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Y3 Med Pharmaceutics sem 1 > Biologics and mAbs recycling > Flashcards

Biologics and mAbs recycling Flashcards

1
Q

What versatility do biologics have?

A

Replaces diseased tissue as well as modify

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2
Q

What is the difference in binding between biologics and small molecules?

A

Small molecules bind unspecifically to molecular structures which can cause toxicity. Biologics (therapeutic proteins) target a specific antigen - highly specific

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3
Q

What is the half life of biologics?

A

in mAbs - half life of 2-3 weeks so less frequent dosing needed. Small molecules have too long/short half life

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4
Q

What circulation times do biologics have compared to small molecules?

A

Long circulation times (weeks vs hours) - less frequent dosing

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5
Q

What are the 7 different types of biologics on the market?

A
  1. Peptides – first on the market
  2. Protein fragments
  3. mAbs – major part of biologics
  4. ADCs – Antibody Drug Conjugates (mAb to which a cytotoxic drug is attached – released into the cell) used in cancer
  5. Viruses
  6. Vaccines – viruses used to create vaccine
  7. New modalities such as LNP (Lipid NanoParticles)
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6
Q

Give an example of a very small molecule

A

Aspirin

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7
Q

What makes up most of mAbs?

A

igG - 10 times larger than small proteins

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8
Q

What are one of the largest particles?

A

Viruses - the larger the particle, the more difficult it is to formulate

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9
Q

What 7 steps are involved in the bioprocess and development of biologics?

A
  1. Immunisation
  2. Preparation of the myeloma cells
  3. Fusion
  4. Clone screening and picking
  5. Functional characterisation
  6. Scale up and wean
  7. Expansion
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10
Q

What is involved in the immunisation step?

A

Mice are immunised with an antigen and later their blood is screened for antibody production. The antibodies produced are splenocytes – isolated for in vitro hybridoma production

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11
Q

What are myeloma cells?

A

Immotilised cells which, once fused with spleen cells, can result in hybridoma, capable of unlimited growth

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12
Q

What happens during fusion?

A

Myeloma cells and isolated spleen cells are fused together to form hybridomas in the presence of polyethene glycol (PEG), which causes the cell membrane to fuse

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13
Q

How does PEG cause the cell membranes fuse?

A

Attracts water molecules, and as such, the membrane of the cells will break (osmolarity)

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14
Q

What happens during clone screening and picking?

A

Clones are screened and selected on the bases of antigen specificity and immunoglobulin class. This confirms and characterises the functionality

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15
Q

What do scale up clones produce?

A

Desired antibodies - leading onto expansion to produce more desired antibody

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16
Q

What 6 steps are involved in the production of biologics ?

A
  1. Cell culture harvest
  2. Protein A chromatography
  3. Viral inactivation
  4. Polishing steps
  5. Viral filtration
  6. Ultrafiltration/ Diafiltratior (uf/df)
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17
Q

What is UF/DF?

A

Batch ultrafiltration/diafiltration (UF/DF) is a very economical, high yield, and robust separation process based on size exclusion that finds application for a wide range of biotherapeutics.

  • UF involves separation of components on the basis of molecular weight/size. It is a pressure-driven process in which soluble macromolecules (e.g., proteins as products) are retained while small molecular-weight particles (e.g., salts, amino acids, mono- or disaccharides), and fluid/water pass through the membrane as waste
  • DF is used to exchange buffer solutions.
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18
Q

What do Globalised manufacturing produce?

A

Globalised manufacturing, produce in multiple sites smaller scale, increase titre, continuous manufacturing, change of cell types, move to no cells.

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19
Q

What was initially used in human antibody development?

A

Started with mouse, as creating human hybridomas was difficult

  • immunogenic clearance
  • lack of Fc effector functions
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20
Q

What has been used in the last 2 decades in regard to human antibody development?

A

Recombinant engineering:

  • Chimeric – mouse variable region (Fv, antigen binding)
  • Humanised - mouse antigen binding loops (CDRs)
  • Fully human antibodies produced via mouse.
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21
Q

What are the therapeutic functions of mAbs?

A

CDC, ADCC, conjugates, apoptosis induction, cell-cell blockade

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22
Q

What is CDC?

A

Complement Dependent Cytotoxicity: antibody will bind to the surface and complement molecules that are attached to it, so this will induce cytotoxicity

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23
Q

What are conjugates?

A

Antibody attached to antigen, cytokines and toxins can also be attached and this leads to cytotoxicity

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24
Q

What is apoptosis induction?

A

Antibody binds to a signal because it binds to a specific receptor and induces a signal of apoptosis (cell kills itself)

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25
Q

What is cell-cell blockade?

A

Antibody blocks receptor/ligand site so ligand cannot bind to receptor on the cell surface - nothing can be produced

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26
Q

What is ADCC?

A

Antibody Dependent Cell-mediated Cytotoxicity: antibody binds to specific antigen on the surface and using Fc receptors, you get neutrophils that will attach to the antibody and induce cytotoxicity

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27
Q

What shape is an antibody?

A

Y shape

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28
Q

What is the bottom of the ‘Y’ antibody and what is it’s relation to Pharmacokinetics?

A

Fc region made out of CH2 and CH3 fragments, binds to FcRn receptor on cells in the body and this induces igG recycling and long half lives are observed

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29
Q

What is the Fab region of the antibody?

A

Top of the Y, binds to antigen - target mediated disposition; region has a lot of charge/PI mediated clearance and could also lead to off-target binding

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30
Q

What is the Glycan receptor?

A

Comes from production of antibody - chain on Fc part of antibody. Need to control presence of glycans

31
Q

How are mAbs administered?

A

Administration either i.v (at low concentrations)., SC (self-administrated – high concentration of antibody) or IM injections

32
Q

What absorption is observed for SC routes?

A

Variable (20-95%) facilitated by lymph system

33
Q

What is the rate of absorption for SC or IM routes?

A

Rate of absorption is slow (maximal plasma concentration between 1-8 days following SC or IM injection) – takes LONGER time than small molecule

34
Q

How are mAbs eliminated?

A

Eliminated by proteolytic catabolism by lysosomal degradation – antibody enters the cells and cannot evade the endosome and lysosomes where it is degraded
- End up with small amino acids are recycled back into the cell

35
Q

What are some other mechanisms of mAbs elimination?

A

Other mechanisms include target mediated clearance, non-specific pynocytosis, Fc gamma receptor (FcR) mediated clearance.

36
Q

What does Target-mediated elimination involve?

A

Target-mediated elimination pathway involves interaction between a mAb and its pharmacological target, and represents the primary route of antibody clearance

37
Q

How are the resulting immune complexes then cleared from the body?

A

Through reticulo-endothelial system (RES)

38
Q

What mediates recycling of albumin and IgG?

A

Neoatal Fc receptor (FcRn)

39
Q

Where does Fc region of IgG travelling in blood bind?

A

Binds to FcRn closed inside endocytic vesicle

40
Q

At what pH does IgG bind?

A

At acidic pH (endosomal pH 6) - changes to 6.4 once inside the vesicle (instead of physiological)

41
Q

Where do unbound proteins move to during mAbs recycling?

A

Lysosomes - where they are destroyed to form just AAs (degredation)

42
Q

What are IgG taken up by in mAbs recycling?

A

Monocytes or endothelial cells through endocytic mechanisms

43
Q

What happens to bound IgG in mAbs recycling?

A

FcRn complexes are sorted and stay attached to membrane - then dissociate at physiological pH

44
Q

What is a critical quality attribute about FcRn binding affinity?

A

Want to have an IgG that binds to the receptor and is released when you want it

45
Q

How can you modify FcRn binding affinity?

A

By changing AA sequence

46
Q

Can the Fv region bind to FcRn?

A

Yes - can happen in mAbs, binding affinity to the FcRn receptor at pH 6 and Isoelectric point are very similar

47
Q

What causes a problem during Fv binding to FcRn?

A

Positive charge patch on Fv fragment of antibody – gives strong affinity to Fc receptor = PROBLEM

48
Q

What half life do Fv-FcRn complexes have?

A

Only dissociates at higher pH - shorter half life. Will want to control when designing mAb

49
Q

Where are antibody and antigen complexes recycled, and why can this be a problem?

A

Recycled through FcRn pathway and this can result in accumulation of bound antigens and extension of half life of antigens (DO NOT WANT THIS)

50
Q

How are antigens removed?

A

Degrade while antibody is recycled

51
Q

What half life do IgG types 1, 2 and 4 have?

A

An average of 21 days

52
Q

What half life does IgG type 3 have?

A

7 days (average)

53
Q

What does molecule weight do to half life?

A

When the molecule has a small weight – the half-life is small

54
Q

What is Glycosylation?

A

Glycosylation is a common post-translational modification for IgG antibodies produced by mammalian cells such as Chinese hamster ovary (CHO) cells, which are frequently used for production.

Glycosylation is the reaction in which a carbohydrate, i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor).

ALL IgGs ARE GLYCOSYLATED

55
Q

What is glycosylation NOT required for?

A

Not required for an IgG antibody’s long half life

56
Q

What lowers the binding affinity of FcRn?

A

the shorter half-life of an Fc-fusion molecule in comparison to the whole IgG has been attributed to the lower binding affinity to FcRn, the glycan mediated disposition and the receptor (of fusion partner) mediated disposition

57
Q

How can PK be improved?

A

Changing the PI of mAbs - something that is more positively charged will have a different ability to bind to the cell (surface of cell receptors are often negatively charged)

58
Q

What is the PI point of mAbs?

A

around 8

59
Q

What features will a modified mAb to have a high PI have?

A

Molecule will be constantly charged and may not be able to feel the pH change

60
Q

What are ADAs and how are they formed?

A

Anti-Drug-Antibodies may be formed upon administration of therapeutic mAbs

61
Q

What do ADAs do?

A
  • ADA bind to the mAb, form immune complexes impacting on PK, PD, safety and efficacy of mAbs.
  • May cause hypersensitivity responses such as anaphylaxis and infusion reactions, and accelerated clearance of drug
  • ADA can be neutralizing (bind to epitopes on mAb needed for biological activity) or non-neutralizing (bind to epitopes not needed for biological activity)
62
Q

How does immunogenicity of mAbs vary?

A

Immunogenicity of mAb varies across species due to different human fraction based on the type of therapeutic antibodies – related to whether its chimeric, humanised or fully human)
o Impact: could test antibodies on animals to see if you would get a response
o No evidence that fully humanised antibodies are any less immunogenic than less humanised ones

63
Q

What is an example of a fully human mAb?

A

Adalimumab - induces ADAs and impacts on efficacy of patient

64
Q

What is done to ensure sufficiently high dose levels achieve the desired exposure?

A

Drop of mAb concentration in blood

65
Q

What can novel antibody formats be clustered in?

A

o Antibody fragments (could be single chain in Fc region)
o Fusion protein,
o Antibody drug conjugates (ADCs)
o Bispecific (modified igGs to which another region has been attached – antibody binds to 2 regions, a lot more specific) – more complex to modify and stabilise
o Multispecific antibodies possessing multiple antigen binding sites

66
Q

How are Novel formats more complex than mAbs?

A

o Different antigen-binding domain in the same molecule,
o Different molecular domains linked through flexible linkers,
o Heterogeneous product through conjugation, synthesis, physical chemical attributes.
o Overall decrease of conformational stability, decreased solubility (when you lose conformational stability, you form aggregates, and they are likely to result in an immune response, also losing a lot of the dose of your mAb)

67
Q

What are antibody fragments?

A

2 parts of Fab fragments: Fab and (Fab)2 E.g. Ranibizuman
(ONE part of wider term)

  • Skip the Fc part of the mAb and keep the rest
  • Most prominent is the fragment antibody binding part (Fab)
68
Q

What are single chain fragments (antibody fragments)?

A

Very small part of Fab Fv (scFv); very unstable and don’t have any Fc fusion, lack of recycling

69
Q

What are single domain antibodies (antibody fragments)?

A

Ab (sdAbs) e.g.: Cephalon GSK available, TNFR1 Dab

70
Q

What are single chain fragments coupled with Fc region, and Fv region coupled with CH region (antibody fragments)?

A

Much more complex, nothing on the market as they lack stability
ScFab, ScFv-Fc, Fv-CH, sc-scFv-sipper

  • ScFv made of VH flexibly linked to VL
71
Q

What are fusion proteins?

A

Fc fusion protein e.g. etanercept

  • Major class of new products
  • Comprise a protein, peptide or receptor exodomain fused to the Fc region of the mAb
  • Fc portion typically contains the hinge region usually along with the conserved N-glycosylation site in the CH2 domain
  • Half-life extended
72
Q

What are albumin fusion proteins (fusion protien)?

A

Modified albumin e.g. Dulaglutide (Trulicity TM)

73
Q

What are ADCs?

A

Ab linked with cytotoxic payload e.g. Transtuzumab, entansine (Kadcyla)

  • No fundamental change in the structure except to add the linkers where you’re going to add the drug. Fc region at the bottom - fusion
  • mAb employed as drug delivery agents with chemotherapeutic drugs, immunotoxins, radioisotopes or cytokines
  • cleavable linker in Lys or Cys residues allowing release of payload
74
Q

What are Multi-functional mAbs?

A

Bispecific e.g. AFM13 (affirmed therapeutics)la)

ALSO, Bispecific Ab-scFv and Diabody; 2 mAbs mutated in the CH3 domain e.g Genmab

  • bispecific or multispecific antibodies contain two or more variable domains with specific affinity to bind to different antigens
  • bispecific formats comprise IgG like and Fab fragment-based constructs

Y3 Med Pharmaceutics sem 1 (5 decks)

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