Charge Detection Mass Spectrometry Reveals Conformational Heterogeneity in Megadalton-Sized Monoclonal Antibody Aggregates.

Aggregation of protein-based therapeutics can occur during development, production, or storage and can lead to loss of efficacy and potential toxicity. Native mass spectrometry of a covalently linked pentameric monoclonal antibody complex with a mass of ∼800 kDa reveals several distinct conformations, smaller complexes, and abundant higher-order aggregates of the pentameric species. Charge detection mass spectrometry (CDMS) reveals individual oligomers up to the pentamer mAb trimer (15 individual mAb molecules; ∼2.4 MDa) whereas intermediate aggregates composed of 6-9 mAb molecules and aggregates larger than the pentameric dimer (1.6 MDa) were not detected/resolved by standard mass spectrometry, size exclusion chromatography (SEC), capillary electrophoresis (CE-SDS), or by mass photometry. Conventional quadrupole time-of-flight mass spectrometry (QTOF MS), mass photometry, SEC, and CE-SDS did not resolve partially or more fully unfolded conformations of each oligomer that were readily identified using CDMS by their significantly higher extents of charging. Trends in the charge-state distributions of individual oligomers provides detailed insight into how the structures of compact and elongated mAb aggregates change as a function of aggregate size. These results demonstrate the advantages of CDMS for obtaining accurate masses and information about the conformations of large antibody aggregates despite extensive overlapping m/z values. These results open up the ability to investigate structural changes that occur in small, soluble oligomers during the earliest stages of aggregation for antibodies or other proteins.

NMR Assessment of Therapeutic Peptides and Proteins: Correlations That Reveal Interactions and Motions.

NMR measurements of rotational and translational diffusion are used to characterize the solution behavior of a wide variety of therapeutic proteins and peptides. The timescales of motions sampled in these experiments reveal complicated intrinsic solution behavior such as flexibility, that is central to function, as well as self-interactions, stress-induced conformational changes and other critical attributes that can be discovery and development liabilities. Trends from proton transverse relaxation (R2 ) and hydrodynamic radius (Rh ) are correlated and used to identify and differentiate intermolecular from intramolecular interactions. In this study, peptide behavior is consistent with complicated multimer self-assembly, while multi-domain protein behavior is dominated by intramolecular interactions. These observations are supplemented by simulations that include effects from slow transient interactions and rapid internal motions. R2 -Rh correlations provide a means to profile protein motions as well as interactions. The approach is completely general and can be applied to therapeutic and target protein characterization.

Development of Anti-CD74 Antibody-Drug Conjugates to Target Glucocorticoids to Immune Cells.

Glucocorticoids (GCs) are excellent anti-inflammatory drugs but are dose-limited by on-target toxicity. We sought to solve this problem by delivering GCs to immune cells with antibody-drug conjugates (ADCs) using antibodies containing site-specific incorporation of a non-natural amino acid, novel linker chemistry for in vitro and in vivo stability, and existing and novel glucocorticoid receptor (GR) agonists as payloads. We directed fluticasone propionate to human antigen-presenting immune cells to afford GR activation that was dependent on the targeted antigen. However, mechanism of action studies pointed to accumulation of free payload in the tissue culture supernatant as the dominant driver of activity and indeed administration of the ADC to human CD74 transgenic mice failed to activate GR target genes in splenic B cells. Suspecting dissipation of released payload, we designed an ADC bearing a novel GR agonist payload with reduced permeability which afforded cell-intrinsic activity in human B cells. Our work shows that antibody-targeting offers significant potential for rescuing existing and new dose-limited drugs outside the field of oncology.

An Integrated Strategy to Identify Tyrosine Sulfation from the Therapeutic Proteins.

Posttranslational modifications (PTMs) are potential critical quality attributes in biotherapeutic development, as they can affect drug efficacy and safety. Tyrosine sulfation plays a critical role in protein-protein interactions and has been found on many surface receptors as well as antibody complementarity-determining regions (CDR). However, the presence and function of tyrosine sulfation in therapeutic proteins have not been broadly investigated due to difficulties in detecting the modification. Here, we establish an integrated strategy to identify tyrosine sulfation in biotherapeutic proteins. In silico prediction was used to estimate possible modification sites, followed by the elucidation with intact LCMS and native SCX-MS. The combination of these three steps takes less than 1 h, which provides quick and confident preliminary detection of potential CQAs. Taking NB1 as an example, three +80 Da mass shifts were observed from intact mass analysis and three acidic peaks were monitored by SCX, allowing confirmation of modification as either phosphorylation or sulfation. Peptide mapping, Fe3+-IMAC enrichment, and dephosphorylation were further conducted to provide improved signal intensity and differentiation of modification such as sulfation or phosphorylation. With this integrated strategy, we were able to identify for the first time both tyrosine sulfation and serine phosphorylation in one therapeutic protein.

Interrogating heterogeneity of cysteine-engineered antibody-drug conjugates and antibody-oligonucleotide conjugates by capillary zone electrophoresis-mass spectrometry.

Production of site-specific cysteine-engineered antibody-drug conjugates (ADCs) in mammalian cells may produce developability challenges, fragments, and heterogenous molecules, leading to potential product critical quality attributes in later development stages. Liquid phase chromatography with mass spectrometry (LC-MS) is widely used to evaluate antibody impurities and drug-to-antibody ratio, but faces challenges in analysis of fragment product variants of cysteine-engineered ADCs and oligonucleotide-to-antibody ratio (OAR) species of antibody-oligonucleotide conjugates (AOCs). Here, for the first time, we report novel capillary zone electrophoresis (CZE)-MS approaches to address the challenges above. CZE analysis of six ADCs made with different parent monoclonal antibodies (mAbs) and small molecule drug-linker payloads revealed that various fragment impurities, such as half mAbs with one/two drugs, light chains with one/two drugs, light chains with C-terminal cysteine truncation, heavy chain clippings, were well resolved from the main species. However, most of these fragments were coeluted or had signal suppression during LC-MS analysis. Furthermore, the method was optimized on both ionization and separation aspects to enable the characterization of two AOCs. The method successfully achieved baseline separation and accurate quantification of their OAR species, which were also highly challenging using conventional LC-MS methods. Finally, we compared the migration time and CZE separation profiles among ADCs and their parent mAbs, and found that properties of mAbs and linker payloads significantly influenced the separation of product variants by altering their size or charge. Our study showcases the good performance and broad applicability of CZE-MS techniques for monitoring the heterogeneity of cysteine-engineered ADCs and AOCs.

A machine learning strategy for the identification of key in silico descriptors and prediction models for IgG monoclonal antibody developability properties.

Identification of favorable biophysical properties for protein therapeutics as part of developability assessment is a crucial part of the preclinical development process. Successful prediction of such properties and bioassay results from calculated in silico features has potential to reduce the time and cost of delivering clinical-grade material to patients, but nevertheless has remained an ongoing challenge to the field. Here, we demonstrate an automated and flexible machine learning workflow designed to compare and identify the most powerful features from computationally derived physiochemical feature sets, generated from popular commercial software packages. We implement this workflow with medium-sized datasets of human and humanized IgG molecules to generate predictive regression models for two key developability endpoints, hydrophobicity and poly-specificity. The most important features discovered through the automated workflow corroborate several previous literature reports, and newly discovered features suggest directions for further research and potential model improvement.

Regenerating islet-derived family member, 4 modulates multiple receptor tyrosine kinases and mediators of drug resistance in cancer.

Regenerating islet-derived family member, 4 (Reg IV) is a secreted protein and member of the C-type lectin superfamily. Expression analyses have characterized Reg IV as a prognostic marker for certain cancers; however, the functional role of Reg IV in cancer, including downstream signaling, has only begun to be elucidated. To investigate the biological role of Reg IV in cancer, phosphorylation events were studied in cancer cell lines in the context of either Reg IV stimulation (HCT116 cells) or knockdown of endogenous Reg IV (PC3 and KM12 cells). In addition to the previously observed impact on epidermal growth factor receptor and Akt phosphorylation, we observed modulation in the phosphorylation of multiple additional receptor tyrosine kinases (RTKs), including insulin receptor, insulin-like growth factor receptor as well as their downstream effectors, mitogen-activated protein kinase and phosphatidylinositol-3-kinase pathways. Furthermore, knockdown of Reg IV impacted the ability of insulin and EGF to stimulate downstream tyrosine phosphorylation. Knockdown of Reg IV in cancer cell lines inhibited anchorage-dependent and anchorage-independent (both soft-agar and spheroid assays) cell growth and induced cell cycle arrest. This was accompanied by upregulation of p21 and p27. Transiently silencing Reg IV in cancer cells induced apoptosis and downregulated Bcl-2. Conversely, stimulation of HCT116 cells with recombinant Reg IV induced Bcl-2. Hsp27, a molecule implicated in drug resistance, was similarly modulated by Reg IV. Consistent with our observations with Reg IV siRNA-mediated knockdown, monoclonal antibodies directed against Reg IV inhibited PC3 and KM12 cell growth. Collectively, Reg IV plays an important role in cancer by modulation of key signaling molecules including Hsp27, Bcl-2 and multiple RTKs.

BioPhi: A platform for antibody design, humanization, and humanness evaluation based on natural antibody repertoires and deep learning.

Despite recent advances in transgenic animal models and display technologies, humanization of mouse sequences remains one of the main routes for therapeutic antibody development. Traditionally, humanization is manual, laborious, and requires expert knowledge. Although automation efforts are advancing, existing methods are either demonstrated on a small scale or are entirely proprietary. To predict the immunogenicity risk, the human-likeness of sequences can be evaluated using existing humanness scores, but these lack diversity, granularity or interpretability. Meanwhile, immune repertoire sequencing has generated rich antibody libraries such as the Observed Antibody Space (OAS) that offer augmented diversity not yet exploited for antibody engineering. Here we present BioPhi, an open-source platform featuring novel methods for humanization (Sapiens) and humanness evaluation (OASis). Sapiens is a deep learning humanization method trained on the OAS using language modeling. Based on an in silico humanization benchmark of 177 antibodies, Sapiens produced sequences at scale while achieving results comparable to that of human experts. OASis is a granular, interpretable and diverse humanness score based on 9-mer peptide search in the OAS. OASis separated human and non-human sequences with high accuracy, and correlated with clinical immunogenicity. BioPhi thus offers an antibody design interface with automated methods that capture the richness of natural antibody repertoires to produce therapeutics with desired properties and accelerate antibody discovery campaigns. The BioPhi platform is accessible at https://biophi.dichlab.org and https://github.com/Merck/BioPhi.

Hexamerization of Anti-SARS CoV IgG1 Antibodies Improves Neutralization Capacity.

The SARS-CoV-2 pandemic and particularly the emerging variants have deepened the need for widely available therapeutic options. We have demonstrated that hexamer-enhancing mutations in the Fc region of anti-SARS-CoV IgG antibodies lead to a noticeable improvement in IC50 in both pseudo and live virus neutralization assay compared to parental molecules. We also show that hexamer-enhancing mutants improve C1q binding to target surface. To our knowledge, this is the first time this format has been explored for application in viral neutralization and the studies provide proof-of-concept for the use of hexamer-enhanced IgG1 molecules as potential anti-viral therapeutics.

Preclinical characterization and clinical translation of pharmacodynamic markers for MK-5890: a human CD27 activating antibody for cancer immunotherapy.

BACKGROUND: Immune checkpoint inhibitors (ICI) have radically changed cancer therapy, but most patients with cancer are unresponsive or relapse after treatment. MK-5890 is a CD27 agonist antibody intended to complement ICI therapy. CD27 is a member of the tumor necrosis factor receptor superfamily that plays a critical role in promoting responses of T cells, B cells and NK cells. METHODS: Anti-CD27 antibodies were generated and selected for agonist activity using NF-кB luciferase reporter assays. Antibodies were humanized and characterized for agonism using in vitro T-cell proliferation assays. The epitope recognized on CD27 by MK-5890 was established by X-ray crystallography. Anti-tumor activity was evaluated in a human CD27 knock-in mouse. Preclinical safety was tested in rhesus monkeys. Pharmacodynamic properties were examined in mouse, rhesus monkeys and a phase 1 dose escalation clinical study in patients with cancer. RESULTS: Humanized anti-CD27 antibody MK-5890 (hIgG1) was shown to bind human CD27 on the cell surface with sub-nanomolar potency and to partially block binding to its ligand, CD70. Crystallization studies revealed that MK-5890 binds to a unique epitope in the cysteine-rich domain 1 (CRD1). MK-5890 activated CD27 expressed on 293T NF-κB luciferase reporter cells and, conditional on CD3 stimulation, in purified CD8+ T cells without the requirement of crosslinking. Functional Fc-receptor interaction was required to activate CD8+ T cells in an ex vivo tumor explant system and to induce antitumor efficacy in syngeneic murine subcutaneous tumor models. MK-5890 had monotherapy efficacy in these models and enhanced efficacy of PD-1 blockade. MK-5890 reduced in an isotype-dependent and dose-dependent manner circulating, but not tumor-infiltrating T-cell numbers in these mouse models. In rhesus monkey and human patients, reduction in circulating T cells was transient and less pronounced than in mouse. MK-5890 induced transient elevation of chemokines MCP-1, MIP-1α, and MIP-1ß in the serum of mice, rhesus monkeys and patients with cancer. MK-5890 was well tolerated in rhesus monkeys and systemic exposure to MK-5890 was associated with CD27 occupancy at all doses. CONCLUSIONS: MK-5890 is a novel CD27 agonistic antibody with the potential to complement the activity of PD-1 checkpoint inhibition in cancer immunotherapy and is currently undergoing clinical evaluation.

Differences in human IgG1 and IgG4 S228P monoclonal antibodies viscosity and self-interactions: Experimental assessment and computational predictions of domain interactions.

Human/humanized IgG4 antibodies have reduced effector function relative to IgG1 antibodies, which is desirable for certain therapeutic purposes. However, the developability and biophysical properties for IgG4 antibodies are not well understood. This work focuses on the head-to-head comparison of key biophysical properties, such as self-interaction and viscosity, for 14 human/humanized, and chimeric IgG1 and IgG4 S228P monoclonal antibody pairs that contain the identical variable regions. Experimental measurements showed that the IgG4 S228P antibodies have similar or higher self-interaction and viscosity than that of IgG1 antibodies in 20 mM sodium acetate, pH 5.5. We report sequence and structural drivers for the increased viscosity and self-interaction detected in IgG4 S228P antibodies through a combination of experimental data and computational models. Further, we applied and extended a previously established computational model for IgG1 antibodies to predict the self-interaction and viscosity behavior for each antibody pair, providing insight into the structural characteristics and differences of these two isotypes. Interestingly, we observed that the IgG4 S228P swapped variants, where the CH3 domain was swapped for that of an IgG1, showed reduced self-interaction behavior. These domain swapped IgG4 S228P molecules also showed reduced viscosity from experiment and coarse-grained simulations. We also observed that experimental diffusion interaction parameter (kD) values have a high correlation with computational diffusivity prediction for both IgG1 and IgG4 S228P isotypes.Abbreviations: AHc, constant region Hamaker constant; AHv, variable region Hamaker constant; CDRs, Complementarity-determining regions; CG, Coarse-grained model; CH1, Constant heavy chain 1; CH2 Constant heavy chain 2; CH3 Constant heavy chain 3; chgCH3 Effective charge on the CH3 region; CL Constant light chain; cP, Centipoise; DLS, Dynamic light scattering; Fab, Fragment antigen-binding; Fc, Fragment crystallizable; Fv, Variable domaing; (r) Radial distribution function; H1 CDR1 of Heavy Chain; H2 CDR2 of Heavy Chain; H3 CDR3 of Heavy Chain; HVI, High viscosity index; IgG1 human immunoglobulin of IgG1 subclass; IgG4 human immunoglobulin of IgG4 subclass; kD, Diffusion interaction parameter; L1 CDR1 of Light Chain; L2 CDR2 of Light Chain; L3 CDR3 of Light Chain; mAb, Monoclonal antibody; MD, Molecular dynamics; PPI Protein-protein interactions; SCM, Spatial charge map; UP-SEC, Ultra-high-performance size-exclusion chromatography; VH, Variable domain of Heavy Chain; VL, Variable domain of Light Chain.

Characterization and Modeling of Reversible Antibody Self-Association Provide Insights into Behavior, Prediction, and Correction.

Reversible antibody self-association, while having major developability and therapeutic implications, is not fully understood or readily predictable and correctable. For a strongly self-associating humanized mAb variant, resulting in unacceptable viscosity, the monovalent affinity of self-interaction was measured in the low µM range, typical of many specific and biologically relevant protein-protein interactions. A face-to-face interaction model extending across both the heavy-chain (HC) and light-chain (LC) Complementary Determining Regions (CDRs) was apparent from biochemical and mutagenesis approaches as well as computational modeling. Light scattering experiments involving individual mAb, Fc, Fab, and Fab'2 domains revealed that Fabs self-interact to form dimers, while bivalent mAb/Fab'2 forms lead to significant oligomerization. Site-directed mutagenesis of aromatic residues identified by homology model patch analysis and self-docking dramatically affected self-association, demonstrating the utility of these predictive approaches, while revealing a highly specific and tunable nature of self-binding modulated by single point mutations. Mutagenesis at these same key HC/LC CDR positions that affect self-interaction also typically abolished target binding with notable exceptions, clearly demonstrating the difficulties yet possibility of correcting self-association through engineering. Clear correlations were also observed between different methods used to assess self-interaction, such as Dynamic Light Scattering (DLS) and Affinity-Capture Self-Interaction Nanoparticle Spectroscopy (AC-SINS). Our findings advance our understanding of therapeutic protein and antibody self-association and offer insights into its prediction, evaluation and corrective mitigation to aid therapeutic development.

Cryo-EM structures of inhibitory antibodies complexed with arginase 1 provide insight into mechanism of action.

Human Arginase 1 (hArg1) is a metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea, and modulates T-cell-mediated immune response. Arginase-targeted therapies have been pursued across several disease areas including immunology, oncology, nervous system dysfunction, and cardiovascular dysfunction and diseases. Currently, all published hArg1 inhibitors are small molecules usually less than 350 Da in size. Here we report the cryo-electron microscopy structures of potent and inhibitory anti-hArg antibodies bound to hArg1 which form distinct macromolecular complexes that are greater than 650 kDa. With local resolutions of 3.5 Å or better we unambiguously mapped epitopes and paratopes for all five antibodies and determined that the antibodies act through orthosteric and allosteric mechanisms. These hArg1:antibody complexes present an alternative mechanism to inhibit hArg1 activity and highlight the ability to utilize antibodies as probes in the discovery and development of peptide and small molecule inhibitors for enzymes in general.

Anterior gradient-2 plays a critical role in breast cancer cell growth and survival by modulating cyclin D1, estrogen receptor-alpha and survivin.

INTRODUCTION: Anterior-gradient 2 (AGR2) is an estrogen-responsive secreted protein. Its upregulation has been well documented in a number of cancers, particularly breast cancer, for which mixed data exist on the prognostic implications of AGR2 expression. Although emerging evidence indicates that AGR2 is associated with poor prognosis, its function and impact on cancer-relevant pathways have not been elucidated in breast cancer. METHODS: To investigate the biologic role of AGR2 in breast cancer, AGR2 was transiently knocked down, by using siRNA, in T47 D and ZR-75-1 (estrogen receptor-alpha (ER)-positive) and MDA-MB-231 and SK-BR-3 (ER-negative) human breast cancer cell lines. The impact of silencing AGR2 was evaluated in both anchorage-dependent and anchorage-independent growth (soft agar, spheroid) assays. Cell-cycle profiles in ER-positive cell lines were determined with BrdU incorporation, and cell death was measured with Annexin V, JC-1, and F7-26 staining. After transiently silencing AGR2 or stimulating with recombinant AGR2, modulation of key regulators of growth and survival pathways was assessed with Western blot. Combination studies of AGR2 knockdown with the antiestrogens tamoxifen and fulvestrant were carried out and assessed at the level of anchorage-dependent growth inhibition and target modulation (cyclin D1, ER). RESULTS: AGR2 knockdown inhibited growth in anchorage-dependent and anchorage-independent assays, with a more-pronounced effect in ER-positive cell lines. Cyclin D1 levels and BrdU incorporation were reduced with AGR2 knockdown. Conversely, cyclin D1 was induced with recombinant AGR2. AGR2 knockdown induced cell death in ZR-75-1 and T47 D cells, and also downregulated survivin and c-Myc. Evidence of AGR2-ER crosstalk was demonstrated by a reduction of ER at the protein level after transiently silencing AGR2. AGR2 knockdown in combination with fulvestrant or tamoxifen did not preclude the efficacy of the antiestrogens, but enhanced it. In addition, p-Src, implicated in tamoxifen resistance, was downregulated with AGR2 knockdown. CONCLUSIONS: Transiently silencing AGR2 in ER-positive breast cancer cell lines inhibited cell growth and cell-cycle progression and induced cell death. Breast cancer drivers (ER and cyclin D1) as well as cancer-signaling nodes (pSrc, c-Myc, and survivin) were demonstrated to be downstream of AGR2. Collectively, the data presented support the utility of anti-AGR2 therapy in ER-positive breast cancers because of its impact on cancer-relevant pathways.

Hyphenation of strong cation exchange chromatography to native mass spectrometry for high throughput online characterization of charge heterogeneity of therapeutic monoclonal antibodies.

Characterization of charge heterogeneity in monoclonal antibodies (mAbs) is needed during developability assessment and downstream development of drug candidates. Charge heterogeneity can come from post-translational modifications like deamidation, isomerization, and sialylation. Elucidation of charge variants with mass spectrometry (MS) has historically been challenging. Due to the nonvolatility and high ionic strength of conventional buffer systems, labor-intensive offline fractionation followed by MS analysis is routinely used. Here, we describe an alternative strategy that directly couples strong cation exchange (SCX) chromatography to high-resolution Orbitrap MS for online native MS analysis (SCX-MS). A combined pH and salt gradient was used for universal separation of mAbs from a wide range of pI values (6.38 ~ 9.2), including infliximab (Remicade®, chimeric IgG1/kappa), NISTmab (humanized IgG1/kappa) and trastuzumab (Herceptin®, humanized IgG1/kappa), without tailoring of chromatographic profiles. Liquid chromatography and MS parameters were optimized to achieve high-quality spectra and enhanced detection of low abundant species under high flow rate conditions. Genedata Expressionist, a vendor agnostic software, was used for data processing. This integrated strategy allows unbiased characterization of numerous charge variant species and low molecular weight fragments (<0.05%) without post-column flow splitting. The application was further expanded with middle-up approaches for subdomain analysis, which demonstrated the versatility of the strategy for analysis of various construct types. With our analysis of mAbs during developability assessment and forced degradation studies, which aimed at assessing potential critical quality attributes in antibody drug molecules, we provide, for the first time, direct visualization of molecular alterations of mAbs at intact level. Furthermore, strong correlation was observed between this novel MS approach and analysis by capillary isoelectric focusing.

Crystal Structure and Characterization of Human Heavy-Chain Only Antibodies Reveals a Novel, Stable Dimeric Structure Similar to Monoclonal Antibodies.

We report the novel crystal structure and characterization of symmetrical, homodimeric humanized heavy-chain-only antibodies or dimers (HC2s). HC2s were found to be significantly coexpressed and secreted along with mAbs from transient CHO HC/LC cotransfection, resulting in an unacceptable mAb developability attribute. Expression of full-length HC2s in the absence of LC followed by purification resulted in HC2s with high purity and thermal stability similar to conventional mAbs. The VH and CH1 portion of the heavy chain (or Fd) was also efficiently expressed and yielded a stable, covalent, and reducible dimer (Fd2). Mutagenesis of all heavy chain cysteines involved in disulfide bond formation revealed that Fd2 intermolecular disulfide formation was similar to Fabs and elucidated requirements for Fd2 folding and expression. For one HC2, we solved the crystal structure of the Fd2 domain to 2.9 Å, revealing a highly symmetrical homodimer that is structurally similar to Fabs and is mediated by conserved (CH1) and variable (VH) contacts with all CDRs positioned outward for target binding. Interfacial dimer contacts revealed by the crystal structure were mutated for two HC2s and were found to dramatically affect HC2 formation while maintaining mAb bioactivity, offering a potential means to modulate novel HC2 formation through engineering. These findings indicate that human heavy-chain dimers can be secreted efficiently in the absence of light chains, may show good physicochemical properties and stability, are structurally similar to Fabs, offer insights into their mechanism of formation, and may be amenable as a novel therapeutic modality.

Predicting Antibody Developability Profiles Through Early Stage Discovery Screening.

Monoclonal antibodies play an increasingly important role for the development of new drugs across multiple therapy areas. The term 'developability' encompasses the feasibility of molecules to successfully progress from discovery to development via evaluation of their physicochemical properties. These properties include the tendency for self-interaction and aggregation, thermal stability, colloidal stability, and optimization of their properties through sequence engineering. Selection of the best antibody molecule based on biological function, efficacy, safety, and developability allows for a streamlined and successful CMC phase. An efficient and practical high-throughput developability workflow (100 s-1,000 s of molecules) implemented during early antibody generation and screening is crucial to select the best lead candidates. This involves careful assessment of critical developability parameters, combined with binding affinity and biological properties evaluation using small amounts of purified material (<1 mg), as well as an efficient data management and database system. Herein, a panel of 152 various human or humanized monoclonal antibodies was analyzed in biophysical property assays. Correlations between assays for different sets of properties were established. We demonstrated in two case studies that physicochemical properties and key assay endpoints correlate with key downstream process parameters. The workflow allows the elimination of antibodies with suboptimal properties and a rank ordering of molecules for further evaluation early in the candidate selection process. This enables any further engineering for problematic sequence attributes without affecting program timelines.

Biophysical and Immunological Characterization and In Vivo Pharmacokinetics and Toxicology in Nonhuman Primates of the Anti-PD-1 Antibody Pembrolizumab.

The programmed cell death 1 (PD-1) pathway represents a major immune checkpoint, which may be engaged by cells in the tumor microenvironment to overcome active T-cell immune surveillance. Pembrolizumab (Keytruda®, MK-3475) is a potent and highly selective humanized mAb of the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. This blockade enhances the functional activity of T cells to facilitate tumor regression and ultimately immune rejection. Pembrolizumab binds to human and cynomolgus monkey PD-1 with picomolar affinity and blocks the binding of human and cynomolgus monkey PD-1 to PD-L1 and PD-L2 with comparable potency. Pembrolizumab binds both the C'D and FG loops of PD-1. Pembrolizumab overcomes human and cynomolgus monkey PD-L1-mediated immune suppression in T-cell cultures by enhancing IL2 production following staphylococcal enterotoxin B stimulation of healthy donor and cancer patient cells, and IFNγ production in human primary tumor histoculture. Ex vivo and in vitro studies with human and primate T cells show that pembrolizumab enhances antigen-specific T-cell IFNγ and IL2 production. Pembrolizumab does not mediate FcR or complement-driven effector function against PD-1-expressing cells. Pembrolizumab displays dose-dependent clearance and half-life in cynomolgus monkey pharmacokinetic and toxicokinetic studies typical for human IgG4 antibodies. In nonhuman primate toxicology studies, no findings of toxicologic significance were observed. The preclinical data for pembrolizumab are consistent with the clinical anticancer activity and safety that has been demonstrated in human clinical trials.

Pharmacokinetic Properties of Humanized IgG1 and IgG4 Antibodies in Preclinical Species: Translational Evaluation.

Assessment of the factors that regulate antibody exposure-response relationships in the relevant animal models is critical for the design of successful translational strategies from discovery to the clinic. Depending on the specific clinical indication, preclinical development paradigms may require that the efficacy or dosing-related attributes for the existing antibody be assessed in various species when cross-reactivity of the lead antibody to the intended species is justified. Additionally, with the success of monoclonal antibodies for management of various human conditions, a parallel interest in therapeutic use of these novel modalities in various veterinary species has followed. The protective role of neonatal Fc receptor (FcRn) in regulation of IgG homeostasis and clearance is now well recognized and the "nonspecific clearance" of antibodies through bone marrow-derived phagocytic and vascular endothelial cells (via lysosomal processes) is modulated by interactions with FcRn receptors. In this study, we have attempted to examine the PK properties of human IgG antibodies in dog and monkey. These studies establish a translational framework for evaluation of IgG antibody PK properties across species.

In Vivo Imaging of the Programmed Death Ligand 1 by 18F PET.

Programmed death ligand 1 (PD-L1) is an immune regulatory ligand that binds to the T-cell immune check point programmed death 1. Tumor expression of PD-L1 is correlated with immune suppression and poor prognosis. It is also correlated with therapeutic efficacy of programmed death 1 and PD-L1 inhibitors. In vivo imaging may enable real-time follow-up of changing PD-L1 expression and heterogeneity evaluation of PD-L1 expression across tumors in the same subject. We have radiolabeled the PD-L1-binding Affibody molecule NOTA-ZPD-L1_1 with 18F and evaluated its in vitro and in vivo binding affinity, targeting, and specificity. Methods: The affinity of the PD-L1-binding Affibody ligand ZPD-L1_1 was evaluated by surface plasmon resonance. Labeling was accomplished by maleimide coupling of NOTA to a unique cysteine residue and chelation of 18F-AlF. In vivo studies were performed in PD-L1-positive, PD-L1-negative, and mixed tumor-bearing severe combined immunodeficiency mice. Tracer was injected via the tail vein, and dynamic PET scans were acquired for 90 min, followed by γ-counting biodistribution. Immunohistochemical staining with an antibody specific for anti-PD-L1 (22C3) was used to evaluate the tumor distribution of PD-L1. Immunohistochemistry results were then compared with ex vivo autoradiographic images obtained from adjacent tissue sections. Results: NOTA-ZPD-L1_1 was labeled, with a radiochemical yield of 15.1% ± 5.6%, radiochemical purity of 96.7% ± 2.0%, and specific activity of 14.6 ± 6.5 GBq/µmol. Surface plasmon resonance showed a NOTA-conjugated ligand binding affinity of 1 nM. PET imaging demonstrated rapid uptake of tracer in the PD-L1-positive tumor, whereas the PD-L1-negative control tumor showed little tracer retention. Tracer clearance from most organs and blood was quick, with biodistribution showing prominent kidney retention, low liver uptake, and a significant difference between PD-L1-positive (percentage injected dose per gram [%ID/g] = 2.56 ± 0.33) and -negative (%ID/g = 0.32 ± 0.05) tumors (P = 0.0006). Ex vivo autoradiography showed excellent spatial correlation with immunohistochemistry in mixed tumors. Conclusion: Our results show that Affibody ligands can be effective at targeting tumor PD-L1 in vivo, with good specificity and rapid clearance. Future studies will explore methods to reduce kidney activity retention and further increase tumor uptake.