By David N. Leff

Editor's note: Science Scan is a roundup of recently published biotechnology-relevant research.

Chalk up two new tumor-strangling entries in the ongoing anti-angiogenesis sweepstakes. A West Coast-East Coast collaboration between human genome librarians and molecular biologists has come up with a pair of challengers to the present front-running blood-vessel ablator, endostatin.

An article in the current, weekly Journal of Biological Chemistry, dated Aug. 13, 1999, bears the title, "METH-1, a human homolog of ADAMTS, and METH-2 are members of a new family of proteins with angio-inhibitory activity." It is co-authored by researchers at the University of California at Los Angeles (UCLA) and at Human Genome Sciences Inc. (HGS) in Rockville, Md.

Their paper defines "METH" as an acronym "due to the novel combination of metalloprotease and thrombospondin domains." Matrix metalloprotease is an angiogenesis promoter. The thrombospondin protein is an early and potent angiogenesis-inhibiting contender. It blocks proliferation of endothelial cells, which form the lining of blood vessels. This molecule owes its anti-angiogenic activity to a stretch of 137 amino acids within its 385 to 522 amino-acid region. The co-authors hypothesized that repeats within this sequence "might be present in other yet unidentified proteins, in a context that enables activity as inhibitors of capillary growth." They therefore initiated a search of HGS databases for novel complementary DNAs, using the anti-angiogenic region of thrombospondin as bait."

That fishing expedition of some 900,000 expressed sequence tags (ESTs) landed several novel cDNAS. Two of these sequences, METH-1 and METH-2, the co-authors reported, "isolated from human heart and lung libraries, were strikingly similar to each other." METH-1 coded for proteins of 950 amino acids; METH-2, 890."

To get a functional antibody handle on these METH molecules, they injected recombinant constructs into the muscles of five guinea pigs and three rabbits. Only two of the guinea pigs generated significant titers of anti-METH-1 antisera. Both of these antibodies recognized recombinant METH-1 protein.

Then, after in vitro testing, the UCLA team put those METH molecules through their putative tumor-starving paces in two in vivo anti-angiogenesis experiments - egg and corneal. One of the body's few tissues that need no blood supply is the smooth white cornea of the eye. In the corneas of anesthetized mice the UCLA team opened a slit or pocket, into which they implanted a pellet containing recombinant METH -1 and METH-2, together with basic fibroblast growth factor (which stimulates blood vessel spread), along with the anti-angiogenesis hopefuls, thrombostatin and endostatin.

In fertilized chicken eggs, angiogenic response involved measuring the number of blood vessels that proliferate on the mid-term chick embryo's chorioallantoic membrane (CAM) - the translucent pellicle just beneath the eggshell's broad end - in the presence of vascular endothelial growth factor (VEGF). To this angiogenesis stimulator, the co-authors added their two METH inhibitors and those same anti-neovascularization contenders - endostatin and thrombostatin. By 24 hours later, VEGF had spread a fine network of blood vessels across the membrane, but METH-1 scotched that proliferation by 65 percent - far more potent than endostatin.

"In measuring the results of this CAM assay," explained cell molecular biologist Luisa Iruela-Arispe of UCLA, the journal paper's co-senior author, "we used 50 nanomolar of protein, normalized for the number of molecules. It's a percent of what the growth factor alone stimulates," she told BioWorld Today. "We have to challenge the blood vessels under the eggshell to grow. When we quantify that growth, it's 100 percent. So the results indicate what percentages of that angiogenesis the inhibitors inhibit. We found in the corneal assay that METH-1 inhibited 65 percent - 100 minus 35 - METH-2, 58 percent, and endostatin 32 percent.

In the mouse corneal assay, Iruela-Arispe continued, the results were even more striking: "In a side-by-side comparison, it took 10 times more molecules of endostatin than of METH-1 or METH-2 to elicit a response. Specifically, it took only 50 nanomolars of METH 1 to completely inhibit blood vessel proliferation, but 500 nanomolars of endostatin. At 100 nanomolars, we saw complete inhibition with thrombospondin, too, and partial inhibition with endostatin."

A Different Horse In Anti-Angiogenic Race Cites 97 Percent Tumor Melt-Down In Mice

Another anti-angiogenesis game in town is Troponin I, which last spring, in the Proceedings of the National Academy of Sciences, dated Mar. 16, 1999, showed high-potency preclinical in vivo results with the Troponin protein. It is synthesized by cartilage - another bloodless tissue. (See BioWorld Today, March 18, 1999, p. 1.)

Now, Boston Life Sciences Inc., of Boston, which has the Troponin I protein under development, announced in an Aug. 5 press release that "it has identified a purified fragment or analogue of Troponin I that is approximately 10 times more potent and soluble than its parent protein. . . [The analogue] was shown to achieve 97 percent metastatic tumor reduction in mice injected with a human sarcoma (a particularly aggressive type of cancer)."