The PPT briefly explains about the fusion proteins, mechanism of action, uses and the pipeline fusion proteins

1. Fusion proteins in
immunotherapy
DR MANJULA MJ

2. Contents
• Introduction to immunotherapy
• Introduction to Fusion proteins
• Construction of fusion proteins
• Advantage/ Mechanism of fusion proteins
• Fusion proteins in immunotherapy
• Other uses of fusion proteins

3. Immunotherapy
• Immunotherapy is used to upregulate or downregulate the
immune system to achieve a therapeutic effect
• Drugs (e.g., immunosuppressors), biologicals (e.g., cytokines,
monoclonal antibodies, and antisera), vitamins and minerals
(e.g., zinc, vitamin C, and vitamin B6), transplantation (e.g.,
bone marrow), and immunizations (e.g., prophylactic and
therapeutic vaccines).

4. Immunotherapy conti….
• Immunodeficiencies
• Hypersensitivity reactions
• Autoimmune diseases
• Tissue and organ transplantations
• Malignancies
• Inflammatory disorders
• Infectious diseases

5. Fusion proteins
A fusion protein is a protein consisting of at least
two domains that are encoded by separate genes
that have been joined so that they are transcribed
and translated as a single unit, producing a single
polypeptide.
Enbrel: Etanercept : TNFR 2: First fusion protein 1998

6. Construction of fusion proteins
• The component proteins
• The linkers
Undesirable outcomes
X Misfolding
X Low yield in protein production
X Impaired bioactivity

7. Linkers
• Maintaining cooperative inter-domain interactions
• Preserving biological activity
Length of the linker
Hydrophobicity
Residual amino acids

8. Introduction of linkers
• Zhou et al: long and flexible glycine --- serine (GS) linker.
• Removed the hinge sequence to form a single chain Fc-dimer
• Human growth hormone (hGH)
• Longer half-life and increased bioactivity than traditional Fc
fusion-based proteins.
https://doi.org/10.1016/j.apsb.2021.05.002

9. https://doi.org/10.1016/j.apsb.2021.05.002

10. • Argos et al: Threonine (Thr), serine (Ser), proline (Pro), glycine (Gly),
aspartic acid (Asp), lysine (Lys), glutamine (Gln), asparagine (Asn), and
alanine (Ala)
• George and Heringa: Pro, arginine (Arg), phenylalanine (Phe), Thr,
glutamic acid (Glu) and Gln
1.Argos P. An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J Mol
Biol. 1990;211:943–958. 2.George R, Heringa J. An analysis of protein domain linkers: their classification and role in protein folding. Protein
Eng. 2002;15:871–879.
Pro: The most restrictive confirmational changes
Natural linkers

11. Common confirmational changes
• Helical, β-strand, coil/bend and turns
• Alpha helical is the most stable form

12. Empirical linkers in Recombinant fusion protein
preparation
Linker Type Functionality
Albumin ANF Flexible Increase Stability/Folding
HBsAg preS1 Flexible Increase Stability/Folding
beta-glucanase-xylanase Rigid Increase Stability/Folding
Virus coat protein Rigid Increase Stability/Folding
hGH-Tf and Tf-hGH Rigid Increase expression
G-CSF-Tf Flexible Improve biological activity
Immunotoxins Clevable Enable targeting
G-CSF-Tf and hGH-Tf
Rigid Alters pharmacokinetic property

13. Structural features

14. Expression and regulation

15. Uses of fusion proteins
• As aids in the purification of cloned genes
• As reporters of expression level and
• As histochemical tags to enable visualization of
the location of proteins in a cell, tissue, or
organism.

17. Increasing therapeutic effect
• Bound to Fc-immunoglobulin G1
• Human serum albumin (HSA)
• Transferrin (Tf)

18. Fusion proteins approved for Immunotherapy
Fusion protein Component Uses
Enbrel -- Etanercept Tumor necrosis factor/Fc-
IgG1
ankylosing spondylitis,
rheumatoid arthritis,
psoriasis, ulcerative colitis,
and Crohn's disease
Arcalyst -- Rilonacept Interleukin-1 Receptor
extracellular domain/ Fc-IgG1
Heriditary inflammatory
disorder
Orencia --- Abatacept Cytotoxic T-Lymphocyte
Antigen-4/Fc-IgG1
Severe Rheumatoid Arthritis

19. Fusion protein Component Uses
Amevive: Alefacept Leukocyte function
antigen-3/Fc-IgG1
moderate to severe
chronic plaque psoriasis
Gleevec (Pro)
Bcl–abl fusion protein Chronic myelogenous
leukemia

20. Fusion proteins : Other uses
doi:10.1016/B978-0-12-374984-0.00565-9
Fusion protein Component Uses
Eloctate Recombinant factor
VIII Fc fusion
protein
Hemophilia A
Nplate Thrombopoietin/Fc-
IgG1
chronic immune thrombocytopenic
purpura (ITP)
Tanzeum Albiglutide – GLP 1
analogue
Type 2 DM
Idelvion Coagulation factor
IX
Hemophilia B

21. Infix letters and their meaning
v → will be used when a multifunctional
fusion protein has multiple and not related
actions;
o → will be used when some other targeting
mechanism (i.e. not antibody, receptor or
antigen) is used in a bifunctional fusion
protein or in a multifunctional fusion protein
with multiple unrelated targeting;
u → will be used when a fusion protein has
multiple actions and no targeting;
h, j, q, w, y → these consonants were excluded
to facilitate the translation.

22. • THANK YOU