Last month’s Immunotherapy and the Basis of Neonatigen Biology blog post focused on neoantigens along with some facets of immunology and cancer that are interrelated. We described in detail how neoantigens and other cancer antigens are generated and classified. We also detailed how neoantigens are recognized by T-cells of the immune system. The post concluded by describing some of the mechanisms by which cancers can differ in their immune nature and how they might evade immune response. With this follow-on blog we are detailing current immunotherapies that leverage neoantigens and other aspects of immune biology to treat various cancers. For a thorough review please consult the Marshall and Djamgoz publication (2018).
Immune checkpoint blockade therapy
As previously discussed in our Immunotherapy and Neoantigen Biology post, one of the most exciting and truly revolutionary recent developments in cancer treatment has been the development of the immune checkpoint inhibitory antibody drugs, also known as immune checkpoint blockade (ICB). Rather than targeting a specific mutation of a particular cancer, these drugs seek to unlock the latent abilities still existing in the patient’s immune system to fight cancer.
- CTLA -4
The cytotoxic T-lymphocyte associated protein 4 (CTLA-4) is expressed on the surface of T-cells, especially regulatory T-cells (Tregs), which in turn are responsible for suppressing the activity of effector T-cells in order to regulate the immune system from reacting too strongly and inflaming healthy tissue (Farkhona et al., 2016). Its binding partners are the CD80 (B7-1) or CD86 (B7-2) proteins normally present on antigen presenting cells (APC). These markers are also present on some cancer cells and in those instances contribute to immune suppression and evasion by tumors. CTLA-4 normally plays its role early on in T-cell maturation when the cells are still present in lymphoid tissue. The antibody drug ipilimumab (Yervoy by Bristol-Myers-Squibb, first FDA approved in 2011) works by binding the CTLA-4 receptor preventing its activity and ultimately depleting the Treg cell population, thus relieving negative regulation of effector T-cells in the tumor microenvironment (TME). Although only a minority of patients receiving ipilimumab achieve a significant response, those that do can experience an extremely durable result (Khalil et al., 2016). Unfortunately, one key drawback of ipilimumab has been the relatively common occurrence of immune related adverse events (irAE) as a side-effect of treatment. This is thought to be due to the central and early role in immune checkpoint progression that CTLA-4 plays. Optimized dosing strategies are employed in the clinic to reduce the severity of these adverse events.
Another inhibitory receptor programmed cell-death protein 1 (PD-1), is expressed on the surface of antigen-stimulated T-cells (Farkhona et al., 2016). Its two normal cognate binding proteins are programmed cell-death protein ligand 1 or 2 (PD-L1 or PD-L2) expressed on APC cells. PD-1 signaling inhibits T-cell proliferation and immune function including cytotoxicity necessary for anti-tumor activity. Tumors can, in many instances, express PDL1 to evade immune recognition. Antibody drugs nivolumab (Cyramza by Eli Lilly) and pembrolizumab (Keytruda by Merck) – both first approved in 2014 – act by binding and blocking activation of PD-1 thus preventing the shutdown of T-cells. Also working on the PD-1/PD-L1 axis is the antibody drug atezolizumab (Tecentriq by Genentech, first FDA approval 2016) which binds PD-L1 to prevent T-cell downregulation.
Image credit: OncoPrescribe
Overall PD-1 blockade has proven to be very effective in the clinic, notably becoming the new standard of care in certain tumor types. Generally these anti PD-1 directed therapeutics lead to higher response rates and lower irAE rates when compared to anti CTLA-4 antibodies, but as with CTLA-4 not all patients respond and side-effects are still a concern. More recently combination cancer immunotherapies such as ipilimumab + nivolumab have been approved by the FDA (2015) by demonstrating higher efficacy and lower side effects than the single agents. It appears that dual CTLA-4/PD-1 blockade is becoming a ‘backbone’ treatment for many cancers (Khalil et al., 2016).
- Other checkpoint receptors
There are several other inhibitory receptors that are part of the larger immune checkpoint mechanism that are now being studied as potential immunotherapy drug targets. Lymphocyte activation gene 3 protein (LAG3), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), T-cell Ig and mucin-domain containing-3 (TIM-3) and V-domain Ig suppressor of T-cell activation (VISTA) fall within this class of viable candidates for the next generation of ICB therapies (Marshall and Djamgoz, 2018, Khalil et al., 2016) and some are currently under intense clinical evaluation both as mono- as well as combination therapy in various indications.
- Co-stimulatory receptors
While there are immune receptors that negatively regulate T-cell activity there are also a host of co-stimulatory receptors that can up-regulate T-cell activity. These include, but are not limited to, receptors 4-1BB (aka CD137 or TNFRSF9), glucocorticoid-induced TNFR family-related protein (GITR) and OX40. Investigations into agonist monoclonal antibodies (mAbs) for potential cancer therapy are relatively advanced for 4-1BB and OX40 (Marshall and Djamgoz, 2018). In a recent report an agonistic antibody to 4-1BB was able to generate significant anti-tumor response in a lymphoma mouse disease model (McKee et al., 2017). Interestingly, this effect was diminished when combined with PD-1 blockade though it is possible that the specific treatment regimen (timing and sequential dosing) might have negatively affected a combination benefit as demonstrated by other studies (Messenheimer et al., 2017). Similarly, an agonist mAb to OX40 was shown to be modulated in its efficacy in mouse tumor models in combination with anti PD-1 by the chosen drug administration strategy (concurrent vs. sequential) (Messenheimer et al., 2017). These examples highlight the complexity of combining even just two immunotherapeutic treatments and the need for an extremely rigorous testing framework which has become a general theme in the field (Morrissey et al., 2016). In particular, efforts are underway to combine even more targeted activities against checkpoint inhibitor (tri-specific, multi-specific) in the context of new and smaller antibody fragment-like modalities. As the clinical success of dual CTLA-4/PD-1 blockade has shown, there is potential increased power and effectiveness in combinations of multiple immunotherapeutic agents that work via different/complementary mechanisms.
As we discussed above and previously in part I of this blog, neoantigens are capable of being targeted by the T-cell based immune system. In addition to targeting specific single mutations shared among multiple cancer patients there is refreshed interest in using broader antigen spectrums for generating specific or even personalized cancer vaccines. A number of potential vaccinating agents derived from cancer tissues have been conceived including actual tumor cells as well as protein, peptide, RNA or DNA sequences derived from such tissue (Barrosoa-Sousa and Ott, 2018). We will discuss some examples of recent progress in this area.
- RNA based vaccine
In one of two recent key Nature papers concerning neoantigen vaccine clinical trials, Sahin et al. (2017) developed personalized vaccines for melanoma patients. Candidate neoantigens were generated by comparative exome and RNA sequencing of tumor biopsies and matching healthy tissue. Candidate neoantigens were screened for predicted human leukocyte antigen (HLA) class I and II binding and ten mutations per patient were engineered into synthetic RNAs encoding five mutations per molecule. Patients were repeatedly vaccinated with the RNA mixtures to induce T-cell response. In this small trial all patients developed T-cell responses to at least some of the vaccine neo-epitopes and several appeared to respond significantly to treatment. One patient in particular mounted a complete response to vaccination in combination with PD-1 blockade.
- Peptide based vaccine
In another Nature paper (Ott et al., 2017), the authors again conducted whole-exome sequencing of matched tumor and normal samples from patients with melanoma, followed by confirmation of neoantigen expression via RNA sequencing of tumor samples. They identified up to 20 tumor neoantigens per patient that were algorithmically predicted to efficiently bind autologous HLA-A or HLA-B. Synthetic long peptides (15-30 amino acids) were mixed with adjuvants and administered to the respective patients over the course of several rounds of vaccinations. Once again, all patients were found to have developed robust CD4+ and CD8+ T-cell responses to at least some of the vaccine neoantigens. In this small but promising study several patients had positive responses, and two who had progressive disease were later successfully treated with anti-PD-1 therapy and experienced complete tumor regression.
The results described in these two vaccine test cases, as well as in other work, highlight the potential of cancer vaccine approaches, as well as the promise of potential successful combination with immune checkpoint inhibitors to achieve higher response rates and improved efficacy.
Adoptive cell therapy
Adoptive cell therapy (ACT) which involves the autologous or compatible-donor transfers of living cells into patients has been in existence for quite some time. In the context of cancer immunotherapy, ACT can occur in three modes: 1) tumor infiltrating lymphocytes (TIL), 2) synthetic T-cell receptors (sTCR) or 3) chimeric antigen receptors into T-cells (CAR-T) (Marshall and Djamgoz, 2018; Khalil et al., 2016).
- Tumor infiltrating lymphocytes (TILs)
In the situations of TILs, the patient’s own T-cells – usually a mixture of CD8+ and CD4+ cells – are isolated from a metastatic tumor sample. The TIL are then grown and expanded ex vivo prior to adoptive transfer. This procedure circumvents immunosuppressive actions in the tumor microenvironment (TME) by expansion of reactive T-cells away from the tumor and infusing them back at higher activity. The treatments are now often accompanied by prior lympho-depletion regimens which is hypothesized to reduce the response-dampening effect of endogenous Tregs and certain cytokines (Farkhona et al., 2016).
- Synthetic T-cell receptors (sTCR)
The construction of sTCR takes advantage of established and evolving technologies in genetic engineering and T-cell culturing to construct cells carrying an engineered TCR that confers a new antigen-specificity. Obviously, application of this technology only makes sense when the target neoantigen(s) are known and expressed on cancer cells in correct context with HLA. This has the benefit that the neoantigen does not need to be independently expressed on the cell surface. Conversely, this facet can be a weakness when the neoantigen alone does not naturally become exposed as this leaves the tumor a possible escape mechanism through loss of HLA genes necessary for presentation (Farkhona et al., 2016).
- Chimeric antigen receptors into T-cells (CAR-T)
CAR-T cells are similar to sTCR carrying cells in that they are subject to genetic modification. In this case it is the transfer of a single-chain variable fragment (scFV) derived from an antibody which is fused to the signaling domain of the TCR complex. This construct typifies the so-called first generation CAR. Second generation CARs have an additional co-stimulatory domain (usually CD28 or 4-1BB) contained within their sequence. Third generation CAR have two different co-stimulatory domains (some combination of CD28 and/or 4-1BB and another domain possibilities including but not necessarily limited to OX40 and ICOS). More recently, so called ‘armoured’ CARs are being investigated where the transgene includes a second protein which confers a survival or cytotoxicity advantage to the resultant T cell (Khalil et al., 2016). Thus far, CAR-T technology has been highly successful in hematological malignancies. Expansion of indications involving solid tumors is still under development. As in the cancer vaccine case there is some initial evidence that combination of CAR-T with PD-1 blockade may boost efficacy.
Image credit: Magee & Snook (2014)
Companies developing immune therapies with a particular emphasis on neoantigens
There are quite a large number of companies working on various aspects of immunotherapy. Some of these companies we recently reviewed. Additional companies are reviewed below with some information on commercial efforts summarized.
BioNTech was founded in 2008, is based in Mainz (Germany) and is currently Europe’s largest privately held biopharmaceutical company. Its founders are clinical scientists Prof. Dr. Ugur Sahin and Prof. Dr. Christoph Huber who are affiliated with the Gutenberg University. The company is investigating individualized mRNA-based medicines for cancer immunotherapy. The company is also working on innovative chimeric antigen receptors and T-cell receptor-based products and novel checkpoint immunomodulators. In a 2017 Nature paper, the authors, including company researchers, posted results from Phase I trial of individualized mutanome RNA based vaccines. Personalized vaccines for additional solid tumors are also in trial evaluation, in collaboration with Genenetech, while other clinical approaches involve shared antigen vaccines to multiple other cancers.
Ziopharm Oncology is a publicly held biotechnology company with headquarters in Boston, founded in 2005, and trading on Nasdaq with the ticker symbol ZIOP. They have two main areas of interest in immune oncology therapy. The first is an adenovirus deliverable IL-12 paired with veledimex regulator which is designed to give controllable expression of the cytokine at tumor sites. This approach can turn immunologically ‘cold’ tumors – ones that exhibit little or no immune system activity – ‘hot’ by calling in an immune response, potentially overcoming some suppressive effects in the TME. This formulation was give FDA Fast Track designation in April of 2019 for the treatment of recurrent glioblastoma multiforme (rGBM) in adults and for which there are few current treatment options. The second area is leveraging the Sleeping Beauty transposon system to create genetically modified CARs and TCRs that target specific tumor-derived antigens which are in Phase I clinical trials in collaboration with MD Anderson and the NCI.
Immatics Biotechnologies is a privately held biopharmaceutical company founded in 2000 as a spin out from H.G. Rammensee’s laboratory at the University of Tübingen, Germany. The Company was founded to develop and commercialize discoveries on the immunopeptidome as targets for all types of T-cell based immunotherapies. In 2015, Immatics and MD Anderson Cancer Center (MDACC) launched Immatics US, Inc. in Houston, Texas as a joint venture to develop transformative Adoptive Cell Therapies. Immatics has two proprietary technologies in immunotherapy. XPRESIDENT® is their platform that uses a combination of transcriptomics and MS of HLA-peptide complexes to identify neoantigens expressed on tumor cells. XCEPTOR® is their platform for discovering high affinity and high specificity TCRs. Leveraging the XPRESIDENT®, ACTolog® and ACTengine® technology they have several adoptive cell therapies (ACTs), using autologous (IMA101) or engineered TCR T-cells (IMA201-3) respectively, in early Phase I clinical trial.
Agenus is a publicly traded company (AGEN) founded by Garo Armen and Pramod Srivastava as Antigenics and headquartered in Lexington MA. Agenus pipeline includes two phase 2 trials of immune checkpoint antagonist PD-1 and CTLA-4 antibodies and a phase 1 trial of an improved CTLA-4 antagonist. They also have phase I trials of other checkpoint antagonists targeting TIM-3 and LAG-3 as well as novel co-stimulatory agonist targets GITR and OX40. Agenus is in phase 2 trial of a cancer vaccine for glioblastoma using Prophage™ which garners its antigen peptides from a patient’s tumor and bound to the heat shock protein gp96 (HSPPC-96). They are also in phase I with AutoSynVax™ vaccine system which attaches predicted neoantigen peptides to heat shock protein 70.
Barrosoa-Sousa and Ott, Transformation of old concepts for a new era of cancer immunotherapy: cytokine therapy and cancer vaccines as combination partners of PD1/PD-L1 inhibitors (2018) Curr Oncol Rep, Nov 15; 21(1).
Bell et al., Oral, Head and Neck Oncology and Reconstructive Surgery, 1st Edition, Elsevier (2017)
Farkhona et al., Cancer immunotherapy: the beginning of the end of cancer? (2016) BMC Med, May 5;1 4:73.
Khalil et al., The future of cancer treatment: immunomodulation, CARs and combination immunotherapy. (2016) Nat Rev Clin Oncol, May13; (5):273-90.
Magee and Snook, Challenges to Chimeric Antigen Receptor (CAR)-T cell therapy for cancer. (2014) Discovery Medicine, Nov 17; 18(100):265-71.
Messenheimer et al., Timing of PD-1 blockade is critical to effective combination immunotherapy with anti-OX40. (2017) Clin Cancer Res, Oct 15; 23(20):6165-6177.
McKee et al., Therapeutic efficacy of 4-1BB costimulation is abrogated by PD-1 blockade in a model of spontaneous B-cell lymphoma. (2017) Cancer Immunol Res, Mar; 5(3):191-197.
Morrissey et al., Immunotherapy and novel combinations in oncology: current landscape, challenges, and Opportunities. (2016) Clin Transl Sci, Apr; 9(2):89-104.
Ott et al., An immunogenic personal neoantigen vaccine for patients with melanoma. (2017) Nature, 547(7662):217-221.
Sahin et al., Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer. (2017) Nature, 547(7662):222-226.