L15. Immune-based Techniques in Research and Medicine 2

Question 1

What did Emil Adolf von Behring (1880s) do?

Answer 1

Used serum from immunised animals for passive immunisation against tetanus and Diptheria.

Question 2

Why did passive immunisation using serum from immunised animals not popular?

Answer 2

Decrease in popularity due to side-effects (serum sickness) and discovery of antibiotics

Question 3

What are 3 examples of diseases where human monoclonal antibodies were used for passive immunotherapy?

Answer 3

Respiratory syncytial virus (RSV), Ebola, COVID

Question 4

What are 2 examples of where conventional antisera (animal antibodies) or human IgG preparations (IVIg/ from blood) still used in some therapies?

Answer 4

1. Neutralise toxins e.g. anti-snake venom can cause serum sickness still though (antisera prepared in animals); anti-tetanus (pooled human IgG)
2. Treat infections caused by emerging pathogens e.g. convalescent sera to treat Ebola and COVID.

Question 5

What are 3 examples of human monoclonal antibodies which target tumour cells, what do they recognise, and what are they used against?

Answer 5

1. e.g. anti-CD52 antibodies (CAMPATH) (CD52 found on most white blood cells)
   >Recognises leukocytes, good activator of complement and ADCC
   >Use in leukaemia, and lymphomas (cancer of white blood cells) to treat white blood cells
   >First humanised (IgG1) antibody used in a clinical trial, shrunk tumour but didn’t cure the tumour but showed humanised monoclonal antibodies can be used to treat cancer.
2. e.g. anti-CD20 antibodies (Rituximab).
   >Recognises B cells, good activator of complement and ADCC (recruiting NK cells, antibody dependent cytotoxicity)
   >Use in leukaemias, lymphomas, and rhymatoid arthiritis
   >Chimeric antibody (mouse variable regions and human constant genes)
3. e.g. anti-Her2 antibodies (Herceptin)
   >Recognise Her2 (receptor tyrosine kinase, expressed at high levels in ~25% breast tumours), antibody blocks the receptor to stop tumour growing.

Question 6

What is a usual way to tell if a drug is a monoclonal antibody?

Answer 6

End with “Mab” (monoclonal antibody, also a short way of writing it).

Question 7

What are 3 ways monoclonal antibodies can modulate an immune response for non-infectious diseases (e.g. auto-immune diseases) and cancer, give examples for all?

Answer 7

1. Monoclonal antibodies for depletion of leukocytes
   >e.g. antibodies to CD52 (CAMPATH) , CD3, CD4 used to purge the organ grafts of their lymphocytes.
   >organ transplantation, graft versus host disease autoimmune disease, where a graft contains some lymphocytes which recognise the host as foreign and attack the host.
2. Inhibiting inflammatory response by blocking of cytokines, cytokine receptors, soluble mediators
   >e.g. antibodies to TNF-α, IL-1, IL-6, complement protein C5 (or their receptors)
   >Inflammatory/ autoimmune disease
   >allergy e.g. antibodies to IgE, cytokines
   >over-reactive response to some infections e.g. COVID
3. Immune checkpoint inhibitors
   >At end of immune response T regulatory cells turn off the immune system, if this is stopped can kill cancers.
   >e.g. antibodies to CTLA-4, PD-1
   >Cancer immunotherapy

Question 8

What are 3 ways cancers can dodge immune responses?

Answer 8

1. Produce cytokines (e.g. IL-10) that induce Tregs
2. CTLA-4 expression induced switching off T cells
3. Express PD-ligand (PDL) switching off T cells

Question 9

Describe how CTLA-4 acts as an immune checkpoint?

Answer 9

> When a T cell is activated by an APC via the T cell receptor (TCR) recognizing an antigen presented by the APC, costimulatory signals are also required for full activation. CD28 is a costimulatory receptor on T cells that binds to B7 on APCs to promote T cell activation and survival.

> CTLA-4, which has a higher affinity for B7 than CD28, acts as a checkpoint to control T cell activation. After initial activation, CTLA-4 is upregulated on the surface of T cells and outcompetes CD28 for binding to B7. This binding sends an inhibitory signal to the T cell to dampen its activity, which is a natural mechanism to prevent overactivation and maintain self-tolerance.

Question 10

How do some cancer exploit CTLA-4 as an immune checkpoint?

Answer 10

Cancer cells can exploit this checkpoint by indirectly influencing the immune environment. For example, cancer cells can induce other cells within the tumor microenvironment to express higher levels of B7, which would preferentially engage CTLA-4 instead of CD28, leading to an inhibition of the T cell response. Additionally, some cancer cells may directly express B7, further engaging CTLA-4 and inhibiting T cells. This is due to competitive binding, as CTLA-4 has higher affinity for B7 than CD28, so will bind to it more often if more B7 is present

Question 11

How can some cancer cells exploit the PD-1 immune checkpoint to evade the immune system?

Answer 11

With PD-L1, cancer cells can directly express this ligand on their surface, and when it binds to PD-1 on T cells, it sends an inhibitory signal to the T cells, thus “turning off” the immune response.

Question 12

How can antibodies stop cancer evading the immune system?

Answer 12

Antibodies that block inhibitory immune checkpoints can reverse immunosuppression.

Question 13

What success have antibodies blocking immune checkpoints already shown?

Answer 13

Antibodies that block immune checkpoints have shown beneficial effects in metastatic melanoma and some types of lung cancer. These are hard to treat cancers that used to mean certain death.

Question 14

What are 2 monoclonal antibodies (mab) that block immune checkpoints against cancer cells?

Answer 14

1) Nivolumab binds PD-1, blocking its interaction with PD-L. T cell reactivated and can recognise the tumour cell

2) Ipilimumab binds CTLA-4, blocking its interaction with B7. CD28 can now interact with B7. T cell reactivated and recognise the tumour cell

Question 15

How can we exploit natural antibody binding/effector functions to directly kill cancer cells?

Answer 15

Cancer cells often have unique molecules on surface that are unique to this cancer cell (as are abnormal cells express weird surface molecules) which can be recognised by antibodies and therefore targeted by NK cells due to ADCC

Question 16

Wy is it important to choose the correct subclass of IgG for intended therapy?

Answer 16

As different types of IgG have different effector functions.

Question 17

What is the order of efficiency of IgG types for 1) Complement activation 2) Fc receptors on phagocytes 3) Fc receptors on NK cells?

Answer 17

1) Complement activation: IgG3 > IgG1 > IgG2 (order of efficiency for activating mechanism)

2) Fc receptors on phagocytes: IgG1=IgG3>IgG4

3) Fc receptors on NK cells: IgG1=IgG3

Question 18

What interaction do we want to promote when administering IgG antibodies and why?

Answer 18

FcRn (determines half-life): all subclasses, interaction of antibodies with this receptor increases half-life (which is wanted). So want to promote this interaction with IgG

Question 19

What is useful about mapping effector sites on antibodies?

Answer 19

> Precise mapping of effector sites can be used to generate “designer antibodies” for use in therapy.

> E.g. We could mutate amino acid residues in domain which activates compliment while keeping the others the same, so NK cells are activated but complement isn’t

Question 20

Give 3 examples of effector sites which have been mapped on the Fc region of an antibody and what innate cell does it interact with?

Answer 20

1) C1q binding occurs at the Hinge/Cγ2 domains

2) FcγRI, FcγRII (these Fc receptors important for phagocytes) bind at the lower hinge

3) FcγRIIIA (receptor for NK cells) at the amino acids: 298, 333, 334

> These can all be mutated for therapy (e.g. switch off some of these but not others for desired activation of cells)

Question 21

What domain determines how active an antibody is and why?

Answer 21

Most Fc receptors and effector functions have sites in CH2 domain (near middle) is important for how active the antibody.

Question 22

What is Glycoengineering?

Answer 22

“Glycoengineering”: engineering of the carbohydrate to alter antibody structure of IgG

Question 23

What is the effect of glycosylation of IgG and why?

Answer 23

GLYCOSYLATION (IgG has 2 N-linked glycans (Asn 297)), in IgG the CH2 domain has a carbohydrate link and if this is lost the effector functions are lost as the conformation of the molecule is lost.

Question 24

Give a specific example Glycoengineering for IgG

Answer 24

Removal of fucose improves from one of the carbohydrates in IgG, improves the ability of IgG interaction with FcγRIII (the FcR on NK cells) so makes more efficient ADCC.

Question 25

What are 3 properties of antibodies that can be improved by protein- or glyco-engineering and an example for each?

Answer 25

1) Antibody engineering to improve half-life (FcRn interaction),

2) Improve or remove effector functions (e.g. Enhance ADCC, complement activation).

3) Alterations to the glycosylation of IgG can improve interaction with FcRn (increases half-life) and interaction with FcR on NK cells (enhances ADCC)

Question 26

As well as relying on intrinsic functions of antibodies what are 3 methods used to treat disease, what is their structure and an example for each?

Answer 26

1) Instead of just relying on intrinsic functions of antibodies, can label antibody with drug/prodrug (activated when antibody binds and internalised by target cell)/toxin/radionuclide
>e.g. Antibody-drug conjugate binds and is internalised, killing the tumour cell.

2) For some applications, antibody fragments are useful (e.g. better tumour penetration as big antibody molecule can’t enter tumour)
>e.g. Fab fragments

3) Bi-specific antibodies with dual specificity link can link tumour binding to “effector” cell proteins e.g. tumour antigen + CD3
>E.g. Antibodies which bind to protein on tumour cell (CD19 on cancer B cells) can be linked to antibody that recognises CD3 on T cells
>Consist of 2 Fabs recognising effector cells close to the cells we want to kill.

Question 27

Why are there a lot of monoclonal antibody therapeutics which target TNF?

Answer 27

A lot of antibodies against TNF as stops inflammation

Question 28

Why were many monoclonal antibody therapeutics ineffective against Covid?

Answer 28

By isolating single B cells from recovered Covid patients were used to generate many Covid monoclonal antibodies, but Covid mutates quickly so ineffective against different variants.

Question 29

How can we combat the mutations rates of a disease like Covid using monoclonal antibody therapies?

Answer 29

Combinations or “cocktails” of monoclonal antibodies may help deal with escape mutants.

Question 30

Describe how a monoclonal antibody therapeutic for Covid-19 was modified to have a longer half life.

Answer 30

Sotrovimab has been engineered to possess an Fc LS mutation (M428L/N434S) that confers enhanced binding to the neonatal Fc receptor[17] resulting in an extended half-life and potentially enhanced drug distribution to the lungs

Question 31

What is Convalescent sera?

Answer 31

Antibodies isolated from recovered patients isolated and given to patients

Question 32

What are the issues when using convalescent sera against Covid-19?

Answer 32

Limited efficacy (polyclonal, time antibodies were isolated after infection critical as response against Covid drops quickly), limited supply, stringent safety requirements

Question 33

What are the advantages and disadvantages of * monoclonal antibodies to SARS Cov-2 e.g. Sotrovimab

Answer 33

1) Advantages over active immunisation:
>Immediate protection (for up to 4 weeks- antibody designed to improve half life)
>Suitable for immunocompromised patients
>May relieve severe symptoms

2) Disadvantages:
>No long-lasting protection
>Not very effective against latest variants
>May promote “escape mutants”, as one antibody reacting to one protein may cause pathogen to survive without this protein.
>Expensive to produce and administer

Question 34

What are 2 future techniques which could be used against Covid to do with monoclonal antibody therapies?

Answer 34

1) Antibodies that recgonised more conserved epitopes.

2) “cocktails” of antibodies to different targets or bi-specifics that recognise different targets on the virus so less easy for pathogen to escape.

Question 35

What do serious Covid cases have?

Answer 35

Excessive inflammation

Question 36

What are 2 monoclonal antibody therapies which can be used to treat excessive inflammation and what do they target?

Answer 36

1) Anti-interleukin 6 receptor (Tocilizumab)

2) Anti- C5a (Vilobelimab) stopping complement activation and associated inflammation.

Question 37

What is an example of a treatment which doesn’t involve antibodies but another immune cell?

Answer 37

CAR-T cells (chimeric antigen receptor)

Question 38

What is an overview of CAR-T therapy?

Answer 38

> T cells “engineered” to recognise a tumour antigen

> Isolate T cells of patient with cancer, engineer them and re-inject into patient

Question 39

Describe an example of CAR-T therapy with Lymphocytic leukaemia in 5 steps

Answer 39

1. T cells are harvested from the blood of a patient with a B-cell tumor.
2. A retrovirus encoding an anti-CD19 CAR infects T cells that are activated with antibodies to CD3 and CD28.
3. Infected T cells express an anti-CD19 CAR to mediate antitumor activity.
4. T cells are infused into patient to mediate antitumor activity
5. Anti-CD19 CAR chimeric receptor can now bind to CD19 on a tumour.

Question 40

Other than leukaemia what is another disease CAR-T has been used against?

Answer 40

Recently also used to treat systemic lupus erythematosus (SLE). CAR-T cells attack self-reactive B cells

Question 41

What is the issue with CAR-T?

Answer 41

Very expensive

Question 42

What are 6 immunotherapies we will have in the future?

Answer 42

1) Soluble T cell receptors (e.g. Kimmtrak)

2) Cancer vaccines
>Inducing immune response against tumour
>The technology for the mRNA vaccine for Covid were developed to combat cancer.

3)Tumour infiltrating lymphocyte therapy (TIL)

4) Gene editing of antibody genes in vivo (CRISPR-Cas9)
>Engineer immune response to produce antibodies of the right type.

5) Modulating innate immune cells

6) Use of γδ T cells for cancer
>These cells are not MHC restrictive.

Question 43

Why might gamma/delta T cells be useful against cancer?

Answer 43

As these cells are not MHC restrictive.

Question 44

Describe the mechanism of action of Kimmtrak for the treatment of uveal melanoma (of the eye) in 4 steps

Answer 44

1. T Cell Receptor (TCR) Targeting:
   >Kimmtrak uses a modified TCR that recognizes a specific peptide from the gp100 protein, which is presented on the surface of melanoma cells via HLA-A*02:01.
2. Phage Display Technique:
   >The TCR is engineered to have increased affinity for the gp100 peptide-HLA-A*02:01 complex on the tumor cells. This is achieved through the use of phage display technology, which allows for the selection of TCRs with high affinity for the target antigen.
3. Bispecific Molecule Construction:
   >This high-affinity TCR is then fused to an anti-CD3 antibody fragment. This creates a bispecific molecule, with one part targeting the tumor antigen (gp100) and the other part targeting CD3 on T cells.
4. Mechanism of Action:
   >The bispecific molecule binds to the gp100 peptide presented by tumor cells on one side and recruits T cells via CD3 on the other. This brings the T cells into close proximity to the tumor cells, activating the T cells and directing them to kill the bound tumor cells.

Question 45

In 2 ways how is Kimmtrak better than CAR-T therapy?

Answer 45

1) Does not require engineering of patient’s cells

2) Permits targeting of intracellular antigens as is processed and presented on surface (e.g. gp100)

Question 46

What is an issue with Kimmtrak?

Answer 46

Does have MHC restriction as is a TCR

Question 47

What is an example of a drug developed against autoimmune disease that went wrong and what happened?

Answer 47

Clinical trials of drug TGN1412 (2006) developed by Tegenero, Volunteers developed a violent reaction within hours of drug administration and ultimately multiple organ failure

Question 48

What were the issues with TGN1412 that caused the organ failures?

Answer 48

> Most immunosuppressive antibodies block activity, whereas TGN1412 binds to CD28 on naïve T cells, inducing stimulation.

> Animal studies indicated selectivity for naïve suppressor T cells (Tregs) BUT in humans memory effector T cells in tissues also express CD28.

> But didn’t take into account that In mice CD28 is only expressed by naive T cells but in humans T memory effector cells also express CD28

> So when administered stimulated memory effector T cells causing a Cytokine storm caused as memory effector T cells activated causing over-production of cytokines (widespread activation of immune system)

> TGN1412 was also an IgG4 antibody, as it doesn’t activate complement so wouldn’t cause inflammation but didn’t realise IgG4 can bind FcR activating phagocytes (inflammation).