Therapeutic Antibody Fragments Research Articles

Automated ELISA for potency measurements of therapeutic antibodies and antibody fragments

Potency assays are essential for the development and quality control of biopharmaceutical drugs, but they are often a time limiting factor due to manual handling steps and consequently low analytical throughput. On the other hand, automation of potency assays can be challenging due to their complexity and the use of biological materials. ELISA (enzyme-linked immunosorbent assay) is widely used for potency determination and is a good candidate for automation as all ELISA types depend on the same basic steps: coating, blocking, sample incubation, detection, and signal measurement. Nevertheless, ELISA for relative potency measurements still require drug-specific development and assay validation thereby complicating automation efforts. To simplify potency testing by ELISA, we first developed a manual protocol generally applicable to different drugs and then adapted this protocol for automated measurements. We identified unexpected critical parameters which had to be adapted to transfer the manual ELISA to an automated liquid handling system and we demonstrated that gravimetric sample dilution is unnecessary with the automated protocol. Both manual and automated protocols were validated and compared using multiple biotherapeutics. The automated protocol showed similar or higher precision and accuracy when compared to the manual method.

Discovery and Development of CD70 As a Cellular Therapy Target in High Risk Multiple Myeloma

Background: Even in the BCMA CAR-T era, multiple myeloma (MM) patients with high-risk genotypes have the poorest outcomes. We thus leveraged MMRF CoMMpass data to identify potential novel surface immunotherapeutic targets in high-risk MM. As described below, we focused on CD70, an immunotherapeutic target known to be upregulated on many hematologic and solid tumors but minimally expressed on normal tissue. While CD70 was previously explored in MM (McEarchern et al, Clin Cancer Res (2008)), it was not further investigated due to heterogeneous expression in newly diagnosed samples. Here we revisit this target to suggest cellular therapies against CD70 could be highly beneficial in high-risk MM patients, currently the greatest unmet need in the field. Methods: Primary patient samples were obtained under an IRB-approved protocol. CAR-T lentiviral transduction was performed on paired CD4/CD8 human T cells from at least 3 healthy donors. Effector function was assessed by cytotoxicity against MM cell lines and CAR-T profiling for memory and exhaustion markers. In vivo efficiency was assessed using bioluminescence. ATAC-seq peaks were called using Promo and HOMER (data from Jin et al. Blood (2018)). Machine learning was performed using XGBoost approach applied to CoMMpass RNA-seq data with 80% of samples for training and 20% as test set. Results: Analyzing CoMMpass mRNA data (release IA19) we first evaluated surface protein encoding-genes upregulated in tumors at relapse compared to diagnosis. Among >100 significantly upregulated genes, we focused on CD70 given known promise as an immunotherapy target. Further analysis across genomic subtypes revealed a specific, marked upregulation of CD70 in t(4;14) and gain 1q ( Fig. 1), as well as R-ISS 3 at diagnosis. Flow cytometry profiling of 8 cell line models, including t(4;14) models KHM11, KMS26, and LP1, revealed consistent CD70 protein expression with antigen density similar to BCMA. Profiling of relapsed primary MM samples confirmed that 19 of 28 samples had CD138+ plasma cells positive for CD70 expression. Of 5 patients in whom both BCMA and CD70 were measured, antigen densities were also similar. To target CD70, we then designed 10 unique anti-CD70 CAR-T constructs. 9 of these were scFv-based binders derived from therapeutic antibody fragments currently in preclinical or clinical development. By a structure-guided design based on the CD70:CD27 co-crystal structure (PDB: 7KX0), we also derived a unique truncated fragment of CD27 as an alternative CD70 binding element. All CD70 CARs were efficacious in in vitro cytotoxicity assays targeting MM cell lines AMO1, MM1.S, LP1, and RPMI8226. We then selected the top 3 performing scFv based-CARs as well as our natural ligand (CD27) CAR-T for in vivo studies. All 4 CD70 CAR-Ts prolonged survival of MM.1S-bearing mice compared to empty CAR ( n=5 mice/arm, p<0.005 by log-rank), similar to BCMA CAR-T controls. Remarkably, the CD27-based CAR showed superior expansion (50-85x, p< 0.0001 by ANOVA) and persistence compared to scFv-based ( Fig. 2). These results underscore that targeting CD70 using a natural ligand-based CAR-T represents a promising preclinical candidate for treatment of relapsed and high risk MM. We further investigated why CD70 may be upregulated in high risk MM. Analysis of ChIP-seq in ENCODE and primary MM tumor ATAC-seq was used to nominate a set of transcription factors that bind the CD70 locus. Extending from CoMMpass data, we built a predictive machine learning model for CD70 expression as a function of transcription factor gene expression, nominating key transcription factors TFAP2A, LEF1, MYB, and PAX5 as CD70 regulators; validation is now underway. Additionally, we showed that knockout of NSD2 in t(4;14) models KMS34 and KMS11 led to significantly decreased CD70 surface expression, implicating this central epigenetic driver in CD70 overexpression in this high-risk MM subtype. Finally, we also demonstrated that treatment with the hypomethylating agent azacytidine can drive increased CD70 expression, similar to that shown in AML models. Conclusion: Taken together, targeting CD70 appears to be a highly promising cellular therapeutic strategy for MM, with particularly high utility in high-risk patients after BCMA CAR-T relapse. We are continuing preclinical and future clinical development of this CAR-T strategy, using our natural ligand CD27 design with extended in vivo persistence.

Protein engineering strategies contribute efficient production of therapeutic antibody fragments

Advances in production methods make antibody fragments a valuable tool in therapeutic applications.

Protein engineering of antibody fragments for pharmaceutical production

Antibody fragments without the Fc region are attracting attention in the pharmaceutical industry due to their high ability to penetrate solid tissues, cost-effective expression using microbial expression systems, and distinctive modes of action compared to those of full-size antibodies. Based on these characteristics, several antibody fragment agents have been approved. However, developing platform engineering methodologies to accelerate their development is important. In this review, we summarize and discuss protein engineering strategies for preparing therapeutic antibody fragments composed of antibody variable domains. Three (introduction of high-solubility tag systems, complementarity-determining region grafting, and domain arrangements) and two (introduction of purification tag systems and mutagenesis studies for protein L- or protein A-binding) protein engineering strategies have been reported for the cultivation and purification processes, respectively. Fusion tags might negatively impact molecular folding, function, immunogenicity, and final yield. If the production behavior of antibody fragments is not improved through complementarity-determining region grafting, domain arrangements, or human sequence-based mutagenesis, using additional fusion tag systems should be considered, with careful attention to the points described above. This summarized knowledge regarding protein engineering strategies for effectively producing antibody fragments will further accelerate therapeutic antibody fragment development.

Shark VNAR phage display libraries: An alternative source for therapeutic and diagnostic recombinant antibody fragments.

The development of recombinant antibody fragments as promising alternatives to full-length immunoglobulins offers vast opportunities for biomedicine. Antibody fragments have important advantages compared with conventional monoclonal antibodies that make them attractive for the biotech industry: superior stability and solubility, reduced immunogenicity, higher specificity and affinity, capacity to target the hidden epitope and cross the blood-brain barrier, the ability to refold after heat denaturation and inexpensive and easy large-scale production. Different antibody formats such as antigen-binding fragments (Fab), single-chain fragment variable (scFv) consisting of the antigen-binding domains of Ig heavy (VH) and light (VL) chain regions connected by a flexible peptide linker, single-domain antibody fragments (sdAbs) like camelid heavy-chain variable domains (VHHs) and shark variable new antigen receptor (VNARs), and bispecific antibodies (bsAbs) are currently being evaluated as diagnostics or therapeutics in preclinical studies and clinical trials. In the present review, we summarize and discuss studies on VNARs, the smallest recombinant antibody fragment, obtained after the screening of different types of phage display antibody libraries. Results published until March 2023 are discussed.

Half-Life Extension and Biodistribution Modulation of Biotherapeutics via Red Blood Cell Hitch-Hiking with Novel Anti-Band 3 Single-Domain Antibodies.

Small therapeutic proteins are receiving increased interest as therapeutic drugs; however, their clinical success has been limited due to their rapid elimination. Here, we report a half-life extension strategy via strategy via red blood cell red blood cell (RBC) hitch-hiking. This manuscript details the development and characterization of novel anti-RBC single-domain antibodies (sdAbs), their genetic fusion to therapeutic antibody fragments (TAF) as bispecific fusion constructs, and their influence on TAF pharmacokinetics and biodistribution. Several sdAbs specific to the band 3 antigen were generated via phage-display technology. Binding affinity to RBCs was assessed via flow cytometry. Affinity maturation via random mutagenesis was carried out to improve the binding affinity of the sdAbs. Bi-specific constructs were generated by fusing the anti-RBC sdAbs with anti-tissue necrosis factor alpha (TNF-α) TAF via the use of a glycine-serine flexible linker, and assessments for binding were performed via enzyme-linked immunosorbent assay and flow cytometry. Pharmacokinetics of anti-RBC sdAbs and fusion constructs were evaluated following intravenous bolus dosing in mice at a 1 mg/kg dose. Two RBC-binding sdAbs, RB12 and RE8, were developed. These two clones showed high binding affinity to human RBC with an estimated KD of 17.7 nM and 23.6 nM and low binding affinity to mouse RBC with an estimated KD of 335 nM and 528 nM for RB12 and RE8, respectively. Two derivative sdAbs, RMA1, and RMC1, with higher affinities against mouse RBC, were generated via affinity maturation (KD of 66.9 nM and 30.3 nM, respectively). Pharmacokinetic investigations in mice demonstrated prolonged circulation half-life of an anti-RBC-TNF-α bispecific construct (75 h) compared to a non-RBC binding control (1.3 h). In summary, the developed anti-RBC sdAbs and fusion constructs have demonstrated high affinity in vitro, and sufficient half-life extension in vivo.

The mosaic puzzle of the therapeutic monoclonal antibodies and antibody fragments - A modular transition from full-length immunoglobulins to antibody mimetics

The use of monoclonal antibodies represents an important and efficient diagnostic and therapeutic tool in disease management and modern science but remains limited by several factors including the uneven distribution in diseased tissues as well as undesired activation of side immune reactions. Major scientific advancements including Recombinant DNA Technology, Hybridoma Technology, and Polymerase Chain Reaction have considerably impacted the use of monoclonal antibodies providing technical and effective solutions to overcome the shortcomings encountered with conventional antibodies. Initially, the introduction of antibody fragments allowed a more uniform and deeper penetration of the targeted tissue and reduced unwanted activation of Fc-mediated immune reactions. On another level, the immunogenicity of murine-derived antibodies was overcome by humanizing their encoding genes with specific sequences of human origin andtransgenic mice able to synthesize fully human antibodies were successfully created. Moreover, the advancement of genetic engineering techniques supported by the modular structure of antibody coding genes paved the way for the development of a new generation of antibody fragments with a wide spectrum of monospecific and bispecific agents. These later could be monovalent, bivalent, or multivalent, and either expressed as a single chain, assembled in multimeric forms or stringed in tandem. This has conferred improved affinity, stability, and solubility to antibody targetting. Lately, a new array of monoclonal antibody fragments was introduced with the engineering of nanobody and antibody mimetics as non-immunoglobulin-derived fragments with promising diagnostic and therapeutic applications. In this review, we decipher the molecular basis of monoclonal antibody engineering with a detailed screening of the antibody derivatives that provides new perspectives to expand the use of monoclonal fragments into previously unexplored fields.

Iminodiacetic Acid (IDA) Cation-Exchange Nonwoven Membranes for Efficient Capture of Antibodies and Antibody Fragments

There is strong need to reduce the manufacturing costs and increase the downstream purification efficiency of high-value therapeutic monoclonal antibodies (mAbs). This paper explores the performance of a weak cation-exchange membrane based on the coupling of IDA to poly(butylene terephthalate) (PBT) nonwoven fabrics. Uniform and conformal layers of poly(glycidyl methacrylate) (GMA) were first grafted to the surface of the nonwovens. Then IDA was coupled to the polyGMA layers under optimized conditions, resulting in membranes with very high permeability and binding capacity. This resulted in IgG dynamic binding capacities at very short residence times (0.1–2.0 min) that are much higher than those achieved by the best cation-exchange resins. Similar results were obtained in the purification of a single-chain (scFv) antibody fragment. As is customary with membrane systems, the dynamic binding capacities did not change significantly over a wide range of residence times. Finally, the excellent separation efficiency and potential reusability of the membrane were confirmed by five consecutive cycles of mAb capture from its cell culture harvest. The present work provides significant evidence that this weak cation-exchange nonwoven fabric platform might be a suitable alternative to packed resin chromatography for low-cost, higher productivity manufacturing of therapeutic mAbs and antibody fragments.

Bacterial molecular mimicry in autoimmune diseases

Bacterial molecular mimicry in autoimmune diseases is one of the leading mechanisms by which microorganisms may induce autoimmunity and survive in the host. The main purpose of the current study was to determine the main microbes that elicit autoimmune reactions through molecular mimicry and identify the most relevant approaches to investigate this mechanism. A classic example is the M protein of Streptococcus pyogenes, which induces antibody cross-reactivity with a cardiac protein and causes rheumatic fever. Another notable example is the protein from Porphyromonas gingivalis that closely resembles the human heat shock protein and accelerates atherosclerotic. There is evidence that antibodies against Helicobacter pylori CagA interact with different parts of smooth muscle and endothelial cells enhancing atherosclerotic vascular disease. Recently, one cause of infertility has been associated with Staphylococcus aureus molecular mimicry that triggers an antibody response that cross-reacts with human spermatozoa proteins. Further examples of bacterial molecular mimicry are associated with Chlamydia pneumoniae, Escherichia coli, Yersinia, and Salmonella. From the literature, the most widely used methods in this field are Basic Local Alignment Search Tool (BLAST), serological assays, and phage display. The subjects of particular concern are vaccine cross-reactivity and immunosuppressive drugs side-effects, therefore alternative approaches are needed. Such an approach is phage display where therapeutic antibody fragments obtained by this technique have been used in the treatment of autoimmune diseases by neutralizing the pathological effects of autoantibodies. Phage display libraries are constructed from the antibody repertoires of autoimmune disease patients. Antibody fragments without the Fc domain can not interact with Fc receptors and proteins of the complement system and trigger autoimmune diseases. Another approach is to block the Fc receptors. In conclusion, this review highlights key aspects of bacterial molecular mimicry to better understand the factors associated with autoimmune diseases and encourage further research in this field.

Machine learning reveals hidden stability code in protein native fluorescence

Conformational stability of a protein is usually obtained by spectroscopically measuring the unfolding melting temperature. However, optical spectra under native conditions are considered to contain too little resolution to probe protein stability. Here, we have built and trained a neural network model to take the temperature-dependence of intrinsic fluorescence emission under native-only conditions as inputs, and then predict the spectra at the unfolding transition and denatured state. Application to a therapeutic antibody fragment demonstrates that thermal transitions obtained from the predicted spectra correlate highly with those measured experimentally. Crucially, this work reveals that the temperature-dependence of native fluorescence spectra contains a high-degree of previously hidden information relating native ensemble features to stability. This could lead to rapid screening of therapeutic protein variants and formulations based on spectroscopic measurements under non-denaturing temperatures only.

Conformational Equilibria of Multimodal Chromatography Ligands in Water and Bound to Protein Surfaces.

Multimodal chromatography uses small ligands with multiple modes of interaction, e.g., charged, hydrophobic or hydrogen bonding, to separate proteins from complex mixtures. The mechanism by which multimodal ligands interact with proteins is expected to be affected by ligand conformations, among other factors. Here, we study conformational equilibria of two commercially used multimodal cation exchange ligands, Capto MMC and Nuvia cPrime, in a range of solvents, a Lennard-Jones (LJ) liquid, ethanol, and water, using molecular dynamics (MD) simulations. By mapping ligand conformations onto two key torsion angles, ω and φ, in these solvents and in low and high dielectric media, we quantify the relative importance of intramolecular and solvent-mediated interactions. In a high dielectric medium, Capto MMC preferentially samples three conformations, which are stabilized by a combination of an intramolecular torsion potential (on ω) and LJ interactions. In an LJ liquid, solvent molecules compete with intramolecular interactions while simultaneously providing an osmotic force, stabilizing both closer and farther distances between ligand sites. This has the overall effect of "flattening out" the conformational landscape. Interestingly, in ethanol and water, hydrogen bonding between the amide hydrogen and solvent molecules stabilizes two additional conformations of Capto MMC in which ω takes on less favorable cis-like configurations. MD simulations of ligands in free solution with three therapeutic antibody fragments show that ligand conformational equilibria remain effectively unchanged upon binding to proteins. Although, there is 20-30% dehydration of the overall ligand upon binding, the hydrogen-bonding sites are dehydrated to a much smaller extent, particularly in cis-like configurations. Conformational preferences of Nuvia cPrime are similar to that of Capto MMC, except for the effect of symmetry arising from the absence of an alkyl thiol tail. Characterizing the conformational equilibria of these two ligands in free solution and bound to a protein provides a foundation for developing a mechanistic understanding of protein-multimodal ligand interactions.

Biological Therapy of Hematologic Malignancies: Toward a Chemotherapy- free Era.

Less than 70 years ago, the vast majority of hematologic malignancies were untreatable diseases with fatal prognoses. The development of modern chemotherapy agents, which had begun after the Second World War, was markedly accelerated by the discovery of the structure of DNA and its role in cancer biology and tumor cell division. The path travelled from the first temporary remissions observed in children with acute lymphoblastic leukemia treated with single-agent antimetabolites until the first cures achieved by multi-agent chemotherapy regimens was incredibly short. Despite great successes, however, conventional genotoxic cytostatics suffered from an inherently narrow therapeutic index and extensive toxicity, which in many instances limited their clinical utilization. In the last decade of the 20th century, increasing knowledge on the biology of certain malignancies resulted in the conception and development of first molecularly targeted agents designed to inhibit specific druggable molecules involved in the survival of cancer cells. Advances in technology and genetic engineering enabled the production of structurally complex anticancer macromolecules called biologicals, including therapeutic monoclonal antibodies, antibody-drug conjugates and antibody fragments. The development of drug delivery systems (DDSs), in which conventional drugs were attached to various types of carriers including nanoparticles, liposomes or biodegradable polymers, represented an alternative approach to the development of new anticancer agents. Despite the fact that the antitumor activity of drugs attached to DDSs was not fundamentally different, the improved pharmacokinetic profiles, decreased toxic side effects and significantly increased therapeutic indexes resulted in their enhanced antitumor efficacy compared to conventional (unbound) drugs. Approval of the first immune checkpoint inhibitor for the treatment of cancer in 2011 initiated the era of cancer immunotherapy. Checkpoint inhibitors, bispecific T-cell engagers, adoptive T-cell approaches and cancer vaccines have joined the platform so far, represented mainly by recombinant cytokines, therapeutic monoclonal antibodies and immunomodulatory agents. In specific clinical indications, conventional drugs have already been supplanted by multi-agent, chemotherapy-free regimens comprising diverse immunotherapy and/or targeted agents. The very distinct mechanisms of the anticancer activity of new immunotherapy approaches not only call for novel response criteria, but might also change fundamental treatment paradigms of certain types of hematologic malignancies in the near future.

Involvement of p38 Activation and Mitochondria in Death of Human Leukemia Cells Induced by an Agonistic Human Monoclonal Antibody Fab Specific to TRAIL Receptor 1.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cancer cell death with minimal damage to normal cells; however, some cancer cells are resistant to TRAIL. TRAIL resistance may be overcome by agonistic antibodies to TRAIL receptors. In this study, we report the toxic effects of a novel recombinant agonistic human anti–TRAIL receptor 1 (DR4) monoclonal antibody Fab fragment, DR4-4, on various TRAIL-resistant and -sensitive cancer cell lines. The mechanisms of DR4-4 Fab–induced cell death in a human T cell leukemia cell line (Jurkat) were investigated using cell viability testing, immunoblotting, immunoassays, flow cytometry, and morphological observation. DR4-4 Fab–induced caspase-independent necrosis was observed to occur in Jurkat cells in association with p38 mitogen-activated protein kinase activation, cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein degradation, decreased mitochondrial membrane potential, and increased mitochondrial reactive oxygen species production. Increased cytotoxic effects of DR4-4 Fab were observed in combination with TRAIL or γ-irradiation. Our results indicate that the novel DR4-4 Fab might overcome TRAIL-resistance and induce death in leukemia cells via cellular mechanisms different from those activated by TRAIL. DR4-4 Fab may have application as a potential therapeutic antibody fragment in single or combination therapy for cancer.

Passive Immunotherapies for Central Nervous System Disorders: Current Delivery Challenges and New Approaches.

Passive immunotherapy, i.e., the administration of exogenous antibodies that recognize a specific target antigen, has gained significant momentum as a potential treatment strategy for several central nervous system (CNS) disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and brain cancer, among others. Advances in antibody engineering to create therapeutic antibody fragments or antibody conjugates have introduced new strategies that may also be applied to treat CNS disorders. However, drug delivery to the CNS for antibodies and other macromolecules has thus far proven challenging, due in large part to the blood-brain barrier and blood-cerebrospinal fluid barriers that greatly restrict transport of peripherally administered molecules from the systemic circulation into the CNS. Here, we summarize the various passive immunotherapy approaches under study for the treatment of CNS disorders, with a primary focus on disease-specific and target site-specific challenges to drug delivery and new, cutting edge methods.

Purification of antibody fragments for the reduction of charge variants using cation exchange chromatography

Recently, antibody fragments have been studied as therapeutic agents because they lack Fc effector function while having affinity similar to their original monoclonal antibody and can be produced using E. coli. Antibody fragments can be purified using affinity chromatography in the capture step, although they need a polishing step because of product-related impurities, mainly charge variants. Unlike monoclonal antibodies, few studies exist regarding the separation of charge variants in antibody variants. In this study, an efficient separation of charge variant method was assessed using a cation exchange chromatography resin with salt and a pH gradient. The SP ImpRes resin and pH gradient exhibited the most effective separation potency using combinations of resin and the separation method. The antibody fragment that did not undergo the charge variant separation process exhibited a difference in the tertiary structure of the protein and in vivo pharmacokinetics. However, the antibody fragment was similar to the reference protein when the charge variant separation process was performed. These results are expected to support efficient charge variant separation of antibody fragments and to be applied to the industrial production of therapeutic antibody fragments.

Nanobodies: Diagnostic and Therapeutic Antibody Fragments

Nanobodies: Diagnostic and Therapeutic Antibody Fragments

NanoLuc Luciferase - A Multifunctional Tool for High Throughput Antibody Screening.

Based on the recent development of NanoLuc luciferase (Nluc), a small (19 kDa), highly stable, ATP independent, bioluminescent protein, an extremely robust and ultra high sensitivity screening system has been developed whereby primary hits of therapeutic antibodies and antibody fragments could be characterized and quantified without purification. This system is very versatile allowing cellular and solid phase ELISA but also homogeneous BRET based screening assays, relative affinity determinations with competition ELISA and direct Western blotting. The new Nluc protein fusion represents a “swiss army knife solution” for today and future high throughput antibody drug screenings.

Mapping the Aggregation Kinetics of a Therapeutic Antibody Fragment.

The analytical characterization of biopharmaceuticals is a fundamental step in the early stages of development and prediction of their behavior in bioprocesses. Protein aggregation in particular is a common issue as it affects all stages of product development. In the present work, we investigate the stability and the aggregation kinetics of A33Fab, a therapeutically relevant humanized antibody fragment at a wide range of pH, ionic strength, and temperature. We show that the propensity of A33Fab to aggregate under thermally accelerated conditions is pH and ionic-strength dependent with a stronger destabilizing effect of ionic strength at low pH. In the absence of added salts, A33Fab molecules appear to be protected from aggregation due to electrostatic colloidal repulsion at low pH. Analysis by transmission electron microscopy identified significantly different aggregate species formed at low and high pH. The correlations between apparent midpoints of thermal transitions (Tm,app values), or unfolded mole fractions, and aggregation rates are reported here to be significant only at the elevated incubation temperature of 65 °C, where aggregation from the unfolded state predominates. At all other conditions, particularly at 4-45 °C, aggregation of A33 Fab was predominantly from a native-like state, and the kinetics obeyed Arrhenius behavior. Despite this, the rank order of aggregation rates observed at 45 °C, 23 and 4 °C still did not correlate well to each other, indicating that forced degradation at elevated temperatures was not a good screen for predicting behavior at low temperature.

Emerging Role and Targeting of Carcinoembryonic Antigen-related Cell Adhesion Molecule 6 (CEACAM6) in Human Malignancies.

Background: Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is a member of the CEA family of cell adhesion proteins that belong to the immunoglobulin superfamily. CEACAM6 is normally expressed on the surface of myeloid (CD66c) and epithelial surfaces. Stiochiomertic expression of members of the CEA family (CEACAM1, 5, 6, 7) on epithelia maintains normal tissue architecture through homo-and hetero-philic interactions. Dysregulated over-expression of CEACAM6 is oncogenic, is associated with anoikis resistance and an invasive phenotype mediated by excessive TGFβ, AKT, FAK and SRC signaling in human malignancies. Methods: Extensive literature review through PubMed was conducted to identify relevant preclinical and clinical research publications regarding CEACAM6 over the last decade and was summarized in this manuscript. Results: CEACAM5 and 6 are over-expressed in nearly 70% of epithelial malignancies including colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDA), hepatobiliary, gastric, breast, non-small cell lung and head/neck cancers. Importantly, CEACAM6 is a poor prognostic marker in CRC, while its expression correlates with tumor stage, metastasis and post-operative survival in PDA. CEACAM6 appears to be an immune checkpoint suppressor in hematologic malignancies including acute lymphoblastic leukemia and multiple myeloma. Several therapeutic monoclonal antibodies or antibody fragments targeting CEACAM6 have been designed and developed as a targeted therapy for human malignancies. A Llama antibody targeting CEACAM6 is being evaluated in early phase clinical trials. Conclusion: This review focuses on the role of CEACAM6 in the pathogenesis and signaling of the malignant phenotype in solid and hematologic malignancies and highlights its potential as a therapeutic target for anti-cancer therapy.

Epidermal growth factor receptor-targeted lipid nanoparticles retain self-assembled nanostructures and provide high specificity.

Next generation drug delivery utilising nanoparticles incorporates active targeting to specific sites. In this work, we combined targeting with the inherent advantages of self-assembled lipid nanoparticles containing internal nano-structures. Epidermal growth factor receptor (EGFR)-targeting, PEGylated lipid nanoparticles using phytantriol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG-maleimide amphiphiles were created. The self-assembled lipid nanoparticles presented here have internal lyotropic liquid crystalline nano-structures, verified by synchrotron small angle X-ray scattering and cryo-transmission electron microscopy, that offer the potential of high drug loading and enhanced cell penetration. Anti-EGFR Fab' fragments were conjugated to the surface of nanoparticles via a maleimide-thiol reaction at a high conjugation efficiency and retained specificity following conjugation to the nanoparticles. The conjugated nanoparticles were demonstrated to have high affinity for an EGFR target in a ligand binding assay.