LIVER CANCER

THE IMMUNOTOX™ PROGRAM AT AGT

Introduction

We are fortunate to be living in the decade of cancer cures. Our understanding of the immune system and how it fights cancer is being translated into clinical products with fewer side effects and greater benefit than conventional cancer therapy. AGT is a part of this revolution as we are creating genetic drugs that will increase treatment potency, reduce long-term worries about modifying the immune system, broaden the types of cancers treated, and offer the greatest benefit to the largest number of patients. AGT is important in the emerging industry of immuno-oncology and seeks to be a leader in this field.

The Landscape of Current Immuno-Oncology Products

What is out there already falls into three types of immuno-oncology products for cancer therapy:

  • Checkpoint inhibitors

    Monoclonal antibody drugs that block the ability of tumors to switch off the immune attack and avoid destruction. Especially for tumors expressing the off signal called PD-L1, checkpoint inhibitor therapy helps the immune system to be more effective for controlling cancer but might trigger long-term problems related to autoimmunity.

  • Chimeric antigen receptor T cells (CAR-T)

    Blood T cells are collected by leukapheresis and modified ex vivo (outside the body) with a lentivirus vector carrying genes for the CAR-T molecule. The CAR is a man-made gene that allows T cells to recognize specific cancer cells. The idea behind CAR-T cell therapy is to overwhelm the cancer by having large numbers of genetically-modified T cells. CAR-T has proven to be effective against lymphoma or myeloma, but the CAR genes used so far recognize targets found in malignant and normal B cells. CAR-T can be very effective against the hematologic cancers but might also deplete normal B cells. Without B cells, treated individuals are lacking one key aspect of the immune system that is responsible for resistance to infectious disease, and persons may require regular infusions of immunoglobulin made from the sera of random blood donors.

  • Oncolytic virotherapy

    Many human viruses grow faster in tumors because cancer is a disease of uncontrolled cell growth and that also helps viruses to replicate and spread. In addition, some viruses carry enzymes that can be exploited for cancer therapy especially when combined with small molecules called pro-drugs that are converted to cellular toxins by the viral enzymes. Partly because most viruses used for oncotherapy have potential to cause their own form of disease, this is a slow developing area.

What Distinguishes AGT’S ImmunoTox?

The ImmunoTox program is AGT’s response to a formidable challenge: Can we develop a genetic drug that targets solid tumors, avoids genetic manipulation of T cells, activates a natural mechanism for tumor destruction, and works against a broad range of tumor types? The answer is YES, and the drug is AGT’s ImmunoTox.

 

ImmunoTox is a genetic medicine that is delivered using a 3rd generation, industrial standard lentivirus vector. Vectors of this type have solid safety records in clinical trials and are the basis of some newly-approved cancer therapies. In AGT’s case, the lentivirus vector is delivered to sites of solid tumor growth where it penetrates tumor cells and alters their normal metabolism. ImmunoTox slows the growth of tumor cells but causes them to look like superfast growing cells because they over-produce a small molecule that is a danger signal that activates γδ T cells (learn more about γδ T cell therapy here) to become potent killers of cancer cells.

 

Unlike checkpoint inhibitor antibodies, the temporary activation of γδ T cells by ImmunoTox vector is expected to resolve once the tumor is eliminated, and we do not expect problems with autoimmunity. Unlike CAR-T, we do not make genetic changes to T cells and do not expect long-term, off-target effects including destruction of normal cells. Unlike oncolytic virotherapy, we use safe and effective lentivirus vectors that do not replicate in the body and have no disease-causing mechanism of their own.

 

The γδ T cells in blood become highly activated by the tumors danger signal and quickly enter the tumor killing mode as part of their normal function. Luckily, the cells have exquisite ability to differentiate normal versus malignant tissue and killing is highly specific for tumor cells. When stimulated by genetically modified tumor cells carrying ImmunoTox, γδ T cells can be directed to attack a broad range of tumor types whether or not the target cells have the ImmunoTox genetic variation. Once cells are activated by ImmunoTox-modified cells, even if the modified cells represent only a fraction of the entire tumor, these charged up γδ T cells can kill all the primary tumor and even secondary tumors or metastases that have never been modified by our lentivirus vector.

Have Others Tried to Develop γδ T cell Therapies for Cancer?

Efforts to understand human γδ T cells and their effects on cancer go back more than 2 decades beginning with the discovery of small molecules (phosphoantigens) that stimulate γδ T cell proliferation and activate their capacity for tumor cell killing. Phosphoantigens are normal cellular metabolites and chemical inhibitors of enzymes acting on phosphoantigens causing them to accumulate in tumors and stimulate γδ T cells. Early studies proceeded with the idea that phosphoantigens or enzyme inhibitors (known as aminobisphosphonate drugs) could be administered systemically through intravenous injection or oral delivery, along with T cell growth factors known as cytokines. The combination would stimulate γδ T cells and activate their tumor killing mechanism.

 

Several clinical trials were conducted in the past 20 years to test the phosphoantigen or aminobisphosphonate plus cytokine methods for activating γδ T cell killing of tumors. In most cases, the studies showed clear evidence for in vivo stimulation of γδ T cells and in some cases showed some evidence for tumor regression. Cancers tested during these studies of positive γδ T cells included breast, prostate, lymphoma, colorectal carcinoma, renal cancer, and myeloma. Here, you can see the breadth of cancer targets that we discuss as a strength of the ImmunoTox program. These studies did not progress to late stage trials and did not produce approved cancer therapies because treatment potencies were not high enough and cytokine injections were very unpleasant with some toxicity. The obstacles of potency and adverse effects of therapy stalled the progress of γδ T cell cancer treatments. AGT saw these difficulties as technical barriers and addressed them through the development of better technology.

How Does AGT’s Technology Work to Improve the γδ T cell Attack on Cancer?

The ImmunoTox vector (in fact, a family of vectors specialized for individual clinical situations) penetrates tumor cells and inserts a new gene into the cancer cell genome. This gene suppresses the expression of enzymes that use up phosphoantigen causing accumulation of the cancer danger signal to very high levels. When a cancer cell has very high levels of the phosphoantigen due to the effects of ImmunoTox, it is potent for stimulating γδ T cells that were passing by in the blood stream. AGT has used many laboratory tests to confirm the activation of γδ T cells that are exposed to cancer сells carrying ImmunoTox. In an early experiment (Figure 1), we used the first ImmunoTox vector (LV-ImmunoTox) to insert the suppressing gene into PC3 human prostate cancer cells or HepG2 liver cancer cells. As a control, we used lentivirus vector carrying a junk sequence with no function. When the genetically modified tumor cells were mixed with γδ T cells fresh from human blood (here labeled as Vδ2), we saw a big increase in the production of a cytokine (TNF-α) that is used as a marker for this type of cellular activity. Comparing the amounts of cells expressing TNF-α after being exposed to control or ImmunoTox-modified tumor cells, we see a large increase in γδ T cell activation (red circled areas) only when tumor cells were modified by ImmunoTox vector.

Figure 1

figure-1

Based on experiments like this one and direct tests for tumor cell killing, AGT developed a model to explain what we expect from the clinical use of ImmunoTox vectors (Figure 2). We know it will be impossible to deliver the ImmunoTox vector to every cell in the tumor. This practical problem has stalled previous strategies for viral vector therapy of cancer. However, the way that ImmunoTox works is different and does not require the blanket coverage. Once ImmunoTox modifies some of the tumor cells, it will initiate a powerful activation of γδ T cells (T), shown in the figure as normal blue tumor cells turning to red modified tumor cells that stimulate γδ T cell proliferation and tumor killing functions (T*). The activated (T*) cells will now attack all cells in the tumor. This is an important key to ImmunoTox function: once the γδ T cell is activated, killing is not restricted to the modified tumor cells but will go after all tumor cells. This results in tumor destruction and, if the original signal is sufficiently strong, T* will circulate, recognize secondary tumors or metastases, and attack them as well. It is important to stress, γδ T cell killing of tumors acts on a broad spectrum of cancer making this a nearly universal approach to malignant disease.

Figure 2

lv-immunotox-3

AGT has tested this concept in mouse models for cancer. Because human γδ T cells only attack human cancer, we used special mice that have no immune system of their own and can be used to study human cancers (the xenotransplant model). For our studies, we concentrated on the PC3 cancer line and tested the effects of several different versions of ImmunoTox.

 

Mice received PC3 tumors that were treated with ImmunoTox lentivirus vectors but only one group received human γδ T cells. The fully treated group (showing 6 representative animals here) were mostly (5 of 6) cured of the tumor and were disease-free when the study ended. Mice that did not receive γδ T cells were euthanized much earlier because of disease progression.

Figure 3

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These very impressive results on the function of ImmunoTox to activate normal γδ T cells and eradicate an established tumor, have been repeated in several studies and with multiple versions of ImmunoTox. These outstanding results from early stage, preclinical studies encouraged AGT to advance this program into the clinic with all due speed.

 

Hepatocellular carcinoma (liver cancer) remains a high priority target for ImmunoTox because liver cancer is often treated by cannulation of the liver to provide direct access to the tumor site. We might exploit this technique for direct delivery of ImmunoTox to the primary tumor. We are also working with advisors and key opinion leaders to guide the clinical development of this program, especially in terms of identifying other cancers that may be particularly suited for early tests of ImmunoTox.

Summary

AGT’s ImmunoTox program is harnessing the exquisite potency and specificity of γδ T cells to attack a broad range of human cancers. We do not need to discover new tumor antigens or generate new recognition molecules because we are activating T cells that naturally attack cancer but need a “booster” to eradicate established tumors. AGT has been granted 2 U.S. patents for ImmunoTox, which is emerging as an important breakthrough technology in the war on cancer, and we look forward to clinical development and testing of these exciting new genetic drugs for cancer therapy.