Immune checkpoint inhibitors: What are the risks?

Immuno-oncology has advanced greatly through T cell-based therapeutic approaches (particularly immune checkpoint inhibitors). However, immune checkpoint inhibitors (ICIs) aren’t efficacious in all patients, and they carry risks. What are these risks? Courtney Ferrebee, PhD, Field Applications Scientist, Taconic Biosciences speaks to Lu Rahman.

Preclinical in vivo models serve as important tools for studying the mechanisms of tumor biology and immuno-oncology (I/O). They also provide researchers and clinicians with a platform to evaluate immunotherapeutic efficacy. Outcomes of many of these studies show the benefits and utility of T-cell based immunotherapy and many physicians have relied on these data to recommend ICIs for clinical use.

ICIs target one or more of the multiple cellular pathways that inhibit immune activation and immunosurveillance. While the clinical utility of these approaches holds a lot of promise in oncology, ICIs are not efficacious for all patients and may pose some risks. In non-responsive patients, there may be multiple pathways preventing tumor immunosurveillance, including a reliance on alternate immune checkpoints, tumour heterogeneity, T cell exhaustion, and impaired cytokine signaling. Given the physiological role of these immunoregulatory pathways, ICIs may induce unintended immune activation. Cytokine release syndrome (CRS) is one major potential adverse event for this class of drugs, leading to an acute systemic and devastating immune activation. Other, less serious adverse events may include symptoms of autoimmune disease or neurotoxicity. The engagement and activation of T cells after target recognition (for example in CAR-T therapy) leads to the release of pro-inflammatory cytokines like IFNγ and TNFα, which can activate macrophages, all of which directly promote CRS. Neurotoxicity is the response that follows CRS and is a result of the overproduction of pro-inflammatory cytokines by activated “bystander” effector myeloid cells in the tumor microenvironment that diffuse into the blood-brain barrier and accumulate and activate microglial cells in the central nervous system.

Other physiological factors may come into play in patient treatment, such as the presence or absence of certain microbes in the microbiome. Publications such as those in Cancer Immunotherapy have documented that favourable and unfavourable gut microbiome profiles can greatly affect the treatment outcomes in patients treated with an ICI, specifically PD-1 checkpoint inhibitors. This is another facet we must consider with the treatment of each individual. It might mean performing a preliminary screen for certain strains of microbes to determine if a patient will respond to the treatment or altering the patient’s microbiome to increase ICI efficacy.

LR: Why do T cell-based immunotherapy approaches pose limitations and how can drug discovery researchers overcome them?

CF: As mentioned, some of the limitations to consider with ICIs are the risks of CRS and neurotoxicity, patient variations which result in responders vs. non-responders, and patient microbiome differences. To overcome these limitations, it is useful to consider a broader range of targeted activation of other immune cell subsets for therapy. Beyond direct T cell activation, researchers should consider exploiting the utility of innate immunity antitumor mechanisms. Dendritic cells (DCs) represent one of several pathways to accomplish this and, for example, are modulated by anti-CTLA-4 therapy. The major purpose of DCs is to present antigens to elicit T cell-dependent responses. Researchers have shown that harnessing this mechanism can generate effective immune responses to tumors in vivo.

Other potential therapeutic strategies include targeting cytokines, manipulating inhibitory receptors such as ITIM for DC activation, directly activating or mobilising DCs, and adoptive transfer of DCs. Adoptive transfer of patient-derived ex vivo manipulated DCs is a promising approach but proves challenging, with efficacy not meeting expectations. Additional considerations include designing DC-based oncolytic vaccines through infusing patients with targeted antigens and adjuvants to stimulate DCs in vivo. Studies using Roferon-A/Interferon-α, which acts as an DC immunostimulatory agent, is efficacious in leukemia and lymphoma patients. More importantly, use of this therapy has set precedence for the development of antigen or cytokine-based vaccines. Clinical trials for the vaccine APCENDEN show some efficacy using tumour cell lysate to activate DCs for treatment against certain malignancies, including prostate and ovarian cancer. Notwithstanding, for some DC-based immunotherapy to be advantageous, researchers need to determine if there is a sufficient population of tumor-associated DCs to exploit these mechanisms. Understanding the functions and the limitations of DC subsets will propel us forward with manipulating this therapy.

Another way to broaden the range of targeted activation is to develop therapeutic modalities focused on natural killer (NK) cells, which can be engineered to target tumor cells in a similar manner through antibody dependent cell cytotoxicity (ADCC) therapies. NK cell-based therapy can also indirectly activate T cells through stimulation by tumour antigens. In addition to this, there are a variety of other approaches to NK cell immunotherapy, including adoptive NK therapy, CAR NK cell therapy, genetically modified or engineered NK cells, and iPSC-derived NK cells. Clinical trials for umbilical cord NK cells for CAR-NK therapy are underway. Liu et al. has shown efficacy and antitumour activity in CAR transduced NK cells for CD19 positive lymphoma. More importantly, the numerous clinical trials for NK therapy were preceded by preclinical experimentation. To explore these and many other possibilities, researchers have the option of using in vivo preclinical models, specifically humanised immune system (HIS) mice, to investigate these therapies and mechanisms for drug discovery and development.

LR: How do therapies like CAR-T and adaptive cell transfer help overcome those limitations? What within this area should the research market be focusing on to develop these therapies successfully?

CF: CAR-T is such a specialised therapy because the technique involves engineering a patient’s own T cells to attack certain cancers. In theory, this should improve the T cell-based therapeutic approach through highly specific autologous cells. To date, CAR-T therapy is approved for blood cancers, such as leukemia and lymphoma. However, this approach poses similar risks and limitations as ICIs, including the inability and challenge of CAR-T cells to target and migrate into tumours.

Preclinical studies in HIS mice have assisted with showing potential efficacy of CAR-T therapeutics. A powerful tool for this is the transgenic hIL2 NOG mouse, which expresses human IL-2 to support human T cell expansion and engraftment. For example, studies published in Cancer Research and Nature Communication determined critical parameters for CAR-T efficacy in hIL2 NOG mice, showing that IL-2 is necessary for CAR-T function and predictive validity of preclinical CAR-T studies. The significance of these investigations is understated because these studies can help inform decisions on patient treatment and are consistent with clinical trial data even in patients who have resistance to ACT therapy with autologous tumor infiltrating T lymphocytes (TILs). Preclinical HIS models including hIL2 NOG expand the realm of possibilities of investigating T cell subset and T cell-based therapies and overcome the drawbacks with these therapies. The focus should be on developing and improving applicable models to test these various therapeutics.

LR: What other cell types are being leveraged effectively for I/O research applications?

CF: Other subsets within the lymphoid lineage (B and NK cells) and those cells that comprise the myeloid lineage are promising for I/O therapeutic development. HIS models that support NK cells, such as the hIL15 NOG, allow for further investigation of these subsets and their contributions to immunotherapy. Katano et al. showed that ADCC against leukaemia xenografts can be demonstrated in hIL15 NOG mice following adoptive NK cell transfer. Tri-specific killer cell engagers (TriKE), also known as NK cell engagers, have been evaluated in preclinical models for NK activation therapies. In vivo studies similar to these are helping researchers to tease out the advantages and limitations of some of these therapies and enhance the design of sustained NK activation strategies, one of which includes clinical trials for GTB-3550.

Models such as the huNOG-EXL and hIL6 NOG allow for exploration of myeloid and monocyte-based I/O applications. PD-1 and CTLA-4 therapies investigated in huNOG-EXL (which allows for improved engraftment of both lymphoid and myeloid lineages) for T cell activation show clear efficacy in various preclinical studies. Due to the model’s support and development of monocytes (including macrophages and dendritic cells), the huNOG-EXL is designed for the investigation of immunotherapy activation of the myeloid lineage. Studies in this model examining treatment with anti-LAG3 antibodies show stimulation of DCs could potentially reduce tumour volume. Additional promising data is also on the horizon in underutilised models such as the hIL6 NOG, which serves as a model for testing antibody drug conjugates that selectively target monocytes in the treatment of cancer.

B cells are widely known as having an important role in the tumour microenvironment and shaping antitumor responses. B cells can even promote chemotherapeutic resistance and appear to be at the crux of tumour progression and immunity. Tumour infiltrating B cells can stimulate immunity through the release of antibodies and cytokines that activate DCs and T cells to slow down tumor progression and development. As a result of these mechanisms, many researchers are considering B cell-based vaccines for immunotherapy. Many of these vaccination strategies rely on patient-derived purified B cells, PBMCs, or TILs and are stimulated via CD40L mechanisms for tumor antigen presentation. CD40 activated B cell vaccines show some preclinical and clinical efficacy with regression of tumor growth and metastases through T cell activation. O’Hara et al. showed that CD40 agonists such as APX005M can activate B cells, DCs, and monocytes for antigen presentation to T cells. While researchers continue to make strides in this area, unfortunately, adaptive immunity in HIS models must improve to further evaluate these mechanisms of B cell applications for immunotherapy. To circumvent some of these issues, researchers may use HLA transgenic mice that “educate” immune cells on human MHC molecules to improve the adaptive response. HLA models developed for other disease applications show a more pronounced and possibly functional B lymphocyte population, suggesting that improvements in these models could be beneficial for future investigations of B cell-based immunotherapy.

LR: What would allow researchers and scientists to consider other immune cell subsets MORE in I/O and immunotherapy research?

CF: If we acknowledge I/O from the immunology standpoint, there are many other immune cells beyond T cells involved. T cells comprise the adaptive immune side, which is acquired and based on antigen-specific immunological memory. However, the innate immune system is just as important in the I/O world and may be underutilised when you think about the clinical therapeutics recommended by physicians and those on the market.

In addition to the importance of the role of NK cells in I/O and immunotherapy applications, as discussed, myeloid lineage cells of the innate immunity serve an equally important role and function. Targeting DCs for immunotherapy is not a new concept. However, with more discovery of the role of DCs in tumour biology, an evolution in targeting and leveraging these cells for immunotherapy is taking place. For example, Li et al. showed that DCs and macrophages within the tumor microenvironment can stimulate T cells through IFNs and other cytokines to elicit anti-tumour responses. Likewise, this crosstalk between immune cells extends to NK cells in the tumor microenvironment, which activates DCs through feedback mechanisms that can also stimulate T cells. Many studies have also shown that tumor-associated macrophages (TAM) play an important role in the tumor microenvironment. Blockade of checkpoint inhibitors such as CD47 show that ICIs can stimulate phagocytosis by TAM and promote immunotherapy efficacy. While this strategy is promising, clinical trials involving blockade of CSF-1 in combination with PD-1 did not show efficacy. Similar to CAR NK and CAR T therapy, CAR macrophages (CAR-Ms) are being proposed as a treatment due to their ability to produce chemokines and cytokines that activate effector T cells. In addition to this, CAR-Ms demonstrate phagocytosis and tumour clearance in vitro and have other antitumour mechanisms including resistance to immunosuppression and enhanced trafficking to and within tumours, presenting additional options for immunotherapy development. Other approaches to modulating macrophages include designing therapeutic vaccines specific for TAMs through GM-CSF signaling. CAR NK cells, as discussed, continue to be a proposed immunotherapy through leveraging the innate immune system. Similar to HIS studies performed for CAR-T in hIL2 NOG mice, studies for CAR NK cells are occurring in hIL15 NOG mice. Overall, in vivo preclinical studies show that each HIS model supports a specific immune cell subset and allows researchers to examine the specific roles of the cells in I/O and immunotherapy.

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