Bryan K. Cho

A Proposed Mechanism for the Induction of Cytotoxic T Lymphocyte Production by Heat Shock Fusion Proteins

A 65 kDa mycobacterial heat shock protein (hsp65), fused to a polypeptide that contains an octapeptide (SIYRYYGL) agonist for a particular T cell receptor (2C TCR), stimulated C57BL/6 mice as well as CD4-deficient mice to produce CD8+ cytolytic T lymphocytes (CTL) to the fusion partners octapeptide. This and other hsp65 fusion proteins but not native hsp65 itself stimulated dendritic cells in vitro and in vivo to upregulate the levels of MHC (class I and II) and costimulatory (B7.2) molecules. The results suggest a mechanism for the general finding that hsp fusion proteins, having fusion partners of widely differing lengths and sequences, elicit CD8 CTL to peptides from the fusion partners without requiring exogenous adjuvants or the participation of CD4+ T cells.

Targeting T cells against brain tumors with a bispecific ligand‐antibody conjugate

High‐affinity receptors expressed on the surface of some tumors can be exploited by chemically conjugating the ligand for the receptor and an antibody against immune effector cells, thus redirecting their cytolytic potential against the tumor. Ovarian carcinomas and some brain tumors express the high‐affinity folate receptor (FR). In this report, a transgenic mouse model that generates endogenously arising choroid plexus tumors was used to show that folate/anti‐ T‐cell receptor antibody conjugates can direct infiltration of T cells into solid brain tumor masses. An engineered single‐chain Fv form of the anti‐T‐cell receptor antibody KJ16 was conjugated with folate, to produce a bispecific agent that was substantially smaller than most previously characterized bispecific antibodies. Folate conjugation to the antibody increased T‐cell infiltration into the tumors by 10‐ to 20‐fold, and significantly prolonged survival of the mice. Int. J. Cancer 76:761–766, 1998.© 1998 Wiley‐Liss, Inc.

Bispecific agents target endogenous murine T cells against human tumor xenografts

A variety of immunological approaches to cancer treatment are currently being explored. These include strategies designed to enhance or redirect the activity of T cells against tumors. Bispecific antibodies comprise a class of agents capable of redirecting T cells by binding to a tumor antigen and the T‐cell receptor (TCR). In vivo pre‐clinical testing of bispecific antibodies against human tumors has to date been limited to the use of immunodeficient mice that receive the bispecific agent, activated human effector T cells, and human tumor cells. In this report, we show that TCR transgenic/RAG‐1 knockout mice (TCR/RAG) serve as a unique model allowing endogenous T cells to be redirected against transplanted human tumors. The findings show that TCR/RAG mice (i) accepted transplants of human tumors, including the folate‐receptor‐positive tumor line KB; (ii) contained endogenous cytotoxic T lymphocytes that could be activated in vivo with an antigenic peptide recognized by the transgenic TCR; (iii) rejected human tumors after treatment with the activating peptide and bispecific agents that contained folic acid co‐valently linked to an anti‐TCR antibody. Successful rejection was achieved with folate conjugates of Fab or scFv fragments. Treatment with activating agents and bispecific conjugates resulted in the complete eradication of freshly transplanted tumors as well as significantly prolonging the survival of mice bearing established solid tumors. Our results highlight the importance of including T‐cell‐activating modalities in combination with bispecific antibodies. Additionally, we introduce a system that allows endogenous T cells to be redirected against human tumor xenografts and in which the T cells may be followed in vivo by use of a clonotypic marker. Int. J. Cancer 83:141–149, 1999.

A Peptide That Antagonizes TCR-Mediated Reactions with Both Syngeneic and Allogeneic Agonists: Functional and Structural Aspects

We identify and consider some characteristics of a peptide antagonist for the Ag-specific receptor on 2C cells (the 2C TCR). The peptide, GNYSFYAL (called GNY), binds to H-2Kb, and a very high-resolution crystal structure of the GNY-Kb complex at 1.35 Å is described. Although the GNY peptide does not bind to Ld, the potency of GNY-Kb as an antagonist is evident from its ability to specifically inhibit 2C TCR-mediated reactions to an allogenic agonist complex (QLSPFPFDL-Ld), as well as to a syngeneic agonist complex (SIYRYYGL-Kb). The crystal structure and the activities of alanine-substituted peptide variants point to the properties of the peptide P4 side chain and the conformation of the Tyr-P6 side chain as the structural determinants of GNYSFYAL antagonist activity.

Targeting tumor cells with bispecific antibodies and T cells

It has been known for some time that mammalian immune systems are capable of eliminating large tumor burdens. Redirecting the immune response of a patient to an established tumor has now become the focus of various therapeutic strategies. In this report, two projects toward this goal are described. The first project involves the development of a transgenic mouse model for T cell directed therapeutics. These mice express specific T cell receptor alpha and beta transgenes on a background in which the recombinational-activating-gene-1 (RAG) has been knocked out. The mice express cytotoxic T cells but not either T helper cells or B cells. Despite these deficiencies, the animals are capable of eliminating tumors that express the appropriate peptide/major histocompatibility complex ligand that is recognized by the alphabeta transgenic T cell receptor. Human tumors grow as transplants in these mice, thereby allowing various agents that redirect the endogenous T cells against human tumors to be tested. The second project involves a description of such agents: bispecific antibodies that simultaneously bind to an immune effector cell and a tumor cell. The bispecific antibody described here consists of folate attached to anti-T cell receptor antibodies, or their fragments. A single-chain Fv coupled with folate can redirect the lysis of human tumor cells that bear the high affinity folate receptor. Preliminary in vivo data showed that the folate/antibody conjugates were also capable of mediating rejection of the human tumor. This transgenic mouse model should now allow the evaluation and optimization of bispecific agents that can redirect a patients own T cell response.

Characterization of a Single-chain Antibody to the β-Chain of the T Cell Receptor

In this report the VH and VL genes of the anti-T cell receptor (TCR) antibody KJ16, which recognizes the TCR Vβ8.1 and Vβ8.2 regions in mice, were cloned and expressed as a single-chain antibody (scFv) in Escherichia coli. A 29-kDa protein was obtained after renaturation from inclusion bodies. The KJ16 scFv had a relative affinity for the native TCR that was slightly higher than KJ16 Fab fragments. The scFv and Fab fragments of the KJ16 antibody, together with monovalent forms of two other anti-TCR antibodies, were evaluated as antagonists of the T cell-mediated recognition of a peptide-class I complex or of a superantigen, Staphylococcus enterotoxin B (SEB) bound to a class II product. Each of the anti-TCR antibodies was efficient at inhibiting the recognition of the SEB-class II complex. In contrast, only the clonotypic antibody, which binds to epitopes on both the Vβ and Vα regions, inhibited the recognition of peptide-class I complex. We conclude that the TCR binding site for the SEB-class II ligand encompasses a larger surface area than the TCR binding site for the peptide-class I ligand.

T Cell Proliferation, Differentiation, and Restoration in Lymphopenic Individuals

When a small number of naive T cells are adoptively transferred into syngeneic lymphopenic hosts, such as mice deficient in recombination activating gene-1 (RAG-/-) or sublethally irradiated normal mice, the transferred T cells proliferate, a phenomenon referred to as homeostasis-driven or lymphopenia-induced proliferation. Although deliberate antigen stimulation is not required, homeostatic T cell proliferation requires i) the engagement of T cell receptor (TCR) with self peptide-major histocompatibility complexes (pepMHC) and ii) the presence of lymphopenia (or “space”) (Tanchot et al.,, 1997, Ernst et al., 1999, Viret et al., 1999, Goldrath and Bevan, 1999, Bender et al., 1999). We have shown that following homeostatic proliferation naive CD8 T cells acquire the phenotype and functional properties characteristic of antigen-induced memory T cells. We have also developed an in vitro system for studying homeostatic T cell proliferation. These findings and their implications for memory T cell development and the stable maintenance of naive and memory T cell compartments are discussed.