Engineered scaffold protein

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 . O ver 100 mAbs have been approved by the FDA as therapeutics . Limitation s: Large size, planar binding interface, constant region, high cost, difficult manipulation.

3. To overcome the limitations of antibodies, N on-antibody binding proteins (protein fragments) A ntibody 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: B inding 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 L igand-specific protein fragments have been engineered using a two-fold strategy: C reation of a library of protein variants via targeted or random mutagenesis of the parent protein. S election 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 ( A dnectins ) A nticalins ( affilins ) A vimers Fynomers Kunitz domains K nottins

6. Category II: ligand-binding amino acids in secondary structure A ffibodies β- hairpin mimetics DARPins

7. Monobodies based on type 3 fibronectin

8. Delivery Agents

9. 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. S mall monobody size overcomes some limitations of antibodies, resulting in greater tumour penetration and limited circulation. 18 F loaded Adnectin , where imaging could be completed on the same day of injection. Adnectin with  64 Cu 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.

10.  Intracellular Applications A core advantage offered by the monobody domain is the absence of a disulphide bond. C urrent immunohistochemistry methods are highly disruptive to the natural environment within a cell, alters the expression and location of endogenous proteins. I ntracellular monobodies were then applied to visualise dynamics in living cells without altering expression or location of the endogenous targets . A nti-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.

11. 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.

12. Fusion to Extend Half-life

13. Combining Monobodies with Antibodies

14. FRET ( Forster Resonance Energy Transfer ) signalling  

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

16. 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. C omposed of up to eight A-domains have been generated and expressed soluble in the cytoplasm of  E .  coli. Avimer C326 (AMG220) - T hree A-domains, engineered to bind IL-6, evaluated in a phase I clinical trial for Crohn’s disease , but further development has been halted.

17. C426 avimer protein ( c-Met antagonist ) A bility 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.

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

19. Cysteine knot m i niproteins

20. Three subfamilies of cystine-knot proteins

21. Ziconotide N aturally derived knottin peptide found in the venom of the fish-eating marine cone snail,  Conus magnus .  A pproved by the FDA in 2004 for the treatment of severe chronic pain. A ntagonizes the N-type voltage-sensitive calcium channels (NVSCCs) abundant in nerves . M ore effective than morphine

22. Linaclotide A pproved 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 .

23. 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. S mall (~7 kDa ), thermostable (T m  ~70°C), and can be easily expressed in bacteria.

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

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

26. Anticalins protein

27. Anticalin proteins are derived from natural human lipocalins (   Retinol-binding protein, apolipoprotein D, neutrophil gelatinase-associated lipocalin ) . R eshaping 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.

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

29. Costimulatory T cell engagement

30. 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. H ydrophobic 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 .

31. 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.

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

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

34. S tructure of DARPins

35. DARPin MP0112 E ngineered to bind VEGF-A T ested in a series of clinical trials for treating age-related macular degeneration (AMD) and diabetic macular edema (DME). E ncouraging 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. C urrently being evaluated in two phase III trials for AMD

36. MP0250 M ulti- DARPin trispecific molecule N eutralize the activities of VEGF and hepatocyte growth factor (HGF) simultaneously. A ble 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.

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

38. Application of DARPins in cancer cell visualization and elimination

39. 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.

40. 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)). C yclic, very small in size (1–2 kDa ) and contain multiple disulfide bonds to stabilize the protein fold.

41. POL5551 A ntagonize 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 inhibito r. POL6326 , an analogue of POL5551, is currently being evaluated for breast cancer treatment in a phase I clinical trial in combination with eribulin .

42. Affibody Molecules

43. D erived from the Z-domain of the Ig-binding region of  Staphylococcus aureus  protein A . T hree-helix bundle motif and contain no cysteine . H igh thermal and proteolytic stability and can be easily expressed in  E .  coli. S mall size - 58 amino acids, 7 kDa   R apid extravasation and rapid tumor penetration and unbound affibodies are quickly cleared from healthy organs and tissues, making them promising reagents for radionuclide imaging .

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

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

46. 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. A n IL-6-specific Affibody molecule employed to target inflammatory disease and soluble disease mediators. 

47. 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.

48. Monomeric Monobodies in Therapeutics and Diagnostics

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

50. Us e 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.