BIOLOGICS – POLYCLONAL/MONOCLONAL ANTIBODIES

Question 1

THERAPEUTIC CONTEXT – SOLID ORGAN
TRANSPLANTATION

Answer 1

*Solid organ transplantation saves lives (prolongs ~4 years/patient)
*Most common solid organ transplant = kidney graft
*Pharmacotherapy is a cornerstone of successful solid organ
transplantation
*Goal:
*Minimize organ rejection
*Minimize adverse effects
*Induction therapy, maintenance therapy, treatment of rejection

Question 2

IMMUNE RESPONSE (QUICK OVERVIEW/REVIEW)

Answer 2

*To differentiate “self” from “non-self”
*Innate immunity (quick action, lack of priming, low affinity) vs.
adaptive immunity (slow in action initially, “acquired”, antigen specific,
requires priming, high affinity, memory)
*Innate immunity: complements, granulocytes, monocytes, macrophages,
mast cells, basophils
*Adaptive immunity: B lymphocytes (generates antibody) and T
lymphocytes (helper, cytolytic, and regulatory)
*B-lymphocyte surface antibodies = receptors/sensors
*T-lymphocytes senses foreign peptide fragments (MHC antigens)
presented by antigen-presenting cells

Question 3

Graft rejection sequence:

Answer 3

*Organ recipient recognizes graft as foreign → activates B and T cells
which differentiate/divide/generate immune mediators (e.g. cytokines)
to upregulate the immune system → host immune system attacks the
graft → graft destruction → eventual graft rejection

Question 4

IMMUNOSUPPRESSION

3 steps

Answer 4

1) T-cell receptor identifies antigen bound
to MHC
2) Co-stimulatory signal is needed for T-cell
activation (CD80/86 – CD28 interaction)
3) Increased interleukin-2 (IL-2) generation.
Feedback amplification

1st step is presentation of antigen in form of protein fragments by APC to the T cell
T cell recognizes foreign antigen presented by MHC

2nd signmal is required and this signal is activated by the binding of CD. 80 and CD. 86 proteins on the antigen presenting cells to the CD 28 protein receptor on the t cell
if we can block CD: 28. We can effectively block the activation of the T cell.

once you have 2 steps, you’re going to activate a cascade of events

• third step is what the third step is actually a
• a feedback amplification.

IL2 made in step 2 travels ouside Tcells to recruit other B and T cells
Also going to travel to surface of Tcepp receptor and activate itself, further amplifies the process

Question 5

THERAPEUTIC CONTEXT – INDUCTION THERAPY

Answer 5

*Purposes: 1) enhance the initial immunosuppressive effects and 2) delay the usage of
nephrotoxic agents (e.g. calcineurin inhibitors)
*Usage: ~60-70% of new transplant patients in the US with high risk of rejection (e.g. 2nd
transplant, sensitized patients, pediatrics). ~ 20% with low risk of rejection.
*Short course, overlapping with maintenance therapy: strong immunosuppression right after
transplantation

*Therapy options:
* Depleting agents (e.g. antithymocyte globulin): depletes T-lymphocytes
* Immune modulators (e.g. basiliximab): inhibits interleukin-2 (IL-2) associated activation of Tlymphocytes
*Tailored induction therapy based on risks of rejection:
* High immunological risk / steroid-sparing: depleting agents
* Low, medium immunological risk: immune modulators
depleting agents and low or medium risk use the immune to the immune modulators

Question 6

POLYCLONAL VS MONOCLONAL ANTIBODIES

Answer 6

*Polyclonal antibodies: collection of antibodies from a variety of B-cells which are capable of
recognizing multiple epitopes on the antigen.
Inject human antigen into rabbit to produce antibodies to recognize all the foreign peptides or epitopes

*Monoclonal antibodies: antibody from a single B-cell and which are only capable of
recognizing a distinctive epitope.

Question 7

ANTITHYMOCYTE GLOBULIN - POLYCLONAL

Answer 7

*Purified gamma globulin from rabbits or horses immunized with human
thymocytes
*ATG (Atgam): equine polyclonal antibody; 10-30 mg/kg/d x 7-14 days (not
commonly used today)
*rATG (Thymoglobulin): rabbit - less immunogenic, more potent (safer); 1-1.5
mg/kg/d for 5-14 days

Question 8

ANTITHYMOCYTE GLOBULIN - MECHANISMS

Answer 8

*1) binds to multiple T-cell surface receptors: CD2, CD3, CD4, CD8,
CD11a,CD18, CD25, CD44, CD45 (50+ targets known to date)
* CD: cluster of differentiation molecules
* Cellular surface markers for the identification and characterization of leukocytes
*2) aggregation ATG on cellular surfaces; induces both complement- and cellmediated cytotoxicity → phagocytosis by macrophages → necrosis/apoptosis
*3) blocks T-cell functions by binding to surface molecules mediating cellular
function → inhibiting cellular signalling → T-cell “hyporesponsiveness”
*e.g. blocks chemokine receptors (e.g. CXCR4) and reduces chemotactic
signalling, minimizing the movement of T-cells to the graft
*4) Can affect B-cells or other leukocyte

• So Atg is going to try to kill the t cells.
  Okay, if you re-suppress the t-cells, you will suppress your adapted immunity.

Question 9

ANTITHYMOCYTE GLOBULIN - KINETICS

Answer 9

*Administration: therapeutic protein, extremely poor oral absorption (molecular
weight, ionization, gastric degradation); only administered by IV infusion
*Distribution: binds to circulating lymphocytes, granulocytes, platelets; poor tissue
distribution
*Elimination: very little urinary excretion; does not undergo typical phase I
(oxidation) or phase II (conjugation) metabolism
*Proteolysis by liver and/or the reticuloendothelial systems (liver/spleen/lymph)
* Endocytosis/pinocytosis → degradation in lysosomes
*Immune reaction to antithymocyte globulins
*Antibodies against antithymocyte globulins:
*ATG: ~78% of subjects
*rATG: ~ 68% of subjects
*PK interactions: minimal (due to lack of renal excretion and conventional
oxidative/conjugative metabolism)

Can’t be given orally, they’re proteins
Your stomach will break them down, proteins are highly charged and can’t cross gi tract membrane
IV only
* They bind to lymphocytes, which makes sense because this is their primary target or mechanism action. They’re going to try to find the t cells, then for sites and bind to them and attack them.
* They do no undergo the typical phase, one and phase, 2 metabolism.
* `dont need to worry about interactions with no hepatic metabolism
* Not excreted so dont worry about renal fxn

Question 10

ANTITHYMOCYTE GLOBULIN - DYNAMICS

Answer 10

*Clinical efficacy (prevention of rejection):
*Reduced rates of acute graft rejection
*Mild and steroid-resistant rejections
*Increased steroid withdrawal - increased chance of steroid withdrawal, so less dependence on cortical steroids.
*Increased rate of hospital discharge
*Little evidence of improved long-term graft survival
*KDIGO (kidney disease improving global outcome) guideline: use of ATG
only in high immunological risk patients

*Clinical efficacy (treatment of rejection):
* Some evidence of effectiveness in T-cell mediated / steroid-resistant acute rejection
*Very little evidence in antibody-mediated acute rejection
*Not considered 1st line agent based on KDIGO guideline

*Toxicity (targets a variety of cell types):
*Frequent dose-limiting myelosuppression (leukopenia, anemia,
thrombocytopenia) – up to 30% incidence
*Anaphylaxis, hypotension, tachycardia, dyspnea, urticaria, rash
*Increased opportunistic infections (e.g. cytomegalovirus disease)
*Increased incidence of cancer & lymphoproliferative disease
*Increased cardiovascular mortality (due to accelerated atherosclerosis or
prolonged lymphopenia) – similar observation in nuclear war survivors

*Serum sickness disease:
*Formation and deposit of antibody-ATG immunocomplex (Anti-Neu5GC) in
tissues
*Arthralgia, painful joints
*Decreased long-term graft survival
*More commonly observed with ATG than rATG

Question 11

ANTITHYMOCYTE GLOBULIN – INFUSIONRELATED SIDE EFFECTS (COMMON), MANAGEMENT

Answer 11

*Chills/fever/itching/erythema: fairly common, possibly due to
increased endogenous pyrogens
*Prophylactic antihistamine, antipyretics (e.g. acetaminophen), slow
infusion (over 4-6 hours)
*Phlebitis:
*Infuse ATG through central vein

Question 12

ANTITHYMOCYTE GLOBULIN – INFUSION-RELATED
SIDE EFFECTS (LESS COMMON), MANAGEMENT

Answer 12

*Anaphylaxis: uncommon, idiosyncratic, more common with horse ATG
* Stop ATG infusion and administer epinephrine, steroids. Do not restart ATG.
*Hemolysis: although clinically significant, also uncommon
* Transfusion, pharmacotherapy (e.g. mannitol – to reduce osmotic swelling of red blood cells), hold
ATG as needed
*Thrombocytopenia: transient in kidney transplant patients
* Platelet count usually return to normal levels without stopping ATG; may require platelet transfusion
in resistant cases
*Respiratory distress: possible sign of anaphylaxis
* May consider holding ATG; administer antihistamine, epinephrine, corticosteroids
*Chest, flank, back pain: possible signs of anaphylaxis/hemolysis
* Management as stated above
*Persistent hypotension: possible sign of anaphylaxis
* Stop ATG infusion and administer epinephrine, steroids. Do not restart ATG; pressors as needed.

Question 13

MONOCLONAL
ANTIBODIES

Answer 13

• You have the Fc. Domain, and this is the domain typically associates or binds to the cells of the receptors, and you have the fabric domain or the variable domain.
• And this this domain contains the that will recognize the specific antigens for epitoles.

Long t1/2
Days or hours
Slow clearance
, sometimes some of these agent you can give it monthly.

Typically 1-2 compartments only

• They don’t want to distribute into the tissues.
• Okay, they want to tend to stay in the within the one compartments.
• Small Vd, stay in one compartment
• Q is the transfer from 1st compartment to peripheral comparments, tend to be very slow as they don’t want to move

Question 14

MONOCLONAL ANTIBODIES- KINETICS

Answer 14

*Administration: therapeutic protein, extremely poor oral
absorption (molecular weight, ionization, gastric degradation);
only administered parenterally
*Distribution: poor and erratic tissue distribution Stay in central compt
*Elimination: very little urinary excretion; does not undergo
typical phase I (oxidation) or phase II (conjugation) metabolism
*Metabolism of IgG: 33% (skin), 24% (muscle), 16% (liver), 12%
(gut)
*Proteolysis by liver and/or the reticuloendothelial systems
(liver/spleen/lymph)
*Endocytosis/pinocytosis → degradation in lysosomes

Question 15

MONOCLONAL ANTIBODIES- KINETICS
*Elimination:

Answer 15

*Non-specific mechanism: proteolysis by liver and reticuloendothelial system
* Macrophages/monocytes
* Binding of the Fc region to the Fc gamma receptor
* Internalization, degradation in lysosomes
* Non-saturable, non-specific

*Targeted elimination:
* Binding of Fv part to target protein - Fv region is specifc to epitope and targeted elim
* Internalization, degradation
* Saturable, non-linear, specific
* AKA “antigen-sink”

*Immune responses to therapeutic monoclonal antibodies
*Depends on how “human” the antibody is
*Completely human antibody can still generate immune reaction-

Question 16

OKT3 (MUROMONAB)

Answer 16

Mechanism of action: * CD 3 is one protein on the t cell receptor on the surface of the t cell.
* and this molecule in the body binds specifically to the CD 3 protein.
Murine monoclonal antibody
Binds to CD3 on the T-cell
receptor complex on the
surface of T-lymphocytes
Inactivates T-cell → eventual
phagocytosis

*Indication:
*Solid organ transplants (withdrawn in Canada)
*Adverse effects:
*Severe cytokine-release syndrome (AKA cytokine storm):
pulmonary edema, acute renal failure, gastrointestinal
disturbances (N/V), myalgia
*Newer, “humanized” anti-CD3 monoclonal antibodies
being developed

• it was used fairly commonly before but now discontinued for solid organ transplants, it’s actually withdrawn in Canada.
• and this is due to it’s. It’s severe AE
  Bad AE because it is from murine and depleting agent
  
  • Blocks the first step in the activation process, and it kills the T cells. What happens if you kill the T cells?
    T cells produce IL2 to interferon. Gamma. A whole bunch of chemicals and sidelines
    Leads to a burst of cytokine release causing AE
    Agent is too potent

Question 17

ALEMTUZUMAB
indication
mech
pk

Answer 17

*Indication:
* Used off-label in solid organ transplantation for induction and treatment of acute rejection
(corticosteroid & antibody-resistant rejection)
* Centre-specific: 30mg as single dose, may be repeated 1-5 days later
*Mechanism:
*Humanized monoclonal antibody against CD52 surface antigen (T and B
lymphocytes; macrophages, monocytes, eosinophils)
* Binding to CD52→antibody-dependent lysis→ T and B cell removal from various
tissues → complete lymphocyte depletion
*Pharmacokinetics:
*Little clinical data in organ transplant patients
*Complete depletion of peripheral blood lymphocytes in “hours” and in lymph nodes
within 2-4 days
* B-lymphocyte depletion lasts 3-12 months
* T-lymphocyte depletion can last up to 3 years

Question 18

ALEMTUZUMAB
di
AE

Answer 18

*Drug interactions:
*not well documented
*Adverse effects:
*Infusion-related: hypotension, fever, shortness of breath,
bronchospasm, rigors.
*Managed with prophylactic antipyretics (e.g. acetaminophen),
corticosteroids, and antihistamines
*Leukopenia, neutropenia, thrombocytopenia, lymphopenia

Question 19

BASILIXIMAB
indication
mech
pk

Basiliximab - chimeric hydrid, not entirely himan

Targets IL-2 receptor, the 3rd pathway
Technically, it’s not involved in the activation process. So so that’s why it’s. It’s a non depleting agent. It’s sort of slow down. It slows down the the process. It blocks the amplification. Doesnt outright kill tcells like other agents

Answer 19

*Indication:
*Induction therapy in kidney transplantation (low or intermediate immunologic
risk patients)
*Allows delayed initiation or low dose calcineurin inhibitor
*20mg IV, on day 0 and day 4 post-transplant
*Mechanism:
*Chimeric monoclonal antibody which binds competitively to the alpha-chain
on the CD25 protein (i.e. IL-2 receptor) on activated T-lymphocytes
*Prevents IL-2 mediated T-cell activation/proliferation
*Immunomodulatory, non-depleting
*Pharmacokinetics:
*t1/2 ~ 7 days
*IL-2 receptor blocking takes effects quickly and may last for 6 weeks postadministration

the effects of a competitive inhibitor can be overcome by high concentrations of IL2.
If you have a IL2 it isn;t going to work as effectively
because these are indicated for low, intermediate, immunoligc risk pt, IL2 conc will tend to be lower too
, it doesn’t really target the first 2 pathways, the activation pathways
AE not as bad as depletng agents

Question 20

BASILIXIMAB
di
AE

Answer 20

*Drug interactions:
*not well documented

*Adverse effects:
*Adverse reaction rates similar to placebo in many trials
*Lack of leukopenia
*Lack of infusion-related reactions
*Lack of cytokine-release syndrome
*Low risk of lymphoproliferative disorder and opportunistic infections
*Low risk of cancer
*May cause hypersensitivity reactions
*< 2% of patients can develop antibodies to basiliximab

Question 21

1
ST APPROVED
MONOCLONAL ANTIBODY
FOR MAINTENANCE
THERAPY
FUSION PROTEIN
BELATACEPT

indication
mech
pk

Answer 21

*Indication:
* Alternative to calcineurin inhibitors for prophylaxis of organ rejection in kidney graft recipients in conjunction with basiliximab induction, mycophenolate mofetil, and steroids
* First monoclonal antibody maintenance treatment in organ transplantation.
* Initial phase (intermittent-IV infusion): 10 mg/kg on days 1, 5, weeks 2, 4, 8, and 12
* Maintenance phase: 5 mg/kg monthly starting on week 16
STEP 2 of pathway, part of activation process, co-stimulation process

*Mechanism:
* T-cell immune response activation requires 2 specific signals: 1) signal 1= antigen binding to
T-cell receptor and 2) co-stimulatory signal mediated by binding of CD28 on T-cells (CD4)
with CD80/CD86 on antigen-presenting cells
* A T-cell co-stimulation inhibitor: binds to CD80/86 on antigen-presenting cells → blocks
interaction with CD28 on T-cells → reduces T-cell activation → T-cell apoptosis

*Pharmacokinetics:
* t1/2 ~ 10 days
* Unlikely to be subjected to metabolism-mediated interactions

Question 22

BELATACEPT
*Adverse effects

Answer 22

*No significant side effects vs. placebo
*Infusion-related reactions uncommon
*Post-transplant lymphoproliferative disorder evident in Epstein-Barr virus
seronegative patients (restricted usage for seropositive subjects)
Seroneg pt may develop lymphoma

Question 23

SIDE EFFECTS SUMMARY – ALL ANTIBODIES

Answer 23

Leukopenia: more common with lymphocyte-depleting agents (e.g. ATG and
alemtuzumab); dose-dependent; Alemtuzumab is associated with longer-term
and much more profound leukopenia

Infections: similar overall infection risk between ATG, IL-2 blockers, and
alemtuzumab; higher incidence of cytomegalovirus infection with depleting
agents

Post-transplant lymphoproliferative disorder / malignancy: up to 50X more
likely in kidney transplant patients in general; induction regimens increase risk
overall; not clear if depleting agents more likely than IL-2 blockers;
alemtuzumab associated with non-Hodgkin’s lymphoma, colorectal, and thyroid
cancer; rATG associated with melanoma

nfusion-related reactions: “cytokine release syndrome” – fever, rash, nausea,
vomiting, and hypotension are commonly associated with ATG and
alemtuzumab. Mitigated with prophylactic acetaminophen, steroids,
antihistamines, and (slow) rate of infusion.

Question 24

THE BENEFIT TRIAL - BELATACEPT

Rationale:
No RCTs available examining the long term (i.e. > 5 years) outcomes of
current immunosuppressive regimens vs. regimens containing cyclosporine
Hypothesis:
 Belatacept can improve long-term outcomes in kidney transplant
recipients by providing adequate immunosuppression without the
nephrotoxicity associated with calcineurin inhibitors

 Study Design:
 Randomized, single-blind, parallel group, controlled
 1:1:1 ratio
 More intensive (MI) belatacept regimen
 Less intensive (LI) belatacept regimen
 Cyclosporine regimen
 Inclusion: adult with kidney transplant from living or deceased donor with cold-ischemic
time < 24 hours (with basiliximab induction, mycophenolate, and steroids)
What are potential confounding factors associated with this study design?

Answer 24

3 main problems
1. Mycophenolate interacts w cyclosporine, cyclosporine affects its kinetics, inhibits its recirculation = increases clearance of mycophenolic acid = shorter t1/2 = less exposure, AUC
- No interactions w belatacept, shouldn’t affect mycophenolate
- For the cyclosporine group, you will have lower mycophenolate conc, control for cyclosporine will look much worse
- Easier to establish beneficial effects of belatacept
- Increase the dose won’t work, supposed to be the same for both arms
- Measure the AUC of mycophenolate, cyclosporine group will have lower AUC in the study if interaction present but wasn’t done in paper

2. Mycophenolic acid is maintenance tx and is pill form given for life, belatacept is IV
-  the cyclo group has cyclo, mycophenolic acid and steroids self-administered by pt themselves
- Belatacept given by nurse once monthly, going to clinic
- Adherence was not checked, you expect cyclosporine group adherence to be lower, may forget to take a pill
- Higher prob of graft failure with less immunosuppression

3. Cyclosporine is no longer used much, tacrolimus is used more (<5% of pop on it)
- Tacrolimus has no interaction w/ mycophenolate, may have diff results
- New pt always given tacro

Question 25

THE BENEFIT TRIAL -
BELATACEPT
outcomes
results

Answer 25

Renal fxn by GFR is not good marker for poor renal fxn, not ideal
- It is an estimation, lose precision and accuracy
- For pt w kidney transplant, they tend to have reduced fxn
- Best marker is 24h urinary granted measurement, but really hard to do cannot be done

by chance you you lose the same number of of subjects in your arm, and they they all have the same
characteristics. You will still be
randomize at the end in theory.
So I was looking for the data, actually demonstrating the the patient demographic toward the end of the study, just to verify that
that the 3 groups were still comparable. But I couldn’t find that information.
in these big trials everything is intention to treat, but I think they also did the for protocol analysis as well.
So if they did the for protocol l analysis.
The loss of subject over time would confound that analysis, more likely to generate error, less sensitive

Biopsy proven rejection higher in the belatacept groups, but also higher survival rates
- this tells you that there’s very poor correlation between biopsy for rejection versus the actual outcome.
this that study demonstrates that this
this method actually does not correlate with the the ultimate patient and graph survival.

So in general there are 2 types of acute rejection. You have the antibody mediated rejection or cell mediated rejection
- They did not differentiate the 2 in their methodolgy

the composite outcome of patient and graph survival. The p value is0.2
For individual outcomes, the p = 0,11 = lose significane
there’s there could be a lot of reasons behind this, but it it could be a power problem.

Question 26

THE BENEFIT TRIAL - BELATACEPT
Limitations:

Answer 26

 In BENEFIT-EXT with extended criteria donors (older age, additional co-existing
conditions), benefits of belatacept over cyclosporine on death and graft loss
were lost.
 BENEFIT-EXT: older age (56.2 vs. 43.2 yrs) and more co-existing conditions.
 Acute rejection (one of the primary endpoints) does not correlate with long term
graft survival. Study did not differentiate between different types of rejection.
 Cyclosporine is not the 1st line calcineurin inhibitor regimen today.
 Cyclosporine (CSA) – mycophenolate (MPA) interaction: cyclosporine increases
the clearance of MPA and decreases MPA exposure. No interaction observed
between belatacept and MPA. No interaction observed between belatacept
and the current 1st line calcineurin inhibitor, tacrolimus.
 Differences in patient compliance between belatacept vs. cyclosporine groups.

Question 27

Select all correct statements about antithymocyte globulins:
A) They are monoclonal antibodies produced from either rabbits or horses immunized
with human thymus
B) They are indicated in maintenance immunosuppression in kidney transplant
recipients
C) They are considered immune modulators blocking the actions of IL-2
D) Rabbit ATG are generally considered to be more immunogenic than horse ATG
E) All are correct
F) All are incorrect

What is the proposed mechanism of action of belatacept?
A) Binds to CD25 protein and prevents IL-2 release from T-lymphocytes
B) Binds to CD52 protein and induces antibody-mediated cell lysis
C) Binds to CD80/CD86 on antigen presenting cells and inhibits the co-stimulatory Tcell activation signal
D) A & B are correct
E) A & C are correct
F) All are incorrec