Abstract
Uncontrolled activation of the innate immune system promotes the deterioration of neurons in different neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). T-cell vaccination (TCV) was developed by Irun Cohen and coworkers at the Weizmann Institute of Science (Israel) during the late 1970s and has been demonstrated to be an effective treatment for human autoimmune diseases and a regulator of macrophage activation in animal models. We treated seven ALS patients with this cell therapy and were able to slow or stop disease progression in the affected individuals. The median survival, which is 3.5 years, was extended to 6 years. They were also treated with autologous adult neural stem cells associated with effector T cells. The observed neurologic improvements after treatment lasted for at least 1 year. Clinical recovery in the treated ALS patients was confirmed by an independent, skilled neurologist using the ALS Functional Rating Scale-Revised (ALSFRS-R). TCV in conjunction with an autologous neural stem cell treatment might be a feasible, minimally invasive, safe, and effective approach to obtain enduring therapeutic effects in ALS patients.
Introduction
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the central nervous system (CNS). It is characterized by the early apoptosis of motoneurons in the anterior horns of the spinal cord and bulb and by the subsequent destruction of motor cortex neurons (3,7).
The onset of ALS is associated with motor dysfunction, most commonly in the upper limbs and occasionally in the lower. The onset and evolution of the disease is asymmetrical, but fatally progressing to both sides affecting all four limbs and the laryngeal and respiratory muscles. These last two motor impairments lead to death in 50% of patients within 2–3 years; 20% survive for 5 years, 10% live for 10 years after onset, and a few individuals have endured 30 years of disease progression (22).
The etiology of this disease is unknown, and the pathogenesis is poorly understood. Nevertheless, we know that some immune-mediated repair processes are activated unsuccessfully. For the past decade, a few reports have shown that replacement of dead neurons by precommitted stem cells may partially restore lost functions and improve the clinical condition of the patient (4,12,13,19).
Injury/repair microglial function appears to play a major role in the pathogenesis of ALS (13). In a previous clinical study, we reported that T-cell vaccination (TCV) treatment is capable of reestablishing control of injury/repair microglial function but failed to induce recovery of lost neural functions (14).
TCV is a proven immunotherapeutic approach that has been demonstrated to effectively repair the autoimmune condition through the development of organ immune tolerance (24). TCV has been successfully applied in patients with multiple sclerosis (MS) since 1991. This protocol was developed during the last two decades of the 20th century by Professor Irum Cohen from the Weizmann Institute of Israel. The tolerance is primarily developed against the upregulation of spleen-generated regulatory T cells and anti-ergotype, cytotoxic cells. Secondarily, there is a macrophage regulatory effect that supports the immune tolerance process (24). In ALS patients, this aspect of the treatment is assumed to be the main therapeutic effect regulating the injury/ repair function of microglia (14).
Using the TCV program, we treated nine patients with ALS. Disease progression had completely stopped in two of the nine patients in the previous 10 years and slowed down in five patients for 3–6 years. In the remaining two individuals, the progression of the disease followed the usual evolution of ALS.
To improve these results, the TCV treatment was complemented by a combined neural repair strategy called BEN (bone marrow mesenchymal stroma cells, effector T cells, and neuroblasts) (15,16).
The BEN protocol is a combined cellular therapy procedure that has been successfully used to repair the spinal cord affected by a chronic, complete functional section (15,16). The rationale behind this therapy is to rebuild the prorepair immune conditions distinctive to the acute phase of CNS trauma to allow the appropriate implant of autologous adult neuroblasts.
TCV is intended to reestablish the injury/repair reaction disrupted by the progression of ALS. The BEN approach might then be able to replace damaged motoneurons in an enduring manner.
The present article addresses the feasibility and safety of the combined TCV-BEN approach and provides an early demonstration of its usefulness in treating ALS patients.
Materials and Methods
Patients
Patients were treated at the Regina Mater Foundation and the Clinic of the Maimonides University, Buenos Aires, Argentina. The treated patients met the internationally accepted diagnostic criteria of ALS (3,7). They had between 3 and 5 years of disease evolution, and none of them required mechanical respiratory assistance. Each patient weighed above 40 kg. Female patients were not pregnant nor breast feeding. None of the patients had a secondary, active neoplasia, including basal cell carcinoma, and none of them suffered from unstable or compromising cardiovascular, respiratory, hepatic, or renal disease. The treated patients signed informed consent forms after the nature and possible consequences of the study were explained to them.
T-Cell Vaccine Preparation
The TCV protocol has been previously described (14). Briefly, patients were apheresed using a Cobe Spectra cell separator machine (Gambro). Approximately 2 blood volumes were processed from each patient using the machine's buffy coat program. The buffy coat suspension obtained had a composition of 85%; mononuclear cells (MNCs), 1.8 × 106/μl red blood cells (RBCs), and 6 × 105/μl platelets. To purify this MNC sample, it was seeded on a Ficoll-Hypaque gradient (1.077 density). The obtained cells were washed in DBSS without Ca2+ and Mg2+. After this procedure, the composition of the ring was approximately 98% MNCs, 0.2 × 106/μl RBCs, and 1 × 104/ml platelets.
This MNC suspension was cultured for 4 days in Dulbecco's minimal essential medium enriched with a partially hydrolyzed bovine brain and spinal cord (Laboratorios Villar, Argentina).
By day 5, the activated MNCs were harvested, and immune-negative T cells were selected. Harvested cells were washed and suspended in PBS plus glycerin at 5%. Aliquots of 5 × 108 cells were preserved in sterile, sealed flasks. The flasks were irradiated with 2500 Rads to inactivate the lymphocytes and kept frozen until use. An aliquot was administered every 28 days. All of the patients were immunized by IV infusion, receiving a total of 10 doses each.
BEN Protocol
The BEN protocol was applied after the third dose of TCV. The methodology has been previously published (15,16). Briefly, patients underwent a bone marrow (BM) harvest, which was performed by an experienced hematologist. BM MNCs in the harvested product were purified and split into two aliquots. One aliquot was given to the patient the same day of bone marrow extraction by selective intralesion infusion into the feeding artery. The second aliquot was used to purify and expand the included mesenchymal stromal cells (MSCs) for 21 days. On day 17 of the therapeutic cycle, the patient underwent a leukoapheresis procedure to obtain a buffy coat sample corresponding to one blood volume. Effector T cells (ETCs) against neural tissues were isolated and expanded for 4 days in the GMP cell processing laboratory (17,23). Then ETCs were harvested and split into two aliquots. The first one was given to the patient by IV infusion, and the second one was cocultured with the patient's MSCs. After 48 h, the MSCs were differentiated into neural stem cells (NSCs) (17). These NSCs were harvested and given to the patient by selective intra-arterial infusion. During the entire cell therapy cycle and thereafter, the patient received a specific neurorehabilitation therapy (9,15,16,18,20).
Quality Control and Quality Assurance of the Cell Therapy Process
All of the cell therapy procedures were performed by highly trained professionals in accordance with AABB standards in a clean room that follows the GTP final rules of the FDA, Part 1271 and cGMP regulations (23).
Safety Evaluations
Safety evaluations were performed in compliance with FDA IND regulations 21CFR 312.32. Controls were performed prior to the administration of either the TCV or BM MNCs, which was 24 h after the administration of the cells: on the day of the IV infusion of ETCs, which was 24 and 96 h after that infusion; and on the day of the intra-arterial infusion of NSCs, which was 24 and 96 h after that infusion. By using these controls, we monitored both early and late reactions.
Adverse events were evaluated by the “Common Terminology Criteria for Adverse Events (AE)” (2004) developed by the NIH. The relevant definitions are posted at http://ctep.cancer.gov/reporting/ctc.html.
If a severe adverse event (SAE) occurred, the treating physician, in consultation with the Internal Review Board (IRB), would determine whether the therapy would be continued (6,23).
Neurorehabilitation
The aim of neurorehabilitation is to orient cells such that they may repair the destroyed motoneurons and neural prolongations. Therefore, a set of techniques previously used for the stimulation of child neurodevelopment was used (Vojta, Kabat, and acupuncture) (9,18). These techniques are similar to those that use adult intellectual capacity information (neural feedback, speech therapy, labor therapy, and psychological support).
Clinical Evolution Assessment
Treatment was evaluated using the ALS functional rating scale (ALSFRS-R) (7), which is widely used to evaluate patients with ALS.
To complete the evaluation, complementary studies were performed: spirometry, hand and thumb finger power using an appropriate dynamometer, and immune reactivity against motoneurons detected in circulating blood MNCs by the lymphocyte proliferation index (14).
Physical evaluations were performed on a monthly basis. Complementary studies were performed at the beginning of treatment before any implant and during the follow-up, every 6–12 months.
Results
Safety
During the exhaustive and intensive monitoring of adverse events, nonserious, adverse events were detected for both the TCV or BEN treatments that were applied to the seven patients.
Adverse events were limited to flu-like symptoms that the patient would experience during the first three to four vaccines. These symptoms did not affect respiratory function in any of the patients (controlled by spirometry test).
Transient reduction of the hematocrit by 1–2 points was detected after bone marrow puncture in two of the seven patients. This condition did not require any treatment. Nonplatelet or white blood cell alterations were detected.
Liver, didney, cardiocirculatory, and metabolic functions were not affected.
Patient number 2 (Table 1) abandoned the treatment for family reasons unrelated to the therapy.
ALS Patients' Clinical Evolution After the TCV-BEN Treatment
ALSFRS-R, amyotrophic lateral sclerosis Functional Rating Scale-Revised. ND, no data.
Alive at the last control.
Withdrawal from treatment by own volition after the third T-cell vaccine (TCV) and the first bone marrow mesenchymal stroma cells, effector T cells, and eeuroblasts (BEN) treatment.
Clinical Evolution
The individual clinical results for the seven treated patients are summarized in Table 1. The patient median survival time, as calculated by Kaplan-Meier analysis, was 72 months; however, four of the seven patients were alive at the end of the study.
Specific tests were performed on a few patients. For patients who started cellular therapy with a diminished vital capacity (patients 1, 3, 6, and 7), an immediate follow-up of respiratory function was performed. None of the patients worsened during the period when they received TCV vaccine or BEN implant. Patients 3, 6, and 7 experienced a progression of respiratory problems 3, 4, and 12 months after completion of TCV, respectively. Patients 3 and 6 were not treated; they also refused to receive mechanical respiratory assistance and died. Patient 7 was treated with a second regimen of TCV and recovered his previous vital capacity.
The immune activity against spinal cord proteins detected in circulating lymphocytes progressively diminished during the TCV treatment. Figure 1 illustrates the autoimmune activity against the spinal cord detected by the lymphocyte proliferation index (LPI) in patient 4. The six serial lymphoplasmaphereses abruptly decreased this index to a value within the normal range. There was then a rebounding reaction that we interpret as a reactivation of the injury/repair process (14,22), as this increase was not related to any clinical deterioration. The LPI returned to normal values and slightly increased after each BEN therapy regimen. After the treatment ended, the LPI abruptly increased when the disease worsened in some of the patients. Patient 7, referred to above, was re-treated with a second TCV protocol. Following this second treatment, he experienced a positive immunological and clinical response and the disease has not progressed for more than a year.
Autoimmune response against the spinal cord detected in peripheral blood by the lymphocyte proliferation index (LPI) of patient 4. LPI indicates the absolute response number of lymphocytes against spinal cord lysate over the absolute response number of nonchallenged lymphocytes. The continuous line with diamond shapes is the curve of the immune response over time. The discontinuous horizontal line marks the limit over which an LPI number is considered a sign of autoimmune activity. The first discontinuous arrow is pointing to the LPI blood testing immediately following the end of the six serial lymphoplasmaphereses and immediately prior to the administration of the first T cell vaccine (TCV) dose. The subsequent continuous arrows indicate the LPI blood testing immediately prior to a TCV dose. The final discontinuous arrow indicates the LPI blood testing a month after the last TCV was administered. Stars are pinpointing the moment when the effector T-cell treatment followed by the neuroblast implants occurred.
The ALSFRS-R recovery that some patients experienced seems to be related to the BEN administration. This clinical gain is sustained as long as the autoimmune activity is under control. Once the autoimmune activity relapsed, clinical deterioration followed. As we demonstrated in patient 7, this phenomenon might be reversed.
Discussion
In the present report, we have demonstrated that it is feasible to treat ALS patients with a combination of two cellular therapy protocols: TCV and BEN. This combination is shown to be safe and, as we described in the immune and clinical observations, might control both the autoimmune process involved and restore some dead motoneurons with clinical significance.
Several authors have proposed that innate immunity dysfunction plays an essential role in ALS pathogenesis. CD4 and CD8 cells are also involved in the pathology and the neuroprotective reaction that counteracts this disease (1,2,5,8,10,21,22,25).
TCV is a well-known, cellular therapeutic approach that has been shown to be effective in the restoration of self-tolerance through several different processes that include the development of an anti-ergotype, lymphocyte network, increasing specific regulatory T cells and macrophage modulation (24). In a previous publication, we produced the first clinical evidence that TCV might be beneficial for patients with ALS (14). The present results support previous findings. Moreover, we also produced evidence that under some conditions, the repetition of this treatment may continue be effective in controlling the relapse in the long run.
The important and long-lasting neurological recovery observed in five of the seven treated patients may be explained by the success of BEN therapy after TCV immunomodulation. Equilibrated immune function appears to be important to achieve an improved, long-lasting neural implant, the modulation of CD4, and macrophage function, which in turn might be the key to achieving these outstanding results with this combined therapy modality (1,2,5,10,25).
The use of autologous adult cells is an additional advantage of this approach and avoids any undesired immune effects that have been observed with the use of embryonic or donor neural stem cells (11). Moreover, the method to produce autologous neuroblasts from the differentiation of MSCs, induced by the ETCs (17), allows for the expansion of the MSCs until the desired number of cells is reached. With this method, the number can be in the billions. Without the use of any recombinant cytokines, coincubation with autologous ETCs provides a fast and highly efficient method of differentiation (more than 70% of the cells became neuroblasts in less than 48 h) (17).
Finally, the intravascular infusion of the BEN combination is the most physiologically relevant way to implant cells, avoiding the poor distribution of local injection (14–16) and/or the highly invasive action of cranial trepanation. Moreover, this method seems to be the safest way to implant large numbers of neuroblasts in the CNS with a wider tissue distribution than other methods.
Conclusion
TCV associated with autologous neural stem cell treatment might be a feasible, minimally invasive, safe and effective approach to obtain enduring therapeutic effects in ALS patients.
Footnotes
Acknowledgments
The highly qualified professional assistance of E. Perez, M.D., E. Galindes, M.D., Professor V. Delgado, M.D., Professor R. Fernandez Viña, M.D., R. Vrsalovic, M.D., and Professor G. S. Shuster, M.D.; the emotional support of Ms. Maricel Brandolino, Ms. B. María Luz Moviglia, and Dr. Ernesto Goberman; and the economic support of Regina Mater and the Scientific Foundation Felipe Fiorellino are all gratefully acknowledged. The authors declare no conflicts of interest.