Blood was drawn from healthy donors . Peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation with Biocoll cell separation solution (Biochrom, Berlin, Germany). The prostate carcinoma cell line LNCaP and the acute lymphatic leukemia cell line Nalm-16 were purchased from the German Collection of Microorganisms and Cell Cultures (DMSZ, Braunschweig, Germany) and were routinely tested negative for mycoplasma. PBMCs and cell lines were kept in RPMI 1640 supplemented as described earlier.12 Dexamethasone (Sigma-Aldrich) was dissolved in RPMI 1640 including 10% FCS and kept at −20°C until use. Dexamethasone (Sigma-Aldrich) was dissolved in RPMI 1640 including 10% FCS and stored at −20°C.

The recombinant bsAb CC-1 (PSMAxCD3 specificity, online supplementary figure 1A) was generated at our institution in the IgGsc format based on the format published by Coloma and Morrison1 (Zekri et al, manuscript submitted December 2019). The PSMA and CD3 binding sites are comprised of the newly generated hybridoma-derived IgG2b antibody 10B3 and a single chain derived from the CD3 antibody UCHT-1, respectively. Fc receptor binding and complement fixation was attenuated by introducing mutations in the CH₂ domain (online supplementary figure S1A).

The humanized anti-IL-6 receptor antibody tocilizumab was purchased in clinical quality from Hoffmann-La Roche (Basel, Switzerland). The bispecific CD19xCD3 BiTE antibody blinatumomab (online supplementary figure S2A) was purchased from Amgen (Thousand Oaks, USA). Flourochrome-labeled antibodies against CD4, CD8, CD10, CD276 and the respective isotype control antibodies were purchased from BioLegend (San Diego, USA). CD45-AmCyan was purchased from BD Biosciences (Franklin Lakes, USA).

For flow cytometry based lysis assays, 50,000 PSMA⁺ LNCaP cells or 100,000 CD19⁺ Nalm-16 cells were incubated in 96 well plates together with 32,500–1,000,000 PBMCs, bsAb at 1 µg/mL and blocking reagents (0,1 µg/mL dexamethasone or 10 µg/mL tocilizumab). After 3 days, flow cytometric analysis was performed. LNCaP cells were defined as CD45^-CD276⁺, Nalm-16 as CD45^-CD10⁺, and T cells as CD45⁺CD4⁺ or CD45⁺CD8. Absolute cell numbers were determined by the acquisition of a defined amount of compensation particles (BD Biosciences) per sample.2 Binding of antibodies to Fc receptors was blocked with Flebogamma DIF (Grifols, Barcelona, Spain) at 50 µg/mL. Data acquisition was performed using a FACSCanto II (BD Biosciences). For data analysis, FlowJo V.10 software (Tree Star, Ashland, OR) was used.

PBMCs from healthy donors (100,000 cells/well) and irradiated (100 Gy) target cells (E:T ratio 1:1) were seeded in triplicates in 96 well plates and incubated with bsAb (1 µg/mL). After 48 hours, cells were pulsed with ³H-methyl thymidine (0.5 µCi/well) and incubated for another 20 hours until they were harvested on filter mats. Incorporated radioactivity was determined using a liquid scintillation counter (MicroBeta2 2450 Microplate counter, PerkinElmer, Waltham, USA).

To observe real-time killing of PSMA +tumor cells, xCELLigence assays were performed. Adherent LNCaP cells (30,000 cells/well) were seeded in a 96 well E-plate and incubated for 20 hours. After constitution of adequate cell indices (>1.5), indices were normalized to 1.0. Then, PBMCs (100,000 cells/well) and a bispecific PSMAxCD3 antibody at 1 µg/mL together with the respective blocking agents were added and cell indices were measured every 15 min to determine viability of the tumor cells.

Animal experiments were performed in accordance with the German Animal Protection Law and after authorization by the competent authority of the state of Baden-Württemberg (Regierungspräsidium Tübingen, authorization number M9/17). Six weeks old NOD.Cg-Prkdc^scid Il2rg^tm1Wjl /SzJ (NSG) mice (Charles River Laboratories, Wilmington, USA) were injected subcutaneously with 1,000,000 PSMA⁺ LNCaP tumor cells into the flank. After formation of large palpable tumors (approximately 4 mm after 7 days), mice were injected intravenously with 5 µg dexamethasone or 200 µg tocilizumab (d1). One hour later, 10,000,000 human PBMCs were applied intraperitoneally as well as 2 µg of a PSMAxCD3 antibody CC-1 (intravenous). Treatment with the same amount of PMBC in combination with CC-1 was repeated at days 8 and 15. Tumor size was measured every 2 days and mice were euthanized if signs of sickness occurred or tumor size exceeded 14 mm.

Patients with castrate-resistant metastatic prostate carcinoma were treated with the bispecific PSMAxCD3 antibody CC-1 and tocilizumab in an individualized experimental approach. CC-1 was produced in compliance with good manufacturing process standards according to section 13 (2b) of the German Medicines Act and applied using an individual dose escalation protocol. Body temperature was monitored at least every 2 hours. For monitoring purposes, IL-6, C reactive protein (CRP) and solubIe IL-2 receptor (sIL-2R) levels were assessed daily using ELISA at the central laboratory of the University Hospital Tübingen. After the first occurrence of fever ≥38.5°C, 400 mg tocilizumab was applied. Patient samples were tested for human antihuman antibodies by ELISA at the University Hospital Tübingen. To this end, (Fab)₂ fragments of CC-1 were coated as capture antibody. After blocking, serum samples of patients were added and specific binding of human antihuman antibodies to (Fab)₂ fragments was detected by addition of goat-antihuman HRP-conjugated secondary antibodies.

Data are displayed as mean±SD. For statistical analysis, Graphpad V.10 was used. If not otherwise stated, Mann-Whitney U tests were used to test for statistical significance in unpaired data sets.

Figure 1A shows T cell proliferation in cocultures of PBMC and irradiated PSMA⁺ tumor cells in the presence of the PSMAxCD3 bsAb CC-1. BsAb induced T cell activation and proliferation was markedly inhibited by increasing concentrations of dexamethasone but not by tocilizumab. Of note, a serum concentration of 1–5 µg/mL of tocilizumab is therapeutically highly effective.13 Serum concentrations of >0,1 µg/mL dexamethasone are easily reached after intravenous application of 16mg,14 the recommended dose for CRS prevention during blinatumomab treatment. We therefore chose this concentration of dexamethasone and compared it to 10 µg/mL of tocilizumab in experiments that assessed bsAb-mediated T cell proliferation and tumor cell killing at different E:T ratios and in several independent assay systems (figure 1B, online supplementary figure1C-D).

Dexamethasone markedly inhibited T cell proliferation, but tumor cell killing was not suppressed at E:T ratios ≥5 (figure 1C, online supplementary figure 2C). This is in line with the findings reported by Brandl et al and Li et al.5 6 At lower ratios, however, that certainly reflect the situation at an established tumor more closely, a marked inhibition of tumor cell killing was observed.

The extent of inhibition depended on the bsAb concentration with more pronounced inhibition at lower, non-saturating concentrations (online supplementary figure 1E). In marked contrast to dexamethasone, tocilizumab inhibited neither bsAb induced T cell proliferation nor tumor cell killing.

To rule out that the observed effects were due to the chosen target antigen or bsAb format, we also performed experiments with blinatumomab (online supplementary figure 2A). Again, a marked inhibition of T cell proliferation and killing of leukemia cells by dexamethasone but not tocilizumab was observed (online supplementary figure 2C-E).

To evaluate whether bsAb-mediated antitumor effects in vivo were affected, an established efficacy model using NSG mice with large established PSMA⁺ expressing tumors was used. Of note, mice of either group did not develop signs of CRS during the treatment. Establishment of CRS in a xenograft model relies on IL-6 production by monocytes15 and can only be achieved by massive tumor loads, for example, in the peritoneal cavity,16 17 which was not the case in our experiments. A single application of 5 µg dexamethasone prior to a 3-week treatment with PBMCs and the PSMAxCD3 bsAb CC-1 led to complete abolition of bsAb activity and almost unrestricted growth of established tumors. In contrast, tocilizumab did not inhibit the rapid and sustained regression of the tumors (figure 1D).

Based on these results, three patients treated with the bsAb CC-1 in individual experimental approaches received tocilizumab to prevent CRS. Application of tocilizumab (400 mg) was initiated at a first fever episode of >38.5°C that occurred at IL-6 levels of 20–100 pg/mL. After tocilizumab application IL-6 levels rose to >4000 pg/mL on further dose escalation, but body temperature declined, and additional antipyretic treatment was not required (figure 2). Profound activation of T cells in the peripheral blood was demonstrated by markedly elevated sIL-2R (figure 2) and CD69 expression levels (Zekri et al, manuscript submitted December 2019). However, CRP levels did not exceed 9 mg/dL (online supplementary figure 3).

Major side effects did not occur in two patients receiving the target bsAb dose of 2.4 mg (figure 2A–B) (American Society for Transplantation and Cellular Therapy (ASTCT) CRS consensus grading 1).18 A third patient (figure 2C) experienced atrial fibrillation and subsequent pulmonary edema and infection at a dose of 1.2 mg (ASTCT CRS grade 3).18 After application of high dose steroids (prednisolone 500 mg), CRS symptoms resolved within days. We assume that these complications were due to the particular history of this patient including bone marrow transplantation and cryptogenic pneumonias after being diagnosed with malignant lymphoma several years ago. We detected human antihuman antibodies against (Fab)₂ fragments of CC-1 that may have facilitated the elimination of tocilizumab in this patient. However, it is also likely that this patient experienced a case of at least partially tocilizumab-refractory CRS that was resolved by application of steroids. This hypothesis is supported by the rapid response to high dose steroid treatment. While steroids are needed for the treatment of severe CRS in some cases, the doses applied for this indication are much higher than the prophylactic dose used prior to bsAb treatment and thus most certainly abrogate therapeutic activity of bsAb. Notably, all patients experienced a rapid and profound (≥50%) reduction of elevated prostate specific antigen (PSA) levels (Zekri et al, manuscript submitted December 2019).