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Dear Editor,

In the course of the last decade, the possibility of employing full-length human alpha-fetoprotein (AFP) (FL-AFP) as a therapeutic agent for autoimmune diseases in clinical patients has become a reality in ongoing clinical trials.1 This is due, in part, to advances in both recombinant protein technology and improved methodologies in the isolation and large scale purification of naturally-occurring proteins. Although it is now possible to produce FL-AFP in scaled-up quantities, does this justify its use as a therapeutic agent (protein) in adult diseases and disorders? Are there safety issues involved with its use in patients, of what do they consist, and what are the possible risks, if any? The present commentary is a call to proceed slowly and with extreme caution in administering an oncofetal protein to human adult patients, a protein whose precise function and physiological roles are not yet fully understood.

Even though the literature is replete with the biological activities ascribed to the FL-AFP, little is known regarding the administration of pharmacologic doses to human adults that normally display scant (5–8 ng/ml) levels in their bloodstream. Unless a patient has liver/germ cell cancer, hepatitis, cirrhosis, or a genetic disorder (i.e., ataxia telangiectasia), low AFP concentrations remain relatively constant throughout life. In contrast, the AFP levels in embryonic and fetal life can range from 20 ug/ml in amniotic fluids to 5 mg/ml in fetal serum.2 These concentrations, however, occur in cells and tissues undergoing frequent cell proliferation, adhesion, migration, differentiation and growth in the constantly changing milieu of the embryonic/fetal organism. Thus, FL-AFP exists and flourishes in fluctuating fetal environments requiring both molecular flexibility and adaptability. This is in dire contrast to albumin which interacts mostly with fully-differentiated cells and tissues of the adult organism.

The potential risks involved with administering FL-AFP to clinical patients have, as root concerns, AFP's ability to transition into multiple conformational variant states depending on its environmental surroundings such as pH, temperature, osmolality, excess ligand concentrations, oxidation and heat/glucose shock.3 The silent danger of treating adult human patients with therapeutic doses of FL-AFP lies in its reversible and transient denaturation (conformational) states which bestow on AFP a rigid-to-flexible vacillation that exits between a compactly-folded form and an extended or open form. HAFP has a remarkably hydrophilic- exposed molecular surface at neutral pH and possesses extensive hydrophobic binding sites located in concealed molecular crevices. The immunochemistry of the FL-AFP molecule has further revealed clusters of five major antigenic epitopes and one major occult epitope which gives rise to open and cryptic forms of AFP depending on its natured versus denatured state, respectively.4, 5 Finally, FL-AFP has also been demonstrated to dimerize with other proteins, such as nuclear receptors (i.e., retinoic receptor), transcription factors and caspases all of which can result in promoting growth of tumor cells.6, 7

FL-AFP has been reported to transition through a molten globule form dependent on extremes of pH, a situation commonly found in the cytoplasm of cells following protein uptake.8 The FL-AFP molecule is known to undergo a slight denaturation and unfolding through a molten globule state, which encompasses a loosening of the tertiary packing while leaving the secondary structure of the molecule intact.9 In contrast, the unfolding–refolding transition states are less common with human albumin, due to its more rigid compact structure resulting from a higher number of disulfide bridges in the molecule. AFP's tertiary form is known to be under ligand binding control; thus, ligand concentration can affect its biological activities.10, 11 Moreover, a relationship exists between the conformational state and the biological activity of AFP as exemplified in a report that tumor and fetal forms of AFP were found to differ in their conformationally-dependent expressions of epitope variants.12 Such transitional variants could conceivably be formed following the injection of FL-AFP into clinical patients and could result in unwanted targeting and aberrant signal transduction of AFP leading to conditions of inappropriate and untimely cell growth inhibition and/or enhancement.

A further potential risk in the therapeutic use of FL-AFP, especially after multiple treatments, is the long-term effects on the growth, development and progression of small tumor foci which may not always be observable during human clinical trials. An effect that might result from extended administration of pharmacologic doses of FL-AFP is the initiation of tumor formation as previously described.13, 14 This induction could result from the transformation of pretumor to tumor cells which have evaded immune surveillance in cancer-susceptible individuals, such as in hepatitis or cirrhotic patents; such groups are at risk for developing hepatomas (see later). FL-AFP has also been reported to promote or up-regulate tumor cell proliferation, cell cycle progression, angiogenesis and the inhibition of apoptosis in human clinical tumors, employing cells derived from hepatocellular carcinoma patients.15 Although FL-AFP has been reported to induce apoptosis in cancer cells under certain conditions,16 the majority of published studies reveal that FL-AFP inhibits apoptosis in multiple cancer cell types thus promoting tumor cell proliferation, growth and progression.7, 17–19

The mechanism of action of tumor growth enhancement by AFP was shown to involve the shielding of human hepatoma cells from tumor necrosis factor (TNF)-induced apoptosis,20 and to promote the escape of tumor cells from lymphocytic attack (immune surveillance) by blocking the caspase apoptotic pathway.21 FL-AFP was further shown to promote cell proliferation in tumor cells and fibroblasts by the up-regulation of K-Ras p21, elevations of cyclic AMP and Protein Kinase A, and raised intracytoplasmic Ca++ levels.22, 23 FL-AFP had previously been found to promote the cell proliferation of human hepatomas, Erlich ascites carcinomas and mammary tumor cells, but not leukemic cells.18, 19, 24 The AFP-induced growth enhancement in some tumor cells ranged from 120 to 150%, while 80 to 200% occurred in the Erlich carcinoma cells.19 Studies of AFP knockdown by means of siRNA was shown to cause a notable delay in the G1 to S-Phase transition of the cell cycle, together with the inhibition of cell proliferation in hepatomas.25, 26 It was further shown that FL-AFP could promote the growth of tumor cells in a dose-dependent fashion; such tumors included hepatomas, lymphoblastomas, Jurket lymphomas and fibroblastomas.26 Finally FL-AFP, in conjunction with its cell surface receptor, was reported to act in synergism with growth factors (EGF, PDGF, IGF-1), cytokines (IFN-alpha, TNF), oncogenes (c-FOS, c-JUN, n-RAS) and transcription factors in promoting the growth of mammary and colon tumor cells.24, 27–31

The concept of FL-AFP enhancing and accelerating the growth of human tumors at first seems to contradict the published findings that high AFP levels during pregnancy has a protective effect against risk of breast cancer in latter life.32, 33 However, upon circumspection, it can be discerned that such reports do not include real-time clinical therapy results, but rather, data encompassing epidemiological findings and trends derived from statistical risk calculations, medical histories, logistic regression models and stored records from population-based data sets. At best, such studies represent statistically significant trends for large cohort groups (populations), but not individual patients' results. Other findings of FL-AFP in association with reduced breast cancer risk are derived from nonhuman (rat) pregnancy models and human cell cultures not linked to actual clinical patient tissue samples.34 These human population-based cohort studies had no means to determine or consider the presence of isoforms or conformational variants of AFP during pregnancy. However, recent clinical reports have described an assay to detect and measure conformationally-transformed AFP (tAFP) concentrations during normal pregnancy; such levels are elevated in conditions of intrauterine growth retardation and threatened preterm labor.35, 36 These authors proposed that some percentage of the AFP molecules become altered or transformed into tAFP via passage through the placenta since the maternal tAFP levels were 10 times greater than those in fetal samples. One can only speculate whether tAFP (a growth inhibitor) may have been involved in those published studies of reduced breast cancer risk. Finally, the presence of hereditary persistance of AFP following pregnancy (serum levels = 20–100 ng/ml) could provide an agrument that this benign autosomal dominant disorder is nonpathological in adult life. However, this condition is not always beign and has been reported to be coincident with tall stature, advanced bone age and growth, testis disease and sometimes germ cell tumors.37

As HAFP is largely a growth promoting molecule, the therapeutic injection of FL-AFP(70 Kd) into normal and/or diseased adults could be potentially hazardous and should require extensive, long-term clinical evaluation. The FL-AFP molecule is bristling with innumerable biologically-active peptide sites,38 some of which may not be fully exposed until AFP is introduced into differing or highly variable biochemical/biophysical microenvironments. Upon stimulation, the biologic response induced by these exposed peptide segments cannot be predicted or controlled and could produce undesired or dangerous side effects. This was precisely the reason why the United States FDA banned the sale and distribution of the human AFP immunoassay reagents as a medical device(kits) in 1971, a ban which endured until 1984.39 Recent examples of uncontrolled or unwanted AFP biologic responses have included reports showing that AFP can inhibit natural killer cell activity; thus AFP can inhibit immunity against tumors.40 AFP has also been reported to enhance cytokine, chemokine, growth factor, H2O2 and nitrite/nitrate levels in human keratinocytes.41 Finally, expression of the fetal AFP transcript and its concomitant DNA synthesis has long been utilized as an indicator for activation of the liver stem cell compartment, while AFP itself is a cell-surface marker of stem cell proliferation.42 It is with more than 50 years of published reports in the field of AFP research that a cancer risk warning can now be issued. Unless under developmental stage control, FL-AFP should be viewed as a biological “loose cannon.” Therefore, function-site specific AFP-derived peptides might offer a safer, more conservative approach for possible future therapy of human clinical patients for diseases such as myasthenia gravis, systemic lupus, Hashomoto's thyroiditis, multiple sclerosis, arthritis and perhaps other inflammatory/autoimmune disorders.

Acknowledgements

The author extends his thanks and gratitude to Dr. Jennifer L. Wright for time expenditure in the typing and processing of the manuscript and references of this report.

Yours sincerely,

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Gerald J. Mizejewski.