The liver is the site of metastases for numerous malignancies, including colorectal cancer. Surgical removal of liver metastases (LM) can cure a small number of patients (Tomlinson et al., 2007). Unfortunately, the overwhelming majority of patients with LM are unable to undergo surgery due to extensive volume of disease. Modern chemotherapy is not curative, and new treatment approaches are needed. The immune system represents a powerful defense against malignant cells. T cells can penetrate virtually every biologic space and have the power to kill malignant cells, as demonstrated by the rare spontaneous remissions of cancer. Many investigators have attempted to apply immunotherapy to treating metastatic tumors. Infusions of activated T cells have yielded promising results. Despite encouraging results with certain diseases such as melanoma, cellular based immunotherapy has failed to achieve reliable success against other solid tumors. Recently, genetic re-engineering of patient T cells has allowed investigators to produce highly specific immunotherapeutic tools for a variety of malignancies (Porter et al., 2017).
We reprogrammed patient T cells by preparing chimeric genes in mammalian expression vectors to yield chimeric antigen receptor modified T cells (CAR-T) from normal patient cells. The chimeric genes will produce a CAR, which will be expressed by the CAR-T. The Ig portion of the CAR-T allows it to recognize tumor antigens and then cytokine signaling induces T cell activation. Prior studies have demonstrated that CARs can direct T cells to attack tumor cells and initiate a self-sustaining immune response (Beaudoin, et al., 2008). The target antigen for these studies is carcinoembryonic antigen (CEA), which is primarily expressed on tumors of the colon and rectum, breast, pancreas and other sites (Midiri et al., 1985). For our studies, we selected CEA as our target, because it is expressed at high levels in the majority of primary and metastatic colorectal cancers, in addition to other tumor types (Midiri et al., 1985). This protein is expressed on normal cells of the colonic epithelium and elsewhere in the gastrointestinal tract. Furthermore, tumor cells frequently express quantitatively much higher levels of CEA, which should enhance discrimination between normal and malignant cells.
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A potential limitation of CAR-T therapy is the difficulty in delivering a sufficient number of cells to the actual tumor sites. To optimize delivery of CAR-T to liver metastases, we have developed the Hepatic Immunotherapy for Metastases (HITM) translational research platform. LM are preferentially supplied by the hepatic artery, whereas normal liver tissue receives most of its blood flow from the portal vein (Archer & Gray, 1989). The HITM platform is based upon infusion of CAR-T directly into the liver via the hepatic artery (Katz et al., 2014). A major advantage of continuous infusion is that it can be performed by portable pumps and undertaken on an outpatient basis. The pumps may be refilled weekly in the clinic, minimizing the need for clinic or hospital visits and nursing interventions. Thus, the continuous infusion was selected as the best option for practicality as well as for therapeutic efficacy.
We aim to demonstrate the safety and clinical activity of CAR-T hepatic artery infusions (HAI) in patients with large volume LM. The HITM trial shall be designed to test the safety and potential increase in tumor killing by using HAI. This novel trial has the potential to generate paradigm-changing data for the management of LM. For the rest of this case study, we shall discuss some of the preclinical activities we conducted that may support our IND submission.
In addition to preclinical pharmacological studies, general toxicity studies, and pharmacokinetic studies, FDA requires characterization testing of cell lines used to produce biologicals and development of a cell bank system (FDA, 1993). We selected PG13 cells known for their ability to produce recombinant viruses like H21G that efficiently kill a variety of human tumor cells to produce our master cell bank (MCB) under Good Manufacturing Practices (GMP). Once generated, the MCB was cryopreserved in aliquots stored in vapor phase of liquid nitrogen. Next, we performed a series of cell line testing to certify our MCB for use in viral vector preparation for Phase I/II clinical trials. Through Pre-IND consultations with the FDA and our contract testing organizations, we screened our MCB for presence of cultivable and non-cultivable mycoplasmas in accordance with United States Pharmacopoeia (USP) and European Pharmacopoeia (EP) guidelines, in vitro adventitious viruses including bovine adventitious viral agents, sterility testing using a direct inoculation method, bacteriostasis and fungistasis, detection of inapparent viruses, and DNA sequencing for species level cell identification. The FDA also recommends testing for retroviruses (FDA, 1993). We opted for ultrastructural evaluation of the cell line for viral particles using transmission electron microscopy (TEM) to detect and characterize retrovirus-like particles. Our PG13 anti-CEA MCB met the acceptance criteria for all tests. Other tests will include viability, sterility, and standard cytotoxicity assays against CEA+ and CEA- targets.
Proposed Study Parameters for IRB Review/Approval
Our in vivo studies have shown acceptable toxicity profiles to doses as high as 1×1011 T cells. Most severe toxicities were related to systemic inflammatory responses. We have found that the regional, HAI approach will minimize the systemic response to CAR-T treatment as evidenced in mouse xenograft models showing good tolerance experienced by the animals, which received systemic doses above the highest cells/kg dose planned in humans.
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For the efficacy parameters, patients will be evaluated as having a complete response, a partial response, stable disease or progressive disease at one month after the final infusion. We will evaluate metabolic response by PET scan and pathologic response by routine histology on LM biopsy samples. Patients shall also be clinically evaluated weekly during therapy but will not be coded for response until one month after the final dose. The criteria of response shall be defined in Appendices of the clinical protocol.
For the pharmacokinetic parameters, circulating levels of modified patient T cells will be monitored by flow cytometry and PCR. Blood samples shall be taken at specific time points and LM and normal liver biopsy specimens will be used to determine the degree of CAR-T tumor intrusion. Biopsy samples will be also analyzed by flow cytometry and/or immunohistochemistry. We will quantify the presence of CAR-T cells by scoring the ratio of MN14 to actin PCR, as specified by Kammula et al. (1999). For pharmacodynamic parameters, samples will be drawn for serum cytokine levels and changes in normal liver T cell populations. We will use ELISA to measure serum interleukin concentrations.
An adverse event is any adverse change from the patient’s baseline (pre-treatment) condition, including concomitant illness after treatment, whether related or not. Adverse events are reported in accordance with FDA criteria for IND safety reporting for unexpected adverse events (21 CFR Part 312.32). IRB and FDA reporting of adverse events is required only after the patient has received an initial dose of any study medication (ICH, 1994). The active treatment period is considered one month following their initial dose. Patients will be followed long-term, until death, for potentially delayed adverse events that may be treatment related. Once the patients are off-treatment, the referring physicians will be requested to provide copies of their routine clinical reports until patient death, and also to notify the study staff if there are any unexpected clinical changes in their patients that might indicate delayed toxicity. Management and evaluation are per routine by the primary referring physician. Clinical adverse events will be graded using the Common Terminology Criteria for Adverse Events (CTCAE), Version 4.0 and reported in detail.
LMs represent a significant cause of death for many types of adenocarcinoma, most commonly colorectal cancer. Current treatment strategies do not cure the vast majority of patients with LM and new therapeutic paradigms are needed. Unfortunately, the majority of patients fail to mount an effective immune response to LM. The immunosuppressive liver microenvironment contributes to this problem. Through our pre-clinical studies, we have demonstrated that a robust T cell response to LM is associated with prolonged survival. CAR-T cells engineered to express receptors specific for CEA bypass the immune system by directly providing specific anti-tumor T lymphocytes. A therapy effective against liver tumors expressing CEA could have a major impact in terms of ameliorating the clinical and financial consequences of cancer in the U.S. We believe our prior laboratory research and completed preclinical and nonclinical studies have been favorable in our pursuit to advance our long-term translational research program devoted to the development of novel treatments for LM.
- FDA. (2018). 21 CFR 312.32—Investigational New Drug Application. IND Safety Reporting.
- Archer, S. G., & Gray, B. N. (1989). Vascularization of small liver metastases. British Journal of Surgery, 76(6), 545–548. https://doi.org/10.1002/bjs.1800760607
- Beaudoin, E. L., Bais, A. J., & Junghans, R. P. (2008). Sorting vector producer cells for high transgene expression increases retroviral titer. Journal of Virological Methods, 148(1–2), 253–259. https://doi.org/10.1016/j.jviromet.2007.12.008
- FDA. (1993). Characterisation of Cell Lines Used to Produce Biologicals. FDA PTC. Retrieved from http://www.fda.gov/cber/gdlns/ptccell.pdf
- International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. (1994). Harmonised Tripartite Guideline: Clinical Safety Data Management: Definitions and Standards for Expedited Reporting E2a. Efficacy Guidelines, (October), 12. Retrieved from http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E2A/Step4/E2A_Guideline.pdf
- Kammula, U.S., Lee, K.H., Riker, A.I., Wang, E., Ohnmacht, G.A., Rosenberg, S.A., et al., (1999) Functional analysis of antigen-specific T lymphocytes by serial measurement of gene expression in peripheral blood mononuclear cells and tumor specimens. Journal of immunology. 163:6867-75
- Katz, S.C., Burga, R., Wang, L., Mooring, W., Davies, R., Stainken, B.F., Assanah, E.O., Khare, P., Ma, Q., Espat, N.J., Junghans, R.P. Hepatic Immunotherapy for Metastases (HITM) – A phase I trial of anti-CEA genetically modified T cells for unresectable adenocarcinoma. Society of Surgical Oncology.
- Midiri, G., Amanti, C., Benedetti, M., Campisi, C., Santeusanio, G., Castagna, G., … Pascal, R. R. (1985). CEA tissue staining in colorectal cancer patients. A way to improve the usefulness of serial serum CEA evaluation. Cancer, 55(11), 2624–2629. https://doi.org/10.1002/1097-0142(19850601)55:11<2624::AID-CNCR2820551115>3.0.CO;2-#
- National Institute of Cancer. (2009). Common Terminology Criteria for Adverse Events (CTCAE), Version 4.0, DCTD, CTI, NIH, DHHS. NIH Publication https://doi.org/10.1080/00140139.2010.489653
- Porter, D. L., Levine, B. L., Kalos, M., Bagg, A., & June, C. H. (2011). Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. The New England Journal of Medicine, 365(8), 725–33. https://doi.org/10.1056/NEJMoa1103849
- Tomlinson, J. S., Jarnagin, W. R., DeMatteo, R. P., Fong, Y., Kornprat, P., Gonen, M., … D’Angelica, M. (2007). Actual 10-year survival after resection of colorectal liver metastases defines cure. Journal of Clinical Oncology, 25(29), 4575–4580. https://doi.org/10.1200/JCO.2007.11.0833
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