Different classes of engineered proteins for their binding epitope geometry and as leads for synthetic secondary and tertiary structure mimics.

1. Engineered protein scaffold as
next generation therapeutics
Dr Mohit Kher
Senior Resident
Pharmacology, LHMC

2. INTRODUCTION
• Antibodies have long been regarded as ‘magic bullets’ in human therapy.
• The first monoclonal antibody (mAb) entered human therapy in 1986.
• Over 100 mAbs have been approved by the FDA as therapeutics.
• Limitations: Large size, planar binding interface, constant region, high cost, difficult
manipulation.

3. • To overcome the limitations of antibodies,
1. Non-antibody binding proteins (protein fragments)
2. Antibody fragments (e.g., single-chain variable fragments (scFv), fragment antigen-
binding (Fab) fragments, and single-domain antibody fragments (nanobodies)) have
been designed and explored as scaffolds for therapeutic applications.
Action of scaffold protein: Binding to ligands, antagonizing receptors & neutralizing
toxins.
Advantages: Small size, High thermostability, Easily produced in microorganism, low
cost of production, Multispecificity and less adverse effects.

4. Display platforms for protein fragment
engineering
• Ligand-specific protein fragments have been engineered using a two-
fold strategy:
1. Creation of a library of protein variants via targeted or random
mutagenesis of the parent protein.
2. Selection of target ligand binders via a phenotypic selection such as
phage display, yeast surface display or ribosome/mRNA display.

5. Category I: ligand-binding amino acids in
exposed loops
• Monobodies based on type 3 fibronectin (Adnectins)
• Anticalins (affilins)
• Avimers
• Fynomers
• Kunitz domains
• Knottins

7. Category II: ligand-binding amino acids in
secondary structure
• Affibodies
• β-hairpin mimetics
• DARPins

9. Monobodies based on type 3 fibronectin

11. Delivery Agents

12. • Use of antibodies to deliver radioisotopes for imaging is limited since the long circulation time
and off-target binding results in reduced tumour penetration while inducing systemic radiation
burden to patients.
• Small monobody size overcomes some limitations of antibodies, resulting in greater tumour
penetration and limited circulation.
• 18F loaded Adnectin, where imaging could be completed on the same day of injection.
• Adnectin with 64Cu where the monobody was able to provide same-day PET visualisation of
tumour cells, resulting in an increase in the monobody uptake in tumours over 24 h post-
injection.
• Centyrin domain evolved to bind EGFR and conjugated with a fluorescent dye.

13. Intracellular Applications
• A core advantage offered by the monobody domain is the absence of a disulphide bond.
• Current immunohistochemistry methods are highly disruptive to the natural environment
within a cell, alters the expression and location of endogenous proteins.
• Intracellular monobodies were then applied to visualise dynamics in living cells without
altering expression or location of the endogenous targets.
• Anti-RAS monobodies specifically targeted K- and H-Ras, are highly specific tools for
monitoring the conformational or mutational state of RAS proteins while also potentially
modulating their signalling pathway.

14. • FN3 domains evolved to bind proteins in the Wnt signalling pathway were
expressed endogenously to block intermolecular interactions of individual
domains within a quaternary protein assembly.
• This approach was taken into mouse models to target WDR5, a component of
mixed lineage leukemia, to effectively suppress leukemogenesis.
• By fusing to domains that confer degradation, such as the Von Hippel–Lindau
oncoprotein suppressor (VHL) or E3 ubiquitin ligases, monobodies can deliver
degradation signals directly to endogenous proteins within a target cell.

15. Fusion to Extend Half-life

16. Combining Monobodies with Antibodies

17. FRET( Forster Resonance Energy Transfer ) signalling

18. Type 3
Fibronectin
Target Indications
CT-322 VEGF receptor Pancreatic cancer
BMS-962476 PCSK9 Dyslipidemia
BMS-986089 Myostatin Duchenne’s
muscular
dystrophy
Trials
Phase 2
Phase 1
Phase 2

19. AVIMER
• Derived from the A-domain of various cell surface receptors such as the low
density-related protein (LRP) and very low density lipoprotein receptor (VLDLR).
• ~35 amino acids (~4 kDa) and stabilized by calcium binding and three pairs of
disulfide bridges.
• Composed of up to eight A-domains have been generated and expressed soluble in
the cytoplasm of E. coli.
• Avimer C326 (AMG220) - Three A-domains, engineered to bind IL-6, evaluated
in a phase I clinical trial for Crohn’s disease, but further development has been
halted.

20. C426 avimer protein (c-Met antagonist)
• Ability to bind the c-Met receptor (receptor tyrosine kinase, RTK).
• Hepatocyte Growth Factor is only a known ligand.
• Deregulation of cMet is found in many neoplasms severity and poor
prognosis makes this RTK an attractive candidate for targeted
anticancer therapy.

21. Avimer Target
C426 cMet
C65 CD40L
C326 IL-6
C2810 CD28
C2 BAFF

22. Cysteine knot miniproteins

23. Three subfamilies of cystine-knot proteins

24. Ziconotide
• Naturally derived knottin peptide found in the venom of the fish-eating marine
cone snail, Conus magnus.
• Approved by the FDA in 2004 for the treatment of severe chronic pain.
• Antagonizes the N-type voltage-sensitive calcium channels (NVSCCs) abundant
in nerves.
• More effective than morphine

25. Linaclotide
• Approved by the FDA in 2012 to treat Irritable Bowel Syndrome with
Constipation (IBS-C) and Chronic Idiopathic Constipation (CIC).
• Activation of GC-C in the intestinal lumen initiates a signal transduction cascade
that results in the secretion of chloride and bicarbonate.

27. Fynomers
• Derived from amino acids 83–156 of the Src-homology 3 (SH3) domain of FYN
tyrosine kinase.
• FYN-SH3 domains are fully conserved between humans, mice, rats, and gibbons.
• Each Fynomer is composed of a pair of anti-parallel beta sheets joined by two flexible
loops.
• Small (~7 kDa), thermostable (Tm ~70°C), and can be easily expressed in bacteria.

28. Fynomer 2C1
• Engineered to bind the proinflammatory cytokine interleukin-17A (IL-
17A) and was able to inhibit the activity of IL-17A in vitro.
• Dimeric 2C1-Fc exhibited >100-fold improved activity against IL-17A
compared to the parent 2C1 molecule.
• Effective in a mouse model of acute inflammation.

29. FynomAb COVA322
• A fusion molecule consisting of an IL-17A-binding Fynomer fused to
the anti-TNF antibody adalimumab.
• Simultaneously inhibit the activity of both TNF and IL-17A for
treatment of rheumatoid arthritis.
• Currently being evaluated in a phase I/II clinical trial

30. Anticalins protein

31. • Anticalin proteins are derived from natural human lipocalins ( Retinol-
binding protein, apolipoprotein D, neutrophil gelatinase-associated
lipocalin).
• Reshaping the binding pockets of natural lipocalins leads to
“Anticalin” proteins.
• Anticalin-based biopharmaceuticals have demonstrated safety and
tolerability in early clinical studies and offer both new treatment
options in immuno-oncology and innovative routes of delivery such as
inhalation.

32. Tear lipocalin (Tlc; Lcn1) and neutrophil-gelatinase
associated lipocalin (NGAL; Lcn2)

34. Costimulatory T cell engagement

36. Kunitz domains
• ~60-amino-acid peptides (~7 kDa) derived from the active motif of Kunitz-type
protease inhibitors such as aprotinin (bovine pancreatic trypsin inhibitor),
Alzheimer’s amyloid precursor protein and tissue factor pathway inhibitor.
• Hydrophobic core of the Kunitz domain is composed of a twisted two-stranded
antiparallel β-sheet and two α-helices stabilized by three pairs of disulfide bonds.

37. Ecallantide (DX-88)
• DX-88 was derived from the Kunitz domain of lipoprotein-associated coagulation
inhibitor (LACI).
• A Kunitz domain-derived inhibitor of kallikrein (a subgroup of serine proteases),
was approved by the FDA in 2012 for treatment of hereditary angioedema (HAE).
• Most HAE is caused by the malfunction of the plasma C1 kallikrein inhibitor
protein.

38. Depelstat (DX890)
• A potent and selective inhibitor of human neutrophil elastase.
• Inflammation mediated by neutrophil elastase contributes to lung
damage in cystic fibrosis.
• Evaluated in a phase II clinical trial for the treatment of cystic fibrosis.

39. DARPin®(Designed ankyrin repeat
domains) Drug Platform

41. Structure of DARPins

43. DARPin MP0112
• Engineered to bind VEGF-A
• Tested in a series of clinical trials for treating age-related macular degeneration (AMD) and diabetic macular
edema (DME).
• Encouraging results in phase I/II studies
• A single intraocular injection of 0.4 mg MP0112 neutralized VEGF in aqueous humor for 8–12 weeks.
• Inflammation was reported in some patients.
• MP0112 was reformulated to contain a PEG molecule and renamed as Abicipar Pegol.
• Currently being evaluated in two phase III trials for AMD

44. MP0250
• Multi-DARPin trispecific molecule
• Neutralize the activities of VEGF and hepatocyte growth factor (HGF)
simultaneously.
• Able to bind human serum albumin (HSA).
• In a phase I clinical trial, MP0250 was found to be well-tolerated after
i.v. infusion at a dose of at least 8 mg/kg.

45. MP0274
• Multimer composed of two DARPins
• Binds to distinct epitopes on the human epidermal growth factor
receptor 2 (HER2) and inhibits downstream HER2- and HER3-
mediated signaling.

46. Application of DARPins in cancer cell visualization
and elimination

47. • Most recently, the DARPin® platform was used to generate extremely
potent viral entry inhibitors against the severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) in the context of the
worldwide coronavirus 2019 (COVID-19) pandemic in just a matter of
weeks, illustrating the advantages of the DARPin® platform when it
comes to very rapid generation of drug candidates.

48. β-Hairpin mimetics
• A single β-hairpin motif designed to reproduce the conformational and
electronic properties of functional native protein epitopes (so-called
protein epitope mimetics (PEM)).
• Cyclic, very small in size (1–2 kDa) and contain multiple disulfide
bonds to stabilize the protein fold.

49. POL5551
• Antagonize CXCR4
• High CXCR4 expression levels also correlate with tumor metastasis.
• POL5551 was later shown to reduce the metastasis of triple-negative breast cancer
in mice when combined with eribulin, a chemotherapeutic microtubule inhibitor.
• POL6326, an analogue of POL5551, is currently being evaluated for breast cancer
treatment in a phase I clinical trial in combination with eribulin.

50. Affibody Molecules

52. • Derived from the Z-domain of the Ig-binding region of Staphylococcus aureus protein A.
• Three-helix bundle motif and contain no cysteine.
• High thermal and proteolytic stability and can be easily expressed in E. coli.
• Small size - 58 amino acids, 7 kDa
• Rapid extravasation and rapid tumor penetration and unbound affibodies are quickly
cleared from healthy organs and tissues, making them promising reagents for
radionuclide imaging.

53. Modes of Action for Affibody Molecules Reported in
Various Therapeutic Efforts

56. Affibody ABY-025
• Engineered to bind HER2.
• In a phase I/II clinical trial, 68Ga-gallium labelled ABY-025
([68Ga]ABY-025) was able to accurately quantify HER2-receptor
status in metastatic breast cancer via PET imaging.

57. AffiMabs
• Affibody molecules fused with antibodies to form functional multispecific
proteins called ‘AffiMabs.
• AffiMabs are more efficient in inhibiting cell growth of the pancreatic cell line
BxPC-3 in vitro and in vivo as compared with the parental cetuximab antibody.
• An IL-6-specific Affibody molecule employed to target inflammatory disease and
soluble disease mediators.

60. REFERENCES
• Simeon R, Chen Z. In vitro-engineered non-antibody protein therapeutics. Protein Cell.
2018;9(1):3-14. doi:10.1007/s13238-017-0386-6
• Shilova ON, Deyev SM. DARPins: Promising Scaffolds for Theranostics. Acta Naturae.
2019;11(4):42-53. doi:10.32607/20758251-2019-11-4-42-53
• Rothe C, Skerra A. Anticalin® Proteins as Therapeutic Agents in Human
Diseases. BioDrugs. 2018;32(3):233-243. doi:10.1007/s40259-018-0278-1
• Kintzing JR, Filsinger Interrante MV, Cochran JR. Emerging Strategies for Developing
Next-Generation Protein Therapeutics for Cancer Treatment. Trends Pharmacol Sci.
2016;37(12):993-1008. doi:10.1016/j.tips.2016.10.005
• Baghban Kohnehrouz B, Talischian A, Dehnad A, Nayeri S. Novel Recombinant Traceable
c-Met Antagonist-Avimer Antibody Mimetic Obtained by Bacterial Expression
Analysis. Avicenna J Med Biotechnol. 2018;10(1):9-14.

67. Monomeric Monobodies in Therapeutics
and Diagnostics

70. Structures of Dimeric Affibody Molecules in Complex
with Aggregation-Prone Peptides

71. • Use of a HER2-targeted Affibody molecule, which had shown excellent tumor uptake in mice, and is in
development for metastasized breast cancer.
• Pseudomonas exotoxin in a truncated version (PE38) has been fused to affibody molecule to create HER2-
targeting Affibody molecule, and the resulting fusion protein was shown to completely eradicate large BT474
tumors in xenograft models.
• An ABD-fused anti-C5 Affibody molecule that could inhibit C5-dependent hemolysis in vitro and potently
block C5 in vivo in a Zymosan-induced peritonitis mouse model was recently tested in healthy volunteers.
• Affibody molecule ZAbeta3 stabilized a β-hairpin of the monomeric amyloid-β peptide to act as an inhibitor
of Aβ fibrillation.
• IL-17-specific Affibody molecules were formatted into a small 18 kDa superior to ixekizumab and
secukinumab in treating psoriasis.