Manipulating the T-Cell Receptor Yields Promising Immunotherapies

Components of the T-cell receptor (TCR) complex, which links antigen recognition with T-cell activity and effector function, are being exploited for several types of cancer immunotherapy. Although these strategies have not received the same level of attention as blockbuster immunotherapies such as checkpoint inhibitors and chimeric antigen receptor (CAR) T-cell therapies, they have made steady progress over the past few years, and many novel agents are undergoing clinical testing.

Broadly speaking, investigational therapies that target the TCR are being developed in 3 categories: TCR-engineered T cells, bispecific antibodies, and immune-mobilizing monoclonal T-cell receptors (ImmTACs).

TCR-engineered T cells are an alternative form of adoptive cell therapy (ACT). These therapies redirect the specificity of the TCR against tumor-associated antigens (TAAs) to target intracellular antigens, where CAR T cells are only just beginning to venture. Stateof- the-art genetic engineering techniques such as CRISPR have the potential to fine-tune these therapies more precisely.

Bispecific antibodies target both tumor cells and T cells via the CD3 component of the TCR complex. Clinical development has yielded FDA approval of the first-in-class drug blinatumomab (Blincyto) for the treatment of patients with acute lymphoblastic leukemia (ALL). Recent expansion of its approved indications offers a new option for patient populations with few therapeutic avenues.

Immune Therapies Exploit T Cells

Researchers also are working on ImmTACs, a new class of bispecific drugs that combines the advantageous properties of targeting intracellular antigens and the benefits of a soluble drug format without the time-consuming and labor-intensive manufacturing requirements of cell-based therapies.As our understanding of the dual role of the immune system in both restraining and promoting cancer has evolved, so too has our ability to harness its components to yield groundbreaking therapies that augment the antitumor immune response and offer the promise of long-lasting tumor regression.

The focus of cancer immunotherapy has been on mobilizing cytotoxic T cells, the central mediators of cell-mediated adaptive immunity. T cells are activated by protein fragments derived from pathogens that are displayed on the surface of antigen-presenting cells (APCs), such as macrophages, bound to major histocompatibility complex (MHC) molecules.

Although cancers are derived from the body’s own cells gone awry and, in theory, should not stimulate an immune response, the accumulated genetic alterations that often drive cancer can generate unique proteins that T cells recognize and by which they are activated. Tumor antigen—specific T cells, known as tumor-infiltrating lymphocytes (TILs), can be identified in most tumor types.

TCR-Mediated T-Cell Activation

In response, cancer cells have evolved a multitude of mechanisms to suppress TIL activity and tip the balance of power to the tumor. The idea of immunotherapy is, therefore, to restimulate the antitumor immune response and wrestle back that control. Two major strategies for manipulating T cells have proved particularly effective: immune checkpoint inhibitors targeting the PD-1/PD-L1 pathway and CTLA-4 and genetically engineered CAR T cells.¹T-cell activation is dependent on the TCR upon its surface, which engages the antigen-MHC complex on APCs, triggering a cascade of signaling molecules within the cell that ultimately promotes the proliferation of T cells and the acquisition of the properties that enable their effector functions.

This downstream signaling is a highly complex branched network of events that is still not fully understood but maintains a precisely set threshold of activation, ensuring that T cells are switched on only at the appropriate time and for only as long as necessary.

The TCR is a member of the immunoglobulin superfamily and consists of a heterodimer of 2 highly variable protein chains (Figure 1). In most cases, those protein chains are encoded by the TRA and TRB genes. In 5% of cases, the TCR instead is composed of TCRδ and γ chains, although these percentages can change in certain circumstances, such as during T-cell development. Each TCR possesses unique antigen specificity thanks to their random assembly of different gene segments.

Figure 1. Basic Structure of the TCR

The constituent protein chains are made up of a variable and constant region that protrudes from the cell, a portion that spans the membrane, and a short tail inside the cell. Unlike most transmembrane receptors, the tail of the TCR has no inherent signaling activity. Instead, the TCR forms a complex with the CD3 protein family consisting of CD3δ, γ, ε, and ζ, which link the antigen recognition abilities of the TCR with the activation of downstream signaling pathways.

Upon binding of the TCR by the antigen, the SRC family tyrosine kinase LCK is activated. LCK phosphorylates key signaling domains within CD3ζ, known as immunoreceptor tyrosine- based activation motifs (ITAMs), which serve as a binding platform for zeta chainassociated protein kinase 70 (ZAP70), which is also phosphorylated and activated by LCK and goes on to phosphorylate a range of adapter proteins, further propagating the signal by triggering a number of key pathways (Figure 2).

T cells require 2 signals to become fully activated, providing another safety check mechanism to prevent damage to healthy tissues. A number of transmembrane receptors play important roles in providing this essential costimulatory signal, including CD28 and CTLA-4, which are among the targets for immune checkpoint inhibitor drugs.^2-5

Figure 2. A View of How the TCR Activates Immune Signaling²

Manipulating the TCR

Alongside immune checkpoint inhibitors at the forefront of cancer immunotherapy, CAR T cells are a form of ACT that involves harvesting the patient’s T cells, culturing them outside the body, and then reinfusing them into the patient. Although they have dominated this field, genetic manipulation of the TCR is also being explored to generate TCR-engineered T cells.

This type of therapy is limited by the fact that the TCRs must be genetically matched to the patient’s immune type but offers a significant advantage over CAR T cells. The latter are antibody based and can recognize only cell surface antigens; the number of unique TAAs expressed on the cancer cell surface is small. TCR-engineered T cells can recognize intracellular proteins that have been processed and presented by APCs, which greatly broadens the scope of potential targets. The TCR can also be manipulated to improve its antigen recognition and binding capabilities.^6-9 It should be noted that CARs targeting intracellular antigens are also being explored.¹⁰

A number of experimental therapies are under evaluation in clinical trials (Table 1). The cancer testis antigens have provided a particularly important source of potential targets; the group of proteins is restricted in expression to the testis, except during cancer development. Among them are the melanomaassociated antigen (MAGE) proteins and NY-ESO-1; the latter holds particular interest because it is one of the most highly immunogenic antigens ever discovered. Other targets that are being explored include melanoma antigen recognized by T cells (MART-1), glycoprotein 100 (gp100), and various virally associated antigens.¹¹

Initial studies have demonstrated early successes in certain tumor types.¹² Six of 20 patients treated with T cells with a highaffinity TCR engineered to recognize MART-1 had tumor regression.¹³ Meanwhile, a study using a high-avidity TCR recognizing the NY-ESO-1 antigen demonstrated objective clinical responses in 4 of 6 patients with synovial sarcomas and 5 of 11 patients with melanoma.¹⁴ In a separate study, in patients with multiple myeloma, NY-ESO-1- specific TCR-engineered T cells elicited clinical responses in 16 of 20 patients.¹⁵

Adaptimmune is developing NY-ESO-1— specific TCR-engineered T cells based on its proprietary SPEAR (specific peptide enhanced affinity receptor) T-cell platform. The results of an ongoing phase I/II study were presented at the 2017 American Society of Hematology (ASH) Annual Meeting. Following autologous stem cell transplant, 25 patients with advanced multiple myeloma were treated with NY-ESO-1 SPEAR T-cells. The objective response rate (ORR) at day 100 was 76%, 1-year progression- free survival (PFS) was 52%, median PFS was approximately 13 months, and median overall survival (OS) around 35 months. The most common adverse events (AEs) included diarrhea, nausea, anemia, decreased appetite, and thrombocytopenia.¹⁶

Table 1. Selected Trials of TCR-Directed T-Cell Therapies in the United States

T Cell-Targeting Antibodies

The use of next-generation genetic engineering technologies, such as CRISPR, is also being explored. The first clinical trial to use CRISPR in the United States involves TCR-engineered T cells designed to target NY-ESO-1. CRISPR will be used to delete the natural TCRs and PD-1 to help bypass some of the immunosuppressive mechanisms that can hinder ACT. The study is recruiting patients with multiple myeloma, melanoma, synovial sarcoma, or myxoid/round cell liposarcoma (NCT03399448). The TCR is being manipulated for cancer therapy for a very different form of immunotherapy. Monoclonal antibodies targeting a variety of TAAs have revolutionized the treatment of numerous cancer types. The oncology research field has been seeking to expand on this success by investigating different antibody formats (Table 2).

Among them are bispecific antibodies, which are engineered to combine single chain variable fragments (scFvs) from 2 different antibodies, allowing them to bind to 2 targets simultaneously. T-cell—targeted bispecific antibodies target a TAA with 1 scFv and the CD3 component of the TCR complex with the other. By binding to a TAA, the antibody can specifically target cancer cells, and binding to CD3 enables it to activate any T cells it encounters, redirecting their activity against the tumor without the need for their TCR to specifically recognize a TAA.

Simultaneous binding of tumor cell and T cell facilitates the formation of an immunological synapse, which results in T-cell activation, proliferation, and expansion at the site of the tumor and the destruction of the target cancer cell.

Although bispecific antibodies are limited to the recognition of cell surface TAAs, they are simpler and less time-consuming and laborintensive to produce than cell-based therapies. A variety of formats have been developed, often named after the proprietary technology platform used by the company developing them. The most advanced in clinical development are the bispecific T-cell engagers (BiTEs).^17,18

Blinatumomab Leads the Charge

Blinatumomab, a BiTE targeting CD19 and CD3, gained FDA approval in 2014 for the treatment of patients with Philadelphia chromosome- negative, relapsed or refractory, precursor B-cell ALL.¹⁹ That accelerated approval was translated into full approval in July 2017, based on the results of the phase II ALCANTARA and phase III TOWER trials. In the former study, blinatumomab was compared with standard-of-care chemotherapy and found to nearly double median OS (7.7 vs 4 months; HR, 0.71; P = .012). In the ALCANTARA trial, the efficacy of blinatumomab was evaluated in patients with Philadelphia chromosome-positive disease, and the efficacy it demonstrated in this population prompted the FDA to expand the indication into a broader range of patients.^20,21

In March, the FDA further expanded the indication to include postremission treatment of patients who have residual disease after their last chemotherapy treatment. This approval was based on data from the phase II BLAST study involving 86 patients in first or second hematologic complete remission 2 or more weeks after chemotherapy. Overall, undetectable minimal residual disease was achieved by 70 patients (81.4%; 95% CI, 71.6%, 89.0%). The median hematological relapse-free survival was 22.3 months.²²

Amgen, which manufactures blinatumomab, is continuing to develop the drug in ALL, diffuse large B-cell lymphoma, and other hematologic malignancies.

Table 2. T Cell-Targeting Bispecific Antibodies in Development

Growing Number of Antibodies in Development

MacroGenics, a big player in the development of bispecific antibodies, is working on a dual affinity retargeting (DART) antibody, flotetuzumab (MGD006), which targets CD123 and CD3. While BiTEs are composed of 2 scFvs fused to form a single polypeptide chain, DARTs are composed of 2 polypeptide chains linked by a disulfide bridge to help stabilize them.

Flotetuzumab was granted orphan drug designation for the treatment of acute myeloid leukemia (AML) in January 2017, and the results from an ongoing first-in-human, phase I trial in patients with AML and myelodysplastic syndrome were presented at the 2017 ASH Annual Meeting. The drug demonstrated acceptable tolerability and promising antitumor activity in the 14 patients evaluable for response treated at the threshold dose of 500 ng/kg/day and beyond who completed at least 1 cycle of therapy. The ORR was 43%, including complete remission or complete remission with incomplete hematologic response in 28% of patients.²³

REGN1979 is a bispecific antibody targeting CD20 and CD3 in development by Regeneron Pharmaceuticals. At the 2017 ASH meeting, the results of a phase I study of this drug in combination with the PD-1 inhibitor cemiplimab in patients with B-cell non-Hodgkin and Hodgkin lymphoma were reported. In the combination arm, 12 patients received 3 mg/kg of cemiplimab every 2 weeks for a minimum of 12 and a maximum of 24 doses, plus REGN1979 administered intravenously weekly for 11 doses, followed by every 2 weeks from week 13 for 11 additional doses. There was 1 partial response (PR), and 4 patients experienced stable disease.²⁴

Roche is developing RO6958688, which targets carcinoembryonic antigen (CEA) in addition to CD3. Preliminary data from 2 phase I studies were presented at the 2017 ESMO World Congress on Gastrointestinal Cancer, both enrolling patients with all types of CEA-positive tumors.

The findings focused on patients with metastatic colorectal cancer; most had microsatellite stable disease and typically would not be expected to respond to immunotherapy. In the first study, the drug was administered as monotherapy, and 80 patients received intravenous doses of 60 to 600 mg weekly. The second study evaluated a combination with the PD-L1 inhibitor atezolizumab (Tecentriq), with 45 patients receiving doses of 5 to 160 mg RO6958688 plus 1200 mg atezolizumab every 3 weeks.

ImmTACs: Best of Both Worlds?

Monotherapy doses of 60 mg and above elicited a PR rate of 6% and a disease control rate (DCR) of 45%. Meanwhile, in combination with atezolizumab, the PR rate ranged from 12% to 18% and the DCR ranged from 52% to 82%, depending on the dose.²⁵ImmTACs are a new class of bispecific with the potential to combine the benefits of targeting intracellular antigens and a soluble format. They are a fusion of a soluble TCR engineered to target a TAA and an scFv that binds to CD3. Like other bispecifics, ImmTACs promote the formation of an immunological synapse by bringing T cells and tumor cells together.²⁶

The lead candidate in this category, IMCgp100, which is being developed by Immunocore, targets the gp100 protein that is highly expressed on melanosomes. It is being evaluated in a phase II clinical trial in patients with uveal melanoma.

Immunocore presented data from 2 phase I clinical trials at the 2017 Society for Immunotherapy of Cancer Annual Meeting. The first trial enrolled patients with late-stage and unresectable melanoma, 16 of whom had uveal melanoma. The second, an ongoing dose-escalation study in patients with advanced uveal melanoma, had 19 patients enrolled at the time of the presentation.

Both studies demonstrated a similar safety profile, with the most frequent AEs including rash, pruritus, and edema. The ORR was 20% and 11% in the first and second trials, respectively. Median PFS was 3.7 months and 5.6 months, respectively, and the 1-year PFS rate and OS rate in the dose-escalation trial were 62% and 73%, respectively.²⁷

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