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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.)

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

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.

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.

Over the past 30 years the incorporation of monoclonal antibody (mAb) treatments into the management of hematologic malignancies has led to significant improvements in patient outcomes. The key limitation of mAb treatments is the necessity for target antigen presentation on major histocompatibility complex (MHC) and costimulatory molecules to elicit a cytotoxic immune response. With the advent of bispecific antibodies (BsAbs), these limitations can be overcome through direct stimulation of cytotoxic T cells, thus limiting tumor cell evasion. BsAbs are rapidly being incorporated into treatment regimens for hematologic malignancies, and there are now seven FDA-approved treatments in this class, six of which have been approved in the past year. In this review we describe the function, complications, and clinical trial data available for CD3 BsAbs in the treatment of lymphoma, myeloma, and leukemia., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Off-the-shelf immunotherapeutics that suppress tumor growth and provide durable protection against relapse could enhance cancer treatment. We report preclinical studies on a CD33 x CD3 bivalent bispecific diabody, AMV564, that not only suppresses tumor growth, but also facilitates memory responses in a mouse model of acute myelogenous leukemia (AML). Mechanistically, a single 5-day treatment with AMV564 seems to reduce tumor burden by redirection of T cells, providing a time window for allogeneic or other T cells that innately recognize tumor antigens to become activated and proliferate. When the concentration of bispecific becomes negligible, the effector: target ratio has also shifted, and these activated T cells mediate long-term tumor control. To test the efficacy of AMV564 in vivo, we generated a CD33+ MOLM13CG bioluminescent human cell line and optimized conditions needed to control these cells for 62 days in vivo in NSG mice. Of note, not only did MOLM13CG become undetectable by bioluminescence imaging in response to infusion of human T cells plus AMV564, but also NSG mice that had cleared the tumor also resisted rechallenge with MOLM13CG in spite of no additional AMV564 treatment. In these mice, we identified effector and effector memory human CD4+ and CD8+ T cells in the peripheral blood immediately prior to rechallenge that expanded significantly during the subsequent 18 days. In addition to the anti-tumor effects of AMV564 on the clearance of MOLM13CG cells in vivo, similar effects were seen when primary CD33+ human AML cells were engrafted in NSG mice even when the human T cells made up only 2% of the peripheral blood cells and AML cells made up 98%. These studies suggest that AMV564 is a novel and effective bispecific diabody for the targeting of CD33+ AML that may provide long-term survival advantages in the clinic., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Eissenberg 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.)

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.)

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: 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.)

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.)

Bispecific HIVxCD3 DART molecules that co-engage the viral envelope glycoprotein (Env) on HIV-1-infected cells and the CD3 receptor on CD3+ T cells are designed to mediate the cytolysis of HIV-1-infected, Env-expressing cells. Using a novel ex vivo system with cells from rhesus macaques (RMs) infected with a chimeric Simian-Human Immunodeficiency Virus (SHIV) CH505 and maintained on ART, we tested the ability of HIVxCD3 DART molecules to mediate elimination of in vitro -reactivated CD4+ T cells in the absence or presence of autologous CD8+ T cells. HIVxCD3 DART molecules with the anti-HIV-1 Env specificities of A32 or 7B2 (non-neutralizing antibodies) or PGT145 (broadly neutralizing antibody) were evaluated individually or combined. DART molecule-mediated antiviral activity increased significantly in the presence of autologous CD8+ T cells. In this ex vivo system, the PGT145 DART molecule was more active than the 7B2 DART molecule, which was more active than the A32 DART molecule. A triple combination of the DART molecules exceeded the activity of the individual PGT145 DART molecule. Modified quantitative virus outgrowth assays confirmed the ability of the DART molecules to redirect RM CD3+ T cells to eliminate SHIV-infected RM CD4+ T cells as demonstrated by the decreased propagation of in vitro infection by the infected cells pre-incubated with DART molecules in presence of effector CD8+ T cells. While mediating cytotoxic activity, DART molecules did not increase proinflammatory cytokine production. In summary, combination of HIVxCD3 DART molecules that have broadly-neutralizing and non-neutralizing anti-HIV-1 Env specificities can leverage the host immune system for treatment of HIV-1 infection but will require appropriate reactivation of the latent reservoir., Competing Interests: JN is employed by MacroGenics and owns MacroGenics stock. JN, BH, and GF have pending patents on some of the molecules. 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 Tuyishime, Dashti, Faircloth, Jha, Nordstrom, Haynes, Silvestri, Chahroudi, Margolis and Ferrari.)

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).)

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.)

We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.

We recently reported the development of a fully-human, CD3-binding bispecific antibody for immunotherapy of malignant glioma. To translate this therapeutic (hEGFRvIII-CD3- bi-scFv) to clinical trials and to help further the translation of other similar CD3-binding therapeutics, some of which are associated with neurologic toxicities, we performed a good laboratory practice (GLP) toxicity study to assess for potential behavioral, chemical, hematologic, and pathologic toxicities including evaluation for experimental autoimmune encephalomyelitis (EAE). To perform this study, male and female C57/BL6 mice heterozygous for the human CD3 transgene (20/sex) were allocated to one of four designated groups. All animals were administered one dose level of hEGFRvIII-CD3 bi-scFv or vehicle control. Test groups were monitored for feed consumption, changes in body weight, and behavioral disturbances including signs of EAE. Urinalysis, hematologic, and clinical chemistry analysis were also performed. Vehicle and test chemical-treated groups were humanely euthanized 48 hours or 14 days following dose administration. Complete gross necropsy of all tissues was performed, and selected tissues plus all observed gross lesions were collected and evaluated for microscopic changes. This included hematoxylin-eosin histopathological evaluation and Fe-ECR staining for myelin sheath enumeration. There were no abnormal clinical observations or signs of EAE noted during the study. There were no statistical changes in food consumption, body weight gain, or final body weight among groups exposed to hEGFRvIII-CD3 bi-scFv compared to the control groups for the 2- and 14-day timepoints. There were statistical differences in some clinical chemistry, hematologic and urinalysis endpoints, primarily in the females at the 14-day timepoint (hematocrit, calcium, phosphorous, and total protein). No pathological findings related to hEGFRvIII-CD3 bi-scFv administration were observed. A number of gross and microscopic observations were noted but all were considered to be incidental background findings. The results of this study allow for further translation of this and other important CD3 modulating bispecific antibodies., Competing Interests: P.C. Gedeon and J.H. Sampson are listed as coinventors of patents regarding the use of fully human bispecific antibodies targeting EGFRvlll which belong to Duke University. This includes granted US patent #9,676,858 entitled “Human bispecific EGFRvIII antibody and CD3 engaging molecules”, granted US patent #10,053,514 entitled “Certain improved human bispecific EGFRvIII antibody engaging molecules”, and international extensions of these patents. Duke University entered a Research Support Services Agreement with Integrated Laboratory Systems, Inc. under which Integrated Laboratory Systems, Inc. functioned as a contractor for the sole purpose of assisting with the completion of the described study. Integrated Laboratory Systems, Inc. holds no stake in the described technology or study outcome. J.H. Sampson reports receiving commercial research grants from Annias and Istari; holds ownership interest (including patents) in Annias, Neuronium, Duke University, and Istari; is a consultant/advisory board member for Bristol Myers Squibb, Medicenna, Insera Health, and Annias. No potential conflicts of interest were disclosed by the other authors. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

T-cell bispecific antibodies (TCBs) crosslink tumor and T-cells to induce tumor cell killing. While TCBs are very potent, on-target off-tumor toxicity remains a challenge when selecting targets. Here, we describe a protease-activated anti-folate receptor 1 TCB (Prot-FOLR1-TCB) equipped with an anti-idiotypic anti-CD3 mask connected to the anti-CD3 Fab through a tumor protease-cleavable linker. The potency of this Prot- FOLR1-TCB is recovered following protease-cleavage of the linker releasing the anti-idiotypic anti-CD3 scFv. In vivo, the Prot-FOLR1-TCB mediates antitumor efficacy comparable to the parental FOLR1-TCB whereas a noncleavable control Prot-FOLR1-TCB is inactive. In contrast, killing of bronchial epithelial and renal cortical cells with low FOLR1 expression is prevented compared to the parental FOLR1-TCB. The findings are confirmed for mesothelin as alternative tumor antigen. Thus, masking the anti-CD3 Fab fragment with an anti-idiotypic mask and cleavage of the mask by tumor-specific proteases can be applied to enhance specificity and safety of TCBs.

Purpose: Multiple myeloma (MM) patients with disease refractory to all available drugs have a poor outcome, indicating the need for new agents with novel mechanisms of action., Experimental Design: We evaluated the anti-MM activity of the fully human BCMA×CD3 bispecific antibody JNJ-7957 in cell lines and bone marrow (BM) samples. The impact of several tumor- and host-related factors on sensitivity to JNJ-7957 therapy was also evaluated., Results: We show that JNJ-7957 has potent activity against 4 MM cell lines, against tumor cells in 48 of 49 BM samples obtained from MM patients, and in 5 of 6 BM samples obtained from primary plasma cell leukemia patients. JNJ-7957 activity was significantly enhanced in patients with prior daratumumab treatment, which was partially due to enhanced killing capacity of daratumumab-exposed effector cells. BCMA expression did not affect activity of JNJ-7957. High T-cell frequencies and high effector:target ratios were associated with improved JNJ-7957-mediated lysis of MM cells. The PD-1/PD-L1 axis had a modest negative impact on JNJ-7957 activity against tumor cells from daratumumab-naïve MM patients. Soluble BCMA impaired the ability of JNJ-7957 to kill MM cells, although higher concentrations were able to overcome this negative effect., Conclusions: JNJ-7957 effectively kills MM cells ex vivo , including those from heavily pretreated MM patients, whereby several components of the immunosuppressive BM microenvironment had only modest effects on its killing capacity. Our findings support the ongoing trial with JNJ-7957 as single agent and provide the preclinical rationale for evaluating JNJ-7957 in combination with daratumumab in MM., (©2020 American Association for Cancer Research.)

Patients with hematologic cancers have improved outcomes after treatment with bispecific antibodies that bind to CD3 on T cells and that redirect T cells toward cancer cells. However, clinical benefit against solid tumors remains to be shown. We made a bispecific antibody that targets both the common prostate tumor-specific antigen PSMA and CD3 (PMSAxCD3) and provide evidence for tumor inhibition in several preclinical solid tumor models. Mice expressing the human extracellular regions of CD3 and PSMA were generated to examine antitumor efficacy in the presence of an intact immune system and PSMA expression in normal tissues. PSMAxCD3 accumulated in PSMA-expressing tissues and tumors as detected by immuno-PET imaging. Although PSMAxCD3 induced T-cell activation and showed antitumor efficacy in mice with low tumor burden, PSMAxCD3 lost efficacy against larger solid tumors, mirroring the difficulty of treating solid tumors in the clinic. Costimulatory receptors can enhance T-cell responses. We show here that costimulation can enhance the antitumor efficacy of PSMAxCD3. In particular, 4-1BB stimulation in combination with PSMAxCD3 enhanced T-cell activation and proliferation, boosted efficacy against larger tumors, and induced T-cell memory, leading to durable antitumor responses. The combination of CD3 bispecific antibodies and anti-4-1BB costimulation represents a therapeutic approach for the treatment of solid tumors., (©2020 American Association for Cancer Research.)

Designing non-natural antibody formats is a practical method for developing highly functional next-generation antibody drugs, particularly for improving the therapeutic efficacy of cancer treatments. One approach is constructing bispecific antibodies (bsAbs). We previously reported a functional humanized bispecific diabody (bsDb) that targeted epidermal growth factor receptor and CD3 (hEx3-Db). We enhanced its cytotoxicity by constructing an Fc fusion protein and rearranging order of the V domain. In this study, we created an additional functional bsAb, by integrating the molecular formats of bsAb and high-affinity mutants previously isolated by phage display in the form of Fv. Introducing the high-affinity mutations into bsDbs successfully increased their affinities and enhanced their cytotoxicity in vitro and in vivo. However, there were some limitations to affinity maturation of bsDb by integrating high-affinity Fv mutants, particularly in Fc-fused bsDb with intrinsic high affinity, because of their bivalency. The tetramers fractionated from the bsDb mutant exhibited the highest in vitro growth inhibition among the small bsAbs and was comparable to the in vivo anti-tumor effects of Fc-fused bsDbs. This molecule shows cost-efficient bacterial production and high therapeutic potential.

Background: DuoBody®-CD3xCD20 (GEN3013) is a full-length human IgG1 bispecific antibody (bsAb) recognizing CD3 and CD20, generated by controlled Fab-arm exchange. Its Fc domain was silenced by introduction of mutations L234F L235E D265A., Methods: T-cell activation and T-cell-mediated cytotoxicity were measured by flow cytometry following co-culture with tumour cells. Anti-tumour activity of DuoBody-CD3xCD20 was assessed in humanized mouse models in vivo. Non-clinical safety studies were performed in cynomolgus monkeys., Findings: DuoBody-CD3xCD20 induced highly potent T-cell activation and T-cell-mediated cytotoxicity towards malignant B cells in vitro. Comparison of DuoBody-CD3xCD20 to CD3 bsAb targeting alternative B-cell antigens, or to CD3xCD20 bsAb generated using alternative CD20 Ab, emphasized its exceptional potency. In vitro comparison with other CD3xCD20 bsAb in clinical development showed that DuoBody-CD3xCD20 was significantly more potent than three other bsAb with single CD3 and CD20 binding regions and equally potent as a bsAb with a single CD3 and two CD20 binding regions. DuoBody-CD3xCD20 showed promising anti-tumour activity in vivo, also in the presence of excess levels of a CD20 Ab that competes for binding. In cynomolgus monkeys, DuoBody-CD3xCD20 demonstrated profound and long-lasting B-cell depletion from peripheral blood and lymphoid organs, which was comparable after subcutaneous and intravenous administration. Peak plasma levels of DuoBody-CD3xCD20 were lower and delayed after subcutaneous administration, which was associated with a reduction in plasma cytokine levels compared to intravenous administration, while bioavailability was comparable., Interpretation: Based on these preclinical studies, a clinical trial was initiated to assess the clinical safety of subcutaneous DuoBody-CD3xCD20 in patients with B-cell malignancies., Funding: Genmab., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

T cell activation is initiated upon binding of the T cell receptor (TCR)/CD3 complex to peptide-major histocompatibility complexes ("signal 1"); activation is enhanced by engagement of a second "costimulatory" receptor, such as the CD28 receptor on T cells binding to its cognate ligand(s) on the target cell ("signal 2"). CD3-based bispecific antibodies act by replacing conventional signal 1, linking T cells to tumor cells by binding a tumor-specific antigen (TSA) with one arm of the bispecific and bridging to TCR/CD3 with the other. Although some of these so-called TSAxCD3 bispecifics have demonstrated promising antitumor efficacy in patients with cancer, their activity remains to be optimized. Here, we introduce a class of bispecific antibodies that mimic signal 2 by bridging TSA to the costimulatory CD28 receptor on T cells. We term these TSAxCD28 bispecifics and describe two such bispecific antibodies: one specific for ovarian and the other for prostate cancer antigens. Unlike CD28 superagonists, which broadly activate T cells and resulted in profound toxicity in early clinical trials, these TSAxCD28 bispecifics show limited activity and no toxicity when used alone in genetically humanized immunocompetent mouse models or in primates. However, when combined with TSAxCD3 bispecifics, they enhance the artificial synapse between a T cell and its target cell, potentiate T cell activation, and markedly improve antitumor activity of CD3 bispecifics in a variety of xenogeneic and syngeneic tumor models. Combining this class of CD28-costimulatory bispecific antibodies with the emerging class of TSAxCD3 bispecifics may provide well-tolerated, off-the-shelf antibody therapies with robust antitumor efficacy., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Bispecific antibodies (bsAb) that bridge tumor cells and CD3-positive effector T cells are being developed against many tumor cell targets. While tumor cell factors other than target expression level appear to play a role in determining the efficacy of CD3 bsAb, the identity of such factors remains largely unknown. Using a co-culture system of primary human T cells and B lymphoma cell lines, we demonstrate a range of sensitivities to CD20xCD3 bsAb that is independent of CD20 surface expression. To identify genes that modulate tumor cell sensitivity to CD3 bsAb, we employed a genome-scale CRISPR activation screen in a CD20xCD3-sensitive human B lymphoma cell line. Among the most highly enriched sgRNAs were those targeting genes with predicted effects on cell-cell adhesion, including sialophorin (SPN). Increased expression of SPN impeded tumor cell clustering with T cells, thereby limiting CD3 bsAb-mediated tumor cell lysis. This inhibitory effect of SPN appeared to be dependent on sialylated core 2 O-glycosylation of the protein. While SPN is not endogenously expressed in the majority of B cell lymphomas, it is highly expressed in acute myeloid leukemia. CRISPR-mediated SPN knockout in AML cell lines facilitated T cell-tumor cell clustering and enhanced CD3 bsAb-mediated AML cell lysis. In sum, our data establish that the cell cross-linking mechanism of CD3 bsAb is susceptible to subversion by anti-adhesive molecules expressed on the tumor cell surface. Further evaluation of anti-adhesive pathways may provide novel biomarkers of clinical response and enable the development of effective combination regimens for this promising therapeutic class.

Although CD3 T cell redirecting antibodies have been successfully utilized for the treatment of hematological malignancies (blinatumomab), the T cell signaling pathways induced by these molecules are incompletely understood. To gain insight into the mechanism of action for T cell redirection antibodies, we created a novel murine CD3xEpCAM bispecific antibody that incorporates a silent Fc to dissect function and signaling of murine CD8 OT1 T cells upon stimulation. T cell-mediated cytotoxicity, cytokine secretion, expression of activation markers, and proliferation were directly induced in T cells treated with the novel CD3xEpCAM bispecific molecule in vitro in the presence of epithelial cell adhesion molecule (EpCAM) expressing tumor cells. Nanostring analysis showed that CD3xEpCAM induced a gene expression profile that resembled antigen-mediated activation, although the magnitude was lower than that of the antigen-induced response. In addition, this CD3xEpCAM bispecific antibody exhibited in vivo efficacy. This is the first study that investigates both in vitro and in vivo murine CD8 T cell function and signaling induced by a CD3xEpCAM antibody having a silent Fc to delineate differences between antigen-independent and antigen-specific T cell activation. These findings expand the understanding of T cell function and signaling induced by CD3 redirection bispecific antibodies and may help to develop more efficacious CD3 redirection therapeutics for cancer treatment, particularly for solid tumors.

Proper evaluation of immunogenicity during clinical development of biotherapeutics is a major challenge to bioanalytical scientists, in part due to matrix interference in anti-drug antibody (ADA) and neutralizing antibody (NAB) assays. If not addressed, matrix interference could confound the immunogenicity assessment of a given biotherapeutic in clinical development. To support clinical development of a B cell maturation antigen (BCMA)-CD3 bispecific antibody, a cell-based NAB assay was developed as part of a tiered approach to evaluating the immunogenicity of the drug. The assay endpoint (T cell activation) was chosen based on its strong association with the mechanism of action of the drug. The BCMA-CD3 bispecific antibody activates T cells through simultaneous binding of CD3 on T cells and BCMA on target cells. In this system, T cell activation was assessed through the measurement of luciferase activity in an engineered Jurkat cell line. In the presence of NAB, the degree of T cell activation measured by the amount of luciferase activity can be reduced. During method development, soluble BCMA (sBCMA) interference in the NAB assay was apparent. The binding of sBCMA to the anti-BCMA domain of the bispecific drug led to reduced T cell activation, which caused false positive results in NAB testing. To mitigate this interference, several strategies to eliminate sBCMA were investigated. Among the procedures tested, a bead-based approach proved most effective in depleting sBCMA, while maintaining robust assay performance and achieving fit-for-purpose sensitivity. Using this sample pretreatment procedure, the NAB assay tolerated sBCMA up to 2 μg/mL, or approximately four times the estimated median sBCMA concentration in serum samples from patients with active multiple myeloma., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: Genetically engineered T cells are powerful anticancer treatments but are limited by safety and specificity issues. We herein describe an MHC-unrestricted modular platform combining autologous T cells, transduced with a targetable synthetic agonistic receptor (SAR), with bispecific antibodies (BiAb) that specifically recruit and activate T cells for tumor killing., Experimental Design: BiAbs of different formats were generated by recombinant expression. T cells were retrovirally transduced with SARs. T-cell activation, proliferation, differentiation, and T-cell-induced lysis were characterized in three murine and human tumor models in vitro and in vivo ., Results: Murine T cells transduced with SAR composed of an extracellular domain EGFRvIII fused to CD28 and CD3ζ signaling domains could be specifically recruited toward murine tumor cells expressing EpCAM by anti-EGFRvIII × anti-EpCAM BiAb. BiAb induced selective antigen-dependent activation, proliferation of SAR T cells, and redirected tumor cell lysis. Selectivity was dependent on the monovalency of the antibody for EGFRvIII. We identified FAS ligand as a major mediator of killing utilized by the T cells. Similarly, human SAR T cells could be specifically redirected toward mesothelin-expressing human pancreatic cancer cells. In vivo , treatment with SAR T cells and BiAb mediated antitumoral activity in three human pancreatic cancer cell xenograft models. Importantly, SAR activity, unlike CAR activity, was reversible in vitro and in vivo ., Conclusions: We describe a novel ACT platform with antitumor activity in murine and human tumor models with a distinct mode of action that combines adoptive T-cell therapy with bispecific antibodies., (©2019 American Association for Cancer Research.)

T cell-retargeting therapies have transformed the therapeutic landscape of oncology. Regardless of the modality, T cell activating therapies are commonly accompanied by systemic cytokine release, which can progress to deadly cytokine release syndrome (CRS). Because of incomplete mechanistic understanding of the relationship between T cell activation and systemic cytokine release, optimal toxicity management that retains full therapeutic potential remains unclear. Here, we report the cell type-specific cellular mechanisms that link CD3 bispecific antibody-mediated killing to toxic cytokine release. The immunologic cascade is initiated by T cell triggering, whereas monocytes and macrophages are the primary source of systemic toxic cytokine release. We demonstrate that T cell-generated tumor necrosis factor-α (TNF-α) is the primary mechanism mediating monocyte activation and systemic cytokine release after CD3 bispecific treatment. Prevention of TNF-α release is sufficient to impair systemic release of monocyte cytokines without affecting antitumor efficacy. Systemic cytokine release is only observed upon initial exposure to CD3 bispecific antibody not subsequent doses, indicating a biological distinction between doses. Despite impaired cytokine release after second exposure, T cell cytotoxicity remained unaffected, demonstrating that cytolytic activity of T cells can be achieved in the absence of cytokine release. The mechanistic uncoupling of toxic cytokines and T cell cytolytic activity in the context of CD3 bispecifics provides a biological rationale to clinically explore preventative treatment approaches to mitigate toxicity., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Although treatment advances over recent decades have significantly improved survival of patients with multiple myeloma, there is still an unmet medical need for more effective treatments. In this study, we identified G-protein-coupled receptor family C group 5 member D (GPRC5D) expression on the surface of malignant cells involved in multiple myeloma, but except for plasma cells and B cells, not at appreciable levels on normal hematopoietic cells and bone marrow progenitors, including hematopoietic stem cells. In addition, we constructed IgG-based anti-GPRC5D/CD3 bispecific T-cell-redirecting antibodies (GPRC5D TRAB), which suppressed the tumor growth of GPRC5D-positive myeloma cells through the activation of T cells in vitro and in vivo in xenograft models. Collectively, these findings suggest that GPRC5D is an antigen specific to multiple myeloma and a potential target of TRAB therapy., (©2019 American Association for Cancer Research.)

T cell redirection mediated by bispecific antibodies (BsAbs) is a promising cancer therapy. Dual antigen binding is necessary for potent T cell redirection and is influenced by the structural characteristics of a BsAb, which are dependent on its IgG subclass. In this study, model BsAbs targeting CD19xCD3 were generated in variants of IgG1, IgG2, and IgG4 carrying Fc mutations that reduce FcγR interaction, and two chimeric IgG subclasses termed IgG1:2 and IgG4:2, in which the IgG1- or IgG4-F(ab) 2 are grafted on an IgG2 Fc. Molecules containing an IgG2 or IgG4-F(ab) 2 domain were confirmed to be the most structurally compact molecules. All BsAbs were shown to bind both of their target proteins (and corresponding cells) equally well. However, CD19xCD3 IgG2 did not bind both antigens simultaneously as measured by the absence of cellular clustering of T cells with target cells. This translated to a reduced potency of IgG2 BsAbs in T-cell redirection assays. The activity of IgG2 BsAbs was fully restored in the chimeric subclasses IgG4:2 and IgG1:2. This confirmed the major contribution of the F(ab) 2 region to the BsAb's functional activity and demonstrated that function of BsAbs can be modulated by engineering molecules combining different Fc and F(ab) 2 domains. Abbreviations: ADCC: Antibody-dependent cellular cytotoxicity; AlphaScreen TM : Amplified Luminescent Proximity Homogeneous Assay Screening; ANOVA: Analysis of variance; BiTE: bispecific T-cell engager; BSA: bovine serum albumin; BsAb: bispecific antibody; cFAE: controlled Fab-arm exchange; CDC: complement-dependent cellular cytotoxicity; CIEX: cation-exchange; CIR: chimeric immune receptor; DPBS: Dulbecco's phosphate-buffered saline; EC 50 value: effective concentration to reach half-maximum effect; EGFR: epidermal growth factor receptor; EI: expansion index (RA t=x /RA t=0 ); FACS: fluorescence-activated cell sorting; FVD: fixable viability dye; HI-HPLC: hydrophobic interaction HPLC; HI-FBS: heat-inactivated fetal bovine serum; HPLC: high-pressure liquid chromatography; IC 50 value: effective concentration to reach half-maximum inhibition; IQ: Inhibition Quotient; IS: immunological synapse; MES: 2-(N-morpholino)ethanesulfonic acid; R-PE: recombinant phycoerythrin; RA: red area in μm 2 /well; RD: receptor density; RFP: red fluorescent protein; R g : radius of gyration; RSV: respiratory syncytial virus; SAXS: small-angle x-ray scattering; scFv: single-chain variable fragment; SD: standard deviation; SPR: surface plasmon resonance; WT: wild-type.

Human CLDN18.2 is highly expressed in a significant proportion of gastric and pancreatic adenocarcinomas, while normal tissue expression is limited to the epithelium of the stomach. The restricted expression makes it a potential drug target for the treatment of gastric and pancreatic adenocarcinoma, as evidenced by efforts to target CLDN18.2 via naked antibody and CAR-T modalities. Herein we describe CLDN18.2-targeting via a CD3-bispecific and an antibody drug conjugate and the characterization of these potential therapeutic molecules in efficacy and preliminary toxicity studies. Anti-hCLDN18.2 ADC, CD3-bispecific and diabody, targeting a protein sequence conserved in rat, mouse and monkey, exhibited in vitro cytotoxicity in BxPC3/hCLDN18.2 (IC 50  = 1.52, 2.03, and 0.86 nM) and KATO-III/hCLDN18.2 (IC 50  = 1.60, 0.71, and 0.07 nM) respectively and inhibited tumor growth of pancreatic and gastric patient-derived xenograft tumors. In a rat exploratory toxicity study, the ADC was tolerated up to 10 mg/kg. In a preliminary assessment of tolerability, the anti-CLDN18.2 diabody (0.34 mg/kg) did not produce obvious signs of toxicity in the stomach of NSG mice 4 weeks after dosing. Taken together, our data indicate that targeting CLDN18.2 with an ADC or bispecific modality could be a valid therapeutic approach for the treatment of gastric and pancreatic cancer.

Bispecific T cell engager (BiTE) antibody constructs are successfully used as cancer therapeutics. We hypothesized that this treatment strategy could also be applicable for therapy of human cytomegalovirus (HCMV) infection, since HCMV-encoded proteins are abundantly expressed on the surface of infected cells. Here we show that a BiTE antibody construct directed against HCMV glycoprotein B (gB) and CD3 efficiently triggers T cells to secrete IFN-γ and TNF upon co-culture with fibroblasts infected with HCMV strain AD169, Towne or Toledo. Titration of gB expression levels in non-infected cells confirmed that already low levels of gB are sufficient for efficient triggering of T cells in presence of the BiTE antibody construct. Comparison of redirecting T cells with the bispecific antibody versus a chimeric antigen receptor (CAR) based on the same scFv showed a similar sensitivity for gB expression. Although lysis of infected target cells was absent, the BiTE antibody construct inhibited HCMV replication by triggering cytokine production. Notably, even strongly diluted supernatants of the activated T cells efficiently blocked the replication of HCMV in infected primary fibroblasts. In summary, our data prove the functionality of the first BiTE antibody construct targeting an HCMV-encoded glycoprotein for inhibiting HCMV replication in infected cells.

A primary barrier to the success of T cell-recruiting bispecific antibodies in the treatment of solid tumors is the lack of tumor-specific targets, resulting in on-target off-tumor adverse effects from T cell autoreactivity to target-expressing organs. To overcome this, we developed an anti-HER2/CD3 T cell-dependent bispecific (TDB) antibody that selectively targets HER2-overexpressing tumor cells with high potency, while sparing cells that express low amounts of HER2 found in normal human tissues. Selectivity is based on the avidity of two low-affinity anti-HER2 Fab arms to high target density on HER2-overexpressing cells. The increased selectivity to HER2-overexpressing cells is expected to mitigate the risk of adverse effects and increase the therapeutic index. Results included in this manuscript not only support the clinical development of anti-HER2/CD3 1Fab-immunoglobulin G TDB but also introduce a potentially widely applicable strategy for other T cell-directed therapies. The potential of this discovery has broad applications to further enable consideration of solid tumor targets that were previously limited by on-target, but off-tumor, autoimmunity., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

T cell-mediated immunotherapy is an attractive strategy for treatment in various disease areas. In this therapeutic approach, the CD3 complex is one of the key molecules to modulate T cell functions; however, in many cases, we cannot evaluate the drug candidates in animal experiments because the therapeutics, usually monoclonal antibodies specific to human CD3, cannot react to mouse endogenous Cd3. Although immunodeficient mice transfused with human hematopoietic stem or precursor cells, known as humanized mice, are available for these studies, mice humanized in this manner are not completely immune competent. In this study we have succeeded in establishing a novel mouse strain in which all the three components of the Cd3 complex - Cd3ε, Cd3δ, and Cd3γ - are replaced by their human counterparts, CD3E, CD3D, and CD3G. Basic immunological assessments have confirmed that this strain of human CD3 EDG-replaced mice are entirely immune competent, and we have also demonstrated that a bispecific antibody that simultaneously binds to human CD3 and a tumor-associated antigen (e.g. ERBB2 or GPC3) can be evaluated in human CD3 EDG-replaced mice engrafted with tumors. Our mouse model provides a novel means to evaluate the in vivo efficacy of human CD3-mediated therapy.

Adoptive transfer of genetically engineered human cells secreting bispecific T-cell engagers has shown encouraging therapeutic effects in preclinical models of cancer. However, reducing the toxicity and improving the effectiveness of this emerging immunotherapeutic strategy will be critical to its successful application. We have demonstrated that for gene-based bispecific antibody strategies, two-chain diabodies have a better safety profile than single-chain tandem scFvs (single-chain variable fragments), because their reduced tendency to form aggregates reduces the risk of inducing antigen-independent T-cell activation. Here, we demonstrate that the incorporation of a 2A self-processing peptide derived from foot-and-mouth disease virus conveying co-translational cleavage into a two-chain anti-CD3 × anti-CEA diabody gene enables near-equimolar expression of diabody chains 1 and 2, and thus increases the final amount of assembled diabody. This was found to maximize diabody-mediated T-cell activation and cytotoxicity against carcinoembryonic antigen-positive tumor cells.

Purpose: Randomized studies with gemtuzumab ozogamicin have validated CD33 as a target for antigen-specific immunotherapy of acute myelogenous leukemia (AML). Here, we investigated the potential of CD33/CD3-directed tandem diabodies (TandAbs) as novel treatment approach for AML. These tetravalent bispecific antibodies provide two binding sites for each antigen to maintain the avidity of a bivalent antibody and have a molecular weight exceeding the renal clearance threshold, thus offering a longer half-life compared to smaller antibody constructs., Experimental Design: We constructed a series of TandAbs composed of anti-CD33 and anti-CD3 variable domains of diverse binding affinities and profiled their functional properties in CD33 + human leukemia cell lines, xenograft models, and AML patient samples., Results: Our studies demonstrated that several CD33/CD3 TandAbs could induce potent, dose-dependent cytolysis of CD33 + AML cell lines. This effect was modulated by the effector-to-target cell ratio and strictly required the presence of T cells. Activation and proliferation of T cells and maximal AML cell cytolysis correlated with high avidity to both CD33 and CD3. High-avidity TandAbs were broadly active in primary specimens from patients with newly diagnosed or relapsed/refractory AML in vitro, with cytotoxic properties independent of CD33 receptor density and cytogenetic risk. Tumor growth delay and inhibition were observed in both prophylactic and established HL-60 xenograft models in immunodeficient mice., Conclusions: Our data show high efficacy of CD33/CD3 TandAbs in various preclinical models of human AML. Together, these findings support further study of CD33/CD3 TandAbs as novel immunotherapeutics for patients with AML. Clin Cancer Res; 22(23); 5829-38. ©2016 AACR., (©2016 American Association for Cancer Research.)

The aim of the present study was to construct a single-chain bispecific antibody (scBsAb) against cervical carcinoma and to investigate its biological activities. The scBsAb was constructed using a genetic cloning technique and antigen binding activities were detected by ELISA. The iodogen method was used to analyze the pharmacokinetics. The Rosette formation test was used to detect the binding ability between peripheral blood lymphocytes (PBLs) and Cs1213 cervical cancer cells. In addition, the MTT method was performed to detect the killing effect of PBLs. The molecular weight of the scBsAb was ~60 kDa. The antigen binding activities of scBsAbs were compared with the anti‑human cervical carcinoma antibody single‑chain Fv fragment (CSAs‑1 scFv) and anti‑cluster of differentiation (CD)3 scFv (P>0.05). In addition, a pharmacokinetics assay demonstrated that compared with the two corresponding scFvs, scBsAbs exhibited a significantly prolonged retention time in the body (P<0.01). In addition, the number of rosettes formed by PBLs and Cs1213 cells in the scBsAb group was markedly greater than that in the scFv groups or the RPMI‑1640 group (P<0.05 and P<0.01, respectively). The killing activity of PBLs against scBsAb‑mediated Cs1213 cells was significantly greater than that mediated by the other antibodies (P<0.05). When the concentration of scBsAb was 40 µg/ml, the killing rate was 64.5%. Thus, anti‑human cervical carcinoma/anti‑CD3 scBsAbs may possess two types of antigen binding activity, prolong the duration in vivo and improve the killing activity of PBLs against cancer cells.

Syndecan-1 (CD138), a heparan sulfate proteoglycan, acts as a co-receptor for growth factors and chemokines and is a molecular marker associated with the epithelial-mesenchymal transition during development and carcinogenesis. In this study, we generated two specific mouse anti-human CD138 monoclonal antibodies (mAbs, clone ID: 480CT5.4.3, 587CT7.3.6.5) using hybridoma technology and identified their immunological characteristics. After hybridoma sequencing, the single-chain variable fragments (ScFvs) cloned from two hybridoma cells were combined with anti-CD3 OKT-3 ScFv to generate two recombinant bispecific antibodies (h-STL002, m-STL002) against CD138 and CD3 molecules, respectively. The bispecific antibodies were able to specifically target CD138 + multiple myeloma (MM) cells and CD3 + T cells, and showed the potent cytotoxicity against MM RPMI-8226 cell line through T cell activation. However, these bispecific antibodies without T cells did not cause toxic side effect on MM cells. Overall, the two hybridoma clones and their bispecific formats have great potential to promote diagnosis and immunotherapy of plasma cell malignancy.

T-cell-directed killing of tumor cells using bispecific antibodies is a promising approach for the treatment of hematologic malignancies. Here we describe our preclinical work with a dual-affinity retargeting (DART) molecule generated from antibodies to CD3 and CD123, designed to redirect T cells against acute myeloid leukemia blasts. The CD3×CD123 DART (also referred to as MGD006/S80880) consists of 2 independent polypeptides, each composed of the VH of 1 antibody in tandem with the VL of the other antibody. The target antigen CD123 (interleukin 3RA) is highly and differentially expressed in acute myeloid leukemia (AML) blasts compared with normal hematopoietic stem and progenitor cells. In this study we demonstrate that the CD3×CD123 DART binds to both human CD3 and CD123 to mediate target-effector cell association, T-cell activation, proliferation, and receptor diversification. The CD3×CD123 DART also induces a dose-dependent killing of AML cell lines and primary AML blasts in vitro and in vivo. These results provide the basis for testing the CD3×CD123 DART in the treatment of patients with CD123(+) AML., (© 2016 by The American Society of Hematology.)

Ephrin receptor A10 (EphA10), a transmembrane receptor that binds to ephrin, is a newly identified breast cancer marker protein that has also been detected in HER2-negative tissue. In this study, we report creation of a novel bispecific antibody (BsAb) binding both EphA10 and CD3, thereby forming a bridge between antigens expressed on both tumor and immune cells and promoting recognition of tumor cells by immune cells and redirection of cytotoxic T cells (CTL). This BsAb (EphA10/CD3) was expressed in supernatants of BsAb gene-transfected cells as monomeric and dimeric molecules. Redirected T-cell lysis was observed when monomeric and dimeric BsAb were added to EphA10-overexpressing tumor cells in vitro. Furthermore, dimeric BsAb (EphA10/CD3) was more cytotoxic than monomeric BsAb, with efficient tumor cell lysis elicited by lower concentrations (≤10(-1) μg/mL) and a lower effector to target (E/T) cell ratio (E/T = 2.5). Dimeric BsAb (EphA10/CD3) also showed significant anti-tumor effects in human xenograft mouse models. Together, these results revealed opportunities to redirect the activity of CTL towards tumor cells that express EphA10 using the BsAb (EphA10/CD3), which could be tested in future clinical trials as a novel and potent therapeutic for breast cancer tumors.

Bispecific antibodies that engage immune cells to kill cancer cells have been actively studied in cancer immunotherapy. In this study, we present a novel bispecific format, S-Fab, fabricated by linking a single-domain anti-carcinoembryonic antigen VHH to a conventional anti-CD3 Fab. In contrast to most bispecific antibodies, the S-Fab bispecific antibody can be efficiently expressed and purified from bacteria. The purified S-Fab is stable in serum and is able to recruit T cells to drive potent cancer cell killing. In xenograft models, the S-Fab antibody suppresses tumor growth in the presence of human immune cells. Our study suggested that the bispecific S-Fab format can be applied to a wide range of immunotherapies.

Background: Bispecific T cell engager (BiTE®) are single-chain bispecific antibody constructs with dual specificity for CD3 on T cells and a surface antigen on target cells. They can elicit a polyclonal cytotoxic T cell response that is not restricted by T cell receptor (TCR) specificity, and surface expression of MHC class I/peptide antigen complexes. Using human EpCAM/CD3-bispecific BiTE® antibody construct AMG 110, we here assessed to what extent surface expression of PD-L1, cytoplasmic expression of indoleamine-2,3-deoxygenase type 1, Bcl-2 and serpin PI-9, and the presence of transforming growth factor beta (TGF-β), interleukin-10 (IL-10) and adenosine in culture medium can impact redirected lysis by AMG 110-engaged T cells., Methods: The seven factors, which are all involved in inhibiting T cell functions by cancer cells, were tested with human EpCAM-expressing Chinese hamster ovary (CHO) target cells at levels that in most cases exceeded those observed in a number of human cancer cell lines. Co-culture experiments were used to determine the impact of the evasion mechanisms on EC50 values and amplitude of redirected lysis by AMG 110, and on BiTE®-induced proliferation of previously resting human peripheral T cells., Findings: An inhibitory effect on redirected lysis by AMG 110-engaged T cells was seen upon overexpression of serpin PI-9, Bcl-2, TGF-β and PD-L1. An inhibitory effect on induction of T cell proliferation was only seen with CHO cells overexpressing IDO. In no case, a single evasion mechanism rendered target cells completely resistant to BiTE®-induced lysis, and even various combinations could not., Conclusions: Our data suggest that diverse mechanisms employed by cancer cells to fend off T cells cannot inactivate AMG 110-engaged T cells, and that inhibitory effects observed in vitro may be overcome by increased concentrations of the BiTE® antibody construct.

The use of a bispecific antibody (BsAb) is a promising and highly specific approach to cancer therapy. In the present study, a fully human recombinant single chain variable fragment BsAb against human epidermal growth factor receptor (HER)2 and cluster of differentiation (CD)3 was constructed with the aim of developing an effective treatment for breast cancer. HER2/CD3 BsAb was expressed in Chinese hamster ovary cells and purified via nickel column chromatography. Flow cytometry revealed that the HER2/CD3 BsAb was able to specifically bind to HER2 and CD3‑positive cells. HER2/CD3 BsAb was able to stimulate T-cell activation and induce the lysis of cultured SKBR‑3 and BT474 cells in the presence of unstimulated T lymphocytes. HER2/CD3 BsAb efficiently inhibited the growth of breast cancer tissue by activating and inducing the proliferation of tumor tissue infiltrating lymphocytes. Therefore, HER2/CD3 BsAb is a potent tool which may be a suitable candidate for the treatment of breast cancer.

Bispecific T cell-engaging (BiTE) antibodies recruit polyclonal cytotoxic T cells (CTL) to tumors. One such antibody is carcinoembryonic antigen (CEA) BiTE that mediates T cell/tumor interaction by simultaneously binding CD3 expressed by T cells and CEA expressed by tumor cells. A widely operative mechanism for mitigating cytotoxic T cell-mediated killing is the interaction of tumor-expressed PD-L1 with T cell-expressed PD-1, which may be partly reversed by PD-1/PD-L1 blockade. We hypothesized that PD-1/PD-L1 blockade during BiTE-mediated T cell killing would enhance CTL function. Here, we determined the effects of PD-1 and PD-L1 blockade during initial T cell-mediated killing of CEA-expressing human tumor cell lines in vitro, as well as subsequent T cell-mediated killing by T lymphocytes that had participated in tumor cell killing. We observed a rapid upregulation of PD-1 expression and diminished cytolytic function of T cells after they had engaged in CEA BiTE-mediated killing of tumors. T cell cytolytic activity in vitro could be maximized by administration of anti-PD-1 or anti-PD-L1 antibodies alone or in combination if applied prior to a round of T cell killing, but T cell inhibition could not be fully reversed by this blockade once the T cells had killed tumor. In conclusion, our findings demonstrate that dual blockade of PD-1 and PD-L1 maximizes T cell killing of tumor directed by CEA BiTE in vitro, is more effective if applied early, and provides a rationale for clinical use.

Bispecific antibodies and antibody fragments in various formats have been explored as a means to recruit cytolytic T cells to kill tumor cells. Encouraging clinical data have been reported with molecules such as the anti-CD19/CD3 bispecific T cell engager (BiTE) blinatumomab. However, the clinical use of many reported T cell-recruiting bispecific modalities is limited by liabilities including unfavorable pharmacokinetics, potential immunogenicity, and manufacturing challenges. We describe a B cell-targeting anti-CD20/CD3 T cell-dependent bispecific antibody (CD20-TDB), which is a full-length, humanized immunoglobulin G1 molecule with near-native antibody architecture constructed using "knobs-into-holes" technology. CD20-TDB is highly active in killing CD20-expressing B cells, including primary patient leukemia and lymphoma cells both in vitro and in vivo. In cynomolgus monkeys, CD20-TDB potently depletes B cells in peripheral blood and lymphoid tissues at a single dose of 1 mg/kg while demonstrating pharmacokinetic properties similar to those of conventional monoclonal antibodies. CD20-TDB also exhibits activity in vitro and in vivo in the presence of competing CD20-targeting antibodies. These data provide rationale for the clinical testing of CD20-TDB for the treatment of CD20-expressing B cell malignancies., (Copyright © 2015, American Association for the Advancement of Science.)

Anti-disialoganglioside GD2 IgG antibodies have shown clinical efficacy in solid tumors that lack human leukocyte antigens (e.g., neuroblastoma) by relying on Fc-dependent cytotoxicity. However, there are pain side effects secondary to complement activation. T-cell retargeting bispecific antibodies (BsAb) also have clinical potential, but it is thus far only effective against liquid tumors. In this study, a fully humanized hu3F8-BsAb was developed, in which the anti-CD3 huOKT3 single-chain Fv fragment (ScFv) was linked to the carboxyl end of the anti-GD2 hu3F8 IgG1 light chain, and was aglycosylated at N297 of Fc to prevent complement activation and cytokine storm. In vitro, hu3F8-BsAb activated T cells through classic immunologic synapses, inducing GD2-specific tumor cytotoxicity at femtomolar EC50 with >10⁵-fold selectivity over normal tissues, releasing Th1 cytokines (TNFα, IFNγ, and IL2) when GD2⁺ tumors were present. In separate murine neuroblastoma and melanoma xenograft models, intravenous hu3F8-BsAb activated T cells in situ and recruited intravenous T cells for tumor ablation, significantly prolonging survival from local recurrence or from metastatic disease. Hu3F8-BsAb, but not control BsAb, drove T cells and monocytes to infiltrate tumor stroma. These monocytes were necessary for sustained T-cell proliferation and/or survival and contributed significantly to the antitumor effect. The in vitro and in vivo antitumor properties of hu3F8-BsAb and its safety profile support its further clinical development as a cancer therapeutic, and provide the rationale for exploring aglycosylated IgG-scFv as a structural platform for retargeting human T cells., (©2014 American Association for Cancer Research.)

Regardless of the important therapeutic advances developed over the last years for the management of cancer, the fact is that many patients still suffer from a tremendous reduction on their quality of life due to lack of complete selectivity of conventionally administered chemotherapeutic drugs. In the search of more efficacious tumor-targeted therapies, the use of bispecific antibodies (bsAbs) capable of simultaneous binding to tumor-associated antigens and to an activating receptor, such as CD3, has emerged as a promising approach. With the intention to complementing and improving this cancer immunotherapy, human HEK-293 cells have been genetically modified ex vivo to secrete a recombinant anti-CEA (carcinoembryonic antigen) × anti-CD3 bsAb. After encapsulation in alginate-poly-l-lysine microcapsules, bsAb-secreting HEK-293 cells were monitorized for several weeks. This system has proved to be feasible for the maintenance of cell growth and recombinant antibody production giving proof-of-concept of its use as immunotherapeutic organoids in cancer treatment.

The EPH receptor A10 (EphA10) is up-regulated in breast cancer but is not normally expressed in healthy tissue, thus it has been suggested that EphA10 may be a useful target for cancer therapy. This study reports a diabody, an antibody derivative binding two different target molecules, EphA10 expressed in tumor cells and CD3 expressed in T cells, which showed T cell dependent-cytotoxicity. The diabody, which has His-tagged and FLAG-tagged chains, was expressed in Escherichia coli and purified in both heterodimer (Db-1) and homodimer (Db-2) formulations by liquid chromatography. Flow cytometry analysis using EphA10-expressing cells showed that binding activity of heterodimers was stronger than that of homodimers. Addition of diabodies to PBMC cultures resulted in T-cell mediated redirected lysis, and the bioactivity was consistent with the stronger binding activity of heterodimeric diabody formulations. Our results indicate that diabodies recognizing both EphA10 and CD3 could have a range of potential applications in cancer therapy, such as breast cancers that express the EPH receptor A10, especially triple negative breast cancer., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Antibody-based immunotherapy has proven efficacy for patients with high-risk neuroblastoma. However, despite being the most efficient tumoricidal effectors, T cells are underutilized because they lack Fc receptors. Using a monovalent single-chain fragment (ScFv) platform, we engineered tandem scFv bispecific antibodies (BsAbs) that specifically target disialoganglioside (GD2) on tumor cells and CD3 on T cells. Structural variants of BsAbs were constructed and ranked based on binding to GD2, and on competency in inducing T-cell-mediated tumor cytotoxicity. In vitro thermal stability and binding measurements were used to characterize each of the constructs, and in silico molecular modeling was used to show how the orientation of the variable region heavy (VH) and light (VL) chains of the anti-GD2 ScFv could alter the conformations of key residues responsible for high affinity binding. We showed that the VH-VL orientation, the (GGGGS)3 linker, disulfide bond stabilization of scFv, when combined with an affinity matured mutation provided the most efficient BsAb to direct T cells to lyse GD2-positive tumor cells. In vivo, the optimized BsAb could efficiently inhibit melanoma and neuroblastoma xenograft growth. These findings provide preclinical validation of a structure-based method to assist in designing BsAb for T-cell-mediated therapy., (© 2014 UICC.)

To harness the potent tumor-killing capacity of T cells for the treatment of CD19(+) malignancies, we constructed AFM11, a humanized tetravalent bispecific CD19/CD3 tandem diabody (TandAb) consisting solely of Fv domains. The molecule exhibits good manufacturability and stability properties. AFM11 has 2 binding sites for CD3 and 2 for CD19, an antigen that is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies. Comparison of the binding and cytotoxicity of AFM11 with those of a tandem scFv bispecific T cell engager (BiTE) molecule targeting the same antigens revealed that AFM11 elicited more potent in vitro B cell lysis. Though possessing high affinity to CD3, the TandAb mediates serial-killing of CD19(+) cells with little dependence of potency or efficacy upon effector:target ratio, unlike the BiTE. The advantage of the TandAb over the BiTE was most pronounced at lower effector:target ratios. AFM11 mediated strictly target-dependent T cell activation evidenced by CD25 and CD69 induction, proliferation, and cytokine release, notwithstanding bivalent CD3 engagement. In a NOD/scid xenograft model, AFM11 induced dose-dependent growth inhibition of Raji tumors in vivo, and radiolabeled TandAb exhibited excellent localization to tumor but not to normal tissue. After intravenous administration in mice, half-life ranged from 18.4 to 22.9 h. In a human ex vivo B-cell chronic lymphocytic leukemia study, AFM11 exhibited substantial cytotoxic activity in an autologous setting. Thus, AFM11 may represent a promising therapeutic for treatment of CD19(+) malignancies with an advantageous safety risk profile and anticipated dosing regimen.

Bispecific antibodies are promising agents for immunotherapy. Here, we describe a quadroma-based trifunctional bispecific antibody binding the chemokine receptor CXCR5 and the T-cell antigen CD3 that efficiently prevents tumor growth in a mouse B-cell lymphoma model. CXCR5 regulates the tissue homeostasis of mature B cells and is highly expressed on B-cell non-Hodgkin and lymphocyte-predominant Hodgkin lymphoma, as well as on a subset of CD4(+) T cells known as follicular T-helper cells. In vitro, the bispecific CXCR5::CD3 antibody efficiently recruited effector T cells to CXCR5 expressing B cells and induced a co-stimulation-independent activation of CD8(+) and CD4(+) T cells as demonstrated by the de novo expression of CD25 and CD69, and secretion of the cytokines IFN-γ, TNF-α, IL-6 and IL-10 by peripheral blood mononuclear cells. Notably, at low antibody concentrations, CXCR5::CD3 displayed a significantly higher cytotoxic activity against autologous B cells than its parental antibodies or rituximab. In vivo imaging revealed that CXCR5::CD3 and its parental CXCR5 antibody efficiently prevent tumor growth in a xenograft model of B-cell lymphoma in mice and prolong their survival. Taken together, our results identify CXCR5 as a promising target for antibody-based therapies in the treatment of B-cell malignancies., (© 2014 UICC.)