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Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or TRAIL-receptor agonistic monoclonal antibodies promote apoptosis in most cancer cells, and the differential expression of TRAIL-R2 between tumor and normal tissues allows its exploitation as a tumor-associated antigen. The use of these antibodies as anticancer agents has been extensively studied, but the results of clinical trials were disappointing. The observed lack of anticancer activity could be attributed to intrinsic or acquired resistance of tumor cells to this type of treatment. A possible strategy to circumvent drug resistance would be to strike tumor cells with a second modality based on a different mechanism of action. We therefore set out to generate and optimize a bispecific antibody targeting TRAIL-R2 and CD3. After the construction of different bispecific antibodies in tandem-scFv or single-chain diabody formats to reduce possible immunogenicity, we selected a humanized bispecific antibody with very low aggregates and long-term high stability and functionality. This antibody triggered TRAIL-R2 in an agonistic manner and its anticancer activity proved dramatically potentiated by the redirection of cytotoxic T cells against both sensitive and resistant melanoma cells. The results of our study show that combining the TRAIL-based antitumor strategy with an immunotherapeutic approach in a single molecule could be an effective addition to the anticancer armamentarium.

T cells are the most potent cells of the immune system; however, they fail in the immunosurveillance of tumors. In previous decades, scientists began studying methods to take advantage of T-cell potency in cancer therapy by redirecting them against tumors independently from the T-cell receptor-defined specificity. Among different approaches, the most promising are the use of bispecific antibodies and T-cell engineering to create chimeric antigen receptors. Bispecific antibodies, by simultaneously recognizing target antigen and an activating receptor on the surface of an immune effector cell, offer an opportunity to redirect immune effector cells to kill cancer cells. The other approach is the generation of chimeric antigen receptors by fusing extracellular antibodies to intracellular signaling domains. Chimeric antigen receptor-engineered T cells are able to specifically kill tumor cells in a MHC-independent way. The efficacy of these reagents in different formats has been clinically validated and will be presented here.

The antitumor specificity of T cells can be induced by gene transfer using a recently developed therapeutic approach (T body). In this work, we genetically conferred anticarbohydrate specificity onto T cells using the variable regions of monoclonal antibody MLuC1, which binds the Lewis(Y) (LeY) tumor-associated antigen that is overexpressed on several human carcinomas. The variable regions of MLuC1, which are in a single-chain Fv (ScFv) configuration, were cloned and spliced in a eukaryotic expression vector with both the gene encoding the signal-transducing gamma-chain of the human Fcgamma receptor and a flexible hinge domain. The chimeric ScFv-gamma gene was expressed in a murine cytotoxic T-cell hybridoma. Transfectants receiving vector only served as a negative control (mock). Screening for functional transfectants was carried out using a tumor growth inhibition assay. The soluble form of MLuC1 ScFv was recovered from bacteria periplasm and tested for binding to LeY-expressing cells by the fluorescence-activated cell sorter analysis. Despite the low binding ability of the soluble MLuC1 ScFv, 7 of 13 genetically engineered cytotoxic T lymphocyte clones inhibited the growth of LeY-positive cells and did not affect growth of LeY-negative cells. None of the mock clones tested specifically inhibited tumor growth. These data indicate that, by chimeric MLuC1 ScFv-gamma gene transfer, it is possible to confer anticarbohydrate specificity onto T cells and extend the applicability of the T-body approach to tumor-associated antigens that are naturally not recognized by T cells.

T cell triggering can be achieved by monoclonal antibodies (mAbs) specific for the CD3/TcR complex. In the presence of appropriate costimulation and/or progression factors, such triggering permits the generation of effector cells for immunotherapy protocols involving the redirection of T cell lysis against tumor cells by mAbs bispecific for anti-CD3/anti-tumor cells (bs-mAbs). Focusing our analysis on the clinically relevant bs-mAb OC/TR, we found that bs-mAbs generated with the same anti tumor specificity, but two other anti-CD3 mAbs, TR66 and OKT3, have the same and a significantly lower lytic potential, respectively, compared with that of OC/TR. To evaluate the relevance of the anti-CD3 component, we examined several anti-CD3 mAbs with respect to binding parameters and the ability to trigger T lymphocytes. Competitive binding assays suggested that all anti-CD3 mAbs recognized the same or overlapping epitopes, although mAbs BMA030 and OC/TR bound with lower avidity than did alpha CD3 (the bivalent anti-CD3 mAb produced by the hybrid hybridoma OC/TR). TR66 and OKT3, as determined by measurement of the affinity constants. In all lymphocyte populations examined, which included resting peripheral blood mononuclear cells (PBMC), activated PBMC and T cell clones, OKT3, BMA033 and OC/TR failed to mobilize Ca2+ without cross-linking, whereas alpha CD3, in both murine and murine-human chimeric versions, TR66 and BMA030, did not require cross-linking. The ability to induce CD3 modulation was associated in part with the induction of Ca2+ fluxes. Despite the differences in the behavior of these mAbs in triggering the events that precede proliferation, all of them ultimately led to expression of the IL-2 receptor and to proliferation in T cells in the presence of accessory cells. Our data suggest that anti-CD3 mAbs that bind more rapidly (strong Ca2+ mobilizers) and more tightly under physiological conditions are good candidates for retargeting T cells in the bs-mAb clinical application.

The high frequency of relapse after induction chemotherapy in advanced ovarian carcinoma patients calls for new therapeutic modalities. Retargeted T cell-mediated lysis can be achieved using the bispecific antibody (BsmAb) OCTR, directed to CD3 on T cells and to the folate receptor on ovarian carcinoma cells. Twenty-eight patients with limited intraperitoneal disease after first-line therapy entered a phase II study. They received two i.p. 5 day cycles of activated PBMC retargeted with OCTR. Despite unfavorable tumor characteristics, 7 of 26 patients (27%) showed complete or partial intraperitoneal responses with strict surgicopathologic evaluation. In most cases, the disease relapsed outside the peritoneal cavity, and in 1 case complete intraperitoneal response was accompanied by progression in retroperitoneal lymph nodes. The morbidity was mild to moderate and transient. Combination of i.v. and i.p. administration of OCTR-retargeted lymphocytes will possibly lead to extraperitoneal cure. Ongoing clinical studies indicate that the i.v. infusion of up to 8 x 10(8) OCTR-retargeted T lymphocytes does not induce a higher toxicity than the i.p. treatment. To avoid PBMC preactivation, new approaches for delivering accessory signals are under investigation. Preliminary results indicate that nonactivated PBMC retargeted by OCTR in the presence of an anti-CD28 monoclonal antibody (mAb) are able to significantly inhibit tumor growth.

Targeting of T lymphocytes against epidermal growth-factor-receptor (EGF-R)+ tumor cells was achieved by constructing a hybrid hybridoma which secretes an anti-EGF-R/anti-CD3 bispecific monoclonal antibody (biMAb) of hybrid isotype (IgG1/IgG2a). Purification of biMAb molecules from parental anti-EGF-R and anti-CD3 MAbs was performed by protein-A chromatography. The purified biMAb was able to trigger the lysis of EGF-R+ tumor cell lines (A431, IGROV-1, MDA-468 and U-87) and of NIH-3T3 transfectants expressing human EGF-R by cytolytic T lymphocytes, but it was ineffective in the case of EGF-R-negative tumor targets. Normal EGF-R+ cells (keratinocytes and endometrial cells) were also susceptible to biMAb-targeted cytolysis. However, the amount of biMAb required to induce half-maximal cytolysis of tumor cells over-expressing the EGF-R molecule (A431) was considerably lower than that required to induce lysis of EGF-R+ tumor or normal cells which express EGF-R at considerably lower density. The ability of such biMAbs to deliver activation signals to T cells was evaluated by Ca++ mobilization and lymphokine production experiments. The soluble anti-EGF-R/anti-CD3 biMAb failed to induce intracellular Ca++ increases, which occurred only after cross-linking induced by an anti-mouse IgG antibody. Secretion of lymphokines (IFN-gamma, TNF-alpha and GM-CSF) was induced by contact of the biMAb-coated effector cells with the relevant tumor target, whereas the soluble biMAb was virtually ineffective. In addition, biMAb-coated effector cells retained the ability to recognize and to lyse EGF-R+ tumor cells for a prolonged period of time. Our data indicate that activation of effector cells targeted by biMAbs can only occur at the tumor site, where cross-linking of surface CD3 molecules is induced by contact with the tumor cells.

To target T lymphocytes against EGF-R+ tumors, we constructed anti-CD3/anti-EGF-R bimAbs either by the generation of a hybrid hybridoma (quadroma) or by a chemical cross-linking method. Analysis of the in vitro functional activity of these two different constructs indicated that the quadroma-secreted bimAb was more efficient in targeting the CD3+8+ clones against EGF-R+ target cells with respect to the bimAb produced by chemical method. In addition, the quadroma-produced bimAb is able to induce cytolysis of EGF-R+ tumor cell lines of PHA-induced lymphoblasts that had been expanded in IL-2-containing medium, whereas tumor cells lacking expression of EGF-R were not lysed. Resting PBL targeted by the bimAb did not display significant cytotoxicity against the relevant tumor. An anti-CD16 hybridoma (IgG1) was fused with an anti-folate-binding protein hybrid (IgG2a) to construct bimAbs to target NK cells against NK-resistant ovarian carcinomas. The hybrid IgG1/IgG2a bimAb triggered the specific lysis of relevant target cells by resting NK cells, but it was ineffective when CD8+TCR alpha/beta+ cultured cell populations were used as effectors. Only marginal increases of cytolytic activity could be induced by the bimAb when IL-2-activated PBL (i.e., LAK cells) were used as effectors due to the high cytolytic activity of these cells against the relevant tumors in the absence of bimAb. The possible use of anti-CD16 or anti-CD3 bimAbs for the development of different cellular immunotherapy strategies against cancer is discussed.

T cells can be induced to specifically lyse tumor cells with bispecific antibodies containing anti-T cell receptor mAbs crosslinked to anti-tumor mAbs. Such "targeted cytolysis" requires that the target cell be bound directly to the cytotoxic cell. In addition, targeted T cells mediate a second activity, the secretion of factors that can block the growth of both tumor target cells and bystander tumor cells. When given to nude mice bearing intraperitoneal human ovarian carcinoma, targeted human T cells cause the rapid removal of most tumor cells from the peritoneum, and markedly prolong the times of survival of treated mice. The efficacy of targeted T cells for treating human cancer is currently being tested in clinical trials.

We have used a relatively new technology to increase the number of human lymphocytes that will react with human ovarian carcinoma cells. This technology, often called "retargeting of the immune system," can temporarily redirect the activity of immune cells that were originally committed to react with foreign substances other than cancer cells. In the example presented here, the antitumor effects of retargeted human T lymphocytes, collected from normal donors, were tested in immunodeficient mice with a human ovarian carcinoma line growing intraperitoneally. We retargeted T cells in vitro with a bispecific antibody that reacted with the T cell receptor complex and with a cell-surface antigen expressed by the ovarian carcinoma cells. Retargeted lymphocytes, injected intraperitoneally into mice 4 days after intraperitoneal injection of the tumor cells, impeded tumor growth and doubled the host survival time. These findings provide support for the concept that treatment of ovarian cancer patients with retargeted T cells could prove beneficial.

MAbs directed against triggering surface molecules expressed by T lymphocytes (CD3, TCR, CD2, CD28) or by NK cells (CD2, CD16) are able to induce the functional program of these cells. These MAbs represent suitable reagents to construct biMAbs directed against TAA, in order to specifically target effector lymphocytes against tumor cells. Anti-CD3/anti-EGF-R biMAbs were constructed to specifically direct T lymphocytes against EGF-R+ tumor cells. Such biMAb are able to induce cytolysis of EGF-R+ tumor cell lines (A431, IGROV, KATO-III and U-87) by cytolytic CD3+ effector lymphocytes while tumor cells having low or absent expression of EGF-R were not lysed. In addition, both cytolytic T (CD8+) cells and non-cytolytic (CD4+) IL-2-expanded lymphocytes were able to secrete lymphokines upon contact with EGF-R+ tumor cells. To target NK cells against NK resistant ovarian carcinomas, we used an anti-CD16 Mab (IgG1) together with an anti-ovarian carcinoma MAb (IgG2a), to construct biMAbs using the hybrid hybridoma technique. The hybrid IgG1/IgG2a biMAb triggered the specific lysis of relevant target cells by resting NK cells and by a subset of NK clones. In addition, some TCR gamma/delta+ clones but not TCR alpha/beta+ clones could be targeted by the biMAb.

A bispecific F(ab')2 fragment with anti-CD3 and antitumor specificity was used to target activated human peripheral blood lymphocytes (PBL) against OVCAR-3 human ovarian carcinoma cells growing i.p. in athymic mice. Mice were given injections of OVCAR-3 cells on day 0 and treated with i.p. injections of activated PBL coated with the [anti-CD3 (TR66) x antitumor (MOv18)] bispecific F(ab')2 on day 4, using an approximate effector:target ratio of 1:1. Treatment was evaluated for the ability either to block tumor growth at 15 days or to prolong survival of tumor-bearing mice. After 15 days, the incidence of mice with tumor growth was 20% among those given PBL coated with bispecific F(ab')2, whereas the incidence among mice untreated or treated with PBL alone or PBL with either parental antibody ranged from 80 to 94%. The mean survival time of tumor-bearing mice treated with PBL and bispecific F(ab')2 was 104 days, which was 3.5 times that of untreated mice and twice that of mice given PBL alone or PBL with either parental antibody. These results provide support for the concept that treatment of ovarian cancer patients with targeted T-cells could prove beneficial.