Patients with hepatocellular carcinoma (HCC) have a poor prognosis and limited therapeutic options. Alpha‐fetoprotein (AFP) is often expressed at high levels in HCC and is an established clinical biomarker of the disease. Expression of AFP in nonmalignant liver can occur, particularly in a subset of progenitor cells and during chronic inflammation, at levels typically lower than in HCC. This cancer‐specific overexpression indicates that AFP may be a promising target for immunotherapy. We verified expression of AFP in normal and diseased tissue and generated an affinity‐optimized T‐cell receptor (TCR) with specificity to AFP/HLA‐A*02+ tumors. Expression of AFP was investigated using database searches, by qPCR, and by immunohistochemistry (IHC) analysis of a panel of human tissue samples, including normal, diseased, and malignant liver. Using in vitro mutagenesis and screening, we generated a TCR that recognizes the HLA‐A*02‐restricted AFP158‐166 peptide, FMNKFIYEI, with an optimum balance of potency and specificity. These properties were confirmed by an extension of the alanine scan (X‐scan) and testing TCR‐transduced T cells against normal and tumor cells covering a variety of tissues, cell types, and human leukocyte antigen (HLA) alleles. Conclusion: We have used a combination of physicochemical, in silico, and cell biology methods for optimizing a TCR for improved affinity and function, with properties that are expected to allow TCR‐transduced T cells to differentiate between antigen levels on nonmalignant and cancer cells. T cells transduced with this TCR constitute the basis for a trial of HCC adoptive T‐cell immunotherapy.

BackgroundThere has been a dramatic increase in T cell receptor (TCR) sequencing spurred, in part, by the widespread adoption of this technology across academic medical centers and by the rapid commercialization of TCR sequencing. While the raw TCR sequencing data has increased, there has been little in the way of approaches to parse the data in a biologically meaningful fashion. The ability to parse this new type of 'big data' quickly and efficiently to understand the T cell repertoire in a structurally relevant manner has the potential to open the way to new discoveries about how the immune system is able to respond to insults such as cancer and infectious diseases.

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Adoptive cell therapy with T cells expressing affinity-enhanced T-cell receptors (TCRs) is a promising treatment for solid tumors. Efforts are ongoing to further engineer these T cells to increase the depth and durability of clinical responses and broaden efficacy toward additional indications. In the present study, we investigated one such approach: T cells were transduced with a lentiviral vector to coexpress an affinity-enhanced HLA class I–restricted TCR directed against MAGE-A4 alongside a CD8α coreceptor. We hypothesized that this approach would enhance CD4+ T-cell helper and effector functions, possibly leading to a more potent antitumor response. Activation of transduced CD4+ T cells was measured by detecting CD40 ligand expression on the surface and cytokine and chemokine secretion from CD4+ T cells and dendritic cells cultured with melanoma-associated antigen A4+ tumor cells. In addition, T-cell cytotoxic activity against 3-dimensional tumor spheroids was measured. Our data demonstrated that CD4+ T cells coexpressing the TCR and CD8α coreceptor displayed enhanced responses, including CD40 ligand expression, interferon-gamma secretion, and cytotoxic activity, along with improved dendritic cell activation. Therefore, our study supports the addition of the CD8α coreceptor to HLA class I–restricted TCR-engineered T cells to enhance CD4+ T-cell functions, which may potentially improve the depth and durability of antitumor responses in patients.

Affinity enhanced T-cell receptors (TCRs) have shown promise in the clinic. Second-generation strategies that enhance T-cell function alongside the TCR may improve the depth and durability of anti-tumor responses. In this second-generation TCR study, we added a CD8α homodimer to our MAGE-A4c1032TCR, a first-generation TCR currently being tested in a clinical trial (NCT03132922). Transduction of HLA class I-restricted, specific peptide enhanced affinity receptor (SPEAR) TCRs into peripheral blood lymphocytes creates both cytotoxic (CD8+) and helper (CD4+) T-cells of the same specificity; however, the lack of CD8 co-receptors on CD4+ T-cells may affect binding avidity of the engineered TCR. The addition of CD8α co-receptor into CD4+ T-cells alongside the engineered TCR (CD8α_MAGE-A4c1032) is anticipated to increase TCR binding avidity and enhance the polyfunctional response of CD4+ T-cells against tumor antigens, thereby widening the immune response to the tumor through dendritic cell (DC) activation and enhanced cytotoxicity. The effect of co-expressing the CD8α co-receptor on the MAGE-A4c1032TCR was assessed by in vitro assays addressing proof of concept for increased potency, focusing on CD4+ function, in parallel with assessment of potential safety issues. In assays involving antigen-positive tumor cell lines co-cultured with T-cells, we demonstrated improved T-cell engagement, as measured by increased CD40L on the T-cell surface in response to antigen. There were modest improvements in T-cell proliferation and cytokine production in response to tumor cells, particularly when isolated CD4+ cells were analyzed. However, when immature DCs were added to the co-culture, a more marked improvement with the second-generation T-cells was seen. DCs and T-cells in conditions containing CD8α_MAGE-A4c1032T-cells produced higher levels of cytokines and chemokines (e.g. IL-12, MIG for DCs, IFNγ, IL-2 for T-cells) than in conditions that contained MAGE-A4c1032T-cells without the CD8α co-receptor. Flow cytometry analyses illustrated T-cell-driven maturation of the DCs during the course of the co-culture. We also saw that second-generation CD4+ T-cells expressing the CD8α homodimer were able to kill antigen-expressing 3D tumor line microspheres, an additional benefit to the improvement of CD4+ helper functions. No changes in TCR specificity, sensitivity, or aberrant cytokine release arose from co-expressing the CD8α co-receptor in T-cells transduced with the MAGE-A4c1032TCR, suggesting no change to the existing safety profile. These data illustrate improved engagement and function in the CD4+ T-cells transduced with CD8α_MAGE-A4c1032, without additional off-target reactivity. The second generation CD8α_MAGE-A4 SPEAR T-cells are expected to improve long term T-cell functions as well as immediate anti-tumor activity in vivo. Citation Format: Victoria E. Anderson, Anika M. Weber, Guy E. Wiedermann, Anette Pachnio, Sumaya Dauleh, Tina Ahmed, Roslin Y. Docta, Adriano Quattrini, George Pope, Laura Quinn, Thomas M. Ashton, Helen M. Tunbridge, Joseph P. Sanderson, Andrew B. Gerry. Enhanced activity of second-generation MAGE-A4 SPEAR T-cells through co-expression of a CD8α homodimer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2313.