TM Residents Journal Club October 2008 Elianna Saidenberg

1. Rituxumab and Intravenous Immune Globulin for Desensitization during Renal TransplantationVo et al NEJM 2008; 359: 242-52 TM Residents Journal Club October 2008 Elianna Saidenberg

2. Objectives • Review relevance of donor specific antibodies in renal allografting • Review methods used in antibody detection in renal transplantation • Review available methods for successful transplantation of sensitized patients • Discuss article and its relevance to us • I WILL NOT: • Provide a comprehensive overview of renal transplantation or transplant immunobiology

3. I don’t know anything about solid organ transplantation. So, why am I doing this to you and to myself? • Earlier in 2008 the Canadian Council for Donation and Transplantation (CCDT) merged with the CBS to create a new national organ and tissue donation and transplantation registry system. • "Canadian Blood Services takes seriously our new responsibilities for donation and transplantation in Canada, and will work closely with organ and tissue stakeholders in moving the work of the CCDT to action," Dr. Graham Sher

4. A brief introduction to renal transplantation • Options for treatment of end stage renal disease (ESRD) include dialysis or kidney transplantation • In 2005, there were 32,375 Canadians on renal replacement therapy • 39% had a functioning transplant, 61% were on dialysis. • Source of organs can be from deceased or living donors and the donor need not be related to the recipient • The donor and recipient are usually immunologically disparate so potent immunosuppressive therapies must be given to prevent the recipient immune system from rejecting the graft • Of the 1,202 kidney transplants performed in 2006, 40% were from living donors • As of December 2006 there were 3,075 Canadians on waiting lists for kidney transplants • Canada has one of the lowest organ donation rates among developed countries • Patients who receive transplantation live longer than similar patients treated with dialysis and have a better quality of life • There are also additional cost savings http://www.kidney.on.ca/english.html

5. Antibody barriers in renal transplant • 2 types of Abs of importance in renal transplantation: • ABO blood group antibodies • Anti-HLA antibodies • Alloantibodies can arise through previous transfusion and pregnancy as well as previous transplants • 33% of females and 17% of males awaiting transplant have DSA • 55% of patients awaiting re-transplantation have DSA compared with 15% of those awaiting 1st transplant Fuggle and Martin Transplantation 2008; 86(3): 384 • Donor specific antibodies (DSA) are known to be involved in the pathogenesis of graft rejection in all 3 phases • Hyperacute rejection occurring within minutes after transplant • Acute rejection occurring within 2 weeks after transplant • Chronic rejection associated with proteinuria and loss of graft function months or years after transplant

6. Tools for detection of DSA • The virtual crossmatch • Presence of HLA Abs is routinely assessed in patients on transplant list • Unacceptable anti-HLA Abs are registered in the National Transplant Database • For every donor the computer will perform a virtual crossmatch on every blood group compatible patient on the list • Deceased donor organs will only be shipped to patients predicted to have a negative crossmatch • This approach requires testing for presence of Abs and assessment of specificity at least quarterly Fuggle and Martin Transplantation 2008; 86(3): 384

7. Tools for detection of DSA: Antibody Identification • Complement Dependent Cytotoxicity (CDC) • Patient serum is tested against a panel of HLA typed leukocytes • Serum is added to wells followed by WBCs • After incubation allowing Ab binding, rabbit serum is added as a source of complement • On further incubation, complement will be activated and WBCs to which Ab has bound will be killed • Cytotoxicity is visualized under microscopy by staining with ethidium bromide which stains only dead cells and acridine orange that stains only living cells

8. Tools for detection of DSA: Antibody Identification-2 • CDC cont’d • If a random panel of donor WBC are used, the percentage of positive reactions or percent panel reactive antigens (%PRA) is an indication of the percentage of potential donors with whom the recipient is likely to have a positive crossmatch • Example: HLA-A2 is present in ~28% of one donor population, so the presence of Abs against this Ag would result in exclusion of 28% of the possible donors • Use of a selected panel of WBCs that do not display common HLA pairings is useful to determine the Ab specificity

9. Tools for detection of DSA: Antibody Identification-3 • CDC cont’d • Disadvantages: • Subjective • Cumbersome • Rigorous control of rabbit reagent required • Only detects complement-fixing Abs • May detect IgM Abs directed against non-HLA Abs whose clinical significance is not clear • Uses: • Donor and recipient crossmatching prior to transplant predicts the risk of hyperacute rejection

10. Tools for detection of DSA: Antibody Identification-4 • Flow Cytometry • Donor cells are incubated with patient serum and then a fluorescent-labeled anti-human IgG is added which will bind any patient Ab that has attached to donor cells. The fluorescent labelled antibody will be detected by the flow cytometer • Use of pooled cells identifies the presence of Abs and the proportion of fluorescence is proportional to the amount of bound Ab and so may be interpreted as the %PRA • Advantages: • More objective • Increased sensitivity of Ab detection • Detects only IgG Abs • Disadvantages • ?Too sensitive • More sensitive tests are less specific at predicting the risk of hyperacute rejection • FC may pick up low titre or low avidity Abs which may indicate an increased risk of rejection over time • Note: The significance of +ve FC crossmatch in the presence of -ve CDC is still debated • However, improved sensitivity is an important goal as prior exposure to an antigen can result in Ab-mediated graft rejection even if the Ab titre is below the level of detection of current assays

11. Tools for detection of DSA: Antibody Identification-5 • Solid phase assays • Methods utilize solubilized or recombinant HLA class I or II molecules coated to a solid matrix • Automated optical detection methods are employed • These methods can be applied as an ELISA test, utilize flow cytometry or utilize newer technology such as Luminex • Solid phase assays offer increased sensitivity and specificity For a nice review of these methods see Fuggle and Martin Transplantation 2008; 86(3): 384

12. Tools for detection of DSA: Antibody Identification-6 • Patient sensitization profile • Results of sequential Ab screening and specification are combined with information about potential sensitizing events to produce a patient sensitization profile • This profile is used to specify those donor HLA Ags which would be unacceptable

13. Impact of DSA • In 2003 ~33% of patients awaiting kidney transplant had a PRA >10% and about 40% of these patients have PRA >80% • Decreased rates since mid-1990s possibly because of use of EPO and therefore fewer transfusions • Impact of universal leukoreduction on HLA sensitization not clear • Median waiting time for transplant increases with increasing PRA and 5-year allograft survival decreases with increasing PRA • Mean waiting time if PRA 0-9% is 857 but is 1620 days if PRA 10-79% and can reach >2200 days if the patient is even more highly sensitized • While awaiting transplantation the patients suffer increasing morbidity and are thus poorer transplant candidates • There is evidence that longer duration of dialysis before transplant worsens post-transplant outcomes Crew and Ratner Seminars in Dialysis 2005; 18(6): 474-81

14. Diagnosing antibody-mediated rejection (AMR) • Previously based on histological assessment • Now staining for C4d is considered the gold standard for evidence of donor-directed IgG binding • Recall: When DSA binds to Ag on grafted kidney the complement system is activated leading to the eventual conversion of C4 to C4b and finally inactivation to C4d which will remain bound to the tissue • Improvements in diagnosis of AMR enable earlier treatment

15. Treatment of AMR • Combination of plasmapheresis and IVIg has been shown to reverse 90-95% of AMR • Other therapies which have been tried include high dose IVIg alone, PLEX and IVIg plus rituximab and PLEX plus ATG

16. Preventing AMR • High dose IVIg (2g/kg) • Thought to work by binding anti-donor Abs and by down-regulating Ab secretion via Fc receptor signalling in plasma cells • NIH IG02 study (Jordan et al J AM Soc Nephrol 2004; 15: 3256-62) • Highly sensitized patients awaiting kidney transplant were randomized to receive either 2g/kg IVIg or placebo (0.1% alumin) monthly for 4 months and additional infusions at 12 and 24 months after entry and were followed to 30 months • Outcomes: • IVIg lowered anti-HLA Ab levels •  rates of transplant in IVIg group (35%0) vs placebo (17%), p=0.02) •  mean waiting time in IVIg group (4.8 years) vs placebo (10.3 years), p=0.03) • 3 year allograft survival 80% in IVIg group vs 70% in placebo group (p not significant)

17. Preventing AMR-2 • Plasmapheresis + IVIg • Zachary et al Transplantation 2003; 1519-25 • Alternate day single-volume plasmapheresis followed by low dose CMV hyperimmune globulin combined with 4 agent immunosuppression (tacrolimus, steroids, MMF and daclizumab) to limit resynthesis of Abs • 31 patients receiving live donor transplants had treatment started before transplant; 12 patients (11 cadaveric transplant recipients) started treatment immediately after transplant as they were not known to have DSA at the time of transplant • At the end of treatment 63% of subjects no longer had DSA; no return of DSA in patients followed for average of 13 months • Acute humoral rejection is reported in ~30-50% of patients treated in PLEX/ IVIg studies; usually reversible with reintroduction of PLEX and IVIg but requirement for as many as 30 treatments has been reported (Holy $$$$ Batman!) • Most feasible in the context of a living donor where the goal is to abrogate a +ve XM to enable transplant

18. Preventing AMR-3 • Rituximab • A chimeric mouse/human monoclonal Ab against CD-20 which is present on pre-B and mature B cells • Rituximab inhibits B cell proliferation and results in rapid B cell depletion • Currently licensed for treatment of NHL and some rheumatologic conditions • Viera et al Transplantation 2004; 77: 542 • Single-dose, dose-escalation phase 1 trial of rituximab in dialysis patients with a PRA >50% • n=9, divided into 3 groups of 3 treated with 50, 150 or 375 mg/m2 rituximab • Primary outcomes: PRA% and Ab titres • At 2 days after treatment there was a significant depletion of CD20+ cells (pre-tx 191+ 137 vs post-tx 12 + 5.6) • 2 subjects: No change in PRA • 1 subject: PRA  from 87% to 51% • 5 subjects: Change in FC histogram structure suggesting a loss of Ab specificity • 1 subject  PRA titre from 1:64 to 1:16 • One of the patients converted to a negative donor XM enabling successful living donor transplant

19. The Trial • “An exploratory, open label, phase 1-2, single centre (Cedars-Sinai, LA) study of the safety and effectiveness of IVIg plus rituximab in reducing levels of anti-HLA antibodies and improving transplant outcomes”

20. Methods • Patients • 20 highly HLA-sensitized patients on waiting list for transplant from live or deceased donors • Mean pre-study PRA 77+19% or had DSA • Treatment • IVIg 2 g/kg on day 0 and day 30 • Rituximab 1g on day 7 and day 22 • Post transplant immunosuppression: alemtuzumab x1 immed after transplant; prednisone, MMF, and tacrolimus • Treatment of rejection episodes: • Cell mediated rejection: methylprednisolone and rabbit ATG • Ab mediated rejection: methylprednisolone, IVIg, rituximab • Grade III AMR or thrombotic microangiopathy: PLEX, IVIg and rituximab • Infection-prophylaxis: ganciclovir/ valganciclovir, nystatin, TMP/SMX

21. Methods-2 • Assessment of DSA • FC XM at study entry, after treatment and before transplant • When donors became available XM performed on serum samples collected after treatment • Acceptable XM = CDC –ve at 1:2 dilution of saline, FC XM –ve • Other data collected on day 0, and weeks 1,2,4 and 6 and at months 3,6 and 12: • Changes in PRA • XM results • Rate of transplantation • Results of transplantation • Complications • All patients were analyzed on an intention-to-treat basis

22. Methods-3 • Statistical analysis • Paired t-tests for analysis of: • PRA status • Graft survival rates • Mean serum creatinine • Transplant status (single vs multiple) • Type of acute rejection (C4d- vs C4d+) • Interaction of transplant status and XM result

23. A moment for the statistically disinclined… • What is a paired t-test? • A t-test is used to compare means on the same or related subject over time or in differing circumstances, for example in a 'before and after' scenario • It is assumed that the observed data are from the same subject or from a matched subject and are drawn from a population with a normal distribution. • Normality can be tested by using a normality test, such as the Shapiro-Wilk and Kolmogorov-Smirnov tests • Once a t value is determined, a p-value can be found using a table of values from Student's t-distribution

24. One more moment for the really disinclined… Figure 3. Formula for the t-test.

25. Results-1

26. Results-2 • 20 patients >18 years of age • 16 (80%) underwent successful transplant • 6 deceased donor transplant, 10 living donor transplants • All recipients at high immunological risk • 63% had ≥1 previous transplant • 69% had +ve XM at time of transplant • 62% had PRA >50% • 4 remaining patients • 3 awaiting deceased donor transplant at time of publication • All have PRA >50%

27. Results-3

28. Brenner: Brenner and Rector's The Kidney, 8th ed.

29. Results-4 • Immunologic factors • All patients had  numbers CD19+ cells after rituximab treatment • No patients developed anti-chimeric Abs • PRA significantly  after treatment • Pre: 77+19% vs post 2nd treatment: 44+30% • Survival rates • Among 16 transplanted patients • 12 month patient survival 100% • 12 month allograft survival 94% • 1 allograft lost when patient’s immunosuppressive therapy reduced • Acute rejection • Occurred in 50% of patients who were transplanted • 31% C4d+ AMR • Most within 1st month after transplant and reversed with therapy • 2 patients had AMR >6 months after transplant related to sub-therapeutic immunosuppressive levels • Other complications • No viral infections • No hospitalizations for infections • No infusion related side effects

30. Authors’ Discussion and Conclusions • Conceptual problems: • Rituximab has no effect on plasma cells and also has no immediate effect on circulating Ab levels • Would limit use of rituximab as sole treatment • Rituximab might interfere with cross-matching assays for B cells • Would limit its use in desensitization protocols and interpretation of B cell XM results for patients awaiting deceased donor transplant • “…data presented here…are encouraging and may support further analysis of this approach.”

31. Editorial Comments • “Despite this study’s limitations, which include a small number of patients, a relatively short follow-up period, and a high rate of early rejection, the implications are important.” • “…observations need to be confirmed and validated by other centres and in larger numbers of patients during longer periods of follow-up. However, their approach may represent a breakthrough in the care of sensitized patients awaiting transplantation” Ron Shapiro NEJM 2008; 359: 305-6

32. My Discussion • What is a “phase 1-2 trial”? • Phase 1=First step in testing a new treatment in humans and test the best way to give a new treatment and the best dose. • The dose is usually increased incrementally in order to find the highest safe dose. • As little is known about the possible risks and benefits of the treatments being tested, usually only a small number of subjects are included • Example: • To evaluate the toxicity, pharmacological and biological properties of ATN-161, 26 adult patients with advanced solid tumours were enrolled in eight sequential dose cohorts (0.1–16 mg kg-1). ATN-161 was well tolerated at all dose levels. Approximately, 1/3 of the patients in the study manifested prolonged stable disease. These findings suggest that ATN-161 should be investigated further as an antiangiogenic and antimetastatic cancer agent alone or with chemotherapy.British Journal of Cancer (2006) 94, 1621–1626

33. Phase 2= Trials done to find out if the treatment works well enough to study in a larger phase 3 trial, which types of conditions the treatment works best for, and more about side effects and best dose • Example: • Trial to determine efficacy and safety of single-agent deforolimus in patients with relapsed or refractory hematologic malignancies.Eligible patients were assigned to one of five disease-specific cohorts and given 12.5 mg deforolimus. Safety, pharmacokinetics, pharmacodynamics, and antitumor response were assessed.55 patients were enrolled and of the 52 evaluable patients, partial responses were noted in five. Hematologic improvement/stable disease was observed in 21. Common treatment-related adverse events were mouth sores, fatigue, nausea, and thrombocytopenia. Decreased levels of 4E-BP1 in 9 of 11 AML/ MDS patients after therapy showed mammalian target of rapamycin inhibition by deforolimus. CONCLUSIONS: Deforolimus was well-tolerated in patients with heavily pretreated hematologic malignancies, and antitumor activity was observed. Further investigation of deforolimus alone and in combination with other therapeutic agents is warranted in patients with selected hematologic malignancies. Clinical Cancer Research. 14(9):2756-62, 2008 May 1.

34. So is this a phase 1, phase 2 or phase 1-2 tiral? • Does it ever make sense to call something a phase 1-2 trial? • How was this study like and unlike a phase 1 trial? How was it like and unlike a phase 2 trial? • It was like a phase 1 trial in that all causes of ESRD were included, a variety of ranges of sensitization were included, both deceased and live donor transplants were included • It was unlike a phase 1 trial as there was no dose escalation in this trial; all patients received the same therapeutic intervention • It was like a phase 2 trial in that its rationale was based on a previous small study (Viera et al) but this trial provided efficacy and safety evidence on a larger number of patients indicating that a larger phase 3 trial might be warranted • It was unlike a phase 2 trial in that no recommendations could be made about which subsets of patients would likely benefit from treatment

35. Is there a better descriptor? • Could it be a proof of concept trial? • PoC trials are often conducted before a formal dose-ranging study and usually represent the first time a drug or device is tested in the intended patient population • Example of a PoC trial compared to this trial • Currently there is no effective treatment available to retard cyst growth and to prevent the progression to end-stage renal failure in patients with ADPKD. • There is no single really effective treatment for HLA-sensitized patients awaiting transplant • Evidence from animal experiments shows that sirolimus markedly slows cyst development and renal functional deterioration. • Evidence from Viera et al indicate that rituximab is safe and probably decreases PRA%; hence this is not the 1st time this agent was tried in this patient population • Based on these promising results in animals we have designed a single center, randomised controlled, open label trial assessing the therapeutic effect, safety and tolerability of sirolimus in patients with ADPKD disease and preserved renal function. • An exploratory, open label, phase 1-2, single centre study of the safety and effectiveness of IVIg plus rituximab in reducing levels of anti-HLA antibodies and improving transplant outcomes • The primary outcome will be the inhibition of kidney volume growth measured by magnetic resonance imaging • Primary outcomes are levels of anti-HLA antibodies and transplant outcomes • The results from this proof-of-concept RCT will for the first time show whether treatment with sirolimus effectively retards cyst growth in patients with ADPKD. • The results of this trial will indicate whether use of rituximab to prevent AMR merits further study BMC Nephrology 2007, 8:13

36. Are the results valid?YES • Not a randomized trial, no need for concealment of therapeutic allocation • Investigators were aware of subjects’ immunologic status at time of data interpretation • Follow up was complete, but short • Paired t-test is appropriate test as long as normality tests were passed • What are the results? • Rituximab is effective at decreasing PRA and no significant complications encountered but high rates of rejection • Significant (p<0.0001) decrease in PRA% pre- vs post-treatment • Excellent rates of patient and allograft survival on short-term follow up • High rates of rejection compared to historical results • Overall rates of acute rejection <15% in USA (Meier-Kriesche Am J Transplant 2004; 4: 378-83) • However, rates of rejection in other trials of highly sensitized patients about the same (see slide #16) • No evidence of significant infectious complications • Can the results be applied to patients? • NO….or maybe • Larger trials needed but there may be few other options for these patients • What do the nephrologists think? Can they even get rituximab for this use in Canada?

37. A possible downside to living donor transplants…