BOX 45 Modulation Of The Clearance Of Therapeutic Proteins By Addition Of Fc Domain Sequences

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In the early 1970s, when recombinant proteins in large quantity first became available in pharmaceutical grade, it was recognized that most proteins less than 50kDa are readily cleared from the circulation if they are not bound to a receptor or do not contain unique amino-acid sequences that provide prolonged residence time in blood. Studies of immunoglobulin biosynthesis and metabolism mapped the important determinants of the half-life of these molecules in plasma, regardless of their antigen specificity. These determinants were located within the Fc domain, which does not participate in antibody-antigen binding interactions. With the availability of the gene or DNA sequences corresponding to the Fc domain of IgG, researchers from a biotechnology company, Genentech, engineered a fusion protein that contains a CD4 linked to Fc domain IgG [32]. The CD4 molecule found on helper T-lymphocytes is one that human immunodeficiency virus recognizes and uses to infect these cells. The recombinant soluble CD4 (55 kDa) is cleared from blood readily, but the fusion protein CD4-Fc provides a much longer residence time in blood. Unfortunately, CD4-Fc removed the HIV gp120 envelope protein from the viral membranes, and thereby failed neutralize the virus, and it remained infectious.

The same principle was later applied by scientists at Immunex to enhance the pharmaceutical properties of the tumor necrosis factor receptor (TNFr), which, like CD4, is rapidly cleared from blood. The hybrid TNFr-Fc molecule (~150kDa), which is now known as Enbrel, is used to reduce the inflammatory responses of rheumatoid arthritis [33,34]. The hybrid TNFr-Fc has extended the plasma half-life of TNFr from several minutes to 190 hours.

■TABLE 4.7. Elucidation of short peptide for immune-stimulatory, but not inflammatory activity of IL-1 b

Responses

Observed Effects

IL-1ß (269 aa) Peptide163-171

Immune stimulation

Immune restoration

+++

++

Vaccine adjuvant activity

+++

+++

Induction of IL-2 and IL-4

+

++

Inflammation

Fever

+++

Corticosteroid induction

+++

PGE2 release by fibroblast

+++

IL-6 induction

+++

IL-1 peptide (163-171)

COOH

VQGEESNDK

Figure 4.8. Schematic representation of amino acid 163-171 IL-1 peptide in relationship to the whole IL-1 b protein.

COOH

VQGEESNDK

Figure 4.8. Schematic representation of amino acid 163-171 IL-1 peptide in relationship to the whole IL-1 b protein.

specific gene with a disease is the result of remarkable progress in the areas of gene sequencing and information technologies [36].

Several thousand molecular targets have been cloned and are available as potential drug discovery targets. These targets include G-protein coupled receptors (GPCRs), ligand-gated ion channels, nuclear receptors and cytokines, and reup-take/transport proteins. A new potential therapeutic approach for the treatment of a known disease is published nearly every week. The sheer volume of genetic information being produced has shifted the emphasis for the generation of novel DNA sequences to the determination of which of these new targets offers biologic function with the greatest opportunity for drug discovery. Consequently target selection and validation have become the most important components of the drug discovery process [36].

An example is a chemokine called myeloid progenitor inhibitor factor-1 (MPIF-1), developed by Human Genome Sciences (HGS) for the treatment of patients with cancer to reduce the toxicity of chemotherapy. It was the first genomics-derived therapeutic product to enter clinical trials [37].

HGS was among the first biotechnology companies to mine large databases of human gene sequences, looking for previously unknown proteins that might have therapeutic value. Rather than attempting to spell out every letter of DNA's code, the scientists at The Institute for Genomic Research (TIGR) identified and characterized messenger RNA (mRNA) copies from active genes. They copied the mRNAs back into DNAs and then spelled out a part of each gene to create expressed sequence tags (ESTs).

Researchers at HSG searched for membrane-bound proteins, including receptors for growth hormones, neuro-transmitters, and cytokines that might serve as drug targets. HSG scientists also looked for protein ligands that might make good drugs themselves. By 1996 they had found about 300 genes that seemed to encode new members of known, secreted protein classes and started making proteins and testing them for activity against diseases. One of these proteins was MPIF-1. The novel chemokine reversibly stopped the proliferation of several types of bone marrow stem cells in culture, suggesting that MPIF-1 might help protect the bone marrow of cancer patients from the toxic effects of chemotherapy, which preferentially kill rapidly dividing cells. Available drugs can boost the number of blood cells after a course of chemotherapy, but there are no drugs to protect such cells from being killed in the first place [38].

Another example is a pharmaceutical agent, imatanib, developed to treat chronic myeloid leukemia (CML). The Philadelphia chromosome, discovered in 1960, was first observed as a consistent cytogenetic abnormality in the tumor cells of CML patients. This abnormal chromosome represents the reciprocal translocation of parts of the long arms of the chromosomes 9 and 22 and was the first cytogenetic evidence of the phenomenon of chromosomal translocation. The important genes in the translocation are the Abl gene on chromosome 9 (a tyrosine kinase) and the Bcr (which stands for "breakpoint cluster region") gene on chromosome 22. The BCR-ABL gene product is sufficient to cause leukemia in animal models, and its cancer-causing ability depends on tyrosine kinase activity.

CML is characterized by massive clonal expansion of cells of the myeloid lineage and progresses through three phases: chronic phase, accelerated phase, and blast phase. Current therapies include allogeneic bone marrow transplantation, which is curative but associated with substantial morbidity and mortality and limited to patients for whom a suitable donor is found, and drug regimens, including interferon-a therapy, which prolongs survival but has considerable toxicity.

In an effort to interfere with CML progression, pharmaceutical scientists at Novartis first cloned and produced recombinant BCR-ABL kinase. With the availability of this enzyme in sufficient quantity and purity, a mass in vitro screen of a series of enzyme inhibitors was implemented to identify drug candidates that produce an optimum pharmaceutical profile. From these, they identified STI571 (Gleevec) as a lead candidate to block the kinase activity of BCR-ABL. STI571 acts as a competitive inhibitor of ATP binding to the enzyme, which leads to the inhibition of tyrosine phosphorylation of proteins involved in BCR-ABL signal transduction [39,40].

Although Gleevec was carefully designed to inhibit the specific tyrosine kinase produced by the Philadelphia chromosome, it also produces unexpected activities. Gleevec blocks c-kit (the receptor for stem cell factor) [40], and the receptor for platelet-derived growth factor [41]. These additional activities could result in a broader array of antitumor activities, in a broader spectrum of toxicities, or both [42].

An example of drug target selection is the pairing of a G-protein coupled receptor, GPR-14, with its neuropeptide ligand, urotensin II, the most potent vasoconstrictor ever identified. GPR-14/urotensin II represents an attractive target for the treatment of disorders related to excessive vasoconstriction, such as hypertension, congestive heart failure, and coronary artery disease [43,44].

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