We’d been hearing the rumors for months. But the simultaneous publication in Nature of three papers describing a critical role for lymphoid structures and B cells in supporting T cell anti-tumor immunity was a remarkable milestone in our evolving understanding of immuno-oncology. Really stunning work. Importantly, these papers fit into a new contextual framework and cap a series of studies that have come out over the last year or so that have enriched our understanding of how the immune system and tumor cell populations interact. This broader and still evolving contextual framework will impact immunotherapy drug development across the immune checkpoint field, the tumor vaccine space, innate immune approaches, the T-cell-directed biologics, and cellular therapies.
But first, these new papers are gorgeous:
The study presented by Petitprez et al. is focused on the response of sarcomas to immunotherapy (https://www.nature.com/articles/s41586-019-1906-8). The soft tissue sarcomas (STS) have mixed clinical responses to immune checkpoint blockade (ICB) treatment, and it is not clear what drives the variable response. In general, STS have been classified as having a low tumor mutational burden (TMB) and are considered non-immunogenic, or ‘cold’, and have little expression of PD-L1. A few STS subtypes are characterized by more complex genetic abnormalities and could potentially have more actionable mutations for the immune system to recognize. Regardless, two of most widely used biomarkers of ICB response (TMB-high or PD-L1-positive) are not generally relevant in STS. In this study, gene expression profiling was used to examine patterns of ICB response in patients across a wide variety of STS subtypes and pathologies. Three distinct genetic classes were identified that match known tumor microenvironments (TME) – immune desert (A), highly vascular (C), and inflamed (E) with two intermediates: B and D. These are well understood classifications and mirror many prior studies of the TME and ICB response and resistance. However, several details that emerged are critical – 1) the inflamed TME (E) and the intermediate form (D) were not associated with any particular STS classification, but were distributed across STS histologies, and 2) the E/D inflamed signature was characterized by a pronounced B cell signature, and by expression of the chemokine CXCL13. These results suggest secondary lymphoid organ development and organization.
A sidebar here: secondary lymphoid organs include spleen, lymph nodes, and Peyers patches and are characterized by critical structural features that include a T cell zone and adjacent B cell follicles that orchestrate coordinated immune responses, for example, to pathogens. Localized lymphoid organs can form in chronically inflamed tissues – these are the tertiary lymphoid structures (TLS) and are classically associated with prolonged inflammation and autoimmunity. The organization of cell types in these structures is controlled in part by chemokines, including CXCL13 and CXCR5.
Using immunohistochemical staining, the authors went searching for TLS in tumor sections, and, as suggested by the gene transcript data, TLS were found in groups E and a bit in D. The presence of TLS in tumors has been noted before, but here the presence of TLS, and of B cells, was associated with patient overall survival. Further in a small cohort of patients (n=47), classes E and D, those most likely to have TLS, responded best to ICB therapy. These observations suggest that B cells and TLS are important for successful ICB therapy.
OK, that’s one study, in an indication in which ICB therapy in general has not worked well. So, are these observations generalizable?
The next paper looked at these features in an indication that is very different from STS. The paper by Cabrita et al. focused on ICB response in melanoma (https://www.nature.com/articles/s41586-019-1914-8). Melanoma is notable for several features, having a very high TMB and being among the most ICB responsive indications. Indeed, in terms of immune-responsive tumor types, melanoma and STS are on the opposite ends of the spectrum. Nonetheless, the analysis of melanoma responsiveness to ICB yielded results strikingly similar to the results of the STS analysis.
Immunofluorescent staining was used to identify T and B cell clusters, and these were associated with the chemokines CXCL13 and CXCR5. The co-occurrence of T and B cells and the identification of a TLS gene signature predicted clinical response to ICB. Mechanistically, tumors that featured TLS and were rich in B cells also had an increased population of naive and/or memory T cells while those tumors without these features had an increased population of exhausted T cells. Notably, the T cell population enriched in the presence of TLS was CD8-positive – the subset associated with cytotoxic anti-tumor immunity. Whether the B cells themselves were also contributing directly to the anti-tumor response via production of anti-tumor antibodies appears less clear. As in the STS study, TMB was not correlated with TLS formation in melanoma.
The final study by Helmink et al. analyzed patients who had enrolled in a phase 2 clinical trial of neoadjuvant ICB therapy for high-risk resectable melanoma (https://www.nature.com/articles/s41586-019-1922-8). In the neoadjuvant setting, ICB therapy is given prior to the surgery that is performed to remove tumors. Gene expression and immune-staining analyses were used to assess TLS and B cell presence in tumors, similar to the prior studies. Of note the results were compared to an analysis of gene signatures in a neoadjuvant trial of ICB treatment of metastatic renal cell carcinoma (RCC). As in the other two studies the presence of B cells and TLS in the tumor prior to therapy was predictive of response to ICB. This was found in patients with metastatic melanoma and in patients with metastatic RCC. In addition, the authors showed that the B cell pool was differentiated, into memory B cells and plasma cells, suggesting that the TLS was productively driving both T and B cell differentiation and perhaps indicating a role for B cell adaptive memory in supporting the anti-tumor immune response.
In these three papers covering three indications (STS, melanoma, RCC), a signature of TLS enriched in B cells was associated with ICB responses and T cell activity, notably of CD8-positive T cells. A useful model for these findings is that organized lymphoid structures like TLS provide an environment in which tumor antigen can be productively displayed to the adaptive immune system, ie. T and B cell mediated immunity. In this setting B cells may have such a strong predictive signature for several reasons: B cells are antigen-presenting cells capable of supporting a persistent T cell response by restimulating memory T cells and by triggering de novo naive T cell responses to tumor antigens. Further, B cells engage in the productive costimulation of T cells via B7/CD28, CD40L/CD40 and adhesion molecule interactions and also produce cytokines that provide activation, proliferation and survival signals to T cells (eg. TNF, IL-2, IL-6, IFNy).
Additional productive areas of investigation would include analyses of dendritic cell populations and, of critical importance I think, the status of tumor-draining lymph nodes (see https://science.sciencemag.org/content/367/6477/eaax0182).
These studies raise some interesting questions. For example, in the context of anti-PD-(L)-1 therapeutics, what is the pattern of PD-L1 expression that accounts for the anti-tumor response? In the current paradigm, PD-L1 expression on the tumor itself is considered the critical target. This has recently been complicated by the finding that PD-L1 expression on myeloid cells in the TME may also be relevant (eg. https://immunology.sciencemag.org/content/5/43/eaay1863/tab-e-letters). And we must recall that PD-1/PD-L1 interaction in lymphoid organ germinal centers regulates T cell / B cell interactions (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2874069/).
It may be that ICB treatment is influencing the anti-tumor immune response in multiple ways.
We’ve seen many novel immunotherapy agents falter and some of those results may reflect this more complex immune-tumor landscape. This leads one to wonder where and how novel agents might function and if they are actually beneficial to anti-tumor immunity. As one example, many PD-L1-based bispecific antibodies are under development. We might hypothesize that an anti-PD-L1/anti-4-1BB bispecific antibody would actually have multiple sites of action, not only in the tumor TME, but also in the TLS and draining lymph nodes. As another example, T cell engagers (BiTEs, DARTs et al), a design that works well in the hematologic cancers, may not work as well in solid tumors unless those tumors have TLS already. Finally, it’s a bit baffling that TMB does not correlate with TLS formation, and we might wonder how this result reflects upon efforts in the neoantigen space. And so on, as we think about tumor vaccines, and innate immune stimulation, and novel checkpoints, etc.
This brings us also to cell therapy and potential lessons for that field, specifically in the solid tumor setting. We have long recognized that CAR T cells that target CD19 (CD19-CARs, eg. Kymriah from Novartis and Yescarta from Kite/Gilead) have dramatically better persistence properties than CARs that target solid tumor antigens. We have hypothesized that the self-renewing nature of the antigen itself is an important aspect of persistence – CD19 is expressed on B cells that are continuously produced by the bone marrow.
In light of these new findings on ICB responses I think we can consider a second feature – the immunological relevance of antigen presentation to the CAR T cell. We have recently seen a rash of efforts to provide artificial and immunologically favorable antigen presentation to CAR T cells. The goal here is to improve CAR T cell fitness by providing the proper immunological niche (eg. a lymph node) and driving persistence. In one example an artificial CAR T ligand was developed that could be injected into a patient who is receiving a CAR T therapy (https://science.sciencemag.org/content/365/6449/162). This artificial CAR-T ligand binds to serum albumin, traffics into lymph nodes and is taken up and displayed by antigen-presenting cells. CAR-T cells trafficking through the lymph node are stimulated both by the displayed antigen and by costimulatory receptors and cytokines, much like the system envisioned in the ICB response/TLS papers described above. Similarly, a nanoparticulate RNA vaccine was used to deliver and express an artificial CAR antigen into a tumor-draining lymph node (https://science.sciencemag.org/content/367/6476/446). Again, this is designed to promote immunologically productive presentation of the target antigen to activate and expand the injected CAR-T cells. Notably, engagement of relevant stimulatory, chemokine and adhesion signals are known to favor the development of T cell memory, a critical element in long term immune protection.
Our in-house technology (www.aletabio.com) uses CD19-targeting by CAR-T cells to leverage persistence and also to take advantage of immunologically relevant antigen presentation. We do this by building CAR T cells to CD19 that simultaneously can target any antigen of interest. We enabled this ‘repurposing’ of CD19-CAR T cells by creating small, highly potent proteins that bridge the CD19-CAR T cell to the tumor antigens we choose, triggering T cell cytotoxicity and killing the tumor cell (https://www.ncbi.nlm.nih.gov/pubmed/31242389). Every bridging protein contains the CD19 protein, so is the target of any CD19-CAR T cell. This bridging protein strategy enables facile multi-antigen targeting because the design is highly modular. We have built CD19-CAR T cells that secrete bridging proteins that recognize CD20, Clec12a, Her2, EGFR and many other different antigens and in some instances multiple antigens simultaneously. Because the CAR T cell is a CAR to CD19, this is a simple, pragmatic and universal solution. And because the bridging protein is secreted by the CD19-CAR T cells themselves, this becomes a simple matter of encoding everything into a lentiviral vector.
CD19-CAR T cell interaction with normal B cells will support production of immunologically relevant stimulatory signals including adhesion interactions, chemokine and cytokine signals, and costimulatory signals, even if the consequence for the B cell is cytotoxic. This organic presentation of immunologically relevant antigen does not require administration of additional agents or exogenous antigen, since B cells are themselves antigen presenting cells, are present in lymphoid organs and in circulation, and represent a self-renewing source of CD19 due to production by the bone marrow.
In the context of solid tumor treatment creating an expanded and persisting CAR T cell pool using CD19-positive B cells as a non-tumor dependent source of antigen becomes very attractive. Our lead solid tumor program uses a CD19-anti-Her2 bridging protein, where the CD19 portion is the CD19 extracellular domain, and the anti-Her2 portion is an anti-Her2 scFv. This bridging protein is encoded into a lentiviral vector downstream of a CD19-CAR sequence, separated by a P2A cleavage site. Thus we have made a ‘Her2-bridging CD19-CAR’ that can bind directly to CD19 on B cells via the CAR domain, and to CD19 painted onto a Her2-positive solid tumor cell, via the bridging protein.
Our lead indication is Her2-positive metastatic breast cancer and metastatic lung cancer, specifically in patients who have relapsed from standard of care therapy by developing CNS metastases. The reasoning is simple: the Her2-bridging CD19-CAR T cells can be injected systemically to become activated by CD19 expressed on normal B cells (and B cell aplasia is a manageable toxicity). The activated CARs will traffic systemically, all the while secreting the small (and short half-life) bridging proteins. Patients can remain on standard of care, including with anti-Her2 antibodies, because we will not need to ‘see’ Her2 in the periphery in order to trigger CAR activation and expansion. And of course, anti-Her2 antibodies like Herceptin can’t cross the blood brain barrier and get into the CNS at all. Once activated CD19-CAR T cells will enter the CNS, as activated T cells are known to do, and the secreted bridging proteins will coat Her2-positive tumor cells with CD19, allowing the CD19-CAR T cells to the attack and kill those cells. Those CAR T cells can also leave, recirculate, become stimulated again by encounter with B cells, and return to the CNS – a trick no direct CAR to Her2 can hope to duplicate. Not to oversell it, but I love this program, and it should work.
The idea that we can repurpose and send off a CD19-CAR T cell to attack any antigen and indication is compelling in its simplicity and modularity. We have already built programs for treating AML and B cell tumors. Our next wave of programs takes advantage of our ability to weave together bridging proteins with two and three antigen-binding domains – and this allows us to contemplate attacking very heterogeneous tumor types with a simple CAR – the CD19-CAR – that has exemplary persistence and fitness characteristics.