Haiqin Li

Viven Denis

Franc ¸ois Gautier Adrien Pelat

Cyril Touzé

Nonlinear energy transfer to improve the acoustic black hole effect

Acoustic Black Hole effect (ABH) describes a passive vibration mitigation technique used to damp out the flexural vibrations of thin structures such as beams. It relies on a wedge profile where the thickness is gradually decreasing, so that wave velocities slows down, potentially decaying to zero without reflection. The device generally shows very interesting damping properties in the mid and high-frequency range, however its efficiency in the low-frequency range still remains limited. In this contribution, the inclusion of a nonlinearity as a way to improve the low-frequency performance of an ABH is investigated. Indeed, the nonlinearity may be used as a vector to transfer energy from the low to the high frequency range, where the damping properties are much more significant. Two different mechanisms are tested, based respectively on geometric and contact nonlinearity.

Acoustic black hole

Acoustic Black Hole (ABH) effect is a passive vibration damping technique without added mass. A common implementation is a plate edge where the thickness is locally reduced with a power law profile and covered with a viscoelastic layer. Such mechanisms have been deeply investigated since the pioneering works by Mironov and Krylov [START_REF] Krylov | Acoustic "black holes" for flexural waves as effective vibration dampers[END_REF]. Nowadays, a number of studies show its efficiency thanks to theoretical, numerical and experimental studies [START_REF] Georgiev | Damping of structural vibrations in beams and elliptical plates using the acoustic black hole effect[END_REF][START_REF] Denis | Modal overlap factor of a beam with an acoustic black hole termination[END_REF][START_REF] Oboy | Damping of flexural vibrations in rectangular plates using the acoustic black hole effect[END_REF][START_REF] Denis | Measurement and modelling of the reflection coefficient of an acoustic black hole termination[END_REF][START_REF] Bowyer | Effect of geometrical and material imperfections on damping flexural vibrations in plates with attached wedges of power law profile[END_REF]. In particular the mitigation properties of a beam with a wedge profile are very important, especially in the mid-and high-frequency range. This is assessed in Fig. 1 where the point mobility (ratio between the vibration velocity and the input exciting force) of a tapered beam (ABH) is compared to that of a uniform beam. Flexural vibrations of the beam are considered, the beam being excited at a single point by a large-band noise excitation. The point mobility of the uniform beam is shown in blue and presents sharp peaks due to the fact that the inner damping is small. The red curve is the point mobility for the ABH, and two different configurations are selected. In Fig. 1(a), the beam is 1.5 m long, with thickness 5 mm. The tapered edge is 20 cm long and the terminal thickness 100 µm. In Fig. 1(b) a shorter beam is considered, with length 1 m and thickness 4 mm. The ABH region is 9 cm long and the terminal thickness 20 µm. In both cases, one can observe that the mobility is severely reduced in the mid and high-frequency range, such that the sharp peaks have been replaced by light bumps, indicating that the damping properties of the beam has been profoundly improved. However, below a certain frequency denoted as the cut-on frequency, the sharp peaks persists and the damping capacity brought by the ABH effect is severely reduced. This cut-on frequency can be roughly estimated around 900 Hz in Fig. 1(a), and 500 Hz in Fig. 1(b).

Improvement due to nonlinearity

The main idea consists in adding a nonlinear mechanism into the vibrational behaviour in order to transfer energy from the low to the high-frequency range, where it is more efficiently dissipated. Two different nonlinearities are tested. The first case consists in using the geometric nonlinearity, due to large amplitude vibrations. Indeed, in the wedge profile where the thickness is decreasing, the amplitude of the vibrations becomes rapidly of the same order as the thickness, hence exciting geometric nonlinearity. The device can then be adapted by using a longer part of the beam with small residual thickness, in order to excite more easily the wave turbulence regime, able to create an energy flux from the large to the small wavelengths [START_REF] Cadot | Wave turbulence in vibrating plates[END_REF][START_REF] Denis | Improvement of the acoustic black hole effect by using energy transfer due to geometric nonlinearity[END_REF]. The second option is to use a unilateral barrier composed of contact points in order to excite a non-smooth nonlinearity. Fig. 2(a) shows the results obtained for the geometric nonlinearity. The power spectral density of the beam shown in Fig. 1(a), excited with a 100-500 Hz white noise shows that, increasing the length of the beam where the thickness is small and constant, energy leakage is clearly observed so that the global amplitudes of the peaks are decreasing in the low-frequency range. Fig. 2(b) shows the results obtained with the contact nonlinearity, and for the beam which mobility is represented in Fig. 1(b). The excitation is a 0-500 Hz white noise, and three different configurations of contact points are used to show how the energy is transfered. Once again, the main peaks in the low-frequency range are broken (see the zoom on the third mode as inset), so that the global energy in this band is decreasing.

Conclusions

Nonlinear energy transfer provides a practical solution to improve the overall efficacy of ABH performance. Two solutions are tested and compared. More detailed results including quantitative comparisons thanks to dedicated indicators allowing for a complete assessment will be provided. In particular, results show that both methods give good result in terms of improving the performance, however the contact nonlinearity seems to be more efficient as being immediately active. On the other hand, the time scales associated to wave turbulence are longer, so that in the case of geometric nonlinearity, energy transfer takes more time to settle down and pump the energy.

Figure 1 :

 1 Figure 1: Point mobility compared between reference (uniform beam, blue line) and ABH beam (red line). (a) Beam of length 1.5 m, terminal thickness at ABH edge 100 µm. (b) Beam of length 1 m, terminal thickness 20 µm.

Figure 2 :

 2 Figure 2: (a) Power spectral density of velocity response to a 100-500 Hz white noise, normalized by the variance of the excitation, in Comparison between 5 different edge termination : E0 (standard ABH with power-law tapered edge) in dashed light blue, E1 (ABH with additional length of constant thickness 50 mm) in brown dash-dotted line, E2 (ABH with added length 100 mm) in red full line, E3 (ABH with added length 150 mm) yellow full line and E4 (added length 200 mm), black thick full line. (b) Velocity spectrum response to a 0-500 Hz white noise compared among different cases of contact condition. Blue line: no contact (standard ABH with power-law tapered edge). Red line: single contact point located at the maximum of eigenmode shape 2. Brown line: 4 unilateral contact points located at the maxima of modes 2-5. Black line: 4 bilateral contact points (up and down the beam).


Sébastien Ferrouillat

Patrice Tochon

Hassan Peerhossaini
email: hassan.peerhossaini@univ-nantes.fr

Micromixing enhancement by turbulence: application to compact heat exchanger-reactors

Keywords: Compact heat exchanger reactor, Micromixing, Metallic foam, Offset strip fins, Continuous system, Multifunctional heat exchanger

Introduction

Initially developed in the 1940s in the aerospace industry to provide compact, light, highly efficient heat exchangers for gas/gas applications, the compact plate-fin heat exchanger has potential applications as a reactor in the chemical industry: heat exchanger reactors (HEX reactors) or multifunctional heat exchangers (MHE). This technology offers many advantages: better reaction control (from the thermal point of view), improved selectivity (through intensified mixing, more isothermal operation and shorter residence time, and sharper residencetime distribution), reduction of byproducts, and greater safety. Indeed, although many traditional designs such as stirred tanks already incorporate heat transfer, in these devices there is a significant distance between the heat-transfer device and the site of the chemical reaction. The aim of the HEX reactor is to eliminate this distance by supplying or removing heat almost as rapidly as it is absorbed or generated by the reaction. The heat-transfer performance comparison of different MHE reactors in Table 1 shows the major advantages of MHE [START_REF] Trambouze | Les réacteurs chimiques de la conception à la mise en oeuvre[END_REF]. Indeed, thanks to its advanced heat-transfer performance and unlike the other technologies, this process should be able to realize strong exothermal reactions without thermal runaway.

However, mixing and in particular micromixing have an important influence on product quality in industrial reactions such as polymerization or precipitation. More precisely, industrial reactions depend on mixing at the molecular scale if their characteristic reaction times are close to the micromixing time. Thus, in order to improve such MHE reactors, it is important to characterize mixing at the molecular scale. Several chemical methods have been proposed to measure the micromixing generated by different geometries inserted in a continuous process [START_REF] Fournier | A new parallel-competing reaction system for assessing micromixing efficiency determination of micromixing time by a simple mixing model[END_REF][START_REF] Bourne | Micromixing and fast chemical reactions in static mixers[END_REF][START_REF] Guichardon | Characterisation of micromixing efficiency by the iodide-iodate reaction system. Part I: experimental procedure[END_REF]. The available literature reports many thermohydraulic data on compact heat exchangers. However, micromixing has almost never been studied in the same process. Therefore, the goal of this work is to quantify the micromixing level generated inside an MHE.

This paper presents an experimental micromixing study realized on different geometries a priori in order to intensify mass transfer in the process. It starts by validating the chemical method used. Then an estimate of the micromixing time generated by different geometries using the incorporation model is presented. In addition to a simple empty duct channel, two other geometries were studied:

-offset strip fins (OSF) -metallic foams This paper evaluates micromixing in each geometry and compares their performance. It also provides a database of the micromixing performances of several geometries so that the design engineer can choose the most appropriate geometry to intensify mass transfer and thus optimise a MHE.

A physicochemical experimental method for determination of micromixing

Experimental data obtained with test reactions show that physical parameters such as impeller speed or feed location can influence the distribution of reaction products in batch processes. Fournier et al. [START_REF] Fournier | A new parallel-competing reaction system for assessing micromixing efficiency: experimental approach[END_REF] reviewed the literature on ways to measure micromixing. Two main chemical methods have been detailed:

-consecutive competing reactions (diazo coupling test) developed by Bourne et al. [START_REF] Baldyga | Simplification of micromixing calculations. I: derivation and application of a new model[END_REF][START_REF] Bourne | Turbulent Mixing and Chemical Reaction[END_REF]:

A + B → R R + B → S
-parallel-competing reactions (iodide-iodate test) developed by Villermaux et al. [START_REF] Fournier | A new parallel-competing reaction system for assessing micromixing efficiency: experimental approach[END_REF][START_REF] Guichardon | Characterization of micromixing efficiency by the iodide-iodate reaction system. Part 1: experimental procedure[END_REF]:

A + B → R C + B → S.
According to Guichardon [START_REF] Guichardon | Characterization of micromixing efficiency by the iodide-iodate reaction system. Part 1: experimental procedure[END_REF][START_REF] Guichardon | Experimental comparison of the iodide-iodate and the diazo coupling micromixing test reactions in stirred reactors[END_REF], the iodide-iodate reaction is preferable for convenience in experimentation and analysis. Consequently, in this work, the micromixing level estimate generated by several geometries was tested using Villermaux et al.'s parallel-competing reaction scheme.

Villermaux et al. proposed a unique parallel-competing reaction system with A, B, R and S as H 2 BO 3 -,H + ,H 3 BO 3 and I 2 , respectively; C denotes the mixture of 5I -+IO 3 -:

H 2 BO 3 -+ H + → H 3 BO 3 quasi -instantaneous, (1)
5I -+ IO 3 -+ 6H + → 3I 2 + 3H 2 O very fast (2)
The redox Eq. ( 2) is fast (in the same range as the micromixing process), but is much slower than the neutralisation reaction (1).

Segregation index

The test procedure consists in adding in a stoichiometric defect, a small quantity of sulphuric acid, to a mixture of iodate, iodide and borate ions. The amount of iodine produced depends on the efficiency of the micromixing process when acid is a limiting reactant, so that the selectivity of iodine is a measure of the segregation state of the fluid. The fraction of acid used in the formation reaction of iodine is a convenient measure of the product distribution and makes possible the calculation of the selectivity of acid Y = 2n I 2 /n H 0 + and the index of molecularscale segregation:

X S = Y Y ST ,
where Y ST = 3C IO 3,0 -/(6C IO 3,0 -+ C H 2 BO 3,0 -) is the selectivity at total segregation. It should be mentioned that this normalized segregation index X s , whose value lies between 0 and 1, decreases with increasing micromixing level. The advantage of X s is that it is closely related to the ratio of two characteristic times: reaction time and micromixing time.

The iodine I 2 formed in reaction (2) can react further with iodide ions I -to yield I 3 -ions according to the quasiinstantaneous equilibrium

I 2 + I -→ I 3 - ( 3
)
The I 3 -concentration can be easily measured by spectrophotometry at 353 nm. Mass balances on iodine atoms yield the expressions

C I -= (C I -) 0 - 5 3 (C I 2 + C I 3 -) -C I 3 - (4)
K B = C I 3 - C I 2 C I - (5)
where K B is the equilibrium constant of reaction [START_REF] Bourne | Micromixing and fast chemical reactions in static mixers[END_REF]. By combining these two equations, we obtain a second-order algebraic equation for iodine concentration:

- 5 3 (C I 2 ) 2 + (C I -) 0 - 8 3 C I 3 -C I 2 - C I 3 - K B = 0 (6)
Resolution of this second-order algebraic equation makes it possible to estimate the iodine concentration and thus the segregation index X S .

Micromixing model

The segregation index is a measure of the influence of hydrodynamics on chemical selectivity. It depends on two processes:

-chemical processes with characteristic times that are directly linked to the kinetics of chemical reactions ( 1) and ( 2), -the physical process of micromixing, with the micromixing time.

For constant initial reactant concentrations, the variation in X S accurately takes into account the variation in micromixing level, namely the micromixing time. Under these conditions, X S can be directly compared for different micromixing conditions, allowing comparison of mixer performance. For nonconstant reaction time and micromixing time conditions, interpretation of the results is more complex and a specific micromixing model based on mixing and reaction-coupled processes must be used. Fournier et al. [START_REF] Fournier | A new parallel-competing reaction system for assessing micromixing efficiency: experimental approach[END_REF] used the incorporation model to estimate the micromixing time t m . Fig. 1 illustrates this model. Two fluids, 1 and 2, are considered. The model is based on the assumption that the fresh fluid 2 (sulphuric acid in the present experiments) is divided into aggregates that are progressively invaded by fluid 1 from the external environment, containing iodide and iodate in some basic medium. The characteristic incorporation time is assumed equal to the micromixing time t m . The volume of the aggregate grows as V 2 = V 2.0 g(t), where g(t) depends on the incorporation mechanism and is less than or equal to V 1,0 + V 2,0 . Two empirical evolution models are proposed: -a linear model: g(t)=1+t/t m , -an exponential model: g(t)=exp(t/t m ).

A constant incorporation flow rate leads to a linear relation time, and flow proportional to instantaneous aggregate volume leads to an exponential expression. The reactions are assumed to occur in aggregate 2, where perfect mixing is assumed achieved. In the reaction volume 2, the concentrations of the j species are given by:

dC j dt = (C j1,0 -C j ) 1 g dg dt + R j (7)
where subscript 1,0 indicates the surrounding fluid and R j is the specific rate of formation of reactant j. From the initial reactant concentrations and an assumed initial segregation index X S , resolving the differential equation system (7) lets us estimate the micromixing time t m .

Experimental test facility

Experiments were carried out in a test section made of a perspex rectangular duct channel of height 6.35 mm and width 40 mm in which various geometries are inserted. Fig. 2 is a schematic diagram of the experimental setup.

The main flow, composed of iodide, iodate and orthoboric acid, is pumped into the test section and sulphuric acid is added by a symmetrical "T" injection with a syringe pump in the core of the geometry. The symmetrical injection is realized by two pipes of diameter 1 mm. The main flow rate is measured by a rotometer and the main initial volume is temperature-regulated at 25 • C. The concentration of I 3 -is measured continuously by spectrophotometry at 353 nm with instantaneous computer data acquisition.

Six geometries (three OSFs and three metallic foams), inserted into the same duct channel of hydraulic diameter D h = 11 mm, height 6.35 mm and width 40 mm, were tested. The three OSFs inserted in the duct channel have the characteristics given in Fig. 3 and Table 2; Table 3 and Fig. 4 give the characteristics of the three metallic foams inserted in the duct channel. Different geometries were tested with the reactant concentrations shown in Table 4 (based on Fang and Lee [START_REF] Fang | Micromixing efficiency in static mixer[END_REF]). Where D H =4whl s /2(wl s + hl s + eh)+ew.

Validation of the physicochemical model

Influence of sulphuric acid injection

In order to quantify the effect of micromixing alone, it is necessary to work in a medium in which macromixing conditions relevant only to micromixing. For the present continuous analysis, the influence of the ratio between the main flow rate and the secondary flow rate on the index segregation X S was tested for the worst micromixing operating conditions, namely in the empty duct channel at Reynolds number 550. Fig. 5 shows the decay of the segregation index with the increase of the ratio between the main flow rate and the secondary flow rate, in other words the decrease of the injection flow rate. The ratio for which the segregation index reaches a constant value is called the critical ratio (Q A /Q B ) critical . For the present conditions, the critical ratio is near 2500. In all subsequent experiments, the ratio (Q A /Q B ) was fixed at 3000 to insure that macromixing plays no significant role during injection.

Influence of reactant concentration

Two different concentrations of sulphuric acid were tested in order to assess their influence on micromixing times. Micromixing analysis depends on two time constants, the characteristic reaction time of the reactions involved and the characteristic micromixing time. Since the chemical systems differ drastically, it is impossible directly to compare the segregation index obtained with these two methods. For this reason, the incorporation model is used to obtain a micromixing time extracted from the segregation index, which depends only on hydrodynamic conditions. Since the experiments were carried out under the same hydrodynamic conditions for both reactant concentrations, the micromixing times obtained should be the same. Fig. 6 shows that for a given flow rate, the micromixing times obtained are of the same order of magnitude, thus verifying that the incorporation model correctly takes into account the effect of the sulphuric acid concentration. Consequently, this method seems well-adapted to comparing micromixing performances generated by different geometries in a continuous process.

Results

The main objective of this work is to evaluate the micromixing generated by different geometries in order to intensify mass transfer in a MHE. Here we summarize and compare micromixing results obtained for the different geometries studied. It also shows that, for a given flow rate, the empty duct channel is clearly the worst in micromixing performance. OSF 1 generates the same order of micromixing level as the empty duct channel, far below that of the other two fins. This observation can be explained by considering OSF hydrodynamics as similar to that of thick flat plates, as suggested by a previous study [START_REF] Michel | Optimisation des échangeurs compacts à ailettes: Étude numérique et expérimentale[END_REF].A characteristic size frequently used in this kind of geometry is the fin half thickness. Many authors [START_REF] Ota | Reattachment length and transition of separated flow over blunt flat plates[END_REF][START_REF] Lane | Leading edge separation from a blunt plate at low Reynolds number[END_REF][START_REF] Sasaki | Three-dimensional vortex structure in a leading-edge separation bubble at moderate Reynolds numbers[END_REF] define several flow regimes around thick flat plates. In order to determine the flow regime, a Reynolds number based on the fin half thickness Re e/2 is defined. These authors agree in proposing three flow regimes, For this reason (see Fig. 8), for a given velocity, the thicker the fin, the higher the Reynolds number Re e/2 and consequently the greater the turbulence and micromixing. The range of Reynolds number Re e/2 is between 5 and 25 for OSF 1, 15 and 65 for OSF 2, and 20 and 75 for OSF 3. This observation suggests a priori that boundary-layer separation and reattachment are laminar and that only OSF 2 and 3 can generate Bénard-Karman vortex shedding. Consequently, OSF 1 generates no more micromixing than the duct channel because of its small fin thickness. However, Michel [START_REF] Michel | Optimisation des échangeurs compacts à ailettes: Étude numérique et expérimentale[END_REF] showed that upstream conditions strongly influence the transition Reynolds number and flow behaviour, e.g. high-intensity turbulence upstream of the fins produces an easier transition to turbulence. Thus upstream conditions can explain the differences between the present results and those in the general fluid mechanics literature.

Offset strip fins

In addition, OSF 3 generates a better micromixing level than OSF 2 even though they have the same fin thickness. This observation suggests that the increase in number of fins per volume unit, as shown in Table 2, induces large flow shearing and then an increase in turbulence and micromixing. It is thus advisable to use OSFs with the largest fin thickness and greatest number of fins per unit volume in order to generate maximum micromixing.

For MHE applications, one must also consider the pumping power, e.g. the pressure drop, which can be obtained by using the correlation in Manglik and Bergles [START_REF] Manglik | Heat transfer and pressure drop correlations for rectangular offset strip fin compact heat exchanger[END_REF] for OSFs geometries and the correlation in Shah and Bhatti [START_REF] Shah | Handbook of Single Phase Convective Heat Transfer[END_REF] for the empty duct channel. Fig. 9, a plot of micromixing time against pumping power, shows that for the same flow rate, the empty duct channel generates the smallest pressure drop. OSFs seem no more attractive in pumping power than the empty duct channel. This approach also shows that the pumping power needed for different OSFs increases with fin thickness and number of fins per unit volume. It is also interesting to see that the micromixing time seems to converge toward the same values for high pumping-power values. The characteristic geometry has a stronger influence in the laminar regime than in the turbulent regime. Indeed, a suitable characteristic geometry decreases the transition to turbulence. Then, when turbulence is established, the geometry has less impact on micromixing.

Metallic foams

As in the OSFs study, Fig. 10 shows that for metallic foams, micromixing time decreases when the flow rate increases, suggesting that the micromixing level depends on flow velocity or turbulence intensity.

First (and unexpectedly), for the same flow rate, micromixing time decreases when the metallic foam pore diameter increases. At first glance this seems contradictory, since strong shearing due to a decrease in pore diameter should normally generate high turbulence intensity. However, if one considers metallic foam as an arrangement of vertical and horizontal tubes, as suggested by Lu et al. [START_REF] Lu | Heat transfer in open-cell metal foams[END_REF], it is possible to consider a metallic foam flow as an external flow around a tube. For that kind of flow, the suitable characteristic length is then the cylinder diameter or metallic foam ligament diameter. As for OSFs, several flow regimes can be defined using a Reynolds number based on cylinder diameter or metallic foam ligament diameter. With this approach, for the same velocity, the larger the ligament diameter, the higher the turbulence intensity. Thus, for a given flow rate, Fig. 10 shows that micromixing time decreases with increasing metallic foam ligament diameter. These results are therefore in agreement with the hypothesis of external flow around a tube. For the same flow rate, metallic foam seems the best geometry for micromixing. As for OSF, micromixing seems to increase with increasing leadingedge effect. Hydrodynamic flows with OSF or metallic foam are similar and can be considered as external flows around a flat plate or a foam ligament.

With the same approach as in the OSF case, Fig. 11 plots micromixing time against pumping power. Unlike the micromixing level, which increases with ligament diameter, the pressure drop increases with decreasing pore diameter. Indeed, the strong shear level induced by the large number of leading edges generates a high singular pressure drop. Consequently, for the 45 ppi SCPS, the pressure drop is a primary drawback for micromix- ing performance. On the other hand, the 20 ppi ERG, which has the largest pore diameter, generates a smaller pressure drop while producing a high micromixing level; thanks to its larger ligament diameter. The micromixing level reflects the ability of the geometry to convert pressure drop into turbulence rather than into viscous friction. Thus in order to produce high micromixing, a metallic foam with a large ligament diameter is recommended-while taking care of the generated pressure drop.

Concluding remarks

For optimal use, heat exchanger reactors must enhance both heat and mass transfer. The integration of inserts that can promote turbulence is of prime importance. This study has shown advantages and drawbacks in using OSFs or metallic foams as fin surfaces for heat exchanger reactors. Since thermohydraulic data on compact heat exchangers are available in open literature but micromixing data are very rare, the challenge of this work has been to quantify micromixing level generated inside an MHE. The present work quantitatively measures the micromixing level generated by several geometries on the basis of the unique parallel-competing reaction scheme proposed by Villermaux et al. Micromixing time was estimated based on incorporation model proposed by Fournier et al. [START_REF] Fournier | A new parallel-competing reaction system for assessing micromixing efficiency: experimental approach[END_REF].

This study showed that for a given flow rate, an empty duct channel generates a reasonable micromixing level without too great pumping power. Nevertheless, the residence time of chemical reactants in an MHE reactor is an important parameter and must be taken into account. When the residence time for 1 m of an MHE reactor is taken into account, Fig. 12 shows that metallic foam is the geometry that generates the best micromixing level for a given residence time. Its isotropic geometry is also an important advantage in creating homogeneous flow in the MHE reactors (perfect plug flow). Indeed, the reaction medium must be as uniform as possible in order to reduce byproduct formation. Furthermore, metallic foam has a large specific area and could be very interesting as a catalyst medium. The pressure drop seems to be the main drawback of the metallic foams. On the other hand, for a given flow rate, OSFs need less pumping power but generate less micromixing than metallic foams. Moreover, for a given micromixing time, the empty duct channel must work at too high velocity, implying an increase in the volume of the MHE. For a given micromixing level, insert geometries let us reduce the volume of the reactor but lead to a pumping-power increase. Thus, it is necessary to take into account in process design the pumping power required for the system, the process compactness, and the micromixing level required.

This work provides a rich database of micromixing performances of several geometries such as OSFs and metallic foams inserted in a duct channel. Moreover, an experimental micromixing method tested and validated with the incorporation model is now available for testing other geometries, such as vortex generators or microchannels, in order to quantify the micromixing level generated in a continuous process.

a

  Results based on metallic foam, 45 ppi at Re = 1000. b Trambouze and Euzen [1].

Fig. 1 .

 1 Fig. 1. Schematic diagram of the incorporation model.

Fig. 2 .

 2 Fig. 2. The experimental setup.

Fig. 3 .

 3 Fig. 3. Offset strip fin geometry.

Fig. 4 .

 4 Fig. 4. Metallic foam photography at the same scales (left to right: 20 ppi ERG, 20 ppi SCPS and 45 ppi SCPS).

Fig. 5 .

 5 Fig. 5. Segregation index vs. ratio between main flow rate and secondary flow rate (empty duct channel, Re = 550).

Fig. 7

 7 Fig. 7 compares micromixing time and flow rate for the empty duct channel and OSFs studied. This figure shows that, whatever the geometry, the micromixing time decreases with the flow rate.It also shows that, for a given flow rate, the empty duct channel is clearly the worst in micromixing performance. OSF 1 generates the same order of micromixing level as the empty duct channel, far below that of the other two fins. This observation can be explained by considering OSF hydrodynamics as similar to that of thick flat plates, as suggested by a previous study[START_REF] Michel | Optimisation des échangeurs compacts à ailettes: Étude numérique et expérimentale[END_REF].A characteristic size frequently used in this kind of geometry is the fin half thickness. Many authors[START_REF] Ota | Reattachment length and transition of separated flow over blunt flat plates[END_REF][START_REF] Lane | Leading edge separation from a blunt plate at low Reynolds number[END_REF][START_REF] Sasaki | Three-dimensional vortex structure in a leading-edge separation bubble at moderate Reynolds numbers[END_REF] define several flow regimes around thick flat plates. In order to determine the flow regime, a Reynolds number based on the fin half thickness Re e/2 is defined. These authors agree in proposing three flow regimes,

Fig. 6 .

 6 Fig. 6. Comparison of micromixing time with two concentration reactants vs. flow rate.

Fig. 7 .

 7 Fig. 7. Micromixing time vs. flow rate for three different offset strip fins and a duct channel.

Fig. 8 .

 8 Fig. 8. Temperature visualization of Bénard-Karman vortices generated by different fin thicknesses. Flow Reynolds number is, respectively, Re = 120 (a); 240 (b); and 360 (c) [11].

Fig. 9 .

 9 Fig. 9. Micromixing time vs. pumping power for three different offset strip fins and a duct channel.

Fig. 10 .

 10 Fig. 10. Micromixing time vs. flow rate for three different metallic foams and a duct channel.

Fig. 11 .

 11 Fig. 11. Micromixing time vs. pumping power for three different metallic foams and a duct channel.

Fig. 12 .

 12 Fig. 12. Micromixing time vs. resident time for 1 m of a heat exchanger reactor for all geometries studied.

Table 1

 1 Heat exchange capability for different reactors

	Process	Compact multifunctional
		heat exchanger (MHE) a

Table 2

 2 Offset strip fins characteristics

	Fin type	OSF 1	OSF 2	OSF 3
	e (mm)	0.1	0.3	0.3
	w (mm)	1.4	3.0	1.2
	l s (mm)	3.2	1.6	3.2
	h s (mm)	6.35	6.35	6.35
	Fin number per volume unit (m -3 )	35.2 × 10 8	32.8 × 10 8	41.0 × 10 8
	D H (mm)	2.2	3.5	1.9

Table 4

 4

	Reactant concentrations
	Reactant	Concentration (mol l -1 )
	H 3 BO 3	0.0121
	NaOH	0.00605
	KIO 3	0.00233
	KI	0.0116
	H 2 SO 4	0.25 or 0.50
	are fulfilled. For rapid sulphuric acid injections, the reactant is
	not well-dispersed in the test section, and macroscopic concen-
	tration gradients can influence the results by generating high
	segregation indices. For this reason, all experiments were car-
	ried out with acid injection time slow enough to prevent macro
	and mesomixing effects. Under these conditions, macromixing
	effects are negligible and the results of chemical test reactions are

Table 3

 3

	Metallic foam characteristics
	Foam type	Materials	Grade (constructor) (ppi)	Pore diameter (mm)	Ligament diameter (mm)	Specific surface (m 2 m -3 )	Porosity (%)
	ERG, 20 ppi	Aluminium	20	1.5	0.4	1720	92
	SCPS, 20 ppi	Copper	20	1.2	0.3	900	97
	SCPS, 45 ppi	Copper	45	0.8	0.2	2400	97

Acknowledgements

This work was partially supported by the "ENERGIE" programme of CNRS and the French Ministry of Research. S.F. acknowledges the financial support of ADEME (French Agency for Energy and the Environment) and CEA (Commissariat of Atomic Energy).


Caroline Rozan

Amélie Cornillon

Corinne Petiard

Martine Chartier

Ghislaine Behar

Charlotte Boix

Brigitte Kerfelec

Bruno Robert

Andre Pelegrin

Corinne Pétiard

André Pèlegrin

Patrick Chames

Jean-Luc Teillaud

Daniel Baty
email: daniel.baty@inserm.fr

Anti-FcγRIII x CEA bispecific Fab-like antibodies 1 Single domain antibody-based and linker-free bispecific antibodies targeting FcγRIII induce potent anti-tumor activity without recruiting regulatory T cells

Keywords: bispecific antibody, CD16, CEA, Fc receptors, nanobody, single domain antibodies, VHH ADCC, antibody-dependent cell-mediated cytotoxicity, bsAbs, bispecific antibodies, bsFabs, Fab-like bispecific antibodies, CEA, carcinoembryonic antigen, FcγRs, Fcγ receptors, IgG, Immunoglobulins G, mAbs, monoclonal antibodies, NK, natural killer cells, NSG, NOD SCID gamma, PBMC, peripheral blood mononuclear cell, sdAb, single domain antibody

Single-domain antibody-based and linker-free bispecific antibodies targeting FcγRIII induce potent antitumor

activity without recruiting regulatory T cells.

INTRODUCTION

Almost 30 monoclonal antibodies (mAbs) are now on the market, including fifteen mAbs for cancer therapy in Europe and the US. Most of these mAbs (9/15) are human Immunoglobulin G1 (IgG1) that trigger various mechanisms, such as target signaling inhibition, apoptosis, activation of the classical complement pathway and/or of immune effector cells expressing Fcγ receptors (FcγRs) [START_REF] Chames | Therapeutic antibodies: successes, limitations and hopes for the future[END_REF][START_REF] Boross | Mechanisms of action of CD20 antibodies[END_REF]. While it is difficult to assess the contribution of each of these mechanisms in their in vivo efficacy, clinical trial results support an important role of antibody-dependent cell-mediated cytotoxicity (ADCC) for both lymphomas and solid tumors [START_REF] Desjarlais | Optimizing engagement of the immune system by anti-tumor antibodies: an engineer's perspective[END_REF]. The affinity between the Fc portion of human IgG1 and FcγRIIIa (CD16a), an activating receptor mostly expressed by natural killer (NK) cells, monocytes and macrophages, has a profound impact on ADCC exerted by antibodies [START_REF] Glorius | The novel tribody [(CD20)(2)xCD16] efficiently triggers effector cell-mediated lysis of malignant B cells[END_REF][START_REF] Umana | Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity[END_REF].

Correlation of clinical responses to mAb therapy with FcγRIIIa polymorphism has been observed with more favorable response in patients homozygous for the high affinity FcγRIIIa (V158) [START_REF] Cartron | Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene[END_REF][START_REF] Musolino | Immunoglobulin G fragment C receptor polymorphisms and clinical efficacy of trastuzumabbased therapy in patients with HER-2/neu-positive metastatic breast cancer[END_REF]. The fact that about 80% of the Caucasian population is homozygous (F/F) or heterozygous (F/V) for low affinity FcγRIIIa (F158) is likely an important issue for antibodybased immunotherapy. Moreover, the in vivo efficacy of therapeutic antibodies is hindered by the presence of fucose residues in the N-glycosylation motif (N297 residue) of the Fc region that markedly decreases their affinity for FcγRIIIa [START_REF] Siberil | FcgammaR: The key to optimize therapeutic antibodies?[END_REF][START_REF] Shinkawa | The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibodydependent cellular cytotoxicity[END_REF][START_REF] De Romeuf | Chronic lymphocytic leukaemia cells are efficiently killed by an anti-CD20 monoclonal antibody selected for improved engagement of FcgammaRIIIA/CD16[END_REF]. Therapeutic antibodies also compete with serum IgG for binding to the high-affinity FcγRI and to the intermediate affinity FcγRIIIa. Strikingly, most antibodies have to be injected at high doses to reach a serum concentration between 10 to 100 µg/ml, while they usually elicit a maximal cytotoxic activity at 10 ng/ml in in vitro ADCC experiments. Competition with patient IgG has been proposed to account for this large difference of concentration [START_REF] Preithner | High concentrations of therapeutic IgG1 antibodies are needed to compensate for inhibition of antibody-dependent cellular cytotoxicity by excess endogenous immunoglobulin G[END_REF]. Finally, the use of therapeutic antibodies may be hampered by their ability to engage the inhibitory FcγRIIb receptor.

FcγRIIb possesses an inhibitory ITIM motif in its cytoplasmic domain, and has been shown to impact the anti-tumor efficacy of therapeutic antibodies in mouse models [START_REF] Clynes | Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets[END_REF]. Several approaches, such as site-directed mutagenesis, computational structure-based design, glycosylation engineering, and selection-based methods, have been used to increase Fc binding to activating receptors (FcγRI, FcγRIIa and FcγRIIIa), and to decrease their interaction with inhibitory FcγRIIb [START_REF] Desjarlais | Optimizing engagement of the immune system by anti-tumor antibodies: an engineer's perspective[END_REF][START_REF] Shields | High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R[END_REF][START_REF] Lazar | Engineered antibody Fc variants with enhanced effector function[END_REF]. Variants possessing up to 100-fold increased affinity for FcγRIIIa, resulting in 100-fold enhanced in vitro ADCC [START_REF] Desjarlais | Optimizing engagement of the immune system by anti-tumor antibodies: an engineer's perspective[END_REF][START_REF] Siberil | Molecular aspects of human FcgammaR interactions with IgG: functional and therapeutic consequences[END_REF] have been selected.

Attractive alternative to recruit and activate effector cells is to use bispecific antibodies (bsAbs) capable of simultaneously binding to a target antigen and to an activating receptor such as FcγRI or FcγRIIIa [START_REF] Michon | In vitro killing of neuroblastoma cells by neutrophils derived from granulocyte colony-stimulating factor-treated cancer patients using an anti-disialoganglioside/anti-Fc gamma RI bispecific antibody[END_REF][START_REF] Muller | Recombinant bispecific antibodies for cellular cancer immunotherapy[END_REF]. Although the in vitro and in vivo anti-tumor efficiencies of bsAbs has been largely demonstrated over the last two decades, their development has been severely hindered by different factors, including immunogenicity and the difficulty to efficiently produce large amounts of active molecules [START_REF] Carter | Bispecific human IgG by design[END_REF]. With the development of antibody engineering, several innovative recombinant formats have been proposed, including diabodies, tandem scFvs and minibodies [START_REF] Muller | Recombinant bispecific antibodies for cellular cancer immunotherapy[END_REF][START_REF] Chames | Bispecific antibodies for cancer therapy[END_REF][START_REF] Chames | Bispecific antibodies for cancer therapy: the light at the end of the tunnel?[END_REF], and some of these molecules are being tested in the clinics [START_REF] Bargou | Tumor regression in cancer patients by very low doses of a T cell-engaging antibody[END_REF]. However, most described recombinant bsAbs heavily rely on the use of peptide linkers. Although these linkers have obvious advantages in terms of antibody engineering, their hydrophilic nature makes them prone to proteolytic cleavage, potentially leading to production issues, poor antibody stability, aggregation and increased immunogenicity [START_REF] Fischer | Bispecific antibodies: molecules that enable novel therapeutic strategies[END_REF]. Llama single domain antibodies (sdAbs), derived from heavy-chain antibodies naturally devoid of light chains [START_REF] Hamers-Casterman | Naturally occurring antibodies devoid of light chains[END_REF] are small (13 kDa), well-expressed and extremely stable fragments. They are highly homologous to the VHIII subset family of human VH [START_REF] Su | Adaptive evolution of variable region genes encoding an unusual type of immunoglobulin in camelids[END_REF][START_REF] Dumoulin | Single-domain antibody fragments with high conformational stability[END_REF], which should result in a low antigenicity in human, if any, and which makes the humanization process easier if needed [START_REF] Vincke | General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold[END_REF]. Due to their small size and single domain nature, sdAbs can recognize epitope usually not accessible to conventional antibodies. These fragments represent ideal molecular building units for bsAb construction [START_REF] Conrath | Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs[END_REF]. In a previous work, we have isolated two sdAbs capable of binding and activating FcγRIIIa, while showing no binding to FcγRI, FcγRIIa or inhibitory receptor FcγRIIb [START_REF] Behar | Isolation and characterization of anti-Fc{gamma}RIII (CD16) llama single-domain antibodies that activate natural killer cells[END_REF] as well as one sdAb able to bind carcinoembryonic antigen (CEA) with high specificity [START_REF] Behar | Llama singledomain antibodies directed against nonconventional epitopes of tumor-associated carcinoembryonic antigen absent from nonspecific cross-reacting antigen[END_REF], a well-characterized tumor marker of interest for mAb-based cancer therapy.

In the present work, we have exploited the natural affinity of human CH1 and Cκ IgG domains as an heterodimerization motif [START_REF] Muller | The first constant domain (C(H)1 and C(L)) of an antibody used as heterodimerization domain for bispecific miniantibodies[END_REF] to produce Fab-like bispecific antibodies (bsFabs), devoid of linker and capable of inducing a strong cytotoxicity against CEA positive tumor cells by human NK cells in vitro, and of inhibiting tumor growth in an in vivo mouse model.

MATERIALS AND METHODS

Expression vector design and generation of recombinant antibodies

Bicistronic expression vectors were designed to express bsFab molecules in periplasm.

(for details see Supplementary Methods).

Production and purification of antibodies

BsFabs were purified from periplasm of E. coli DH5α and sdAb C17-Fc from culture supernatant of HEK293T cells (for details see Supplementary Methods).

Cell lines

Jurkat (ATCC TIB-152), LS174T (ATCC CL-188), BXPC3 (ATCC CRL-1687), LAN-1 [START_REF] Michon | In vitro killing of neuroblastoma cells by neutrophils derived from granulocyte colony-stimulating factor-treated cancer patients using an anti-disialoganglioside/anti-Fc gamma RI bispecific antibody[END_REF], HT29 (ATCC HTB-38) and HEK293T (ATCC CRL-11268) cells purchased from ATCC were cultured in RPMI 1640+ Glutamax-I medium (Invitrogen) supplemented with 10 % FBS (PAA). SKOV3 (ATCC HTB-77) and CO115 (an established CEA-negative subclone from a human colon carcinoma cell line) [START_REF] Carrel | Establishment of a cell line (Co-115) from a human colon carcinoma transplanted into nude mice[END_REF][START_REF] Leconte | Involvement of circulating CEA in liver metastases from colorectal cancers re-examined in a new experimental model[END_REF] were cultured in DMEM + Glutamax-I medium (Invitrogen) supplemented with 10 % FBS (PAA). MC38 and C15.4.3.AP (MC38-CEA) cells are murine colorectal cancer cells [START_REF] Mizobata | Induction of cytotoxic T cells and their antitumor activity in mice transgenic for carcinoembryonic antigen[END_REF]. MC38-CEA cells are MC38 cells transfected with a cDNA encoding CEA. MC38 were cultured in DMEM + Glutamax-I medium supplemented with 10 % FBS and MC38-CEA in DMEM + Glutamax-I medium supplemented with 10 % FBS and 0.5 mg/ml of geneticin. Jurkat-huFcγRIIIa/γ cells are Jurkat lymphoma T cells transfected with a cDNA encoding the extracellular domain of FcγRIIIa fused to the [START_REF] Vivier | Signaling function of reconstituted CD16: zeta: gamma receptor complex isoforms[END_REF]. These cells were cultured in RPMI 1640 + Glutamax-I medium supplemented with 10 % FBS and 0.5 mg/ml of geneticin. SKOV3-CEA-Luc are SKOV3 human ovarian carcinoma transfected with a cDNA encoding the extracellular domain of CEA and with a cDNA encoding luciferase; cells were cultured in DMEM + Glutamax-I medium supplemented with 10 % FBS, 0.5 mg/ml of geneticin and 0.4 mg/ml hygromycin B. All cell lines were grown in a humidified 37°C incubator containing 5% CO 2 .

All cell lines purchased from ATCC were not cultured for more than 2 months. Other cell lines used in this study have not been authenticated.

Human NK cell purification

Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh peripheral blood of healthy donors (Etablissement Français du Sang, Marseille and Hôtel-Dieu hospital, Paris, France) by Ficoll LSM 1077 (PAA) gradient centrifugation. Cells were counted and tested for viability by trypan blue exclusion assay. NK cells were isolated by depleting non-NK cells using the NK cell isolation kit (Miltenyi Biotec) as described by the manufacturer.

Flow cytometry assays

Increasing concentrations of bsFabs (1.2 to 100 nM) were used for staining 2x10 5 Jurkat-huFcγRIIIa/γ or 2x10 5 MC38-CEA cells in 50 µl PBS supplemented with 2% bovine serum albumin at 4°C for 1 h. Bound bsFabs were detected by incubating cells with either an anti-flag mAb M2 (4 µg/ml) (Sigma Aldrich) or anti-c-myc mAb 9E10 (10 µg/ml) (Santa Cruz Biotechnology) for 30 min at 4°C followed by a FITC-conjugated goat anti-mouse IgG F(ab') 2 (10 µg/ml) (Jackson ImmunoResearch) incubated 30 min at 4°C. After washing, labeled cells were analyzed by flow cytometry using a FACS Calibur (BD biosciences) or MACSQuant (Miltenyi Biotec) flow cytometers.

For serum stability assays, bsFabs were incubated at 1 µM in 90% human serum (not heat-inactivated) (Sigma Aldrich) at 37°C for various periods of time (7 to were collected at different time points, frozen and kept at -20°C. The remaining binding activity of the samples after storage was determined by flow cytometry using non-saturating bsFab concentrations (10 nM for bsFab C21 and 100 nM for bsFab C28 against Jurkat-huFcγRIIIa/γ, and 100 nM for both against MC38-CEA).

For binding competition assays with serum IgG, Jurkat-huFcγRIIIa/γ cells were preincubated with increasing concentrations of human serum (12.5 to 100 %) at 4°C for 1 h then incubated with a constant amount of bsFab C21 (4 nM) or bsFab C28 (36 nM) or biotinylated sdAb C17-Fc (200 nM). Bound antibodies were detected using either mouse anti-flag mAb M2 (4 µg/ml) (Sigma Aldrich) followed by an incubation with FITC-conjugated goat antimouse IgG F(ab') 2 (10 µg/ml) (Jackson ImmunoResearch Laboratories) for bsFab labeling or streptavidin-PE diluted 1:10 (Beckman Coulter) for biotinylated sdAb C17-Fc labeling. In another setting, Jurkat-huFcγRIIIa/γ cells were pre-incubated with constant concentrations of human serum (90 %) or PBS 1X at 4°C for 1 h and then incubated with various amounts of bsFab C21 or bsFab C28. Bound antibodies were detected as described previously.

For CEA detection, cells were pre-incubated with anti-CEA sdAb C17 (10 µg/ml) for 1 h at 4°C. Bound antibody was detected using mouse anti-6his mAb (1 µg/ml) (Novagen) followed by incubation with FITC-conjugated goat anti-mouse IgG F(ab') 2 (10 µg/ml) (Jackson ImmunoResearch).

Rosette formation assay

5×10 4 MC38-CEA and 25×10 4 Jurkat-huFcγRIIIa/γ were co-cultured in RPMI 1640+ Glutamax-I medium supplemented with 10 % FBS and 0.5 mg/ml of geneticin with or without bsFabs (2 µg/ml) at 37°C for 4h. Rosette formation was then observed by optical microscopy.

IL-2 secretion assay

Jurkat-huFcγRIIIa/γ cells (10 6 ) (or non-transfected Jurkat cells as negative control) were incubated for 18 h in EMS medium, containing 5% FBS, 10 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma Aldrich) and bsFabs (50 nM) or anti-FcγRIII mAb 3G8 [START_REF] Fleit | Human neutrophil Fc gamma receptor distribution and structure[END_REF] 50 nM as a positive control. In some experiments, anti-flag M2 mAb was added (4 µg/ml) to promote bsFab dimerization. In some experiments, 10 5 LS174T cells (or LAN-1 cells used as a negative control) were used as target cells. After incubation, cells were centrifuged for 5 min at 1200 x g and human IL-2 present in the culture medium was measured by ELISA using READY-SET-GO human IL-2 kit (eBioscience).

ADCC assay

Target cells (BxPC3, HT29, LS174T, SKOV3-CEA-Luc or SKOV3; 5x10 3 cells/well) were mixed with 5x10 4 freshly isolated human NK cells (Effector/Target ratio: 10:1). Variable concentrations of bsFabs or sdAb C17-Fc were added to the cells in a final volume of 200 µl.

All procedures were done in triplicate with different donors. Following overnight incubation at 37°C, target cell viability was quantified with Cell titer Glo viability assay (Promega) according to manufacturer's protocol. The formula used for % lysis calculation was: % lysis= To investigate the effect of soluble CEA, target and effector cells were incubated as previously described with variable concentrations of bsFab and constant concentration (0.1 µg/ml or 1 µg/ml) of soluble CEA (Chemicon). FcγRIIIa genotyping of donors was performed as described [START_REF] Dall'ozzo | Rapid single-step FCGR3A genotyping based on SYBR Green I fluorescence in real-time multiplex allele-specific PCR[END_REF]. Dose response curves were treated by non-linear regression analysis using Prism software (GraphPad Software). Data were expressed as mean ± SEM.

Biodistribution assay.

Swiss female nude mice (Charles River Laboratories, France) bearing human colon carcinoma LS174T (CEA positive) or CO115 (for CEA negative control) tumors on the right and left flanks respectively were injected i.v. with 125 I labeled bsFab C21 (2.8 10 5 Bq, 3 µg) 14 days after s.c. tumor cell injection. Four groups of three mice were sacrificed at 3, 6, 15 or 24 h post-bsFab injection. Blood was collected and tissues and organs were weighted and dissected at each time point. Radioactivity was quantified using a γ-counter COBRAII (Packard) and results were expressed as % Injected Dose/g tissue (% ID/g). Mice housed in individual cages with laboratory chow and water. All experiments were performed according to the French Animal Protection Law with the permission from the local authorities.

Tumor growth inhibition.

Seven-week-old female NOD SCID gamma (NSG) mice (Charles River Laboratories, France) (n=8/group) were xenografted subcutaneously with human pancreatic carcinoma CEA positive BxPC3 (2x10 6 cells/mice) on the right flank and were injected with freshly purified human PBMCs (30x10 6 /mice) (i.p.) and bsFab C21 (100 µg/mice) or PBS (i.p.) at Day 0. At Days 1 and 2, mice were injected i.p. with bsFab C21 (50 µg/mouse) or PBS. Tumor growth was measured with a Vernier caliper during 28 days to permit calculation of tumor volumes (V=(L×W 2 )/2, where L and W were length and width, respectively). All animals were sacrificed at the end of experiment in accordance with institutional guidelines. Mice housed in individual cages with laboratory chow and water. All experiments were performed according to the French Animal Protection Law with the permission from the local authorities.

Statistical analysis.

Data are presented as mean ± SEM. Cytotoxicity assays were statistically analyzed using a one-way ANOVA test. In vivo studies were analyzed using Student's t test. For all tests, P< 0.05 was considered statistically significant.

RESULTS

BsFab design and production.

For construction of bsFabs, we designed a bicistronic expression vector allowing the coexpression of one sdAb directly fused to the human Cκ chain and another sdAb directly fused to the human CH1 chain in the periplasm of E. coli (Fig. 1A). We used the anti-CEA sdAb C17 previously characterized (Kd: 8 nM) [START_REF] Behar | Llama singledomain antibodies directed against nonconventional epitopes of tumor-associated carcinoembryonic antigen absent from nonspecific cross-reacting antigen[END_REF] for targeting CEA positive cells and two different anti-FcγRIII sdAbs for targeting FcγRIIIa positive effector cells. The anti-FcγRIII sdAb C28 was previously shown [START_REF] Behar | Isolation and characterization of anti-Fc{gamma}RIII (CD16) llama single-domain antibodies that activate natural killer cells[END_REF] to display an apparent affinity for FcγRIII (Kd: 82 nM) in the range of the Fc portion of human IgG1 (>100 nM) whereas the anti-FcγRIII sdAb C21 displays a higher affinity of Kd: 10 nM for FcγRIII [START_REF] Behar | Isolation and characterization of anti-Fc{gamma}RIII (CD16) llama single-domain antibodies that activate natural killer cells[END_REF]. The resulting bispecific Fab-like fragments were named bsFab C21 and bsFab C28, respectively (Fig. 1B). BsFabs were produced in E. coli periplasm and affinity-purified by anti-CH1 followed by anti-Cκ columns.

As shown in Figure 1C, SDS-PAGE analyses of the second step of bsFab C21 purification reveal, under non reducing conditions, one band in the expected range of size (50-55 kDa) due to heterodimerization.

In parallel, we developed a human Fc fusion protein, sdAb C17-Fc, by fusing anti-CEA sdAb C17 to the hinge, CH2 and CH3 domains of IgG1 (Fig. 1B). Anti-CEA sdAb C17-Fc was produced by transient transfection in HEK293T cells and purified from culture supernatant by affinity chromatography on a G-protein column. This construction was designed to provide a molecule with the same epitope specificity of bsFabs, but triggering ADCC via a conventional Fc/FcγR interaction.

Binding analysis of bsFabs.

Binding specificities of bsFabs to FcγRIIIA and CEA were analyzed by flow cytometry using Jurkat-huFcγRIIIA/γ and MC38-CEA cells, respectively. As shown in Figure 2A, both bsFabs efficiently bound to these cells in a dose-dependent manner. To ensure that signals were generated by heterodimers, bsFab binding to MC38-CEA cells was assessed using the c- myc tag fused to the chain bearing the anti-FcγRIIIA sdAb and vice-versa. A marked shift in mean fluorescence intensity was observed when comparing bsFab C21 and bsFab C28 binding to Jurkat-huFcγRIIIA/γ, likely related to the 8-fold difference of affinity between these anti-FcγRIIIa sdAbs [START_REF] Behar | Isolation and characterization of anti-Fc{gamma}RIII (CD16) llama single-domain antibodies that activate natural killer cells[END_REF]. As expected, bsFab C21 and C28, both bearing the same anti-CEA sdAb C17 exhibited a similar binding profile to MC38-CEA cells. No binding to MC38 and Jurkat cells was observed with any of these bsFabs (data not shown).

The ability of bsFabs to simultaneously bind to MC38-CEA and Jurkat-huFcγRIIIa/γ cells was demonstrated by rosette formation between the two types of cells. The addition of bsFabs in MC38-CEA and Jurket-huFcγRIIIa/γ co-culture led to rosette formation between Jurkat-huFcγRIIIa/γ and MC38-CEA cells (up to nine Jurkat-huFcγRIIIa/γ cells bound to one MC38-CEA cell have been observed) (Fig. 2B). In the absence of bsFab, no rosette formation was observed.

In vitro stability of bsFabs in human serum.

In vitro stability was analyzed by incubation of each bsFab in human serum at 37°C for up to 168 h and subsequent examination of CEA and FcγRIIIa binding by flow cytometry. Figure 2C shows that bsFab C21 and bsFab C28 retain their full binding on both target and effector cells, even after 168 h of incubation, demonstrating a remarkable stability. SDS-PAGE and Western-blot analyses showed the absence of breakdown products of both bsFabs (data not shown), further confirming that both bsFabs are stable up to 168 h in physiological conditions.

Competition with human IgG for FcγRIIIa binding.

The potential impact of serum, containing high amount of endogenous IgG, on FcγRIIIa binding by bsFabs was explored. Jurkat-huFcγRIIIa/γ cells were pre-incubated in the presence of various volumes of human serum and then stained with bsFabs. SdAb C17-Fc was used as control. As shown in Supplementary Fig. 1A, both bsFabs retain binding in the presence of human serum while sdAb C17-Fc binding is, as expected, strongly inhibited. Competition assays were also performed on Jurkat-huFcγRIIIa/γ cells in the presence of 90% human serum and various concentrations of bsFabs (Supplemental Fig. 1B). The interaction of bsFabs with FcγRIIIa was not hindered by the presence of human IgG, even at low bsFab concentrations. The same results were observed with purified human polyclonal IgG in a competition assay (data not shown). These results are in agreement with our previous data [START_REF] Behar | Isolation and characterization of anti-Fc{gamma}RIII (CD16) llama single-domain antibodies that activate natural killer cells[END_REF] showing that sdAbs C21 and C28 recognize FcγRIIIa epitopes different from those bound by human IgG Fc portion.

IL-2 release assays.

One possible side effect of antibodies targeting activating receptors is the activation of effector cells in the absence of target cells, potentially leading to a systemic inflammatory response (cytokine storm). To evaluate this possibility, Jurkat and Jurkat-huFcγRIIIa/γ cells were cultured in the presence of monovalent bsFab. Bivalent anti-FcγRIIIa mAb 3G8, able to crosslink the receptor and trigger activation was used as positive control. Monovalent bsFabs only led to a marginal IL-2 secretion, indicating that FcγRIIIa positive cells cannot be activated in the absence of target cells (Fig. 3), unlike bivalent anti-FcγRIIIa mAb 3G8. The low difference in IL-2 secretion observed between the two bsFabs could be explained by their different affinity for FcγRIIIa and/or different epitopes. The addition of a monoclonal antibody targeting the Flag tag fused to the C-terminus of Ck domain and thus leading to bsFab dimerization led to IL-2 secretion as expected (Fig. 3). These results demonstrate that monovalent bsFab do not induce effector cell activation in the absence of target cells.

However, the co-culture of Jurkat-huFcγRIIIa/γ with CEA positive LS174T tumor cells in the presence of bsFab C21 or C28 could lead to IL-2 secretion. This activation was not seen in the absence of bsFab, or when using non-transfected Jurkat cells or CEA negative LAN-1 human tumor cells in the presence of bsFab, as negative controls (Fig. 3). These results demonstrate in vitro that the efficient activation of effector cells by monovalent bsFabs is dependent on the presence of cells expressing the tumor antigen, and does not lead to unwanted cytokine release by FcγRIII positive effector cells in the absence of tumor cells.

BsFab-dependent NK cell-mediated cytotoxicity.

The cell-mediated cytotoxicity of bsFabs was investigated using a panel of four different human tumor cells expressing various levels of CEA, as shown in Figure 4A. Freshly purified human NK cells were used as effector cells at an E:T ratio of 10:1. Dose-response curves allowed the determination of EC 50 values (Fig. 4B) and percentages of maximal lysis. Both bsFabs triggered a strong cytotoxicity against SKOV3-CEA cells in a dose-dependent manner but were ineffective when CEA-negative SKOV3 cells were tested (Fig. 4B). Cells from all CEA positive tumor cell lines were killed with a similar efficiency, leading to EC 50 in the 10 pM range using three different donors. In all three cases, a slightly higher efficiency was observed with bsFab C21, potentially due to its higher affinity for FcγRIIIa (Table 1).

Sensitivity to FcγRIIIa polymorphism.

Since the efficiency of ADCC is affected by FcγRIIIa polymorphism at position 158, experiments were performed using SKOV3-CEA cells as target cells and purified NK cells from FcγRIIIa 158V/V, F/F or V/F donors and bsFab C21 or sdAb C17-Fc. As shown in Figure 5A, the cytotoxic activity triggered by sdAb C17-Fc was lower than that of bsFab C21, both in terms of EC 50 (values in the nM range) and of maximal lysis (< 50%), despite its bivalent binding to CEA. This result is likely due to the weaker interaction of the Fc portion of sdAb C17-Fc with FcγRIIIa, as compared to bsFab C21. More importantly the lytic activity of sdAb C17-Fc varied according to FcγRIIIa polymorphism, NK cells from 158F/F donor being significantly less efficient than NK cells from 158V/V and 158V/F donors. In contrast, the cytotoxic activity of bsFab C21 was very similar whatever the FCGR3 genotype of the donor. These results confirm that the epitope recognized by sdAb C21 is not affected by the nature of residue 158, and demonstrate that bsFab can trigger highly potent tumor killing at very low doses, independently of FcγRIIIa polymorphism. It is well known that CEA can be released by phospholipases from the cell surface through cleavage of its glycosyl-phosphatidyl-inositol-linkage, leading to the appearance of a soluble form in the blood [START_REF] Sack | Release of carcinoembryonic antigen from human colon cancer cells by phosphatidylinositol-specific phospholipase C[END_REF]. It represents a valuable tumor marker because its serum level correlates disease progression. However, this soluble form of CEA could also interfere with antibody-based immunotherapy by competing with membrane-bound CEA for antibody binding. Thus, we performed ADCC assays using BxPC3 tumor cells in presence of intermediate (0.1 µg/ml) or high (1 µg/ml) concentrations of soluble CEA. As shown on Figure 5B, soluble CEA has a detectable but moderate effect on the anti-tumor efficacy of bsFab C21. EC 50 values increased to around 100 pM at the highest CEA concentration, although it did not affect the maximal lysis value. These results suggest that circulating CEA should not critically impact the effect of bsFab therapy, in particular in clinical settings where levels of soluble CEA are low.

Sensitivity to the presence

Biodistribution of bsFab in xenografted mice.

The biodistribution of bsFab C21, which exhibited the most favorable in vitro characteristics in terms of cell binding and cytotoxicity, was studied using xenografted nude mice. BsFab C21 was labeled with 125 I and was injected i.v. in mice bearing both LS174T cells (CEA positive colorectal tumor) (right flank) and CO115 cells (CEA negative colorectal tumor) (left flank, as negative control). Mice were sacrificed 3, 6, 15, 24 h post bsFab injection to quantify the presence of bsFab in blood, various organs and tumors. Best tumor-to-normal organ ratios were observed 15 h post-injection (Fig. 6A) with more than 4.5% ID/g of bsFab being localized in the CEA positive tumor, whereas less than 0.8% ID/g remained in the blood or other organs and CEA negative tumor, except for kidneys (around 1%). Non-tumor infiltrated organs with noticeable level of bsFab were kidneys, probably due to bsFab clearance.

BsFab in vivo efficacy in xenografted mice.

To determine if the potent in vitro

DISCUSSION

Redirecting the cytotoxic potential of leukocytes to eliminate tumor cells has been a major impulse for the development of bsAbs for cancer immunotherapy. FcγRIIIa is an attractive candidate to recruit effector cells. It is strongly expressed by NK cells that play a critical role in the induction of ADCC, one of the major modes of action of anti-tumor antibodies. In addition, FcγRIIIa is also expressed on monocytes and macrophages that are important actors of anti-tumor immunity. Anti-FcγRIIIa bsAbs have the potential to bypass several important limitations faced by therapeutic antibodies. However, difficulties in producing sufficient amounts of functional and stable bsAbs at reasonable costs have strongly hampered their development for many years, although early clinical trials had been promising [START_REF] Weiner | Phase I trial of 2B1, a bispecific monoclonal antibody targeting c-erbB-2 and Fc gamma RIII[END_REF][START_REF] Hartmann | Treatment of refractory Hodgkin's disease with an anti-CD16/CD30 bispecific antibody[END_REF]. Molecular constructs, such as bispecific diabodies, single chain diabodies, tandem scFvs and F(ab') obtained by recombinant DNA technology, might circumvent the above limits. These formats can be expressed in bacteria or mammalian cells, and may show benefits due to their small size, although they can present manufacturing challenges related to their production and in vitro and in vivo stability. Notably, the variable and unpredictable expression yields observed for different scFvs hamper their widespread use. Moreover, the linker connecting the VH and VL in the scFv and that between different scFvs can provoke aggregation of the molecule. Shortening the linkers to produce diabodies does not always guarantee success, since the linkers can induce an erroneous angle between the VH-VL pair, thus forming poorly functional antibodies [START_REF] Lawrence | Orientation of antigen binding sites in dimeric and trimeric single chain Fv antibody fragments[END_REF].

An important motivation of the work reported herein was therefore to design a new linkerfree bispecific format to avoid aggregation and stability issues and easy to produce. Thus, we have studied a format relying on the use of llama sdAbs that possess valuable functional and structural properties and of human CH1/Cκ domains as heterodimerization motif, to produce Fab-like bispecific antibody fragments.

As a proof of concept, we constructed bsFabs targeting CEA positive tumor cells and human FcγRIIIa positive immune effector cells, using previously isolated anti-FcγRIIIa sdAbs binding epitopes located outside the Fc binding site, and an anti-CEA sdAb chosen for its affinity comparable to conventional anti-CEA mAbs, despite its lack of bivalency. These molecules were easily produced in E. coli with routine yield ranging from 0.5 and 2 mg/l of culture in non-optimal shaker flask conditions in an academic laboratory, which compares favorably with other formats such as tandem scFv (the BiTE format) requiring eukaryotic cells for production [START_REF] Baeuerle | Bispecific T-cell engaging antibodies for cancer therapy[END_REF]. The high stability of these bsFabs conferred by the absence of linkers is illustrated by the fact that they retained their full binding activity at least for 7 days in human serum at 37°C, even at low concentration.

One of the key issues with bsAbs is their capacity to trigger an efficient cell-mediated cytotoxicity. Our in vitro data demonstrated a strong and specific NK cell-mediated lysis of CEA expressing tumor cells (from pancreatic and colorectal cancers) at picomolar concentrations, i.e., orders of magnitude lower than conventional mAbs [START_REF] De Romeuf | Chronic lymphocytic leukaemia cells are efficiently killed by an anti-CD20 monoclonal antibody selected for improved engagement of FcgammaRIIIA/CD16[END_REF][START_REF] Horton | Potent in vitro and in vivo activity of an Fc-engineered anti-CD19 monoclonal antibody against lymphoma and leukemia[END_REF]. These results suggest that a high affinity for FcγRIIIa translates into improved cytotoxic activity of effector cells in vitro, a finding also described with mutated and glyco-engineered antibodies [START_REF] Repp | Combined Fc-protein-and Fc-glyco-engineering of scFv-Fc fusion proteins synergistically enhances CD16a binding but does not further enhance NK-cell mediated ADCC[END_REF][START_REF] Nordstrom | Anti-tumor activity and toxicokinetics analysis of MGAH22, an anti-HER2 monoclonal antibody with enhanced Fcgamma receptor binding properties[END_REF]. As opposed to classical mAbs such as rituximab, target antigen density did not A possible limitation of anti-CEA bsFabs might be the presence of shed extracellular domains of CEA in the serum of cancer patients, possibly blocking the anti-CEA binding site.

Here, we demonstrate that bsFab-dependent cytotoxicity is only slightly impacted by concentration of soluble CEA exceeding concentrations found in the majority of cancer patients [START_REF] Booth | Serum carcinoembryonic antigen in clinical disorders[END_REF]. Thus, as already suggested by some clinical studies using anti-CEA antibodies [START_REF] Behr | Phase I/II clinical radioimmunotherapy with an iodine-131-labeled anti-carcinoembryonic antigen murine monoclonal antibody IgG[END_REF][START_REF] Juweid | Phase I/II trial of (131)I-MN-14F(ab)2 anti-carcinoembryonic antigen monoclonal antibody in the treatment of patients with metastatic medullary thyroid carcinoma[END_REF], soluble CEA should not have a significant impact on the clinical efficacy of the bsAb.

Another important issue faced by therapeutic antibody fragments devoid of Fc portion is their pharmacokinetics property. As a likely consequence of their small size and of their lack of interaction with FcRn, Fab fragments have a mean retention time in the body 35-fold lower than full-length IgG [START_REF] Covell | Pharmacokinetics of monoclonal immunoglobulin G1, F(ab')2, and Fab' in mice[END_REF]. Despite this disadvantage, it has been shown that their smaller size as compared to whole IgG limits their retention in non-targeted organs and increases their tumor penetration. Consistent with those data, biodistribution experiments performed in nude mice bearing CEA positive tumor xenografts revealed a rapid increase of CEA positive to CEA negative tumor ratio up to almost 7-fold at 15 h, demonstrating a specific and efficient tumor targeting of bsFabs. Nevertheless, the bsFab format authorizes the linker-free addition of single domain antibodies at the C-terminus of CH1 or Cĸ domains (data not shown). It can therefore be envisaged to construct tri-specific formats by adding an anti-human serum albumin sdAb, a method that has been shown to significantly increase tumor retention and half-life [START_REF] Holt | Anti-serum albumin domain antibodies for extending the half-lives of short lived drugs[END_REF][START_REF] Tijink | Improved tumor targeting of anti-epidermal growth factor receptor Nanobodies through albumin binding: taking advantage of modular Nanobody technology[END_REF]. In conclusion, we have designed a new class of single domain-based bispecific antibodies that combines easy production, high stability and potent in vitro ADCC activity irrespective of FcγRIIIa polymorphism, glycosylation and competing endogenous IgG issues. Altogether, these properties associated to a significant cytotoxic activity in a preclinical in vivo model confer to this bispecific Fab-like antibody format the potential to lead to a new generation of highly active therapeutic antibodies for tumor therapy. A) BsFabs were incubated with antigen positive cells at various concentrations. Binding on MC38-CEA and Jurkat-huFcγRIIIa/γ was detected using anti-c-myc or anti-flag antibodies, respectively, followed by goat anti-mouse labeled antibodies. Filled black: isotype control. Rosette formation was observed by optical microscopy. C) BsFab stability in human serum.

bsFabs were incubated at 1 µM in 90% serum for up to 168 h. Binding experiments were next performed by flow cytometry using non saturating concentrations (10 nM for bsFab C21 and 100 nM for bsFab C28 against Jurkat-huFcγRIIIa/γ, and 100 nM for both against MC38-CEA) . Filled black: isotype control. Open black: bsFab incubated for various times at 37°C in PBS. Filled grey: bsFab incubated for various times at 37°C in 90% human serum. CEA: MC38-CEA cell line. FcγRIIIa: Jurkat-huFcγRIIIa/γ cell line.

[

  T-(TEAb-E)]/[T-Tdead]×100 (T=Target, E= effector, Tdead= Target lysed with 1% triton solution, TEAb=Target + Effector + Antibody luminescent signal).

  activity of bsFab would translate into inhibition of tumor growth in vivo, an adoptive transfer model was used. At Day 0, NOD SCID gamma (NSG) on July 14, 2013. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 11, 2013; DOI: 10.1158/1535-7163.MCT-12-1012 mice were engrafted s.c. with CEA positive human pancreatic cancer BxPC3 cells and with human PBMCs from healthy donors and treated with 100 µg of bsFab C21 by i.p injection. Injections of bsFab C21 (50 µg) were performed the two following days (cumulative dose/mouse= 200 µg). Mice treated only with either PBS, bsFab C21 or PBMC + PBS were used as control groups (n=8). As shown in Figure 6B, a significant tumor growth inhibition was visible in mice treated with PBMCs and bsFab C21 as compared with PBS or bsFab treatment alone. A slight reduction of tumor growth was observed with human PBMCs treatment alone. on July 14, 2013. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 11, 2013; DOI: 10.1158/1535-7163.MCT-12-1012

  significantly impact bsFab-mediated ADCC as shown by the use of tumor cell lines expressing different amounts of CEA. The significant inhibition of tumor growth observed upon human PBMCs adoptive transfer to CEA positive tumor-bearing NSG mice supports the in vivo efficacy of the bsFab format. The low level of INF-γ secretion by NK cells induced by bsFab in the absence of target cells in vitro suggests in addition that bsFabs should not induce any systemic activation of NK cells in vivo and should not provoke cytokine storm. Importantly, by carefully choosing the anti-FcγRIII sdAb, we have constructed bsFabs whose action is insensitive to FcγRIIIa polymorphism and that do not bind inhibitory FcγRIIb. Because of their high efficiency and the absence of competition with serum IgG, these molecules should be clinically active at much lower concentrations than those used for conventional mAbs as already reported for the bispecific T-cell engager format (21). These properties, added to the possibility to produce easily these fragments in E. coli in large on July 14, 2013. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 11, 2013; DOI: 10.1158/1535-7163.MCT-12-1012 amounts, have the potential to substantially reduce the high manufacturing costs associated with mAb therapy. The retargeting of FcγRIII expressing cells has another advantage over CD3 + T cell retargeting by bsAb. Anti-CD3 bsAbs have been shown to lead to the recruitment and the activation of regulatory T cells (Treg) (45) possibly leading to a downregulation of the antitumor response. FcγRIII retargeting does not activate the Treg subset, and does not trigger immunosuppression.

Figure 1 .

 1 Figure 1. Antibody formats. A) A biscistronic construct was designed to allow the production of bsFab in the periplasm of E. coli. Dark grey: portion of hinge (H) and Cκ and CH1-3 constant domains of human IgG1. Light grey: sdAbs. Ptac: tac promoter. RBS1 and RBS2: ribosome binding sites. 55: Optimized PelB signal sequence. WT: PelB wild-type signal sequence. F: flag tag. C: c-myc tag. 6H: hexahis tag. Pß-act: chicken beta-actin promoter. Kozac: ribosome binding site. µp: micro phosphatase signal sequence. B) Scheme of antibody formats used in this study. C) Analysis of purified bsFabs by Coomassie blue stained SDS-PAGE after affinity chromatography on IgG-CH1 matrix followed on LC-kappa matrix. MW: molecular weight ladder, FT: flow through.

Figure 2 .

 2 Figure 2. BsFab binding analysis.

Figure 3 .

 3 Figure 3. IL-2 release assays. Jurkat or Jurkat-huFcγRIIIa/γ cells were pre-stimulated with PMA and incubated with 50 nM of bsFab or anti-FcγRIIIa bivalent mAb 3G8. In some case, anti-Flag tag mAb was added to induce bsFab dimerization. The addition of CEA positive LS174T tumor cells could induce IL-2 secretion while CEA negative LAN-1 human tumor cells did not. IL-2 secreted in the medium was quantified by ELISA. Error bars represent the standard deviation of experiments performed in triplicates.

Figure 4 .Figure 5 .

 45 Figure 4. BsFab-dependent NK cytotoxicity. A) CEA expression of various tumor cell lines was assessed by flow cytometry (open black: isotype control, filled grey: anti-CEA sdAb C17) on SKOV3, SKOV3-CEA, BxPC3, LS174T or HT29 cells. B) Human NK cells were mixed at ratio 10:1 with CEA positive cells in the presence of indicated concentrations of bsFabs. Target cell viability was measured by Cell titer Glo viability assay after overnight incubation at 37°C in the presence of indicated concentrations of bsFab C21 (open circle), bsFab C28 (open square), except for SKOV3 cells for which bsFab C21 is represented by open triangle and bsFab C28 by open diamond. A table summarizes EC 50 (pM) of each bsFab determined for every tumor cell line. Error bars represent the standard deviation of experiments performed in triplicates.

Figure 6 .

 6 Figure 6. BsFab in xenografted mice. A) Biodistribution: 125 I-labeled bsFab C21 was injected i.v. into LS174T (CEA positive) and Co115 (CEA negative) xenografted nude mice. Mice (n=3/group) were sacrificed at indicated times 3 h (black), 6 h (dark grey), 15 h (light grey), 24 h (white) and radioactivity of each organ was measured and plotted as % injected dose/g of tissue. The significance of bsFab retention in CEA positive tumor versus CEA negative tumor was compared at each time. Asterisks indicate significant difference between groups: two asterisks= p< 0.005, one asterisk= p< 0.05; and n.s= non significant. Error bars represent the standard deviation of experiments performed in triplicates. B) Tumor growth inhibition assay: At Day 0, NSG mice

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 2 Figure 1

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  Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 11, 2013; DOI: 10.1158/1535-7163.MCT-12-1012 transmembrane and intracellular domains of the γ chain (generous gift of Prof. E. Vivier,

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  Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Author Manuscript Published OnlineFirst on June 11, 2013; DOI: 10.1158/1535-7163.MCT-12-1012

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  on July 14, 2013. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 11, 2013; DOI: 10.1158/1535-7163.MCT-12-1012

Table 1 .

 1 Half maximal effective concentration of bsFabs in ADCC assay

	Cell lines	bsFab C21	EC 50 (pM)	bsFab C28
	SKOV3-CEA	4 ± 0.2		6 ± 0.5
	LS174T	3 ± 1.5		4 ± 0.4
	BxPC3	8 ± 3		17 ± 6
	HT29	5 ± 1.5		27 ± 10

  Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.Author Manuscript Published OnlineFirst on June 11, 2013; DOI: 10.1158/1535-7163.MCT-12-1012

Filled grey: bsFab C21. Open black: bsFab C28. B) MC38-CEA (black arrow) and Jurkat-huFcγRIIIa/γ (dotted arrow) were co-cultured with or without bsFabs C21 or bsFab C28. on July 14, 2013. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

on July 14, 2013. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 11, 2013; DOI: 10.1158/1535-7163.MCT-12-1012

ACKNOWLEDGMENTS

We would like to thank Sabrina Seddik, Stéphanie Charles for skilled technical assistance and Isabelle Teulon, Jacques Barbet, Hervé Watier, Séverine Barth and Mathieu Collin for helpful discussion.

GRANT SUPPORT

This work was supported by INSERM, INSERM-Transfert (ProCop). C. Boix was supported by a grant from the Pôle de Compétitivité Méditech Santé, Ile-de-France (Immucan project). C. Pétiard was supported by a grant from Inserm-Transfert (ProCop). on July 14, 2013. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 11, 2013; DOI: 10.1158/1535-7163.MCT-12-1012