Symbiosis between cyanobacteria and plants: from molecular studies to agronomic applications.

Nitrogen-fixing cyanobacteria from the order Nostocales are able to establish symbiotic relationships with diverse plant species. They are promiscuous symbionts, as the same strain of cyanobacterium is able to form symbiotic biological nitrogen-fixing relationships with different plants species. This review will focus on the different types of cyanobacterial-plant associations, both endophytic and epiphytic, and provide insights from a structural viewpoint, as well as our current understanding of the mechanisms involved in the symbiotic crosstalk. In all these symbioses, the benefit for the plant is clear; it obtains from the cyanobacterium fixed nitrogen and other bioactive compounds, such as phytohormones, polysaccharides, siderophores, or vitamins, leading to enhanced plant growth and productivity. Additionally, there is increasing use of different cyanobacterial species as bio-inoculants for biological nitrogen fixation to improve soil fertility and crop production, thus providing an eco-friendly, alternative, and sustainable approach to reduce the over-reliance on synthetic chemical fertilizers.

Assessment of KRAS G12C Target Engagement by a Covalent Inhibitor in Tumor Biopsies Using an Ultra-Sensitive Immunoaffinity 2D-LC-MS/MS Approach.

KRAS is one of the most frequently mutated oncogenes, with KRAS G12C recently becoming an actionable target for small molecule intervention. GDC-6036 is an investigational KRAS G12C inhibitor that acts by irreversibly binding to the switch II pocket of KRAS G12C when in the inactive GDP-bound state, thereby blocking GTP binding and activation. Assessing target engagement is an essential component of clinical drug development, helping to demonstrate mechanistic activity, guide dose selection, understand pharmacodynamics as it relates to clinical response, and explore resistance. Here, we report the development of an ultra-sensitive approach for assessing KRAS G12C engagement. Immunoaffinity enrichment with a commercially available anti-RAS antibody was combined with a targeted 2D-LC-MS/MS technique to quantify both free and GDC-6036-bound KRAS G12C proteins. A KRAS G12C-positive non-small cell lung cancer xenograft model was dosed with GDC-6036 to assess the feasibility of this assay for analyzing small core needle biopsies. As predicted, dose-dependent KRAS G12C engagement was observed. To date, a sensitivity of 0.08 fmol/µg of total protein has been achieved for both free and GDC-6036-bound KRAS G12C with as little as 4 µg of total protein extracted from human tumor samples. This sub-fmol/µg level of sensitivity provides a powerful potential approach to assess covalent inhibitor target engagement at the site of action using core needle tumor biopsies from clinical studies.

A homogeneous high-DAR antibody-drug conjugate platform combining THIOMAB antibodies and XTEN polypeptides.

The antibody-drug conjugate (ADC) is a well-validated modality for the cell-specific delivery of small molecules with impact expanding rapidly beyond their originally-intended purpose of treating cancer. However, antibody-mediated delivery (AMD) remains inefficient, limiting its applicability to targeting highly potent payloads to cells with high antigen expression. Maximizing the number of payloads delivered per antibody is one key way in which delivery efficiency can be improved, although this has been challenging to carry out; with few exceptions, increasing the drug-to-antibody ratio (DAR) above ∼4 typically destroys the biophysical properties and in vivo efficacy for ADCs. Herein, we describe the development of a novel bioconjugation platform combining cysteine-engineered (THIOMAB) antibodies and recombinant XTEN polypeptides for the unprecedented generation of homogeneous, stable "TXCs" with DAR of up to 18. Across three different bioactive payloads, we demonstrated improved AMD to tumors and Staphylococcus aureus bacteria for high-DAR TXCs relative to conventional low-DAR ADCs.

Genetic and lipidomic analyses suggest that Nostoc punctiforme, a plant-symbiotic cyanobacterium, does not produce sphingolipids.

Sphingolipids, a class of amino-alcohol-based lipids, are well characterized in eukaryotes and in some anaerobic bacteria. However, the only sphingolipids so far identified in cyanobacteria are two ceramides (i.e., an acetylsphingomyelin and a cerebroside), both based on unbranched, long-chain base (LCB) sphingolipids in Scytonema julianum and Moorea producens , respectively. The first step in de novo sphingolipid biosynthesis is the condensation of l-serine with palmitoyl-CoA to produce 3-keto-diyhydrosphingosine (KDS). This reaction is catalyzed by serine palmitoyltransferase (SPT), which belongs to a small family of pyridoxal phosphate-dependent α-oxoamine synthase (AOS) enzymes. Based on sequence similarity to molecularly characterized bacterial SPT peptides, we identified a putative SPT (Npun_R3567) from the model nitrogen-fixing, plant-symbiotic cyanobacterium, Nostoc punctiforme strain PCC 73102 (ATCC 29133). Gene expression analysis revealed that Npun_R3567 is induced during late-stage diazotrophic growth in N. punctiforme . However, Npun_R3567 could not produce the SPT reaction product, 3-keto-diyhydrosphingosine (KDS), when heterologously expressed in Escherichia coli . This agreed with a sphingolipidomic analysis of N. punctiforme cells, which revealed that no LCBs or ceramides were present. To gain a better understanding of Npun_R3567, we inferred the phylogenetic position of Npun_R3567 relative to other bacterial AOS peptides. Rather than clustering with other bacterial SPTs, Npun_R3567 and the other cyanobacterial BioF homologues formed a separate, monophyletic group. Given that N. punctiforme does not appear to possess any other gene encoding an AOS enzyme, it is altogether unlikely that N. punctiforme is capable of synthesizing sphingolipids. In the context of cross-kingdom symbiosis signalling in which sphingolipids are emerging as important regulators, it appears unlikely that sphingolipids from N. punctiforme play a regulatory role during its symbiotic association with plants.

The impact of chitosan on the early metabolomic response of wheat to infection by Fusarium graminearum.

BACKGROUND: Chitosan has shown potential for the control of Fusarium head blight (FHB) disease caused by Fusarium graminearum. The objective of this study was to compare the effect of chitosan hydrochloride applied pre- or post-fungal inoculation on FHB and to better understand its' mode of action via an untargeted metabolomics study. RESULTS: Chitosan inhibited fungal growth in vitro and, when sprayed on the susceptible wheat cultivar Remus 24 hours pre-inoculation with F. graminearum, it significantly reduced the number of infected spikelets at 7, 14 and 21 days post-inoculation. Chitosan pre-treatment also increased the average grain weight per head, the number of grains per head and the 1000-grain weight compared to the controls sprayed with water. No significant impact of chitosan on grain yield was observed when the plants were sprayed 24 hours post-inoculation with F. graminearum, even if it did result in a reduced number of infected spikelets at every time point. An untargeted metabolomic study using UHPLC-QTOF-MS on wheat spikes revealed that spraying the spikes with both chitosan and F. graminearum activated known FHB resistance pathways (e.g. jasmonic acid). Additionally, more metabolites were up- or down-regulated when both chitosan and F. graminearum spores were sprayed on the spikes (117), as compared with chitosan (51) or F. graminearum on their own (32). This included a terpene, a terpenoid and a liminoid previously associated with FHB resistance. CONCLUSIONS: In this study we showed that chitosan hydrochloride inhibited the spore germination and hyphal development of F. graminearum in vitro, triggered wheat resistance against infection by F. graminearum when used as a pre-inoculant, and highlighted metabolites and pathways commonly and differentially affected by chitosan, the pathogen and both agents. This study provides insights into how chitosan might provide protection or stimulate wheat resistance to infection by F. graminearum. It also unveiled new putatively identified metabolites that had not been listed in previous FHB or chitosan-related metabolomic studies.

Experimental comparison of two methods to study barley responses to partial submergence.

BACKGROUND: Crop yield is dependent on climate conditions, which are becoming both more variable and extreme in some areas of the world as a consequence of global climate change. Increased precipitation and flooding events are the cause of important yield losses due to waterlogging or (partial) submergence of crops in the field. Our ability to screen efficiently and quickly for varieties that have increased tolerance to waterlogging or (partial) submergence is important. Barley, a staple crop worldwide, is particularly sensitive to waterlogging. Screening for waterlogging tolerant barley varieties has been ongoing for many years, but methods used to screen vary greatly, from the type of soil used to the time at which the treatment is applied. This variation makes it difficult to cross-compare results. RESULTS: Here, we have devised a scoring system to assess barley tolerance to waterlogging and compare two different methods when partial submergence is applied with either water or a starch solution at an early developmental stage, which is particularly sensitive to waterlogging or partial submergence. The use of a starch solution has been previously shown to result in more reducing soil conditions and has been used to screen for waterlogging tolerance. CONCLUSIONS: Our results show that the two methods provide similar results to qualitatively rank varieties as tolerant or sensitive, while also affecting plants differently, in that application of a starch solution results in stronger and earlier symptoms than applying partial submergence with water.

Calicheamicin Antibody-Drug Conjugates with Improved Properties.

Calicheamicin antibody-drug conjugates (ADCs) are effective therapeutics for leukemias with two recently approved in the United States: Mylotarg (gemtuzumab ozogamicin) targeting CD33 for acute myeloid leukemia and Besponsa (inotuzumab ozogamicin) targeting CD22 for acute lymphocytic leukemia. Both of these calicheamicin ADCs are heterogeneous, aggregation-prone, and have a shortened half-life due to the instability of the acid-sensitive hydrazone linker in circulation. We hypothesized that we could improve upon the heterogeneity, aggregation, and circulation stability of calicheamicin ADCs by directly attaching the thiol of a reduced calicheamicin to an engineered cysteine on the antibody via a disulfide bond to generate a linkerless and traceless conjugate. We report herein that the resulting homogeneous conjugates possess minimal aggregation and display high in vivo stability with 50% of the drug remaining conjugated to the antibody after 21 days. Furthermore, these calicheamicin ADCs are highly efficacious in mouse models of both solid tumor (HER2+ breast cancer) and hematologic malignancies (CD22+ non-Hodgkin lymphoma). Safety studies in rats with this novel calicheamicin ADC revealed an increased tolerability compared with that reported for Mylotarg. Overall, we demonstrate that applying novel linker chemistry with site-specific conjugation affords an improved, next-generation calicheamicin ADC.

Cyanobacteria-Derived Proline Increases Stress Tolerance in Arabidopsis thaliana Root Hairs by Suppressing Programmed Cell Death.

Nitrogen-fixing heterocystous cyanobacteria are used as biofertilizer inoculants for stimulating plant growth but can also alleviate plant stress by exometabolite secretion. However, only a small number of studies have focused on elucidating the identity of said bioactives because of the wide array of exuded compounds. Here, we used the root hair assay (RHA) as a rapid programmed cell death (PCD) screening tool for characterizing the bioactivity of cyanobacteria Nostoc muscorum conditioned medium (CM) on Arabidopsis thaliana root hair stress tolerance. We found that heat-stressed A. thaliana pre-treated with N. muscorum CM fractions exhibited significantly lower root hair PCD levels compared to untreated seedlings. Treatment with CM increased stress tolerance by suppressing PCD in root hairs but not necrosis, indicating the bioactive compound was specifically modulating the PCD pathway and not a general stress response. Based on documented N. muscorum exometabolites, we identified the stress-responsive proline as a compound of interest and strong evidence from the ninhydrin assay and HPLC indicate that proline is present in N. muscorum CM. To establish whether proline was capable of suppressing PCD, we conducted proline supplementation experiments. Our results showed that exogenous proline had a similar effect on root hairs as N. muscorum CM treatment, with comparable PCD suppression levels and insignificant necrosis changes. To verify proline as one of the biologically active compounds in N. muscorum CM, we used three mutant A. thaliana lines with proline transporter mutations (lht1, aap1 and atprot1-1::atprot2-3::atprot3-2). Compared with the wild-type seedlings, PCD-suppression in lht1and aap1 mutants was significantly reduced when supplied with low proline (1-5 µM) levels. Similarly, pre-treatment with N. muscorum CM resulted in elevated PCD levels in all three mutant lines compared to wild-type seedlings. Our results show that plant uptake of cyanobacteria-derived proline alters their root hair PCD sensitivity threshold. This offers evidence of a novel biofertilizer mechanism for reducing stress-induced PCD levels, independent of the existing mechanisms documented in the literature.

Anti-Lymphocyte Antigen 6 Complex, Locus E-Seco-Cyclopropabenzindol-4-One-Dimer Antibody-Drug Conjugate That Forms Adduct with α1-Microglobulin Demonstrates Slower Systemic Antibody Clearance and Reduced Tumor Distribution in Animals.

Anti-Ly6E-seco-cyclopropabenzindol-4-one dimer antibody-drug conjugate (ADC) has been reported to form an adduct with α1-microglobulin (A1M) in animal plasma, but with unknown impact on ADC PK and tissue distribution. In this study, we compared the PK and tissue distribution of anti-Ly6E ADC with unconjugated anti-Ly6E mAb in rodents and monkeys. For PK studies, animals received an intravenous administration of anti-Ly6E ADC or unconjugated anti-Ly6E mAb. Plasma samples were analyzed for total antibody (Tab) levels and A1M adduct formation. PK parameters were generated from dose-normalized plasma concentrations. Tissue distribution was determined in tumor-bearing mice after a single intravenous dosing of radiolabeled ADC or mAb. Tissue radioactivity levels were analyzed using a gamma counter. The impact of A1M adduct formation on target cell binding was assessed in an in vitro cell binding assay. The results show that ADC Tab clearance was slower than that of mAb in mice and rats but faster than mAb in monkeys. Correspondingly, the formation of A1M adduct appeared to be faster and higher in mice, followed by rats, and slowest in monkeys. Although ADC tended to show an overall lower distribution to normal tissues, it had a strikingly reduced distribution to tumors compared with mAb, likely due to A1M adduct formation interfering with target binding, as demonstrated by the in vitro cell binding assay. Together, these data 1) demonstrate that anti-Ly6E ADC that forms A1M adduct had slower systemic clearance with strikingly reduced tumor distribution and 2) highlight the importance of selecting an appropriate linker-drug for successful ADC development. SIGNIFICANCE STATEMENT: Anti-lymphocyte antigen 6 complex, locus E, ADC with seco-cyclopropabenzindol-4-one-dimer payload formed adduct with A1M, which led to a decrease in systemic clearance but also attenuated tumor distribution. These findings demonstrate the importance of selecting an appropriate linker-drug for ADC development and also highlight the value of a mechanistic understanding of ADC biotransformation, which could provide insight into ADC molecule design, optimization, and selection.

Systematic Variation of Pyrrolobenzodiazepine (PBD)-Dimer Payload Physicochemical Properties Impacts Efficacy and Tolerability of the Corresponding Antibody-Drug Conjugates.

Cytotoxic pyrrolobenzodiazepine (PBD)-dimer molecules are frequently utilized as payloads for antibody-drug conjugates (ADCs), and many examples are currently in clinical development. In order to further explore this ADC payload class, the physicochemical properties of various PBD-dimer molecules were modified by the systematic introduction of acidic and basic moieties into their chemical structures. The impact of these changes on DNA binding, cell membrane permeability, and in vitro antiproliferation potency was, respectively, determined using a DNA alkylation assay, PAMPA assessments, and cell-based cytotoxicity measurements conducted with a variety of cancer lines. The modified PBD-dimer compounds were subsequently incorporated into CD22-targeting ADCs, and these entities were profiled in a variety of in vitro and in vivo experiments. The introduction of a strongly basic moiety into the PBD-dimer scaffold afforded a conjugate with dramatically worsened mouse tolerability properties relative to ADCs derived from related payloads, which lacked the basic group.

Improved translation of stability for conjugated antibodies using an in vitro whole blood assay.

For antibody-drug conjugates to be efficacious and safe, they must be stable in circulation to carry the payload to the site of the targeted cell. Several components of a drug-conjugated antibody are known to influence stability: 1) the site of drug attachment on the antibody, 2) the linker used to attach the payload to the antibody, and 3) the payload itself. In order to support the design and optimization of a high volume of drug conjugates and avoid unstable conjugates prior to testing in animal models, we wanted to proactively identify these potential liabilities. Therefore, we sought to establish an in vitro screening method that best correlated with in vivo stability. While traditionally plasma has been used to assess in vitro stability, our evaluation using a variety of THIOMABTM antibody-drug conjugates revealed several disconnects between the stability assessed in vitro and the in vivo outcomes when using plasma. When drug conjugates were incubated in vitro for 24 h in mouse whole blood rather than plasma and then analyzed by affinity capture LC-MS, we found an improved correlation to in vivo stability with whole blood (R2 = 0.87, coefficient of determination) compared to unfrozen or frozen mouse plasma (R2 = 0.34, 0.01, respectively). We further showed that this whole blood assay was also able to predict in vivo stability of other preclinical species such as rat and cynomolgus monkey, as well as in human. The screening method utilized short (24 h) incubation times, as well as a custom analysis software, allowing increased throughput and in-depth biotransformation characterization. While some instabilities that were more challenging to identify remain, the method greatly enhanced the process of screening, optimizing, and lead candidate selection, resulting in the substantial reduction of animal studies.

Antibody-mediated delivery of chimeric protein degraders which target estrogen receptor alpha (ERα).

Chimeric molecules which effect intracellular degradation of target proteins via E3 ligase-mediated ubiquitination (e.g., PROTACs) are currently of high interest in medicinal chemistry. However, these entities are relatively large compounds that often possess molecular characteristics which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. Accordingly, we explored whether conjugation of chimeric degraders to monoclonal antibodies using technologies originally developed for cytotoxic payloads might provide alternate delivery options for these novel agents. In this report we describe the construction of several degrader-antibody conjugates comprised of two distinct ERα-targeting degrader entities and three independent ADC linker modalities. We subsequently demonstrate the antigen-dependent delivery to MCF7-neo/HER2 cells of the degrader payloads that are incorporated into these conjugates. We also provide evidence for efficient intracellular degrader release from one of the employed linkers. In addition, preliminary data are described which suggest that reasonably favorable in vivo stability properties are associated with the linkers utilized to construct the degrader conjugates.

Plant Cell Biology: UVA on Guard.

Stomata are pores on the surfaces of leaves that function to regulate loss of water for cooling while at the same time facilitating the uptake of carbon dioxide for photosynthesis. A new study shows how stomatal guard cells can sense ultraviolet-A radiation via cGMP signalling to inhibit the opening of these pores in order to reduce transpirational water loss in the short-term.

Antibody-Drug Conjugates Derived from Cytotoxic seco-CBI-Dimer Payloads Are Highly Efficacious in Xenograft Models and Form Protein Adducts In Vivo.

This work discloses the first examples of antibody-drug conjugates (ADCs) that are constructed from linker-drugs bearing dimeric seco-CBI payloads (duocarmycin analogs). Several homogeneous, CD22-targeting THIOMAB antibody-drug conjugates (TDCs) containing the dimeric seco-CBI entities are shown to be highly efficacious in the WSU-DLCL2 and BJAB mouse xenograft models. Surprisingly, the seco-CBI-containing conjugates are also observed to undergo significant biotransformation in vivo in mice, rats, and monkeys and thereby form 1:1 adducts with the Alpha-1-Microglobulin (A1M) plasma protein from these species. Variation of both the payload mAb attachment site and length of the linker-drug is shown to alter the rates of adduct formation. Subsequent experiments demonstrated that adduct formation attenuates the in vitro antiproliferation activity of the affected seco-CBI-dimer TDCs, but does not significantly impact the in vivo efficacy of the conjugates. In vitro assays employing phosphatase-treated whole blood suggest that A1M adduct formation is likely to occur if the seco-CBI-dimer TDCs are administered to humans. Importantly, protein adduct formation leads to the underestimation of total antibody (Tab) concentrations using an ELISA assay but does not affect Tab values determined via an orthogonal LC-MS/MS method. Several recommendations regarding bioanalysis of future in vivo studies involving related seco-CBI-containing ADCs are provided based on these collective findings.

Growth under cold conditions in a wide perennial ryegrass panel is under tight physiological control.

BACKGROUND: Perennial ryegrass is a cool-season grass species from the family Poaceae and is widely cultivated in temperate regions because it exhibits rapid growth and establishment, and possesses high forage quality. The extension of the growing season in Ireland in spring and autumn is a breeding target to make farming more profitable since a grass-fed diet based on grazing is the cheapest way of nutrition for ruminants. METHODS: Fifty-seven perennial ryegrass accessions were screened for their ability to grow under typical Irish spring conditions as taken from long term temperature records in controlled climate chambers. They were grown in low temperature (8 °C/2 °C day/night) and control conditions (15 °C/8 °C day/night) in three consecutive independent experiments. Fresh weight, height, chlorophyll content and electrolyte leakage were measured, and these parameters were used to rank plant performance under low temperature growth conditions. RESULTS: The results showed that height, yield and electrolyte leakage are excellent measures for the impact of cold stress tolerance. Little variation in growth was seen under cold stress, but a wide variety of responses were observed under control conditions. DISCUSSION: Our results suggest that cold stress is under tight physiological control. Interestingly, the various genotypes responded differentially to more amenable control conditions, indicating that a quick response to more amenable growth conditions is a better target for breeding programmes.

Exploration of Pyrrolobenzodiazepine (PBD)-Dimers Containing Disulfide-Based Prodrugs as Payloads for Antibody-Drug Conjugates.

A number of cytotoxic pyrrolobenzodiazepine (PBD) monomers containing various disulfide-based prodrugs were evaluated for their ability to undergo activation (disulfide cleavage) in vitro in the presence of either glutathione (GSH) or cysteine (Cys). A good correlation was observed between in vitro GSH stability and in vitro cytotoxicity toward tumor cell lines. The prodrug-containing compounds were typically more potent against cells with relatively high intracellular GSH levels (e.g., KPL-4 cells). Several antibody-drug conjugates (ADCs) were subsequently constructed from PBD dimers that incorporated selected disulfide-based prodrugs. Such HER2 conjugates exhibited potent antiproliferation activity against KPL-4 cells in vitro in an antigen-dependent manner. However, the disulfide prodrugs contained in the majority of such entities were surprisingly unstable toward whole blood from various species. One HER2-targeting conjugate that contained a thiophenol-derived disulfide prodrug was an exception to this stability trend. It exhibited potent activity in a KPL-4 in vivo efficacy model that was approximately three-fold weaker than that displayed by the corresponding parent ADC. The same prodrug-containing conjugate demonstrated a three-fold improvement in mouse tolerability properties in vivo relative to the parent ADC, which did not contain the prodrug.

Modulating Antibody-Drug Conjugate Payload Metabolism by Conjugation Site and Linker Modification.

Previous investigations on antibody-drug conjugate (ADC) stability have focused on drug release by linker-deconjugation due to the relatively stable payloads such as maytansines. Recent development of ADCs has been focused on exploring technologies to produce homogeneous ADCs and new classes of payloads to expand the mechanisms of action of the delivered drugs. Certain new ADC payloads could undergo metabolism in circulation while attached to antibodies and thus affect ADC stability, pharmacokinetics, and efficacy and toxicity profiles. Herein, we investigate payload stability specifically and seek general guidelines to address payload metabolism and therefore increase the overall ADC stability. Investigation was performed on various payloads with different functionalities (e.g., PNU-159682 analog, tubulysin, cryptophycin, and taxoid) using different conjugation sites (HC-A118C, LC-K149C, and HC-A140C) on THIOMAB antibodies. We were able to reduce metabolism and inactivation of a broad range of payloads of THIOMAB antibody-drug conjugates by employing optimal conjugation sites (LC-K149C and HC-A140C). Additionally, further payload stability was achieved by optimizing the linkers. Coupling relatively stable sites with optimized linkers provided optimal stability and reduction of payloads metabolism in circulation in vivo.

High-Throughput Cysteine Scanning To Identify Stable Antibody Conjugation Sites for Maleimide- and Disulfide-Based Linkers.

THIOMAB antibody technology utilizes cysteine residues engineered onto an antibody to allow for site-specific conjugation. The technology has enabled the exploration of different attachment sites on the antibody in combination with small molecules, peptides, or proteins to yield antibody conjugates with unique properties. As reported previously ( Shen , B. Q. , et al. ( 2012 ) Nat. Biotechnol. 30 , 184 - 189 ; Pillow , T. H. , et al. ( 2017 ) Chem. Sci. 8 , 366 - 370 ), the specific location of the site of conjugation on an antibody can impact the stability of the linkage to the engineered cysteine for both thio-succinimide and disulfide bonds. High stability of the linkage is usually desired to maximize the delivery of the cargo to the intended target. In the current study, cysteines were individually substituted into every position of the anti-HER2 antibody (trastuzumab), and the stabilities of drug conjugations at those sites were evaluated. We screened a total of 648 THIOMAB antibody-drug conjugates, each generated from a trastuzamab prepared by sequentially mutating non-cysteine amino acids in the light and heavy chains to cysteine. Each THIOMAB antibody variant was conjugated to either maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E (MC-vc-PAB-MMAE) or pyridyl disulfide monomethyl auristatin E (PDS-MMAE) using a high-throughput, on-bead conjugation and purification method. Greater than 50% of the THIOMAB antibody variants were successfully conjugated to both MMAE derivatives with a drug to antibody ratio (DAR) of >0.5 and <50% aggregation. The relative in vitro plasma stabilities for approximately 750 conjugates were assessed using enzyme-linked immunosorbent assays, and stable sites were confirmed with affinity-capture LC/MS-based detection methods. Highly stable conjugation sites for the two types of MMAE derivatives were identified on both the heavy and light chains. Although the stabilities of maleimide conjugates were shown to be greater than those of the disulfide conjugates, many sites were identified that were stable for both. Furthermore, in vitro stabilities of selected stable sites translated across different cytotoxic payloads and different target antibodies as well as to in vivo stability.

Development of Efficient Chemistry to Generate Site-Specific Disulfide-Linked Protein- and Peptide-Payload Conjugates: Application to THIOMAB Antibody-Drug Conjugates.

Conjugation of small molecule payloads to cysteine residues on proteins via a disulfide bond represents an attractive strategy to generate redox-sensitive bioconjugates, which have value as potential diagnostic reagents or therapeutics. Advancement of such "direct-disulfide" bioconjugates to the clinic necessitates chemical methods to form disulfide connections efficiently, without byproducts. The disulfide connection must also be resistant to premature cleavage by thiols prior to arrival at the targeted tissue. We show here that commonly employed methods to generate direct disulfide-linked bioconjugates are inadequate for addressing these challenges. We describe our efforts to optimize direct-disulfide conjugation chemistry, focusing on the generation of conjugates between cytotoxic payloads and cysteine-engineered antibodies (i.e., THIOMAB antibody-drug conjugates, or TDCs). This work culminates in the development of novel, high-yielding conjugation chemistry for creating direct payload disulfide connections to any of several Cys mutation sites in THIOMAB antibodies or to Cys sites in other biomolecules (e.g., human serum albumin and cell-penetrating peptides). We conclude by demonstrating that hindered direct disulfide TDCs with two methyl groups adjacent to the disulfide, which have heretofore not been described for any bioconjugate, are more stable and more efficacious in mouse tumor xenograft studies than less hindered analogs.