Teaching immune cells how to kill, and other things I learned in 2020
Therapeutics and targets mentioned: 4-1BB, Bispecific-engagers, CAR-T, CD39/CD73/A2AR, CD47, FcαRI, FcγRIIa, Flt3L, GM-CSF, IL-2, Immune Checkpoints, LILBR2/ILT-4, OX40, PD-1, Siglec10/CD24, STING, TIGIT/DNAM-1, TIL, TLR7/8 & 9.
Companies mentioned: Agenus, Aleta Biotherapeutics, Alkermes, Alligator, Apexigen, AstraZeneca, Celldex, GSK, IgM Biosciences, I-Mab, Immune-Onc, Iovance, Jounce, Merck, Nektar, Seagen, Roche.
Two talks given at SITC 2019 session set me thinking about the quality of immune cell interactions, the outcomes for the interacting cells and the implications for cancer immunotherapy. These talks, by Ron Germain and Michael Dustin, presented the lives of immune cells in a series of diverse locations with a complex cast of characters. Learnings regarding immune geography and cell:cell contact are increasingly important as we consider how best to advance cell therapies for diverse hematologic malignancies and solid tumors (www.aletabio.com).
These investigators work to understand the cell biology that supports a productive immune encounter, and this depends in part on location as much as it does on cell type. The bio-pharma field has focused on T cells as the major target cell type for cancer immunotherapy, but it is clear that B cells, myeloid cells, dendritic cells, NK cells and neutrophils can play unique and critical roles. Immunology insights gained in 2020 will influence how we think about immune-checkpoint therapeutics, cell therapeutics and tumor resistance to therapy. Historically, we can link these lessons back to two of the very earliest “applied” immune-therapeutics, the cytokines IL-2 and GM-CSF, that trigger distinct subsets of immune cells.
Part 1: Location, location, location.
In January 2020 four papers were published that described the correlation between the presence of tertiary lymphoid organs and B cells with successful immune checkpoint therapy in diverse cancer indications (see here). This was an interesting finding and one that I think remains under-appreciated by the immuno-oncology drug development field.
These papers raised an interesting question – why are tertiary lymphoid structures (TLS) and by extension, secondary lymphoid organs such as lymph nodes, spleen, and Peyers patches, important for successful immune checkpoint blockade therapy (ICB)? Aren’t we just waking up exhausted T cells, or moving T cells from the tumor margin into the tumor bed? Isn’t that how anti-PD-1/anti-PD-L1 antibodies work? Why should you need a TLS or lymph node?
These questions compel us to once again deconstruct the tumor and its surroundings. One might start with the immediate tumor microenvironment (TME) under direct control by tumor cells, stroma and stroma-embedded fibroblasts and myeloid cells. A second view might consider the vascularized tumor bed, with access to blood vessels and lymphatics. A third view: the invasive tumor margin, where tumor cells are invading normal tissue. A fourth: sites within the tumor where immune cells are present, either active or immobilized. Fifth: associated lymphoid tissues and organs. And so on, although it won’t help to make things too complicated. Not by coincidence the list overlaps with the phases of the tumor-immunity cycle (Chen & Mellman, 2013).
As to why you need a TLS or lymph node, the answer probably lies in the quality of the T cell pool. As we learned from the work of many labs (reviewed here: https://doi.org/10.1038/s41577-019-0221-9) T cell exhaustion is a complex state, with subsets of cells having distinct functionality and fates. Indeed, ‘exhaustion’ may be too broad a term. For example, we know from Stephen Rosenberg’s work that TILs can be isolated from bulk tumor tissue, expanded using IL-2, and thereby “re-animated” ex vivo. Therefore, TILs are not always terminally exhausted. Iovance has successfully exploited these findings and shown efficacy in late-stage clinical trials using patient-derived TILs to treat melanoma and cervical cancer.
These efforts can be traced back to the approval of high dose IL-2 for the treatment of renal cell carcinoma in 1992 and metastatic melanoma in 1998. That 1992 date is notable, as IL-2 was discovered only 16 years earlier in Dr Robert Gallo’s lab (link). Those approvals also are the basis of extensive efforts to produce less toxic variants of IL-2 by engineering selective IL-2 receptor engagement, as exemplified by the drug development work of Nektar, Alkermes, Roche and many others. IL-2 is also used in the expansion of NK cells, indicating the pleiotropic activities of this cytokine.
Of note, TILs expanded in the presence of IL-2 can exhibit a differentiated phenotype that can shorten their long-term persistence and survival in vivo. Recent analyses of successful TIL therapy have stressed the importance of a “stem-like” T cell population that has both proliferative and self-renewal capacity and fosters the development of long-lived memory T cells (Rosenberg lab: here). I note in passing that their analyses suggest that strategies aimed at the CD39/CD73/A2AR pathway may have limited clinical impact. A similar population of T cells has been associated with successful ICB therapy (discussed: link) and may play a role in productive CAR-T cell expansion.
A specific type of dendritic cell (DC) has been identified as a critical component of ICB therapy and this brings us back to lymph nodes and to TLS. The cDC1 dendritic cell subset is implicated in the support of T cell mediated anti-tumor immunity (discussed by Gajewski & Cron here). These are interesting cells that can be found in lymphoid organs, in inflamed tissues and within tumors. Tumor antigens can make their way into lymphoid tissues by direct antigen drainage (review) with specific regions within lymph nodes supporting distinct DC populations and supporting distinct T cell responses (it turns out that B cells help with this spatial organization). Tumor antigens can also be carried from the tumor into the lymph nodes by cDC1 themselves (link). So now we have a narrative that accounts for the benefit of having lymphoid tissue in the context of anti-PD-1/PD-L1 therapy – this organized lymphoid tissue amplifies any existing anti-tumor response with a de novo response, sending additional T cell soldiers to the tumor front lines.
There are additional puzzles hidden within this narrative. Possibly the one that bothers me the most is seeming failure of therapies that target T cell agonist pathways – notably 4-1BB and OX40 – to improve the response unleashed by ICB therapy. Without burrowing deep into an immunology rabbit hole, I propose that anti-4-1BB and anti-OX40 agonist antibodies fail because they amplify signals in the wrong place or at the wrong time. The immune system is tightly regulated and unkind to inappropriate signals. Along these lines it is worth noting that completely blocking PD-1 will also backfire, as has been shown in disparate experimental systems (example). This is translationally important, as PD-1-knockout CAR-T cells were eliminated in patients, either by active elimination or due to competitive disadvantage (paper, and presentation by Carl June, ASGCT 2020). In contrast, signals that activate the DC compartment – GM-CSF, Flt3L and agonists that target CD40 (see Roche, Apexigen, Alligator, Seagen, Celldex and others) – do appear to augment anti-tumor immunity, and this may be the ideal way to think about boosting ICB therapies and perhaps CAR T cell therapies (hint). A historical note: GM-CSF expression is a critical component of the T-VEC oncolytic viral therapy approved in 2015, just about 20 years after the first amino acid sequence data became available from the labs of Metcalf, Burgess, Dunn and colleagues during 1984-5 (here is a history by Glenn Dranoff).
Part 2: Knocking on other doors.
If location is critical, perhaps it’s time to move back to the TME. I’ve thought for a long time that some TME-directed efforts are misguided. I suspect several cell types commonly associated with the TME are epiphenomena that perhaps amplify, but do not create, the immunosuppressive microenvironment. T-regulatory cells (T-regs) are one such cell type, and suppressive myeloid cells may be another. The immuno-oncology drug development field has, to date, fallen short in attempts to deplete or alter these cell types for clinical benefit.
This should be surprising since T-regs and myeloid suppressor cells are abundant in TMEs across indications, but I would argue that tumor cells themselves and associated cell types in the tumor stroma, notably fibroblasts, are dominant. ICB resistance signatures include VEGF, beta-catenin and TGF-beta – these factors appear to create the immunosuppressive milieu and subvert incoming immune cells. Depleting T-regs or attempting to convert immunosuppressive myeloid cells (eg. ‘M2s’) to pro-inflammatory myeloid cells (eg. ‘M1s’) does not address the underlying immunosuppressive TME, which has arisen as a result of selective pressure on the tumor cell population. I’ve discussed ICB resistance previously (see here and here).
However, the immunosuppressive TME and its attendant cell types can be upended, most notably by triggering evolutionarily ancient pathways that trump the immunosuppressive signals. Many of these pathways are well known – the TLR7/8 and TLR9 agonists, the STING agonists, and the CD47 pathway inhibitors being prosecuted by many companies (see eg. AstraZeneca’s MEDI9197, a TLR7/8 agonist, Glaxo’s GSK3745417 STING agonist, I-Mab’s CD47 program, among many others). Of note, localization of agonist signaling is critical in this space as well. For example, TLR signaling is generally targeted at tumor cells directly, whereas it is debated whether STING agonists should target myeloid-lineage cells within the TME, tumor cells themselves, or both.
I particularly like the idea of engineering CD47 antagonism into other modalities, eg. T cell engagers. Indeed, blocking CD47 to induce myeloid cell phagocytic activity is an active field, and this has encouraged a search for similar signals, for example, the Siglec10/CD24 pathway. Moving even further afield we encounter quite novel myeloid cell signals and can consider pathways that are not as widely targeted. One is the ILT (aka LILBR) system, where most activity is centered on antibodies to ILT2 and ILT4. Here we begin to intersect with multiple cell types, as ILT2 is expressed by monocytes, macrophages, DC, B cells, and subsets of T cells and NK cells, and ILT4 is expressed by neutrophils, myeloid cells and DCs. These proteins have inhibitory signaling domains that are triggered by MHC binding, including to the HLA-G protein, normally expressed on myeloid lineage antigen-presenting cells (macrophages, DCs) where expression serves to immune-suppress interacting cells. HLA-G is also overexpressed on many tumor cell types. Thus, the ILT/HLA-G system appears to be another immune checkpoint, perhaps with a broader range of activity than the PD-1 system. Merck has shown early positive clinical data using an antagonist anti-ILT4 antibody, MK-4830 (from Agenus), in combination with pembrolizumab (anti-PD-1) in heavily pretreated cancer patients (presented at ESMO 2020). Jounce Therapeutics and Immune-Onc showed preclinical data at SITC 2020 on their anti-LILBR2 (ILT-4) programs, and there are additional efforts underway. I suspect this field will grow quickly, and perhaps match the TIGIT/DNAM-1 space in interest and complexity.
Part 3. Fc-hacking immune responses.
As mentioned above, the immune system has strict rules and regulations, and can be resistant to having these over-ridden by therapeutics. Hacks are possible of course, as shown by the success of CAR-T cells and the T-cell engager bispecifics. Along these lines, decades of work on the Fc-domains of antibodies has allowed fine tuning of biologic therapies. We are all familiar with optimization of ADCC and CDC activity (up or down), but more recent advances are less widely known. I want to explore two examples – one will bring us back to LN and cDC1 activation, the other will advance the discussion on myeloid cell activation and will introduce the interaction of myeloid cells and neutrophils as a novel component of the anti-cancer immune response.
Jeffrey Ravitch’s lab recently published a method for Fc engineering of IgG antibodies for selective high-affinity binding to the activating Fcγ receptor FcγRIIa (paper). In a viral respiratory model (in mice having human FcγRs) this Fc-hack resulted in an enhanced ability to prevent or treat lethal viral respiratory infection, with increased maturation of dendritic cells and the induction of anti-viral CD8+ T cell responses. Specifically, they noted up-regulation of CD40 expression in the cDC1 subset—the dendritic cell population specialized for cross-presentation and CD8 T cell stimulation in the lung virus model, and the very same DC subset we discussed earlier in the context of TLS and LN-mediated anti-tumor responses. Just to close the circle, Fumito Ito and colleagues used irradiation, Flt3L, TLR and CD40 stimulation to demonstrate cDC1 induction of stem-cell line CD8+ T cells in a variety of murine tumor models (linked here). It follows that engineering antibodies with the selectivity demonstrated in the Ravetch paper will find utility in the anti-tumor field.
I started off by referencing presentations from Ron Germain and Michael Dustin at SITC 2019, over a year ago. Dr Germain presented a story that really struck a chord for me (see Uderhardt et al. 2019). In tissue injury and pathogen infection models, neutrophils comprise the first line of defense, as innate immune signals cause them to swarm at the affected site. Early infiltrating neutrophils undergo activation induced cell death, which can drastically amplify the response and potentially cause tissue damage. In order to terminate this potent immune response tissue-resident macrophages rapidly sense neutrophil activity and cell death and extend membrane processes to limit the damage. This ‘‘cloaking’’ mechanism thus limits neutrophil activation. Of note, neutrophils can be abundant in tumors where they have been linked to diverse activities ranging from potent anti-tumor immunity to immune-suppression. Neutrophils, like myeloid cells and NK cells, can be hacked using Fc-receptor engagement. Neutrophils express FcγRIIA, just discussed in the context of cDC1 activation, and therefore it will be interesting to examine the activation of these (and other the FcγRIIA-expressing cells) in the context of IgG Fc-engineering. Neutrophils and myeloid cells also express FcαRI, a very interesting receptor that when engaged by IgA-isotype antibodies triggers targeted cell killing. Neutrophils will engage in phagocytosis, degranulation and reactive oxygen production to mediate killing after FcαRI engagement, while myeloid cells will be triggered to engulf targeted cells. The specific responses induced depend on the valency of IgA (monomeric, dimeric, aggregated) but it seems likely that the Fc-domain can be hacked in order to optimize productive engagement. With a recent spotlight shown on IgM as an Fc-engaging platform (see IgM Biosciences) we can anticipate accelerated drug development across all of these diverse Ig-classes.
To wrap up – as we move forward in the related disciplines of immuno-oncology and cell therapy, we should consider these principles: optimizing T cell/DC interactions, localizing immune checkpoint therapy to lymphoid tissues, and engaging additional cells to bring the full power of the immune system to the anti-tumor battle.