Bispecific antibodies tethering innate receptors induce human tolerant-dendritic cells and regulatory T cells.

There is an urgent need for alternative therapies targeting human dendritic cells (DCs) that could reverse inflammatory syndromes in many autoimmune and inflammatory diseases and organ transplantations. Here, we describe a bispecific antibody (bsAb) strategy tethering two pathogen-recognition receptors at the surface of human DCs. This cross-linking switches DCs into a tolerant profile able to induce regulatory T-cell differentiation. The bsAbs, not parental Abs, induced interleukin 10 and transforming growth factor ß1 secretion in monocyte-derived DCs and human peripheral blood mononuclear cells. In addition, they induced interleukin 10 secretion by synovial fluid cells in rheumatoid arthritis and gout patients. This concept of bsAb-induced tethering of surface pathogen-recognition receptors switching cell properties opens a new therapeutic avenue for controlling inflammation and restoring immune tolerance.

Design and characterization of protective pan-ebolavirus and pan-filovirus bispecific antibodies.

Monoclonal antibodies (mAbs) are an important class of antiviral therapeutics. MAbs are highly selective, well tolerated, and have long in vivo half-life as well as the capacity to induce immune-mediated virus clearance. Their activities can be further enhanced by integration of their variable fragments (Fvs) into bispecific antibodies (bsAbs), affording simultaneous targeting of multiple epitopes to improve potency and breadth and/or to mitigate against viral escape by a single mutation. Here, we explore a bsAb strategy for generation of pan-ebolavirus and pan-filovirus immunotherapeutics. Filoviruses, including Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV), cause severe hemorrhagic fever. Although there are two FDA-approved mAb therapies for EBOV infection, these do not extend to other filoviruses. Here, we combine Fvs from broad ebolavirus mAbs to generate novel pan-ebolavirus bsAbs that are potently neutralizing, confer protection in mice, and are resistant to viral escape. Moreover, we combine Fvs from pan-ebolavirus mAbs with those of protective MARV mAbs to generate pan-filovirus protective bsAbs. These results provide guidelines for broad antiviral bsAb design and generate new immunotherapeutic candidates.

Combination of T cell-redirecting strategies with a bispecific antibody blocking TGF-ß and PD-L1 enhances antitumor responses.

T cell-based immunotherapies for solid tumors have not achieved the clinical success observed in hematological malignancies, partially due to the immunosuppressive effect promoted by the tumor microenvironment, where PD-L1 and TGF-ß play a pivotal role. However, durable responses to immune checkpoint inhibitors remain limited to a minority of patients, while TGF-ß inhibitors have not reached the market yet. Here, we describe a bispecific antibody for dual blockade of PD-L1 and TFG-ß, termed AxF (scFv)2, under the premise that combination with T cell redirecting strategies would improve clinical benefit. The AxF (scFv)2 antibody was well expressed in mammalian and yeast cells, bound both targets and inhibited dose-dependently the corresponding signaling pathways in luminescence-based cellular reporter systems. Moreover, combined treatment with trispecific T-cell engagers (TriTE) or CAR-T cells significantly boosted T cell activation status and cytotoxic response in breast, lung and colorectal (CRC) cancer models. Importantly, the combination of an EpCAMxCD3×EGFR TriTE with the AxF (scFv)2 delayed CRC tumor growth in vivo and significantly enhanced survival compared to monotherapy with the trispecific antibody. In summary, we demonstrated the feasibility of concomitant blockade of PD-L1 and TGF-ß by a single molecule, as well as its therapeutic potential in combination with different T cell redirecting agents to overcome tumor microenvironment-mediated immunosuppression.

Development of ISB 1442, a CD38 and CD47 bispecific biparatopic antibody innate cell modulator for the treatment of multiple myeloma.

Antibody engineering can tailor the design and activities of therapeutic antibodies for better efficiency or other advantageous clinical properties. Here we report the development of ISB 1442, a fully human bispecific antibody designed to re-establish synthetic immunity in CD38+ hematological malignancies. ISB 1442 consists of two anti-CD38 arms targeting two distinct epitopes that preferentially drive binding to tumor cells and enable avidity-induced blocking of proximal CD47 receptors on the same cell while preventing on-target off-tumor binding on healthy cells. The Fc portion of ISB 1442 is engineered to enhance complement dependent cytotoxicity, antibody dependent cell cytotoxicity and antibody dependent cell phagocytosis. ISB 1442 thus represents a CD47-BsAb combining biparatopic targeting of a tumor associated antigen with engineered enhancement of antibody effector function to overcome potential resistance mechanisms that hamper treatment of myeloma with monospecific anti-CD38 antibodies. ISB 1442 is currently in a Phase I clinical trial in relapsed refractory multiple myeloma.

Haemophilia in the era of novel therapies: Where do inhibitors feature in the new landscape?

INTRODUCTION: The advent of therapeutic recombinant factor VIII (FVIII) and factor IX (FIX) protein infusions revolutionized the care of persons with haemophilia in the 1990s. It kicked off an era with the increasing use of prophylactic factor infusions for patients and transformed conversations around the ideal trough activity levels as well as the ultimate goals in tailored, individualized care. Our knowledge surrounding the immunologic basis of inhibitor development and treatment derives from a time when patients were receiving frequent factor infusions and focused on immune tolerance induction following inhibitor development. DISCUSSION: More recently, care was revolutionized again in haemophilia A with the approval of emicizumab, a bispecific antibody mimicking activated FVIII function, to prevent bleeding. The use of emicizumab prophylaxis has resulted in a significantly slower accumulation of factor exposure days and continued effective prophylaxis in the case of inhibitor development. While emicizumab is effective at reducing the frequency of bleeding events in patients with haemophilia A, management of breakthrough bleeds, trauma, and surgeries still requires additional treatment. Ensuring that FVIII is a therapeutic option, particularly for life-threatening bleeding events and major surgeries is critical to optimizing the care of persons with haemophilia A. Other novel non-factor concentrate therapies, including rebalancing agents, will dramatically change the landscape for persons with haemophilia B with inhibitors. CONCLUSION: This review discusses the changing landscape regarding the timing of inhibitor development and management strategies after inhibitor development, stressing the importance of education across the community to continue to vigilantly monitor for inhibitors and be prepared to treat persons with inhibitors.

Molecular assessment of intratumoral immune cell subsets and potential mechanisms of resistance to odronextamab, a CD20×CD3 bispecific antibody, in patients with relapsed/refractory B-cell non-Hodgkin lymphoma.

BACKGROUND: Patients with relapsed/refractory B-cell non-Hodgkin lymphoma (R/R B-NHL) have a significant need for effective treatment options. Odronextamab is an Fc-silenced, human, CD20×CD3 bispecific antibody that targets CD20-expressing cells via T-cell-mediated cytotoxicity independent of T-cell/major histocompatibility complex interaction. Phase I results in patients with R/R B-NHL demonstrated that odronextamab monotherapy could achieve deep and durable responses with a generally manageable safety profile (ELM-1; NCT02290951). As part of a biomarker analysis of the same study, we investigated potential biomarkers and mechanisms of resistance to odronextamab. METHODS: Patients with R/R B-NHL enrolled in ELM-1 received one time per week doses of intravenous odronextamab for 4×21 day cycles, then doses every 2 weeks thereafter. Patient tumor biopsies were obtained at baseline, on-treatment, and at progression. Immune cell markers were analyzed by immunohistochemistry, flow cytometry, single-cell RNA sequencing, and whole genome sequencing. RESULTS: Baseline tumor biopsies showed that almost all patients had high proportions of B cells that expressed the CD20 target antigen, whereas expression of other B-cell surface antigens (CD19, CD22, CD79b) was more variable. Responses to odronextamab in patients with diffuse large B-cell lymphoma were not related to the relative level of baseline CD20 expression, cell of origin, or high-risk molecular subtype. A potential link was observed between greater tumor programmed cell death-ligand 1 expression and increased likelihood of response to odronextamab. Similarly, a trend was observed between clinical response and increased levels of CD8 T cells and regulatory T cells at baseline. We also identified an on-treatment pharmacodynamic shift in intratumoral immune cell subsets. Finally, loss of CD20 expression through inactivating gene mutations was identified as a potential mechanism of resistance in patients who were treated with odronextamab until progression, as highlighted in two detailed patient cases reported here. CONCLUSIONS: This biomarker analysis expands on clinical findings of odronextamab in patients with R/R B-NHL, providing verification of the suitability of CD20 as a therapeutic target, as well as evidence for potential mechanisms of action and resistance.

A novel conditional active biologic anti-EpCAM x anti-CD3 bispecific antibody with synergistic tumor selectivity for cancer immunotherapy.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that plays several roles in cancer biology. EpCAM is an attractive therapeutic target because of its expression in most solid tumors. However, targeting EpCAM has been challenging because it is also highly expressed in normal epithelial tissues. Initial attempts to develop EpCAM-specific T-cell engagers were unsuccessful due to severe cytokine release effects, as well as serious on-target, off-tumor drug-related toxicities. We developed novel, conditionally active biological (CAB) bispecific antibodies that bind to both EpCAM and CD3 in an acidic tumor microenvironment. In healthy tissues, binding to EpCAM and CD3 is greatly reduced by a novel, dual CAB selection, where each binding domain is independently blocked by the presence of physiological chemicals known as Protein-associated Chemical Switches (PaCS). The CAB anti-EpCAM T-cell engagers displayed the anticipated bispecific binding properties and mediated the potent lysis of EpCAM-positive cancer cell lines through the recruitment of T cells in the tumor microenvironment. Xenograft studies showed that the efficacy of CAB bispecific antibodies is similar to that of a non-CAB anti-EpCAM bispecific antibody, but they have markedly reduced toxicity in non-human primates, indicating an unprecedentedly widened therapeutic index of over 100-fold. These preclinical results indicate that the dual CAB bispecific antibody is potentially both a powerful and safe therapeutic platform and a promising T cell-engaging treatment for patients with EpCAM-expressing tumors.

Development of a novel conditionally active EpCAM-specific T-cell engager with enhanced safety and tolerability for treatment of solid tumors.

Development of Plant-Derived Bispecific Monoclonal Antibody Targeting PD-L1 and CTLA-4 against Mouse Colorectal Cancer.

Checkpoint blockade immunotherapy has revolutionized cancer treatment, with monoclonal antibodies targeting immune checkpoints, yielding promising clinical benefits. However, with the advent of resistance to immune checkpoint inhibitor treatment in clinical trials, developing next-generation antibodies with potentially increased efficacy is critical. Here, we aimed to generate a recombinant bispecific monoclonal antibody for dual inhibition of programmed cell death protein 1/programmed cell death ligand 1 and cytotoxic T-lymphocyte-associated protein 4 axes. The plant system was used as an alternative platform for bispecific monoclonal antibody production. Dual variable domain immunoglobulin atezolizumab × 2C8 is a plant-derived bispecific monoclonal antibody that combines both programmed cell death ligand 1 and cytotoxic T-lymphocyte-associated protein 4 blockade into a single molecule. Dual variable domain immunoglobulin atezolizumab × 2C8 was transiently expressed in Nicotiana benthamiana and the expression level was determined to be the highest after 4 days of infiltration. The size and assembly of the purified bispecific monoclonal antibody were determined, and its function was investigated in vitro and in vivo. The molecular structures of plant-produced dual variable domain immunoglobulin atezolizumab × 2C8 are as expected, and it was mostly present as a monomer. The plant-produced dual variable domain immunoglobulin atezolizumab × 2C8 showed in vitro binding to programmed cell death ligand 1 and cytotoxic T-lymphocyte-associated protein 4 proteins. The antitumor activity of plant-produced bispecific monoclonal antibody was tested in vivo by treating humanized Balb/c mice bearing a CT26 colorectal tumor. Plant-produced dual variable domain immunoglobulin atezolizumab × 2C8 significantly inhibited tumor growth by reducing tumor volume and weight. Body weight changes indicated that the plant-produced bispecific monoclonal antibody was safe and tolerable. Overall, this proof of concept study demonstrated the viability of plants to produce functional plant-based bispecific immunotherapy.

Dual Targeting of Glioblastoma Cells with Bispecific Killer Cell Engagers Directed to EGFR and ErbB2 (HER2) Facilitates Effective Elimination by NKG2D-CAR-Engineered NK Cells.

NKG2D is an activating receptor of natural killer cells that recognizes stress-induced ligands (NKG2DL) expressed by many tumor cells. Nevertheless, NKG2DL downregulation or shedding can still allow cancer cells to evade immune surveillance. Here, we used lentiviral gene transfer to engineer clinically usable NK-92 cells with a chimeric antigen receptor (NKAR) which contains the extracellular domain of NKG2D for target recognition, or an NKAR, together with the IL-15 superagonist RD-IL15, and combined these effector cells with recombinant NKG2D-interacting bispecific engagers that simultaneously recognize the tumor-associated antigens epidermal growth factor receptor (EGFR) or ErbB2 (HER2). Applied individually, in in vitro cell-killing assays, these NKAB-EGFR and NKAB-ErbB2 antibodies specifically redirected NKAR-NK-92 and NKAR_RD-IL15-NK-92 cells to glioblastoma and other cancer cells with elevated EGFR or ErbB2 levels. However, in mixed glioblastoma cell cultures, used as a model for heterogeneous target antigen expression, NKAR-NK cells only lysed the EGFR- or ErbB2-expressing subpopulations in the presence of one of the NKAB molecules. This was circumvented by applying NKAB-EGFR and NKAB-ErbB2 together, resulting in effective antitumor activity similar to that against glioblastoma cells expressing both target antigens. Our results demonstrate that combining NK cells carrying an activating NKAR receptor with bispecific NKAB antibodies allows for flexible targeting, which can enhance tumor-antigen-specific cytotoxicity and prevent immune escape.

Enhancing anti-CD274 (PD-L1) targeting through combinatorial immunotherapy with bispecific antibodies and fusion proteins: from preclinical to phase II clinical trials.

INTRODUCTION: Immune checkpoint inhibitors have achieved great success in the treatment of many different types of cancer. Programmed cell death protein ligand 1 (PD-L1, CD274) is a major immunosuppressive immune checkpoint and a target for several already approved monoclonal antibodies. Despite this, novel strategies are under development, as the overall response remains low. AREAS COVERED: In this review, an overview of the current biomarkers for response to PD-L1 inhibitor treatment is given, followed by a discussion of potential novel biomarkers, including tumor mutational burden and circulating tumor DNA. Combinatorial immunotherapy is a potential novel strategy to increase the response to PD-L1 inhibitor treatment and currently, several interesting bispecific antibodies as well as bispecific fusion proteins are undergoing early clinical investigation. We focus on substances targeting PD-L1 and a secondary target, and a secondary immunomodulatory target like CTLA-4, TIGIT, or CD47. EXPERT OPINION: Overall, the presented studies show anti-tumor activity of these combinatorial immunotherapeutic approaches. However, still relatively low response rates suggest a need for better biomarkers.

Measurement of recombinant porcine factor VIII in patients with congenital haemophilia A and inhibitors in the presence of emicizumab.

INTRODUCTION: Recombinant porcine factor VIII (rpFVIII) is a treatment option for break-through bleeds in patients with congenital haemophilia A with inhibitors (CHAwI) on emicizumab. However, there are limited data about the measurement of rpFVIII in the presence of emicizumab. AIM: To analyse whether rpFVIII can be measured with a chromogenic assay with bovine component (bCSA) in plasma from CHAwI on emicizumab treatment. METHODS: In the first part of the study, FVIII deficient plasma was spiked with rpFVIII, in the second part, commercial plasma from CHAwI was spiked with emicizumab and rpFVIII, and in the third part, plasma from CHAwI on emicizumab treatment was spiked with rpFVIII. FVIII was then measured with bCSA and a chromogenic assay with human component (hCSA). Thrombin generation (TG) and clot-waveform analysis (CWA) were also carried out. RESULTS: The recovery of rpFVIII measured with bCSA is approximately 80% and is further influenced by the presence of an anti-porcine inhibitor. rpFVIII assessed with hCSA was influenced by emicizumab. CWA and TG showed a weak correlation with baseline emicizumab concentration, but peak thrombin and CWA correlated well with increasing emicizumab concentrations and rpFVIII activities. CONCLUSION: This study indicates that rpFVIII can be measured in the presence of emicizumab with a bCSA. A calibration curve for the measurement of rpFVIII with bCSA should be established.

A bispecific anti-PD-1 and PD-L1 antibody induces PD-1 cleavage and provides enhanced anti-tumor activity.

Combinatorial strategies, such as targeting different immune checkpoint receptors, hold promise to increase the breadth and duration of the response to cancer therapy. Here we describe the preclinical evaluation of CTX-8371, a protein construct which combines PD-1 and PD-L1 targeting in one bispecific, tetravalent antibody. CTX-8371 matched or surpassed the activity of anti-PD-1 and PD-L1 benchmark antibodies in several in vitro T cell activation assays and outperformed clinically approved benchmarks in the subcutaneous MC38 colon and the B16F10 lung metastasis mouse tumor models. Investigation into the mechanism of action revealed that CTX-8371 co-engagement of PD-1 and PD-L1 induced the proteolytic cleavage and loss of cell surface PD-1, which is a novel and non-redundant mechanism that adds to the PD-1/PD-L1 signaling axis blockade. The combination of CTX-8371 and an agonistic anti-CD137 antibody further increased the anti-tumor efficacy with long-lasting curative therapeutic effect. In summary, CTX-8371 is a novel checkpoint inhibitor that might provide greater clinical benefit compared to current anti-PD-1 and PD-L1 antibodies, especially when combined with agents with orthogonal mechanisms of action, such as agonistic anti-CD137 antibodies.

Impact of tissue penetration and albumin binding on design of T cell targeted bispecific agents.

Bispecific agents are a rapidly growing class of cancer therapeutics, and immune targeted bispecific agents have the potential to expand functionality well beyond monoclonal antibody agents. Humabodies⁎ are fully human single domain antibodies that can be linked in a modular fashion to form multispecific therapeutics. However, the effect of heterogeneous delivery on the efficacy of crosslinking bispecific agents is currently unclear. In this work, we utilize a PSMA-CD137 Humabody with an albumin binding half-life extension (HLE) domain to determine the impact of tissue penetration on T cell activating bispecific agents. Using heterotypic spheroids, we demonstrate that increased tissue penetration results in higher T cell activation at sub-saturating concentrations. Next, we tested the effect of two different albumin binding moieties on tissue distribution using albumin-specific HLE domains with varying affinities for albumin and a non-specific lipophilic dye. The results show that a specific binding mechanism to albumin does not influence tissue penetration, but a non-specific mechanism reduced both spheroid uptake and distribution in the presence of albumin. These results highlight the potential importance of tissue penetration on bispecific agent efficacy and describe how the design parameters including albumin-binding domains can be selected to maximize the efficacy of bispecific agents.

T-cell stimulating vaccines empower CD3 bispecific antibody therapy in solid tumors.

CD3 bispecific antibody (CD3 bsAb) therapy is clinically approved for refractory hematological malignancies, but responses in solid tumors have been limited so far. One of the main hurdles in solid tumors is the lack of sufficient T-cell infiltrate. Here, we show that pre-treatment vaccination, even when composed of tumor-unrelated antigens, induces CXCR3-mediated T-cell influx in immunologically 'cold' tumor models in male mice. In the absence of CD3 bsAb, the infiltrate is confined to the tumor invasive margin, whereas subsequent CD3 bsAb administration induces infiltration of activated effector CD8 T cells into the tumor cell nests. This combination therapy installs a broadly inflamed Th1-type tumor microenvironment, resulting in effective tumor eradication. Multiple vaccination formulations, including synthetic long peptides and viruses, empower CD3 bsAb therapy. Our results imply that eliciting tumor infiltration with vaccine-induced tumor-(un)related T cells can greatly improve the efficacy of CD3 bsAbs in solid tumors.

A bispecific antibody targeting HER2 and CLDN18.2 eliminates gastric cancer cells expressing dual antigens by enhancing the immune effector function.

Gastric cancer (GC) is widely regarded as one of the toughest cancers to treat. Trastuzumab, which targets the human epidermal growth factor receptor 2 (HER2) for GC treatment, has demonstrated clinical success. However, these patients have a high likelihood of developing resistance. Additionally, Claudin18.2 (CLDN18.2) is a promising emerging target for GC treatment. Therefore, therapies that simultaneously target both HER2 and CLDN18.2 targets are of great significance. Here, we constructed a bispecific antibody targeting both HER2 and CLDN18.2 (HC-2G4S; BsAb), which displayed satisfactory purity, thermostability and enhancing antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In a tumor spheroids model of GC, BsAb demonstrated greater therapeutic efficacy than monoclonal antibodies (mAb) or combination treatment strategies. We propose that the enhanced anti-tumor potency of BsAbs in vivo is due to the monovalent binding of single-chain antibodies to more targets due to weaker affinity, resulting in a more potent immune effect function. Therefore, HC-2G4S could be a productive agent for treating GC that is HER2-positive, CLDN18.2-positive, or both, with the potential to overcome trastuzumab resistance and provide significant clinical benefits and expanded indications.

T cell-Dependent Bispecific Therapy Enhances Innate Immune Activation and Antibody-Mediated Killing.

T cell-retargeting therapies have transformed the therapeutic landscape for hematologic diseases. T cell-dependent bispecific antibodies (TDB) function as conditional agonists that induce a polyclonal T-cell response, resulting in target cell destruction and cytokine release. The relationship between this response and its effects on surrounding innate immune populations has not been fully explored. Here we show that treatment with mosunetuzumab in patients results in natural killer (NK) cell activation in the peripheral blood. We modeled this phenomenon in vitro and found that TDB-mediated killing activated NK cells, increasing NK function and antibody-dependent cellular cytotoxicity (ADCC), and enhanced the capability of macrophages to perform antibody-dependent cellular phagocytosis (ADCP). This enhancement was triggered by cytokines released through TDB treatment, with IL2 and IFNγ being major drivers for increased ADCC and ADCP, respectively. Surprisingly, cytolytic ability could be further augmented through neutralization of IL10 for NK cells and TNFα for macrophages. Finally, we showed that TDB treatment enhanced the efficacy of Fc-driven killing to an orthogonal solid tumor target in vivo. These results provide rationale for novel antibody therapy combinations that take advantage of both adaptive and innate immune responses.

A CD3 humanized mouse model unmasked unique features of T-cell responses to bispecific antibody treatment.

ABSTRACT: T-cell bispecific antibodies (T-BsAbs) such as blinatumomab hold great promise for cancer immunotherapy. A better understanding of the in vivo immune response induced by T-BsAbs is crucial to improving their efficacy and safety profile. However, such efforts are hindered by the limitations of current preclinical models. To address this, we developed a syngeneic murine model with humanized CD3 and target antigen (CD20). This model enables the development of disseminated leukemia with a high tumor burden, which mirrors clinical findings in human patients with relapsed/refractory acute lymphoblastic leukemia. Treatment of this model with T-BsAbs results in cytokine release syndrome, with cytokine profiles and levels reflecting observations made in human patients. This model also faithfully recapitulates the dynamics of T-cell activation seen in human patients, including the temporary disappearance of T cells from the bloodstream. During this phase, T cells are sequestered in secondary lymphoid organs and undergo activation. Clinical correlative studies that rely primarily on peripheral blood samples are likely to overlook this critical activation stage, leading to a substantial underestimation of the extent of T-cell activation. Furthermore, we demonstrate that surface expression of the T-BsAb target antigen by leukemia cells triggers a swift immune response, promoting their own rejection. Humanizing the target antigen in the recipient mice is crucial to facilitate tolerance induction and successful establishment of high tumor burden. Our findings underscore the importance of meticulously optimized syngeneic murine models for investigating T-BsAb-induced immune responses and for translational research aimed at improving efficacy and safety.

Regulatory T cells hamper the efficacy of T-cell-engaging bispecific antibody therapy.

T-cell-engaging bispecific antibodies (T-BsAb) have produced impressive clinical responses in patients with relapsed/refractory B-cell malignancies, although treatment failure remains a major clinical challenge. Growing evidence suggests that a complex interplay between immune cells and tumor cells is implicated in the mechanism of action and therefore, understanding immune regulatory mechanisms might provide a clue for how to improve the efficacy of T-BsAb therapy. Here, we investigated the functional impact of regulatory T (Treg) cells on anti-tumor immunity elicited by T-BsAb therapy. In a preclinical model of myeloma, the activation and expansion of Treg cells in the bone marrow were observed in response to anti-B-cell maturation antigen (BCMA) T-BsAb therapy. T-BsAb triggered the generation of induced Treg cells from human conventional CD4 cells after co-culture with tumor cells. Moreover, T-BsAb directly activated freshly isolated circulating Treg cells, leading to the production of interleukin-10 and inhibition of T-BsAb-mediated CD8 T-cell responses. The activation of Treg cells was also seen in bone marrow samples from myeloma patients after ex vivo treatment with T-BsAb, further supporting that T-BsAb have an impact on Treg homeostasis. Importantly, transient ablation of Treg cells in combination with T-BsAb therapy dramatically improved effector lymphocyte activities and disease control in the preclinical myeloma model, leading to prolonged survival. Together, this information suggests that therapy-induced activation of Treg cells critically regulates anti-tumor immunity elicited by T-BsAb therapy, with important implications for improving the efficacy of such treatment.