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Clinical Cancer Research Vol. 12, 1229-1236, February 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Clinical

Adoptive Transfer of Allogeneic Cytotoxic T Lymphocytes Equipped with a HLA-A2 Restricted MART-1 T-Cell Receptor: A Phase I Trial in Metastatic Melanoma

Lone Duval1, Henrik Schmidt1, Keld Kaltoft3, Kirsten Fode1, Jens Jorgen Jensen2, Steen Mellerup Sorensen2, Michael I. Nishimura4 and Hans von der Maase1

Authors' Affiliations: Departments of 1 Oncology and 2 Radiology, Aarhus University Hospital; 3 Department of Human Genetics, University of Aarhus, Aarhus, Denmark; and 4 Department of Surgery, University of Chicago, Illinois

Requests for reprints: Lone Duval, Department of Oncology, Aarhus University Hospital, Noerrebrogade 44, 8000 Aarhus C, Denmark. Phone: 45-8949-2639; Fax: 45-8619-7109; E-mail: duval{at}as.aaa.dk.


   Abstract
Top
 Abstract
Patients and Methods
 Results
 Discussion
 References
 
Purpose: We did a phase I dose-escalation trial to evaluate the feasibility and safety of intratumoral injections of C Cure 709, an allogeneic, continuous CTL cell line that, restricted by HLA-A2, recognizes MART-1-positive tumor cells through transduction with a T-cell receptor encoding gene.

Experimental Design: Cells were administered intratumorally in four dose levels ranging from 108 to 109 cells/d on days 1, 4, 7, 10, 14, and 28 of each treatment cycle to patients with metastatic melanoma. Main inclusion criteria were HLA-A2 tissue type, MART-1-positive tumor cells, and metastases suitable for ultrasound-guided injections. Patients were assessed for toxicity and response. Three to six patients were treated per dose level. Patients without progressive disease were offered up to three treatment cycles.

Results: Fifteen patients received a total of 24 treatment cycles with a total of 266 injections of C Cure 709. Toxicity was minor to moderate and most common injection site reactions were fever, fatigue, nausea/vomiting, and arthralgia/myalgia. Side effects disappeared in general within 24 hours. Toxicity was not dose dependent. One patient obtained a partial response, encompassing both metastases used and not used for intratumoral injections. Remaining patients did not achieve an overall response. In addition, we observed local regression of metastases used for injection in two patients and of metastases not used for injection in one patient.

Conclusion: Intratumoral injections of C Cure 709 are feasible, safe, and capable of inducing tumor regression. Further investigation in a phase II setting is warranted.

Adoptive immunotherapy in cancer has shown promising clinical results by selection and amplification of autologous, antigen-specific lymphocytes before reinjection into patients (1–4). Specificity of the transferred lymphocytes relies on the T-cell receptor that equips each lymphocyte clone with reactivity against one and only one antigen (5). Further development within this field has been by assignment of specificity to T lymphocytes via experimental introduction of T-cell receptor encoding gene sequences and challenge of these lymphocytes against target cells (6, 7). Preclinical studies suggest that the use of genetic receptor transduction in lymphocytes is feasible and may eradicate target cells in a variety of cancers (8–13). Passive immunization (i.e., the transfer of cells that are specific in their recognition of tumor cells as well as initially activated) theoretically implies several advantages compared with attempts dependent on active immunization to induce tumor cell kill [i.e., injection of antigens (14), cytokines (15, 16), or dendritic cells (17)]. First, patients with metastatic cancer are often immune compromised. Next, transfer of activated cells hypothetically circumvents the problem of tolerance. Tolerance is understood as the presence of T lymphocytes rendered ignorant toward antigen recognition due to former antigen encounter without suitable cofactors (18, 19). Obstacles in the use of autologous tumor infiltrating cells consist of the need of enough tumor tissue, time to grow cells, and lack of guarantee that a sufficient number of cells will have specificity against tumor antigens. The limited efficacy of some of the previous trials using tumor infiltrating lymphocytes might rely on these caveats (20, 21). Another problem may be barriers in the tumor microenvironment, which may hamper admission of T lymphocytes from the blood to the interior of tumors (22).

We present here the first clinical experience with C Cure 709, a continuous, allogeneic CTL cell line with an inserted T-cell receptor encoding gene sequence. C Cure 709 recognizes HLA-A2 MART-1 antigen–bearing cells in vitro with high specificity and sensitivity. The T-cell receptor encoding sequence was identified by Cole et al. (23) and was shown to confer T lymphocytes with antitumor reactivity (24). MART-1 is a tumor-associated antigen present in melanoma cells lines and melanocytes. Recognition of MART-1 by C Cure 709 is restricted by HLA-A2, which makes testing in an animal model impossible. To augment the likelihood of T-cell receptor/antigen encounter as well as bypass of inhibitory factors in the microenvironment (22), we have used a strategy of intratumoral injections of cells. Although severe graft-versus-host disease has been reported with the use of allogeneic cells (25), it seems that the induced immunologic reactions may contribute to the efficacy of transferred cells and that transfer is feasible (26, 27).

Our main hypothesis was that local administration of C Cure 709 would induce regression of metastases; i.e., it would promote tumor cell kill at sites of injection, hopefully leading to systemic immunologic reactions toward tumor cells through liberation of tumor antigens, production of cytochemokines, and proliferation of T lymphocytes with further specificities against tumor associated antigens.


   Patients and Methods
Top
 Abstract
 Patients and Methods
Results
 Discussion
 References
 
C Cure 709 construction. The melanoma-specific T-cell line C Cure 709 originates from the normal T-cell line C Cure 707, which was transduced with a melanoma-specific T-cell receptor. C Cure 707 was established in the presence of high cytokine concentration (28) from a skin biopsy from a patient with Sezary's Syndrome (29). The phenotype of C Cure 709 is illustrated by flow cytometric analyses in Fig. 1. Isotype controls are usually used to avoid nonspecific binding to cells via Fc-immunoglobulin G receptors like CD16. As can be seen from the flow cytometric analysis, C-Cure 709 cells are devoid of CD16 expression, implicating lack of Fc-immunoglobulin G receptors on C-Cure 709. All the monoclonal antibodies in the flow diagram are mouse immunoglobulin G{kappa} antibodies. In conclusion, CD16 and CD56 are thus isotype controls for the other monoclonals used in the flow diagram and CD16 and CD56 have no Fc receptor binding and no binding to C-Cure 709.


Figure 1
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  		Fig. 1. Phenotype of C Cure 709. As evidenced by flow cytometric analyses, the C Cure 709 cell line is a T cell expressing the T-cell receptor TCR-2 (TCR-{alpha}/ß). C Cure 709 expresses the subfamily BV 12 of the {alpha}/ß T-cell receptor. The cell line has CD8+ expression, which characterizes cytotoxic T cells, but not CD16 and CD56, which are found on natural killer cells. In addition, there is a high expression of the adhesion molecule complex CD11/CD18, which binds to CD54 (intercellular adhesion molecule 1). CD54 is also expressed on the C Cure 709 cell line. The expression of CD49a shows that the lymphocytes are activated T cells.

 
Like other described immortal T-cell lines (28, 30), C Cure 709 does not contain human T-cell lymphotrophic virus sequences. The A7 retroviral vector coding for the complete tumor-infiltrating lymphocytes TIL 5, T-cell receptor A7, which is restricted by HLA-A2 and recognizes the MART-127-35 peptide (AAGIGILTV), was described in detail by Clay et al. (23). This is illustrated in Fig. 2. They showed that the TIL 5 T-cell receptor can be transferred to normal human T lymphocytes and thereby recognize melanoma cells expressing both HLA-A2 and MART-1 (24). We have transduced the TIL 5 T-cell receptor to C Cure 707 by addition of the supernatant of the A7/PG13 packaging cell line followed by selection in G418 as previously described (24). This leads to the T-cell line C Cure 709, which shows stable expression of the TIL 5 T-cell receptor as outlined in Fig. 3 and Tables 1 and 2.


Figure 2
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  		Fig. 2. T-cell receptor encoding retroviral vector. Schematic presentation of the T-cell receptor encoding retroviral vector. LTR, long terminal repeat originating from Moloney murine leukemia virus. {alpha} and ß chains, {alpha} and ß chains of the melanoma-specific A7 T-cell receptor. The expression of the T-cell receptor chain is driven by the 5' LTR promoter, which also drives the expression of the neomycin phosphotransferase gene (neo) via an internal ribosomal entry site (IRES). A hybrid human T-cell lymphotrophic virus, type I/SV40 SR promoter drives the ß chain expression. The A7 vector encodes the BV 7.3 subfamily of the T-cell receptor. {psi}+, package signal; SD, splice donor site; SA, splice acceptor site; pA, polyadenylation signal. Arrows, transcription sites.

 

Figure 3
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  		Fig. 3. Specificity of C Cure 709. C Cure 709 recognizes MART-1 bearing cells in vitro with increasing production of IFN-{gamma} along with increasing concentration of cells. The amount of IFN-{gamma} was measured after 20 hours. T2, a transporter associated protein–deficient HLA-A2+ T/B hybrid lymphoblastoid cell line.

 

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  		Table 1. HLA-A2 restricted tumor cell kill of the melanoma cell line 888 by C Cure 709 after 4 hours

 

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  		Table 2. Cytokine production induced by C Cure 709 antigen recognition and kill of melanoma cell line

 
Figure 3 shows that C Cure 709 (106 cells/mL) specifically recognizes T2 cells (in the ratio 1:1) pulsed with the MART-127-35 peptide. T2 is a transporter associated protein–deficient HLA-A2+ T/B hybrid lymphoblastoid cell line (i.e., a cell line that cannot transport endogenous peptides to the surface but can bind exogenous peptides like MART-127-35; ref. 31).

To investigate whether C Cure 709, in addition to the MART-1 specificity, is also able to kill cells, the HLA-A2 melanoma cell line 888-A2 (target 105 cell/mL) was added to C Cure 709 as shown in Table 1. This table shows melanoma cell killing within 4 hours of the HLA-A2+, MART-1+ tumor cell line 888-A2 by the effector cell line C Cure 709, whereas C Cure 709 does not kill the melanoma cell line 888, which expresses MART-1 but not HLA-A2 (32). The parental cell line C Cure 707 does not kill 888-A2 (data not shown). Killing of the 888-A2 melanoma cell line is associated with cytokine production. Table 2 shows the constitutive and induced cytokine production of C Cure 709 with and without stimulation by the melanoma cell line 888-A2.

For the present protocol, C Cure 709 was cultured in a medium composed of RPMI 1640 with 2.5% human serum and 100 ng/mL interleukin 2 (IL-2; Proleukin, Chiron, the Netherlands).

Study design. The clinical evaluation of C Cure 709 was done as an open-labeled, nonrandomized, dose-escalation phase I trial in which cells were injected intratumorally to patients with metastatic melanoma. The primary objective of the study was to assess the feasibility and safety of intratumoral injections of C Cure 709. Secondary objective was to assess clinical efficacy. Criteria of eligibility were histologically verified metastatic malignant melanoma; HLA-A2 tissue type; MART-1 positivity as examined by immunohistochemistry (Melan A, clone A103, DAKO Cytomation) in a recent tumor biopsy; Eastern Cooperative Oncology Group performance status of ≤2; measurable disease; accessible sites of disease for ultrasound guided injections; and sufficient renal, liver, and bone marrow functions. Previous immune modulating treatment was allowed if terminated ≥4 weeks before starting treatment with C Cure 709. Criteria of exclusion were brain metastases, severe cardiovascular, autoimmune or psychiatric disease, and treatment-demanding infections. The national and the local ethics committee as well as the Danish Health Authorities approved the trial. A written informed consent was obtained from each patient. The study was carried out in accordance with criteria of Good Clinical Practice.

Dose levels. We lacked knowledge of the amount of cells that, in the human organism, could (a) initiate tumor cell kill and (b) confer development of systemic antitumoral reactions. Therefore, our fourth and maximal dose level (109 cells per treatment day) was planned based on the highest possible concentration of T lymphocytes in the solution and the decision of using a volume of ~1 mL for injections. This volume was considered appropriate for injected cells to remain within the lesions used for injection. The first dose level (108 cells per treatment day) was arbitrarily chosen a factor of 10 lower than the fourth dose level and the second (2 x 108 cells per treatment day) and third dose level (4 x 108 cells per treatment day) in between the first and fourth dose level.

Treatment schedule and injection of cells. T lymphocytes were irradiated with 60 Gy 1 hour before injection to avoid cell division in vivo. The radiation dose was preclinically shown not to affect the capability of tumor cell kill/cytokine production for 1 to 2 days. Thus, the first five injections were given every fourth day (days 1, 4, 7, 10, and 14) followed by a sixth injection after 2 weeks (day 28). These 28 days represented a treatment cycle. An eventual new treatment cycle was initiated after evaluation at day 38. Irradiated cells were brought from the Department of Human Genetics to the Department of Radiology in 30-mL vials. Cell suspension was withdrawn and the volume for injection accurately measured in 2.0-mL plastic syringes. The cells were injected, ultrasonically guided, and directly into tumor lesions using biopsy needles with a diameter of 0.9 mm and a length between 120 and 200 mm. For s.c. metastases, needles for i.m. injections with a diameter of 0.8 mm and a length of 80 mm were used. Local anesthetics were applied before injection into visceral metastases.

Lesions for injections. In light of our hypothesis of inducing systemic immunologic antitumor reactions by injecting specific, activated T lymphocytes locally into metastases, it was obvious that injections could and should only be given to a limited amount of metastatic lesions. However, one could imagine that not all lesions contained the same amount of immune cells necessary for antigen uptake and antigen presentation. We therefore aimed to use two metastases for injection and, if possible, to distribute injections between visceral and s.c./lymphoid lesions. Metastases present in lungs or spleen were not used for injection because of the high risk of complications. Metastases for injection were chosen among measurable lesions identified by computer-assisted tomography scans done at a maximum of 4 weeks before initiation of treatment. Besides our attempt to distribute injections between visceral and s.c./lymphoid lesions whenever possible and considerations of safety, no other criteria were used in the selection of metastases.

Evaluation of toxicity. Patients were enrolled consecutively in cohorts of three patients according to the dose-escalation plan. Toxicity of the first cycle was evaluated in all three patients before enrollment of patients at the next dose level. In case of clinically significant grade 3 to 4 adverse events, another three patients were required at the same dose level. Toxicity was assessed at baseline and days 7, 14, 28, and 38 using Common Toxicity Criteria Version 2.0 from the NIH.

Evaluation of efficacy. All patients had computer-assisted tomography scans of thorax, abdomen, and pelvis or, alternatively, magnetic resonance imaging before and after each treatment cycle. The overall response to treatment was assessed at day 38 according to Response Evaluation Criteria in Solid Tumors (33). In absence of progressive disease, patients were offered up to three treatment cycles. A significant local regression, not classifying for an overall response, was defined as an at least 30% reduction of the longest perpendicular diameter of a metastatic lesion either used or not used for injections of C Cure 709. Response was reconfirmed after a period of at least 4 weeks and subsequently validated by an independent review. Data on survival and response were updated October 2005. Median survival was calculated by use of Kaplan-Meier statistics.


   Results
Top
 Abstract
 Patients and Methods
 Results
Discussion
 References
 
Fifteen patients were included between November 2002 and May 2004. Median age was 54 years (range, 24-63 years). Three patients had metastases limited to lymph nodes and skin whereas the remaining patients had involvement of visceral metastases including liver and lungs. Three patients had received prior systemic treatment for metastatic disease (IL-2) and one patient had received IFN-{alpha} in the adjuvant setting. Table 3 summarizes patient characteristics. A total of 24 treatment cycles with a total of 266 intratumoral injections were administered. Apart from one serious adverse event, the administration of the ultrasound guided injections was feasible and safe without complications or major discomfort. Three patients were treated at each of dose levels I, II, and IV and six patients at dose level III due to a grade 4 toxicity in patient no. 8.


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  		Table 3. Patient characteristics

 
The median survival time was 6.7 months (range, 1-25 months) with two patients still alive at the time of analysis at 17+ and 29+ months of follow-up, respectively.

Toxicity. Symptoms not present at baseline or worsening of symptoms already present were considered as adverse events and registered as the worst grade observed throughout treatment cycles. Table 4 presents the number of patients in which a given adverse event was observed as distributed among dose levels and according to minor to moderate (grade 1-2) and severe (grade 3-4) adverse events. Toxicity was in general limited to grade 1-2 events, besides most common injection site reactions of fever, fatigue, nausea/vomiting, and arthralgia/myalgia. Side effects disappeared in general within 24 hours. Representing possible reactivity against self-antigens and autoimmunity, we observed two incidences of vitiligo and one possible worsening of hyperthyroidism. Two serious events were registered. Patient no. 3 (dose level I) had a thrombophlebitis of the deep veins in the left arm, which had no effect on the amount of treated patients at the first dose level as it occurred late in the second treatment cycle, and thus patients were already ongoing at the second dose level. No other patients experienced thromboembolic complications. Patient no. 8 (dose level III) experienced septicemia after an injection through the gut into a retroperitoneal lymph node metastasis (fifth injection in the first treatment cycle). This patient had simultaneously pronounced progression of metastases in the abdominal cavity. The serious adverse event was registered as possibly related to the study treatment and, consequently, another three patients were included at the third dose level. No further serious adverse events were observed. The toxicity between dose levels was not significantly different (Table 4).


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  		Table 4. Number of patients who experienced adverse events throughout treatment

 
Efficacy. All patients were evaluable for response. One patient (no. 15) experienced a partial response. The remaining patients did not achieve an overall treatment response.

Additional observations. Two patients (no. 6 and 10) had a significant local tumor regression of metastases used for injections and one patient (no. 9) had a significant local tumor regression of metastases not used for injections. These observations are described in the following.

Patient no. 6. This patient had lung and liver metastases. The first treatment cycle was administered in one of two liver metastases. Early in the second cycle, lack of previously observed symptoms, such as fever and chills, made it questionable whether the metastasis used for injection any longer contained target cells. To ensure the likelihood of C Cure 709 and tumor cell encounter, we chose to use the other liver metastasis for injections. Computer-assisted tomography scans done 1 year later remained without signs of active disease in the liver. However, the patient developed new lung metastases and by that had progressive disease.

Patient no. 9. This patient had lung metastases as well as retroperitoneal and inguinal lymph node metastases. The C Cure 709 cells were injected into retroperitoneal and inguinal lymph node metastases. At evaluation 4 weeks after the third treatment cycle, the patient had progressive disease due to new lymph node metastases in the left supraclavicular region. In one of the lung metastases present at baseline, a significant local regression was observed.

Patient no. 10. This patient was treated with two cycles of C Cure 709 administered in lymph node metastases of the left axilla and supraclavicular region. Magnetic resonance imaging scans done after the first treatment cycle showed a significant local regression of the metastasis used for injection located in the left axilla. The patient developed brain metastases (progressive disease) during the second treatment cycle.

Patient no. 15. This patient had three treatment cycles for bilateral neck lymph node metastases. Right and left angular lymph node metastases were chosen for injections (Fig. 4A) during the first treatment cycle, in which the patient experienced fever, injection site reactions, and fatigue. Symptoms emerged after the third injection. Magnetic resonance imaging done after the first cycle visualized huge edema in injected regions, weaker delineation of the injected angular lymph node metastases, and shrinkage of right submandibular lymph node metastases not used for injections (Fig. 4B). As observed in patient no. 6, the lack of previously observed symptoms during the second treatment cycle made it questionable whether the metastases used for injection any longer contained target cells. To ensure the likelihood of C Cure 709 and tumor cell encounter, we chose to use two other metastases for injections. At evaluation 4 weeks after the third treatment cycle, the patient had a partial response, which was reconfirmed with further regression of the metastases 1 month later (Fig. 4C). Four months after termination of treatment, the patient developed progressive disease by growth of metastases in the right parotic region and at left subclavian lymph nodes.


Figure 4
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  		Fig. 4. Magnetic resonance imaging scans before, during, and after treatment with C Cure 709 in patient no. 15. A, right and left angular lymph node metastases used for injections (arrows) before initiation of the first treatment cycle. x, right submandibular lymph node metastases, which were not used for injections. B, evaluation after the first treatment cycle disclosed edema in the treated regions, less sharp delineation of the angular lymph node metastases used for injections of C Cure 709 and shrinkage of right submandibular lymph node metastases (x) not used for injections. C, evaluation 8 weeks after termination of the third treatment cycle shows significant regression of both angular and submandibular lymph node metastases.

 

   Discussion
Top
 Abstract
 Patients and Methods
 Results
 Discussion
References
 
We have investigated the feasibility, safety, and efficacy of intratumoral injections of an allogeneic T lymphocyte cell line transducted with a receptor encoding gene sequence. This treatment principle has, to our knowledge, not previously been investigated in cancer patients.

We observed one serious adverse event, which may have been associated with the injection of C Cure 709 cells through the gut into a retroperitoneal lymph node metastasis. On the other hand, we applied a total of further 265 injections to different tumor sites without complications or major discomfort. Thus, we consider the procedure feasible and safe. Toxicity was generally mild and no significant difference in toxicity between dose levels was observed. The fourth and highest dose level is considered appropriate for further investigation in a future phase II trial. Apart from two incidences of vitiligo and one possibly worsening of hyperthyroidism, we observed no signs of autoimmune reactions or graft-versus-host disease, the former being one of the greatest concerns for use of T-cell receptor gene transfer because of recognition of self-antigens (34) and the latter a well-known complication to the transfer of allogeneic cells (25). One of the patients, who experienced vitiligo, also had a local response. The observed toxicity is comparable to or less than described in other studies of adoptive cell transfer (1–4). The higher level of toxicity in these studies probably relies on the combination with chemotherapy and/or IL-2.

As for efficacy, one patient had a partial response lasting for 4 months. Interestingly, the partial response encompassed regression of metastases both used and not used for intratumoral injections of C Cure 709 cells. In addition, we observed local regression of metastases used for injection in further two patients and of metastases not used for injection in one patient. The observed findings suggest that intratumoral injections of C Cure 709 promote reactions against tumor cells abroad where the cells are initially injected, hypothetically through accelerated proliferation and circulation of host T lymphocytes, as elucidated in animal studies (12, 35). The systemic effect may also indicate that the cytokine production, related to the C Cure 709–induced tumor cell kill observed in vitro, also takes place in vivo. As opposed to specific tumor cell kill mediated directly by C Cure 709, one could hypothesize this observation as an adjuvant reaction due to injection of any cell in media. To prove that this was not the case would have demanded far more patients randomized to receive treatment with receptor-specific and nonspecific cells, a study not considered appropriate or ethically defendable at this early stage. Overall, the clinical findings support, but do not prove, that C Cure 709 induces a specific tumor cell kill through receptor-antigen interaction. To examine this, we collected tumor biopsies before and during treatment optimally from both injected and noninjected metastases and from peripheral blood samples. These analyses are ongoing and will be published separately.

Our intent was to inject C Cure 709 cells into one or two metastases throughout the treatment period for each patient. However, as aforementioned, in two patients, we suspected that the lesions used for injections no longer harbored further vital tumor cells during the second treatment cycle due to lack of symptoms related to injections. Subsequently, injections were administered in alternative tumor lesions, which again caused symptoms and further tumor regression. Thus, repetitive injections in the same metastases may hamper further induction of antitumor reactions, and this should be taken into consideration in future studies of this treatment. Furthermore, the observation speaks against regression of metastases as merely induced by host-versus-graft reactions due to injections of allogeneic cells. Had this been the case, we would not have expected symptoms to disappear nor emerge again after the shift in metastases used for injection.

It would have been tempting to investigate the efficacy of C Cure 709 in combination with other compounds from the very beginning. However, we found it important to examine the efficacy as well as the toxicity of C Cure 709 alone before proceeding to combination studies. On the other hand, animal studies have elucidated that transferred specific T lymphocytes need addition of growth factors as well as slightly modified antigens to efficiently overcome problems with host tolerance and destruction of transferred cells (36). Especially IL-2 has been shown to render T lymphocytes more cytotoxic (37) and to live longer (35). These preclinical experiences have been confirmed clinically without resulting in substantial toxicity (38). Addition of dendritic cells is another approach that has been shown to improve efficacy of transferred T lymphocytes in preclinical studies (39). Thus, it seems obvious to examine C Cure 709 in a combined schedule as the next step of the development of this new treatment principle.

In conclusion, intratumoral injections of C Cure 709 are feasible, safe, and capable of inducing tumor regression. Further investigation in a phase II setting is warranted.


   Acknowledgments
 
We thank the staff members at the Departments of Oncology and Radiology, Aarhus University Hospital, for their careful management of the patients.


   Footnotes
 
Grant support: The Danish Cancer Society, "Max and Inger Woerzners foundation," "Frits, Georg og Marie Cecilie Gluds foundation," and NIH grants CA90873 and CA100240.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Keld Kaltoft owns CellCure that produces the C Cure 709 cell line. During the study, he was involved in handling and delivering the C Cure cells but was otherwise not involved in the treatment of the patients or evaluation of the study.

Received 7/11/05; revised 10/27/05; accepted 11/30/05.


   References
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 Abstract
 Patients and Methods
 Results
 Discussion
 References
 

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