Types of protein and peptide therapeutics

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Chapter: Pharmaceutical Drugs and Dosage: Protein and peptide drug delivery

Antibody is a protein produced by β-lymphocytes in response to substances recognized as foreign (antigens).

Types of protein and peptide therapeutics


Antibody is a protein produced by β-lymphocytes in response to substances recognized as foreign (antigens). Antibodies recognize and bind to antigens, resulting in their inactivation or opsonization (binding of antibody to the membrane surface of invading pathogen, thus marking it for phagocytosis) or complement-mediated destruction. Antibodies are also known as immu-noglobulins (abbreviated Ig) because they are immune-response proteins that are globular proteins (compact with higher orders of structure and hydrophilic surface making them soluble; as against fibrous proteins, which have predominantly secondary structure and are insoluble). Of the five major types of antibodies (Table 25.3), IgG is preferred for therapeutic application due to its wide distribution and function. Structurally, Ig is commonly represented in a typical Y-arm structure (Figure 25.5) consisting of two large/heavy and two small/light polypeptide chains joined by disul-fide bridges. Antibody fragments consist of a constant region (designated, Fc) and a variable, antigen-binding region (designated, Fab). Antibodies that recognize multiple sites of an antigen are termed polyclonal, whereas anti-bodies that target only a specific site are monoclonal. Identical immune cells make monoclonal antibodies, whereas polyclonal antibodies are produced by a mass of immune cells that may produce antibodies against different regions of the antigen. In industrial application, monoclonal antibodies are prepared by recombinant DNA technology in cell cultures. For human clinical applications, generally monoclonal antibodies are pre-ferred. Polyclonal antibodies are utilized for diagnostic and lab use such as immunohistochemistry.

Table 25.3 Types of antibodies

Figure 25.5 Typical structure of an antibody.

A number of immunoglobulin (Ig) G products have been developed for therapeutic use in various immune disorders (Table 25.1). Due to their specificity, there is a growing interest in the use of monoclonal anti-bodies and their modifications as therapeutics. For example, antibodies whose Fab fragment segments have been reduced in size to the small-est known antigen-binding fragments are known as domain antibodies. Also, antibodies that can bind two different antigens are called bispecific antibodies.

The usefulness of antibodies was limited by the immune response gener-ated by the host to the administered antibodies, especially when the anti-bodies were generated by antigen injection in foreign animal species, such as mouse. The antibodies generated in mouse were named with the suffix ~momab. The use of humanized/human monoclonal antibodies with the use of recombinant DNA technology has helped to overcome these limitations.

·           Chimeric and humanized antibodies are the antibodies produced from nonhuman species whose protein sequences have been modified to increase their similarity to the antibody variants that are naturally found in humans.

·           Chimeric antibodies consist of murine variable regions fused with human constant regions, resulting in ~65% human amino acid sequence. This reduces immunogenicity and increases plasma half-life. These antibodies are named with the suffix ~ximab. For example, rituximab is a chimeric antibody.

·           Humanized antibodies are made by grafting the murine variable amino acid domains (which determine antigen specificity) onto human antibodies, resulting in ~95% human amino acid sequence. These, however, have lower antigen-binding affinity than murine antibodies. These antibodies are named with the suffix ~zumab. For example, bevacizumab (Avastin®) is a humanized antibody that targets vascular endothelial growth factor (VEGF) and is rec-ommended as first-line therapy in advanced colorectal cancer in combination with other drugs.

·           Human monoclonal antibodies can be produced using phage display or transgenic mice. Transferring the human Ig genes into the mouse genome can produce these antibodies. These antibod-ies are named with the suffix ~mumab. For example, ipilimumab is a human mAb that inhibits the checkpoint receptor cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and is recommended for advanced-stage melanoma.

Most therapeutic antibodies exert their therapeutic effects by binding to selected cellular targets, which are then destroyed by physiological mech-anisms activated by the effector functions of the antibody. In addition, antibodies can also be used as drug delivery and targeting vehicles. Active research and development is being pursued on customized antibodies conju-gated to toxins, radioisotopes, small drugs, enzymes, and genes for selectively destroying harmful cells in the body. For example, several ADCs have been developed for the treatment of cancer that utilizes a toxin, which is a small molecule attached to an antibody. For example, Adcetris® and Kadcyla® are ADCs for tumor treatment.

Hormones and physiological proteins

Protein therapeutics to replace or supplement endogenous protein mol-ecules are used for several diseases such as diabetes (insulin), growth hormone deficiency (growth hormone), and hemophilia (factors VIII and IX). Table 25.1 lists some protein therapeutics and their clinical applications.

Chemically modified proteins and peptides

Chemical modifications of proteins are carried out to either

·           Increase target specificity, for example, abatacept (Table 25.1) and conjugation to sugars.

·           Increase therapeutic ability, for example, radiolabeled antibodies and ADCs (Table 25.1).

·           Increase plasma half-life, for example, by PEGylation of antibodies.

Conjugation with sugars

Conjugation of sugars, such as sucrose, mannose (mannosylation), or lactose (lactosylation), to proteins can be used to provide targeted deliv-ery of proteins. For example, receptors for carbohydrates, such as the asialoglycoprotein receptor on hepatocytes, and the mannose receptor on macrophages, such as Kupffer cells, recognize corresponding sugars. Mannosylated bovine serum albumin (Man-BSA) and galactosylated BSA (Gal-BSA) preferentially bind to alveolar macrophages and hepatocytes, respectively. Galactosylated and mannosylated recombinant human super-oxide dismutase (Gal-SOD, Man-SOD) exhibited inhibitory effects supe-rior to native SOD against hepatic ischemia-perfusion injury.


Proteins may be conjugated to Polyethylene glycol (PEG), a nonimmunogenic, nontoxic, and FDA-approved polymer, to increase their plasma half-life. The process of conjugation with PEG is called PEGylation, and the protein after the conjugation is called the PEGylated protein. PEG consists of a flexible polyether chain that provides a hydrophilic surface, thus shielding hydropho-bic groups and minimizing nonspecific interactions. Attachment of PEG on protein surface also increases the hydrodynamic diameter of proteins. Either straight chain or branched PEG can be used for PEGylation. The flexibility of the side chain allows the PEGylated protein to interact with the target.

PEGylation can increase biocompatibility, reduce immune response, increase in vivo stability, delay clearance by the reticuloendothelial system, and prevent protein adsorption on the surface of the delivery device, such as syringe.


Interferon (IFN)-2α has a low plasma half-life and needs daily injections. However, IFN-2α conjugated to branched PEG 40 (i.e., PEG of 40 kDa average molecular weight) provides sustained plasma concentrations upon once a week injection. Other examples of PEG-modification to modulate clearance rate of proteins include PEG-adenosine deaminase (PEG-ADA), PEG-asparaginase, PEG-rIL2, and PEG-interferon. Native ADA is not effective due to its short half-life (<30 min) and is immunogenic due to bovine source, whereas PEGylated ADA (Adagen®) is quite effective, has long half-life, and is nonimmunogenic.


PEG has two hydroxyl groups at each end of the linear chain. PEGylation is often done by creating a reactive electrophilic intermediate with succinimide (thus producing N-hydroxysuccinimide, NHS), which undergoes electrophilic substitution by an amine group of the protein (Figure 25.6). 

Figure 25.6 PEGylation of proteins using N-hydroxysuccinimide (NHS) derivative of methoxy PEG.

The NHS ester groups primarily react with the α-amines at the N-terminals and the ε–amines of lysine side chains. Two hydroxyl groups—one at either end—make the natural PEG bifunctional. To prevent the potential for cross-linking and polymerization with the natural bifunctional polymer, monofunctional PEG polymer can be used. To make PEG monofunctional, one end of the chain is blocked with a methyl ether (methoxy) group. Such a monofunctional PEG is termed monomethoxyPEG (mPEG). Thus, mPEG contains only one hydroxyl group per chain, thus limiting activation and coupling to one site.


PEGylation usually reduces binding affinity of the protein to its target. PEGylation also increases the viscosity of protein formulations, which may limit the development of concentrated solutions for injection. Protein reac-tion with PEG generally has low efficiency and is difficult to optimize. In addition, PEG often contains peroxide impurities, which can lead to oxida-tive protein degradation during shelf life storage.

Other protein conjugation approaches

Proteins can also be conjugated to hydroxyethyl starch (HESylation) or to polysialic acid (PSAylation) using similar chemistry to increase their plasma half-life. PEGylation remains the most common protein modification.

Antibody drug conjugates

In recent years, several mAb-based therapeutics that have a small molecule conjugated to the antibody—the ADCs—have been commercialized, such as Kadcyla® and Adcentris®. Most current ADCs are developed for oncology indications and utilize a high potency cytotoxic drug called payload attached through a covalent linker to a monoclonal antibody that serves as a targeting moiety. The discovery and development of ADCs follow unique paradigms that overlap both small and large molecule drug discovery and development but have unique distinctions. For example, the attachment of hydrophobic drug on the mAb changes mAb surface properties and conformational stabil-ity. It can increase protein aggregation and surface hydrophobicity.

Chemistry of conjugation of small molecule drug to the antibody is con-stantly evolving. In general, the conjugation can be random (through, e.g., lysine or cysteine residues) or site specific (through, e.g., engineered antibod-ies that have specific amino acid residues). One needs to pay attention to the selection of mAb, payload, and linker for an effective ADC. Currently, several ADCs are in clinical trials as monotherapies or in combination with other anticancer drugs.

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