Isolation of human MHC class II-restricted T cell receptors from the autologous T-cell repertoire with potent anti-leukaemic reactivity

Summary Adoptive transfer of T cells genetically modified with tumour-specific T-cell receptors (TCR) is a promising novel approach in the treatment of cancer. We have previously isolated an allorestricted MHC class I-restricted TCR with specificity for Formin-like protein 1 (FMNL1) with potent activity against chronic lymphocytic leukaemia cells. CD4 + T cells have been described to be highly important for tumour elimination although TCR derived from CD4 + T cells with anti-tumour reactivity have been only rarely described. In this study we aimed to isolate MHC class-II-restricted CD4 + T cells and TCR with specificity for leukaemia antigens. We used professional antigen-presenting cells pulsed with the leukaemiaassociated and tumour-associated antigen FMNL1 for stimulation of autologous T cells in vitro. We isolated two CD4 + HLA-DR-restricted T-cell clones and T-cell-derived TCR with so far unknown specificity but high reactivity against lymphoma cells and native malignant cells derived from HLA-matched patients with diverse leukaemias. Moreover, characterization of the TCR after TCR gene transfer revealed that specific characteristics of isolated TCR as reactivity in response to Toll-like receptors were transferable on effector cells. Our results have a major impact on the development of novel immunotherapies. They demonstrate that TCR with potent HLA-DR-restricted anti-leukaemic reactivity against so far undefined self-restricted antigens can be isolated from the healthy autorestricted CD4 + T-cell repertoire and these TCR are highly interesting candidate tools for novel immunotherapies

Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry

Although mutations may represent attractive targets for immunotherapy, direct identification of mutated peptide ligands isolated from human leucocyte antigens (HLA) on the surface of native tumour tissue has so far not been successful. Using advanced mass spectrometry (MS) analysis, we survey the melanoma-associated immunopeptidome to a depth of 95,500 patient-presented peptides. We thereby discover a large spectrum of attractive target antigen candidates including cancer testis antigens and phosphopeptides. Most importantly, we identify peptide ligands presented on native tumour tissue samples harbouring somatic mutations. Four of eleven mutated ligands prove to be immunogenic by neoantigen-specific T-cell responses. Moreover, tumour-reactive T cells with specificity for selected neoantigens identified by MS are detected in the patient's tumour and peripheral blood. We conclude that direct identification of mutated peptide ligands from primary tumour material by MS is possible and yields true neoepitopes with high relevance for immunotherapeutic strategies in cancer

Identification and Characterization of Neoantigens As Well As Respective Immune Responses in Cancer Patients

Cancer immunotherapy has recently emerged as a powerful tool for the treatment of diverse advanced malignancies. In particular, therapeutic application of immune checkpoint modulators, such as anti-CTLA4 or anti-PD-1/PD-L1 antibodies, have shown efficacy in a broad range of malignant diseases. Although pharmacodynamics of these immune modulators are complex, recent studies strongly support the notion that altered peptide ligands presented on tumor cells representing neoantigens may play an essential role in tumor rejection by T cells activated by anti-CTLA4 and anti-PD-1 antibodies. Neoantigens may have diverse sources as viral and mutated proteins. Moreover, posttranslational modifications and altered antigen processing may also contribute to the neoantigenic peptide ligand landscape. Different approaches of target identification are currently applied in combination with subsequent characterization of autologous and non-self T-cell responses against such neoantigens. Additional efforts are required to elucidate key characteristics and interdependences of neoantigens, immunodominance, respective T-cell responses, and the tumor microenvironment in order to define decisive determinants involved in effective T-cell-mediated tumor rejection. This review focuses on our current knowledge of identification and characterization of such neoantigens as well as respective T-cell responses. It closes with challenges to be addressed in future relevant for further improvement of immunotherapeutic strategies in malignant diseases

Paving the Way to Solid Tumors: Challenges and Strategies for Adoptively Transferred Transgenic T Cells in the Tumor Microenvironment

T cells are important players in the antitumor immune response. Over the past few years, the adoptive transfer of genetically modified, autologous T cells—specifically redirected toward the tumor by expressing either a T cell receptor (TCR) or a chimeric antigen receptor (CAR)—has been adopted for use in the clinic. At the moment, the therapeutic application of CD19- and, increasingly, BCMA-targeting-engineered CAR-T cells have been approved and have yielded partly impressive results in hematologic malignancies. However, employing transgenic T cells for the treatment of solid tumors remains more troublesome, and numerous hurdles within the highly immunosuppressive tumor microenvironment (TME) need to be overcome to achieve tumor control. In this review, we focused on the challenges that these therapies must face on three different levels: infiltrating the tumor, exerting efficient antitumor activity, and overcoming T cell exhaustion and dysfunction. We aimed to discuss different options to pave the way for potent transgenic T cell-mediated tumor rejection by engineering either the TME or the transgenic T cell itself, which responds to the environment

Adoptive Cellular Therapy for Multiple Myeloma Using CAR- and TCR-Transgenic T Cells: Response and Resistance

Despite the substantial improvement of therapeutic approaches, multiple myeloma (MM) remains mostly incurable. However, immunotherapeutic and especially T cell-based approaches pioneered the therapeutic landscape for relapsed and refractory disease recently. Targeting B-cell maturation antigen (BCMA) on myeloma cells has been demonstrated to be highly effective not only by antibody-derived constructs but also by adoptive cellular therapies. Chimeric antigen receptor (CAR)-transgenic T cells lead to deep, albeit mostly not durable responses with manageable side-effects in intensively pretreated patients. The spectrum of adoptive T cell-transfer covers synthetic CARs with diverse specificities as well as currently less well-established T cell receptor (TCR)-based personalized strategies. In this review, we want to focus on treatment characteristics including efficacy and safety of CAR- and TCR-transgenic T cells in MM as well as the future potential these novel therapies may have. ACT with transgenic T cells has only entered clinical trials and various engineering strategies for optimization of T cell responses are necessary to overcome therapy resistance mechanisms. We want to outline the current success in engineering CAR- and TCR-T cells, but also discuss challenges including resistance mechanisms of MM for evading T cell therapy and point out possible novel strategies

Cuboid and grid representations of the T–cell receptor geometries.

<p>(A) Localisation of the considered Vα and Vβ variable domains within the ternary TCR:pMHC complex. A TCR consists of two chains, the α and the β chain (blue and red). Each chain is partitioned into two domains, the constant domain (Cα and Cβ shown transparently) and a variable domain (Vα and Vβ, here surrounded by cuboids). The Vα and Vβ domains form the binding interface to the major histocompatibility complex (MHC) molecule (green) presenting an antigenic peptide (magenta) to the TCR. This work focuses on the variable domains. (B) Superimposition of the TCR variable domains. (i) The TCR structures were superimposed on the Vα domains leading to displaced Vβ domains. (ii) Cuboids were placed around the superimposed Vα and Vβ domains. This unified description of the different domains allows a quantitative analysis of the displacement. (C) Preparation of the cuboid placement templates. Vα (blue) and Vβ (red) domains of the structure 2bnu are used as reference structure. Both chains are surrounded with cuboids of the size of their spatial extent. Residues considered for superimposition are determined in an iterative process (unused residues are depicted transparently). These residues are used to compute the angular displacement of the Vβ domain relative to the Vα domain. (D) Center of Rotation (CoR). (i) Different geometries of (only three for clearness) β-cuboid geometries (red), superimposed on the α-cuboids (blue). (ii) Grids were fit into the β-cuboids. (iii) For each grid point <i>i</i>, the sum of pairwise distances and the variance was computed according to Formula 2. (iv) The residues at the center of rotation (CoR, green sphere) were investigated. For most of the structures, a conserved pair hydrogen bond interaction between the α and the β chain is located directly at the CoR. These hydrogen bonds are established by conserved Q residues.</p

Pairwise Euler Angle Distances [°] of the bound and free 1G4 TCR variants.

<p>The structures were superimposed to the α variable domains. All Euler angle distances given in degrees. Averaged angle distances: inter unbound: 2.5°, Inter bound (bold): 2.1°, bound vs. unbound (underlined): 8.0°.</p><p>^a) Subtypes: W = wild type, V = AV-wt, A = c5c1, B = c49c50, C = c58c62, D = C58c61</p><p>^b) Ligands: u = unbound, v = SLLMWITQV+HLA-A*0201, c = SLLMWITQC+HLA-A*0201.</p><p>Pairwise Euler Angle Distances [°] of the bound and free 1G4 TCR variants.</p

Pairwise Euler Angle Distances [°] of the bound and free 2C TCR variants.

<p>The structures were superimposed to the α variable domains. All Euler angle distances are given in degrees in respect to averaged geometries of all biological units. Unlike other 2C T7 variants (m6, T7-wt-s, m13) the m67 variant (underlined) affiliates to cluster (C) 6 occupied by the 2C wt TCRs bound to a different ligand. The two clusters are emphasized by bold typesetting.</p><p>^a) Cluster affiliation.</p><p>^b) Subtypes.</p><p>^c) Ligands: U = unbound, <i>K</i>E = H2-K1^b+EQYKFYSV, <i>K</i>S = H2-K1^b+SIYRYYG, <i>L</i>Q = H2-L^d+QLSPFPFDL. The ligand main type (MHC) is indicated by the first letter in italics.</p><p>^d) MHC mutation: (F9Y)(V12T)(I23T).</p><p>Pairwise Euler Angle Distances [°] of the bound and free 2C TCR variants.</p