IC2: Biopharmaceutical products derived from endocrine &immune sys 1 Flashcards
What are two types of immunotherapy
Activation type: augment and/or reestablish immune system’s ability to prevent, fight disease
suppression type: to reduce/suppress immune system
What are the 4 therapeutic strategies in immunotherapy
- Cytokines 2. Antibodies 3. T cells (TIL, TCR-T, CAR-T, immune checkpoint inhibition) 4. Cancer vaccines
Name the classes of cytokines (5) and their use
- interferons (IFN alpha, beta, gamma) –> produced by cells in response to viral infections, tumors and other biological tumors ; promote antiviral state in other neighbouring cells
- interleukins (IL2,11) –> produced by leukocytes mainly. Affect growth and differentiation of hemapoietic & immune cell. regulate hematopoiesis, immunity, inflammation
- Colony stimulating factors (CSF,EPO) –> stimulate cell division and differentiation of blood cells from bone marrow precursor
- chemokines –> stimulate leukocyte chemotaxis
- others eg TNF –> only present in small amounts under constitutive conditions. increased production by macrophage in response to inflam condition (cytokine storm). considered bad guy due to pro-inflam + pro-apoptosis action. aka adipokine produced by adipocytes , contribute to insulin resistance in type 2 DM
Are cytokines big or small proteins
small, usually <20 kDa
Describe the properties of cytokines (5)
- small proteins
- short half life
- are glycoproteins
- usually act at short range in an autocrine (i.e. on the cells that produce them) or paracrine (i.e. on cells nearby) manner 4. binds specifically to corresponding cytokine receptor
Describe the principles of TIL therapy
- Isolation of autologous tumor-infiltrating lymphocytes from patient’s excised tumor masses which consist of T and NK cells. IMPT: isolated T cells are polyclonal + diverse antigen specificity (due to gene arrangement @ Fab region)
- Isolated TILs are primed and expanded ex vivo by a rapid expansion process (REP) - TILs are exposed to a cocktail [high dose IL 2 + anti-CD3 antibody (To activate CD3 in TCR) + irridiated feeder cells (usually autologous PBMCs obtained from pt) ]
- Large quantities produced, TILs infused back into patients –> re-infiltrate tumor, recognise tumor antigens + attack cancer cells
How does the high dose IL2 + anti-CD3 antibody + irradiated feeder cells (PBMC) cocktail allow expansion of TILs in TIL therapy?
- IL2 -> IL2 is a cytokine produced by helper T cells (CD4+) which binds to IL2 receptors found on immature and mature T cells, causing T cell activation. Introducing a high dose of IL2 thus allows higher T cell activation
- Anti- CD3 antibody -> activates CD3 which is abundant on T cells surface
- Irradiated feeder cells -> produce lots of cytokines to allow T cell activation and expansion
What are the basic principles of TCR-T and CAR-T therapies
- T cells are isolated from patient’s peripheral blood (and expanded if quantities not high enough for genetic manipulation)
- They are genetically modified under in vitro lab conditions (require vector system) to develop tumor antigen specific T cells
- Genetically engineered T cells infused back to pt -> recognise tumor antigen and attack cancer cells
What are the advantages of TIL therapy
TIL therapy is generally safe among all ACT techniques
What are the limitations of TIL therapy
- excised tumor masses are devoid of or contain very low quantities of TILs.
- expanded naturally occurring tumor-specific T cells are heterogeneous = possessing varying antigen specificities. TIL reinfusion may not be lethal enough to attack and eradicate cancer cells.
- limited or none of the tumor antigen- specific T cells possess high affinity.
How is the Tcell genetically modified in TCR-T therapy?
- Genes encoding Valpha and Vbeta within the alpha and beta chains that make up (TCR) are tumor antigen-specific.
- These genes are cloned in retro-/lentiviral vectors, used to transduce Tcells from pt’s peripheral blood
- genetically modified T cells are less heterogeneous
Why is retro/lentiviral vectors used for TCR-T for genetic manipulation of T cells?
It has high transduction efficiency, ensures reinfused T cells possess high tumor antigen specificity
List the advantages of TCR-T therapy (2)
- Possess full TCR complex –> can recognize antigens expressed at both cell surface/tumor surface and within tumor cell/tumor mass –> can penetrate tumors –> effective against solid and hematological tumors.
- More effective than CAR-T as it uses full TCR complex for antigen recog and signal transduction (by CD3 proteins) : 1. can activate fully @ low target cell antigen densities 2. onset of signalling is slow but longer duration 3. execute extended killing
List the disadvantages of TCR-T therapy (2)
- Engineered TCR-T cells express particular tumor-specific TCR –> ONLY limited for use in pt subpopulation carrying MHC/HLA allele that is recog by TCR. (MHC is polygenic (made up of diff types of genes) and polymorphic (among genes, diff allele))
- Less safe than TIL due to
- On target OFF TUMOR toxicity: normal tissues may express same antigen (eg gp100 & MART-1 in both normal melanocytes and melanoma cells
- OFF TARGET toxicity: TCR-T cells not specific + cross react w/ other antigenic fragments
- CYTOKINE-release syndrome: infusion of TCR-T cells induce cytokine storm
Describe how CAR-T therapy works
- Construction of chimeric antigen receptor (CAR): genetic sequence encoding for specific antigen-binding sites within VH and VL in the Fab domain of an identified antibody is cloned into a retro/lentiviral vector
- T cells from pt peripheral blood are transduced by vectors carrying CAR gene
- Transduced T cells express CAR on surface: becomes CAR-T cells
- In vitro expansion of CAR-T cells
- Cells are infused into the patient to bind to antigens on cancer cells to kill them
What is the design of generations of CAR-T cells based on?
Structure of intracellular domain (which is different for every gen)
What is the same across all generations of CAR-T cells?
Extracellular domain: scFv @ Fab arm of Ig. responsible for antigen binding and recognition
What constitutes the 1st gen CAR-T cell design?
Intracellular domain: only 1 signalling domain (CD3ζ).
Signalling not strong enough to sustain CAR-T cell expansion and in vivo survival. Fail to hv potent antitumor activity clinically
What constitutes the 2nd gen CAR-T cell design?
Intracellular domain: 2 signalling domains (CD3ζ + additional co-stimulatory CD28 OR 4-1 BB domain) –> 2nd activation signal upon target antigen recognition.
Stronger signalling = lasting invitro proliferation + potent antitumor activity
What constitutes the 3rd gen CAR-T cell design?
Intracellular domain: 3 signalling domains (CD3ζ + additional co-stimulatory CD28 + 4-1 BB domain
Stronger activation signal upon target antigen recognition for lasting in vitro proliferation and potent antitumor activity
What constitutes the 4th gen CAR-T cell design?
Intracellular domain: 3 signalling domains (CD3ζ + additional co-stimulatory CD28 + 4-1 BB domain) AND transgene
Upon target antigen binding, trigger strong CAR signalling + activate transgene to express cytokines eg IL12 which exerts autocrine and/or paracrine effect on T cells @ target site –> activate more T cells to eliminate cancer cells
Antigen negative cancer cells can be eliminated by 4th gen CAR-T cells. T/F?
T. Addition of transgene allows the production of IL12 once activated. IL12 is not specifically target to cancer cells rather it just activates immune cells to participate in killing of cells`
What are the advantages of CAR-T cells (2)
- CAR-T cells recognize and bind to unprocessed tumor surface antigens without MHC processing. possess full TCR complex –> can recognize antigens without MHC proteins
- scFv domain only binds to cell surface antigen –>effective against hematological tumor like acute lymphoid leukemia. Not effective against solid tumors
Hematological tumor = tumor in blood
What are the disadvantages of CAR-T therapy (3)
1, scFv may guide CAR-T cells to an antigen-independent mech –>failed therapy
- less effective than TCR-T as CAR-T only activated when there is high expression of antigen on cancer cells (higher target cell surface antigen densities) + Only one subunit (scFv) binding to target cell surface antigen –> weaker CAR signaling and activation –> execute faster killing function but lack extended killing.
- AE
- on target OFF TUMOR toxicity [limited to B cell aplasia] –> due to CD19-specific CAR-T cells attacking and killing normal B cells also expressing CD19 like the malignant CD19+ B cells)
- OFF TARGET toxicity (not highly reported)
- Cytokine release syndrome (more than TCR-T)
Checkpoint proteins are expressed on T cell surface to trigger activation of T cell activity. T/F?
F. They trigger suppression of T cell activity
What is the native purpose of checkpoint proteins on T cells?
To counter OVERSTIMULATION of T cell activity + prevent autoimmune response
Two checkpoint proteins explored for therapeutics?
CD28 & PD-1`
They are expressed on the surface of activated T cells
What molecule does CTLA-4 compete with for binding on CD80/CD86 expressed on APC cell surface?
CD28
What happens when CD28 on activated T cells binds to CD80/CD86 on APC
Increase T cell activation –> activated T cells execute cell killing effect
Binding of CTLA-4 on CD80/CD86 reduces CD80/CD86-CD28 interaction and `causes T cell inhibition. T/F
T
Binding of CTLA-4 on CD80/CD86 reduces CD80/CD86-CD28 interaction and `causes T cell inhibition. T/F
T
Fill in the blanks. PD-1 is expressed on ___(1)____ while PD-L1 and/or PD-L2 is expressed on ___(2)_
(1) activated T cells
(2) other molecules: DC, tumor associated macrophage, fibroblast, tumor cells`
PD-L1/PD-L2 binds to PD-1 to ___ T cell activity
suppress
Approved mab PD-1 inhibitor?
pembrolizumab, nivolumab
Approved mab PD-L1 inhibitor?
Atezolizumab,Avelmab, Durvalumab
Limitations of checkpoint inhibitors (3)
- not all cancer pts respond well
- efficacy is transient
- development of immune-related AE during treatment
Name the 3 categories of cancer vaccine
- cell 2. protein/peptide 3. nucleic acid (RNA/DNA)
Tumor cell vaccines contains what?
antigens expressed by tumor cells to induce T cells in patient
Dendritic cell vaccines contain what?
Tumor antigenic proteins/peptides or tumor cells that are loaded on DCs. Subsequently, DCs administered into cancer pt for tumor antigen to induce T cells
How does provenge (a cancer vaccine) work for prostrate treatment?
3-step Treatment:
1. Leukocyte fraction extracted from patient’s peripheral blood (DCs are the
main APCs in product).
2. DCs cultured ex vivo with a fusion protein consisting of :
(a) antigen prostatic acid phosphatase (present in 95% of prostate cancer cells).
(b) immune signalling factor GM-CSF for APC maturation.
3. Activated APCs re-infused into patient. → Evoke immune response against
cancer cells carrying antigen.
Nucleic acid vaccine contains DNA/RNA that code for tumor associated-antigenic proteins/peptides. T/F
T
Main consideration for DNA vaccine?
Nucleic acid gets incorporated into host cell genome
–> more lasting expression of antigenic proteins/peptides
risk of inducing carcinogenicity if insertion of gene causes insertional mutagenesis and switch on oncogenes.
Main consideration of RNA vaccines?
No risk of carcinogenicity caused by insertional mutagenesis since RNA not incorporated into host cell genome.
Main concern is RNA stability in formulation and upon administration
IFNs interfere with viral replication and are produced by virally infected cells thus establishing ______
anti viral state
Biological effects of IFN? (3)
- Induction of cellular resistance to viral attack
- regulate most aspects of immune function
- regulate growth and differentiation
Type 1 IFN are _(1)__ while Type 2 IFN are _(2)__
- IFN alpha, beta
2. IFN gamma
What is the purpose of Recombinant IFN alpha
for upregulation of immune system for antiviral and/or anticancer therapy
IFN-B is effective for treatment of which disease?
multiple sclerosis (MS)
- inhibits IFN-gamma activity.
- slows growth of attacking immune cells.
- stops production of myelin-destroying compounds.
iFN-Y is a _____ cytokine that activates resting macrophages and monocytes to increase their phagocytic activity and induces macrophages to express cytokines (IL-2, TNF-alpha) MHC proteins and Ig Fc receptors → immunostimulation.
immunomodulatory
Main properties of IL
- Mostly glycosylated
- Involved in regulation of immune cell growth, differentiation and maturation.
- Short circulation times; production regulated by positive and negative loops.
IL2 is also known as
T cell growth factor
stimulates growth, differentiation and activation of T cells, B cells and NK cells
IL2 acts on all cells. T/F
F. Only acts on cells expressing IL2 receptors.
IL11 is produced by?
fibroblasts and bone marrow stromal cells
Describe the action of IL11
Stimulates proliferation of hematopoietic stem cells and induces megakaryocyte maturation → results in increased platelet formation.
Platelets last 8-10 days.
Hematopoietic growth factors (CSF, EPO, ILs) help restore severe deficiency of hematopoietic cells resulted from chemotherapy or radiation treatment. T/F
T
Does antiserum contain mono or polyclonal antibodies and what’s the purpose
Polyclonal. Given to patients for passive immunisation
Describe the production of antiserum
- Whole blood from immunized animal collected → left to clot or add coagulant → clotting factors removed → serum obtained as supernatant after centrifugation to separate cellular components
- Raw serum further purified by eliminating serum proteins and enriching the fraction of Ig that reacts with the target Ag (by protein A/G purification (only affinity for Fc domain) or immunoaffinity column chromatography)
Limitations of murine mabs?
- Induce immunogenicity
- fail to trigger a number of effector functions
- shorter half life (30-40h)
Limitations of chimeric mabs (~75% human)?
- Constant regions Ch and Cl in Fc and Fab domains contribute to immunogenicity
- Antigen-binding sequences in VH and VL fragments are still conserved, thus chimeric Mab retains antigen selectivity and affinity similar to parent murine Mab
What stays constant in humanised mab (90% human) from chimeric mabs?
Hypervariable CDR region of Ig which reside within Vh and VL fragments.
All the other mouse amino acid sequences in chimeric mabs are replaced to form >90% human mab
In what application is Fc domain NOT needed?
- Antagonism of enzyme actions by binding to enzyme active
site –> achieve enzyme inhibition. - To neutralize receptor ligands such as hormones or cytokines. To counteract overproduction of cytokines (cytokine storm).
- To neutralize toxins. Eg. Snake venom
Other than binding to Fc receptors on effector cells, what function can an Fc domain have when added to a biopharmaceutical product
allows for longer in-vivo half life
What is an Ig conjugate?
Radioisotope, cytotoxic cytokine or toxin that is conjugated to full-length antibody.
How does ig conjugate work?
When antibody binds to surface molecule of a cell, complex further gets taken up by the cell. The conjugated radioisotope, cytokine or toxin then exerts lethal effect on cell
ScFv contains both heavy and light chains of the variable domain of a Fab arm in a single polypeptide chain. T/F?
T. It is a single chain variable fragment, contains AA seq of CDRs in VH and VL of Fab arm
How are F(ab’)2 [2 fab arm] and Fab [1 fab arm] formed
Pepsin and papain proteases cleave peptide bonds to yield them
Complete the sentence: Bispecific T cell engagers have 2 distinct __(1)__ and are capable to bind to 2 distinct _(2)__. It lacks the (3) domain.
(1) Fab domain
(2) epitopes
(3) Fc
What are the function of trimabs?
bind to 2 different antigens and possesses an Fc domain, thus have the capacity to mediate Fc-dependent effector functions. Possess a Fc domain.
Example: Catumaxomab
Naturally occuring IgG are not fucosylated. T/F
F. They are.
Fc domain is N-linked glycosylated (two N-linked oligosaccharide
chains bound to the Fc region).
- N-glycans attached to Asn297 in Fc domain in IgG are linked to
fucose.
Fucosylation at Fc domain reduces or increases affinity of the Fc domain to bind to a subtype of activating Fc receptor (FcyRIII)?
reduces
Once Fc domain of naturally occuring IgG binds to subtype of Fc receptor (FcyRIII), what happens next?
ADCC induction by effector cells. However due to fucosylation, there is reduced ADCC induction by effector cells –> reduced affinity of fucosylated Ab
Why do therapeutic Ab register reduced clinical efficacy
Endogenous fucosylated IgG competes with therapeutic Ab for binding to effector cells –> inhibit elicitation of ADCC
How does defucosylated Abs work?
- Removal of fucose from the N-glycans attached to Asn297.
- Enhanced affinity of defucosylated Abs towards FcyRIII –>
preferential interaction with FcyRIII than endogenous IgG with FcyRIII –> increased ADCC induction by effector cells –> increased efficacy of defucosylated Abs
