Utilizing the BiTE (bispecific T-cell engager) platform for immunotherapy of cancer

Abstract

Various approaches of T-cell-based cancer immunotherapy are currently under investigation, among these are BiTE® (bispecific T-cell engager) antibody constructs, which have a unique design and mechanism of action. They are constructed by genetically linking onto a single polypeptide chain the minimal binding domains of monoclonal antibodies for tumor-associated surface antigens and for the T-cell receptor-associated molecule CD3. Concurrent engagement of the target cell antigen and CD3 leads to activation of polyclonal cytotoxic T-cells, resulting in target cell lysis. Blinatumomab, a BiTE targeting CD19, is being investigated in a broad range of B-cell malignancies and has recently been approved in the USA by the US FDA for Philadelphia chromosome-negative relapsed/refractory B-acute lymphoblastic leukemia under the trade name BLINCYTO™. The BiTE platform is one of the clinically most advanced T-cell immunotherapy options.

Keywords:

• bispecific antibody
• BiTE®
• blinatumomab
• cancer immunotherapy
• T-cells

1. Introduction

The immune system has an innate ability to target tumor cells, with evidence suggesting that immune surveillance plays a critical role in the control of tumorigenesis. Epidemiologic data from patients with congenital or acquired T-cell defects show that they have an increased risk of cancer, and histologic evidence of T-cell infiltration in tumor tissue has been positively correlated with prognosis. However, the antitumor response of T-cells is often insufficient in part because of cancer cells subverting mechanisms of T-cell control under selective pressure. Mechanisms of evasion of immune surveillance include but are not limited to: i) modification of the tumor microenvironment by cytokines; ii) loss of MHC class I or other co-stimulatory molecules on tumor cells; iii) engagement of immune checkpoint proteins such as CTLA-4 or PD-1; and iv) up-regulation of anti-apoptotic proteins [1]. Cell-based immunotherapeutic approaches such as allogeneic hematopoietic cell transplantation (alloHCT) or expanded autologous tumor-infiltrating lymphocytes have demonstrated efficacy. However, there are several limitations in their use. Graft versus host disease increases nonrelapse mortality following alloHCT and necessitates immunosuppression [2]. Ex vivo expansion of tumor-infiltrating lymphocytes is laborious and often fails to generate a sufficient target cell dose [3].

Increased understanding of the mechanisms of evasion of immune surveillance has led to various novel approaches of T-cell based immunotherapy. A broad range of these approaches are currently under investigation for the treatment of cancer, including oncolytic immunotherapy with vaccines [4] or autologous antigen-presenting cells activated by recombinant infusion proteins [5]. Checkpoint inhibitors, such as antibodies targeting CTLA-4 or PD-1, prevent the downstream activation of inhibitory phosphatases that dampen T-cell receptor signaling cascades [6]. Other approaches use engineered T-cells expressing chimeric antigen receptors (CAR), which redirect T-cell antigen specificity to cell surface proteins [7]. BiTE antibody constructs (a registered trade mark of Amgen Inc.) are designed from monoclonal antibodies and have a unique mechanism of action, linking T-cells with tumor cells, ultimately leading to apoptosis of the tumor cell [8]. Our discussion focuses on the BiTE platform, highlighting key features and reviewing clinical data for the most advanced molecule in this class, blinatumomab. An outlook on additional BiTE antibody constructs in earlier phases of clinical development along with future perspectives for the platform is provided.

2. Features of BiTE technology

BiTE antibody molecules are constructed by genetically linking the minimal binding domains (single-chain fragment variables; scFvs) of monoclonal antibodies for CD3ϵ on T-cells and surface molecules on targeted cancer cells. ScFvs are connected via a non-immunogeneic, 5 amino-acid repetitive linker (Figure 1). This results in a non-glycosylated protein with an approximate molecular weight of 55 kDa. Once bound to the target antigen expressed on the surface of the respective target cells, binding of CD3 leads to activation and polyclonal expansion of cytotoxic T cells. Linking T-cells and target cells leads to the formation of an immunological synapse. Activation of the T-cell is achieved only in the presence of target cells independent of TCR specificity, co-stimulation, or peptide antigen presentation. Following synapse formation, T-cells release perforines and granzymes, followed by activation of target cell caspases and apoptosis (Figure 2) [9]. Video microscopy experiments have demonstrated that BiTE antibody constructs enable serial lysis of tumor cells by engaged T-cells [10].

Figure 1. BiTE antibody constructs are generated by genetically linking minimal binding domains of mAbs for CD3 on T-cells and for surface antigens on target cancer cells onto a single polypeptide chain [8].

Figure 2. Engagement by BiTE antibody constructs leads to activation and polyclonal expansion of T-cells. The activation of T-cells requires the presence of target cells. Upon binding of the BiTE antibody construct to both CD3 on T-cells and the tumor-associated antigen on target cells, the formation of an immunological synapse is forced, thereby bypassing MHC/antigen-dependent activation of T-cells. Activation is achieved independently of TCR specificity, costimulation, or peptide antigen presentation. Subsequent to formation of the immunological synapse, apoptosis of the target cell is induced [8,9,27,28].

3. Blinatumomab

The first BiTE approved in the USA by the US FDA, blinatumomab, targets CD19, a cell surface antigen expressed on both healthy and neoplastic B-cells, while absent on hematopoietic stem cells and plasma cells.

Blinatumomab was first administered as a short-term intravenous infusion in patients with non-Hodgkin’s lymphoma. Signals of biological activity included polyclonal T-cell activation with transient production of proinflammatory cytokines. Accordingly, the most frequent adverse events (AEs) were pyrexia, rigor, and fatigue. In addition, neurologic events were seen. However, no objective responses were detected. This may have been due to the relatively short half-life of blinatumomab (∼2 h), which prevented continuous exposure to the drug [9]. Therefore, the next trial administered blinatumomab as a continuous intravenous infusion (civ) [9]. Study MT103–104 [11] was conducted to define a maximum-tolerated dose (MTD) of blinatumomab civ as 4 – 8 week cycles and to investigate antilymphoma activity in 76 patients with relapsed non-Hodgkin’s lymphoma of both indolent and aggressive histologies, including follicular, mantle cell and diffuse large B-cell lymphoma (DLBCL). B-cell depletion and objective responses were observed starting at 15 µg/m²/d. Dose-limiting toxicities were neurologic events; the MTD was 60 µg/m²/d. The most common AEs during the study were pyrexia, fatigue, headache, weight increase, and weight decrease. Step-dose escalation and corticosteroid premedication were instituted to minimize the incidence and severity of AEs, particularly cytokine release syndrome (CRS) and neurologic events. At the target dose of 60 µg/m²/d, the overall response rates were 75 and 55% in indolent lymphoma and DLBCL, respectively, resulting in the continued development of blinatumomab in a broad range of B-cell malignancies (Table 1) [12].

Table 1. Disease settings investigated with blinatumomab, a CD19-specific BiTE antibody construct.

Patient population	ClinicalTrials.gov identifier study code/acronym	Phase/study type	Study status*	N	Ref.
B-precursor ALL
Adult R/R B-precursor ALL	NCT01209286 MT103-206	2, Open label	Ongoing	36	[16]
Adult Philadelphia chromosome-negative R/R B-precursor ALL	NCT01466179 MT103-211	2, Open label, pivotal	Ongoing	189	[17]
Adult patients with R/R B-precursor ALL	NCT02013167 20120311/TOWER	3, Randomized, open label	Recruiting	n.a.	n.a.
Adult R/R Philadelphia chromosome-positive B-precursor ALL	NCT02000427 20120216/Alcantara	2, Open label	Recruiting	n.a.	n.a.
Pediatric and adolescent R/R B-precursor ALL	NCT01471782 MT103-205	1/2, Open label	Ongoing	93	[18]
Younger (1-30 years) patients with relapsed B-cell ALL	NCT02101853 COGAALL1331	3, Risk stratified, randomized,	Recruiting	n.a.	n.a.
Newly diagnosed BCR-ABL-negative B lineage ALL	NCT02003222 EGOG1910	3, Randomized, open label	Recruiting	n.a.	n.a.
Elderly newly diagnosed ALL	NCT02143414 SWOG S1318	2, Open label	Not yet recruiting	n.a.	n.a.
Adult MRD of B-precursor ALL	NCT00560794 MT103-202	2, Open label	Completed	21	[13,14]
Adult MRD of B-precursor ALL	NCT01207388 MT103-203/BLAST	2, Open label, EU pivotal	Ongoing	116	[15]
NHL
Patients with relapsed NHL	NCT00274742 MT103-104	1, Cohort	Completed	76	[11]
Patients with R/R DLBCL	NCT01741792 MT103-208	2, Open label	Ongoing	25	[19]

*According to clinicaltrials.gov.

Adapted with permissions from Zimmerman et al. [12].

ALL: Acute lymphoblastic leukemia; BiTE: Bispecific T-cell engager; DLBCL: Diffuse Large B-Cell Lymphoma; MRD: Minimal residual disease; NHL: Non-Hodgkin’s lymphoma R/R: Relapsed/refractory.

The first trial in adult B-precursor acute lymphoblastic leukemia (ALL) was in patients with minimal residual disease (MRD) receiving blinatumomab civ at a dose of 15 µg/m²/d for up to five 4–week cycles. Study MT103–202 reported 80% MRD response, including durable responses in four of six Philadelphia chromosome-negative patients. One patient discontinued treatment for a grade 3 neurologic event [13,14]. A confirmatory Phase II study MT103–203 with 116 patients treated represents the largest to date in MRD-positive disease. Seventy-eight percent of patients achieved the primary endpoint of complete MRD response rate (no amplification by PCR) after one cycle. Reponses were similar across all subgroups including older patients. No predictive factor for MRD complete response was identified. Among the occurring neurologic events, 81% were of worst grade of ≤ 2; 10 patients discontinued treatment for these events. Forthcoming analysis of this trial will determine whether high MRD complete response rate translates into extended relapse-free (RFS) and overall survival (OS) [15].

Topp et al. [16], reported the results of a Phase II study (n = 36), including a dose-finding and an expansion cohort, in adult patients with relapsed/refractory (R/R) ALL (study MT103–206). The dosing selected for this setting was 5 µg/m²/d for the first week of a 4–week treatment cycle, followed by 15 µg/m²/d for the remainder of the cycle and all subsequent cycles. Twenty-five patients (69%) achieved a CR/CRh (partial hematologic recovery: platelets > 50,000/µl, hemoglobin > 7g/dl, and absolute neutrophil count > 500/µl). Median OS was 9.8 months, and median RFS was 7.6 months. Treatment was discontinued in two and one patients for reoccurring neurologic AEs and grade 4 CRS, respectively. Based on these results, a confirmatory Phase II study (MT103–211) enrolled adult patients with Philadelphia chromosome-negative R/R ALL. Eligibility criteria limited the study to patients with the most aggressive form of R/R ALL by excluding those in first salvage with a first remission duration of > 1 year. One hundred and eight-nine patients received blinatumomab civ (9µg/d for the first 7 days and 28 µg/d thereafter, equivalent to prior dosing in µg/m²/d) > 4 weeks for up to five cycles. After two cycles, 43% of patients achieved the primary endpoint of CR/CRh with a median RFS of 5.9 months (95% CI 4.8 – 8.3). Median OS in all patients was 6.1 months (95% CI 4.2 – 7.5). Two patients had grade 4 CRS. Neurologic events of grade 3 or 4 occurred in 20 (11%) and four (2%) patients, respectively [17]. Subsequently, blinatumomab received accelerated approval in the USA by the U.S. FDA for the treatment of Philadelphia chromosome-negative R/R B-cell precursor ALL under the trade name of BLINCYTO™.

Complementary to these studies, the pediatric study MT103–205 aimed to define blinatumomab dosing and to assess antileukemia activity and AEs in pediatric patients in second or greater relapse or who relapsed after alloHCT. Forty-one patients with R/R B-precursor ALL were evaluated in the Phase I dose-escalation part. The MTD was 15 μg/m²/day. Although CRS was dose-limiting, stepwise dosing of 5 – 15 μg/m²/d was effective in ameliorating CRS. Thirty-two percent of patients achieved CR [18]. Results of the Phase II portion will be forthcoming.

Finally, in a Phase II study in R/R DLBCL (25 patients), blinatumomab stepwise dosing (9/28/112 µg/d) was associated with antitumor activity: overall response rate was 42.9% for evaluable patients, and the CR rate was 19.0%. Response duration ranged from 26 to 533 days. The most common AE was tremor (52%). Grade 3 neurologic AEs were reported in 28% of patients, with five patients discontinuing treatment for neurologic events [19].

In light of the antitumor activity demonstrated in advanced leukemia and lymphoma, ongoing and planned trials will evaluate blinatumomab earlier in the treatment course of ALL and in more lymphoma histologies.

4. Other BiTE antibody constructs in clinical development

The BiTE antibody construct platform can be adapted to bind a wide range of cell surface target antigens by incorporating scFvs with distinct specificities [8]. AMG330, a BiTE antibody construct targeting the myeloid-cell antigen CD33, enables T-cells to kill primary acute myeloid leukemia cells in vitro, suggesting therapeutic potential [20].

In addition to hematologic indications, BiTE antibody constructs targeting solid tumors are also under investigation. A Phase I study of the anti-EpCAM BiTE AMG110 (MT-110, NCT00635596) has recently completed recruitment, and analysis is ongoing [21-23]. Meanwhile, two other molecules targeting carcinoembryonic antigen and prostate-specific membrane antigen (PSMA), respectively, have entered Phase I clinical trials based on promising preclinical results [24,25]. Investigation of the carcinoembryonic antigen CEA BiTE is ongoing in two different studies as short-term infusion (NCT01284231) and as civ (NCT02291614) in advanced gastrointestinal adenocarcinoma with the aim to define the respective MTDs. The PSMA BiTE is being investigated as a subcutaneous injection in a dose-escalation study in patients with castrate-resistant prostate cancer (NCT01723475).

5. Expert opinion

T-cell based immunotherapy has emerged as a promising approach to cancer treatment. Blinatumomab, a first-in-class BiTE, is among the first of these immunotherapies to receive regulatory approval in the USA. The modular design of the BiTE platform allows its adaptation to numerous target cell types, and multiple clinical trials are ongoing to determine safety and efficacy of BiTE antibody constructs in different disease settings. Their short half-life is both a potential benefit and disadvantage. In the case of blinatumomab, toxicities that relate to T-cell activation, i.e., CRS, as well as those with unclear pathophysiology, namely neurologic events, are typically reversible by treatment discontinuation and administration of corticosteroids. However, continuous infusion may be challenging for many patients, and their acceptance of the inconvenience is dependent on the severity of the clinical situation and the availability of alternative therapies. Among various approaches to increase the convenience of BiTE therapy, subcutaneous administration of the PSMA BiTE is being tested in the clinical setting. With a broader range of BiTE antibody constructs entering clinical trials, more will be learned about toxicities that are target antigen-dependent (e.g., the neurologic events of blinatumomab have also been reported for CD19–specific CAR-T-cells [26]) and those that are generalizable to T-cell activation.

The novel mechanism of action of BiTE antibody constructs may explain why blinatumomab has shown comparable antitumor activity in patients with ALL who received one or multiple lines of prior therapy. Whereas mutations associated with cross-resistance to conventional chemotherapy have been described, the mechanisms of resistance to blinatumomab await to be elucidated. The loss of CD19 on target cells is a known phenomenon but it is observed relatively infrequently. Resistance may occur either at the cancer cell or the T-cell part of the tumor/immune cell interaction. For example, T-cells may become exhausted by chronic antigen stimulation, opening up the possibility of adding immune-modulators to increase potency. Additionally, lower tumor burden is correlated with higher likelihood of response, which may be an in vivo correlate of a higher effector: target ratio.

As we enter a new age of immunotherapy, a great wealth of clinical data will emerge from patients treated with BiTE antibody constructs and other T-cell-targeted agents. We are optimistic that, collectively, the field will identify biomarkers predicting response to T–cell-based therapies, and that these biomarkers may also pave the way to potential combination therapies as has been the case with conventional chemotherapy. Finally, we hope that further improvement in the management of toxicities, combined with long-term survival benefits, will create enthusiasm to bring these agents to the frontline of cancer therapy.

Acknowledgments

The authors would like to thank Craig Kiefer (Amgen Inc.), for art work design and Beate Quednau, PhD (Amgen Inc.), for editorial support.

Declaration of interest

This paper has been funded by Amgen, Inc. J Stieglmaier is an employee of Amgen Research (Munich) GmbH and a shareholder in Amgen Inc. D Nagorsen is an employee of Amgen Inc., an inventor on blinatumomab-related patents and shareholder in Amgen Inc. J Benjamin is an employee of Amgen Inc. and a shareholder in Amgen Inc. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Notes