Gene and Vaccine Therapy Funded Projects

CLL is a highly variable disease. At diagnosis some patients have “early stage” disease, with a small amount of CLL in their bodies and no symptoms related to CLL. From this point the disease could remain stable for years to decades, or grow quickly and require treatment. We have an algorithm that can predict how soon treatment will be needed (i.e. identify “high-risk” early stage patients).

Even patients with “early-stage” CLL have immune dysfunction, increasing their risk of infection and development of other cancers. Treatment at an early stage may be more effective for patients and have fewer side effects. Therefore, earlier treatment in high-risk early-stage patients may be superior to the traditional approach of treating only when CLL is causing symptoms.

Other patients with CLL are receiving ibrutinib treatment, a tablet that effectively controls, but does not eradicate, the CLL. Patients take ibrutinib life-long. We hope that adding “consolidation” therapy to ibrutinib will eradicate the CLL and allow us to stop ibrutinib therapy; this will reduce cost, side-effects, and the likelihood that the ibrutinib will stop working.

This is a phase II (designed to assess how effective a treatment is) clinical trial of INVAC-1 in 2 groups, early stage, high-risk and ibrutinib consolidation. INVAC-1 is a new type of immune therapy. It works by teaching the immune system to attack a protein called hTERT that is used by CLL cells during their growth and division, but not used by normal cells. Patients will receive injections of INVAC-1 once/month for 6 months, then have a bone marrow aspiration to determine whether the treatment has been effective in eradicating CLL. Injections are into the skin with a device that produces a high-pressure water jet (i.e. needle-free). There have been no significant side effects in previous Phase I studies (designed to assess side effects and dose of medication).

CLL is currently considered incurable with standard treatments. Complete remission can be achieved in the majority of previously untreated patients with the first treatment; however, their disease will eventually return. To make further progress in treatment of patients with CLL, new treatments with novel and complementary mechanisms of action to chemotherapy are needed.

Immune-based therapy holds great potential as a new treatment and is the premise of this project. In this immune-based therapy, leukemia cells from patients with CLL are taken and exposed in the laboratory to a genetically altered virus that carries a gene for a protein (ISF35) that can function as an immune-stimulating protein.

Upon infection with this virus, the CLL cells express the ISF35 on their surface and in doing so, acquire the ability to stimulate the immune system to react against the leukemia cells. These cells are given back to the patient as a vaccine to stimulate the patients’ own immune system to react against and eliminate the leukemia. We have done a clinical trial with a single dose of modified cells and showed that this is a safe and active strategy for treatment.

We have planned a clinical trial with repeated infusions of the modified cells to evaluate the effectiveness of this treatment strategy in treating patients with CLL. In this trial, 20 patients will receive a total of 5 infusions of modified cells. The working hypothesis is that clinically significant, sustained remissions will be observed in patients treated with repeated doses of ISF35-modified leukemia cells that result from immune-mediated responses against the leukemia cells.

We originally proposed to conduct the phase II clinical trial of repeated doses of the ISF35 CLL vaccine for the Alliance project. Unfortunately, the pharmaceutical company that was sponsoring the clinical trial, Memgen, terminated the study before we were able to open it, even though we have been through all the regulatory steps for approval. Memgen reportedly had no financial resources for further ISF35 development at this time and therefore is not able to release ISF35, even for investigator-initiated studies.

Therefore, we focused our efforts on studying T cells from patients with CLL in order to understand the abnormalities in these cells that prevent effective immunization in these patients and prevent or dampen immune responses that are induced by vaccines, such as ISF35. By understanding these defects we may be able to develop strategies to bypass the defects or augment T cells responses in order to have a more productive T cell immune response. We have also been evaluating the T cell responses against leukemia cells in patients treated on the phase I ISF35 clinical trial.

We discovered that the leukemia cells of all patients with CLL have a protein on their surface called ROR1. This protein is an enzyme that is normally present only on cells in fetal life and is not found on normal lymphcytes, including the lymphocytes from which the CLL cells are believed to have originated.

We made this discovery by examining the anti-leukemia antibodies made by patients who had received infusions of their own CLL cells, which were genetically engineered to make an immune stimulant (called CD154) in gene therapy studies conducted at UCSD. The anti-leukemia antibodies made by some of these patients reacted with leukemia cells, but not normal lymphocytes. Some of these antibodies reacted with ROR1 to shut off the capacity of this enzyme to promote leukemia-cell survival. As such, ROR1 represents a leukemia-specific antigen that can be targeted by the immune system to generate an anti-leukemia immune response in patients with CLL.

We are developing novel cancer vaccines that can induce specific immune responses against ROR1. These vaccines will be tested in the laboratory and in experimental animals to determine which of these vaccines is/are most effective in inducing an immune response against the leukemia cells of patients with CLL. The best candidate vaccine(s) will be made in sufficient quantities to perform preclinical studies that are required by the Federal Food and Drug Administration to allow for clinical studies in patients with CLL. It is projected that at the end of the two-year period of funding, we will have an optimized vaccine for clinical studies in patients with CLL.

We generated several different types of vaccines intended to induce immune responses against ROR1, a protein that we found was made by leukemia cells of patients with CLL but not on normal adult cells. We examined the structure of these vaccines and tested for their capacity to make the desired protein. We used these vaccines to immunize experimental animals and compared them in their ability to elicit antibody responses against ROR1.

We are comparing vaccines that involve simple pieces of DNA, or more elaborate systems, that use an inactive cold virus to insert the vaccine into cells. We are comparing the relative efficiency of each to determine which vaccine best can be used in planned clinical trials involving patients with CLL.

Various acquired functional abnormalities are responsible for the behaviour of malignant cells. Members of the receptor tyrosine kinase (RTK) families contribute to the malignant cell behaviour; these enzymes are important structures to be explored for new therapeutic interventions.

RTK has not been previously described in CLL. However, in a recent report on the gene expression profile of CLL, we noticed that the RTK ROR1 was markedly increased compared to normal B-cells. Based on this preliminary observation, we have initiated a large project to characterize ROR1 in CLL and to develop agents targeting ROR1.

There is not much information on the function of ROR1 and few tools are available for analyses e.g. monoclonal antibodies. An important part of the study is to raise antibodies against ROR1 for analytical purposes.

Expression patterns of ROR1 protein will be measured in CLL as well as other leukemias and in normal cells. Activated forms of ROR1 will also be evaluated in these samples. Modifications of the ROR1 protein that can be seen in similar proteins with abnormal function will also be studied.

ROR1 is present on the surface of CLL cells; monoclonal antibodies will be generated against different parts of the surface ROR1. Preliminary results are promising, and the antibodies will be tested to see if they kill CLL cells. The events which are evoked by these antibodies leading to cell death will be analysed. When a therapeutic candidate has been selected, collaboration with a biotech company will be initiated for the production of a human monoclonal antibody for treatment.

ROR1 is a promising target for specific treatments of CLL, not only antibodies but also later small molecules and vaccines.

We have identified a new potential target for CLL therapy – the tyrosine kinase receptor ROR1. During the last year, 13 different mouse monoclonal antibodies against the external part of ROR1 have been produced and tested for specificity and their capability to induce specific cell death of CLL cells.

Three of the antibodies are excellent in inducing a specific leukemia cell death alone (comparable to rituximab but targeting another receptor). The antibodies induce cleavage of the apoptotic protein, PARP, indicating activation of an apoptotic pathway and supporting the produced antibodies in killing the leukemic cells. We have also identified a few small molecule TK inhibitors specific for ROR1 kinase activity.

In summary, three very promising CLL specific antibodies and a few small molecules have been produced which will be further explored and developed with the aim of bringing them to clinical testing for therapy. These unique antibodies and molecules against ROR1 may result in novel targeted therapeutics in CLL.

CLL treatment is becoming increasingly effective and resulting in longer disease-free survival but is currently not curative and still needs improvement. Most treatment approaches now include one or more therapeutic antibodies. Ideally, the target of a therapeutic antibody is a protein that is present at a high level on the malignant cells but is not present on normal cells. As of yet, there are no therapeutic antibodies with this profile available for CLL treatment.

Large-scale studies of gene expression have identified a number of candidate genes which code for proteins that are potential antibody targets, but few of these candidates have been validated at the protein level. We will use new technology that allows relatively cheap, small-scale preparation of reagents that will be used to validate these candidate protein targets. Targets that are also present on normal cells, and therefore unsuitable for therapeutic antibodies, can be used to improve the specificity and sensitivity of laboratory-based assays for diagnosis and monitoring of disease, and in turn, these assays can help optimize treatment.

No update is available at this time. Please check back soon.

There is evidence from cell culture experiments that CLL is in part caused by an impaired immune system of the individual patient, i.e. non-functional T-lymphocytes that are not able to attack the tumor cell. In recent years, different strategies were established to overcome this so called T-cell anergy, or lack of energy.

One promising approach with initial evidence of clinical efficacy is the use of gene-modified CLL cells, i.e. transferring co-stimulatory molecules in the CLL cells by gene vectors to make the tumor cell recognizable as a target for T-lymphocytes. Within the CLL Alliance, we will participate in gene therapy trials using different vector systems (adenovirus, adeno-associated virus) to genetically modify the CLL cell.

Our specific aim is to assess the immune reaction of the patients towards these vaccines. In the context of clinical trials, we will look for immune responses against molecules and peptides presented at the surface of the CLL cells. This analysis will be based on previous work where we identified several of these immunogenic structures on the CLL cells in lab experiments (fibromodulin, CD229, MDM2 etc.).

We will also check the profile of genes expressed in immune cells, i.e. in T-lymphocytes, before and after vaccination and correlate this with the clinical outcome of the patients. This will give us insight as to which important genes are necessary for an effective immune therapy.

In the future, we might be able to predict treatment outcomes for the individual patient. Once we are able to define what the critical targets are on the CLL cells and we know the immunologic profile of patients who benefit from this kind of gene therapy intervention, we might be able to further design more tailored vaccination therapies for patients with CLL.

No update is available at this time. Please check back soon.E