Small modular immunopharmaceutical

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Small modular immunopharmaceuticals, or SMIPs for short, are artificial proteins that are intended for use as pharmaceutical drugs. They are largely built from parts of antibodies (immunoglobulins), and like them have a binding site for antigens that could be used for monoclonal antibody therapy. SMIPs have similar biological half-life and, being smaller than antibodies, are reasoned to have better tissue penetration properties. They were invented by Trubion and are now being developed by Emergent BioSolutions, which acquired Trubion in 2010. [1]

Protein biological molecule consisting of chains of amino acid residues

Proteins are large biomolecules, or macromolecules, consisting of one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli, providing structure to cells and organisms, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in protein folding into a specific three-dimensional structure that determines its activity.

Binding site region on a protein or piece of DNA or RNA to which ligands (specific molecules and/or ions) may form a chemical bond. An equilibrium exists between unbound ligands and bound ligands

In biochemistry and molecular biology, a binding site is a region on a macromolecule such as a protein that binds to another molecule with specificity. The binding partner of the macromolecule is often referred to as a ligand. Ligands may include other proteins, enzyme substrates, second messengers, hormones, or allosteric modulators. The binding event is often, but not always, accompanied by a conformational change that alters the function of the protein. Binding to protein binding sites is most often reversible, but can also be covalent reversible or irreversible.

Antigen molecule capable of inducing an immune response (to produce an antibody) in the host organism

In immunology, antigens (Ag) are structures specifically bound by antibodies (Ab) or a cell surface version of Ab ~ B cell antigen receptor (BCR). The term antigen originally described a structural molecule that binds specifically to an antibody only in the form of native antigen. It was expanded later to refer to any molecule or a linear molecular fragment after processing the native antigen that can be recognized by T-cell receptor (TCR). BCR and TCR are both highly variable antigen receptors diversified by somatic V(D)J recombination. Both T cells and B cells are cellular components of adaptive immunity. The Ag abbreviation stands for an antibody generator.

Contents

Structure

Sketch of a SMIP monomer consisting of four domains. The antigen binding site is on the left edge. scFv: modified single-chain variable fragment Fc: one half of a fragment crystallizable region VH: heavy chain variable domain VL: light chain variable domain CH2: constant domain 2 CH3: constant domain 3 (effector domain) Arrows: linker peptide (left) and hinge region (right) N: N-terminus C: C-terminus Small modular immunopharmaceutical.svg
Sketch of a SMIP monomer consisting of four domains. The antigen binding site is on the left edge.
scFv: modified single-chain variable fragment
Fc: one half of a fragment crystallizable region
VH: heavy chain variable domain
VL: light chain variable domain
CH2: constant domain 2
CH3: constant domain 3 (effector domain)
Arrows: linker peptide (left) and hinge region (right)
N: N-terminus
C: C-terminus

SMIPs are single-chain proteins that comprise one binding region, one hinge region as a connector, and one effector domain. The binding region is a modified single-chain variable fragment (scFv), and the rest of the protein can be constructed from the fragment crystallizable region (Fc) and the hinge region of an immunoglobulin G 1 (IgG1). Genetically modified cells produce SMIPs as antibody-like dimers, which are about 30% smaller than real antibodies. [2]

Single-chain variable fragment

A single-chain variable fragment (scFv) is not actually a fragment of an antibody, but instead is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. The image to the right shows how this modification usually leaves the specificity unaltered.

Fragment crystallizable region

The fragment crystallizable region is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains; IgM and IgE Fc regions contain three heavy chain constant domains in each polypeptide chain. The Fc regions of IgGs bear a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is essential for Fc receptor-mediated activity. The N-glycans attached to this site are predominantly core-fucosylated diantennary structures of the complex type. In addition, small amounts of these N-glycans also bear bisecting GlcNAc and α-2,6 linked sialic acid residues.

Immunoglobulin G (IgG) is a type of antibody. Representing approximately 75% of serum antibodies in humans, IgG is the most common type of antibody found in blood circulation. IgG molecules are created and released by plasma B cells. Each IgG has two antigen binding sites.

Like ordinary monoclonal antibodies, SMIPs are monospecific, meaning they recognize and attach to a single antigen target to initiate their biological activity. SMIP drug candidates are intended to target antigens with the same specificity and predictable biological activity as monoclonal antibodies. Examples are TRU-015, a CD20 targeting SMIP under research for rheumatoid arthritis, [3] and TRU-016, a CD37 targeting potential treatment for chronic lymphocytic leukemia and other B-cell cancers. [4] [5]

Monospecific antibodies are antibodies whose specificity to antigens is singular in any of several ways: antibodies that all have affinity for the same antigen; antibodies that are specific to one antigen or one epitope; or antibodies specific to one type of cell or tissue. Monoclonal antibodies are monospecific, but monospecific antibodies may also be produced by other means than producing them from a common germ cell. Regarding antibodies, monospecific and monovalent overlap in meaning; both can indicate specificity to one antigen, one epitope, or one cell type. However, antibodies that are monospecific to a certain tissue, or all monospecific to the same tissue because clones, can be polyvalent in their epitope binding.

CD20 protein-coding gene in the species Homo sapiens

B-lymphocyte antigen CD20 or CD20 is an activated-glycosylated phosphoprotein expressed on the surface of all B-cells beginning at the pro-B phase and progressively increasing in concentration until maturity.

Rheumatoid arthritis An arthritis that is an autoimmune disease which attacks healthy cells and tissue located in joint.

Rheumatoid arthritis (RA) is a long-term autoimmune disorder that primarily affects joints. It typically results in warm, swollen, and painful joints. Pain and stiffness often worsen following rest. Most commonly, the wrist and hands are involved, with the same joints typically involved on both sides of the body. The disease may also affect other parts of the body. This may result in a low red blood cell count, inflammation around the lungs, and inflammation around the heart. Fever and low energy may also be present. Often, symptoms come on gradually over weeks to months.

Production

A monoclonal antibody targeting the desired antigen can be developed the classical way, using hybridoma technology. The scFv is then constructed from the antibody's variable regions. A large number of different hinge regions and effector domains are taken from libraries of immunoglobulins, and the combined proteins are produced in genetically modified (transfected) cells and screened for clones with useful properties like high binding specificity. The selected protein is multiplied in transfected cells suitable for medium- or large-scale production, for example Chinese hamster ovary cells, and purified by chromatography. [2]

Hybridoma technology

Hybridoma technology is a method for producing large numbers of identical antibodies. This process starts by injecting a mouse with an antigen that provokes an immune response. A type of white blood cell, the B cell, produces antibodies that bind to the injected antigen. These newly produced antibodies are then harvested from the mouse. These isolated B cells are in turn fused with immortal B cell cancer cells, a myeloma, to produce a hybrid cell line called a hybridoma, which has both the antibody-producing ability of the B-cell and the exaggerated longevity and reproductivity of the myeloma. The hybridomas can be grown in culture, each culture starting with one viable hybridoma cell, producing cultures each of which consists of genetically identical hybridomas which produce one antibody per culture (monoclonal) rather than mixtures of different antibodies (polyclonal). The myeloma cell line that is used in this process is selected for its ability to grow in tissue culture and for an absence of antibody synthesis. In contrast to polyclonal antibodies, which are mixtures of many different antibody molecules, the monoclonal antibodies produced by each hybridoma line are all chemically identical.

High-throughput screening

High-throughput screening (HTS) is a method for scientific experimentation especially used in drug discovery and relevant to the fields of biology and chemistry. Using robotics, data processing/control software, liquid handling devices, and sensitive detectors, high-throughput screening allows a researcher to quickly conduct millions of chemical, genetic, or pharmacological tests. Through this process one can rapidly identify active compounds, antibodies, or genes that modulate a particular biomolecular pathway. The results of these experiments provide starting points for drug design and for understanding the noninteraction or role of a particular location.

Chinese hamster ovary cell cell line

Chinese hamster ovary (CHO) cells are an epithelial cell line derived from the ovary of the Chinese hamster, often used in biological and medical research and commercially in the production of therapeutic proteins. They have found wide use in studies of genetics, toxicity screening, nutrition and gene expression, particularly to express recombinant proteins. CHO cells are the most commonly used mammalian hosts for industrial production of recombinant protein therapeutics.

Related Research Articles

Antibody large Y-shaped protein produced by B-cells, used by the immune system; large, Y-shaped protein produced mainly by plasma cells that is used by the immune system to neutralize pathogens such as pathogenic bacteria and viruses

An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein produced mainly by plasma cells that is used by the immune system to neutralize pathogens such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the pathogen, called an antigen, via the Fab's variable region. Each tip of the "Y" of an antibody contains a paratope that is specific for one particular epitope on an antigen, allowing these two structures to bind together with precision. Using this binding mechanism, an antibody can tag a microbe or an infected cell for attack by other parts of the immune system, or can neutralize its target directly. Depending on the antigen, the binding may impede the biological process causing the disease or may activate macrophages to destroy the foreign substance. The ability of an antibody to communicate with the other components of the immune system is mediated via its Fc region, which contains a conserved glycosylation site involved in these interactions. The production of antibodies is the main function of the humoral immune system.

CD32 surface receptor protein

CD32 is a surface receptor protein and part of a large population of B cell co-receptors, which act to modulate signaling.

Humanized antibodies are antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans. The process of "humanization" is usually applied to monoclonal antibodies developed for administration to humans. Humanization can be necessary when the process of developing a specific antibody involves generation in a non-human immune system. The protein sequences of antibodies produced in this way are partially distinct from homologous antibodies occurring naturally in humans, and are therefore potentially immunogenic when administered to human patients. There are other types of antibodies developed. The International Nonproprietary Names of humanized antibodies end in -zumab, as in omalizumab.

Protein A surface protein in bacteria cell wall known for its ability to bind many mammal immunoglobulins

Protein A is a 42 kDa surface protein originally found in the cell wall of the bacteria Staphylococcus aureus. It is encoded by the spa gene and its regulation is controlled by DNA topology, cellular osmolarity, and a two-component system called ArlS-ArlR. It has found use in biochemical research because of its ability to bind immunoglobulins. It is composed of five homologous Ig-binding domains that fold into a three-helix bundle. Each domain is able to bind proteins from many mammalian species, most notably IgGs. It binds the heavy chain within the Fc region of most immunoglobulins and also within the Fab region in the case of the human VH3 family. Through these interactions in serum, where IgG molecules are bound in the wrong orientation, the bacteria disrupts opsonization and phagocytosis.

Monoclonal antibody therapy

Monoclonal antibody therapy is a form of immunotherapy that uses monoclonal antibodies (mAb) to bind monospecifically to certain cells or proteins. The objective is that this treatment will stimulate the patient's immune system to attack those cells. Alternatively, in radioimmunotherapy a radioactive dose localizes a target cell line, delivering lethal chemical doses. More recently antibodies have been used to bind to molecules involved in T-cell regulation to remove inhibitory pathways that block T-cell responses. This is known as immune checkpoint therapy.

Biological response modifiers (BRMs) are substances that modify immune responses. They can be both endogenous and exogenous, and they can either enhance an immune response or suppress it. Some of these substances arouse the body's response to an infection, and others can keep the response from becoming excessive. Thus they serve as immunomodulators in immunotherapy, which can be helpful in treating cancer and in treating autoimmune diseases, such as some kinds of arthritis and dermatitis. Most BRMs are biopharmaceuticals (biologics), including monoclonal antibodies, interleukin 2, interferons, and various types of colony-stimulating factors. "Immunotherapy makes use of BRMs to enhance the activity of the immune system to increase the body's natural defense mechanisms against cancer", whereas BRMs for rheumatoid arthritis aim to reduce inflammation.

Abatacept is a drug used to treat autoimmune diseases like rheumatoid arthritis, by interfering with the immune activity of T cells. It is a modified antibody.

CD16, also known as FcγRIII, is a cluster of differentiation molecule found on the surface of natural killer cells, neutrophil polymorphonuclear leukocytes, monocytes and macrophages. CD16 has been identified as Fc receptors FcγRIIIa (CD16a) and FcγRIIIb (CD16b), which participate in signal transduction. The most well-researched membrane receptor implicated in triggering lysis by NK cells, CD16 is a molecule of the immunoglobulin superfamily (IgSF) involved in antibody-dependent cellular cytotoxicity (ADCC). It can be used to isolate populations of specific immune cells through fluorescent-activated cell sorting (FACS) or magnetic-activated cell sorting, using antibodies directed towards CD16.

Isotype (immunology)

In immunology, the immunoglobulin (Ig) isotype (class) is encoded by the constant region segments of the immunoglobulin gene which form the Fc portion of an antibody. The expression of a specific isotype determines the function of an antibody via the specific binding to Fc receptor molecules on different immune effector cells. Isotype expression reflects the maturation stage of a B cell. Naive B cells express IgM and IgD isotypes with unmutated variable genes, which are produced from the same initial transcript following alternative splicing. Expression of other antibody isotypes occurs via a process of class-switch recombination (CSR) after antigen exposure. Class-switching is mediated by the AID enzyme and only occurs after the B cell binds an antigen through its B cell receptor, and is further activated through interaction with a T helper cell.

Protein L

Protein L was first isolated from the surface of bacterial species Peptostreptococcus magnus and was found to bind immunoglobulins through L chain interaction, from which the name was suggested. It consists of 719 amino acid residues. The molecular weight of Protein L purified from the cell walls of Peptostreptoccus magnus was first estimated as 95kD by SDS-PAGE in the presence of reducing agent 2-mercaptoethanol, while the molecular weight was determined to 76kD by gel chromotography in the presence of 6 M guanidine HCl. Protein L does not contain any interchain disulfide loops, nor does it consist of disulfide-linked subunits. It is an acidic molecule with a pI of 4.0. Unlike Protein A and Protein G, which bind to the Fc region of immunoglobulins (antibodies), Protein L binds antibodies through light chain interactions. Since no part of the heavy chain is involved in the binding interaction, Protein L binds a wider range of antibody classes than Protein A or G. Protein L binds to representatives of all antibody classes, including IgG, IgM, IgA, IgE and IgD. Single chain variable fragments (scFv) and Fab fragments also bind to Protein L.

Trubion was a publicly held biopharmaceutical company that was focused on creating a pipeline of protein-based therapeutic product candidates to treat autoimmune and inflammatory diseases and cancer. Trubion was acquired by Emergent BioSolutions on October 28, 2010. Trubion was established in 1999 in the State of Washington as an early stage development company, and was later reincorporated in October 2002 in the State of Delaware.

Atacicept is a recombinant fusion protein designed to inhibit B cells, thereby suppressing autoimmune disease. The designer protein combines the binding site for two cytokines that regulate maturation, function, and survival of B cells - B-lymphocyte stimulator (BLyS) and A proliferation-inducing ligand (APRIL), with the constant region of immunoglobin. Atacicept blocks activation of B cells by the tumor necrosis factor receptor superfamily member 13B, a transmembrane receptor protein found predominantly on the surface of B cells. Like the monoclonal antibody belimumab, atacicept blocks the binding of BLyS, but it also blocks APRIL. Binding of these TACI ligands induces proliferation, activation, and longevity of B cells and thus their production of autoantibodies. Atacicept is thought to selectively impair mature B cells and plasma cells with less impact on progenitor cells and memory B cells.

Gene expression profiling has revealed that diffuse large B-cell lymphoma (DLBCL) is composed of at least 3 different sub-groups, each having distinct oncogenic mechanisms that respond to therapies in different ways. Germinal Center B-Cell like (GCB) DLBCLs appear to arise from normal germinal center B cells, while Activated B-cell like (ABC) DLBCLs are thought to arise from postgerminal center B cells that are arrested during plasmacytic differentiation. The differences in gene expression between GCB DLBCL and ABC DLBCL are as vast as the differences between distinct types of leukemia, but these conditions have historically been grouped together and treated as the same disease.

Ocaratuzumab is a humanized monoclonal antibody designed for the treatment of cancer and autoimmune disorders. The antibody is engineered for enhanced affinity to the CD20 antigen on B-lymphocytes, increased antibody-dependent cell-mediated cytotoxicity (ADCC), and for improved treatment of low-affinity FcγRIIIa allotypes.

ImmTAC

ImmTACs are a class of bispecific biological drug being investigated for the treatment of cancer and viral infections which combines engineered cancer-recognizing TCRs with immune activating complexes. ImmTACs target cancerous or virally infected cells through binding human leukocyte antigen (HLA) presented peptide antigens and redirect the host's cytotoxic T cells to recognise and kill them.

Recombinant antibodies are antibody fragments produced by using recombinant antibody coding genes. They mostly consist of a heavy and light chain of the variable region of immunoglobulin. Recombinant antibodies have many advantages in both medical and research applications, which make them a popular subject of exploration and new production against specific targets. The most commonly used form is the single chain variable fragment (scFv), which has shown the most promising traits exploitable in human medicine and research. In contrast to monoclonal antibodies produced by hybridoma technology, which may lose the capacity to produce the desired antibody over time or the antibody may undergo unwanted changes, which affect its functionality, recombinant antibodies produced in phage display maintain high standard of specificity and low immunogenicity.

MOR103

MOR103 is a so-called fully human antibody which has been developed by the biotechnology company MorphoSys. It can also be referred to as HuCAL antibody, HuCAL standing for Human Combinatorial Antibody Library and being a technology used to generate monoclonal antibodies. MOR103 is directed against the granulocyte-macrophage colony stimulating factor (GM-CSF), a monomeric glycoprotein functioning as a cytokine promoting both proliferation and activation of macrophages and neutrophils.

References

  1. ↑ TruEmergent: SMIP Therapeutics Archived 2011-08-22 at the Wayback Machine .
  2. 1 2 Zhao, X.; Lapalombella, R.; Joshi, T.; Cheney, C.; Gowda, A.; Hayden-Ledbetter, M. S.; Baum, P. R.; Lin, T. S.; Jarjoura, D.; Lehman, A.; Kussewitt, D.; Lee, R. J.; Caligiuri, M. A.; Tridandapani, S.; Muthusamy, N.; Byrd, J. C. (2007). "Targeting CD37-positive lymphoid malignancies with a novel engineered small modular immunopharmaceutical". Blood. 110 (7): 2569–2577. doi:10.1182/blood-2006-12-062927. PMC   1988922  Lock-green.svg. PMID   17440052.
  3. ↑ Rubbert-Roth, A. (2010). "TRU-015, a fusion protein derived from an anti-CD20 antibody, for the treatment of rheumatoid arthritis". Current Opinion in Molecular Therapeutics. 12 (1): 115–123. PMID   20140823.
  4. ↑ Robak, T.; Robak, P.; Smolewski, P. (2009). "TRU-016, a humanized anti-CD37 IgG fusion protein for the potential treatment of B-cell malignancies". Current Opinion in Investigational Drugs. 10 (12): 1383–1390. PMID   19943209.
  5. ↑ TruEmergent: TRU-016 for the Treatment of B-Cell Malignancies Archived 2011-08-22 at the Wayback Machine .
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