Mesenchymal Stem Cells Infusion in Patients With Autoimmune Diseases

The goal of this study is to learn if mesenchymal stem cell therapy (treatment group) can effectively treat autoimmune diseases, when compared to normal saline (given to placebo group). The primary outcome measures will be clinical improvement based on the respective disease specific clinical scores, normalization of T-lymphocyte subsets and \> 50% reduction in disease specific antibody titres. The study will also document the type and frequency of any adverse event or side effects, reported by or seen in any of the trial participants.

Patients in treatment group will receive single session of MSC therapy and placebo group will receive 0.9% saline solution. The participants will be followed at 3 and 6 months.

The autoimmune disorders are a spectrum of diseases ranging from organ-specific, in which antibodies and T cells react to self-antigens localized in a specific tissue, to systemic, which are characterized by reactivity against a specific antigen(s) spread throughout various tissues in the body. The incidence of different autoimmune diseases is variable, according to the Centers for Disease Control and Prevention, 22.7% have rheumatic diseases. Although no population-based epidemiological study has been carried out in Pakistan purely on autoimmune diseases; a study by Mohsin Z et al. reports the combined prevalence of rheumatic diseases at a tertiary hospital in Karachi to be 17.3%. Autoimmune diseases and their treatment modalities are the prime cause of disability in developing countries and have the highest incapacitating rates in health-related quality of life (HRQoL) and daily functioning.

The 21st century has seen rapid advances in the treatment of diseases of immune dysfunction. One such treatment modality is stem cell therapy which is believed to repair and regenerate tissues. In particular, mesenchymal stem cells (MSCs) have been applied to treat diseases associated with age, changing lifestyle, immune dysfunction and stroke. MSC therapy has promise to treat various autoimmune disorders like refractory systemic lupus erythematosus (SLE), Crohn's disease, systemic sclerosis (SS), rheumatoid arthritis (RA), multiple sclerosis (MS), graft versus host disease, diabetes mellitus, thyroiditis and even different types of neurological disorders. At present, nearly a thousand clinical trials have used MSC-based therapies. Among those around one hundred trials have been conducted for treatment of immune-mediated disorders, the first one being more than fifteen years ago (table 1).

The interest surrounding field of MSCs was initially based on their inherent capacity for self-renewal and regeneration with a potential to form cells of mesodermal origin (adipocytes, osteocytes, chondrocytes, hepatocytes, neurons, muscle cells and epithelial cells) depending on the surrounding microenvironment. Later on, due to their abilities to home to inflamed areas and exert immunomodulatory effects, therapies with MSCs extended to treatment of autoimmune and chronic inflammatory processes. Multiple studies have also demonstrated that MSCs have intrinsic immunomodulatory and anti-inflammatory properties.

Table 1: Clinical trials in which mesenchymal stem cells are being used as therapeutic modality.

Immune mediated disorder Number of Clinical trials Year of first Clinical trial Reference Graft vs. host disease 49 2004 Inflammatory bowel disease 23 2006 Multiple sclerosis 29 2006 Systemic lupus erythematosus 10 2007 Type I diabetes 26 2008 Primary Sjögren syndrome 1 2009 Type II diabetes 13 2010 Autoimmune hepatitis 2 2011 Ankylosing spondylitis 2 2011 Chronic urticarial 1 2017 Refractory autoimmune thrombocytopenia 1 2019

Source of MSCs MSCs were initially identified in the BM and are commonly isolated by gradient centrifugation to separate nucleated cells, followed by in vitro culture and serial passages. The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) has designated the term 'multipotent mesenchymal stromal cells' for the plastic-adherent cells found under standard culture conditions. The immunophenotype of these cells as per ISCT criteria is positive for cell-surface markers CD73, CD90 and CD105 and negative for surface CD14 or CD11b, CD45, CD34, CD79 or CD19, and HLA-DR. MSCs have also been obtained from adipose tissue, placenta, amniotic fluid, umbilical cord blood (UCB), connective tissues of skeletal muscle and dermis,dental tissue, and fetal tissues such as lung and blood. Mobilized peripheral blood cells have also been reported as a source of MScs. Although gene expression studies demonstrate that MSC populations obtained from different tissue sources are highly heterogeneous, their ability to renew, differentiate, and major functional properties, such as regulation of immunological tolerance, wound healing, inflammation and fibrosis, are common to all MSCs.

1. Effects on cells of immune system MSCs suppress T-cell proliferation induced by alloantigens or mitogens via increasing the number of regulatory T cells and lessen complications of GVHD after HSCT and immune-mediated disease. In addition, MSCs inhibit function of B cells, natural killer cells and dendritic cells. The main immunosuppressive function of MSCs is by production of induced soluble factors; however, these cells can also exert immunosuppressive effects by direct cell-to-cell interaction. The immunosuppressive capacity of MSCs is enhanced under inflammatory conditions in the presence of the proinflammatory cytokines interferon (IFN)-g, tumor necrosis factor-alpha and interleukin (IL)-6.Under immunologically quiescent conditions, MSCs promote T-cell survival and can induce the activation and proliferation of CD4 positive regulatory T cells.
2. Production of immunomodulatory soluble factors MSCs constitutively produce inducible soluble factors, like transforming growth factor-β (TGF-β), hepatocyte growth factor, nitric oxide, HLA-G59 and indoleamine 2,3-dioxygenase that mediate their effects. Large amounts of IL-6 andIL-8, and the chemokine CCL-2 are also produced. MSCs treated with IFN-γ, secrete ICAM-1, CXCL-10 and CCL-8, whereas IL-8 production is decreased. This phenomenon suggests that MSCs target neutrophils and monocytes under non-inflammatory conditions, but attract monocytes, dendritic cells, T cells and natural killer cells under inflammatory conditions. A number of studies reported TGF-βas a key mediator of immunomodulation by MSCs.
3. Proinflammatory "licensing" of MSCs MSCs have been dubbed as ''smart" immune modulators since their suppressive effects require a previous licensing step that occurs in the presence of an inflammatory environment. If MSCs are transplanted during acute inflammation, the microenvironment containing polarized M1 macrophages 'licenses' MSCs to inhibit effector T, B, natural killer and dendritic cells. In contrast, if MSCs are licensed after the polarization of M2 macrophages by Th2-type cytokines as occurs during chronic inflammation, the microenvironment provides alternative licensing and recruits MSCs to the fibrosis process.
4. MSC paracrine factors in the repair mechanism Cellular regeneration of an ischemic tissue necessitates massive cell supply, on the order of a billion for an infarcted heart, for example. Experimental studies and clinical trials have revealed that MSC-mediated therapeutic benefit might largely rely on the contribution of the secreted amounts of growth factors and cytokines rather than on their potential for differentiation into cardiomyocytes, vascular or renal cells as shown by previous study. The panel of regulatory and trophic factors secreted by MSCs include a large number of growth factors, cytokines and chemokines.

Translation of MSC knowledge into clinical application Cell therapy indeed appears to be an applicable translational strategy for autoimmune diseases.

1. Graft versus Host Disease (GVHD) In humans, the most studied application for MSCs is GVHD, a complication of hematopoietic stem cell transplantation. In 2004, a 9-year-old boy with severe treatment-resistant acute GVHD of the gut and liver was treated with third-party haploidentical mother-derived MSCs. Phase II clinical trials in patients with steroid-resistant severe acute GVHD showed a 70% initial response rate that was not related to age or HLA match. However, durable complete responses or other primary endpoints in these trials are still lacking.
2. Crohn´s Disease (CD) The first report of a phase I clinical trial of cell therapy using autologous adipose-derived MSCs in CD was published in 2005. Local injection led to healing of fistulas (6/8) with no adverse effects These results were confirmed by the same group in 2009 in a phase II multicenter in a randomized controlled trial \[24\]. Currently, there is a phase III, multicenter, placebo-controlled, randomized and blind study to evaluate the safety and efficacy of allogeneic BM-MSCs, conducted by Osiris Therapeutics. (http://www.clinicaltrials. gov/ct2/show/NCT00482092).
3. Multiple Sclerosis (MS) Phase I/II studies in patients with refractory MS has confirmed the absence of adverse effects during follow-up (6-28 months). An increase in the proportion of CD4+CD25+ regulatory T cells with decreased proliferative responses of lymphocytes and activation markers on dendritic cells was detected hours after MSC transplantation. Patients improved on measures of visual function, without evidence of significant adverse events. Progression of general disability was also reduced after treatment.
4. Systemic Lupus Erythematosus (SLE) Perhaps the most remarkable results of human MSC therapy emerge now from clinical trials aimed at severe, treatment refractory SLE. Patients with active disease and lupus nephritis that was unresponsive to monthly i.v. cyclophosphamide and oral prednisone (≥ 20 mg/day) have shown improved outcomes when treated with MSCs. The Disease Activity Index (SLEDAI) in these patients improved significantly at one, six and twelve months follow-up, as did urinary protein. In some of the trials, patients with high SLEDAI received one infusion of allogeneic BM-MSCs from passage 3-5 from non HLA matched healthy family members. Their follow-up has reached a median of 17.2 (3-36) months, with no adverse effects, deaths or ensuing GVHD. Quite surprisingly, 24 h proteinuria decreased significantly as early as one week after MSC therapy, even preceding changes in anti-dsDNA antibodies, which decreased significantly at one month and three months post MSC dose. T regulatory (Treg) cells, found to be quantitatively and qualitatively deficient in active SLE, were restored at week one as judged by the percentage of CD4+ Foxp3+ cells among peripheral blood mononuclear cells. Other trials although with shorter follow-up also demonstrate significant improvement, verified for SLEDAI score, serum albumin, 24 h urinary protein, serum creatinine, serum complement and anti-dsDNA antibodies. Undoubtedly MSC therapy must be further explored in SLE.
5. Systemic Sclerosis (SS) SS is an immune mediated disease with a prominent vascular and microvascular component often leading to ischemic complications. Since MSCs can differentiate to endothelial cells in vitro and also participate in blood vessel formation in adult tissues, therapy both with autologous and haploidentical third party donor MSCs has been reported, leading to striking improvement in two separate case reports.
6. Rheumatoid Arthritis (RA) Literature search reveals that more than a hundred preclinical studies on RA animal model and nine clinical trials on human patients have been completed on use of MSC-based therapy in RA and establish its safety and efficacy. Whereas, nine clinical trials are still ongoing. In two-thirds of these patients a single dose of allogeneic MSCs at about a dose between 1-10 x106/kg were used. It is believed that MSCs targets the pathogenic memory T cells and halt the progression of disease course in RA patients.
7. Polymyositis/Dermatomyositis (PM/DM) Polymyositis/dermatomyositis (PM/DM) is an autoimmune disease characterized by weakness of proximal skeletal muscles and obvious skin manifestations, and is known to affect multiple organs, such as muscles, lungs, and kidneys. Currently, the etiology is not known. Several studies have suggested that T helper (Th) cells are involved in the pathogenesis of PM/DM, since Th cell-related cellular dysfunction plays an important role in the occurrence and development of PM or DM. Therefore, MSCT could provide a new therapeutic strategy for the treatment of PM and DM. Several studies have demonstrated that this approach has promising clinical outcomes. In another study 32 PM and DM patients were injected intravenously with 1 × 106/kg MSCs. The results of the 9-year follow-up study demonstrated that the symptoms and serological indicators of patients improved, showing the effectiveness and safety of MSCT in PM and DM, while 11 patients died due to reasons not related to transplantation.

In another study, 81 patients with PM/DM were randomly divided into two groups: 44 patients in the control group were individually treated with glucocorticoids and immunosuppressants for 6 months, while 37 patients in the transplantation group were injected intravenously with 3.5-5.2 × 107 UC-MSCs. The results of that study showed that the creatine kinase values in both groups were significantly decreased; however, the transplantation group had better results than the control group at several time points, and the lung function was significantly improved in the transplantation group. One patient died after transplantation and no transplantation-related complications occurred. Currently, there are only a few studies investigating PM/DM, and large-scale and randomized clinical studies are needed to evaluate the long-term effectiveness and safety of MSCT in PM/DM patients, including the risks of tumors and infections, as well as the optimal transplantation dose and schedule.

Since the clinical outcome in case reports and phase I-II trials seem occasionally striking, there exists a dire need to perform structured and preferably controlled multicenter trials and document results in our own population.

Eligibility Criteria:
Inclusion Criteria:

A- RA

* 18 Years and older
* Diagnosis of RA, as defined by fulfilling 2010 American College of Rheumatology (ACR) criteria
* Positive for rheumatoid factor (RF) and/or anticyclic citrullinated peptide (CCP)
* The presence of arthritis symptoms for more than 6 weeks but less than 5 year
* Active RA, as defined as DAS ESR\>3.1 (as defined by moderate and sever RA )
* Willing to adhere to the study requirements
* Willing to use acceptable effective forms of contraception

Exclusion Criteria:

* • Allergy to methotrexate (MTX)

  * Previous exposure to anti-CD20 monoclonal antibody (mAb) or other type(s) of mAb therapy
  * Receipt of intra-articular injections within 4 weeks prior to study entry
  * Unwilling to stop drinking alcohol (ETOH)
  * History of alcohol or substance abuse
  * Active infection, or chronic or persistent infection that might worsen with immunosuppressive treatment (e.g., Human Immunodeficiency Virus \[HIV\], hepatitis B virus \[HBV\], hepatitis C virus \[HCV\], tuberculosis \[TB\])
  * Interstitial lung disease observed by chest x-ray \[chest radiograph\]
  * Known coronary artery disease or significant cardiac arrhythmias or severe congestive heart failure (New York Heart Association \[NYHA\] classes III or IV)
  * Definitive diagnosis of another autoimmune rheumatologic disease (e.g., systemic lupus erythematosus \[SLE\], scleroderma, primary Sjögren's syndrome, primary vasculitis)
  * History of immunoglobulin E (IgE)-mediated or non-IgE-mediated hypersensitivity or known anaphylaxis to mouse proteins
  * History of cancer. Exception: participants with previous resected basal or squamous cell carcinoma, treated cervical dysplasia, or treated in situ Grade I cervical cancer within 5 years prior to study entry are not excluded from study eligibility
  * History of positive purified protein derivative (PPD) test (i.e., positive tuberculosis \[TB\] test or mantoux test) without treatment for TB infection or chemoprophylaxis for TB exposure
  * Live vaccine within 3 months of study entry
  * Any psychiatric disorder that would prevent a participant from providing informed consent
  * Pregnancy or breastfeeding women.
  * HIV-positive patients.
  * Patients receiving any other investigational/disease modifying agents within 4 weeks of study entry.
  * History of allergic reactions attributed to compounds of similar biologic composition to mesenchymal stem cells.
  * Psychiatric, addictive or any other disorder that compromises ability to give a truly informed consent and perform all study assessments.
  * Active Malignancy
  * Any other serious medical illness that might preclude safe participation in the study.

Inclusion criteria for SLE

* Adults aged at least 18 years old
* Active musculoskeletal SLE diagnosed by SLICC criteria.
* No contraindication to the use of IV methylprednisolone, biosimilar rituximab, or any other required medications such as antipyretics and antihistamines
* Willing to use appropriate contraception if at risk of pregnancy
* Disease activity that is refractory to hydroxychoroquine and one DMARD, or patients unable to take hydroxychoroquine due to contra-indication or prior toxicity Exclusion criteria for SLE Severe "critical" SLE flare defined as: (i) BILAG 2004 A flare in CNS system; (ii) BILAG 2004 A flare in the renal system; or (iii) any other SLE manifestation requiring more immunosuppression than allowed within the protocol in the physician's opinion

  * Pregnancy
  * Breast Feeding
  * Malignancy
  * Receipt of intravenous immunoglobulin, plasma exchange or cyclophosphamide within the last 3 months
  * Rituximab within the past 18 months or other biologic therapies within the past 6 months
  * Active infections, including but not limited to the human immunodeficiency virus, hepatitis B (including prior infection as judged by positive Hepatitis B core antibody) or hepatitis C
  * Receipt of a live attenuated vaccine within 3 months prior to study enrolment
  * History of cancer in the past 5 years except for squamous or basal cell carcinoma that has been completely excised or treated cervical carcinoma in situ
  * Planned surgery within the study period that is expected to require overnight hospital admission
  * Any other concomitant medical condition that, in the investigator's opinion, or after discussion with the CI, places the participant at risk by participating in this study Inclusion criteria for systemic sclerosis
* Adult patients, \>/= 18 years of age
* Systemic sclerosis, as defined by American College of Rheumatology (1980) criteria
* Disease duration upto 5 years (defined as time from first non-Raynaud phenomenon manifestation)
* \>/= 15 and \</= 40 mRSS units at screening
* Active disease, as defined by protocol
* Negative pregnancy test for a female subject of childbearing potential Exclusion criteria
* Major surgery (including joint surgery) within 8 weeks prior to and/or during study enrollment
* Rheumatic autoimmune disease other than systemic sclerosis
* Previous treatment with tocilizumab
* History of severe allergic or anaphylactic reactions to human, humanized, or murine monoclonal antibodies
* Severe cardiopulmonary disease
* Known active current or history of recurrent infections
* Use of any investigational, biologic, or immunosuppressive therapies .
* Primary or secondary immunodeficiency Inclusion criteria for ankylosing spondlitis Male or non-pregnant, non-lactating female patients at least 18 years of age

Diagnosis of moderate to severe AS with prior documented radiologic evidence (x-ray or radiologist's report) fulfilling the Modified New York criteria for AS:

* Active AS assessed by BASDAI ≥4 (0-10) at Baseline
* Spinal pain as measured by BASDAI question #2 ≥ 4 cm (0-10 cm) at Baseline
* Total back pain as measured by VAS ≥ 40 mm (0-100 mm) at Baseline Patients should have had inadequate response or failure to respond to at least 2 NSAIDs at an approved dose for a minimum of 4 weeks in total and a minimum of 2 weeks for each NSAID prior to randomization, or less than 4 weeks if therapy had to be withdrawn due to intolerance, toxicity or contraindications Patients who are regularly taking NSAIDs (including COX-1 or COX-2 inhibitors) as part of their AS therapy are required to be on a stable dose for at least 2 weeks before randomization patients who have been on a TNFα inhibitor (not more than one) must have experienced an inadequate response to previous or current treatment given at an approved dose for at least 3 months prior to randomization or have been intolerant to at least one administration of an anti-TNFα agent Exclusion criteria for ankylosing spondlitis

  * Chest X-ray or MRI with evidence of ongoing infectious or malignant process
  * Previous exposure to secukinumab or any other biologic drug directly targeting IL-17 or IL-17 receptor
  * Pregnant or nursing (lactating) women Inclusion criteria for dermatomyositis
* 5 to 50 Years of age
* Adults with definite or probable dermatomyositis or polymyositis and pediatric patients five years of age and over with definite or probable juvenile dermatomyositis by Bohan and Peter criteria. Diagnosis of JDM based on an age of onset (i.e., first symptom of myositis or dermatomyositis rash) is less 16 years of age
* Refractory myositis, defined by intolerance to or inadequate response to corticosteroids plus an adequate regime of at least one other immunosuppressive agent. Intolerance is defined as side effects that require discontinuation of the medication or an underlying condition that precludes further use of the medication.
* Baseline manual muscle testing which is based on a maximum MMT-8 (Manual Muscle Test) score of 150:Adult subjects with dermatomyositis (DM) or polymyositis (PM) must have a score that is no greater than 125/150 in conjunction with 2 other abnormal core set measures.

Subjects with a diagnosis of Juvenile Dermatomyositis (JDM) must meet either of the following criteria:

1. An MMT-8 (Manual Muscle Test) score that is no greater than 125/150 in conjunction with 2 other abnormal core set measures.

   OR
2. If MMT (Manual Muscle Test) score is greater than 125/150 the patient MUST meet at least 3 abnormal core set measures.

   * Background therapy with at least 1 non-corticosteroid immunosuppressive agent at a stable dose for at least 6 weeks prior to screening
   * Able and willing to complete self-report questionnaires. Parents of pediatric participants will be required to complete the questionnaires on behalf of their children.
   * Willing to use acceptable forms of contraception for the duration of the study for patients of reproductive potential.
   * Parent willing to provide informed consent, if applicable
   * Willing to forgo immunization with a live vaccine for the duration of the study Exclusion for dermatomyositis

     * Drug-induced myositis. Patients who have myositis or myopathic syndromes caused by taking medications known to induce myositis-like syndromes, including but not limited to statin agents, fibric acid derivatives, colchicine, and hydroxychloroquine.
     * Inclusion body myositis
     * Cancer-associated myositis, defined as the diagnosis of myositis within 2 years of the diagnosis of cancer. Patients with basal or squamous cell skin cancer or carcinoma in situ of the cervix are not excluded, if it has been at least 5 years since excision.
     * Myositis in overlap with another connective tissue disease that may preclude the accurate assessment of a treatment response
     * Live viral vaccine within 4 weeks prior to study entry
     * Any joint disease or other musculoskeletal condition that may interfere with muscle strength testing
     * Known hypersensitivity to mouse proteins
     * Any concomitant or life-threatening non-myositis illness that, in the opinion of the investigator, may interfere with the study
     * Known or suspected history of drug or alcohol abuse within the last 6 months prior to study entry, as determined by medical record or patient interview
     * Anticipated poor compliance with study requirements
     * Participation in another clinical trial within 30 days prior to screening
     * Any history or evidence of any severe illness or other condition that, in the opinion of the investigator, may interfere with the study
     * Previously received rituximab
     * Evidence of prior infection with hepatitis B or hepatitis C virus
     * Initiation of an exercise program within 4 weeks of screening OR initiation of an exercise program during the study
     * Consumed any creatine-containing, over-the-counter products in the form of dietary supplements 30 days prior to screening visit and for the duration of the study