Immunotherapy is a treatment which, if it triggers a positive response, can have a miraculous effect on the body’s ability to fight cancer.
Amrik Singh is an immunology fellow at Cell Signaling Technology (CST). A qualified biochemist trained at Boston University Medical Center and Beth Israel Deaconess Medical Center, his role at CST involves helping to develop and manage the adoptive cancer cell therapy product portfolio, with a specific interest in developing antibodies and assays to characterize CAR-T cells.
In this conversation with Fierce Biotech’s Stephanie Butler, Amrik dives into the work the company is conducting in this highly specialized field.
“CAR-T cell therapies have truly revolutionized the treatment of life-threatening blood cancers,” he tells us. “Patients previously unresponsive to other conventional treatments have gone into remission with CAR-T therapy, with some patients essentially being cured. Indeed, numerous patients receiving CAR-T therapies targeting the tumor associated antigens, such as CD19 and BCMA, have achieved deep durable responses.”
CST has made significant strides over the past year, especially around the development of novel antibodies to facilitate the detection of surface expressed chimeric antigen receptors. Amrik continues the conversation by outlining the significance of this progress, while hinting what the future might have in store for the use of these antibodies to treat patients.
To discover more, listen to the full interview with Amrik today.
Stephanie Butler: Hi. I'm delighted to be here with Amrik Singh, who is an immunology fellow at Cell Signaling Technology. So welcome, Amrik.
Amrik Singh: Hi Stephanie, thank you, it's a pleasure to be here. Thank you for inviting me.
Stephanie Butler: Excellent. I'm really interested to hear more about what your company is doing and what you're working on. So, I kind of wanted to start with kind of an opening question: What are chimeric antigen receptors (CARs), and how are they advancing cancer therapies?
Amrik Singh: So, I'll start by saying that the overarching goal of cancer immunotherapy is to leverage the host immune system to direct an anti-tumor response. In some autologous platforms of adoptive cell transfer, which leverage a novel technology that's based upon chimeric antigen receptors, a patient's own immune cells are collected, and these are typically T cells. They're genetically modified to target cancer cells and then are reintroduced back into the patient, where they circulate through the bloodstream and have the ability to home in on malignant cells. Then, they initiate an attack and killing program. CAR T-cell therapies have truly revolutionized the treatment of life-threatening blood cancers, and patients previously unresponsive to other conventional treatments have essentially gone into remission with CAR-T therapy, with some patients essentially being cured. Indeed, numerous patients receiving CAR-T therapies targeting the tumor-associated antigens, such as CD19 and BCMA, have achieved deep, durable responses.
At the molecular level, CARs are considered a novel class of signaling immune receptors, and they're derived, essentially, from components of multiple immune receptor types. For example, the antigen recognition and signaling components of CARs often reside on the same polypeptide chain. And they are designed to recognize native surface antigens on the surface of tumor cells, independent of MHC restriction. This is an important consideration given that components of the Class I MHC presentation pathway are very often compromised in many types of human cancers. In terms of signaling, CARs generally utilize either a CD28- or 4-1BB-derived signaling domain for co-stimulation, and they are fused in frame with the tyrosine residue-rich ITAM motifs derived from the CD3-zeta chain of the T-cell receptor signaling complex. These signaling modules essentially drive the intracellular signaling cascades, which are engaged upon antigen recognition by the CAR, that govern T-cell effector functions, including cell proliferation as well as cytotoxicity.
Stephanie Butler: So why, then, is CAR detection important, and what challenges do researchers encounter when they're detecting the CARs?
Amrik Singh: CAR transgenes can be delivered to cells via either viral or non-viral methods. The ability to employ analytical methods that are designed to directly monitor CAR protein expression provides a robust readout for determining the percentage of CAR positive cells obtained in a culture. Because it's unlikely that 100% of the cell population is going to express the CAR transgene of interest, it's important to leverage methods as well as reagents that can provide accurate and reproducible readouts of CAR expression, and this has enormous implications; for example, in calculating a dosing regimen within clinical and commercial manufacturing protocols.
In terms of methods to detect the presence of a CAR transgene in an engineered cell, there are many that are used in different scenarios. For example, genomic and transcriptomic methods, while they are sensitive, lack the ability to inform one of the status of CAR protein expression and functionality. Some existing reagents that enable the detection of CAR protein surface expression include protein L, anti-idiotype antibodies, and anti-Fc antibodies, as well as CAR target antigens. They're designed to target the antigen recognition component of the CAR, and that's because these regions can be used to interrogate CAR expression on live cells. However, there are drawbacks to some of these detection reagents, which are centered around the fact that not all CAR designs incorporate the structural elements that these reagents target. In some cases, there's a lack of commercial availability. Some reagents can only detect a CAR-defined specificity, and then some reagents, such as protein L, are not easily incorporated into multiparametric flow panels, which is an important attribute of an efficient detection reagent because one is often doing extensive phenotyping of the engineered cells.
Stephanie Butler: So you had kind of mentioned antibodies, and I kind of want to move on to ask you: How do novel antibodies developed by Cell Signaling Technology aid in the detection of CARs, and how are those antibodies then validated?
Amrik Singh: Within the last year, as you mentioned, we've developed some novel antibodies to facilitate detection of the surface expression of chimeric antigen receptors. And in light of the challenges that I've just described with respect to the existing methods of CAR detection, we asked whether there are other structural elements within the extracellular domain of scFv-based CARs that can be exploited for the discovery of novel detection reagents. So, it's important before I get into how we develop these antibodies and what their utilities are… it's important to mention that for scFv-based CARs, the length as well as the amino acid composition of the peptide linker sequence within the scFv is critical for proper folding, stability, and alignment, of the variable heavy and variable light domains. This region of the receptor can form a competent confirmation for target binding.
Most scFv-based CARs utilize linkers that have either multiple repeats of four glycines and one serine; three repeats of this pentapeptide is the most common, or what's called a Whitlow linker peptide. And because these two linker sequences are fairly ubiquitous in scFv-based CARs, we wanted to raise monoclonal antibodies against them in the hopes of generating a versatile tool for CAR detection. And indeed, we generated two recombinant rabbit monoclonal antibodies against each unique linker sequence that I just mentioned. We characterized their specificity using a live cell flow cytometry assay. And these two monoclonal antibodies are ideally suited to detect surface-expressed CARs of varying antigen specificity and model systems such as primary human T cells and NK cells, and they're able to do so with a high degree of specificity and sensitivity. CST currently offers these two clones in multiple RUO formats that are research-use only, including biotin conjugates, as well as fluorophore conjugates. And this expansive offering affords the end user with the flexibility to incorporate these directly conjugated antibodies into multi-parametric flow panels, which can then be used to delve into extensive phenotypic characterization of CAR-engineered cells.
Stephanie Butler: So, it's really interesting technology, and I'm curious what are the future plans for these antibodies, and are there new applications and uses?
Amrik Singh: The versatility of these antibodies and their ability to detect multiple CARs, agnostic of antigen specificity, has really opened the door to ask questions surrounding what other attributes these antibodies have and then what other applications can they be leveraged in. And at CST, we are actively engaged in expanding our portfolio of products to support the characterization of CAR-engineered cells. For example, we'd like to ask if these antibodies can be leveraged in magnetic bead-based immuno-affinity enrichment protocols. We'd also like to identify new clones that can be used to monitor the presence of CAR-engineered cells in formalin fixed and paraffin-embedded tissue. And this will have enormous implications, I think, for advancing the CAR technology to attack solid tumors. Lastly, we're also considering the possibility of converting these antibodies into scFv fragments, which can be potentially used for in vivo tracking of CAR-engineered cells. So we have a lot going on in our internal antibody discovery engine, and in the future, we hope to offer these antibodies, or derivatives of these antibodies, to address new and powerful questions to help advance this field forward.
Stephanie Butler: It's really exciting. I'm really looking forward to seeing where this is going in the future. So, thank you so much to Amrik and Cell Signaling Technology for your time today and for sharing your insights. Looking forward to seeing where you go next.