Targeting of tumor cells by lymphocytes engineered to express chimeric receptor genes

Abstract

Adoptive cellular immunotherapy of cancer has been limited to date mostly due to the poor immunogenicity of tumor cells, the immunocompromised status of cancer patients in advanced stages of their disease, and difficulties in raising sufficient numbers of autologous tumor-specific T lymphocytes. On the other hand, the slow tumor penetration and short half-life of exogenously administered tumor-specific monoclonal antibodies have provided major obstacles for an effective destruction of tumor cells by the humoral effector arm of the immune system. Attempts to improve the efficacy of adoptive cellular cancer immunotherapy have led to the development of novel strategies that combine advantages of T cell-based (i.e., efficient tumor penetration, cytokine release and cytotoxicity) and antibody-based (high specificity for tumor-associated antigens) immunotherapy by grafting cytotoxic T lymphocytes (CTLs) with chimeric receptors composed of antibody fragments (which recognize tumor-cell antigens) and a cellular activation motif. Antigen recognition is therefore not restricted by major histocompatibility genes, as the physiological T-cell receptor, but rather is directed to native cell surface structures. Since the requirements of major histocompatibility complex (MHC) restriction in the interaction of effector cells with target cells are bypassed, the tumor cell-binding of CTLs grafted with chimeric receptors is not affected by down-regulation of HLA class I antigens and by defects in the antigen-processing machinery. Ligand binding by the chimeric receptor triggers phosphorylation of immunoglobulin tyrosine activation motifs (ITAMs) in the cytoplasmic region of the molecule and this activates a signaling cascade that is required for the induction of cytotoxicity, cytokine secretion and proliferation. Here, the authors discuss the potential of lymphocytes grafted with chimeric antigen receptors in the immunotherapy of malignant disease.

Fig. 1

Construction of single-chain Fv fragment. mRNA was isolated from the murine hybridoma cells HB8696 producing anti-HER-2/neu mAb, followed by synthesis of first strand cDNA. PCR amplification was performed for both Ig heavy and light chains with the use of cDNA as a template. Sets of sense and antisense PCR primers were designed to amplify the V_H and V_L regions. After the first round of PCR, V_H and V_L genes were purified and on a second round PCR these were linked by a (Gly₄ Ser)₃ linker. The assembled scFv PCR products were ligated into the pHEN1 phagemid, which also contained the c-myc tag peptide as a recognition marker. E. Coli transformed with the pHEN1-scFv produced soluble scFv, which was isolated and purified from culture supernatants

Fig. 2

Schematic representation of a scFv (anti-HER-2/neu)-ζ or -γ construct. The Moloney murine leukemia virus 5′ long terminal repeat (LTR) controls the expression of the CAR scFv (HB8696) ζ or γ, which consists of an N-terminal heavy chain leader peptide (L), the HER-2/neu–specific single-chain antibody scFv (hb8696), an myc-tag (shown in Fig. 1), the hinge region of CD8 α chain, and the CD3 ζ or FcɛRI γ chain. The neomycin-resistance gene for G418 selection of transduced cells is driven by the SV40 early promoter

Fig. 3

Examples of CARs. CARs interact with their antigen (Ag)-binding regions of Ig (scFv-α-Ag) that consist of V_H and V_L chains linked by a short peptide linker. CD8 α chain functions in many cases as a flexible linker. CARs also incorporate the transmembrane (TM) and intracellular (IC) signaling domains of the Fcγ, TCR, and CD28, as shown