Preclinical and translational pharmacology of afucosylated anti-CCR8 antibody for depletion of tumour-infiltrating regulatory T cells.

BACKGROUND AND PURPOSE: RO7502175 is an afucosylated antibody designed to eliminate C-C motif chemokine receptor 8 (CCR8)+ Treg cells in the tumour microenvironment through enhanced antibody-dependent cellular cytotoxicity (ADCC). EXPERIMENTAL APPROACH: We report findings from preclinical studies characterizing pharmacology, pharmacokinetics (PK)/pharmacodynamics (PD) and safety profile of RO7502175 and discuss the translational PK/PD approach used to inform first-in-human (FiH) dosing strategy and clinical development in solid tumour indications. KEY RESULTS: RO7502175 demonstrated selective ADCC against human CCR8+ Treg cells from dissociated tumours in vitro. In cynomolgus monkeys, RO7502175 exhibited a biphasic concentration-time profile consistent with immunoglobulin G1 (IgG1) antibodies, reduced CCR8+ Treg cells in the blood, induced minimal and transient cytokine secretion, and was well tolerated with a no-observed-adverse-effect level (NOAEL) of 100 mg·kg-1 . Moreover, RO7502175 caused minimal cytokine release from peripheral blood mononuclear cells (PBMCs) in vitro. A quantitative model was developed to capture surrogate anti-murine CCR8 antibody PK/PD and tumour dynamics in mice and RO7502175 PK/PD in cynomolgus monkeys. Subsequently, the model was used to project RO7502175 human PK and receptor occupancy (RO) in patients. Because traditional approaches resulted in a low FiH dose for this molecule, even with its superior preclinical safety profile, an integrated approach based on the totality of preclinical data and modelling insights was used for starting dose selection. CONCLUSION AND IMPLICATIONS: This work demonstrates a translational research strategy for collecting and utilizing relevant nonclinical data, developing a mechanistic PK/PD model and using a comprehensive approach to inform clinical study design for RO7502175.

Application of N-Terminal Site-Specific Biotin and Digoxigenin Conjugates to Clinical Anti-drug Antibody Assay Development.

Biotin- and digoxigenin (DIG)-conjugated therapeutic drugs are critical reagents used for the development of anti-drug antibody (ADA) assays for the assessment of immunogenicity. The current practice of generating biotin and DIG conjugates is to label a therapeutic antibody with biotin or DIG via primary amine groups on lysine or N-terminal residues. This approach modifies lysine residues nonselectively, which can impact the ability of an ADA assay to detect those ADAs that recognize epitopes located at or near the modified lysine residue(s). The impact of the lysine modification is considered greater for therapeutic antibodies that have a limited number of lysine residues, such as the variable heavy domain of heavy chain (VHH) antibodies. In this paper, for the first time, we report the application of site-specifically conjugated biotin- and DIG-VHH reagents to clinical ADA assay development using a model molecule, VHHA. The site-specific conjugation of biotin or DIG to VHHA was achieved by using an optimized reductive alkylation approach, which enabled the majority of VHHA molecules labeled with biotin or DIG at the desirable N-terminus, thereby minimizing modification of the protein after labeling and reducing the possibility of missing detection of ADAs. Head-to-head comparison of biophysical characterization data revealed that the site-specific biotin and DIG conjugates demonstrated overall superior quality to biotin- and DIG-VHHA prepared using the conventional amine coupling method, and the performance of the ADA assay developed using site-specific biotin and DIG conjugates met all acceptance criteria. The approach described here can be applied to the production of other therapeutic-protein- or antibody-based critical reagents that are used to support ligand binding assays.

Structural basis of antibody inhibition and chemokine activation of the human CC chemokine receptor 8.

The C-C motif chemokine receptor 8 (CCR8) is a class A G-protein coupled receptor that has emerged as a promising therapeutic target in cancer. Targeting CCR8 with an antibody has appeared to be an attractive therapeutic approach, but the molecular basis for chemokine-mediated activation and antibody-mediated inhibition of CCR8 are not fully elucidated. Here, we obtain an antagonist antibody against human CCR8 and determine structures of CCR8 in complex with either the antibody or the endogenous agonist ligand CCL1. Our studies reveal characteristic antibody features allowing recognition of the CCR8 extracellular loops and CCL1-CCR8 interaction modes that are distinct from other chemokine receptor - ligand pairs. Informed by these structural insights, we demonstrate that CCL1 follows a two-step, two-site binding sequence to CCR8 and that antibody-mediated inhibition of CCL1 signaling can occur by preventing the second binding event. Together, our results provide a detailed structural and mechanistic framework of CCR8 activation and inhibition that expands our molecular understanding of chemokine - receptor interactions and offers insight into the development of therapeutic antibodies targeting chemokine GPCRs.

High-Throughput Analyses of Therapeutic Antibodies Using High-Field Asymmetric Waveform Ion Mobility Spectrometry Combined with SampleStream and Intact Protein Mass Spectrometry.

Intact protein mass spectrometry (MS) coupled with liquid chromatography was applied to characterize the pharmacokinetics and stability profiles of therapeutic proteins. However, limitations from chromatography, including throughput and carryover, result in challenges with handling large sample numbers. Here, we combined intact protein MS with multiple front-end separations, including affinity capture, SampleStream, and high-field asymmetric waveform ion mobility spectrometry (FAIMS), to perform high-throughput and specific mass measurements of a multivalent antibody with one antigen-binding fragment (Fab) fused to an immunoglobulin G1 (IgG1) antibody. Generic affinity capture ensures the retention of both intact species 1Fab-IgG1 and the tentative degradation product IgG1. Subsequently, the analytes were directly loaded into SampleStream, where each injection occurs within ∼30 s. By separating ions prior to MS detection, FAIMS further offered improvement in signal-overnoise by ∼30% for denatured protein MS via employing compensation voltages that were optimized for different antibody species. When enhanced FAIMS transmission of 1Fab-IgG1 was employed, a qualified assay was established for spiked-in serum samples between 0.1 and 25 µg/mL, resulting in ∼10% accuracy bias and precision coefficient of variation. Selective FAIMS transmission of IgG1 as the degradation surrogate product enabled more sensitive detection of clipped species for intact 1Fab-IgG1 at 5 µg/mL in serum, generating an assay to measure 1Fab-IgG1 truncation between 2.5 and 50% with accuracy and precision below 20% bias and coefficient of variation. Our results revealed that the SampleStream-FAIMS-MS platform affords high throughput, selectivity, and sensitivity for characterizing therapeutic antibodies from complex biomatrices qualitatively and quantitatively.

A novel, label-free, pre-equilibrium assay to determine the association and dissociation rate constants of therapeutic antibodies on living cells.

BACKGROUND AND PURPOSE: Monoclonal antibodies (Ab) represent the fastest growing drug class. Knowledge of the biophysical parameters (kon , koff and KD ) that dictate Ab:receptor interaction is critical during the drug discovery process. However, with the increasing complexity of Ab formats and their targets, it became apparent that existing technologies present limitations and are not always suitable to determine these parameters. Therefore, novel affinity determination methods represent an unmet assay need. EXPERIMENTAL APPROACH: We developed a pre-equilibrium kinetic exclusion assay using recent mathematical advances to determine the kon , koff and KD of monoclonal Ab:receptor interactions on living cells. The assay is amenable to all human IgG1 and rabbit Abs. KEY RESULTS: Using our novel assay, we demonstrated for several monoclonal Ab:receptor pairs that the calculated kinetic rate constants were comparable with orthogonal methods that were lower throughput or more resource consuming. We ran simulations to predict the critical conditions to improve the performance of the assays. We further showed that this method could successfully be applied to both suspension and adherent cells. Finally, we demonstrated that kon and koff , but not KD , correlate with in vitro potency for a panel of monoclonal Abs. CONCLUSIONS AND IMPLICATIONS: Our novel assay has the potential to systematically probe binding kinetics of monoclonal Abs to cells and can be incorporated in a screening cascade to identify new therapeutic candidates. Wide-spread adoption of pre-equilibrium assays using physiologically relevant systems will lead to a more holistic understanding of how Ab binding kinetics influence their potency.

Combined PD-L1/TGFß blockade allows expansion and differentiation of stem cell-like CD8 T cells in immune excluded tumors.

TGFß signaling is associated with non-response to immune checkpoint blockade in patients with advanced cancers, particularly in the immune-excluded phenotype. While previous work demonstrates that converting tumors from excluded to inflamed phenotypes requires attenuation of PD-L1 and TGFß signaling, the underlying cellular mechanisms remain unclear. Here, we show that TGFß and PD-L1 restrain intratumoral stem cell-like CD8 T cell (TSCL) expansion and replacement of progenitor-exhausted and dysfunctional CD8 T cells with non-exhausted T effector cells in the EMT6 tumor model in female mice. Upon combined TGFß/PD-L1 blockade IFNγhi CD8 T effector cells show enhanced motility and accumulate in the tumor. Ensuing IFNγ signaling transforms myeloid, stromal, and tumor niches to yield an immune-supportive ecosystem. Blocking IFNγ abolishes the anti-PD-L1/anti-TGFß therapy efficacy. Our data suggest that TGFß works with PD-L1 to prevent TSCL expansion and replacement of exhausted CD8 T cells, thereby maintaining the T cell compartment in a dysfunctional state.

Mini-PBPK-Based Population Model and Covariate Analysis to Assess the Complex Pharmacokinetics and Pharmacodynamics of RO7449135, an Anti-KLK5/KLK7 Bispecific Antibody in Cynomolgus Monkeys.

RO7449135, an anti-kallikrein (KLK)5/KLK7 bispecific antibody, is in development as a potential therapy against Netherton's syndrome (NS). In cynomolgus monkey studies, RO7449135 bound to KLK5 and KLK7, causing considerable accumulation of total KLKs, but with non-dose-proportional increase. To understand the complex PKPD, a population model with covariate analysis was developed accounting for target binding in skin and migration of bound targets from skin to blood. The covariate analysis suggested the animal batch as the categorical covariate impacting the different KLK5 synthesis rates between the repeat-dose study and single-dose study, and the dose as continuous covariate impacting the internalization rate of the binary and ternary complexes containing KLK7. To comprehend the mechanism underlying, we hypothesized that inhibition of KLK5 by RO7449135 prevented its cleavage of the pro-enzyme of KLK7 (pro-KLK7) and altered the proportion between pro-KLK7 and KLK7. Besides the pro-KLK7, RO7449135 can interact with other proteins like LEKTI through KLK7 connection in a dose-dependent manner. The different high-order complexes formed by RO7449135 interacting with pro-KLK7 or LEKTI-like proteins can be subject to faster internalization rate. Accounting for the dose and animal batch as covariates, the model-predicted free target suppression is well aligned with the visual target engagement check. The population PKPD model with covariate analysis provides the scientific input for the complex PKPD analysis, successfully predicts the target suppression in cynomolgus monkeys, and thereby can be used for the human dose projection of RO7449135.

Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action.

Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase that plays a major role in developmental processes and metabolism. The dysregulation of FGFR1 through genetic aberrations leads to skeletal and metabolic diseases as well as cancer. For this reason, FGFR1 is a promising therapeutic target, yet a very challenging one due to potential on-target toxicity. More puzzling is that both agonistic and antagonistic FGFR1 antibodies are reported to exhibit similar toxicity profiles in vivo, namely weight loss. In this study, we aimed to assess and compare the mechanism of action of these molecules to better understand this apparent contradiction. By systematically comparing the binding of these antibodies and the activation or the inhibition of the major FGFR1 signaling events, we demonstrated that the molecules displayed similar properties and can behave either as an agonist or antagonist depending on the presence or the absence of the endogenous ligand. We further demonstrated that these findings translated in xenografts mice models. In addition, using time-resolved FRET and mass spectrometry analysis, we showed a functionally distinct FGFR1 active conformation in the presence of an antibody that preferentially activates the FGFR substrate 2 (FRS2)-dependent signaling pathway, demonstrating that modulating the geometry of a FGFR1 dimer can effectively change the signaling outputs and ultimately the activity of the molecule in preclinical studies. Altogether, our results highlighted how bivalent antibodies can exhibit both agonistic and antagonistic activities and have implications for targeting other receptor tyrosine kinases with antibodies.

Development of an 18F-labeled anti-human CD8 VHH for same-day immunoPET imaging.

PURPOSE: Cancer immunotherapies (CITs) have revolutionized the treatment of certain cancers, but many patients fail to respond or relapse from current therapies, prompting the need for new CIT agents. CD8+ T cells play a central role in the activity of many CITs, and thus, the rapid imaging of CD8+ cells could provide a critical biomarker for new CIT agents. However, existing 89Zr-labeled CD8 PET imaging reagents exhibit a long circulatory half-life and high radiation burden that limit potential applications such as same-day and longitudinal imaging. METHODS: To this end, we discovered and developed a 13-kDa single-domain antibody (VHH5v2) against human CD8 to enable high-quality, same-day imaging with a reduced radiation burden. To enable sensitive and rapid imaging, we employed a site-specific conjugation strategy to introduce an 18F radiolabel to the VHH. RESULTS: The anti-CD8 VHH, VHH5v2, demonstrated binding to a membrane distal epitope of human CD8 with a binding affinity (KD) of 500 pM. Subsequent imaging experiments in several xenografts that express varying levels of CD8 demonstrated rapid tumor uptake and fast clearance from the blood. High-quality images were obtained within 1 h post-injection and could quantitatively differentiate the tumor models based on CD8 expression level. CONCLUSION: Our work reveals the potential of this anti-human CD8 VHH [18F]F-VHH5v2 to enable rapid and specific imaging of CD8+ cells in the clinic.

Dual antibody inhibition of KLK5 and KLK7 for Netherton syndrome and atopic dermatitis.

The epidermis is a barrier that prevents water loss while keeping harmful substances from penetrating the host. The impermeable cornified layer of the stratum corneum is maintained by balancing continuous turnover driven by epidermal basal cell proliferation, suprabasal cell differentiation, and corneal shedding. The epidermal desquamation process is tightly regulated by balance of the activities of serine proteases of the Kallikrein-related peptidases (KLK) family and their cognate inhibitor lymphoepithelial Kazal type-related inhibitor (LEKTI), which is encoded by the serine peptidase inhibitor Kazal type 5 gene. Imbalance of proteolytic activity caused by a deficiency of LEKTI leads to excessive desquamation due to increased activities of KLK5, KLK7, and KLK14 and results in Netherton syndrome (NS), a debilitating condition with an unmet clinical need. Increased activity of KLKs may also be pathological in other dermatoses such as atopic dermatitis (AD). Here, we describe the discovery of inhibitory antibodies against murine KLK5 and KLK7 that could compensate for the deficiency of LEKTI in NS. These antibodies are protective in mouse models of NS and AD and, when combined, promote improved skin barrier integrity and reduced inflammation. To translate these findings, we engineered a humanized bispecific antibody capable of potent inhibition of human KLK5 and KLK7. A crystal structure of KLK5 bound to the inhibitory Fab revealed that the antibody binds distal to its active site and uses a relatively unappreciated allosteric inhibition mechanism. Treatment with the bispecific anti-KLK5/7 antibody represents a promising therapy for clinical development in NS and other inflammatory dermatoses.

Structure of the core human NADPH oxidase NOX2.

NOX2 is the prototypical member of the NADPH oxidase NOX superfamily and produces superoxide (O2•-), a key reactive oxygen species (ROS) that is essential in innate and adaptive immunity. Mutations that lead to deficiency in NOX2 activity correlate with increased susceptibility to bacterial and fungal infections, resulting in chronic granulomatous disease. The core of NOX2 is formed by a heterodimeric transmembrane complex composed of NOX2 (formerly gp91) and p22, but a detailed description of its structural architecture is lacking. Here, we present the structure of the human NOX2 core complex bound to a selective anti-NOX2 antibody fragment. The core complex reveals an intricate extracellular topology of NOX2, a four-transmembrane fold of the p22 subunit, and an extensive transmembrane interface which provides insights into NOX2 assembly and activation. Functional assays uncover an inhibitory activity of the 7G5 antibody mediated by internalization-dependent and internalization-independent mechanisms. Overall, our results provide insights into the NOX2 core complex architecture, disease-causing mutations, and potential avenues for selective NOX2 pharmacological modulation.

Genome-wide association study identifies kallikrein 5 in type 2 inflammation-low asthma.

BACKGROUND: Clinical studies of type 2 (T2) cytokine-related neutralizing antibodies in asthma have identified a substantial subset of patients with low levels of T2 inflammation who do not benefit from T2 cytokine neutralizing antibody treatment. Non-T2 mechanisms are poorly understood in asthma but represent a redefined unmet medical need. OBJECTIVE: We sought to gain a better understanding of genetic contributions to T2-low asthma. METHODS: We utilized an unbiased genome-wide association study of patients with moderate to severe asthma stratified by T2 serum biomarker periostin. We also performed additional expression and biological analysis for the top genetic hits. RESULTS: We identified a novel protective single nucleotide polymorphism at chr19q13.41, which is selectively associated with T2-low asthma and establishes Kallikrein-related peptidase 5 (KLK5) as the causal gene mediating this association. Heterozygous carriers of the single nucleotide polymorphisms have reduced KLK5 expression. KLK5 is secreted by human bronchial epithelial cells and elevated in asthma bronchial alveolar lavage. T2 cytokines IL-4 and IL-13 downregulate KLK5 in human bronchial epithelial cells. KLK5, dependent on its catalytic function, induces epithelial chemokine/cytokine expression. Finally, overexpression of KLK5 in airway or lack of an endogenous KLK5 inhibitor, SPINK5, leads to spontaneous airway neutrophilic inflammation. CONCLUSION: Our data identify KLK5 to be the causal gene at a novel locus at chr19q13.41 associated with T2-low asthma.

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.

Development of an SPR-based binding assay for characterization of anti-CD20 antibodies to CD20 expressed on extracellular vesicles.

Characterization of anti-CD20 antibody binding to CD20 is critical to development of anti-CD20 therapeutics. While SPR is widely used to characterize binding of therapeutics to their targets, its application to the characterization of anti-CD20 therapeutics has been limited by the challenges of obtaining recombinant or native full-length CD20 suitable for ligand binding assays. Extracellular vesicles (EVs) are nanoparticles naturally released from cells that provide a favorable microenvironment for membrane proteins such as CD20 to maintain proper conformation and activity. Here, we report a novel SPR-based assay that enables elucidation of binding kinetics and affinity measurements for anti-CD20 antibody binding to EV-expressed CD20. Our SPR assay is label-free, easy to perform, and demonstrates specific interaction of rituximab and obinutuzumab to CD20 expressed on EVs. The SPR assay revealed that rituximab and obinutuzumab have different binding kinetics and mechanisms to CD20 although both bind to CD20 with high affinity. Our results are consistent with existing literature and verified the validity of this method. The detailed binding kinetics information may also contribute to a better understanding of the interaction between these two antibodies and CD20. Moreover, our method provides a platform with which to characterize other therapeutic antibodies binding to EV-expressed membrane proteins.

Conformation-locking antibodies for the discovery and characterization of KRAS inhibitors.

Small molecules that stabilize inactive protein conformations are an underutilized strategy for drugging dynamic or otherwise intractable proteins. To facilitate the discovery and characterization of such inhibitors, we created a screening platform to identify conformation-locking antibodies for molecular probes (CLAMPs) that distinguish and induce rare protein conformational states. Applying the approach to KRAS, we discovered CLAMPs that recognize the open conformation of KRASG12C stabilized by covalent inhibitors. One CLAMP enables the visualization of KRASG12C covalent modification in vivo and can be used to investigate response heterogeneity to KRASG12C inhibitors in patient tumors. A second CLAMP enhances the affinity of weak ligands binding to the KRASG12C switch II region (SWII) by stabilizing a specific conformation of KRASG12C, thereby enabling the discovery of such ligands that could serve as leads for the development of drugs in a high-throughput screen. We show that combining the complementary properties of antibodies and small molecules facilitates the study and drugging of dynamic proteins.

Antibody toolkit reveals N-terminally ubiquitinated substrates of UBE2W.

The ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the N-termini of proteins, although its substrate repertoire remains unclear. To identify endogenous N-terminally-ubiquitinated substrates, we discover four monoclonal antibodies that selectively recognize tryptic peptides with an N-terminal diglycine remnant, corresponding to sites of N-terminal ubiquitination. Importantly, these antibodies do not recognize isopeptide-linked diglycine (ubiquitin) modifications on lysine. We solve the structure of one such antibody bound to a Gly-Gly-Met peptide to reveal the molecular basis for its selective recognition. We use these antibodies in conjunction with mass spectrometry proteomics to map N-terminal ubiquitination sites on endogenous substrates of UBE2W. These substrates include UCHL1 and UCHL5, where N-terminal ubiquitination distinctly alters deubiquitinase (DUB) activity. This work describes an antibody toolkit for enrichment and global profiling of endogenous N-terminal ubiquitination sites, while revealing functionally relevant substrates of UBE2W.

Imaging Reveals Importance of Shape and Flexibility for Glomerular Filtration of Biologics.

Advances in antibody engineering have enabled the construction of novel molecular formats in diverse shapes and sizes, providing new opportunities for cancer immunotherapeutic drug discovery while also revealing limitations in knowledge of structure-activity relationships. The current understanding of renal filtration originates largely from data reported for dextrans, IgG, albumin, and selected globular proteins. For a one-armed IgG-based T-cell imaging agent, we observed higher renal signal than typically observed for bivalent IgGs, prompting us to explore the factors governing renal filtration of biologics. We constructed a small representative library of IgG-like formats with varied shapes and hinge flexibilities falling broadly into two categories: branched molecules including bivalent IgG and (scFv)2Fc, and nonbranched molecules including one-armed IgG, one-armed IgG with stacked Fab, and one-armed IgG with a rigid IgA2 hinge. Transmission electron microscopy revealed Y-shaped structures for the branched molecules and pseudo-linear structures for the nonbranched molecules. Single-photon emission CT imaging, autoradiography, and tissue harvest studies demonstrated higher renal uptake and catabolism for nonbranched molecules relative to branched molecules. Among the nonbranched molecules, the one-armed IgG with rigid IgA2 hinge molecule demonstrated higher kidney uptake and decreased systemic exposure relative to molecules with a more flexible hinge. Our results show that differences in shape and hinge flexibility drive the increased glomerular filtration of one-armed relative to bivalent antibodies and highlight the practical advantages of using imaging to assess renal filtration properties. These findings are particularly relevant for T-cell-dependent bispecific molecules, many of which have nonstandard antibody structures.

Homeostatic functions of monocytes and interstitial lung macrophages are regulated via collagen domain-binding receptor LAIR1.

Myeloid cells encounter stromal cells and their matrix determinants on a continual basis during their residence in any given organ. Here, we examined the impact of the collagen receptor LAIR1 on myeloid cell homeostasis and function. LAIR1 was highly expressed in the myeloid lineage and enriched in non-classical monocytes. Proteomic definition of the LAIR1 interactome identified stromal factor Colec12 as a high-affinity LAIR1 ligand. Proteomic profiling of LAIR1 signaling triggered by Collagen1 and Colec12 highlighted pathways associated with survival, proliferation, and differentiation. Lair1-/- mice had reduced frequencies of Ly6C- monocytes, which were associated with altered proliferation and apoptosis of non-classical monocytes from bone marrow and altered heterogeneity of interstitial macrophages in lung. Myeloid-specific LAIR1 deficiency promoted metastatic growth in a melanoma model and LAIR1 expression associated with improved clinical outcomes in human metastatic melanoma. Thus, monocytes and macrophages rely on LAIR1 sensing of stromal determinants for fitness and function, with relevance in homeostasis and disease.

Discovery of a caspase cleavage motif antibody reveals insights into noncanonical inflammasome function.

Inflammasomes sense a number of pathogen and host damage signals to initiate a signaling cascade that triggers inflammatory cell death, termed pyroptosis. The inflammatory caspases (1/4/5/11) are the key effectors of this process through cleavage and activation of the pore-forming protein gasdermin D. Caspase-1 also activates proinflammatory interleukins, IL-1ß and IL-18, via proteolysis. However, compared to the well-studied apoptotic caspases, the identity of substrates and therefore biological functions of the inflammatory caspases remain limited. Here, we construct, validate, and apply an antibody toolset for direct detection of neo-C termini generated by inflammatory caspase proteolysis. By combining rabbit immune phage display with a set of degenerate and defined target peptides, we discovered two monoclonal antibodies that bind peptides with a similar degenerate recognition motif as the inflammatory caspases without recognizing the canonical apoptotic caspase recognition motif. Crystal structure analyses revealed the molecular basis of this strong yet paradoxical degenerate mode of peptide recognition. One antibody selectively immunoprecipitated cleaved forms of known and unknown inflammatory caspase substrates, allowing the identification of over 300 putative substrates of the caspase-4 noncanonical inflammasome, including caspase-7. This dataset will provide a path toward developing blood-based biomarkers of inflammasome activation. Overall, our study establishes tools to discover and detect inflammatory caspase substrates and functions, provides a workflow for designing antibody reagents to study cell signaling, and extends the growing evidence of biological cross talk between the apoptotic and inflammatory caspases.

Preclinical optimization of Ly6E-targeted ADCs for increased durability and efficacy of anti-tumor response.

Early success with brentuximab vedotin in treating classical Hodgkin lymphoma spurred an influx of at least 20 monomethyl auristatin E (MMAE) antibody-drug conjugates (ADCs) into clinical trials. While three MMAE-ADCs have been approved, most of these conjugates are no longer being investigated in clinical trials. Some auristatin conjugates show limited or no efficacy at tolerated doses, but even for drugs driving initial remissions, tumor regrowth and metastasis often rapidly occur. Here we describe the development of second-generation therapeutic ADCs targeting Lymphocyte antigen 6E (Ly6E) where the tubulin polymerization inhibitor MMAE (Compound 1) is replaced with DNA-damaging agents intended to drive increased durability of response. Comparison of a seco-cyclopropyl benzoindol-4-one (CBI)-dimer (compound 2) to MMAE showed increased potency, activity across more cell lines, and resistance to efflux by P-glycoprotein, a drug transporter commonly upregulated in tumors. Both anti-Ly6E-CBI and -MMAE conjugates drove single-dose efficacy in xenograft and patient-derived xenograft models, but seco-CBI-dimer conjugates showed reduced tumor outgrowth following multiple weeks of treatment, suggesting that they are less susceptible to developing resistance. In parallel, we explored approaches to optimize the targeting antibody. In contrast to immunization with recombinant Ly6E or Ly6E DNA, immunization with virus-like particles generated a high-affinity anti-Ly6E antibody. Conjugates to this antibody improve efficacy versus a previous clinical candidate both in vitro and in vivo with multiple cytotoxics. Conjugation of compound 2 to the second-generation antibody results in a substantially improved ADC with promising preclinical efficacy.