The systemic stability of the antibody−drug linker is crucial for delivery of an intact antibody−drug conjugate (ADC) to target-expressing tumors. Linkers stable in circulation but readily processed in the target cell are necessary for both safety and potency of the delivered conjugate. Here, we report a range of stabilities for an auristatin-based payload site-specifically attached through a cleavable valine-citrulline-p-aminobenzylcarbamate (VC-PABC) linker across various sites on an antibody. We demonstrate that the conjugation site plays an important role in determining VC-PABC linker stability in mouse plasma, and that the stability of the linker positively correlates with ADC cytotoxic potency both in vitro and in vivo. Furthermore, we show that the VC-PABC cleavage in mouse plasma is not mediated by Cathepsin B, the protease thought to be primarily responsible for linker processing in the lysosomal degradation pathway. Although the VC-PABC cleavage is not detected in primate plasma in vitro, linker stabilization in the mouse is an essential prerequisite for designing successful efficacy and safety studies in rodents during preclinical stages of ADC programs. The divergence of linker metabolism in mouse plasma and its intracellular cleavage offers an opportunity for linker optimization in the circulation without compromising its efficient payload release in the target cell.

Listeria monocytogenes causes a serious food-borne disease due to its ability to spread from the intestine to other organs, a process that is poorly understood. In this study we used 20 signature-tagged wild-type clones of L. monocytogenes in guinea pigs in combination with extensive quantitative data analysis to gain insight into extraintestinal dissemination. We show that L. monocytogenes colonized the liver in all asymptomatic animals. Spread to the liver occurred as early as 4 h after ingestion via a direct pathway from the intestine to the liver. This direct pathway contributed significantly to the bacterial load in the liver and was followed by a second wave of dissemination via the mesenteric lymph nodes (indirect pathway). Furthermore, bacteria were eliminated in the liver, whereas small intestinal villi provided a niche for bacterial replication, indicating organ-specific differences in net bacterial growth. Bacteria were shed back from intestinal villi into the small intestinal lumen and reinfected the Peyer's patches. Together, these results support a novel dissemination model where L. monocytogenes replicates in intestinal villi, is shed into the lumen, and reinfects intestinal immune cells that traffic to liver and mesenteric lymph nodes, a process that occurs even during asymptomatic colonization.

The efficacy of an antibody-drug conjugate (ADC) is dependent on the properties of its linker-payload which must remain stable while in systemic circulation but undergo efficient processing upon internalization into target cells. Here, we examine the stability of a non-cleavable Amino-PEG6-based linker bearing the monomethyl auristatin D (MMAD) payload site-specifically conjugated at multiple positions on an antibody. Enzymatic conjugation with transglutaminase allows us to create a stable amide linkage that remains intact across all tested conjugation sites on the antibody, and provides us with an opportunity to examine the stability of the auristatin payload itself. We report a position-dependent degradation of the C terminus of MMAD in rodent plasma that has a detrimental effect on its potency. The MMAD cleavage can be eliminated by either modifying the C terminus of the toxin, or by selection of conjugation site. Both approaches result in improved stability and potency in vitro and in vivo. Furthermore, we show that the MMAD metabolism in mouse plasma is likely mediated by a serine-based hydrolase, appears much less pronounced in rat, and was not detected in cynomolgus monkey or human plasma. Clarifying these species differences and controlling toxin degradation to optimize ADC stability in rodents is essential to make the best ADC selection from preclinical models. The data presented here demonstrate that site selection and toxin susceptibility to mouse plasma degradation are important considerations in the design of non-cleavable ADCs, and further highlight the benefits of site-specific conjugation methods.

Alpha toxin (AT) is the major virulence factor of Clostridium septicum that is a proteolytically activated pore-forming toxin belonging to the aerolysin-like family of toxins. AT is predicted to be a three-domain molecule based on functional and sequence similarity with aerolysin, for which the crystal structure has been solved. In the present study we have substituted the entire primary structure of AT with alanine or cysteine in order to identify those amino acids that comprise functional domains involved in receptor binding, oligomerization and pore formation. These studies revealed that receptor binding is restricted to domain 1 of the AT structure, whereas domains 1 and 3 are involved in oligomerization. These studies also revealed the presence of a putative functional region of AT proximal to the receptor-binding domain, but distal from the pore-forming domain that is proposed to regulate the insertion of the transmembrane β-hairpin of the prepore oligomer.Clostridium septicum is a major cause of non-traumatic gas gangrene in humans, a fulminant form of myonecrosis that can progress to a fatal infection in less than 24 hours with reported mortality rates ranging from 67-100% (1-4). Of its many virulence factors, alpha toxin (AT) is the only lethal factor secreted by this organism (5) and it has recently been shown to be absolutely required for virulence of C. septicum (6). AT is classified as a pore-forming toxin and is a member of the aerolysin-like family of pore-forming toxins. AT and aerolysin share a great deal of structural and sequence similarity (72%) (5), which has allowed for the development of a molecular model of AT using the previously solved crystal structure of aerolysin ( Fig. 1) (7,8). Aerolysin is a two lobed protein in which the small lobe is comprised of domain 1 (D1) and the large lobe is comprised of domains 2-4 (D2-D4, Fig. 1). The primary structure of AT exhibits similarity with the large lobe of aerolysin but it lacks the small lobe structure of about 83 amino acids. Both toxins appear to follow a similar ordered path to form pores in cell membranes (5). Following secretion, they bind to receptors on the cell membrane where they are cleaved into their active form by cell surface proteases, usually furin. Once activated, the toxins oligomerize on the cell surface into a prepore complex followed by insertion of a transmembrane β-barrel into the membrane (8).

Recombinant Listeria monocytogenes is a promising intracellular live vaccine vector for delivering immunogens and for inducing vaccine-elicited immune responses and immunity to infections or tumors. Compared to other bacterial vectors, such as Mycobacterium bovis BCG and Salmonella, recombinant L. monocytogenes appears to elicit CD8 ϩ T-cell responses more readily. The gram-positive facultative intracellular organism L. monocytogenes can invade a wide range of host cells, including phagocytes and nonphagocytic cells, and can escape from the phagolysosomes to the cytoplasm by means of the pore-forming cholesterol-dependent cytolysin listeriolysin O (LLO). The ability of L. monocytogenes to escape from the endosome to the cytosol after entry into host cells allows vaccine antigens to enter both major histocompatibility complex class I and II pathways of antigen processing and presentation and therefore to elicit both CD4 and CD8 T-cell responses (13,20,39,56). In addition, both wild-type and attenuated recombinant L. monocytogenes strains appear to be gastrointestinal tract-tropic and oropharyngeal mucosa-tropic, enabling mucosa-targeted delivery of vaccine immunogens. Finally, underlying immunity to the L. monocytogenes vector itself does not prevent priming or boosting of immune responses to foreign immunogens (6,52,53).Recent progress in bacterial genetics has facilitated the development of genetically manipulated L. monocytogenes strains as attenuated vaccine vectors (2,8,10,16,34,41,56,57). Some of these strains have been shown to induce protective immune responses to infections and cancers (7,34,38,40,57), whereas others are currently being tested in clinical trials (2). In particular, deletion of the actA gene in L. monocytogenes results in remarkable attenuation since the actin-assembling protein encoded by actA is necessary for L. monocytogenes to utilize actin-based motility for cell-to-cell spread or transmission (14,19,26,55). In fact, an attenuated L. monocytogenes ⌬actA vaccine strain has been shown to exhibit diminished pathogenicity and toxicity in vivo but to maintain immune potency (8). The current efforts to develop recombinant L. monocytogenes vaccines appear to focus on at least three aspects: (i) increasing the success rate for making recombinant L. monocytogenes constructs and augmenting expression of various foreign immunogens in attenuated L. monocytogenes strains; (ii) enhancing the ability of L. monocytogenes vaccine vectors to elicit immunogen-specific cellular immune responses; and (iii) exploiting the ability of L. monocytogenes vaccine vectors to elicit humoral immune responses.Molecular approaches targeting increased activity of L. monocytogenes transcriptional regulatory factors may make it * Corresponding author. Mailing address: MC790, 909 S. Wolcott Ave., Chicago, IL 60612.

Clostridium septicum alpha-toxin is a ␤-barrel pore-forming cytolysin that is functionally similar to aerolysin. Residues important in receptor binding, oligomerization, and pore formation have been identified; however, little is known about the activity of the toxin in an infection, although it is essential for disease. We have now shown that deletion of a small portion of the transmembrane domain, so that the toxin is no longer able to form pores, completely abrogates its ability to contribute to disease, as does replacement of the sole cysteine residue with leucine. However, although previous biochemical and cytotoxicity assays clearly indicated that mutations in residues important in oligomerization, binding, and prepore conversion greatly reduced activity or rendered the toxin inactive, once the mutated toxins were overexpressed by the natural host in the context of an infection it was found they were able to cause disease in a mouse model of myonecrosis. These results highlight the importance of testing the activity of virulence determinants in the normal host background and in an infectious disease context and provide unequivocal evidence that it is the ability of alpha-toxin to form a pore that confers its toxicity in vivo.

Applications of microbial transglutaminase (mTGase) produced from Streptomyces mobarensis (S. mobarensis) were recently extended from food to pharmaceutical industry. To use mTGase for clinical applications, like generation of site specific antibody drug conjugates, it would be beneficial to manufacture mTGase in Escherichia coli (E. coli). To date, attempts to express recombinant soluble and active S. mobarensis mTGase have been largely unsuccessful. mTGase from S. mobarensis is naturally expressed as proenzyme and stepwise proteolytically processed into its active mature form outside of the bacterial cell. The pro-domain is essential for correct folding of mTGase as well as for inhibiting activity of mTGase inside the cell. Here, we report a genetically modified mTGase that has full activity and can be expressed at high yields in the cytoplasm of E. coli. To achieve this we performed an alanine-scan of the mTGase pro-domain and identified mutants that maintain its chaperone function but destabilize the cleaved pro-domain/ mTGase interaction in a temperature dependent fashion. This allows proper folding of mTGase and keeps the enzyme inactive during expression at 208C, but results in full activity when shifted to 378C due to loosen domain interactions. The insertion of the 3C protease cleavage site together with pro-domain alanine mutants Tyr14, Ile24, or Asn25 facilitate high yields (30-75 mg/L), and produced an enzyme with activity identical to wild type mTGase from S. mobarensis. Site-specific antibody drug conjugates made with the E .coli produced mTGase demonstrated identical potency in an in vitro cell assay to those made with mTGase from S. mobarensis.

ABSTRACTListeriolysin O (LLO) is a pore-forming toxin of the cholesterol-dependent cytolysin (CDC) family and a primary virulence factor of the intracellular pathogenListeria monocytogenes. LLO mediates rupture of phagosomal membranes, thereby releasing bacteria into the growth-permissive host cell cytosol. Several unique features of LLO allow its activity to be precisely regulated in order to facilitate phagosomal escape, intracellular growth, and cell-to-cell spread. To improve our understanding of the multifaceted contribution of LLO to the pathogenesis ofL. monocytogenes, we developed a screen that combined saturation mutagenesis and signature tags, termedinvivoanalysis bysaturation mutagenesis andsignature tags (IVASS). We generated a library of LLO mutant strains, each harboring a single amino acid substitution and a signature tag, by using the previously described pPL2 integration vector. The signature tags acted as molecular barcodes, enabling high-throughput, parallel analysis of 40 mutants in a single animal and identification of attenuated mutants by negative selection. Using the IVASS technique we were able to screen over 90% of the 505 amino acids present in LLO and identified 60 attenuated mutants. Of these, 39 LLO residues were previously uncharacterized and potentially revealed novel functions of the toxin during infection. The mutants that were subsequently analyzedin vivoeach conferred a 2- to 4-orders of magnitude loss in virulence compared to wild type, thereby validating the screening methods. Phenotypic analysis of the LLO mutant library using commonin vitrotechniques suggested that the functional contributions of some residues could only have been revealed throughin vivoanalysis.