed interface libraries and selected them for improved stability or affinity. Among the variants identified, one conservative substitution (F45βY) was most prevalent. Further analysis of F45βY showed that it enhanced thermostability and increased affinity by 60-fold. Thus, introducing a single hydroxyl group at the Vα/Vβ interface, at a significant distance from the TCR·peptide·MHC-binding site, remarkably affected ligand binding. The variant retained a high degree of specificity for MART-1·HLA-A2, indicating that our approach provides a general strategy for engineering improvements in either soluble or cell-based TCRs for therapeutic purposes.