INTRODUCTION:

Between 2000 and 2003, 30 new protein drugs were approved by regulatory authorities in the US and Europe, while 80 new small-molecule drugs were also approved¹. This means that protein drugs made up a quarter of the new drug approvals during that time. The number of approved protein drugs is expected to increase as more of the proteins currently in clinical trials reach the market. This is partly because biopharmaceuticals generally move through development stages faster and have a higher success rate than small-molecule drugs².

In 2003, sales of protein drugs exceeded US $30 billion and were predicted to surpass US $50 billion by 2010³, indicating a growing acceptance of these drugs in the pharmaceutical industry. However, many first-generation protein drugs were developed without the advanced strategies used for small-molecule drugs, such as high-throughput screening of compound libraries, lead optimization, and structure-activity relationship (SAR) development. This review covers the next generation of biopharmaceuticals, both currently available and in development, that have been engineered to improve upon native proteins in various drug properties. It also discusses new drug discovery strategies and tools being used to develop these innovative protein drugs.

From Native Proteins To Improved Recombinant Biopharmaceuticals:

Early protein drugs focused on replacement (e.g., erythropoietin, insulin, HGH, IFNs) or antagonist therapies (blocking proteins with antibodies)⁴. Many top-selling drugs, like recombinant erythropoietin and G-CSF, use native proteins^3,5. Monoclonal antibodies (mAbs) and decoy receptors effectively target cell-surface and soluble proteins with fewer off-target effects, as seen in CD20-targeting mAbs for B-cell lymphomas^6-8. Limitations included reliance on natural protein functions and minimal modification for drug-like properties. Native proteins often lack key drug attributes like solubility, stability, potency, and reduced immunogenicity⁹.

Medicinal Biology Strategies To Transform Native Proteins Into Optimized Drugs:

Protein drug development lacks systematic screening, SAR creation, and extensive optimization seen in small-molecule drugs. Modifying just five amino acids creates over three million (20⁵) candidates, each requiring cloning, expression, and screening¹¹. Display technologies, like de novo antibody selection, have expanded diversity—Humira® (adalimumab) was the first drug developed this way¹². Computational protein analysis and HTS of recombinant libraries also aid optimization¹³. This process, termed "medicinal biology," enhances native proteins for better drug properties (Figure 1). The next sections cover marketed drugs and key optimization techniques (Table 1).

Amino Acid Engineering Tools:

Amino acid engineering is the process of altering a protein's basic sequence. It might involve small-scale modifications, such as changing a single residue, or more extensive manipulations, like creating chimeric or humanised mAbs. Engineering is done to modify many different physical features, decrease immunogenicity, increase solubility, change half-life, and increase recombinant expression (Figure 1.1).

(Figure 1.1. Transforming protein into drug with improved a physical properties and biological activities.)

Mutations of Selected Amino Acids:

Minor amino acid modifications impact protein drugs' pharmacokinetics and stability. Replacing surface Cys prevents oxidation (Proleukin®, Betaseron®)^14,15. His additions regulate receptor binding (G-CSF)¹⁶, while Fc modifications enhance antibody binding and half-life¹⁷. HGH and G-CSF stability improved via core redesign^18,19. Insulin analogs (Humalog®, Novolog®, Lantus®) adjust solubility and multimer formation^20-22. Fc-engineered mAbs (Herceptin®, Rituxan®) boost cytotoxic activity^23,24.

Chimeric And Humanized Mabs:

The main goal is to increase the human protein content and reduce the mouse protein fraction in mAbs. Most marketed mAbs are either humanized or chimeric. Chimeric antibodies are created by grafting a mouse variable region onto a human constant region, while humanized antibodies like Herceptin® and Tysabri® further reduce murine content by grafting only mouse CDRs onto a human variable region framework (Table 1.1).A promising alternative is fully human antibodies, generated through two main technologies. One uses human antibody libraries with selection methods like phage display²⁵, though even fully human antibodies, such as Humira®, can trigger immune responses in some patients. Another approach involves transgenic mice carrying human immunoglobulin loci, allowing the production of fully human variable regions²⁶. While several are in clinical trials, none have been approved yet (Figure 1.2).

Figure 1.2. Transition from mouse to human MABS

Table 1.1. Selected engineered protein drugs in preclinical/clinical development.

Name

Company

Original Protein

Engineering Rationale

A) Amino acid engineering

(i) Multimer state

Humalog® (insulin lispro)

Eli Lilly & Co Ltd

Insulin

Rapid release [20]

NovoLog® (insulin aspart)

Novo Nordisk A/S

Insulin

Rapid release [21]

α1-Antitrypsin, other .serpins

Sejong University

α1-Antitrypsin, antithrombin III

Stabilization of serine protease inhibitors [55]

(ii) Solubility and stability

Modified G-CSF

Xencor Inc

G-CSF

Improved shelf-life, half-life [19]

Modified HGH

Xencor Inc

HGH

Improved thermal stability [18]

Proleukin® (aldesleukin)

Chiron Corp

IL-2

Rapid release [14]

(iii) Selectivity and potency

EvIL-12

Maxygen Inc

Seven shuffled mammalian IL-12s

More potent than native IL-12 [11]

TNKase® (tenecteplase)

Genentech Inc

Tissue plasminogen-activator

Increased serum half-life, specificity for fibrin [58]

(iv) Protein fusions

Enbrel® (etanercept)

Amgen Inc/Immunex Corp

Soluble TNFR2

Greater half-life (IgG1-Fc fusion) [27]

Amevive® (alefacept)

Biogen Idec Inc

LFA-3

Greater half-life (IgG1-Fc fusion) [59]

(v) Protein truncations

Retavase® (reteplase)

F Hoffmann-La Roche AG/ Centocor Inc

Tissue.plasminogen-activator

Increased half-life, non-glycosylated [60]

ReFacto®(antihemophilic factor)

Wyeth BioPharma

Factor VIII

Ease of manufacturing [61]

(vi) Chimeric antibodies

ReoPro® (abciximab)

Centocor Inc/Eli Lilly

& Co Ltd

Anti-GPIIb/IIIa mAb

Reduced immunogenicity, improved half-life [64]

Simulect® (basiliximab)

Novartis AG

Anti-IL-2 receptor mAb

Reduced immunogenicity [65]

(B) Post-translational and post-production modifications

(i) Glycosylation

Antibodies (various)

Glycart Biotechnology Inc/

Antibodies (general)

Afucosylated to improve effector function [38,39]

Aranesp® darbepoetin.alfa

Amgen Inc/Kirin Brewery Co Ltd

EPO

Hyperglycosylated to increase half-life [36]

Cerezyme® (imiglucerase)

Genzyme Corp

β-Glucocerebrosidase

Desialylated to increase half-life [73]

(ii) Pegylation

Neulasta® (pegfilgrastim)

Amgen Inc/Roche Holding AG

G-CSF

Increased serum half-life [75]

Oncaspar® (pegaspargase)

Enzon Pharmaceuticals Inc/

Asparaginase

Increased serum half-life [76]

IL Interleukin, IgG immunoglobulin G, EPO erythropoietin, EPOR erythropoietin receptor, TNF tumor necrosis factor, TNFR tumor necrosis factor receptor, EGF epidermal growth factor, VEGF vascular endothelial growth factor, RSV respiratory syncytial virus, BAFF B-cell activating factor, BCMA B-cell maturation antigen, TACI transmembrane activator and CAML-interactor, LFA lymphocyte function-associated antigen, CTLA cytotoxic T-lymphocyte-associated antigen, MOG myelin oligodendrocyte glycoprotein, BChE butyrylcholinesterase, MMAE monomethyl auristatin E.

Protein-Protein Fusions:

Protein fusion is a key strategy for improving biopharmaceuticals. Enbrel® (etanercept) enhances stability and FcRn binding by fusing IgG1 with soluble TNF receptor II²⁷, while abatacept (CTLA4-Ig) uses an IgG4 fusion to minimize ADCC effects²⁸.

Fusion proteins also aid cell targeting, as seen in MOGxanti-CD3, which depletes autoreactive B-cells in autoimmune diseases by recruiting T-cells²⁹. Additionally, IgG1 Fc fusion enables systemic drug delivery, with erythropoietin achieving effective pulmonary absorption in cynomolgus monkeys, suggesting a less invasive delivery method³⁰.

Introduction of Unnatural Amino Acids:

New techniques enable the incorporation of unnatural amino acids into proteins, expanding functional diversity. This has been achieved in E. coli³¹ and recently in mammalian cells, where 5-hydroxytryptophan was introduced into a specific protein³², providing a potential modification site.

Post-Translational Modification Tools:

Protein glycosylation can be modified by selecting different expression systems (E. coli, yeast, insects, plants, or mammalian cells) and adjusting expression conditions³⁵. This alters both the quantity and type of carbohydrate structures present.

Hyperglycosylation:

Hyperglycosylation is a strategy to enhance the pharmacokinetics of protein drugs by adding extra glycosylation sites through amino acid engineering.

Elliott et al. demonstrated this approach by increasing the number of N-linked glycosylation sites from three to five, resulting in Aranesp® (darbepoetin alfa),a version of erythropoietin with a longer serum half-life³⁶. The same research team showed that this method of hyperglycosylation can be applied to various proteins and has further refined the structural motifs for N-linked glycosylation in erythropoietin³⁷.

Figure 1.3. Aranesp® (darbepoetin alfa)

Hypoglycosylation:

Producing antibodies or Fc fusion drugs with afucosylated Fc domains enhances FcγRIIIa binding and ADCC, improving tumor cell killing^38,39. Unlike amino acid engineering, this preserves the original sequence but poses manufacturing challenges due to glycosylation variability and batch inconsistencies⁴⁰. To address this, aglycosylated antibodies have been engineered by removing the Asn297 glycosylation site while maintaining antigen and Fc receptor binding, preserving ADCC activity⁴¹. Combining amino acid and glycoform engineering optimizes antibody function, pharmacokinetics, and production.

Post-Production Modification Tools:

Protein drugs can be chemically altered after production to enhance pharmacokinetics and potency, even though modifications to their structure and function can also be achieved through changes in amino acid sequences and glycosylation patterns. The most common post-production modifications for marketed biopharmaceuticals include the formation of drug conjugates with polyethylene glycol (PEG), toxins, or radioisotopes.

Pegylation:

Pegylation, the chemical attachment of polyethylene glycol (PEG) to proteins, extends serum half-life, often targeting short-lived proteins like interferons (IFNs)⁴³ and tumor necrosis factors (TNF)⁴⁴, rather than antibodies or Fc fusion proteins due to their favorable pharmacokinetics¹⁷. Pegylated drugs, such as Pegasys® and Peg-Intron® (IFNα) and Somavert® (HGH), reduce dosing frequency⁴².

While pegylation can lower immunogenicity, it may still trigger immune responses, as seen with Amgen’s pegylated MGDF, which caused thrombocytopenia in clinical trials⁴⁵. It can also reduce potency—Pegasys® has a 70-fold longer half-life than Roferon®-A but is 93% less potent⁴³. Beyond non-specific lysine pegylation, site-specific pegylation using maleimide chemistry allows PEG attachment to engineered cysteine residues. This method improved TNF pharmacokinetics by introducing a single cysteine (Arg31→Cys31) while removing two others (Cys69→Val69, Cys101→Asp101), creating a stable TNF homotrimer with enhanced half-life and no potency loss⁴⁴.

Toxins And Radioisotopes Conjugates:

Beyond Fc optimization, a direct tumor-targeting approach involves antibody-drug conjugates (ADCs) and fusion proteins carrying cytotoxic payloads. Examples include Mylotarg® (anti-CD33 linked to calicheamicin)⁴⁶, Ontak® (IL-2 fused to diphtheria toxin)⁴⁷, and Zevalin® (anti-CD20 conjugated to 111In or 90Y)⁷. Zevalin-111In enables imaging, followed by Zevalin-90Y for targeted β-irradiation. Despite complex manufacturing, these drugs align with the "magic bullet" concept for tumor-specific therapy.

Future Challenges For Developing Protein Drugs:

Despite advancements in protein engineering, significant challenges remain in optimizing protein drugs compared to small-molecule drugs.

Oral Absorption Issues:

Protein drugs have poor oral absorption and typically require intravenous or subcutaneous administration. While pulmonary delivery has shown promise for drugs like erythropoietin³⁰ and insulin⁵², improving oral bioavailability remains a major challenge.

Immunogenicity Concerns:

All therapeutic proteins can trigger neutralizing antibodies, and predicting immunosafety before clinical trials is impossible⁵³. Humanization and minimal modifications help but don’t eliminate immunogenicity, especially after subcutaneous injection^{40, 45, 53}. Strategies to reduce immune responses include enhancing solubility and removing MHC class II agretopes, making proteins less visible to antigen-presenting cells^53,54.

CONCLUSION:

Numerous drug optimization strategies now enhance native proteins, including computational protein design, amino acid and glycoform engineering, pegylation, and other conjugation techniques. These strategies can optimize properties such as solubility, stability, potency, pharmacokinetics, and manufacturability, improving the safety, efficacy, and cost of new drugs.

More significantly, biopharmaceutical development is no longer limited to natural biological activities. For example, antagonists can be created from native protein agonists, cytokines can be designed with novel receptor selectivity, and enzymes can be engineered to act on new substrates. Modern medicinal biology tools have established a new paradigm for protein development, where compound libraries are designed and screened, hits are identified, and leads are optimized to create the next generation of innovative protein drugs.

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Received on 31.08.2024 Revised on 16.01.2025

Accepted on 15.05.2025 Published on 19.06.2025

Available online from June 23, 2025

Asian J. Research Chem.2025; 18(3):179-184.

DOI: 10.52711/0974-4150.2025.00029

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