Lecture 5 - mAbs Flashcards by Eleanor Nicklason

Which cell type produces Ig and is used in the lab as an ‘Ig producing factory’?

Plasma cells

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What is a monoclonal antibody?

A population of Ab’s from a single B cell clone

i.e., they all have the same specificity

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What factors give mAbs great therapeutic potential?

Well tolerated (come from us)
Very high specificity
Long-lived

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What is the half-life of Ab?

Several weeks

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Which types of diseases are mAbs mainly used for at the moment?

Malignancies

* Auto-immune conditions

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What are ‘The Big 5’ mAbs?

Adalimumab (Anti-TNF)
Infliximab (Anti-TNF)
Trastizumab (Anti-HER2)
Bevacizumab (Anti-VEGFA)
Rituximab (Anti-CD20)

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What is the name of Anti-TNF?

Remicade (Infliximab)

Humira (Adalimumab)

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What is the name for Anti-CD20?

Rituximab

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What is the name for Anti-VEGFA?

Avastin / Bevacizumab

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What is the name for Anti-HER2?

Herceptin / Trastuzumab

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What recognition did the researchers who came up with mAbs receive?

Nobel Prize for Medicine in 1984

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Describe the process of harvesting mAbs from mice

Mice spleen cells + myeloma cells
Fusion
Hybridomas
Culture in drug
Selection of positive cells
Harvest of mAbs

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What are the shortcomings of mAbs from mice?

They are recognised as foreign and thus have a short half life
The ‘mice’ constant region means that they are lacking some effector functions

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Describe ‘Humanisation’ of mice mAb

CDR grafting in vitro
• Mouse variable region grafted onto human constant region
• Ig are fully human apart from the CDRs
• Longer half life in serum (than fully mouse)

2. Transgenic mice
 • Have human Ig genes
 • Challenge mice with antigen
 • Mice produce human Ig
 • Very good half life

Harvesting from humans
• Harvest B cells from immune individual
• Fuse with EBV to immortalise cells
• Screening to select for the desired specificity
• Ig isolation

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How good are humanised mAbs raised in vitro?

Still fairly crude

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Describe mAbs raised in transgenic mice

Transgenic mice:
• have human Ig genes (C or V & C)

These mAbs are really good because they contain barely any mice parts

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Describe mAb generation from humans

Infected human with high affinity, IgG
Memory cells collected
Immortalisation: Memory cells infected with EBV
Screening
mAbs harvesting

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What are the advantages and disadvantages of mAbs from infected humans?

Pros:
• not rejected by patients
• mAbs have specificity that was effective at clearing the infection

Cons:
• Specificities limited to foreign immunogens

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What are human mAbs most often used for?

Passive immunisation

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What are the pros of passive immunisation?

Useful when a very quick immune response is needed (HIV, SARS, influenza)

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What is ‘naked mAb’ useful for?

ADCC: Antibody dependent cell-mediated cytotoxicity
CDC: cell dependent cytotoxicity

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What are some ways we can ‘arm’ mAbs?

Multistep targeting:
 • Bispecific mAb
Immunoconjugates:
 • Radioimmunoconjugate
 • Immunocytokine
 • Immunotoxin
 • Immunoliposome
 • Cellular immunoconjugates

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Describe Radioimmunoconjugates

Radioactive substance conjugates to a mAb which is specific for a tumour cell

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Describe Immunocytokines

Cytokine conjugated to a mAb specific for a tumour cell

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Describe Cellular immunoconjugates

mAb bispecific for tumour cell and killer cell | This brings the killer cell right to the tumour cell

What are the mechanisms of action of mAbs?

1. Ligand blockade 2. Receptor blockade 3. Target cell depletion 4. Target cell activation

Which mAb is used against Rheumatoid arthritis, psoriasis & Crohn's disease?

Anti-TNFa: • Remicade (Infliximab) • Humira (Adalimumab)

Which mAb is used for anti-angiogenesis and cancer therapy?

Anti-VEGFA: | • Avastin (Bevacizumab)

Which mAb is used for Breast cancer?

Anti-HER2: | • Herceptin / trastuzumab

Which mAb is used for Rheumatoid arthritis and non-Hodgkins lymphoma?

Anti-CD20: | • Rituximab

Describe the mechanism of action of anti-TNFa

* "Ligand blockade" * mAb specific for TNF (pro-inflammatory cytokine) * TNF bound by mAb, so it can't bind to TNF-R on inflammatory cells

Describe improvement in Rheumatoid arthritis with Remicade

Increased function of joints compared to patients not taking the drug

What is the role of TNF-a in Rheumatoid arthritis?

* Strong pro-inflammatory cytokine | * Leads to joint destruction

Describe the success of Avastin / Bevacizumab

mAb used in cancer, anti-angiogenesis agent * Better survival rates compared to conventional therapy * However, benefit is not that great * Not a cure

Describe the function of anti-RANKL mAbs

Denozumab * "Ligand blockade" * Specific for RANKL, which is a factor that increases the action of osteoclasts * By blocking RANK-L, we decrease bone resorption in Multiple Myeloma

Which cancer cells produce lots of RANK-L?

Myeloma cells

Where is RANK?

Osteoclasts

What are some examples of mAb ligand blockade?

* Anti-TNF-α (Adalimumab, Infliximab) * Anti-RANK-L (Denosumab) * Anti-VEGFA (Bevacizumab)

Describe receptor down-modulation

When receptors are engaged a lot, they are taken back into the cell and degraded, in order to down regulate the response

What are some examples of mAb receptor blockade or down-regulation?

Receptor down regulation: Anti-HER2 • HER2 is a receptor in malignant breast cancer cells • Tumour growth is suppressed by blocking the receptor

What is HER-2? | In which disease is it overactive?

* Epidermal GF receptor | * Breast cancer

What are some ways that depletion occurs?

Anti-CD20 • MAC formation • ADCC • Phagocytosis

Which cells express CD20?

All B cells

Describe the action of Rituximab

* mAb specific for CD20 | * Brings about transient depletion of ALL B cells

Describe the effectiveness of Rituximab

50% remission of B cell lymphoma (eg. non-Hogdkins lymphoma) patients

What is combination therapy?

Conventional treatment + mAb

What was Rituximab's initial use, and what is it used for now?

Started off as a cancer therapy | Now used for auto-immune conditions

What is Rituximab specific for?

CD20

What is Herceptin specific for?

HER2 | aka Trastuzumab

What is Remicade specific for?

TNFα | aka Infliximab

What is Avastin specific for?

VEGFA

Describe mAbs used for cell activation What is significant about this? Give examples

``` • mAbs specific for parts of T cells • When engaged, lead to activation of T cells (**T cell activation without antigen!**) → • more T cell cytotoxicity • more T cell help eg. ``` 1. Anti-CD3 2. Anti-CD28 → this didn't end well 3. Anti-CTLA4

What happened with the first in man trials of Anti-CD28?

Massive cytokine storm and organ failure in the 6 healthy subjects

What is Ipilimumab, and what disease is it used for?

Anti-CTLA4 | Used in metastatic melanoma

What is CTLA4?

Expressed on the surface of T cells after activation by DCs to prevent further activation

What is the effect of Anti-CTLA4?

Blockage of inhibition of T cell activation | → More T cell activation

What are some limitations of mAbs as therapeutics?

* Anti-CD28 disaster * Cardiotoxicity * infections (in immune cell depletions) * Anti-mAb antibodies (loss of efficacy)

What is an orphan disease?

Rare diseases with limited treatment development

Describe the structure of CARs

Chimeric antigen receptors * External Ab 'for specificity) * Intracellular T cell activation section

Describe how CARs could be used to treat disease

(Chimeric antigen receptor) 1. T cells isolated from patient 2. Viral vector delivers anti-CD19 CAR gene to T cells 3. T cells in culture express anti-CD19 CAR 4. Lymphocytes killed in patient 5. CAR expressing T cells reinstated in patient 6. CAR expressing T cells recognise B cells (through CD19) and induce CTL killing 7. Depletion of B cells Used for CLL & Follicular lymphoma

How are the CARs put into T cells?

Viral vector

What is the effect of CAR therapy in B cell cancers?

Complete depletion of B cells

On which cells is CD19 expressed?

All lymphocytes

What is IL-1β?

Pro-inflammatory cytokine ``` Brings about systemic features of inflammation: • fever • pain • malaise • tissue damage ```

Which diseases is Anti-IL1β used for? What is the mechanism of action? How has it been received in patients?

Used for CAPS: a collection of rare autoimmune disorders Mechanism of action: • Ligand blockade ``` In patients: • Complete response within 7 days • Well tolerated • Long lasting • No side effects ```

What are 'off-label' trials?

A drug becomes licensed for one rare disease, and then is use in trials for more common diseases. This is what happened with Rituximab and now for Anti-IL1β mAbs

What other disorders is Anti-IL1β being used for?

• Juvenile idiopathic arthritis • Gout • Late onset Type 2 Diabetes (chronic inflammatory conditions)

Describe the mechanism of B cell depletion with Anti-CD20 (Rituximab)

→ mAb binds to CD20 1. ADCC • IgG(1) binds to CD20 • NK cells bind IgG with IgγR I • NK cells induce apoptosis of B cell ``` 2. Complement activation • IgM binds to CD20 • Activation of Cq1 • Complement cascade • Formation of membrane attack complex ``` ``` 3. Phagocytosis • Ig binds to CD20 • Phagocytes binds Ab with FcR • Phagocytosis • Intracellular killing in phagosome ```

What is the mechanism of action of Anti-VEGFA?

Ligand blockade

Which class are monoclonal antibodies?

IgG1

What is the nomenclature for the following: • Chimaeric mAbs • CDR grafted (humanised) mAbs • Fully human mAbs?

Chimaeric mAbs: -xi- CDR grafted (humanised): -zu- Fully human: -u-

Lecture 5 - mAbs Flashcards

(71 cards)