Cancer immunomodulation using bispecific aptamers

Abstract

Evasion of immune destruction is a major hallmark of cancer. Recent US Food and Drug Administration (FDA) approvals of various immunomodulating therapies underline the important role that reprogramming the immune system can play in combating this disease. However, a wide range of side effects still limit the therapeutic potential of immunomodulators, suggesting a need for more precise reagents with negligible off-target and on-target/off-tumor effects. Aptamers are single-chained oligonucleotides that bind their targets with high specificity and affinity owing to their three-dimensional (3D) structures, and they are one potential way to address this need. In particular, bispecific aptamers (bsApts) have been shown to induce artificial immune synapses that promote T cell activation and subsequent tumor cell lysis in various in vitro and in vivo pre-clinical models. We discuss these advances here, along with gaps in bsApt biology at both the cellular and resident tissue levels that should be addressed to accelerate their translation into the clinic. The broad application, minimal production cost, and relative lack of immunogenicity of bsApts give them some ideal qualities for manipulating the immune system. Building upon lessons from other novel therapies, bsApts could soon provide clinicians with an immunomodulating toolbox that is not only potent and efficacious but exercises a wide therapeutic index.

Keywords: antibody; bispecific; clinical translation; immunomodulation; molecular design; oligonucleotide therapy; oncolytic.

Graphical abstract

Figure 1

Aptamer selection (SELEX) Simplified overview of the aptamer selection process using SELEX. Starting with the box labeled “Target” and moving clockwise. Target: choose a target of interest, most commonly a recombinant protein but can be a peptide, cell line, or animal model. Aptamer Library: library consisting of ∼10¹²⁻¹⁵ sequence of DNA or RNA oligonucleotides. Library sequences are flanked by 5′ and -3′ constant regions called primer binding sites. If RNA oligonucleotides are desired, in vitro transcription (IVT) can be done prior to the next step. Partition: library is incubated with target of interest and species that are unbound are removed via multiple wash steps. Bound sequences are then eluted for the next step. Negative Selection: subtractive step using a control protein, peptide, cell line, or animal model. There is typically at least one negative selection per protocol. Amplification: the library is amplified via PCR. If RNA oligonucleotides were used, an additional reverse transcription (RT) step is required prior to amplification. Repeat and Analyze: the partitioning and optional subtraction steps are repeated 6–15 more times. The library is then sequenced and analyzed using various computational and laboratory methods.

Figure 2

Defining valency and specificity (A) Multispecific aptamers can be characterized using the [m + n] nomenclature where the [m] represents the tumor-targeting aptamer and [n] represents the immune-cell-targeting aptamer. (B and C) Immunomodulating aptamers may be defined by valency (monovalent or multivalent) or specificity (monospecific or multispecific). Multivalent aptamers can bind the same cell (in cis) or two different cells (in trans). Immunomodulating trispecific aptamers have not yet be described in the literature but can follow [m + n] nomenclature wherein m or n is expanded by parenthesis. For example, a trispecific reagent that embodies two aptamers that bind two different cancer cell targets ([m] = 1 + 1) and one aptamer that bind one immune cell target ([n] = 1) would be denoted as [(1 + 1) + 1]. (D) bsApts can by synthesized as a single product or as two separate products that are hybridized. These constructs may then be circularized by enzymatic or chemical ligation. Gray denotes hybridized bases. Arrowheads denote ligation sites.

Figure 3

Immunomodulating bsApts Published immunomodulating bsApts covered in this review. Immune-cell-targeting aptamers (blue) and tumor-targeting aptamers (black). Boxes include target names (bold) and aptamer and linker features such as composition (e.g., DNA versus RNA) and synthesis method (e.g., hybridization versus single PCR product). If hybridized, method of linker extension is noted in the box (e.g., 3′ end of each aptamer). Circularized aptamers were hybridized and then ligated using DNA ligase. Linker sequences provided in gray below boxes. Conserved nucleotide sequences between linkers are in italics.

Figure 4

Considerations during bsApt development Highlights important factors to consider during the bsApt development process. This includes selection and post-selection molecular engineering. Relevant populations to consider include the tumor cell, immune cell, and TME.