Purpose: We generated a humanized antibody, HuLuc63, which specifically targets CS1 (CCND3 subset 1, CRACC, and SLAMF7), a cell surface glycoprotein not previously associated with multiple myeloma. To explore the therapeutic potential of HuLuc63 in multiple myeloma, we examined in detail the expression profile of CS1, the binding properties of HuLuc63 to normal and malignant cells, and the antimyeloma activity of HuLuc63 in preclinical models. Experimental Design: CS1 was analyzed by gene expression profiling and immunohistochemistry of multiple myeloma samples and numerous normal tissues. HuLuc63-mediated antimyeloma activity was tested in vitro in antibody-dependent cellular cytotoxicity (ADCC) assays and in vivo using the human OPM2 xenograft model in mice.Results: CS1mRNA was expressed in >90% of 532 multiple myeloma cases, regardless of cytogenetic abnormalities. Anti-CS1antibody staining of tissues showed strong staining of myeloma cells in all plasmacytomas and bone marrow biopsies. Flow cytometric analysis of patient samples using HuLuc63 showed specific staining of CD138+ myeloma cells, natural killer (NK), NK-like Tcells, and CD8+ Tcells, with no binding detected on hematopoietic CD34+ stem cells. HuLuc63 exhibited significant in vitro ADCC using primary myeloma cells as targets and both allogeneic and autologous NK cells as effectors. HuLuc63 exerted significant in vivo antitumor activity, which depended on efficient Fc-CD16 interaction as well as the presence of NK cells in the mice. Conclusions: These results suggest that HuLuc63 eliminates myeloma cells, at least in part, via NK-mediated ADCC and shows the therapeutic potential of targeting CS1with HuLuc63 for the treatment of multiple myeloma.

The botulinum neurotoxins (BoNTs) cause the paralytic human disease botulism and are one of the highest-risk threat agents for bioterrorism. To generate a pharmaceutical to prevent or treat botulism, monoclonal antibodies (mAbs) were generated by phage display and evaluated for neutralization of BoNT serotype A (BoNT͞A) in vivo. Although no single mAb significantly neutralized toxin, a combination of three mAbs (oligoclonal Ab) neutralized 450,000 50% lethal doses of BoNT͞A, a potency 90 times greater than human hyperimmune globulin. The potency of oligoclonal Ab was primarily due to a large increase in functional Ab binding affinity. The results indicate that the potency of the polyclonal humoral immune response can be deconvoluted to a few mAbs binding nonoverlapping epitopes, providing a route to drugs for preventing and treating botulism and diseases caused by other pathogens and biologic threat agents. monoclonal antibody ͉ immunotherapy ͉ antibody engineering ͉ vaccine ͉ phage display

Steric and hydrophobic effects on substrate specificity were probed by protein engineering of subtilisin. Subtilisin has broad peptidase specificity and contains a large hydrophobic substrate binding cleft. A conserved glycine (Gly(166)), located at the bottom of the substrate binding left, was replaced by 12 nonionic amino acids by the cassette mutagenesis method. Mutant enzymes showed large changes in specificity toward substrates of increasing size and hydrophobicity. In general, the catalytic efficiency (k(cat)/K(m)) toward small hydrophobic substrates was increased (up to 16 times) by hydrophobic substitutions at position 166 in the binding cleft. Exceeding the optimal binding volume of the cleft ( approximately 160 A(3)), by enlarging either the substrate side chain or the side chain at position 166, evoked precipitous drops in catalytic efficiency (k(cat)/K(m)) (up to 5000 times) as a result of steric hindrance.

Protein engineering of electrostatic interactions between charged substrates and complementary charged amino acids, at two different sites in the substrate binding cleft of the protease subtilisin BPN', increases kct/Km toward complementary charged substrates (up to 1900 times) and decreases kcat/Km toward similarly charged substrates. From kinetic analysis of 16 mutants of subtilisin and the wild type, the average free energies for enzyme-substrate ion-pair interactions at the two different sites are calculated to be -1.8 ± 0.5 and -2.3 + 0.6 kcal/mol (1 cal = 4.18 J) [at 250C in 0.1 M Tris-HCl (pH 8.6)]. The combined electrostatic effects are roughly additive. These studies demonstrate the feasibility for rational design of charged ligand binding sites in proteins by tailoring of electrostatic interactions.Protein engineering by in vitro mutagenesis of cloned genes (1) has been successfully applied to improve the thermal stability (2, 3) and oxidative stability (4-6) of various proteins and to produce enzymes with altered substrate specificities (7)(8)(9)(10) Position 166 mutants were prepared by cassette mutagenesis (23) as described (7). Fragment 3, containing the carboxyl-terminal portion of the subtilisin gene including the desired position 166 codon, was isolated as a 610-base-pair Sac I/BamHI fragment. Fragment 2 was a duplex synthetic DNA cassette that properly restored the coding sequence except at codon 156. The top strand of the cassette was synthesized to contain a glutamine codon, and the complementary bottom strand coded for serine at position 156. Ligation of heterophosphorylated cassettes leads to a large and favorable bias for the phosphorylated over the nonphosphorylated oligonucleotide sequence in the final segregated plasmid product (23). To obtain glutamine-156, the top strand was phosphorylated, annealed to the nonphosphorylated bottom strand (serine-156), and ligated with fragments 1 and 3. Mutant sequences that were isolated after ligation and transformation were confirmed by restriction analysis and DNA sequencing (27). The double 156/166 mutants were prepared by ligation of the 4.6-kilobase Sac I to BamHI fragment from the relevant position 156 mutant plasmid, and tTo whom reprint requests should be addressed. 1219The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.