Peptide ligands for the universal purification of exosomes by affinity chromatography.

Exosomes are gaining prominence as vectors for drug delivery, vaccination, and regenerative medicine. Owing to their surface biochemistry, which reflects the parent cell membrane, these nanoscale biologics feature low immunogenicity, tunable tissue tropism, and the ability to carry a variety of payloads across biological barriers. The heterogeneity of exosomes' size and composition, however, makes their purification challenging. Traditional techniques, like ultracentrifugation and filtration, afford low product yield and purity, and jeopardizes particle integrity. Affinity chromatography represents an excellent avenue for exosome purification. Yet, current affinity media rely on antibody ligands whose selectivity grants high product purity, but mandates the customization of adsorbents for exosomes with different surface biochemistry while their binding strength imposes elution conditions that may harm product's activity. Addressing these issues, this study introduces the first peptide affinity ligands for the universal purification of exosomes from recombinant feedstocks. The peptides were designed to (1) possess promiscuous biorecognition of exosome markers, without binding process-related contaminants and (2) elute the product under conditions that safeguard product stability. Selected ligands SNGFKKHI and TAHFKKKH demonstrated the ability to capture of exosomes secreted by 14 cell sources and purified exosomes derived from HEK293, PC3, MM1, U87, and COLO1 cells with yields of up to 80% and up-to 50-fold reduction of host cell proteins (HCPs) upon eluting with pH gradient from 7.4 to 10.5, recommended for exosome stability. SNGFKKHI-Toyopearl resin was finally employed in a two-step purification process to isolate exosomes from HEK293 cell fluids, affording a yield of 68% and reducing the titer of HCPs to 68 ng/mL. The biomolecular and morphological features of the isolated exosomes were confirmed by analytical chromatography, Western blot analysis, transmission electron microscopy, nanoparticle tracking analysis.

Chemical synthesis and immunological evaluation of cancer vaccines based on ganglioside antigens and α-galactosylceramide.

iNKT cells - often referred as the "Swiss Army knife" of the immune system - have emerged as central players in cancer vaccine therapies. Glycolipids activating iNKT cells, such as α-galactosylceramide (αGalCer), can enhance the immune response against co-delivered cancer antigens and have been applied in the design of self-adjuvanting anti-tumor vaccines. In this context, this work focuses on the chemical synthesis of ganglioside tumor-associated carbohydrate antigens (TACAs), namely GM3 and (Neu5Gc)GM3 antigens, their conjugation to αGalCer, and their formulation into liposomes as an efficient platform for their in vivo delivery. Liposomes containing GM3-αGalCer, (Neu5Gc)GM3-αGalCer, and equimolar amounts of the two conjugates have been fully characterized and their ability to activate iNKT cell has been confirmed ex vivo in mouse and human cell assays. The candidates were tested in in vivo immunization studies, demonstrating an ability to induce both TH1 and TH2 cytokines leading to the production of all subclasses of IgG antibodies. Notably, the study also demonstrated that serum antibodies raised against the two TACAs, alone and in combination, were cross-reactive. This finding has consequences for future vaccine designs - even if a highly tumor-selective antigen is chosen, the resulting antibody response may be broader than anticipated.

Cell-based glycoengineering of extracellular vesicles through precise genome editing.

Engineering of extracellular vesicles (EVs) towards more efficient targeting and uptake to specific cells has large potentials for their application as therapeutics. Carbohydrates play key roles in various biological interactions and are essential for EV biology. The extent to which glycan modification of EVs can be achieved through genetic glycoengineering of their parental cells has not been explored yet. Here we introduce targeted glycan modification of EVs through cell-based glycoengineering via modification of various enzymes in the glycosylation machinery. In a "simple cell" strategy, we modified major glycosylation pathways by knocking-out (KO) essential genes for N-glycosylation (MGAT1), O-GalNAc glycosylation (C1GALT1C1), glycosphingolipids (B4GALT5/6), glycosaminoglycans (B4GALT7) and sialylation (GNE) involved in the elongation or biosynthesis of the glycans in HEK293F cells. The gene editing led to corresponding glycan changes on the cells as demonstrated by differential lectin staining. Small EVs (sEVs) isolated from the cells showed overall corresponding glycan changes, but also some unexpected differences to their parental cell including enrichment preference for certain glycan structures and absence of other glycan types. The genetic glycoengineering did not significantly impact sEVs production, size distribution, or syntenin-1 biomarker expression, while a clonal influence on sEVs production yields was observed. Our findings demonstrate the successful implementation of sEVs glycoengineering via genetic modification of the parental cell and a stable source for generation of glycoengineered sEVs. The utilization of glycoengineered sEVs offers a promising opportunity to study the role of glycosylation in EV biology, as well as to facilitate the optimization of sEVs for therapeutic purposes.

Novel genetically glycoengineered human dendritic cell model reveals regulatory roles of α2,6-linked sialic acids in DC activation of CD4+ T cells and response to TNFα.

Dendritic cells (DCs) are central for the initiation and regulation of appropriate immune responses. While several studies suggest important regulatory roles of sialoglycans in DC biology, our understanding is still inadequate primarily due to a lack of appropriate models. Previous approaches based on enzymatic- or metabolic-glycoengineering and primary cell isolation from genetically modified mice have limitations related to specificity, stability, and species differences. This study addresses these challenges by introducing a workflow to genetically glycoengineer the human DC precursor cell line MUTZ-3, described to differentiate and maturate into fully functional dendritic cells, using CRISPR-Cas9, thereby providing and validating the first isogenic cell model for investigating glycan alteration on human DC differentiation, maturation, and activity. By knocking out (KO) the ST6GAL1 gene, we generated isogenic cells devoid of ST6GAL1-mediated α(2,6)-linked sialylation, allowing for a comprehensive investigation into its impact on DC function. Glycan profiling using lectin binding assay and functional studies revealed that ST6GAL1 KO increased the expression of important antigen presenting and co-stimulatory surface receptors and a specifically increased activation of allogenic human CD4 + T cells. Additionally, ST6GAL1 KO induces significant changes in surface marker expression and cytokine response to TNFα-induced maturation, and it affects migration and the endocytic capacity. These results indicate that genetic glycoengineering of the isogenic MUTZ-3 cellular model offers a valuable tool to study how specific glycan structures influence human DC biology, contributing to our understanding of glycoimmunology.

Design and engineering of bispecific antibodies: insights and practical considerations.

Bispecific antibodies (bsAbs) have attracted significant attention due to their dual binding activity, which permits simultaneous targeting of antigens and synergistic binding effects beyond what can be obtained even with combinations of conventional monospecific antibodies. Despite the tremendous therapeutic potential, the design and construction of bsAbs are often hampered by practical issues arising from the increased structural complexity as compared to conventional monospecific antibodies. The issues are diverse in nature, spanning from decreased biophysical stability from fusion of exogenous antigen-binding domains to antibody chain mispairing leading to formation of antibody-related impurities that are very difficult to remove. The added complexity requires judicious design considerations as well as extensive molecular engineering to ensure formation of high quality bsAbs with the intended mode of action and favorable drug-like qualities. In this review, we highlight and summarize some of the key considerations in design of bsAbs as well as state-of-the-art engineering principles that can be applied in efficient construction of bsAbs with diverse molecular formats.

Deep mining of antibody phage-display selections using Oxford Nanopore Technologies and Dual Unique Molecular Identifiers.

Antibody phage-display technology identifies antibody-antigen interactions through multiple panning rounds, but traditional screening gives no information on enrichment or diversity throughout the process. This results in the loss of valuable binders. Next Generation Sequencing can overcome this problem. We introduce a high accuracy long-read sequencing method based on the recent Oxford Nanopore Technologies (ONT) Q20 + chemistry in combination with dual unique molecular identifiers (UMIs) and an optimized bioinformatic analysis pipeline to monitor the selections. We identified binders from two single-domain antibody libraries selected against a model protein. Traditional colony-picking was compared with our ONT-UMI method. ONT-UMI enabled monitoring of diversity and enrichment before and after each selection round. By combining phage antibody selections with ONT-UMIs, deep mining of output selections is possible. The approach provides an alternative to traditional screening, enabling diversity quantification after each selection round and rare binder recovery, even when the dominating binder was > 99% abundant. Moreover, it can give insights on binding motifs for further affinity maturation and specificity optimizations. Our results demonstrate a platform for future data guided selection strategies.

Structural trends in antibody-antigen binding interfaces: a computational analysis of 1833 experimentally determined 3D structures.

Antibodies are attractive therapeutic candidates due to their ability to bind cognate antigens with high affinity and specificity. Still, the underlying molecular rules governing the antibody-antigen interface remain poorly understood, making in silico antibody design inherently difficult and keeping the discovery and design of novel antibodies a costly and laborious process. This study investigates the characteristics of antibody-antigen binding interfaces through a computational analysis of more than 850,000 atom-atom contacts from the largest reported set of antibody-antigen complexes with 1833 nonredundant, experimentally determined structures. The analysis compares binding characteristics of conventional antibodies and single-domain antibodies (sdAbs) targeting both protein- and peptide antigens. We find clear patterns in the number antibody-antigen contacts and amino acid frequencies in the paratope. The direct comparison of sdAbs and conventional antibodies helps elucidate the mechanisms employed by sdAbs to compensate for their smaller size and the fact that they harbor only half the number of complementarity-determining regions compared to conventional antibodies. Furthermore, we pinpoint antibody interface hotspot residues that are often found at the binding interface and the amino acid frequencies at these positions. These findings have direct potential applications in antibody engineering and the design of improved antibody libraries.

Clinical Staphylococcus aureus inhibits human T-cell activity through interaction with the PD-1 receptor.

IMPORTANCE: Therapies that target and aid the host immune defense to repel cancer cells or invading pathogens are rapidly emerging. Antibiotic resistance is among the largest threats to human health globally. Staphylococcus aureus (S. aureus) is the most common bacterial infection, and it poses a challenge to the healthcare system due to its significant ability to develop resistance toward current available therapies. In long-term infections, S. aureus further adapt to avoid clearance by the host immune defense. In this study, we discover a new interaction that allows S. aureus to avoid elimination by the immune system, which likely supports its persistence in the host. Moreover, we find that blocking the specific receptor (PD-1) using antibodies significantly relieves the S. aureus-imposed inhibition. Our findings suggest that therapeutically targeting PD-1 is a possible future strategy for treating certain antibiotic-resistant staphylococcal infections.

A window into the human immune system: comprehensive characterization of the complexity of antibody complementary-determining regions in functional antibodies.

The human immune system uses antibodies to neutralize foreign antigens. They are composed of heavy and light chains, both with constant and variable regions. The variable region has six hypervariable loops, also known as complementary-determining regions (CDRs) that determine antibody diversity and antigen specificity. Knowledge of their significance, and certain residues present in these areas, is vital for antibody therapeutics development. This study includes an analysis of more than 11,000 human antibody sequences from the International Immunogenetics information system (IMGT). The analysis included parameters such as length distribution, overall amino acid diversity, amino acid frequency per CDR and residue position within antibody chains. Overall, our findings confirm existing knowledge, such as CDRH3's high length diversity and amino acid variability, increased aromatic residue usage, particularly tyrosine, charged and polar residues like aspartic acid, serine, and the flexible residue glycine. Specific residue positions within each CDR influence these occurrences, implying a unique amino acid type distribution pattern. We compared amino acid type usage in CDRs and non-CDR regions, both in globular and transmembrane proteins, which revealed distinguishing features, such as increased frequency of tyrosine, serine, aspartic acid, and arginine. These findings should prove useful for future optimization, improvement of affinity, synthetic antibody library design, or the creation of antibodies de-novo in silico.

Exploration of cell state heterogeneity using single-cell proteomics through sensitivity-tailored data-independent acquisition.

Single-cell resolution analysis of complex biological tissues is fundamental to capture cell-state heterogeneity and distinct cellular signaling patterns that remain obscured with population-based techniques. The limited amount of material encapsulated in a single cell however, raises significant technical challenges to molecular profiling. Due to extensive optimization efforts, single-cell proteomics by Mass Spectrometry (scp-MS) has emerged as a powerful tool to facilitate proteome profiling from ultra-low amounts of input, although further development is needed to realize its full potential. To this end, we carry out comprehensive analysis of orbitrap-based data-independent acquisition (DIA) for limited material proteomics. Notably, we find a fundamental difference between optimal DIA methods for high- and low-load samples. We further improve our low-input DIA method by relying on high-resolution MS1 quantification, thus enhancing sensitivity by more efficiently utilizing available mass analyzer time. With our ultra-low input tailored DIA method, we are able to accommodate long injection times and high resolution, while keeping the scan cycle time low enough to ensure robust quantification. Finally, we demonstrate the capability of our approach by profiling mouse embryonic stem cell culture conditions, showcasing heterogeneity in global proteomes and highlighting distinct differences in key metabolic enzyme expression in distinct cell subclusters.

Expanding the CRISPR toolbox for Chinese hamster ovary cells with comprehensive tools for Mad7 genome editing.

The production of high-value biopharmaceuticals is dominated by mammalian production cells, particularly Chinese hamster ovary (CHO) cells, which have been widely used and preferred in manufacturing processes. The discovery of CRISPR-Cas9 significantly accelerated cell line engineering advances, allowing for production yield and quality improvements. Since then, several other CRISPR systems have become appealing genome editing tools, such as the Cas12a nucleases, which provide broad editing capabilities while utilizing short guide RNAs (gRNAs) that reduce the complexity of the editing systems. One of these is the Mad7 nuclease, which has been shown to efficiently convey targeted gene disruption and insertions in several different organisms. In this study, we demonstrate that Mad7 can generate indels for gene knockout of host cell proteins in CHO cells. We found that the efficiency of Mad7 depends on the addition of protein nuclear localization signals and the gRNAs employed for genome targeting. Moreover, we provide computational tools to design Mad7 gRNAs against any genome of choice and for automated indel detection analysis from next-generation sequencing data. In summary, this paper establishes the application of Mad7 in CHO cells, thereby improving the CRISPR toolbox versatility for research and cell line engineering.

Generation of robust bispecific antibodies through fusion of single-domain antibodies on IgG scaffolds: a comprehensive comparison of formats.

Bispecific antibodies (bsAbs) enable dual binding of different antigens with potential synergistic targeting effects and innovative therapeutic possibilities. The formation of bsAbs is, however, often dependent on complex engineering strategies with a high risk of antibody chain mispairing leading to contamination of the final product with incorrectly assembled antibody species. This study demonstrates formation of bsAbs in a generic and conceptually easy manner through fusion of single-domain antibodies (sdAbs) onto IgG scaffolds through flexible 10 amino acid linkers to form high-quality bsAbs with both binding functionalities intact and minimal product-related impurities. SdAbs are attractive fusion partners due to their small and monomeric nature combined with antigen-binding capabilities comparable to conventional human antibodies. By systematically comparing a comprehensive panel of symmetric αPD-L1×αHER2 antibodies, including reversely mirrored antigen specificities, we investigate how the molecular geometry affects production, stability, antigen binding and CD16a binding. SdAb fusion of the heavy chain was generally preferred over light chain fusion for promoting good expression and high biophysical stability as well as maintaining efficient binding to both antigens. We find that N-terminal sdAb fusion might sterically hinder antigen-binding to the Fv region of the IgG scaffold, whereas C-terminal fusion might disturb antigen-binding to the fused sdAb. Our work demonstrates a toolbox of complementary methods for in-depth analysis of key features, such as in-solution dual antigen binding, thermal stability, and aggregation propensity, to ensure high bsAb quality. These techniques can be executed at high-throughput and/or with very low material consumption and thus represent valuable tools for bsAb screening and development.

Eliminating OFF-frame clones in randomized gene libraries: An improved split ß-lactamase enrichment system.

Large, randomized libraries are a key technology for many biotechnological applications. While genetic diversity is the main parameter most libraries direct their resources on, less focus is devoted to ensuring functional IN-frame expression. This study describes a faster and more efficient system based on a split ß-lactamase complementation for removal of OFF-frame clones and increase of functional diversity, suitable for construction of randomized libraries. The gene of interest is inserted between two fragments of the ß-lactamase gene, conferring resistance to ß-lactam drugs only upon expression of an inserted IN-frame gene without stop codons or frameshifts. The preinduction-free system was capable of eliminating OFF-frame clones in starting mixtures of as little as 1% IN-frame clones and enriching to about 70% IN-frame clones, even when their starting rate was as low as 0.001%. The curation system was verified by constructing a single-domain antibody phage display library using trinucleotide phosphoramidites for randomizing a complementary determining region, while eliminating OFF-frame clones and maximizing functional diversity.

Immobilization-Free Binding and Affinity Characterization of Higher Order Bispecific Antibody Complexes Using Size-Based Microfluidics.

Simultaneous targeting of different antigens by bispecific antibodies (bsAbs) is permitting synergistic binding functionalities with high therapeutic potential, but is also rendering their analysis challenging. We introduce flow-induced dispersion analysis (FIDA) for the in-depth characterization of bsAbs with diverse molecular architectures and valencies under near-native conditions without potentially obstructive surface immobilization. Individual equilibrium dissociation constants are determined in solution, even in higher-order complexes with both antigens involved, hereby allowing the analysis of binding cooperativity and elucidation of a potential interference between the interactions. We further illustrate bispecific binding functionality as incremental increases in complex sizes when the bsAbs are exposed to one or two antigens. The possibility for comprehensive binding analysis with low material consumption and high matrix tolerability irrespective of molecular format and with little optimization renders FIDA a versatile tool for format selection and characterization of complex bi/multispecific protein therapeutics throughout the drug development and biomanufacturing pipeline.

Fully human glyco-optimized recombinant FSH (follitropin epsilon) - a randomized, comparator-controlled phase II clinical trial.

RESEARCH QUESTION: The study aimed to determine the standard treatment dose of follitropin epsilon for ovarian stimulation in the context of IVF treatment. DESIGN: A total of 247 women aged 18-37 years were treated with either 52.5, 75, 112.5 or 150 IU follitropin epsilon daily, or 150 IU every other day, or 150 IU follitropin alfa daily in a long gonadotrophin-releasing hormone agonist protocol. The study was performed as a randomized, assessor-blinded, comparator-controlled, six-armed phase II trial in eight fertility clinics in two European countries. RESULTS: The primary results were as follow. First, none of the doses of follitropin epsilon showed superiority for the main outcome measure, i.e. number of follicles ≥12 mm in size. Follitropin epsilon 75 IU produced results most similar to those of follitropin alfa 150 IU. In terms of secondary results, stronger effects of follitropin epsilon 112.5 IU compared with follitropin alfa 150 IU were seen for secondary outcome measures such as hormone concentrations (oestradiol, inhibin B and progesterone) and oocyte number. CONCLUSIONS: Follitropin epsilon 75 IU daily results in a similar ovarian response to a standard dose of 150 IU follitropin alfa. This dose could be tested in a phase III trial.

A novel scale-down mimic of perfusion cell culture using sedimentation in an automated microbioreactor (SAM).

Continuous upstream processing in mammalian cell culture for recombinant protein production holds promise to increase product yield and quality. To facilitate the design and optimization of large-scale perfusion cultures, suitable scale-down mimics are needed which allow high-throughput experiments to be performed with minimal raw material requirements. Automated microbioreactors are available that mimic batch and fed-batch processes effectively but these have not yet been adapted for perfusion cell culture. This article describes how an automated microbioreactor system (ambr15) can be used to scale-down perfusion cell cultures using cell sedimentation as the method for cell retention. The approach accurately predicts the viable cell concentration, in the range of about 1 × 107 cells/mL for a human cell line, and cell viability of larger scale cultures using a hollow fiber based cell retention system. While it was found to underpredict cell line productivity, the method accurately predicts product quality attributes, including glycosylation profiles, from cultures performed in bioreactors with working volumes between 1 L and 1,000 L. The spent media exchange method using the ambr15 was found to predict the influence of different media formulations on large-scale perfusion cultures in contrast to batch and chemostat experiments performed in the microbioreactor system. The described experimental setup in the microbioreactor allowed an 80-fold reduction in cell culture media requirements, half the daily operator time, which can translate into a cost reduction of approximately 2.5-fold compared to a similar experimental setup at bench scale.

Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts.

The programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis plays a central role in suppression of anti-tumor immunity. Blocking the axis by targeting PD-L1 with monoclonal antibodies is an effective and already clinically approved approach to treat cancer patients. Glyco-engineering technology can be used to optimize different properties of monoclonal antibodies, for example, binding to FcγRs. We generated two glycosylation variants of the same anti-PD-L1 antibody: one bearing core fucosylated N-glycans in its Fc part (92%) and its de-fucosylated counterpart (4%). The two glycosylation variants were compared to a non-glycosylated commercially available anti-PD-L1 antibody in various assays. No differences were observed regarding binding to PD-L1 and blocking of this interaction with its counter receptors PD-1 or CD80. The de-fucosylated anti-PD-L1 antibody showed increased FcγRIIIa binding resulting in enhanced antibody dependent cellular cytotoxicity (ADCC) activity against PD-L1+ cancer cells compared to the "normal"-glycosylated variant. Both glycosylation variants showed no antibody-mediated lysis of B cells and monocytes. The non-glycosylated reference antibody showed no FcγRIIIa engagement and no ADCC activity. Using mixed leukocyte reaction it was observed that the de-fucosylated anti-PD-L1 antibody induced the strongest CD8 T cell activation determined by expression of activation markers, proliferation, and cytotoxicity against cancer cells. The systematic comparison of anti-PD-L1 antibody glycosylation variants with different Fc-mediated potencies demonstrates that our glyco-optimization approach has the potential to enhance CD8 T cell-mediated anti-tumor activity which may improve the therapeutic benefit of anti-PD-L1 antibodies.

Phase I study of TrasGEX, a glyco-optimised anti-HER2 monoclonal antibody, in patients with HER2-positive solid tumours.

PURPOSE: TrasGEX is a second-generation monoclonal antibody of trastuzumab, glyco-optimised to enhance antibody-dependent cellular cytotoxicity while fully retaining trastuzumab's antigen-binding properties to human epidermal growth factor receptor 2 (HER2). A phase I dose-escalation study was conducted to establish the optimal TrasGEX dose and regimen for phase II studies and to define the safety, pharmacokinetics (PK) and preliminary antitumour activity of TrasGEX. PATIENTS AND METHODS: A total of 37 patients with advanced HER2-positive carcinomas and progressive disease received TrasGEX intravenously every 3 weeks until disease progression in doses of 12-720 mg in a three-plus-three dose escalation design, including an expansion cohort at the highest dose. RESULTS: No dose limiting toxicity was observed, and no maximum tolerated dose was reached. Drug-related adverse events were mainly infusion-related reactions occurring during the first infusion in 51% of patients; all but two were mild-to-moderate. Compared with trastuzumab, the PK parameters were dose dependent, with a mean terminal half-life (t1/2) of 263±99 hours for the 720 mg dose. Clinical benefit in 15 out of 30 (50%) evaluable patients included one ongoing complete response, two partial remissions lasting 16 and 77 weeks and disease stabilisation (SD) in 12 patients lasting a median (range) of 17 (7-26) weeks; three of them had SD of 24, 25 and 26 weeks, respectively. CONCLUSION: TrasGEX was safe, well-tolerated and showed antitumour activity in 50% of evaluable patients, all with progressive disease at study entry. Infusions at an interval of 2-3 weeks should achieve clinically relevant trough levels for future studies (NCT01409343).

Phase I study of tomuzotuximab, a glycoengineered therapeutic antibody against the epidermal growth factor receptor, in patients with advanced carcinomas.

BACKGROUND: Changes in glycosylation of the constant domain (Fc) of monoclonal antibodies (mAbs) enhance antibody-dependent cell-mediated cytotoxicity independently of downstream effects following receptor blockade by the antibody, thus extending their indication. We investigated the safety, pharmacokinetics, pharmacodynamics and antitumour activity of tomuzotuximab, an IgG1 glycoengineered mAb against the epidermal growth factor receptor with enhanced tumour cytotoxicity in a phase I dose-escalation study (NTC01222637). PATIENTS AND METHODS: Forty-one patients with advanced solid tumours refractory to standard therapies received tomuzotuximab weekly (12-1370 mg) or two-weekly (990 mg) on a three-plus-three dose escalation design. RESULTS: A maximum tolerated dose was not reached. The most frequent treatment-related adverse events were infusion-related reactions in 31 (76%) patients (grade 3, 12%), mainly confined to the first dose, and skin toxicities (grade 1 or 2) in 30 (73%) patients. Hypomagnesaemia was observed in 9 out of 23 evaluable patients (39%). Similar to cetuximab, tomuzotuximab concentrations increased proportionally to dose from doses≥480 mg with a median terminal half life (t½) of 82 hours, range 55-113 hours. Antitumour activity included one complete response ongoing since more than 4.5 years in a patient with non-small-cell lung cancer and one partial response lasting 353 days in a patient with colorectal cancer. Twelve patients achieved stable disease (median, 166 days, range, 71-414 days) and two patients had prolonged control (>1 year) of their non-measurable disease. CONCLUSION: Tomuzotuximab was safe and showed promising antitumour activity in heavily pretreated patients with advanced metastatic disease. A phase IIb trial of chemotherapy and weekly tomuzotuximab or cetuximab followed with maintenance therapy with the corresponding mAb in patients with recurrent or metastatic head and neck squamous cell carcinoma is ongoing.