Immunocytokines with target cell-restricted IL-15 activity for treatment of B cell malignancies.

Despite the advances in cancer treatment achieved, for example, by the CD20 antibody rituximab, an urgent medical need remains to optimize the capacity of such antibodies to induce antibody-dependent cellular cytotoxicity (ADCC) that determines therapeutic efficacy. The cytokine IL-15 stimulates proliferation, activation, and cytolytic capacity of NK cells, but broad clinical use is prevented by short half-life, poor accumulation at the tumor site, and severe toxicity due to unspecific immune activation. We here report modified immunocytokines consisting of Fc-optimized CD19 and CD20 antibodies fused to an IL-15 moiety comprising an L45E-E46K double mutation (MIC+ format). The E46K mutation abrogated binding to IL-15Rα, thereby enabling substitution of physiological trans-presentation by target binding and thus conditional IL-15Rßγ stimulation, whereas the L45E mutation optimized IL-15Rßγ agonism and producibility. In vitro analysis of NK activation, anti-leukemia reactivity, and toxicity using autologous and allogeneic B cells confirmed target-dependent function of MIC+ constructs. Compared with Fc-optimized CD19 and CD20 antibodies, MIC+ constructs mediated superior target cell killing and NK cell proliferation. Mouse models using luciferase-expressing human NALM-6 lymphoma cells, patient acute lymphoblastic leukemia (ALL) cells, and murine EL-4 lymphoma cells transduced with human CD19/CD20 as targets and human and murine NK cells as effectors, respectively, confirmed superior and target-dependent anti-leukemic activity. In summary, MIC+ constructs combine the benefits of Fc-optimized antibodies and IL-15 cytokine activity and mediate superior NK cell immunity with potentially reduced side effects. They thus constitute a promising new immunotherapeutic approach shown here for B cell malignancies.

An Fc-modified monoclonal antibody as novel treatment option for pancreatic cancer.

Pancreatic cancer is a highly lethal disease with limited treatment options. Hence, there is a considerable medical need for novel treatment strategies. Monoclonal antibodies (mAbs) have significantly improved cancer therapy, primarily due to their ability to stimulate antibody-dependent cellular cytotoxicity (ADCC), which plays a crucial role in their therapeutic efficacy. As a result, significant effort has been focused on improving this critical function by engineering mAbs with Fc regions that have increased affinity for the Fc receptor CD16 expressed on natural killer (NK) cells, the major cell population that mediates ADCC in humans. Here we report on the preclinical characterization of a mAb directed to the target antigen B7-H3 (CD276) containing an Fc part with the amino acid substitutions S239D/I332E to increase affinity for CD16 (B7-H3-SDIE) for the treatment of pancreatic cancer. B7-H3 (CD276) is highly expressed in many tumor entities, whereas expression on healthy tissues is more limited. Our findings confirm high expression of B7-H3 on pancreatic cancer cells. Furthermore, our study shows that B7-H3-SDIE effectively activates NK cells against pancreatic cancer cells in an antigen-dependent manner, as demonstrated by the analysis of NK cell activation, degranulation and cytokine release. The activation of NK cells resulted in significant tumor cell lysis in both short-term and long-term cytotoxicity assays. In conclusion, B7-H3-SDIE constitutes a promising agent for the treatment of pancreatic cancer.

Protocol of a first-in-human clinical trial to evaluate the safety, tolerability, and preliminary efficacy of the bispecific CD276xCD3 antibody CC-3 in patients with colorectal cancer (CoRe_CC-3).

Introduction: Colorectal cancer (CRC) is the third most common cancer worldwide in men and women. In the metastasized stage, treatment options and prognosis are limited. To address the high medical need of this patient population, we generated a CD276xCD3 bispecific antibody termed CC-3. CD276 is expressed on CRC cells and on tumor vessels, thereby allowing for a "dual" anticancer effect. Methods and analysis: This first-in-human clinical study is planned as a prospective multicenter trial, enrolling patients with metastatic CRC after three lines of therapy. During the dose-escalation part, initially, an accelerated titration design with single-patient cohorts is employed. Here, each patient will receive a fixed dose level (starting with 50 µg for the first patient); however, between patients, dose level may be increased by up to 100%, depending on the decision of a safety review committee. Upon occurrence of any adverse events (AEs) grade ≥2, dose-limiting toxicity (DLT), or reaching a dose level of ≥800 µg, the escalation will switch to a standard 3 + 3 dose design. After maximum tolerated dose (MTD) has been determined, defined as no more than one of the six patients experiencing DLT, an additional 14 patients receive CC-3 at the MTD level in the dose-expansion phase. Primary endpoints are incidence and severity of AEs, as well as the best objective response to the treatment according to response evaluation criteria in solid tumors (RECIST) 1.1. Secondary endpoints include overall safety, efficacy, survival, quality of life, and pharmacokinetic investigations. Ethics and dissemination: The CD276xCD3 study was approved by the Ethics Committee of the Medical Faculty of the Heinrich Heine University Düsseldorf and the Paul-Ehrlich-Institut (P00702). Clinical trial results will be published in peer-reviewed journals. Trial registration numbers: ClinicalTrials.cov Registry (NCT05999396) and EU ClinicalTrials Registry (EU trial number 2022-503084-15-00).

Phase I study evaluating the Fc-optimized FLT3 antibody FLYSYN in AML patients with measurable residual disease.

BACKGROUND: About half of AML patients achieving complete remission (CR) display measurable residual disease (MRD) and eventually relapse. FLYSYN is an Fc-optimized antibody for eradication of MRD directed to FLT3/CD135, which is abundantly expressed on AML cells. METHODS: This first-in-human, open-label, single-arm, multicenter trial included AML patients in CR with persisting or increasing MRD and evaluated safety/tolerability, pharmacokinetics and preliminary efficacy of FLYSYN at different dose levels administered intravenously (cohort 1-5: single dose of 0.5 mg/m2, 1.5 mg/m2, 5 mg/m2, 15 mg/m2, 45 mg/m2; cohort 6: 15 mg/m2 on day 1, 15 and 29). Three patients were treated per cohort except for cohorts 4 and 6, which were expanded to nine and ten patients, respectively. Primary objective was safety, and secondary efficacy objective was ≥ 1 log MRD reduction or negativity in bone marrow. RESULTS: Overall, 31 patients were treated, of whom seven patients (22.6%) experienced a transient decrease in neutrophil count (two grade 3, others ≤ grade 2). No infusion-related reaction or dose-limiting toxicity was observed. Adverse events (AEs) were mostly mild to moderate, with the most frequent AEs being hematologic events and laboratory abnormalities. Response per predefined criteria was documented in 35% of patients, and two patients maintained MRD negativity until end of study. Application of 45 mg/m2 FLYSYN as single or cumulative dose achieved objective responses in 46% of patients, whereas 28% responded at lower doses. CONCLUSIONS: FLYSYN monotherapy is safe and well-tolerated in AML patients with MRD. Early efficacy data are promising and warrant further evaluation in an up-coming phase II trial. Trial registration This clinical is registered on clinicaltrials.gov (NCT02789254).

IgG-based B7-H3xCD3 bispecific antibody for treatment of pancreatic, hepatic and gastric cancer.

T cell-based immunotherapy has significantly improved treatment options for many malignancies. However, despite these and other therapeutic improvements over the last decades, gastrointestinal cancers, in particular pancreatic, hepatic and gastric cancer, are still characterized by high relapse rates and dismal prognosis, with an accordingly high unmet medical need for novel treatment strategies. We here report on the preclinical characterization of a novel bispecific antibody in an IgG-based format termed CC-3 with B7-H3xCD3 specificity. In many cancer entities including pancreatic, hepatic and gastric cancers, B7-H3 (CD276) is overexpressed on tumor cells and also on the tumor vasculature, the latter allowing for improved access of immune effector cells into the tumor site upon therapeutic targeting. We demonstrate that CC-3 induces profound T cell reactivity against various pancreatic, hepatic and gastric cancer cell lines as revealed by analysis of activation, degranulation and secretion of IL2, IFNγ as well as perforin, resulting in potent target cell lysis. Moreover, CC-3 induced efficient T cell proliferation and formation of T cell memory subsets. Together, our results emphasize the potential of CC-3, which is presently being GMP-produced to enable clinical evaluation for treatment of pancreatic, hepatic and gastric cancer.

Novel ACE2 fusion protein with adapting activity against SARS-CoV-2 variants in vitro.

Despite the successful development of vaccines and neutralizing antibodies to limit the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerging variants prolong the pandemic and emphasize the persistent need to develop effective antiviral treatment regimens. Recombinant antibodies directed to the original SARS-CoV-2 have been successfully used to treat established viral disease. However, emerging viral variants escape the recognition by those antibodies. Here we report the engineering of an optimized ACE2 fusion protein, designated ACE2-M, which comprises a human IgG1 Fc domain with abrogated Fc-receptor binding linked to a catalytically-inactive ACE2 extracellular domain that displays increased apparent affinity to the B.1 spike protein. The affinity and neutralization capacity of ACE2-M is unaffected or even enhanced by mutations present in the spike protein of viral variants. In contrast, a recombinant neutralizing reference antibody, as well as antibodies present in the sera of vaccinated individuals, lose activity against such variants. With its potential to resist viral immune escape ACE2-M appears to be particularly valuable in the context of pandemic preparedness towards newly emerging coronaviruses.

An optimized IgG-based B7-H3xCD3 bispecific antibody for treatment of gastrointestinal cancers.

T cell-based immunotherapy has revolutionized oncological treatment. However, many patients do not respond to treatment, and long-term remissions remain rare, particularly in gastrointestinal cancers like colorectal cancer (CRC). B7-H3 is overexpressed in multiple cancer entities including CRC on both tumor cells and tumor vasculature, the latter facilitating influx of effector cells into the tumor site upon therapeutic targeting. We generated a panel of T cell-recruiting B7-H3xCD3 bispecific antibodies (bsAbs) and show that targeting a membrane-proximal B7-H3 epitope allows for a 100-fold reduction of CD3 affinity. In vitro, our lead compound CC-3 showed superior tumor cell killing, T cell activation, proliferation, and memory formation, whereas undesired cytokine release was reduced. In vivo, CC-3 mediated potent antitumor activity in three independent models using immunocompromised mice adoptively transferred with human effector cells with regard to prevention of lung metastasis and flank tumor growth as well as elimination of large established tumors. Thus, fine-tuning of both target and CD3 affinities as well as binding epitopes allowed for the generation of a B7-H3xCD3 bsAbs with promising therapeutic activity. CC-3 is presently undergoing good manufacturing practice (GMP) production to enable evaluation in a clinical "first-in-human" study in CRC.

B7-H3-targeting Fc-optimized antibody for induction of NK cell reactivity against sarcoma.

Natural killer (NK) cells largely contribute to antibody-dependent cellular cytotoxicity (ADCC), a central factor for success of monoclonal antibodies (mAbs) treatment of cancer. The B7 family member B7-H3 (CD276) recently receives intense interest as a novel promising target antigen for immunotherapy. B7-H3 is highly expressed in many tumor entities, whereas expression on healthy tissues is rather limited. We here studied expression of B7-H3 in sarcoma, and found substantial levels to be expressed in various bone and soft-tissue sarcoma subtypes. To date, only few immunotherapeutic options for treatment of sarcomas that are limited to a minority of patients are available. We here used a B7-H3 mAb to generate chimeric mAbs containing either a wildtype Fc-part (8H8_WT) or a variant Fc part with amino-acid substitutions (S239D/I332E) to increase affinity for CD16 expressing NK cells (8H8_SDIE). In comparative studies we found that 8H8_SDIE triggers profound NK cell functions such as activation, degranulation, secretion of IFNγ and release of NK effector molecules, resulting in potent lysis of different sarcoma cells and primary sarcoma cells derived from patients. Our findings emphasize the potential of 8H8_SDIE as novel compound for treatment of sarcomas, particularly since B7-H3 is expressed in bone and soft-tissue sarcoma independent of their subtype.

IgG-Based Bispecific Anti-CD95 Antibodies for the Treatment of B Cell-Derived Malignancies and Autoimmune Diseases.

Antibodies against the B cell-specific antigens CD20 and CD19 have markedly improved the treatment of B cell-derived lymphoma and autoimmune diseases by depleting malignant and autoreactive B cells. However, since CD20 and CD19 are also expressed on healthy B cells, such antibodies lack disease specificity. Here, we optimize a previously developed concept that uses bispecific antibodies to induce apoptosis selectively in malignant and autoreactive B cells that express the death receptor CD95. We describe the development and characterization of bispecific antibodies with CD95xCD20 and CD95xCD19 specificity in a new IgG-based format. We could show that especially the CD95xCD20 antibody mediated a strong induction of apoptosis in malignant B cells in vitro. In vivo, the antibody was clearly superior to the previously used Fabsc format with identical specificities. In addition, both IgGsc antibodies depleted activated B cells in vitro, leading to a significant reduction in antibody production and cytokine secretion. The killing of resting B cells and hepatocytes that lack CD95 and CD20/CD19, respectively, was marginal. Thus, our results imply that bispecific anti-CD95 antibodies in the IgGsc format are an attractive tool for a more selective and efficient depletion of malignant as well as autoreactive B cells.

BCR::ABL1 tyrosine kinase inhibitors hamper the therapeutic efficacy of blinatumomab in vitro.

PURPOSE: Acute B-lymphoblastic leukemia (B-ALL) is a malignant disease characterized by accumulation of clonal immature lymphocytes in the bone marrow and peripheral blood. The approval of BCR::ABL1 tyrosine kinase inhibitors (TKI) such as imatinib, dasatinib, nilotinib and ponatinib marked a milestone in targeted therapy only for a subset of patients carrying the translocation t(9;22)(q34;q11). Immunotherapy with the bispecific antibody (bsAb) blinatumomab targeting CD19xCD3 revolutionized treatment of all B-ALL cases. The combination of both TKI and bsAb, so-called "dual targeting", is currently under clinical investigation, although TKI might influence T cell effects. METHODS: We here investigated the combination of different TKI and blinatumomab in BCR::ABL1+ and BCR::ABL1- B-ALL cell lines and primary samples regarding T cell proliferation, differentiation, cytokine release and killing of tumor cells. RESULTS: In vitro analysis revealed profound reduction of T cell proliferation, differentiation, cytokine release and killing of tumor cells upon application of BCR::ABL1 TKI with blinatumomab. Inhibition was more pronounced with dasatinib and ponatinib compared to nilotinib and imatinib. T cell signalling after CD3 stimulation was impaired by TKI mirrored by inhibition of LCK phosphorylation. This known off-target effect might influence the efficacy of bsAb therapy when combined with BCR::ABL1 TKI. CONCLUSION: In conclusion, we propose that nilotinib and imatinib might also be suitable substances for combination with blinatumomab and suggest evaluation in clinical trials.

A novel IgG-based FLT3xCD3 bispecific antibody for the treatment of AML and B-ALL.

BACKGROUND: In lymphoid malignancies, the introduction of chimeric antigen receptor T (CAR-T) cells and bispecific antibodies (bsAbs) has achieved remarkable clinical success. However, such immunotherapeutic strategies are not yet established for acute myeloid leukemia (AML), the most common form of acute leukemia in adults. Common targets in AML such as CD33, CD123, and CLEC12A are highly expressed on both AML blasts and on normal myeloid cells and hematopoietic stem cells (HSCs), thereby raising toxicity concerns. In B-cell acute lymphoblastic leukemia (B-ALL), bsAbs and CAR-T therapy targeting CD19 and CD22 have demonstrated clinical success, but resistance via antigen loss is common, motivating the development of agents focused on alternative targets. An attractive emerging target is FLT3, a proto-oncogene expressed in both AML and B-ALL, with low and limited expression on myeloid dendritic cells and HSCs. METHODS: We developed and characterized CLN-049, a T cell-activating bsAb targeting CD3 and FLT3, constructed as an IgG heavy chain/scFv fusion. CLN-049 binds the membrane proximal extracellular domain of the FLT3 protein tyrosine kinase, which facilitates the targeting of leukemic blasts regardless of FLT3 mutational status. CLN-049 was evaluated for preclinical safety and efficacy in vitro and in vivo. RESULTS: CLN-049 induced target-restricted activation of CD4+ and CD8+ T cells. AML cell lines expressing a broad range of surface levels of FLT3 were efficiently lysed on treatment with subnanomolar concentrations of CLN-049, whereas FLT3-expressing hematopoietic progenitor cells and dendritic cells were not sensitive to CLN-049 killing. Treatment with CLN-049 also induced lysis of AML and B-ALL patient blasts by autologous T cells at the low effector-to-target ratios typically observed in patients with overt disease. Lysis of leukemic cells was not affected by supraphysiological levels of soluble FLT3 or FLT3 ligand. In mouse xenograft models, CLN-049 was highly active against human leukemic cell lines and patient-derived AML and B-ALL blasts. CONCLUSIONS: CLN-049 has a favorable efficacy and safety profile in preclinical models, warranting evaluation of its antileukemic activity in the clinic.

Platelets subvert antitumor efficacy of T cell-recruiting bispecific antibodies.

T cell-based immunotherapy, for example, with T cell-recruiting bispecific antibody (bsAb), has revolutionized oncological treatment. However, many patients do not respond to treatment, and long-term remissions are still rare. Several tumor immune evasion mechanisms have been reported to counteract efficiency of T cell-engaging therapeutics. Platelets largely affect cancer pathophysiology by mediating tumor invasion, metastasis, and immune evasion. On treatment of patients in a clinical trial with a PSMA×CD3 bsAb (NCT04104607), we observed profound treatment-associated platelet activation, mirrored by a decrease of total platelet count. On modeling the treatment setting, we found that platelet activation significantly reduced bsAb-mediated CD4+ and CD8+ T-cell reactivity as revealed by impaired T-cell degranulation, secretion of perforin, and ultimately, inhibition of target cell lysis. This effect occurred in a transforming growth factor beta (TGF-ß)-dependent manner and was not restricted to PSMA×CD3 bsAb, but rather observed with various CD3-directed bispecific constructs, including the approved CD19×CD3 bsAb blinatumomab. BsAb-mediated T-cell reactivity could be restored by platelet inhibition and specifically by blocking the TGF-ß axis. Together, our findings demonstrate that platelets undermine the efficacy of T cell-recruiting bsAb and identify modulation of platelet function as a means to reinforce the effectiveness of bsAb treatment.

Identification of CD105 (endoglin) as novel risk marker in CLL.

Several genetic and clinical markers are established as prognostic factors in chronic lymphocytic leukemia (CLL). However, additional markers are needed for risk stratification. Flow cytometric analysis is a mainstay of CLL diagnostics, thus identification of novel prognostic surface markers can improve risk assessment without increasing burden for patients and physicians. Furthermore, surface molecules preferentially expressed in high-risk cases could serve as therapeutic targets for immunotherapy. CD105 (endoglin) is a TGF-beta coreceptor and activates endothelial cells in healthy tissues and cancer. In addition, it is expressed on healthy hematopoietic precursors as well as lymphoid and myeloid leukemias. In acute myeloid leukemia (AML), a CD105 antibody is successfully applied in clinical studies. In CLL, mRNA expression of the CD105 gene ENG reportedly correlates with other risk factors but failed to show significant correlation with overall survival. However, CD105 protein expression in CLL has never been studied. We here analyzed CD105 surface expression on CLL cells from 71 patients by flow cytometry and report for the first time that substantial levels of CD105 are detectable on CLL cells in 70.4% of patients. Using receiver operating characteristics, we established a cutoff of 5.99% positive cells to distinguish between low and high CD105 levels, the latter correlating with decreased time to first treatment and overall survival. High CD105 expression further correlates with CD38 expression. Our study identified membrane expression of CD105 as a potential risk marker and therapeutic target in high-risk CLL. However, multivariant analyses of large cohorts should be performed in confirmatory studies.

CD18 Antibody Application Blocks Unwanted Off-Target T Cell Activation Caused by Bispecific Antibodies.

T cell-recruiting bispecific antibodies (bsAbs) are successfully used for the treatment of cancer. However, effective treatment with bsAbs is so far hampered by severe side effects, i.e., potentially life-threatening cytokine release syndrome. Off-target T cell activation due to binding of bispecific CD3 antibodies to T cells in the absence of target cells may contribute to excessive cytokine release. We report here, in an in vitro setting, that off-target T cell activation is induced by bsAbs with high CD3 binding affinity and increased by endothelial- or lymphoid cells that act as stimulating bystander cells. Blocking antibodies directed against the adhesion molecules CD18/CD54 or CD2/CD58 markedly reduced this type of off-target T cell activation. CD18 blockade-in contrast to CD2-did not affect the therapeutic activity of various bsAbs. Since CD18 antibodies have been shown to be safely applicable in patients, blockade of this integrin holds promise as a potential target for the prevention of unwanted off-target T cell activation and allows the application of truly effective bsAb doses.

Bispecific NKG2D-CD3 and NKG2D-CD16 Fusion Proteins as Novel Treatment Option in Advanced Soft Tissue Sarcomas.

Soft tissue sarcoma (STS) constitutes a rare group of heterogeneous malignancies. Effective treatment options for most subtypes of STS are still limited. As a result, especially in metastatic disease, prognosis is still dismal. The ligands for the activating immunoreceptor NKG2D (NKG2DL) are commonly expressed in STS, but generally absent in healthy tissues. This provides the rationale for utilization of NKG2DL as targets for immunotherapeutic approaches. We here report on the preclinical characterization of bispecific fusion proteins (BFP) consisting of the extracellular domain of the NKG2D receptor fused to Fab-fragments directed against CD3 (NKG2D-CD3) or CD16 (NKG2D-CD16) for treatment of STS. After characterization of NKG2DL expression patterns on various STS cell lines, we demonstrated that both NKG2D-CD16 and NKG2D-CD3 induce profound T and NK cell reactivity as revealed by analysis of activation, degranulation and secretion of IFNγ as well as granule associated proteins, resulting in potent target cell lysis. In addition, the stimulatory capacity of the constructs to induce T and NK cell activation was analyzed in heavily pretreated STS patients and found to be comparable to healthy donors. Our results emphasize the potential of NKG2D-CD3 and NKG2D-CD16 BFP to target STS even in an advanced disease.

Mass spectrometry for quality control of bispecific antibodies after SDS-PAGE in-gel digestion.

Recombinant bispecific antibodies (bsAbs) are increasingly included in regimens for cancer therapy. Strict good manufacturing practice (GMP) compliant quality control measures are required to ensure quality and safety of these innovative biologicals. Gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) and size exclusion chromatography (SEC) are the cornerstones of quality control methods. BsAbs are often prone to aggregation or incomplete synthesis due to their artificial nature. In addition, host cell proteins and host cell DNA as well as impurities from the purification process itself constitute potential contaminants. Such impurities may then appear as additional, unexpected bands or peaks on SDS-PAGE gels and SEC, respectively. Here we describe a standardized protocol for rapid analysis of recombinant antibodies by mass spectrometry (MS) after tryptic digestion of bands excised from SDS-PAGE gels. We have used this protocol to characterize unexpected "contaminating bands" that were observed during the clinical development of a novel bsAb with PSMAxCD3 specificity, either during the production of the protein itself or during the development of a surrogate molecule for evaluation in syngeneic mouse models. MS analysis allowed us to precisely determine the origin of these bands, which resulted from artifacts or from incomplete protein synthesis. The combined utilization of SDS-PAGE und MS can therefore substantially support GMP-compliant production of recombinant proteins.

An Fc-Optimized CD133 Antibody for Induction of NK Cell Reactivity against B Cell Acute Lymphoblastic Leukemia.

In recent decades, antibody-dependent cellular cytotoxicity (ADCC)-inducing monoclonal antibodies (mAbs) have revolutionized cancer immunotherapy, and Fc engineering strategies have been utilized to further improve efficacy. A promising option is to enhance the affinity of an antibody's Fc-part to the Fc-receptor CD16 by altering the amino acid sequence. Herein, we characterized an S239D/I332E-modified CD133 mAb termed 293C3-SDIE for treatment of B cell acute lymphoblastic leukemia (B-ALL). Flow cytometric analysis revealed CD133 expression on B-ALL cell lines and leukemic cells of 50% (14 of 28) B-ALL patients. 293C3-SDIE potently induced NK cell reactivity against the B-ALL cell lines SEM and RS4;11, as well as leukemic cells of B-ALL patients in a target antigen-dependent manner, as revealed by analysis of NK cell activation, degranulation, and cytotoxicity. Of note, CD133 expression did not correlate with BCR-ABL, CD19, CD20, or CD22, which are presently used as therapeutic targets in B-ALL, which revealed CD133 as an independent target for B-ALL treatment. Increased CD133 expression was also observed in MLL-AF4-rearranged B-ALL, indicating that 293C3-SDIE may constitute a particularly suitable treatment option in this hard-to-treat subpopulation. Taken together, our results identify 293C3-SDIE as a promising therapeutic agent for the treatment of B-ALL.

Bispecific Antibodies in Prostate Cancer Therapy: Current Status and Perspectives.

Prostate carcinoma (PC) is the second most common cancer in men. When the disease becomes unresponsive to androgen deprivation therapy, the remaining treatment options are of limited benefit. Despite intense efforts, none of the T cell-based immunotherapeutic strategies that meanwhile have become a cornerstone for treatment of other malignancies is established in PC. This refers to immune checkpoint inhibition (CI), which generally reinforces T cell immunity as well as chimeric antigen receptor T (CAR-T) cells and bispecific antibodies (bsAbs) that stimulate the T cell receptor/CD3-complex and mobilize T cells in a targeted manner. In general, compared to CAR-T cells, bsAb would have the advantage of being an "off the shelf" reagent associated with less preparative effort, but at present, despite enormous efforts, neither CAR-T cells nor bsAbs are successful in solid tumors. Here, we focus on the various bispecific constructs that are presently in development for treatment of PC, and discuss underlying concepts and the state of clinical evaluation as well as future perspectives.

An IgG-based bispecific antibody for improved dual targeting in PSMA-positive cancer.

The prostate-specific membrane antigen (PSMA) has been demonstrated in numerous studies to be expressed specifically on prostate carcinoma cells and on the neovasculature of several other cancer entities. However, the simultaneous expression of PSMA on both, tumor cells as well as tumor vessels remains unclear, even if such "dual" expression would constitute an important asset to facilitate sufficient influx of effector cells to a given tumor site. We report here on the generation of a PSMA antibody, termed 10B3, which exerts superior dual reactivity on sections of prostate carcinoma and squamous cell carcinoma of the lung. 10B3 was used for the construction of T-cell recruiting bispecific PSMAxCD3 antibodies in Fab- and IgG-based formats, designated Fabsc and IgGsc, respectively. In vitro, both molecules exhibited comparable activity. In contrast, only the larger IgGsc molecule induced complete and durable elimination of established tumors in humanized mice due to favorable pharmacokinetic properties. Upon treatment of three patients with metastasized prostate carcinoma with the IgGsc reagent, marked activation of T cells and rapid reduction of elevated PSA levels were observed.

Protocol of a prospective, multicentre phase I study to evaluate the safety, tolerability and preliminary efficacy of the bispecific PSMAxCD3 antibody CC-1 in patients with castration-resistant prostate carcinoma.

INTRODUCTION: Prostate cancer is the second most common cancer in men worldwide. When the disease becomes resistant to androgen-deprivation therapy, treatment options are sparse. To address the high medical need in castration-resistant prostate cancer (CRPC), we generated a novel PSMAxCD3 bispecific antibody termed CC-1. CC-1 binds to prostate-specific membrane antigen that is expressed on prostate cancer cells and tumour vessels, thereby allowing a dual anticancer effect. METHODS AND ANALYSIS: This first in human clinical study is a prospective and multicentre trial which enrols patients with metastatic CRPC after failure of established third-line therapy. CC-1 is applied after prophylactic interleukin-6 receptor blockade with tocilizumab (once 8 mg/kg body weight). Each patient receives at least one cycle of CC-1 over a time course of 7 days in an inpatient setting. If clinical benefit is observed, up to five additional cycles of CC-1 can be applied. The study is divided in two parts: (1) a dose escalation phase with intraindividual dose increase from 28 µg to the target dose of 1156 µg based on a modified fast titration design by Simon et al to determine safety, tolerability and the maximum tolerated dose (MTD) as primary endpoints and (2) a dose expansion phase with additional 14 patients on the MTD level of part (1) to identify first signs of efficacy. Secondary endpoints compromise overall safety, tumour response, survival and a translational research programme with, among others, the analysis of CC-1 half-life, the induced immune response, as well as the molecular profiling in liquid biopsies. ETHICS AND DISSEMINATION: The PSMAxCD3 study was approved by the Ethics Committee of The University Hospital Tübingen (100/2019AMG1) and the Paul-Ehrlich-Institut (3684/02). Clinical trial results will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBERS: ClinicalTrials.gov Registry (NCT04104607) and ClinicalTrials.eu Registry (EudraCT2019-000238-20).