CIMT 2016: Mechanisms of efficacy in cancer immunotherapy — Report on the 14th Annual Meeting of the Association for Cancer Immunotherapy May 10–12 2016, Mainz, Germany

Increasing functional avidity of TCR-redirected T cells by removing defined N-glycosylation sites in the TCR constant domain

Adoptive transfer of T lymphocytes transduced with a T cell receptor (TCR) to impart tumor reactivity has been reported as a potential strategy to redirect immune responses to target cancer cells (Schumacher, T.N. 2002. Nat. Rev. Immunol. 2:512-519). However, the affinity of most TCRs specific for shared tumor antigens that can be isolated is usually low. Thus, strategies to increase the affinity of TCRs or the functional avidity of TCR-transduced T cells might be therapeutically beneficial. Because glycosylation affects the flexibility, movement, and interactions of surface molecules, we tested if selectively removing conserved N-glycoslyation sites in the constant regions of TCR alpha or …

Increasing Functional Avidity of T Cell Receptor (TCR)-Redirected T Cells by Removing Defined N-Glycoslyation Sites in the Constant Domain of Introduced TCR Chains.

Abstract Adoptive transfer of T lymphocytes transduced with a TCR to impart tumor reactivity has been reported as potential strategy to redirect immune responses to target cancer cells. However, the affinities of most TCRs specific for shared tumor antigens that can be isolated are usually low, in part reflecting the nature of the targeted tumor antigens which are self-proteins. Thus strategies that can increase the affinity or functional avidity of TCRs to be used in therapy to transduce T cells might be therapeutically beneficial. However, current strategies for increasing TCR affinity require extensive and usually random mutagenesis followed by screening the many derived mutations, and m…

Designing TCR for Cancer Immunotherapy

Reprogramming T-cell populations by T-cell receptor (TCR) gene transfer is a new therapeutic tool for adoptive tumor immunotherapy. Gene transfer of human leukocyte antigen (HLA)-transgenic mice-derived TCR into human T-cells allows the circumvention of tolerance to tumor-associated (self) antigens (TAA). This chapter reports on the identification of the alpha and beta chains of the heterodimeric TCR derived from a mouse T-cell clone. The related DNA fragments are inserted into a retroviral vector for heterologous expression of the TAA-specific TCR in human T-cells. Polymerase chain reaction (PCR)-based cloning protocols are provided for the tailor-made customization of murine TCR. We descr…

Cytotoxicity of tumor antigen specific human T cells is unimpaired by arginine depletion.

Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8(+) T cell activation…

Circumventing tolerance to a human MDM2-derived tumor antigen by TCR gene transfer

We identified a tumor-associated cytotoxic T lymphocyte (CTL) epitope derived from the widely expressed human MDM2 oncoprotein and were able to bypass self-tolerance to this tumor antigen in HLA-A*0201 (A2.1) transgenic mice and by generating A2.1-negative, allo-A2.1-restricted human T lymphocytes. A broad range of malignant, as opposed to nontransformed cells, were killed by high-avidity transgenic mouse and allogeneic human CTLs specific for the A2.1-presented MDM2 epitope. Whereas the self-A2.1-restricted human T cell repertoire gave rise only to low-avidity CTLs unable to recognize the natural MDM2 peptide, human A2.1+ T lymphocytes were turned into efficient MDM2-specific CTLs upon exp…

Cooperation of Human Tumor-Reactive CD4+ and CD8+ T Cells after Redirection of Their Specificity by a High-Affinity p53A2.1-Specific TCR

Abstract Efficient immune attack of malignant disease requires the concerted action of both CD8 + CTL and CD4 + Th cells. We used human leukocyte antigen (HLA)-A*0201 (A2.1) transgenic mice, in which the mouse CD8 molecule cannot efficiently interact with the α3 domain of A2.1, to generate a high-affinity, CD8-independent T cell receptor (TCR) specific for a commonly expressed, tumor-associated cytotoxic T lymphocyte (CTL) epitope derived from the human p53 tumor suppressor protein. Retroviral expression of this CD8-independent, p53-specific TCR into human T cells imparted the CD8 + T lymphocytes with broad tumor-specific CTL activity and turned CD4 + T cells into potent tumor-reactive, p53…

Facilitating matched pairing and expression of TCR chains introduced into human T cells.

AbstractAdoptive transfer of T lymphocytes is a promising treatment for a variety of malignancies but often not feasible due to difficulties generating T cells that are reactive with the targeted antigen from patients. To facilitate rapid generation of cells for therapy, T cells can be programmed with genes encoding the α and β chains of an antigen-specific T-cell receptor (TCR). However, such exogenous α and β chains can potentially assemble as pairs not only with each other but also with endogenous TCR α and β chains, thereby generating αβTCR pairs of unknown specificity as well as reducing the number of exogenous matched αβTCR pairs at the cell surface. We demonstrate that introducing cy…

Redirection of T cells by delivering a transgenic mouse-derived MDM2 tumor antigen-specific TCR and its humanized derivative is governed by the CD8 coreceptor and affects natural human TCR expression.

Retroviral transfer of T cell antigen receptor (TCR) genes selected by circumventing tolerance to broad tumor- and leukemia-associated antigens in human leukocyte antigen (HLA)-A*0201 (A2.1) transgenic (Tg) mice allows the therapeutic reprogramming of human T lymphocytes. Using a human CD8 x A2.1/Kb mouse derived TCR specific for natural peptide-A2.1 (pA2.1) complexes comprising residues 81-88 of the human homolog of the murine double-minute 2 oncoprotein, MDM2(81-88), we found that the heterodimeric CD8 alpha beta coreceptor, but not normally expressed homodimeric CD8 alpha alpha, is required for tetramer binding and functional redirection of TCR- transduced human T cells. CD8+T cells that…

Molecular Design of the Cαβ Interface Favors Specific Pairing of Introduced TCRαβ in Human T Cells

Abstract A promising approach to adoptive transfer therapy of tumors is to reprogram autologous T lymphocytes by TCR gene transfer of defined Ag specificity. An obstacle, however, is the undesired pairing of introduced TCRα- and TCRβ-chains with the endogenous TCR chains. These events vary depending on the individual endogenous TCR and they not only may reduce the levels of cell surface-introduced TCR but also may generate hybrid TCR with unknown Ag specificities. We show that such hybrid heterodimers can be generated even by the pairing of human and mouse TCRα- and TCRβ-chains. To overcome this hurdle, we have identified a pair of amino acid residues in the crystal structure of a TCR that …

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

Genetic engineering of T cells with a T cell 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 and therapeutic efficiency of an optimized single-chain TCR (scTCR) specific for an HLA-A2.1-restricted (non-mutated) p53(264–272) peptide in adoptive T cell 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 T cells does not induce on-tar…

Crystal structure of the bifunctional soybean Bowman-Birk inhibitor at 0.28-nm resolution. Structural peculiarities in a folded protein conformation.

The Bowman-Birk inhibitor from soybean is a small protein that contains a binary arrangement of trypsin-reactive and chymotrypsin-reactive subdomains. In this report, the crystal structure of this anticarcinogenic protein has been determined to 0.28-nm resolution by molecular replacement from crystals grown at neutral pH. The crystal structure differs from a previously determined NMR structure [Werner, M. H. & Wemmer, D. E. (1992) Biochemistry 31, 999-1010] in the relative orientation of the two enzyme-insertion loops, in some details of the main chain trace, in the presence of favourable contacts in the trypsin-insertion loop, and in the orientation of several amino acid side chains. The p…