Full-length Antibodies Research Articles

A Cell-free Expression Pipeline for the Generation and Functional Characterization of Nanobodies.

Cell-free systems are well-established platforms for the rapid synthesis, screening, engineering and modification of all kinds of recombinant proteins ranging from membrane proteins to soluble proteins, enzymes and even toxins. Also within the antibody field the cell-free technology has gained considerable attention with respect to the clinical research pipeline including antibody discovery and production. Besides the classical full-length monoclonal antibodies (mAbs), so-called “nanobodies” (Nbs) have come into focus. A Nb is the smallest naturally-derived functional antibody fragment known and represents the variable domain (VHH, ∼15 kDa) of a camelid heavy-chain-only antibody (HCAb). Based on their nanoscale and their special structure, Nbs display striking advantages concerning their production, but also their characteristics as binders, such as high stability, diversity, improved tissue penetration and reaching of cavity-like epitopes. The classical way to produce Nbs depends on the use of living cells as production host. Though cell-based production is well-established, it is still time-consuming, laborious and hardly amenable for high-throughput applications. Here, we present for the first time to our knowledge the synthesis of functional Nbs in a standardized mammalian cell-free system based on Chinese hamster ovary (CHO) cell lysates. Cell-free reactions were shown to be time-efficient and easy-to-handle allowing for the “on demand” synthesis of Nbs. Taken together, we complement available methods and demonstrate a promising new system for Nb selection and validation.

Structural Characterization of the Full-Length Anti-CD20 Antibody Rituximab.

Rituximab, a murine–human chimera, is the first monoclonal antibody (mAb) developed as a therapeutic agent to target CD20 protein. Its Fab domain and its interaction with CD20 have been extensively studied and high-resolution atomic models obtained by X-ray diffraction or cryo-electron microscopy are available. However, the structure of the full-length antibody is still missing as the inherent protein flexibility hampers the formation of well-diffracting crystals and the reconstruction of 3D microscope images. The global structure of rituximab from its dilute solution is here elucidated by small-angle X-ray scattering (SAXS). The limited data resolution achievable by this technique has been compensated by intensive computational modelling that led to develop a new and effective procedure to characterize the average mAb conformation as well as that of the single domains. SAXS data indicated that rituximab adopts an asymmetric average conformation in solution, with a radius of gyration and a maximum linear dimension of 52 Å and 197 Å, respectively. The asymmetry is mainly due to an uneven arrangement of the two Fab units with respect to the central stem (the Fc domain) and reflects in a different conformation of the individual units. As a result, the Fab elbow angle, which is a crucial determinant for antigen recognition and binding, was found to be larger (169°) in the more distant Fab unit than that in the less distant one (143°). The whole flexibility of the antibody has been found to strongly depend on the relative inter-domain orientations, with one of the Fab arms playing a major role. The average structure and the amount of flexibility has been studied in the presence of different buffers and additives, and monitored at increasing temperature, up to the complete unfolding of the antibody. Overall, the structural characterization of rituximab can help in designing next-generation anti-CD20 antibodies and finding more efficient routes for rituximab production at industrial level.

A Novel Full-length IgG Recombinant Antibody Highly Specific to Clothianidin and Its Application in Immunochromatographic Assay.

The toxicity of clothianidin to non-target organisms has gradually attracted world-wide attention. It is essential to develop reliable methods for the on-site detection of clothianidin residue. In this study, analogue-based heterologous ic-ELISAs were designed to rapidly screen desirable hybridomas, which could be used for the construction of recombinant antibodies (RAbs) against clothianidin. Based on the antibody variable region genes, two full-length IgG RAbs (1F7-RAb and 5C3-RAb) were produced by the mammalian cell expression system. The performance of the two RAbs was characterized and compared by heterologous ic-ELISAs and non-competitive surface plasmon resonance (SPR) assays. Using heterologous ic-ELISAs, the 1F7-RAb exhibited highly specific and sensitive recognition to clothianidin with an IC50 of 4.62 μg/L, whereas the 5C3-RAb could bind to both clothianidin and dinotefuran. The results of the non-competitive SPR assay further verified that the 1F7-RAb had a higher specificity and affinity to clothianidin than the 5C3-RAb. Finally, a gold immunochromatographic assay based on the novel antibody, 1F7-RAb, was developed for rapid detection of clothianidin with high sensitivity (visual detection limit of 2.5 μg/L), specificity, and good reproducibility, which can be used as an effective supervision tool for clothianidin residue in agricultural and environmental samples.

Exploring carbohydrate binding module fusions and Fab fragments in a cellulose-based lateral flow immunoassay for detection of cystatin C

This paper presents a lateral flow assay (LFA) for the quantitative, fluorescence-based detection of the kidney biomarker cystatin C that features conjugates of capture antibodies and fusions of carbohydrate binding modules (CBM) with ZZ domains anchored on cellulose deposited over nitrocellulose (NC). The ZZ-CBM3 fusion provides a biomolecular interface between the cellulose layer and the Fc portion of the capture antibodies. By resorting to detection Fab fragments that lack the Fc portion we overcome the observed interference of full-length detection antibodies with the ZZ-CBM3 fusion at the test lines. Using the new LFA architecture, a linear concentration–response relationship was observed in the 0–10 ng/mL cystatin C concentration range, which is compatible with the clinically normal (5–120 ng/mL) and abnormal (> 250 ng/mL) levels of cystatin C, as long as proper dilutions are made. An inter assay CoV of 0.72% was obtained. Finally, mock urine samples characteristic of normal (100 ng/mL) and kidney tubular disease (4000 ng/mL) patients were successfully analyzed. Overall, we demonstrate an innovative LFA architecture that combines NC strips with layered cellulose, ZZ-CBM3 fusions and fluorescently labeled Fab fragments.

Cover Feature: Investigating a Boronate‐Affinity‐Guided Acylation Reaction for Labelling Native Antibodies (Chem. Eur. J. 17/2022)

A boronic acid based biorthogonal probe in combination with an acyl donor has been introduced that enables site-specific incorporation of azide group at the Ser and Tyr residues of nonengineered full-length antibody. Further derivatization of this functional site was accomplished through click reaction, yielding well-defined immunoconjugates. The generality of this boronate-affinity method has been demonstrated on a number of monoclonal antibodies, all with different binding specificities. More information can be found in the Research Article by W.-S. Wayne Chang, C.-C. Lin et al. (DOI: 10.1002/chem.202104178).

Abstract OT2-15-01: Updated analysis of MCLA-128 (zenocutuzumab), trastuzumab, and vinorelbine in patients (pts) with HER2 positive/amplified (HER2+) metastatic breast cancer (MBC) who progressed on previous anti-HER2 ADCs

Abstract Background Zenocutuzumab is a humanized bispecific full-length IgG1 antibody targeting both HER2 and HER3 with enhanced ADCC activity. The unique Dock & Block mechanism inhibits HER3 from interacting with its ligands by targeting HER2 at a different epitope than trastuzumab, that optimally positions it to block HER2/HER3 dimerization and downstream PI3K/AKT/mTOR signaling. In MBC, HER3 overexpression and/or HER3 ligand upregulation are important drivers of carcinogenesis leading to trastuzumab resistance, indicating a potential role for zenocutuzumab. Preclinical activity was seen in HER2+ breast cancer models when zenocutuzumab was combined with trastuzumab. Single-agent zenocutuzumab showed consistent antitumor activity in heavily pretreated HER2+ MBC pts. This phase 2, open-label study explored zenocutuzumab/trastuzumab/vinorelbine in MBC. Methods This open-label study planned to enroll up to 40 evaluable pts with HER2+ MBC progressing after up to 5 anti-HER2 lines of therapy including trastuzumab, pertuzumab, and an anti-HER2 ADC. This sample size with a clinical benefit rate (CBR) of 45% would provide adequate precision to exclude 30% (lower limit of 90% CI > 30%). The threshold for the CBR rate at 24 w was based on the assumption that progression-free survival (PFS) follows an exponential distribution with a median of 5 months (clinically relevant) and 3.5 months (not clinically relevant).Pts received zenocutuzumab (750 mg, 2h IV), trastuzumab (8 mg/kg loading, then 6 mg/kg), and vinorelbine (25 mg/m², D1 and 8), q3w. A safety run-in of zenocutuzumab + trastuzumab ± vinorelbine was performed. The primary endpoint of the study was CBR at 24 w (tumor assessment [TA] by RECIST 1.1, per investigator), secondary endpoints include CBR at 24 w (TA by RECIST 1.1, per central review), overall response rate (ORR), safety, biomarkers, and pharmacokinetics. Cutoff date for the efficacy endpoints was 31Mar2021. This is an updated analysis of the 2020 ASCO abstract after all patients had completed at least 6 months of treatment or discontinued. Result total of 39 pts with a median of 3 lines (range 2-5) of prior anti-HER2 therapy including TDM1 received a median of 6 (range 1-23) cycles of zenocutuzumab. In the 37 pts evaluable for efficacy and with locally confirmed HER2 overexpression (3+ IHC or 2+ IHC confirmed by FISH), CBR at 24 w per investigator was 49% (90% CI: 34-63%); ORR was 27% (95% CI: 15-42%). The CBR at 24 w was consistent across different methods of HER2 overexpression/amplification detection (local vs central laboratory) and response assessment (investigator and central independent radiological review; see table below). As of a 12Jan2021 safety data update, the most common related AEs (all grades; G3-4) were neutropenia/neutrophil count decrease (61%; 46%), diarrhea (61%; 4%), asthenia/fatigue (46%; 0), and nausea (29%; 0). The PK half-life was 117h. The correlation of HER2-HER3 pathway activation at baseline with best ORR, duration of response, PFS, and overall survival was analyzed and will be presented in the poster. Conclusion Updated analyses confirm that the efficacy of zenocutuzumab combinations with trastuzumab/vinorelbine in heavily pretreated, HER2+ MBC, with progression after TDM-1, met prespecified protocol criteria for success. The regimen is safe and well tolerated with AEs mostly related to chemotherapy. CBR with zenocutuzumab, trastuzumab, and vinorelbinePopulationNCBR at 24 wks, %. (90% CI)HER2+ by local test/TA by RECIST1.1 per investigator3749 (34-63)HER2+ by central test/TA by RECIST1.1 per investigator2955.1 (38-71)HER2+ by local test/TA by RECIST1.1 by central independent radiologist review3644 (30-59)HER2+ by central test/TA by RECIST1.1 by central independent radiologist review2850.0 (33-67) Citation Format: Erika Hamilton, Thierry Petit, Barbara Pistilli, Anthony Goncalves, Ana Alexandra Ferreira, Florence Dalenc, Fatima Cardoso, Monica M Mita, Luis Manso, Syed M Karim, Francois-Clement Bidard, Philippe Aftimos, Santiago Escriváa-de-Romaníi, Noemia Afonso, Ernesto Wasserman, Kees Bol, Viktoriya Stalbovskaya, Anastasia Vliet, Anastasia Murat, Mohamed Bekradda, Thomas Bachelot. Updated analysis of MCLA-128 (zenocutuzumab), trastuzumab, and vinorelbine in patients (pts) with HER2 positive/amplified (HER2+) metastatic breast cancer (MBC) who progressed on previous anti-HER2 ADCs [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-15-01.

Evaluation of Cellular Targeting by Fab' vs Full-Length Antibodies in Antibody-Nanoparticle Conjugates (ANCs) Using CD4 T-cells.

Targeted delivery of chemotherapeutic drugs can improve their therapeutic efficiency by localizing their toxic effects at the diseased site. This is often achieved either by direct conjugation of drugs to antibodies targeting overexpressed receptors on cancer cells (antibody-drug conjugates/ADCs) or by conjugating antibodies to nanoparticles bearing drugs (antibody-nanoparticle conjugates/ANCs). Here, we report a platform for utilizing hinge cysteines on antigen-binding fragment (Fab') of an anti-CD4 antibody for site-specific conjugation to nanoparticles giving rise to anti-CD4 Fab'-nanoparticle conjugates (Fab'-NCs). We demonstrate a convenient route for obtaining functional anti-CD4 Fab' from full-length antibody and examine the targeted delivery efficiencies of anti-CD4 Fab'-NCs vs ANCs for selective delivery to CD4high mT-ALL cells. Our results indicate that higher avidity of full-length anti-CD4 antibody, i.e., protein alone translated to higher binding ability to CD4high mT-ALL cells in comparison with anti-CD4 Fab' alone. However, the targeted delivery efficiency of anti-CD4 Fab'-NCs was comparable to ANCs indicating that the avidity of Fab' is restored in a nanoparticle-conjugate format. Fab'-NCs are equally capable of achieving targeted drug delivery to CD4high T-cells as ANCs and are a versatile alternative to ANCs by offering site-selective modification strategy while retaining their advantages.

Co-Expression of Chaperones for Improvement of Soluble Expression and Purification of An Anti-HER2 scFv in Escherichia Coli.

Single-chain fragment variable (scFv) is one of the most commonly used antibody fragments. They offer some advantages over full-length antibodies, including better penetration to target tissues. However, their functional production has been a challenge for manufacturers due to the potential misfolding and formation of inclusion bodies. Here we evaluated the soluble expression and purification of molecular chaperone co-expression. E. coli BL21(DE3) cells were co-transformed with the mixture of plasmids pKJE7 and pET22b-scFv by the electroporation method. First, L-arabinose was added to induce the expression of molecular chaperones, and then IPTG was used as an inducer to start the expression of anti-HER2 scFv. The effect of cultivation temperature and IPTG concentration on soluble expression of the protein with or without chaperones was evaluated. The soluble expressed protein was subjected to native purification using the Ni-NTA affinity column. SDS-PAGE analysis confirmed the successful co-expression of anti-HER2-scFv and DnaK/DnaJ/GrpE chaperones. Co-expression with chaperones and low-temperature cultivation synergistically improved the soluble expression of anti-HER2 scFv. Co-expression with chaperone also exhibited an approximately four-fold increase in the final yield of purified soluble protein. The combination of co-expression with chaperones and low temperature presented in this work may be useful for the improvement of commercial production of other scFvs in E. coli as functionally bioactive and soluble form.

The Evolution of Targeted Radionuclide Diagnosis of HER2-Positive Breast Cancer.

This review examines the evolution of the radionuclide diagnosis of HER2-positive breast cancer using various compounds as a targeting module in clinical practice: from full-length antibodies to a new group of small synthetic proteins called alternative scaffold proteins. This topic is of especial relevance today in view of the problems attendant to the detection of breast cancer with HER2/neu overexpression, which, in most cases, introduce errors in the treatment of patients. The results of clinical studies of radiopharmaceuticals based on affibody molecules, ADAPTs, and DARPins for SPECT and PET have demonstrated good tolerability of the compounds, their rapid excretion from the body, and the possibility to differentiate tumor sites depending on the HER2/neu status. This indicates that targeted radionuclide diagnosis holds promise and the need to continue research in this direction.

Optimal Efficacy and Safety of Humanized Anti-Scg3 Antibody to Alleviate Oxygen-Induced Retinopathy.

The retinopathy of prematurity (ROP), a neovascular retinal disorder presenting in premature infants, is the leading causes of blindness in children. Currently, there is no approved drug therapy for ROP in the U.S., highlighting the urgent unmet clinical need for a novel therapeutic to treat the disease. Secretogranin III (Scg3) was recently identified as a disease-selective angiogenic factor, and Scg3-neutralizing monoclonal antibodies were reported to alleviate pathological retinal neovascularization in mouse models. In this study, we characterized the efficacy and safety of a full-length humanized anti-Scg3 antibody (hAb) to ameliorate retinal pathology in oxygen-induced retinopathy (OIR) mice, a surrogate model of ROP, by implementing histological and functional analyses. Our results demonstrate that the anti-Scg3 hAb outperforms the vascular endothelial growth factor inhibitor aflibercept in terms of efficacy and safety to treat OIR mice. Our findings support the development of anti-Scg3 hAb for clinical application.

Bispecific Antibodies for IFN-β Delivery to ErbB2+ Tumors.

The main aim of our work was to create a full-length bispecific antibody (BsAb) as a vehicle for the targeted delivery of interferon-beta (IFN-β) to ErbB2+ tumor cells in the form of non-covalent complex of BsAb and IFN-β. Such a construct is a CrossMab-type BsAb, consisting of an ErbB2-recognizing trastuzumab moiety, a part of chimeric antibody to IFN-β, and human IgG1 Fc domain carrying knob-into-hole amino acid substitutions necessary for the proper assembly of bispecific molecules. The IFN-β- recognizing arm of BsAb not only forms a complex with the cytokine but neutralizes its activity, thus providing a mechanism to avoid the side effects of the systemic action of IFN-β by blocking IFN-β Interaction with cell receptors in the process of cytokine delivery to tumor sites. Enzyme sandwich immunoassay confirmed the ability of BsAb to bind to human IFN-β comparable to that of the parental chimeric mAb. The BsAb binds to the recombinant ErbB2 receptor, as well as to lysates of ErbB2+ tumor cell lines. The inhibition of the antiproliferative effect of IFN-β by BsAb (IC50 = 49,3 µg/mL) was demonstrated on the HT29 cell line. It can be proposed that the BsAb obtained can serve as a component of the immunocytokine complex for the delivery of IFN-β to ErbB2-associated tumor cells.

Fully human recombinant antibodies against EphA2 from a multi-tumor patient immune library suitable for tumor-targeted therapy

ABSTRACT Enhanced EphA2 expression is observed in a variety of epithelial-derived malignancies and is an important target for anti-tumor therapy. Currently, Therapeutic monoclonal antibodies against immune checkpoints have shown good efficacy for tumor treatment. In this study, we constructed an immune single-chain fragment variable (scFv) library using peripheral blood mononuclear cells (PBMCs) from 200 patients with a variety of malignant tumors. High affinity scFvs against EphA2 can be easily screened from the immune library using phage display technology. Anti-EphA2 scFvs can be modified into any form of recombinant antibody, including scFv-Fc and full-length IgG1 antibodies, and the recombinant antibody affinity was improved following modification. Among the modified anti-EphA2 antibodies the affinity of 77-IgG1 was significantly increased, reaching a pmol affinity level (10−12). We further demonstrated the binding activity of recombinant antibodies to the EphA2 protein, tumor cells, and tumor tissues using macromolecular interaction techniques, flow cytometry and immunohistochemistry. Most importantly, both the constructed scFvs-Fc, as well as the IgG1 antibodies against EphA2 were able to inhibit the growth of tumor cells to some extent. These results suggest that the immune libraries from patients with malignant tumors are more likely to screen for antibodies with high affinity and therapeutic effect. The constructed fully human scFv immune library has broad application prospects for specific antibody screening. The screened scFv-Fc and IgG1 antibodies against EphA2 can be used for the further study of tumor immunotherapy.

Intra-articular delivery of full-length antibodies through the use of an in situ forming depot

Monoclonal antibodies (mAbs) are large size molecules that have demonstrated high therapeutic potential for the treatment of cancer or autoimmune diseases. Despite some excellent results, their intravenous administration results in high plasma concentration. This triggers off-target effects and sometimes poor targeted tissue distribution. To circumvent this issue, we investigated a local controlled-delivery approach using an in situ forming depot technology. Two clinically relevant mAbs, rituximab (RTX) and daratumumab (DARA), were formulated using an injectable technology based on biodegradable PEG-PLA copolymers. The stability and controlled release features of the formulations were investigated. HPLC and mass spectrometry revealed the preservation of the protein structure. In vitro binding of formulated antibodies to their target antigens and to their cellular FcγRIIIa natural killer cell receptor was fully maintained. Furthermore, encapsulated RTX was as efficient as classical intravenous RTX treatment to inhibit the in vivo tumor growth of malignant human B cells in immunodeficient NSG mice. Finally, the intra-articular administration of the formulated mAbs yielded a sustained local release associated with a lower plasma concentration compared to the intra-articular delivery of non-encapsulated mAbs. Our results demonstrate that the utilization of this polymeric technology is a reliable alternative for the local delivery of fully functional clinically relevant mAbs.

Trial in Progress: A Multicentre, Parallel Arm, Open-Label Trial of Frontline R-CHOP/Polatuzumab Vedotin-RCHP and Glofitamab in Younger Patients with Higher Risk Diffuse Large B Cell Lymphoma (COALITION)

Background:R-CHOP remains a standard frontline treatment for patients with DLBCL and high-grade B-cell lymphoma (HGBL). A significant proportion of patients will have refractory disease or subsequently relapse, particularly those with high-risk features such as an elevated IPI score or rearrangements of MYC and BCL2 and/or BCL6 (double/triple hit (DH/TH)). This population remains in need of improved induction treatments that can reduce the requirement for subsequent therapies which are associated with significant toxicities and diminishing response rates.Rationale:Glofitamab is a novel full-length bispecific antibody with a unique 2:1 configuration (two CD20 binding domains and one CD3 binding domain). In combination with a single pre-dose of obinutuzumab, glofitamab has demonstrated >70% complete remission in aggressive B-cell lymphoma at the recommended target dose in a phase 1 trial (Carlo-Stella, EHA 2021). Pre-clinical studies suggest that glofitamab's activity is retained in the presence of concomitant cytotoxic and CD20 antibody therapies, making it an attractive agent for combination with R-CHOP-like induction. Polatuzumab vedotin (pola) is an antibody-drug conjugate approved for R/R DLBCL in combination with BR, and is currently in evaluation for the front-line treatment of DLBCL in combination with RCHP in a randomised trial.The safety and preliminary efficacy of glofitamab in combination with R-CHOP, or pola-RCHP as a front-line treatment for high risk DLBCL is being evaluated.Study Design and Methods:This is a parallel-arm phase Ib/II trial. Treatment consists of an initial cycle of R-CHOP, followed by 5 cycles of combination induction treatment and 2 cycles of consolidation glofitamab monotherapy. Key inclusion criteria are: age 18-65 years, a diagnosis of DLBCL or HBGL, high-risk features (IPI ≥3 or NCCN-IPI ≥4 or presence of DH/TH), treatment naïve or after 1 cycle of R-CHOP, ECOG 0-3. The primary endpoint is the safety of the combination and secondary endpoints include complete response rates at interim and end of treatment FDG-PET assessments by Lugano criteria, progression free survival and overall survival. Correlative studies assessing baseline immunologic profiles, tumour phenotype and potential resistance mechanisms are planned.Approximately 40 patients will be treated in each arm across 12-14 Australian sites. The trial commenced recruitment in July 2021 (NCT04914741). The ability to recruit prior to either cycle 1 or cycle 2 allows seamless cross-referral from non-trial sites. [Display omitted] DisclosuresMinson: Hoffman La Roche: Research Funding; Novartis: Research Funding. Hamad: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Seymour: AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sunesis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Morphosys: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mei Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Research Funding, Speakers Bureau. Dickinson: Celgene: Research Funding; Amgen: Honoraria; Takeda: Research Funding; MSD: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead Sciences: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Other: travel, accommodation, expenses, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Stress-dependent Flexibility of a Full-length Human Monoclonal Antibody: Insights from Molecular Dynamics to Support Biopharmaceutical Development

After several decades of advancements in drug discovery, product development of biopharmaceuticals remains a time- and resource-consuming endeavor. One of the main reasons is associated to the lack of fundamental understanding of conformational dynamics of such biologic entities, and how they respond to various stresses encountered during manufacturing, storage, and shipping. In this work, we have studied the conformational dynamics of human IgG1κ b12 monoclonal antibody (mAb) using molecular dynamics simulations. The hundreds of nanoseconds long trajectories reveal that b12 mAb is highly flexible. Its variable domains show greater conformational fluctuations than the constant domains. Additionally, it collapses towards a more globular shape in response to thermal stress, leading to decrease in the total solvent exposed surface area and radius of gyration. This behavior is more pronounced for the deglycosylated b12 mAb, and it appears to correlate with increase in inter-domain contacts between specific regions of the antibody. Conformational fluctuations also cause transient formation and disruption of hydrophobic and charged patches on the antibody surface, which is particularly important for the prediction of CMC properties during development phases of antibody-based biotherapeutics. The insights gained through these simulations may help the development of biologic drugs.

Future Perspectives for Antiangiogenic Therapy in Retinal Diseases

The World Health Organization considers eye disorders as the serious problem of our time [1]. According to world statistics, the number of people with visual impairment is 1.3 billion, most of this number are people over 50 years old [2]. Over the past 20 years, developments in the treatment of AMD and fundus diseases have advanced and include drugs such as vascular endothelial growth factor inhibitors. The molecular structures of drugs intended for intravitreal use range from RNA aptamers (pegaptanib) to full-length monoclonal antibodies (mAb: bevacizumab) to Fab fragments (ranibizumab) and an antibody conjugate (aflibercept). In addition, single-chain variable fragment (scFv: brolucizumab), bispecific monoclonal antibody (faricimab) and DARPin (abigar pegol) show promising results in clinical trials.[6],[7] Brolucizumab (RTH258) was developed by ESBATech (ES-BATech AG — Schlieren ZH, Switzerland) originally under the name ESBA1008, an inhibitor of the humanized single chain antibody fragment (scFv) of all isoforms of vascular endothelial growth factor-A (VEGF-A). [6],[7],[11]. The Faricimab (ROCHE, Switzerland) molecule is characterized by the presence of a bispecific antibody that simultaneously binds to both VEGF-A and Ang-2; the drug consists of an anti-Ang-2 antigen-binding fragment (Fab), an anti-VEGF-A Fab and a crystallizing modified fragment (Fc region) with a total size of 150 kDa. This “crossover” effect provided high affinity for both targets while also maintaining a good stability profile compared to natural antibodies [8]. Abicipar Pegol (Abicipar, Allergan. Dublin, Ireland) is a DARPin aimed at binding all VEGF-A isoforms, like ranibizumab. It has a higher affinity and a longer half-life from the eye than ranibizumab (>13 days versus 7.2 days), making it a potential drug with a longer duration of action and the need for less frequent injections [15]. In this article, we tried to summarize the literature data on new anti-VEGF drugs being developed and ready for release. We hope that the appearance of these drugs on the market will make it possible to reduce the injection load on the patient and optimize their material costs.

An oncolytic virus expressing a full-length antibody enhances antitumor innate immune response to glioblastoma

Oncolytic herpes simplex virus-1 is capable of lysing tumor cells while alerting the immune system. CD47, in collaboration with SIRPα, represents an important immune checkpoint to inhibit phagocytosis by innate immune cells. Here we show locoregional control of glioblastoma by an oncolytic herpes virus expressing a full-length anti(α)-human CD47 IgG1 or IgG4 antibody. The antibodies secreted by the virus-infected glioblastoma cells block the CD47 ‘don’t eat me’ signal irrespective of the subclass; however, αCD47-IgG1 has a stronger tumor killing effect than αCD47-IgG4 due to additional antibody-dependent cellular phagocytosis by macrophages and antibody-dependent cellular cytotoxicity by NK cells. Intracranially injected αCD47-IgG1-producing virus continuously releases the respective antibody in the tumor microenvironment but not into systemic circulation; additionally, αCD47-IgG1-producing virus also improves the survival of tumor-bearing mice better than control oncolytic herpes virus combined with topical αCD47-IgG1. Results from immunocompetent mouse tumor models further confirm that macrophages, and to a lesser extent NK cells, mediate the anti-tumor cytotoxicity of antibody-producing oncolytic herpesviruses. Collectively, oncolytic herpes simplex virus-1 encoding full-length antibodies could improve immune-virotherapy for glioblastoma.

A comparative analysis of recombinant Fab and full‐length antibody production in Chinese hamster ovary cells

Monoclonal antibodies are the leading class of biopharmaceuticals in terms of numbers approved for therapeutic purposes. Antigen-binding fragments (Fab) are also used as biotherapeutics and used widely in research applications. The dominant expression systems for full-length antibodies are mammalian cell-based, whereas for Fab molecules the preference has been an expression in bacterial systems. However, advances in CHO and downstream technologies make mammalian systems an equally viable option for small- and large-scale Fab production. Using a panel of full-length IgG antibodies and their corresponding Fab pair with different antigen specificities, we investigated the impact of the IgG and Fab molecule format on production from Chinese hamster ovary (CHO) cells and assessed the cellular capability to process and produce these formats. The full-length antibody format resulted in the recovery of fewer mini-pools posttransfection when compared to the corresponding Fab fragment format that could be interpreted as indicative of a greater overall burden on cells. Antibody-producing cell pools that did recover were subsequently able to achieve higher volumetric protein yields (mg/L) and specific productivity than the corresponding Fab pools. Importantly, when the actual molecules produced per cell of a given format was considered (as opposed to mass), CHO cells produced a greater number of Fab molecules per cell than obtained with the corresponding IgG, suggesting that cells were more efficient at making the smaller Fab molecule. Analysis of cell pools showed that gene copy number was not correlated to the subsequent protein production. The amount of mRNA correlated with secreted Fab production but not IgG, whereby posttranscriptional processes act to limit antibody production. In summary, we provide the first comparative description of how full-length IgG and Fab antibody formats impact on the outcomes of a cell line construction process and identify potential limitations in their production that could be targeted for engineering increases in the efficiency in the manufacture of these recombinant antibody formats.

Facile Generation of Potent Bispecific Fab via Sortase A and Click Chemistry for Cancer Immunotherapy.

Simple SummaryThe formats of bispecific antibody have been investigated for many years to enhance the stability of the structure and anti-tumor efficacy. One of the formats combining two Fabs at their C termini provides unmodified variable region and comparable activity to other fragment-based bispecific antibodies that are usually combined in a head-to-tail manner. However, the current strategy to produce the BiFab molecule is limited to a semisynthetic method that introduces unnatural amino acid to antibodies’ sequences during production. To improve the application of BiFab format in investigational biodrugs, we have applied sortase A-mediated “bio-click” chemistry to generate BiFab, for facile assembly of Fab molecules that have been expressed and stored as BiFab module candidates. The BiFabs made by our method stimulate T cell proliferation and activation with favorable in vitro and in vivo anti-tumor activit. Our results indicate that BiFab made by sortase A-mediated click chemistry could be used to efficiently generate various BiFabs with high potency, which further supports personalized tumor immunotherapy in the future.Bispecific antibodies (BsAbs) for T cell engagement have shown great promise in cancer immunotherapy, and their clinical applications have been proven in treating hematological malignance. Bispecific antibody binding fragment (BiFab) represents a promising platform for generating non-Fc bispecific antibodies. However, the generation of BiFab is still challenging, especially by means of chemical conjugation. More conjugation strategies, e.g., enzymatic conjugation and modular BiFab preparation, are needed to improve the robustness and flexibility of BiFab preparation. We successfully used chemo-enzymatic conjugation approach to generate bispecific antibody (i.e., BiFab) with Fabs from full-length antibodies. Paired click handles (e.g., N3 and DBCO) was introduced to the C-terminal LPETG tag of Fabs via sortase A mediated transpeptidation, followed by site-specific conjugation between two click handle-modified Fabs for BiFab generation. Both BiFabCD20/CD3 (EC50 = 0.26 ng/mL) and BiFabHer2/CD3 exhibited superior efficacy in mediating T cells, from either PBMC or ATC, to kill target tumor cell lines while spared antigen-negative tumor cells in vitro. The BiFabCD20/CD3 also efficiently inhibited CD20-positive tumor growth in mouse xenograft model. We have established a facile sortase A-mediated click handle installation to generate homogeneous and functional BiFabs. The exemplary BiFabs against different targets showed superior efficacy in redirecting and activating T cells to specifically kill target tumor cells, demonstrating the robustness of sortase A-mediated “bio-click” chemistry in generating various potent BiFabs. This approach also holds promise for further efficient construction of a Fab derivative library for personalized tumor immunotherapy in the future.

ABCL-360: Glofitamab Step-Up Dosing (SUD): Updated Efficacy Data Show High Complete Response Rates in Heavily Pretreated Relapsed/Refractory (R/R) Non-Hodgkin Lymphoma (NHL) Patients (Pts)

<h3>Context</h3> Glofitamab, a T-cell-engaging, bispecific, full-length antibody, allows bivalent binding to CD20 (B-cells) and monovalent binding to CD3 (T-cells). In NP30179 (NCT03075696), an ongoing multicenter, phase I, dose-escalation and expansion study, glofitamab SUD, in addition to Gpt, allowed dose escalation ≤30 mg to maximize efficacy while mitigating cytokine release syndrome (CRS) (Hutchings <i>et al.</i> J Clin Oncol 2021). <h3>Objective</h3> To present updated efficacy data from glofitamab monotherapy SUD cohorts. <h3>Methods</h3> Gpt (1000 mg) was given 7 days pre-glofitamab initial dose. Intravenous glofitamab SUD was given on day (D) 1 and 8 of cycle (C) 1, then at target dose from C2D1 (2.5/10/16 mg or 2.5/10/30 mg); treatment continued for ≤12 cycles, every 21 days. Response rates were based on the Lugano criteria (Cheson <i>et al.</i> J Clin Oncol 2014). <h3>Results</h3> As of December 1, 2020, 52 pts received glofitamab SUD; 17 and 35 pts received 2.5/10/16 mg and 2.5/10/30 mg, respectively. Twenty-eight pts (53.8%) had aggressive NHL (aNHL) and 24 pts had indolent NHL (iNHL). Median age: 68 (44–85) years; median 3 (1–12) prior lines of therapy; 40 (76.9%) and 38 (73.1%) pts were refractory to their most recent and any prior CD20 therapy, respectively. Median follow-up duration: 6.3 months. Best overall response (OR) and complete metabolic response (CMR) rates for aNHL cohort: 64.3% and 57.1%, respectively. 4/5 pts (80%) with mantle cell lymphoma (2.5/10/16 mg, n=2; 2.5/10/30 mg, n=2) had CMR. For aNHL, 13/16 CMRs are ongoing, with 8 CMRs lasting >3 months. OR and CMR rates for pts with iNHL: 79.2% and 70.8%, respectively, with 14/17 CMRs ongoing and 10 CMRs lasting >3 months. As of August 3, 2020, common adverse events were CRS (63.5%), neutropenia (38.5%), and pyrexia (32.7%), with CRS mostly confined to C1. Grade (Gr) 1 and 2 CRS was reported in 18 (34.6%) and 12 (23%) pts, respectively; 3 pts had Gr 3 CRS; none had Gr 4/5 events (defined by ASTCT 2019 criteria). <h3>Conclusions</h3> Updated data for glofitamab monotherapy SUD show higher preliminary response rates than previously reported in pts with R/R NHL who have failed multiple lines of therapy. CRS was mostly manageable, low-grade, and confined to C1.