Tumour immunology and immunotherapy of cancer

Question 1

What will a real T-cell have on its surface

Answer 1

Protrusions and tensicles to attack cells (tumour) /pathogens

Question 2

What is truncal ataxia

Answer 2

Truncal ataxia (or trunk ataxia) is a wide-based “drunken sailor” gait characterised by uncertain starts and stops, lateral deviations and unequal steps. It is an instability of the trunk and often seen during sitting. It is most visible when shifting position or walking heel-to-to

Question 3

Describe how breast cancer may present

Answer 3

• severe vertigo
• unintelligible speech
• truncal and appendicular ataxia (abnormal movements)

07-03-97: unable to sit, stand, use hands

Question 4

How can breast cancer be linked to the following symptoms: severe vertigo, unintelligible speech, truncal and appendicular ataxia?

Answer 4

Paraneoplastic cerebellar degeneration

The antibody involved is CDR2
CDR2 = cerebellum degeneration-related antigen 2

Question 5

Give a timeline for a case of a patient with paraneoplastic cerebellar degeneration

Answer 5

07-03-97: unable to sit, stand, use hands
21-03-97: detection of anti-CDR2 antibody in the serum
27-03-97: detection of breast cancer

Question 6

How can you detect the CDR2 antigen

Answer 6

Take a section of breast tissue (biopsy)
Add the antibody
Antibody contains a peroxidase (peroxidase-Ig conjugate)- when bound to the antigen will become brown upon addition of the dye.
So if CDR2 is expressed- the section will be brown
If not expressed- it will be a normal pinky colour.

Question 7

Explain how breast cancer can lead to degeneration of the cerebellum

Answer 7

The antigen (CDR2) that the immune response is directed against is normally expressed in neural tissue (Purkynje fibres)

It is only expressed in breast tissue when there is a tumour

The abnormal expression of this antigen in the breast was noticed and an immune response was mounted, which then also reacted with the normal antigens in the neural tissue à destruction of purkinje cells in the cerebellum
The result of this humoral immune response against the tumour antigen is auto-immune neurologic disease

Question 8

What are Purkinje cells

Answer 8

Purkinje cells: type of motor neuron in cerebellum

Question 9

What does this example regarding cerebellar degeneration seen in breast cancer patients teach us about tumour immunology

Answer 9

1. At least certain tumours can express antigens that are absent from (or not detectable in) corresponding normal tissues.
2. The immune system can, in principle, detect such abnormally expressed antigens and, as a result, launch an attack against the tumour.
3. In certain cases, this may result in auto-immune destruction of normal somatic tissues.

Question 10

Describe the cirumstantial evidence of immune control of tumours in humans

Answer 10

1. Autopsies of accident victims have shown that many adults have microscopic colonies of cancer cells, with no symptoms of disease. Immune control?
2. Patients treated for melanoma, after many years apparently free of disease, have been used as donors of organs for transplantation. Transplant recipients have developed tumours. Donor had developed ‘immunity’ to the melanoma, but the transplant recipients had no such ‘immunity’.
3. Deliberate immunosuppression (e.g. in transplantation) increases risk of malignancy
4. Men have twice as great chance of dying from malignant cancer as do women (women typically mount stronger immune responses)

Question 11

Explain the concept of tumour ‘immunosurveillance’

Answer 11

Concept of tumour ‘immunosurveillance’: malignant cells are generally controlled by the action of the immune system.

Question 12

Ultimately, what is the purpose of immunotherapy

Answer 12

Immunotherapy tries to enhance immune responses to cancer.

Question 13

Summarise T cells

Answer 13

T-cells: MHC restricted - Class I/II
Alpha beta TCRs
Gamma delta TCRs

Question 14

Summarise B cells

Answer 14

BCR- cell surface antibody

can bind to a vast range of molecules e.g virus neutralisation

Question 15

Describe the cancer-immunity cycle

Answer 15

Release of cancer cell antigens (cancer cell death)
Cancer antigen presentation (dendritic cells/APCs)
Priming and activation of T cells in lymph nodes (APCs and T cells)
Trafficking of T cells to tumours (CTLs)
Infilltration of T cells into Tumours (CTLs, endothelial cells)
Recognition of cancer cells by T cells ( CTLs, cancer cells)
Killing of cancer cells (immune and cancer cells)

Question 16

What are the T cells that infiltrate the tumour called

Answer 16

a. TIL = Tumour Infiltrating Lymphocytes.

They infiltrate the endothelium of blood vessels to enter the tissues.

Question 17

Describe the selection pressure exerted by the immune system on the cancer

Answer 17

7. T-cells kill tumour.
   a. Cycle then repeats as cellular contents released.
   b. This will result in immune selection pressure which can result in loss of tumour MHC expression – like how bacteria avoid antibiotics.

The cancer cells that can avoid immune system destruction will be selected for.

Question 18

When will T cells be activated in the cancer immunity cycle

Answer 18

If the environment is sufficiently inflammatory and there is enough costimulation then you will get activation of the T cell response

Question 19

Ultimately, what regulates each stage of the cancer immunity cycle

Answer 19

Stimulatory and inhibitory factors
For example, cancer cell antigens will only be released in immunogenic cell death and not tolerogenic cell death.

IL-10,4,-13 are inhibitory in cancer cell presentation
VEGF and endothelin B receptors are inhibitory in the infiltration of T cells into tumours.
Reduced pMHC on cancer cells will reduce the recognition of cancer cells by T cells.

Question 20

Which inhibitory factors are targeted in the immune checkpoint blockade

Answer 20

Can boost immune response to tumours either by increasing stimulatory factors or decreasing inhibitory factors. The latter can be achieved by blocking the function of PDL-1 which inhibits the priming and activation of T cells as well as the killing of cancer cells.

Question 21

Describe the effect of PD-1, PDL-1 signalling on the T cell response

Answer 21

When a T cell has been exposed to an antigen several times, it starts to express PD-1 receptors
Tumour cells the upregulate expression of the PDL-1 ligand, which can bind to the PD-1 receptor and downregulate the T cell response
Blockade of the PD1-PDL1 interaction could help stimulate the T cell response

Question 22

What does the initiation of cancer usually result from

Answer 22

Initiation of cancer usually results frommultiple sporadic events over time

For example. irradiation, chemical mutagens, spontaneous errors during DNA replication and tumour virus-induced changes in genome can lead to the induction of mutations in cellular DNA.

Question 23

Describe the consequences of aberrant regulation of the cell cycle and apoptosis

Answer 23

Mutations in genes encoding proteins regulating the cell cycle or apoptosis can lead to the aberrant regulation of these processes. As a result the tumour cells will proliferate and grow.

Question 24

What is the consequence of tumour growth

Answer 24

The tumour continues to grow until it produces inflammatory signals ( small clusters of tumours won’t be big enough to cause inflammation).
This will lead to the recruitment of cells of the innate immunity (dendritic cells, macrophages and NK cells)
These cells (particularly the dendritic cells) will present the cancer antigens by draining the lymph node
Leading to subsequent activation of the B cells and T cells involved in adaptive, antigen-specific immunity.

Question 25

What are the requirements for the activation of an adaptive, anti-tumour immune response

Answer 25

Local inflammation in the tumour (“danger signal”)- costimulation

2. Expression and recognition of tumour antigens

Question 26

Why are small tumours problematic in terms of immune surveillance

Answer 26

o Small tumours don’t cause a lot of inflammation and is a problem (whereas viruses instantly start the immune response).
o If there is no co-stimulation coming from inflammation, there may be anergy of the immune cells.

Question 27

Describe two problems in the immune surveillance of cancer

Answer 27

1. It takes a while for a tumour to cause local inflammation.
2. Antigenic differences between normal and tumour cells can be very subtle and hard to pick up on by the APCs (don’t want adaptive response against self cells)
   So, if the above conditions are not met or fail to spontaneously active the adaptive immune response, we can “teach” the adaptive immune response to selectively detect and destroy tumour cells. CANCER IMMUNOTHERAPY.

Question 28

How can cancer immunotherapy be used clinically

Answer 28

Cancer Immunotherapy

-Potential alternative/supplement to conventional therapies (surgery, chemotherapy, radiotherapy)

Question 29

What do most cancerous mutations result in and what is the consequence of this for immune surveillance.

Answer 29

Most cancerous mutations result in mutations in intracellular proteins- therefore they will be expressed on MHC and recognised by T cells as T cells can ‘see’ inside cells, and canrecognise tumour-specific antigens

However, some tumours (breast cancers) will express something on the surface that can be recognised by antibodies too.

Question 30

What is the function of MHC

Answer 30

‘Display’ contents of cell for surveillance
by T cells: infection, carcinogenesis

MHC 1- displays endogenous peptides.

Question 31

What is meant by a tumour specific antigen

Answer 31

An antigen only found in the tumour cells -ideal target for cancer immunotherapy

Question 32

Describe the different types of tumour specific antigens

Answer 32

Viral Proteins:
EBV, HPV

Mutated Cellular Proteins
TGF-B receptor III
Chromosomal translocations (BCR-ABL)- generates new sequence- generation of novel protein

Question 33

Describe how viruses can cause cancers

Answer 33

cause deregulation of cell cycle and contain oncogenes/proteins leading to uncontrolled cell growth

Question 34

Describe viral opportunistic malignancies

Answer 34

Opportunistic malignancies: Immunosuppression

• EBV-positive lymphoma: Post-transplant immunosuppression - also CMV
• HHV8-positive Kaposi sarcoma: HIV (now treatment is effective at preventing this risk)

Question 35

Describe cancers of viral origin in immunocompetent in individuals

Answer 35

• HTLV1-associated leukaemia/lymphoma
• HepB virus- and HepC virus-associated hepatocellular carcinoma
• Human papilloma virus-positive genital tumours

Question 36

What cancer can HPV cause

Answer 36

Cervical cancer - HPV 16

Tumour cells express
viral antigens!
Vaccination successful

Question 37

Describe HPV 16

Answer 37

Internal proteins of the virus cause deregulation of the cell cycle and thus aberrant cell growth (E6 and E7 oncoproteins)
These are intracellular antigens- peptides from them are expressed on the cell surface (MHC I) and can be recognised by T cells.

Question 38

Describe the target antigens for prevenative HPV vaccination

Answer 38

· We can target these antigens (E6, E7) for a preventative HPV vaccine!

Surface proteins, incorporated into Virus-Like Particles (VLPs) The vaccine contains no DNA- so no risk of transfer of oncogenes

Early genes -E1-5
Late genes- L1-2

Question 39

Give an example of a HPV vaccine

Answer 39

Gardasil 9 ( 9 recombinant viral surface proteins)
Helps protect girls aged 9-26 against cervical, vaginal, vulvar and anal cancers and genital warts caused by 9 types of HPV

Helps protect boys aged 9-26 against anal cancers and genital warts against those same 9 HPV types.

Extremely effective in reducing the incidence of cervical cancer.

Question 40

Describe the potential progression of HPV16 infection

Answer 40

> 99% will have strong immunity and clear the HPV infection and develop immunological memory
However, a small minority will have immune failure, potentially leading to cervical neoplasia (50% NO IMMUNITY, 50% non-functional immunity)

Question 41

How can the HPV vaccine be used

Answer 41

Preventative vaccination (before the disease) 
Therapeutic vaccination (try to control the disease once it has occurred)- in those who didn't receive the prophylactic vaccine and went on to develop cervical cancer.

Question 42

What is meant by tumour associated antigens

Answer 42

Tumour-associated antigens (TAA) are normal cellular proteins which are aberrantly expressed (timing, location or quantity).

Because they are normal self proteins, for an immune response to occur tolerance may need to be overcome

Question 43

What is key to remember about tumour associated antigens

Answer 43

They are not mutated

Question 44

Describe some ectopically expressed auto-antigens that are tumour associated antigens

Answer 44

• Cancer-testes antigens (developmental antigens): Silent in normal adult tissues except male germ cells (some expressed in placenta).
  e. g. MAGE family: Melanoma associated antigens. Identified in melanoma also expressed in other tumours.

Question 45

Describe some other tumour associated antigens

Answer 45

Human epidermal growth factor receptor 2 (HER2): overexpressed in some breast carcinomas

Mucin 1 (MUC-1): membrane-associated glycoprotein, overexpressed in very many cancers

Carcinoembryonic antigen (CEA): normally only expressed in foetus/embryo, but overexpressed in a wide range of carcinomas

PROSTATE: prostate-specific antigen (PSA)
prostate-specific membrane antigen (PSMA)
prostatic acid phosphatase (PAP)

Question 46

Summarise tolerance

Answer 46

§ Tolerance induction is via negative selection in the thymus (central tolerance).
§ This central tolerance is not perfect and we all have some potential auto-reactive T-cells in our repertoire.
o Some of these cells have some use in immunotherapy in tumour treatment against TAAs if we can inhibit the tolerance or stimulate their expression.

Question 47

Summarise T cell maturation in the thymus

Answer 47

Population of DP thymocytes
Some of these will die by neglect – leading to pre-selection repertoire
Negative selection
Positive selection- exported to the periphery and will be self-MHC restricted and self tolerant
However central tolerance is not perfect and so some T cells will be autoreactive.

Question 48

When is p53 considered a tumour association antigen and when is it considered a tumour specific antigen

Answer 48

Tumour-associated antigen – when it is over-expressed

Tumour specific antigen – when it becomes mutated

Question 49

Describe the potential problem with differentiation (lineage-specific) auto-antigens

Answer 49

§ Tyrosinase is a differentiation-type antigen.
§ Lots of people have poor tolerance of this Tyrosinase and so lots of people have T-cells that can recognise peptides from Tyrosinase.
o Tyrosinase is involved in skin pigmentation.
o In treatment of melanoma, the t-cells target the cancer cells and the Tyrosinase and so there is a loss of skin pigmentation – vitiligo.
§ Immune response to cancer leads to some symptoms of autoimmunity.

Question 50

What are the two main problems in targeting tumour-associated auto-antigens for T-cell mediated immunotherapy

Answer 50

Two major problems:

Auto-immune responses against normal tissues

2. Immunological tolerance
   - Normal tolerance to auto-antigens
   - Tumour-induced tolerance (cancers may downregulate immune response)

Question 51

Describe the issues associated with overcoming tolerance

Answer 51

o Overcoming any immunological tolerance:
§ Normal tolerance means you cannot use that antigen for immunotherapy.
§ Sometimes the tumour cells expressing the antigens can induce tolerance as they might not cause inflammation so the presentation of the antigens without co-stimulation could make the T-cells anergic and induce tolerance

Question 52

List the different approaches being used and developed for tumour immunotherapy

Answer 52

Antibody-based therapy (of a single specificity)
Therapeutic vaccination
Immune checkpoint blockade
Adoptive transfer of immune cells
Combinations of 1) to 4) above

Question 53

Describe the naked monoclonal antibody-based therapy

Answer 53

“Naked”
e.g. Trastuzumab (Herceptin®) anti HER2,
anti CD20, anti CD52, anti EGFR

HER2- BREAST CANCER
CD20, CD52- B CELL lymphomas
EGFR- COLORECTAL CANCERS

Question 54

Described conjugated monoclonal antibody-based therapies

Answer 54

Conjugated- linked to something that will aid it’s deliver to the tumour

radioactive particle e.g. Ibritumomab tioxetan (Zevalin®), anti CD20 linked to yttrium-90

drug e.g. Trastuzumab emtansine (Kadcyla®), anti 	HER2 linked to cytotoxic drug

Question 55

Describe the bi-specific mono-clonal antibodies

Answer 55

Genetic engineering- split the two Fab fragments- two different targets

Genetically engineered to combine 2 specificities, e.g. anti CD3 and anti CD19 (Blinatumomab, approved for use in patients with B cell tumours)

Question 56

What is a key issue with monoclonal antibodies

Answer 56

They are expensive

Question 57

Describe therapeutic cancer vaccination

Answer 57

There is one FDA approved vaccine to treat cancer (also licensed for sale in the UK, but not NICE approved):

Provenge® (sipuleucel-T) for advanced prostate cancer

Patient’s own WBC are treated with a fusion protein between prostatic acid phosphatase (PAP) and the cytokine GM-CSF (patients own cells are removed and then put back in- adoptive transfer)

Stimulates DC maturation and enhances PAP-specific T cell responses

Question 58

Describe personalised tumour specific cancer vaccines

Answer 58

Extract DNA from tumour cell- then use whole exome sequencing to identify its somatic mutations and expression confirmation of mutated genes
Then compare this to the WES of the normal cell to identify mutations and compare to HLA typing of normal cell to determine which mutant proteins could bind to HLA
Use this information to create candidate neoantiges and add adjuvant (to potentiate immune response) to create personalised vaccine.

This is an expensive technique

Question 59

What is the ultimate function of immune checkpoint blockade

Answer 59

Rather than directly stimulate responses, this approach seeks to reduce/remove negative regulatory controls of existing T cell responses
Particularly useful in melanoma
Useful method- as it is general- no need for TSAs or TAAs and so widely applicable.

Question 60

Which pathways may be targeted in the immune checkpoint blockade

Answer 60

Targets CTLA-4 and PD-1 pathways:
CTLA-4 is expressed on activated and regulatory T cells, binds to CD80/86 (costimulatory molecules on APC)
PD-1 is expressed on activated T cells, binds to PD-L1/L2 (complex expression patterns, may be upregulated on tumours)

e.g. Ipilimumab (anti CTLA-4), Nivolumab (anti PD-1), antagonistic antibodies

Question 61

Describe adoptive transfer of cells

Answer 61

T-Cell source- blood or TILs removed in surgery

§ Extract the TILs and then multiple the number of TILs and reinfuse the TILs into the patient. (non-specific TIL expansion)
o They can be expanding with use of cytokines as well ( antigen-specific expansion using IL-2)- CULTURE IN FLASK- EXPAND AND then re-infuse into patient
o Genetic engineering techniques can also be used to express chimeric antigen receptors (CARs) (see below)

Question 62

Describe chimeric antigen receptors

Answer 62

Genetic engineering of adopted T cells to change their specificity.
CAR is usually Fab part of antibody that has been fuses with a linker protein — single chain fragment variable (scFv)
This will bind to the antigen (i.e CD20 in B cell lymphoma)
This will bind to transmembrane part of TCR, and will also contain co-stimulatory protein I and ZETA chain- from CD3 polypeptides- so that when CAR binds to antigen- T-cells will be activated by co-stimulatory part and tyrosine phosphorylation part (CD3 domains)- three-license model for activation.

All T cells transfected will contain this receptor and attack the cancer cells- very general and applicable- just change specificity of CAR

Question 63

What is key to remember about cancer immunotherapy

Answer 63

Two major problems in developing immunotherapy for tumours are autoimmune reactions against normal self tissue, and breaking immunological tolerance against self. These problems don’t apply for viral tumour antigens.
Even when responses are made against tumours, immune selection pressure may lead to the outgrowth of cells that have down-regulated or have mutations in proteins required for antigen presentation, e.g. MHC molecules.