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Odronextamab, a CD20×CD3 bispecific antibody that engages cytotoxic T cells to destroy malignant B cells, has demonstrated encouraging activity across multiple subtypes of relapsed/refractory (R/R) B-cell non-Hodgkin lymphoma. This phase II study (ELM-2; NCT03888105) evaluated odronextamab in patients with R/R follicular lymphoma after two or more lines of systemic therapy. Patients received intravenous odronextamab in 21-day cycles, with step-up dosing in cycle 1 to help mitigate the risk of cytokine release syndrome, until disease progression or unacceptable toxicity. The primary endpoint was objective response rate by independent central review. Among 128 patients evaluated, 95% completed cycle 1, and 85% completed four or more cycles. At 20.1 months' efficacy follow-up, objective response rate was 80.0% and complete response rate was 73.4%. Median duration of complete response was 25.1 months. Median progression-free survival was 20.7 months, and median overall survival was not reached. Discontinuation of odronextamab due to adverse events occurred in 16% of patients. The most common treatment-emergent adverse events were cytokine release syndrome [56%; grade ≥3 1.7% (1/60) with 0.7/4/20 mg step-up], neutropenia (39%), and pyrexia (38%). Odronextamab achieved high complete response rates with generally manageable safety in patients with heavily pretreated R/R follicular lymphoma. • Outcomes for patients with follicular lymphoma (FL) worsen with each relapse, representing a clear medical unmet need. • Odronextamab achieved high rates of deep response with durability in R/R FL, with 73% of patients attaining complete response. • Grade ≥3 cytokine release syndrome was seen in one patient with the optimized step-up regimen, and safety was generally manageable with odronextamab treatment. [ABSTRACT FROM AUTHOR]

Characterization and understanding of protein higher order structure (HOS) is essential at all stages of biologics development. Here, two folding variants of a bispecific monoclonal antibody, the correctly folded form and an alternative configuration with reduced potency, were characterized by several HOS characterization techniques. Specifically, differential scanning calorimetry (DSC), circular dichroism (CD), Fourier-transform infrared spectroscopy (FTIR), Raman and Raman optical activity (ROA) spectroscopy were used together to elucidate the impacts of disulfide bond scrambling in the fused scFv domains on the structure and thermal stability of the antibody. This study illustrates the importance of selecting appropriate biophysical characterization techniques based on the nature and magnitude of the HOS change. [ABSTRACT FROM AUTHOR]

• A comprehensive forced stress study involving different sources of stress and analysis by various analytical and characterization techniques elucidates the product stability, degradation products, and pathways of asymmetric monovalent bispecific IgG1 monoclonal antibody produced using duet mAb technology, which to our knowledge has not been reported so far. • The duet mAb exhibits structural integrity, degradation products, and overall stability identical to a standard IgG1 mAb. • The study demonstrates the prominent stability of an asymmetric bispecific IgG1 mAb format as a stable biotherapeutic platform. The developed asymmetric monovalent bispecific IgG1 or Duet monoclonal antibody (Duet mAb) has two distinct fragment antigen-binding region (Fab) subunits that target two different epitope specificities sequentially or simultaneously. The design features include unique engineered disulfide bridges, knob-into-hole mutations, and kappa and lambda chains to produce Duet mAbs. These make it structurally and functionally complex, so one expects challenging developability linked to instability, degradation of products and pathways, and limited reports available. Here, we have treated the product with different sources of extreme stress over a lengthy period, including varying heat, pH, photo stress, chemical oxidative stress, accelerated stress in physiological conditions, and forced glycation conditions. The effects of different stress conditions on the product were assessed using various analytical characterization tools to measure product-related substances, post-translational modifications (PTMs), structural integrity, higher-order disulfide linkages, and biological activity. The results revealed degradation products and pathways of Duet mAb. A moderate increase in size, charge, and hydrophobic variants, PTMs, including deamidation, oxidation, isomerization, and glycation were observed, with most conditions exhibiting biological activity. In addition, the characterization of fractionated charge variants, including deamidated species, showed satisfactory biological activity. This study demonstrated the prominent stability of the Duet mAb format comparable to most marketed mAbs. [ABSTRACT FROM AUTHOR]

To evaluate 2-year efficacy, durability, and safety of the bispecific antibody faricimab, which inhibits both angiopoietin-2 and VEGF-A. TENAYA (ClinicalTrials.gov identifier, NCT03823287) and LUCERNE (ClinicalTrials.gov identifier, NCT03823300) were identically designed, randomized, double-masked, active comparator-controlled phase 3 noninferiority trials. Treatment-naive patients with neovascular age-related macular degeneration (nAMD) 50 years of age or older. Patients were randomized (1:1) to intravitreal faricimab 6.0 mg up to every 16 weeks (Q16W) or aflibercept 2.0 mg every 8 weeks (Q8W). Faricimab fixed dosing based on protocol-defined disease activity at weeks 20 and 24 up to week 60, followed up to week 108 by a treat-and-extend personalized treatment interval regimen. Efficacy analyses included change in best-corrected visual acuity (BCVA) from baseline at 2 years (averaged over weeks 104, 108, and 112) and proportion of patients receiving Q16W, every 12 weeks (Q12W), and Q8W dosing at week 112 in the intention-to-treat population. Safety analyses included ocular adverse events (AEs) in the study eye through study end at week 112. Of 1326 patients treated across TENAYA/LUCERNE, 1113 (83.9%) completed treatment (n = 555 faricimab; n = 558 aflibercept). The BCVA change from baseline at 2 years was comparable between faricimab and aflibercept groups in TENAYA (adjusted mean change, +3.7 letters [95% confidence interval (CI), +2.1 to +5.4] and +3.3 letters [95% CI, +1.7 to +4.9], respectively; mean difference, +0.4 letters [95% CI, −1.9 to +2.8]) and LUCERNE (adjusted mean change, +5.0 letters [95% CI, +3.4 to +6.6] and +5.2 letters [95% CI, +3.6 to +6.8], respectively; mean difference, −0.2 letters [95% CI, −2.4 to +2.1]). At week 112 in TENAYA and LUCERNE, 59.0% and 66.9%, respectively, achieved Q16W faricimab dosing, increasing from year 1, and 74.1% and 81.2%, achieved Q12W or longer dosing. Ocular AEs in the study eye were comparable between faricimab and aflibercept groups in TENAYA (55.0% and 56.5% of patients, respectively) and LUCERNE (52.9% and 47.5% of patients, respectively) through week 112. Treat-and-extend faricimab treatment based on nAMD disease activity maintained vision gains through year 2, with most patients achieving extended dosing intervals. Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article. [ABSTRACT FROM AUTHOR]

Ichnos has developed a multi-specific antibody platform based on the BEAT® (Bispecific engagement by antibodies based on the T-cell receptor) interface. The increased complexity of the bi- and multi-specific formats generated with this platform makes these molecules difficult-to-express proteins compared to standard monoclonal antibodies (mAbs). This report describes how expression limitations of a bi-specific bi-paratopic BEAT antibody were improved in a holistic approach. An initial investigation allowed identification of a misbalance in the subunits composing the BEAT antibody as the potential root cause. This misbalance was then addressed by a signal peptide optimization, and the overall expression level was increased by the combination of two vector design elements on a single gene vector. Further improvements were made in the selection of cell populations and an upstream (USP) platform process was applied in combination with a cell culture temperature shift. This allowed titer levels of up to 6 g/L to be reached with these difficult-to-express proteins. Furthermore, a high-density seeding process was developed that allowed titers of around 11 g/L for the BEAT antibody, increasing the initial titer by a factor of 10. The approach was successfully applied to a tri-specific antibody with titer levels reaching 10 g/L. In summary, a platform process for difficult-to-express proteins was developed using molecular biology tools, cell line development, upstream process optimization and process intensification. • Holistic development overcoming expression limits in bi-/multispecific antibodies via mol. bio., cell line dev, process opt. • Molecular constructs in article enable very high expression levels, particularly beneficial for challenging proteins. • Report of a process allowing titers > 10 g/L (product) for bi- and multi-specific antibody formats. [ABSTRACT FROM AUTHOR]

Bispecific antibodies (BsAbs) are an effective treatment used in relapsed or refractory multiple myeloma. Despite a well-tolerated safety profile, infectious events appear to be frequent in clinical trials. Real-world data on epidemiology, characteristics, risk factors, and outcomes of infections in patients treated with BsAb are still needed. A retrospective, multicentre study in BsAb-treated patients with multiple myeloma was performed in 14 French centres from December 2020 to February 2023. The primary objective was to describe the incidence of infections that required hospitalization, specific treatment, or adaptation in BsAb administration. Among 229 patients with multiple myeloma treated with BsAb, 153 (67%) received teclistamab, 47 (20%) received elranatamab, and 29 (13%) talquetamab. We reported a total of 234 infections, including 123 (53%) of grade of ≥3. Predominant infections affected the respiratory tract (n = 116, 50%) followed by bacteraemias (n = 36, 15%). The hospitalization rate was 56% (n = 131), and 20 (9%) infections resulted in death. Global cumulative incidence of the first infection was 70% in all patients, 73% in patients treated with B-cell maturation antigen-targeting, and 51% with GPRC5D-targeting BsAb. In univariate analyses, corticosteroids for cytokine release syndrome (CRS)/immune effector cell-associated neurotoxicity syndrome (ICANS) were associated with a higher risk of first infection (HR = 2.13; 95% CI, 1.38–3.28), whereas GPRC5D-targeting BsAb and anti-bacterial prophylaxis were associated with a lower risk (HR = 0.53; 95% CI, 0.3–0.94 and HR = 0.65; 95% CI, 0.46–0.9). Fine and Gray multivariate model found that only corticosteroids for CRS/ICANS were correlated with a higher risk of first infection (HR = 2.01; 95% CI, 1.27–3.19). The implementation of preventive measures that aim to mitigate the risk of infection under BsAb is pivotal, notably in patients who received corticosteroids for CRS/ICANS. [ABSTRACT FROM AUTHOR]

Despite an important therapeutic arsenal targeting the ErbB family, patients inevitably develop drug resistance, mediated by acquired mutations and crosstalk pathway activation. Bispecific antibodies emerge as enticing ErbB therapeutics, mainly due to their multitarget involvement to help overcome drug resistance. Targeted protein degradation (TPD) strategies are promising new approaches to restrict ErbB family oncogenicity. Peptides and nanobodies are ever-evolving fields in oncology, offering small molecules that bypass major constraints in tumors and representing suitable biotherapeutics and diagnostic tools for ErbB-driven cancer treatment. EGF-like domain-containing proteins within the tumor microenvironment have recently received attention due to their direct interaction with ErbB family and involvement in cancer progression, rendering them promising anticancer targets. The epidermal growth factor receptor (EGFR) family is a class of transmembrane proteins, highly regarded as anticancer targets due to their pivotal role in various malignancies. Standard cancer treatments targeting the ErbB receptors include tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAbs). Despite their substantial survival benefits, the achievement of curative outcomes is hindered by acquired resistance. Recent advancements in anti-ErbB approaches, such as inhibitory peptides, nanobodies, targeted-protein degradation strategies, and bispecific antibodies (BsAbs), aim to overcome such resistance. More recently, emerging insights into the cell surface interactome of the ErbB family open new avenues for modulating ErbB signaling by targeting specific domains of ErbB partners. Here, we review recent progress in ErbB targeting and elucidate emerging paradigms that underscore the significance of EGF domain-containing proteins (EDCPs) as new ErbB-targeting pathways. [ABSTRACT FROM AUTHOR]

T cell-redirecting bispecific antibodies are specifically designed to bind to tumor-associated antigens, thereby engaging with CD3 on the T cell receptor. This linkage between tumor cells and T cells actively triggers T cell activation and initiates targeted killing of the identified tumor cells. These antibodies have emerged as one of the most promising avenues within tumor immunotherapy. However, despite success in treating hematological malignancies, significant advancements in solid tumors have yet to be explored. In this review, we aim to address the critical challenges associated with T cell-redirecting bispecific antibodies and explore novel strategies to overcome these obstacles, with the ultimate goal of expanding the application of this therapy to include solid tumors. This review summarizes the developments of T cell-redirecting bispecific antibodies, with a focus on therapy-associated key toxicities, challenges and future perspectives. [Display omitted] [ABSTRACT FROM AUTHOR]

• Activated factor VIII (FVIIIa) functions as a cofactor for FIXa in the tenase complex. • Emicizumab is a humanized bispecific monoclonal antibody acts as a FVIIIa-mimetic. • We examined the binding affinity among emicizumab, FIXa and FX by FRET. • Emicizumab orientates FIXa and FX in close proximity on phospholipid membranes. [ABSTRACT FROM AUTHOR]

• NXT007 is a bispecific IgG engineered from emicizumab to potentiate the activity. • The natural anticoagulant function of AT is unaffected by the presence of emicizumab. • NXT007 was here found not to affect the AT-function despite its enhanced FX-binding. • The results suggest that NXT007 would not increase the AT-related thrombotic risk. [ABSTRACT FROM AUTHOR]

Multiple myeloma remains an incurable disease, despite the development of numerous drug classes and combinations that have contributed to improved overall survival. Immunotherapies directed against cancer cell-surface antigens, such as chimeric antigen receptor (CAR) T-cell therapy and T-cell-redirecting bispecific antibodies, have recently received regulatory approvals and shown unprecedented efficacy. However, these immunotherapies have unique mechanisms of action and toxicities that are different to previous treatments for myeloma, so experiences from clinical trials and early access programmes are essential for providing specific recommendations for management of patients, especially as these agents become available across many parts of the world. Here, we provide expert consensus clinical practice guidelines for the use of bispecific antibodies for the treatment of myeloma. The International Myeloma Working Group is also involved in the collection of prospective real-time data of patients treated with such immunotherapies, with the aim of learning continuously and adapting clinical practices to optimise the management of patients receiving immunotherapies. [ABSTRACT FROM AUTHOR]

Bispecific antibody‒drug conjugates (BsADCs) represent an innovative therapeutic category amalgamating the merits of antibody‒drug conjugates (ADCs) and bispecific antibodies (BsAbs). Positioned as the next-generation ADC approach, BsADCs hold promise for ameliorating extant clinical challenges associated with ADCs, particularly pertaining to issues such as poor internalization, off-target toxicity, and drug resistance. Presently, ten BsADCs are undergoing clinical trials, and initial findings underscore the imperative for ongoing refinement. This review initially delves into specific design considerations for BsADCs, encompassing target selection, antibody formats, and the linker–payload complex. Subsequent sections delineate the extant progress and challenges encountered by BsADCs, illustrated through pertinent case studies. The amalgamation of BsAbs with ADCs offers a prospective solution to prevailing clinical limitations of ADCs. Nevertheless, the symbiotic interplay among BsAb, linker, and payload necessitates further optimizations and coordination beyond a simplistic "1 + 1" to effectively surmount the extant challenges facing the BsADC domain. In comparison to traditional ADCs, bispecific antibody drug conjugates present distinct design considerations and properties as the next-generation ADCs. [Display omitted] [ABSTRACT FROM AUTHOR]

Antibodies are potent biopharmaceuticals used to treat severe diseases, including cancers. During the past decade, more complex modalities have been developed including bispecific T-cell engager (BiTE®) molecules, e.g. by Amgen. However, non-natural and complex molecule formats often prove to be difficult-to-express (DTE), which is the case for BiTE® molecules. Due to the growing importance of multispecific modalities such as half-life extended (HLE) BiTE® and HLE dual-targeting bispecific T-cell engager (dBiTE) molecules, this artificial class of therapeutic proteins was investigated for molecular bottlenecks in stable production cell lines, by analyzing all relevant steps of recombinant protein production. As a result, drastically reduced intracellular BiTE® molecule-encoding mRNA levels were identified as a potential production bottleneck. Using in vitro transcription (IVT), the transcription rate of the BiTE® molecule-encoding mRNA was identified as the root cause for reduced amounts of intracellular mRNA. In an attempt to improve the transcription rate of a BiTE® molecule, it could be demonstrated that the artificial and special structure of the BiTE® molecule was not the rate limiting step for reduced IVT rate. However, modulation of the primary DNA sequence led to significant improvement of IVT rate. The analyses presented provide insight into the HLE BiTE® / HLE d(BiTE®) class of DTE proteins and perhaps into other classes of DTE proteins, and therefore may lead to identification of further production bottlenecks and optimization strategies to overcome manufacturability challenges associated with various complex therapeutics. • Bispecific T cell engager (BiTE®) molecules proved to be difficult-to-express. • Low intracellular BiTE mRNA levels were identified as potential production bottleneck. • Transcription rate was the root cause for reduced intracellular BiTE mRNA levels. • Artificial or repetitive structures of the BiTE® molecule were not the rate limiting factor. • Modulation of primary DNA sequence led to significantly improved transcription rate. [ABSTRACT FROM AUTHOR]

Cancer immunotherapy, where a patient's immune system is harnessed to eradicate cancer cells selectively, is a leading strategy for cancer treatment. However, successes with immune checkpoint inhibitors (ICI) are hampered by reported systemic and organ-specific toxicities and by two-thirds of the patients being non-responders or subsequently acquiring resistance to approved ICIs. Hence substantial efforts are invested in discovering novel targeted immunotherapies aimed at reduced side-effects and improved potency. One way is utilizing the dual targeting feature of bispecific antibodies, which have made them increasingly popular for cancer immunotherapy. Easy and predictive screening methods for activation ranking of candidate drugs in tumor contra non-tumor environments are however lacking. Herein, we present a cell-based assay mimicking the tumor microenvironment by co-culturing B cells with engineered human embryonic kidney 293 T cells (HEK293T), presenting a controllable density of platelet-derived growth factor receptor β (PDGFRβ). A target density panel with three different surface protein levels on HEK293T cells was established by genetic constructs carrying regulatory elements limiting RNA translation of PDGFRβ. We employed a bispecific antibody-affibody construct called an AffiMab capable of binding PDGFRβ on cancer cells and CD40 expressed by B cells as a model. Specific activation of CD40-mediated signaling of immune cells was demonstrated with the two highest receptor-expressing cell lines, Level 2/3 and Level 4, while low-to-none in the low-expressing cell lines. The concept of receptor tuning and the presented co-culture protocol may be of general utility for assessing and developing novel bi-specific antibodies for immuno-oncology applications. [Display omitted] • Novel method for tuning cancer receptor surface density using regulatory element constructs. • Swift co-culture protocol allows combination of tuned cancer receptor cells and leukocytes. • Presented platform for co-culturing and receptor tuning allows for evaluation of bispecifics. [ABSTRACT FROM AUTHOR]

Recently developed multi-specific antibody formats enable new therapeutic concepts. Conveniently, formats with an Fc domain allow purification in well-established mAb platform processes. However, due to the structural complexity of the formats, the assembled molecules may be sensitive to extreme pH commonly used for viral inactivation. An alternative to low pH incubation for virus inactivation is the use of a mixture of tri-n-butyl phosphate (TnBP, solvent) and Polysorbate 80 (PS80, detergent). While TnBP is toxic, this combination has a long history of use in the manufacturing of human plasma-derived products that are sensitive to low or high pH incubation. Data are provided demonstrating that the solvent/detergent (S/D) treatment using TnBP and PS80 can be successfully used for pH-sensitive, multi-specific antibody formats in the clarified cell culture fluid (CCCF). A different placement of the S/D within the purification process, namely during the capture by Protein A (PA), has been evaluated. This alternative placement allows effective viral inactivation by S/D while preserving the viral reduction and viral inactivation achieved through the PA step itself, enabling the cumulation of these effects. Furthermore, the process alternative simplifies the liquid handling by reducing the added volumes of the required S/D liquids, thus reducing the amount of toxic TnBP to a minimum. Data are shown demonstrating a complete removal of TnBP and PS80 in the process. • S/D treatment allows efficient inactivation of enveloped viruses during the manufacturing process of pH sensitive multi-specific antibodies. • A novel placement of S/D incubation on the Protein A column reduces the amount of S/D required and simplifies the required liquid handling. • The novel placement ensures efficient virus inactivation, acceptable product quality and removal of S/D components without additional purification steps. [ABSTRACT FROM AUTHOR]

High-risk neuroblastoma has poor survival due to treatment failure and off-target side effects of therapy. Small molecule inhibitors have shown therapeutic efficacy at targeting oncogenic cell cycle dysregulators, such as polo-like kinase 1 (PLK1). However, their clinical success is limited by a lack of efficacy and specificity, causing off-target toxicity. Herein, we investigate a new treatment strategy whereby a bispecific antibody (BsAb) with dual recognition of methoxy polyethylene glycol (PEG) and a neuroblastoma cell-surface receptor, epidermal growth factor receptor (EGFR), is combined with a PEGylated small interfering RNA (siRNA) lipid nanoparticle, forming BsAb-nanoparticle RNA-interference complexes for targeted PLK1 inhibition against high-risk neuroblastoma. Therapeutic efficacy of this strategy was explored in neuroblastoma cell lines and a tumor xenograft model. Using ionizable lipid-based nanoparticles as a low-toxicity and clinically safe approach for siRNA delivery, we identified that their complexing with EGFR-PEG BsAb resulted in increases in cell targeting (1.2 to >4.5-fold) and PLK1 gene silencing (>2-fold) against EGFR+ high-risk neuroblastoma cells, and enhancements correlated with EGFR expression on the cells (r > 0.94). Through formulating nanoparticles with PEG-lipids ranging in diffusivity, we further identified a highly diffusible PEG-lipid which provided the most pronounced neuroblastoma cell binding, PLK1 silencing, and significantly reduced cancer growth in vitro in high-risk neuroblastoma cell cultures and in vivo in a tumor-xenograft mouse model of the disease. Together, this work provides an insight on the role of PEG-lipid diffusivity and EGFR targeting as potentially relevant variables influencing the therapeutic efficacy of siRNA nanoparticles in high-risk neuroblastoma. [Display omitted] • High-risk neuroblastoma, an aggressive pediatric cancer, requires treatment strategies that are selective and effective. • PLK1 silencing using siRNA-loaded PEGylated lipid nanoparticles reduces viability and expansion of high-risk neuroblastoma. • EGFR targeting of siRNA nanoparticles using a bispecific antibody improves therapeutic potency of PLK1 silencing in vitro. • Formulating targeted siRNA nanoparticles with highly diffusible DMG-PEG lipid decreases neuroblastoma tumour growth in vivo. [ABSTRACT FROM AUTHOR]

Tissue regulatory T cells (Tregs) exert pivotal functions in both immune and metabolic regulation, maintaining local tissue homeostasis, integrity, and function. Accordingly, Tregs play a crucial role in controlling obesity-induced inflammation and supporting efficient muscle function and repair. Depending on the tissue context, Tregs are characterized by unique transcriptomes, growth, and survival factors and T cell receptor (TCR) repertoires. This functional specialization offers the potential to selectively target context-specific Treg populations, tailoring therapeutic strategies to specific niches, thereby minimizing potential side effects. Here, we discuss challenges and perspectives for niche-specific Treg targeting, which holds promise for highly efficient and precise medical interventions to combat metabolic disease. Becker and Dirschl et al. discuss challenges and perspectives for Treg-mediated control of metabolic tissues, highlighting the functional specialization of tissue Tregs. This specialization holds the future potential for precise interventions in metabolic diseases through niche-specific targeting of Tregs. [ABSTRACT FROM AUTHOR]

CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily of receptors expressed on a variety of cell types. The CD40–CD40L interaction gives rise to many immune events, including the licensing of dendritic cells to activate CD8+ effector T cells, as well as the facilitation of B cell activation, proliferation, and differentiation. In malignant cells, the expression of CD40 varies among cancer types, mediating cellular proliferation, apoptosis, survival and the secretion of cytokines and chemokines. Agonistic human anti-CD40 antibodies are emerging as an option for cancer treatment, and early-phase clinical trials explored its monotherapy or combination with radiotherapy, chemotherapy, immune checkpoint blockade, and other immunomodulatory approaches. In this review, we present the current understanding of the mechanism of action for CD40, along with results from the clinical development of agonistic human CD40 antibodies in cancer treatment (selicrelumab, CDX-1140, APX005M, mitazalimab, 2141-V11, SEA-CD40, LVGN7409, and bispecific antibodies). This review also examines the safety profile of CD40 agonists in both preclinical and clinical settings, highlighting optimized dosage levels, potential adverse effects, and strategies to mitigate them. [Display omitted] • CD40L-CD40 signaling plays an intricate role in regulation of immune and malignant cells in the tumor microenvironment. • CD40 is linked to both biological and immunological functions. • Preclinical studies and clinical development of CD40 agonism therapies underscore its significance in cancer treatment. • Careful management of dosage optimization and adverse effects is crucial for future design of CD40 agonism cancer therapy. • Cytokine therapies, RAS/RAF/PI3K pathway targeted therapies, and CART therapies can potentially be incorporated with CD40 agonism therapy. [ABSTRACT FROM AUTHOR]

The poor penetration of monoclonal antibodies (mAb) across the blood-brain barrier (BBB) impedes the development of regenerative therapies for neurological diseases. For example, Nogo-A is a myelin-associated protein highly expressed in the central nervous system (CNS) whose inhibitory effects on neuronal plasticity can be neutralized with direct administration of 11C7 mAb in CNS tissues/fluids, but not with peripheral administrations such as intravenous injections. Therefore, in the present study, we engineered a CNS-penetrating antibody against Nogo-A by combining 11C7 mAb and the single-chain variable fragment (scFv) of 8D3, a rat antibody binding transferrin receptor 1 (TfR) and mediating BBB transcytosis (11C7-scFv8D3). The binding of 11C7-scFv8D3 to Nogo-A and to TfR/CD71 was validated by capture ELISA and Biolayer Interferometry. After intravenous injection in mice, capture ELISA measurements revealed fast plasma clearance of 11C7-scFv8D3 concomitantly with brain and spinal cord accumulation at levels up to 19 fold as high as those of original 11C7 mAb. 11C7-scFv8D3 detection in the parenchyma indicated effective blood-to-CNS transfer. A single dose of 11C7-scFv8D3 induced stronger activation of the growth-promoting AkT/mTOR/S6 signaling pathway than 11C7 mAb or control antibody. Taken together, our results show that BBB-crossing 11C7-scFv8D3 engages Nogo-A in the mouse CNS and stimulates neuronal growth mechanisms. [Display omitted] • 11C7-scFv8D3 is a bispecific antibody crossing the BBB and neutralizing the neuronal plasticity inhibitor Nogo-A in the mouse CNS. • After intravenous injection in mice, 11C7-scFv8D3 is rapidly cleared from the plasma and accumulates in CNS tissues compared with 11C7 mAb. • 11C7-scFv8D3 is a more potent inducer of the growth-associated mTOR signaling in CNS neurons than original 11C7 mAb. [ABSTRACT FROM AUTHOR]

The bispecific antibody gefurulimab (also known as ALXN1720) was developed to provide patients with a subcutaneous treatment option for chronic disorders involving activation of the terminal complement pathway. Gefurulimab blocks the enzymatic cleavage of complement component 5 (C5) into the biologically active C5a and C5b fragments, which triggers activation of the terminal complement cascade. Heavy-chain variable region antigen-binding fragment (VHH) antibodies targeting C5 and human serum albumin (HSA) were isolated from llama immune-based libraries and humanized. Gefurulimab comprises an N-terminal albumin-binding VHH connected to a C-terminal C5-binding VHH via a flexible linker. The purified bispecific VHH antibody has the expected exact size by mass spectrometry and can be formulated at greater than 100 mg/mL. Gefurulimab binds tightly to human C5 and HSA with dissociation rate constants at pH 7.4 of 54 pM and 0.9 nM, respectively, and cross-reacts with C5 and serum albumin from cynomolgus monkeys. Gefurulimab can associate with C5 and albumin simultaneously, and potently inhibits the terminal complement activity from human serum initiated by any of the three complement pathways in Wieslab assays. Electron microscopy and X-ray crystallography revealed that the isolated C5-binding VHH recognizes the macroglobulin (MG) 4 and MG5 domains of the antigen and thereby is suggested to sterically prevent C5 binding to its activating convertase. Gefurulimab also inhibits complement activity supported by the rare C5 allelic variant featuring an R885H substitution in the MG7 domain. Taken together, these data suggest that gefurulimab may be a promising candidate for the potential treatment of complement-mediated disorders • Gefurulimab selectively binds to human complement component C5 and serum albumin. • Gefurulimab binds to the macroglobulin 4 and 5 domains of C5. • Gefurulimab completely inhibits terminal complement activity. • Binding to albumin confers the potential for an extended circulatory half-life. [ABSTRACT FROM AUTHOR]

Molecular biological methods have emerged as inevitable tools to accompany the process of cell line development for the generation of stable and highly productive manufacturing cell lines in the biopharmaceutical industry. PCR-based methods are especially useful for screening and characterization of cell lines due to their low cost, scalability, precision and propensity for multidimensional read-outs. In this study, the diverse applications of droplet digital PCR (ddPCR) as a molecular biological tool for cell line development are demonstrated. Specifically, it is shown that ddPCR can be used to enable precise, sensitive and reproducible absolute quantification of genomically integrated transgene copies during cell line development and cell bank characterization. Additionally, an amplitude multiplexing approach is applied to simultaneously run multiple assays on different genetic targets in a single reaction and advance clonal screening by measuring gene expression profiles to predict the assembly and homogeneity of difficult-to-express (DTE) proteins. The implementation of ddPCR-based assays during cell line development allows for early screening at a transcriptional level, particularly for complex, multidomain proteins, where balanced polypeptide chain ratios are of primary importance. Moreover, it is demonstrated that ddPCR-based genomic characterization improves the robustness, efficiency and comparability of absolute transgene copy number quantification, an essential genetic parameter that must be demonstrated to regulatory authorities during clinical trial and market authorization application submissions to support genetic stability and consistency of the selected cell substrate. • Gene expression analysis by ddPCR can predict the assembly of bispecific antibodies. • Amplitude-multiplexing allows for a simultaneous quantification of several genes. • Precise and sensitive absolute quantification of transgene copy numbers. • High correlation between RNA sequencing, ddPCR and capillary gel electrophoresis. [ABSTRACT FROM AUTHOR]

Temperature cycling stability studies can be appropriately designed and utilized to ensure that drug product quality, efficacy, and safety are not compromised when materials are subjected to short term temperature excursions from intended storage that may occur during e.g., shipping, transport, or patient use. Some countries, such as Australia and Brazil, impose specific regulations that specify the need to conduct stability studies that are supportive of "real world" excursions as part of licensing approval requirements. These temperature cycling stability studies extend beyond what is described in ICH Guidelines Q1A(R2) and Q5C, and companies may be challenged in designing studies that not only satisfy country specific regulations, but also satisfy all global regulatory health authority expectations. This article focuses on responses to a cross-industry survey conducted within the International Consortium for Innovation and Quality (iqconsortium.org) member companies, regarding practices related to temperature cycling stability studies, in order to determine how these requirements are being interpreted and met. The results indicate that while there is no one-size-fits-all approach to performing temperature cycling stability studies, there are common and best practices that can be followed to satisfy global health authority regulatory guidelines and requirements. The purpose of this paper is to describe the outcome of an industry survey and common/best practices on temperature cycling stability studies performed on drug product (DP) to satisfy the requirements established for marketing authorizations in Australia and Brazil or any other countries that may have similar requirements. The framework is proposed within the context of late phase and commercial development of common biological and/or large molecule modalities, such as monoclonal antibodies (mAbs, including bispecific antibodies), fusion proteins, complex proteins, oligonucleotides, and antibody-drug conjugates (ADCs), but many of the general principles involved may be applied to other therapeutics, such as Virus Like Particles (VLP), gene or cell therapies (GTx or CTx), or vaccines. For the purposes of this paper, temperature cycling stability studies refer to studies that are designed, in part, to support short term temperature excursions that drug product may be subjected to during shipping and storage activities and is outside of the labeled storage condition of the product. [ABSTRACT FROM AUTHOR]

Bispecific T cell engagers (bsTCEs) are promising but challenging-to-develop therapeutics that harbor narrow therapeutic indices. Quantitative systems pharmacology (QSP) modeling can integrate nonclinical, preclinical, and clinical data to support the development of bsTCEs. QSP has been increasingly used to bridge data gaps, generate mechanistic hypotheses, identify optimal doses, and predict safety and efficacy in heterogeneous patient populations. Recent marketing approvals of bsTCEs highlight the utility and growing familiarity of stakeholders with QSP-based dose optimization. Bispecific T cell engagers (bsTCEs) have emerged as a promising class of cancer immunotherapy. Several bsTCEs have achieved marketing approval; dozens more are under clinical investigation. However, the clinical development of bsTCEs remains rife with challenges, including nuanced pharmacology, limited translatability of preclinical findings, frequent on-target toxicity, and convoluted dosing regimens. In this opinion article we present a distinct perspective on how quantitative systems pharmacology (QSP) can serve as a powerful tool for overcoming these obstacles. Recent advances in QSP modeling have empowered developers of bsTCEs to gain a deeper understanding of their context-dependent pharmacology, bridge gaps in experimental data, guide first-in-human (FIH) dose selection, design dosing regimens with expanded therapeutic windows, and improve long-term treatment outcomes. We use recent case studies to exemplify the potential of QSP techniques to support future bsTCE development. [ABSTRACT FROM AUTHOR]

Prodrug design is a promising approach for reducing the off-target effects of therapeutic antibodies, particularly bispecific antibodies (bsAbs) that recruit T cells for activation; this design uses masking sequences that inhibit antibody binding until they reach the tumor microenvironment, where they are removed. In this study, we propose PAS, a polypeptide sequence composed of repeated Pro, Ala, and Ser residues, as a universal masking sequence. PAS has no specificity, but can inhibit antibody binding through steric hindrance caused by its large fluid dynamic radius and disordered structure; additionally, its length can be adjusted. We fused PAS to the N-terminus of an anti-CD3 single-chain variable fragment (scFv) and a bsAb, that targets both the epidermal growth factor receptor and CD3, via a recognition sequence cleaved by cancer-related proteases. PAS integration inhibited anti-CD3 scFv binding with higher efficacy than the epitope sequence, and the extent of inhibition was proportional to the length of the PAS sequence. For masked bsAbs, T cell-binding ability, cancer growth inhibition effects, and T cell activation effects were also reduced depending on the length of PAS and were fully restored upon removing PAS sequences using protease. The masking procedure using PAS was successfully applied to another scFv. The provision to adjust the masking effects of PAS by tuning its length, makes PAS fusion a valuable tool for the universal design of prodrug antibodies. • A masking sequence, PAS, was used to inhibit antibody binding. • Fusion of PAS inhibited anti-CD3 scFv binding in a length-dependent manner. • Fusion of PAS inhibited the functions of T cell-engaging bispecific antibodies. • Antibody function was recovered by removing the PAS sequence. • Masking procedure using PAS was successfully applied to another scFv. [ABSTRACT FROM AUTHOR]

Bispecific biotherapeutics offer potent and highly specific treatment options in oncology and immuno-oncology. However, many bispecific formats are prone to high levels of aggregation and instability, leading to prolonged development timelines, inefficient manufacturing, and high costs. The novel class of Mabcalin™ molecules consist of Anticalin® proteins fused to an IgG and are currently being evaluated in pre-clinical and clinical studies. Here, we describe a robust high-yield manufacturing platform for these therapeutic fusion proteins providing data up to commercially relevant scales. A platform upstream process was established for one of the Mabcalin bispecifics and then applied to other clinically relevant drug candidates with different IgG target specificities. Process performance was compared in 3 L bioreactors and production was scaled-up to up to 1000 L for confirmation. The Mabcalin proteins' structural and biophysical similarities enabled a downstream platform approach consisting of initial protein A capture, viral inactivation, mixed-mode anion exchange polishing, second polishing by cation exchange or hydrophobic interaction chromatography, viral filtration, buffer exchange and concentration by ultrafiltration/diafiltration. All three processes met their target specifications and achieved comparable clearance of impurities and product yields across scales. The described platform approach provides a fast and economic path to process confirmation and is well comparable to classical monoclonal antibody approaches in terms of costs and time to clinic. • Mabcalin proteins are novel bispecifics consisting of IgG-Anticalin protein fusions. • Scalable upstream and downstream manufacturing processes for bispecific molecules. • Platform for faster development timelines of bispecifics with reduced costs. • Establishing a high-titer bispecific platform yielding consistent product quality. [ABSTRACT FROM AUTHOR]

A therapeutic system with the ability to target more than one protein is an important aim of cancer therapy since tumor growth is accompanied by dysregulation of many biological pathways. G-quadruplexes (G4s) are non-canonical structures formed by guanine-rich DNA or RNA oligonucleotides, with the ability to bind to different targets. In this study, we constructed ten novel bispecific G-quadruplex conjugates based on AT11, TBA, T40214 and T40231 aptamer structures, with the ability to bind two different targets at once in cancer cells. We analyzed the physicochemical aspects and the anticancer properties of novel molecules relating them with the single G-quadruplex unit and attempted to comprehend the correlation between the structures of bispecific G-quadruplexes with their biological activity. Our studies uncovered conjugates with considerable antiproliferative potential in HeLa and MCF-7 cancer cell lines, however with relatively low thermal stability or low nuclease resistance. Three conjugates among all studied oligonucleotides possess improved antiproliferative activity in MCF-7 cell line in comparison to their single G-quadruplex units leading to up to 90% inhibition of cancer cells growth, but their inhibitory potential is rather comparable to the effect observed for mix of two separate G-quadruplex units. Importantly, the conjugation enhances oligonucleotides enzymatic stability leading to the improvement of their therapeutic profile. The comprehensive studies presented herein indicate new approach for possibly effective cancer therapy and for the design of G4-based drugs. • Novel bispecific G-quadruplex conjugates containing two domains are developed. • G-quadruplexes have the ability to bind to two different targets. • Bispecific G-quadruplexes were characterized physicochemically and biologically. • Novel molecules have considerable anticancer activity in HeLa and MCF-7 cell lines. • The conjugation enhances oligonucleotides enzymatic stability. [ABSTRACT FROM AUTHOR]

The development of Immune checkpoint blockade(ICB) therapy and BRAF- and MEK-targeted therapies has reshaped the survival outcomes of the patients with advanced melanoma. PD-1/PD-L1 blockade was an approved strategy in melanoma treatment. Here we design a PD-L1 xCD3 nanobody as a novel bispecific T cell engager (BiTE) in treating PD-L1 overexpression melanoma. BiTE PD-L1 × CD3 Nb was predicted to bind near a large acidic surface on CD3-ε similar to UCHT1-scFv antibody based on alpha-fold and molecular docking. BiTE PD-L1 × CD3 Nb and anti-CD3 Nb retained the ability to activate T cells to produce TNF-α and IFN-γ in a dose-dependent manner. The IC 50 value of BiTE PD-L1 × CD3 Nb was 4.208 μ g/mL. BiTE PD-L1 × CD3 Nb showed obvious cytotoxic activity on both A375WT and A375PD-L1 related to PD-L1 expression level. • A bispecific PD-L1xCD3 nanobody was designed as a novel Bispecific T-cells engager(BiTE). • BiTE PD-L1 × CD3 Nb was predicted to bind near a large acidic surface on CD3-ε similar to UCHT1-scFv antibody based on Alpha-fold and molecular docking. • A specific PD-L1 overexpression A375 cell line was constructed as a humanized melanoma model. • BiTE PD-L1 × CD3 Nb could disrupt PD-1/PD-L1 engagement and activate T cells to produce cytokines. [ABSTRACT FROM AUTHOR]

The enzymatic browning behavior of a model ground tissue, banana peel, influenced by the frequency variable of pulsed electric field (PEF), has been visualized and studied. The tissue was PEF-treated with 15 µs pulse at 3.3 kV/cm using trains of 5, 15, and 30 pulses, each varying from 0.1 Hz to 10 Hz; images and electrical conductivity were recorded intermittently throughout the incubation. The degree of browning was quantified with a browning index (BI) term, and the uniformity of browning was visually assessed by inspecting the pattern of pigment distribution. Moreover, the degree of tissue disintegration was calculated using a conductivity-based tissue disintegration index (DI) and compared to the BI and specific energy input. Browning uniformity was found to be significantly affected and increased with decreasing frequency. The effect was most noticeable up to disintegration degree of about 63, DI, or when the frequency of 5 or 15 pulses was reduced from 1 Hz to 0.1 Hz. Remarkably, the decrease at 5 pulses resulted in a significant increase in BI without a substantial increase in DI. The observed effect has been attributed to increased electrophoretic mobility of browning agents along the extracellular path during 0.1 Hz pulsation. Furthermore, lowering the frequency to 0.1 Hz reduced the energy required for a browning level close to saturation by about 57% compared to 10 Hz. [ABSTRACT FROM AUTHOR]

At least 0.5% of people in the Western world develop inflammatory bowel disease (IBD). While antibodies that block tumor necrosis factor (TNF) α and Interleukin (IL-)23 have been approved for the treatment of IBD, IL-6 antibodies failed in the phase II clinical trial due to non-tolerable side effects. However, two clinical phase II studies suggest that inhibiting IL-6/soluble IL-6R (sIL-6R)-induced trans-signaling via the cytokine receptor gp130 benefit IBD patients with fewer adverse events. Here we develop inhibitors targeting a combination of IL-6/sIL-6R and TNF or IL-12/IL-23 signaling, named cs130-TNFVHHFc and cs130-IL-12/23VHHFc. Surface plasmon resonance experiments showed that recombinant cs130-TNFVHHFc and cs130- IL-12/23VHHFc bind with high affinity to IL-6/sIL-6R complexes and human TNFα (hTNFα) or IL-12/IL-23, respectively. Immunoprecipitation experiments have verified the higher ordered complex formation of the inhibitors with IL-6/sIL-6R and IL-12. We demonstrated that cs130-TNFVHHFc and cs130-IL12/23VHHFc block IL-6/sIL-6R trans-signaling-induced proliferation and STAT3 phosphorylation of Ba/F3-gp130 cells, as well as hTNFα- or IL-23-induced signaling, respectively. In conclusion, cs130-TNFVHHFc and cs130-IL-12/23VHHFc represent a class of dimeric and bispecific chimeric cytokine inhibitors that consist of a soluble cytokine receptor fused to anticytokine nanobodies. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitors targeting PD-1 or CTLA-4 individually have shown substantial clinical benefits in the treatment of malignancies. We aimed to assess the safety and antitumour activity of cadonilimab monotherapy, a bispecific PD-1/CTLA-4 antibody, in patients with advanced solid tumours. This multicentre, open-label, phase 1b/2 trial was conducted across 30 hospitals in China. Patients aged 18 years or older with histologically or cytologically confirmed, unresectable advanced solid tumours, unsuccessful completion of at least one previous systemic therapy, and an Eastern Cooperative Oncology Group performance status of 0 or 1 were eligible for inclusion. Patients who had previously received anti-PD-1, anti-PD-L1, or anti-CTLA-4 treatment were not eligible for inclusion. In the dose escalation phase of phase 1b, patients received intravenous cadonilimab at 6 mg/kg and 10 mg/kg every 2 weeks. In the dose expansion phase of phase 1b, cadonilimab at 6 mg/kg and a fixed dose of 450 mg were given intravenously every 2 weeks. In phase 2, cadonilimab at 6 mg/kg was administered intravenously every 2 weeks in three cohorts: patients with cervical cancer, oesophageal squamous cell carcinoma, and hepatocellular carcinoma. The primary endpoints were the safety of cadonilimab in phase 1b and objective response rate in phase 2, based on the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1. The safety analysis was done in all patients who received at least one dose of cadonilimab. Antitumour activity was assessed in the full analysis set for the cervical cancer cohort, and in all patients with measurable disease at baseline and who received at least one dose of cadonilimab in the oesophageal squamous cell carcinoma and hepatocellular carcinoma cohorts. The study is registered on ClinicalTrial.gov , NCT03852251 , and closed to new participants; follow-up has been completed. Between Jan 18, 2019, and Jan 8, 2021, 240 patients (83 [43 male and 40 female] in phase 1b and 157 in phase 2) were enrolled. Phase 2 enrolled 111 female patients with cervical cancer, 22 patients with oesophageal squamous cell carcinoma (15 male and seven female), and 24 patients with hepatocellular carcinoma (17 male and seven female). During dose escalation, no dose-limiting toxicities occurred. Grade 3–4 treatment-related adverse events occurred in 67 (28%) of 240 patients; the most frequent grade 3 or worse treatment-related adverse events were anaemia (seven [3%]), increased lipase (four [2%]), decreased bodyweight (three [1%]), decreased appetite (four [2%]), decreased neutrophil count (three [1%]), and infusion-related reaction (two [1%]). 17 (7%) patients discontinued treatment due to treatment-related adverse events. 54 (23%) of 240 patients reported serious treatment-related adverse events, including five patients who died (one due to myocardial infarction; cause unknown for four). In phase 2, in the cervical cancer cohort, with a median follow-up of 14·6 months (IQR 13·1–17·5), the objective response rate was 32·3% (32 of 99; 95% CI 23·3–42·5). In the oesophageal squamous cell carcinoma cohort, with a median follow-up of 17·9 months (IQR 4·0–15·1), the objective response rate was 18·2% (four of 22; 95% CI 5·2–40·3). In the hepatocellular carcinoma cohort, with a median follow-up of 19·6 months (IQR 8·7–19·8), the objective response rate was 16·7% (four of 24; 95% CI 4·7–37·4). Cadonilimab showed an encouraging tumour response rate, with a manageable safety profile, suggesting the potential of cadonilimab for the treatment of advanced solid tumours. Akeso Biopharma. For the Chinese translation of the abstract see Supplementary Materials section. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) remains one of the most common and highly fatal malignancies in humans worldwide with increasing prevalence and limited therapeutic options. For many decades, many researchers have attempted to find effective curative methods for HCC and great strides have been made. GPC3 is overexpressed in HCC, but not in normal liver, making it a rational immunotherapeutic target for HCC. GC33, a humanized mAb directed against GPC3, is a safe and well-tolerated therapy choice for patients with HCC, which tested in a phase I trial in advanced HCC patients. Phase II trials of GC33 to evaluate its efficacy and safety in advanced or metastatic HCC, showed no significant differences in overall survival and progression-free survival compared with the placebo. Retrospective analysis indicates that high drug exposure and high CD16 expression may contribute to the clinical efficacy of GC33. Chugai Pharmaceutical has restarted its Phase I trial of GC33, continuing to explore its clinical value targeting GPC3 in solid tumors. To enhance the antitumor potency of GC33, we designed a GPC3/CD16A bispecific antibody (QDEB). In this study, we obtained QDEB at high purity and assessed its effectiveness in the therapy of HCC compared with GC33. In vitro cytotoxicity assays and in vivo experiments demonstrated that QDEB could enhance anti-tumor efficacy compared with GC33. CD16A activation and increased cytokines release were associated with higher anti-tumor activity. In conclusion, this bispecific antibody may possibly help develop new therapeutic strategies for HCC and develop new treatment options in the future. [Display omitted] • QDEB is a quadrivalent bispecific NK-cell engager targeting CD16A on NK cells and GPC3 on HCC tumor cells. • QDEB possesses better antitumor potential than their parental antibody GC33. • QDEB showed an enhanced ability to activate NK cells and stronger antitumor activity in vitro and in vivo compared to GC33. [ABSTRACT FROM AUTHOR]

Antibody-based cancer therapies have been evolving at a rapid pace in the pharmaceutical market. Bispecific antibody-drug conjugates that engage immune cells to target and kill cancer cells with precision have inspired the development of immunotherapy. Miniaturized antibody fragments such as diabodies, nanobodies, or single-chain variable fragments (scFvs) hold great promise as antibody-drug conjugates as they specifically target tumor tissue and can penetrate it. Here, we optimized the soluble periplasmic expression of the scFv OKT3 comprising the variable V H and V L domains of the mouse anti-human CD3 antibody muromonab-CD3 (trade name Orthoclone OKT3) in E. coli. By an expansion of the genetic code, we site-specifically incorporated the reactive non-canonical amino acid Nε-((2-azidoethoxy)carbonyl)- L -lysine (AzK) into scFv OKT3 using an orthogonal pyrrolysyl-tRNA synthetase/tRNA CUA pair. To confirm the AzK incorporation and to demonstrate the accessibility of the reactive azide group, we conjugated a fluorophore to scFv OKT3 AzK variants by copper-free strain-promoted alkyne-azide cycloaddition ('click chemistry'). The scFv OKT3 wild type and the AzK variants bound T cells at nanomolar concentrations. In this study, a 'ready-to-click' scFv OKT3 was successfully developed for future applications, e.g. as controlled anti-T cell antibody-drug conjugate or bispecific T cell engager and for imaging immune T cell migration in cancers. • Developed a 'ready-to-click' muromonab single-chain variable fragment. • Muromonab-CD3 single-chain variable fragment tolerates non-canonical amino acid incorporation. • Selectively conjugated Cyanine-3 to muromonab single-chain variable fragment. • Osmotic shock with 2 mM magnesium salts is efficient to extract periplasmic protein. [ABSTRACT FROM AUTHOR]

T cell-redirecting bispecific antibodies (bsAbs) have been under development as a new class of biotherapeutics for cancer immunotherapy. T cell-redirecting bsAbs simultaneously bind tumor-associated antigens on tumor cells and CD3 on T cells, resulting in T cell-mediated cytotoxicity against tumor cells. In this study, we prepared a tandem scFv-typed bsAb targeting HER2 and CD3 (HER2-CD3), and evaluated the impact of aggregation of HER2-CD3 on the in vitro immunotoxicity. A cell-based assay using CD3-expressing reporter cells revealed that the aggregates of HER2-CD3 directly activated CD3-expressing immune cells in the absence of target antigen (HER2)-expressing cells. Comparison of the aggregates generated under various stress conditions indicated the possibility that insoluble protein particles, which were detected by qLD analysis and contained non-denatured functional domains, contributed to the activation of CD3-expressing immune cells. In addition, HER2-CD3 aggregates stimulated hPBMCs and strongly induced the secretion of inflammatory cytokines and chemokines. The cytokine/chemokine-release profiles suggested that the aggregates could induce inflammatory responses not only by CD3-mediated T cell activation but also by other immune cell activations. These results indicated the potential risk of aggregation of T cell-redirecting bsAbs, which could induce unwanted immune cell activation and inflammation and thereby immune-mediated adverse reactions. [ABSTRACT FROM AUTHOR]

• Immunogenicity is a recurring concern across all therapies for Hemophilia A. It impacts the safety and efficacy of new and old recombinant Factor VIII products, the long-acting FVIII versions, as well as other clinical options such as bypassing agents and gene-based therapies. • The risk of anti-drug antibody development for any given Hemophilia A patient remains under investigation but a multitude of factors, related to the patient's genetic mutations, health status, and treatment or product history, have been recognized. • Gene replacement and gene editing therapies for Hemophilia A represent new treatment options for generating long-term production of factor VIII in patients, but pre-existing and de novo immunity against the vector capsids and transgene products pose significant impediments in their development. The introduction and development of biologics such as therapeutic proteins, gene-, and cell-based therapy have revolutionized the scope of treatment for many diseases. However, a significant portion of the patients develop unwanted immune reactions against these novel biological modalities, referred to as immunogenicity, and no longer benefit from the treatments. In the current review, using Hemophilia A (HA) therapy as an example, we will discuss the immunogenicity issue of multiple biological modalities. Currently, the number of therapeutic modalities that are approved or recently explored to treat HA, a hereditary bleeding disorder, is increasing rapidly. These include, but are not limited to, recombinant factor VIII proteins, PEGylated FVIII, FVIII Fc fusion protein, bispecific monoclonal antibodies, gene replacement therapy, gene editing therapy, and cell-based therapy. They offer the patients a broader range of more advanced and effective treatment options, yet immunogenicity remains the most critical complication in the management of this disorder. Recent advances in strategies to manage and mitigate immunogenicity will also be reviewed. [ABSTRACT FROM AUTHOR]

The complex structure of bispecific antibody molecules and the rapid increase in upstream expression pose a significant challenge for downstream purification. Compared to mAbs, bsAbs typically contain a higher proportion of aggregates and fragments that are difficult to remove. Extensive study and optimization are often required because the properties of impurities are close to the target molecules. In this investigation, ion exchange resins and mixed-mode resins were used to evaluate the effectiveness in removing byproducts. Mixed-mode anion chromatography was found to be effective in polishing step of bsAbs with strong hydrophobicity. Hydroxyapatite chromatography using arginine hydrochloride as an eluent provided excellent separation performance compared to that of cation exchange resins and conventional mixed-mode resins. The separation process was optimized and verified in a 200 L pilot batch. The final products were obtained with excellent purity (99.0 %) and yield (80.70 %). This work provides a new idea for high-yield polishing in downstream processing, which improves the economics of large-scale production. [Display omitted] • By-products can be removed through hydrophobic and ionic interactions. • Hydroxyapatite offers excellent resolution eluted with an Arg-HCl gradient. • Hydroxyapatite can effectively remove non-covalently bound aggregates. • Yield and purity show a high degree of consistency when scaled up to 200 L. [ABSTRACT FROM AUTHOR]

In recent years, the development of bispecific antibodies (bsAbs) has been rapid, with many new structures and target combinations being created. The boom in bsAbs has led to the successive issuance of industry guidance for their development in the US and China. However, there is a high degree of similarity in target selection, which could affect the development of diversity in bsAbs. This review presents a classification of various bsAbs for cancer therapy based on structure and target selection and examines the advantages of bsAbs over monoclonal antibodies (mAbs). Through database research, we have identified the preferences of available bsAbs combinations, suggesting rational target selection options and warning of potential wastage of medical resources. We have also compared the US and Chinese guidelines for bsAbs in order to provide a reference for their development. This review summarizes the developments of bispecific antibodies, with a focus on structure, target selection and regulatory guidance. [Display omitted] [ABSTRACT FROM AUTHOR]

Bispecific antibodies have recently received approval for relapsed refractory B-cell lymphomas (r/r BCL). Little is known about the safety of radiation (RT) delivered with bispecific therapy (BT). We sought to assess the toxicity of combination therapy with BT and RT. We retrospectively identified patients (pts) at a single institution receiving BT and RT between 2018 and 2023. The temporal relationship of RT with respect to BT was defined as follows: "Pre-BT" RT 3 months (mo) prior to and up to the first dose of BT, "Peri-BT" 1 or more RT fractions between the first and last bispecific dose, "Post-BT" RT after the last BT dose through 3 mo post-BT. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were graded per ASTCT, and all other toxicities per CTCAE version 5. Nineteen patients underwent 20 courses of BT and received RT to 34 lesions. BT included mosunetuzumab (8), blinatumomab (7), epcoritamab (4), and glofitamab (1), with median BT treatment duration of one mo (0-6). Median follow up post RT and post BT was 2 mo (0-48) and 1 mo (0-48), respectively. Baseline characteristics were as follows: median age 61 (45-86), 25% female, 55% ECOG 0-1, histology DLBCL 65%, FL 25%, Grey zone 5%, Burkitt 5%, prior treatments 4.5 (1-12), 45% prior CAR T, 85% advanced stage, 80% extranodal, and 15% bulky disease (> 10 cm). Of the radiated lesions, 56% were Pre-BT (38% within 4 weeks of BT start), 21% Peri-BT, and 26% Post-BT. Sites treated included 29% spine/paraspinal, 21% abdomen/pelvis, 18% axilla, 15% head/neck, 6% skin/subcutaneous, 6% thorax, 6% CNS. Eighty-eight percent of lesions received RT with palliative intent. 16% of pts received comprehensive RT to all active lesions (5% bridging pre-BT, 11% salvage). Median CTV was 174 cc (10-4376). Median total dose was 20 Gy (4-45). Among DLBCL pts, the most common regimens included 20 Gy/5 fx (64%), 8 Gy/2 fx (16%), and 30 Gy/10 fx (12%). Common regimens among FL pts included 4 Gy/2 fx (50%), 8 Gy/2 fx (17%), and 30 Gy/10 fx (17%). CRS and ICANS events were infrequent, and all low grade (4 (20%) Gr 1-2; 3 (15%) Gr 1-2, respectively). Radiation-related toxicities were similarly all low grade. Of all lesions treated, 35% were associated with Gr 1 toxicities, and 24% with Gr 2. Toxicities included fatigue (29%), dermatitis (21%), nausea (9%), GI (15%), pain (3%) and dry mouth (3%). No patients were hospitalized for bispecific related toxicity. RT with BT appears safe, with acceptable rates of low grade, RT-related toxicity. CRS and ICANS events were infrequent and all low grade, suggesting no obvious exacerbation by RT. The safety and efficacy of combination RT and BT warrants further study in larger cohorts. [ABSTRACT FROM AUTHOR]

Despite advances in genetic characterization of Chinese hamster ovary (CHO) cell lines regarding identification of integration sites using next generation sequencing, e.g. targeted locus amplification sequencing (TLA-seq), the concatemer structure of the integrated vectors remains elusive. Here, the entire integration locus of two CHO manufacturing cell lines was reconstructed combining CRISPR/Cas9 target enrichment, nanopore sequencing and the Canu de novo assembly pipeline. An IgG producing CHO cell line integrated 3 vector copies, which were near full-length and contained all relevant vector elements such as transgenes and their promoters on each of the vector copies. In contrast, a second CHO cell line producing a bivalent bispecific antibody integrated 7 highly fragmented vector copies in different orientations leading to head-to-head and tail-to-tail fusions. The size of the vector fragments ranged from 3.0 to 11.4 kbp each carrying 1–3 transgenes. The breakpoints of the genome-vector and vector-vector junctions were validated using Sanger sequencing and Southern blotting. A comparison to TLA-seq data confirmed the genomic breakpoints, but most of the breakpoints of the vector-vector fusions were missed by TLA-seq. For the first time, the complete transgene locus of CHO manufacturing cell lines could be deciphered. Strikingly, the application of the nanopore long-read sequencing technology led to novel insights into the complexity of genomic transgene integrations of CHO manufacturing cell lines generated via random integration. • Nanopore sequencing resolves complete concatemer structure of randomly integrated vectors. • Assembly of concatemer structure reveals highly fragmented and depleted genetic architecture. • Cas9-based enrichment leads to 37-fold increase of expression vector coverage. • Complete concatemer structure allows for a thorough interpretation of complex Southern blot data. [ABSTRACT FROM AUTHOR]

HER2 is overexpressed or amplified in a subset of biliary tract cancer. Zanidatamab, a bispecific antibody targeting two distinct HER2 epitopes, exhibited tolerability and preliminary anti-tumour activity in HER2-expressing or HER2 (also known as ERBB2)-amplified treatment-refractory biliary tract cancer. HERIZON-BTC-01 is a global, multicentre, single-arm, phase 2b trial of zanidatamab in patients with HER2 -amplified, unresectable, locally advanced, or metastatic biliary tract cancer with disease progression on previous gemcitabine-based therapy, recruited at 32 clinical trial sites in nine countries in North America, South America, Asia, and Europe. Eligible patients were aged 18 years or older with HER2 -amplified biliary tract cancer confirmed by in-situ hybridisation per central testing, at least one measurable target lesion per Response Evaluation Criteria in Solid Tumours (version 1.1), and an Eastern Cooperative Oncology Group performance status of 0 or 1. Patients were assigned into cohorts based on HER2 immunohistochemistry (IHC) score: cohort 1 (IHC 2+ or 3+; HER2-positive) and cohort 2 (IHC 0 or 1+). Patients received zanidatamab 20 mg/kg intravenously every 2 weeks. The primary endpoint was confirmed objective response rate in cohort 1 as assessed by independent central review. Anti-tumour activity and safety were assessed in all participants who received any dose of zanidatamab. This trial is registered with ClinicalTrials.gov , NCT04466891 , is ongoing, and is closed to recruitment. Between Sept 15, 2020, and March 16, 2022, 87 patients were enrolled in HERIZON-BTC-01: 80 in cohort 1 (45 [56%] were female and 35 [44%] were male; 52 [65%] were Asian; median age was 64 years [IQR 58–70]) and seven in cohort 2 (five [71%] were male and two [29%] were female; five [71%] were Asian; median age was 62 years [IQR 58–77]). At the time of the data cutoff (Oct 10, 2022), 18 (21%) patients (17 in cohort 1 and one in cohort 2) were continuing to receive zanidatamab; 69 (79%) discontinued treatment (radiographic progression in 64 [74%] patients). The median duration of follow-up was 12·4 months (IQR 9·4–17·2). Confirmed objective responses by independent central review were observed in 33 patients in cohort 1 (41·3% [95% CI 30·4–52·8]). 16 (18%) patients had grade 3 treatment-related adverse events; the most common were diarrhoea (four [5%] patients) and decreased ejection fraction (three [3%] patients). There were no grade 4 treatment-related adverse events and no treatment-related deaths. Zanidatamab demonstrated meaningful clinical benefit with a manageable safety profile in patients with treatment-refractory, HER2-positive biliary tract cancer. These results support the potential of zanidatamab as a future treatment option in HER2-positive biliary tract cancer. Zymeworks, Jazz, and BeiGene. [ABSTRACT FROM AUTHOR]

Monospecific antibodies have been utilised increasingly for anti-cancer drug targeting owing to their ability to minimise off-target toxicity by binding specifically to a tumour epitope, hence selectively delivering drugs to the tumour cells. Nevertheless, the monospecific antibodies only engage a single cell surface epitope to deliver their drug payload. Hence, their performance is often unsatisfactory in cancers where multiple epitopes need to be engaged for optimal cellular internalisation. In this context, bispecific antibodies (bsAbs) that simultaneously target two distinct antigens or two distinct epitopes of the same antigen offer a promising alternative in antibody-based drug delivery. This review describes the recent advances in developing bsAb-based drug delivery strategies, encompassing the direct conjugation of drug to bsAbs to form bispecific antibody-drug conjugates (bsADCs) and the surface functionalisation of nanoconstructs with bsAbs to form bsAb-coupled nanoconstructs. The article first details the roles of bsAbs in enhancing the internalisation and intracellular trafficking of bsADCs with subsequent release of chemotherapeutic drugs for an augmented therapeutic efficacy, particularly among heterogeneous tumour cell populations. Then, the article discusses the roles of bsAbs in facilitating the delivery of drug-encapsulating nanoconstructs, including organic/inorganic nanoparticles and large bacteria-derived minicells, that provide a larger drug loading capacity and better stability in blood circulation than bsADCs. The limitations of each type of bsAb-based drug delivery strategy and the future prospects of more versatile strategies (e.g., trispecific antibodies, autonomous drug delivery systems, theranostics) are also elaborated. [Display omitted] [ABSTRACT FROM AUTHOR]

PSMA expression is preserved throughout all stages of prostate cancer. In this phase 1 study of JNJ-63898081, a bispecific antibody binding to PSMA-expressing tumor cells and CD3-expressing T cells, patients with metastatic castration-resistant prostate cancer had frequent cases of cytokine release syndrome, which were manageable, and transient declines in PSA. Treatment-related deaths and radiographic responses were not observed. PSMA is a potential therapeutic target for T-cell redirection in prostate cancer. Introduction: Cancer immunotherapies have limited efficacy in prostate cancer due to the immunosuppressive prostate microenvironment. Prostate specific membrane antigen (PSMA) expression is prevalent in prostate cancer, preserved during malignant transformation, and increases in response to anti-androgen therapies, making it a commonly targeted tumor associated antigen for prostate cancer. JNJ-63898081 (JNJ-081) is a bispecific antibody targeting PSMAexpressing tumor cells and CD3-expressing T cells, aiming to overcome immunosuppression and promoting antitumor activity. Patients and Methods: We conducted a phase 1 dose escalation study of JNJ-081 in patients with metastatic castration-resistance prostate cancer (mCRPC). Eligible patients included those receiving =1 prior line treatment with either novel androgen receptor targeted therapy or taxane for mCRPC. Safety, pharmacokinetics, and pharmacodynamics of JNJ-081, and preliminary antitumor response to treatment were evaluated. JNJ-081 was administered initially by intravenous (IV) then by subcutaneous (SC) route. Results: Thirty-nine patients in 10 dosing cohorts received JNJ-081 ranging from 0.3 µg/kg to 3.0 µg/kg IV and 3.0 µg/kg to 60 µg/kg SC (with step-up priming used at higher SC doses). All 39 patients experienced =1 treatment-emergent AE, and no treatment-related deaths were reported. Dose-limiting toxicities were observed in 4 patients. Cytokine release syndrome (CRS) was observed at higher doses with JNJ-081 IV or SC; however, CRS and infusion-related reaction (IRR) were reduced with SC dosing and step-up priming at higher doses. Treatment doses > 30 µg/kg SC led to transient PSA decreases. No radiographic responses were observed. Antidrug antibody responses were observed in 19 patients receiving JNJ-081 IV or SC. Conclusion: JNJ-081 dosing led to transient declines in PSA in patients with mCRPC. CRS and IRR could be partially mitigated by SC dosing, step-up priming, and a combination of both strategies. T cell redirection for prostate cancer is feasible and PSMA is a potential therapeutic target for T cell redirection in prostate cancer. [ABSTRACT FROM AUTHOR]

T-cell redirecting bispecific antibodies (BsAbs) and chimeric antigen receptor T cells (CAR T cells) have revolutionised multiple myeloma therapy, but adverse events such as cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS), cytopenias, hypogammaglobulinaemia, and infections are common. This Policy Review presents a consensus from the European Myeloma Network on the prevention and management of these adverse events. Recommended measures include premedication, frequent assessing for symptoms and severity of cytokine release syndrome, step-up dosing for several BsAbs and some CAR T-cell therapies; corticosteroids; and tocilizumab in the case of cytokine release syndrome. Other anti-IL-6 drugs, high-dose corticosteroids, and anakinra might be considered in refractory cases. ICANS often arises concomitantly with cytokine release syndrome. Glucocorticosteroids in increasing doses are recommended if needed, as well as anakinra if the response is inadequate, and anticonvulsants if convulsions occur. Preventive measures against infections include antiviral and antibacterial drugs and administration of immunoglobulins. Treatment of infections and other complications is also addressed. [ABSTRACT FROM AUTHOR]

• Amivantamab was the first approved agent for post-platinum EGFR ex20ins NSCLC. • IRRs with amivantamab were low grade with proactive management and split dosing. • IRRs primarily are first-infusion events, rarely occurring with ensuing doses. • Amivantamab administration should include IRR monitoring and as-needed intervention. Amivantamab, a fully humanized EGFR-MET bispecific antibody, has antitumor activity in diverse EGFR- and MET-driven non-small cell lung cancer (NSCLC) and a safety profile consistent with associated on-target activities. Infusion-related reaction(s) (IRR[s]) are reported commonly with amivantamab. We review IRR and subsequent management in amivantamab-treated patients. Patients treated with the approved dose of intravenous amivantamab (1050 mg, <80 kg; 1400 mg, ≥80 kg) in CHRYSALIS—an ongoing, phase 1 study in advanced EGFR-mutated NSCLC—were included in this analysis. IRR mitigations included split first dose (350 mg, day 1 [D1]; remainder, D2), reduced initial infusion rates with proactive infusion interruption, and steroid premedication before initial dose. For all doses, pre-infusion antihistamines and antipyretics were required. Steroids were optional after the initial dose. As of 3/30/2021, 380 patients received amivantamab. IRRs were reported in 256 (67%) patients. Signs/symptoms of IRR included chills, dyspnea, flushing, nausea, chest discomfort, and vomiting. Most of the 279 IRRs were grade 1 or 2; grade 3 and 4 IRR occurred in 7 and 1 patients, respectively. Most (90%) IRRs occurred on cycle 1, D1 (C1D1); median time-to-first-IRR onset during C1D1 was 60 min; and first-infusion IRRs did not compromise subsequent infusions. Per protocol, IRR was mitigated on C1D1 with holding of infusion (56% [214/380]), reinitiating at reduced rate (53% [202/380]), and aborting infusion (14% [53/380]). C1D2 infusions were completed in 85% (45/53) of patients who had C1D1 infusions aborted. Four patients (1% [4/380]) discontinued treatment due to IRR. In studies aimed at elucidating the underlying mechanism(s) of IRR, no pattern was observed between patients with versus without IRR. IRRs with amivantamab were predominantly low grade and limited to first infusion, and rarely occurred with subsequent dosing. Close monitoring for IRR with the initial amivantamab dose and early intervention at first IRR signs/symptoms should be part of routine amivantamab administration. [ABSTRACT FROM AUTHOR]

Activation of PD-1 by anchoring it to Antigen Receptor (AR) components or associated co-receptors represents an attractive approach to treat autoimmune conditions. In this study, we provide evidence that CD48, a common lipid raft and Src kinase-associated coreceptor, induces significant Src kinase-dependent activation of PD-1 upon crosslinking, while CD71, a receptor excluded from these compartments, does not. Functionally, using bead-conjugated antibodies we demonstrate that CD48-dependent activation of PD-1 inhibits proliferation of AR-induced primary human T cells, and similarly, PD-1 activation using PD-1/CD48 bispecific antibodies inhibits IL-2, enhances IL-10 secretion, and reduces NFAT activation in primary human and Jurkat T cells, respectively. As a whole, CD48-dependent activation of PD-1 represents a novel mechanism to fine tune T cell activation, and by functionally anchoring PD-1 with receptors other than AR, this study provides a conceptual framework for rational development of novel therapies that activate inhibitory checkpoint receptors for treatment of immune-mediated diseases. • Programmed cell death protein 1 (PD-1) is a validated immune checkpoint inhibitory receptor in health and disease. • Induction of PD-1 activation represents a therapeutic strategy with potential to ameliorate immune-mediated diseases. • PD-1 activation by forced co-localization to kinase-rich lipid rafts via an anchor molecule achieves T cell inhibition. • This study outlines a novel therapeutic approach for treatment of immune-mediated disease via inhibitory receptor activation. [ABSTRACT FROM AUTHOR]

Abundant cancer-associated fibroblasts (CAFs) in highly fibrotic breast cancer constitute an immunosuppressive barrier for T cell activity and are closely related to the failure of immune checkpoint blockade therapy (ICB). Inspired by the similar antigen-processing capacity of CAFs to professional antigen-presenting cells (APCs), a "turning foes to friends" strategy is proposed by in situ engineering immune-suppressed CAFs into immune-activated APCs for improving response rates of ICB. To achieve safe and specific CAFs engineering in vivo , a thermochromic spatiotemporal photo-controlled gene expression nanosystem was developed by self-assembly of molten eutectic mixture, chitosan and fusion plasmid. After photoactivatable gene expression, CAFs could be engineered as APCs via co-stimulatory molecule (CD86) expression, which effectively induced activation and proliferation of antigen-specific CD8 + T cells. Meanwhile, engineered CAFs could also secrete PD-L1 trap protein in situ for ICB, avoiding potential autoimmune-like disorders caused by "off-target" effects of clinically applied PD-L1 antibody. The study demonstrated that the designed nanosystem could efficiently engineer CAFs, significantly enhance the percentages of CD8+ T cells (4-folds), result in about 85% tumor inhibition rate and 83.3% survival rate at 60 days in highly fibrotic breast cancer, further inducing long-term immune memory effects and effectively inhibiting lung metastasis. [Display omitted] • No-destructive and precise photoactivatable gene expression. • In situ engineering immune-suppressed CAFs as immune-activated APCs. • In situ blockading of PD-1/PD-L1 interaction. [ABSTRACT FROM AUTHOR]

Described as a "don't eat me" signal, CD47 becomes a vital immune checkpoint in cancer. Its interaction with signal regulatory protein alpha (SIRP α) prevents macrophage phagocytosis. In recent years, a growing body of evidences have unveiled that CD47-based combination therapy exhibits a superior anti-cancer effect. Latest clinical trials about CD47 have adopted the regimen of collaborating with other therapies or developing CD47-directed bispecific antibodies, indicating the combination strategy as a general trend of the future. In this review, clinical and preclinical cases about the current combination strategies targeting CD47 are collected, their underlying mechanisms of action are discussed, and ideas from future perspectives are shared. CD47-based combination strategy may represent the future trend of CD47-targeted therapy. Different combinational regimens, their underlying mechanisms, and some future perspectives are summarized in this review. [Display omitted] [ABSTRACT FROM AUTHOR]