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Certain immune-checkpoint inhibitors have a narrow therapeutic window (TW) as cancer therapeutics, and engineered dual-/multi-targeting agents could potentially widen the TW to bring true clinical benefits. We report a new rationally-designed bispecific-antibody (BsAb), HX009, simultaneously targeting PD1 and CD47 to improve both the efficacy and safety over the respective single-targeting agents by grafting the extracellular domain of SIRPα onto the parental anti-PD1-monoclonal antibody, HX008. This resulted in an IgG4-based "2 × 2" symmetric structure but with an intentionally-reduced CD47-binding affinity, suggesting a novel candidate cancer immunotherapy. Specifically, HX009 has binding affinity constant of 8.951 × 10–9 M for human PD1 and 2.557 × 10–8 M for human CD47, respectively, where the CD47 binding is significantly weaker as compared to the binding affinity of HX008 to PD1 as well as the binding affinity of SIRPα-Fc to CD47, leading to little binding to RBCs and platelets and is contrasting to many CD47-agents in development. However, HX009 effectively and simultaneously binds to the PD1 and CD47 on PD1+CD47+ T-cells via cis-binding and elicits enhanced T cell activation compared to the parental HX008. HX009 caused little cytokine-release in human peripheral blood mononuclear cells. HX009 cross-species binds to cynomolgus monkey PD1/CD47 but not to rodents, making cynomolgus monkeys the choice of species to investigate the pharmacokinetics (PK) and toxicology of HX009. HX009's anti-tumor activities were confirmed in several humanized preclinical mouse models by determining either its anti-PD1 (humanized hu-CD47-MC38 models) or anti-CD47 (HuT-102 lymphoma CDX and three PDX-AML models) functions, although limited available humanized models have hindered broadly demonstration of enhanced anti-tumor activities contributed from the dual targeting of the BsAb. The expanded DLBCL-PDX trial data suggested that both EBV-status and OX40 expression could potentially be two positive predictors for response to HX009. An intravenous (IV) infusion PK study in cynomolgus monkey revealed its largely vasculature distribution, terminal half-life (T1/2) of ~ 50 h, and dose-proportional exposure without accumulation. The anti-drug antibody (ADA) was observed in all monkeys as expected, affecting the PK parameters of repeated administration. The IV single-dose toxicology study with a 14-day observation revealed a maximum tolerated dose of 150 mg/kg, while the repeated-dose (once weekly for 4 weeks, 5 doses in total) study showed a highest non-severely toxic dose (HNSTD) of 15 mg/kg. The desired preclinical PK and safety profiles, along with its antitumor activity, support HX009's candidacy for its clinical development. [ABSTRACT FROM AUTHOR]

Abstract Certain immune-checkpoint inhibitors have a narrow therapeutic window (TW) as cancer therapeutics, and engineered dual-/multi-targeting agents could potentially widen the TW to bring true clinical benefits. We report a new rationally-designed bispecific-antibody (BsAb), HX009, simultaneously targeting PD1 and CD47 to improve both the efficacy and safety over the respective single-targeting agents by grafting the extracellular domain of SIRPα onto the parental anti-PD1-monoclonal antibody, HX008. This resulted in an IgG4-based “2 × 2” symmetric structure but with an intentionally-reduced CD47-binding affinity, suggesting a novel candidate cancer immunotherapy. Specifically, HX009 has binding affinity constant of 8.951 × 10–9 M for human PD1 and 2.557 × 10–8 M for human CD47, respectively, where the CD47 binding is significantly weaker as compared to the binding affinity of HX008 to PD1 as well as the binding affinity of SIRPα-Fc to CD47, leading to little binding to RBCs and platelets and is contrasting to many CD47-agents in development. However, HX009 effectively and simultaneously binds to the PD1 and CD47 on PD1+CD47+ T-cells via cis-binding and elicits enhanced T cell activation compared to the parental HX008. HX009 caused little cytokine-release in human peripheral blood mononuclear cells. HX009 cross-species binds to cynomolgus monkey PD1/CD47 but not to rodents, making cynomolgus monkeys the choice of species to investigate the pharmacokinetics (PK) and toxicology of HX009. HX009’s anti-tumor activities were confirmed in several humanized preclinical mouse models by determining either its anti-PD1 (humanized hu-CD47-MC38 models) or anti-CD47 (HuT-102 lymphoma CDX and three PDX-AML models) functions, although limited available humanized models have hindered broadly demonstration of enhanced anti-tumor activities contributed from the dual targeting of the BsAb. The expanded DLBCL-PDX trial data suggested that both EBV-status and OX40 expression could potentially be two positive predictors for response to HX009. An intravenous (IV) infusion PK study in cynomolgus monkey revealed its largely vasculature distribution, terminal half-life (T1/2) of ~ 50 h, and dose-proportional exposure without accumulation. The anti-drug antibody (ADA) was observed in all monkeys as expected, affecting the PK parameters of repeated administration. The IV single-dose toxicology study with a 14-day observation revealed a maximum tolerated dose of 150 mg/kg, while the repeated-dose (once weekly for 4 weeks, 5 doses in total) study showed a highest non-severely toxic dose (HNSTD) of 15 mg/kg. The desired preclinical PK and safety profiles, along with its antitumor activity, support HX009’s candidacy for its clinical development.

ABSTRACTBispecific antibodies (bsAbs) are a class of antibodies that can mediate novel mechanisms of action compared to monospecific monoclonal antibodies (mAbs). Since the discovery of mAbs and their adoption as therapeutic agents in the 1980s and 1990s, the development of bsAbs has held substantial appeal. Nevertheless, only three bsAbs (catumaxomab, blinatumomab, emicizumab) were approved through the end of 2020. However, since then, 11 bsAbs received regulatory agency approvals, of which nine (amivantamab, tebentafusp, mosunetuzumab, cadonilimab, teclistamab, glofitamab, epcoritamab, talquetamab, elranatamab) were approved for the treatment of cancer and two (faricimab, ozoralizumab) in non-oncology indications. Notably, of the 13 currently approved bsAbs, two, emicizumab and faricimab, have achieved blockbuster status, showing the promise of this novel class of therapeutics. In the 2020s, the approval of additional bsAbs can be expected in hematological malignancies, solid tumors and non-oncology indications, establishing bsAbs as essential part of the therapeutic armamentarium.

Simple Summary: Chronic Lymphocytic Leukemia (CLL) is one of the most frequent leukemia in the west countries for adult patients. It's typically a slow-growing disease and the majority of patients do not need immediate treatment at diagnosis. Advanced stages or symptomatic disease require therapy. The treatment landscape of CLL has changed considerably in the last decades with the introduction of new targeted agents leading to improved outcome for patients with CLL compared to standard chemo-immunotherapy, especially for those with high-risk features, as del17p13, TP53 mutations and unmutated immunoglobulin heavy chain (IGHV) genes. In this narrative review, we comprehensively summarized and discussed all the new approaches currently investigated in completed and on-going clinical trials, both with single new agents and in combination strategies to pursuing not only a disease control but the eradication of the leukemic clone. In the last few years, several agents targeting molecules that sustain the survival and the proliferation of chronic lymphocytic leukemia (CLL) cells have become clinically available. Most of these drugs target surface proteins, such as CD19 or CD20, via monoclonal or bispecific monoclonal antibodies (BsAbs), CAR T cells, intracellular proteins like BTK by using covalent or non-covalent inhibitors or BCL2 with first or second generation BH3-mimetics. Since the management of CLL is evolving quickly, in this review we highlighted the most important innovative treatments including novel double and triple combination therapies, CAR T cells and BsAbs for CLL. Recently, a large number of studies on novel combinations and newer strategic options for CLL therapy have been published or presented at international conferences, which were summarized and linked together. Although the management of treatment with a single continuous agent is easier, the emergence of protein mutations, long-term toxicities and costs are important concerns that favor the use of a fixed duration therapy. In the future, a measurable residual disease (MRD)-guided treatment cessation and MRD-based re-initiation of targeted therapy seems to be a more feasible approach, allowing identification of the patients who might benefit from continuous therapy or who might need a consolidation with BsAbs or CAR T cells to clear the neoplastic clone. [ABSTRACT FROM AUTHOR]

The tumor suppressor p53 is the most common mutated gene in cancer, with the R175H as the most frequent p53 missense mutant. However, there are currently no approved targeted therapies or immunotherapies against mutant p53. Here, we characterized and investigated a monoclonal antibody (mAb) that recognizes the mutant p53-R175H for its affinity, specificity, and activity against tumor cells in vitro. We then delivered DNA plasmids expressing the anti-R175H mAb or a bispecific antibody (BsAb) into mice to evaluate their therapeutic effects. Our results showed that the anti-R175H mAb specifically bound to the p53-R175H antigen with a high affinity and recognized the human mutant p53-R175H antigen expressed on HEK293T or MC38 cells, with no cross-reactivity with wild-type p53. In cultured cells, the anti-R175H mAb showed higher cytotoxicity than the control but did not induce antibody-dependent cellular cytotoxicity. We made a recombinant MC38 mouse cell line (MC38-p53-R175H) that overexpressed the human p53-R175H after knocking out the endogenous mutant p53 alleles. In vivo, administration of the anti-R175H mAb plasmid elicited a robust anti-tumor effect against MC38-p53-R175H in mice. The administration of the anti-R175H BsAb plasmid showed no therapeutic effects, yet potent anti-tumor activity was observed in combination with the anti-PD-1 antibody. These results indicate that targeting specific mutant epitopes using DNA-delivered mAbs or BsAbs presents a form of improved natural immunity derived from tumor-infiltrating B cells and plasma cells against intracellular tumor antigens.

Mucin1 (MUC1) is abnormally glycosylated and overexpressed in a variety of epithelial cancers and plays a critical role in tumor progression. MUC1 has received remark attention as an oncogenic molecule and is considered a valuable tumor target for immunotherapy, while many monoclonal antibodies (mAbs) targeting MUC1-positive cancers in clinical studies lack satisfactory results. It would be highly desirable to develop an effective therapy against MUC1-expressing cancers. In this study, we constructed a novel T cell-engaging bispecific antibody (BsAb) targeting MUC1 and CD3 with the Fab-ScFv-IgG format. A high quality of MUC1-CD3 BsAb can be acquired through a standard method. Our study suggested that this BsAb could specifically bind to MUC1- and CD3-positive cells and efficiently enhance T cell activation, cytokine release, and cytotoxicity. Furthermore, our study demonstrated that this BsAb could potently redirect T cells to eliminate MUC1-expressing tumor cells in vitro and significantly suppress MUC1-positive tumor growth in a xenograft mouse model. Thus, T cell-engaging MUC1/CD3 BsAb could be an effective therapeutic approach to combat MUC1-positive tumors and our MUC1/CD3 BsAb could be a promising candidate in clinical applications for the treatment of MUC1-positive cancer patients. [ABSTRACT FROM AUTHOR]

Simple Summary: Tumor cells from the same specimen are functionally heterogeneous. Cancer stem cells (CSCs) are populations of tumor cells with self-renewal and differentiation properties. CSCs are found in nearly all solid and hematological tumors and are characterized by various surface or intracellular markers. These markers can be used to develop tumor-specific antibodies, cytotoxic immune cells, vaccines, and direct immune responses to the tumor cells, including CSC populations. This review discusses the emerging CSC-directed immunotherapies, the current state of their clinical development, the approaches to improve their safety and efficacy, and future strategies to strengthen anti-CSC immunotherapy. The generally accepted view is that CSCs hijack the signaling pathways attributed to normal stem cells that regulate the self-renewal and differentiation processes. Therefore, the development of selective targeting strategies for CSC, although clinically meaningful, is associated with significant challenges because CSC and normal stem cells share many important signaling mechanisms for their maintenance and survival. Furthermore, the efficacy of this therapy is opposed by tumor heterogeneity and CSC plasticity. While there have been considerable efforts to target CSC populations by the chemical inhibition of the developmental pathways such as Notch, Hedgehog (Hh), and Wnt/β-catenin, noticeably fewer attempts were focused on the stimulation of the immune response by CSC-specific antigens, including cell-surface targets. Cancer immunotherapies are based on triggering the anti-tumor immune response by specific activation and targeted redirecting of immune cells toward tumor cells. This review is focused on CSC-directed immunotherapeutic approaches such as bispecific antibodies and antibody-drug candidates, CSC-targeted cellular immunotherapies, and immune-based vaccines. We discuss the strategies to improve the safety and efficacy of the different immunotherapeutic approaches and describe the current state of their clinical development. [ABSTRACT FROM AUTHOR]

T cell-redirecting strategies have emerged as effective cancer immunotherapy approaches. Bispecific antibodies (bsAbs) are designed to specifically recruit T cells to the tumor microenvironment and induce the assembly of the immunological synapse (IS) between T cells and cancer cells or antigen-presenting cells. The way that the quality of the IS might predict the effectiveness of T cell-redirecting strategies, including those mediated by bsAbs or by chimeric antigen receptors (CAR)-T cells, is currently under discussion. Here we review the organization of the canonical IS assembled during natural antigenic stimulation through the T cell receptor (TCR) and to what extent different bsAbs induce T cell activation, canonical IS organization, and effector function. Then, we discuss how the biochemical parameters of different formats of bsAbs affect the effectivity of generating an antigen-induced canonical IS. Finally, the quality of the IS assembled by bsAbs and monoclonal antibodies or CAR-T cells are compared, and strategies to improve bsAb-mediated T cell-redirecting strategies are discussed. [ABSTRACT FROM AUTHOR]

Size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) is an analytical method routinely used for assessing aggregation content in protein samples. As SEC-HPLC separates analytes based on their hydrodynamic radius, it generally lacks the capability of differentiating species that are similar in size. Recently while purifying a bispecific antibody (bsAb), we noticed that SEC-HPLC can provide certain degree of resolution between the target bsAb and a disulfide scrambled form, although these two species were identical in molecular weight. In seeing the unexpected potential of SEC-HPLC at resolving species with similar size, we further tested Zenix SEC-300, a mixed-mode SEC-HPLC column from Sepax, which was reported to be capable of separating protein analytes based on other factors besides size. The Zenix column indeed provided resolution much better than the regular SEC-HPLC column. Upon further optimization, the Zenix column allowed close to baseline separation of the correctly folded and the disulfide scrambled species. The current study, as a complement to the previous reports, further demonstrates that mix-mode SEC-HPLC is capable of separating protein analytes that are close in size but are different in conformation and/or surface characteristics. • During the purification of a bsAb, one byproduct identified was a disulfide scrambled form. • The native and scrambled species were well resolved on native gel, suggesting that they adopted different conformations. • Regular SEC-HPLC provided limited resolution between the native and the scrambled species, which are identical in size. • Mixed-mode SEC-HPLC provided much better resolution between the correctly folded and the disulfide scrambled species. [ABSTRACT FROM AUTHOR]

Abstract Cancer immunotherapy has made remarkable progress in the past decade. Bispecific antibodies (BsAbs) have acquired much attention as the next generation strategy of antibody-target cancer immunotherapy, which overwhelmingly focus on T cell recruitment and dual receptors blockade. So far, BsAb drugs have been proved clinically effective and approved for the treatment of hematologic malignancies, but no BsAb have been approved in solid tumors. Numerous designed BsAb drugs for solid tumors are now undergoing evaluation in clinical trials. In this review, we will introduce the formats of bispecific antibodies, and then update the latest preclinical studies and clinical trials in solid tumors of BsAbs targeting EpCAM, CEA, PMSA, ErbB family, and so on. Finally, we discuss the BsAb-related adverse effects and the alternative strategy for future study.

Advancement in sequencing technologies allows for the identification of molecular pathways involved in tumor progression and treatment resistance. Implementation of novel agents targeting these pathways, defined as targeted therapy, significantly improves the prognosis of cancer patients. Targeted therapy also includes the use of monoclonal antibodies (mAbs). These drugs recognize specific oncogenic proteins expressed in cancer cells. However, as with many other types of targeting agents, mAb-based therapy usually fails in the long-term control of cancer progression due to the development of resistance. In many cases, resistance is caused by the activation of alternative pathways involved in cancer progression and the development of immune evasion mechanisms. To overcome this off-target resistance, bispecific antibodies (bsAbs) were developed to simultaneously target differential oncogenic pathway components, tumor-associated antigens (TAA) and immune regulatory molecules. As a result, in the last few years, several bsAbs have been tested or are being tested in cancer patients. A few of them are currently approved for the treatment of some hematologic malignancies but no bsAbs are approved in solid tumors. In this review, we will provide an overview of the state-of-the-art of bsAbs for the treatment of solid malignancies outlining their classification, design, main technologies utilized for production, mechanisms of action, updated clinical evidence and potential limitations. [ABSTRACT FROM AUTHOR]

Bispecific antibodies (bsAbs) are artificial antibodies with two distinct antigenbinding sites that can bind to different antigens or different epitopes on the same antigen. Based on a variety of technology platforms currently developed, bsAbs can exhibit different formats and mechanisms of action. The upgrading of antibody technology has promoted the development of bsAbs, which has been effectively used in the treatment of tumors. So far, 7 bsAbs have been approved for marketing in the world, and more than 200 bsAbs are in clinical and preclinical research stages. Here, we summarize the development process of bsAbs, application in tumor treatment and look forward to the challenges in future development. [ABSTRACT FROM AUTHOR]

Tumour therapy has entered the era of immunotherapy. Monoclonal antibodies (mAb), immune checkpoint inhibitors, chimeric antigen receptor T‐cell (CAR‐T), cytokine‐induced killer (CIK), tumour‐infiltrating lymphocytes (TILs) and other cellular immunotherapies have become the focus of current research. The CD47/SIRPα target is becoming another popular tumour immunotherapy target following the PDCD1/CD247(PD1/PD‐L1) checkpoint inhibitor. In recent years, a large number of CD47/SIRPα mAbs, fusion proteins, and CD47/SIRPα‐based bispecific antibodies (BsAbs) are undergoing preclinical and clinical trials and have good curative effects in the treatment of haematological tumours and solid tumours. They bring new vitality and hope for the treatment of patients with advanced tumours. This review summarizes the research progress of CD47/SIRPα‐based BsAbs with different targets for tumour treatment. There are 12 and 9 BsAbs in clinical trials and pre‐clinical research, respectively. We report on the mechanism of 15 BsAb molecules with different target and analyse the efficacy and safety of preclinical and clinical trials, discuss the issues that may be faced in the development of CD47‐based BsAbs, and dual‐target molecules, and summarize their development prospects. This review provides a reference for the safety and effectiveness of BsAbs in clinical application and in the future development of antibodies. [ABSTRACT FROM AUTHOR]

Intratumoral regulatory T cells (Tregs) express high levels of CD25 and TIGIT, which are also recognized as markers of effector T cell (Teff) activation. Targeting these molecules each alone with monoclonal antibodies (mAbs) poses a risk of concurrently depleting both Teffs and peripheral Tregs, thereby compromising the effectiveness and selectivity of intratumoral Treg depletion. Here, leveraging the increased abundance of CD25 + TIGIT + double-positive Tregs in the solid tumor microenvironment (but not in peripheral tissues), we explore the feasibility of using a CD25×TIGIT bispecific antibody (bsAb) to selectively deplete intratumoral Tregs. We initially constructed a bsAb co-targeting mouse CD25 and TIGIT, NSWm7210, and found that NSWm7210 conferred enhanced intratumoral Treg depletion, Teff activation, and tumor suppression as compared to the parental monotherapies in mouse models. We subsequently constructed a bsAb co-targeting human CD25 and TIGIT (NSWh7216), which preferentially eliminated CD25 + TIGIT + double-positive cells over single-positive cells in vitro. NSWh7216 exhibited enhanced anti-tumor activity without toxicity of peripheral Tregs in CD25 humanized mice compared to the parental monotherapies. Our study illustrates the use of CD25×TIGIT bsAbs as effective agents against solid tumors based on selective depletion of intratumoral Tregs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.)

Mucin1 (MUC1) is abnormally glycosylated and overexpressed in a variety of epithelial cancers and plays a critical role in tumor progression. MUC1 has received remark attention as an oncogenic molecule and is considered a valuable tumor target for immunotherapy, while many monoclonal antibodies (mAbs) targeting MUC1-positive cancers in clinical studies lack satisfactory results. It would be highly desirable to develop an effective therapy against MUC1-expressing cancers. In this study, we constructed a novel T cell-engaging bispecific antibody (BsAb) targeting MUC1 and CD3 with the Fab-ScFv-IgG format. A high quality of MUC1-CD3 BsAb can be acquired through a standard method. Our study suggested that this BsAb could specifically bind to MUC1- and CD3-positive cells and efficiently enhance T cell activation, cytokine release, and cytotoxicity. Furthermore, our study demonstrated that this BsAb could potently redirect T cells to eliminate MUC1-expressing tumor cells in vitro and significantly suppress MUC1-positive tumor growth in a xenograft mouse model. Thus, T cell-engaging MUC1/CD3 BsAb could be an effective therapeutic approach to combat MUC1-positive tumors and our MUC1/CD3 BsAb could be a promising candidate in clinical applications for the treatment of MUC1-positive cancer patients.

PACE generates an active TriFab T cell engager from two inactive prodrugs By crosslinking the T cell receptor on T lymphocytes and tumor cells, TCBs induce potent T cell-mediated cytotoxicity at the tumor site ([22]). Keywords: bsAb; immunotherapy; prodrug; T cell engager EN bsAb immunotherapy prodrug T cell engager 495 508 14 04/12/22 20220401 NES 220401 Introduction The therapeutic applicability of numerous antibody derivatives in clinical development is limited by unfavorable properties such as safety or pharmacokinetics ([17]; [38]; [39]). Total antibody-like protein (kappa(+)) was detected by capturing with a biotinylated anti-human kappa light chain Fab (Clone M-1.7.10), coated onto a Streptavidin microtiter plate (SA-MTP, MicroCoat Biotechnologie GmbH, Bernried) and detection was performed with a digoxigenin-labeled anti human Ch1 antibody (Clone M-1.19.31) Biotin-binding antibodies (formed if two prodrugs assemble into a functional -TriFab) were detected by capturing the antibody with biotinylated albumin, coated to a streptavidin microtiter plate. [Extracted from the article]

Cancer immunotherapy has made remarkable progress in the past decade. Bispecific antibodies (BsAbs) have acquired much attention as the next generation strategy of antibody-target cancer immunotherapy, which overwhelmingly focus on T cell recruitment and dual receptors blockade. So far, BsAb drugs have been proved clinically effective and approved for the treatment of hematologic malignancies, but no BsAb have been approved in solid tumors. Numerous designed BsAb drugs for solid tumors are now undergoing evaluation in clinical trials. In this review, we will introduce the formats of bispecific antibodies, and then update the latest preclinical studies and clinical trials in solid tumors of BsAbs targeting EpCAM, CEA, PMSA, ErbB family, and so on. Finally, we discuss the BsAb-related adverse effects and the alternative strategy for future study. [ABSTRACT FROM AUTHOR]

T cell-redirecting strategies have emerged as effective cancer immunotherapy approaches. Bispecific antibodies (bsAbs) are designed to specifically recruit T cells to the tumor microenvironment and induce the assembly of the immunological synapse (IS) between T cells and cancer cells or antigen-presenting cells. The way that the quality of the IS might predict the effectiveness of T cell-redirecting strategies, including those mediated by bsAbs or by chimeric antigen receptors (CAR)-T cells, is currently under discussion. Here we review the organization of the canonical IS assembled during natural antigenic stimulation through the T cell receptor (TCR) and to what extent different bsAbs induce T cell activation, canonical IS organization, and effector function. Then, we discuss how the biochemical parameters of different formats of bsAbs affect the effectivity of generating an antigen-induced canonical IS. Finally, the quality of the IS assembled by bsAbs and monoclonal antibodies or CAR-T cells are compared, and strategies to improve bsAb-mediated T cell-redirecting strategies are discussed.

Advancement in sequencing technologies allows for the identification of molecular pathways involved in tumor progression and treatment resistance. Implementation of novel agents targeting these pathways, defined as targeted therapy, significantly improves the prognosis of cancer patients. Targeted therapy also includes the use of monoclonal antibodies (mAbs). These drugs recognize specific oncogenic proteins expressed in cancer cells. However, as with many other types of targeting agents, mAb-based therapy usually fails in the long-term control of cancer progression due to the development of resistance. In many cases, resistance is caused by the activation of alternative pathways involved in cancer progression and the development of immune evasion mechanisms. To overcome this off-target resistance, bispecific antibodies (bsAbs) were developed to simultaneously target differential oncogenic pathway components, tumor-associated antigens (TAA) and immune regulatory molecules. As a result, in the last few years, several bsAbs have been tested or are being tested in cancer patients. A few of them are currently approved for the treatment of some hematologic malignancies but no bsAbs are approved in solid tumors. In this review, we will provide an overview of the state-of-the-art of bsAbs for the treatment of solid malignancies outlining their classification, design, main technologies utilized for production, mechanisms of action, updated clinical evidence and potential limitations.

Oncolytic viruses have emerged as a promising modality for cancer treatment due to their unique abilities to directly destroy tumor cells and modulate the tumor microenvironment. Bispecific T-cell engagers (BsAbs) have been developed to activate and redirect cytotoxic T lymphocytes, enhancing the antitumor response. To take advantage of the specific infection capacity and carrying ability of exogenous genes, we generated a recombinant herpes simplex virus type 1 (HSV-1), HSV-1dko-B7H3nb/CD3 or HSV-1dko-B7H3nb/mCD3, carrying a B7H3nb/CD3 or B7H3nb/mCD3 BsAb that replicates and expresses BsAb in tumor cells in vitro and in vivo. The new generation of oncolytic viruses has been genetically modified using CRISPR/Cas9 technology and the cre-loxp system to increase the efficiency of HSV genome editing. Additionally, we used two fully immunocompetent models (GL261 and MC38) to assess the antitumor effect of HSV-1dko-B7H3nb/mCD3. Compared with the HSV-1dko control virus, HSV-1dko-B7H3nb/mCD3 induced enhanced anti-tumor immune responses and T-cell infiltration in both GL261 and MC38 models, resulting in improved treatment efficacy in the latter. Furthermore, flow cytometry analysis of the tumor microenvironment confirmed an increase in NK cells and effector CD8+ T cells, and a decrease in immunosuppressive cells, including FOXP3+ regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and CD206+ macrophages (M2). Overall, our study identified a novel camel B7H3 nanobody and described the genetic modification of the HSV-1 genome using CRISPR/Cas9 technology and the cre-loxp system. Our findings indicate that expressing B7H3nb/CD3 BsAb could improve the antitumor effects of HSV-1 based oncolytic virus. • We conducted screening for a new camelid B7H3 nanobody. • We elucidated the genetic modification of the HSV-1 genome employing CRISPR/Cas9 technology and the cre-loxp system. • We developed an oncolytic HSV-1 expressing a novel B7H3nb/CD3 BsAb selectively altering the immunosuppressive microenvironment. [ABSTRACT FROM AUTHOR]

For a certain number of mAbs, bispecific antibodies (bsAbs) and Fc-fusion proteins that we worked on, the Protein A capture step experienced low yield (i.e., ∼80%). A previous case study suggested that non-binding aggregate formed in cell culture was the root cause of low Protein A step yield. In the current work, we selected five projects with the low Protein A yield issue to further illustrate this phenomenon. In all cases, existence of non-binding aggregates was confirmed by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) analysis of Protein A load and flow-through. In addition, we demonstrated that aggregates failed to bind to Protein A resin mainly due to their large sizes, which prevented them from entering the resin beads. As the data suggested, SEC-HPLC analysis of Protein A load and flow-through, although not a standard procedure, can provide information that is critical for understanding the unexpected performance of Protein A chromatography in cases like those being presented here. Thus, SEC-HPLC analysis of Protein A load and flow-through is highly recommended for antibodies/Fc-fusions suffering from low Protein A yield. • Low Protein A step yield has been observed for certain mAbs, bsAbs and Fc-fusions. • SDS-PAGE analysis of Protein A flow-through suggested abnormal binding in these cases but missed important information. • SEC-HPLC analysis of column load and flow-through revealed that poor Protein A performance was due to non-binding aggregates. • Aggregates failed to bind mainly due to their large sizes, which prevented the aggregates from entering the resin beads. • For projects experiencing low Protein A yield, SEC-HPLC analysis of load and flow-through is highly recommended. [ABSTRACT FROM AUTHOR]

The manufacturability assessment and optimization of bispecific antibodies (bsAbs) during the discovery stage are crucial for the success of the drug development process, impacting the speed and cost of advancing such therapeutics to the Investigational New Drug (IND) stage and ultimately to the market. The complexity of bsAbs creates challenges in employing effective evaluation methods to detect developability risks in early discovery stage, and poses difficulties in identifying the root causes and implementing subsequent engineering solutions. This study presents a case of engineering a bsAb that displayed a normal solution appearance during the discovery phase but underwent significant precipitation when subjected to agitation stress during 15 L Chemistry, Manufacturing, and Control (CMC) production Leveraging analytical tools, structural analysis, in silico prediction, and wet-lab validations, the key molecular origins responsible for the observed precipitation were identified and addressed. Sequence engineering to reduce protein surface hydrophobicity and enhance conformational stability proved effective in resolving agitation-induced aggregation. The refined bsAb sequences enabled successful mass production in CMC department. The findings of this case study contribute to the understanding of the fundamental mechanism of agitation-induced aggregation and offer a potential protein engineering procedure for addressing similar issues in bsAb. Furthermore, this case study emphasizes the significance of a close partnership between Discovery and CMC teams. Integrating CMC's rigorous evaluation methods with Discovery's engineering capability can facilitate a streamlined development process for bsAb molecules., (© The Author(s) 2024. Published by Oxford University Press on behalf of Antibody Therapeutics.)

Abstract Cancer immunotherapy is the most exciting advancement in cancer therapy. Similar to immune checkpoint blockade and chimeric antigen receptor T cell (CAR-T), bispecific antibody (BsAb) is attracting more and more attention as a novel strategy of antitumor immunotherapy. BsAb not only offers an effective linkage between therapeutics (e.g., immune effector cells, radionuclides) and targets (e.g., tumor cells) but also simultaneously blocks two different oncogenic mediators. In recent decades, a variety of BsAb formats have been generated. According to the structure of Fc domain, BsAb can be classified into two types: IgG-like format and Fc-free format. Among these formats, bispecific T cell engagers (BiTEs) and triomabs are commonly investigated. BsAb has achieved an exciting breakthrough in hematological malignancies and promising outcome in solid tumor as showed in various clinical trials. In this review, we focus on the preclinical experiments and clinical studies of epithelial cell adhesion molecule (EpCAM), human epidermal growth factor receptor (HER) family, carcinoembryonic antigen (CEA), and prostate-specific membrane antigen (PSMA) related BsAbs in solid tumors, as well as discuss the challenges and corresponding approaches in clinical application.

Bispecific antibodies (bsAbs) are a class of antibodies that can mediate novel mechanisms of action compared to monospecific monoclonal antibodies (mAbs). Since the discovery of mAbs and their adoption as therapeutic agents in the 1980s and 1990s, the development of bsAbs has held substantial appeal. Nevertheless, only three bsAbs (catumaxomab, blinatumomab, emicizumab) were approved through the end of 2020. However, since then, 11 bsAbs received regulatory agency approvals, of which nine (amivantamab, tebentafusp, mosunetuzumab, cadonilimab, teclistamab, glofitamab, epcoritamab, talquetamab, elranatamab) were approved for the treatment of cancer and two (faricimab, ozoralizumab) in non-oncology indications. Notably, of the 13 currently approved bsAbs, two, emicizumab and faricimab, have achieved blockbuster status, showing the promise of this novel class of therapeutics. In the 2020s, the approval of additional bsAbs can be expected in hematological malignancies, solid tumors and non-oncology indications, establishing bsAbs as essential part of the therapeutic armamentarium.

Purpose: Bi-specific antibody (BsAb) is an emerging novel format of antibody. We aimed to develop the natural killer (NK) cell receptor NK group 2, member D (NKG2D)-mediated, immune surveillance system. In this system, the NKG2D ligand MHC class I-related chain A (MICA) was fused with BsAb, which targeted a cluster of differentiation 24 (CD24), a tumor-initiating cell marker that is over-expressed on hepatocellular carcinoma (HCC). Methods: The Homo MICA extracellular domains (hMICA) were fused to the end of the heavy chain of cG7 with the flexible pentapeptide (Gly-Gly-Gly-Gly-Ser; G4S), which formed the cG7-MICA that was further identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting (WB). The targeting specificity was characterized using the Surface Plasmon Resonance (SPR) technology and a flow cytometry assay. Furthermore, the design of BsAb cG7-MICA that targeted CD24 and NKG2D was proven to enhance antibody-dependent, cell-mediated cytotoxicity (ADCC) in vitro by the CytoTox 96 Nonradioactive Cytotoxicity assay. Degranulation and a cytokine production assay of NK cells demonstrated that NK cells were activated effectively by cG7-MICA. Further, in HCC-bearing nude mice, the anti-tumor effects of cG7-MICA combined with sorafenib were verified again. Results: We purified cG7-MICA successfully, and it has a high affinity. In vivo, cG7-MICA recruited NK cells to the tumor site and improved the anti-tumor efficacy of sorafenib. cG7-MICA also activated NK cells to release interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α), and it increased the CD107a expression on the surface of the NK cells in vitro. Conclusion: NK cells play a major role in the natural, innate immune system, and they have the function of identifying and killing target cells. cG7-MICA remodels the function of MICA molecules to activate NK cells, which provides a possible strategy for HCC-targeting immunotherapy. [ABSTRACT FROM AUTHOR]

Bispecific antibodies (BsAbs), capable of directing T cells to kill specific cancer cells by transiently binding the two cell types, have emerged as one class of promising cancer immunotherapies. However, their wide clinical application might be hampered by two deficiencies: high cost and inconvenience in drug administration. This study presents concept-proving data that these problems could be bypassed by using an enhanced nonviral DNA vector minicircle (MC) to produce BsAb in vivo. It was found that the anti-CD3/CD20 produced from the minicircle (MC.CD20) could effectively mediate the T-cell killing of multiple CD20-positive human B-cell lymphoma cell lines in vitro. More importantly, it was demonstrated that delivery of 5 μg of MC.CD20 to mouse liver via hydrodynamic injection resulted in both the expression of a therapeutic level of anti-CD3/CD20 throughout the 32-day experiment and effective anticancer activity in a B-cell lymphoma xenograft mouse model. The data suggest that MC encoding the BsAbs may become an attractive cancer immunotherapy modality based on its excellent features of safety, efficacy, and convenience in both preparation and use, and its affordability once the delivery technology matures. [ABSTRACT FROM AUTHOR]

Poly ADP ribose polymerase (PARP) inhibitors are mainly used in treating BRCA-mutant cancers, and their application in novel therapies to expand their benefit is of interest in personalized medicine. A recent report showed that pharmacological targeting of PARP increases the sensitivity of cancer cells to EGFR inhibition, but the therapeutic value of this combination has not been fully determined. We propose a strategy of combining PARP inhibitors with bispecific antibodies that target both EGFR and Notch signalling, highlighting the difficulties posed by deregulation of Notch signalling and the enrichment of cancer stem cells (CSCs) during therapy. In the present study, we showed that although PARP plus EGFR targeting led to more penetrant and durable responses in the non-small cell lung cancer (NSCLC) PDX model, it influenced the enrichment of stem-like cells and their relative proportion. Stem-like cells were significantly inhibited in vitro and in vivo by EGFR/Notch-targeting bispecific antibodies. These bispecific antibodies were effective in PDX models and showed promise in cell line models of NSCLC, where they delayed the development of acquired resistance to cetuximab and talazoparib. Moreover, combining EGFR/Notch-targeting bispecific antibodies and talazoparib had a more substantial antitumour effect than the combination of talazoparib and cetuximab in a broad spectrum of epithelial tumours. EGFR/Notch bispecific antibodies decrease the subpopulation of stem-like cells, reduce the frequency of tumour-initiating cells, and downregulate mesenchymal gene expression. These findings suggest that combining EGFR and Notch signalling blockade can potentially increase the response to PARP blockade., Competing Interests: Competing Interests: J.Z. is a shareholder at KOCHKOR Biotech Inc., Shanghai., (© The author(s).)

The tumor suppressor p53 is the most common mutated gene in cancer, with the R175H as the most frequent p53 missense mutant. However, there are currently no approved targeted therapies or immunotherapies against mutant p53. Here, we characterized and investigated a monoclonal antibody (mAb) that recognizes the mutant p53-R175H for its affinity, specificity, and activity against tumor cells in vitro . We then delivered DNA plasmids expressing the anti-R175H mAb or a bispecific antibody (BsAb) into mice to evaluate their therapeutic effects. Our results showed that the anti-R175H mAb specifically bound to the p53-R175H antigen with a high affinity and recognized the human mutant p53-R175H antigen expressed on HEK293T or MC38 cells, with no cross-reactivity with wild-type p53. In cultured cells, the anti-R175H mAb showed higher cytotoxicity than the control but did not induce antibody-dependent cellular cytotoxicity. We made a recombinant MC38 mouse cell line (MC38-p53-R175H) that overexpressed the human p53-R175H after knocking out the endogenous mutant p53 alleles. In vivo , administration of the anti-R175H mAb plasmid elicited a robust anti-tumor effect against MC38-p53-R175H in mice. The administration of the anti-R175H BsAb plasmid showed no therapeutic effects, yet potent anti-tumor activity was observed in combination with the anti-PD-1 antibody. These results indicate that targeting specific mutant epitopes using DNA-delivered mAbs or BsAbs presents a form of improved natural immunity derived from tumor-infiltrating B cells and plasma cells against intracellular tumor antigens., (© 2023 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.)

T cell-redirecting strategies have emerged as effective cancer immunotherapy approaches. Bispecific antibodies (bsAbs) are designed to specifically recruit T cells to the tumor microenvironment and induce the assembly of the immunological synapse (IS) between T cells and cancer cells or antigen-presenting cells. The way that the quality of the IS might predict the effectiveness of T cell-redirecting strategies, including those mediated by bsAbs or by chimeric antigen receptors (CAR)-T cells, is currently under discussion. Here we review the organization of the canonical IS assembled during natural antigenic stimulation through the T cell receptor (TCR) and to what extent different bsAbs induce T cell activation, canonical IS organization, and effector function. Then, we discuss how the biochemical parameters of different formats of bsAbs affect the effectivity of generating an antigen-induced canonical IS. Finally, the quality of the IS assembled by bsAbs and monoclonal antibodies or CAR-T cells are compared, and strategies to improve bsAb-mediated T cell-redirecting strategies are discussed.

Bispecific antibodies (bsAbs) are artificial antibodies with two distinct antigen-binding sites that can bind to different antigens or different epitopes on the same antigen. Based on a variety of technology platforms currently developed, bsAbs can exhibit different formats and mechanisms of action. The upgrading of antibody technology has promoted the development of bsAbs, which has been effectively used in the treatment of tumors. So far, 7 bsAbs have been approved for marketing in the world, and more than 200 bsAbs are in clinical and preclinical research stages. Here, we summarize the development process of bsAbs, application in tumor treatment and look forward to the challenges in future development., 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 © 2022 Kang, Sun and Zhang.)

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, difficult-to-diagnose, highly aggressive myeloid malignancy with poor prognosis and no standard of care. The interleukin-3 receptor α, or CD123, is highly expressed in patients with myeloid malignancies, particularly acute myeloid leukemia and BPDCN. CD123 is overexpressed on leukemic stem cells compared with normal hematopoietic stem cells, suggesting CD123 as an attractive immunotherapeutic target. To date, multiple CD123-targeted therapeutic avenues have been explored to treat BPDCN and other CD123 + hematologic malignancies. This review summarizes immunotherapies targeting CD123 for the treatment of BPDCN and related neoplasms., Competing Interests: Disclosure The authors have nothing to disclose. Conflict of Interest The authors disclose no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A bispecific antibody (bsAb) is able to bind two different targets or two distinct epitopes on the same target. Broadly speaking, bsAbs can include any single molecule entity containing dual specificities with at least one being antigen-binding antibody domain. Besides additive effect or synergistic effect, the most fascinating applications of bsAbs are to enable novel and often therapeutically important concepts otherwise impossible by using monoclonal antibodies alone or their combination. This so-called obligate bsAbs could open up completely new avenue for developing novel therapeutics. With evolving understanding of structural architecture of various natural or engineered antigen-binding immunoglobulin domains and the connection of different domains of an immunoglobulin molecule, and with greatly improved understanding of molecular mechanisms of many biological processes, the landscape of therapeutic bsAbs has significantly changed in recent years. As of September 2019, over 110 bsAbs are under active clinical development, and near 180 in preclinical development. In this review article, we introduce a system that classifies bsAb formats into 30 categories based on their antigen-binding domains and the presence or absence of Fc domain. We further review the biology applications of approximately 290 bsAbs currently in preclinical and clinical development, with the attempt to illustrate the principle of selecting a bispecific format to meet biology needs and selecting a bispecific molecule as a clinical development candidate by 6 critical criteria. Given the novel mechanisms of many bsAbs, the potential unknown safety risk and risk/benefit should be evaluated carefully during preclinical and clinical development stages. Nevertheless we are optimistic that next decade will witness clinical success of bsAbs or multispecific antibodies employing some novel mechanisms of action and deliver the promise as next wave of antibody-based therapeutics., (© The Author(s) 2020. Published by Oxford University Press on behalf of Antibody Therapeutics.)