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Bispecific antibodies (bsAb) and multispecific antibodies (msAb) encompass a diverse variety of formats that can concurrently bind multiple epitopes, unlocking mechanisms to address previously difficult-to-treat or incurable diseases. Early assessment of candidate developability enables demotion of antibodies with low potential and promotion of the most promising candidates for further development. Protein-based therapies have a stringent set of developability requirements in order to be competitive (e.g. high-concentration formulation, and long half-life) and their assessment requires a robust toolkit of methods, few of which are validated for interrogating bsAbs/msAbs. Important considerations when assessing the developability of bsAbs/msAbs include their molecular format, likelihood for immunogenicity, specificity, stability, and potential for high-volume production. Here, we summarize the critical aspects of developability assessment, and provide guidance on how to develop a comprehensive plan tailored to a given bsAb/msAb.

Aim: To demonstrate the importance of critical reagent characterization for immunogenicity assay development for multi-specific drugs using two case studies. Methods: Bridging anti-drug antibody (ADA) assay with acid-dissociated samples were used for both cases. Results: In the first case study, the unexpected interference in an ADA assay from clinical samples was identified; a model was created to replicate the issue, and an anti-target antibody was identified to mitigate the target interference. In the second case study, an issue due to non-specific binding of a domain-specific confirmatory reagent was identified, and various mitigation techniques were evaluated. Conclusion: A thorough characterization of the critical reagents helped identify the issues with these ADA case studies and provided strategies for resolving them.

In contrast to natural antibodies that rely mainly on the heavy chain to establish contacts with their cognate antigen, we have developed a bispecific antibody format in which the light chain (LC) drives antigen binding and specificity. To better understand epitope-paratope interactions in this context, we determined the X-ray crystallographic structures of an antigen binding fragment (Fab) in complex with human CD47 and another Fab in complex with human PD-L1. These Fabs contain a κ-LC and a λ-LC, respectively, which are paired with an identical heavy chain (HC). The structural analysis of these complexes revealed the dominant contribution of the LCs to antigen binding, but also that the common HC provides some contacts in both CD47 and PD-L1 Fab complexes. The anti-CD47 Fab was affinity optimized by diversifying complementary-determining regions of the LC followed by phage display selections. Using homology modeling, the contributions of the amino acid modification to the affinity increase were analyzed. Our results demonstrate that, despite a less prominent role in natural antibodies, the LC can mediate high affinity binding to different antigens and neutralize their biological function. Importantly, Fabs containing a common variable heavy (VH) domain enable the generation of bispecific antibodies retaining a truly native structure, maximizing their therapeutic potential.

Natural killer (NK) cells are key players in human innate immunity. Cell engager antibody formats that recruit and activate NK cells more effectively have emerged as a promising immunotherapy approach to target cancer cells through more effective antibody-dependent cell-mediated cytotoxicity (ADCC). Monoclonal antibody drugs with ADCC activity have shown clinical benefit and improved outcomes for patients with certain types of cancer. CD16a, a Fc gamma III receptor, is the major component that is responsible for the ADCC activity of NK cells. Screening AvantGen's yeast displayed human antibody libraries led to the isolation of 2 antibody clones, #1A2 and #2-2A2, that selectively recognize both isoforms (F and V) of CD16a on primary NK cells with high affinity, yet minimally (#1A2) or do not (#2-2A2) cross-react with both allelotypes of CD16b (NA1 and NA2) expressed by neutrophils. Epitope mapping studies revealed that they bind to an epitope dependent on residue Y158 of CD16a, since mutation of Y158 to the corresponding CD16b residue H158 completely abolishes binding to CD16a. When formatted as bispecific antibodies targeting CD16a and a tumor-associated antigen (TAA, e.g. CD19), they exhibit specific binding to NK cells and induce potent NK cell activation upon encountering tumor cells, resulting in effective tumor cell killing. Notably, these bispecific antibody engagers stimulate NK cell cytokine release during co-culture with target cells, resulting in target cell cytotoxicity. These anti-CD16a antibody clones are promising candidates for combination with any TAA of interest, offering the potential for novel NK cell engager-based cancer therapeutics that are minimally affected by the high concentrations of human IgG in the circulation.

The development of bispecific antibodies that bind at least two different targets relies on bringing together multiple binding domains with different binding properties and biophysical characteristics to produce a drug-like therapeutic. These building blocks play an important role in the overall quality of the molecule and can influence many important aspects from potency and specificity to stability and half-life. Single-domain antibodies, particularly camelid-derived variable heavy domain of heavy chain (VHH) antibodies, are becoming an increasingly popular choice for bispecific construction due to their single-domain modularity, favorable biophysical properties, and potential to work in multiple antibody formats. Here, we review the use of VHH domains as building blocks in the construction of multispecific antibodies and the challenges in creating optimized molecules. In addition to exploring traditional approaches to VHH development, we review the integration of machine learning techniques at various stages of the process. Specifically, the utilization of machine learning for structural prediction, lead identification, lead optimization, and humanization of VHH antibodies.

T-cell engaging (TCE) bispecific antibodies are potent drugs that trigger the immune system to eliminate cancer cells, but administration can be accompanied by toxic side effects that limit dosing. TCEs function by binding to cell surface receptors on T cells, frequently CD3, with one arm of the bispecific antibody while the other arm binds to cell surface antigens on cancer cells. On-target, off-tumor toxicity can arise when the target antigen is also present on healthy cells. The toxicity of TCEs may be ameliorated through the use of pro-drug forms of the TCE, which are not fully functional until recruited to the tumor microenvironment. This can be accomplished by masking the anti-CD3 arm of the TCE with an autoinhibitory motif that is released by tumor-enriched proteases. Here, we solve the crystal structure of the antigen-binding fragment of a novel anti-CD3 antibody, E10, in complex with its epitope from CD3 and use this information to engineer a masked form of the antibody that can activate by the tumor-enriched protease matrix metalloproteinase 2 (MMP-2). We demonstrate with binding experiments and in vitro T-cell activation and killing assays that our designed prodrug TCE is capable of tumor-selective T-cell activity that is dependent upon MMP-2. Furthermore, we demonstrate that a similar masking strategy can be used to create a pro-drug form of the frequently used anti-CD3 antibody SP34. This study showcases an approach to developing immune-modulating therapeutics that prioritizes safety and has the potential to advance cancer immunotherapy treatment strategies.

Bispecific antibodies, including bispecific IgG, are emerging as an important new class of antibody therapeutics. As a result, we, as well as others, have developed engineering strategies designed to facilitate the efficient production of bispecific IgG for clinical development. For example, we have extensively used knobs-into-holes (KIH) mutations to facilitate the heterodimerization of antibody heavy chains and more recently Fab mutations to promote cognate heavy/light chain pairing for efficient in vivo assembly of bispecific IgG in single host cells. A panel of related monospecific and bispecific IgG 1 antibodies was constructed and assessed for immunogenicity risk by comparison with benchmark antibodies with known low (Avastin and Herceptin) or high (bococizumab and ATR-107) clinical incidence of anti-drug antibodies. Assay methods used include dendritic cell internalization, T cell proliferation, and T cell epitope identification by in silico prediction and MHC-associated peptide proteomics. Data from each method were considered independently and then together for an overall integrated immunogenicity risk assessment. In toto , these data suggest that the KIH mutations and in vitro assembly of half antibodies do not represent a major risk for immunogenicity of bispecific IgG 1 , nor do the Fab mutations used for efficient in vivo assembly of bispecifics in single host cells. Comparable or slightly higher immunogenicity risk assessment data were obtained for research-grade preparations of trastuzumab and bevacizumab versus Herceptin and Avastin, respectively. These data provide experimental support for the common practice of using research-grade preparations of IgG 1 as surrogates for immunogenicity risk assessment of their corresponding pharmaceutical counterparts.

Purpose: Accumulating analyses of pro-oncogenic molecular mechanisms triggered a rapid development of targeted cancer therapies. Although many of these treatments produce impressive initial responses, eventual resistance onset is practically unavoidable. One of the main approaches for preventing this refractory condition relies on the implementation of combination therapies. This includes dual-specificity reagents that affect both of their targets with a high level of selectivity. Unfortunately, selection of target combinations for these treatments is often confounded by limitations in our understanding of tumor biology. Here, we describe and validate a multipronged unbiased strategy for predicting optimal co-targets for bispecific therapeutics., Experimental Design: Our strategy integrates ex vivo genome-wide loss-of-function screening, BioID interactome profiling, and gene expression analysis of patient data to identify the best fit co-targets. Final validation of selected target combinations is done in tumorsphere cultures and xenograft models., Results: Integration of our experimental approaches unambiguously pointed toward EGFR and EPHA2 tyrosine kinase receptors as molecules of choice for co-targeting in multiple tumor types. Following this lead, we generated a human bispecific anti-EGFR/EPHA2 antibody that, as predicted, very effectively suppresses tumor growth compared with its prototype anti-EGFR therapeutic antibody, cetuximab., Conclusions: Our work not only presents a new bispecific antibody with a high potential for being developed into clinically relevant biologics, but more importantly, successfully validates a novel unbiased strategy for selecting biologically optimal target combinations. This is of a significant translational relevance, as such multifaceted unbiased approaches are likely to augment the development of effective combination therapies for cancer treatment. See related commentary by Kumar, p. 2570., (©2023 The Authors; Published by the American Association for Cancer Research.)

The PD-1/PD-L1 signaling pathway plays a crucial role in cancer immune evasion, and the use of anti-PD-1/PD-L1 antibodies represents a significant milestone in cancer immunotherapy. However, the low response rate observed in unselected patients and the development of therapeutic resistance remain major obstacles to their clinical application. Accumulating studies showed that overexpressed TGF-β is another immunosuppressive factor apart from traditional immune checkpoints. Actually, the effects of PD-1 and TGF-β pathways are independent and interactive, which work together contributing to the immune evasion of cancer cell. It has been verified that blocking TGF-β and PD-L1 simultaneously could enhance the efficacy of PD-L1 monoclonal antibody and overcome its treatment resistance. Based on the bispecific antibody or fusion protein technology, multiple bispecific and bifunctional antibodies have been developed. In the preclinical and clinical studies, these updated antibodies exhibited potent anti-tumor activity, superior to anti-PD-1/PD-L1 monotherapies. In the review, we summarized the advances of bispecific antibodies targeting TGF-β and PD-L1 in cancer immunotherapy. We believe these next-generation immune checkpoint inhibitors would substantially alter the cancer treatment paradigm, especially in anti-PD-1/PD-L1-resistant patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li, Wang, Niu, Wang, Zhou, Wu and Yi.)

Background: Due to their ability to recruit immune cells to kill tumor cells directly, bispecific T cell engager antibodies (BiTE) hold great potential in T cell redirecting therapies. BiTE is able to activate T cells through CD3 and target them to tumor-expressed antigens. However, there are many components in the tumor microenvironment (TME) such as mesenchymal stem cells (MSCs) that may interfere with BiTE function. Herein, we designed an anti-PDL1-BiTE that targets programmed death ligand 1 (PDL1) and CD3 and investigated its effect on PDL1pos cancer cells in the presence or absence of adipose-derived MSCs (ASCs)., Method: Our anti-PDL1-BiTE comprises of VL and VH chains of anti-CD3 monoclonal antibody (mAb) linked to the VL and VH chains of anti-PDL1 mAb, which simultaneously bind to the CD3 ε subunit on T cells and PDL1 on tumor cells. Flow cytometry was employed to assess the strength of binding of anti-PDL1-BiTE to tumor cells and T cells. Cytotoxicity, proliferation, and activation of peripheral blood lymphocyte (PBLs) were evaluated by CFSE assay and flow cytometry after using anti-PDL1-BiTE in the presence or absence of ASCs and their conditioned media (C.M.)., Results: Anti-PDL1-BiTE had the ability to induce selective lysis of PDL1pos U251-MG cancer cells while PDL1neg cells were not affected. Also, anti-PDL1-BiTE significantly stimulated peripheral blood lymphocyte (PBL) proliferation and CD69 expression. ASCs/C.M. did not show a significant effect on the biological activity of anti-PDL1-BiTE., Conclusion: Overall, anti-PDL1-BiTE selectively depletes PDL1pos cells and represents a new immunotherapeutic approach. It would increase the accumulation of T cells and can improve the prognosis of PDL1pos cancers in spite of the immunomodulatory effects of ASCs and C.M., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2023 Leila Moeinzadeh et al.)

Immunotherapies have shown remarkable, albeit tumor-selective, therapeutic benefits in the clinic. Most patients respond transiently at best, highlighting the importance of understanding mechanisms underlying resistance. Herein, we evaluated the effects of the engineered immunocytokine PD1-IL2v in a mouse model of de novo pancreatic neuroendocrine cancer that is resistant to checkpoint and other immunotherapies. PD1-IL2v utilizes anti-PD-1 as a targeting moiety fused to an immuno-stimulatory IL-2 cytokine variant (IL2v) to precisely deliver IL2v to PD-1 + T cells in the tumor microenvironment. PD1-IL2v elicited substantial infiltration by stem-like CD8 + T cells, resulting in tumor regression and enhanced survival in mice. Combining anti-PD-L1 with PD1-IL2v sustained the response phase, improving therapeutic efficacy both by reprogramming immunosuppressive tumor-associated macrophages and enhancing T cell receptor (TCR) immune repertoire diversity. These data provide a rationale for clinical trials to evaluate the combination therapy of PD1-IL2v and anti-PD-L1, particularly in immunotherapy-resistant tumors infiltrated with PD-1 + stem-like T cells., Competing Interests: Declaration of interests D.H. is a Director of Opna Bio; S.W. declares employment and stock with Molecular Partners; and L.C.D., C.K., and P. U. declare employment, patents, and stock with Roche. Roche has filed patent applications 15/943,237 and US20220072103 relevant to this work., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The prognosis for patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) is poor. Glofitamab is a bispecific antibody that recruits T cells to tumor cells., Methods: In the phase 2 part of a phase 1-2 study, we enrolled patients with relapsed or refractory DLBCL who had received at least two lines of therapy previously. Patients received pretreatment with obinutuzumab to mitigate cytokine release syndrome, followed by fixed-duration glofitamab monotherapy (12 cycles total). The primary end point was complete response according to assessment by an independent review committee. Key secondary end points included duration of response, survival, and safety., Results: Of the 155 patients who were enrolled, 154 received at least one dose of any study treatment (obinutuzumab or glofitamab). At a median follow-up of 12.6 months, 39% (95% confidence interval [CI], 32 to 48) of the patients had a complete response according to independent review. Results were consistent among the 52 patients who had previously received chimeric antigen receptor T-cell therapy (35% of whom had a complete response). The median time to a complete response was 42 days (95% CI, 42 to 44). The majority (78%) of complete responses were ongoing at 12 months. The 12-month progression-free survival was 37% (95% CI, 28 to 46). Discontinuation of glofitamab due to adverse events occurred in 9% of the patients. The most common adverse event was cytokine release syndrome (in 63% of the patients). Adverse events of grade 3 or higher occurred in 62% of the patients, with grade 3 or higher cytokine release syndrome in 4% and grade 3 or higher neurologic events in 3%., Conclusions: Glofitamab therapy was effective for DLBCL. More than half the patients had an adverse event of grade 3 or 4. (Funded by F. Hoffmann-La Roche; ClinicalTrials.gov number, NCT03075696.)., (Copyright © 2022 Massachusetts Medical Society.)

Background: G protein-coupled receptor, family C, group 5, member D (GPRC5D) is an orphan receptor expressed in malignant plasma cells. Talquetamab, a bispecific antibody against CD3 and GPRC5D, redirects T cells to mediate killing of GPRC5D-expressing myeloma cells., Methods: In a phase 1 study, we evaluated talquetamab administered intravenously weekly or every other week (in doses from 0.5 to 180 μg per kilogram of body weight) or subcutaneously weekly, every other week, or monthly (5 to 1600 μg per kilogram) in patients who had heavily pretreated relapsed or refractory multiple myeloma that had progressed with established therapies (a median of six previous lines of therapy) or who could not receive these therapies without unacceptable side effects. The primary end points - the frequency and type of dose-limiting toxic effects (study part 1 only), adverse events, and laboratory abnormalities - were assessed in order to select the recommended doses for a phase 2 study., Results: At the data-cutoff date, 232 patients had received talquetamab (102 intravenously and 130 subcutaneously). At the two subcutaneous doses recommended for a phase 2 study (405 μg per kilogram weekly [30 patients] and 800 μg per kilogram every other week [44 patients]), common adverse events were cytokine release syndrome (in 77% and 80% of the patients, respectively), skin-related events (in 67% and 70%), and dysgeusia (in 63% and 57%); all but one cytokine release syndrome event were of grade 1 or 2. One dose-limiting toxic effect of grade 3 rash was reported in a patient who had received talquetamab at the 800-μg dose level. At median follow-ups of 11.7 months (in patients who had received talquetamab at the 405-μg dose level) and 4.2 months (in those who had received it at the 800-μg dose level), the percentages of patients with a response were 70% (95% confidence interval [CI], 51 to 85) and 64% (95% CI, 48 to 78), respectively. The median duration of response was 10.2 months and 7.8 months, respectively., Conclusions: Cytokine release syndrome, skin-related events, and dysgeusia were common with talquetamab treatment but were primarily low-grade. Talquetamab induced a substantial response among patients with heavily pretreated relapsed or refractory multiple myeloma. (Funded by Janssen Research and Development; MonumenTAL-1 ClinicalTrials.gov number, NCT03399799.)., (Copyright © 2022 Massachusetts Medical Society.)

New therapeutics and combination regimens have led to marked clinical improvements for the treatment of a subset of colorectal cancer. Immune checkpoint inhibitors have shown clinical efficacy in patients with mismatch-repair-deficient or microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC). However, patients with microsatellite-stable (MSS) or low levels of microsatellite instable (MSI-L) colorectal cancer have not benefited from these immune modulators, and the survival outcome remains poor for the majority of patients diagnosed with mCRC. In this article, we describe the discovery of a novel T-cell-dependent bispecific antibody (TDB) targeting tumor-associated antigen LY6G6D, LY6G6D-TDB, for the treatment of colorectal cancer. RNAseq analysis showed that LY6G6D was differentially expressed in colorectal cancer with high prevalence in MSS and MSI-L subsets, whereas LY6G6D expression in normal tissues was limited. IHC confirmed the elevated expression of LY6G6D in primary and metastatic colorectal tumors, whereas minimal or no expression was observed in most normal tissue samples. The optimized LY6G6D-TDB, which targets a membrane-proximal epitope of LY6G6D and binds to CD3 with high affinity, exhibits potent antitumor activity both in vitro and in vivo. In vitro functional assays show that LY6G6D-TDB-mediated T-cell activation and cytotoxicity are conditional and target dependent. In mouse xenograft tumor models, LY6G6D-TDB demonstrates antitumor efficacy as a single agent against established colorectal tumors, and enhanced efficacy can be achieved when LY6G6D-TDB is combined with PD-1 blockade. Our studies provide evidence for the therapeutic potential of LY6G6D-TDB as an effective treatment option for patients with colorectal cancer., (©2022 The Authors; Published by the American Association for Cancer Research.)

COVID-19 pathogen SARS-CoV-2 has infected hundreds of millions and caused over 5 million deaths to date. Although multiple vaccines are available, breakthrough infections occur especially by emerging variants. Effective therapeutic options such as monoclonal antibodies (mAbs) are still critical. Here, we report the development, cryo-EM structures, and functional analyses of mAbs that potently neutralize SARS-CoV-2 variants of concern. By high-throughput single cell sequencing of B cells from spike receptor binding domain (RBD) immunized animals, we identify two highly potent SARS-CoV-2 neutralizing mAb clones that have single-digit nanomolar affinity and low-picomolar avidity, and generate a bispecific antibody. Lead antibodies show strong inhibitory activity against historical SARS-CoV-2 and several emerging variants of concern. We solve several cryo-EM structures at ~3 Å resolution of these neutralizing antibodies in complex with prefusion spike trimer ectodomain, and reveal distinct epitopes, binding patterns, and conformations. The lead clones also show potent efficacy in vivo against authentic SARS-CoV-2 in both prophylactic and therapeutic settings. We also generate and characterize a humanized antibody to facilitate translation and drug development. The humanized clone also has strong potency against both the original virus and the B.1.617.2 Delta variant. These mAbs expand the repertoire of therapeutics against SARS-CoV-2 and emerging variants., (© 2022. The Author(s).)

The global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires effective therapies against coronavirus disease 2019 (COVID-19), and neutralizing antibodies are a promising therapy. A noncompeting pair of human neutralizing antibodies (B38 and H4) blocking SARS-CoV-2 binding to its receptor, ACE2, have been described previously. Here, we develop bsAb15, a bispecific monoclonal antibody (bsAb) based on B38 and H4. bsAb15 has greater neutralizing efficiency than these parental antibodies, results in less selective pressure and retains neutralizing ability to most SARS-CoV-2 variants of concern (with more potent neutralizing activity against the Delta variant). We also selected for escape mutants of the two parental mAbs, a mAb cocktail and bsAb15, demonstrating that bsAb15 can efficiently neutralize all single-mAb escape mutants. Furthermore, prophylactic and therapeutic application of bsAb15 reduced the viral titer in infected nonhuman primates and human ACE2 transgenic mice. Therefore, this bsAb is a feasible and effective strategy to treat and prevent severe COVID-19., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Monoclonal antibodies constitute a promising class of targeted anticancer agents that enhance natural immune system functions to suppress cancer cell activity and eliminate cancer cells. The successful application of IgG monoclonal antibodies has inspired the development of various types of therapeutic antibodies, such as antibody fragments, bispecific antibodies, and antibody derivatives (e.g., antibody-drug conjugates and immunocytokines). The miniaturization and multifunctionalization of antibodies are flexible and viable strategies for diagnosing or treating malignant tumors in a complex tumor environment. In this review, we summarize antibodies of various molecular types, antibody applications in cancer therapy, and details of clinical study advances. We also discuss the rationale and mechanism of action of various antibody formats, including antibody-drug conjugates, antibody-oligonucleotide conjugates, bispecific/multispecific antibodies, immunocytokines, antibody fragments, and scaffold proteins. With advances in modern biotechnology, well-designed novel antibodies are finally paving the way for successful treatments of various cancers, including precise tumor immunotherapy, in the clinic., (© 2022. The Author(s).)

Bispecific antibodies (BsAb) refer to a class of biomacromolecules that are capable of binding two antigens or epitopes simultaneously. This can elicit unique biological effects that cannot be achieved with either individual antibody or two unlinked antibodies. Bispecific antibodies have been used for targeting effector cells to tumor cells, preferential targeting of cells expressing two target biomarkers over cells expressing either target biomarker individually, or to couple two molecular targets on the same cell surface to trigger unique intracellular signaling pathways. Here, we present two related methods that enable direct, rapid assembly of bispecific antibodies from any two "off-the-shelf" Immunoglobulin G (IgG) antibodies, in as little as 1 day. Both workflows can be summarized into two steps: (1) attach a small photoreactive antibody binding domain (pAbBD) fused to SpyCatcher or SpyTag (peptide-protein partners derived from the S. pyogenes fibronectin-binding protein FbaB) to each component IgG, respectively; (2) assemble the BsAb through the spontaneous isopeptide bond formation that occurs between SpyTag and SpyCatcher. These approaches enable production of BsAbs from any two IgG molecules without the need to elucidate their amino acid sequences or genetically alter their structure. Binding assays and T cell-mediated cytolysis assays were performed to validate the binding and functional properties of Trastuzumab × Cetuximab BsAb and Cetuximab × OKT3 BsAb, respectively. This approach enables rapid, low-cost production of highly homogeneous tetravalent BsAbs in a modular fashion, presenting an opportunity to quickly evaluate antibody pairs in a BsAb format for unique or synergistic functionalities.

A subset of antibodies found in cattle comprises ultralong CDR-H3 regions of up to 70 amino acids. Interestingly, this type of immunoglobulin usually pairs with the single germline VL gene, V30 that is typically very conserved in sequence. In this work, we have engineered ultralong CDR-H3 common light chain bispecific antibodies targeting Epidermal Growth Factor Receptor (EGFR) on tumor cells as well as Natural Cytotoxicity Receptor NKp30 on Natural Killer (NK) cells. Antigen-specific common light chain antibodies were isolated by yeast surface display by means of pairing CDR-H3 diversities following immunization with a single V30 light chain. After selection, EGFR-targeting paratopes as well as NKp30-specific binders were combined into common light chain bispecific antibodies by exploiting the strand-exchange engineered domain (SEED) technology for heavy chain heterodimerization. Biochemical characterization of resulting bispecifics revealed highly specific binding to the respective antigens as well as simultaneous binding to both targets. Most importantly, engineered cattle-derived bispecific common light chain molecules elicited potent NK cell redirection and consequently tumor cell lysis of EGFR-overexpressing cells as well as robust release of proinflammatory cytokine interferon-γ. Taken together, this data is giving clear evidence that bovine bispecific ultralong CDR-H3 common light chain antibodies are versatile for biotechnological applications., Competing Interests: Authors SZ, LP, SK, PA, and BV are employees of Merck Healthcare KGaA. Author DK was taking part in an internship at Merck Healthcare KGaA at the time of this study. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest, (Copyright © 2022 Klewinghaus, Pekar, Arras, Krah, Valldorf, Kolmar and Zielonka.)

Antibodies binding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike have therapeutic promise, but emerging variants show the potential for virus escape. This emphasizes the need for therapeutic molecules with distinct and novel neutralization mechanisms. Here we describe the isolation of a nanobody that interacts simultaneously with two RBDs from different spike trimers of SARS-CoV-2, rapidly inducing the formation of spike trimer-dimers leading to the loss of their ability to attach to the host cell receptor, ACE2. We show that this nanobody potently neutralizes SARS-CoV-2, including the beta and delta variants, and cross-neutralizes SARS-CoV. Furthermore, we demonstrate the therapeutic potential of the nanobody against SARS-CoV-2 and the beta variant in a human ACE2 transgenic mouse model. This naturally elicited bispecific monomeric nanobody establishes an uncommon strategy for potent inactivation of viral antigens and represents a promising antiviral against emerging SARS-CoV-2 variants., (© 2022. The Author(s).)

Rationale: The CEA-CD3 T cell bispecific antibody cibisatamab (CEA-TCB) is currently undergoing clinical trials. Here we study its performance against three-dimensional tumor organoids in cocultures with T cells as compared to a higher affinity CEACAM5-CD3 (CEACAM5-TCB) bispecific antibody using time-lapse confocal microscopy. Methods: Pre-labelled spheroids derived from colon cancer cell lines and primary organoids derived from four colorectal cancer surgical specimens, which expressed different graded levels of CEA, were exposed in cocultures to T lymphocytes. Cocultures were treated with CEA-CD3 T-cell engagers and were followed by live confocal microscopy. Caspase 3 activation detected in real-time was used as an indicator of tumor cell death. Co-cultures were also set up with autologous tumor-associated fibroblasts to test the co-stimulatory effect of a fibroblast activated protein (FAP)- targeted 4-1BBL bispecific antibody fusion protein currently undergoing clinical trials. Results: Tumor-cell killing of 3D colon carcinoma cultures was dependent on the levels of surface CEA expression, in such a way that the lower affinity agent (CEA-TCB) did not mediate killing by human preactivated T cells below a certain CEA expression threshold, while the high affinity construct (CEACAM5-TCB) remained active on the low CEA expressing organoids. Modelling heterogeneity in the levels of CEA expression by coculturing CEA high and low organoids showed measurable but weak bystander killing. Cocultures of tumor organoids, autologous fibroblasts and T cells allowed to observe a costimulatory effect of anti-FAP-4-1BBL both to release IFNγ and to attain more efficacious tumor cell killing. Conclusion: Three-dimensional tumor cocultures with T cells using live confocal microscopy provide suitable models to test the requirements for colon-cancer redirected killing as elicited by CEA-targeted T-cell engagers undergoing clinical trials and treatment allow combinations to be tested in a relevant preclinical system., Competing Interests: Competing Interests: IM reports receiving commercial research grants from BMS, Bioncotech, Alligator, Pfizer, Leadartis and Roche; has received speakers bureau honoraria from MSD; and is a consultant or advisory board member for BMS, Roche, Genmab, F-Star, Bioncotech, Bayer, Numab, Pieris, Alligator, and Merck Serono. PU, VK, MC, CK and MB are employed by Roche and declare stock ownership and patents with Roche. All other authors declare no competing interests., (© The author(s).)

Anti-drug antibodies (ADA) can limit the efficacy and safety of therapeutic antibodies. However, determining the exact nature of ADA interactions with the target drug via epitope mapping is challenging due to the polyclonal nature of the IgG response. Here, we demonstrate successful proof-of-concept for the application of hydroxyl radical footprinting (HRF)-mass spectrometry for epitope mapping of ADAs obtained from goats that were administered a knob-into-hole bispecific antibody (BsAb1). Subsequently, we performed epitope mapping of ADAs obtained from cynomolgus (cyno) monkeys that were administered BsAb1 as we described in a recently published paper. Herein, we provide the first data to demonstrate the feasibility of using HRF for ADA epitope mapping, and show that both goat and cyno-derived ADAs specifically target the complementary-determining regions in both arms of BsAb1, suggesting that the ADA epitopes on BsAb1 may be species-independent.

Early humoral immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are dominated by IgM and IgA antibodies, which greatly contribute to virus neutralization at mucosal sites. Given the essential roles of IgM and IgA in the control and elimination of SARS-CoV-2 infection, the mucosal immunity could be exploited for therapeutic and prophylactic purposes. However, almost all neutralizing antibodies that are authorized for emergency use and under clinical development are IgG antibodies, and no vaccine has been developed to boost mucosal immunity for SARS-CoV-2 infection. In addition to IgM and IgA, bispecific antibodies (bsAbs) combine specificities of two antibodies in one molecule, representing an important alternative to monoclonal antibody cocktails. Here, we summarize the latest advances in studies on IgM, IgA and bsAbs against SARS-CoV-2. The current challenges and future directions in vaccine design and antibody-based therapeutics are also discussed.

The immune checkpoint cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is an inhibitory regulator of T-cell mediated responses that has been investigated as target of monoclonal antibodies (mAbs) for cancer immunotherapy. The anti-CTLA-4 mAb ipilimumab represents the first immune checkpoint inhibitor that significantly improved overall survival in patients with unresectable/metastatic melanoma. The subsequent approved indications (often in the first-line setting) for melanoma and other advanced/metastatic solid tumors always require ipilimumab combination with nivolumab, an anti-programmed cell death protein 1 (PD-1) mAb. However, the improved clinical efficacy of the mAb combination is associated with increased immune-related adverse events, which might require treatment discontinuation even in responding patients. This drawback is expected to be overcome by the recent development of anti-CTLA-4 probodies proteolitycally activated in the tumor microenvironment and bispecific molecules targeting both CTLA-4 and PD-1, whose co-expression is characteristic of tumor-infiltrating T cells. These molecules would preferentially stimulate immune responses against the tumor, reducing toxicity toward normal tissues., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Monoclonal antibodies are coming of age as powerful tools for the prevention of infectious diseases. In recent years, the rate of antibody discovery has accelerated, and the coronavirus disease 2019 (COVID-19) pandemic has shone a spotlight on the role of these antibodies in combating pathogens. However, questions remain about the utility of monoclonal antibodies, especially when effective vaccines are also available. In this article, I discuss the role of monoclonal antibodies and briefly describe the effort to identify potent human monoclonal antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including our study on bispecific antibodies that neutralize SARS-CoV-2 variants of concern.

Since its discovery 35 years ago, there have been no therapeutic interventions shown to enable full HIV-1 remission. Combined antiretroviral therapy (cART) has achieved the sustained control of HIV-1 replication, however, the life-long treatment does not eradicate long-lived latently infected reservoirs and can result in multiple side effects including the development of multidrug-resistant escape mutants. Antibody-based treatments have emerged as alternative approaches for a HIV-1 cure. Here, we will review clinical advances in coreceptor-targeting antibodies, with respect to anti-CCR5 antibodies in particular, which are currently being generated to target the early stages of infection. Among the Env-specific antibodies widely accepted as relevant in cure strategies, the potential role of those targeting CD4-induced (CD4i) epitopes of the CD4-binding site (CD4bs) in eliminating HIV-1 infected cells has gained increasing interest and will be presented. Together, with approaches targeting the HIV-1 replication cycle, we will discuss the strategies aimed at boosting and modulating specific HIV-1 immune responses, highlighting the harnessing of TLR agonists for their dual role as latency reverting agents (LRAs) and immune-modulatory compounds. The synergistic combinations of different approaches have shown promising results to ultimately enable a HIV-1 cure.

AMG 966 is a bi-specific, heteroimmunoglobulin molecule that binds both tumor necrosis factor alpha (TNFα) and TNF-like ligand 1A (TL1A). In a first-in-human clinical study in healthy volunteers, AMG 966 elicited anti-drug antibodies (ADA) in 53 of 54 subjects (98.1%), despite a paucity of T cell epitopes observed in T cell assays. ADA were neutralizing and bound to all domains of AMG 966. Development of ADA correlated with loss of exposure. In vitro studies demonstrated that at certain drug-to-target ratios, AMG 966 forms large immune complexes with TNFα and TL1A, partially restoring the ability of the aglycosylated Fc domain to bind FcγRIa and FcγRIIa, leading to the formation of ADA. In addition to ADA against AMG 966, antibodies to endogenous TNFα were also detected in the sera of subjects dosed with AMG 966. This suggests that the formation of immune complexes between a therapeutic and target can cause loss of tolerance and elicit an antibody response against the target., Competing Interests: All authors were employees of Amgen during the time the study was being conducted. The authors declare this study received funding from Amgen. The funder was responsible for study design, interpretation of data, and the writing of this article. AMG 966 is disclosed in patent application WO2017/106383., (Copyright © 2021 Kroenke, Barger, Hu, Miller, Kalenian, He, Hsu, Bartley, Chow, Teixeira dos Santos, Sullivan, Cheng, Parnes, Padaki, Kuhns and Mytych.)

Background: Checkpoint inhibitors and currently approved cellular products for metastatic castration-resistant prostate cancer have not resulted in revolutionary changes in outcomes compared to other solid tumors. Much of this lack of progress is attributed to the unique tumor microenvironment of prostate cancer that is often immunologically cold and immunosuppressive. These unique conditions emphasize the need for novel therapeutic options. In this review, we will discuss progress made in design of T- and NK cell immune engagers in addition to chimeric antigen receptor products specifically designed for prostate cancer that are currently under investigation in clinical trials., Methods: We searched peer-reviewed literature on the PubMed and the ClinicalTrials.gov databases for active clinical trials using the terms "bispecific T-cell engager," "bispecific killer engager," "trispecific killer engager," "chimeric antigen receptor," "metastatic castration-resistant prostate cancer," and "neuroendocrine prostate cancer.", Results: Ten bispecific T-cell engager studies and nine chimeric antigen receptor-based products were found. Published data were compiled and presented based on therapeutic class., Conclusions: Multiple immune engagers and cell therapies are in the development pipeline and demonstrate promise to address barriers to better outcomes for metastatic castration-resistant prostate cancer patients., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Similar to pediatric acute myeloid leukemia (AML) the subgroup of biphenotypic acute lymphoblastic leukemia (ALL) is a rare complex entity with adverse outcome, characterized by the surface expression of CD33. Despite novel and promising anti-CD19 targeted immunotherapies such as chimeric antigen receptor T cells and bispecific anti-CD19/CD3 antibodies, relapse and resistance remain a major challenge in about 30% to 60% of patients. To investigate the potential role of the fully humanized bispecific antibody CD16 × CD33 (BiKE) in children with CD33 + acute leukemia, we tested whether the reagent was able to boost NK cell effector functions against CD33 + AML and biphenotypic ALL blasts. Stimulation of primary NK cells from healthy volunteers with 16 × 33 BiKE led to increased cytotoxicity, degranulation and cytokine production against CD33 + cell lines. Moreover, BiKE treatment significantly increased degranulation, IFN-γ and TNF-α production against primary ALL and AML targets. Importantly, also NK cells from leukemic patients profited from restoration of effector functions by BiKE treatment, albeit to a lesser extent than NK cells from healthy donors. In particular, those patients with low perforin and granzyme expression showed compromised cytotoxic function even in the presence of BiKE. In patients with intrinsic NK cell deficiency, combination therapy of CD16xCD33 BiKE and allogeneic NK cells might thus be a promising therapeutic approach. Taken together, CD16xCD33 BiKE successfully increased NK cell effector functions against pediatric AML and biphenotypic ALL blasts and constitutes a promising new option for supporting maintenance therapy or "bridging" consolidation chemotherapy before hematopoietic stem cell transplantation., (© 2021. The Author(s).)

Assembly of IgG-like asymmetric bispecific antibodies (bsAbs) requires heavy chain heterodimerization and cognate heavy-light chain pairings. Multiple strategies have been developed to solve these chain association issues. While these strategies greatly promote correct chain pairing, they normally cannot prevent low amount of chain mispaired byproducts from being generated. Besides, byproducts can also be generated as a result of discordant chain expression. The existence of various byproducts poses considerable challenges to downstream processing during the production of recombinant IgG-like bsAbs. In many cases, yield is greatly compromised for purity improvement. This mini review introduces eight IgG-like bsAb platforms, which share a common feature: they all contain built-in purification-facilitating elements in addition to chain pairing control designs. These platforms, by simultaneously providing solutions to the two issues associated with bsAb production (i.e., correct chain pairing and efficient purification), improve both efficiency and robustness of bsAb production., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Staphylococcal enterotoxin B (SEB) simultaneously crosslinks MHC class II antigen and TCR, promoting proliferation of T cells and releasing a large number of toxic cytokines. In this report, we computationally examined the possibility of using a single-chain biparatopic bispecific antibody to target SEB and prevent TCR binding. The design was inspired by the observation that mixing two anti-SEB antibodies 14G8 and 6D3 can block SEB-TCR activation, and we used 14G8-6D3-SEB tertiary crystal structure as a template. Twelve simulation systems were constructed to systematically examine the effects of the designed bispecific scFV MB102a, including isolated SEB, MB102a with different linkers, MB102a-SEB complex, MB102a-SEB-TCRβ complex, MB102a-SEB-TCR-MHC II complex, and MB102a-SEB-MHC II. Our all atom molecular dynamics simulations (total 18,900 ns) confirmed that the designed single-chain bispecific antibody may allosterically prevent SEB-TCRβ chain binding and inhibit SEB-TCR-MHC II formation. Subsequent analysis indicated that the binding of scFV to SEB correlates with SEB-TCR binding site motion and weakens SEB-TCR interactions., Competing Interests: Author BM was employed by company Molcell Biodesign, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bai, Ge, Su, Chen, Zeng, Lu and Ma.)

The objective of this manuscript is to provide the reader with a hypothetical case study to present an immunogenicity risk assessment for a multi-specific therapeutic as part of Investigational New Drug (IND) application. In order to provide context for the bioanalytical strategies used to support the multi-specific therapeutic presented herein, the introduction focuses on known immunogenicity risk factors. The subsequent hypothetical case study applies these principles to a specific example HC-12, based loosely on anti-TNFα and anti-IL-17A bispecific molecules previously in development, structured as an example immunogenicity risk assessment for submission to health authorities. The risk of higher incidence and safety impact of anti-drug antibodies (ADA) due to large protein complexes is explored in the context of multi-specificity and multi-valency of the therapeutic in combination with the oligomeric forms of the targets., (© 2021. The Author(s).)

Expression of the cell surface receptor CD137 has been shown to enhance anti-cancer T cell function via engagement with its natural ligand 4-1BBL. CD137 ligation with engineered ligands has emerged as a cancer immunotherapy strategy, yet clinical development of agonists has been hindered by either toxicity or limited efficacy. Here we show that a CD137/PD-1 bispecific antibody, IBI319, is able to overcome these limitations by coupling CD137 activation to PD-1-crosslinking. In CT26 and MC38 syngeneic mouse tumour models, IBI319 restricts T cell co-stimulation to PD-1-rich microenvironments, such as tumours and tumour-draining lymph nodes, hence systemic (liver) toxicity arising from generalised T cell activation is reduced. Besides limiting systemic T cell co-stimulation, the anti-PD-1 arm of IBI319 also exhibits checkpoint blockade functions, with an overall result of T and NK cell infiltration into tumours. Toxicology profiling in non-human primates shows that IBI319 is a well-tolerated molecule with IgG-like pharmacokinetic properties, thus a suitable candidate for further clinical development., (© 2021. The Author(s).)

Salinomycin (SAL) and lasalocid (LAS) are widely used as ionophore antibiotics for coccidiosis control. However, their common use as feed additives has led to the occurrence of feed cross-contamination, which has toxic effects on non-target animals. There have been few reports on multiple-residue detection for SAL and LAS in recent years. In this study, two single-chain antibody fragments (scFvs) capable of specifically recognizing SAL and LAS were constructed. Using LAS-scFv and SAL-scFv as parent antibodies, a complete bispecific single-chain diabody (scDb) against both LAS and SAL was built using splicing by overlap extension polymerase chain reaction (SOE-PCR). In addition, the key amino acid sites and interaction energy of antibody variable regions for small-molecule recognition were preliminarily studied by homology modeling and molecular docking. Finally, IC 50 values of 12.9 and 8.6 ng/mL, with a linear range of 6.9-24.0 and 4.7-16.0 ng/mL, were obtained for LAS-scFv and SAL-scFv, respectively. An indirect competitive enzyme-linked immunosorbent assay (icELISA) method was established using scDb to obtain an IC 50 of 3.5 ng/mL for LAS and 4.1 ng/mL for SAL, which showed better sensitivity and specificity than those of the parent scFv antibodies. The recoveries of LAS and SAL in chicken liver were 89.2-92.7%(CV<4.7%) and 88.6-90.2% (CV<6.8%)), respectively., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Bispecific T-cell engagers are an established therapeutic strategy for the treatment of hematologic malignancies but face several challenges when it comes to their application for the treatment of solid tumors, including on-target off-tumor adverse events. Employing an avidity-mediated specificity gain by introducing an additional binding moiety for the tumor-associated antigen can be achieved using formats with a 2+1 stoichiometry., Methods: Besides biochemical characterization and validation of target cell binding to cancer cells with different HER3 expression, we used in vitro co-culture assays with human peripheral blood mononuclear cells (PBMCs) and HER3-expressing target cells to determine T-cell activation, T-cell proliferation and PBMC-mediated cancer cell lysis of HER3-positive cell lines by the trivalent, bispecific antibodies., Results: In this study, we developed trivalent, bispecific antibodies comprising a silenced Fc region for T-cell retargeting to HER3-expressing tumor cells, combining a bivalent single-chain diabody (scDb) fused to a first heterodimerizing Fc chain with either an Fab or scFv fused to a second heterodimerizing Fc chain. All these HER3-targeting T-cell engagers comprising two binding sites for HER3 and one binding site for CD3 mediated target cell killing. However, format and orientation of binding sites influenced efficacy of target cell binding, target cell-dependent T-cell activation and T-cell-mediated target cell killing. Beneficial effects were seen when the CD3 binding site was located in the scDb moiety. These molecules showed efficient killing of medium HER3-expressing cancer cells with very low induction of cytokine release, while sparing target cells with low or undetectable HER3 expression., Conclusion: Our study demonstrates that these trivalent, bispecific antibodies represent formats with superior interdomain spacing resulting in efficient target cell killing and a potential advantageous safety profile due to very low cytokine release., Competing Interests: Competing interests: NA, OS and RK are named inventors on patent applications covering the HER3 antibody and scDb-based T-cell engagers., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Evasion of the immune response is a hallmark of cancer, and programmed cell death 1 (PD-1) and PD-1 ligand 1 (PD-L1) are major mediators of this immunosuppression. Chitinase 3-like 1 (CHI3L1) is induced in many cancers, where it portends a poor prognosis and contributes to tumor metastasis and spread. However, the mechanism(s) that CHI3L1 uses in metastasis have not been defined. Here we demonstrate that CHI3L1 regulates the expression of PD-L1, PD-L2, PD-1, LAG3, and TIM3 and plays a critical role in melanoma progression and lymphatic spread. CHI3L1 also contributed to IFN-γ-stimulated macrophage PD-L1 expression, and RIG-like helicase innate immunity suppressed CHI3L1, PD-L1, and melanoma progression. Individual antibodies against CHI3L1 or PD-1 had discrete antitumor effects and additive antitumor responses in metastasis models and T cell-tumor cell cocultures when administered simultaneously. Synergistic cytotoxic tumor cell death was seen in T cell-tumor cell cocultures, and significantly enhanced antitumor responses were seen in in vivo tumor models treated with bispecific antibodies that simultaneously target CHI3L1 and PD-1. CHI3L1 contributes to tumor progression by stimulating the PD-1/PD-L1 axis and other checkpoint molecules. The simultaneous targeting of CHI3L1 and the PD-1/PD-L1 axis with individual and, more powerfully, with bispecific antibodies represents a promising therapy for pulmonary metastasis and progression.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern threatens the efficacy of existing vaccines and therapeutic antibodies and underscores the need for additional antibody-based tools that potently neutralize variants by targeting multiple sites of the spike protein. We isolated 216 monoclonal antibodies targeting SARS-CoV-2 from plasmablasts and memory B cells collected from patients with coronavirus disease 2019. The three most potent antibodies targeted distinct regions of the receptor binding domain (RBD), and all three neutralized the SARS-CoV-2 Alpha and Beta variants. The crystal structure of the most potent antibody, CV503, revealed that it binds to the ridge region of SARS-CoV-2 RBD, competes with the angiotensin-converting enzyme 2 receptor, and has limited contact with key variant residues K417, E484, and N501. We designed bispecific antibodies by combining nonoverlapping specificities and identified five bispecific antibodies that inhibit SARS-CoV-2 infection at concentrations of less than 1 ng/ml. Through a distinct mode of action, three bispecific antibodies cross-linked adjacent spike proteins using dual N-terminal domain–RBD specificities. One bispecific antibody was greater than 100-fold more potent than a cocktail of its parent monoclonals in vitro and prevented clinical disease in a hamster model at a dose of 2.5 mg/kg. Two bispecific antibodies in our panel comparably neutralized the Alpha, Beta, Gamma, and Delta variants and wild-type virus. Furthermore, a bispecific antibody that neutralized the Beta variant protected hamsters against SARS-CoV-2 expressing the E484K mutation. Thus, bispecific antibodies represent a promising next-generation countermeasure against SARS-CoV-2 variants of concern.

The dramatically expanding coronavirus disease 2019 (COVID-19) needs multiple effective countermeasures. Neutralizing nanobodies (Nbs) are a potential therapeutic strategy for treating COVID-19. Here, we characterize several receptor binding domain (RBD)-specific Nbs isolated from an Nb library derived from an alpaca immunized with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (S); among them, three Nbs exhibit picomolar potency against SARS-CoV-2 live virus, pseudotyped viruses, and circulating SARS-CoV-2 variants. To improve their efficacy, various configurations of Nbs are engineered. Nb 15 -Nb H -Nb 15 , a trimer constituted of three Nbs, is constructed to be bispecific for human serum albumin (HSA) and RBD of SARS-CoV-2. Nb 15 -Nb H -Nb 15 exhibits single-digit ng/ml neutralization potency against the wild-type and Delta variants of SARS-CoV-2 with a long half-life in vivo. In addition, we show that intranasal administration of Nb 15 -Nb H -Nb 15 provides effective protection for both prophylactic and therapeutic purposes against SARS-CoV-2 infection in transgenic hACE2 mice. Nb 15 -Nb H -Nb 15 is a potential candidate for both the prevention and treatment of SARS-CoV-2 through respiratory administration., Competing Interests: Declaration of interests The authors declared no competing interests. X.W. and Z.W. are listed as inventors on a China patent (CN202110120326.8) related with the Nbs for the treatment of SARS-CoV-2 infection., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Combinations of immune checkpoint therapies show encouraging results in the treatment of many human cancers. However, the higher costs and greater side effects of such combinations compared with single-agent immunotherapies limit their further applications. In this work, a novel smart agent, KN046@ 19 F-ZIF-8, is developed to overcome these limitations. KN046 is a novel recombinant humanized PD-L1/CTLA-4 bispecific single-domain antibody-Fc fusion protein, which can bind to both PD-L1 and CTLA-4 effectively. ZIF-8 is a smart delivery system, which can safely and effectively deliver KN406 to a tumor. In vitro and in vivo results demonstrate that the smart agent KN046@ 19 F-ZIF-8 not only improves the immune response rate of the antibody drug in treatment of tumors but also reduces its toxic side effects, thereby achieving excellent antitumor efficacy. This study provides an engineering strategy for clinical applications of a more effective immunotherapy., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

CAR-T cells originate from two different approaches, cellular immunotherapy based on tumor immunosurveillance by T lymphocytes, combined with molecular engineering of bispecific antibodies and antibody fragments. The latter makes it possible to retarget immune effector cytotoxic cells (such as NK cells and T lymphocytes) to tumor cells through the binding to tumor-associated antigens. We present herein the history of bispecific antibodies, highlighting how such antibodies played a major role in CAR-T cell development. We will first evoke how antibody engineering led to the construction of various bispecific formats, in particular using the single chain Fv fragment (scFv) which has been used as the initial building block to generate chimeric bi-, tri- or multifunctional molecules. We will also describe how bispecific antibodies, either full IgG or as scFv or F(ab') 2 format, directed against Fcγ receptors or CD3ɛ and against tumor-associated antigens, induce a potent anti-tumor cytotoxicity following the recruitment and activation of immune effector cells, including CD3 + T lymphocytes. These anti-tumor effects have been translated into the clinics, especially to treat malignant hemopathies. At last, recently generated bispecific CAR-T cells suggest that the embrace between cell therapy and bispecific antibodies is not over and that we are yet to witness further discoveries enabling these cells to be even more efficient., (Copyright © 2021. Published by Elsevier Masson SAS.)

Bispecific therapies targeting CD3, so-called T-cell engagers (TCE), belong to the new spectrum of anti-tumor immunotherapies stimulating T-lymphocytes. TCE are unique constructs targeting the MHC-independent CD3 epsilon subunit (CD3e) and a tumor antigen. To date, only blinatumomab have reached market agreements in lymphoid malignancies with constructs targeting CD3exCD19. Other TCE are in advances development, with promising results targeting CD20 and BSMA in lymphoma and myeloma. These successes have relaunched the development of TCE in solid tumors, bringing mixed results so far (notably in terms of tolerance). Still, TCE pave the way to new immunotherapy in tumors considered to be refractory to inhibitors of immune checkpoints such as prostate cancer or colorectal cancer., (Copyright © 2021 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

T-cell-dependent bispecific antibodies (TDB) have been a major advancement in the treatment of cancer, allowing for improved targeting and efficacy for large molecule therapeutics. TDBs are comprised of one arm targeting a surface antigen on a cancer cell and another targeting an engaging surface antigen on a cytotoxic T cell. To impart this function, the antibody must be in a bispecific format as opposed to the more conventional bivalent format. Through in vitro and in vivo studies, we sought to determine the impact of changing antibody valency on solid tumor distribution and catabolism. A bivalent anti-HER2 antibody exhibited higher catabolism than its full-length monovalent binding counterpart in vivo by both invasive tissue harvesting and noninvasive single photon emission computed tomography/X-ray computed tomography imaging despite similar systemic exposures for the two molecules. To determine what molecular factors drove in vivo distribution and uptake, we developed a mechanistic model for binding and catabolism of monovalent and bivalent HER2 antibodies in KPL4 cells. This model suggests that observed differences in cellular uptake of monovalent and bivalent antibodies are caused by the change in apparent affinity conferred by avidity as well as differences in internalization and degradation rates of receptor bound antibodies. To our knowledge, this is the first study to directly compare the targeting abilities of monovalent and bivalent full-length antibodies. These findings may inform diverse antibody therapeutic modalities, including T-cell-redirecting therapies and drug delivery strategies relying upon receptor internalization., (©2021 American Association for Cancer Research.)

Advances in antibody engineering have enabled the construction of novel molecular formats in diverse shapes and sizes, providing new opportunities for cancer immunotherapeutic drug discovery while also revealing limitations in knowledge of structure-activity relationships. The current understanding of renal filtration originates largely from data reported for dextrans, IgG, albumin, and selected globular proteins. For a one-armed IgG-based T-cell imaging agent, we observed higher renal signal than typically observed for bivalent IgGs, prompting us to explore the factors governing renal filtration of biologics. We constructed a small representative library of IgG-like formats with varied shapes and hinge flexibilities falling broadly into two categories: branched molecules including bivalent IgG and (scFv) 2 Fc, and nonbranched molecules including one-armed IgG, one-armed IgG with stacked Fab, and one-armed IgG with a rigid IgA 2 hinge. Transmission electron microscopy revealed Y-shaped structures for the branched molecules and pseudo-linear structures for the nonbranched molecules. Single-photon emission CT imaging, autoradiography, and tissue harvest studies demonstrated higher renal uptake and catabolism for nonbranched molecules relative to branched molecules. Among the nonbranched molecules, the one-armed IgG with rigid IgA 2 hinge molecule demonstrated higher kidney uptake and decreased systemic exposure relative to molecules with a more flexible hinge. Our results show that differences in shape and hinge flexibility drive the increased glomerular filtration of one-armed relative to bivalent antibodies and highlight the practical advantages of using imaging to assess renal filtration properties. These findings are particularly relevant for T-cell-dependent bispecific molecules, many of which have nonstandard antibody structures., (©2021 American Association for Cancer Research.)

Diamond MMC Mustang is a relatively new mixed-mode resin, which mediates both cation exchange and hydrophobic interactions. In this work, we evaluated this resin using Cytiva's Capto MMC ImpRes, a well-established mixed-mode resin with similar properties, as a benchmark. The data suggest that in comparison with Capto MMC ImpRes, Diamond MMC Mustang exhibits comparable binding capacity and resolution. In addition, the resin under evaluation shows good lot-to-lot consistency. The information provided in this study allows users to have additional options when selecting mixed-mode resin for intermediate purification or final polishing, which is favourable especially at the present time when the supply chains of many manufacturers are negatively impacted by the coronavirus pandemic., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Bispecific antibodies are novel approaches of immunotherapy engaging immune cells to destroy tumor cells. Their structure is variable and underlies their pharmacocinetic properties. These coumpounds are now being evaluated across multiple hematological malignancies. The anti-CD3/CD19 antibody blinatumomab is the first in class and have been approved for the treatment of patients with Ph-negative B-cell acute lymphoblastic leukemia. Other emerging applications are lymphoma, multiple myeloma and acute myeloid leukemia. The safety profile of bispecific antibodies is acceptable while limited by neurotoxicity and cytokine-release syndrome. The present review aims to depict the landscape of emerging bispecific antibodies currently in development for hematological malignancies., (Copyright © 2021 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

Immunotherapies have recently emerged as potential game changers in the treatment of multiple myeloma (MM). Those include monoclonal antibodies (targeting CD38 or CS1), bispecific antibodies (BsAb, mainly targeting BCMA, GPRC5D or FcRH5), antibody-drug conjugate (mainly targeting BCMA) and CAR-T cells (mainly targeting BCMA). BsAb have the capacity to bind two different antigens, one at the tumor cell surface and one on T cells (CD3), recreating the immune synapse. In this article, we discuss the main clinical data on BsAb in MM, as well as their different constructs and the potential mechanism of resistance., (Copyright © 2021 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

Natural killer (NK) cells serve as key innate effectors and their activity has been considered a prognostic biomarker in diverse human diseases. Currently, NK cell functional assays have several problems primarily related to adequate preparation, labeling, or treatment of target cells, which are cumbersome and often hamper consistent sensitivity for NK cells. Here, bispecific antibodies (BsAb's) targeting NKG2D and 2B4 receptors, whose combination mounts selective cytotoxicity and IFN-γ production of NK cells, are developed as acellular, consistent, and easy-to-use strategies for assessing NK cell functions. These NK cell activator BsAb's (NKABs) are constructed in symmetric dual bivalent formats with different interdomain spacings [immunoglobulin G (IgG)-single-chain variable fragment (scFv) and dual-variable domain (DVD)-Ig] and kappa constant (Cκ)-scFv format linking two scFv's with a Cκ domain. These NKABs are specific and superior to a combination of monospecific antibodies for NK cell activation. NKAB elicits both direct cytotoxicity and IFN-γ production via integration of NKG2D and 2B4 signals. Moreover, stimulation with NKAB IgG-scFv and Cκ-scFv reveals defective NK cell functions in X-linked lymphoproliferative disease involving 2B4 dysfunction in NK cells and multiple myeloma in peripheral blood mononuclear cells and whole blood, respectively. Hence, this work provides a proof of concept that NKAB facilitates the reliable and comprehensive measurement of NK cell function in clinical settings for diagnostic and prognostic purposes.