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July 01, 2018 (Vol. 38, No. 13)

CAR T-Cell Therapies with a Bispecific Twist

To Bypass Antigen Escape, TanCAR and NanoCAR Therapies Take the Bispecific Route

An illustration of a T cell’s chimeric antigen receptor (CAR) binding to a cancer cell’s antigen. The CAR depicted here incorporates a single-chain variable fragment (scFv) and is monomeric, like the CAR in Kymriah, the first approved CAR T-cell therapy. A monomeric CAR may target only one epitope of a tumor-associated antigen. [Meletios Verras / Getty Images]

  • Click Image To Enlarge +
    Schematic representation of nanobody and antibody domains. Human and rodent IgGs are composed of six constant heavy (CH) regions, two constant light (CL) regions, two variable heavy (VH) regions, and two variable light (VL) regions. Antibody fragments are built similarly, where scFvs are composed of a VH region and a VL region connected by a short peptide linker, and single domain antibodies are simply a VH or a VL region. To generate camelid nanobodies, researchers isolate one of the two variable heavy (VHH) domains present in camelid IgGs, commonly termed heavy chain–only antibodies because they are composed only of four CH regions and two VHH domains. [The diagram, which originally appeared in Theranostics (2014; 4(4): 386–398), is adapted from a diagram that appeared in Nat. Biotechnol. (2005; 23: 1126–36).]]

    Chimeric antigen receptor (CAR) T-cell therapy has matured rapidly. What used to be the “drug of the future” is suddenly the “drug of the now.” The first CAR T-cell therapeutic, Kymriah, was approved by the FDA about 10 months ago, and there are at least 300 different CAR T-cell-based clinical trials being conducted across the globe. Even more numerous are the CAR T-cell projects currently in the discovery or preclinical phases of development. In these projects, multitudes of cancer types, targets, and formats are being evaluated.

    All this activity makes for a robust CAR T-cell market. Currently worth an estimated $168.7 million, the CAR T-cell market is expected to maintain a compound annual growth rate of 46.2% from 2019 to 2028. These enviable figures are partly due to Kymriah’s demonstrated efficacy in treating acute lymphoblastic leukemia.

    Kymriah targets CD19, the most prevalent marker of B-cell malignancies, and shows a high overall response rate. After treatment commences, about 83% of patients experience remission within three months. In up to half of all treated patients, however, remission is only temporary because antigen escape occurs, allowing cancer to recur.

    Recognizing Antigen Escape

    Antigen escape is one of the key challenges for monomeric CAR T-cell therapies. It can be defined as the loss of a tumor-associated antigen (TAA), particularly after administration of TAA targeting therapeutics. Despite the strong effect antigen escape has on CAR T-cell efficiency, there have not been many studies carried out to elucidate its cause.

    Research efforts led by Andrei Thomas-Tikhonenko, Ph.D., a professor of pathology and laboratory medicine at the Children’s Hospital of Philadelphia, demonstrate that leukemias that reemerge after CD19-directed therapy have a higher rate of CD19 splice variation and CD19 mutation. In these leukemias, the CD19 epitope is eliminated, such that newly derived leukemia cells prove invulnerable to any CD19-based therapeutic.

    Although antigen escape is not yet fully understood, it is recognized as a challenge that must be (and can be) addressed. In the case of CD19 loss, Dr. Thomas-Tikhonenko and colleagues speculated in 2015 that targeting alternative CD19 ectodomains could improve patient outcomes. Subsequently, other investigators suggested that broader immune activation might prevent outgrowth of tumor antigen escape variants following targeted therapies. Currently, researchers are combining CD19-based CAR T-cell approaches with bispecific antibody (bsAb) technologies to counter antigen escape.

  • Taking bsAbs on Board

    Antibody therapeutics that rely on bsAbs simultaneously target two different antigens, leading to the co-stimulation of different metabolic pathways within the same or different cell types. In terms of immuno-oncology, bsAb therapies resemble CAR T-cell therapies. Like CAR T-cell therapies, bsAb therapies can redirect various T cells to attack and kill tumors. In addition, bsAb therapies can direct other immunological effector cells such as natural killer cells, macrophages, and monocytes.

    Currently, there are more than 200 bsAb-based therapeutics entering or currently in clinical trials. Over 80% of these therapeutics induce a cytolytic synapse between immune cells and targeted cancer cells.

    The first bsAb-based therapeutic approved by the FDA was blinatumomab, a bispecific T-cell engager, or BiTE. Blinatumomab contains two different single-chain fragment variables (scFvs)—one targeting CD19, and one targeting CD3—to treat acute lymphoblastic leukemia. This therapeutic has demonstrated efficacy, suggesting that bsAbs have promise. Nonetheless, like other therapeutics, bsAbs present difficulties.

    A major problem with using bsAbs to treat cancers is that the distance between the antigen-binding domains tends to be rather small due to the natural structure of bsAbs. In BiTEs, for example, this distance is around 60 kDa. Therefore, if one bsAb is to simultaneously bind a cancer cell and an effector cell, the cells must be close to one another.

    To circumvent this issue, researchers began developing bispecific CAR T-cell formats. Some of these formats looked promising initially but were eventually found to be wanting. For example, one bispecific CAR T-cell format involved the mixing of two T-cell lines, each expressing a different scFv on its surface. Unfortunately, this approach led to preferential growth of one cell line over the other, basically resulting in a traditional monomeric CAR.

    Another bispecific CAR T-cell format involved the simultaneous transduction into T cells of two different scFv-based CAR plasmids. Although this approach was conceptually sound, it proved to be impractical. In the laboratory, attempts to transduce two CAR cassettes into one viral vector failed simply because sufficiently roomy vectors were unavailable.

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  • Making Progress with TanCARs

    One of the more successful bispecific CAR T-cell formats is called tandem CAR (TanCAR). It involves transducing a T cell with one CAR plasmid that expresses two different scFvs. TanCAR-based therapeutics look promising because their scFv-based CARs possess bispecific functionality.

    The TanCAR approach, unlike the mixing or combining methods, generates a CAR T cell with even distribution of two distinct antigen-binding scFvs. TanCARs also exhibit Boolean-like activity, meaning that a full T-cell response can be activated when either scFv becomes engaged.

    A highly effective TanCAR was developed by scientific team led by Yvonne Y. Chen, Ph.D., an assistant professor of chemical and biomolecular engineering at the University of California, Los Angeles. This TanCAR was the first to successfully target different epitopes expressed on the same cancer cell.

    Dr. Chen’s motivation for creating this construct was to eliminate the risk of CD19 antigen escape. Her approach involved introducing an additional scFv, one targeting CD20, another biomarker of B-cell cancers. This way, any reemerging CD19-negative cancerous tissue could still be targeted—and killed—without the patient having to go through another, separate round of CAR T-cell therapy.

    Although TanCARs are of demonstrated efficacy, they have a built-in weakness: they keep their scFvs close together. When the scFvs are multiplexed, they tend to aggregate with one another; therefore, scFv-based CAR T-cells can aggregate and lead to CAR CD3 domain phosphorylation, tonic T-cell activation, and T-cell exhaustion. To avoid such problems, some developers are constructing non-scFv-based CARs using nanobodies.

  • Exploring NanoCAR Options

    Nanobodies are composed of one heavy-chain variable domain from a TAA-targeting full-length antibody. Nanobodies can be used on their own because they contain all three complementary determining regions required to bind with an antigenic epitope. Nanobodies can also be incorporated into a modified TanCAR format. For example, tandem nanobodies may replace a bispecific CAR’s tandem scFvs.

    This approach, which goes by the name nanoCAR, is being explored in the laboratory of Bart Vandekerckhove, M.D., Ph.D., a professor of clinical chemistry, microbiology, and immunology at the University of Ghent and a group leader at Cancer Research Institute Ghent. According to Dr. Vandekerckhove and colleagues, nanoCAR technology can generate CAR T cells that evenly distribute two nanobodies and are therefore capable of simultaneously stimulating two cancer-killing pathways.

    Using nanobodies rather than scFv-based CARs or bsAbs may present several advantages:

    • First, nanobodies are so small they do not interact or overlap with each other, allowing them to be built upon one another to form multitarget therapeutics while avoiding the common side effects of scFv aggregation.
    • Second, mouse-made nanobodies contain a human-like VH domain, allowing them to be weakly immunogenic compared with mouse or humanized scFv- or IgG-based therapeutics.
    • Third, recent clinical trial data has shown that nanobody-based therapeutics develop minimal antidrug antibodies compared with other common forms of antibody therapeutics.
    • Fourth, and most important, monomeric nanobody-based CARs show almost identical efficacy as scFv-based CARs targeting the same TAA in clinical trials.

    A recent publication from the Vandekerckhove team highlights the strong efficacy of a nanoCAR that targets both CD20 and Her2. To construct this nanoCAR, which was based on a previously generated scFv CAR targeting the same antigens, the team selectively replaced the two scFvs with nanobodies.

    The nanoCAR was able to selectively bind to either or both antigens and elicit a T-cell response equivalent to that of its TanCAR predecessor. Unfortunately, the team has yet to generate a bispecific nanobody CAR targeting the same cell type; however, it may be only a matter of time before this format is generated and tested in the clinic. What remains clear is that CAR T-cell therapeutics are only at the beginning of charge into the immuno-oncology scene. If their potential curative powers are realized, these therapeutics stand to benefit a great many cancer patients.

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