Adoptive T-cell therapy represents one of the major recent breakthroughs in anticancer therapy. The evidence that T-cell-mediated graft versus tumor (GVT) or graft versus leukemia (GVL) effect can cure cancer comes from multiple studies, showing that patients who develop graft versus host disease (GVHD) post allogeneic stem cell transplant (SCT) have better disease control than those who do not have GVHD [1,2] [1,2]. Furthermore, induction of GVHD in patients with donor lymphocyte infusion has also been shown to have antitumor activity in various hematologic malignancies including leukemia, lymphoma, and multiple myeloma [3,4] [3,4] through a GVT effect.
Given that the allogeneic SCT remains a relatively toxic therapy with multiple side effects including severe GVHD, multiple different methods of adoptive T-cell therapy have been evaluated to achieve the antitumor activity without increasing clinical toxicities. These have included use of tumor-infiltrating lymphocytes [5,6] [5,6], anti-CD3-activated T cells (ATC) , and anti-CD3/anti-CD28 coactivated T cells , which demonstrated activity in various preclinical models but failed to show significant clinical utility. More recently, excellent outcomes in B-cell malignancies have been reported with chimeric antigen receptor (CAR) T cells [9,10] [9,10]. These represent genetically engineered autologous patient-derived T cells, which are directed toward tumor-associated antigens (TAAs) such as CD19 . Clinical trials have reported excellent responses with these cells in B-cell acute lymphoblastic leukemia [11,12] [11,12] and chronic lymphocytic leukemia (CLL)  and non-Hodgkin's lymphoma (NHL) . The production of these cells requires use of autologous T cells or allogeneic human leukocyte antigen-identical sibling donor T cells and then transducing the gene for the CAR into the T cells , a process which is labor intensive and time consuming.
In this review, we discuss another method of creating artificial antibody receptors by arming the T cells with bispecific antibodies (BiAbs), which bind to anti-CD3 ATC and TAA at the same time leading to increased perforin/granzyme-mediated non-MHC-restricted specific antitumor cytotoxicity by the armed T cells.
The concept of BiAbs was first introduced in 1980s as a method to target multiple antigens by a single antibody . The recombinant BiAbs are classified into two types such as the Fc-containing BiAbs and BiAb derivatives without Fc regions . The mechanism of action of BiAbs includes binding to the tumor cells on one side through the Fab portion of the antibody against the tumor-specific antigen (such as CD19, HER2, EGFR, or GD2) and to the immune effector cells such as T cells and NK cells, which leads to activation of those immune effector cells and Fc-receptor bearing phagocytic cells such as monocytes/macrophages that can also mediate direct lysis of the tumor cells .
Multiple preclinical studies and animal models have shown good efficacy of BiAbs constructed with anti-CD3 × anti-CD19 components [19–21] [19–21] [19–21]. The studies showed that the cytotoxicity induced by the BiAb was rapid, effective, and specific to the CD19-positive B cells. Earliest clinical trial of such BiAb was conducted in CD19-expressing NHL and patients with CLL using the anti-CD3 × anti-CD19 BiAb . The treatment was well tolerated, although no significant clinical benefit was seen in the patients. Failure was thought to be related to rapid clearance of BiAb.
More recently, Blinatumomab, which is a is a Bi-specific T-cell engager antibody construct with dual specificity for CD19 and CD3, has shown impressive activity in relapsed/refractory B-cell acute lymphoblastic leukemia in multiple clinical trials [23 ▪ ,24] [23 ▪ ,24] as a single agent. In a large clinical trial , 189 patients with relapsed/refractory B-cell ALL were treated with Blinatumomab, 43% of the patients were able to achieve a complete remission and among these patients a significant number (82%) were able to achieve a negative test for minimal residual disease as well. Cytokine release syndrome can be a side-effect of the Blinatumomab therapy that was addressed by the continuous infusion administration successfully. Clinical trials to evaluate the efficacy of Blinatumomab in NHL and CLL are currently underway. BiAbs have been developed for multiple targets (Table 1) and are currently in clinical trials.
USE OF ACTIVATED T CELLS ARMED WITH BISPECIFIC ANTIBODIES
The critical element of a BiAb is that it takes advantage of the binding specificities of two antibodies and combines them with the powerful effector functions of cytotoxic immune cells [35,36] [35,36]. Thus, arming the ATC with TAA antigen-specific BiAbs converts every cytotoxic T cell into a cytotoxic T lymphocyte directed at only tumor cells . Figure 1 shows the mechanism of action of these armed ATC.
Preclinical evidence of the efficacy of bispecific antibodies armed ATC in various malignancies
In an earlier study using peripheral blood mononuclear cells from normal donors or a patient with history of B cell lymphoma , ATC armed with CD20Bi showed enhanced cytotoxicity against CD20-positive lymphoma cell lines as compared with ATC alone (Fig. 2). BiAbs were produced by chemical heteroconjugation of OKT3 (a murine IgG2a anti-CD3 monoclonal antibody) and rituximab (a chimeric anti-CD20 IgG1). After this, the ATC were armed with the BiAb using an optimal concentration of BiAb (50 ng/106 ATC) with anti-CD3 × anti-CD20 BiAb[CD20Bi]. Furthermore, the ATC armed with BiAb were able to kill ARH-77, which is a rituximab-resistant cell line and was able to kill CD20-positive cell lines in the presence of 1–1000 μg/ml of rituximab. A rituximab level of 1000 μg/ml of was required to inhibit cytotoxicity by 50%.
To determine whether, activated umbilical cord-derived T cells armed with CD20Bi would mediate specific cytotoxicity against the CD20-positive lymphoma cell lines, cord blood T cells were expanded and activated from cord blood mononuclear cells using 20 ng/ml OKT3 and 100 IU/ml of interleukin (IL)-2 for 14 days . The study showed that T cells obtained from either the fresh or cryopreserved umbilical cords can be consistently expanded by 20- to 50-fold with the use of anti-CD3 monoclonal antibody and IL-2. T cells armed with an optimal concentration of CD20Bi showed about 30% cytotoxicity against the B-cell lymphoma cell lines. The cytotoxicity of armed T cells peaked at day 10 after coculture with the lymphoma cell lines. These cytotoxic responses were specific to the CD20-positive cells and the CD20Bi armed T cells did not show significant reactivity to other non-CD20-positive cell lines. Similar specific cytotoxicity was seen against HER2-positive breast cancer cell lines when ATC were armed with anti-CD3 × anti-HER2 BiAb (HER2Bi) armed ATC .
Excellent cytotoxicity was seen in malignant glioblastoma cell lines  when treated with ATC armed with HER2Bi or anti-CD3 × anti-EGFR (EGFRBi). EGFRBi-armed ATC killed up to 85% of U87, U118, and U251 targets at E:T ratios ranging from 1 : 1 to 25 : 1. EGFRBi armed ATC exhibited 50–80% cytotoxicity against four primary glioblastoma lines and a temozolomide-resistant variant of U251 cell line.
Similarly, neuroblastoma cell lines  were killed by ATC armed with anti-CD3 × anti-GD2 (GD2Bi). All of the neuroblastoma cell lines tested except one (LAN-6) expressed high levels of GD2. The results showed that GD2Bi-armed ATC exhibited specific killing of GD2-positive neuroblastoma cell lines significantly above unarmed ATC. Armed ATC secreted increased levels of Th1 cytokines when they specifically engaged tumor targets and in addition, chemokines such as RANTES, MIP-1α, and MIP-1β were significantly upregulated by GD2-armed ATC upon engagement with tumor cells.
In a SCID mouse model with PC-3 (prostate cancer cell line) xenografts, ATC armed HER2Bi constructs were able to induce remissions when injected directly into the tumors. Intravenously administered HER2Bi-armed ATC localized to PC-3 xenografts mediated cytotoxicity toward tumor cells and produced significant tumor growth delay of PC-3 tumors .
Similarly, good preclinical efficacy was seen in ovarian cancer cell lines and a mouse model  when treated with ATC armed with BiAb constructs containing HER2Bi and anti-CD3 × anti-CA125 BiAbs. The cytotoxicity measured by 51Cr release assay increased to 89% with armed cytokine induced killer (CIK) cells as compared with unarmed CIK cells. In a xenograft SCID mouse model, real-time tumor regression and remission induction was seen with the activated cells infusion. Mice treated with activated killer cells showed significant reduction in the tumor burden and improved survival.
The above preclinical work clearly demonstrates the activity of ATC armed with BiAbs against multiple tumor targets.
Clinical use of activated T cells armed with bispecific antibodies
Earliest clinical use of T cells armed with BiAb was reported in 1990 in patients with glioma . Lymphokine-activated killer cells treated with anti-CD3 × anti-glioma BiAb were injected directly into the brain tumors of 10 patients. Clinical responses were seen in 8 of 10 patients with regression of complete eradication of the tumors. In the early 1990s, several phase I and II studies were conducted in which patients with ovarian carcinoma were treated with intraperitoneal injections of ATC armed with anti-CD3 × anti-Mov28 (ovarian carcinoma-associated antigen) or anti-CD3 × antifolate receptor, with encouraging clinical responses [45–47] [45–47] [45–47].
CLINICAL USE IN NON-HODGKIN'S LYMPHOMA
In a small pilot clinical trial  containing three patients with history of heavily pretreated diffuse large B-cell lymphoma (two high-risk and one refractory) with CD20Bi armed ATC with IL-2 were given post autologous SCT. These patients receive 15 infusions post SCT (three infusions per week for 3 weeks followed by once weekly infusions for 6 weeks). This study showed that it was feasible to grow T cells collected from patients exposed to multiple lines of chemotherapy and multiple infusions could be given safely without major toxicities augmenting transplant-related toxicities.
In a phase 1 clinical trial  we treated 12 patients with relapsed/refractory CD20-positive diffuse large B-cell lymphoma post autologous SCT. Peripheral blood mononuclear cells (PBMC) were collected from the patients, activated with anti-CD3, and expanded in IL-2 as described above. After patients underwent autologous SCT with a conditioning regimen of R-BEAM followed by infusion of stem cell graft. CD20Bi targeted ATC were infused as early as day +1 after SCT. The median total dose of armed ATC delivered was 6.7 × 1010. Infusions of the ATC were well tolerated and no dose-limiting toxicity was observed. The most frequent side effects included fever, chills, malaise, nausea, and/or vomiting, tachycardia, hypotension, headache, transient hypoxia, hypertension, and dyspnea.
Cytotoxicity assays were performed on PBMC collected from the patients post infusion and showed significant higher cytotoxicity against NHL cell lines as compared with preinfusion PBMC. Infusions of CD20Bi armed ATC induced a Th1 cytokine pattern in the serum that was associated with increased chemokine levels. The clinical efficacy of the ATC could not be assessed because of the small sample size and concurrent use of transplantation but the median overall survival (OS) for the entire group was not achieved at a median follow-up of 24 months. The trial established the safety of the CD20Bi armed ATC in patients with resistant/refractory NHL.
CLINICAL USE OF ACTIVATED T CELLS ARMED WITH BISPECIFIC ANTIBODIES IN SOLID TUMORS
We conducted a phase 1 study [50▪▪] of using ATC armed with HER2Bi in patients with metastatic breast cancer. Patients with both HER2-positive and HER2-negative breast cancer were allowed on the study based on preclinical evidence that HER2-directed therapies can kill both high and very low HER2-expressing breast cancer cells . A total of 23 patients with metastatic breast cancer were enrolled and treated with eight infusions of HER2Bi armed ATC in combination with low-dose IL-2 and granulocyte monocyte colony-stimulating factor (GM-CSF). At a median follow up of 14.5 weeks, one patient showed tumor regression with partial remission, 11 patients had stable disease, and nine patients had progressive disease. The median OS for 23 patients is 36.2 months, 57.4 months for the HER2 3+ group, and 27.4 months for the HER2 0–2+ group. The infusions were well tolerated and MTD was not reached in the study. Cytotoxicity assays done on the PBMC obtained posttreatment showed that armed ATC infusions induced both specific anti-SK-BR-3 and innate endogenous antitumor immune responses. These cytotoxic responses persisted up to 4 months after the last infusion. We also demonstrated the trafficking of the armed ATC to the tumor sites. In summary, although we did not see significant immediate tumor shrinkage responses with the therapy, the OS of these patients was impressive given that they had received multiple lines of chemotherapy in the past, suggesting that the immunotherapy may have antitumor activity even without visible tumor shrinkage. One patient who progressed went to hospice and came out of the hospice after several months without any additional treatment.
The same treatment regimen was used in a phase 1 study  of eight patients with metastatic castrate-resistant prostate cancer. HER2Bi armed ATC in combination with IL-2 and GM-CSF. Patients were treated with two infusions per week for a total of 4 weeks and received 2.5, 5, or 10 × 109 armed ATC per infusion. There were no dose-limiting toxicities and there was one partial responder (prostate specific antigen decreased by >50% for 4 months) and three of seven patients had significant decreases in their prostate specific antigen (PSA) levels and their pain scores. Immune evaluations of fresh PBMC in two patients before and after immunotherapy showed increases in interferon-gamma EliSpot responses and Th1 serum cytokines. We are currently conducting a phase 1 clinical trial of ATC armed with anti-CD3 × anti-GD2 BiAb for treatment of relapsed/refractory neuroblastoma.
CLINICAL USE OF ARMED UMBILICAL CORD ACTIVATED T CELLS
For patients with hematologic malignancies or patients with solid tumors whose T cells cannot be expanded, multiple armed cord blood ATC preparations that have been irradiated could potentially be used to provide a GVL or GVT effect. Cryopreserved irradiated armed ATC could be banked for thawing when needed to provide anti-GVL as a TAA-specific cytotoxic T lymphocyte infusion post SCT or post chemotherapy.
Use of ex-vivo bispecific antibody armed activated T cells requires significantly less amount of bispecific antibodies to be used and decreases infusional toxicities
The total amount of BiAb required  is much less when used with armed ATC (up to 200 times less) as compared with when the BiAb is used as a single agent, which can reduce the side effects such as cytokine release syndrome commonly seen with BiAbs used as single agents. In addition, the BiAb attached to an effector cell also has less clearance than BiAb used as single agent. No dose-limiting toxicities were seen in patients receiving up to 160 × 109 armed ATC.
Activated T cells armed with bispecific antibodies can vaccinate host immune system against the tumor antigen
Our preclinical and clinical studies demonstrate that armed ATC secrete various cytokines (interferon gamma, tumor necrosis factor alpha, GM-CSF, IL-4, IL-6, and IL-10), leading to shift toward a Th1 immune profile , which can immunize the host immune system to TAAs (Fig. 1).
Methods to improve the clinical efficacy of the armed activated T cells
Although we do not see immediate clinical activity that could be masked in solid tumor patients and the true endpoint being OS as opposed to time to progression that could be confused with tumor flare reactions in our early clinical trials, the following approaches could help us improve and measure the clinical efficacy of the BiAb-targeted ATC.
These have been employed in CAR T cells by inserting receptors such as CD28 and CD80, which help increase the cytotoxicity and cytokine secretion by CAR T cells . Similar strategies have been used with BiAb with use of anti-CD28, or other costimulator monoclonal antibodies to enhance the cytotoxicity of armed ATC [53,54] [53,54].
Depletion of regulatory/inhibitory T cells with use of cyclophosphamide can help enhance the activity of BiAb armed ATC as shown in preclinical models . This strategy could help enhance the clinical efficacy of armed ATC.
Binding affinity of bispecific antibodies
Changing the binding affinity of BiAbs can help improve the clinical efficacy by enhanced binding both to the TAA and the effector immune cells.
Inhibition of checkpoint inhibitors
With the success of nivoluminab, pembrolizumab, and ipilimumab, there will be future opportunities to combine BiAb armed ATC with infusions of checkpoint inhibitors to optimize the antitumor effect of the T cells.
Improved imaging and staging strategies to optimize local tumor responses
There is a need to be able to evaluate with appropriate sensitivity whether there is infiltration of the tumor site with the infused armed T cells and/or the endogenous immune response. The assessment of progression needs to be re-evaluated in solid tumor patients. The Wolchok criteria  should be applied and scans should be done later than 3 months and repeated to assess tumor progression since tumor flare, which is a significant inflammatory response, is likely to confuse the assessment of tumor size
Platform adapted for generic cytotoxic T cells
ATC grown from normal donors, patients, and cord bloods can be armed, irradiated (to prevent GVHD), and cryopreserved, and retrieved for clinical therapies as needed for any malignancy if the monoclonal antibody equivalent is available to produce the BiAb construct targeting the TAA.
Use of ATCs armed with BiAbs for redirecting immune system toward cancer cells shows promising activity. Further success will depend on identifying the right tumor antigens, ability to increase the affinity of the BiAb to the tumor and the effector immune cells and identifying the right mode, frequency, and duration of therapy.
Financial support and sponsorship
Conflicts of interest
L.L. has received support from the Department of Human and Health Service, R01 CA 092344, R01 CA 140314, and R01 CA 182526, the Leukemia and Lymphoma Society Translational Grants TRP #6066-06 and TRP #6092-09, a Michigan Life Sciences Grant #1819, and startup funds from Karmanos Cancer Institute. L.L. is a cofounder of Transtarget. The rest of the authors have no conflicts of interest.
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