ABSTRACT
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.
Subject(s)
Aminobenzoates/pharmacokinetics , Drug Carriers/pharmacokinetics , Oligopeptides/pharmacokinetics , Aminobenzoates/administration & dosage , Aminobenzoates/chemistry , Animals , Antibodies/administration & dosage , Drug Carriers/administration & dosage , Drug Carriers/chemistry , Drug Stability , Female , HEK293 Cells , Humans , Macaca fascicularis , Mice, SCID , Oligopeptides/administration & dosage , Oligopeptides/chemistry , RatsABSTRACT
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.
Subject(s)
Aminobenzoates/chemistry , Antibodies, Monoclonal/chemistry , Antineoplastic Agents/chemistry , Immunoconjugates/chemistry , Oligopeptides/chemistry , Pancreatic Neoplasms/drug therapy , Aminobenzoates/blood , Aminobenzoates/pharmacokinetics , Aminobenzoates/pharmacology , Animals , Antineoplastic Agents/blood , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Carbamates/chemistry , Cathepsin B/chemistry , Cathepsin B/metabolism , Cell Line, Tumor , Dipeptides/chemistry , Drug Delivery Systems/methods , Drug Stability , Female , Humans , Immunoconjugates/blood , Immunoconjugates/pharmacokinetics , Immunoconjugates/pharmacology , Mice , Mice, Nude , Models, Molecular , Oligopeptides/blood , Oligopeptides/pharmacokinetics , Oligopeptides/pharmacology , Pancreatic Neoplasms/blood , Pancreatic Neoplasms/pathology , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
Listeriolysin O (LLO) is a pore-forming toxin of the cholesterol-dependent cytolysin (CDC) family and a primary virulence factor of the intracellular pathogen Listeria 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 of L. monocytogenes, we developed a screen that combined saturation mutagenesis and signature tags, termed in vivo analysis by saturation mutagenesis and signature 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 analyzed in vivo each 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 common in vitro techniques suggested that the functional contributions of some residues could only have been revealed through in vivo analysis.
Subject(s)
Bacterial Toxins/metabolism , Heat-Shock Proteins/metabolism , Hemolysin Proteins/metabolism , Listeria monocytogenes/pathogenicity , Alanine , Amino Acid Sequence , Animals , Bacterial Toxins/genetics , DNA, Bacterial , Female , Gene Expression Regulation, Bacterial/physiology , Heat-Shock Proteins/genetics , Hemolysin Proteins/genetics , Listeria monocytogenes/genetics , Mice , Models, Molecular , Mutagenesis, Site-Directed , Mutation , Phenotype , Polymerase Chain Reaction , Protein Conformation , Protein Structure, TertiaryABSTRACT
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.
Subject(s)
Intestines/microbiology , Listeria monocytogenes , Listeriosis/microbiology , Liver/microbiology , Lymph Nodes/microbiology , Animals , Bacterial Load , Female , Guinea Pigs , Listeriosis/pathology , Spleen/microbiology , Time FactorsABSTRACT
While recombinant Listeria monocytogenes strains can be explored as vaccine candidates, it is important to develop attenuated but highly immunogenic L. monocytogenes vaccine vectors. Here, prfA* mutations selected on the basis of upregulated expression of L. monocytogenes PrfA-dependent genes and proteins were assessed to determine their abilities to augment expression of foreign immunogens in recombinant L. monocytogenes vectors and therefore enhance vaccine-elicited immune responses (a prfA* mutation is a mutation that results in constitutive overexpression of PrfA and PrfA-dependent virulence genes; the asterisk distinguishes the mutation from inactivation or stop mutations). A total of 63 recombinant L. monocytogenes vaccine vectors expressing seven individual viral or bacterial immunogens each in nine different L. monocytogenes strains carrying wild-type prfA or having prfA* mutations were constructed and investigated. Mutations selected on the basis of increased PrfA activation in recombinant L. monocytogenes prfA* vaccine vectors augmented expression of seven individual protein immunogens remarkably. Consistently, prime and boost vaccination studies with mice indicated that the prfA(G155S) mutation in recombinant L. monocytogenes DeltaactA prfA* strains enhanced vaccine-elicited cellular immune responses. Surprisingly, the prfA(G155S) mutation was found to enhance vaccine-elicited humoral immune responses as well. The highly immunogenic recombinant L. monocytogenes DeltaactA prfA* vaccine strains were as attenuated as the recombinant parent L. monocytogenes DeltaactA vaccine vector. Thus, recombinant attenuated L. monocytogenes DeltaactA prfA* vaccine vectors potentially are better antimicrobial and anticancer vaccines.
Subject(s)
Bacterial Vaccines/immunology , Listeria monocytogenes/genetics , Mutation , Peptide Termination Factors/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Animals , Antibodies, Bacterial/blood , Antibodies, Viral/blood , Bacterial Vaccines/genetics , Enzyme-Linked Immunosorbent Assay , Female , Immunization, Secondary , Interferon-gamma/biosynthesis , Listeria monocytogenes/immunology , Mice , Mice, Inbred BALB C , T-Lymphocytes/immunology , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunologyABSTRACT
Alpha toxin (AT) is the major virulence factor of Clostridium septicum that is a proteolytically activated pore-forming toxin that belongs to the aerolysin-like family of toxins. AT is predicted to be a three-domain molecule on the basis of its functional and sequence similarity with aerolysin, for which the crystal structure has been determined. In this study, we have substituted the entire primary structure of AT with alanine or cysteine 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 beta-hairpin of the prepore oligomer.