The HtrA serine protease has been shown to be essential for bacterial virulence and for survival after exposure to many types of environmental and cellular stresses. A Listeria monocytogenes 10403S htrA mutant was found to be sensitive to oxidative and puromycin-induced stress at high temperatures, showed a reduced ability to form biofilms, and was attenuated for virulence in mice.

No abstract

Anti-CD30 diabodies were engineered with two cysteine mutations for site-specific drug conjugation in each chain of these homodimeric antibody fragments. Diabodies were conjugated with f4 equivalents of the anti-tubulin drugs, monomethyl auristatin E or F, via a protease-cleavable dipeptide linker, to create the conjugates, diabody-vcE4 and diabody-vcF4, respectively. Diabody conjugation had only minor (<3-fold) effects on antigen binding. DiabodyvcF4 was potently cytotoxic against the antigen-positive cell lines, Karpas-299 (34 pmol/L IC 50 ) and L540cy (22 pmol/L IC 50 ), and was 8-and 21-fold more active than diabody-vcE4 against these cell lines, respectively. Clearance of diabody-vcF4 (99-134 mL/d/kg) was 5-fold slower than for the nonconjugated diabody in naive severe combined immunodeficient mice. Diabody-vcF4 had potent and dose-dependent antitumor activity against established Karpas-299 xenografts and gave durable complete responses at well-tolerated doses. Biodistribution experiments with diabody-[ 3 H]-vcF4 (0.72-7.2 mg/kg) in tumorbearing mice showed a dose-dependent increase in total auristatin accumulation in tumors (V520 nmol/L) and decrease in relative auristatin accumulation (V8.1 %ID/g), with peak localization at 4 to 24 h after dosing. Diabody-vcF4 had f4-fold lower cytotoxic activity than the corresponding IgG1-vcF4 conjugate in vitro. A similar potency difference was observed in vivo despite 25-to 34-fold faster clearance of diabody-vcF4 than IgG1-vcF4.

B-cell maturation antigen (BCMA) is expressed on normal and malignant plasma cells and represents a potential target for therapeutic intervention. BCMA binds to two ligands that promote tumor cell survival, a proliferation inducing ligand (APRIL) and B-cell activating factor. To selectively target BCMA for plasma cell malignancies, we developed antibodies with ligand blocking activity that could promote cytotoxicity of multiple myeloma (MM) cell lines as naked antibodies or as antibody-drug conjugates. We show that SG1, an inhibitory BCMA antibody, blocks APRIL -dependent activation of nuclear factor-KB in a dose-dependent manner in vitro. Cytotoxicity of SG1 was assessed as a naked antibody after chimerization with and without Fc mutations that enhance Fc;RIIIA binding. The Fc mutations increased the antibody-dependent cellmediated cytotoxicity potency of BCMA antibodies against MM lines by f100-fold with a z2-fold increase in maximal lysis. As an alternative therapeutic strategy, anti-BCMA antibodies were endowed with direct cytotoxic activity by conjugation to the cytotoxic drug, monomethyl auristatin F. The most potent BCMA antibody-drug conjugate displayed IC 50 values of V130 pmol/L for three different MM lines. Hence, BCMA antibodies show cytotoxic activity both as naked IgG and as drug conjugates and warrant further evaluation as therapeutic candidates for plasma cell malignancies.

An anti-CD70 antibody conjugated to monomethylauristatin F (MMAF) via a valine-citrulline dipeptide containing linker has been shown previously to have potent antitumor activity in renal cell cancer xenograft studies. Here, we generated a panel of humanized anti-CD70 antibody IgG variants and conjugated them to MMAF to study the effect of isotype (IgG1, IgG2, and IgG4) and Fc; receptor binding on antibody-drug conjugate properties. All IgG variants bound CD70 + 786-O cells with an apparent affinity of f1 nmol/L, and drug conjugation did not impair antigen binding. The parent anti-CD70 IgG1 bound to human Fc;RI and Fc;RIIIA V158 and mouse Fc;RIV and this binding was not impaired by drug conjugation. In contrast, binding to these Fc; receptors was greatly reduced or abolished in the variant, IgG1v1, containing the previously described mutations, E233P:L234V:L235A. All conjugates had potent cytotoxic activity against six different antigen-positive cancer cell lines in vitro with IC 50 values of 30 to 540 pmol/L. The IgGv1 conjugate with MMAF displayed improved antitumor activity compared with other conjugates in 786-O and UMRC3 models of renal cell cancer and in the DBTRG05-MG glioblastoma model. All conjugates were tolerated to z40 mg/kg in mice. Thus, the IgG1v1 MMAF conjugate has an increased therapeutic index compared with the parent IgG1 conjugate. The improved antitumor activity of the IgG1v1 auristatin conjugates may relate to increased exposure as suggested by pharmacokinetic analysis. The strategy used here for enhancing the therapeutic index of antibody-drug conjugates is independent of the antigen-binding variable domains and potentially applicable to other antibodies.