The generation of tumor-specific lymphocytes and their use in adoptive immunotherapy is limited to a few malignancies because most spontaneous tumors are very weak or not at all imunogenic. On the other hand, many anti-tumor antibodies have been described which bind tumorassociated antigens shared among tumors ofthe same histology. Combining the variable regions (Fv) of an antibody with the constant regions of the T-cell receptor (TCR) chains results in chimeric genes endowing T lymphocytes with antibody-type specificity, potentially allowing cellular adoptive immunotherapy against types of tumors not previously possible. To generalize and extend this approach to additional lymphocyteactivating molecules, we designed and constructed chimeric genes composed of a single-chain Fv domain (scFv) of an antibody linked with v or Cchains, the common signaltransducing subunits of the immunoglobulin receptor and the TCR. Such chimeric genes containing the Fv region of an anti-trinitophenyl antibody could be expressed as functional surface receptors in a cytolytic T-cell hybridoma. They triggered interleukin 2 secretion upon encountering antigen and mediated non-major-histocompatibility-complex-restricted hapten-specific target cell lysis. Such chimeric receptors can be exploited to provide T cells and other effector lymphocytes, such as natural killer cells, with antibody-type recognition directly coupled to cellular activation.Many clinical attempts to recruit the humoral or cellular arms of the immune system for passive anti-tumor immunotherapy have not fulfilled expectations. While it has been possible to obtain anti-tumor antibodies, their therapeutic potential has been limited so far to blood-borne tumors (1, 2), primarily because solid tumors are inaccessible to sufficient amounts of antibodies (3). On the other hand, the use of effector lymphocytes in adoptive immunotherapy, although effective in selected solid tumors, suffers from a lack of specificity [such as in the case of lymphokine-activated killer cells (LAK cells) (4)] or from the difficulty in recruiting tumor-infiltrating lymphocytes (TILs) and expanding such specific T cells for most malignancies (5). Yet, the observations that TILs can be obtained in melanoma and renal cell carcinoma tumors, that they can be effective in selected patients, and that foreign genes can function in these cells (6) demonstrate the therapeutic potential embodied in these cells.A strategy which we and others have recently developed (7-11) allows us to combine the advantage of the antibody's specificity with the homing, tissue penetration, and targetcell destruction of T lymphocytes and to extend, by ex-vivo genetic manipulations, the spectrum of anti-tumor specificity of T cells. In this approach a chimeric T-cell receptor (cTCR) To overcome these problems and to extend the applicability of the "T-body" approach to other cells and receptor molecules, we developed a single-chain approach to the cTCR. It is based on the demonstrated ability to express in bacteria an ...

Purpose: A phase I study was conducted to assess the safety of adoptive immunotherapy using gene-modified autologousTcells for the treatment of metastatic ovarian cancer. Experimental Design: T cells with reactivity against the ovarian cancer^associated antigen a-folate receptor (FR) were generated by genetic modification of autologous T cells with a chimeric gene incorporating an anti-FR single-chain antibody linked to the signaling domain of the Fc receptor g chain. Patients were assigned to one of two cohorts in the study. Eight patients in cohort 1received a dose escalation of Tcells in combination with high-dose interleukin-2, and six patients in cohort 2 received dual-specificTcells (reactive with both FR and allogeneic cells) followed by immunization with allogeneic peripheral blood mononuclear cells.

To design and direct at will the specificity of T cells in a non-major histocompatibility complex (MHC)-restricted manner, we have generated and expressed chimeric T-cell receptor (TcR) genes composed of the TcR constant (C) domains fused to the antibody's variable (V) domains. Genomic expression vectors have been constructed containing the rearranged gene segments coding for the V region domains of the heavy (VH) and light (VL) chains of an anti-2,4,6-trinitrophenyl (TNP) antibody (SP6) spliced to either one of the C-region gene segments of the a or (3 TcR chains. Following transfection into a cytotoxic T-cell hybridoma, expression of a functional TcR was detected. The chimeric TcR exhibited the idiotope of the Sp6 anti-TNP antibody and endowed the T cells with a non-MHC-restricted response to the hapten TNP. The transfectants specifically killed and produced interleukin 2 in response to TNP-bearing target cells across strain and species barriers. Moreover, such transfectants responded to immobilized TNPprotein conjugates, bypassing the need for cellular processing and presentation. In the particular system employed, both the TNP-binding site and the Sp6 idiotope reside almost exclusively in the VH chain region. Hence, introduction into T cells of TcR genes containing only the VHSp6 fused to either the Ca or C(3 was sufficient for the expression of a functional surface receptor. Apparently, the VHCa or VHCP chimeric chains can pair with the endogenous ( or a chains ofthe recipient T cell to form a functional a(3 heterodimeric receptor. Thus, this chimeric receptor provides the T cell with an antibody-like specificity and is able to effectively transmit the signal for T-cell activation and execution of its effector function.meric TcR" (cTcR) would contain the extracellular C region, the transmembrane segment, and the cytoplasmic domains of normal TcRs and should therefore be able to function normally to induce T-cell proliferation, interleukin production, and target cell lysis.Spontaneous transcription of an aberrantly joined IgVH gene and a TcR JaCa gene resulting from site-specific chromosome 14 inversion in human T-cell tumors was reported (4-6); however, no protein product was detected. Chimeric fusion proteins have also been produced in myelomas by the introduction of the TcR C exons between the VK and CK exons (7). More recent reports have shown that a chimeric protein containing the TcR V,, domain and the immunoglobulin C domain can be synthesized in myeloma cells. This protein associates with normal L chains to form a secreted tetramer (8). Attempts to assemble and secrete similar chimeric protein containing the VtC,-, and VC3CK have not been successful (9).The studies described above all reported the construction of nonfunctional chimeric Ig-TcR proteins. In this paper we describe the construction and functional expression in T cells of chimeric TcR genes made by recombining the immunoglobulin VH and VL rearranged gene segments to the C-region exons of the TcR a and 13 chains. The resulting cTcR...

To generate chimeric Ag receptors (CARs) for the adoptive immunotherapy of cancer patients with ErbB2-expressing tumors, a single-chain Ab derived from the humanized mAb 4D5 Herceptin (trastuzumab) was initially linked to T cell signaling domains derived from CD28 and the CD3ζ to generate a CAR against ErbB2. Human PBLs expressing the 4D5 CAR demonstrated Ag-specific activities against ErbB2+ tumors. However, a gradual loss of transgene expression was noted for PBLs transduced with this 4D5 CAR. When the CD3ζ signaling domain of the CAR was truncated or mutated, loss of CAR expression was not observed, suggesting that the CD3ζ signaling caused the transgene decrease, which was supported by the finding that T cells expressing 4D5 CARs with CD3ζ ITAM mutations were less prone to apoptosis. By adding 4–1BB cytoplasmic domains to the CD28-CD3ζ signaling moieties, we found increased transgene persistence in 4D5 CAR-transduced PBLs. Furthermore, constructs with 4–1BB sequences demonstrated increased cytokine secretion and lytic activity in 4D5 CAR-transduced T cells. More importantly, PBLs expressing this new version of the 4D5 CAR could not only efficiently lyse the autologous fresh tumor digests, but they could strongly suppress tumor growth in a xenogenic mouse model.

Primary human activated T lymphocytes were genetically grafted with chimeric T cell receptors (TCR). Three domain single chain (sc-) TCR as well as two chain (tc-) TCR gene constructs were derived from the melanoma-specific cytotoxic human T cell (CTL) clone 82/30, and linked to the CD3-signaling element. Chimeric TCR ␣ and ␤ receptor genes were structurally designed to prevent pairing with endogenous TCR ␣ and ␤ chains in order to prevent the generation of unpredictable immune specificities. After transduction of polyclonally activated human peripheral blood lymphocytes