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Novel T-cell immunotherapies such as bispecific T-cell engagers (BiTEs) are emerging as promising therapeutic strategies for prostate cancer. BiTEs are engineered bispecific antibodies containing two distinct binding domains that allow for concurrent binding to tumor-associated antigens (TAAs) as well as immune effector cells, thus promoting an immune response against cancer cells. Prostate cancer is rich in tumor associated antigens such as, but not limited to, PSMA, PSCA, hK2, and STEAP1 and there is strong biologic rationale for employment of T-cell redirecting BiTEs within the prostate cancer disease space. Early generation BiTE constructs employed in clinical study have demonstrated meaningful antitumor activity, but challenges related to drug delivery, immunogenicity, and treatment-associated adverse effects limited their success. The ongoing development of novel BiTE constructs continues to address these barriers and to yield promising results in terms of efficacy and safety. This review will highlight some of most recent developments of BiTE therapies for patients with advanced prostate cancer and the evolving data surrounding BiTE constructs undergoing clinical evaluation.

Monoclonal neutralizing antibodies (mAbs) are considered an important prophylactic against SARS-CoV-2 infection in at-risk populations and a strategy to counteract future sarbecovirus-induced disease. However, most mAbs isolated so far neutralize only a few sarbecovirus strains. Therefore, there is a growing interest in bispecific antibodies (bsAbs) which can simultaneously target different spike epitopes and thereby increase neutralizing breadth and prevent viral escape. Here, we generate and characterize a panel of 30 novel broadly reactive bsAbs using an efficient controlled Fab-arm exchange protocol. We specifically combine some of the broadest mAbs described so far, which target conserved epitopes on the receptor binding domain (RBD). Several bsAbs show superior cross-binding and neutralization compared to the parental mAbs and cocktails against sarbecoviruses from diverse clades, including recent SARS-CoV-2 variants. BsAbs which include mAb COVA2-02 are among the most potent and broad combinations. As a result, we study the unknown epitope of COVA2-02 and show that this mAb targets a distinct conserved region at the base of the RBD, which could be of interest when designing next-generation bsAb constructs to contribute to a better pandemic preparedness.

Multiple myeloma (MM), a cancer of plasma cells, remains difficult to treat due to its incurability and high recurrence rates. Recent advancements in immunotherapies, such as CAR T cells, bispecific antibodies, and bispecific T cell engagers (BITEs) targeting B-cell maturation antigen (BCMA), have improved treatment options for relapsed and refractory MM (RRMM). However, these therapies face challenges, including complex manufacturing, high cost, and severe side effects. In this study, we developed a stable cell line that produces anti-BCMA × anti-CD3 BITEs and generated BITE-armed T cells (BATs) as a novel MM treatment approach. These αBCMA × αCD3 BATs specifically targeted BCMA-expressing cells, promoting T cell activation, proliferation, and cytotoxicity. BATs demonstrated superior cytotoxicity compared to unarmed T cells, likely due to enhanced antigen specificity and targeting efficiency, even at low effector-to-target ratios. The antitumor activity of BATs against BCMA-expressing cells was antigen-specific and dose-dependent. BATs also triggered T cell expansion and significant cytokine release (IL-2, TNF-α, IFN-γ) without increasing IL-6, suggesting a lower risk of cytokine release syndrome (CRS). Our findings indicate that BCMA-targeting BATs offer a promising and accessible therapeutic strategy for MM, with a simple, rapid, and cost-effective production process. These results support future development of BITE-armed T cells as a novel cancer treatment to enhance therapeutic outcomes for MM patients., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: All experiments were conducted at the Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. The authors declare that the research was carried out without any commercial or financial relationships that could have influenced the reported findings., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

An enzyme-linked immunosorbent assay (ELISA) based anti-drug antibody (ADA) assay was developed to support the clinical development of a bispecific antibody biotherapeutic anti-A/B. This anti-A/B clinical ADA Version 1 (V1) assay was successfully validated initially using commercial samples from the target indication. However, applying the validation cut point factors (CPFs) led to a high untreated ADA positive rate in the Phase 1 study baseline sample analysis. While implementing the in-study CPFs was effective to mitigate the high baseline prevalence, this led to unfavorable assay sensitivity with no drug tolerance, which necessitated an assay re-optimization. The re-optimized Version 2 assay (V2) was able to mitigate the matrix interference observed in the clinical sample testing using the V1 assay, proven to be a more suitable method. The V2 assay optimization work was discussed, and the performance of the V1 and V2 assays during validation and clinical sample analysis was compared. Preliminary sample testing results generated using the two versions of the assay were compared and the ADA clinical impact was discussed. Our experience insinuates that a successfully validated method does not guarantee to be appropriate for sample testing. Adjustments of the method may be required to ensure that it performs as expected during sample testing and throughout the assay's lifecycle. This work highlights the importance of verifying the assay suitability during clinical sample testing and making appropriate adjustments as needed, especially in the first clinical study and the first study for a new indication., Competing Interests: Declarations. Conflict of interest: All authors are employees of Genentech Inc., a member of the Roche group and Roche shareholders., (© 2024. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

Ubamatamab, a Mucin 16 (MUC16) × cluster of differentiation 3 (CD3) bispecific antibody that promotes T-cell-mediated cytotoxicity of MUC16-expressing cells, is being investigated for the treatment of ovarian cancer. Intravenous administration of ubamatamab, with or without the anti-programmed cell death-1 inhibitor cemiplimab, is being evaluated in a first-in-human study in patients with recurrent ovarian cancer. In vitro cytotoxicity and cytokine data and projected ubamatamab human pharmacokinetic (PK) profiles scaled with monkey PK parameters enabled starting-dose selection in humans. Mouse tumor regression studies identified ubamatamab effective concentrations. Preclinical and clinical PK, cytokine, safety, and efficacy data from dose escalation were integrated to determine expansion regimens. A starting dose of 0.1 mg was selected, which showed acceptable safety in patients. A step-up dosing approach was used to effectively manage cytokine release syndrome. Mouse tumor regression models suggested an ubamatamab efficacious concentration range of 0.4-50 mg/L, consistent with clinical activity observed at ubamatamab trough concentrations ≥5 mg/L. Integrating preclinical and clinical data determined a target trough concentration range of 5-30 mg/L, which supports evaluation of ubamatamab 250 mg with or without cemiplimab and 800 mg monotherapy once every 3 weeks in expansion cohorts. Preclinical data (cytokine release, tumor regression, monkey PK) had translational value in supporting regimen selection in dose escalation and subsequently in dose expansion after integration with patient data from dose escalation., (© 2024 The Author(s). Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Tumor immunotherapy includes bispecific antibodies (BsAbs), immune checkpoint inhibitors (ICIs), vaccines, and adoptive cell immunotherapy. BsAbs belong to the family of antibodies that can specifically target two or more different antigens and are a promising option for tumor immunotherapy. Immune checkpoints are antibodies targeting PD-1, PD-L1, and CTLA4 and have demonstrated remarkable therapeutic efficacy in the treatment of hematological and solid tumors, whose combination therapies have been shown to synergistically enhance the antitumor effects of BsAbs. In addition, the clinical efficacy of existing monoclonal antibodies targeting PD-1 (e.g., ipilimumab, nivolumab, pembrolizumab, and cemiplimab) and PD-L1 (e.g., atezolizumab, avelumab, and durvalumab) could also be enhanced by conjugation to small drugs as antibody-drug conjugates (ADCs). The development of truly effective therapies for patients with treatment-resistant cancers can be achieved by optimizing the various components of ADCs., (© 2024 John Wiley & Sons Ltd.)

Human coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2 have recurrently emerged as significant pathogens, causing severe respiratory illnesses and presenting challenges to monoclonal antibody therapeutics due to their rapid evolution, particularly the diverse variants of SARS-CoV-2. In this study, we utilized "Knob-into-Hole" and "IgG-scFv" technologies to engineer bispecific antibodies (bsAbs) that target both the viral receptor and spike protein, enhancing their neutralization breadth and potency. Our bsAbs, combining anti-SARS-CoV-2 or anti-MERS-CoV antibodies with an anti-ACE2 antibody, demonstrated effective neutralization across a range of SARS-CoV-2 variants, SARS-CoV and MERS-CoV in both pseudovirus and authentic virus assays. Notably, the "IgG-scFv" bsAbs format exhibited superior binding and neutralization capabilities compared to the "Knob-into-Hole" configurations. The most effective of these, "IgG-scFv" H11B11_m336, displayed exceptional neutralization potency against a panel of 24 pseudotyped Beta-Coronaviruses, with IC 50 values ranging from 0.001-0.183 μg/mL. Overall, our findings underscore the potential of bsAbs as an effective strategy to meet the immediate challenges posed by existing and emerging pathogens, thereby enhancing global pandemic preparedness.

In 2022, the monkeypox virus (mpox virus, MPXV) exhibited global dissemination across six continents, representing a notable challenge owing to the scarcity of targeted antiviral interventions. Passive immunotherapy, such as the use of monoclonal antibodies (mAbs) and bispecific antibodies (bsAbs), has emerged as a promising option for antiviral regimens. Here, we generated several mAbs against M1R and B6R of MPXV, and subsequently characterized the antiviral activity of these antibodies both in vitro and in vivo . Two neutralizing mAbs, M1H11 and M3B2, targeting M1R, and one B6R-specific mAb, B7C9, were identified. They exhibited varying antiviral efficacy against vaccinia virus (VACV) in vitro and in vivo . A cocktail comprising M1H11 and M3B2 demonstrated a superior protective effect in vivo . A bsAb, Bis-M1M3, was engineered by conjugating the fragment crystallizable (Fc) region of the human-mouse chimeric engineered M1H11 with the single-chain fragment variable (scFv) of M3B2. In mice challenged with MPXV, Bis-M1M3 showed a notable protective effects. Analysis of neutralization mechanism showed that these mAbs and Bis-M1M3 exerted virus-neutralizing effects before the virus infects cells. In vivo pharmacokinetic experiments showed that Bis-M1M3 has a long half-life in rhesus macaques. This study provides crucial insights for further research on broad-spectrum antiviral drugs against MPXV and other orthopoxviruses.

Purpose: NB12 is a bispecific antibody that consists of two anti-programmed cell death-ligand 1 (PD-L1) nanobodies and two anti-programmed cell death-ligand 2 (PD-L2) nanobodies. The aim of this study was to design a novel tracer, [ 124 I]I-NB12, targeting PD-L1/2 and perform preclinical evaluations to dynamically monitor PD-L1/2 expression for determining cancer patient responsiveness to ICI therapy., Methods: NB12 was labelled with the radionuclide 124 I at room temperature (RT). An in vitro binding assay was performed to assess the affinity of [ 124 I]I-NB12 for PD-L1 and PD-L2. Cellular uptake, pharmacokinetic, and biodistribution experiments were performed to evaluate the biological properties. Micro-PET/CT imaging with [ 124 I]I-NB12 was conducted at different time points. Immunohistochemical and haematoxylin and eosin (HE) staining experiments were carried out using tumour tissues. Routine blood, biochemical indices and major organ pathology were used to evaluate the biosafety of the tracers., Results: The radiochemical yield of [ 124 I]I-NB12 was 84.62 ± 3.90%, and the radiochemical purity (RCP) was greater than 99%. [ 124 I]I-NB12 had a high affinity for the PD-L1 (Kd = 19.82 nM) and PD-L2 (Kd = 2.93 nM). Cellular uptake experiments confirmed that the uptake of [ 124 I]I-NB12 by A549-PDL1/2 cells was greater than that by A549 cells. The half-lives of the distribution phase and elimination phase were 0.26 h and 4.08 h, respectively. Micro-PET/CT showed significant [ 124 I]I-NB12 uptake in the tumour region of A549-PDL1/2 tumour-bearing mice compared with A549 tumour-bearing mice 24 h postinjection. Immunohistochemical and HE staining experiments confirmed that tumour-bearing mice was successfully constructed., Conclusion: We constructed a bispecific antibody that targets PD-L1 and PD-L2, namely, [ 124 I]I-NB12. Biological evaluation revealed its specificity and affinity for PD-L1/2, and micro-PET/CT confirmed the feasibility of visualizing tumour PD-L1/2 in vivo. Using [ 124 I]I-NB12 may be a promising strategy for identifying cancer patients that can potentially benefit from ICI therapy., Competing Interests: Declarations. Ethics approval: All animal experiments were performed according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the Animal Care and Ethics Committee of Peking University Cancer Hospital (Approval No. EAEC 2022-01). Conflict of interest: The authors have no relevant financial or non-financial interests to disclose., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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

SARS-CoV-2 has been evolving into a large number of variants, including the highly pathogenic Delta variant, and the currently prevalent Omicron subvariants with extensive evasion capability, which raises an urgent need to develop new broad-spectrum neutralizing antibodies. Herein, we engineer two IgG-(scFv) 2 form bispecific antibodies with overlapping epitopes (bsAb1) or non-overlapping epitopes (bsAb2). Both bsAbs are significantly superior to the parental monoclonal antibodies in terms of their antigen-binding and virus-neutralizing activities against all tested circulating SARS-CoV-2 variants including currently dominant JN.1. The bsAb1 can efficiently neutralize all variants insensitive to parental monoclonal antibodies or the cocktail with IC 50 lower than 20 ng/mL, even slightly better than bsAb2. Furthermore, the cryo-EM structures of bsAb1 in complex with the Omicron spike protein revealed that bsAb1 with overlapping epitopes effectively locked the S protein, which accounts for its conserved neutralization against Omicron variants. The bispecific antibody strategy engineered from overlapping epitopes provides a novel solution for dealing with viral immune evasion.

Background: Bispecific T-cell engagers (BiTEs) are recombinant bispecific proteins designed to stimulate polyclonal T-cell immunity. In recent years, B7H3, a pan-cancer antigen, has been considered a promising target for future immunotherapy. However, the B7H3-targeting BiTE faces the challenge of systemic toxicity. Oncolytic viruses (OVs) represent a new class of cancer immunotherapeutics and serve as an appropriate platform for locoregional delivery of therapeutic genes. In this study, we designed an oncolytic adenovirus (OAd) encoding BiTE targeting human B7H3. We hypothesized that OVs encoding B7H3 BiTE deliver this molecule persistently to the tumor site while mediating polyclonal T-cell activation and redirecting it to tumor cells., Methods: B7H3-targeting BiTE was constructed by linking a single-chain variable fragment (scFv) that recognizes human B7H3 to an scFv that recognizes human CD3. B7H3 BiTE was inserted into OAd to construct OAd-B7H3-BiTE. The function of the OV-delivered B7H3 BiTE was detected via co-culturing B7H3 + target cells and peripheral blood mononuclear cells. A humanized immune system mouse model was used to evaluate the therapeutic effects in vivo., Results: B7H3 is highly expressed in a high proportion of human malignancies. OV-delivered BiTEs bind to T cells and target cells. We observed a series of phenomena reflecting T-cell activation induced by OAd-B7H3-BiTE, including cell clustering, cell size, activation markers, cytokine secretion, and proliferation. Furthermore, T-cell activation was mirrored by the corresponding cytotoxicity against B7H3 + tumor cells. In vivo, B7H3 BiTE was persistently expressed in tumors and enhanced the antitumor T-cell immune response., Conclusions: Using an OV for the local expression of B7H3 BiTE maximizes the local concentration of BiTE while reducing systemic exposure. OV also provides a relatively "hot" T-cell immune environment for the function of BiTE. Because of its capacity to activate polyclonal T cells, BiTE has the potential to redirect virus-specific T cells to tumors. Our study provides new opportunities for the exploitation of B7H3-BiTE-armed OVs as therapeutic agents for the treatment of B7H3-positive malignancies., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Immunotherapy has changed the treatment landscape of cancer in the past decade. Amongst various forms of immunotherapy, manipulating the T cell has shown promise in past few years. T cell manipulation can be done by CAR-T cell therapy or bispecific antibodies. These 2 therapeutic modalities have been studied and shown efficacy in hematologic malignancies in relapsed refractory setting. They have not been compared to each other in randomized control trials; hence, the adequate sequencing is not known. Although cross-trial comparison is not ideal, available evidence indicates toward similar efficacy and better tolerability of BiTEs compared to CAR-T cell therapy. Bispecific antibodies, though, have an advantage of being able to be used in an "off the shelf" manner considering these are not MHC specific and can be administered to all patients regardless of HLA type. There has been an explosive growth in the indications for bispecific antibodies in the recent past. In this article, we have reviewed recent approvals, indication and literature regarding efficacy and adverse effects of bispecific antibodies in all available indications. This will be a useful read for clinical practitioners to understand the mechanism of action and place of all available bispecific antibodies in the current oncology landscape., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s).)

Background: In the tumor microenvironment (TME), the transforming growth factor-β (TGF-β) and programmed cell death receptor 1 (PD-1)/programmed death ligand 1 (PD-L1) signaling axes are complementary, nonredundant immunosuppressive signaling pathways. Studies have revealed that active TGF-β is mainly released from the glycoprotein A repetitions predominant (GARP)-TGF-β complex on the surface of activated regulatory T cells (Tregs), B cells, natural killer (NK) cells, and tumor cells. The currently available antibodies or fusion proteins that target TGF-β are limited in their abilities to simultaneously block TGF-β release and neutralize active TGF-β in the TME, thus limiting their antitumor effects., Methods: We designed and constructed a bispecific, trifunctional antibody, namely, BPB-101, that specifically targets the GARP-TGF-β complex and/or small latent complex (SLC), active TGF-β, and PD-L1. The binding ability of BPB-101 to the different antigens was determined by ELISA, FACS, and biolayer interferometry (BLI). The blocking ability of BPB-101 to the TGF-β and PD-1/PD-L1 signaling axes was determined by reporter gene assay (RGA). The antitumor effect and biosafety of BPB-101 were determined in a transgenic mouse tumor model and cynomolgus monkeys, respectively. Stability assessments, including stability in serum, after exposure to light, after repeated freeze-thaw cycles, and after high-temperature stress tests had been completed to evaluate the stability of BPB-101., Results: BPB-101 bound efficiently to different antigenic proteins: the GARP-TGF-β complex and/or SLC, active TGF-β, and PD-L1. Data showed that BPB-101 not only effectively inhibited the release of TGF-β from human Tregs, but also blocked both the TGF-β and PD-1/PD-L1 signaling pathways. In an MC38-hPD-L1 tumor-bearing C57BL/6-hGARP mouse model, BPB-101 at a dose of 5 mg/kg significantly inhibited tumor growth, with a complete elimination rate of 50%. Stability assessments confirmed the robustness of BPB-101. Furthermore, BPB-101 showed a favorable safety profile in nonhuman primate (NHP) toxicity studies., Conclusion: BPB-101 is a potentially promising therapeutic candidate that may address unmet clinical needs in cancer immunotherapy, thus, BPB-101 warrants further clinical investigation., Competing Interests: The authors are employees of Betta Pharmaceuticals Co., Ltd., which also provided funding for this study, thereby presenting a potential conflict of interest., (Copyright © 2024 Xu, Xu, Li, Xu, Cheng, Zheng, Zhang, Liu, Ye, Jiang, Yu, Wang and Ding.)

Crucial for cell activities, ion channels are key drug discovery targets. Although small-molecule and peptide modulators dominate ion channel drug discovery, antibodies are emerging as an alternative modality. However, challenges persist in generating potent antibodies, especially for channels with limited extracellular epitopes. We herein present a bi-epitopic crosslinking strategy to overcome these challenges, focusing on Na V 1.7, a potential analgesic target. Aiming to crosslink two non-overlapping epitopes on voltage-sensing domains II and IV, we construct bispecific antibodies and ligand-antibody conjugates. Enhanced affinity and potency are observed in comparison to the monospecific controls. Among them, a ligand-antibody conjugate (1080-PEG 7 -ACDTB) displays a two-orders-of-magnitude improvement in potency (IC 50 of 0.06 ± 0.01 nM) and over 1,000-fold selectivity for Na V 1.7. Additionally, this conjugate demonstrates robust analgesic effects in mouse pain models. Our study introduces an approach to developing effective antibodies against Na V 1.7, thereby initiating a promising direction for the advancement of pain therapeutics., Competing Interests: Declaration of interests J.D., Y.Z., and R.Z. have a patent pending to Tsinghua University., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The redirection of T lymphocytes against tumor-associated or tumor-specific antigens, using bispecific antibodies or chimeric antigen receptors (CAR), has shown therapeutic success against certain hematological malignancies. However, this strategy has not been effective against solid tumors. Here, we describe the development of CAR T cells targeting p95HER2, a tumor-specific antigen found in HER2-amplified solid tumors. These CAR T cells display robust activity against p95HER2-expressing cell lines but demonstrate limited efficacy against patient-derived xenografts. As p95HER2 is invariably detectable on tumor cells that overexpress HER2, but not those that express HER2 at normal levels, we arm p95HER2-specific CAR T cells with affinity-tuned bispecific antibodies against HER2 and CD3 in order to redirect them only to HER2-amplified cells. The combination of p95HER2.CAR T cells and HER2 x CD3 bispecific antibodies lead to a complete regression in three HER2-positive, patient-derived mouse xenografts tumor models. This combination represents a promising strategy to redirect T cells against a subset of HER2-positive tumors., Competing Interests: Competing interests J.A. has received research funds from Roche, Byondis, Menarini and Molecular Partners and consultancy honoraria from Menarini, Mnemo and ARKIN. J.A. is an inventor of patent applications EP22382294, EP20382457, EP16191933.7, EP0930183.5 and P200801652. M.R.A., I.R.R., C.K., E.M., are inventors of patent application EP20382457. M.R.A., S.D., A.G.E., I.R.R., V.N. are inventors of patent application EP22382294. C.K. declares employment, patents and stock ownership with Roche. E.M. declares employment with Roche. E.G. reports: research agreements with Novartis, Roche, Thermo Fisher, AstraZeneca, Taiho, BeiGene, Janssen; consultant/advisor at Roche, Ellipses Pharma, Boehringer Ingelheim, Janssen Global Services, Seattle Genetics, Thermo Fisher, MabDiscovery, Anaveon, F-Star Therapeutics, Hengrui, Sanofi, Incyte; and payment or honoraria for speakers’ bureaus from Merck Sharp & Dohme, Roche, Thermo Fisher, Lilly, Novartis, SeaGen. The remaining authors declare no competing interests., (© 2024. The Author(s).)

Recent advancements in cancer immunotherapy have spotlighted the PD-1/PD-L1 pathway, crucial for its role in immune checkpoint regulation. Traditional inhibitors, though successful, face challenges like resistance and adverse effects. Bispecific antibodies targeting PD-1 and PD-L1 present a ground-breaking solution by simultaneously engaging multiple immune regulatory molecules. Developments in PD-1/PD-L1 bispecific antibodies up to now have been summarized, and the latest findings from the 2024 ASCO conference are presented, revealing that bispecific antibodies exhibit robust efficacy in treating various types of cancers, marking a significant step forward in cancer treatment., Competing Interests: Declarations Conflicts of interest The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Antibody drugs targeting single inflammatory cytokines have revolutionized the treatment of immune-mediated inflammatory diseases. To investigate whether dual targeting interleukin-17 (IL-17) and IL-36 enhances anti-inflammatory activity, bispecific Ab HB0043 was generated by linking the single chain fragment variables (scFvs) from humanized anti-IL-36R antibody (HB0034) to the C-terminus of the heavy chain of anti-IL-17A IgG1 (HB0017) Fc using a flexible peptide linker. HB0043 largely maintained the binding affinities and biological activities of the two parent monoclonal antibodies (mAbs) in vitro . IL-17 and IL-36 cooperated to amplify the expression of pro-inflammatory and pro-fibrotic genes in normal human dermal fibroblasts (NHDF). However, HB0043 more effectively blocked IL-6 and IL-8 production in NHDF stimulated by IL-17A and IL-36 compared to two monoclonal antibodies. In a mouse model of Oxazolone (OXA)-induced atopic dermatitis and Imiquimod (IMQ)-induced skin inflammation, administration of both anti-IL17A mAb HB0017 and anti-mouse IL-36R surrogate antibody HB0034SA showed improved effectiveness in alleviating skin thickening and inflammation based on histological assessment. Further, in cynomolgus monkeys, HB0043 showed no enhanced target-related toxicity compared with the two parental mAbs in vivo and with a moderate increase in production of anti-drug antibodies. Together, dual blockade of IL-17A and IL-36R in the form of a bispecific antibody may have advantages in blocking the overlapping and non-overlapping functions of these two cytokines in skin inflammation that could not optimally be curtailed with single mAbs. In conclusion, as monotherapy may reach therapeutic celling for certain difficult-to-treat inflammatory and fibrotic diseases, dual targeting could potentially pave a way to combat these diseases., Competing Interests: XM, SZ, XR, JYX, JGX, HL, SC, YW, HJ, YF, SX, XPC, XFC, XP, SW, HY, XW, ML, QQ, XZ and YZ are employees of Huaota Biopharmaceutical Co. Ltd. The remaining author 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. The authors declare that this study received funding from Huaota Biopharmaceutical Co. Ltd. The funder had the following involvement in the study: study design, collection, analysis, interpretation of data, the writing of this article, and the decision to submit it for publication. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Ma, Zhang, Ren, Feng, Li, Chen, Xu, Wang, Peng, Yan, Jia, Xia, Cui, Chen, Pan, Wang, Yu, Wei, Li, Liu, Xu, Qian, Zhu, Zhan and Lu.)

Bispecific antibodies (bsAbs) and multispecific antibodies (msAbs) represent a promising frontier in therapeutic antibody development, offering unique capabilities not achievable with traditional monoclonal antibodies. Despite their potential, significant challenges remain due to their increased molecular complexity. One prominent challenge is the correct assembly of light and heavy chains, as improper pairing leads to mispaired or incompletely assembled species that lack therapeutic efficacy and possess undesired properties, impairing the developability, manufacturability, and safety. There is a critical need for rapid, sensitive analytical tools to monitor and control these undesired species and ensure the quality assessment of bsAbs and msAbs. To address this need, we present a novel high-throughput, format-agnostic intact mass workflow that significantly enhances the efficiency of detecting and quantifying biotherapeutic products and related impurities. This workflow integrates automated sample preparation, novel high-resolution rapid mass detection powered by SampleStream-MS, and an advanced data analysis pipeline. It offers increased throughput and data quality while substantially reducing analysis turnover time and labor. This was demonstrated in a pilot program where ∼800 multispecific antibodies were processed in 10 working days. The article details the evaluation and validation of our method, demonstrating its repeatability and intermediate precision in terms of measurement accuracy and relative quantification of various product-related species. We underscore the transformative potential of this end-to-end high-throughput workflow in expediting bispecific and multispecific antibody discovery, optimizing production processes, and ensuring high-quality development and manufacturing for therapeutic antibodies.

The concern of COVID-19 persists due to the continuous emergence of variants and the potential spillover of animal coronaviruses. The broad-spectrum neutralizing antibodies play a pivotal role in the prevention and treatment of coronavirus (CoV) infections. Here, we constructed 18 bi-specific antibodies (bsAbs) using 9 antibodies isolated from COVID-19 convalescents and vaccinated individuals, designed as dual variable domain immunoglobulin (DVD-Ig). A bsAb 5-HI showed a high binding capability to the S1 subunit of spike and exhibited breadth and potency against pseudotyped SARS-CoV-2 variants of concerns (VOCs) and SARS-related-CoVs (SARSr-CoVs), with half maximal effective concentration (EC 50 ) of 0.028-3.444 nM and 50% inhibitory concentration (IC 50 ) of 0.008-0.800 nM. In addition, it retained neutralization potency against the peudotyped virus of recently prevalent JN.1 strain (IC 50 , 12.74 nM). We found that the parental antibodies showed weak or no binding to the receptor binding domain (RBD) of the SARS-CoV, EG.5.1, and JN.1. However, the 5-HI maintained the binding with RBD and prevented the binding between hACE2 and RBD (IC 50 for the RBD of SARS-CoV, 1.067 nM; EG.5.1, 0.423 nM; JN.1, 0.223 nM). In neutralization assays with the authentic virus, we found that the 5-HI effectively neutralized Omicron variants XBB.1.5 (IC 50 , 0.308 nM), EG.5.1 (IC 50 , 0.129 nM), and JN.1 (IC 50 , 13.692 nM), while its parental antibodies showed weakened or no neutralization. Therefore, the 5-HI represents a promising candidate for further development in the treatment and prevention of ongoing evolved SARS-CoV-2 VOCs and other SARSr-CoVs that potentially emerge in the future., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Current antibody-based immunotherapy depends on tumor antigen shedding for proper T cell priming. Here we select a novel human CD40 agonistic drug candidate and generate a bispecific antibody, herein named BiA9*2_HF, that allows for rapid antibody-peptide conjugate formation. The format is designed to facilitate peptide antigen delivery to CD40 expressing cells combined with simultaneous CD40 agonistic activity. In vivo, the selected bispecific antibody BiA9*2_HF loaded with peptide cargos induces improved antigen-specific proliferation of CD8 + (10-15 fold) and CD4 + T cells (2-7 fold) over control in draining lymph nodes. In both virus-induced and neoantigen-based mouse tumor models, BiA9*2_HF demonstrates therapeutic efficacy and elevated safety profile, with complete tumor clearance, as well as measured abscopal impact on tumor growth. The BiA9*2_HF drug candidate can thus be utilized to tailor immunotherapeutics for cancer patients., (© 2024. The Author(s).)

Siglec-9 is a promising immune checkpoint molecule, and therapeutics targeting Siglec-9 have the potential to augment anti-tumor immunity. Here, we generated a bispecific antibody, named as aSE4-1-Fc, by fusing two distinct alpaca derived nanobodies, which can simultaneously target the extracellular Ig variable (V)-set domain and C2-set domains of murine Siglec-9 (also known as Siglec-E) with high affinity. In vivo studies showed that aSE4-1-Fc was better than its component antibodies in inhibiting tumor growth/metastasis, and Siglec-E blockade using aSE4-1-Fc generated protective anti-tumor T cell memory. Furthermore, the combination of aSE4-1-Fc with anti-PD-L1 therapy greatly improved the antitumor effects by augmenting both T and NK cells. Taken together, this study emphasizes the importance of Siglec-9 as a potential cancer therapeutic target, demonstrates the synergistic effect of co-inhibition of Siglec-9 and PD-L1, and may have implications for developing engineered antibodies targeting Siglec-9 with enhanced therapeutic efficacy., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Tengchuan Jin has patent pending to Tengchuan Jin, Dan Zhao. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bispecific antibodies (bsAbs) are emerging immune-therapeutics, and many formats exist that differ considerably in structure. However, little systematic data exist about how the spatial organization of their components influences activity, requiring innovative approaches combining empirical and quantitative frameworks. This study presents a modular DNA nanotechnology platform to generate numerous bsAbs with surrogate geometries that span the structural features of the BiTE, IgG-like, and IgG-conjugate platforms to screen for T-cell engagement. Results highlight interesting structure-activity relationships regarding bsAb potency and selectivity and raise questions regarding the molecular phenomena underlying activity. To elucidate some effects, the platform was paired with a simple mathematical model. This work is thus one of the first to systematically investigate and reveal the importance of the spatial organization of bsAb components on activity and equally provides an accessible and convenient tool for rapidly mapping out such trends for other combinations of target epitopes.

ABL602 2 + 1, a bispecific antibody with two distinct domains binding to CLL-1 on leukemias and CD3 on T cells, exhibits superior T cell activation and tumour lysing activity. Treatment outcomes of bispecific antibody rely on acute myeloid leukemia cell replication and antibody induced tumour lysing, but their quantitative relationship was unknown. Mathematical models are employed to quantitatively investigate HL-60 cell kinetics determined by bispecific antibody and tumour burden. First, we analysed cytotoxicity assay data testing HL-60 cell against bispecific antibody and T cells, and found efficiency of bispecific antibody induced tumour lysing increases but saturates with increase of HL-60 cell, T cell and bispecific antibody concentration. As a result, their interaction leads to bistable HL-60 cell kinetics; namely, at a given bispecific antibody and T cell concentration interval, HL-60 cell kinetics with small tumour burdens are inhibited but refractory to large tumour burdens. T cell concentration is strong negatively correlated with HL-60 cell concentration. With bispecific antibody clearance, observed bistable HL-60 cell kinetics still exists. Our finding explains observed phenomenon that bispecific antibody was less efficacious at high tumour burden even with enough activated cytotoxic CD8 + T cells. Maintaining high antibody concentration and preventing T-cell exhaustion are equivalently important to sustain long-term control., (© 2024. The Author(s).)

Multiple myeloma (MM) is a complex hematological malignancy characterized by abnormal antibody production from plasma cells. Despite advances in the treatment, many patients experience disease relapse or become refractory to treatment. G-protein-coupled receptor class C group 5 member D (GPRC5D), an orphan GPCR predominantly expressed in MM cells, is emerging as a promising target for MM immunotherapy. Talquetamab, a Food and Drug Administration-approved T-cell-directing bispecific antibody developed for treatment of MM, targets GPRC5D. Here, we elucidate the structure of GPRC5D complexed with the Fab fragment of talquetamab, using cryo-electron microscopy, providing the basis for recognition of GPRC5D by the bispecific antibody. GPRC5D forms a symmetric homodimer with the interface between transmembrane helix (TM) 4 of one protomer and TM4/5 of the other protomer. A single talquetamab Fab interacts with the GPRC5D dimer with its orientation toward the dimer interface. All six complementarity-determining regions of talquetamab engage with extracellular loops and TM3/5/7. In particular, the side-chain of an arginine residue from the antibody penetrates into a shallow pocket on the extracellular surface of GPRC5D. The structure offers insights for optimizing antibody design against GPRC5D for relapsed or refractory MM therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

B-cell depletion therapy (BCDT) has been employed to treat autoimmune disease for ~20 years. Immunoglobulin G1 (IgG1) monoclonal antibodies targeting CD20 and utilizing effector function (eg, antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis) to eliminate B cells have historically been the predominant therapeutic approaches. More recently, diverse BCDT approaches targeting a variety of B-cell surface antigens have been developed for use in hematologic malignancies, including effector-function-enhanced monoclonal antibodies, chimeric antigen receptor T-cell (CAR-T) treatment, and bispecific T-cell engagers (TCEs). The latter category of antibodies employs CD3 engagement to augment the killing of target cells. Given the improvement in B-cell depletion observed with CAR-T and TCEs compared with conventional monospecific antibodies for treatment of hematologic malignancies and the recent case reports demonstrating therapeutic benefit of CAR-T in autoimmune disease, there is potential for these mechanisms to be effective for B-cell-mediated autoimmune disease. In this review, we discuss the various BCDTs that are being developed in autoimmune diseases, describing the molecule designs, depletion mechanisms, and potential advantages and disadvantages of each approach as they pertain to safety, efficacy, and patient experience. Additionally, recent advances and strategies with TCEs are presented to help broaden understanding of the potential for bispecific antibodies to safely and effectively engage T cells for deep B-cell depletion in autoimmune diseases., Competing Interests: WR has received consulting fees from IGM Biosciences. DF has received consulting fees from IGM Biosciences, Kyverna Therapeutics, Argenx, Priovant Therapeutics, Pfizer, Biogen, Bristol Meyers Squibb, and Serono. LC has received consulting fees from IGM Biosciences, Kyverna Therapeutics, Mitsubishi Tanabe, Lilly, Janssen, Mediar Therapeutics, and Genentech. LM serves as a member of independent data safety monitoring boards for Boehringer Ingelheim and Celltrion. MD, MH, CS, and RK are employees of IGM Biosciences., (Copyright © 2024 Robinson, Fiorentino, Chung, Moreland, Deodhar, Harler, Saulsbery and Kunder.)

Despite effective countermeasures, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persists worldwide because of its ability to diversify and evade human immunity. This evasion stems from amino acid substitutions, particularly in the receptor binding domain (RBD) of the spike protein that confers resistance to vaccine-induced antibodies and antibody therapeutics. To constrain viral escape through resistance mutations, we combined antibody variable regions that recognize different RBD sites into multispecific antibodies. Here, we describe multispecific antibodies, including a trivalent trispecific antibody that potently neutralized diverse SARS-CoV-2 variants and prevented virus escape more effectively than single antibodies or mixtures of the parental antibodies. Despite being generated before the appearance of Omicron, this trispecific antibody neutralized all major Omicron variants through BA.4/BA.5 at nanomolar concentrations. Negative stain electron microscopy suggested that synergistic neutralization was achieved by engaging different epitopes in specific orientations that facilitated binding across more than one spike protein. Moreover, a tetravalent trispecific antibody containing the same variable regions as the trivalent trispecific antibody also protected Syrian hamsters against Omicron variants BA.1, BA.2, and BA.5 challenge, each of which uses different amino acid substitutions to mediate escape from therapeutic antibodies. These results demonstrated that multispecific antibodies have the potential to provide broad SARS-CoV-2 coverage, decrease the likelihood of escape, simplify treatment, and provide a strategy for antibody therapies that could help eliminate pandemic spread for this and other pathogens.

Targeting multiple viral proteins is pivotal for sustained suppression of highly mutable viruses. In recent years, broadly neutralizing antibodies that target the influenza virus hemagglutinin and neuraminidase glycoproteins have been developed, and antibody monotherapy has been tested in preclinical and clinical studies to treat or prevent influenza virus infection. However, the impact of dual neutralization of the hemagglutinin and neuraminidase on the course of infection, as well as its therapeutic potential, has not been thoroughly tested. For this purpose, we generated a bispecific antibody that neutralizes both the hemagglutinin and the neuraminidase of influenza viruses. We demonstrated that this bispecific antibody has a dual-antiviral activity as it blocks infection and prevents the release of progeny viruses from the infected cells. We show that dual neutralization of the hemagglutinin and the neuraminidase by a bispecific antibody is advantageous over monoclonal antibody combination as it resulted an improved neutralization capacity and augmented the antibody effector functions. Notably, the bispecific antibody showed enhanced antiviral activity in influenza virus-infected mice, reduced mice mortality, and limited the virus mutation profile upon antibody administration. Thus, dual neutralization of the hemagglutinin and neuraminidase could be effective in controlling influenza virus infection., Competing Interests: Declaration of interests A provisional patent on BiAb is currently being filed, in which Y.B.-O. and R.M. are inventors., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

This paper describes the synthesis, characterization, and functional activity of 26 MegaMolecule-based bispecific antibody mimics for T-cell redirection toward HER2+ cancer cells. The work reports functional bispecific MegaMolecules that bind both receptor targets, and recruit and activate T-cells resulting in lysis of the target tumor cells. Changing the orientation of linkage between Fabs against either HER2 or CD3ε results in an approximately 150-fold range in potency. Increasing scaffold valency from Fab dimers up to tetramers improves the potency of the antibody mimics up to 5-fold, but with diminishing returns in effective dose beyond trimeric formats. Antibody mimics that present either one or two Fabs against either receptor target allows for initial engagement of one cell type over the other. Finally, the antibody mimics significantly reduce HER2+ tumor volumes in a humanized xenograft model of breast cancer.

With the development of antibody engineering, a diverse range of structures for T cell-dependent bispecific antibodies targeting CD3 have been devised to meet the needs of various clinical therapies. T cell-dependent bispecific antibodies can be categorized into IgG-like and non-IgG-like formats based on the presence or absence of an Fc domain. The most common difficulty in manufacturing bispecific antibodies is the mispairing between light and heavy chains. To overcome this challenge, several techniques, such as knob-into-hole and CrossMab technologies, have been implemented for structural design. Given that the mechanism of action of T cell-dependent bispecific antibodies is to redirect T cells to tumor cells to induce a killing effect, abolishment of Fc domain effector function, the affinity design of the target, and the distance design of immune synapse formation are all focal points in the structural design of T cell-dependent bispecific antibodies. This review aims to provide an overview of recent advances in the structural design of T cell-dependent bispecific antibodies.

Bispecific antibodies (BsAbs) have emerged as a major class of antibody therapeutics owing to their substantial potential in disease treatment. While several BsAbs have been successfully approved in recent years, ongoing development efforts continue to focus on optimizing various BsAbs tailored to particular antigens and action mechanisms, aiming to achieve favorable physicochemical properties. BsAbs generally encounter challenges due to their unfavorable physicochemical characteristics and poor manufacturing efficiencies, highlighting the need for optimization to achieve reliable productivity and developability. Herein, we describe the development of a novel symmetric BsAb, REGULGENT™ (N-term/C-term), comprising two Fab domains, using a common light chain. The heavy chain fragment encoded two antigen-binding determinants in one chain. The design and production of REGULGENT™ (N-term/C-term) are simple owing to the use of the same light chain, which does not induce heavy and light chain mispairing, frequently observed with the asymmetric BsAb format. REGULGENT™ (N-term/C-term) exhibited high expression and low aggregation characteristics during cell culture and stress treatment under low pH conditions. Differential scanning calorimetric data indicated that REGULGENT™ molecules had high conformational stability, similar to that of stabilized monoclonal antibodies. Surface plasmon resonance data showed that REGULGENT™ (N-term/C-term) could bind to two antigens simultaneously and exhibited a high affinity for two antigens. In summary, the symmetric BsAb format of REGULGENT™ confers its desirable IgG-like physicochemical properties, thus making it an excellent candidate for commercial development. The findings demonstrate a novel BsAb with substantial development potential for clinical applications., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Bispecific antibodies (bsAbs) are engineered to target 2 different epitopes simultaneously. About 75% of the 16 clinically approved bsAbs have entered the clinic internationally since 2022. Hence, research on biomedical imaging of various radiolabeled bsAb scaffolds may serve to improve patient selection for bsAb therapy. Here, we provide a comprehensive overview of recent advances in radiolabeled bsAbs for imaging via PET or SPECT. We compare direct targeting and pretargeting approaches in preclinical and clinical studies in oncologic research. Furthermore, we show preclinical applications of imaging bsAbs in neurodegenerative diseases. Finally, we offer perspectives on the future directions of imaging bsAbs based on their challenges and opportunities., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Bispecific antibodies (bsAbs) are engineered molecules designed to target two different epitopes or antigens. The mechanism of action is determined by the bsAb molecular targets and structure (or format), which can be manipulated to create variable and novel functionalities, including linking immune cells with tumor cells, or dual signaling pathway blockade. Several bsAbs have already changed the treatment landscape of hematological malignancies and select solid cancers. However, the mechanisms of resistance to these agents are understudied and the management of toxicities remains challenging. Herein, we review the principles in bsAb engineering, current understanding of mechanisms of action and resistance, data for clinical application, and provide a perspective on ongoing challenges and future developments in this field., Competing Interests: Declaration of interests None declared by authors., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Immunotherapies, including chimeric antigen receptor (CAR) T cells and bispecific antibodies (BsAbs), encounter several challenges in the management of acute myeloid leukemia (AML), including limited persistence of these treatments, antigen loss and resistance of leukemia stem cells (LSCs) to therapy., Methods: Here, we proposed a novel dual-targeting approach utilizing engineered anti-IL10R CAR-T cells to secrete bispecific antibodies targeting CD33. This innovative strategy, rooted in our previous research which established a connection between IL-10 and the stemness of AML cells, designed to improve targeting efficiency and eradicate both LSCs and AML blasts., Results: We first demonstrated the superior efficacy of this synergistic approach in eliminating AML cell lines and primary cells expressing different levels of the target antigens, even in cases of low CD33 or IL10R expression. Furthermore, the IL10R CAR-T cells that secret anti-CD33 bsAbs (CAR.BsAb-T), exhibited an enhanced activation and induction of cytotoxicity not only in IL10R CAR-T cells but also in bystander T cells, thereby more effectively targeting CD33-positive tumor cells. Our in vivo experiments provided additional evidence that CAR.BsAb-T cells could efficiently redirect T cells, reduce tumor burden, and demonstrate no significant toxicity. Additionally, delivering bsAbs locally to the tumor sites through this strategy helps mitigate the pharmacokinetic challenges typically associated with the rapid clearance of prototypical bsAbs., Conclusions: Overall, the engineering of a single-vector targeting IL10R CAR, which subsequently secretes CD33-targeted bsAb, addresses the issue of immune escape due to the heterogeneous expression of IL10R and CD33, and represents a promising progress in AML therapy aimed at improving treatment outcomes., (© 2024. Springer Nature Switzerland AG.)

Targeting immune receptors on T cells is a common strategy to treat cancer and autoimmunity. Frequently, this is accomplished through monoclonal antibodies targeting the ligand binding sites of stimulatory or inhibitory co-receptors. Blocking ligand binding prevents downstream signalling and modulates specific T cell functions. Since 1985, the FDA has approved over 100 monoclonal antibodies against immune receptors. This therapeutic approach significantly improved the care of patients with numerous immune-related conditions; however, many patients are unresponsive, and some develop immune-related adverse events. One reason for that is the lack of consideration for the localization of these receptors on the cell surface of the immune cells in the context of the immune synapse. In addition to blocking ligand binding, changing the location of these receptors on the cell surface within the different compartments of the immunological synapse could serve as an alternative, efficient, and safer approach to treating these patients. This review discusses the potential therapeutic advantages of altering proteins' localization within the immune synapse and summarizes published work in this field. It also discusses the novel use of bispecific antibodies to induce the clustering of receptors on the cell surface. It presents the rationale for developing novel antibodies, targeting the organization of signalling receptor complexes on the cell surface. This approach offers an innovative and emerging technology to treat cancer patients resistant to current immunotherapies., (© 2024 John Wiley & Sons Ltd.)

B-cell lymphoma, clinically, comprises a heterogeneous group of malignancies that encompass various subtypes. CD20 is an optimal target for therapeutic antibodies in B-cell lymphoma immunotherapy since approximately 90% of B-cell malignancies typically exhibit CD20 expression on their surface, while its presence is limited in normal tissues. In this study, we have developed a series of novel non-IgG-like T cell-dependent bispecific antibodies by constructing Fab-FabCH3, referred to as Tandem Antigen-binding Fragment 002 (TFAB002), which specifically target CD20 for the treatment of malignant B-cell lymphoma. TFAB002s display strong binding affinity with CD20 and moderate binding affinity with CD3, thereby triggering target-specific T-cell activation, cytokine release, and tumor cell lysis in vitro. Furthermore, TFAB002s exhibit potent cytotoxicity against B-cell malignancies that express varying levels of CD20. Besides, the TFAB002s show potent pharmacodynamic activity in vivo in the WIL2-S cells CDX mouse model. Collectively, these results underscore the potential of TFAB002s as a highly promising therapeutic approach for selectively depleting CD20-positive B cells, thereby warranting further clinical evaluation as a viable treatment option for CD20-expressing B-cell malignancies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Kirsten Rat Sarcoma viral oncogene homolog (KRAS) is one of the most frequent oncogenes. However, there are limited treatment options due to its intracellular expression. To address this, we developed a novel bispecific T-cell engager (BiTE) antibody targeting HLA-A2/KRAS G12V complex and CD3 (HLA-G12V/CD3 BiTE). We examined its specific binding to tumor cells and T cells, as well as its anti-tumor effects in vivo. HLA-G12V/CD3 BiTE was expressed in Escherichia coli and its binding affinities to CD3 and HLA-A2/KRAS G12V were measured by flow cytometry, along with T-cell activation. In a xenograft pancreatic tumor model, the HLA-G12V/CD3 BiTE's anti-tumor effects were assessed through tumor growth, survival time, and safety. Our results demonstrated specific binding of HLA-G12V/CD3 BiTE to tumor cells with an HLA-A2/KRAS G12V mutation and T cells. The HLA-G12V/CD3 BiTE also activated T-cells in the presence of tumor cells in vitro. HLA-G12V/CD3 BiTE in vivo testing showed delayed tumor growth without severe toxicity to major organs and prolonged mouse survival. This study highlights the potential of constructing BiTEs recognizing an HLA-peptide complex and providing a novel therapy for cancer treatment targeting the intracellular tumor antigen.

Purpose: ATG-101, a bispecific antibody that simultaneously targets the immune checkpoint PD-L1 and the costimulatory receptor 4-1BB, activates exhausted T cells upon PD-L1 crosslinking. Previous studies demonstrated promising anti-tumour efficacy of ATG-101 in preclinical models. Here, we labelled ATG-101 with 89 Zr to confirm its tumour targeting effect and tissue biodistribution in a preclinical model. We also evaluated the use of immuno-PET to study tumour uptake of ATG-101 in vivo., Methods: ATG-101, anti-PD-L1, and an isotype control were conjugated with p-SCN-Deferoxamine (Df). The Df-conjugated antibodies were radiolabelled with 89 Zr, and their radiochemical purity, immunoreactivity, and serum stability were assessed. We conducted PET/MRI and biodistribution studies on [ 89 Zr]Zr-Df-ATG-101 in BALB/c nude mice bearing PD-L1-expressing MDA-MB-231 breast cancer xenografts for up to 10 days after intravenous administration of [ 89 Zr]Zr-labelled antibodies. The specificity of [ 89 Zr]Zr-Df-ATG-101 was evaluated through a competition study with unlabelled ATG-101 and anti-PD-L1 antibodies., Results: The Df-conjugation and [ 89 Zr]Zr -radiolabelling did not affect the target binding of ATG-101. Biodistribution and imaging studies demonstrated biological similarity of [ 89 Zr]Zr-Df-ATG-101 and [ 89 Zr]Zr-Df-anti-PD-L1. Tumour uptake of [ 89 Zr]Zr-Df-ATG-101 was clearly visualised using small-animal PET imaging up to 7 days post-injection. Competition studies confirmed the specificity of PD-L1 targeting in vivo., Conclusion: [ 89 Zr]Zr-Df-ATG-101 in vivo distribution is dependent on PD-L1 expression in the MDA-MB-231 xenograft model. Immuno-PET with [ 89 Zr]Zr-Df-ATG-101 provides real-time information about ATG-101 distribution and tumour uptake in vivo. Our data support the use of [ 89 Zr]Zr-Df-ATG-101 to assess tumour and tissue uptake of ATG-101., (© 2024. The Author(s).)

In recent studies, we have established the unique adapter chimeric antigen receptor (CAR) platform RevCAR which uses, as an extracellular CAR domain, a peptide epitope instead of an antibody domain. RevCAR adapters (termed RevCAR target modules, RevTMs) are bispecific antibodies that enable the reversible ON/OFF switch of the RevCAR system, improving the safety compared to conventional CARs. Here, we describe for the first time its use for retargeting of both T and NK-92 cells. In addition, we describe the development and preclinical validation of a novel RevTM for targeting of the fibroblast growth factor-inducible 14 (Fn14) surface receptor which is overexpressed on Glioblastoma (GBM) cells, and therefore serves as a promising target for the treatment of GBM. The novel RevTM efficiently redirects RevCAR modified T and NK-92 cells and leads to the killing of GBM cells both in vitro and in vivo. Tumor cell killing is associated with increased IL-2, TNF-α and/or IFN-γ secretion. Hence, these findings give an insight into the complementary potential of both RevCAR T and NK-92 systems as a safe and specific immunotherapeutic approach against GBM., (© 2024. The Author(s).)

Bispecific antibodies (bsAbs) are engineered immunoglobulins that combine two different antigen-binding sites in one molecule. BsAbs can be divided into two molecular formats: IgG-like and non-IgG-like antibodies. Structural elements of each format have implications for engaging the immune system. T cell engager antibodies (TCEs) are bsAbs designed to engage T cells with target cells. TCEs can be applied not only in cancer but also in infectious disease therapy to activate T-cell responses. In this review, we focus on current literature on the design and use of bsAbs as an innovative strategy to enhance adaptive antiviral immune responses. We summarized the novel T cell-related immunotherapies with a focus on TCEs, that are developed for the treatment of chronic hepatitis B. Chronic infection with the hepatitis B virus (HBV) had a death toll of 1.1 million humans in 2022, mainly due to liver cirrhosis and hepatocellular carcinoma developing in the more than 250 million humans chronically infected. A curative treatment approach for chronic hepatitis B is lacking. Combining antiviral therapy with immune therapies activating T-cell responses is regarded as the most promising therapeutic approach to curing HBV and preventing the sequelae of chronic infection. Attracting functionally intact T cells that are not HBV-specific and, therefore, have not yet been exposed to regulatory mechanisms and activating those at the target site in the liver is a very interesting therapeutic approach that could be achieved by TCEs. Thus, TCEs redirecting T cells toward HBV-positive cells represent a promising strategy for treating chronic hepatitis B and HBV-associated hepatocellular carcinoma., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: T-cell redirecting bispecific antibodies (BsAbs), targeting B-cell maturation antigen (BCMA) or G-protein - coupled receptor class C group 5 member D (GPRC5D), are efficacious new agents for the treatment of patients with relapsed or refractory MM., Areas Covered: This review discusses the pharmacokinetic properties, efficacy, and safety profile of T-cell redirecting BsAbs in MM, with a special focus on their optimal dosing schedule, resistance mechanisms and future strategies to enhance efficacy, reduce toxicity, and maximize duration of response., Expert Opinion: To further improve the efficacy of BsAbs, ongoing studies are investigating whether combination therapy can enhance depth and duration of response. An important open question is also to what extent response to BsAbs can be improved when these agents are used in earlier lines of therapy. In addition, more evidence is needed on rational de-intensification strategies of BsAb dosing upon achieving a sufficient response, and if (temporary) treatment cessation is possible in patients who have achieved a deep remission (e.g. complete response or minimal residual disease-negative status).

Off-the-shelf T-cell-redirecting bispecific antibodies targeting BCMA, GPRC5D, and FcRH5 have high activity in multiple myeloma with a manageable toxicity profile. However, not all patients respond to bispecific antibodies and patients can develop bispecific antibody resistance after an initial response. Mechanisms that contribute to bispecific antibody resistance are multifactorial and include tumour-related factors, such as high tumour burden, expression of T-cell inhibitory ligands, and antigen loss. Resistance due to antigen escape can be prevented by simultaneously targeting two tumour-associated antigens with a trispecific antibody or a combination of two bispecific antibodies. There is also increasing evidence that primary resistance to bispecific antibodies is associated with impaired baseline T-cell function. Long-term exposure to bispecific antibodies with chronic T-cell stimulation further aggravates T-cell dysfunction, which could contribute to failure of disease control. Therapeutic interference with T-cell exhaustion by targeting inhibitory or costimulatory pathways could improve bispecific antibody-mediated antitumour activity. The immunosuppressive microenvironment also contributes to bispecific antibody resistance. CD38-targeting antibodies hold promise as combination partners for bispecific antibodies because of their potential to eliminate CD38 + immune suppressor cells. In conclusion, a better understanding of the mechanisms underlying the absence of disease response has provided novel insights to optimise T-cell activity and bispecific antibody efficacy in multiple myeloma., Competing Interests: Declaration of interests NWCJvdD has received research support, paid to his institution, from Janssen Pharmaceuticals, AMGEN, Celgene, Novartis, Cellectis, and BMS and has been on advisory boards for Janssen Pharmaceuticals, AMGEN, Celgene, BMS, Sanofi, Takeda, Roche, Novartis, Bayer, Adaptive, Kite Pharma, Merck, Pfizer, AbbVie, and Servier. AC has received research support from Janssen Pharmaceuticals; has received honoraria for lectures from Antengene; and has been a consultant or advisor for AbbVie, Adaptive, Amgen, Antengene, Bristol Myers Squibb, Forus, Genentech–Roche, GlaxoSmithKline, Janssen Pharmaceuticals, Karyopharm Therapeutics, Millennium–Takeda, and Sanofi–Genzyme. MVM has been on advisory boards for Amgen, Bristol Myers Squibb–Celgene, GlaxoSmithKline, Janssen Cilag, Kite Pharma, Oncopeptides, Pfizer, Roche, Sanofi, and Stemline and is a speaker bureau member of Janssen Cilag., (Copyright © 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

NKG2D is an activating receptor expressed by natural killer (NK) cells and other cytotoxic lymphocytes that plays a pivotal role in the elimination of neoplastic cells through recognition of different stress-induced cell surface ligands (NKG2DL). To employ this mechanism for cancer immunotherapy, we generated NKG2D-engaging bispecific antibodies that selectively redirect immune effector cells to cancer cells expressing the tumor-associated antigen ErbB2 (HER2). NKG2D-specific single chain fragment variable (scFv) antibodies cross-reactive toward the human and murine receptors were derived by consecutive immunization of chicken with the human and murine antigens, followed by stringent screening of a yeast surface display immune library. Four distinct species cross-reactive (sc) scFv domains were selected, and reformatted into a bispecific engager format by linking them via an IgG4 Fc domain to a second scFv fragment specific for ErbB2. The resulting molecules (termed scNKAB-ErbB2) were expressed as disulfide-linked homodimers, and demonstrated efficient binding to ErbB2-positive cancer cells as well as NKG2D-expressing primary human and murine lymphocytes, and NK-92 cells engineered with chimeric antigen receptors derived from human and murine NKG2D (termed hNKAR and mNKAR). Two of the scNKAB-ErbB2 molecules were found to compete with the natural NKG2D ligand MICA, while the other two engagers interacted with an epitope outside of the ligand binding site. Nevertheless, all four tested scNKAB-ErbB2 antibodies were similarly effective in redirecting the cytotoxic activity of primary human and murine lymphocytes as well as hNKAR-NK-92 and mNKAR-NK-92 cells to ErbB2-expressing targets, suggesting that further development of these species cross-reactive engager molecules for cancer immunotherapy is warranted., Competing Interests: KS, JH, AM, PO, HK and WW are named as inventors on patents and patent applications in the field of cancer immunotherapy owned by their respective academic institutions. 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 © 2024 Pfeifer Serrahima, Schoenfeld, Kühnel, Harwardt, Macarrón Palacios, Prüfer, Kolaric, Oberoi, Kolmar and Wels.)

Current medical therapies for treating acute myeloid leukemia (AML) remain unmet, and AML patients may benefit from targeted immunotherapy approaches that focus on specific tumor antigens. GRP78, which is upregulated in various malignant tumors such as AML, is partially expressed as cell surface GRP78 (csGRP78) on the cell membrane, making it an ideal target for redirecting T cells, including T-cell engagers. However, considering the conventional approach of using two scFv segments to construct a bispecific T-cell engager (BiTE), we have undertaken the development of a novel BiTE that utilizes a cyclic peptide ligand to specifically target csGRP78, which we refer to as GRP78-CD3/BiTE. We studied the effects of GRP78-CD3/BiTE on treatments for AML in vitro and in vivo and assessed the pharmacokinetics of this engager. Our findings demonstrated that GRP78-CD3/BiTE could not only effectively mediate the cytotoxicity of T cells against csGRP78-expressing AML cells but also specifically eliminate primary AML tumor cells in vitro. Furthermore, GRP78-CD3/BiTE exhibited a longer half-life despite having a lower molecular weight than CD19-CD3/BiTE. In a xenograft mouse model of AML, treatment with GRP78-CD3/BiTE prolonged the survival time of the mice. Our findings demonstrate that GRP78-CD3/BiTE is effective and selective for eliminating csGRP78-expressing AML cells and suggest that this approach to targeted immunotherapy could lead to effective new treatments for AML., (© 2024. The Author(s).)

The growing clinical success of bispecific antibodies (bsAbs) has led to rapid interest in leveraging dual targeting in order to generate novel modes of therapeutic action beyond mono-targeting approaches. While bsAbs that bind targets on two different cells (trans-targeting) are showing promise in the clinic, the co-targeting of two proteins on the same cell surface through cis-targeting bsAbs (cis-bsAbs) is an emerging strategy to elicit new functionalities. This includes the ability to induce proximity, enhance binding to a target, increase target/cell selectivity, and/or co-modulate function on the cell surface with the goal of altering, reversing, or eradicating abnormal cellular activity that contributes to disease. In this review, we focus on the impact of cis-bsAbs in the clinic, their emerging applications, and untangle the intricacies of improving bsAb discovery and development., Competing Interests: Declaration of interests R.C.O., P.M.H., S.A.L., and O.O.F. are employees of InduPro., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The mechanism of action of bispecific antibodies (bsAbs) directing T-cell immunity to solid tumors is incompletely understood. Here, we screened a series of CD3xHER2 bsAbs using extracellular matrix (ECM) embedded breast cancer tumoroid arrays exposed to healthy donor-derived T-cells. An initial phase of random T-cell movement throughout the ECM (day 1-2), was followed by a bsAb-dependent phase of active T-cell recruitment to tumoroids (day 2-4), and tumoroid killing (day 4-6). Low affinity HER2 or CD3 arms were compensated for by increasing bsAb concentrations. Instead, a bsAb binding a membrane proximal HER2 epitope supported tumor killing whereas a bsAb binding a membrane distal epitope did not, despite similar affinities and intra-tumoroid localization of the bsAbs, and efficacy in 2D co-cultures. Initial T-cell-tumor contact through effective bsAbs triggered a wave of subsequent T-cell recruitment. This critical surge of T-cell recruitment was explained by paracrine signaling and preceded a full-scale T-cell tumor attack., (© 2024. The Author(s).)

Anti-CTLA-4 antibodies faced challenges due to frequent adverse events and limited efficacy, which spurred the exploration of next-generation CTLA-4 therapeutics to balance regulatory T cells (Tregs) depletion and CD8 T cells activation. CCR8, identified primarily on tumor-infiltrating Tregs, has become a target of interest due to the anti-tumor effects demonstrated by CCR8 antibody-mediated Tregs depletion. Single-cell RNA sequencing analysis reveals that CCR8-positive Tregs constitute a small subset, with concurrent expression of CCR8 and CTLA-4. Consequently, we proposed a novel bispecific antibody targeting CCR8 and CTLA-4 that had the potential to enhance T cell activation while selectively depleting intratumor Tregs. The candidate molecule 2MW4691 was developed in a tetravalent symmetric format, maintaining a strong binding affinity for CCR8 while exhibiting relatively weaker CTLA-4 binding. This selective binding ability allowed 2MW4691 to target and deplete tumor-infiltrating Tregs with higher specificity. In vitro assays verified the antibody's capacity for antibody-dependent cellular cytotoxicity (ADCC) to Tregs with high level of CTLA-4 expression, but not CD8 T cells with relatively low level of CTLA-4 on cell surface. Also, 2MW4691 inhibited the CTLA-4 pathway and enhanced T cell activation. The in vivo therapeutic efficacy of 2MW4691 was further demonstrated using hCCR8 or hCTLA-4 humanized mouse models and hCCR8/hCTLA-4 double knock-in mouse models. In cynomolgus monkeys, 2MW4691 was well-tolerated, exhibited the anticipated pharmacokinetic profile, and had a minimal impact on the peripheral T cell population. The promising preclinical results supported the further evaluation of 2MW4691 as a next-generation Treg-based therapeutics in clinical trials., (© 2024. The Author(s).)

Anti-cluster of differentiation (CD) 3 × α programmed death-ligand 1 (PD-L1) bispecific T-cell engager (BsTE)-bound T-cells (BsTE:T) are a promising new cancer treatment agent. However, the mechanisms of action of bispecific antibody-armed activated T-cells are poorly understood. Therefore, this study aimed to investigate the anti-tumor mechanism and efficacy of BsTE:T. The BsTE:T migration was assessed in vivo and in vitro using syngeneic and xenogeneic tumor models, flow cytometry, immunofluorescence staining, transwell migration assays, microfluidic chips, Exo View R100, western blotting, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology. In murine B16 melanoma, MC38 colon cancer, and human multiple myeloma cells, BsTE:T exhibited superior tumor elimination relative to that of T-cells or BsTE alone. Moreover, BsTE:T migration into tumors was significantly enhanced owing to the presence of PD-L1 in tumor cells and secretion of PD-L1-containing exosomes. Furthermore, increased infiltration of CD44 high CD62L low effector memory CD8 + T-cells into tumors was closely associated with the anti-tumor effect of BsTE:T. Therefore, BsTE:T is an innovative potential anti-tumor therapy, and exosomal PD-L1 plays a crucial role both in vitro and in vivo in the anti-tumor activity of BsTE:T., (© 2024. The Author(s).)

Introduction: Since the approval of the bispecific antibody blinatumomab in 2017 for the treatment of acute lymphoblastic leukemia in relapse, the development of numerous bispecific antibody constructs has dramatically expanded in hematologic malignancies. Many have recently received Food Drug Administration and European Medicines Agency approvals in various stages of treatment for lymphomas, leukemias, and multiple myeloma., Areas Covered: The purpose of this review is to provide an overview of bispecific antibody treatment including the mechanisms leading to effector T cells targeting tumor-associated antigens, the treatment indications, efficacies, toxicities, and challenges of the different constructs. A literature search was performed through access to PubMed and clinicaltrials.gov., Expert Opinion: While there has been substantial success in the treatment of NHL, MM, and ALL, there are still hematologic malignancies such as AML where there has been limited progress. It is important to continue to investigate new designs, tumor antigen targets, and further refine where current approved bispecific antibodies fit in terms of sequencing of therapy. Hopefully, with the knowledge gained in recent years and the explosion of these therapies, patients with blood cancers will continue to benefit from these treatments for years to come.

In the last decade, there has been a surge in developing immunotherapies to enhance the immune system's ability to eliminate tumor cells. Bispecific antibodies known as T cell engagers (TCEs) present an attractive strategy in this pursuit. TCEs aim to guide cytotoxic T cells toward tumor cells, thereby inducing a strong activation and subsequent tumor cell lysis. In this study, we investigated the activity of different TCEs on both conventional alpha-beta (αβ) T cells and unconventional gamma delta (γδ) T cells. TCEs were built using camelid single-domain antibodies (VHHs) targeting the tumor-associated antigen CEACAM5 (CEA), together with T cell receptor chains or a CD3 domain. We show that Vγ9Vδ2 T cells display stronger in vitro antitumor activity than αβ T cells when stimulated with a CD3xCEA TCE. Furthermore, restricting the activation of fresh human peripheral T cells to Vγ9Vδ2 T cells limited the production of protumor factors and proinflammatory cytokines, commonly associated with toxicity in patients. Taken together, our findings provide further insights that γδ T cell-specific TCEs hold promise as specific, effective, and potentially safe molecules to improve antitumor immunotherapies., (© 2024 The Authors. European Journal of Immunology published by Wiley‐VCH GmbH.)