Antibody therapy in cancer Flashcards
LO
Part A
Immunotherapy
Antibodies
Monoclonal antibodies
Part B
Therapeutic antibodies
Effector mechanisms
Part C
Examples of therapeuci mAb
Anti-Id; CD20; Herceptin and cetuximab; immunomodulatory
Part D
Optional- how we try to define effector mechanisms
Why do we need new therapies?
Increasing intensity doesnt equal better outcomes
Who was the father of immunotherapy?
William B. Coley
Explain the work that William B. Coley undertook, the experiments he did and the outcome
- For many the father of immunotherapy was William B. Coley.
- An outstanding New York Surgeon who claimed to cure inoperable tumours using an extract of various bacteria.
- He was trained at Harvard medical school on a three-year programme.
- In fact, he entered straight into the second year on the basis that he had spent a summer as an assistant to his uncle travelling round on a horse and trap visiting sick patients.
- After just one year he had a summer job as Junior Assistant Surgeon in a New York Hospital and after his first week wrote to his girlfriend saying that he had had a fantastic week having in his first week sewed on a patient finger.
- So, I am sure that our Professor of Surgery will agree that Surgical training has changed little.
- However, he has helpless in the face of the many advanced cancers he saw and started to investigate alternative approaches.
- He became fascinated by reports of rare spontaneous regressions in very advanced disease.
- What he called ‘Natures little miracles.
- He was also intrigued by the observation that certain cancers would regress quite markedly during a fever followed infection.
- As a result, in the late 1900 century Coley and other physicians started to deliberately infect cancer patient’s various strains of streptococcus bacteria or applying gangrene carrying bandages to the cancer.
- This could be achieved by moving the patient into the bed of a patient already carrying such an infection.
- Unfortunately, and despite claiming some improvements in disease, patients were apt of die from infections, so the practice did not gain support.
- These were described as ‘practical difficulties. What Coley did observe was that the infection and the therapeutic activity were separate events and he believed that a ‘factor’ or ‘active principle’ existed that might be separated from the infective part of the treatment.
- The mixture he extracted became known as Coley’s toxin and he claimed huge considerable success. But science is all about time and place and William Coley’s time had not arrived.
- Others could not repeat his success and the X-ray machine was about to arrive which gave Oncologists an easy-to-use treatment that brought instant success. So what was Coley’s toxin doing?
- It is difficult to know how much Coley appreciate about what he was doing, but now we have a much clearer picture it involves the immune system. The body’s immune system has evolved to cope with infection and by stimulating a profound response Coley was probably producing beneficial side effects of boosting immune response against the cancer.
- The same kind of response that occasionally produced natures little miracles. that we are only now starting to understand. was success later claimed to have performed 360 hernia operations with a single infection-related death.
From William B. Coleys experiments, what did we learn?
- A tumour is genetically unstable
- Continuously generates new variants of itself
- The immune system kills most
- ‘successful’ variants survive
- Many of these hijacked immune control systems or checkpoints
- Tumour evades immune rejection
What is some evidence of cancer immune surveillance?
- Spontaneous regression in patients diagnosed with melanoma, RCC
- Cancer incidence high in immune suppressed host
E.g., human transplant recipients
Immune deficient mice
- Immune escape
Ag loss (stealth tactics)
- Active local immune evasion
What do TIL (tumour-infiltrating lymphocytes) mediate and what does this correlate with?
The TIL mediate tumour rejection and correlate with survival
Immunotherapy uses the immune system to fight cancer. What aspects of the immune system are used to do this?
Whats the structure of an Immunoglobulin?
- Y shaped glycoprotein
- 2 identical heavy chains (50-70 kDa) and 2 identical light chains (25 kDa)
- Fab (fragment antigen binding) bind antigen
- Fc (fragment crystallisable) bind to Fc receptors (changes structure depending on class- IgM, IgD, IgG, IgE, IgA)
- Theres a sugar in the middle of the antibody that (in the Fc regions)
The antibody revolution: approved* Ab therapeutics since 1997
Tell me the stages to monoclonal Ab technology
- Mouse is immunised with antigen X, and mouse spleen produced plasma cells that secrete antibodies against the antigen
- Myeloma cells unable to produce antibodies or HGPRT are selected
- Mouse spleen is removed. Plasma cells from spleen are isolated and mixed with myeloma cells. cell fusion is induced to produce hybridomas
- Cells are transferred to HAT medium
- Hybridomas that produce antibodies specific to antigen X are selected and grown into bulk
Why are monoclonal Ab used?
- The same every time so easier to work with
- Can generate forever. Once generated the hybridoma it is immortal and continues to generate the antibody forever
- Myeloma cells are immortal cancer cells that used to be plasma cells
What are some key developments in antibody immunotherapy?
What are some of the advantages of this?
Chimerisation and humanisation of antibodies
Advantages:
- Reduced immunogenicity
- Improved effector engagement
- Improved half-life
Modular recombination is a key development in antibody immunotherapy. Tell me some of the formats of this and what they are
- F(ab) which is one binding arm on its own
- Single chain FV (scFV) which is an even smaller part of variable domain
- Bispecific: one arm binds to one thing and the other to something else (recent development in cancer therapeutics)
- Certain receptors where ligands are trimers (try to mimic with a three arm antibody)
What are some considerations for mAb alternatives?
Do we want effector functions (Fab2/Fab)
Do we want multiple specificities? (BsAb)
Do we want the mAb to circulate for a long period of time? (No Fc – rapid half-life)
Can we improve on tissue penetration (smaller size, the better)
What is associated with greater tumour-targeting specificity than IgG?
Diabody
Currently approved mAbs for cancer
Many oncology antibodies in the clinic are directed to the same target
What was the first mAb approved for the treatment of malignant disease?
Rituximab
Rituximab the archetypal direct targeting mAb
- Rituximab was the first monoclonal antibody approved for the treatment of malignant disease
- On introduction has had one of the most significant impacts on patient responses in 30 years (in combination with chemotherapy)
- Since treated several million people
How do mAb work?
- A MAB works by recognising and finding specific proteins on cells. Some work on cancer cells, others target proteins on cells of the immune system.
- Each MAB recognises one particular protein. They work in different ways depending on the protein they are targeting.
- MABs work as an immunotherapy in different ways. Some MABs work in more than one way.
- They can:
- trigger the immune system to attack and kill cancer cells
- act on cells to help the immune system attack cancer cells
Complement dependent cytotoxicity
What are some signalling properties of monoclonal antibodies and give an example of a mAb that undertakes each one
Signalling properties
- Induce signals resulting in growth inhibition/ death of the cell
E.g., anti-Idiotype
- Prevent signals necessary for tumour cell survival
e. g., Herceptin - Prevent signals necessary for tumour cell proliferation
e. g., anti-growth factor receptors (e.g., avastin)
Direct cell death of tumour cells
How do mAbs work?
Effector cells engage a family of related receptors: FcgammaRs
What are some consequences of FCgammaR ligation?
- NK cells- only express activator R (CD16)
- Activatory FcgammaR signal through ITAMs on associated gamma chain
- B cells express only inhibitory FcgammaR (CD32)- inhibits response
- Other cells express both activatory and inhibitory FCgammaR (DC, mono, macro, etc.)
How do CD32b signal?
What effects does this lead to?
via an ITIM
leads to…
- decreased proliferation
- decreased Ca2+ influx
- Apoptosis
Phagocytosis of tumour cells
Modulation of the FCgammaR response leads to what?
Cellular activation (complement factors, cytokines, TLR etc)
Genetics (FcR expression and function)
FCgammaR are polymorphic and have CNV
Tell me about the modulation of the FCgammaR response
- Cellular activation (complement factors, cytokines, TLR etc)
- Genetics (FcR expression and function)
- Ab isotype
What does the Ab isotype dictate?
FCgammaR engagement
Tell me the different human IgG subclasses and their effect on complement activation and FcR binding.
This is looking at the effect of isotype on mAb effector function
What is the evidence for the importance for FCgammaR in mAb therapy?
- Activating FcR required for immunotherapy
- Immunotherapy improves in CD32 KO
- Immunotherapy in patients correlates with higher affinity FcR alleles
What are the ways to improve the effector functions of mAb?
- Engineering of residues involved in binding FcRn in order to modify antibody half-life
- Identify residues in Fc that alter complement activation
- Identification of a range of Fc mutations that enhance ADCC and tumour cell killing
How do you improve the effector functions of fucosylation?
- Human IgG antibodies are heavily fucosylated (sugar residues bound to the Fc region.)
- The ability of mAb to evoke ADCC (through FcR) is dependent on the amount of fucose and is dramatically enhanced by a reduction in fucose.
- Non-fucosylated therapeutic antibodies show more potent efficacy than their fucosylated counterparts both in vitro and in vivo
- (Done by expressing mAb in cells defective in sugar adding enzymes)
Summary L1
- Fc engineering can be used to modify the biological activities of antibodies
- Mutations have been identified that alter the relative
- binding to different classes of human FcR
- Some mutations giving reduced activation may be more appropriate for use in Antibodies intended for inhibitory action
- Other mutations have been identified that enhance
- biological activity and these may offer improvements in antibodies intended for disorders such as cancer
Manipulation for FCgammaR for immunotherapy…
whats the balance for tumour destruction?
Whats an anti-idiotype?
What is their function?
anti-idiotype is specific for individual tumours (targets the CDR region of the BCR expressed on the tumour)
Was the first successful mAb used
Induced regression in B cell tumour- very successful
Treatment with anti-Id Ab resulted in what?
How does it work?
Treatment with anti-Id Ab resulted in antigenic modulation and inhibited delivery of Id to the cell surface- no role for effectors?
Probably works by mimicking the normal signals of the BCR when it encounters antigen – B cells at certain stages of development are triggered to die through apoptosis when they recognise self-antigen
So why do we not use anti-Id?
- Demands individual mAb generation for each patient
- Expensive, not scalable
- Idiotype can mutate – tumour escape
- Was an important proof of concept that mAb can work!
What is HER2/neu?
How does it function?
What does it transmit?
Member of the EGFR protein family also known as ErbB-2 and Human Epidermal growth factor Receptor-2
Approximately 15-20 percent of breast cancers have an amplification of the gene or over-expression of the protein
Functions by dimerising with other members of the ErbB – family (eg EGFR, ErbB-1; Her3, ErbB-3 etc)
Transmits survival and growth signals to the cancer cell resulting in higher aggressiveness in breast cancers
Tell me about Herceptin and how does it work?
- Herceptin attaches to a HER2+ cancer cell and tells your body’s defence system to target the HER2+ cencer cell
- Herceptin may also stop the HER2+ cancer cells from telling itself to grow and divide into more cancer cells
Tell me about the EGFR mAb…
Over-expression of EGFR common in solid tumours
50-90% of NSCLC over-express it
10% have an activating mutation
Also, high prevalence in head and neck cancer
Good target – lots of drug company interest
Developed several mAb
Panitumumab and Cetuximab
Compare Panitumumab Vs Cetuximab
Although they both target the EGFR, panitumumab (IgG2) and cetuximab (IgG1) differ in their isotype
They might differ in their mechanism of action.
Human IgG1 isotype activates the complement pathway and mediates ADCC better
Lots of other similar mAb (Nimotuzumab, Matuzumab, Zalutumumab, Ipilimumab, IMC A12 all IgG1; Tremelimumab and CP-751,871 IgG2)
Now some fully human versions (zalutumumab (HuMax-EGFr®)
What are some questions for mAb design?
Target specificity – unique; cell specific; tissue specific; stem cells?
Dose limiting toxicity – reason for most therapeutics to fail. mAb generally quite good (eg 375 mg/kg Ritux PLUS chemo) but not always (eg. Zalutumumab 16 mg/kg – dose limiting rash)
Often cytokine mediated
Unexpected effects – Northwick park (TGN1412) anti-CD28 super-agonist
What effector systems do we want? – CDC/ADCC/Signalling?
With immunomodulatory mAb whats the 4th effector mechanism?
Engaging cells of the immune system rather than to target cells directly
What do immunomodulatory mAb work to boost?
anti-cancer immune responses
What do immunomodulatory mAb provoke?
Tumour specific polyclonal T cell responses
What do Immunomodulatory mAb offer?
The promise of anti-cancer ‘immunity’
Immunomodulatory mAb
Numerous potential targets of mAb
What do cancer cells do to escape immune rejection?
They adapt
Are there multiple levels of immune evasion?
Yes
What are the two classes of immunomodulatory mAbs and provide examples for each
Activating receptors
- CD28
- OX40
- GITR
- CD137
- CD27
- HVEM
Inhibitory receptors
- CTLA-4
- PD-1
- TIM-3
- BTLA
- VISTA
- LAG-3
What can both classes of immunomodulatory mAbs do?
They can both be therapeutic
What is needed for full T cell activation?
Tell me what these are?
Co-stimulatory signals are needed for full T cell activation
e.g., 4-1BB, OX40
Whats often missing in the tumour environment?
What response does this lead to?
Co-stimulatory signals
This often leads to an ineffective immune response
What are some of the events that occur as a result of tumour effects?
- Down-regulation of MHC molecules on tumour cells
- Insufficient cross-presentation of tumour antigens
- Immunosuppressive cytokines
- Inadequate CD4+ T cell help
- Immature antigen presenting cells lacking co-stimulatory molecules
- immuoregulation
What can monoclonal antibodies do when it comes to T cell activation?
Provide any examples where appropriate
What type of response does this lead to?
They can provide surrogate co-stimulatory signals
Can block inhibitor signals
Potential synergy with both…
Ipilimumab (CTLA4): early results
Ipilimumab is a type of cancer treatment called immunotherapy. You might have ipilimumab to treat: melanoma that can’t be removed with surgery or has spread to another part of the body (advanced melanoma) advanced kidney cancer (renal cell carcinoma) with another drug called nivolumab.
Tell me the steps to Ipilimumab: cutting the brakes
Ipilimumab + Nivolumab: change in target lesions
What are the purpose of immune checkpounds and give examples of some and explain the mechanism for one of the examples…
Immune checkpoints regulate different components in the evolution of an immune response
CTLA-4 checkpoint is induced in T-cells at the time of initial antigen response
(level of CTLA-4 response is proportional to the amplitude of the TCR-mediated signaling)
After the TCR encounters antigen – CTLA-4 is transported to the cell surface in intracellular vesicles
CTLA-4 acts as signal dampner
Food for thought: ipilimumab- more than just a blocker?
Why are we excited about the clinical results of immunomodulatory mABs?
They show that you can stimulate an immune response, even with relatively straightforward treatment
It looks as though you can get long-lasting effects in some cases
We are at the beginning of this area of research…
It is complex, and we do not fully understand the mechanisms of action, even for the licensed agents
We really need to understand the predictors of response: a lot of work is going on in this area
We are going to be busy working out how to combine treatments, and how to make them more selective
Summary
- mAb have been the most successful immunotherapy agents to date
- They employ multiple effector mechanisms to do it
- The importance of each effector mechanism depends on the antigen targeted
- Difficult to determine the critical ones in vivo (and in humans)
- Only by understanding this can we improve on current mAb
When dissecting the mAb mechanisms, what were the problems of prior analysis?
- Wrong mAb isotype (Hu versus mouse)
- Effector functions
- Half-life
- Potential MAHA
- Cell-lines not primary cells
- In virtro not in vivo
- Problems with xenografts
- Small numbers of cells
- Human-mouse reaction
- No protection of human cells from mouse complement
What is the B cell depletion model?
CD20 mAB depletion of circulating B cells
MlgG2a optimal (>> mlgG1)
FCgammaRII is detrimental
Direct targeting mAb in mice…
Direct targeting mAb
e.g., Rituximab (anti-CD20)
In mouse:
IgG2a most effective
Best A:I ratio
Rituximab summary
- Complement and apoptosis are not important for rituximab deletion but FcgR are in mice
- These FcgR seem to be on macrophages
- Dissecting mechanisms in vivo is complex (how close does the mouse mimic humans; how closely do normal B cells mimic their malignant counterparts?)
- Growing evidence in humanised mice and with intravital imaging
