Arun Chawla, MD

INDUCTION THERAPY WITH AUTOLOGOUS MESENCHYMAL STEM CELLS IN LIVING

RELATED KIDNEY TRANSPLANTS

OVERVIEW• What are mesenchymal stem cells?

• Immunomodulatory function of those Cells

• Clinical use thus far (Bench to Bedside)

• Trial

• Use of an induction antibody has grown; in 2009, 58% of patients received a T-cell depleting antibody, 21.2% an interleukin-2 receptor antagonist (IL2-RA), and 3.6% both a T-cell depleting antibody and an IL2-RA; only 17.2% did not receive induction

WHAT ARE MSCS• Mesenchymal stem or stromal cells (MSCs) are multipotent cells, which can be isolated

from various types of tissue, such as bone marrow, adipose tissue and multiple others.

IMMUNOMODULATORY EFFECTS OF MSC

ANIMAL MODELS• studies have demonstrated that skin and heart transplant survival can be prolonged by

intravenous infusion of MSCs

• Organ injury models – like improved cardiac function after MI and improved lung function in rats in a COPD model suggesting MSCs have a regenerative effect on injured organs

MECHANISMS OF ACTION• act by differentiating into functional cells

• Paracrine manner - Via the secretion of cytokines, growth factors and prostaglandins with immune-modulatory and regenerative function

• MSCs express several chemokine and growth factor receptors, including CXCR4, and the PDGF, HGF and bFGF receptors , suggesting they are capable of migration in response to inflammation induced chemokine release, and tissue injury.

• MSCs secrete a range of anti-inflammatory factors, including IL-10, TGFb , hepatocyte growth factor (HGF), nitric oxide etc.

• Via HGF – they may inhibit fibrosis

• Direct regenerative function by targeting resident progenitor cells via the secretion of bFGF, and VEGF

EFFECTS ON T CELLS

• able to suppress T lymphocyte activation and proliferation in vitro – Le Blanc et al Scand J Immunol 2003

• have been reported to inhibit the cytotoxic effects of antigen-primed cytotoxic T cells – Potian et al J Immunol 2003

• Di Nicola et al, who found that neutralizing antibodies against TGF-β and HGF restored the proliferative response of T cells.

• Ge et al. showed that administration of MSCs resulted in allograft tolerance as a result of regulatory T-cell generation in a mouse kidney transplant model

• The induction of regulatory T cells was dependent upon the expression of indolamine 2,3-dioxygenase (IDO) by MSCs

• DCs generated in the presence of MSCs were impaired in their response to maturation signals and exhibited no expression of costimulatory molecules.

• Altered cytokine production pattern, ie decreased production of proinflammatory cytokines tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-12 and increased production of the anti-inflammatory cytokine IL-10

• Not only do they block proliferation & differentiation but also antibody production and chemotactic behavior of B cells was affected by MSCs.

• It has been suggested that MSCs suppress IL-2 or IL-15 driven NK-cell proliferation and IFN- production

• MSCs exert an inhibitory effect on the NK-cell cytotoxicity

Immunomodulatory effects of MSCs. CTL indicates cytotoxic T cell; HGF, hepatocyte growth factor; IDO, indoleamine 2,3-dioxygenase; PGE2, prostaglandin E2; and TGF-β, transforming growth factor

β.

Nauta A J , Fibbe W E Blood 2007;110:3499-3506

©2007 by American Society of Hematology

FABRY’S DISEASE

• X-linked genetic disorder - deficiency of lysosomal enzyme alpha-galactosidase

• Using patients own MSC

• Transduced with a functional galactosidase gene

• Return MSC to the patient

• Correction of deficiency (Osiris, 2000)

OSTEOGENESIS IMPERFECTA

• Horwitz et al 1999 reported 3 children transplanted with allogeneic MSC from HLA-compatible siblings

• New lamellar bone formation, improved osteogenesis with fewer fractures

• Engrafted MSC were shown to differentiate into osteoblasts

• Multicenter - Between October, 2001, and January, 2007, 55 patients were treated.

• 39 of 55 patients with steroid-resistant, severe, acute GVHD responded to treatment with mesenchymal stem cells (allogeneic).

• Over half of patients with a complete response were alive at 2 years.

FIGURE 1. Challenges in solid-organ transplantation and possible applicability of mesenchymal stem cells (MSCs).

The immunomodulatory and regenerative capacities of MSCs are suggested to be beneficial to ameliorate transplantation related ischemia–reperfusion injury, prevent or treat rejection and target allograft pathology either directly by immunomodulation or indirectly by decreasing the need for immunosuppressive medication.

• purpose of the study was to establish the safety and clinical feasibility of cell-based therapy with MSCs in the context of kidney transplantation.

• Patients were given T cell– depleting induction therapy and maintenance immunosuppression with cyclosporine and mycophenolate mofetil. On day 7 posttransplant, MSCs were administered intravenously.

• Clinical and immuno-monitoring of MSC-treated patients was performed up to day 360 postsurgery.

• In the two patients given MSCs, but not in the control transplant recipients, the percentage of memory CD8 T cells markedly decreased posttransplant

• Despite the two patients given MSCs were receiving CsA, known to prevent the development of Treg by the inhibition of IL-2 (10,47), a progressive expansion of Treg was documented posttransplant.

AIM

• examining the effect of autologous MSC infusion as an alternative to anti-IL-2 receptor antibody for induction therapy in adults undergoing living-related donor kidney transplants.

METHODS

• Single-site prospective, randomized study

• all donors had a documented linear blood relationship with their respective recipient (eg, parent to children and siblings with the same parents)

• Transplants were performed according to ABO blood compatibility and negative HLA crossmatch results

• Blood transfusions were never used before or after the transplants.

• mean time patients undergo dialysis before transplant is 6.8 or fewer months

• Patients enrolled from February 2008 through May 2009

EXCLUSION CRITERIA• systemic infections,

• prior treatment for cancer, were

• Pregnant,

• Obese ([BMI > 28) ,

• abnormal blood chemistries (ie, total cholesterol 300 mg/dL, triglycerides 400 mg/dL, WBC 3000/μL, or platelets 75k/μL).

Treatment groups were identified as groups A, B, or C to preserve blinding during statistical analysis.

All groups received similar doses of mycophenolate mofetil and steroids.

AUTOLOGOUS MSC CULTURES• Bone marrow cell aspirates (60-80 mL) were obtained while patients were under local

anesthesia from the posterior iliac crest of the kidney recipient 1 month before the transplant.

• MSC cultures were tested negative for endotoxin, HCV, hepatitis B virus, HIV, syphilis, fungus, Mycoplasma species, and Chlamydia before infusion.

• G-banding karyotype analysis was performed to confirm absence of chromosomal aberrations in the final cellular product.

• MSC suspensions of 1 ×106/mL were transferred into 20-mL syringes for intravenous infusion over 15 to 20 minutes.

• Each participant received autologous MSC infusion (1–2 ×106/kg each) 10 minutes before the graft’s vein and artery were unclamped, and 2 weeks posttransplantation.

IMMUNOSUPPRESSION REGIMEN• Only the control group received 20 mg of anti–IL-2 receptor antibody intravenously within

2 hours of surgery and 4 days after surgery.

• Tacrolimus was initiated at 0.12 mg/kg, targeting trough levels of 8 to 12 mg/kg for the first trimester, 5 to 8 mg/kg for the second, and 3 to 7ng/mL for the third trimester and beyond.

• Cyclosporine was initiated at 6.5 mg/kg with its target levels (concentration 2 hours after dose, C2) of 1000 to 1200 ng/mL in the first trimester, 800 to 1000 ng/mL in the second trimester, and 600 to 800 ng/mL in the third trimester and beyond.

• MMF at either 2.0 g/d for patients who weighed 80 kg or more or 1.5 g/d for those who weighed less than 80 kg.

• Those in MSC low-dose CNI group received reduced 80% of the standard CNI dose

IMMUNOSUPPRESSION REGIMEN• Immediately post op and through day 3, patients received 6 mg/kg of methylprednisolone

intravenously, 240 mg/d on day 4, 160 mg/d on day 5, and 80 mg/d on day 6.

• On days 7 through 14, patients received 30 mg/d of prednisone.

• Doses were tapered to 20 mg/d for the first trimester, 10 to 15 mg/d for the second trimester and 5 to 10 mg/d for the third trimester and beyond.

• Acute rejection episodes were treated with methylprednisolone pulse therapy.

• Glucorticoid- resistant rejection was treated with ATG

FOLLOW-UP• weekly for the first trimester and monthly thereafter for 1year.

• Acute rejection was defined as an increase of 0.3 mg/dL of serum creatinine (nadir creatinine), confirmed by renal biopsy within 24 hours of initiation of antirejection therapy.

• Biopsies were read and the severity of lesions was scored by a blinded pathologist and were classified according to Banff 97criteria. Acute humoral rejection was confirmed by complement C4d immune staining in peritubular capillaries.

• Three participants (2 in the control group and 1 in the autologous MSC low-dose CNI group) were lost to follow-up after emigrating from China. They were excluded from analysis

• All participants remained in the study group to which they were assigned, and there were no missing values from the 12-month follow-up.

END POINTS• The primary outcome was the incidence of biopsy-confirmed acute rejection and

estimated glomerular filtration rate (eGFR) within the first year (as per MDRD).

• The secondary outcome was 1-year patient and graft survival and the incidence of adverse events, including opportunistic infections.

STATISTICAL ANALYSIS• “Power and sample size considerations assume a 30% incidence of acute rejection within

the first year after kidney transplant in China and a reduction to 7.5% with MSC therapy based on the preliminary pilot study performed at our center (data unpublished)”

• adequate power to detect this assumed difference with 53 patients per group (type I error, 0.05; 80% power)

• In considering early renal function recovery measured by eGFR, a mean (SD) difference in eGFR levels between those treated with MSCs and those treated with anti–IL-2 receptor antibodies of more than 10mL/min (11 mL/min) would be adequately powered with approximately 20 patients per group

Tan J, Qiu J, Lu T, et al. Thirty years of kidney transplantation in two Chinese centers. Clin Transpl. 2005:203-207.

RESULTS• Recipients had a mean BMI of 21 (range, 14-31) and were a mean age of 38 years

(range, 18-61 years).

• Less than 3% tested positive for cytomegalovirus.

• The 1-year patient survival rate was100% in all groups

• One participant in the control group died of pneumonia at 399 days after surgery.

ACUTE REJECTION RATES

DELAYED GRAFT FUNCTION RATES WERE SIMILAR AMONG GROUPS

ADVERSE EFFECTS• Lower adverse events were seen in both autologous MSC groups than in the control

group

• The incidence of opportunistic infection was significantly lower in the MSC low dose CNI group than in the control group but was not lower in the standard- dose group compared with the control group

• No difference in hematuria, proteinuria, lymphocele, delayed wound healing etc complications after transplant

Significantly decreased risk of opportunistic infection in the low-dose CNI group than in the control group (hazard ratio, 0.28; 95% CI, 0.10-0.76; P=.01

Combined analysis of MSC-treated groups revealed significantly decreased risk of opportunistic infection than the control group (hazard ratio, 0.42; 95% CI0.20-0.85; P=.02

ADVERSE EFFECTS• Mal-differentiation, promotion of tumor growth, and malignant transformation have also

been suggested

• So far, however, tumor-promoting events of MSCs have never been observed in any of more than 500 patients who received this cell therapy

CONCLUSION• Among patients undergoing LRRT, the use of autologous MSCs compared with anti-IL-2

receptor– induction therapy resulted in a lower incidence of acute rejection, decreased risk of opportunistic infection, and better estimated renal function at 1 year.

• Importantly, autologous MSC inoculum was not associated with adverse events nor did it compromise kidney transplant survival

DISCUSSION• Additional potential benefits of autologous MSCs include improved recovery from

ischemia or reperfusion injury, a recognized important risk factor for graft failure and acute rejection

• Early function is a favorable prognostic factor for long-term graft survival

• reduction in CNIs

• Opportunistic infection’s occurring mostly during the first 3 to 6 months after transplant procedures are associated with the highest mortality rate in kidney transplant recipients in China

EBM USER’S GUIDES TO THERAPY