19th century illustration published in the Natural History of the Animal Kingdom. Image courtesy of ancestryimages.com.
Image courtesy of ancestryimages.com.

Research into the immunobiology of jawless vertebrates has identified an adaptive immune system of clonally diverse lymphocytes expressing the variable lymphocyte receptor (VLR), distinct from the immunoglobulin (Ig)-based adaptive immune system of jawed vertebrates.

We caught up with Dr. Goetz Ehrhardt, Assistant Professor in the Department of Immunology, whose lab is using VLR antibodies (generated by immunizing sea lamprey larvae) to detect B cell surface markers that may not be recognized by conventional (mammalian) antibodies. Because of the unique structure of VLRs and the evolutionary distance between lampreys and mammals, VLRs may have the potential to complement existing research tools.

IMMpress: You previously worked with Max Cooper as part of the team that originally characterized VLRs in sea lampreys. How did you become interested in VLRs, and how did you decide to work on this project?

[During my PhD at UBC], I was very biochemically-oriented. At some point, I developed an interest in immunology, and applied for a postdoc position in Max Cooper’s lab and started working on memory B cells. But because of my background in signal transduction and molecular biology, I basically entered into the VLR project for technical reasons.

I was actually quite glad because it is truly fascinating. The key person that actually worked on this (in addition to Max) was a postdoc named Zeev Pancer, who was very, very experienced in the study of leucine-rich repeat molecules from research he did at CalTech. He was the right guy, in the right lab, at the right time.

The major focus of my [own] lab is the regulation of memory B cell responses, and the problem with memory B cells, as with many other cells, is that they are by and large functionally defined. In many ways, you identify a memory B cell based on what it is not – it is not a naïve B cell, not a germinal centre cell, not a plasma cell. So the question is: can we actually identify something that is specific for memory B cells? That is where my interest in VLRs came in.

IMMpress: What makes VLRs a good candidate for biomarker research?

There are several characteristics for why we think lamprey antibodies (VLR antibodies) are very, very promising. On the one hand, it is the different protein architecture. VLRs may be able to interact with antigens in manners not seen with conventional antibodies. Thus, the first point is: can we make VLR antibodies to targets that conventional antibodies cannot interact with because of structural limitations?

A key feature of the mammalian adaptive immune system is its ability to discriminate self from non-self. Thus, the mammalian immune repertoire is limited by certain constraints to maintain tolerance. Because the jawless vertebrates branched off from jawed vertebrates about 450 million years ago, it is very likely that the tolerogenic constraints are quite distinct from the ones that acted on our immunoglobulin repertoire. So the second point is: can we generate VLR antibodies to targets excluded by conventional antibodies due to tolerogenic constraints?

IMMpress: How are VLR antibodies generated in the lamprey and in vitro, and what are some challenges to large-scale production of VLR antibodies?

One of the key differences between mammalian antibodies and the VLR antibody system is that VLRs are secreted as decameric or octameric molecules. The secreted form of the antibody is generated by iteration of the same gene product, which means it is only a single chain. This is in contrast to mammalian antibodies, which need a matching pair of heavy and light chains – that is where hybridoma technology comes in very handy. We actually clone the antibody by PCR-based isolation of the sequence, and then transfect the recombinant VLR into HEK293T cells. These cells generate and secrete the antibody, so we do not have to develop hybridoma technology for lamprey lymphocytes.

It is certainly true that production of large quantities of these VLR antibodies is a problem that is not completely solved. Not all of the VLR antibodies that are secreted by HEK293T cells are actually the multimeric version. We are mostly interested in the high molecular weight multimers, because VLR antibodies usually display avidity-based binding. Nonetheless, in our latest batch we got milligram quantities of VLR antibodies. It is less than what a good hybridoma line can secrete, and I do not know if you could call that “large-scale”, but for many applications, a few milligrams’ worth of antibody is quite sufficient.

The VLRA crystal structure. Sequence variability within select leucine-rich repeats is indicated by the color gradation from red (the most variable positions) to blue (the least variable positions). Image credit: Modified from Kanda et al 2014 under CC BY 4.0.
The VLRA crystal structure. Sequence variability within select leucine-rich repeats is indicated by the colour gradation from red (the most variable positions) to blue (the least variable positions). Image credit: Modified from Kanda et al 2014 under CC BY 4.0.

IMMpress: What hurdles would you foresee that other scientists may have in the adoption of VLRs over conventional monoclonal antibodies in their research?

I would not characterize lamprey antibodies as either superior or inferior to conventional antibodies – they are different. They very well have a place to complement existing antibody repertoires without the aim of replacing them. One of the characteristics of VLR antibodies is that they tend to be high avidity and low affinity, which can be a drawback if your application of choice requires strong binding by individual subunits. There is no evidence so far that affinity maturation exists in the lamprey. On the other hand, the solenoid structure of the VLR antibody does not argue against generating high affinity VLR antibodies per se. You can certainly, by random mutagenesis in vitro, affinity mature VLR antibodies to very high affinity (picomolar), which is pretty much what a good conventional antibody is able to achieve.

IMMpress: You are currently developing VLR antibodies for detection of cancerous cells. What can you tell us about that?

We isolated several VLRs that show binding patterns to primary lymphocytes, which are inconsistent with any conventional antibody, suggesting that we actually have antigens that these VLR antibodies recognize that conventional antibodies do not. One of our VLRs recognizes specifically plasma cells and nothing else, which is not only interesting for many academic- and research-based questions, but also has potential clinical applications. There are very few characterized antigens that are expressed only on plasma cells. Syndecan-1 is typically used as a plasma cell marker, but it is also expressed on many epithelial cells, which makes it not a very attractive therapeutic target. The antibody that we have isolated (and we are in the process of identifying the antigen) recognizes an antigen distinct from Syndecan-1. Furthermore, this antibody detects ⅔ of malignant plasma cells in bone marrow aspirates from multiple myeloma patients.

Can we get any clinical information that correlates with binding or with absence of binding of this antibody? Does it correlate with certain cytogenetic abnormalities? Does it correlate with a response to treatment? Does it correlate with the predicted course of disease? Is this a malignant form or is this an indolent form of the disease? As for prognostic or diagnostic tools, the potential for application is more immediate than any therapeutic application.

IMMpress: What about the potential for VLRs to be used in the clinic?

You have to be careful when it comes to potential clinical or therapeutic applications of VLR antibodies. Conventional antibodies are probably the most rapidly growing segment in biopharmaceuticals. However, you could expect that the VLR itself would be recognized as foreign so you would have to find a way to ‘humanize’ it. Whether that is possible or not is a different question. We are starting collaborations with investigators at McMaster to see whether we can use the chimeric antigen receptor (CAR) system together with VLRs as the targeting tool for specific cell populations. Usually in these CARs, the extracellular domain of the fusion product is a single chain antibody, which we want to instead replace with a VLR (for example, our VLR that recognizes all plasma cells, including all malignant ones that have been tested so far). Can we actually use this new CAR system for potential clinical applications? The stage we are at is called “drawing board”, so that is really earmarked for the future.

IMMpress: What do you think the landscape of VLR technology will look like 10 years from now? What is “next” in the field?

Personally, I think it will expand. There is published data out there that suggest VLRs are good at recognizing carbohydrate antigens; as we know, it is very hard to generate anti-carbohydrate conventional antibodies. I think once there are more published monoclonal VLR antibodies targeting cell populations of broader interest, the system will become more widely used. At the moment, we are still standing at the very, very beginning. The number of monoclonal VLR antibodies that are actually out there is extremely limited. We hope to change this. VLR antibodies can be used in basically any application technique that conventional antibodies can be used, like flow, ELISA, Western, IP, arrays, etc. So what we have here is a novel reagent for which we do not need novel technology in order to use – you could plug it into most existing assays. Once more VLR antibodies are available, I do not see any reason why they should not receive broad recognition.

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