Adoptive T-cell therapy is a novel cancer treatment strategy in which T cells (a type of white blood cells) are isolated from a cancer patient, genetically modified to express tumor-targeting receptors, expanded in the laboratory, and then re-infused into the same patient.  

Several clinical trials have shown that T cells expressing chimeric antigen receptors (CARs) that target the B-cell marker CD19 can eradicate advanced B-cell leukemia and lymphoma for patients who had been unsuccessfully treated by conventional therapies. CD19 CAR-T cells’ remarkable clinical outcomes highlight the transformative potential of CAR–T-cell therapy for cancer. However, the CD19 CAR remains the only CAR that has consistently achieved robust clinical efficacy, and the basis of its functional superiority remains unclear. Incomplete understanding of the parameters that critically influence CAR functionality has limited the rational design of novel CARs. As a result, CAR development remains dependent on trial-and-error approaches that are costly and inefficient.  

To address this important limitation, we propose a high-throughput, performance-based method to generate robustly functional CARs. Products of the screening process will be systematically analyzed to determine whether specific molecular properties dictate CAR functionality.  

Results of these studies will enhance our ability to efficiently engineer novel CAR–T-cell therapeutics. CARs are synthetic proteins that bind to specific disease markers (antigens) via an extracellular sensing domain consisting of a single-chain variable fragment (scFv).  

Previous studies have shown that the scFv domain is not only critical to targeting specificity, but also affect the overall effectiveness of the CAR molecules and the T cells in which the CARs are expressed. Here, we describe a novel screening method by which CAR molecules with diverse scFv sequences will be expressed in human T cells and selected based on their ability to (1) prevent premature T-cell exhaustion and (2) promote T-cell proliferation upon exposure to antigens.  

The high-performance CARs generated through this process will be analyzed for specific properties—including tendency for spontaneous receptor clustering, scFv-antigen binding affinity and kinetics, and potential synergy between a given scFv sequence and particular co-stimulatory domains incorporated in the CAR. Results of these analyses will elucidate whether any of these properties can be specifically engineered to enhance CAR functionality.  

CAR–T-cell therapy has been hailed as one of the most promising breakthroughs in cancer therapy, and its full potential beyond the treatment of B-cell malignancies remains to be realized. Successful completion of the proposed research will significantly improve the efficiency by which highly functional CARs targeting new disease markers can be generated, thus facilitating the development of novel therapeutics for diseases that await better treatment options.  

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia and is still considered incurable, mandating development of improved treatment strategies. While most CLL patients respond to initial chemotherapy, approximately 10-20% of newly diagnosed patients and more than 50% of relapsed patients have limited or no response to cytotoxic agents.  

These chemotherapy resistant patients frequently have CLL cells that have high-risk cytogenetic abnormalities, such as deletions in the short arm of chromosome 17 (17p-) and/or dysfunctional TP53. Patients with 17p- and/or dysfunctional TP53 respond poorly to chemoimmunotherapy and have a short median survival relative to that of patients with functional TP53. As such, the therapeutic options for patients with 17p- and/or dysfunctional TP53 are limited and usually involve high-risk treatments or allogeneic stem cell transplant, which has a high risk of mortality.  

Our proposal provides a potential solution to this problem. We discovered that adenovirus vector transduction of CLL cells with ISF35 (a recombinant CD154) could enhance in vitro and in vivo the sensitivity of leukemia cells to drugs such as fludarabine in a TP53-independent manner (Dicker F. et al, Blood 2006. Castro JE. et al; ASH-2009 meeting abstract). This results from activation of another member of the TP53 family, namely P73. We found CD40 ligation activates P73 in both transduced and bystander leukemia cells and induces the CLL cells to become efficient antigen presenting cells that are capable of inducing host anti-leukemia immune responses. Activation of this alternative pathway can circumvent the resistance of TP53-deficient CLL cells to anticancer drug therapy.  

As such, cell-gene immune therapy provides for a novel strategy to re-sensitize resistant TP53-deficient leukemia cells. Patients will receive 3 infusions of autologous CLL cells transduced with ISF35 followed by only 3 cycles of chemoimmunotherapy with FCR (fludarabine, cyclophosphamide and rituximab).  

In preliminary work we have found that infusion of Ad-ISF35 transduced autologous leukemia cells causes systemic activation of bystander leukemia cells, rendering the entire leukemia-cell population sensitive to anti-cancer treatments.  

To date, two patients with refractory TP53-deficient leukemia have completed therapy on this protocol and have achieved a complete remission. Alliance for Cancer Gene Therapy funds will be used to continue this important work and to perform correlative science studies and clinical monitoring of patients who will enroll in this clinical study. The results generated in this proposal may allow for eventual approval for such gene therapy and offer new hope for patients with intractable leukemia.  

This Alliance for Cancer Gene Therapy Research Fellow is funded in part by Swim Across America.

Efficacious cancer immunotherapies will likely require combinations of strategies that enhance tumor antigen presentation and antagonize negative immune regulatory circuits. We demonstrated that vaccination with irradiated, autologous tumor cells engineered to secrete granulocyte-macrophage colony stimulating factor (GM-CSF) followed by antibody blockade of cytotoxic T lymphocyte associated antigen-4 (CTLA-4) accomplishes clinically significant tumor destruction with minimal toxicity in a majority of stage IV metastatic melanoma patients and some advanced ovarian carcinoma patients. 

The extent of tumor necrosis in post-treatment biopsies was linearly related to the natural logarithm of the ratio of infiltrating CD8+ effector T cells to FoxP3+ Tregs, suggesting that further Treg inhibition might increase the frequency of clinical responses. Through an analysis of cytokine deficient mice, we delineated a critical role for GM-CSF in Treg homeostasis.  

GM-CSF is required for the expression of milk fat globule epidermal growth factor-8 (MFG-E8), a phosphatidylserine binding protein, in antigen presenting cells, whereby the uptake of apoptotic cells by phagocyte-derived MFG-E8 maintains peripheral Treg numbers through TGF-beta, MHC class II, and CCL22. In wild type mice, MFG-E8 restrains the potency of GM-CSF secreting tumor cell vaccines through Treg induction, while a dominant negative MFG-E8 mutant (RGE) intensifies therapeutic immunity through Treg inhibition, resulting in regressions of established tumors. 

Furthermore, an orthologous human RGE dominant negative mutant similarly manifests immunostimulatory capabilities in cultures of human peripheral blood mononuclear cells. Together, these findings suggest that combinations of GM-CSF and MFG-E8 blockade (through an RGE mutant) might improve the potency of cancer vaccines and complement the activity of CTLA-4 blockade. Here, we propose to develop a standardized platform for the cellular co-delivery of human RGE and GM-CSF for use in clinical vaccination trials for patients with diverse solid and hematologic malignancies. 

Our overall approach will be to admix irradiated K562 cells engineered by plasmid mediated gene transfer to secrete RGE or GM-CSF with irradiated autologous tumor cells. Under an investigator held IND (BB-IND 11923, Glenn Dranoff, Sponsor), we already are performing Phase I trials of vaccination with lethally irradiated, GM-CSF secreting K562 cells admixed with irradiated, autologous tumor cells in several advanced tumors. We plan to manufacture K562 cells engineered by plasmid mediated gene transfer to secrete RGE, using comparable techniques as for the GM-CSF secreting K562 cells, and then to obtain an investigator sponsored IND that will support clinical evaluation of this combinatorial vaccination strategy in advanced cancer patients.

Mantle cell lymphoma carries the worst prognosis of any lymphoma. Treatment includes high-dose chemotherapy followed by stem cell transplant, which eliminates most of the lymphoma, but temporarily wipes out a patient’s immune system. Even with this aggressive therapy, residual lymphoma cells remain and cause the patients to recur within a few years. Novel approaches are needed to eliminate those residual lymphoma cells.  

Gene therapy is amongst the most promising approaches being studied for the elimination of cancer, but a primary obstacle is the difficulty in delivering the therapeutic gene to a sufficient proportion of cancer cells. Our approach of using a gene-engineered lymphoma vaccine induces the patient’s immune system to eliminate residual lymphoma cells, so that widespread gene-delivery is not needed.  

Earlier trials of such an approach have demonstrated its feasibility and some impressive initial results. The innovative aspects of our study are that it utilizes a novel, non-viral gene transfer technology and takes advantage of the unique immunologic setting of patients who have undergone stem cell transplantation. Prior to stem cell transplant, the patient’s immune system accepts lymphoma, allowing it to progress. After a patient’s immune system is wiped clean during stem cell transplant, giving an anti-lymphoma vaccine may cause it to be re-established in such a way that it is no longer accepting of lymphoma and can eliminate those few remaining cells.

Studies in recent years have documented the lytic effects of various viruses on many human cancers, and the study and re-engineering of oncolytic viruses is intensifying. Recombinant measles viruses [MV] appear to be ideal vectors for lymphoma treatment, as wild-type MV infection occasionally induces lymphoma regression in humans.  

We plan to produce viruses that replicate selectively in transformed lymphocytes, viruses with modulatable cytotoxicity, and viruses with a targeted envelope. Eventually we will combine the characteristics of the most effective viruses into a ‘second generation’ virus that is armed and targeted.

The myelodysplastic syndromes (MDS) are a group of clonal neoplastic hematologic disorders characterized by varying degrees of bone marrow failure, abnormal hematopoiesis, and proliferation of myeloid blast cells. Impaired maturation of hematopoietic progenitors is manifest clinically by peripheral cytopenias and morphologic abnormalities in the marrow (“dysplasia”). Thought to be disorders of hematopoietic stem cells, clonal cytogenetic abnormalities are frequently identified. Although the disease can evolve toward acute leukemia, morbidity and mortality most frequently result from a marrow failure syndrome.  

Evidence exists that immune activation against hematopoietic elements frequently occurs in MDS patients, based on the identification of lymphocytic infiltrates in the marrow, oligoclonal expansion of T cells, and excessive production of tumor necrosis factor alpha. Whether this represents a secondary event in response to cell injury and the generation of neo-antigens, or an initiating event inducing immunopathology, remains controversial. Nevertheless, MDS are thought to be immunologically responsive diseases, as immunomodulatory drugs can induce remissions, and allogeneic bone marrow transplantation can be curative in the small fraction of patients for whom this is an option.  

Recently, three classes of therapeutic agents have been shown to have activity in MDS; a) DNA methyltransferase inhibitors, b) histone deacetylase inhibitors (HDACi), and c) immunomodulatory derivatives (IMiDs) of thalidomide. Central to this proposal is the observation that all three classes of drugs augment discrete elements of host immunity, making their integration with therapeutic cancer vaccines ripe for exploration.  

We have developed a genetically modified tumor cell vaccine for the treatment of myeloid malignancies. The human erythroleukemia cell line K562 has been stably transfected to secrete GM-CSF. K562 cells express many of the antigens shown to be overexpressed in myeloid leukemias and MDS. In early phase clinical trials for both acute and chronic myeloid leukemias, we have observed the induction of anti-tumor immunity and associated clinical responses following K562/GM-CSF vaccination (see preliminary data). In this proposal, we seek to evaluate the integration of K562/GM-CSF vaccination with systemic therapies for MDS that alter host immunity and/or hematopoietic cellular differentiation. Specifically, we will:  

1. Examine the in vivo effect of: a) DNA methyltransferase inhibitors, b) HDACi, and c) IMiDs on the response to GM-CSF tumor vaccines in a mouse model (year 1).  

2. Conduct a clinical trial in MDS testing K562/GM-CSF vaccination integrated with the systemic agent(s) identified in aim 1 as being most active in combination with GM-CSF tumor vaccines (years 2 and 3). 

3. Evaluate immune responses specific for autologous MDS cells as well for as candidate antigens overexpressed in MDS using pre and post vaccination blood and marrow samples (years  

An ultimate goal of cancer immunotherapy is to activate tumor-specific T cells through therapeutic vaccinations to eradicate pre-established tumor. However, tumor-specific T cell tolerance remains one of the major barriers in cancer immunotherapy. Thus, to elicit effective anti-tumor immunity, it is necessary to develop vaccine strategies capable of overcoming T cell tolerance.  

In the previous application funded by Alliance for Cancer Gene Therapy, we have demonstrated an essential role of the innate immune system in shaping adaptive immune responses. In a series of 10 peer-reviewed publications, we have identified several parameters that are critical for the potency of a vaccine in overcoming T cell tolerance: 1) the ability of the vaccine to activate multiple innate immune pathways, leading to production of both type I interferons (IFNs) and pro-inflammatory cytokines; 2) the ability to activate both plasmacytoid dendritic cells (pDCs) and conventional DCs (cDCs); and 3) the ability to activate other innate immune cells such as NK cells, which further enhances adaptive immune responses.  

Based on these important parameters, we have demonstrated in a murine model of pre-established lymphoma that DC vaccines co-administered with the TLR9 ligand, CpG in vivo are effective in activating tumor-specific T cell response and treating pre-established lymphoma. This is probably related to the ability of CpG to activate both cDCs and pDCs and to produce pro-inflammatory cytokines and type I IFNs, respectively. In addition, CpG can also activate NK cells. 

 In this application, we will test the central hypothesis that DC vaccines co-administered with CpG in vivo are effective in activating tumor-specific CD8+ T cell response in patients with lymphoma through the following three specific aims: 1) To perform and analyze FDA required bio-distribution and toxicology studies in mice; 2) To obtain full regulatory approval and GMP manufacturing of DC vaccines to support the trial; and 3) To conduct a pilot phase I to study the safety and immunological efficacy of Epstein-Barr virus (EBV)-derived tumor antigen (LMP2) loaded DC vaccines in patients with EBV-associated lymphoma. We plan to administer LMP2-loaded DC vaccines twice intravenously.  

The boost vaccination will be administered 4 weeks after the first vaccination. GMP-grade CpG will be given intramuscularly with each vaccination. We will determine the safety and feasibility of the treatment by determining 1) clinical toxicology; 2) tumor antigen-specific immune responses; 3) although a secondary goal, anti-tumor effect will also be measured. In summary, this will be the first clinical trial designed to enhance anti-tumor immunity using a combined strategy of tumor. 

Natural killer (NK) cells mediate natural cytotoxicity against virus-infected or transformed cells. Alloreactive NK cells derived from haplotype mismatched hematopoietic stem cell (HSC) transplantation donors are used to successfully treat patients with high-risk acute myeloid leukemia (AML) without causing graft versus host disease (GVHD). However, equal benefit is not afforded to patients with B-cell acute lymphoblastic leukemia (B-ALL), suggesting that alloreactive NK cells fail to control B-ALL.  

This failure may be due to inhibitory signal-mediated resistance caused by B-ALL. To address this problem, mature NK cells can be genetically modified to express chimeric antigen receptors (CARs) specific for tumor antigen, thus harnessing their ability to kill B-ALL blasts. To date, adoptive transfer of ex vivo expanded mature NK cells has not shown therapeutic benefit in hematological malignancies, in part from the lack of target specificity and the short period of persistence after infusion.  

We hypothesize that CAR modified NK progenitors/precursors derived from CD34+ HSCs combined with HSC transplantation may provide enhanced anti-B-ALL-specific NK effectors with long-term persistence, thus likely increasing the efficacy of NK cell therapy for B-ALL. To test this hypothesis, two specific aims are proposed.  

Aim 1: To evaluate specific and enhanced killing of B-ALL in vitro and in immunodeficient mice by genetically modified NK progenitors/precursors. We will use the non-viral Sleeping Beauty (SB) transposon system to achieve integration and stable expression of CAR for CD19 antigen in cord blood-derived CD34+ cells. Transfected CD34+ cells will subsequently be differentiated into NK cells using the feeder free Glycostem clinical grade bioreactor system. As controls, we will use stromal cell co-culture derived NK progenitors and peripheral blood NK cells that are similarly modified by SB. Ex vivo generated NK cells will be evaluated for transgene expression, surface phenotype, cytotoxicity and cytokine production against B-ALL cells and patient blasts. NK cell expansion, persistence and anti-B-ALL activity in mice will be determined.  

Aim 2: To establish conditions for the production of genetically modified NK progenitors/precursors in a good manufacturing practice (GMP) facility. GMP grade NK cells will be evaluated for in vitro function and in vivo anti-leukemia efficacy. We are well positioned to complete these studies considering the extensive experience of our team in SB-mediated HSC gene transfer and NK cell therapeutics. Our strong preliminary data also support the likelihood of accomplishing the proposed aims.  

Our study is significant and innovative because our work can rapidly lead to a clinical trial for high-risk B-ALL using SB modified NK progenitors/precursors as universal “off-the-shelf” immunotherapy and can potentially be applied to the treatment of other hematological malignancies, solid tumors and viral infections. 

This clinical translational proposal aimed to assess the therapeutic potential of genetically targeted T cells in the treatment of leukemia and lymphoma. The introduction of genes that code for tumor-specific receptors into T lymphocytes, which are cells of the immune system that mediate tumor elimination, was a novel approach to cancer therapy.

Dr. Sadelain demonstrated that human peripheral blood T cells could be effectively redirected to recognize the CO19 antigen and eradicate established, systemic human B cell tumors borne by immunodeficient mice, This was the first publication showing that genetically modified human T cells can induce durable remissions in an in vivo setting.

Importantly, he found that T cell stimulation with the cytokine lnterleukln-15, but not the widely used lnterteukin-2, is critical for sustaining the therapeutic activity of infused CO19-spedflc T cells. Having examined the nature of the signals required by the T cells for their own survival and sustained functionality, Dr. Sadelain designed improved antigen receptors that provide both activating and co-stimulatory signals to the tumor-targeted T cell.

The specific alms of the 2004 application included:

  1. Demonstrating the therapeutic efficacy of leukemia and lymphoma patients’ cos+ T lymphocytes transduced with the 19/28z receptor In tumor-bearing mice
  2. Establishing clinical conditions for patient T cell purification, transduction and expansion, for review by the FDA
  3. Investigating, in a phase I clinical trial In patients with relapsed B cell leukemias, the safety, persistence and therapeutic efficacy of autologous IL15-actlvated CDS+ T cells that express 19/2Sz

Relapsed or drug-resistant leukemia and/or lymphoma are very difficult to cure using the traditional work horses of cancer therapy, surgery, radiation and chemotherapy. New classes of therapies need to be developed. In my laboratory we are using the patient’s immune system to directly attack the disease. This therapy harnesses the power of T cells, a type of immune cell, and uses gene therapy to redirect the function of the T cell so that it can target a patient’s leukemia and/or lymphoma. These are exciting times for immune-based therapy and we expect that the experiments funded by ACGT will provide critical insights into designing new ways of using T-cells to treat patients.

ACGT funds were used to genetically modify T cells to redirect their specificity to desired tumor-associated antigens (TAA).  This was accomplished by the development of a chimeric antigen receptor (CAR) that redirects specificity to TAA displayed on the cell surface independent of the major histocompatibility complex (MHC).  Over the course of our funding we developed a 1st generation CAR that relies solely on CD3-ζ endodomain to activate T cells for CAR-dependent lysis (major finding #1).  This resulted in our first gene therapy trial infusing CD19-specific CAR+ T cells after lymphodepleting chemotherapy (major finding #2).  This trial showed that the design of the CAR was insufficient to fully-activate T cells for a fully competent signal and that the presence of bacterial and viral transgenes in the expression vector led to deleterious immune-mediated clearance.  Therefore, a 2nd generation CAR was designed that could not only lyse CD19+ tumor targets, but could activate the T cells for sustained proliferation by signaling through CD28 as well as CD3-ζ (major finding #3).  We developed a new non-viral gene transfer strategy based on the Sleeping Beauty (SB) system (major finding #4) to improve the efficiency of CAR integration and together with the development of CD19+ artificial antigen presenting cells (aAPC, major finding #5), we developed an approach to generate T cells without the need to express immunogenic transgenes.  A next-generation clinical trial for first-in-human application of the SB system and CD19+ aAPC has been favorably reviewed by NIH-OBA (major finding #6) and is currently being reviewed by the FDA (major finding #7).  This upcoming trial will infuse autologous 2nd generation CD19-specific T cells in patients who are undergoing autologous hematopoietic stem-cell transplantation.  In addition to developing the scientific rational for targeted therapy using CAR+ T cells we also developed the infrastructure to translate basic immunology into applied practice by establishing a laboratory within a laboratory that operates in compliance with current good laboratory practices. This group works seamlessly with the manufacturing team to generate clinical-grade T cells in compliance with current good manufacturing practice. Furthermore, we established a clinical trials team to be able to conduct gene therapy trials, including a regulatory affairs group to handle the administrative burden of reporting to institutional and federal regulatory oversight bodies.  And, we put in place a group to undertake correlative studies to help understand the biologic impact of adoptive transfer of T cells.  In aggregate, with the help of ACGT funding we were able to develop a program to genetically modify T cells with desired specificity for CD19+ tumors and initiate a series of gene therapy trials to test the safety and feasibility of infusing CAR+ T cells.