T cell-based immunotherapy for cancer: a virtual reality?
Authors: L. G. Lum
Journal: CA A Cancer Journal for Clinicians (1999)

Advances in T cell, molecular, and cancer immunobiology have stimulated new immunotherapeutic approaches to cancer treatment, a number of which use the T cell as a therapeutic platform. This review provides historical and current perspectives on immunotherapy with emphasis on the use of T cells for adoptive immunotherapy trials and will focus on strategies that utilize T cells as cytotoxic effectors, mobile minicytokine factories, redirected cytotoxic effectors using bispecific antibodies (biAb), and cytotoxic effectors that target tumor associated antigen (TAA) using genetically engineered chimeric receptors. Both antigen (Ag)-specific and Ag-non-specific systems will be covered. Because immune responses to tumor are dependent upon network interactions between cellular elements of the immune system, studies involving natural killer (NK) cells, lymphokine activated natural killer (LAK) cells, and dendritic cells (DC) will also be reviewed. Finally, we will explore the use of autologous or allogeneic stem cell transplant after high-dose chemotherapy (HDC) in combination with immunotherapy to optimize antitumor activity. Immunotherapy has been administered to cancer patients alone, in combination with non-ablative chemotherapy, and after myeloablative chemoradiotherapy with autologous or allogeneic bone marrow transplantation (BMT). A number of key observations have accrued on the basis of these experiences. These include: 1) The effect of adoptively transferred T cells is dose-dependent; 2) Tumors can suppress the ability of the host immune system to detect and/or develop anti-tumor responses; 3) Tumor immunogenicity varies; 4) Memory T cells from an immune donor can transfer anti-tumor activity to a recipient; 5) Interleukin 2 (IL-2) can augment the in vivo therapeutic effect of T cells; 6) Donor-derived HLA-identical allogeneic T cells are more effective than autologous T cells in providing anti-tumor effects in BMT; and 7) HDC can enhance the effectiveness of T cell infusions. With early detection, most malignancies can be cured by conventional surgery, chemotherapy, or radiotherapy. In contrast, it is nearly impossible for the immune system to reject bulky or metastatic disease. The challenge for the immunotherapist, therefore, is to identify Ag-specific or non-specific systems that will improve clinical responses in the treatment of advanced cancers and hematologic malignancies. Decreased Class I major histocompatibility complex (MHC) expression on the tumor cells, as well as down-regulatory cytokines, prostaglandins, or other factors secreted by tumors, can induce a state of anergy or non-responsiveness.1 Recently, a preclinical model using transgenic T cells from tumor-bearing mice suggests that Ags expressed on tumor cells can fail to induce immune responses, even in the presence of increased numbers of cytotoxic T lymphocyte (CTL) precursors.2 Successful immunotherapeutic approaches will need to include strategies that address these and other issues. Antigen Presentation to CD4+ Cell The Search for Antigens Much has been learned over the past 25 years about the immune system's ability to discriminate between self and non-self Ags. The immune response to foreign Ag proceeds through a series of highly regulated, complex steps leading to Ag-specific responses and the establishment of immune memory, so that re-exposure to Ag leads to recall and amplification of specific immune responses (Figs. 1 and 2). The development of monoclonal antibodies (mAbs), which provided the tools for identifying new Ags, and the identification of tumor-Ags—as well as the elucidation of the mechanisms that led to the development of specific antibody or CTL responses in preclinical models—have provided the foundation for the development of therapeutic applications. Unfortunately, most "tumor-associated antigens" are over-expressed self Ags. The key challenge in immunotherapy is to induce the immune system of a cancer patient to make a specific immune response to autologous tumor. A few tumor-specific Ags, such as HER-2/neu,3 malignant melanoma,4-6 and p53,7 are very well characterized and are known to induce in vitro and in vivo specific immune responses. These Ags have been used in vaccine studies as well as for the generation of Ag-specific CTL. Antigen Presentation to the CD8+ Cell Native, humanized, toxin-conjugated, radionuclide-conjugated, and anti-idiotypic mAbs have been used clinically in the cancer setting with varying degrees of success. In the adjuvant setting, significant clinical responses were achieved with 17–1A mAb directed at colon carcinoma Ag in a large follow-up series of patients with colon carcinoma.8 Anti-CD20 mAbs conjugated with radionuclides after peripheral blood stem cell transplantation (PBSCT) for non-Hodgkin's lymphomas (NHL)9, 10 and anti-idiotypic mAbs directed at the idiotype of the patient's B cell lymphoma have yielded impressive clinical responses in patients with lymphomas.11 Other recent examples of mAbs used clinically include Rituxan (Ritxumab, humanized anti-CD20) and Herceptin (Trastuzumab, humanized anti-HER-2/neu), which were approved by the FDA for the treatment of low-grade NHL12 and HER-2/neu+ metastatic breast cancer,13 respectively. Additionally, Zenapax (Daclizumab, humanized anti-IL-2R mAb) was approved for use in combination with immunosuppressive regimens for the treatment of renal graft rejection.14 Although the development of mAb therapy directed at solid tumors has not been as rapid as anticipated, the success of Herceptin therapy for HER-2/neu+ stage IV breast cancer suggests that appropriately engineered antibodies soon will become more prominent. These successes also provide the rationale for developing approaches that combine antibody and cell therapy, a combination that may lead to further improvements in remission rates or survival among high-risk patients. Interactions between the CD8+ Cell and Tumor Although adoptively transferred T cells can eliminate or reduce lethal tumor burdens in animals,15-17 adapting this principle in humans has been problematic. Moreover, while dramatic clinical responses have been observed in some patients with renal cell carcinoma (RCC) and malignant melanoma (MM) who received treatment with tumor infiltrating lymphocytes (TIL),18, 19 the success of murine models did not translate into higher cure rates in large trials of patients with RCC and MM. The reasons for these disappointing results are discussed later in this review. Cytotoxic CD8+ cells depend upon the presence of MHC Class I on the tumor target for killing to take place (Fig. 3). Consequently, tumor cells that lack Class I expression escape from the Class-I-mediated killing.20 The use of biAbs for retargeting the lytic activity of CTL,16, 21 or of a T cell with a chimeric receptor containing a portion of a targeting mAb (T-body) that targets specific tumor associated antigens22-25 represent efforts to overcome this barrier by redirecting the T cells in a non-MHC restricted manner. Both approaches can increase the precursor frequency of tumor specific CTL. The type of T cell used in immunotherapy may enrich immune responses and augment anti-tumor effect. Based on murine models, there appear to be two distinct populations of CD4+ cells (Th1 and Th2) that can be distinguished by their cytokine profiles rather than by phenotyping. Th1 cells secrete cytokines interferon γ(IFNγ) and tumor necrosis factor α (TNFα) that mediate responses to viruses, bacteria, and protozoans; and Th2 cells secrete cytokines that help B cells proliferate and differentiate.26 Th1 responses lead to increased cellular immune responses to tumor, as well as increased secretion of tumoricidal IFNγ and TNFα.26 Th1 and Th2 cell types have been identified in human systems. Recent studies show that the coactivation of human T cells with anti-CD3/anti-CD28 mAbs can generate a Th1 functional phenotype.27-29 Furthermore, the addition of IL-12 to cultures of coactivated T cells enhances the levels of IFNγ secretion.30 Similar functional profiles have been described in the CD8+ subset analogous to Th1 and Th2 profiles seen in CD4+ cells;26, 31-33 they have been designated Tc1 and Tc2 profiles, respectively. Costimulation of T cells may provide highly active CTL populations for immunotherapy. Clinical trials of adoptive immunotherapy began in the mid-1980s. Today's successes with different types of immune cells, and the variety of clinical settings in which these approaches are being applied, have resulted from more than 10 years of basic research and preclinical testing. NK exposed to high concentrations of IL-2 become LAK that lyse both Daudi cells—a LAK target—and fresh tumor specimens.34-37 NK cells, which are CD16+, CD56+, mostly CD2+, and CD3-cells, are responsible for tumor surveillance.38-41 Preclinical studies and murine trials led to human clinical trials using IL-2 alone and combinations of LAK and IL-2.42-45 Human trials using LAK and high dose IL-2 for the treatment of RCC and MM34, 42-44, 46-51 have reported response rates up to 20%. Although most of the responses seen were due to the administration of IL-2, the need for large numbers of LAK demonstrated that cell expansion cultures could be scaled up to produce LAK in quantity. An intuitively satisfying approach was to expand TIL that display cytotoxicity directed at autologous tumor using IL-2 and to reinfuse the TIL into patients with RCC and MM. TIL are CD3+ cells that display LAK activity, but are more effective killers than LAK on a per cell basis.18 TIL have been reported to traffic to metastatic melanoma lesions.52 Trials using TIL and high dose IL-2 in patients with advanced RCC, MM, and other advanced tumors have achieved clinical responses ranging from 13% to 60%,53 with most reports ranging between 15% and 20%.54-57 The wide range of responses may be explained by differences in patient selection, as well as by laboratory processing differences. One limitation of TIL therapy is the toxicity associated with high-dose IL-2 infusion, which restricts its use in patients who have poor performance status.58-60 The major toxicities of IL-2 are fluid gain and capillary leak leading to respiratory distress and hypotension often requiring vasopressor support and ICU monitoring.58 Other side effects include fever, chills, malaise, diarrhea, increased creatinine, mental status changes, cardiac arrhythmias, and rashes.59, 60 Although high doses of TIL alone can be infused without toxicities,61 TIL efficacy is believed to be linked to co-administration of high-dose IL-2. Subsequent studies suggest that high dose IL-2 alone is equivalent to high dose IL-2 in combination with TIL therapy. Another promising application has been the use of TIL to treat ovarian carcinomas. For example, TIL from ovarian carcinomas stimulated with anti-CD3 and IL-2 were used to treat 12 patients after surgery and chemotherapy.62 This approach uses anti-CD3 as a nonspecific activator of existing "tumor specific lymphocytes" infiltrating the ovarian carcinoma. After a median follow up of 22 to 23 months, the treatment group had a 100% survival by Kaplan-Meier, whereas the two-year survival for patients with progressive epithelial ovarian cancer is reported as between 47% and 63%.63, 64 Long-term follow up is needed to confirm the value of this approach. Although the experience of most investigators suggests that the combination of TIL and high dose IL-2 may be clinically useful, responses are still unacceptably low. Unfortunately, the antitumor activity exhibited by TIL has not been consistent in larger clinical series.18 Although the reasons for this remain unclear, several studies suggest that inconsistent anti-tumor activity may be due to impaired T cell receptor (TCR) signaling functions.65-67 Approaches that overcome such defects in TIL or other T cell preparations may improve clinical responses. Lymph nodes that drain tumors contain sensitized but not fully functional pre-effector T cells that participate in the generation of Ag-specific CTL. In preclinical studies, adoptively transferred anti-CD3 stimulated lymph node T cells cultured in IL-2 (2–10 IU/ml) for two days could mediate the regression of established metastases in a murine sarcoma model.68 This approach takes advantage of the likelihood that the precursor frequency of pre-effector T cells that facilitate the development of tumor-specific CTL would be highest in tumor-draining lymph nodes. Theoretically, in vitro anti-CD3/IL-2 activation would overcome blunted antitumor responses and permit the expansion of tumor specific CTL. It is important to note that in vivo anti-tumor activity did not correlate with in vitro cytotoxicity assays.68 In a clinical study using these lymph node-derived cells— in 11 patients with RCC and 11 patients with MM—one of 11 MM patients had a partial response and six of 11 RCC patients had clinical responses.69 Furthermore, five of seven responders developed delayed type hypersensitivity (DTH) reactivity to autologous tumor. In another study, tumor-specific DTH detected by skin testing and tumor responses were enhanced by vaccinating patients with tumor cells mixed with bacille Calmette-Guérin. Subsequently, T cells were obtained from regional lymph nodes for ex vivo expansion and reinfusion into patients together with IL-2 infusions.70 In the latter study, there was one partial response in 11 MM patients, and two complete and two partial responses in 12 RCC patients. This approach is also encouraging and needs further evaluation. ALT involves infusions of autologous peripheral blood mononuclear cells (PBMC) produced by cultures containing extracts of autologous tumor and conditioned media (CM) derived from OKT3-stimulated PBMC.71 Preclinical studies using tumor extracts from lung carcinoma and melanoma showed that murine splenocytes can respond to tumor challenge.72, 73 ALT-generated cytotoxic cells could be obtained from PBMC grown in CM produced by OKT3-stimulated PBMC instead of autologous tumor. ALT cells are indirectly activated via supernatants from OKT3-stimulated PBMC, whereas T cell receptor activated T cells (TRAC) are prepared by directly cross linking the T cell receptor (TCR) with OKT3 followed by expansion in low dose IL-2 (see TRAC). Anti-CD3 MAb Activation of T Cells ALT was used in 90 patients with metastatic RCC who were randomized to treatment with cimetidine alone or cimetidine plus ALT. Six doses of 109 ALT were given monthly without toxicity.71 Survival in the ALT group was 2.5 times that for the cimetidine group alone (p=0.008), and patients who were exposed to more than 500 pg of IL-1 in the CM had a six-fold survival advantage (p<0.00005). ALT was safe, and ex vivo ALT mediated an anti-tumor effect without IL-2 infusions. These results were confirmed in a 355-patient, multi-institutional study.74 Nevertheless, follow-up studies did not confirm significant differences compared to treatment with interferon, and the ALT studies were discontinued. Cross linking of the TCR with anti-CD3 (OKT3) results in T cell proliferation, cytokine synthesis, and immune responses.75-78 TRAC are produced by OKT3 and IL-2 (100 IU/ml) stimulation of PBMC (Fig. 4). TRAC have LAK- and NK-like cytotoxic properties and produce cytokines, such as IFN, TNFα, or granulocyte macrophage colony stimulating factor (GM-CSF), which may provide anti-tumor effects. They also can serve as vehicles to deliver targeting antibodies or gene products. In preclinical models, TRAC exerted anti-tumor or antilymphoma effects. For example, TRAC reduced liver metastases due to MCA-38-LD adenocarcinoma more effectively than the same number of LAK cells,79 produced 20-fold higher levels of cytotoxicity directed at a syngeneic mastocytoma than LAK cells,80 and were effective in preventing death due to tumor in a model in which TRAC were injected into severe combined immunodeficient mice with a human carcinoma.81 Moreover, TRAC infused at the same time as BMT increased the survival of mice preinjected with syngeneic lymphoma.82 Anti-CD3/Anti-CD28 Coactivation of T Cells TRAC can be expanded from PBMC or bone marrow of normal subjects, and from patients with malignancy, to mediate non-MHC restricted cytotoxicity.83-93 In vitro studies showed that human TRAC exhibit non-MHC restricted cytotoxicity against Daudi cells (LAK targets), K562 cells (NK targets), leukemic blasts,94, 95 neuroblastomas,84 and autologous plasma cells in multiple myeloma.96 A clinical trial using TRAC in solid tumor patients with RCC and MM has been reported.97 PBMC activated with OKT3 for 18 hours were given with IL-2 infusions. The central principle involved in the TRAC trials was to use in vitro anti-CD3 activation and the patient as his own bioreactor for in vivo expansion of TRAC. This therapy led to a marked lymphocytosis (50,000 cells/μl) with mild and tolerable toxicities that were likely due to IL-2. A recent murine study showed that infusing the CD4+ T cells during the nadir in white blood cell counts after cyclophosphamide and infusion IL-2 is important for obtaining clinical responses.98 The phase I clinical trial using anti-CD3 activated CD4+ cells and IL-2 after 300 or 1,000 mg/m2 IV cyclophosphamide showed promise with the induction of one complete responder, two partial responders, and eight minor responders in a group of 31 patients with advanced cancers and NHL.99 Cross linking of the TCR with anti-CD3 triggers a signaling cascade resulting in T cell proliferation, cytokine synthesis, and immune responses.75-78 Optimal activation and proliferation, however, require costimulation of CD28 on T cells with anti-CD28 mAb or the B7.1 and B7.2 molecules (CD80 and CD86).100-104 Coactivation of T cells is depicted in Fig. 5. These interactions enhance proliferation and stabilization of mRNAs for IL-2, IFNγ, TNFα, and GM-CSF.105 Costimulation of the CD28 receptor also leads to enhanced production of beta chemokines RANTES, MIP1-α, and MIP1- β.106 The enhanced secretion of chemokines at the tumor site may augment recruitment of effector cells. COACTS exhibit in vitro anti-tumor activity directed at a variety of tumor cell lines.29 COACTS generate Th1-type cytokine profiles100, 107 and may survive longer in vivo due to induction of the cell survival gene Bcl-x1, which confers resistance to apoptosis.108, 109 In a B16 melanoma murine model, the use of T cells from draining lymph nodes, costimulated with anti-CD28, resulted in higher levels of IFNγ secretion and specific cytotoxicity.110 Anti-CD28 costimulation could overcome blunted anti-CD3 stimulated proliferative responses of lymph node lymphocytes in patients with head and neck squamous cell carcinoma.111 Such studies suggest that anti-CD28 costimulation may overcome local or systemic anergy.112-114 We recently completed a phase I dose-escalation study using infusions of autologous ex vivo expanded COACTS for the treatment of refractory cancer patients.115 The technical limits of ex vivo COACTS expansion, the in vivo localization and trafficking of COACTS, and immune effects induced by COACTS infusions in the patients, were evaluated. Infusions of COACTS were safe, induced detectable serum levels of IFNy, GM-CSF, and TNFα, and significantly enhanced the ability of freshly isolated PBMC to secrete IFNγ and GM-CSF upon in vitro anti-CD3/anti-CD28 costimulation. These data suggest that the immune systems of these patients were modulated by the COACTS infusions. Follow-up studies are in progress to evaluate COACTS in combination with chemotherapy and biologic response modifiers. The use of COACTS after CD34-selected PBSCT for intermediate grade NHL appears promising.116 Eighteen patients have been enrolled in a study, and 15 have completed therapy. The median follow up after dose-intensive chemotherapy and CD34-selected stem cell reinfusion is 408 days, with a range from 77 to 569 days. The median time to progression-free and overall survival was longer than that expected with dose-intensive chemotherapy alone. Some patients who achieved complete responses exhibited clinical evidence of immune responses against their own lymphoma. The analysis of T cell activation responses before and after adoptive transfer of the COACTS suggests that coactivation may correct T cell activation defects present prior to therapy. In vitro coactivation studies on PBMC from autologous and allogneneic BMT recipients showed that coactivation significantly enhanced depressed anti-CD3-induced proliferative responses or IL-2 production.117 The IL-2 secreted by T cells from three autologous and three allogeneic recipients was enhanced 0.9- to 25-fold by coactivation. Coactivation of PBMC from selected recipients increased T cell proliferation into the normal range and increased IL-2 secretion. These studies suggest that infusions of COACTS may reconstitute nonspecific and specific anti-tumor immune responses in cancer patients. An elegant approach to adoptive immunotherapy is the development of Ag-specific CTL directed at viruses or tumor-specific Ags.118-121 The development of CTL directed at cytomegalovirus (CMV)122, 123 and EBV-lymphoproliferative disease (EBV-LPD),124 for example, are helping to pave the way for developing tumor specific CTL. First, infusions of CMV-specific CTL could prevent the development of CMV pneumonia in seropositive allogeneic BMT recipients. Second, EBV-specific CTL have been produced by stimulating bone marrow donor T cells with EBV-transformed B cell lines from the recipient for treatment of EBV-LPD after T-cell depleted allogeneic BMT. The generation of CTL directed at p21 ras,125 p53,7, 126-129 and HER-2/neu130 have been reported. Clinical trials using p21 ras vaccination show that immune responses can be induced by vaccination strategies.131 We can reasonably anticipate that there will soon be reports of adoptive transfer of ex vivo expanded CTL directed at mutated oncogene products produced by in vitro priming. Although the culture and expansion of Ag-specific CTL is labor intensive, new culture strategies may reduce the efforts required for growing adequate quantities of CTL for clinical use. Development and refinement of such strategies will have a considerable impact on this area of immunotherapy. Essential to the development of tumor specific responses are "professional" Ag-presenting cells (APC) or DC. DC are the most effective presenters of Ag in the immune system and can powerfully trigger T cell responses after encountering Ag.132-134 DC internalize, process, and present Ag to T cells135-139 and respond to Ag-encounter by upregulating expression of MHC molecules, costimulatory molecules, and cytokines.140 The ex vivo culture of DC followed by peptide loading for DC immunization protocols or manipulation of cultured DC to optimize vaccine strategies are being actively pursued by many groups. Pulsing DC with tumor peptides and then infusing DC into the patient uses the patient's own immune system as a bioreactor to educate and expand tumor specific CTL in vivo.132, 134, 141-143 DC can be expanded from PBMC or bone marrow and loaded with peptides or tumor lysates for clinical use.134, 139, 141-146 One elegant vaccination approach uses infusions of DC loaded with tumor-specific idiotype protein to stimulate host anti-tumor immunity.147 In four patients with follicular B-cell lymphoma, for example, one had complete regression, one had a partial remission, and a third had molecular evidence of resolution of disease. These data suggest that clinically relevant specific tumor responses can be achieved with DC under the appropriate in vivo conditions. This type of approach would obviate the need to expand large numbers of T cells for immunotherapy. For example, in a phase I study, autologous DC from HLA-A2+ and A2- refractory prostate cancer patients were pulsed with prostate specific membrane antigen (PSMA) or peptides thereof (PSM-P1 or PSM-P2) and infused into men to induce immune responses to their prostate cancer.148 There were no clinical toxicities related to peptide or DC infusions, and seven subjects demonstrated partial responses based on National Prostate Cancer Project criteria.148 In a phase II trial, several men immunized with DC pulsed with PSMA peptides showed PSMA-peptide-specific responses or IFNγ secretion upon restimulation with PSMA peptide.149 Although this approach needs refinement, it illustrates the potential of using DC loaded with peptides or proteins to induce immune system antitumor responses. Bispecific Antibody Targeting of Tumor Peptide vaccination takes advantage of endogenous DC and T cells to upregulate systemic responses to the immunogen. Although classical vaccination cannot be characterized as adoptive immunotherapy, the principle of inducing Ag-specific responses without performing ex vivo manipulation is an attractive strategy. Such was the case in a recent vaccination trial using HLA-A2 restricted immunodominant peptides from the gp100 melanoma-associated Ag. The peptides were identified and used to vaccinate 31 patients with metastatic MM; IL-2 was also given to patients to enhance in vivo T cell functions.150 Thirteen of 31 (42%) patients had objective responses and four had mixed or minor responses. Other active immunotherapy approaches using carcinoembryonic antigen (CEA) or peptides thereof have also been developed for clinical trials in colon and breast cancer. Additional studies are needed to determine whether tumor cell lysates,151 whole tumor proteins, the HLA-A2 restricted peptides,146, 150, 152 RNA from tumors,153 or DNA154, 155 are the best immunogens for optimizing host responses. The good news is that roughly 50% of the Caucasian population is HLA-A2+; unfortunately, the remaining 50% of the Caucasian population, as well as the non-white population, is HLA-A2-. Other active immunotherapy strategies include enhancing the immunogenicity of the tumor by gene modification to augment recognition by transducing cytokines or costimulatory ligands into the tumors.156 The development of biAbs combines mAb targeting specificity with the cytotoxicity of T cells or other effector cells. The specificities of two mAbs are combined into one protein molecule—the biAb—so that it can bind and redirect the cytotoxicity of the effector cell to a tumor associated antigen on the tumor cell (Fig. 6). BiAbs can be produced by chemical heteroconjugation,157 hybrid hybridomas,158 or recombinant technology.159 One mAb binds to the T cell and the other targets a tumor Ag. In this manner, arming of nonspecific polyclonally activated T cells can redirect the non-MHC restricted cytotoxicity exhibited by TRAC to artificially increase the precursor frequency of CTL directed at specific tumor Ags. Treatment with biAbs could lead to specific binding and enrichment of effector cells at the tumor site as well as the augmentation of tumoricidal activity. BiAbs have been used for targeting drugs, pro-drug activation, and immune recruitment strategies.21 It has been more than 12 years since biAbs were first constructed by chemically conjugating two inAbs.160-163 The Table highlights some of the preclinical/clinical studies that utilized anti-CD3 and a second mAb directed at tumor targets. Recombinant technology has allowed the rapid and reliable cloning of mAb variable regions and the generation of recombinant single chain antibody fragments (scFv). These advancements have led, in turn, to the development of biAb fusion proteins. For example, recombinant anti-CD3 × anti-L6 (carcinoma Ag) was produced by fusing the binding domains of anti-L6 and anti-CD3 single-chain molecules.179 The anti-CD3 × anti-L6 fusion protein mediated adhesion between T cells and L6+ tumor cells, stimulated proliferation, and enhanced cytotoxicity directed at L6+ tumor cells. The use of recombinant technology will undoubtedly lead to the continued development of useful fusion proteins for tumor targeting and has already resulted in the development of humanized or human reagents that avoid the development of human anti-mouse antibody responses during therapy. Recently, researchers have taken advantage of targeting and coactivating T cells by using two biAbs. The first biAb combination contains anti-CD3 and antitumor Ag mAbs to activate T cells and bind to the tumor target. The second biAb combination contains anti-CD28 and anti-tumor Ag mAbs to coactivate T cells and also target tumor Ag. This approach provides a coactivation signal (via CD28 receptor stimulation) to the T cells, as well as enhanced binding of the T cells to the tumor, by using two targeting biAbs. Using this system, costimulation with anti-CD28 alone or anti-CD28 (anti-TAA) resulted in enhanced signaling,180, 181 cytokine production,182 potency of killer activity in lymphoma/leukemia models,172, 183, 184 and cytotoxicity directed at colon carcinoma lines.158 Clinical studies in which biAbs were used to arm NK, granulocytes, or monocytes show promise. NK-mediated cytotoxicity can be redirected with anti-CD16 (anti-Fcγ R) to target human melanoma cells with antibody (96.5)161 and neutrophils can be redirected with an anti-disialoganglioside × anti-FC(γ)RI biAb to kill targets with disialoganglioside.185 In 27 patients with breast cancer, for instance, infusions of 2B1 biAb (which targets c-erbB-2 and Fcγ RIII) resulted in two partial and three minor responses.186 In another trial, MDX-H210 (anti-CD64 × HER-2/neu), which redirects FcRI-positive monocytes and macrophages to kill tumors that overexpress HER-2/neu, was used to treat 10 women with breast cancer;187 there was one partial response and one mixed response. Using a similar approach, several groups have developed second- and third-generation humanized biAbs reactive to T cells and HER-2/neu+ tumors.188-190 These reagents may prove to be quite effective in clinical trials. To gene