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Special Report on Immunology & Autoimmunity: Turn of the coin
Tumors and infectious microbes have been remarkably successful at evading the human immune system, co-opting molecular mechanisms to dampen the ability of immune cells to see them as anything other than 'self.'
At least for the former, this understanding has led to an explosion of efforts to reverse this cloaking device, to help the immune system identify and fight the tumor cells as unwelcome foreigners. Therapeutic antibodies, cytokines, chimeric antigen receptor T (CAR-T) cells and checkpoint inhibitors have been leveraged to make inroads against various cancers, giving birth to the field of immuno-oncology.
At the same time, many of the mechanisms being perturbed to treat cancer are the same mechanisms the body uses to avoid self-harm in the form of reactivity to autoantigens and the development of autoimmune diseases like rheumatoid arthritis, psoriasis, multiple sclerosis and lupus.
“If we look at autoimmune and inflammatory disorders in terms of global incidence, it is extraordinarily significant,” says Anish Suri, chief scientific officer of Cue Biopharma, a company working in both the autoimmune and immuno-oncology spaces, suggesting that about 10 to 12 percent of the global population suffers from one or more autoimmune disorders.
Yes, you need a diverse immune repertoire because you cannot predict the nature of the next Ebola or West Nile outbreak. The price one pays for that diversity, however, is the ever-present possibility of self-reactivity, which Suri sees reflected in our experiences with immuno-oncology.
Whether you see them as two sides of a coin or two edges of a sword, immuno-oncology has been instrumental in informing the autoimmune landscape, in terms of both the technological insights into the immune system as well as the pathophysiological yin-yang that each condition represents.
“To me, the learnings from immuno-oncology have been significant in autoimmunity because of two fundamental observations,” adds Suri.
“One is that every major adverse event when you have practiced checkpoint therapy has been self-reactive in nature,” he explains. “What that tells me, as an immunologist, is that all or most of us may harbor self-reactive T cells within us, and that they are held in check by the similar mechanisms that the tumor is co-opting for antitumor T cells.”
In other words, he reframes, everybody has the potential to break tolerance. Thus, for those patients who develop cancer and receive treatments that might block the mechanisms of peripheral tolerance like anti-CTLA-4 or anti-PD-1, there is a risk of developing an autoimmune disease.
“And that’s what happens when you look at the knockout phenotypes,” Suri says. “In a CTLA-4 knockout in the experimental animals setting, the pups are dead by three to four weeks of age from systemic inflammation.”
“The other thing that is important is that the onset of the autoimmune adverse event has no bearing on whether you have a tumor response or not,” he presses. “Those are independent events.”
“From the patient’s perspective, that may not be desirable, because not only are you taking a gamble with hoping that you raise an antitumor response, but you may actually end up worse if you only get the autoimmune aspect with no benefit from the antitumor aspect.”
So, how do you develop a therapeutic platform that allows you to activate the immune system when fighting cancer, while also tightly controlling the immune system when it goes awry?
For many companies and research groups, the answer arises from immuno-oncology and the development of modular immunotherapy platforms.
“If you target a receptor to activate something, you could very well envision blocking it to inhibit something,” explains Jane Gross, chief scientific officer of Aptevo Therapeutics. “You could actually target the same receptors, just differently for treatment of the different cancers or autoimmune diseases.”
One molecule, two components
According to Dan Passeri, Cue Biopharma’s CEO, the company’s Immuno-STAT platform is both conceptually and etymologically tied to the idea of a rheostat, something with the ability to control up and down by exploiting, in their case, the same biology.
The platform, which the company is developing for both autoimmune and immuno-oncology indications, involves two components: one that targets the cell of interest via its antigen (or autoantigen) and a second that serves to stimulate or inhibit target cells depending on the disease you are treating.
Both Suri and Passeri are quick to credit the system design to Albert Einstein College of Medicine’s Steve Almo, who is a protein engineer and not an immunologist.
“The reason that is relevant is he was not contaminated with preconceived notions of how to combat cancer, looking at the tumor microenvironment, etc.,” Passeri reasons. “He just looked at the question of how to modulate the immune system via T cells, taking advantage of the exquisite selectivity that is there with the T cell receptor to design a molecule that can dock at the T cell receptor and deliver a co-stimulatory molecule concurrently.”
This emulates the way that antigen-presenting cells interface with T cells.
“It is a very modular approach that allows us to have a tremendous amount of versatility in terms of dialing-in and dialing-out certain features so that we can design molecules and optimize them based on what we’re trying to achieve,” Passeri enthuses.
Last summer, Cue announced it had generated the first autoimmune Immuno-STAT molecule as part of its collaboration with Merck.
As Suri explains, Merck had expressed interest in two select indications, but he is not at liberty to elaborate on what those are, other than to say that they are “well-recognized autoimmune diseases where there is a very strong MHC association.”
Once you’ve targeted the offending T cell, he continues, you can either try to purge them from the immune repertoire or, as is the case at Cue, convert them in vivo from a pathogenic to regulatory phenotype.
This approach, he argues, provides “selective disease-specific regulation without broad dampening of the system as one would see potentially with anti-cytokine antibodies or anti-CD3 or things like that which just dampen the whole compartment.”
Other groups, he suggests, are attempting the Treg (regulatory T cell) approach but ex vivo, removing T cells from the body, converting their phenotype and then reintroducing them, but, as he warns, with no idea of T cell receptor specificity.
Aptevo Therapeutics is taking a similar approach to Cue’s with its ADAPTIR platform, where the co-stimulatory component is the cytokine IL-10.
As Jane Gross explains, cytokines are pleiotropic in that they can both up- and down-regulate various activities in the immune system, making them both attractive but potentially risky targets.
“What people have started to realize in the last four or five years is that you can create a bispecific that targets the cytokine in a special way,” she says.
“Some have attached cytokines to antibodies,” she reports. “Other people have PEGylated them in a particular way, so they target a specific set of receptors and they increase the half-life.”
“We prefer the strategy of recombinantly fusing a cytokine to a structure that has the antibody-like modality,” she notes. “The antibody again gives you the increased half-life, the ability to manufacture in a particular way, and the targeting arm targets it to a specific cell type, or you could target to a specific receptor.”
This approach is the basis of the company’s autoimmune lead APVO210.
“With APVO210, what that drug has allowed us to do is target IL-10 to a particular set of cell types, which are more appropriate for down-regulating the immune system,” Gross explains.
”APVO210 targets the myeloid and dendritic cell lineages, and not lymphocytes,” she continues. “The myeloid and dendritic cell lineages are the relevant population to shut down the pathogenesis of the disease.
“The lymphocytes are the ones that would rev up the immune system, so by eliminating that, it is beneficial for autoimmune disease.”
Another part of that equation is a specific type of regulatory T cells known as Tr1, which are dedicated to maintaining immune tolerance.
As a first step, Stanford University’s Maria Grazia Roncarolo and colleagues demonstrated last year that APVO210 was able to differentiate CD14+ monocytes in vitro into tolerogenic dendritic cells. These dendritic cells were able to induce Tr1 cells, which inhibited primary T cell proliferation.
“The specific ability of APVO210 to deliver IL-10 to CD86+ cells, as compared to IL-10 [alone], which has a pleiotropic effect, may have significant advantages for in vivo use,” the authors wrote. “The systemic administration of IL-10 in vivo has indeed been limited by the development of adverse effects that are due to its stimulatory functions on CD8+ T cells and B cells.”
They further added, “We hypothesize that the in vivo use of this molecule could decrease the risk of triggering specific T- and B-cell responses associated with IL-10 systemic delivery, and, therefore, lead to more targeted and safe control of undesired inflammatory and autoimmune responses.”
Similar to Aptevo’s cytokine approach, VIB-Ugent’s Jan Tavernier, cofounder of Orionis Biosciences, and colleagues recently described a targeted approach to controlling immune cells, but in this case, the cytokine was type 1 interferon (IFN) rather than IL-10.
Their platform, known as AcTaferons (AFNs) or activity-on-target IFNs, consists of a mutant IFN with reduced receptor affinity coupled either to a camelid antibody or a ligand that selectively recognizes a cell-specific surface marker.
The researchers tested their platform in experimental autoimmune encephalitis (EAE), a mouse model of multiple sclerosis, noting that although wildtype mouse IFN significantly delayed onset and progression of disease, it caused significant mortality and hematological deficits in the mice.
Targeting their AFN to Clec9A+ dendritic cells, in contrast, significantly protected the mice without the noted side effects. In fact, Clec9A-AFN offered long-term protection from progression and the development of paralysis even when mice were dosed after disease onset.
They also noted that Treg cell numbers increased in Clec9A-AFN-treated EAE mice and that the percentage of Tregs producing TGFβ and IL-10 increased with both Clec9A-AFN and another AFN targeting SiglecH.
“Our results indicate that dissecting positive and negative IFN effects via cell-specific targeting may result in better and safer IFN-based MS therapy,” the researchers concluded.
Furthermore, they suggested their platform may be a safer alternative to those interested in developing cell-based therapies that accomplish similar endpoints (more below).
“The use of autologous, ex vivo generated tolerogenic DCs (tolDCs) was recently advocated as a promising novel therapy for MS and other autoimmune diseases, and clinical trials are being set up,” they wrote, acknowledging data supporting this approach.
“Needless to say, these therapeutic cell-based strategies are extremely laborious, time-consuming, and entirely personalized, and challenging obstacles and pitfalls are associated with the ex vivo generation of tolDCs,” they cautioned. “Hence, we speculate that targeting IFN activity to DCs in patients using AFNs may induce systemic tolerization and thus provide an in vivo, generic, safe and easy means to dampen MS, in contrast to cell-based DC transduction.”
For JDRF/Wellcome Diabetes and Inflammation Laboratory’s Linda Wicker and colleagues, the cytokine of interest was IL-2, taking a nod from recent studies showing that low-dose recombinant human IL-2 (Proleukin) reduced disease activity in patients with systemic lupus erythematosus (SLE).
Like the other efforts, to optimize the desired effects of the cytokine, the researchers mutated IL-2 to significantly reduce its ability to activate CD4+ and CD8+ effector T cells and natural killer cells, while only modestly reducing its ability to activate Tregs. They then coupled this IL-2 mutein, as they described it, to an effector-silent human IgG1, evaluating the platform in vitro in human blood and in vivo in cynomolgus monkeys and humanized mice.
The STAT5 assay confirmed that the mutein was more selective for Treg activation when compared to the more pleiotropic effects of wildtype IL-2. Tests in both monkeys and mice confirmed this, and researchers found that they could dose the monkeys less frequently and get greater dose-dependent Treg expansion with the IL-2 mutein than Proleukin.
Immunogenicity issues, unfortunately, prevented the researchers from testing their construct vs. autoimmune disease models. That said, they were hopeful that the IL-2 mutein had the potential to correct Treg deficiencies and to restore the balance between Tregs and effector T cells that goes awry in autoimmune disease.
“Our results in nonhuman primates demonstrate the feasibility of developing a long-lived IL-2 mutein with reduced binding to the intermediate affinity IL-2Rβγ receptor that selectively expands Treg cells in vivo,” the authors concluded.
“Most notable was the lack of expansion of eosinophils and NK cells by the IL-2 mutein (N88D) at doses where substantial expansion of Treg cells occurred, a specificity not observed with either short- or long-lived wildtype IL-2 molecules that increased eosinophils and NK cells as well as Treg cells,” they continued. “IgG-(IL-2N88D)2 treatment of patients with autoimmune diseases should provide benefit due to this preferential expansion of Treg cells that will in turn suppress autoreactive effector T cells.”
Another aspect that makes these biologics attractive and where advances in immuno-oncology have largely led the way is the decades of experience in therapeutic antibody manufacture. (See also the October 2018 DDNews “Special Report on Cell Biology: Well CHOsen?”)
“What’s wonderful about [the Immuno-STAT] platform is that the COGs and manufacturing, both upstream and downstream, is exactly like an antibody molecule,” Suri enthuses. “We just made our first GMP batch for our Phase 1 studies that we plan to initiate this year in CHO cell lines, in line with CMOs that make antibodies.”
“Immuno-STAT is an antibody molecule,” he reframes. “Essentially it’s an Fc except that the antigen-binding site has been replaced with a specific peptide HLA and either a co-stimulatory or co-inhibitory to only go after those T cells that bind to those rather than every T cell, as would be the case with a CAR-T or that sort of approach.”
Gross echoes these sentiments.
“If your protein’s not the issue, then you’re going to have a better chance of success,” she says. “We worked very hard on the manufacturability, the stability and the half-life. That allows you to then have better delivery, have better stability, which always leads to better success whether clinically or from a safety profile.”
Perhaps the greatest opportunity in many of the targeted molecules described above, however, is the modularity of their design.
“The attractive nature of a bispecific and the immune system is that once you get a platform technology developed, you can use it in different ways and exploit the knowledge of basic research to tweak either positively or negatively,” says Gross. “We have entertained the idea of now taking cytokines and fusing them to an ADAPTIR structure for oncology.”
“I think the applicability is potentially to block a checkpoint inhibitor and induce an activation at the same time,” she says. “And you can do it either in trans or in cis, so either between two cells or on one cell.”
But just as immuno-oncology has not limited itself to protein therapeutics, neither has the autoimmunity field, which is also exploring cell-based approaches to reversing autoantigen reactivity.
Late last year, Zhenhua Dai and colleagues at Guangdong Provincial Hospital of Chinese Medicine reviewed the current state of efforts to use CAR-T approaches to Tregs in the hope of inducing immunological tolerance.
They recounted one effort showing that CAR-Tregs specific for trinitrophenol (TNP), an antigen commonly used to generate mouse models of colitis, was able to suppress effector T cell proliferation whereas control Tregs could not.
“When colitis was induced with 2,4,6-trinitrobenzene sulphonic acid (TNBS), the mortality rate of TNP-CAR-transgenic mice significantly decreased in comparison with wildtype mice, indicating that TNP-CAR Tregs may have protected mice from colitis,” the authors noted. “Similarly, transfer of TNP-CAR Tregs, but not unmodified Tregs, to wildtype mice with TNBS colitis alleviated the disease and prolonged mouse survival. In situ fluorescent microendoscopic evaluation verified that TNP-CAR Tregs localized to inflamed colonic mucosa.”
What proved particularly interesting, they continued, was that the same specific cells offered protection or “bystander suppression” from other forms of induced colitis.
“Thus, Tregs redirected to inflamed tissue can exert protective effects with a specificity that differs from that of pathogenic T cells,” they noted.
They also highlighted another study that examined the impact of CAR-Tregs with specificity to myelin oligodendrocyte glycoprotein (MOG) in EAE mice.
“MOG-CAR Tregs efficiently homed to various regions in the brain after intranasal cell delivery, as demonstrated by examination of horizontal cryosections of the brain, and suppressed ongoing encephalomyelitis, as evidenced by reduced disease symptoms and decreased mRNA levels of IL-12 and IFN-g in the brain tissue,” the authors described. “Moreover, EAE mice treated with MOG-CAR Tregs were protected from a second EAE challenge, indicating a sustained effect of the engineered CAR-Tregs.”
Surveying the landscape, Dai and colleagues suggested that antigen-directed CAR-Tregs were likely to generate fewer side effects than blanket immunosuppression and were obviously more specific than polyclonal Tregs, but they also cautioned that antigen selection could be significantly challenging. Furthermore, incidences of cytokine storm or neuronal toxicity have been noted with anti-tumor CAR-T cells.
Pharmicell’s Mi-Young Park and colleagues took cellular intervention one step broader, looking at the application of myeloid-derived precursor cells (MDSCs) to treat psoriasis in the imiquimod- (IMQ-) induced mouse model. MDSCs are known to release many immunosuppressive mediators such as IL-10, TGF-β and prostaglandin E2 as well as induce Treg cells.
They noted that MDSC injection significantly reduced inflammatory symptoms in IMQ-treated mice and inhibited the release of proinflammatory cytokines associated with psoriasis. Likewise, there was a dose-dependent increase in Tregs and a concomitant decrease in immunostimulatory T helper cells (Th1, Th17).
The findings are consistent with similar work in murine models of arthritis and SLE.
“Taken together,” the authors concluded, “these results imply that MDSCs have immunomodulatory and immunosuppressive effects on disease progression in a murine model of psoriasis and that MDSCs could be used in preventive or therapeutic strategies for the management of autoimmune inflammatory skin disorders, such as psoriasis.”
Coping with treatment
Another way in which immuno-oncology and autoimmunity can be viewed as opposites comes in the expectations of patients receiving treatment in terms of willingness to deal with adverse events.
Marco Silleni, vice president and rheumatology and autoimmune disorder therapeutic area lead at ICON plc, suggests that because autoimmune disorders are largely chronic conditions that a patient may have to face for his or her entire life, patients are much less likely to accept what is accepted by cancer patients undergoing treatment; e.g., hair loss, nausea and other gastrointestinal symptoms.
Cue Biopharma’s Suri concurs.
“I used to study type 1 diabetes when I had my lab at Washington University, and it is very clear with kids that manifest with this disease early on that you’re looking at trying to accomplish a healthy life span for the child,” he recounts. “Those considerations are very different than what may be in a patient with a Stage IV cancer. Not to minimize one over the other, but the thinking is very different.”
“You don’t want to be on these drugs for the rest of your life,” adds Aptevo’s Gross, citing both the costs of treatment and quality-of-life perspectives.
“Most people are looking for something that doesn’t just shut down your immune system or revs it up temporarily, they want to change the environment completely, so that you now could go off the drug and in a very idealistic term, be cured,” she presses. “That is the ultimate desire.”
“If you can generate a Treg population that’s long-lived, that sees the pathogenic antigens, then you can potentially take people off the drug and you have a cure,” she offers. “You’re resetting or reprogramming the immune environment so that you’re not in your disease state anymore.”
According to Silleni, this potential for extended therapy means that regulatory authorities are much more focused on chronic toxicities and require long-term safety data.
“Often open-label extensions, ranging from one to five years, are advisable to collect long-term safety data and to make trials more attractive to investigators and to patients,” he continues.
This conservatism, Silleni presses, means that regulatory agencies are less likely to look at biomarkers or surrogate endpoints in clinical trials. He offers the examples of DAS24 and ACR20 in rheumatoid arthritis—based on counts of swollen and tender joints, as well as some patient- and physician-reported outcomes—and the PASI and PGA in psoriasis as clinical measures.
“With regard to clinical monitoring, for such chronic diseases and lengthy trials, it can be a burden and a cost,” he says, layering on that “the qualifications and the skills of assessors are paramount.”
Newcastle University’s Chijijoke Mosanya and John Isaacs recently reflected on these challenges in a review of efforts to use cellular therapies to induce tolerance, which they suggest should mean life-time drug-free remission.
“Tolerance takes time to develop and tolerogenic therapies may not reduce symptoms in the short-term, necessitating the temporary continuation of more conventional therapies,” they wrote. “Careful clinical trial designs will therefore be fundamental in order to identify, robustly, tolerance induction.”
“In the short term,” they continued, “this is likely to require immune monitoring, for example, using autoantibody arrays and MHC-peptide tetramers, in order to track and interrogate the quality and quantity of the autoantigen-specific response.”
Other factors that need to be considered, they argue, are:
And that is ultimately where it comes back to the patient.
For someone potentially facing a life of pain and discomfort, how those costs are defined—financially or emotionally—is just one more coin with two sides.
Microbes and mimicry
As Cue Biopharma Chief Scientific Officer Anish Suri suggests, one of the prices of having a healthy and diverse immune repertoire may be the presence of autoreactive T cells that are held in check to maintain tolerance, but which can be triggered by a mechanism like checkpoint inhibition in cancer treatment to induce autoimmune disease.
Increasingly, researchers are also scanning the microbes that exist naturally within us or invade our bodies for clues to autoimmune triggers.
“A field that is emerging but is not our core area of focus is the interplay between the microbiome and the immune response, be it autoimmunity or cancer immunotherapy,” Suri explains. (See also the January 2019 DDNews “Special Report on Microbiomics: Army of One.”)
In particular, he points to the concept of molecular mimicry, where a microbial epitope cross-reacts with a natural epitope (self) in the host. Thus, when the body responds to infection, the normally quiescent autoreactive T cells are activated and attack not only the infectious microbes, but also any tissues expressing the autoantigen.
Volker Stadler, CEO of PEPperPRINT, points to the Immune Epitope Database for quick examples of molecular mimicry.
“If you search for a sequence like GRGRGR—so a poly-glycine-arginine stretch—you’ll find hits in Epstein Barr virus (EBV)—in its nuclear antigen 1—and you can find exactly the same sequence in a small ribonucleoprotein called SMB1, which is a known autoantigen in humans and mouse models,” he explains. “Given that there is a nearly identical sequence in the virus and in an autoantigen, you can easily imagine that such an autoantibody response was raised by a viral infection.”
He suggests that there is a growing body of evidence linking EBV infection with rheumatoid arthritis, for example, and that his company has started to explore this area.
“Not only EBV, but there are also papers that describe a link to a bacterial infection with Porphyromonas gingivalis,” he continues. “This is a bacterium that can be found in the mouth flora and is also being discussed as being linked to rheumatoid arthritis.”
“I wouldn’t be surprised if there are many more, varying from disease to disease.”
Discovering such instances of molecular mimicry is a perfect example of the application of PEPperPRINT’s central platform, a peptide microarray.
“What we can do with our peptide microarrays is narrow down the binding site of an antibody to its antigen, not only down to the epitope with single amino acid resolution, but also to identify conserved and variable amino acid positions,” Stadler explains. “And from there, deduce the cross-reactivity of an antibody. The more conserved or essential amino acid positions there are, the higher the target specificity.”
Late last year, Keren LLC’s David Dreyfus and colleagues described their efforts to identify epitopes from EBV proteins that reacted with antibodies in human serum, whether from a healthy control subject or one with scleroderma.
They synthesized an overlapping series of 15-mer peptides on the chip, each peptide overlapping the next by 13 amino acids, progressively shifting along the protein sequence in 2-amino-acid increments. They then incubated the chips with serum and identified reactive antibodies via anti-IgG probes.
The researchers identified IgG reactivity to conserved A/T hook domains within the EBV proteins EBNA-1 and BALF-2 recombinase, which are related to host DNA-binding proteins such as RAG-1 recombinase and histones, as well as reactivity to the EBV-encoded virokine BCRF-1, an IL-10 homologue.
“Preliminary data obtained from the immune response to some shared gene-encoded proteins reported in this work suggests that while some shared gene-encoded proteins such as BZLF-1 and EBNA-1 are highly antigenic and thus trigger IgG against self-proteins, other EBV-encoded shared genes such as virokines may be poorly antigenic, permitting the viral-encoded projects to function as antagonists or partial agonists of the host immune response,” the authors noted.
Beyond the specific reactivities, however, the researchers also wondered about the implications of such proteomic assays for disease diagnosis and treatment monitoring.
“Using inexpensive and highly automated molecular fingerprints, it might be possible in the future to identify patients at risk of autoimmune syndromes prior to development of symptoms based solely on their response to specific epitopes in viral proteins and shared host proteins,” they concluded.
Stadler sees the diagnostic opportunities for the peptide microarray as slowly evolving.
“From a scientific point of view, you can identify way more detail and get way more information with a peptide microarray compared to an ELISA or a western blot,” he says, but then cautions that this increased information comes at the cost of a more technically sophisticated platform.
Rather, he sees the peptide microarray and ELISA as interconnectable.
“What we see on the microarray can usually be translated into a standard ELISA format,” he explains.
Stadler also echoes the opportunities for patient stratification afforded by an understanding of epitopes and reactivity, offering an example from immuno-oncology.
“You can generate peptide microarrays that contain a number of tumor-related antigens translated into overlapping peptides for epitope mapping,” he says. “What you could do in addition is translate the sequencing data from a patient into peptide pairs with the wildtype peptide and the mutant peptide next to each other and add this to the microarray.”
“You can use the antigens and the neoepitopes to see if there is an increase in the antibody response against the tumor-related antigen or even the patient-specific neoepitopes,” he enthuses.