Once a rarely used subset of medical treatments, protein therapeutics have increased dramatically in number and frequency of use since the introduction of the first recombinant protein therapeutic--human insulin--25 years ago. Protein therapeutics already have a significant role in almost every field of medicine, but this role is still only in its infancy. This article overviews some of the key characteristics of protein therapeutics, summarizes the more than 130 protein therapeutics used currently and suggests a new classification of these proteins according to their pharmacological action.

Protein therapeutics: a summary and pharmacological classification

It has recently been demonstrated that in vivo administration of murine interleukin 12 (IL-12) to mice results in augmentation of cytotoxic natural killer (NK)/lymphocyte-activated killer cell activity, enhancement of cytolytic T cell generation, and induction of interferon gamma secretion. In this study, the in vivo activity of murine IL-12 against a number of murine tumors has been evaluated. Experimental pulmonary metastases or subcutaneous growth of the B16F10 melanoma were markedly reduced in mice treated intraperitoneally with IL-12, resulting in an increase in survival time. The therapeutic effectiveness of IL-12 was dose dependent and treatment of subcutaneous tumors could be initiated up to 14 d after injection of tumor cells. Likewise, established experimental hepatic metastases and established subcutaneous M5076 reticulum cell sarcoma and Renca renal cell adenocarcinoma tumors were effectively treated by IL-12 at doses which resulted in no gross toxicity. Local peritumoral injection of IL-12 into established subcutaneous Renca tumors resulted in regression and complete disappearance of these tumors. IL-12 was as effective in NK cell-deficient beige mice or in mice depleted of NK cell activity by treatment with antiasialo GM1, suggesting that NK cells are not the primary cell type mediating the antitumor effects of this cytokine. However, the efficacy of IL-12 was greatly reduced in nude mice suggesting the involvement of T cells. Furthermore, depletion of CD8+ but not CD4+ T cells significantly reduced the efficacy of IL-12. These results demonstrate that IL-12 has potent in vivo antitumor and antimetastatic effects against murine tumors and demonstrate as well the critical role of CD8+ T cells in mediating the antitumor effects against subcutaneous tumors.

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Antitumor and Antimetastatic Activity of Interleukin 12 against Murine Tumors

Cytokines are soluble proteins that allow for communication between cells and the external environment. Interferon (IFN) alpha, the first cytokine to be produced by recombinant DNA technology, has emerged as an important regulator of growth and differentiation, affecting cellular communication and signal transduction pathways as well as immunological control. This review focuses on the biological and clinical activities of the cytokine. Originally discovered as an antiviral substance, the efficacy of IFN-alpha in malignant, viral, immunological, angiogenic, inflammatory, and fibrotic diseases suggests a spectrum of interrelated pathophysiologies. The principles learned from in vivo studies will be discussed, particularly hairy cell leukemia, chronic myelogenous leukemia, certain angiogenic diseases, and hepatitis. After the surprising discovery of activity in a rare B-cell neoplasm, IFN-alpha emerged as a prototypic tumor suppressor protein that represses the clinical tumorigenic phenotype in some malignancies capable of differentiation. Regulatory agencies throughout the world have approved IFN-alpha for treatment of 13 malignant and viral disorders. The principles established with this cytokine serve as a paradigm for future development of natural proteins for human disease.

https://www.pnas.org/content/pnas/91/4/1198.full.pdf

Cytokine therapeutics: lessons from interferon alpha.

https://www.ibms.sinica.edu.tw/%7Esroff/anti-PEG/bailon2001.pdf

Rational design of a potent, long-lasting form of interferon: a 40 kDa branched polyethylene glycol-conjugated interferon alpha-2a for the treatment of hepatitis C.

The interferon system. A bird's eye view of its biochemistry.

The interferons (IFN) are one of the body's natural defensive responses to such foreign components as microbes, tumors, and antigens. The IFN response begins with the production of the IFN proteins (α, β, and γ), which then induce the antiviral, antimicrobial, antitumor, and immunomodulatory actions of IFN. Recent advances have led to Food and Drug Administration approval of five clinical indications for IFN. Interferon alfa is approved for hairy-cell leukemia, condyloma acuminatum, Kaposi's sarcoma in the acquired immunodeficiency syndrome, and non-A, non-B (type C) viral hepatitis. Interferon gamma has properties distinctive from those of IFNs α and β and is approved as an immunomodulatory treatment for chronic granulomatous disease. Promising clinical results with IFNs have also been reported for basal cell carcinoma, chronic myelogenous leukemia, cutaneous squamous cell carcinoma, early human immunodeficiency virus infection, hepatitis B, and laryngeal papillomatosis. Future clinical uses of IFNs may emphasize combination therapy with other cytokines, chemotherapy, radiation, surgery, hyperthermia, or hormones. (JAMA. 1991;266:1375-1383)

The interferons. Mechanisms of action and clinical applications.

Human lymphocytes were treated with human alpha (IFN-alpha), beta (IFN-beta), or recombinant gamma (IFN-gamma) interferons separately or in combination to determine their ability to enhance natural killing against mouse L cell targets. Our results showed that recombinant IFN-gamma was approximately 50 times more active per unit of antiviral activity than either IFN-alpha or IFN-beta. Moreover, the levels of natural killing by lymphocytes treated with combinations of IFN-alpha and IFN-beta were additive, whereas combinations of recombinant IFN-gamma and IFN-alpha or recombinant IFN-gamma and IFN-beta were synergistic. The development of natural killing in lymphocytes treated with recombinant IFN-gamma did not occur more rapidly but reached higher levels (62%) than that observed with lymphocytes treated with IFN-alpha or IFN-beta (15%). The results suggest the importance of IFN-gamma and mixtures of IFN-gamma with IFN-alpha or IFN-beta in the enhancement of natural killing activity against virus infections and neoplasia.

Potentiation of lymphocyte natural killing by mixtures of alpha or beta interferon with recombinant gamma interferon.

Interferons (IFN) have been approved for a number of clinical uses. The accepted routes of administration are intramuscular, subcutaneous, and intravenous. Recently, interferons administered by the oral route have been shown to exert a systemic effect. Oral administrations of IFN-alpha, IFN-beta, and IFN-gamma have been shown to cause a suppression of the peripheral white blood cell (WBC) count in mice. This study investigates the mechanism by which this suppression occurs. The results show that, in contrast to their intraperitoneal administration, oral administration of rHuIFN-alpha A/D or rMuIFN-gamma does not result in the presence of detectable levels of interferons in the blood. In addition, although the presence of circulating specific antibody to interferon blocks the peripheral WBC suppressive effects of intraperitoneally administered MuIFN-beta or rMuIFN-gamma, the presence of those antibodies does not block the peripheral WBC suppressive effects of the orally administered interferons. The peripheral WBC suppressive effect of orally administered rHuIFN-alpha A/D and rMuIFN-gamma can be transferred by injection of blood from oral interferon-treated donor mice to recipient mice. Recipient mice receiving plasma from donor mice showed no peripheral WBC suppression. Recipient mice receiving blood cells from donor mice showed significant peripheral WBC suppression. No effect of orally administered rHuIFN-alpha A/D on the relative percentages of lymphocytes, neutrophils, and monocytes was noted. These results indicate that the mechanism by which orally administered interferons exert their WBC suppressive effect differs from that of intraperitoneally administered interferons. WBC suppression resulting from orally administered interferons may involve cell to cell transfer of the interferons' effects, rather than the systemic distribution of the interferons in the blood. These studies further suggest that there may be a role for oral administration as a new route of interferon administration and provide a glimpse into the mechanism by which the orally administered interferons exert their systemic effects.

Orally administered interferons exert their white blood cell suppressive effects via a novel mechanism.

The ability of interferons (IFN) to exert a systemic effect following their oral administration was evaluated. One systemic effect of parenteral interferon administration has been shown to be a suppression of the number of peripheral white blood cells both in man and in mouse models. Using the mouse model of peripheral white blood cell suppression, the relative systemic effects of orally and subcutaneously administered interferons were determined. Murine IFN-beta, murine IFN-gamma and cross-reactive recombinant human IFN-alpha A/D were examined. The oral administrations of each of the three interferons were found to cause a dose-dependent suppression of the peripheral white blood cell counts. Significant levels of suppression were seen with as little as 5 units/day of murine IFN-beta and with 500 units/day of recombinant human IFN-alpha A/D and murine IFN-gamma. The dose-response curves obtained with orally administered interferons were much more shallow than those obtained with subcutaneously administered interferons. The results demonstrate that oral administration of interferons can provide a significant systemic effect. Further, the results support the possibility that the oral administration of interferons may have therapeutic potential.

Modulation of peripheral leukocyte counts in mice by oral administration of interferons.

Mouse B16 melanoma cells rapidly develop resistance to the antiproliferative effects of interferon α (IFNα) and interferon β (IFNβ) when they are exposed to the interferons in vitro. This resistance was characterized to be non-genetic and dose-dependent, and does not alter other IFN-induced effects such as antiviral effects and elevation of 2′,5′-oligoadenylate synthetase activity in IFN-treated cells. The study of these IFN-resistant cells has been extended to an in vivo tumor model. Resistance, if it occurred in vivo, did not adversely affect the survival of IFN-treated mice. Further, IFN-treated mice inoculated with B16 cells that were resistant in vitro (B16αres cells) survived significantly longer than IFN-treated mice inoculated with B16 cells that were sensitive in vitro. The IFN-treated B16αres-inoculated mice had a significantly higher cure rate as well. The prolonged survival of the mice bearing B16αres cell tumors did not seem to be caused by the slower growth rate of the B16αres cells, since experiments performed with a tenfold higher B16αres cell inoculum and a tenfold lower B16 cell inoculum did not show any change in the survival pattern. It is clear that in vitro resistant B16αres cells are more sensitive to antitumor effects induced by IFN in vivo than in vitro sensitive B16 cells.

Fleischmann Wr

Papers

The interferons. Mechanisms of action and clinical applications.

Potentiation of lymphocyte natural killing by mixtures of alpha or beta interferon with recombinant gamma interferon.

Orally administered interferons exert their white blood cell suppressive effects via a novel mechanism.

Modulation of peripheral leukocyte counts in mice by oral administration of interferons.

Enhanced in vivo sensitivity to interferon with in vitro resistant B16 tumor cells in mice.