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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.

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.)

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.)

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).)

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).)

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.

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.

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.)

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 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).)

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.

Introduction: Bispecific antibodies (BsAbs) can simultaneously target two epitopes of different antigenic targets, bringing possibilities for diversity in antibody drug design and are promising tools for the treatment of cancers and other diseases. T-cell engaging bsAb is an important application of the bispecific antibody, which could promote T cell-mediated tumor cell killing by targeting tumor-associated antigen (TAA) and CD3 at the same time., Methods: This study comprised antibodies purification, Elisa assay for antigen binding, cytotoxicity assays, T cell activation by flow cytometry in vitro and xenogenic tumor model in vivo ., Results: We present a novel bsAb platform named PHE-Ig technique to promote cognate heavy chain (HC)-light chain (LC) pairing by replacing the CH1/CL regions of different monoclonal antibodies (mAbs) with the natural A and B chains of PHE1 fragment of Integrin β2 based on the knob-in-hole (KIH) technology. We had also verified that PHE-Ig technology can be effectively used as a platform to synthesize different desired bsAbs for T-cell immunotherapy. Especially, BCMA×CD3 PHE-Ig bsAbs exhibited robust anti-multiple myeloma (MM) activity in vitro and in vivo ., Discussion: Moreover, PHE1 domain was further shortened with D14G and R41S mutations, named PHE-S, and the PHE-S-based BCMA×CD3 bsAbs also showed anti BCMA + tumor effect in vitro and in vivo , bringing more possibilities for the development and optimization of different bsAbs. To sum up, PHE1-based IgG-like antibody platform for bsAb construction provides a novel strategy for enhanced T-cell immunotherapy., Competing Interests: Author JL is employed by Shanghai Synvida Biotechnology Co. 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 Wang, Jiang, Yin, Liang, Li, Ding, Yang, Zhang, Liu and Xu.)

Bispecific and multispecific agents have become increasingly utilized in cancer treatment and immunotherapy, yet their complex design parameters present a challenge in developing successful therapeutics. Bispecifics that crosslink receptors on two opposing cells can provide specific activation of a receptor only when these cells are in close spatial proximity, such as an immune cell and cancer cell in a tumor. These agents, including T cell activating bispecifics, can avoid off-tumor toxicity through activation only in the tumor microenvironment by utilizing a tumor target to cluster T-cell receptors for a selective costimulatory signal. Here, we investigate a panel of PD-1/CD137 targeted Humabody V H  domains to determine the key factors for T cell activation, such as affinity, valency, expression level, domain orientation, and epitope location. Target expression is a dominant factor determining both specificity and potency of T cell activation. Given an intrinsic expression level, the affinity can be tuned to modulate the level of activation and IC 50 and achieve specificity between low and high expression levels. Changing the epitope location and linker length showed minor improvements to activation at low expression levels, but increasing the valency for the target decreased activation at all expression levels. By combining non-overlapping epitopes for the target, we achieved higher receptor activation at low expression levels. A kinetic model was able to capture these trends, offering support for the mechanistic interpretation. This work provides a framework to quantify factors for T cell activation by cell-crosslinking bispecific agents and guiding principles for the design of new agents., (© 2024. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

An appropriately designed pharmacokinetic (PK) assay that is sensitive for anti-drug antibody (ADA) impact on relevant exposure is an alternative strategy to understand the neutralizing potential of ADAs. However, guidance on how to develop such PK assays and how to confirm the functional ADA impact on exposure is missing. Here, the PK assay of a T-cell-engaging bispecific antibody, cibisatamab, was developed based on its mechanism of action (MoA). Using critical monoclonal anti-idiotypic (anti-ID) antibody positive controls as ADA surrogates, the impact on exposure was evaluated pre-clinically. In a phase I clinical trial (NCT02324257), initial data suggest that the combination of ADA and PK assays for correlation of the ADA response with cibisatamab exposure. To understand the neutralizing potential of patient-derived ADAs on drug activity, advanced ADA characterization has been performed. Structural binding analysis of ADAs to antibody domains of the drug and its impact on targeting were assessed. For this purpose, relevant patient ADA binding features were identified and compared with the specific monoclonal anti-ID antibody-positive controls. Comparable results of target binding inhibition and similar impacts on exposure suggest that the observed reduction of Cmax and Ctrough levels in patients is caused by the neutralizing potential of ADAs and allows a correlation between ADA response and loss of exposure. Therefore, the described study provides important functional aspects for the development of an appropriately designed PK assay for bispecific antibodies as an alternative option towards understanding the neutralizing ADA impact on exposure., Competing Interests: CK is employed at F. Hoffman-La Roche AG and holds ownership of stocks and patents of the company. All authors were employees of Roche Diagnostics GmbH or F. Hoffman-La Roche AG during the time this study and associated analyses were being conducted. Authors might hold shares or patents of the company., (Copyright © 2024 Lotz, Lutz, Martin-Facklam, Hansbauer, Schick, Moessner, Antony, Stuchly, Viert, Hosse, Freimoser-Grundschober, Klein, Schäfer, Ritter and Stubenrauch.)

Human cytomegalovirus is a ubiquitous herpesvirus that, while latent in most individuals, poses a great risk to immunocompromised patients. In contrast to directly acting traditional antiviral drugs, such as ganciclovir, we aim to emulate a physiological infection control using T cells. For this, we constructed several bispecific T-cell engager (BiTE) constructs targeting different viral glycoproteins of the murine cytomegalovirus and evaluated them in vitro for their efficacy. To isolate the target specific effect without viral immune evasion, we established stable reporter cell lines expressing the viral target glycoprotein B, and the glycoprotein complexes gN-gM and gH-gL, as well as nano-luciferase (nLuc). First, we evaluated binding capacities using flow cytometry and established killing assays, measuring nLuc-release upon cell lysis. All BiTE constructs proved to be functional mediators for T-cell recruitment and will allow a proof of concept for this treatment option. This might pave the way for strikingly safer immunosuppression in vulnerable patient groups.

Bispecific T-cell-engaging antibodies are a growing class of therapeutics with numerous molecules being tested in clinical trials and, currently, seven of them have received market approval. They are structurally complex and function as adaptors to redirect the cytotoxicity of T cells to kill tumor cells. T-cell-engaging bispecific antibodies can be generally divided into two categories: IgG/IgG-like and non-IgG-like formats. Different formats may have different intrinsic potencies and physiochemical properties, and comprehensive studies are needed to gain a better understanding of how the differences in formats impact on structural and functional characteristics. In this study, we designed and generated bispecific T-cell-engaging antibodies with IgG-like (DVD-Ig) and non-IgG (BiTE) formats. Both target the same pair of antigens (EGFR and CD3) to minimize the possible influence of targets on functional characterization. We performed a side-by-side comparison to assess differences in the physiochemical and biological properties of these two bispecific T-cell-engaging antibodies using a variety of breast and ovarian cancer cell-based functional assays to delineate the structural-functional relationships and anti-tumor activities/potency. We found that the Fc portion of T-cell-engaging bispecific antibodies can significantly impact antigen binding activity, potency, and stability in addition to eliciting different mechanisms of action that contribute the killing of cancer cells., 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 © 2024 Mohan, Ayinde, Peng, Dutta, Shen, Falkowski, Biel, Ju and Wu.)

The broader clinical use of bispecific T cell engagers for inducing anti-tumour toxicity is hindered by their on-target off-tumour toxicity and the associated neurotoxicity and cytokine-release syndrome. Here we show that the off-tumour toxicity of a supramolecular bispecific T cell engager binding to the T cell co-receptor CD3 and to the human epidermal growth factor receptor 2 on breast tumour cells can be halted by disengaging the T cells from the tumour cells via the infusion of the small-molecule drug amantadine, which disassembles the supramolecular aggregate. In mice bearing human epidermal growth factor receptor 2-expressing tumours and with a human immune system, high intravenous doses of such a 'switchable T cell nanoengager' elicited strong tumour-specific adaptive immune responses that prevented tumour relapse, while the infusion of amantadine restricted off-tumour toxicity, cytokine-release syndrome and neurotoxicity. Supramolecular chemistry may be further leveraged to control the anti-tumour activity and off-tumour toxicity of bispecific antibodies., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Neural cell adhesion molecule L1 (L1CAM) is a cell-surface glycoprotein involved in cancer occurrence and migration. Up to today, L1CAM-targeted therapy appeared limited efficacy in clinical trials although quite a few attempts by monoclonal antibody (mAb) or chimeric antigen receptor T-cell therapy (CAR-T) have been reported. Therefore, the development of new effective therapies targeting L1CAM is highly desirable. It has been demonstrated that T cell-engaging bispecific antibody (TCE) plays an effective role in cancer immunotherapy by redirecting the cytotoxic activity of CD3 + T cells to tumor cells, resulting in tumor cell death. In this study, we designed and characterized a novel bispecific antibody (CE7-TCE) based on the IgG-(L)-ScFv format, which targets L1CAM and CD3 simultaneously. In vitro, CE7-TCE induced specific killing of L1CAM-positive tumor cells through T cells. In vivo, CE7-TCE inhibited tumor growth in human peripheral blood mononuclear cell/tumor cell co-grafting models. To overcome the adaptive immune resistance (AIR) that impairs the efficacy of TCEs, we conducted a combination therapy of CE7-TCE with Pembrolizumab (anti-PD1 mAb), which enhanced the anti-tumor activity of CE7-TCE. Our results confirmed the feasibility of using L1CAM as a TCE target for the treatment of solid tumors and revealed the therapeutic potential of CE7-TCE combined with immune checkpoint inhibitors., 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 Masson SAS.. All rights reserved.)

Background: EGFR and/or HER2 expression in pancreatic cancers is correlated with poor prognoses. We generated homodimeric (EGFRxEGFR or HER2xHER2) and heterodimeric (EGFRxHER2) T cell-engaging bispecific antibodies (T-BsAbs) to direct polyclonal T cells to these antigens on pancreatic tumors., Methods: EGFR and HER2 T-BsAbs were constructed using the 2 + 2 IgG-[L]-scFv T-BsAbs format bearing two anti-CD3 scFvs attached to the light chains of an IgG to engage T cells while retaining bivalent binding to tumor antigens with both Fab arms. A Fab arm exchange strategy was used to generate EGFRxHER2 heterodimeric T-BsAb carrying one Fab specific for EGFR and one for HER2. EGFR and HER2 T-BsAbs were also heterodimerized with a CD33 control T-BsAb to generate 'tumor-monovalent' EGFRxCD33 and HER2xCD33 T-BsAbs. T-BsAb avidity for tumor cells was studied by flow cytometry, cytotoxicity by T-cell mediated 51 Chromium release, and in vivo efficacy against cell line-derived xenografts (CDX) or patient-derived xenografts (PDX). Tumor infiltration by T cells transduced with luciferase reporter was quantified by bioluminescence., Results: The EGFRxEGFR, HER2xHER2, and EGFRxHER2 T-BsAbs demonstrated high avidity and T cell-mediated cytotoxicity against human pancreatic ductal adenocarcinoma (PDAC) cell lines in vitro with EC50s in the picomolar range (0.17pM to 18pM). They were highly efficient in driving human polyclonal T cells into subcutaneous PDAC xenografts and mediated potent T cell-mediated anti-tumor effects. Both EGFRxCD33 and HER2xCD33 tumor-monovalent T-BsAbs displayed substantially reduced avidity by SPR when compared to homodimeric EGFRxEGFR or HER2xHER2 T-BsAbs (∼150-fold and ∼6000-fold respectively), tumor binding by FACS (8.0-fold and 63.6-fold), and T-cell mediated cytotoxicity (7.7-fold and 47.2-fold), while showing no efficacy against CDX or PDX. However, if either EGFR or HER2 was removed from SW1990 by CRISPR-mediated knockout, the in vivo efficacy of heterodimeric EGFRxHER2 T-BsAb was lost., Conclusion: EGFR and HER2 were useful targets for driving T cell infiltration and tumor ablation. Two arm Fab binding to either one or both targets was critical for robust anti-tumor effect in vivo. By engaging both targets, EGFRxHER2 heterodimeric T-BsAb exhibited potent anti-tumor effects if CDX or PDX were EGFR + HER2 + double-positive with the potential to spare single-positive normal tissue., (© 2024. The Author(s).)

Predicting the toxicity of cancer immunotherapies preclinically is challenging because models of tumours and healthy organs do not typically fully recapitulate the expression of relevant human antigens. Here we show that patient-derived intestinal organoids and tumouroids supplemented with immune cells can be used to study the on-target off-tumour toxicities of T-cell-engaging bispecific antibodies (TCBs), and to capture clinical toxicities not predicted by conventional tissue-based models as well as inter-patient variabilities in TCB responses. We analysed the mechanisms of T-cell-mediated damage of neoplastic and donor-matched healthy epithelia at a single-cell resolution using multiplexed immunofluorescence. We found that TCBs that target the epithelial cell-adhesion molecule led to apoptosis in healthy organoids in accordance with clinical observations, and that apoptosis is associated with T-cell activation, cytokine release and intra-epithelial T-cell infiltration. Conversely, tumour organoids were more resistant to damage, probably owing to a reduced efficiency of T-cell infiltration within the epithelium. Patient-derived intestinal organoids can aid the study of immune-epithelial interactions as well as the preclinical and clinical development of cancer immunotherapies., (© 2023. The Author(s).)

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

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

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

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

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

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

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

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

Although T cell-recruiting CD3-binding bispecific antibodies (BiMAb) have been proven to be clinically effective for hematologic malignancies, the success of BiMAb targeting solid tumor-associated antigens (TAA) in carcinomas so far remains poor. We reasoned that provision of co-stimulatory BiMAb in combination with αTAA-αCD3 BiMAb would boost T cell activation and proliferative capacity, and thereby facilitate the targeting of weakly or heterogeneously expressed tumor antigens. Various αTAA-αCD3 and αTAA-αCD28 BiMAb in a tetravalent IgG1-Fc based format have been analyzed, targeting multiple breast cancer antigens including HER2, EGFR, CEA, and EpCAM. Moreover, bifunctional fusion proteins of αTAA-tumor necrosis factor ligand (TNFL) superfamily members including 4-1BBL, OX40L, CD70 and TL1A have been tested. The functional activity of BiMAb was assessed using co-cultures of tumor cell lines and purified T cells in monolayer and tumor spheroid models. Only in the presence of tumor cells, αTAA-αCD3 BiMAb activated T cells and induced cytotoxicity in vitro , indicating a strict dependence on cross-linking. Combination treatment of αTAA-αCD3 BiMAb and co-stimulatory αTAA-αCD28 or αTAA-TNFL fusion proteins drastically enhanced T cell activation in terms of proliferation, activation marker expression, cytokine secretion and tumor cytotoxicity. Furthermore, BiMAb providing co-stimulation were shown to reduce the minimally required dose to achieve T cell activation by at least tenfold. Immuno-suppressive effects of TGF-β and IL-10 on T cell activation and memory cell formation could be overcome by co-stimulation. BiMAb-mediated co-stimulation was further augmented by immune checkpoint-inhibiting antibodies. Effective co-stimulation could be achieved by targeting a second breast cancer antigen, or by targeting fibroblast activation protein (FAP) expressed on another target cell. In tumor spheroids derived from pleural effusions of breast cancer patients, co-stimulatory BiMAb were essential for the activation tumor-infiltrating lymphocytes and cytotoxic anti-tumor responses against breast cancer cells. Taken together we showed that co-stimulation significantly potentiated the tumoricidal activity of T cell-activating BiMAb while preserving the dependence on TAA recognition. This approach could provide for a more localized activation of the immune system with higher efficacy and reduced peripheral toxicities., Competing Interests: The authors declare that the research was conducted in the absence or any commercial of financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Warwas, Meyer, Gonçalves, Moldenhauer, Bulbuc, Knabe, Luckner-Minden, Ziegelmeier, Heussel, Zörnig, Jäger and Momburg.)

HER3 is a member of the EGF receptor family and elevated expression is associated with cancer progression and therapy resistance. HER3-specific T-cell engagers might be a suitable treatment option to circumvent the limited efficacy observed for HER3-blocking antibodies in clinical trials. In this study, we developed bispecific antibodies for T-cell retargeting to HER3-expressing tumor cells, utilizing either a single-chain diabody format (scDb) with one binding site for HER3 and one for CD3 on T-cells or a trivalent bispecific scDb-scFv fusion protein exhibiting an additional binding site for HER3. The scDb-scFv showed increased binding to HER3-expressing cancer cell lines compared to the scDb and consequently more effective T-cell activation and T-cell proliferation. Furthermore, the bivalent binding mode of the scDb-scFv for HER3 translated into more potent T-cell mediated cancer cell killing, and allowed to discriminate between moderate and low HER3-expressing target cells. Thus, our study demonstrated the applicability of HER3 for T-cell retargeting with bispecific antibodies, even at moderate expression levels, and the increased potency of an avidity-mediated specificity gain, potentially resulting in a wider safety window of bispecific T-cell engaging antibodies targeting HER3.

Adoptive T cell therapies for solid tumors is challenging. We generated metabolically enhanced co-activated-T cells by transducing intracellular co-stimulatory (41BB, ICOS or ICOS-27) and CD3ζ T cell receptor signaling domains followed by arming with bispecific antibodies (BiAbs) to produce armed "Headless CAR T cells" (hCART). Various hCART armed with BiAb directed at CD3ϵ and various tumor associated antigens were tested for: 1) specific cytotoxicity against solid tumors targets; 2) repeated and dual sequential cytotoxicity; 3) survival and cytotoxicity under in vitro hypoxic condition; and 4) cytokine secretion. The 41BBζ transduced hCART (hCART 41BBζ ) armed with HER2 BiAb (HER2 hCART 41BBζ ) or armed with EGFR BiAb (EGFR hCART 41BBζ ) killed multiple tumor lines significantly better than control T cells and secreted Th 1 cytokines/chemokines upon tumor engagement at effector to target ratio (E:T) of 2:1 or 1:1. HER2 hCART serially killed tumor targets up to 14 days. Sequential targeting of EGFR or HER2 positive tumors with HER2 hCART 41BBζ followed by EGFR hCART 41BBζ showed significantly increased cytotoxicity compared single antigen targeting and continue to kill under in vitro hypoxic conditions. In summary, metabolically enhanced headless CAR T cells are effective serial killers of tumor targets, secrete cytokines and chemokines, and continue to kill under in vitro hypoxic condition., Competing Interests: LL is co-founder of Transtarget Inc. and serves on the SAB for Rapa Therapeutics, CJ is a co-founder of Tmunity Therapeutics, Inc. and AT is co-founder of AlphaImmunePlatform LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Thakur, Scholler, Kubicka, Bliemeister, Schalk, June and Lum.)

Various antibody-redirected immunotherapeutic approaches, including antibody-drug conjugates (ADCs), bispecific antibodies (bsAbs), and chimeric antigen receptor-T (CAR-T) cells, have been devised to produce specific activity against various cancer types. Using genetically encoded unnatural amino acids, we generated a homogeneous Her2-targeted ADC, a T cell-redirected bsAb, and a FITC-modified antibody capable of redirecting anti-FITC CAR-T (switchable CAR-T; sCAR-T) cells to target different Her2-expressing breast cancers. sCAR-T cells showed activity against Her2-expressing tumor cells comparable to that of conventional anti-Her2 CAR-T cells and superior to that of ADC- and bsAb-based approaches. To prevent antigen escape, we designed bispecific sCAR-T cells targeting both the Her2 receptor and IGF1R, which showed an overall improved activity against cancer cells with low Her2 expression. This study increases our understanding of various explored cancer therapeutics and underscores the efficient application of sCAR-T cells as a promising therapeutic option for breast cancer patients with low or heterogeneous antigen expression.

Background: Acute myeloid leukemia (AML) remains one of the most challenging hematological malignancies. Despite progress in therapeutics, majority of patients succumb to this neoplasm. CD33 is a proven therapeutic target, given its expression on most AML cells. Almost all anti-CD33 antibodies target the membrane distal immunoglobulin V (IgV) domain of the CD33 extracellular domain., Methods: In this manuscript, we present data on three bispecific antibodies (BsAbs) against the CD33 IgV and membrane proximal immunoglobulin C (IgC) domains. We use in vitro binding and cytotoxicity assays to show the effect of these BsAbs on AML cell lines. We also use immunodeficient mice-bearing leukemias from cell lines and patient-derived xenografts to show the effect of these BsAbs in vivo., Results: In vitro, the IgV-targeting BsAb had higher binding to AML cell lines using flow cytometry and delivered more potent cytotoxicity in T-cell-dependent cytotoxicity assays; importantly, the IgC domain-targeting outperformed the IgV domain-targeting BsAb in medullary and extramedullary leukemia animal models., Conclusions: These data support further clinical development of this BsAb for first-in-human phase I clinical trial., Competing Interests: Competing interests: SSH and N-KVC were named as co-inventors in patents on CD33 bispecific antibodies filed by Memorial Sloan Kettering Cancer Center (MSKCC). BC133 and BC275 were licensed to Y-mAbs Therapeutics by MSKCC. Both MSKCC and N-KVC have financial interest in Y-mAbs. SSH and MV are employees of Y-mAbs Therapeutics. The remaining authors declare no competing financial interests., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Chimeric antigen receptors (CARs) or bispecific antibodies (bsAbs) redirected T cell against tumors is one of the most promising immunotherapy approaches. However, insufficient clinical outcomes are still observed in treatments of both solid and non-solid tumors. Limited efficacy and poor persistence are two major challenges in redirected T cell therapies. The immunological synapse (IS) is a vital component during the T cell response, which largely determines the clinical outcomes of T cell-based therapies. Here, we review the structural and signaling characteristics of IS formed by natural T cells and redirected T cells. Furthermore, inspired by the elaborate natural T cell receptor-mediated IS, we provide potential strategies for higher efficacy and longer persistence of redirected T cells., 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 © 2021 Gao, Wang, Luo and Chu.)

Rationale: Endoglin, also known as CD105, is a homo-dimeric membrane glycoprotein required for angiogenesis and serves as a marker for cancer vasculature. In this study, we constructed a bispecific T-cell engager (BiTE) antibody that targets human endoglin and CD3 (hEND-CD3/BiTE). We examined BiTE binding to endoglin-expressing cells and its effects on the cytolytic activity of T cells and cancer development. Methods: The in vitro effects of hEND-CD3/BiTE, including binding to target cells, T-cell activation, proliferation, and cytotoxicity, were examined in endoglin-expressing 293T cells, human umbilical vascular endothelial cells, tumor-derived endothelial cells, and CD3 + T cells. An in vivo xenograft tumor model was established using A549 human lung cancer cells. The therapeutic efficacy of hEND-CD3/BiTE was assessed by monitoring tumor growth, angiogenesis, and mouse survival. Results: hEND-CD3/BiTE specifically bound to endoglin-expressing cells and CD3 + T cells in vitro and stimulated T-cell activation, proliferation, and Th1 cytokine secretion, and promoted T-cell-mediated cytolysis of endoglin-expressing cells. The hEND-CD3/BiTE in vivo caused minimal toxicity to major organs, reduced tumor neoangiogenesis, inhibited tumor growth, and significantly improved mouse survival. Conclusions: Our study demonstrated the therapeutic potential of hEND-CD3/BiTE and provided a novel approach to clinical cancer treatment., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Cancer therapies benefit from accelerated development of biotechnology, and many immunotherapeutic strategies spring up including vaccines, the immune checkpoint blockade, chimeric antigen receptor T cells, and bispecific antibodies (BsAbs). Glypican-3 (GPC3) is a member of the heparan sulfate proteoglycan family of proteins and is highly expressed in hepatocellular carcinoma (HCC) cell membranes. Here, the authors describe a new tetravalent BsAb h8B-BsAb targeting GPC3 and CD3 antigens and studied its antitumor activities against HCC. h8B-BsAb was designed based on immunoglobulin G with a fragment variable fused to the light chain, whose biophysical stabilities including degradation resistance and thermostability were improved by introducing disulfide bonds. In vitro activity of h8B-BsAb showed potent T-cell recruitment and activation for HCC cell lysis by the presence of peripheral blood mononuclear cells, but no specific killing in GPC3-negative cells. In HCC xenograft mouse studies, h8B-BsAb induced robust regression of tumors. In summary, we engineered a highly stable and efficacious BsAb as a potential candidate for HCC treatment., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose of Review: Both chimeric antigen receptor (CAR) T cells and T cell-engaging antibodies (BiAb) have been approved for the treatment of hematological malignancies. However, despite targeting the same antigen, they represent very different classes of therapeutics, each with its distinct advantages and drawbacks. In this review, we compare BiAb and CAR T cells with regard to their mechanism of action, manufacturing, and clinical application. In addition, we present novel strategies to overcome limitations of either approach and to combine the best of both worlds., Recent Findings: By now there are multiple approaches combining the advantages of BiAb and CAR T cells. A major area of research is the application of both formats for solid tumor entities. This includes improving the infiltration of T cells into the tumor, counteracting immunosuppression in the tumor microenvironment, targeting antigen heterogeneity, and limiting off-tumor on-target effects. BiAb come with the major advantage of being an off-the-shelf product and are more controllable because of their half-life. They have also been reported to induce less frequent and less severe adverse events. CAR T cells in turn demonstrate superior response rates, have the potential for long-term persistence, and can be additionally genetically modified to overcome some of their limitations, e.g., to make them more controllable.

Background: Programmed death-ligand 1 (PD-L1) is an important immune checkpoint protein that can be regarded as a pan-cancer antigen expressed by multiple different cell types within the tumor. While antagonizing PD-L1 is well known to relieve PD-1/PD-L1-mediated T cell suppression, here we have combined this approach with an immunotherapy strategy to target T cell cytotoxicity directly toward PD-L1-expressing cells. We developed a bi-specific T cell engager (BiTE) crosslinking PD-L1 and CD3ε and demonstrated targeted cytotoxicity using a clinically relevant patient-derived ascites model. This approach represents an immunological 'volte-face' whereby a tumor immunological defense mechanism can be instantly transformed into an Achilles' heel for targeted immunotherapy., Methods: The PD-L1 targeting BiTE comprises an anti-PD-L1 single-chain variable fragment (scFv) or nanobody (NB) domain and an anti-CD3 scFv domain in a tandem repeat. The ability to activate T cell cytotoxicity toward PD-L1-expressing cells was established using human carcinoma cells and PD-L1-expressing human ('M2') macrophages in the presence of autologous T cells. Furthermore, we armed oncolytic herpes simplex virus-1 (oHSV-1) with PD-L1 BiTE and demonstrated successful delivery and targeted cytotoxicity in unpurified cultures of malignant ascites derived from different cancer patients., Results: PD-L1 BiTE crosslinks PD-L1-positive cells and CD3ε on T cells in a 'pseudo-synapse' and triggers T cell activation and release of proinflammatory cytokines such as interferon-gamma (IFN-γ), interferon gamma-induced protein 10 (IP-10) and tumour necrosis factor-α (TNF-α). Activation of endogenous T cells within ascites samples led to significant lysis of tumor cells and M2-like macrophages (CD11b+CD64+ and CD206+/CD163+). The survival of CD3+ T cells (which can also express PD-L1) was unaffected. Intriguingly, ascites fluid that appeared particularly immunosuppressive led to higher expression of PD-L1 on tumor cells, resulting in improved BiTE-mediated T cell activation., Conclusions: The study reveals that PD-L1 BiTE is an effective immunotherapeutic approach to kill PD-L1-positive tumor cells and macrophages while leaving T cells unharmed. This approach activates endogenous T cells within malignant ascites, generates a proinflammatory response and eliminates cells promoting tumor progression. Using an oncolytic virus for local expression of PD-L1 BiTE also prevents 'on-target off-tumor' systemic toxicities and harnesses immunosuppressive protumor conditions to augment immunotherapy in immunologically 'cold' clinical cancers., Competing Interests: Competing interests: None., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Adoptive transfer of Bispecific antibody Armed activated T cells (BATs) showed promising anti-tumor activity in clinical trials in solid tumors. The cytotoxic activity of BATs occurs upon engagement with tumor cells via the bispecific antibody (BiAb) bridge, which stimulates BATs to release cytotoxic molecules, cytokines, chemokines, and other signaling molecules extracellularly. We hypothesized that the release of BATs Induced Tumor-Targeting Effectors (TITE) by this complex interaction of T cells, bispecific antibody, and tumor cells may serve as a potent anti-tumor and immune-activating immunotherapeutic approach. In a 3D tumorsphere model, TITE showed potent cytotoxic activity against multiple breast cancer cell lines compared to control conditioned media (CM): Tumor-CM (T-CM), BATs-CM (B-CM), BiAb Armed PBMC-CM (BAP-CM) or PBMC-CM (P-CM). Multiplex cytokine analysis showed high levels of Th 1 cytokines and chemokines; phospho-protein signaling array data suggest that the prominent JAK1/STAT1 pathway may be responsible for the induction and release of Th 1 cytokines/chemokines in TITE. In xenograft breast cancer models, IV injections of 10× concentrated TITE (3×/week for 3 weeks; 150 μl TITE/injection) was able to inhibit tumor growth significantly (ICR/scid, p < 0.003; NSG p < 0.008) compared to the control mice. We tested the key components of the TITE for immune activating and anti-tumor activity individually and in combinations, the combination of IFN-γ, TNF-α and MIP-1β recapitulates the key activities of the TITE. In summary, master mix of active components of BATs-Tumor complex-derived TITE can provide a clinically controllable cell-free platform to target various tumor types regardless of the heterogeneous nature of the tumor cells and mutational tumor.

Haematology has been at the vanguard of cancer immunotherapy. Immune checkpoint inhibitors (ICIs), bispecific T-cell engagers (BiTEs), allogeneic haematopoietic stem cell transplantation (allo-HSCT) and donor lymphocyte infusion (DLI), as well as adoptive T-cell therapies outside the setting of allo-HSCT, have been approved for distinct haematologic malignancies producing durable responses in otherwise untreatable patients. Despite recent advances, immunotherapies do not benefit most patients, due to resistance or lack of response, and are only approved in specific settings. Moreover, immunotherapies are expensive and may produce severe immune related adverse reactions. Combination therapy complicates the picture and requires further evaluation. This review considers the current status and future perspectives of ICIs and BiTEs approved for haematological malignancies by analysing their strengths, weaknesses, opportunities and threats (SWOT). The biological rationale for anti-cancer mechanisms, clinical data for specific haematological cancers, efficacy, toxicity, response and resistance profiles, novel strategies to improve these characteristics as well as the potential targets to enhance or expand the application of ICIs and BiTEs are also discussed., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Immunotherapy has raised high expectations in the treatment of virtually every cancer. Many current efforts are focused on ensuring the efficient delivery of active cytotoxic cells to tumors. It is assumed that, once these active cytotoxic cells are correctly engaged to cancer cells, they will unfailingly eliminate the latter, provided that inhibitory factors are in check. T cell bispecific antibodies (TCBs) and chimeric antigen receptors (CARs) offer an opportunity to test this assumption. Using TCB and CARs directed against HER2, here we show that disruption of interferon-gamma signaling confers resistance to killing by active T lymphocytes. The kinase JAK2, which transduces the signal initiated by interferon-gamma, is a component repeatedly disrupted in several independently generated resistant models. Our results unveil a seemingly widespread strategy used by cancer cells to resist clearance by redirected lymphocytes. In addition, they open the possibility that long-term inhibition of interferon-gamma signaling may impair the elimination phase of immunoediting and, thus, promote tumor progression.