Transcript Rituxumab and Intravenous Immune Globulin for

Rituxumab and Intravenous
Immune Globulin for
Desensitization during Renal
Transplantation
Vo et al NEJM 2008; 359: 242-52
TM Residents Journal Club
October 2008
Elianna Saidenberg
1
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
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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
3
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
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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
5
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
6
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
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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
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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
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Tools for detection of DSA:
Antibody Identification-4
•
Flow Cytometry
– Donor cells are incubated with patient serum and then a fluorescent-labeled antihuman 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
10
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
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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
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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
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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
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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
15
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)
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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
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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
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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”
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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
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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
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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
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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
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One more moment for the really
disinclined…
Figure 3. Formula for the t-test.
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Results-1
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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%
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Results-3
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Brenner: Brenner and Rector's The Kidney, 8th ed.
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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 subtherapeutic immunosuppressive levels
Other complications
– No viral infections
– No hospitalizations for infections
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– No infusion related side effects
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.”
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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
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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 ATN161 should be investigated further as an antiangiogenic and
antimetastatic cancer agent alone or with chemotherapy.
British Journal of Cancer (2006) 94, 1621–162632
• 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.
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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
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• 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
35
BMC Nephrology 2007, 8:13
•
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?
36
A possible downside to living donor
transplants…
37