Date of Award

12-2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Microbiology

Advisor

Chen, Wen Y

Committee Member

Wagner , Thomas E

Committee Member

Ellis , Steven E

Committee Member

Larcom , Lyndon L

Abstract

Potential indications for human prolactin (hPRL) and human growth hormone (hGH) are the support and sustainment of lactation and the enhancement or reconstitution of the immune system when under dysregulation. A prolactin receptor (PRLR) antagonist, hPRL-G129R (G129R), and a PRLR and growth hormone receptor (GHR) antagonist, hGH-G120R (G120R), have been developed which may be useful for the treatment of breast cancer since PRLR is elevated in a majority of human breast tumors and makes cancer cells highly sensitive to the mitogenic and anti-apoptotic activity of hPRL and hGH.

As a single agent, G129R is more cytostatic than cytotoxic to human breast cancer cells xenografted into nude mice; when combined with common chemotherapies (e.g. Tamoxifen or Herceptin) G129R has additive effects. In Chapter 2, we propose to enhance the cytotoxicity of G129R by fusing it to a toxin that potently inhibits protein synthesis. We demonstrate that the fusion protein specifically targets the PRLR and drastically enhances the cytotoxicity of G129R in vitro and that it can be administered safely to mice in vivo.

The low molecular weight of hPRL and G129R make it difficult to maintain therapeutic levels in the plasma. To increase the plasma half-life we conjugated hPRL and G129R with hydroxyethyl starch (HES); unfortunately, the ligands lost their intrinsic activities. In Chapter 3, we propose to increase the plasma half-life of hPRL, hGH, and their respective antagonists by fusing them to a small serum albumin binding peptide. We demonstrate that the serum albumin binding peptide extends the plasma half-life of the ligands and enhances the bioactivity of hPRL and hGH in vivo.

In Chapter 4, we propose to increase the plasma half-life and make hPRL and hGH more amendable to chemical modifications such as HESylation by fusing two molecules of hPRL and hGH in tandem (PRL-PRL and GH-GH). We demonstrate that their intrinsic activity is unaffected and that the plasma half-life of PRL-PRL is extended. We also fused two molecules of G129R or G120R in tandem (G129R-G129R and G120R-G120R) and unexpectedly ended up with agonists. This indicates that the PRLR is flexible and that the receptor binding sites on PRL-PRL and GH-GH can potentially interact with receptors in three different ways, reducing the probability that chemical modifications will affect their crucial receptor binding sites.

hGH can mediate its effects via a PRLR homodimer or a GHR homodimer and there is no reason to believe it might not also mediate its effects via a PRLR/GHR heterodimer. Unfortunately, there is no easy way to discern which receptor hGH exploits. In Chapter 5, we extend the findings of Chapter 4 (i.e. that the fusion of two antagonists together results in an agonist) and propose to make two unique ligands that are only capable of binding to a PRLR/GHR heterodimer. We confirm that PRLR/GHR heterodimers either pre-exist or can be induced, something which has previously only been shown to occur in rudiments. Also we made a GHR agonist, characterized it, and confirmed that it is a pure GHR agonist. This molecule can be used in place of hGH if one only wants to observe somatogenic effects in human cell lines.


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