Laura Bies

High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4 + T cells in human melanoma

Tumor-specific neo-antigens that arise as a consequence of mutations are thought to be important for the therapeutic efficacy of cancer immunotherapies. Accumulating evidence suggests that neo-antigens may be commonly recognized by intratumoral CD8+ T cells, but it is unclear whether neo-antigen–specific CD4+ T cells also frequently reside within human tumors. In view of the accepted role of tumor-specific CD4+ T-cell responses in tumor control, we addressed whether neo-antigen–specific CD4+ T-cell reactivity is a common property in human melanoma.

Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy

The transfer of T cell receptor (TCR) genes can be used to induce immune reactivity toward defined antigens to which endogenous T cells are insufficiently reactive. This approach, which is called TCR gene therapy, is being developed to target tumors and pathogens, and its clinical testing has commenced in patients with cancer. In this study we show that lethal cytokine-driven autoimmune pathology can occur in mouse models of TCR gene therapy under conditions that closely mimic the clinical setting. We show that the pairing of introduced and endogenous TCR chains in TCR gene-modified T cells leads to the formation of self-reactive TCRs that are responsible for the observed autoimmunity. Furthermore, we demonstrate that adjustments in the design of gene therapy vectors and target T cell populations can be used to reduce the risk of TCR gene therapy–induced autoimmune pathology.

Altered peptide ligands revisited: vaccine design through chemically modified HLA-A2-restricted T cell epitopes.

Virus or tumor Ag–derived peptides that are displayed by MHC class I molecules are attractive starting points for vaccine development because they induce strong protective and therapeutic cytotoxic T cell responses. In thus study, we show that the MHC binding and consequent T cell reactivity against several HLA-A*02 restricted epitopes can be further improved through the incorporation of nonproteogenic amino acids at primary and secondary anchor positions. We screened more than 90 nonproteogenic, synthetic amino acids through a range of epitopes and tested more than 3000 chemically enhanced altered peptide ligands (CPLs) for binding affinity to HLA-A*0201. With this approach, we designed CPLs of viral epitopes, of melanoma-associated Ags, and of the minor histocompatibility Ag UTA2-1, which is currently being evaluated for its antileukemic activity in clinical dendritic cell vaccination trials. The crystal structure of one of the CPLs in complex with HLA-A*0201 revealed the molecular interactions likely responsible for improved binding. The best CPLs displayed enhanced affinity for MHC, increasing MHC stability and prolonging recognition by Ag-specific T cells and, most importantly, they induced accelerated expansion of antitumor T cell frequencies in vitro and in vivo as compared with the native epitope. Eventually, we were able to construct a toolbox of preferred nonproteogenic residues with which practically any given HLA-A*02 restricted epitope can be readily optimized. These CPLs could improve the therapeutic outcome of vaccination strategies or can be used for ex vivo enrichment and faster expansion of Ag-specific T cells for transfer into patients.

Blockade of TGF-β Signaling Greatly Enhances the Efficacy of TCR Gene Therapy of Cancer

TCR gene therapy is a promising approach for the treatment of various human malignancies. However, the tumoricidal activity of TCR-modified T cells may be limited by local immunosuppressive mechanisms within the tumor environment. In particular, many malignancies induce T cell suppression in their microenvironment by TGF-β secretion. In this study, we evaluate whether blockade of TGF-β signaling in TCR-modified T cells enhances TCR gene therapy efficacy in an autochthonous mouse tumor model. Treatment of mice with advanced prostate cancer with T cells genetically engineered to express a tumor-reactive TCR and a dominant-negative TGF-β receptor II induces complete and sustained tumor regression, enhances survival, and leads to restored differentiation of prostate epithelium. These data demonstrate the potential to tailor the activity of TCR-modified T cells by additional genetic modification and provide a strong rationale for the clinical testing of TGF-β signaling blockade to enhance TCR gene therapy against advanced cancers.

RNAi-mediated TCR Knockdown Prevents Autoimmunity in Mice Caused by Mixed TCR Dimers Following TCR Gene Transfer

Genetically modified T cells that express a transduced T cell receptor (TCR) α/β heterodimer in addition to their endogenous TCR are used in clinical studies to treat cancer. These cells express two TCR-α and two TCR-β chains that do not only compete for CD3 proteins but also form potentially self-reactive mixed TCR dimers, composed of endogenous and transferred chains. To overcome these deficits, we developed an RNAi-TCR replacement vector that simultaneously silences the endogenous TCR and expresses an RNAi-resistant TCR. Transduction of the virus-specific P14 TCR without RNAi resulted in unequal P14 TCR-α and -β chain surface levels, indicating heterodimerization with endogenous TCR chains. Such unequal expression was also observed following TCR gene optimization. Equal surface levels of the introduced TCR chains were however achieved by silencing the endogenous TCR. Importantly, all mice that received cells transduced with the native or optimized P14 TCR developed lethal TCR gene transfer-induced graft-versus-host-disease (TI-GVHD) due to formation of mixed TCR dimers. In contrast, TI-GVHD was almost completely prevented when using the RNAi-TCR replacement vector. Our data demonstrate that RNAi-assisted TCR replacement reduces the formation of mixed TCR dimers, and thereby significantly reduces the risk of TI-GVHD in TCR gene therapy.

Erratum: Corrigendum: High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4+ T cells in human melanoma

Nat. Med. 21 81–85 (2015); published online 22 December 2014; corrected after print 18 August 2016 In the version of this article initially published, the article did not mention some restrictions on the availability of reagents. The following text has been added to the HTML and PDF versions of the paper: “The retroviral vectors containing BCL-6 and BCL-xL have been generated by a for-profit company, AIMM Therapeutics, which makes the plasmids available.

Abstract B044: Patient-specific immunotherapy through TCR gene transfer

Adoptive cell transfer (ACT) of autologous ex vivo expanded tumor-infiltrating lymphocytes has shown to mediate significant tumor regression in metastatic melanoma. This finding has demonstrated the capacity of endogenous T cells to mount an immune response against and eliminate cancer cells. Two clinically relevant approaches to adoptive cell transfer are ACT of expanded TIL and ACT of gene-modified peripheral blood lymphocytes (PBLs). The former approach consists of isolation of tumor-infiltrating lymphocytes from tumor biopsies, ex vivo expansion and re-infusion after treatment with a lymphodepleting regimen. In the latter, PBLs are engineered to express T-cell receptors (TCRs) recognizing common melanoma tumor antigens. While both approaches have shown different extents of clinical benefit, several limitations hamper clinical efficacy. T cell specificity, phenotype and differentiation state of ex vivo expanded TIL are largely unknown, while ACT of gene-modified lymphocytes is limited by the use of TCRs recognizing common tumor antigens, which can be differentially expressed between patients. Furthermore, the use of these TCRs is often limited to a handful of HLA types. These factors can lead to reduced therapeutic efficacy and applicability. Here we aim to compare the tumor control of T cells expressing ‘personalized’ tumor-reactive TCRs and expanded tumor-infiltrating lymphocytes after adoptive cell transfer in a xenograft mouse model. To this end, highly tumor-reactive T cell receptors are isolated from TIL on a per-patient basis. These TCRs are introduced into donor peripheral blood lymphocytes, forming a population of tumor-reactive cells tailored to the patient tumor. These cells are subsequently tested for efficacy in a xenograft mouse model bearing autologous (patient-derived) tumor. Using the outlined approach, we aim to identify determinants of therapy efficacy. Preliminary results have shown dramatic tumor regression after ACT of patient-specific gene-modified T cells, leading to complete remission of tumors in all treated mice. In contrast, tumors of mice treated with TIL or control T cells did not show significant reduction of tumor burden, when compared to untreated controls. Citation Format: Lorenzo F. Fanchi, Laura Bies, Ji-Ying Song, Carsten Linnemann, Ton Schumacher. Patient-specific immunotherapy through TCR gene transfer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B044.

Abstract 4704: Neo-antigen enriched TIL therapy mediates superior tumor eradication in a patient-derived xenograft model of human melanoma

The mutational load in melanoma is high relative to that in most other human malignancies, resulting in the possible expression of large numbers of patient-specific mutated antigens. This may, in part, explain the immunogenicity of this disease and the high rate of responsiveness to immunotherapeutic strategies such as tumor-infiltrating lymphocyte (TIL) therapy. Indeed, recent data have shown that cytotoxic T-cell reactivity targeting neo antigens may be common in human melanoma TILs. Importantly though, the clinical relevance of neo-antigen specific T cell populations remains uncertain. To directly address the tumoricidal potential of defined neo-antigen specific T cell populations, we first identified two neo-antigen specific T-cell populations within a bulk melanoma TIL culture by the combination of exome sequencing and MHC multimer-based T cell screens. Subsequently, we generated TIL products that are highly enriched for these neo-antigen reactivities and we compared the anti-tumor activity of these neo-antigen enriched TIL with that of ‘standard’ bulk TIL in NSG mice bearing the autologous tumor. We observed outgrowth of the tumors in mice treated with standard TIL. In contrast, tumors in mice treated with enriched TILs were controlled long-term. Additional experiments showed that this superior activity of neo-antigen enriched TIL was caused by the increased numbers of T cells with a high anti-tumor activity, rather than the depletion of cell populations with inhibitory activity. Furthermore, once tumors eventually recurred these were still recognized in vitro by cells from the initially infused TIL culture. Together, these preclinical data demonstrate that neo-antigen reactive T cells within bulk TIL cultures form a critical component of anti-tumor reactivity and provide a further basis to target mutated antigens with cancer immunotherapy. Citation Format: Sander Kelderman, Laura Bies, Marit M. Van Buuren, Nienke Van Rooij, John Haanen, Pia Kvistborg, Ton Schumacher. Neo-antigen enriched TIL therapy mediates superior tumor eradication in a patient-derived xenograft model of human melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4704. doi:10.1158/1538-7445.AM2015-4704