mAb Fragments Research Articles

Targeted hematopoietic stem cell depletion through SCF-blockade

BackgroundHematopoietic stem cell transplantation (HSCT) is a curative treatment for many diverse blood and immune diseases. However, HSCT regimens currently commonly utilize genotoxic chemotherapy and/or total body irradiation (TBI) conditioning which causes significant morbidity and mortality through inducing broad tissue damage triggering infections, graft vs. host disease, infertility, and secondary cancers. We previously demonstrated that targeted monoclonal antibody (mAb)-based HSC depletion with anti(α)-CD117 mAbs could be an effective alternative conditioning approach for HSCT without toxicity in severe combined immunodeficiency (SCID) mouse models, which has prompted parallel clinical αCD117 mAbs to be developed and tested as conditioning agents in clinical trials starting with treatment of patients with SCID. Subsequent efforts have built upon this work to develop various combination approaches, though none are optimal and how any of these mAbs fully function is unknown.MethodsTo improve efficacy of mAb-based conditioning as a stand-alone conditioning approach for all HSCT settings, it is critical to understand the mechanistic action of αCD117 mAbs on HSCs. Here, we compare the antagonistic properties of αCD117 mAb clones including ACK2, 2B8, and 3C11 as well as ACK2 fragments in vitro and in vivo in both SCID and wildtype (WT) mouse models. Further, to augment efficacy, combination regimens were also explored.ResultsWe confirm that only ACK2 inhibits SCF binding fully and prevents HSC proliferation in vitro. Further, we verify that this corresponds to HSC depletion in vivo and donor engraftment post HSCT in SCID mice. We also show that SCF-blocking αCD117 mAb fragment derivatives retain similar HSC depletion capacity with enhanced engraftment post HSCT in SCID settings, but only full αCD117 mAb ACK2 in combination with αCD47 mAb enables enhanced donor HSC engraftment in WT settings, highlighting that the Fc region is not required for single-agent efficacy in SCID settings but is required in immunocompetent settings. This combination was the only non-genotoxic conditioning approach that enabled robust donor engraftment post HSCT in WT mice.ConclusionThese findings shed new insights into the mechanism of αCD117 mAb-mediated HSC depletion. Further, they highlight multiple approaches for efficacy in SCID settings and optimal combinations for WT settings. This work is likely to aid in the development of clinical non-genotoxic HSCT conditioning approaches that could benefit millions of people world-wide.

Advancements in colorectal cancer detection: The role of immuno‐positron emission tomography, immuno‐single‐photon emission computed tomography, and machine learning applications

AbstractColorectal cancer (CRC) ranks as the world's second most prevalent cancer and third in mortality. Detection and diagnosis are crucial in research and clinical settings. While colonoscopy and computed tomographic colonography are widely used for identifying organic lesions, positron emission tomography (PET) and single‐photon emission computed tomography (SPECT) offer superior visualization of molecular changes. These immuno‐PET and immuno‐SPECT techniques surpass conventional [18F] Fluorodeoxyglucose PET/CT in specificity and sensitivity, improving CRC diagnostics and supporting therapeutic strategies. This review emphasizes the role of immuno‐PET/SPECT in CRC diagnosis and establishing a foundation for therapeutic strategies, facilitating hierarchical management through the identification of treatment‐responsive populations, prediction of therapeutic outcomes, and support for intraoperative imaging. This review introduces the preclinical and clinical utility of immunoconjugates for detecting colorectal adenomas, and primary, metastatic, or recurrent CRC, focusing on specific CRC cell targets like the epidermal growth factor receptor and carcinoembryonic antigen. The review also covers various mAb‐based immunoconjugates and engineered mAb fragments, including diabodies and minibodies. Finally, it looks into the great promise of machine learning in PET or SPECT and it addresses the challenges of translating preclinical successes into clinical practice for colorectal adenoma diagnosis, proposing potential solutions and directions for future research.

TCR-like bispecific antibodies toward eliminating infected hepatocytes in HBV mouse models

Therapeutics for eradicating hepatitis B virus (HBV) infection are still limited and current nucleos(t)ide analogs (NAs) and interferon are effective in controlling viral replication and improving liver health, but they cannot completely eradicate the hepatitis B virus and only a very small number of patients are cured of it. The TCR-like antibodies recognising viral peptides presented on human leukocyte antigens (HLA) provide possible tools for targeting and eliminating HBV-infected hepatocytes. Here, we generated three TCR-like antibodies targeting three different HLA-A2.1-presented peptides derived from HBV core and surface proteins. Bispecific antibodies (BsAbs) were developed by fusing variable fragments of these TCR-like mAbs with an anti-CD3ϵ antibody. Our data demonstrate that the BsAbs could act as T cell engagers, effectively redirecting and activating T cells to target HBV-infected hepatocytes in vitro and in vivo. In HBV-persistent mice expressing human HLA-A2.1, two infusions of BsAbs induced marked and sustained suppression in serum HBsAg levels and also reduced the numbers of HBV-positive hepatocytes. These findings highlighted the therapeutic potential of TCR-like BsAbs as a new strategy to cure hepatitis B.

Novel purification platform based on multimodal preparative scale separation of mAb fragments and aggregates

Monoclonal antibodies (mAbs) continue to dominate the biopharmaceutical industry. Certain mAbs are prone to fragmentation and clipping and in these cases, adequate removal of these species is critical during manufacturing. Fragments can be generated during fermentation, purification, storage, formulation, and administration. Their addition to the acidic charge-variant of the purified mAb has been reported to decrease stability and potency of the final product. However, contrary to mAb aggregation, manufacturers have not given much attention to removal of fragments and clipped species and as a result most conventional mAb platforms offer at best limited capabilities for their removal. In this study, we propose a novel purification platform that uses multimodal chromatography and achieves complete removal of a range of mAb fragments and clipped products (25–120 kDa). The utility of the platform has been successfully demonstrated for 2 IgG1s and 2 IgG4s. Further, adequate removal of the various host cell impurities such as host cell proteins (<10 ppm) and host cell DNA (<5 ppb) has been achieved. Finally, the platform was able to deliver adequate removal of high molecular weight impurities (<1 %) and a 30 % clearance of the acidic charge variant. The proposed single step has been shown to deliver what the polishing chromatography and intermediate purification chromatography steps deliver in a traditional mAb platform.

Supramolecular Heterodimer Peptides Assembly for Nanoparticles Functionalization.

Nanoparticle (NP) surface functionalization with proteins, including monoclonal antibodies (mAbs), mAb fragments, and various peptides, has emerged as a promising strategy to enhance tumor targeting specificity and immune cell interaction. However, these methods often rely on complex chemistry and suffer from batch-dependent outcomes, primarily due to limited control over the protein orientation and quantity on NP surfaces. To address these challenges, a novel approach based on the supramolecular assembly of two peptides is presented to create a heterotetramer displaying VHHs on NP surfaces. This approach effectively targets both tumor-associated antigens (TAAs) and immune cell-associated antigens. In vitro experiments showcase its versatility, as various NP types are biofunctionalized, including liposomes, PLGA NPs, and ultrasmall silica-based NPs, and the VHHs targeting of known TAAs (HER2 for breast cancer, CD38 for multiple myeloma), and an immune cell antigen (NKG2D for natural killer (NK) cells) is evaluated. In in vivo studies using a HER2+ breast cancer mouse model, the approach demonstrates enhanced tumor uptake, retention, and penetration compared to the behavior of nontargeted analogs, affirming its potential for diverse applications.

Physical origin of the peak tailing of monoclonal antibodies in size-exclusion chromatography using bio-compatible systems and columns.

The analysis of mixtures containing monoclonal antibody (mAb) (approximately 150 kDa molecular weight) and sub-unit impurities (approximately 100 kDa) is challenging, even when adopting the latest ultra-high-pressure liquid chromatography (UHPLC) columns (4.6 mm [Formula: see text] 150mm coated hardware, 1.7 [Formula: see text]m 250 BEH[Formula: see text] Surface-modified Particles) and systems (ACQUITY[Formula: see text] UPLC[Formula: see text] I-class Bio Plus). The main issue still encountered is a persistent tail of the mAb peak. Here, the physical origin(s) of such peak tailing in size-exclusion chromatography (SEC) are investigated from both fundamental and practical approaches. Up to five relevant physical origins are analyzed: sample heterogeneity (glycoforms), UHPLC system dispersion, strong residual binding of the mAb to the SEC particles (via hydrophobic and/or electrostatic interactions) and to the stainless steel column/system hardware, slow escape kinetics of the mAb from the SEC particles, and flow heterogeneity caused by the non-ideal slurry packing of SEC columns. Experiments (testing sample heterogeneity, system dispersion, and strong residual interactions) and calculations (predicting the transient absorption/escape kinetics in a single SEC particle and the two-dimensional peak concentration profiles) altogether unambiguously demonstrate that the observed mAb peak tailing is caused primarily by the long-range velocity biases across the SEC column combined with the slow transverse dispersion of mAbs. Therefore, improvement in the resolution between mAb and sub-unit fragment impurities can only be achieved by increasing the column length, e.g., by applying recycling chromatography at acceptable pressures.

DEVELOPMENT OF A METHOD FOR PRODUCING FAB/F(AB’)2 FRAGMENTS FROM A FULL-LENGTH MONOCLONAL ANTIBODY FOR BIOANALYTICAL ASSAYS

Abstract Background Monoclonal antibodies (mAb) comprise of two Fab fragments and one Fc fragment or one F(ab)’2 fragment and one Fc fragment. While a full-length mAb is frequently used as an assay reagent for bioanalysis, mAb fragments are required in certain cases. For example, to build a sandwich assay for detection of anti-drug antibodies (ADA) for therapeutic antibodies, Fab or F(ab)’2 fragment is used instead of a full-length mAb as capture reagent. This is because therapeutic antibodies, either humanized or fully human, are in many ways indistinguishable from the ADA generated in patients, especially in Fc fragment. When ADA detection methods utilizes anti-human Fc antibodies as the detection reagent, the full-length mAb drug will be directly bound by the detection reagent, causing interference. Preparation of a Fab or F(ab)’2 fragment is therefore needed. Methods and results We are developing a method for enzymatic digestion of therapeutic antibodies to generate monovalent Fab or bivalent F(ab’)2 fragments in this study. With such reagents becoming available, a sandwich ADA assay formats can be expanded to allow anti-human Fc antibodies as detection regents. To standardize the method, we explored various enzymatic conditions, including type of enzymes (i.e., pepsin, papain, and IdeS Protease), digestion-time (1, 2, 4, and 6 h), enzyme to antibody ratio (1:10, 1:20, and 1:40 w/w), IgG species and isotypes (human IgG1-κ, IgG1-λ, and IgG4-κ). The enzymatic hydrolysates were quantified by NanoDrop and purified by dialysis (10K MWCO) and Protein A/G/L magnetic bead methods. The effective recovery of truncated antibodies was &amp;gt; 90%, as assessed by reduced/non-reduced SDS-PAGE and ELISA analysis. Digestion of human IgG1 and IgG4 with pepsin resulted into a complete cleavage into F(ab')2 fragments and degradation of Fc fragments. While IdeS Protease produced an equivalent quantity of F(ab’)2 and Fc fragments with a similar efficiency, removal of the intact Fc fragment was required as an additional step. If the Fab fragments were desired, papain could be used with yield being over 90%. We have subsequently utilized either Fab or F(ab’)2 as a capture reagent for ADA detection. Conclusion We have successfully developed the enzymatic digestion method to prepare Fab or F(ab’)2 fragments. The optimized conditions described here are broadly applicable to different IgG isotypes across many therapeutic antibodies.

Resolution limits of size exclusion chromatography columns identified from flow reversal and overcome by recycling liquid chromatography to improve the characterization of manufactured monoclonal antibodies

The flow reversal (FR) technique consists of reversing the flow direction along a chromatographic column. It is used to reveal the origin (such as poor column packing, active sites, or slow absorption/escape kinetics) for the resolution limit of 4.6 mm × 150 mm long columns packed with 1.7 μm 200 Å Bridge-Ethylene-Hybrid (BEHTM) Particles. These columns are used to separate manufactured monoclonal antibodies (mAb, ∼ 150 kDa) from their close impurities (or IdeS fragments, ∼ 100 kDa) by size exclusion chromatography (SEC). FR unambiguously demonstrates that the resolution limit of these SEC columns is primarily due to long-range flow velocity biases covering distances of at least 500 μm across the column diameter. This confirms the existence of center-to-wall flow heterogeneities which cause undesirable tailing for the mAb peak. Because the transverse dispersion coefficient (Dt=1.1 × 10−6 cm2/s) of mAbs across the column diameter is intrinsically low, the bandspreading of the mAb in a single flow direction is in part reversible upon reversing the flow direction. For the very same residence time in the column, the column efficiency is found to increase by +85% relative to that observed under conventional elution mode. The observed peak tailing of the mAb and its sub-units is not caused by active surface sites or by slow absorption/escape from the BEH Particles. Therefore, the most critical mAb impurities (hydrolytic degradation Fab/c and IdeS F(ab′)2 fragments) can only be successfully separated and quantified with acceptable accuracy by adopting alternate pumping recycling liquid chromatography (APRLC). APRLC enables the full baseline separation of the mAb and 100 kDa mAb fragments and partial separation of Fab/c and F(ab′)2 fragments after increasing the number of cycles to ten. It was made possible to accurately measure the relative abundances of the mAb (99.0 ± 0.1%), F(ab′)2 fragment (0.88 ± 0.03%), and Fab/c immunogenic fragment (0.13 ± 0.02%) in less than 45 min for a total mAb sample load of only 5 μg. Still, further improvements are needed to increase the sensitivity of the APRLC method and to reduce the solvent consumption by adopting narrow-bore 2.1 mm i.d. SEC columns.

Optimization and Characterization of Novel ALCAM-Targeting Antibody Fragments for Transepithelial Delivery.

Activated leukocyte cell adhesion molecule (ALCAM) is a cell adhesion molecule that supports T cell activation, leukocyte migration, and (lymph)angiogenesis and has been shown to contribute to the pathology of various immune-mediated disorders, including asthma and corneal graft rejection. In contrast to monoclonal antibodies (mAbs) targeting ALCAM's T cell expressed binding partner CD6, no ALCAM-targeting mAbs have thus far entered clinical development. This is likely linked with the broad expression of ALCAM on many different cell types, which increases the risk of eliciting unwanted treatment-induced side effects upon systemic mAb application. Targeting ALCAM in surface-exposed tissues, such as the lungs or the cornea, by a topical application could circumvent this issue. Here, we report the development of various stability- and affinity-improved anti-ALCAM mAb fragments with cross-species reactivity towards mouse, rat, monkey, and human ALCAM. Fragments generated in either mono- or bivalent formats potently blocked ALCAM-CD6 interactions in a competition ELISA, but only bivalent fragments efficiently inhibited ALCAM-ALCAM interactions in a leukocyte transmigration assay. The different fragments displayed a clear size-dependence in their ability to penetrate the human corneal epithelium. Furthermore, intranasal delivery of anti-ALCAM fragments reduced leukocyte infiltration in a mouse model of asthma, confirming ALCAM as a target for topical application in the lungs.

Monitoring oxidation in recombinant monoclonal antibodies at subunit level through two-dimensional liquid chromatography coupled with mass spectrometry

Monoclonal antibodies (mAbs) are highly susceptible to chemical/post-translational modifications (PTMs), a crucial concern for chemical and biological safety during mAb production and development. Modifications in different mAb domains have different consequences on mAb stability and activity. Hence, characterization and monitoring of domain-specific modification in mAbs is of significant interest so as to be able to assure consistency in product quality. Generally, mAb samples are enzymatically digested and analyzed with traditional reversed-phase chromatography for domain-specific characterization. However, conventional analytical methods do not provide complete resolution and identification of mAb modifications. Here, a novel workflow of two-dimensional liquid chromatography with mass spectrometry interfacing has been proposed to overcome the limitations of conventional one-dimensional analytical methods and provide complete information on oxidized mAb domains. The proposed method successfully analysed the oxidized mAb samples and the control sample at the subunit level, simultaneously separating oxidized variants that are otherwise not detected by one-dimensional methods. The proposed 2D WCX-RPC-MS setup uses 2D multiple heart-cut method that identified 10 oxidation variants in mAb A, 6 in the Fab fragment and 4 in the Fc fragment, of which 3 were exclusive and not identified by the conventional 1D analysis. Further the 2D method takes only 7–13 h in sample preparation and analysis to anticipate oxidation in mAb fragments. Hence, the proposed workflow offers a faster and more convenient platform for routine monitoring and domain-specific mAb characterization with mass spectrometry coupling.

Monoclonal antibodies (mAbs) optical detection by coupling innovative imprinted biopolymers and magnetic beads: The case of therapeutic mAb anti-myostatin detection

Immunotherapy has revolutionized modern medicine, becoming the largest part of the growing bio-drugs market. Along with the progress in this direction, the need for reliable monoclonal antibodies (mAbs) monitoring methods is a current foremost issue. In this scenario, the goal of this study was to design a straightforward beads-based plasmonic approach that combines magnetic beads (MBs) with a polynorepinephrine imprinted biopolymer (IBP) for real-time mAbs detection in biological matrices. Specifically, MBs-encoded by a specific antigen were exploited not only for the selective capture of the target mAb from human serum, but also MBs are directly involved in the molecular architecture of a sandwich assay. The mAb extraction from the real matrix occurred in a site-oriented manner by exploiting a paratope-epitope specific recognition, while leaving the constant mAb fragment (Fc) free to interact with an IBP specific for the mAb Fc portion. Anti-MYO-029, a recombinant monoclonal IgG1 antibody developed for myostatin inhibition and muscular-wasting disorders’ treatment in clinical settings, was addressed as biological target to evaluate the analytical performances of the beads-based sensing assay. Myostatin-neutralizing antibodies may be misused as performance-enhancing drugs in sports competitions and their effective quantification has a central role also in the anti-doping control field. The optimization of the assay conditions led to establishing an assay able to achieve very good analytical performances in terms of repeatability and sensitivity, with negligible cross-reactivity with other Ig classes and IgG subclasses. In this frame, we proposed an extremely modulable assay, with potential large applicability, for multiplexing mAbs detection.

CRISPR/Cas9-based Engineering of Immunoglobulin Loci in Hybridoma Cells.

Development of the hybridoma technology by Köhler and Milstein (1975) has revolutionized the immunological field by enabling routine use of monoclonal antibodies (mAbs) in research and development efforts, resulting in their successful application in the clinic today. While recombinant good manufacturing practices production technologies are required to produce clinical grade mAbs, academic laboratories and biotechnology companies still rely on the original hybridoma lines to stably and effortlessly produce high antibody yields at a modest price. In our own work, we were confronted with a major issue when using hybridoma-derived mAbs: there was no control over the antibody format that was produced, a flexibility that recombinant production does allow. We set out to remove this hurdle by genetically engineering antibodies directly in the immunoglobulin (Ig) locus of hybridoma cells. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and homology-directed repair (HDR) to modify antibody's format [mAb or antigen-binding fragment (Fab')] and isotype. This protocol describes a straightforward approach, with little hands-on time, leading to stable cell lines secreting high levels of engineered antibodies. Parental hybridoma cells are maintained in culture, transfected with a guide RNA (gRNA) targeting the site of interest in the Ig locus and an HDR template to knock in the desired insert and an antibiotic resistance gene. By applying antibiotic pressure, resistant clones are expanded and characterized at the genetic and protein level for their ability to produce modified mAbs instead of the parental protein. Finally, the modified antibody is characterized in functional assays. To demonstrate the versatility of our strategy, we illustrate this protocol with examples where we have (i) exchanged the constant heavy region of the antibody, creating chimeric mAb of a novel isotype, (ii) truncated the antibody to create an antigenic peptide-fused Fab' fragment to produce a dendritic cell-targeted vaccine, and (iii) modified both the constant heavy (CH)1 domain of the heavy chain (HC) and the constant kappa (Cκ) light chain (LC) to introduce site-selective modification tags for further derivatization of the purified protein. Only standard laboratory equipment is required, which facilitates its application across various labs. We hope that this protocol will further disseminate our technology and help other researchers. Graphical abstract.

Clip Formation in the Complementarity Determining Region of Bevacizumab Lowers Monomer Stability and Affinity for Both FcRn and FcγR: A Comprehensive Characterization of the Clipped Variant Including its Higher Order Structure

The presence of monoclonal antibody (mAb) fragments in pharmaceutical mAb products is a critical quality attribute and should be controlled for safety. Several mAb fragments derived from clip formation in the complementarity determining regions (CDRs), as well as from cleavage in the hinge region, have been reported. However, the properties of CDR-clipped variants are not fully understood because of difficulties in separating them from intact mAbs under non-denaturing conditions due to similarities in size. We have established a method for separating CDR-clipped variants under non-denaturing conditions using an appropriate size exclusion chromatography column.1 In this report, we provide a comprehensive characterization of a CDR-clipped variant from bevacizumab. The variant exhibited a lower pI, a higher tendency to form dimers, and a lower affinity for both neonatal Fc receptor (FcRn) and Fcγ receptor (FcγR). The effects of clip formation in CDR H3 on the higher order structure were analyzed by hydrogen/deuterium exchange mass spectrometry, and the observed changes in the structures of the VH, CH2, and VL domains were in agreement with the lowered affinity for antigen, FcRn, and FcγR. These findings suggest that clip formation in the CDR may affect the efficacy, safety, and pharmacokinetics of pharmaceutical mAbs.

SARS-CoV-2-neutralising monoclonal antibodies to prevent COVID-19.

SARS-CoV-2-neutralising monoclonal antibodies to prevent COVID-19.

Design of a Novel Fab-Like Antibody Fragment with Enhanced Stability and Affinity for Clinical use.

With increasing interest in applying recombinant monoclonal antibodies (mAbs) in human medicine, engineered mAb fragments with reduced size and improved stability are in demand to overcome current limitations in clinical use. Herein, a novel Fab-like antibody fragment generated via an in silico-based engineering approach where the CH1 and CL domains of Fab are replaced by the IgG1 CH3 domains is described. This construct, designated as FabCH3, maintains the natural N-terminus and C-terminus of IgG antibody, can be expressed at a high level in bacterial cells and, importantly, exhibits much higher stability and affinity than the parental Fab when tested in a mesothelin-specific Fab m912, as well as a vascular endothelial growth factor A (VEGFA)-specific Fab Ranibizumab (in vivo). The high-resolution crystal structures of m912 FabCH3 and m912 Fab are determined, and the comparative analysis reveals more rigid structures in both constant domains and complementarity-determining regions of FabCH3, explaining its enhanced stability and affinity. Overall, the stabilized FabCH3 described in this report provides a versatile platform for engineering Fab-like antibody fragments with higher stability and antigen-binding affinity that can be used as a distinct class of antibody therapeutics.

Radioimmunotherapy in Oncology: Overview of the Last Decade Clinical Trials.

Simple SummaryMonoclonal antibody-bearing radionuclides have been under clinical investigation over the last two decades for their use in theranostic (diagnostic and therapeutic) applications in cancer. However, despite the numerous trials that have been conducted, only two radioimmunotherapies (RIT) have been approved by the FDA for the targeted therapy of hematologic tumors expressing CD20 antigens. Moreover, RIT applications for solid cancers faced major issues—such as radiotoxicity due to low antibodies penetrance requiring substantial curative dose—where new discoveries concerning antibody engineering or radionuclides are trying to overcome. Here, we performed an overview of the last 11-year clinical trials involving RIT for solid and non-solid cancers conducted either with full antibodies or antibody fragments. We discussed the low-to-moderate efficiency of RIT compared to conventional therapies and described the last advances in clinic for antibodies carriers (F(ab′)2, Fab′, ScFv). Finally, we discussed about the complexity of RIT as a therapy and depicted both the issues and the prospects of such a strategy. The specific irradiation of tumors with selective radiolabeled antibodies constitutes an attractive therapeutic approach. Consequent preclinical research has been conducted by both biologists to identify pertinent targets and to select corresponding antibodies (mAb) and by radiochemists to radiolabel mAbs. These numerous preclinical investigations have ascertained the therapeutic interest of radioimmunotherapy (RIT) protocols in mice models. Here, we summarize the clinical studies that have been performed the last decade, including clinical trials (phases I, II, and III), prospective and retrospective studies, and cases series. We thereby reported 92 clinical studies. Among them, 62 concern the treatment of hematological malignancies, and 30 concern solid tumors. For hematologic diseases, the analysis was complex due to the high discrepancy of therapeutic strategies (first-line therapy, consolidation, stem cell transplantation conditioning) as well as the high variety of malignancies that were treated. The clinical studies from the last decade failed to expand anti-CD20 RIT indications but confirmed that RIT using radiolabeled anti-CD20 remains a pertinent choice for patients with relapse follicular lymphomas. For solid tumors, the positive benefit of RIT is more mitigated, apart for few malignancies that can be treated locally. Clinical trials also demonstrated the potential of some antibody formats, such as F(ab′)2, which has already been approved by the China State FDA under the trend name Licartin®. Despite disparate results, mAb fragments are an interesting prospect for the improvement of RIT efficiency as well as for pretargeted strategies that delay the injection of radioactive treatments from the mAb ones.

Systematic workflow for studying domain contributions of bispecific antibodies to selectivity in multimodal chromatography.

In this article, a systematic workflow was formulated and implemented to understand selectivity differences and preferred binding patches for bispecific monoclonal antibodies (mAbs) and their parental mAbs on three multimodal cation exchange resin systems. This workflow incorporates chromatographic screening of the parent mAbs and their fragments at various pH followed by surface property mapping and protein footprinting using covalent labeling followed by liquid chromatography-mass spectrometry analysis. The chromatography screens on multimodal resins with the intact mAbs indicated enhanced selectivity as compared to single-mode interaction systems. While the bispecific antibody (bsAb) eluted between the two parental mAbs on most of the resins, the retention of the bispecific transitioned from co-eluting with one parental mAb to the other parental mAb on Capto MMC. To investigate the contribution of different domains, mAb fragments were evaluated and the results indicated that the interactions were likely dominated by the Fab domain at higher pH. Protein surface property maps were then employed to hypothesize the potential preferred binding patches in the solvent-exposed regions of the parental Fabs. Finally, protein footprinting was carried out with the parental mAbs and the bsAb in the bound and unbound states at pH 7.5 to identify the preferred binding patches. Results with the intact mAb analysis supported the hypothesis that interactions with the resins were primarily driven by the residues in the Fab fragments and not the Fc. Furthermore, peptide mapping data indicated that the light chain may be playing a more important role in the higher binding of Parent A as compared with Parent B in these resin systems. Finally, results with the bsAb indicated that both halves of the molecule contributed to binding with the resins, albeit with subtle differences as compared to the parental mAbs. The workflow presented in this paper lays the foundation to systematically study the chromatographic selectivity of large multidomain molecules which can provide insights into improved biomanufacturability and expedited downstream bioprocess development.

Identification and Characterization of a Monoclonal Antibody Variant Species with a Clipping in the Complementarity Determining Region Isolated by Size Exclusion Chromatography Under Native Conditions.

The content of monoclonal antibody (mAb) fragments in pharmaceutical mAb products is a critical quality attribute and should be controlled for safety. Peptide bonds in the hinge region of mAbs are susceptible to hydrolysis, generating Fc-Fab fragments, which are associated with lower efficacy than the intact antibody. Fc-Fab fragments can be separated from intact antibody molecules under native conditions by size exclusion chromatography (SEC). Although several fragments generated by a clip in the complementarity determining region (CDR) have been reported, their efficacies have not been analyzed. This is because these fragments could not be separated from intact antibodies under native conditions owing to their similar molecular sizes. Here, we report that bevacizumab variant with clipping in the CDR, with the resulting fragments remaining intact in the variant, can be isolated under native conditions by selecting an adequate SEC column.

A phase 1 dose-escalation study of a PD-L1xCD27 bispecific antibody CDX-527 in patients with advanced malignancies.

2585 Background: CDX-527 is a bispecific antibody (BsAb) targeting PD-L1 and CD27 that is designed to block immune checkpoint PD-L1/PD-1 interactions while providing immune costimulation through CD27 signaling. CD27 is a key immunostimulatory molecule that enhances T cell activation, effector function, and survival. Combining anti-PD-L1 and anti-CD27 mAbs synergize in preclinical studies, activating complementary cytotoxic and proliferative gene expression profiles, respectively. Clinical studies demonstrated the safety and biological activity of combining varlilumab, an agonist anti-CD27 mAb, with nivolumab or atezolizumab, along with modest clinical activity of the combinations. CDX-527 is a novel human BsAb containing a neutralizing, high affinity IgG1k PD-L1 mAb and the single chain Fv fragment (scFv) of an agonist anti-CD27 mAb genetically attached to the C-terminus of each heavy chain, thereby making CDX-527 bivalent for each target. Pre-clinical studies demonstrated enhanced T cell activation by CDX-527 and anti-tumor activity of a surrogate bispecific compared to individual mAb combinations. Methods: CDX527-01 is a phase 1 first-in-human, open-label, multi-center, dose-escalation (DE) and expansion study evaluating safety, pharmacokinetics (PK), pharmacodynamics (PD), and clinical activity of CDX-527 in patient with advanced solid tumors that have progressed on standard-of-care therapy. The primary study objective is to characterize the safety and tolerability of CDX-527. CDX-527 is administered intravenously Q2W with doses ranging from 0.03 mg/kg up to 10.0 mg/kg or until the maximum tolerated dose. The first 2 cohorts of the DE phase initiate with single patients and subsequent DE cohorts will be conducted in 3+3 manner. Tumor-specific expansion cohorts may be enrolled to further characterize the safety, PK, PD, and efficacy of CDX-527. Tumor assessments are performed Q8W by the investigator per iRECIST. Biomarker assessments include characterizing the effects on peripheral blood immune cells and cytokines, and for the expansion cohorts, the impact of CDX-527 on the tumor microenvironment in paired tumor biopsies. Results: To date, 8 patients have received CDX-527 in doses ranging from 0.03 mg/kg to 1 mg/kg and 3 are still on treatment. There has been no drug related SAEs, DLTs or discontinuations due to an AE. Most common treatment related AEs were influenza-like illness, fatigue, and arthralgia (all at 25%). All drug related AEs have been grade 1 or 2. Conclusions: Preliminary results indicate that the novel anti-PD-L1xCD27 bispecific antibody CDX-527 up to and including the 1 mg/kg dose level has been well tolerated. Additional data will be presented, including the safety profile at higher dose levels along with clinical activity, as well as PK and PD data. Clinical trial information: NCT04440943.

SARS-CoV-2-neutralising monoclonal antibodies to prevent COVID-19

Objectives: This is a protocol for a Cochrane Review (intervention) The objectives are as follows: To assess the effectiveness and safety of SARS-CoV-2-neutralising mAbs, including mAb fragments, to prevent infection with SARS-CoV-2 causing COVID-19;and to maintain the currency of the evidence, using a living systematic review approach Copyright © 2021 The Cochrane Collaboration Published by John Wiley & Sons, Ltd