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Protein Expression and Purification

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Mabfilin and Fabfilin - New antibody-scaffold fusion formats formultispecific targeting concepts

Mathias Kahl, Florian Settele, Paul Knick, Ulrich Haupts, Eva Bosse-Doenecke∗

Navigo Proteins GmbH, Formerly Scil Proteins GmbH, Heinrich-Damerow-Straße 1, D-06120 Halle Saale, Germany

A R T I C L E I N F O

Keywords:AffilinScaffoldBispecificAntibody formatTargetingTransient expression

A B S T R A C T

Protein based binding molecules have a broad applicability from therapeutic to technical use. Monoclonal an-tibodies represent the major class of this type of agents complemented by innovative approaches using scaffoldproteins with tailor-made properties. Various concepts for new formats combining antibody chains or antibodyfragments and fusions with other entities have been developed recently. This strategy opens up options to designmolecules with biophysical, biochemical and pharmacological characteristics in a broad range while simulta-neously addressing several targets or epitopes. The demand for such compounds is still growing as reflected bythe literature and further new ideas are expected. In this context we developed so called Mabfilin and Fabfilinmolecules. The formats synergistically bring together the classical antibody or fragments thereof supplementedwith additional binding moieties, the Affilin® molecules. These are based on the scaffold ubiquitin endowed withnovel targeting properties by local randomization and selection from combinatorial libraries. Mab-/Fabfilinvariants show advantageous biochemical properties and open a new route for the development of multispecificcompounds for flexible applications.

1. Introduction

Monoclonal antibodies are widely used in therapeutics and diag-nostics. For many scenarios binding of only one epitope or one target isinsufficient. Bie or multispecific compounds are recent developmentsto improve efficacy and fulfill unmet medical needs. The simultaneousbinding of two or more interaction sites paves the way for innovativetherapeutic concepts and synergistic modes of action. Complex path-ways in living cells can be addressed and rescue mechanisms preventingthe sustained activity of conventional mono-mechanistic therapeuticscan be avoided at the same time. Strikingly, hitting more than onetarget frequently improves potency and specificity thereby renderingtherapies more efficient with little side effects [1–4]. Moreover, twodifferent cell types can be cross-linked, for example immune cells andcancer cells to accomplish immunotherapeutic strategies [5–8].

During the last years the concepts described above have been oftenpursued with bispecific antibody formats. Pairing heavy and lightchains of different origin is an inefficient process often leading to het-erogeneous and unstable products limiting commercialization. Some ofthe inherent problems with antibody pairing have been solved bytechniques such as the knobs-into-holes amino acid exchange [9],complementary strand exchange, SEED (strand exchange engineereddomains), Fab arm exchange [10], additional disulphide bridges, single

point mutations for electrostatic interactions [11], mixtures thereof[12] and combinations of antibody fragments amongst others [13–19].More than 100 different formats are described in the literature (for acomprehensive review see Brinkmann and Kontermann 2017 [20]).

Despite these efforts there is still further need for multi-specificdrug-like compounds [21]. Recently, this demand has been approachedwith alternative fusion proteins of an antibody and one or more addi-tional entities comprising so called scaffold binders. Examples for thisnew class are the FynomAbs [22], AffiMabs [23], antibody DARPinfusions [24] and antibody Anticalin fusion proteins [25]. These com-pounds bring together the favorable properties of both the antibody andthe scaffold world. Affilin molecules are based on the scaffold of one ortwo genetically connected ubiquitin moieties with acquired affinity forselected targets [26]. The combinations presented in this work arechosen based on three general therapeutic principles. First, the classicalbispecificity was generated combining Cetuximab (anti-EGFR) with ananti-HER2 Affilin opening the possibility to address two targets, in thiscase related growth factor receptors, involved in cancer and often lo-cated on the same cell or also on different cells [27]. Second, a multi-epitope scenario was set up with an anti-EGFR Affilin fused to Cetux-imab. Via binding to two different sites on the EGFR potentialsynergistic effects could be pursued caused by allosteric regulation oralterations in homo- or hetero-dimerization propensity of the receptor

https://doi.org/10.1016/j.pep.2018.04.013Received 16 October 2017; Received in revised form 16 March 2018; Accepted 19 April 2018

∗ Corresponding author.E-mail address: eva.bosse-doenecke@navigo-proteins.com (E. Bosse-Doenecke).

Protein Expression and Purification 149 (2018) 51–65

Available online 25 April 20181046-5928/ © 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

T

[28,29]. The third well known concept is the therapeutic crosslinking ofcancer and immune cells represented by the classical anti-CD3/anti-HER2 pairing [30–33] using combinations of anti-HER2 Affilin and theCD3 targeting antibody Okt3.

2. Material and methods

2.1. Cloning and expression

Fusions of Affilin molecules to antibody chains or Fab fragmentchains were achieved by constructing an expression-cassette for every

combination (Ne or C-terminal fusion to heavy or light chain of theCetuximab antibody or Fab fragment). Suitable DNA stretches weresynthesized by GeneART@ Strings™ DNA Fragments (Thermo FisherScientific). The resulting expression cassette is illustrated in Fig. 1A andcomprises a Kozak sequence, a signal peptide, a sequence for the heavyor light chain of the monoclonal antibody or Fab fragment, a 15 aminoacid linker consisting of glycine and serine ((Gly4Ser)3), a suitable tagfor purification and immunological detection and two stop codons (2×Stop). Each expression cassette is flanked by a BamH1 restriction site forcloning into a plasmid. A BsaI restriction site was inserted for cloningdifferent Affilin variants to the Ne or C-terminus of the respective

Fig. 1. Outline of antibody-Affilin formats. (A) Expression cassette assembled of the Kozak-sequence, the signal peptide of the respective antibody chain, the antibodychain, an N-terminal tag sequence and two stop codons. For N- or C-terminal Affilin fusion, a 15 amino acid (Gly4Ser)3-linker is attached N- or C-terminal of theantibody chain. Cloning of the ubiquitin or Affilin was via BsaI restriction sites, cloning into the vector via BamHI. (B) Schematic drawing of monoclonal antibody-Affilin-fusion proteins, called Mabfilin, or Fab-fragment-Affilin-fusion proteins, called Fabfilin. Red circles represent Affilin molecules or ubiquitin as control joined todifferent sites of an antibody or Fab-fragment. Light blue boxes specify the fusion site of the proteins and the orange boxes the nomenclature of the respective fusionproteins. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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antibody or antibody Fab fragment chain.Mammalian expression vector pCEP4 (Thermo Fisher Scientific) was

chosen for expression of Mabfilin and Fabfilin proteins. The expressioncassette was cloned into pSCIL008b (proprietary vector Scil Proteins)via BamH1 as an intermediate. Seamless insertion of Affilin cDNA wasrealized using BsaI. The complete cassette was then integrated intopCEP4 via BamH1.

For expression of Cetuximab-Mabfilin molecules, 15×106

FreeStyle™ 293-F cells were seeded with 30mL FreeStyle™ 293 ex-pression medium (Thermo Fisher Scientific) into a 250mL Erlenmeyerflask (Corning) and cultured for 24 h at 37 °C, 8% CO2, 95% humidity at200 rpm. For transfection, 22.5 μg of plasmid DNA of each antibodychain were mixed with 60 μg polyethylenimine in 3mL of OptiPRO™

SFM medium (Thermo Fisher Scientific), incubated for 15min at roomtemperature and finally applied to the cell culture. After 24 h, 30mLFreeStyle™ 293-F expression medium were added. Cell culture super-natant was harvested 144 h after transfection. Vitality of cells andgrowth were assessed by Neubauer improved hemocytometer usingBromphenol blue staining (Sigma Aldrich). Expression was monitoredby SDS-PAGE analysis of cell culture supernatant. For scaling up/down,the same ratios of each ingredient were used.

To improve expression yields, the Expi293-expression system(Thermo Fisher Scientific) was used for the manufacturing ofCetuximab-Fabfilin fusion proteins. 50× 106 Expi293F™-cells wereseeded in 17mL Expi293F™ expression medium in 50mL Falcon tubes.For transfection, 10 μg plasmid of each Fab fragment chain were used.

Fig. 2. Expression, purification and HPLC analysis of Mabfilin Cetuximab-CL-141926. (A) SDS-PAGE analysis of cell culture supernatant and purified fractions (viaProtein A affinity chromatography and gelfiltration) of Cetuximab-CL-141926 expressed in HEK293F™ cells. Protein of interest is marked by arrows. (B) Sizeexclusion profile of Cetuximab-CL-141926 using a Superdex™ 200 1/500 Tricorn column and PBS running buffer. (C) Reversed phase profile using a Grace Vydaccolumn (C4, 214MS54, 4.6 × 250 mm, 5 μm, 300 Å). Constant line represents UV profile at 280 nm and dotted line illustrates solvent composition as rate of 100%Acetonitrile +0.1% TFA (solvent A) and 100% H2O + 0.1% TFA (solvent B).

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Transfection was performed according to the instructions of the man-ufacturer. Cell culture supernatant was harvested 144 h after transfec-tion.

OKT3-Mabfilin and OKT3-Fabfilin molecules were produced inhigher scales by keeping constant the ratios of described ingredients.Vitality of cells and growth were measured by propidium iodide (PI)staining using a cell counting system (Guava® easyCyte HT, Merck-Millipore). Expression was monitored by SDS-PAGE analysis of cellculture supernatant.

2.2. Purification

Cetuximab-Mabfilin molecules were purified using an ÄKTAxpresspurification system (GE-Healthcare) including affinity interaction (AIC)and size exclusion chromatography (SEC). For the AIC step a HiTrapProtein A FF 1mL column (GE Healthcare) was run with 20mM

NaH2PO4 (pH 7.0) binding buffer. The proteins were eluted with 0.1Mcitrate buffer (pH 3.0), directly loaded to a gelfiltration column(Superdex 200 16/600 pg, GE Healthcare) and run with PBS (pH 7.3).

Cetuximab-Fabfilin proteins were purified by one step purificationusing 300 μL of KappaSelect matrix packed into spin column cartridges(Sigmaprep spin columns, Sigma Aldrich). 5mL of cell culture super-natant were added successively to the spin columns followed by 4washing steps with PBS. The proteins were eluted in 4 steps with 0.1Mglycine buffer (pH 2.5). The maximum applied volume to the columnwas 600 μL. Centrifugation steps were performed for 1min at 200× gat room temperature.

OKT3 Mabfilin and Fabfilin molecules were purified by immobilizedmetal affinity chromatography (IMAC) and size exclusion chromato-graphy (SEC). For the AIC step a HisTrap HP 1ml column (GEHealthcare) was run with 50mM NaH2PO4, 300mM NaCl and 20mMimidazole (pH 8.0) binding buffer. The proteins were eluted with

Fig. 3. Expression, purification and HPLC analysis of Fabfilin Cetuximab Fab-CL-141926. (A) SDS-PAGE analysis of cell culture supernatant and purified fractions ofCetuximab Fab-CL-141926 expressed in the Expi293™ expression system. For purification, KappaSelect (GE Healthcare) affinity matrix was packed into spin columns.Protein of interest is marked with arrows. (B) Size exclusion profile of Cetuximab Fab-CL-141926 using a Superdex 200 1/500 Tricorn column and PBS runningbuffer. Different buffer composition of the sample induces baseline deflection at ∼9min. (C) Reversed phase profile using a Grace Vydac column (C4, 214MS54,4.6 × 250 mm, 5 μm, 300 Å). Constant line represents UV profile at 280 nm and dotted line illustrates solvent composition as rate of 100% Acetonitrile +0.1% TFA(solvent A) and 100% H2O + 0.1% TFA (solvent B).

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increased imidazole concentration at 500mM in the running buffer anddirectly loaded to a gelfiltration column (Superdex 200 16/600 pg, GEHealthcare) run with PBS (pH 7.3).

2.3. EGFR expression and purification

Extracellular domain of epidermal growth factor receptor (EGFR,GenBank NCBI: CAA25240.1 [Met1 – Ser645]) was genetically fused tohuman IgG1-Fc fragment (hIgG1-Fc, GenBank NCBI: AAC82527.1[Pro99 – Lys329]) via an IEGRMD amino acid linker and cloned into themultiple cloning site of pCMV6 vector using HindIII/NotI restrictionsites.

For expression of EGFR-Target, 5× 107 FreeStyle™ 293-F cells wereseeded in 100mL FreeStyle™ 293 expression medium (Thermo FisherScientific) in a 500mL Erlenmeyer flask (Corning) and cultured for24 h at 37 °C, 8% CO2, 95% humidity at 135 rpm. For transfection,100 μg of plasmid DNA of each antibody chain were mixed with 200 μgpolyethylenimine in 10mL of OptiPRO™ SFM medium (Thermo FisherScientific), incubated for 15min at room temperature and finally ap-plied to the cell culture. After 24 h, cell suspension was transferred intoa 1 L Erlenmeyer flask (Corning) and 100mL FreeStyle™ 293-F ex-pression medium were added. Cell culture supernatant was harvested

192 h after transfection. Vitality of cells and growth were assessed byNeubauer improved hemocytometer using bromphenol blue staining(Sigma Aldrich). Expression was monitored by SDS-PAGE analysis ofcell culture supernatant. For scaling up/down, the same ratios of eachcomponent were used.

The target was purified via affinity chromatography using a HiTrapProtein A FF 1mL column (GE Healthcare) running with 20mMNaH2PO4 (pH 7.0) binding buffer. The protein was eluted with 0.1Mcitrate buffer (pH 4.0) and immediately neutralized with 1M TRISbuffer (pH 9.0) to pH 7.3. Further purification was done by a gelfil-tration column (Superdex 200 16/600 pg, GE Healthcare) run with PBS(pH 7.3).

2.4. Analytical biochemical methods

SDS-PAGE analysis was performed with Novex NuPAGE™ bis-tris(4–12%) (Thermo Fischer Scientific) and Mini-Protean®TGX™ (BioRad)precast gel systems according to the instructions of the manufacturer.Samples were loaded directly into gel-slots after adding of samplebuffer without boiling.

For size exclusion chromatography (SEC) a Superdex™ 200 5/100 GL column (GE Healthcare) was run on a Dionex HPLC system(P680 HPLC-Pump, AS1-100 Automated Sample Injector and UVD 170U). Samples were injected at maximally 0.2 mg/mL in 50 μL total vo-lume. Analysis was performed with PBS running buffer (0.3 mL/min) atroom temperature for 20min.

Detection of impurities and folding intermediates by reversed phasechromatography (RP-HPLC) was established using an Ultimate 3000RS-System (Dionex) and a Grace Vydac (214MS54 C4, 4.6× 250mm,5 μm, 300 Å) column. Mobile phase was a mixture of 100% acetonitrile(buffer A) and 100% water for HPLC (buffer B) containing 0.1% TFArespectively. Flow rate was set at 1.5mL/min, column was heated to55 °C and 10 μL protein sample was injected. Buffer gradient profile isrepresented in the respective chromatogram.

Differential scanning fluorimetry (DSF). Samples with 0.1mg/mLprotein and 10 times Sypro®Orange (Sigma Aldrich) in a final volume of30 μL were transferred to a RT-PCR 96 well plate. The samples wereheated stepwise 1 °C/min up to 90 °C in a ViiA-7 (Applied Biosystems).Fluorescence was constantly measured at an excitation wavelength of520 nm and emission wavelength of 623 nm. All samples were mea-sured in triplicate and the derived fluorescence maximum was used fordetermination of mean thermal transition point using Protein ThermalShift™ Software v1.3 (Applied Biosystems).

Surface Plasmon resonance (SPR). Binding affinities were de-termined with a Biacore 3000 system on CM5 sensor chips. Antibodyand Affilin targets were coupled via standard NHS/EDC chemistry.hIgG-Fc (Athens Research & Technologies, Cat.No. 16-16-090707) wascoupled to respective reference flow cells. Binding curves were eval-uated with BiaEvaluation software (GE Healthcare) using Langmuir 1:1binding model.

2.5. Cell binding

Cell lines. For analysis of cellular EGFR and HER2 binding, CHO-K1(ACC-110, DSMZ) were transfected with the respective target cDNA inthe vector pCMV6-Entry (RC 217384 and RG212583, Origene), selectedwith G418 and single clones/lines isolated by limiting dilution. For CD3binding of HER2/CD3 bispecific proteins and respective control pro-teins, CD3 positive Jurkat cell line (ACC-282) and CD3 negative cellline K562 (ACC-10) from DSMZ were used.

Flow cytometry. Target overexpressing cells and control cells wereused. Adherent cells were detached from cell culture flask usingTrypsin/EDTA solution (Gibco), suspension cells harvested by cen-trifugation and diluted in pre-cooled FACS buffer (1× PBS, 3% FCS(Gibco)). Cell numbers were determined with a Neubauer improvedhemocytometer, adjusted to 1×106 cells/ml and cells were seeded into

Table 1Melting temperatures of Mabfilin and Fabfilin molecules and reference proteinsdetermined by differential scanning fluorimetry using SYPRO® Orange proteinstain.

Protein Melting temperature 1[°C]

Melting temperature 2[°C]

Cetuximab (MerckSerono)

68.6 /

Cetuximab (Scil Proteins) 69.0 /Cetuximab-CL-139819 65.8 69.7Cetuximab-CL-Ubiquitin 68.3 /Cetuximab-NL-139819 69.0 /Cetuximab-NL-Ubiquitin 68.9 /Cetuximab-CH-139819 67.9 /Cetuximab-CH-Ubiquitin 69.3 /Cetuximab-NH-139819 68.6 /Cetuximab-NH-Ubiquitin 69.0 /Cetuximab-NH-139819-CL-

13979165.8 70.0

Cetuximab-CL-141926 63.9 71.0Cetuximab-NL-141926 67.9 /Cetuximab-CH-141926 67.4 /Cetuximab-NH-141926 67.5 /

Cetuximab Fab (ScilProteins)

72.1 /

Cetuximab Fab-CL-139819 71.4 /Cetuximab Fab-CL-

Ubiquitin72.1 /

Cetuximab Fab-NL-139819 71.0 /Cetuximab Fab-NL-

Ubiquitin71.0 /

Cetuximab Fab-CL-141926 71.7 /Cetuximab Fab-CL-139090 71.8 /Cetuximab Fab-NL-141926 70.2 /Cetuximab Fab-NL-139090 70.7 /Cetuximab Fab-CH-141926 71.7 /Cetuximab Fab-CH-139090 72.1 /Cetuximab Fab-NH-

14192671.4 /

Cetuximab Fab-NH-139090

72.1 /

OKT3 69.8 /OKT3-CL-142628 62.6 68.1OKT3-CL-139090 68.8 /

OKT3 Fab 69.0 /OKT3 Fab-CL-142628 68.1 /OKT3 Fab-CL-139090 69.1 /

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a 96-well plate (Greiner, U-shape) in triplicate for each cell line.Different concentrations of binding proteins were diluted in FACSbuffer, added to the cells and incubated for 45min at 4 °C. Cells werewashed with 100 μL buffer/well and centrifuged for 3min at 800× g.For the detection of Cetuximab-Mabfilin and Cetuximab-Fabfilin mo-lecules, the supernatants were removed and goat anti-human IgG AlexaFluor 488 antibody (Invitrogen, Cat.-No.: A-11013) 1:1000 diluted inFACS blocking buffer was added and the samples incubated for30min at 4 °C. Binding of OKT3-Mabfilin and OKT3-Fabfilin moleculeswas detected by Rabbit Anti-Strep-Tag antibody (GenScript, Cat.-No.:ABIN1573895) 1:300 diluted followed by goat anti rabbit IgG AlexaFluor 488 antibody (Invitrogen, Cat.-No.: A-11008) 1:1000 diluted inFACS blocking buffer. Each antibody was incubated for 30min at 4 °C.After washing, cells were fixed for 10min using 1% PFA solution at4 °C, resuspended in 100 μL PBS buffer and measured on the Guava®

easyCyte HT device (Merck Millipore) at excitation wavelength 499 nmand emission wavelength 520 nm.

2.6. Anti-proliferation assay

To determine a potential antiproliferative effect of Cetuximab-antiEGFR-Affilin molecules (Mabfilin and Fabfilin), the EGFR over-expressing cell line A431 (ACC-93, DSMZ, Braunschweig, Germany)was used. Adherent cells were detached from cell culture flask usingTrypsin/EDTA solution (Gibco) and cell number was determined withNeubauer improved hemocytometer, adjusted to 200 cells/ml in RPMI-1640 medium (Lonza) without any supplements. 4000 cells weretransferred to each well of a 96 well tissue culture plate and incubatedfor 24 h at 95% humidity, 5% CO2 and 37 °C. Cetuximab-Mabfiline andCetuximab-Fabfilin proteins were diluted into RPMI-1640 mediumcontaining 6mM EGF (BioVision, Cat.-No.: 4022-500) and 50 μL weretransferred to the wells. EGF-positive control includes only 6mM EGF,negative control was RPMI-1640 medium without any supplement.Cells were incubated for 72 h at 95% humidity, 5% CO2 and 37 °C.Proliferation of cells was determined by adding 10 μL/well WST-1-re-agent (Roche) and measuring the absorption 450 nm/620 nm on a platereader (Tecan Infinite M200 Pro) after 10min incubation at roomtemperature.

Fig. 4. Biacore sensorgrams of Cetuximab based proteins. (A) Sensorgram comparing binding of inhouse produced Cetuximab antibody (Scil Proteins) to commercialantibody available from Merck Serono to immobilized recombinant extracellular domain of EGFR as Fc fusion (1440 RU) at concentrations of 25 nM–0.39 nM in 1:4dilution series. (B) Binding curves of Mabfilin Cetuximab-CL-141926 versus immobilized extracellular domain of EGFR (Cat.# EGR-H5222, ACRO Biosystems) (1474RU) at the indicated concentrations. (C) Binding curves of Cetuximab-CL-141926 versus immobilized extracellular domain of HER2 as Fc fusion (Cat.# 1129-ER-050,R&D Systems) at the indicated concentrations. (D) Simultaneous binding of Cetuximab-CL-141926 and Cetuximab-NL-141926 Mabfilin variants at a concentration of50 nM. Proteins were injected to a chip with immobilized HER2-Fc (Cat.# 1129-ER-050, R&D Systems) (1033 RU), after 350 s, extracellular domain EGFR-Fc(recombinantly expressed, Scil Proteins) was injected at concentrations between 100 nM and 25 nM in a 1:2 serial dilution.

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3. Results and discussion

3.1. Production and characterization of Cetuximab-HER2 Affilin fusionproteins (bispecific formats)

Mabfilin and Fabfilin molecules were each expressed as separatechains on two different plasmids. Constructs were developed in amodular way providing antibody chain segments with appropriatesignal peptide sequences, tags and separating linkers. A cloning cassettewas engineered into the plasmids for variable fusion of selected Affilinmolecules and the antibody chain in all possible orientations as de-picted in Fig. 1. The well characterized chimeric antibody Cetuximabtargeting the EGFR receptor was chosen for EGFR/HER2 bispecificformats. These fusion proteins comprise the antibody or Fab fragmentchains connected at one of the four possible ends to an Affilin targetingthe HER2 receptor (141926). For each construct, ubiquitin wildtype atthe respective fusion position and the antibody itself served as controls.All proteins could be transiently expressed in the 293F or Expi293system (Thermo Fisher Scientific) in sufficient amounts although withclear difference between the mAb- and the Fab-fusions. The yields ofCetuximab-141926 Mabfilin variants from non-optimized small scaleexpression cultures of 60mL were in the range of 5–23mg/L and thoseof the corresponding Fabfilin variants in the range of 74–143mg/L cellculture supernatant (Table S1). This outcome correlates well with thenumbers reported for standard antibodies or Fab fragments in the lit-erature. Thus, the Affilin fusion has no impact on the expression effi-ciency compared to the control proteins Cetuximab and Cetuximab-Fab.

Appropriate purification strategies and protocols were developed toprovide lab scale amounts of proteins for further characterization.

60mL culture supernatant with Mabfilin were applied to Protein A af-finity chromatography followed by a size exclusion column. After thetwo step purification routine variants were obtained with good qualityas shown by SDS-PAGE (Fig. 2A) and analytical SE- and RP-HPLC(Fig. 2B and C, for variant Cetuximab-CL-141926). The proteins arewell expressed, secreted to cell culture supernatant and extracted by thechosen purification strategy. In the SE-HPLC analysis the variants showsome interaction with the column matrix, but no aggregation or frac-tionation. Peaks from RP-HPLC runs are monomodal demonstratinghigh purity.

Supernatants with expressed Fabfilins were purified in KappaSelectequipped spin columns yielding sufficiently pure protein as shown forCetuximab Fab-CL-141926 (Fig. 3). A distinct expression band andenrichment on KappaSelect can be visualized by SDS-PAGE Fig. 3A).HPLC analysis shows single monomodal peaks pointing to high proteinpurity and homogeneity (Fig. 3B and C).

The stability of the proteins was assessed by DSF. Thermal transitionwas measured for most Cetuximab-141926 Mabfilin variants with onetransition point between 64 and 68 °C (Table 1). Two transition pointswere induced by fusion of an Affilin molecule but not ubiquitin to the C-terminus of the light chain of Cetuximab, see for example Cetuximab-CL-141926 (Fig. S1 and Table 1). This behavior is typical for differentialunfolding of the Fc and the Fab parts of antibodies, respectively, andmight point to a slight effect of this combination on the overall struc-ture of the molecule. However, this analysis cannot discriminate if thepositioning phenomenon leads to altered unfolding of the Fab or that ofthe Affilin part of the fusion protein. There seems to be no negativeimpact of this property on the function of the respective molecules asthey show the same or slightly better performance compared to the

Table 2Affinity parameters of Mabfilin and Fabfilin molecules.

Protein name target antibody/Fab-fragment target Affilin Affinity antibody/Fab-fragment target Affinity Affilin target

KD [nM] Rmax [RU] KD [nM] Rmax [RU]

Cetuximab (Merck Serono) EGFR ≤ 0.2 84Cetuximab (Scil Proteins) EGFR ≤ 0.2 80 ←Cetuximab CL-139819 EGFR EGFR ≤ 0.1 208 ←Cetuximab CL-Ubiquitin EGFR control ≤ 0.2 96 /Cetuximab NL-139819 EGFR EGFR ≤ 0.6 96 ←Cetuximab NL-Ubiquitin EGFR control ≤ 0.4 29 /Cetuximab CH-139819 EGFR EGFR ≤ 0.3 149 ←Cetuximab CH-Ubiquitin EGFR control ≤ 0.2 79 /Cetuximab NH-139819 EGFR EGFR ≤ 0.8 120 ←Cetuximab NH-Ubiquitin EGFR control 0.5 51 /Cetuximab NH-139819-CL-139791 EGFR EGFR ≤ 0.3 149 ←Cetuximab CL-141926 EGFR (mono) HER2 1.0 117 1.4 142Cetuximab NL-141926 EGFR (mono) HER2 2.4 11 ≤ 3.1 80Cetuximab CH-141926 EGFR (mono) HER2 ≤ 0.6 37 6.6 129Cetuximab NH-141926 EGFR (mono) HER2 1.1 16 3.4 120

Cetuximab Fab (Scil Proteins) EGFR 1.3 46 ←Cetuximab Fab-CL-139819 EGFR EGFR ≤ 0.3 116 ←Cetuximab Fab-CL-Ubiquitin EGFR control 1 46 ←Cetuximab Fab-NL-139819 EGFR EGFR ≤ 0.6 85.1 ←Cetuximab Fab-NL-Ubiquitin EGFR control 1.3 36,6 ←Cetuximab Fab-CL-141926 EGFR HER2 0.9 48 26 145Cetuximab Fab-CL-139090 EGFR control 1.1 51 /Cetuximab Fab-NL-141926 EGFR HER2 2.2 32 237 171Cetuximab Fab-NL-139090 EGFR control 1.3 38 /Cetuximab Fab-CH-141926 EGFR HER2 1.8 44 99.8 142Cetuximab Fab-CH-139090 EGFR control 1.6 50 /Cetuximab Fab-NH-141926 EGFR HER2 3.1 36 195 148Cetuximab Fab-NH-139090 EGFR control 1.9 41 /

OKT3 CD3 Binding detectedOKT3 CL-142628 CD3 HER2 ≤ 0.5 305OKT3 CL-139090 CD3 control /

OKT3 Fab CD3 Binding detectedOKT3 Fab-CL-142628 CD3 HER2 1.1 216OKT3 Fab-CL-139090 CD3 control /

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other combinations (see binding analyses below). Whether this iscaused by an enhancement specifically based on the CL-fusion andconcomitant structural flexibility indicated by a second unfolding pro-cess is speculative at the moment. It might also be that the other fusionsites are affected. Tm values around 71 °C were observed for Cetuximab-Fab and Cetuximab-Fabfilin fusion proteins (Table 1).

Binding to the respective targets was analyzed by surface plasmonresonance spectroscopy (Fig. 4). Cetuximab produced in house was setas reference as its binding properties were comparable to the com-mercially available antibody (Fig. 4A).The Mabfilin constructs showedbinding to the extracellular domain of EGFR in the same range knownand actually measured for Cetuximab. Due to a very slow dissociation,the reliable determination of the koff rates was difficult for immobilizedextracellular domain EGFR-Fc (ED-EGFR-Fc) and monomeric ED-EGFRas ligands (for example, see CL-141926 in Fig. 4B). Binding parametersfor Cetuximab-141926 Mabfilin variants are given in Table 2. All C-terminal fusions of Affilin or ubiquitin yielded higher Rmax values thanthe N-terminal fusions, in particular. Mabfilin CL-141926 with tenfoldincreased value compared to Mabfilin NL-141926. This could indicatean inhibition of the binding of the variable antibody domains to thetarget in the presence of a further binding domain in their closeproximity.

To evaluate the second specificity, the Mabfilin proteins were testedfor their binding to the ED- HER2-Fc (Cetuximab CL-141926 in Fig. 4C).Fusion of an Affilin molecule in either one of the four possible positionsresulted in nanomolar affinities for HER2-Fc (Table 2). Furthermoresimultaneous engagement of both targets was clearly detectable forMabfilin fusion proteins (Fig. 4D). The two step binding of the CL andthe NL variant reflect the results of the single target experiments, withCL-141926 being superior and NL-141926 showing the weakest overallsignal.

Hints for possible effects of the relative orientation of the fusion ofthe Affilin to the antibody were also observed in the correspondingCetuximab-141926 Fabfilin analyses. SPR binding parameters for theinteraction with the ED-EGFR-Fc are given in Table 2 and the bindingcurves of Cetuximab Fab CL-141926 are shown exemplarily in Fig. 5A.Fusions of di-ubiquitin (139090) to the Ne or C-terminus of eitherantibody chain did not induce significant changes in the apparent KD ofthe Fab fragment. The same effect was detected for the C-terminal an-chorage of anti-HER2 Affilin 141926. However, its fusion to the N-terminus of the heavy or light chains resulted in slightly lower affinitiescompared to the Fab fragment alone, maybe induced by interference ofAffilin 141926 with the binding moiety of the Fab fragment. CetuximabFab-NH- or -NL-141926 variants showed higher KD values for bindingthe target HER2 than Cetuximab Fab-CH-141926 and -CL-141926variants (Table 2, binding curve of Cetuximab Fab-CL-141926 inFig. 5B). Thus, positioning of the Affilin at the C-terminus of the lightchain was advantageous in case of these fusion proteins. Simultaneousbinding of both targets could be demonstrated for all Fabfilin variantstested (see for example Cetuximab Fab-CL-141926 in Fig. 5C).

Recombinantly produced extracellular parts of the receptor tyrosinekinases EGFR and HER2 were used to determine target binding in SPRmeasurements. However, cellular full length receptors are presented ina more natural environment. For unequivocal analysis, all targets wereexpressed in the neutral background of CHO cells. Binding of theMabfilin and Fabfilin variants to the respective target overexpressingCHO cells was determined by flow cytometry at different concentra-tions. Fig. 6A shows the concentration dependent binding of Cetux-imab-141926 Mabfilin proteins to CHO-EGFR cells. Cellular binding isdetectable for all variants with slight differences between the C-term-inal and N-terminal fusion, indicating a negative effect of the mod-ification at the N-terminus of the antibody, which is a region involvedin the main interaction of Cetuximab and its target. This was supportedby similar findings for wildtype di-ubiquitin control proteins with thesame tendency arguing for an unspecific hindrance and against anunfavorable property of a specific Affilin. Cetuximab-141926 Mabfilin

Fig. 5. Biacore sensorgrams of Fabfilin Cetuximab Fab-CL-141926. (A) Bindingcurves of Cetuximab Fab-CL-141926 versus EGFR-Fc (Scil Proteins) (1440 RU)and (B) versus HER2-Fc (Cat.# 1129-ER-050, R&D Systems) (1103 RU) im-mobilized on a CM5 Chip (GE Healthcare) at the indicated concentrations. (C)For determining simultaneous binding Cetuximab Fab-CL-141926 was injectedat a concentration of 50 nM to a HER2-Fc (Cat.# 1129-ER-050, R&D Systems)(1103 RU) immobilized chip. After 350 s, extracellular domain EGFR-Fc (ScilProteins) was injected at concentrations between 100 nM and 1.24 nM in a 1:3dilution series.

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(caption on next page)

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fusion proteins bound to CHO-HER2 cells with only minor differences inefficiency between the CL- and NHe variants (Fig. 6B). No binding onCHO control cells transfected with empty vector was measurable.

Cetuximab Fabfilin variants were assessed for cellular binding(Fig. 6C and D) and bound specifically to the respective target cells butnot to control cells. C-terminal fusions of Affilin or ubiquitin to Ce-tuximab Fab showed the tendency to result in higher overall MFIs onCHO-EGFR cells (Fig. 6C). The N-terminally fused counterparts withlower binding signals point to a preference to exclude the variable re-gion of the antibody from modification. In case of binding to CHO-HER2 cells (Fig. 6D), the favorable position of the Affilin in the mole-cular context of the Fabfilin also seems to be the C-terminus suggestinga mutual influence of the EGFR and the HER2 binding in the case ofnear proximity.

3.2. Production and characterization of Cetuximab-EGFR Affilin fusions(bi-epitope formats)

EGFR-binding Affilin variants fused to Cetuximab chains at differentpositions were produced and tested for functionality to validate themulti-epitope targeting concept. Expression and purification of Mabfilinand Fabfilin variants yielded amounts of 4–30mg/L for Mabfilin and83–90mg/L for Fabfilin molecules (Table S1). Protein analytics forpurity and uniformity revealed well-behaved proteins with monomodalcharacteristics in the analytical SE- and RP-HPLC runs (Fig. S2 A-D:example Cetuximab Fab-CL/NL-139819). Thermal stability determinedby DSF showed Tm values between 66 and 69 °C for Mabfilin andaround 71 °C for Fabfilin molecules (Table 1). All analyzed Mabfilin andFabfilin variants bound their target EGFR in SPR analyses at least asefficient as the parent antibody or Fab fragment. The exact KD ofMabfilin variants for EGFR could not be determined because of theirvery slow dissociation and limitations of the used SPR instrument. TheKD determination for the Fabfilin format lacking the avidity effect fromFc pairing was assessable. In Fig. 7B binding curves of SPR measure-ments by Biacore on ED-EGFR-Fc are shown for Cetuximab Fab-CL-139819 as example. The corresponding KD values are given in Table 2comparing Cetuximab Fab-CL-139819 and Cetuximab Fab-NL-139819with the related control proteins. Affilin 139819 contributes notably tobinding affinity in this format irrespective of its point of attachment.Compared to the unmodified Cetuximab Fab shown in Fig. 7A an atleast twofold lower dissociation constant can be calculated frombinding kinetics for the NL fusion protein and at least threefold affinityenhancement for the CL fusion protein. The latter might be caused byless interference of the fused Affilin with the target binding region ofthe Fab. Accordingly, the additional EGFR binding moiety 139819 at-tached to the Ne or C-terminus of the light chain led to subnanomolarKD values mainly triggered by better koff characteristics.

Concentration dependent binding of all Mabfilin and Fabfilin var-iants to EGFR expressing cells was confirmed (Fig. 7B and C). Moreover,to assess the functional impact of compounds with multi-domainbinding, growth inhibition was measured for bi-paratopic Cetuximab-139819 Mabfilin and Fabfilin molecules in comparison to Cetuximaband its Fab fragment, respectively. The Cetuximab-CL-139819 Mabfilinhaving the preferred architecture showed a concentration dependentnegative influence on the proliferation of EGFR overexpressingA431 cells (Fig. 8A) exceeding the effect of Cetuximab and Cetuximab-NH-139819 Mabfilin. Fusions with ubiquitin at the same positions wereused as control. These results are consistent with the Biacore binding

data. In the Fabfilin format, both Cetuximab Fab-CL-139819 and Ce-tuximab Fab-NL-139819 led to a marked proliferation inhibition(Fig. 8B). This lack of differentiation might merely arise from thesmaller size of the Fabfilin molecules or a different molecular geometryas in the Mabfilin.

We also tested a format with two different Affilin variants with thesame specificity fused to Cetuximab at opposite attachment points,yielding Mabfilin Cetuximab NH-139819-CL-139791. This conceptworked in terms of manufacturability and functionality; however it wasnot evaluated in greater depth or tested in more detail. The proteincould be produced in similar amounts as the mono-substituted mole-cules (Table S1) and was equally stable (Table 1) and binding compe-tent (Table 2). This suggests an even broader flexibility in the design ofthese multifunctional entities.

3.3. Production and characterization of OKT3-HER2 Affilin fusions (bi-specific formats for immunotherapy)

To validate the described formats in a different context, we en-gineered Mabfilin and Fabfilin variants based on CD3 specific murineantibody OKT3 and anti-HER2-Affilin variants. HER2-specific Affilin142628 was fused to the putative most tolerable position CL and thefusion protein was produced in mammalian cells concomitant with thematching controls. The constructs were cloned as described above.Expression tests showed moderate levels of the parental antibody OKT3and consistently lower yields of the respective Affilin fusion proteinscompared to the analogues with Cetuximab. Two cell lines, CHO-S orExpi293 were used for transient expression to account for species dif-ferences. However, yields in these cells were similar between 3.7 and70mg/L (Table S1). All proteins could be expressed and highly purifiedvia Ni-NTA affinity chromatography and gel filtration. Tm values ofaround 63 °C for the Mabfilin and 69 °C for the Fabfilin indicate goodoverall stabilities (Table 1). Again the fusion of the Affilin to the C-terminus of the light chain induced a biphasic melting behavior. SPRmeasurements prove preservation of the binding characteristics of theparental antibody and Affilin respectively (Fig. 9A and B, Table 2) andsimultaneously (Fig. 9C). CD3 is a complex multidomain entity en-tangled in the T-cell receptor formation. It is not possible to exactlyreconstitute this entity biochemically in vitro. Therefore, SPR analysiswas performed with the epsilon domain of CD3 carrying the OKT3epitope, additionally stabilized or complemented by the delta subunit.This analysis suffices for a first estimate of putative CD3 binding but itis mandatory to further evaluate the binding in more physiologic cel-lular context. For CD3 recognition of HER2/CD3 bispecific proteins,CD3 positive Jurkat cell line and CD3 negative control cell line K562were used. A specific interaction with the target expressing Jurkat cellscould be observed by flow cytometry (Fig. 10A). Likewise, binding tocellular HER2 was confirmed in a similar way as for the Cetuximab-Affilin-fusion proteins although the Affilin lost some of its affinity inthis context (Fig. 10B).

4. Conclusion

Introducing multispecificity into therapeutic or diagnostic bindingmolecules holds great potential for creative applications in medical ortechnical contexts [34]. The challenge is to develop flexible and stableformats preferably accessible in short time periods. The prototype for thisapproach is a bifunctional antibody [35], bearing certain limitations.

Fig. 6. Flow cytometry of Mabfilin and Fabfilin molecules binding to EGFR or HER2 overexpressing cell lines. Cetuximab-141926 Mabfilin variants and Cetuximab(Scil Proteins) were tested on (A) EGFR- and (B) HER2-overexpressing cell lines. Cetuximab-141926 Fabfilin molecules were tested in the same way on (C) EGFR and(D) HER2 overexpressing lines at the indicated concentrations. PBS column represents buffer blank and pENTRY column exemplary binding of in house producedCetuximab (Scil Proteins) (A+ B), Cetuximab Fab-CL-141926 (C) and Cetuximab Fab-NH-141926 (D) to control cell line at highest measured concentration.Wildtype column (D) represents binding of Cetuximab Fabfilin variants with di-ubiquitin wildtype Affilin-139090 at 50 nM (exemplarily Cetuximab Fab-CL-139090)as control. No unspecific binding of the described controls was detected. Error bars represent SD of triplicates.

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Fig. 7. SPR and flow cytometry analysis of Cetuximab-139819 Mabfilin and Fabfilin molecules. (A) Sensorgrams with binding curves of Cetuximab Fab fragment and(B) Fabfilin Cetuximab Fab-CL-139819 versus immobilized extracellular domain of EGFR-Fc (Scil Proteins) (1440 RU). Proteins were injected in different con-centrations from 25 nM to 0.4 nM. Concentration dependent cell binding of (C) Mabfilin and (D) Fabfilin molecules on EGFR overexpressing cell lines is shown. PBScolumn represents buffer blank and pENTRY column exemplary binding of Cetuximab-CL-139819 (C) and Cetuximab Fab-NL-139819 (D) molecules to control cellline at highest measured concentration. No unspecific binding was detected in the controls. Error bars represent SD of triplicates.

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Many variations on the theme have been put into practice and someproducts already entered the clinic [36,37]. The combination of antibodyand non-antibody binding molecules opens up an attractive way to ac-celerate development time by starting with well characterized antibodycandidates and adding a new binding component. Scaffold binders areideal for this task because of their small size, favorable biochemicalproperties, and quick generation times. We used Affilin molecules toendow known antibodies with additional properties. To be furthermoreflexible in size and optimal fit for the desired application, whole anti-bodies and Fab fragments thereof were utilized for different showcasecombinations. All tested examples could be produced and retained theirspecific binding properties to recombinant or cellular targets. Propensityfor aggregation or fragmentation was very low throughout the formats.In the presented data a tendency for preferred coupling sites could benoticed in some instances. Although a general recommendation con-cerning this cannot be given, different behavior of the respective con-structs opens up even further space for modulation of distinct properties.

Efficacy, specificity, mode of action and other parameters describing thepharmacodynamics and pharmacokinetics might vary and could possiblybe influenced by means of the molecular design. This has to be estab-lished from case to case and would have to be finally validated through invivo testing for the individual situation. For example, the putative thirdfunctionality of the Mabfilin constructs, the Fc part holds the potential tobe influenced by the combination with a specific Affilin in the desireddirection, i.e. preservation or weakening of physiological Fc interactions.The presented concept could be easily extended to tri- and multispecificformats with different Affilin entities connected at eligible positions.Examples for such complex scenarios have also been tested by us andproved to be comparably technically feasible as the variants shownabove. Along these lines we open up a new flexible route for cutting-edgetherapy.

Fig. 8. Proliferation assay of Cetuximab-139819 Mabfilin and Fabfilin molecules on EGFR overexpressing cell line A431. Cells were seeded into a 96-well tissueculture plate in RPMI-1640 medium without any supplement and incubated for 24 h. (A)Mabfilin and (B) Fabfilin proteins and controls were diluted into RPMI-1640medium containing EGF and added to the wells at the indicated concentrations. After incubation for 72 h WST-1 reagent was added and absorption measured. Errorbars represent SD of triplicates. The assay was independently repeated twice.

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Fig. 9. Biacore sensorgrams of Mabfilin OKT3-CL-142628 versus HER2-Fc or CD3εδ and simultaneous binding to both targets. (A) Binding curves of OKT3-CL-142628 versus immobilized extracellular domain of HER2-Fc (Cat.# 1129-ER-050, R&D Systems) (2531 RU) at the indicated concentrations. (B) Binding curvesversus immobilized CD3εδ-Fc (Cat.# CT026-H0323H, Sino Biological Inc.) (2461 RU) at the indicated concentrations. (C) For simultaneous binding, OKT3-CL-142628 at 50 nM was injected to a chip with immobilized HER2-Fc (2531 RU), followed by injection of extracellular domain CD3εδ-Fc at concentrations between1 μM and 197.5 nM in 1:1.5 dilution series.

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Author contributions

Conceived and designed the experiments: MK FS UH EBD. Performedthe experiments: MK FS PK. Analyzed the data: MK FS PK EBD. Wrote thepaper: MK EBD. Made substantive contributions to the manuscript: FS.Reviewed the final manuscript: MK FS PK UH EBD. Edited the manuscript:EBD.

Conflicts of interest

The authors declare that they have no conflict of interest.

Acknowledgement

We thank Anja Eicke and Anett Swoboda for expert technical as-sistance and Kerstin Haucke for helpful contributions to the manuscript.

This research did not receive any specific grant from fundingagencies in the public, commercial, or not-for-profit sectors.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.pep.2018.04.013.

Fig. 10. Flow cytometry of OKT3 Mabfilin and Fabfilin molecules binding to HER2 or CD3 expressing cell lines. (A) Binding of OKT3-142628 Mabfilin and Fabfilinmolecules and of inhouse produced OKT3 antibody and Fab-fragment to CD3 expressing Jurkat cell line tested at 33.3 nM. K562 column represents exemplarybinding of OKT3-CL-142628 to non CD3 expressing control cell line. Buffer (PBS) control is shown for both cell lines. (B) Concentration dependent binding of OKT3-CL-142628 Mab- or Fabfilin to HER2 overexpressing cell line CHO-K1. Affilin-142628, OKT3 and OKT3 Fab-fragment were used as controls at 50 nM. PBS columnsrepresent buffer blank for both cell lines and pENTRY column the CHO empty vector control with exemplary binding of 50 nM OKT3-CL-142628. Error bars representSD of triplicates.

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