Immunotherapy

Information on this page may also be found on the DCVax page, the ICT-107 page, and on the Currently Recruiting Trials pages – Glioblastoma, High Grade Glioma, Low Grade Glioma.

In some ways, therapies which stimulate a cancer-fighting immune response are the ideal way to treat the disease. Non-invasive, selective, without toxicity to healthy cells, a properly trained immune system can act as an elite search-and-destroy team.

The bodies of each one of us contains cells in various degrees of pre-malignancy. It is mainly the proper functioning of the immune system which detects these rogue cells and destroys them before they can multiply into an active tumour. The immune system is a complex team composed of many different cell types with various, even opposite, functions. Additionally, immune cells may have differing functions depending on the cytokines and other chemical signals existing in the cellular microenvironment. Due to this plasticity of function, cancer cells have the ability to reprogram, or re-educate tumour infiltrating immune cells such as macrophages, changing their activity from cancer-fighting to cancer-promoting.

See the Re-educating the Immune System page for strategies involving commercially available supplements and repurposed prescription drugs. The current page focuses on immunotherapies, such as vaccines, available in clinical trials for brain cancer.

A Selection of Completed Trials

Autologous dendritic cells pulsed with tumor lysate

Dendritic cells are immune cells which circulate in the blood, whose main function is to present antigens to immune effector cells. Autologous means “from the same patient” and refers to the fact that both the dendritic cells and the tumor lysate which together make the vaccine, are derived from the same patient that receives the finished vaccine.

Dendritic cell based vaccine therapies require sufficient fresh tumour material from the patient receiving the vaccine, as well as the collection of dendritic cells from the patient, by leukapheresis. The dendritic cells are multiplied and co-cultured with the tumour lysate and later the DC cells, now capable of presenting tumour antigens to immune effector cells, are re-injected into the patient’s bloodstream.

A personalized dendritic cell vaccine therapy called DCVax, being developed by Northwest Biotherapeutics, is currently being tested in a phase III trial for glioblastomas, with estimated primary completion date of September 2015.

In a small preliminary trial including 23 newly diagnosed and recurrent glioblastoma patients, the vaccine showed excellent results, with the 15 newly diagnosed patients having a median survival time of 35.9 months, which is roughly double the median survival time for glioblastomas receiving the standard radiation and TMZ protocol. Patients who received additional immune stimulation with topical imiquimod cream did particularly well in this study.

Dendritic Cell Vaccine prepared with glioma-associated antigens (peptides)

Another form of dendritic cell vaccine utilizes synthetic peptides for glioma-associated antigens, rather than using tumor lysate prepared from the patient’s tumor. Outstanding results for glioblastomas have also been seen using this method.

In a phase I trial [4], the ICT-107 vaccine (ImmunoCellular Therapeutics Ltd.), a dendritic cell vaccine prepared with six synthetic peptides from tumor-associated antigens, was administered to 16 newly diagnosed glioblastoma patients. Progression-free survival of these patients was an impressive 16.9 months, and median overall survival was 38.4 months, comparable to the survival results seen in the phase I DCVax trial. A recent update revealed that 5 year survival was 50% (in other words, half of the patients were still alive at 5 years).

Wilms Tumor 1 peptide vaccine (phase 1)

March 17, 2015
Results of a phase 1 trial at the University of Osaka (Japan) were published online several days ago. The trial was testing a Wilms tumor 1 (WT1) peptide vaccine combined with standard of care RT/TMZ for newly diagnosed glioblastoma. Wilms tumor 1 is a tumor-associated antigen commonly overexpressed in many types of cancer. GBM patients in this trial were given up to 24 monthly cycles of TMZ, which is the standard at this institution. Seven patients were included in this trial (4 had total resections, 2 had partial resections, and one had biopsy only). Remarkably, at the time of analysis all seven patients were still alive, and five of these patients were disease-free at 3 or more years. Only one patient in the trial experienced disease progression. Median time to progression and overall survival have not yet been reached, with a median follow up time of 43.5 months (about 3 and a half years). No patient was positive for IDH1 mutation. At this institution, median PFS and OS with up to 24 cycles of TMZ without the vaccine is 10.7 and 21 months. In this small trial, median PFS and OS is more than 43.5 months, which was the median follow-up time at the time of analysis. Further trials of Wilms tumor 1 peptide vaccine plus TMZ are now being planned.
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Improving the Efficacy of Immunotherapy

See the Re-educating the Immune System page for information on the immune-stimulating properties of various supplements and prescription drugs.

PD-1 antibodies (nivolumab and pembrolizumab)

Late in 2014, the FDA approved two novel drugs for metastatic melanoma. These two drugs, pembrolizumab (Keytruda) and nivolumab (Opdivo) are both antibodies against PD-1, an immune checkpoint found on the surface of immune cells, that leads to anergy (deactivation) or death of immune effector cells when in contact with PD-L1 (ligand for PD-1) expressed on other cells. Though the PD-1/PD-L1 immune checkpoint system evolved as an important means to prevent autoimmunity conditions, the system is clearly co-opted by tumors to escape targeting by host immune cells. As of this writing in July 2016, there are no less than 13 trials currently recruiting glioblastoma patients for therapy involving nivolumab or pembrolizumab, and one trial testing a novel PD-L1 antibody (MEDI4736). The first trial for high grade gliomas to combine a dendritic cell vaccine with a PD-1 antibody is the AVERT trial at Duke University, which opened in early 2016 (NCT02529072). A second trial called CAPTIVE, opened in June 2016, combines the DNX-2401 adenovirus with pembrolizumab (NCT02798406). These two trials are likely the leading edge of the future of immunotherapy: combination of vaccine or oncolytic virotherapy with immune checkpoint blockade.

Preclinical evidence supporting this combination in high grade glioma comes in the form of a study published online in July 2016, by a team of UCLA researchers [14]. In this study, mice were treated with subcutaneous injections of a lysate-pulsed dendritic cell vaccine prepared from murine progenitor bone marrow cells and GL261 mouse glioma cell lysate. Mice were treated with vaccines at either the first day of intracranial GL261 glioma implantation, or at day 3 after tumors were established. Dendritic cell (DC) vaccination significantly increased survival times only in the group receiving early vaccination. In contrast, no prolongation of survival was seen in mice receiving later vaccination, in spite of tumor infiltration by glioma-reactive CD3+ lympocytes in these mice.

In this latter group, PD-1 was increased on tumor-infiltrating lymphocytes in both untreated and DC vaccinated mice compared to splenic lymphocytes, indicating a localized immunosuppression in the tumor environment. Following DC vaccination, PD-1 was the only marker of immune inhibition increased in the tumor-infiltrating lymphocyte population. All other markers examined, including CTLA-4 (target of the drug ipilimumab) and TGF-beta, were downregulated following vaccination.

These observations led the researchers to treat mice with established gliomas with the combination of DC vaccination plus PD-1 monoclonal antibody (similar in function to the FDA-approved antibodies nivolumab and pembrolizumab). Remarkably, while no survival benefit was observed in mice treated with either vaccine or PD-1 antibody alone, 40% of mice treated with the combination survived at least to day 60 (none of the mice in the other arms survived to day 40).

The survival benefit of the combined therapy was found to be completely dependent on CD8+ cytotoxic T-lymphocytes, as depletion of these cells in the mice wiped out any benefit of treatment. Though mice in both DC vaccine treated, and DC vaccine plus PD-1 antibody treated groups had increased infiltration of CD8+ lymphocytes into their tumors, only combination treated mice had increased proportions of activated CD8+ CD25+ T-cells, showing that PD-1 antibody in addition to DC vaccine was necessary to maintain an activated population of CD8+ T-cells in the tumor environment.

Combination treatment also increased populations of tumor-infiltrating memory T-cells, and when surviving mice from the combination treatment group were re-challenged with a second injection of GL261 glioma cells in the contralateral brain hemisphere at day 60, they again survived significantly longer than untreated controls, though they had no additional treatments beyond the initial treatment at the time of the first tumor cell injections.

GBM samples taken from clinical trial patients before and after treatment with DC vaccines at UCLA showed both abundant expression of PD-L1 (the ligand for PD-1) in the tumor environment, as well as increased PD-1 expression on CD8+ tumor-infiltrating lymphocytes following dendritic cell vaccination. This demonstrates that, as seen in mice, PD-1 expression is a mechanism of immunosuppression following DC vaccination in human patients, a means of tumor resistance to the treatment.

Ex vivo, tumor-infiltrating lymphocytes taken from GBM patients undergoing resection showed greatly increased cytotoxicity against tumor cells when PD-1 antibody was added to the co-culture.

Significance: this study clearly shows that tumor lysate-pulsed dendritic cell vaccination is not sufficient to increase survival in mice with established gliomas, and that additional reversal of local immunosuppression with PD-1 antibody is also required. Human GBM tissue showed the same increase in PD-1 expression following dendritic cell vaccination, as a means of tumor resistance to treatment. Pending demonstration of the safety of combining dendritic cell vaccinations with PD-1 antibodies in current trials for high grade glioma, future vaccine trials should logically include such antibodies (eg. nivolumab or pembrolizumab), especially for patients with significant residual tumor post-resection. This combination treatment is likely necessary to bring the benefits of anti-tumor vaccines to a patient population that otherwise might not significantly benefit – those with significant residual tumor burden.

COX-2 inhibitors
Parecoxib plus glioma cell immunization in vivo

A study [8] published in Journal of Neuroimmunology (July 2014, online) shows that the addition of a selective COX-2 inhibitor to immunization with irradiated whole glioma cells is curative for rats implanted intracranially with syngeneic rat glioma cells. Monotherapy with either irradiated glioma cell immunization, or COX-2 inhibition alone were not curative.

Fischer 344 rats were first implanted with the syngeneic N32 rat glioma cells. On days 1, 15, and 29, the rats were immunized subcutaneously with irradiated (80 Gy) N32 cells into the right thigh, in the attempt to arouse an immune response against the grafted brain tumours. Additionally, parecoxib (a selective COX-2 inhibitor) was pumped into two groups of rats intraperitoneally for either the first 28 days, or on days 7-13 and 17-23.

According to Figure 1, all untreated tumour-bearing rats died before day 30. Parecoxib-treated rats did not have improved survival compared to untreated controls. Rats immunized with irradiated glioma cells had a statistically significant improvement in survival, with several rats surviving to around day 40.

Figure 1 Eberstal 2014

Dramatically, the combination of the COX-2 inhibitor parecoxib and immunization led to long-term survival (beyond day 160) and apparent cures in the majority of rats. The groups of rats treated with continuous parecoxib (day 1-28) plus immunization had a 60% cure rate, while the rats receiving intermittent parecoxib plus immunization had a 50% cure rate.

At day 170, the surviving rats were challenged again with tumour cells in the opposite brain hemisphere, but given no further treatment. 89% of the surviving rats that had previously received immunotherapy plus continuous parecoxib survived the tumour re-challenge (to at least day 270), while 43% of the rats that had previously received immunotherapy plus intermittent parecoxib survived the tumour-rechallenge.

When the blood of the various groups of rats was analyzed, the combination-treated rats were found to have much increased levels of interferon-gamma (one of the key cytokines involved in type 1 immunity) as well as increased numbers of CD8+ effector memory T cells. Rat brains from tumour re-challenged (and previously combination-treated) rats were shown to have massively increased infiltration of TCR (T cell-receptor) and CD8 positive cells, mediating the anti-tumour response.

Parecoxib is an injectable COX-2 inhibitor available in Europe as a treatment for post-operative pain. The drug was refused approval in 2005 by the FDA. Perhaps the most commonly prescribed selective COX-2 inhibitor is the orally administered celecoxib (Celebrex). Several clinical trials and retrospective studies have looked at Celebrex as an addition to standard therapies for malignant glioma patients, though the outcomes were inconclusive. As the COX-2 enzyme and its downstream products, especially prostaglandin E2 (PGE2) play a large role in cancer-induced immunosuppression, it is likely that COX-2 inhibitors such as Celebrex will be found most useful as an addition to cancer immunotherapy, such as dendritic cell vaccines.

Celecoxib plus tumor-lysate pulsed dendritic cells in vivo

In this study [9] published in 2013, Wistar rats were implanted intracranially with C6 rat glioma cells, followed by one of the following treatments: a) untreated. b) unpulsed dendritic cells. c) C6 glioma lysate pulsed dendritic cells. d) celecoxib (Celebrex) fed in the diet. e) celecoxib plus C6 lysate pulsed dendritic cells. Dendritic cells were injected subcutaneously on days 3, 10, and 17, while celecoxib was given daily in the diet.

Median survival in the untreated control group was 21 days. Nonpulsed dendritic cell treatment prolonged median survival to 30 days. C6 pulsed dendritic cell vaccination alone, or celecoxib treatment alone both prolonged median survival further to around 40 days. Finally, the combination of celecoxib plus C6 pulsed dendritic cells prolonged median survival to around 55 days, or a 2.6-fold increase in survival time versus untreated controls. In this last group, half the rats were still alive at 60 days when the experiment was ended.

Figure 2 Zhang 2013

The combination treatment also had the greatest effect in reducing tumour blood vessel density, increasing apoptosis, reducing immunosuppressive cytokines such as IL-10 and PGE2, increasing the type 1 immune cytokine IL-12, and increasing the cytotoxic activity of rat splenocytes.

Notably, the dose of celecoxib (Celebrex) orally fed to the rats in this study, when converted to a human equivalent dose, is within the therapeutic dose range prescribed to humans (200 mg per day, 400 mg per day max).

Imiquimod (Aldara) cream

Imiquimod (trade name Aldara) is an immune response modifier which acts through toll-like receptor 7 on immune cells. Imiquimod is FDA approved for skin conditions such as actinic keratosis, external genital warts, and as of 2004, superficial basal cell carcinoma (a type of skin cancer). At least two trials testing dendritic cell vaccines for glioma patients are adding imiquimod or the related investigational drug resiquimod to the vaccine therapy as adjuvants (see NCT01792505 and NCT01204684).

Unexpectedly, a preclinical study [10] with mice conducted at the University of Minnesota found that imiquimod as a single agent inhibits the growth of intracranial gliomas in the syngeneic GL261 mouse model. In this experiment, mice were divided into 4 groups after implantation of GL261 tumours and received treatment with either a) GL261 cell lysate as immunization to stimulate an immune response b) GL261 cell lysate injected into an Aldara topical application site c) Aldara alone d) saline control. Though the mice given Aldara topical application alone were intended as a control group, their median survival was increased by 50% relative to the saline only group (from 28 days to 42 days). By day 21, tumour burden was reduced 3-fold in the Aldara alone group relative to the saline control group.

Further experiments were conducted to determine Aldara’s mechanism in the treated mice. GL261 tumour-bearing mice were treated every 7 days with topical Aldara cream and immune cells were analyzed on day 22. Aldara treatment depleted T-cell and B-cell populations in the blood with an increase in circulating natural killer and dendritic cells. In contrast, in the cervical lymph nodes (in the neck, which are the draining lymph nodes of the brain) Aldara significantly increased the absolute number and percentage of CD4 T-cells, CD8 T-cells and dendritic cells.

The effect of Aldara was then investigated in brain infiltrating leukocytes at the tumour site. Aldara did not effect B-cell or natural killer cell infiltration. Remarkably, treatment with Aldara nearly doubled the number of dendritic cells and CD4 T-cells in the brain, tripled the number of CD8 (cytotoxic) T-cells, and quadrupled the number of CD8 T cells which displayed CD107a cell surface mobilization in response to GL261 antigen. CD107a is a marker of lytic granules, which cytotoxic CD8 T-cells use to destroy their targets. Immunosuppressive Tregs were depleted in the brain by Aldara treatment.

In further testing, brain-infiltrating leukocytes from saline treated mice did not produce interferon-gamma, while BILs from Aldara treated mice produced interferon-gamma in a GL261 antigen-dependent manner.

Caveats: the ability of imiquimod cream (Aldara) to reproduce these impressive effects in humans is unknown. The authors of this study note that rodents and humans have differing thickness of skin and differing subsets of cells which express toll-like receptor 7. Also, the relative surface area of the skin to which Aldara was applied in the study was larger than commonly applied to humans. The authors conclude that further study with Aldara as a single agent in human cancer trials is warranted and also suggest testing low-dose systemic application of imiquimod or similar drugs (as opposed to topical).

Poly-ICLC

Poly-ICLC is a stabilized double-stranded RNA viral mimic, which stimulates the innate and adaptive immune response. In glioma trials, it is injected into muscle tissue. While a small preliminary trial [6] with Poly-ICLC showed impressive results for 11 anaplastic astrocytoma patients (most of whom were also treated with CCNU chemotherapy), a larger more recent trial of Poly-ICLC for 45 recurrent anaplastic gliomas did not replicate that success [7]. In the larger trial, 51% of patients had response or stable disease following Poly-ICLC monotherapy, while only 24% were still progression-free at six months. The greater promise of Poly-ICLC is likely as an addition to dendritic cell vaccine therapies.

Low-dose and intermittent chemotherapy as anti-tumour immune stimulant

Metronomic chemotherapy is the use of low daily doses of standard chemotherapy agents. This is in contrast with maximum tolerated dosing strategies in which short bursts of high-dose chemotherapy are followed by a recovery period, due to the toxicity of these high-dose regimens. There are several rationales for metronomic chemotherapy, including anti-angiogenic effects and surprisingly, anti-tumour immunological effects. In this section I will discuss the immunological effects of low dose and intermittent chemotherapy.

In one of the most striking studies [11] of the immunological effects of ultra-low dose temozolomide, approximately 70% of mice survived long term (over 100 days) when given a combination of daily low dose TMZ (2.5 mg per kilogram mouse body weight) with a tumor lysate-pulsed dendritic cell vaccine. In contrast, no mice survived to 100 days when given low-dose TMZ alone, and less than 20% survived long term when given the dendritic cell vaccine alone. This model consisted of intracranial implantation of syngeneic GL26 mouse glioma cells, intraperitoneal delivery of TMZ on days 2-6 after tumour implantation, and subcutaneous DC vaccinations on days 4, 11 and 18.

Figure 3 Kim 2010

To investigate the mechanisms behind the increased efficacy of combined dendritic cell vaccination and low-dose temozolomide, splenocytes were harvested from some of the mice in each group on day 35. At a 40:1 immune effector cell to tumour cell ratio, the immune cells from mice treated with either TMZ alone, dendritic cell vaccination alone, or TMZ plus unaltered dendritic cells all had a tumour cell killing efficacy (specific lysis) of approximately 30%. Immune cells from mice treated with combined TMZ and lysate-pulsed dendritic cells had a killing efficacy of nearly 50% and this cell killing was specific to the target GL26 glioma cells. In further testing, CD4+ and CD8+ T-cells from mice treated with both TMZ monotherapy and lysate-pulsed dendritic cell vaccine monotherapy had increased expression of interferon-gamma (a cytokine stimulating type 1 immune responses), with maximum expression of interferon-gamma in the combined TMZ and dendritic cell vaccinated mice. Furthermore, increased infiltration of CD4+ and CD8+ T-cells were observed in the brains of combination treated mice versus mice treated with either TMZ or lysate-pulsed dendritic cells alone.

In vitro, high concentrations of TMZ (100 to 400 micromolar) led to surface expression of calreticulin on the GL26 glioma cells at 48 hours, a sign of immunogenic cell death. In a final experiment, splenocytes from tumour-bearing mice treated and not treated with TMZ were examined at various time points and the frequency of immunosuppressive regulatory T-cells (Tregs) was determined. By days 22, 29 and 36, the TMZ-treated mice had suppressed Treg frequency (similar to non tumour-bearing mice), while the untreated tumour-bearing mice showed a marked increase in the frequency of immunosuppressive Tregs.

In summary, very low dose daily temozolomide (a mouse dose which would be allometrically scaled to a human dose of approximately 10 mg per square meter of body surface) seems to increase the tumour killing efficacy of T-cells and infiltration of those T-cells into mouse brain tumours, which may be directly related to the depletion of immunosuppressive Tregs by the low-dose TMZ treatment. High concentrations of TMZ in vitro led to immunogenic cell death of GL26 mouse glioma cells, though this was not demonstrated in vivo in this study. Of note, a prior study [12] had also shown the depletion of Tregs in rats bearing RG2 rat gliomas with even lower doses of temozolomide (0.5 mg per kilogram rat body weight). However, survival was slightly but not significantly prolonged in this study by the low-dose TMZ treatment, indicating that the very low-dose metronomic TMZ schedule may work best in combination with other immunotherapies such as dendritic cell vaccines.

Immunotherapy trial listings may be found on the Currently Recruiting Trials pages.

References
  1. Effector T-cell infiltration positively impacts survival of glioblastoma patients and is impaired by tumor-derived TGF-beta. Lohr et al. 2011.
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  2. Gene expression profile correlates with T-cell infiltration and relative survival in glioblastoma patients vaccinated with dendritic cell immunotherapy. Prins et al. 2011.
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  3. Resection and immunotherapy for recurrent grade III glioma. Elens et al. 2012.
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  4. Phase I trial of a multi-epitope-pulsed dendritic cell vaccine for patients with newly diagnosed glioblastoma. Phuphanich et al. 2013.
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  5. Induction of CD8+ T-cell responses against novel glioma-associated peptides and clinical activity by vaccinations with alpha-type 1 polarized dendritic cells and polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose in patients with recurrent malignant glioma. Okada et al. 2011.
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  6. Activating the host natural defense: POLY-ICLC and malignant brain tumors. Salazar.
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  7. A North American brain tumor consortium phase II study of poly-ICLC for adult patients with recurrent anaplastic gliomas. Butowski et al. 2009.
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  8. Immunizations with unmodified tumor cells and simultaneous COX-2 inhibition eradicate malignant rat brain tumors and induce a long-lasting CD8+ T cell memory. Eberstal et al. 2014.
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  9. Enhancement of Antitumor Activity by Combination of Tumor Lysate-Pulsed Dendritic Cells and Celecoxib in a Rat Glioma Model. Zhang et al. 2013.
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  10. Topical imiquimod has therapeutic and immunomodulatory effects against intracranial tumors. Xiong and Ohlfest, 2011.
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  11. Immunological factors relating to the antitumor effect of temozolomide chemoimmunotherapy in a murine glioma model. Kim et al. 2010.
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  12. Treg depletion with a low-dose metronomic temozolomide regimen in a rat glioma model. Banissi et al. 2009.
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  13. A phase II, multicenter trial of rindopepimut (CDX-110) in newly diagnosed glioblastoma: the ACT III study. Schuster et al. 2015.
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  14. PD-1 blockade enhances the vaccination-induced immune response in glioma. Antonios et al. 2016.
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