Loss of the intracellular enzyme QPCTL limits chemokine function and reshapes myeloid infiltration to augment tumor immunity.

Tumor-associated macrophages are composed of distinct populations arising from monocytes or tissue macrophages, with a poorly understood link to disease pathogenesis. Here, we demonstrate that mouse monocyte migration was supported by glutaminyl-peptide cyclotransferase-like (QPCTL), an intracellular enzyme that mediates N-terminal modification of several substrates, including the monocyte chemoattractants CCL2 and CCL7, protecting them from proteolytic inactivation. Knockout of Qpctl disrupted monocyte homeostasis, attenuated tumor growth and reshaped myeloid cell infiltration, with loss of monocyte-derived populations with immunosuppressive and pro-angiogenic profiles. Antibody targeting of the receptor CSF1R, which more broadly eliminates tumor-associated macrophages, reversed tumor growth inhibition in Qpctl-/- mice and prevented lymphocyte infiltration. Modulation of QPCTL synergized with anti-PD-L1 to expand CD8+ T cells and limit tumor growth. QPCTL inhibition constitutes an effective approach for myeloid cell-targeted cancer immunotherapy.

LRRC15+ myofibroblasts dictate the stromal setpoint to suppress tumour immunity.

Recent single-cell studies of cancer in both mice and humans have identified the emergence of a myofibroblast population specifically marked by the highly restricted leucine-rich-repeat-containing protein 15 (LRRC15)1-3. However, the molecular signals that underlie the development of LRRC15+ cancer-associated fibroblasts (CAFs) and their direct impact on anti-tumour immunity are uncharacterized. Here in mouse models of pancreatic cancer, we provide in vivo genetic evidence that TGFß receptor type 2 signalling in healthy dermatopontin+ universal fibroblasts is essential for the development of cancer-associated LRRC15+ myofibroblasts. This axis also predominantly drives fibroblast lineage diversity in human cancers. Using newly developed Lrrc15-diphtheria toxin receptor knock-in mice to selectively deplete LRRC15+ CAFs, we show that depletion of this population markedly reduces the total tumour fibroblast content. Moreover, the CAF composition is recalibrated towards universal fibroblasts. This relieves direct suppression of tumour-infiltrating CD8+ T cells to enhance their effector function and augments tumour regression in response to anti-PDL1 immune checkpoint blockade. Collectively, these findings demonstrate that TGFß-dependent LRRC15+ CAFs dictate the tumour-fibroblast setpoint to promote tumour growth. These cells also directly suppress CD8+ T cell function and limit responsiveness to checkpoint blockade. Development of treatments that restore the homeostatic fibroblast setpoint by reducing the population of pro-disease LRRC15+ myofibroblasts may improve patient survival and response to immunotherapy.

On Demand Bioorthogonal Switching of an Antibody-Conjugated SPECT Probe to a Cytotoxic Payload: from Imaging to Therapy.

Antibody-drug conjugates (ADCs) for the treatment of cancer aim to achieve selective delivery of a cytotoxic payload to tumor cells while sparing normal tissue. In vivo, multiple tumor-dependent and -independent processes act on ADCs and their released payloads to impact tumor-versus-normal delivery, often resulting in a poor therapeutic window. An ADC with a labeled payload would make synchronous correlations between distribution and tissue-specific pharmacological effects possible, empowering preclinical and clinical efforts to improve tumor-selective delivery; however, few methods to label small molecules without destroying their pharmacological activity exist. Herein, we present a bioorthogonal switch approach that allows a radiolabel attached to an ADC payload to be removed tracelessly at will. We exemplify this approach with a potent DNA-damaging agent, the pyrrolobenzodiazepine (PBD) dimer, delivered as an antibody conjugate targeted to lung tumor cells. The radiometal chelating group, DOTA, was attached via a novel trans-cyclooctene (TCO)-caged self-immolative para-aminobenzyl (PAB) linker to the PBD, stably attenuating payload activity and allowing tracking of biodistribution in tumor-bearing mice via SPECT-CT imaging (live) or gamma counting (post-mortem). Following TCO-PAB-DOTA reaction with tetrazines optimized for extra- and intracellular reactivity, the label was removed to reveal the unmodified PBD dimer capable of inducing potent tumor cell killing in vitro and in mouse xenografts. The switchable antibody radio-drug conjugate (ArDC) we describe integrates, but decouples, the two functions of a theranostic given that it can serve as a diagnostic for payload delivery in the labeled state, but can be switched on demand to a therapeutic agent (an ADC).

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.

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.

Carfilzomib Is Not an Appropriate Payload of Antibody-Drug Conjugates Due to Rapid Inactivation by Lysosomal Enzymes.

Carfilzomib (CFZ) is a proteasome inhibitor used for oncology indications including treating multiple myeloma. CFZ is a potent cytotoxic agent with an IC50 value in the nanomolar range in various cancer cell lines and was considered as a potential payload for antibody drug conjugates (ADCs); however, the conjugated CFZ to anti-CD22 or anti-HER2 antibody totally abolishes the in vitro potency. This was a surprise since with other payloads such as monomethyl auristatin E (MMAE), where potent antiproliferation efficacy was retained as MMAE alone or as a payload in an ADC. Further investigations were conducted using CFZ alone, CFZ with a linker, and CFZ-ADC with tissue matrices including lysosomal enzymes. With CFZ linked to the ADC, cathepsin B (a lysosomal enzyme) was efficient in liberating CFZ from the ADC by cleavage of the valine-citrulline linker. At the same time, the liberated CFZ in the lysosome was inactivated due to further metabolism by lysosomal enzymes. The products from epoxide and amide hydrolysis were identified from these incubations. These results suggested that the CFZ-ADC upon uptake and internalization specifically delivers CFZ payload to the lysosomes, where CFZ was inactivated. On the other hand, CFZ by itself is not as vulnerable and could reach its target. Therefore, lysosomal stability is an important criterion in the selection of a payload for making the next generation of potent ADC therapeutics.

Exposure-Efficacy Analysis of Antibody-Drug Conjugates Delivering an Excessive Level of Payload to Tissues.

Antibody-drug conjugates (ADCs) contain a disease-receptor antibody and a payload drug connected via a linker. The payload delivery depends on both tumor properties and ADC characteristics. In this study, we used different linkers, attachment sites, and doses to modulate payload delivery of several ADCs bearing maytansinoids (e.g., DM1), auristatins (e.g., MMAE), and DNA alkylating agents [e.g., pyrrolo[2,1-c][1,4]benzodiazepine-dimer (PBD)] as payloads in HER2- or CD22-expressing xenograft models. The tumor growth inhibition and ADC stability and exposure data were collected and analyzed from these dosed animals. The trend analysis suggests that intratumoral payload exposures that directly related the combination of conjugate linker and dose correlate with the corresponding efficacies of three payload types in two antigen-expressing xenograft models. These preliminary correlations also suggest that a minimal threshold concentration of intratumoral payload is required to support sustained efficacy. In addition, an ADC can deliver an excessive level of payload to tumors that does not enhance efficacy ("Plateau" effect). In contrast to tumor payload concentrations, the assessments of systemic exposures of total antibody (Tab) as well as the linker, dose, site of attachment, plasma stability, and drug-to-antibody ratio changes of these ADCs did not consistently rationalize the observed ADC efficacies. The requirement of a threshold payload concentration for efficacy is further supported by dose fractionation studies with DM1-, MMAE-, and PBD-containing ADCs, which demonstrated that single-dose regimens showed better efficacies than fractionated dosing. Overall, this study demonstrates that 1) the linker and dose together determine the tissue payload concentration that correlates with the antitumor efficacy of ADCs and 2) an ADC can deliver an unnecessary level of payload to tumors in xenograft models.

Catalytic Cleavage of Disulfide Bonds in Small Molecules and Linkers of Antibody-Drug Conjugates.

In cells, catalytic disulfide cleavage is an essential mechanism in protein folding and synthesis. However, detailed enzymatic catalytic mechanism relating cleavage of disulfide bonds in xenobiotics is not well understood. This study reports an enzymatic mechanism of cleavage of disulfide bonds in xenobiotic small molecules and antibody conjugate (ADC) linkers. The chemically stable disulfide bonds in substituted disulfide-containing pyrrolobenzodiazepine (PBD, pyrrolo[2,1-c][1,4]benzodiazepine) monomer prodrugs in presence of glutathione or cysteine were found to be unstable in incubations in whole blood of humans and rats. It was shown the enzymes involved were thioredoxin (TRX) and glutaredoxin (GRX). For a diverse set of drug-linker conjugates, we determined that TRX in the presence of TRX-reductase and NADPH generated the cleaved products that are consistent with catalytic disulfide cleavage and linker immolation. GRX was less rigorously studied; in the set of compounds studied, its role in the catalytic cleavage was also confirmed. Collectively, these in vitro experiments demonstrate that TRX as well as GRX can catalyze the cleavage of disulfide bonds in both small molecules and linkers of ADCs.

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.

Immolation of p-Aminobenzyl Ether Linker and Payload Potency and Stability Determine the Cell-Killing Activity of Antibody-Drug Conjugates with Phenol-Containing Payloads.

The valine-citrulline (Val-Cit) dipeptide and p-aminobenzyl (PAB) spacer have been commonly used as a cleavable self-immolating linker in ADC design including in the clinically approved ADC, brentuximab vedotin (Adcetris). When the same linker was used to connect to the phenol of the cyclopropabenzindolone (CBI) (P1), the resulting ADC1 showed loss of potency in CD22 target-expressing cancer cell lines (e.g., BJAB, WSU-DLCL2). In comparison, the conjugate (ADC2) of a cyclopropapyrroloindolone (CPI) (P2) was potent despite the two corresponding free drugs having similar picomolar cell-killing activity. Although the corresponding spirocyclization products of P1 and P2, responsible for DNA alkylation, are a prominent component in buffer, the linker immolation was slow when the PAB was connected as an ether (PABE) to the phenol in P1 compared to that in P2. Additional immolation studies with two other PABE-linked substituted phenol compounds showed that electron-withdrawing groups accelerated the immolation to release an acidic phenol-containing payload (to delocalize the negative charge on the anticipated anionic phenol oxygen during immolation). In contrast, efficient immolation of LD4 did not result in an active ADC4 because the payload (P4) had a low potency to kill cells. In addition, nonimmolation of LD5 did not affect the cell-killing potency of its ADC5 since immolation is not required for DNA alkylation by the center-linked pyrrolobenzodiazepine. Therefore, careful evaluation needs to be conducted when the Val-Cit-PAB linker is used to connect antibodies to a phenol-containing drug as the linker immolation, as well as payload potency and stability, affects the cell-killing activity of an ADC.

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.

Conjugation of Indoles to Antibodies through a Novel Self-Immolating Linker.

A novel strategy to attach indole-containing payloads to antibodies through a carbamate moiety and a self-immolating, disulfide-based linker is described. This new strategy was employed to connect a selective estrogen receptor down-regulator (SERD) to various antibodies in a site-selective manner. The resulting conjugates displayed potent, antigen-dependent down-regulation of estrogen receptor levels in MCF7-neo/HER2 and MCF7-hB7H4 cells. They also exhibited similar antigen-dependent modulation of the estrogen receptor in tumors when administered intravenously to mice bearing MCF7-neo/HER2 tumor xenografts. The indole-carbamate moiety present in the new linker was stable in whole blood from various species and also exhibited good in vivo stability properties in mice.

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.

High-Resolution Accurate-Mass Mass Spectrometry Enabling In-Depth Characterization of in Vivo Biotransformations for Intact Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) represent a promising class of therapeutics for the targeted delivery of highly potent cytotoxic drugs to tumor cells to improve bioactivity while minimizing side effects. ADCs are composed of both small and large molecules and therefore have complex molecular structures. In vivo biotransformations may further increase the complexity of ADCs, representing a unique challenge for bioanalytical assays. Quadrupole-time-of-flight mass spectrometry (Q-TOF MS) with electrospray ionization has been widely used for characterization of intact ADCs. However, interpretation of ADC biotransformations with small mass changes, for the intact molecule, remains a limitation due to the insufficient mass resolution and accuracy of Q-TOF MS. Here, we have investigated in vivo biotransformations of multiple site-specific THIOMAB antibody-drug conjugates (TDCs), in the intact form, using a high-resolution, accurate-mass (HR/AM) MS approach. Compared with conventional Q-TOF MS, HR/AM Orbitrap MS enabled more comprehensive identification of ADC biotransformations. It was particularly beneficial for characterizing ADC modifications with small mass changes such as partial drug loss and hydrolysis. This strategy has significantly enhanced our capability to elucidate ADC biotransformations and help understand ADC efficacy and safety in vivo.

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.

Design, synthesis, and biological evaluation of pyrrolobenzodiazepine-containing hypoxia-activated prodrugs.

The ability of various pyrrolobenzodiazepine(PBD)-containing cytotoxic compounds to function as hypoxia-activated prodrugs was assessed. These molecules incorporated a 1-methyl-2-nitro-1H-imidazole hypoxia-activated trigger (present in the clinically evaluated compound TH-302) in a manner that masked a reactive imine moiety required for cytotoxic activity. Incubation of the prodrugs with cytochrome P450-reductase under normoxic and hypoxic conditions revealed that some, but not all, were efficient substrates for the enzyme. In these experiments, prodrugs derived from PBD-monomers underwent rapid conversion to the parent cytotoxic compounds under low-oxygen conditions while related PBD-dimers did not. The ability of a given prodrug to function as an efficient cytochrome P450-reductase substrate correlated with the ratio of cytotoxic potencies measured for the compound against NCI460 cells under normoxic and hypoxic conditions.

Chemical Structure and Concentration of Intratumor Catabolites Determine Efficacy of Antibody Drug Conjugates.

Despite recent technological advances in quantifying antibody drug conjugate (ADC) species, such as total antibody, conjugated antibody, conjugated drug, and payload drug in circulation, the correlation of their exposures with the efficacy of ADC outcomes in vivo remains challenging. Here, the chemical structures and concentrations of intratumor catabolites were investigated to better understand the drivers of ADC in vivo efficacy. Anti-CD22 disulfide-linked pyrrolobenzodiazepine (PBD-dimer) conjugates containing methyl- and cyclobutyl-substituted disulfide linkers exhibited strong efficacy in a WSU-DLCL2 xenograft mouse model, whereas an ADC derived from a cyclopropyl linker was inactive. Total ADC antibody concentrations and drug-to-antibody ratios (DAR) in circulation were similar between the cyclobutyl-containing ADC and the cyclopropyl-containing ADC; however, the former afforded the release of the PBD-dimer payload in the tumor, but the latter only generated a nonimmolating thiol-containing catabolite that did not bind to DNA. These results suggest that intratumor catabolite analysis rather than systemic pharmacokinetic analysis may be used to better explain and predict ADC in vivo efficacy. These are good examples to demonstrate that the chemical nature and concentration of intratumor catabolites depend on the linker type used for drug conjugation, and the potency of the released drug moiety ultimately determines the ADC in vivo efficacy.

Antibody Drug Conjugates Differentiate Uptake and DNA Alkylation of Pyrrolobenzodiazepines in Tumors from Organs of Xenograft Mice.

Pyrrolobenzodiazepine (PBD)-dimer is a DNA minor groove alkylator, and its CD22 THIOMAB antibody drug conjugate (ADC) demonstrated, through a disulfide linker, an efficacy in tumor reduction for more than 7 weeks with minimal body weight loss in xenograft mice after a single 0.5-1 mg/kg i.v. dose. The DNA alkylation was investigated here in tumors and healthy organs of mice to understand the sustained efficacy and tolerability. The experimental procedures included the collection of tumors and organ tissues of xenograft mice treated with the ADC followed by DNA isolation/hydrolysis/quantitation and payload recovery from reversible DNA alkylation. PBD-dimer formed a considerable amount of adducts with tissue DNA, representing approximately 98% (at 24 hours), and 99% (at 96 hours) of the total PBD-dimer in tumors, and 78-89% in liver and lung tissues, suggesting highly efficient covalent binding of the released PBD-dimer to tissue DNA. The amount of PBD-DNA adducts in tumor tissues was approximately 24-fold (at 24 hours) and 70-fold (at 96 hours) greater than the corresponding amount of adducts in liver and lung tissues. In addition, the DNA alkylation levels increased 3-fold to 4-fold from 24 to 96 hours in tumors [41/106 base pairs (bp) at 96 hours] but remained at the same level (1/106 bp) in livers and lungs. These results support the typical target-mediated cumulative uptake of ADC into tumors and payload release that offers an explanation for its sustained antitumor efficacy. In addition, the low level of DNA alkylation in normal tissues is consistent with the tolerability observed in mice.

Analogues of DNA minor groove cross-linking agents incorporating aminoCBI, an amino derivative of the duocarmycins: Synthesis, cytotoxicity, and potential as payloads for antibody-drug conjugates.

A Pd-catalysed amination method is used to convert seco-CBI, a synthetic analogue of the alkylating subunit of the duocarmycin natural products, from the phenol to amino form. This allows efficient enantioselective access to the more potent S enantiomer of aminoCBI and its incorporation into analogues of DNA minor groove cross-linking agents. Evaluation in a panel of nine human tumour cell lines shows that the bifunctional agents containing aminoCBI are generally less cytotoxic than their phenolCBI analogues and more susceptible to P-glycoprotein-mediated resistance. However, all bifunctional agents are potent cytotoxins, some in the sub-pM IC50 range, with in vitro properties that compare favourably with established microtubule-targeted ADC payloads.

The cryptophycins as potent payloads for antibody drug conjugates.

The cryptophycins are a potent class of cytotoxic agents that were evaluated as antibody drug conjugate (ADC) payloads. Free cryptophycin analog 1 displayed cell activity an order of magnitude more potent than approved ADC payloads MMAE and DM1. This potency increase was also reflected in the activity of the cryptophycin ADCs, attached via a either cleavable or non-cleavable linker.