An overview of site-specific methods for achieving antibody drug conjugates with homogenous drug to antibody ratio.

INTRODUCTION: Antibody drug conjugates (ADCs) have emerged as a potent tool in cancer treatment, where cytotoxic drugs are linked to antibodies targeting specific antigens. While conventional ADC synthesis methods have seen success as commercials therapeutics, there is a growing interest in next-generation ADCs, looking at homogeneity of the drug-to-antibody ratio. AREAS COVERED: The article provides a high-level overview for achieving said homogeneity by site-directed conjugations via encompassing engineered amino acids, enzyme-mediated strategies, peptide sequences, affinity peptides, and beyond. As the field rapidly evolves with multiple ADCs in clinical trials and the advent of biosimilars, the article explores the benefits and challenges in both conventional and non-platform ADC technologies. EXPERT OPINION: The choice of site selection approach must be based on multiple criteria as discussed in this report. Two ADCs made from conjugation to engineered cysteines have been approved by regulatory agencies which have contributed to the excitement in this space. For the others, though successful as proof-of-concept, the true test of merit will be determined as these technologies advance into the clinic. The promise of improving the therapeutics index and decreasing toxicities will continue to drive progress in this area.

Functional epitopes and neutralizing antibodies of vaccinia virus.

Smallpox is an infectious disease caused by the variola virus, and it has a high mortality rate. Historically it has broken out in many countries and it was a great threat to human health. Smallpox was declared eradicated in 1980, and Many countries stopped nation-wide smallpox vaccinations at that time. In recent years the potential threat of bioterrorism using smallpox has led to resumed research on the treatment and prevention of smallpox. Effective ways of preventing and treating smallpox infection have been reported, including vaccination, chemical drugs, neutralizing antibodies, and clinical symptomatic therapies. Antibody treatments include anti-sera, murine monoclonal antibodies, and engineered humanized or human antibodies. Engineered antibodies are homologous, safe, and effective. The development of humanized and genetically engineered antibodies against variola virus via molecular biology and bioinformatics is therefore a potentially fruitful prospect with respect to field application. Natural smallpox virus is inaccessible, therefore most research about prevention and/or treatment of smallpox were done using vaccinia virus, which is much safer and highly homologous to smallpox. Herein we summarize vaccinia virus epitope information reported to date, and discuss neutralizing antibodies with potential value for field application.

[Single chain antibody fragment display systems: a review].

Single chain antibody fragment (scFv) is a small molecule composed of a variable region of heavy chain (VH) and a variable region of light chain (VL) of an antibody, and these two chains are connected by a flexible short peptide. scFv is the smallest functional fragment with complete antigen-binding activity, which contains both the antibody-recognizing site and the antigen-binding site. Compared with other antibodies, scFv has the advantages of small molecular weight, strong penetration, low immunogenicity, and easy expression. Currently, the most commonly used display systems for scFv mainly include the phage display system, ribosome display system, mRNA display system, yeast cell surface display system and mammalian cell display system. In recent years, with the development of scFv in the field of medicine, biology, and food safety, they have also attracted much attention in the sectors of biosynthesis and applied research. This review summarizes the advances of scFv display systems in recent years in order to facilitate scFv screening and application.

Preclinical in vitro evaluation of immune suppression induced by GYM329, Fc-engineered sweeping antibody.

Fc-engineering is commonly used to improve the therapeutic potency of antibody (Ab) treatments. Because FcγRIIb is the only inhibitory FcγR that contains an immunoreceptor tyrosine-based inhibition motif (ITIM), Fc-engineered Abs with enhanced binding affinity to FcγRIIb might provide immune suppression in clinical contexts. GYM329 is an anti-latent myostatin Fc-engineered Ab with increased affinity to FcγRIIb which is expected to improve muscle strength in patients with muscular disorders. Cross-linking of FcγRIIb by immune complex (IC) results in phosphorylation of ITIM to inhibit immune activation and apoptosis in B cells. We examined whether the IC of Fc-engineered Abs with enhanced binding affinity to FcγRIIb causes phosphorylation of ITIM or B cell apoptosis using GYM329 and its Fc variant Abs in human and cynomolgus-monkey (cyno) immune cells in vitro. IC of GYM329 with enhanced binding affinity to human FcγRIIb (×5) induced neither ITIM phosphorylation nor B cell apoptosis. As for GYM329, FcγRIIb should work as an endocytic receptor of small IC to sweep latent myostatin, so it is preferable that GYM329 induces neither ITIM phosphorylation nor B cell apoptosis to prevent immune suppression. In contrast, IC of myo-HuCy2b, the Ab with enhanced binding affinity to human FcγRIIb (×4), induced ITIM phosphorylation and B cell apoptosis. The result of the present study demonstrated that Fc-engineered Abs with similar binding affinity to FcγRIIb had different effects. Thus, it is important to also investigate FcγR-mediated immune functions other than binding to fully understand the biological effects of Fc-engineered Abs.

HED, a Human-Engineered Domain, Confers a Unique Fc-Binding Activity to Produce a New Class of Humanized Antibody-like Molecules.

Our laboratory has identified and developed a unique human-engineered domain (HED) structure that was obtained from the human Alpha-2-macroglobulin receptor-associated protein based on the three-dimensional structure of the Z-domain derived from Staphylococcal protein A. This HED retains µM binding activity to the human IgG1CH2-CH3 elbow region. We determined the crystal structure of HED in association with IgG1's Fc. This demonstrated that HED preserves the same three-bundle helix structure and Fc-interacting residues as the Z domain. HED was fused to the single chain variable fragment (scFv) of mAb 4D5 to produce an antibody-like protein capable of interacting with the p185Her2/neu ectodomain and the Fc of IgG. When further fused with murine IFN-γ (mIFN-γ) at the carboxy terminus, the novel species exhibited antitumor efficacy in vivo in a mouse model of human breast cancer. The HED is a novel platform for the therapeutic utilization of engineered proteins to alleviate human disease.

Chemical Biology Approaches toward Precise Structure Control of IgG-Based Antibody-Oligonucleotide Conjugates.

Antibody-oligonucleotide conjugates (AOCs) are important tools for drug development and biochemical analysis. However, the structural heterogeneity of AOCs synthesized through conventional coupling methods raises reproducibility and safety concerns in clinical trials. To address these issues, different covalent coupling approaches have been developed to synthesize AOCs with precise site-specificity and degree of conjugation. This Concept article categorizes these approaches as linker-free or linker-mediated and provides details on their chemistry and potential applications. Several factors, including site-specificity, conjugation control, accessibility, stability, and efficiency, are highlighted when evaluating the pros and cons of these approaches. The article also discusses the future of AOCs, including the development of better conjugation approaches to ensure stimuli-responsive release and the application of high-throughput methods to facilitate their development.

Inhalable neutralizing antibodies - promising approach to combating respiratory viral infections.

Monoclonal antibodies represent an exciting class of therapeutics against respiratory viral infections. Notwithstanding their specificity and affinity, the conventional parenteral administration is suboptimal in delivering antibodies for neutralizing activity in the airways due to the poor distribution of macromolecules to the respiratory tract. Inhaled therapy is a promising approach to overcome this hurdle in a noninvasive manner, while advances in antibody engineering have led to the development of unique antibody formats which exhibit properties desirable for inhalation. In this Opinion, we examine the major challenges surrounding the development of inhaled antibodies, identify knowledge gaps that need to be addressed and provide strategies from a drug delivery perspective to enhance the efficacy and safety of neutralizing antibodies against respiratory viral infections.

Single chain antibody fragment display systems: a review / 生物工程学报

Single chain antibody fragment (scFv) is a small molecule composed of a variable region of heavy chain (VH) and a variable region of light chain (VL) of an antibody, and these two chains are connected by a flexible short peptide. scFv is the smallest functional fragment with complete antigen-binding activity, which contains both the antibody-recognizing site and the antigen-binding site. Compared with other antibodies, scFv has the advantages of small molecular weight, strong penetration, low immunogenicity, and easy expression. Currently, the most commonly used display systems for scFv mainly include the phage display system, ribosome display system, mRNA display system, yeast cell surface display system and mammalian cell display system. In recent years, with the development of scFv in the field of medicine, biology, and food safety, they have also attracted much attention in the sectors of biosynthesis and applied research. This review summarizes the advances of scFv display systems in recent years in order to facilitate scFv screening and application.

Construction, expression and functional characterization of an engineered antibody against tumor antigen MUC-1C.

Minibodies (single-chain Fv-CH3) are fusion proteins of a single-chain variable fragment (scFv) to the human IgG1 CH3 domain. They exhibit superior properties as compared to whole antibodies due to their smaller size and less complex composition, and also as compared to scFvs due to the two antigen-binding domains, for immunotherapy and imaging of various carcinomas including breast cancer. In the current study, efficient production of the recombinant anti-MUC-1 minibody for its dominant format (VH-VL) was obtained in the periplasmic space of the Escherichia coliBL21 (DE3) expression system. The active recombinant protein was successfully purified from soluble fraction. Functional assays presented the in vitro targeting properties and specificity of the expressed anti-MUC-1 HL minibody in the MUC-1 positive cell lines compared to normal cell.

Engineered human antibodies for the opsonization and killing of Staphylococcus aureus.

Gram-positive organisms with their thick envelope cannot be lysed by complement alone. Nonetheless, antibody-binding on the surface can recruit complement and mark these invaders for uptake and killing by phagocytes, a process known as opsonophagocytosis. The crystallizable fragment of immunoglobulins (Fcγ) is key for complement recruitment. The cell surface of S. aureus is coated with Staphylococcal protein A (SpA). SpA captures the Fcγ domain of IgG and interferes with opsonization by anti-S. aureus antibodies. In principle, the Fcγ domain of therapeutic antibodies could be engineered to avoid the inhibitory activity of SpA. However, the SpA-binding site on Fcγ overlaps with that of the neonatal Fc receptor (FcRn), an interaction that is critical for prolonging the half-life of serum IgG. This evolutionary adaptation poses a challenge for the exploration of Fcγ mutants that can both weaken SpA-IgG interactions and retain stability. Here, we use both wild-type and transgenic human FcRn mice to identify antibodies with enhanced half-life and increased opsonophagocytic killing in models of S. aureus infection and demonstrate that antibody-based immunotherapy can be improved by modifying Fcγ. Our experiments also show that by competing for FcRn-binding, staphylococci effectively reduce the half-life of antibodies during infection. These observations may have profound impact in treating cancer, autoimmune, and asthma patients colonized or infected with S. aureus and undergoing monoclonal antibody treatment.

The influence of antibody engineering on Fc conformation and Fc receptor binding properties: Analysis of FcRn-binding engineered antibodies and an Fc fusion protein.

Therapeutic immunoglobulin G (IgG) antibodies have comparatively long half-lives because the neonatal Fc receptor (FcRn) binds to the IgG Fc at acidic pH in the endosome and protects IgG from degradation. To further prolong the half-lives, amino acid-substituted antibodies having high affinity to FcRn are being developed, and one such therapeutic antibody (ravulizumab) has been approved. In this study, we investigated the binding property to FcγR and the conformation of seven FcRn affinity-modulated adalimumab variants to clarify the impact of the amino acid substitutions on the function and conformation of IgG Fc. The amino acid substitutions in T254-P261 caused a change in deuterium uptake into some regions of Fc in HDX-MS analysis, but those at T311, M432 and N438 did not cause such a change. The conformations around F245-L255 (FLFPPKPKDTL) were particularly influenced by the amino acid substitution in M256-P261, and the conformational changes of this region were correlated with the decrease of the affinity to FcγRIIIa. Additionally, we investigated the conformational difference of Fc between a Fc fusion protein (etanercept) and a native IgG (adalimumab). Although the Fc fusion proteins were expected to have similar FcRn affinity to IgGs, the affinity of etanercept to FcRn was lower than that of adalimumab, and its half-life was shorter than those of the IgG antibodies. Differences in deuterium uptakes were observed in the two regions where they were also detected in the adalimumab variants, and the conformational differences appeared to be an important factor for the low FcRn affinity of etanercept.

Murlentamab, a Low Fucosylated Anti-Müllerian Hormone Type II Receptor (AMHRII) Antibody, Exhibits Anti-Tumor Activity through Tumor-Associated Macrophage Reprogrammation and T Cell Activation.

AMHRII, the anti-Müllerian hormone receptor, is selectively expressed in normal sexual organs but is also re-expressed in gynecologic cancers. Hence, we developed murlentamab, a humanized glyco-engineered anti-AMHRII monoclonal antibody currently in clinical trial. Low-fucosylated antibodies are known to increase the antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) potency of effector cells, but some preliminary results suggest a more global murlentamab-dependent activation of the immune system. In this context, we demonstrate here that the murlentamab opsonization of AMHRII-expressing ovarian tumor cells, in the presence of unstimulated- or tumor-associated macrophage (TAM)-like macrophages, significantly promotes macrophage-mediated ADCC and shifts the whole microenvironment towards a pro-inflammatory and anti-tumoral status, thus triggering anti-tumor activity. We also report that murlentamab orients both unstimulated- and TAM-like macrophages to an M1-like phenotype characterized by a strong expression of co-stimulation markers, pro-inflammatory cytokines and chemokines, favoring T cell recruitment and activation. Moreover, we show that murlentamab treatment shifts CD4+ Th1/Th2 balance towards a Th1 response and activates CD8+ T cells. Altogether, these results suggest that murlentamab, through naïve macrophage orientation and TAM reprogrammation, stimulates the anti-tumor adaptive immune response. Those mechanisms might contribute to the sustained clinical benefit observed in advanced cancer patients treated with murlentamab. Finally, the enhanced murlentamab activity in combination with pembrolizumab opens new therapeutic perspectives.

Microcystin-LR heterologous genetically engineered antibody recombinant and its binding activity improvement and application in immunoassay.

Microcystin-LR (MC-LR) is a high-toxic biohazard that pollutes ecological environment and agroproducts. In this study, a newly recombined genetically engineered antibody (AVHH-MVH) with higher thermal stability and binding activity was designed by chain shuffling and based on our previously obtained anti-MC-LR scFv and nanobody. Based on AVHH-MVH template, a capacity of 8.99 × 105 CFU/mL of phage display AVHH-MVH mutagenesis library was constructed by site-directed mutagenesis in MVH-CDR3 region, and then used for ultrasensitive mutants screening. Afterwards, a total of five positive AVHH-MVH mutants were isolated from the mutagenesis library, and their binding activity was higher than AVHH-MVH for MC-LR. The AVHH-MVH mutant 3 was cloned into pET-25b vector for soluble expression, and the concentration of target protein expressed in culture system was 43.5 mg/L. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was established based on purified AVHH-MVH mutant 3 protein, and it showed ultrasensitive binding activity for MC-LR with the detection limit of 0.0075 µg/L, which was far below the maximum residue limit standard of 1.0 µg/L in drinking water proposed by World Health Organization. The established IC-ELISA shows good accuracy, repeatability, stability and applicability for MC-LR spiked samples, and it is promising for MC-LR ultrasensitive monitoring.

Macromolecules and Antibody-Based Drugs.

Macromolecule drugs particularly antibody drugs are very powerful therapies developing rapidly in the recent 20 years, providing hopes for many patients diagnosed with "incurable" diseases in the past. They also provide more effective and less side effects for many afflicting diseases, and greatly improve the survival rate and life quality of patients. In the last two decades, the proportion of US Food and Drug Administration (FDA) approved macromolecules and antibody drugs are increasing quickly, especially after the discovery of immune checkpoints. To crown all, the 2017 Nobel prize in physiology or medicine was given to immunotherapy. In this chapter, we would like to summarize the current situation of macromolecule and antibody drugs, and what effort scientists and pharmaceutical industry have made to discover and manufacture better antibody drugs.

Ion Channel Targeting with Antibodies and Antibody Fragments for Cancer Diagnosis.

The antibody era has greatly impacted cancer management in recent decades. Indeed, antibodies are currently applied for both cancer diagnosis and therapy. For example, monoclonal antibodies are the main constituents of several in vitro diagnostics, which are applied at many levels of cancer diagnosis. Moreover, the great improvement provided by in vivo imaging, especially for early-stage cancer diagnosis, has traced the path for the development of a complete new class of antibodies, i.e., engineered antibody fragments. The latter embody the optimal characteristics (e.g., low renal retention, rapid clearance, and small size) which make them ideal for in vivo applications. Furthermore, the present review focuses on reviewing the main applications of antibodies and antibody fragments for solid cancer diagnosis, both in vitro and in vivo. Furthermore, we review the scientific evidence showing that ion channels represent an almost unexplored class of ideal targets for both in vitro and in vivo diagnostic purposes. In particular, we review the applications, in solid cancers, of monoclonal antibodies and engineered antibody fragments targeting the voltage-dependent ion channel Kv 11.1, also known as hERG1.

Artificial immunoglobulin light chain with potential to associate with a wide variety of immunoglobulin heavy chains.

Immunoglobulins play important roles in antigen recognition during the immune response, and the complementarity-determining region (CDR) 3 of the heavy chain is considered as the critical antigen-binding site. We previously developed a statistical protocol for the extensive analysis of heavy chain variable region repertoires and the dynamics of their immune response using next-generation sequencing (NGS). The properties of important antibody heavy chains predicted in silico by the protocol were examined by gene synthesis and antibody protein expression; however, the corresponding light chain that matches with the heavy chain could not be predicted by our protocol. To understand the dynamics of the heavy chain and the effect of light chain pairing on it, we firstly tried to obtain an artificial light chain that pairs with a broad range of heavy chains and then analyzed its effect on the antigen binding of heavy chains upon pairing. During the pre-B cell stage, the surrogate light chain (SLC) could pair with the nascent immunoglobulin µ heavy chains (Ig-µH) and promote them to function in the periphery. On the basis of this property, we designed several versions of genetically engineered "common light chain" prototypes by modifying the SLC structure. Among them, the mouse-derived VpreB1λ5Cκ light chain showed acceptable matching property with several different heavy chains without losing specificity of the original heavy chains, though the antigen affinities were variable. The extent of matching depended on the heavy chain; surprisingly, a specific heavy chain (IGHV9-3) could match with two different conventional Vκs (IGKV3-2*01 and IGKV10-96*01) without losing the antigen affinities, whereas another heavy chain (IGHV1-72) completely lost its antigen affinities by the same matching. Thus, the results suggested that the antigen recognition of the heavy chain is variably affected by the paired light chain, and that the artificial light chain, Mm_VpreB1λ5Cκ, has the potential to be a "common light chain", providing a novel system to analyze the effects of light chains in antigen recognition of heavy chains.

Generation of human monoclonal antibody Fab fragment to Zika Virus by Phage Display Technology / 中华实验和临床病毒学杂志

Objective@#In this study, phage display technology was used to construct the human anti-Zika virus(ZIKV), phage antibody library and to obtain and express the monoclonal antibody. The aim was to master the preparation and expression of human phage antibody library screening method for highly specific antibodies.@*Methods@#The whole blood samples of Zika patients were collected and the lymphocytes were isolated. The RT-PCR method was used to amplify the antibody light chain and heavy chain Fab gene from lymphocyte Ig mRNA. The pComb3H system was used to construct the gene with genetic diversity Preparation of human anti-ZIKV phage antibody library. The purified antibody library was screened by using the purified ZIKV and the obtained ZIKV E protein antigen.@*Results@#The monoclonal antibody Fab fragment gene was successfully obtained for the ZIKV E protein antigen. The gene can be efficiently expressed in Escherichia coli.@*Conclusions@#According to the sequence analysis, this study showed that the monoclonal antibody was a new human genetically engineered antibody against ZIKV, which laid the foundation for the early diagnosis of ZIKV, and obtain a specific monoclonal antibody to ZIKV for human treatment of ZIKV infection.

Effect of linker flexibility and length on the functionality of a cytotoxic engineered antibody fragment.

Engineered antibody fragments often contain natural or synthetic linkers joining the antigen-binding domain and multimerization regions, and the roles of these linkers have largely been overlooked. To investigate linker effects on structural properties and functionality, six bivalent cytotoxic antibody fragments with of linkers of varying flexibility and length were constructed: (1) 10-AA mouse IgG3 upper hinge region, (2) 20-AA mouse IgG3 upper hinge region repeat, (3) 10-AA glycine and serine linker, (4) 20-AA glycine and serine linker repeat, (5) 21-AA artificial linker, and (6) no-linker control. Interestingly, a higher cytotoxicity was observed for fragments bearing the rigid short linkers compared to the flexible longer linkers. More importantly, amino acid composition related to the rigidity/flexibility was found to be of greater importance upon cytotoxicity than linker length alone. To further study the structure-function relationship, molecular modelling and dynamics simulation were exploited. Resultantly, the rigid mouse IgG3 upper hinge region was predicted to enhance structural stability of the protein during the equilibrium state, indicating the improved cytotoxicity over other combinations of fragments. This prediction was validated by measuring the thermal stability of the mouse IgG3 upper hinge as compared to the artificial linker, and shown to have a higher melting temperature which coincides with a higher structural stability. Our findings clearly suggest that appropriate linker design is required for enhancing the structural stability and functionality of engineered antibody fragments.

Antibody targeting of HER2/HER3 signaling overcomes heregulin-induced resistance to PI3K inhibition in prostate cancer.

Dysregulated expression and/or mutations of the various components of the phosphoinositide 3-kinase (PI3K)/Akt pathway occur with high frequency in prostate cancer and are associated with the development and progression of castration resistant tumors. However, small molecule kinase inhibitors that target this signaling pathway have limited efficacy in inhibiting tumor growth, primarily due to compensatory survival signals through receptor tyrosine kinases (RTKs). Although members of the epidermal growth factor receptor (EGFR), or HER, family of RTKs are strongly implicated in the development and progression of prostate cancer, targeting individual members of this family such as EGFR or HER2 has resulted in limited success in clinical trials. Multiple studies indicate a critical role for HER3 in the development of resistance against both HER-targeted therapies and PI3K/Akt pathway inhibitors. In this study, we found that the growth inhibitory effect of GDC-0941, a class I PI3K inhibitor, is markedly reduced in the presence of heregulin. Interestingly, this effect is more pronounced in cells lacking phosphatase and tensin homolog function. Heregulin-mediated resistance to GDC-0941 is associated with reactivation of Akt downstream of HER3 phosphorylation. Importantly, combined blockade of HER2 and HER3 signaling by an anti-HER2/HER3 bispecific antibody or a mixture of anti-HER2 and anti-HER3 antibodies restores sensitivity to GDC-0941 in heregulin-treated androgen-dependent and -independent prostate cancer cells. These studies indicate that the combination of PI3K inhibitors with HER2/HER3 targeting antibodies may constitute a promising therapeutic strategy for prostate cancer.

Engineered antibodies for molecular imaging of cancer.

Antibody technology has transformed drug development, providing robust approaches to producing highly targeted and active therapeutics that can routinely be advanced through clinical evaluation and registration. In parallel, there is an emerging need to access similarly targeted agents for diagnostic purposes, including non-invasive imaging in preclinical models and patients. Antibody engineering enables modification of key properties (immunogenicity, valency, biological inertness, pharmacokinetics, clearance route, site-specific conjugation) in order to produce targeting agents optimized for molecular imaging. Expanded availability of positron-emitting radionuclides has led to a resurgence of interest and applications of immunoPET (immuno-positron emission tomography). Molecular imaging using engineered antibodies and fragments provides a general approach for assessing cell surface phenotype in vivo and stands to play an increasingly important role in cancer diagnosis, treatment selection, and monitoring of molecularly targeted therapeutics.