The overall goal of the proposed studies is to utilize human gene therapy approach using human embryonic stem cells to direct our body’s defenses to specifically attack melanoma tumor cells. Current technologies try to accomplish this by genetically manipulating certain circulating T lymphocytes, such that they will target tumor cells. T lymphocytes are the major cell type of our body’s immune system. However it is likely that this type of approach will not result in the presence of stable, lifelong genetically modified T cells. In contrast, a potentially more long-lasting approach would be to genetically modify human embryonic stem cells with the same therapeutic gene. Stem cells have the ability to form any type of blood cell, including T cells. Importantly, stem cells can persist for the life of the individual, and thus have the potential to produce genetically modified T cells for many years. In addition, these new tumor specific cells should expand in the body in response to the presence of the tumor, thus a large supply of tumor-fighting cells should be available as long as needed. This project proposes to develop novel means to introduce the anti-cancer gene into human embryonic stem cells. These stem cells will then be differentiated to generate tumor specific T cells utilizing animal model systems. We will then use several laboratory and mouse models to determine if the T cells derived from these genetically modified stem cells have anti-tumor activity. If successful, we will have provided proof-of principle that long-lived stem cells have the potential be utilized as a means of producing anti-cancer T cells. In the long run, these results could provide important information for design of future clinical trials designed to produce life-long improved anti-cancer immune responses.
We propose to use human embryonic stem cells to develop a novel, yet potentially very effective method to treat invasive melanoma. Melanoma is a serious type of skin cancer which, if not removed early, spreads internally and is usually fatal. Overall melanoma is the 6th most common cancer in males and 7th in females and the incidence of this form of cancer is currently increasing at an epidemic rate. Although melanomas may occur in areas of skin that are not normally exposed to sunlight, sun exposure is believed to be a factor in about 70% of new cases. California’s mild winters and high number of sunny days provide opportunities for a number of occupational and recreational outdoor activities, and people in California are exposed to more than average levels of solar radiation. Consequently, there is a higher risk of developing this disease. As a matter of fact, California is one of the five US states with the highest predicted incidence of new cases of melanoma. According to the California Cancer Registry, each year 4,700 new cases of invasive melanoma and over 800 deaths related to this disease are reported in California, with the incidence rate increasing by 15% over the last decade. While the white population is at the greatest risk of developing this disease, it was recently reported that the rates of invasive melanoma have risen substantially in Hispanic people living in California as well. If our proposal is successful, our work could pave the way to the development of a new and effective form of melanoma therapy, one which would clearly benefit all the people of California affected by this disease.
SYNOPSIS: This is a well focused and developed proposal from a junior investigator to pursue 2 specific aims:
Aim 1: Generate hESC-derived transgenic T-cells expressing a high affinity T-cell receptor for MART1 melanoma antigen. The investigator has cloned a T-cell receptor (TCR) specific for the MART-1 melanoma-associated antigen. They have assembled the coding sequences for the TCR into a multi-cistronic lentiviral vector which also includes the coding sequences for a truncated tk to visualize tumor cells in vivo and EGFP for selection of the transduced hESC population. Dr. Galic has recently published a description of two-step T-cell differentiation protocol which involves generation of CD34+ from hESC in vitro with further differentiation of these cells into mature T-lymphocytes following injection into immunodeficient mice having human Thy/Liver grafts.
Aim 2: Assess functionality of tumor specific naïve T cells in vitro and in an in vivo tumor challenge model. Mature, tumor specific T cells generated through the protocol described in Aim 1 will be infused into animals bearing a xenograph of ahuman melanoma. Localization of the lymphocytes to the tumor and potential tumor regression will be used as end points to evaluate the functionality of the genetically engineered T-lymphocytes derived from hESC.
SIGNIFICANCE AND INNOVATION: This is a highly innovative proposal by a relatively junior, albeit established, young investigator who has assembled an experienced group of collaborators to help with this project. It represents a new approach to deriving tumor specific T-lymphocytes that is well worth pursuing. Melanoma do indeed represent a clinical challenge of increasing frequency and therefore the results of this research should be highly significant.
Tumor immunotherapy has recently focused on genetic transduction of peripheral T cells to express anti-tumor T cell receptors (TCRs) and adoptive transfer of these effector cells to patients. Besides generation of autoreactive T cells (since the adoptively transferred T cells have not undergone thymic selection) and ensuing autoimmune disease, insertion of TCR cDNA into T cells already expressing endogenous TCR chains will generate and TCR chain mispairings and a reduced yield of functional and specific anti-tumor TCRs. Further, because of the finite, relatively short lifespan of peripheral T cells, stable (ie life-long) anti-tumor immunity will not result. The current application proposes to circumvent these serious limitations by genetically engineering human embryonic stem cells (hESCs) (not pre-existing T cells) to express anti-tumor TCRs against the MART-1 melanoma antigen (as well as the reporter/suicide gene for herpes simplex virus thymidine kinase (HSV tk)), expanding and differentiating the transduced hESCs into hematopoietic stem cells (HSCs) in vitro, and then allowing positive and negative thymic selection in SCID-hu mice bearing human thymic implants; mice bearing human fetal liver implants will provide negative-control cells. The hESC-derived MART-1-specific anti-tumor T cells isolated from the foregoing animals will then be adoptively transferred to syngeneic SCID mice bearing MART-1-expressing melanoma xenografts, with the anti-tumor activity assessed in terms of reduction in size of the targeted tumor. In addition, in vivo trafficking of the hESC-derived anti-tumor T cells will be tracked by PET using an HSV tk substrate. The significance of this proposal thus lies in circumventing the serious problems of potential autoimmune disease, low yield of functional anti-tumor effector cells, and short-term anti-tumor immunity associated with tumor immunotherapy based on pre-existing T cells genetically transduced to express anti-tumor TCRs.
The work proposed in this application is also highly innovative. The innovation of this proposal lies in using hESCs rather pre-existing T cells to generate anti-tumor TCR-expressing effector cells for adoptive immunotherapy of cancer.
-Well developed proposal in which the investigator exhibits experience with a methodology and cognizance of the various challenges both technical and biological.
-Rich research environment with senior collaborators, both intra- and inter-institutional (signed letters of collaboration are provided), having extensive expertise who are dedicated to the success of this project.
-Significant preliminary data establishing the potential feasibility of proposed approaches.
-Highly responsive to the CIRM SEED Grant RFA.
-A relatively junior, but very promising, Principal Investigator.
-Highly significant - the proposal seeks to develop a new, effective treatment for melanoma, an important and difficult cancer.
-Highly innovative - the Proposal seeks to generate TCR-specific T cells from hESCs; according to the applicant, this has not been previously accomplished.
-Ex vivo confirmation of T cell penetration into tumors - treated tumors will be harvested and analyzed for T cell content for human CD3 by PCR and/or immunohistochemistry.
-Incorporation of the viral tk gene into the vector not only to provide a means of tracking the transduced cells in vivo by PET but also to serve as a “suicide gene” and thus safeguard (through the use of anti-virals such as ganciclovir) against the possibility of malignant transformation of the adoptively transferred cells.
WEAKNESSES: Specific Aim 2 is contingent on success in Specific Aim 1. Although the generation of tumor specific T-lymphocytes with the proposed approach is a reasonable probability, failure would preclude being able to perform the 2nd aim.
Target-cell homing of adoptive transfer cells is notoriously inefficient (less than 5% following intravenous administration, with a large proportion of T cells being trapped in the pulmonary capillaries (Koehne et al. Nature Biotechnology 21: 405-413, 2003).
There is no provision in the research plan for “titration” of the adoptively transferred T cells, that is, administration of increasing numbers of T cells to different cohorts of tumor-bearing animals to determine the administered cell numbers at which tumor regression begins and then becomes maximal.
The in vivo assay of the anti-tumor activity of the TCR-specific T cells is based on size reduction (by caliper measurements?) of pre-existing tumor grafts. However, because immunotherapy of cancer is generally rather ineffective against such bulky disease, this approach may actually underestimate the effectiveness of the therapeutic approach being evaluated.
Notable lack of detail on the PET aspects of the proposal. For example, the PET scanner, the PET radionuclide and radiotracer, the administered activity, the timing of administration of the radiotracer relative to the adoptive transfer of the hESCs etc are not specified.
Possible difficulties and alternative plans not adequately described.
Although the work proposed includes PET and PET radiotracers, “Radioisotopes” are not checked, “Yes,” in the “Biosafety” section (page 12).
The total of the Proposed Budget, $642,501, appears to exceed the specified limit of $500,000 of the CIRM SEED Grants (2 years at $200,000 per year maximum Direct Costs plus $50,000 (25%) per year maximum Indirect Costs). Although the Supplies budget (Item 6. Imaging) includes “contrast agents for CT scans,” CT scanning is not described in the narrative of the proposal.
The proposal could benefit from a modification of the in vivo tumor-regression model by: 1) administering the hESCs at the same time as (or even before) adoptive transfer of the hESCs so that the tumor-cell burden would not abrogate a potentially effective (ie measurable) immunotherapeutic response and 2) genetically transducing the targeted tumor cells (and not the immune effector cells) to express FLuc and thus provide a sensitive (ie more sensitive than palpation), quantitative measure of tumor burden and therapeutic response.
DISCUSSION: This is a highly innovative proposal, highly responsive to the RFA, from a junior investigator. The work is highly significant as well, however the technical issues concerning the xenograft model tempered enthusiasm. In the tumorigenicity model, pre-formed tumors are targeted that in general are not well treated by therapeutics. There is no test of T-cell dosage (i.e., no titration of the anti-tumor T cells) which might give clues as to the inefficiencies of targeting. Titration is likely to be essential for success.
There was discussion over whether this work could be done with cells other than hESCs, but this PI has a track record in hES work and the therapeutic potential of the work was thought to be well-supported in the application.