A major barrier to successful antitumor vaccination is tolerance of high-avidity T cells specific to tumor antigens. In keeping with this notion, HER-2/neu (neu)-targeted vaccines, which raise strong CD8+ T cell responses to a dominant peptide (RNEU420-429) in WT FVB/N mice and protect them from a neu-expressing tumor challenge, fail to do so in MMTV-neu (neu-N) transgenic mice. However, treatment of neu-N mice with vaccine and cyclophosphamide-containing chemotherapy resulted in tumor protection in a proportion of mice. This effect was specifically abrogated by the transfer of neu-N–derived CD4+CD25+ T cells. RNEU420-429-specific CD8+ T cells were identified only in neu-N mice given vaccine and cyclophosphamide chemotherapy which rejected tumor challenge. Tetramer-binding studies demonstrated that cyclophosphamide pretreatment allowed the activation of high-avidity RNEU420-429-specific CD8+ T cells comparable to those generated from vaccinated FVB/N mice. Cyclophosphamide seemed to inhibit regulatory T (T reg) cells by selectively depleting the cycling population of CD4+CD25+ T cells in neu-N mice. These findings demonstrate that neu-N mice possess latent pools of high-avidity neu-specific CD8+ T cells that can be recruited to produce an effective antitumor response if T reg cells are blocked or removed by using approaches such as administration of cyclophosphamide before vaccination.

Iron–dextran nanoparticles functionalized with T cell activating proteins have been used to study T cell receptor (TCR) signaling. However, nanoparticle triggering of membrane receptors is poorly understood and may be sensitive to physiologically regulated changes in TCR clustering that occur after T cell activation. Nano-aAPC bound 2-fold more TCR on activated T cells, which have clustered TCR, than on naive T cells, resulting in a lower threshold for activation. To enhance T cell activation, a magnetic field was used to drive aggregation of paramagnetic nano-aAPC, resulting in a doubling of TCR cluster size and increased T cell expansion in vitro and after adoptive transfer in vivo. T cells activated by nano-aAPC in a magnetic field inhibited growth of B16 melanoma, showing that this novel approach, using magnetic field-enhanced nano-aAPC stimulation, can generate large numbers of activated antigen-specific T cells and has clinically relevant applications for adoptive immunotherapy.

Artificial antigen presenting cells (aAPC), which deliver stimulatory signals to cytotoxic lymphocytes, are a powerful tool for both adoptive and active immunotherapy. Thus far, aAPC have been synthesized by coupling T cell activating proteins such as CD3 or MHC-peptide to micron-sized beads. Nanoscale platforms have different trafficking and biophysical interaction properties and may allow development of new immunotherapeutic strategies. We therefore manufactured aAPC based on two types of nanoscale particle platforms: biocompatible iron-dextran paramagnetic particles (50–100 nm in diameter) and avidin-coated quantum dot nanocrystals, (~30 nm). Nanoscale aAPC induced antigen-specific T cell proliferation from mouse splenocytes and human peripheral blood T cells. When injected in vivo, both iron-dextran particles and quantum dot nanocrystals enhanced tumor rejection in a subcutaneous mouse melanoma model. This is the first description of nanoscale aAPC that induce antigen-specific T cell proliferation in vitro and lead to effective T cell stimulation and inhibition of tumor growth in vivo.

Adoptive immunotherapy (AIT) can mediate durable regression of cancer, but widespread adoption of AIT is limited by the cost and complexity of generating tumor-specific T cells. Here we develop an Enrichment + Expansion strategy using paramagnetic, nanoscale artificial Antigen Presenting Cells (aAPC) to rapidly expand tumor-specific T cells from rare naïve precursors and predicted neo-epitope responses. Nano-aAPC are capable of enriching rare tumor-specific T cells in a magnetic column and subsequently activating them to induce proliferation. Enrichment + Expansion resulted in greater than 1000-fold expansion of both mouse and human tumor-specific T cells in one week, with nano-aAPC based enrichment conferring a proliferation advantage during both in vitro culture and after adoptive transfer in vivo. Robust T cell responses were not only seen for shared tumor antigens, but also for computationally predicted neo-epitopes. Streamlining the rapid generation of large numbers of tumor-specific T cells in a cost-effective fashion through Enrichment + Expansion can be a powerful tool for immunotherapy.

SummaryThe interaction of the T cell receptor (TCR) with its cognate peptide-major histocompatibility complex (MHC) on the surface of antigen presenting cells (APCs) is a primary event during T cell activation. Here we used a dimeric IE k -MCC molecule to study its capacity to activate antigen-specific T cells and to directly analyze the role of CD4 in physically stabilizing the TCR-MHC interaction. Dimeric IE k -MCC stably binds to specific T cells. In addition, immobilized dimeric IE k -MCC can induce TCR downregulation and activate antigen-specific T cells more efficiently than anti-CD3. The potency of the dimeric IE k -MCC is significantly enhanced in the presence of CD4. However, CD4 does not play any significant role in stabilizing peptide-MHC-TCR interactions as it fails to enhance binding of IE k -MCC to specific T cells or influence peptide-MHC-TCR dissociation rate or TCR downregulation. Moreover, these results indicate that dimerization of peptide-MHC class II using an IgG molecular scaffold significantly increases its binding avidity leading to an enhancement of its stimulatory capacity while maintaining the physiological properties of cognate peptide-MHC complex. These peptide-MHC-IgG chimeras may, therefore, provide a novel approach to modulate antigen-specific T cell responses both in vitro and in vivo.

and Eisai. TAC has served as an advisor for Bristol Myers Squibb, Illumina, Eisai, and An2H. Under a licensing agreement between NexImmune and the Johns Hopkins University, JPS is entitled to shares of royalty received by the university on sales of artificial antigen-presenting cell products described in this article. He also owns NexImmune stock, which is subject to certain restrictions under university policy. JPS is a member of the company's Scientific Advisory Board. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its conflict-of-interest policies. JPS acknowledges grant funding from AstraZeneca.

While activated T cells are known to have enhanced biological responses to antigen stimulation, the biophysical basis of this increased sensitivity remains unknown. Here, we show that, on activated T cells, the TCR avidity for peptide-MHC complexes is 20- to 50-fold higher than the TCR avidity of naive T cells. This increased avidity for peptide-MHC depends on TCR reorganization and is sensitive to the cholesterol content of the T cell membrane. Analysis of the binding data indicates the enhanced avidity is due to increases in cross-linking of TCR on activated T cells. Activation-induced membrane (AIM) changes in TCR avidity represent a previously unrecognized means of increasing the sensitivity of activated T cells to small amounts of antigen in the periphery.

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