Preclinical evaluation of a diabody-based 177Lu-radioimmunoconjugate for CD22-directed radioimmunotherapy in a non-Hodgkin lymphoma mouse model

ARTICLE IN PRESS Cancer Letters ■■ (2016) ■■–■■

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

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Q2 Original Articles

Preclinical evaluation of a diabody-based 177 Lu-radioimmunoconjugate for CD22-directed radioimmunotherapy in a non-Hodgkin lymphoma mouse model

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Q1 Tobias Weber a,1, Benedikt Bötticher a,1, Michaela A.E. Arndt a,b, Walter Mier c,

Max Sauter c,d, Evelyn Exner a, Armin Keller a, Susanne Krämer c, Karin Leotta c, Artjom Wischnjow c, Ludger Grosse-Hovest e, Dirk Strumberg f, Dirk Jäger a, Hermann-Josef Gröne g, Uwe Haberkorn c,d, Gottfried Brem h, Jürgen Krauss a,*

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a

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Department of Medical Oncology, National Center for Tumor Diseases, Heidelberg University Hospital, Im Neuenheimer Feld 460, 69120 Heidelberg, Germany Immunotherapy Program, National Center for Tumor Diseases, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany Department of Nuclear Medicine, Heidelberg University Hospital, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany d Clinical Cooperation Unit Nuclear Medicine, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany e Department of Immunology, University of Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany f Department of Hematology and Medical Oncology, Marienhospital Herne, University of Bochum, Hölkeskampring 40, 44625 Herne, Germany g Division of Cellular and Molecular Pathology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany h Institute of Animal Breeding and Genetics, VUW, Veterinärplatz 1, Wien 1210, Austria b c

A R T I C L E

I N F O

Article history: Received 6 May 2016 Received in revised form 15 July 2016 Accepted 9 August 2016 Keywords: CD22 Diabody Lymphoma Radioimmunotherapy Dual-targeting PEGylation

A B S T R A C T

Radioimmunotherapy is considered as treatment option in recurrent and/or refractory B-cell nonHodgkin lymphoma (B-NHL). To overcome the dose limiting bone marrow toxicity of IgG-based radioimmunoconjugates (RICs), we modified a humanized diabody with 5-, 10-, or 20-kDa polyethylene glycol (PEG) for CD22-targeted radioimmunotherapy using the low-energy β-emitter lutetium-177 (177Lu). Favorable pharmacokinetics was observed for the 10-kDa-PEG-diabody in nude mice being xenografted with subcutaneous human Burkitt lymphoma. Even at high doses of 16 MBq this diabody RIC was well tolerated by NOD Rag1null IL2rγnull (NRG) mice and did not reveal signs of organ long-term toxicity 80 days post injection. Combination therapy of the diabody RIC with unconjugated anti-CD20 Rituximab demonstrated therapeutic efficacy in established disseminated mantle cell lymphoma xenograft models. When compared with the combination of the IgG formatted 177Lu anti-CD22 antibody and Rituximab, dual targeted therapy with the diabody RIC achieved an improved reduction of disease burden in the first nine days following treatment. The data indicate that the PEGylated anti-CD22 diabody may have potential for extending the repertoire of radiopharmaceuticals for the treatment of patients with B-NHL. © 2016 Elsevier Ireland Ltd. All rights reserved.

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Introduction Abbreviations: AUC, area under the curve; BLI, bioluminescence imaging; B-NHL, B-cell non-Hodgkin lymphoma; CHX-A″-DTPA, N-[(R)-2-amino-3-(p-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N′,N″,N″-pentaacetic acid; 64 Cu, copper-64; CYS, cystatin C; Db, diabody; FITC, fluorescein isothiocyanate; HE, hematoxylin and eosin; 124I, iodine-124; IgG, immunoglobulin G; 111In, indium111; ITLC, instant thin layer chromatography; 177Lu, lutetium-177; mAb, monoclonal antibody; MFImax, maximum median fluorescence intensity; MTD, maximum tolerated dose; NRG mice, non-obese diabetic-recombination activating gene-1 (NODRag1null) interleukin (IL)-2 receptor common gamma chain (IL2rγnull) null mice; PAS, periodic acid–Schiff; PBS, phosphate-buffered saline; PEG, polyethylene glycol; RIC, radioimmunoconjugate; RIT, radioimmunotherapy; SATA, N-succinimidyl S-acetylthioacetate; scFv, single-chain variable fragment; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEC, size exclusion chromatography; 90Y, yttrium-90; %IA/g, percentage of injected activity per gram tissue. * Corresponding author. Fax: +49 6221 565317. E-mail address: [email protected] (J. Krauss). 1 These authors contributed equally to this work.

CD20-directed 90Y-based radioimmunotherapy (RIT) has been established as treatment option for patients suffering from relapsed or treatment-refractory B-cell non-Hodgkin lymphoma (B-NHL). In contrast to unlabeled monoclonal antibodies (mAbs), such as CD20directed Rituximab, the tissue penetration range of β-radiation allows also the destruction of adjacent tumor cells that are not immediately targeted by the radioimmunoconjugate (RIC) [1]. Anti-CD20 90 Y-ibritumomab (Zevalin®) has demonstrated high treatment efficacy in both Rituximab naïve [2] and Rituximab refractory patients [3]. In comparison to 90Y the lanthanide 177Lu exhibits a reduced β-radiation path length (1.5 mm; Emax 0.496 MeV versus 12.0 mm; Emax 2.3 MeV) [4]. Consequently, 177Lu-based RICs exhibit lower ‘crossfire effects’ and may thus confer less non-specific tissue toxicity when

http://dx.doi.org/10.1016/j.canlet.2016.08.007 0304-3835/© 2016 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: Tobias Weber, et al., Preclinical evaluation of a diabody-based Hodgkin lymphoma mouse model, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.08.007

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Lu-radioimmunoconjugate for CD22-directed radioimmunotherapy in a non-

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compared with 90Y-labeled RICs [5,6]. However, despite demonstrated efficacy across several lymphoma entities in a phase I/II study in patients with indolent B-NHL, dose limiting bone marrow toxicity of 177Lu-Rituximab still constitutes a narrow therapeutic window of IgG-based 177Lu-RICs as well [7]. As a result, patients with ≥25% bone marrow involvement have to be generally excluded from RIT approaches [2,7,8]. Anti-CD20 radioimmunotherapy involves pre-dosing of patients with unlabeled anti-CD20 IgG antibody for improving biodistribution and tumor targeting of the subsequently administered radiolabeled antibody. Competitive binding of unlabeled and radiolabeled antibodies to the same target antigen, however, may limit therapeutic efficacy [9]. Therefore, dual-targeting of different B-cell antigens in combined immunotherapy/radioimmunotherapy approaches is currently under investigation [9,10]. The currently best defined antigenic target for dual targeting B-NHL approaches is the internalizing CD22 receptor being expressed in the majority of B-cell malignancies [11]. After the anti-CD22 RIC 90Y-epratuzumab achieved high rates of durable responses in clinical trials when administered as single agent [12] combination with unconjugated CD20-directed veltuzumab demonstrated encouraging therapeutic activity in patients with relapsed/ refractory aggressive NHL [8]. We recently demonstrated therapeutic efficacy of another CD22-directed IgG1 (huRFB4) conjugated to 177Lu. This RIC likewise mediated superior efficacy when combined with unlabeled Rituximab in a xenografted subcutaneous human Burkitt lymphoma tumor model [13]. Due to their long residence times in the human body classically IgG formatted RICs may, however, not represent the optimal configuration for RIT approaches. Several investigators have therefore employed smaller sized antibody fragments for RIC development. Diabodies with an intermediate size of approximately 55 kDa [14] are rapidly cleared from the circulation through the kidneys but in comparison to full-size IgG antibodies (150 kDa) exhibit improved diffusion rates and tumor penetration [15,16]. Thus far radiolabeled diabodies have been mainly employed for immunoimaging (PET/SPECT) of solid tumors [17–20] but also of B-cell lymphoma with 124I-labeled anti-CD20 diabodies [21]. Although the small size of diabodies holds also potential for therapeutic interventions by attenuating hematologic toxicity of antiCD20 IgG-RICs [3,22,23], this format has thus far not been investigated for B-NHL radioimmunotherapy. Major obstacle for the clinical translation of such antibody formats for targeted radiotherapy is the sizedependent renal retention of chelate–metal complex [24] resulting in dose limiting nephrotoxicity [25]. The objective of the present study was to evaluate the efficacy of a humanized anti-CD22 diabody derivative of huRFB4 for 177Lu-radioimmunotherapy in a clinically relevant mouse model of disseminated growing mantle cell lymphoma xenografts. To keep the blood residence and bone marrow exposure time of the diabody-formatted anti-CD22 177Lu-RIC as short as possible and at the same reduce renal accumulation the molecular weight of the diabody was successively increased through conjugation of polyethylene glycol (PEG) chains of 5, 10, and 20 kDa, respectively. These size-modified anti-CD22 177Lu-diabodies were analyzed in preclinical studies with regard to biodistribution and toxicity. The anti-tumor efficacy of the 10 kDa-PEGylated 177Lu-diabody was evaluated in combination with anti-CD20 mAb therapy using disseminated mantle cell lymphoma xenografts in NRG mice as a model system.

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Materials and methods

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Tumor cell lines

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The human Burkitt lymphoma cell line Raji (ATCC, Manassas, USA) was cultured as previously described [13]. The human mantle cell lymphoma cell line Granta519 (Leibniz Institute DSMZ GmbH, Braunschweig, Germany) was stably transfected with the luciferase reporter vector pGL4.51[luc2/CMV/Neo] (Promega GmbH, Mannheim, Germany) and maintained in DMEM AQMedia supplemented with 10%

fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma-Aldrich, St. Louis, USA) and 0.8 mg/ml G418 (Carl Roth GmbH, Karlsruhe, Germany). Recombinant protein production The humanized anti-CD22 IgG1 (huRFB4) was purified from cell culture supernatants of stably transfected Sp2/0-Ag14 mouse myeloma cells as reported previously [13]. The corresponding diabody (huRFB4 Db) derived from the stability-engineered humanized diabody SGIIIM [26] was cloned into a mammary gland specific expression vector for production into the milk of transgenic rabbits [27,28]. The purified protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analytical size exclusion chromatography (SEC) as described previously [29]. The extinction coefficient and the molecular weight of recombinant proteins were calculated using Gene Inspector 1.6 (Textco BioSoftware, West Lebanon, USA). Size modification of huRFB4 Db by PEGylation HuRFB4 Db (1.0 mg/ml, PBS, pH 7.4) was incubated with an 8-fold molar excess of the heterobifunctional crosslinking agent N-succinimidyl S-acetylthioacetate (SATA; Thermo Fisher Scientific, Waltham, USA) for 1 h at room temperature. Excess SATA was removed by dialysis against PEGylation buffer (100 mM Na2HPO4, 150 mM NaCl, 10 mM EDTA, pH 7.4) at 4 °C overnight. SATA-modified diabody was deacetylated by adding 100 μl of a 0.5 M hydroxylamine solution (0.1 M sodium phosphate, 10 mM EDTA, pH 7.4) per ml diabody solution. After incubation for 2 h at room temperature, excess hydroxylamine was removed using disposable PD MiniTrap G-25 columns (GE Healthcare, Little Chalfont, UK) preequilibrated with PEGylation buffer. α-Methoxyω-maleinimide polyethylene glycols (Rapp Polymere GmbH, Tübingen, Germany) with an average molecular weight of either 5, 10, or 20 kDa were freshly dissolved in PEGylation buffer (50 mg/ml) for subsequent thiol-selective coupling. The thiolmodified diabody was reacted with 30-fold molar excess of respective polyethylene glycols (PEGs) at 4 °C overnight. Upon preparative SEC in PBS (pH 7.4) using a HiLoad 16/60 Superdex 200 prep grade column (GE Healthcare, Little Chalfont, UK) eluted column fractions were analyzed by SDS-PAGE to determine non-, mono- and multiPEGylated diabody derivatives. Fractions containing mono-PEGylated diabody were pooled, concentrated and sterile-filtrated. Purity of concentrated mono-PEGylated diabody derivatives was analyzed by SDS-PAGE and analytical SEC as described previously [29]. Conjugation of CHX-A″-DTPA and radiolabeling Conjugation of the acyclic chelating agent N-[(R)-2-amino-3-(p-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N′,N″,N″-pentaacetic acid (CHX-A″-DTPA; Macrocyclics, Dallas, USA) and radiolabeling with 177Lu (PerkinElmer, Waltham, USA) was performed using established methods [13]. The required molar excess of CHX-A″-DTPA to yield an average attachment of one mole CHX-A″-DTPA per mole antibody was determined by isotopic dilution titration as previously described [13]. Radiochemical stability of 177Lu-labeled mAbs was assessed by incubation in human blood serum in the presence of excess DTPA as reported earlier [13]. Cell binding analysis

148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195

Biodistribution

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Biodistribution studies were carried out with female athymic nude-Foxn1nu mice (Harlan Laboratories, Indianapolis, USA) at 6–8 weeks of age as previously reported [13]. Groups of 3–4 mice with palpable Raji xenografts were injected with 1 MBq of 177Lu-labeled antibodies via a tail vein and tissue samples were collected 1 h, 6 h, 24 h, and 4 days after injection of the RICs. The tissue samples were blotted dry, weighed and radioactivity was measured along with a sample of the injection solution to calculate the percentage of injected activity per gram tissue (%IA/g) and tumor-to-blood ratios. To estimate the time course of residence of the 177Lu-labeled IgG and diabodies in blood, kidney, and subcutaneously B-NHL xenografted tumor, the following calculations were made: (i) cumulative radioactivity was calculated by fitting a biexponential curve to the non-decay corrected biodistribution data. The AUCs (kBq × h per MBq of injected RICs) were determined by integrating the bi-exponential function to infinity. (ii) The comparison of the respective activity concentration was obtained as time course of the kBq × h values. For this purpose blood was assumed to account for 7.9% of the total weight of the mice, the average tumor mass at the time of injection was assessed (50 mg) and the mass of the kidney was experimentally determined.

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The immunoreactivity of huRFB4 Db derivatives was determined by flow cytometry using Raji cells as previously described [13]. Bound diabody derivatives were detected with a mouse anti-(HIS)6 IgG (Dianova GmbH, Hamburg, Germany) followed by two washing steps and subsequent incubation with a FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, USA).

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Toxicity studies

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Groups of 5 female NMRI mice at 8 weeks of age (Janvier Labs, Saint-Berthevin Cedex, France) received a single dose of 9.5 MBq of 177Lu-labeled antibodies (huRFB4 Db, its 10 kDa PEG derivative, or huRFB4 IgG) or PBS by tail vein injection to investigate long-term toxicity. In case of 177 Lu-huRFB4 IgG, mice were coinjected intraperitoneally with an irrelevant murine IgG2a (Bio X Cell, West Lebanon, USA) as reported previously [13]. RIC blood samples of anesthetized mice 80 days post injection were acquired by terminal heart puncture and tissues were collected. To assess potential long-term nephrotoxicity, serum creatinine, cystatin C and urea levels were determined. Statistical analysis was performed by one-way analysis of variance using GraphPad Prism 5.0 (GraphPad Software, La Jolla, USA). Tissues were fixed using 4% buffered formalin and stained with hematoxylin and eosin (HE), or periodic acid–Schiff (PAS) for histopathologic analysis.

Q4

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Radioimmunotherapy

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We have previously shown that non-obese diabetic-recombination activating gene-1 (NOD-Rag1null) interleukin (IL)-2 receptor common gamma chain (IL2rγnull) null (NRG) mice are well-suited as an immunodeficient radioresistant model to evaluate the efficacy of radioimmunotherapy of lymphoma xenografts [13]. Female mice at 6–8 weeks of age were obtained from The Jackson Laboratory (Bar Harbor, USA). Cohorts of 2 non-tumor-bearing mice were injected with escalating doses (8, 12, or 16 MBq) of the 177Lu-labeled huRFB4 Db or its 10 kDa PEG derivative via a tail vein to determine the maximum tolerated dose (MTD). Body weight and general appearance were monitored at least twice weekly until the end of the experiment (45 days). Mice that exhibited a decrease in total body weight of >20% were euthanized. For therapy studies NRG mice were injected with 5 × 106 luciferase-transfected Granta-519 cells via a tail vein to obtain disseminated human mantle cell lymphoma xenografts. Mice were randomized into groups of 9–10 each. All therapies were administered in 100 μl PBS via a tail vain. For dual-targeted therapy, animals were pretreated with unlabeled Rituximab (Roche Pharma AG, Grenzach-Wyhlen, Germany) 48 h post xenotransplantation (1.0 mg/mouse). RICs were given at day 5 post tumor cell inoculation using an activity of 16 MBq (177Lu-huRFB4 Db-10-kDaPEG) or 9.5 MBq (177Lu-huRFB4 IgG) per mouse. Control animals were only injected with Rituximab or PBS 48 h post xenotransplantation. In case of 177Lu-huRFB4 IgG, mice were coinjected intraperitoneally with an irrelevant murine IgG2a as described earlier [13]. Mice were monitored for general appearance and weighed at least every other day. Animals exhibiting beginning paralysis of the hind leg or >20% loss of initial body weight were euthanized. Tumor progression was monitored by in vivo bioluminescence imaging at weekly intervals starting at day 7 after xenotransplantation. Mice were injected intraperitoneally with 3 mg Luciferin-EF (Promega GmbH, Mannheim, Germany) and anesthetized with 1.5% isoflurane through an O2 flow modulator. 10 min after injection, emitted photons were counted for 2 min using a photon imager system (Biospace Lab, Paris, France). At the end of every acquisition, a photographic body image was superimposed upon the bioluminescence signal. The number of photons per animal was determined by drawing a rectangular region of interest (ROI) and evaluated as photon count per minute (CPM). Statistical analysis (GraphPad Prism 5.0) was performed using a Kruskal–Wallis pretest and an unpaired two-tailed Mann–Whitney test (confidence interval: 95%). Survival studies were analyzed by log-rank test.

Results In vitro characterization of huRFB4 Db and diabody-derived RICs The humanized anti-CD22 diabody huRFB4 Db was produced in the milk of transgenic rabbits because only low production yields could be obtained from both periplasmic expressions in bacteria [26] and stably transfected mammalian cells (data not shown). Production yields of homogenously purified huRFB4 Db were 52.8 mg per liter transgenic rabbit milk on average. The affinity constant (KD) of 0.16 nM corresponded well to that of the parental huRFB4 IgG (0.22 nM) [13]. To determine the optimal size of the huRFB4 Db as a RIC for therapeutic application in B-NHL the hydrodynamic volume was successively increased by employing 5, 10, or 20 kDa PEG, respectively. Retention times on analytical SEC of mono-PEGylated diabody derivatives decreased (5 kDa: 13.4 ml; 10 kDa: 12.2 ml; 20 kDa: 11.3 ml) when compared to unmodified diabody (15.7 ml) and correlated well with the increasing size (Fig. 1A). Following purification non-covalently bound monoPEGylated diabodies migrated in denaturing SDS-PAGE according to the molecular weight of one monomer with approximately 28 kDa. Bands of PEGylated monomers shifted according to higher molecular weights with increasing PEG size (Fig. 1B). To achieve an average attachment of 0.7–1.1 moles CHX-A″-DTPA per mole huRFB4 Db or monoPEGylated

3

derivatives, a 250–300-fold molar excess of CHX-A″-DTPA was needed and adjusted by isotopic dilution titration. Size modification by PEGylation and conjugation of CHX-A″-DTPA via accessible amino groups did not result in significant impairment of antigen binding affinity (Fig. 1C). Determined apparent equilibrium dissociation constants (KD) were 0.92, 0.96, and 2.15 nM for PEGylated huRFB4 Dbs carrying 5, 10, and 20 kDa PEGs, respectively, and 0.53 nM for the CHX-A″-DTPAmodified non-PEGylated diabody. 177Lu-labeled diabody derivatives exhibited exceptionally high stability in human blood serum (Fig. 1D).

293 294 295 296 297 298 299 300 301 302 303 Biodistribution in mice 304 305 To evaluate the impact of the antibody format and PEGylation 306 on biodistribution (Fig. 2), groups of 3–4 athymic nude mice with 307 palpable subcutaneous Raji xenograft tumors received 1 MBq of 177Lu308 labeled huRFB4 diabody derivatives intravenously. Biodistribution 309 of the corresponding 177Lu-labeled anti-CD22 IgG has previously been 310 described for the same animal model [13]. Serum concentrations 311 of diabody-based RICs were lower than that of the full-size IgG RIC 312 at 24 h (14.4%ID/g) and 4 d (10.8%ID/g) post injection [13]. Kidney 313 uptake of huRFB4 Db was maximal 24 h post injection (125.0%ID/g) 314 and PEGylation resulted in a reduction by 3.5-fold for the 5 kDa PEG 315 Db, 7.0-fold for the 10 kDa PEG Db, and 17.1-fold for the 20 kDa PEG 316 Db, respectively. Tumor uptake of diabody constructs was highest 317 for the 10 kDa PEG diabody with a maximum of 6.0%ID/g at 24 h 318 post injection which is 2.1-fold higher than that of the non319 PEGylated diabody. Although the IgG format exhibited superior 320 tumor uptake with a maximum of 26.5%ID/g at day 4 post injec321 tion [13], non-PEGylated Db, 5 kDa PEG Db, and 10 kDa PEG Db RICs 322 demonstrated superior tumor-to-blood ratios. Tumor-to-blood ratio 323 improved 3.7-fold for the 10 kDa PEG Db when compared with the 324 IgG (8.8 versus 2.4) at day 4 post injection, respectively [13]. 325 To estimate the time course of activity in critical organs, the res326 idence times of the IgG- and diabody-based RICs were calculated 327 as kBq × h values (Table 1). Even with the limited number of time 328 points, the results of the biexponential fit yielded a very good ad329 aptation of the measured values and therefore allowed a good 330 prediction of the organ exposure. Compared to the non-PEGylated 331 Db exposure of the tumor for both the 5 kDa PEG Db and the 10 kDa 332 PEG Db doubled (2.3-fold and 2.1-fold, respectively), whereas for 333 the 20 kDa PEG Db only a 1.5-fold increase was observed. The res334 idence time of 177Lu-huRFB4 IgG in the kidneys was only 10% as 335 compared to the 177Lu-huRFB4 Db. PEGylation of the diabody reduced 336 the exposure of the kidneys from 27% (177Lu-Db-5-kDa PEG) to 14% 337 (177Lu-Db-10-kDa PEG) to 6% (177Lu-Db-20-kDa PEG), respectively. Based on the biodistribution data, the intermediate-sized 10 kDa Q5 338 339 PEG Db was selected as a candidate with favorable pharmacoki340 netics and in addition to the unPEGylated diabody was therefore 341 employed in further animal experiments. 342 343 Toxicity 344 345 We have previously shown that NRG mice are particularly well 346 suited for studying radioimmunotherapeutic approaches for B-NHL 347 since this mouse strain is relatively radioresistant and a good 348 349 350

Table 1 Residence times in critical organs per MBq of RICs injected. Construct

Blood (kBq × h)

Kidney (kBq × h)

Tumor (kBq × h)

351 352

177Lu-huRFB4

565 3,085 3,524 3,535 64,300

22,477 6,063 3,041 1,401 2,158

123 289 261 192 3,801

353 354 355 356 357

Db Lu-huRFB4 Db-5-kDa-PEG 177Lu-huRFB4 Db-10-kDa-PEG 177Lu-huRFB4 Db-20-kDa-PEG 177 Lu-huRFB4 IgG 177

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Fig. 1. Modification of huRFB4 diabody for radioimmunotherapy. Purified mono-PEGylated huRFB4 diabody derivatives were assessed by (A) analytical SEC on a Superdex 200 10/300 GL column and (B) SDS-PAGE followed by Coomassie Brilliant Blue staining. Note that huRFB4 Db (52.8 kDa) is separated into two scFv monomers (26.4 kDa) under denaturing conditions. Lane L: Spectra Multicolor Broad Range Protein Ladder; lane 1: unmodified huRFB4 Db; lanes 2–4: mono-PEGylated huRFB4 Db using 5, 10, or 20 kDa PEG. (C) Equilibrium binding curves were determined by flow cytometry after chemical conjugation of CHX-A″-DTPA. Measurements were done in triplicates and bars indicate standard deviations of mean values. MFImax, maximum median fluorescence intensity. (D) Radiochemical stability of 177Lu-labeled huRFB4 Db derivatives was analyzed by incubation in human blood serum at 37 °C in the presence of excess DTPA. Protein bound radioactivity was determined by ITLC at indicated time points. d, days; h, hour(s).

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acceptor for human lymphoma xenotransplants [13]. We therefore employed this mouse strain also for the preclinical in vivo evaluation of the diabody RICs in the present study. In this set of experiments administration of 8, 12, and even 16 MBq 177 Lulabeled huRFB4 Db (Fig. 3A) or its 10 kDa PEGylated derivative (Fig. 3B) was well tolerated and all animals survived until the end of the experiment (45 days). Because a temporary decrease in body weight of >10% occurred in both animals treated with 16 MBq of the PEGylated Db this dose was determined as MTD, which is 1.7fold higher than the MTD for the corresponding 177Lu-labeled huRFB4 IgG [13]. The MTD of the non-PEGylated Db was not reached at the doses tested. To comparatively evaluate organ toxicity of the huRFB4 diabody, its 10 kDa PEG derivative, and huRFB4 IgG in a head-to-head fashion we next employed the MTD of the IgG format (9.5 MBq) for all constructs, followed by 80 days of observation after injection and sacrifice of mice for subsequent histopathological analyses. None of the animals exhibited histopathologic signs of renal damage, such

as inflammation, sclerosis or hyaline material deposits in the glomeruli, respectively (Fig. 4). In addition, histopathologic analyses did not show RIC induced alterations of liver, spleen, lung, heart, and bone marrow. Serum creatinine and urea levels of RIC-treated mice did not increase after treatment (Table 2). Because creatinine and

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Table 2 Evaluation of long-term nephrotoxicity. Treatment (n = 5)

Serum creatinine (mg/dl)

Serum urea (mg/dl)

Cystatin C (mg/l)

392 393 394

Buffer treated control Lu-huRFB4 Db 177Lu-huRFB4 Db-10-kDa-PEG 177 Lu-huRFB4 IgG

<0.2 <0.2 <0.2 <0.2

36.0 ± 7.3 30.8 ± 3.0 34.8 ± 7.9 31.6 ± 1.7

0.16 ± 0.04 0.20 ± 0.03** 0.15 ± 0.03 0.17 ± 0.03

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Blood analysis was performed at day 80 post injection of 177Lu-RICs. Values represent the mean value ± standard deviation. Significance between treatment group and control group is indicated as follows: **, P < 0.015.

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Fig. 2. Biodistribution experiments. Groups of 3–4 athymic nude mice with subcutaneous Raji tumors were injected intravenously with 1 MBq of 177Lu-labeled huRFB4 Db derivatives. Organ uptake at indicated time points is shown as mean value ± standard deviation. %IA/g, percentage of injected activity per gram tissue.

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urea may not be sensitive enough for appropriately assessing long term kidney damage [30], we additionally determined serum cystatin C (CYS), a reliable and early marker for impaired kidney function in mice [31]. We could show that CYS levels (p = 0.015) at day 80 did increase for 177 Lu-huRFB4 Db, whereas CYS values of mice treated with either IgG or 10 kDa PEGylated diabody 177Luradioimunoconjugates remained within the normal range (Table 2). Treatment efficacy To assess the therapeutic efficacy of the RIC constructs in a clinically relevant setting we established a novel disseminated mantle cell lymphoma NRG mouse model. Disseminated growth and progression of lymphoma cells upon RIT were quantitatively monitored by in vivo bioluminescence imaging (Fig. 5A and B). Without treatment all animals developed rapid disseminated lymphoma as indicated by high in vivo luciferase activity (Fig. 5B) and had to be euthanized within 22 days post xenotransplantation (Fig. 5C). Monotherapy with unlabeled CD20-specific Rituximab (1 mg/ mouse) at day 2 after inoculation of CD20/CD22-positive LucGranta-519 cells led to moderate tumor growth inhibition (Fig. 5B) and extended median survival from 20 days to 28 days when compared to the untreated control group (Fig. 5C). Combination of

Rituximab and CD22-specific 177Lu-huRFB4 Db-10-kDa-PEG (16 MBq, MTD) demonstrated more pronounced tumor growth inhibition (Fig. 5B) resulting in a significantly increased median survival of 36 days when compared to Rituximab monotherapy (p < 0.05) (Fig. 5C). Dual-targeted therapy applying the 177 Lu-labeled huRFB4 IgG (9.5 MBq, MTD) and Rituximab resulted in the longest overall survival of 47 days (Fig. 5C). Importantly, the anti-CD22 diabodybased RIT displayed a significantly better reduction in luciferase activity than both anti-CD20 Rituximab monotherapy and antiCD22 IgG RIT at day 14 (Fig. 5D). Discussion We previously reported the generation of the humanized diabody SGIIIM, derived from the humanized scFv SGIII with grafted specificity of the murine anti-CD22 mAb RFB4 and stability-optimized by site directed mutagenesis of a single critical VL framework residue (VL-36) at the VH–VL interface [26,32]. In this study, the antiCD22 diabody (huRFB4 Db) was developed on the basis of SGIIIM for mammary gland specific expression and production in transgenic rabbits. In comparison with larger mammalian species, such as goats or cows, transgenic rabbits are particularly suitable for the production of acceptable quantities of recombinant proteins at

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Fig. 3. Dose escalation study. Groups of 2 NRG mice were injected via a tail vein with indicated doses of 177Lu-labeled huRFB4 Db derivatives. Body weight was monitored over a period of 45 days. Lines represent individual mice. Dashed line, weight loss >10% of the initial weight. (A) 177Lu-huRFB4 Db; (B) 177Lu-huRFB4 Db-10-kDa-PEG.

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relatively low cost [33,34]. The yield of purified anti-CD22 diabody per liter rabbit milk was in the present study 621-fold higher than production rates in bacterial periplasm [26]. These data do not only demonstrate that for difficult-to-produce recombinant antibody fragments generation of transgenic rabbits may represent a valuable alternative but in the present case do in fact provide the basis for the possibility for moving the compound further into potential clinical applications. The anti-CD20 IgG RIC Zevalin® provides a treatment option for patients with relapsed or treatment-refractory B-NHL [2,3]. However, bone marrow toxicity limits clinical applicability of IgG-based RICs in general [2,7,8] and smaller antibody fragment derivatives with

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Fig. 4. Renal histopathology. Groups of 5 NMRI mice were injected with PBS or 9.5 MBq of 177Lu-RICs. Kidney sections were PAS stained and examined for signs of renal damage 80 days after treatment. For each treatment group, one kidney section is illustrated in an exemplary manner. (A) Buffer treated control; (B) 177Lu-huRFB4 Db; (C) 177Lu-huRFB4 Db-10-kDa-PEG; (D) 177Lu-huRFB4 IgG.

shorter half-lives have been proposed as alternatives since many years ago [15,16,35]. Nonetheless there are only few preclinical studies addressing the suitability of diabody-based RICs for RIT approaches [24,36]. Since radiometal-labeled diabodies have been shown to accumulate in the kidney [25], the usage of β-emitting radiometals may result in renal damage as demonstrated for e.g. a 90 Y-labeled anti-HER2/neu diabody in long-term toxicity experiments [24]. We have previously demonstrated superior tumor uptake and growth inhibition of the 177Lu-labeled CD22-specific IgG1 huRFB4 in comparison with 177Lu-labeled CD20-directed Rituximab in xenografted subcutaneous human Burkitt lymphoma tumor models [13]. Due to the shorter tissue penetration range of β-radiation in comparison with 90Y [4], the employment of a 177Lu-labeled diabody was considered to not only reduce hematologic toxicity but also renal toxicity in comparison with 90Y-labeled diabodies [24]. Since preclinical experiments have previously also demonstrated that kidney uptake of 111 In- or 64 Cu-labeled diabodies can be reduced by PEGylation [25,37], we employed this technology for further attenuating potential nephrotoxicity of the 177Lu-labeled diabody. As anticipated, PEGylation significantly attenuated renal retention and additionally improved tumor uptake of the diabodybased RIC. Although most likely due to longer residence time in blood, the previously described IgG format exhibited superior tumor uptake [13], and diabody-based RICs demonstrated superior tumorto-blood ratios. Serum half-life of diabody-formatted RICs was most likely not only reduced because of their small molecular size but also due to the lack of Fc domains [35] that prevents interaction with the neonatal Fc receptor as in case of IgG antibodies [38]. Since the 10 kDa PEG diabody represented the best compromise between kidney and tumor uptake, this intermediate-sized diabody was selected for further preclinical efficacy assessment. We have previously reported that for 177Lu-labeled IgG antibodies the MTD was reached at 9.5 MBq per NRG mouse [13]. Because of its lower hematologic toxicity the MTD of the PEGylated 177LuhuRFB4 Db in the present study was 1.7-fold higher. As the small size of diabodies leads to their rapid renal ultrafiltration kidneys

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Fig. 5. Radioimmunotherapy experiment. NRG mice were injected intravenously with 5 × 106 luciferase-transfected Granta-519 cells per mouse and randomized into groups of 9–10 each (day 0). Rituximab was injected at day 2 and 177Lu-RICs were administered at day 5. Tumor progression was monitored by in vivo bioluminescence imaging (BLI) at weekly intervals starting at day 7. (A) Representative BLI pictures shown for one mouse of the 177Lu-huRFB4 IgG treated group. (B) Tumor progression assessed by BLI for individual mice at indicated time points. cpm, photon count per minute. (C) Survival was significantly higher for mice treated either with Rituximab plus 177LuhuRFB4 Db-10-kDa-PEG or Rituximab plus 177Lu-huRFB4 IgG compared with mice treated with Rituximab alone (p < 0.05). (D) Statistical analysis of in vivo luciferase activity by Mann–Whitney test at day 14. Bars represent standard deviations (SDs) of mean values. ***, p < 0.001; ns, not significant.

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exhibit the highest degree of retention of radioactivity when compared to other organs. The present study shows that the absorbed renal dose could be reduced by almost 87% for the 10 kDa PEGylated 177 Lu-huRFB4 Db. As a result, serum cystatin C values remained in the normal range for the PEGylated diabody until mice were sacrificed at day 80 whereas mice treated with the non-PEGylated diabody RIC showed elevated cystatin C levels as a sign for renal damage. It was very recently reported that renal toxicity of radiofolates with manifestation of functional and morphological changes of kidneys may occur at even significantly longer postexposure times than 80 days [30]. We therefore cannot completely rule out the possibility that renal toxicity of the PEGylated diabodyRIC may have manifested even after the chosen observation time of 80 days. This important safety issue clearly remains to be addressed in the future development of the PEGylated diabody RIC. Diabody-based 177Lu-RIT may be particularly suitable for patients with low or easily accessible tumor burdens in disseminated disease settings. However, bone marrow toxicity of RIT in SCID mice strains has thus far hampered evaluation of this treatment modality in this clinically important setting [5,39]. In the present study, we therefore have established such a xenograft model of B-NHL by employing NRG mice being more tolerant to ionizing radiation [40]. 177 Lu-RICs were evaluated in combination with unlabeled Rituximab because CD20- or CD22-directed RIT is usually preceded by CD20specific mAb-therapy in the clinical situation [2,7,8]. We could show that the therapeutic efficacy of CD20-directed mAb-therapy was significantly improved by combination with the PEGylated 177LuhuRFB4 Db. However, anti-tumor activity was lower than that of the corresponding 177Lu-huRFB4 IgG alone. This is most likely caused by the shorter blood circulation of the PEGylated 177Lu-huRFB4 since RICs were comparable in both binding affinity and number of attached CHX-A″-DTPA molecules [13]. In summary, we have created a novel diabody-based 177Lu-RIC for RIT of CD22-positive hematologic malignancies. We showed that the RIC was improved by PEGylation resulting in decreased kidney accumulation and increased tumor uptake while preserving its rapid clearance from the blood circulation. Because high doses of 177LuhuRFB4 Db-10-kDa-PEG were administered without causing significant toxicity in animals we believe that further optimization of the dosing schedule as well as careful selection of the radioisotope (e.g. short-lived alpha-emitters) may expand the range of clinical applicabilities for the PEGylated diabody in comparison with conventional IgG-based 177Lu-RICs [7].

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Acknowledgments This study was funded by the Deutsche José Carreras LeukämieStiftung (DJCLS R 12/16). The authors declare that they have no conflict of interest. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

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