One approach has been to forgo the selection of tumor reactive T cells entirely and begin TIL rapid expansion immediately after they are isolated. Another approach involves the selection of tumor-specific T cells expressing activation markers. This suggests that these markers could be used to enrich TIL for melanoma-reactive T cells. At some centers, TIL are now being expanded in gas-permeable flasks rather than traditional T-flasks and bags.
TIL can be grown at a greater density in gas-permeable flasks which results in the use of less media [14]. TIL rapid expansion in gas-permeable flasks rather than T-flask and bags requires approximately 5- to 8-fold less media and media supplements including cytokines and AB serum.
This results in a significant reduction in the cost of TIL production. An additional difficulty with the typical methods used to expand TIL is that irradiated pooled allogeneic peripheral blood leukocytes PBL collected from healthy subjects are used as feeder cells to stimulate TIL rapid expansion.
It would be desirable to have an alternate off- the-shelf, widely available product for TIL expansion. Artificial antigen presenting cells are being evaluated as a substitute for allogeneic PBL feeder cells for TIL rapid expansion.
They are using K cells that are genetically engineered to express Fc receptors and costimulatory ligands, such as BBL. The degree of expansion and cell characteristic were similar to those expanded with allogeneic PBL but at lower TIL:APC ratios, meaning fewer feeder cells are required.
As discussed by multiple presenters during the meeting, several TIL characteristics that are associated with better clinical outcomes and likely a more potent product have now been identified. New work is aimed at identifying the functional attributes of persisting TIL [16]. The infusion of greater number of TIL and longer intravascular persistence have also been associated with improved clinical outcome [17]. Since results emerging from TIL therapy trials suggest that clinical outcome is dependent in part on the number of TIL infused, efforts are being made to increase the quantity of TIL produced from each patient.
Interestingly, as pointed out by Pamela S. Together, findings such as these highlight the great advancements being made in immunology and technology which makes it more feasible than ever to efficiently generate autologous tumor-reactive TIL products of high quality and quantity for use in adoptive immunotherapy of cancer.
Genetically engineered T cells In order to improve ACT therapy, T cells are being genetically engineered in a number of ways. Investigators lead by Steven A. Conferring the ability of tumor antigen-specific T cells to produce IL greatly improves their function in a variety of pre-clinical studies [23,24].
Since IL can cause systemic toxicity, they have been attempting to focus IL production and prevent systemic toxicity by including a nuclear factor of activated T cells NFAT transcription factor in addition to IL in the vector used to engineer TIL.
Autologous T cells are also being engineered to express high affinity TCRs. One of the challenges of this approach is to target antigens that have enough specificity to the tumor so that cancer regresses without causing off target toxicity and complications.
The preliminary results of early phase clinical trials of T cells that have been engineered to express chimeric antigen receptors CAR have been very promising and research in this area is growing rapidly.
Three centers have reported the results of anti-CD19 CAR T cell clinical trials treating B cell lymphomas and leukemias and while the result at all three centers have been promising, there were several differences among the trials []. Leukocyte depletion is important for the success of this therapy [29] and the pre- treatment leukoreduction protocols also differed among the three centers.
To limit off target toxicity, tumor sensing T cell are being developed [30,31]. One approach is to engineer T cells so that they recognize two antigens, thereby being less likely to cause off target cytotoxicity. Another approach to limit toxicity is to use transiently expressed RNA vectors rather than permanently expressed lentiviral or retroviral vectors.
This precocious differentiation was TCR-ligation dependent and required cell-to-cell contact. These studies suggest that the removal of more differentiated TEM and TEFF cells from adoptive cellular therapies is necessary to prevent the corruption of the full potential of less differentiated anti-tumor T cells.
TCM have a unique capacity for self-renewal, proliferation, persistence and an ability to differentiate into effector T cells. Philip D. Greenberg and colleagues University of Washington, Seattle, WA, USA are using a novel combination of cytokines to maintain a young phenotype during T cell culture and expansion. Another approach to obtaining less differentiated cells for adoptive cell transfer appears to be blockage of T cell glycolysis. They found that TN cells which relay on fatty acid oxidation as a primary source for ATP generation, shifted to a glucose metabolism following antigen stimulation and effector differentiation.
Blockade of glycolysis during T cell priming by 2-deoxyglucose 2-DG prevented differentiation and the treatment of tumor-bearing mice with 2-DG treated T cells resulted in increased tumor-infiltration, cytokine release and tumor regression than control cells.
Dendritic cell therapy DC therapy is showing promising results in several different types of cancer. The vaccine appeared to have been of some clinical benefit in five patients that displayed no evidence of residual DCIS and HER-2 expression was undetectable in 11 patients. DC vaccines have also been used to treat patients with metastatic melanoma and the availability of an autologous melanoma cell line. The tumor cells or DCs were given once weekly for 3 weeks and then monthly for 5 months.
Sipuleucel-T is an autologous DC-like immunotherapy approved for the treatment of castrate resistant prostate cancer. Recent studies have shown that this therapy affects the immune cell infiltrate in the prostate cancer microenvironment.
They found that sipuleucel-T increased infiltration of T and B cells in the microenvironment. They also documented systemic T and B cell activation.
The oncogenic virus human papilloma virus HPV causes cervical cancer, vulvar cancer, anal cancer and head and neck cancer. A peptide vaccine was developed to avoid the high costs associated with the production of DCs. The strength of vaccine- induced HPV T cell specific responses correlated with significant clinical response [41,42].
The vaccine is currently being tested in combination with chemotherapy. Disease stabilization of 12 months or longer was observed in 9 of 22 accrued patients. Oncolytic viruses Vaccinia viruses infect and lyse some tumors. Recent clinical trials have found these oncolytic viruses to be safe and in some cases they have demonstrated tumor selectivity. Vaccinia infects most mammalian cell types and causes minor illness in humans.
Analysis of the mononuclear cell subset tropism of several oncolytic vaccinia virus constructs found that they preferentially infected monocytes and activated T cells, but were much less likely to infect B cells, NK cells and resting T cells. However, the viral amplification and cytotoxicity were greater in control cancer cells than in the mononuclear cells.
Oncolytic vaccinia is now being used to treat patients with chronic myelomonocytic leukemia CMML in an early phase clinical trial. Vaccinia has been engineered to express GM-CSF in order to modulate the tumor microenvironment and enhance anti-tumor immunity and has been used as an anti-tumor vaccine [46,47]. The recombinant fowlpox vector is being tested in patients with bladder cancer in a phase I clinical trial. PanVac is being given intratumorally to patients with pancreatic cancer.
PanVac has been given to 11 patients with locally advanced disease and although 10 patients in the trial remained metastasis-free, they all died of local disease progression.
This virus is being tested for the treatment of cervical cancer using a murine model. The vaccine is given with cisplatin chemotherapy in order to change the tumor microenvironment by causing tumor lysis which increases antigen uptake by DCs. Vesicular stomatitis virus VSV is also being used in cancer immune therapy [48]. The VSV was administered as an intratumoral injection. Studies of tumor bearing mice have found that the mechanism by which VSV induces tumor lysis involves the induction of strong innate immune response when injected into tumors which likely is responsible for killing both infected and non-infected tumor cells.
VSV is also being used to encode tumor associated antigens in order to prime strong anti-tumor T cell responses. Bacteria as cancer vaccines Bacteria are also being used for cancer vaccines.
Listeria monocytogenes has been engineered to express antigens and used to treat cancers [50]. Listeria preferentially infects monocytes and generates a strong innate immune response. ADXS has been used in a phase II clinical trial of patients with recurrent and refractory cervical cancer that had been treated with chemotherapy or radiotherapy. Some patients experienced complete responses and partial responses, but no improvement in responses were noted in patients that received cisplatin chemotherapy in addition to ADXS Combining immunotherapy and targeted therapy for melanoma Antoni Ribas University of California Los Angeles, CA, USA reported that traditional cancer chemotherapy or novel targeted therapies are being tested in combination to improve the effects of tumor immunotherapies.
Several agents have been used to overcome these problems including a proteasome inhibitor bortezomib which sensitizes NK cells, a Bcl-2 inhibitor ABT which sensitizes T cells by making cancer cells sensitive to granzyme B induced apoptosis [51] and the HDAC inhibitor LAQ which improves antigen presentation by tumor cells and enhances the function of T cells [52]. Initial studies of vemurafenib have been promising. Clinically, vemurafenib has been found to increase the quantity of T cell infiltrates in regressing melanomas.
Studies of pmel-1 murine melanoma model have found that when given with ACT, vemurafenib increases immune cell function and possibly modulates the tumor microenvironment. Specifically vemurafenib paradoxically increased MAPK signaling, in vivo cytotoxic activity, and intratumoral cytokine secretion by adoptively transferred cells [53]. Many cancers have RAS mitogen-activated protein kinase MAPK oncogene mutations that lead to tumor immune evasion in part due to immune escape. Recently, it has been discovered that the inhibition of BRAF increases the activity of immunotherapies.
Antoni Ribas and colleagues demonstrated that in mouse models, BRAF inhibitors increase the in vivo cytotoxic activity and intratumoral cytokine secretion by adoptively transferred cells, without altering their expansion, distribution, or tumor accumulation. In a second clinical trial, following surgical resection of GIST, patients were randomized to receive either imatinib or no additional therapy.
Disease recurrence and progression was delayed in patients treated with Imatinib, but it did not result in improved long-term disease free survival. The number of TAM decreased in patients sensitive to imatinib but develop a type 2 phenotype post treatment.
In contrast, the number of TAM increase and retain a type M1 phenotype in patients resistant to imatinib [57]. These NK cells and T cells are activated and produce Th1 cytokines.
In these studies, increased levels of sB7-H6 and decreased levels of sMIC were associated with poor prognosis. After cetuximab treatment their numbers increased further. Radiotherapy to enhance immunotherapy Radiation therapy is reported to be able to convert the tumor into an in situ vaccine by inducing tumor cell death and a pro-inflammatory microenvironment.
The fact that this is a rare event suggests that radiotherapy usually has an immunosuppressive effect. Single cell high throughput technologies An important aspect of cancer immunotherapy is measuring the immune response. Several advances have been made in high throughput single cell analysis. SCNP is a multiparametric flow cytometry-based analysis that simultaneously measures, at the single- cell level, both extracellular surface markers and changes in intracellular signaling proteins in response to extracellular modulators.
This approach allows for simultaneous functional measurements from multiple cell subpopulations without the need for prior cell separation [61,62]. Another approach to evaluate immune cells is single cell mass cytometry which is a multiparametric approach that combines single cell analysis with mass spectrometry. As discussed by Gary Nolan Stanford School of Medicine, Stanford, CA, USA , cells are first labeled with antibodies conjugated with elemental isotopes, nebulized to free ions with high temperature K and then analyzed with mass cytometer.
This approach allows the analysis of over 45 different parameters at the single cell level. These guidelines have been drafted and have been posted on SITC web site and are open for comment. Future guidelines will address immunotherapy of genital-urinary tract malignancies and hematological malignancies.
FDA update Raj K. Conclusions Immunotherapy of cancer continues to grow. The success of TIL therapy is being documented at several centers and TIL production is becoming simpler and less expensive. Methods to produce T cells with stem cell characteristics are being developed for use in ACT in order to improve the survival and proliferation adoptively transferred T cells. Several DC therapies have proven to reliably induce peripheral blood T cell responses and T cell and B cell infiltration into the tumor microenvironment.
Initial studies of immunotherapy combinations with targeted therapies have been promising, as have vaccines making use of oncolytic viruses, Listeria monocytogenes and mRNA.
Showing all 3 of 4 Collapse View all. Return to homepage. Biography: The overall goal of my research is to advance the integration of immunotherapy within comprehensive cancer care, as a complementary modality to surgery, chemo and radiotherapy. Pawel Kalinski. Cancer Innovations: Making immunotherapy work for more patients.
Meet TeamRoswell: Dr. T-cell priming by type-1 and type-2 polarized dendritic cells: the concept of a third signal. Immunol Today. PMID: Alpha-type-1 polarized dendritic cells: a novel immunization tool with optimized CTL-inducing activity.
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