Category Archives: oncology

A few things I Iearned in 2020: an immune-oncology perspective

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.

Stay tuned.

T cell fitness and genetic engineering

This is a subject we have been thinking about in great detail and this publication in Cell was a trigger for me to start organizing those thoughts. Here is the full reference to the paper discussed: In press, Roth et al., Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies, Cell (2020).

My thanks to Mark Paris from Daiichi Sankyo for his tip to read this paper.

Screen Shot 2020-05-04 at 9.01.39 AM

This publication (https://doi.org/10.1016/j.cell.2020.03.039) is by Theodore Roth and colleagues from Alexander Marson’s lab at UCSF.  They present a nice technological advance, the development of a process by which a pool of genes are knocked into a locus, allowing one to examine the consequence of altering the responsiveness of a cell, in this case, a T cell. This type of work springs from a long lineage of genetic manipulation strategies, from random mutagenesis, to random then targeted gene knockouts (in cells and animals) and gene knockins (what we once called transgenics) and elegant gene-editing technologies (gene therapy, CRISPR/Cas-9, cell therapy, gene-delivery) and so on.

The focus in this paper is on optimizing T cell activity in the setting of solid tumors, something we think about every waking hour at Aleta Biotherapeutics (www.aletabio.com). So, let’s see what we’ve got here.

The pooled knockin strategy relies on two key elements – DNA barcoding, a well-developed technology that has its roots in high throughput library screening technologies, and locus targeting via HDR, which can be achieved using CRISPR/Cas9 and guide templates. Put these two things together and you now have the ability to mix and match genes of interest (following these via their specific barcodes) and place then into the desired locus – here that locus is the TRAC (the TCR locus). They also knocked in a defined TCR (for NY-ESO-1). So, this is a nice system with a known TCR and various immune modifications. There are some limitations. Only 2000-3000 base pairs will fit into the targeting vector (here using a non-viral method). It appears that only a fraction of the targeted T cells are functionally transfected (around 15% per Figure C and note that not every knocked-in cell has both the TCR and the extra gene). The expression level in primary human T cells is high, but I’m guessing expression is of limited duration (although at least 10 days, Figure S5). This is used here as a screening tool, where the goal is to identify critical pathways that reduce or enhance T cell activities (proliferation, effector function, release from immunosuppression).

The authors used a pooling approach to introduce one or two coding sequences from a short list of proteins implicated in T cell biology. Some sequences were modified to be dominant-negative or to be “switch receptors”, where the extracellular domain of the receptor is coupled to a T cell-relevant signaling component (eg. FAS-CD28, TGFβRII-4-1BB). Here are the components they used for their library:

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As we can see from the list there are interesting immune checkpoints, death receptors, cytokine receptors and signaling components that can be mixed and matched. The pool is made and transfected into primary T cells that are then put under selective pressure. The T cells that are enriched under that selective pressure are then analyzed by barcode sequencing to see who the “winners” are, as shown in this schematic from Figure 1A:

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The first screen was simple TCR stimulation (anti-CD3/anti-CD28) which rather robustly showed that a FAS truncation allowed for better cell proliferation (Figure 3B in the paper). This is an expected result – activated T cells undergo FAS-mediated cell death (activation-induced cell death, AICD) that is triggered by FAS-ligand expression, ie. activated T cells kill each other using this pathway. Since there are only T cells in this TCR stimulation culture a lot of other pathways are rendered irrelevant and therefore don’t appear (PD-1 for example):

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The key data are on the far right, showing a 2-4 fold increase in T cell number relative to input. The knockins in light blue showed a statistically meaningful increase vs. input number, across 4 different donor T cells (each circle is a different donor).

The second selective pressure was to stimulate the T cells in the presence of soluble TGFβ (see Figure 3D). As one might guess, the TGFβRII dominant-negative (dn) and switch receptors now come into play: TGFβRII-MyD88, TGFβRII-4-1BB, TGFβRII-dn. The FAS-dn and switch receptors are also represented as are two T cell proliferative components: the IL2RA and TCF-7 (aka TCF-1). These latter hits suggest that amping up T cell proliferation can allow the pool to outrun TGFβ-mediated immunosuppression, at least in vitro. Again, refer to Figure 3D in the paper for the results.

Several other selective pressures were applied in vitro, including tumor cytotoxicity using the NY-ESO-expressing melanoma cell line A375. Of more interest, the A375 cell line was used to establish a xenograft tumor in immunodeficient NSG mice, and the knockin pools of transfected T cells were injected into the mice after the tumor had established. A technical note here – 10 million T cells were injected, of which approximately 1 million were transfected – and 5 days later the tumors were removed and the TIL (tumor-infiltrating lymphocytes) were isolated by screening for the TCR. Bar-code analysis of the TCR-positive TIL allowed the team to identify which transfected T cells got in and expanded. This is tricky, because you’ve allowed time for extensive proliferation (so T cells that are dividing quickly will dominate) and you don’t know what you lost when the T cell pool encountered NY-ESO-positive tumor cells (did some die or did some traffic out of the tumor?). We should expect these data to be noisy and they are, but clear “winners” emerge, namely the TCF-7 transfectants, the TGFβRII-dn, and TGFβRII switch receptors with 4-1BB and also with the TLR signaling component MyD88. Since A375 melanoma cells do not make TGFβ (as far as I know) we have to assume that the T cells themselves are making this, and this is the TGFβ that is triggering these potent (NF-κB triggering) signaling components.

The TGFβRII-dn and switch receptors supported increased IL-2 and IFNγ production – note that IFNγ should have induced PD-L1 on the melanoma cells, but none of the PD-1 based cassettes had any notable effect (from Figure 6B):

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As with the PD-1 pathway, neither the FAS switch receptors nor the FAS-dn construct seemed to play a role in this setting. It’s not clear if FAS-L was upregulated in the tumor model, so that might explain the result.

There was a stark difference in T cell phenotype induced by TCF-7 versus the TGFβRII synthetic constructs. They are in fact polar opposites in some ways (CCR7 expression, Granzyme B expression, IFNγ expression – see Figure 6 E in the paper). Finally, the authors made a bona fide, polycistronic, TCR construct expressing the TGFβRII-4-1BB cassette or the TCF-7 sequence, used this to transduce donor T cells and then tested these for anti-tumor efficacy in vivo (Figure 7). T cells expressing the NY-ESO TCR and the TGFβRII-4-1BB cassette were able to clear the tumor completely. So that’s a very nice result.

Let’s put this into broader context. The table below is a small representation of the literature on genes associated with T cell anti-tumor responses, presented in no particular order. In the left column is the technology used to do the work, then the target, the result, the DOI if you want to read more and then some notes where applicable. I left off a lot of papers, my apologies to those labs.

Technology Target Result notes Reference
dominant-negative transgene FAS increased T cell persistence  and anti-tumor activity 10.1172/JCI121491
transgene overexpression c-Jun reversed tonic-signal induced exhaustion in T cells AP-1 driven 10.1038/s41586-019-1805-z
knockout  Reginase-1 increased T cell persistence, fitness, and anti-tumor activity > Batf and < PTPN2, SOCS1 10.1038/s41586-019-1821-z
knockout PTPN2 increased Lck, STAT5 signaling, and anti-tumor responses multiple papers 10.15252/embj.2019103637
disruption by random integration TET-2 improved CAR-CD19 clinical outcome   10.1172/JCI130144
CRISPR screen (CD8) Dhx37 increased tumor infiltration and effector function multiple papers 10.1016/j.cell.2019.07.044
dominant-negative transgene TGFβRII increased T cell proliferation, effector function, persistence, and anti-tumor activity multiple papers 10.1016/j.ymthe.2018.05.003
integration site association TGFβRII associated with positive clinical outcomes many other sites also identified 10.1172/JCI130144
pooled shRNA screen PP2r2d increased TCR activation, cytokine secretion, T cell trafficking into tumor   10.1038/nature12988
knockout NR4a complex increased CD8 effector T cell function and solid tumor control linked to Nf-kB, AP-1 activity, multiple papers 10.1038/s41586-019-0985-x
T cell profiling Tcf1/TCF-7 increased T cell stemness and anti-tumor activity (with anti-PD-1) multiple papers 10.1016/j.immuni.2018.11.014

I won’t go through all these but there are a few things to note here. One is the appearance of the three pathways we just discussed in the context of the pooled KI paper: FAS, TGFβRII and TCF-7. As mentioned earlier the FAS/FAS-L connection to AICD has been known for a long time, and that information has already been exploited in the context of CAR T cell engineering. Elaboration of the roles of TGFβ in mediating tumor resistance to immune therapy is a more recent advance, but now well established. As noted above I think one interesting question raised by this paper is the source of the TGFβ in the in vitro and in vivo tumor models. I’ve assumed this is T cell derived and understanding the trigger for TGFβ activation in these settings would be very interesting. The role of Tcf1 (aka TCF-7) in anti-tumor immunity has recently been explored in detail in the context of T cell “stemness” leading to the hypothesis that anti-PD-(L)-1 therapeutics work by releasing these T cell with stem-like properties, and allowing their maturity into effector T cell populations (see 10.1016/j.immuni.2018.12.021 and 10.1016/j.immuni.2018.11.014 for examples). It seems that in this knockin, enforcing TCF-7 (Tcf1) expression locked the T cells into a sort of limbo, proliferating, homing into the tumor, but failing to mature into effector cells with anti-tumor functions. A very interesting result. Development of a model in which canonical PD-1/PD-L1 immunosuppressive biology could be examined in order to probe for synergies would be a welcome next step.

Finally, word or two about some of the other targets. As shown in the paper, and as recently shown in the clinical setting (10.1126/science.aba7365), knockins are, at this time, an imperfect tool. Some of the targets listed in the table are associated with autoimmunity (eg. PTPN2) or T cell leukemia (eg. c-Jun, NR4a) and so care is needed when exploiting these targets. Safely engineering specific targets for improved cellular therapeutics will be an important advance on the road to durable and curative solid tumor therapy.

Stay tuned.

THE NEXT GREAT DRUG HUNT: Integrins, TGF-beta and Drug Development in Oncology and Fibrosis

PART 1: Integrin αvβ8

Advances in our understanding of the regulation and function of TGF-β is driving novel drug development for the treatment of diverse diseases. This is a field I’ve followed for a long time and of course in the development of cell therapeutics we (www.aletabio.com) always have an eye on immunosuppressive pathways – indeed, the immunotherapy and cell therapy fields cross-fertilize often and productively (see http://www.sugarconebiotech.com/?m=202002).

Several new papers in this space have caught my eye and I’m keen to share some key findings. This will be a multi-part post and today I want to talk about an integrin.

Long time readers will appreciate the importance of alpha v-integrin-mediated regulation of TGF-β release from the latent complex (http://www.sugarconebiotech.com/?p=1073). The model that first emerged around 2010 was elegant: various signaling pathways triggered GPCRs that could activate an integrin beta strand (paired with an alpha v integrin) and coordinate the release of TGF-β from the cell surface. Soluble TGF-β, free from restraint, could diffuse across nearby cells and trigger TGF-β-receptor activation. Three integrins have been linked to the regulation of TGF-β release: αvβ6, αvβ8 and αvβ3. The mechanism for releasing TGF-β from the latent complex on the cell surface requires a conformation change in the integrin structure. From this insight emerged diverse drug development efforts targeting specific integrins, targeting the ligands for specific GPCRs and so on. Notable examples include the anti-αvβ6 antibody STX-100 (Biogen), the autotaxin inhibitor GLPG1690 (Galapagos), small molecule inhibitors designed to block integrin conformational change, and isoform-specific anti-TGF-β biologics, among many others. The mechanism of action of these drugs includes reduction of free, active TGF-β and therefore reduced TGF-β-receptor signaling. STX-100 was withdrawn from clinical development due to toxicity – more on this another time. GLPG1690 is now in a Phase III trial (in IPF) having shown anti-fibrotic activity in earlier clinical trials – this drug has had an interesting life, originally partnered by Galapagos with Johnson & Johnson, later returned, and now part of the mega-partnership with Gilead. I’ve previously discussed these and many other drugs in development in the context of fibrosis pathogenesis (http://www.sugarconebiotech.com/?p=1073). We’ll look at novel TGF-β-directed antagonists and their role in immune-oncology in part 2, as part of a long-running thread (http://www.sugarconebiotech.com/?m=201811).

So back to integrins. The dogma that emerged based on work from disparate labs was that an activated integrin was required to release TGF-β from the latent (inactive) complex on cell surfaces, allowing for precise regulation of TGF-β activity. More specifically, this model refers to the release of two of the three isoforms of TGF-β – isoforms 1 and 3. Isoform 2 regulation is different and relies on physical force acting directly on cells to trigger release. Of note, isoform 2 antagonism contributes to the toxicity associated with pan-TGF-β blockade but does not appear to contribute significantly to disease pathology either in fibrosis or in oncology. Therefore, specifically antagonizing TGF-β-1/3 without antagonizing TGF-β-2 is ideal – and the model we’ve just outlined allows for this specificity by targeting specific integrins.

The model that alpha v integrins mediated release of free, active TGF-β has held firm for nearly a decade. Now however there is a fascinating update to this model that involves the αvβ8 integrin. Work from the labs of Yifan Cheng and Steve Nishimura at UCSF has revealed a novel mechanism of TGF-β regulation that has interesting implications for drug development (https://doi.org/10.1016/j.cell.2019.12.030). Uniquely, integrin αvβ8 lacks critical intracellular binding domains that allow an integrin to anchor to actin fibers within the cell. As a result, binding to αvβ8 does not cause the release of TGF-β from the latent complex on the cell surface but rather presents an active form of TGF-β on that cell surface, without release from the latent complex. Importantly the complex formed between αvβ8 and TGF-β is conformationally stable and relies (in their experimental system) on trans-interaction between one cell expressing αvβ8 and a second cell expressing TGF-β as displayed on a latent protein complex (here, containing the GARP protein), and expressing the TGF-β receptors. In this system TGF-β remains anchored to the GARP-complex, but the conformational rotation caused by αvβ8 binding allows anchored TGF-βto interact with TGF-β-RII, thereby recruiting TGF-β-RI and inducing signaling.

The focus on GARP (aka LRRC32) relates to this groups long-standing interest with T-regulatory cells, which uniquely express GARP. Biotech investors will recall the Abbvie/Argenx deal on this target, which is in clinical development (https://clinicaltrials.gov/ct2/show/NCT03821935). A related protein called LRRC33 has been discovered on myeloid lineage cells.

More important, in my view, is that αvβ8 is expressed widely on tumor cells and has been variably reported to correlate with metastases (depending on the indication). This suggests that one means that tumor cells have of inducing TGF-β activation on interacting cells (eg. lymphocytes, myeloid cells and perhaps stromal cells) is via αvβ8 activity. The dependent hypothesis would be that such activation is immunosuppressive for those tumor-interacting cells. This is consistent with the known effects of TGF-β on immune cells in particular, but also stromal cells like fibroblasts. As an aside I like this model as one way of accounting for the appearance of T-regulatory cells and myeloid lineage suppressor cells in the tumor microenvironment as result of, rather than the cause of, immunosuppression, that is, these cells may be epi-phenomena of broad TGF-β-mediated immunosuppression. This may in turn explain why targeting such cells as T-regs and MDSCs has been largely unsuccessful to date as a therapeutic strategy for cancer.

There are some other implications. As the authors point put, the integrin/TGF-β complex is stable, and the binding domain that mediates the interaction is buried with the protein complex. It is unclear whether anti-TGF-β antagonists that target the canonical integrin binding cleft would be able to access this site within the complex. It’s possible that some of these drugs (whether antibodies or small molecules) can’t work in this setting. On the other hand, antibodies to αvβ8 clearly prevent the complex from forming and should block TGF-β-mediated immunosuppressive signaling in settings where αvβ8 expression is dominant. An anti-αvβ8 antibody strategy is being pursued by Venn Therapeutics (disclosure: I sit on Venn’s SAB). Further, the structural features identified in the paper include well-defined pockets that might be suitable for small molecule drugs. Indeed, one of the structural features in the b8 protein, consisting of hydrophobic residues, appears to account for the differential binding of various integrins (β6, β1, β2, β4, β7) to TGF-β, a remarkable finding. Analyses of the differences between the structure of β8 and other β integrins has been extensive across laboratories (see https://www.nature.com/articles/s41467-019-13248-5 for another important paper). Small molecule drug discovery is well underway in this field (see for example Pliant Therapeutics and Morphic Therapeutics) and one might imagine that these novel results found an interested audience in many bio-pharma labs.

Next: what has Scholar Rock been up to, and what can we learn from their work?

Stay tuned.

Radical optimism: considering the future of immunotherapy

I wrote recently about the sense of angst taking hold in the next-generation class of immuno-therapeutics – those targets that have come after the anti-CTLA4 and anti-PD-(L)-1 classes, and raised the hope that combination immunotherapy would broadly raise response rates and durability of response across cancer indications.

There are diverse next-generation immuno-therapeutics including those that target T cells, myeloid cells, the tumor stromal cells, innate immune cells and so on. A few examples are given here (and note that only a few programs are listed for each target):

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There are of course many other therapeutic targets – OX40/CD134, Glutaminase, ICOS, TIM-3, LAG-3, TIGIT, RIG-1, the TLRs, various cytokines, NK cell targets, etc.

In the last year – since SITC 2017 – there has been a constant stream of negative results in the next generation immuno-therapy space, with few exceptions. Indeed, each program listed in the table has stumbled in the clinic, with either limited efficacy or no efficacy in the monotherapy setting or the combination therapy setting, typically with an anti-PD-(L)-1 (ie. an anti-PD-1 or an anti-PD-L-1  antibody). This is puzzling since preclinical modeling data (in mouse models and with human cell assays) and in some cases, translation medicine data (eg. target association with incidence, mortality, or clinical response to therapy), suggest that all of these targets should add value to cancer treatment, especially in the combination setting. I’ve discussed the limitations of these types of data sets here, nonetheless the lack of success to date has been startling.

With SITC 2018 coming up in a few days (link) I think it is a good time to step back and ask: “what are we missing?”

One interesting answer comes from the rapidly emerging and evolving view of tumor microenvironments (TME), and the complexity of those microenvironments across cancer indications, within cancer indications and even within individual patient tumors. TME complexity has many layers, starting with the underlying oncogenic drivers of specific tumor types, and the impact of those drivers on tumor immunosuppression. Examples include activation of the Wnt-beta catenin pathway and MYC gain of function mutations, which mediate one form of immune exclusion from the tumor (see below), and T cell immunosuppression, respectively (review). In indications where both pathways can be operative (either together or independently, eg. colorectal cancer, melanoma and many others) it is reasonable to hypothesize that different strategies would be needed for combination immuno-therapy to succeed, thereby producing clinical responses above anti-CTLA4 or anti-PD-(L)-1 antibody treatment alone.

A second and perhaps independent layer of complexity is TME geography, which has been roughly captured by the terms immune infiltrated, immune excluded, and immune desert (review). These TME types are illustrated simply here:

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The different states would appear to be distinct and self-explanatory: there are immune cells in the tumor (infiltrated), or they are pushed to the periphery (excluded), or they are absent (desert). The latter two states are often referred to as “cold” as opposed to the “hot” infiltrated state. It is common now to propose as a therapeutic strategy “turning cold tumors hot”. The problem is that these illustrated states are necessary over-simplifications. Thus, immune infiltration might suggest responsiveness to immune checkpoint therapy with anti-PD-(L)-1 antibodies, and indeed, one biomarker of tumor responsiveness is the presence of CD8+ T cells in the tumor. But in reality, many tumors are infiltrated with T cells that fail to respond to immune checkpoint therapy at all. The immune excluded phenotype, alluded to above with reference to the Wnt-beta catenin pathway, can be driven instead by TGF beta signaling, or other pathways. The immune desert may exist because of active immune exclusion, lack of immune stimulation (eg. MHC-negative tumors) or because of physical barriers to immune infiltration. Therefore, all three states represent diverse biologies within and across tumor types. Further, individual tumors have different immune states in different parts of the tumor, and different tumors within the patient can also have diverse phenotypes.

There are yet other layers of complexity: in the way tumors respond to immune checkpoint therapy (the “resistance” pathways, see below), the degree to which immune cells responding to the tumor cells are “hardwired” (via epigenetic modification), the metabolic composition of the TME, and so on. Simply put, our understanding remains limited. The effect of this limited understanding is evident: if we challenge tumors with a large enough immune attack we can measure a clinical impact – this is what has been achieved, for example, with the anti-PD-(L)-1 class of therapeutics. With a lesser immune attack we can see immune correlates of response (so something happened in the patient that we can measure as a biomarker) but the clinical impact is less. This is what has happened with nearly all next-generation immuno-therapeutics. As a side note, unless biomarker driven strategies are wedded to a deep understanding of specific tumor responsiveness to the therapeutic they can be red herrings - one example may be ICOS expression, although more work is needed there. Understanding specific tumor responsiveness is critical regardless of biomarker use, due to the layered complexity of each indication, and even each patient’s tumors within a given indication.

So why should we be optimistic?

I propose that some of the next generation immuno-therapeutics will have their day, and soon, due to several key drivers: first, for some of these classes, improved drugs are moving through preclinical and early clinical pipelines (eg. A2AR, STING). Second, the massive amount of effort being directed toward understanding the immune status of diverse tumors ought to allow more specific targeting of next generation immuno-therapeutics to more responsive tumor types. The TGF beta signature presents a particularly interesting example. Genentech researchers recently published signatures of response and resistance to atezolizumab (anti-PD-L1) in bladder cancer (link). In bladder cancer about 50% of tumors have an excluded phenotype, and about 25% each have an immune infiltrated or immune desert phenotype. The response rate to treatment with atezolizumab was 23% with a complete response rate of 9% (note that responses did not correlate with PD-L1 expression but did correlate with both tumor mutational burden and a CD8+ T cell signature). Non-responding patients were analyzed for putative resistance pathways. One clear signature of resistance emerged – the TGF beta pathway, but only in those patients whose tumor showed the immune excluded phenotype. The pathway signature was associated with fibroblasts, but not myeloid cells, in multiple tumor types. The T cells were trapped by collagen fibrils produced by the fibroblasts:

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(The image is a screenshot from Dr Turley’s talk at CICON18 last month).

It follows that a combination of a TGF beta inhibitor and a PD-(L)-1 inhibitor for the treatment of bladder and perhaps other cancers should be used in patients whose tumors show the immune excluded TME phenotype, and perhaps also show a fibroblast signature in that exclusion zone. Indeed, in a recent paper, gene expression profiling of melanoma patients was used to demonstrate that a CD8-related gene signature could predict response to immuno-therapy – but only if the TGF beta signature was low (link).

There are other immunotherapy resistance pathways – some we know and some are yet to be discovered. We should eventually be able in future to pair specific pathway targeting drugs to tumors whose profile includes that pathway’s signature – this has been done, retrospectively, with VEGF inhibitors and anti-PD-(L)-1 therapeutics. This will require a more comprehensive analysis of biopsy tissue beyond CD8+ T cell count and PD-1 or PD-L1 expression – perhaps immunohistochemistry and gene transcript profiling – but these are relatively simple technologies to develop, and adaptable for a hospital clinical lab settings. Not every next generation immuno-therapeutic will succeed as the clinical prosecution becomes more targeted, but some certainly will (we might remain hopeful about adenosine pathway inhibitors, STING agonists, and oncolytic virus therapeutics, to name a few examples).

Another driver of success will be cross-talk with other technologies within immuno-oncology – notably cell therapy (eg. CAR-T) and oncolytic virus technologies. We have already seen the successful adaptation of cytokines, 4-1BB signaling, OX40 signaling and other T cell stimulation pathways into CAR T cell designs, and the nascent use of PD-1 and TGF beta signaling domains in cell therapy strategies designed to thwart immuno-suppression (we should note here that CAR T cells, like tumor infiltrating T cells, will face  barriers to activity in different tumor indications). The example of local (and potentially safer) cytokine secretion by engineered CAR-T cells has helped drive the enormous interest in localized cytokine technologies. Most recently, the combination of CAR-T, oncolytic virus and immune checkpoint therapy has shown remarkable preclinical activity.

SITC 2018 – #SITC18 on Twitter – will feature sessions on  immunotherapy resistance and response, the tumor microenvironment, novel cytokines and other therapeutics, cell-based therapies, and lessons from immuno-oncology trials (often, what went wrong). We can expect lots of new information, much of it now focused on understanding how better to deploy the many next generation immuno-therapeutics that have been developed.

So, I would argue that “radical optimism” for next generation immunotherapy and immunotherapy combinations is warranted, despite a year or more of clinical setbacks. Much of the underlying science is sound and it is targeted clinical translation that is often lagging behind. Progress will have to come from sophisticated exploratory endpoint analysis (who responded, and why), sophisticated clinical trial inclusion criteria (who to enroll, and why) and eventually, personalized therapeutic application at the level of the indication and eventually the patient.

In the meantime, stay tuned.

Novel Immunotherapeutic Approaches to the Treatment of Cancer: Drug Development and Clinical Application

Our new immunotherapy book has been published by Springer:

http://www.springer.com/us/book/9783319298252

I want to take a moment to acknowledge the stunning group of authors who made the book a success. I’d also like to promote our fund raising effort in memory of Holbrook Kohrt, to whom the volume is dedicated – 5% of net sales will be donated by me, on behalf of all of our authors, the the Cancer Research Institute in New York. So please consider buying the book or just the chapters you want (they can be purchased individually through the link given above.

Now, the authors:

from Arlene Sharpe and her lab (Harvard Medical School, Boston):

Enhancing the Efficacy of Checkpoint Blockade Through Combination Therapies

from Taylor Schreiber (Pelican Therapeutics, Heat Biologics):

Parallel Costimulation of Effector and Regulatory T Cells by OX40, GITR, TNFRSF25, CD27, and CD137: Implications for Cancer Immunotherapy

from Russell Pachynski (Washington University St Louis) and Holbrook Kohrt (Stanford University Medical Center)

NK Cell Responses in Immunotherapy: Novel Targets and Applications

from Larry Kane and Greg Delgoffe (University of Pittsburgh School of Medicine):

Reversing T Cell Dysfunction for Tumor Immunotherapy

from Josh Brody and Linda Hammerich (Icahn School of Medicine, Mt Sinai, NYC)

Immunomodulation Within a Single Tumor Site to Induce Systemic Antitumor Immunity: In Situ Vaccination for Cancer

From Sheila Ranganath and AnhCo (Cokey) Nguyen (Enumeral Inc, Cambridge MA)

Novel Targets and Their Assessment for Cancer Treatment

From Thomas (TJ) Cradick, CRISPR Therapeutics, Cambridge MA):

Cellular Therapies: Gene Editing and Next-Gen CAR T Cells

From Chris Thanos (Halozyme Inc, San Diego) and myself:

The New Frontier of Antibody Drug Conjugates: Targets, Biology, Chemistry, Payload

and a second topic covered by Chris Thanos (Halozyme):

Targeting the Physicochemical, Cellular, and Immunosuppressive Properties of the Tumor Microenvironment by Depletion of Hyaluronan to Treat Cancer

and finally, my solo chapter (and representing Aleta Biotherapeutics, Natick MA and SugarCone Biotech, Holliston MA):

Novel Immunomodulatory Pathways in the Immunoglobulin Superfamily

Please spread the word that all sales benefit cancer research and more specifically, cancer clinical trial development and execution through the Cancer research Institute, and as I said, consider buying the book, or the chapters you want to read.

cheers-

Paul

International Cancer Immunotherapy Conference, quick take: tumor antigens

The sessions yesterday were dominated by discussions of the role of tumor mutations in driving anti-tumor immunity. Tumor mutations can be abundant or rare depending on the indication, and this has an impact on the utility of anti-immune checkpoint therapeutics, as one example. But the question of tumor immunogenicity – can the immune system “see” the tumor – touches multiple therapeutic modalities, among them cellular therapies (TIL and engineered TCR-T cells) and the tumor vaccine field.

Two themes emerged that were not readily compatible. One theme, elegantly on display in the talk by Dr Rosenberg (NCI), is how rare and unique immune activating tumor mutations actually are, when you query patient tumors (or peripheral blood cells) for T cells that can respond to identified tumor mutations. The biology is complex, involving both CD4+ and CD8+ T cells (and the corresponding antigen recognition machinery) on the one hand and variable HLA haplotypes for peptide expression on the other hand. Only when the peptide/MHC (I or II) complex can be recognized by the TCR on a CD4 or CD8 cell can the T cell productively respond. Dr Rosenberg presented analyses of diverse tumor types, making the argument that tumor mutations that can induce T cells responses (thus tumor neoantigens), are unique across patients even within the same indication. Therefore he reasoned that expanding tumor infiltrating lymphocytes (TIL) derived from tumors (using specific cell surface markers) would give one the best chance of finding the right T cell reactivity – after all, that’s why the T cells are within the tumor. Dr Rosenberg has shown very impressive clinical results obtained exploiting these TIL. Such work may also inform efforts by cellular therapeutic companies that use TIL or TCR technology (Lion Bio, Kite, Juno etc).

A different theme emerged later in the session, focusing not on rare tumor antigens but rather on more common tumor antigens. Talks by Dr Sahin (Univ Mainz and BIONTECH Inc) and Dr Rammensee (Univ Tubingen) fell firmly in this camp, with the effort focussed on methods to identify neoantigens that could serve as vaccine components. Much of this work was preclinical, but also some interesting technology validation and early clinical application. This work has broad implications for the tumor vaccine field and perhaps the cell therapeutic modalities, as mentioned above.

So, these two themes clash over the concept the tumor neoantigens are either rare, or more common. This is a puzzle. As always, the details matter. In discussion over dinner, Taylor Schreiber, Anil Goyal and George Fromm from Heat Biologics and Amit Chaudhuri from Medgenome offered possible reasons for the discrepancies:

1) Cell selection – different methods were used to identify the specific populations of T cells to study

2) Antigen analysis – different methods were used to characterize tumor mutations and putative tumor neoantigens

3) Different algorithms – some bioinformatics tools may miss some mutations based on how they distinguish signal from noise (the cancer/testis families were offered as an example here)

So, time to go back and reread the literature.

stay tuned

SugarCone Biotech will be at the International Cancer Immunotherapy Conference: Translating Science into Survival 2015

Paul Rennert, Founder & Principal of SugarCone Biotech LLC, will be attending the joint CRI/AACR event: International Cancer Immunotherapy Conference: Translating Science into Survival 2015. SugarCone Biotech LLC is a consulting firm specializing in biotech strategy and investment.

The conference is in NYC Sept 16-19 and promises to be the key immunotherapy meeting of the fall conference season. Please reach out to connect with Paul at rennertp@sugarconebiotech.com.

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SugarCone Biotech will be at Sach’s Biopartnering 2015

Sach’s is a premier international biopartnering event, a highly focused 2-day conference spanning multiple disciplines and therapeutic modalities. Paul Rennert, Founder & Principal of SugarCone Biotech LLC, will be in attendance, to introduce novel programs and companies to interested investors, and conversely, to connect up and coming biotechs and academic spin-outs to the investment community. Please reach out to Paul during the conference using the one-on-one meeting app or via rennertp@sugarconebiotech.com.

We hope to see you there. Here is the conference web site:

http://www.sachsforum.com/15th-annual-biotech-in-europe-forum-for-global-partnering–investment-29-30-september-2015-congress-center-basel.html

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“Combination Cancer Immunotherapy and New Immunomodulatory Targets” published in Nature Reviews Drug Discovery

Part of the Article Series from Nature Reviews Drug Discovery, our paper hit the press today

Combination cancer immunotherapy and new immunomodulatory targets. Nature Reviews Drug Discovery 14, 561–584. 2015.  doi:10.1038/nrd4591

by Kathleen Mahoney, Paul Rennert, Gordon Freeman.

a prepublication version is available here: nrd4591 (1)

#ASCO15

ASCO 2015 is just weeks away and it’s time to get the calendar straightened out. This will be a busy and exciting meeting, and chaotic as always.

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If you would like to meet at ASCO please contact me ASAP at rennertp@sugarconebiotech.com. I love discussing what we do, how we do it, and will happily explain our outstanding breadth of expertise and remarkably synergistic interactions with the biotech, pharma and investor communities.