1. Fuerst, Mark L.

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BOSTON-New research in cancer immunology shows the promise of using T lymphocytes, the warriors of the immune system, to combat cancer in patients with metastatic disease.

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Cell transfer therapy, the administration of autologous lymphocytes, to cancer patients has many advantages, said Steven A. Rosenberg, MD, PhD, Chief of Surgery at the National Cancer Institute (NCI).


These advantages include administration of high numbers of highly selected cells with high avidity for tumor antigens; the cells can be activated outside the body to exhibit anti-tumor effects; and researchers can identify exact cell subpopulations and the functions required for cancer regression.


"Most importantly, we can manipulate the host prior to cell transfer to provide an altered environment for transferred cells," Rosenberg said, adding that "because the immune agents are taken from the body, we can eliminate negative influences so the cells we give are more effective."


Adoptive Cell Therapy

NCI researchers recently completed accrual of a trial looking at adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes with two lymphodepleting regimens. Patients were randomized to non-myeloablative chemotherapy with cyclophosphamide and fludarabine that eliminated all circulating infiltrating lymphocytes for eight days or the chemotherapy plus total body irradiation for more complete lymphodepletion.


Response rates in the 101 metastatic melanoma patients, many of them refractory to other treatments, ranged from 45-60 percent. Complete responses (CR) were achieved by 24 percent in each group, with 23 of the 24 CRs in both groups now ongoing from 14 to 53 months. "The therapy extended survival out to three or four years in metastatic melanoma patients," Rosenberg said.


Some 194 consecutive patients have received the lymphodepletion therapy. After a median follow-up of five years, 44 patients achieved CR, with only two recurrences; 42 of the CR patients are ongoing for 14 to 137 months. "This is likely a curative regimen for about one-quarter of patients who develop metastatic melanoma," Rosenberg said. "This is another example of the power of immune lymphocytes in the treatment of cancer patients."


There was no relationship between the bulk of disease or sites of disease and the likelihood of CR. Lymphocytes eliminate cancer at multiple sites, and it happens fast-the tumor may disappear before the patient leaves the hospital, some within 12 days, he said.


Only two patients received more than one treatment. "The lymphocytes administered are living cells and can manifest ongoing reactivity," he said, pointing out that most chemotherapies are excreted within minutes to hours after administration.


The researchers have seen regression in brain metastases, he noted.


TILs appear to recognize something unique to tumors. Now researchers are investigating whether mutations can be used as targets for immunotherapy. They are mining the cancer exome to identify immunogenic cancer mutations. Only rare mutations have the likelihood of being immunogenic, Rosenberg said.


"We have developed a blueprint for the generation of mutation reactive T cells from an individual patient with a common cancer. From the tumor, we do whole exome sequencing to identify mutations. Then we sequence and synthesize tandem minigenes that contain all mutations," he said.


Mutated antigens are recognized by T cells from melanoma patients. "In 25 patients with metastatic melanoma, the majority of them with complete durable regressions, we have used this technique to identify 64 different somatic mutations. All mutations were unique to that patient. There was no duplication of any antigens recognized in complete regressors from one patient to another," Rosenberg said.


Virtually none of the intracellular proteins are shared. "This is probably why cancer vaccines have not worked. No shared antigens are recognized by the immune system," he said.


Rosenberg said "ACT can mediate complete, durable, likely curative regressions of metastatic melanoma based on the recognition of immunogenic cancer mutations. Recognition of random somatic mutations is the final common pathway explaining cancer regression from most immunotherapies for solid cancers."


He noted that "IL-2, anti-CTLA4, anti-PD-1, anti-CD40, and TIL each unleash immune reactions against unique mutations in each individual patient."


The next step is to utilize this tandem minigene approach to treat common epithelial cancers. "In 22 consecutive patients with epithelial cancer, we found 57 different somatic mutations recognized by autonomous TILs," he said. The cancers included cholangiocarcinoma, and cancers of the colon, rectum, pancreas, esophagus, lung, breast, and cervix.


When the researchers looked at the cellular level with minute detail, each patient was unique. Only one gene was found in two patients, the KRAS oncogene, one of the most common oncogenes. This highlights the uniqueness of what the immune system recognizes, he said.


To treatment epithelial cancers in this way, researchers need to identify the mutation and grow T cells from fresh tumor specimens, and then purify anti-tumor cells. "Can we obtain tumor reactive cells in peripheral blood through enrichment of tumor reactive cells in TIL based on expression of PD-1?" he asked.


They have begun to look at ways to do just that using so-called exhaustion markers expressed on the surface of lymphocytes, including PD-1, LAG-3, TIM-3, and 41BB. "The selection of PD-1-positive lymphocytes from TILs and from peripheral blood can enable direct enrichment of tumor reactive cells. Peripheral blood cells with these markers can be screened with a high throughput genomic approach. Mutation-specific cells detected in TILs are most often in this population and easy to isolate," he said.


He added: "We have a way to purify cells in the peripheral blood. Any intracellular protein can become a target of therapy. It's ironic in a sense. It's possible that the Achilles' heel of cancer will be the result of the very genes that caused the cancer in the first place. To cause cancer, genes have to mutate. We may be able to take advantage of those genes to treat the patient."


The absolute frequency of T-cell receptors might be a clue to anti-tumor activity and be an easier way to find T-cell receptors with anticancer activity. "In 12 melanomas tested, within the first four the most frequent T-cell receptors had anti-cancer activity. This might be a simple way to isolate cells with substantial anti-tumor activity without doing deep sequencing and screening," he said.


Targeting Treatment

Rosenberg said "cell transfer therapy can mediate durable regressions in patients who are refractory to other treatments. T cells that recognize unique somatic mutations can be found in TILs and in the peripheral blood. Identification and targeting of mutations unique to each cancer has the potential to extend cell therapy to patients with common epithelial cancers."


This approach, although daunting, may lead to highly personalized cancer therapy. "You have to create a new drug for every patient. We have to obtain T cells, identify the unique mutation present in that cancer that is capable of binding to unique MAC molecule, and use it for therapy," Rosenberg said. "Once we have a reactive T cell, it can destroy any solid tumor."