Biological therapies

Question 1

Why talk about cancer

Answer 1

• Biggest investment (most money spent in healthcare)
• Carries the biggest emotional burden
• Kills people
• Has a lot of unmet needs
• Fastest progression in oncology
- In terms of therapeutic area (biggest expanding)
• Need for new cancer therapies

Question 2

Definition of biological therapies

Answer 2

A biological medicine is a medicine that is derived from a biological (living) source, such as bacteria or viruses, blood, tissues or living cells in culture’ (MHRA 2017)
•

Question 3

Biologic vs small molecule

Answer 3

Biologic is produced by a living organism 
-	Whereas small molecules are chemical entities 
•	Examples of biologics include:
-	Monoclonal antibody (mAb)
-	Gene therapy
-	RNA therapeutics
-	 Somatic cell therapy
-	Blood coagulation factors
-	Vaccines
-	Peptides (e.g. insulin)
•	Small molecules move intracellularly 
-	Small molecules are significantly smaller than biologics

Question 4

Anti-cancer treatment

Answer 4

1. Surgery
   – Early stage, low risk patients (patients less likely to die)
   – Surgery doesn’t benefit people with curative intent
   – Whereas the other treatments are done for curative intent + prolonging therapy
2. Radiotherapy
   – Ionising radiation – X ray, γ-ray, neutrons, electron and protons
   – External/Internal
3. Systemic therapy
   – Cytotoxic chemotherapy
   – Hormonal therapy – Breast/prostate
   – Targeted therapy
   – Immunotherapy
   • Systemic therapy used to small molecules (chemotherapy)
   – This is the backbone of cancer, but now there are different types of therapy
   • Anti-cancer treatment has made tremendous progression in the last 20yrs

Question 5

traditional chemotherapy

Answer 5

• Maximum tolerated dose
- Single ascending dose
- You want to give the highest dose to kill the cancer cells (but you can also kill healthy cells that are actively dividing)
- so you need a dose that won’t cause too much harm & kill normal cells
- maximum tolerated dose is very important (do not want cytotoxicity)
- Treatment cycles seen a lot in chemotherapy
• Administered as treatment cycles (e.g. 3 weeks)
– Balance recovery time of normal tissues, amount of cancer cell death and adverse reactions
– Patients need time to recover (allowing healthy cells to recover but achieve maximum kill of cancerous cells)
• Chemotherapy regimens often consist of multiple agents need multiple treatments)
– working synergistically targeting different phases of the cell cycle to↑ cell death
– e.g. alkylating agents work around different phases of cell cycle, but toxoids only work on G2 & M phase (they work on different phases of cell cycle to get max kill)
• ↓ therapeutic resistance and toxicities (normal dividing cells)
- Cancer cells can become resistant due to mutations
- The same patients have very different genetic makeups & cancers create their own microenvironments
- Chemotherapy agents work on different parts of the cell (can lead to toxicities)
– Multiple treatments can lead to toxicities such as:
• Gastrointestinal tract – N&V
• Bone marrow - myelosuppression
• Peripheral neuropathy
• Systemic: fatigue and anorexia
 Note: response rate is variable, approximately 15-20% for a single agent
- Using single agent = lower response rate (big unmet need)

Question 6

Targeted therapy in cancer

Answer 6

Definition: same systemic treatment targeting specific molecular pathways, such as:
• Cell proliferation
• Protein synthesis
• Angiogenesis (growth & formation of new blood vessels)
• Immune cells regulations
Companion diagnostics:
• Driver mutations within the somatic genome that enhance cancer cell growth in cancer cells
• Companion diagnostics – identify oncogenes that drugs can target
- Need to identify the driver mutation via a test to identify the main oncogene involved - so drugs can target oncogene
- Targeted therapy (need companion diagnostics for it)
- Not only do you design a drug but you design a good pathway that is identified for further research (increases complexity in drug development process)

Question 7

Schemes for targeted therapy

Answer 7

• Trastuzumab/Pertuzumab = most successful oncology drug in the last 20yrs
- They target HER2 receptor
• Hormones = old school targeted therapy
- Intracellular receptors (Usually small molecules)
• Growth factor receptors & tumour microenvironments = biologics
- Biologics target extracellularly need soluble receptor that exists around the blood / on cell surface)
- too big for intracellular targeting ( cannot cross the cell membrane )
• Monoclonal biologics are more specific
• You would get less off target effects with biologics
- Off target pharmacology = 2nd pharmacology / pharmalogical effects that you would not expect)
Toxicity is drug dependent – targeted therapy does not reduce chance and severity of potential toxicities

Question 8

Immunotherapy

Answer 8

• Type of targeted therapy
• Tumour infiltrating lymphocytes (usually T cells – physiologically identify & kill cancer cells)
– Identify and destroys abnormal/cancer cells
– Natural Immune system response to cancer
– Cancer cells have multiple mechanism evading immune response e.g.
– Mutations – evasion of cancer cells identification (can’t be identified)
– Cell surface protein inactivating T-cells
– Alteration in tumour microenvironment to prevent interactions with immune cells

• Tumour heterogeneity – genetic and phenotypical variations between cancers cells of the same patient  treatment response ↓
- Difficult to treat cancer with curative intent (hard to completely eradicate cancer cells)

• Immunotherapy activates systemic tumour-specific CD8+ cytotoxic T lymphocyte (CTL) responses against cancer cells
– Overcoming immune evasion
– Bypass tumour heterogeneity
 In tumour heterogeneity – once the immune system is activated = you make it more active
- Achieve higher cancer kill
 Why is there a problem with making immune system more immunogenic (more activated)?
- = increased risk of autoimmune disease
- Autoimmunity is a big problem in immunotherapy

Question 9

• 3 types of immunotherapies:

Answer 9

• Antibody-based ‘checkpoint’ inhibition
• Adoptive T cell therapy
• Therapeutic vaccination (least advanced – in terms of bringing it into clinical practice

Question 10

comparison of biologics and small molecules

Answer 10

• Biologics can only hit surface receptor (extracellularly)
• Small molecules can hit surface receptor & intracellular pathways at different time points
• Bispecific antibody – can target 2 things specifically, not just 1 like other antibodies
• Antibody drug conjugate (ADC) – new technology
- Antibody is loaded with cytotoxic drug & upon binding with the right receptor
- = cytotoxic drug gets released & undergoes it’s mechanism of action
• Gene therapy = still under development
- The ASO (antisense oligonucleotide) (small pieces of DNA or RNA which can bind to specific molecules of RNA and block the ability of RNA to make a protein) / RNA therapeutics are getting more common
- They need to be inside the cell with the RNA (intracellular)
• Newer developments will mean that biologics might be able to target intracellularly

Question 11

Monoclonal antibodies

Answer 11

• Fab (fragment antigen binding portion) is where the antigen is
- Monoclonal = so recognises 1 antigen
• Most mAb are IgG
• Need to be injected (problem delivering to patients)
- Too big to pass through if swallowed
- Novel mAbs can be swallowed but only works topically in the guts & it can get into the system

Question 12

Monoclonal (mAb) advantages

Answer 12

• Most mAbs are mostly IgG
- It follows IgG does in the body & the Pk of that mAb antibody
- It’s a starting point for mAb & then it can be modified
• Advantages
– Single target antigen specific, limited or no off-target effects
• Toxicity relates to on-target, off tumour toxicities
- If you have immunotherapy; you get immune system that targets cancer but immune system also targets other parts of the body (on target – off target tumour toxicities)
• Drug interactions: mostly pharmacodynamic
– Long acting (because its monoclonal

Question 13

Monoclonal (mAb) disadvantages

Answer 13

– Require cell surface or soluble receptor targets
– Parenteral – oral administration has limited systemic applicability
– Long acting (If you have a problem – you need to wait for the drug to clear)
– Immunogenicity
- If you inject antibody into the system, the immune system will react to the monoclonal making it unpredictable in terms of PK
- Immunogenicity can also cause immune reactions
- For biologics immunogenicity is the biggest problem
- For small molecules immunogenicity is not a problem

Question 14

what is immune CHECKPOINT`

Answer 14

• Immune checkpoint molecules are expressed on cell surface of the adaptive immune system, particularly on T cells, and of the innate immune system
• They are crucial for maintaining the self-tolerance (prevent autoimmunity) and modulating the duration and magnitude of immune responses of effectors in different tissues to minimise the tissue damage
- Crucial to avoid auto-immune reactions

Question 15

checkpoint inhibitors - mAB

Answer 15

• Checkpoint inhibitors = allow tumour cells to get immune response
- Inactivation of the checkpoint
- Tumour cells kill by deactivating the immune system (inhibition of PD1 – with PDL1)
- Therefore, T-cells are not activated to kill tumour cells
• Checkpoint inhibition via mAbs allow reactivation of T helper cells reactivation
• Checkpoint inhibitors is a game changing drug that has become standard of car for lung cancer
• However, Immune related adverse reactions

Question 16

Monoclonal antibodies vs chemical drugs:

Answer 16

weight:
production:

Question 17

Antibody drug conjugate (ADC)

Answer 17

• ADCs are most effective when the target cell surface antigens are highly expressed in tumour cells but not in healthy tissues
• The cytotoxic ‘payload’, which is the ultimate effector part of the ADC, is highly potent and characterised
• The linker to the payload is crucial to the therapeutic window - stability vs ease of release
• Systemic toxicity is related to small molecule payload

• Payload / warhead = once it binds to cell surface, it gets released
• Most important component of ADC is the linker
• You need to have a good understanding of the cytotoxic drug that you want to use as a payload
- as it needs it will a very specific cellular target & need a very small dose
• you use a small dose to target the cell you want = larger effect with smaller dose & less likely to get systemic toxicity
- This is why people are interested in ADC
• However in reality it doesn’t work like that !!!
- If the linker is attached too tight to payload = cytotoxic drug is not released & does not kill cancerous cells
- If the linker is attached too weakly to payload = cytotoxic drug is released everywhere = increased toxicity (+ you get chemotherapy as well)
- ADC has to deal with toxicity
• ADC linker technology is very important for ADCs to work effectively

Question 18

ADC mechanism of action

Answer 18

• ADC work by:
- Binding to antigen
- Undergo ADC receptor mediated internalisation via endocytosis
- Apoptosis of drug target cell
- Degradation of ADC in lysosomes
- Payload release of cytosol
• All of these lead to cytotoxic mechanism = leads to fall of cancerous cell cycle

Question 19

ADC - Ado-trastuzumab emtansine

Answer 19

• ADC that conjugates trastuzumab to the microtubule inhibitor DM1 (one of the most successful ADC)
– Trastuzumab is a mAb binding the human epidermal growth factor receptor 2 (HER2)  standard of care in HER2 breast cancer (15-20%) for 20 years
– Trastuzumab silenced intracellular signalling and stimulated immune responses, but resistance is common
– DM1 is added as a cytotoxic payload
• Trastuzumab is already very successful in breast cancer – adding cytotoxic payload – increase efficacy
• However, cytotoxicity is a issue because of linker technology

Question 20

Bispecific antibodies

Answer 20

• Bispecific antibodies are capable of binding two target antigens
– Simultaneously modulate two signalling pathways in the same disease target (e.g. dilpacimab, an antiangiogenic agent targeting both the delta-like ligand four and vascular endothelial growth factor pathwaysss DIAGRAM B)
– Encourage the binding of both cells and proteins (bridging cells) e.g. blinatumomab promoting binding of T cells to tumour cells in oncology
– Blinatumomab = most advanced (used in myeloid leukaemia & haemophilia)
– Monoclonal are only specific to 1 antigen
• Bispecific antibodies have a variable construct:
– Ranging from a whole IgG-like molecule to a smaller molecule with just a single-chain variable fraction. The PK is dependent on size and therefore more variable than mAb
– Used 40/50 constructs = variety in co-receptor to conceptualise the mechanism (but vey useful)
• Only cell surface or soluble target
• Diagram:
- A = bispecific antibodies bridge the cell
- You can get cancer cell & bridge it to an effector cell (e.g. immune cells)
- B = activate 2 pathways they might not be activated at the same time point but you bind them together
- Activation leads to down streaming events = receptor inhibition
• Useful tool as you can simultaneously block two useful pathways

Question 21

Adoptive T-cell therapy

Answer 21

• A ‘living’ drug – T-cell based immunotherapy (actual living cells e.g. T-cell transfusion)

1: Tumour-Infiltrating Lymphocyte (TIL) Therapy
• Selection of pre-existing TIL in patients
- Concentrate the T-cells and give them to the patient (it’s what they naturally produce in their body)
- Identify what can kill cancer & grow it and give it back to the patient

2: Engineered T Cell Receptor (TCR) Therapy
• Engineered T-cell receptor insertion to bind to tumour antigen presentation via MHC
- Overcome T cells with a lack of receptor to identify the cancer
- The T cells don’t identify the receptor naturally ( so you insert T-cell receptor you want to identify) & give it back to the patient

3: Chimeric Antigen Receptor (CAR) T Cell Therapy
• Chimeric antigen receptor insertion to bind to tumour even without antigen presentation via MHC
- Create an antigen receptor insertion
- Creates an extra receptor without relying on antigen presentation via MHC cells
- You can activate T cells independent of MHC antigen presentation
• Adverse events: cytokine release syndrome (CRS)
- CAR T-cell therapy is a very powerful form of immunotherapy & can read to CRs (massive inflammatory reaction)

Question 22

Problems with T-cell therapy

Answer 22

• The problem with these therapies is the duration of response
- They don’t work forever (develop resistance)
- They are still experimental (need to get good quality results consistently)
• Lymphodepletion chemotherapy has been used recently
- Very powerful if it works but some cells may not respond
- Red dot = stage where you can lymphodepletion chemotherapy before give the Adoptive T-cell therapies back to the patients
- It has been used to decrease myeloid suppressor cells (cells that can suppress immune system) & T cells
- So, the T-cells that you induce get a lot of immunotherapies
- However, drawback = increased cytokine release syndrome (CRS)

Question 23

CAR-T cell therapy

Answer 23

Engineered CAR-T cell receptor, which is independent of antigen presentation
- Therefore a lot more flexible
• However, very individualised for every cancer cell
- Need new receptor needs to be produced every time
• CAR-T receptor many not always bind as cancer cells can be very heterogenous in terms of receptor presentation
- They can have mutations

Question 24

RNA therapeutics

Answer 24

• ASO = antisense oligonucleotides =
Small pieces of DNA or RNA that can bind to specific molecules of RNA. This blocks the ability of the RNA to make a protein or work in other ways.

• RNA therapeutics used to silence gene / mutation
• Once they have done this, the effect will last a very long time
• If you do not want a protein to be produced in cancerous cell
- You can use a piece of RNA to bind to the gene of that protein = no production of that protein
• siRNA = small interference RNA
- same concept but double stranded
- so, it is a bit more stable
• However, for RNA therapeutics the technology isn’t mature yet
- The problem is sending the RNA to the right place to silence the gene (no specificity)
- But once the RNA is there = long duration of action (advantage)

Question 25

Immunogenicity

Answer 25

• Immunogenicity = the ability for an foreign substance to provoke an immune response

• May affect PK (clearance), PD (efficacy and adverse effects)
- Biggest problem in biologics = it can affect PK & PD
- Therefore, affecting efficacy and causing adverse events
• Large variability with regard to incidence and impact of ADA formation among different biologics
• The biggest problem with immunogenicity is that you have an antibody against your antibody for cancer
- Leading to unpredictable effects (you don’t know what can happen)
- Although sometimes it will be fine, with no immunogenic response

• Not all ADAs affect the efficacy and/or safety of the biologics. Various characteristics of the ADA determine this:
• (Some tests that identify any ADAs developed, so you can understand what they do properly):
– The ADA titre
– binding affinity
– ability to neutralize the mAb’s activity
• The incidence reporting for immunogenicity is highly variable and dependent on the assay measuring the antidrug–antibody. There is no standard unit of quantification and results are assay dependent. Data on each mAb continues to emerge and evolve (e.g. adalimumab)
• Immunogenicity is the biggest problem for biologics
- ADAs are a huge problem & cause loss of action of a drug

Question 26

contributory factors of immunogenecity

Answer 26

• Fraction of foreign sequence on mAB
• Duration of therapy
• Dose
• Previous treatment with similar mab
• Route of administration subcutaneous
• Genetic predisposition + underlying disease

Question 27

cancer epidemiology

Answer 27

cancer cases are expected to increase yearly

age is biggest risk factor

Question 28

cancer is a genetic disease

Answer 28

1: Something wrong with the genetic code
2: Structural alterations = pieces of chromosome that go from 1 chromosome to the other
3: Numerical abnormalities = abnormal amount no. of chromosomes

Question 29

Tumours have multiple layers of genomic diversity

Answer 29

• Tumours have lots of genomic alterations – some are functional (aka drivers), whilst others are not (aka passengers).
 10s-10,000s different alterations / mutations per tumour.
- Some are driver / passenger mutations
- Drivers = carry high burden of the disease
- passengers = mutations that are present but don’t contribute much to tumour
• Many different genomic alterations in each tumour (no 2 patients have the same cancer analogy)
• Many different combinations of alterations.
• Thus, new tools that allows us to alter the genome can accelerate our ability to understand the driving forces of cancer.

Question 30

CRISPR bacteria example

Answer 30

• Part of a CRISPR locus within the bacterial genome
• CRISPR cas9 is an adaptive immune response in bacteria
- Image shows CRISPR in the bacterial cell (made up of repat, spacer, repeat, spacer … segments)
• Repeat segment is exactly the same for each repeat segment – it is a short segment of DNA
• Spacer comes from a virus, when it infects a bacterium
• When the virus infects the bacteria, the bacteria incorporates a small part of viral DNA into the bacterial CRISPR locus
- The orange bit is from the viral DNA that is incorporated into the bacteria
•
• This is how bacteria can vaccinate themselves from future viral infection
• Guide RNA (gRNA) made up of repeat – spacer segment
• Cas9 = molecular scissor that cut DNA
• gRNA recognises the sequence that needs to be edited
• gRNA guides cas9 to viral genetic code (this is where gRNA matches the sequence in the virus)
• gRNA tells cas9 where to cut the viral RNA in the bacterium
• Allowing cas9 to obliterate the virus, which is then degraded

Question 31

What is CAS9

Answer 31

Nuclease - a protein that cut nucleid acids
cas9 makes a double strand break
directed to a sequence by a rna - in bacteria the rna is produced from crispr loci

Question 32

crispr/cas9 gene editing tool

Answer 32

• If you want to make a cut in the human genome, you need to design a gRNA that matches the sequence of the human genome that you want to cut.
• The gRNA takes Cas9 to the specific part of the genome that you want to cut
- = double strand break in the DNA
You can make CRISPR Cs9 a cut & paste tool for genome editing

Question 33

CRISPR/Cas9: two kinds of editing

Answer 33

• Non-homologous end joining (introduces knockout)
• Once you have cut the DNA = disrupts the gene completely
- Cuts out the gene completely = knockout
• Non-homologous end joining will just repair the DNA
- Ligates the bit where the DNA was cut

• Homologous recombination
• You give the cell instructions
• When you make a cut, you want the doner DNA to replace it
• Instead of just ligating the cut together
- You do the same thing, but you introduce a cut & give it instructions
- So when the DNA is broken, you can get doner DNA to ligate the sequence

Question 34

CRISPR Cas9 has important implications in research as well as cancer

Answer 34

CRISPR baby snails: discovering the gene that controls shell coiling direction
- Most baby snails’ coil to the right hand side (are right handed)
- Used CRISPR Cas9 to change direction, so that the snails coil to the left hand side
Agriculture: Enhancing crop yields
- You can control how much yield can be produced by a tomato plant
- With CRISPR Cas9 added to these plants = can control specific genomic regions
- They found yields were 2/3x higher & very good quality
- This has huge implications around the world considering climate change
• CRISPR Cas9 is very versatile & has lots of implications (uses)

Question 35

somatic vs germ line genome editing

Answer 35

• Somatic cell = fully differentiated (e.g. me & you)
- Changes made here stay with the individual, as these changes are not made in the germ line
• Germ cell e.g. sperm, egg, embryonic cells
- Passed down generations

• CCR5 is very important for people with HIV
• Normal version = wildtype with HIV
• Disrupted (normal population) = we have 32 deletions that make us resistant from having HIV
• A scientist introduced CRISPR to these HIV twin embryos
- Found that the deletion did not happen at the right bit for both twins
• CRISPR can have off target effects (not very specific)
- Sometimes CRISPR Cas9 cannot be very targeted & may not be effective at what it is supposed to do
• Huge controversy why this experiment was done

Question 36

DNA hypermethylation in cancer

Answer 36

• Normal cells = tumour suppressor genes are not methylated - methylation leads to gene inactivation / silencing)
• Cancer cells = their tumour suppressor genes are heavily methylated
- Therefore, tumour suppressor genes are inactivated
- = no suppression of tumour

Question 37

targeting p16 with methylation

Answer 37

• P16 = tumour suppressor gene
• You can take Cas9, with an enzyme responsible for causing methylation (e.g. DNMT enzyme) and gRNA
- Found that you can introduce methylation to p16 (tumour suppressor gene)
- = leads to inactivation of p16

• Found that when p16 was inactivated, the cells proliferated & divided uncontrollably
• As time went on the no. of these cells increased
- Uncontrollable cell division is an important phenotype for cancer
• We can use Cas9 to induce cancer
- Imagine if we can do this to oncogenes (cells can proliferate very fast)
- You can therefore target oncogenes & reduce cell proliferation

Question 38

Challenges with CRISPR/Cas9

Answer 38

• Potential off-target effects that gives rise to chromosomal rearrangements and can induce mutations.
- Roughly 20 nucleotide long (not very long)
- Hard for Cas9 to bind to that specific space & causing them to bind somewhere else
- Therefore, potential cause of mutation
• Lack of efficiency
- Doesn’t always work
• Immunogenicity of CRISPR components.
- Can elicit a response from immune system
• Needs to be personalised (as each patient can have numerous mutations)
- Need specific gRNA to make the cut with Cas9
- However, you would not target all the mutations
- need to identify the driver mutation (mutation with highest to the disease)
• Advantage: CRISPR Cas9 is very cost effective
• A lot of changes are being made to Cas9 to help modify it (look at papers)