Method to screen high affinity antibody

Abstract

The current invention reports a method for producing an antibody comprising the steps of a) providing a plurality of hybridoma cells each expressing an antibody, b) determining the time dependent amount of said antibody bound to the respective antigen by surface plasmon resonance at different temperatures and different antibody concentrations, c) calculating with the time dependent amount determined in b) based on equations (II) to (XIII) at least the thermodynamic parameters (i) standard association binding entropy (ΔS°‡ass), (ii) standard dissociation binding entropy (ΔS°‡diss), (iii) standard binding entropy (ΔS°), (iv) free standard binding enthalpy (ΔG°), (v) standard dissociation free binding enthalpy (ΔG°‡diss), (vi) standard association free binding enthalpy (ΔG°‡ass), (vii) −TΔS°, (viii) dissociation rate constant k.sub.d, (ix) equilibrium binding constant K.sub.D, and (x) association rate constant k.sub.a, d) selecting a hybridoma cell producing an antibody with at least two of the following: i) a standard association binding entropy of less than 10 J/K*mol, ii) an absolute standard dissociation binding entropy of 100 J/mol*K or more, iii) an absolute standard binding entropy of 100 J/mol*K or more, e) producing an antibody by cultivating said selected cell under conditions suitable for the expression of said antibody and recovering said antibody from the cells or/and the cultivation medium.

Claims

1. A method for producing an antibody comprising the following steps: a) i) generating a plurality of cells or hybridomas, each expressing an antibody, whereby the cells collectively produce a plurality of different antibodies that bind to the same antigen, ii) cultivating each of the cells or hybridomas in a separate volume, b) performing surface plasmon resonance on each of the antibodies at a plurality of different temperatures and different antibody concentrations to determine a time dependent amount of the antibody bound to the antigen by surface plasmon resonance at the different temperatures and the different antibody concentrations, c) calculating with the time dependent amount determined in b) based on equations (II) to (XIII)
ΔG°=ΔH°−T*ΔS°  (II)
ΔG°=−R*T*ln K.sub.D  (III)
ln K.sub.D=−1/T*(ΔH°/R)/slope−(ΔS°/R)/intercept  (IV)
R*T*ln K.sub.D=ΔH°.sub.T0−T*ΔS°.sub.T0+ΔC°.sub.p(T−T.sub.0)−T*ΔC.sub.p° ln(T/T.sub.0)  (V)
k.sub.a=(k.sub.b*T/h)*e.sup.(−ΔG°‡/R*T)  (VI)
ln k.sub.a/T=−1/T*(ΔH°‡/R)/slope+(ΔS°‡*R+ln k.sub.b/h)/intercept  (VII)
k.sub.a=A*e.sup.−Ea/R*T  (VIII)
ln k.sub.a=ln A/intercept−(1/T*Ea/R)/slope  (IX)
k.sub.d=(k.sub.b*T/h)*e.sup.(−ΔG°‡/R*T)  (X)
ln k.sub.d/T=−1/T*(ΔH°‡/R)/slope+(ΔS°‡/R+ln k.sub.B/h)/intercept  (XI)
k.sub.d=A*e.sup.−Ea/R*T  (XII)
ln k.sub.d=ln A/intercept−(1/T*Ea/R)/slope  (XIII) at least the thermodynamic parameters (i) standard association binding entropy (ΔS°‡ass) (ii) standard dissociation binding entropy (ΔS°‡diss) (iii) standard binding entropy (ΔS°) d) selecting one of the cells or hybridomas producing one of the antibodies that meets at least two of the following: i) a standard association binding entropy (ΔS°‡ass) of less than 10 J/K*mol, ii) an absolute standard dissociation binding entropy (|ΔS°‡diss|) of 100 J/mol*K or more, iii) an absolute standard binding entropy (|ΔS°|) of 100 J/mol*K or more, e) producing or having produced the antibody associated with the selected cell or hybridoma by cultivating the selected cell or hybridoma under conditions suitable for the expression of the antibody and recovering the antibody from the cells, hybridomas, or/and the cultivation medium.

2. The method according to claim 1 further comprising one or both of the following additional steps: after a) and before b): al) cultivating the cells of a) and providing culture supernatants each containing antibodies expressed by the cells, after step d) and before step e): dl) isolating the nucleic acid encoding the antibody from the selected cell, providing based on the isolated nucleic acid a further nucleic acid encoding a chimeric, CDR-grafted, T-cell epitope depleted and/or humanized variant of the antibody, providing an expression plasmid containing the modified nucleic acid in an expression cassette, and transfecting a CHO cell, a NS0 cell, a SP2/0 cell, a HEK293 cell, a COS cell, or a PER.C6 cell with the expression plasmid.

3. The method according to claim 1, wherein selecting the cell further comprises producing the antibody with (i) the standard association binding entropy (ΔS°‡ass) of less than 10 J/K*mol, and (ii) the absolute standard dissociation binding entropy (|ΔS°‡diss|) of 100 J/mol*K or more.

4. The method according to claim 1, wherein selecting the cell further comprises producing the antibody with (i) the standard association binding entropy (ΔS°‡ass) of less than 10 J/K*mol, and (iii) the absolute standard binding entropy (|ΔS°|) of 100 J/mol*K or more.

5. The method according to claim 1, wherein selecting the cell further comprises producing the antibody with (ii) the absolute standard dissociation binding entropy (|ΔS°‡diss|) of 100 J/mol*K or more, and (iii) the absolute standard binding entropy (|ΔS°|) of 100 J/mol*K or more.

6. The method according to claim 1, wherein selecting the cell further comprises producing the antibody with (i) the standard association binding entropy (ΔS°‡ass) of less than 10 J/K*mol, (ii) the absolute standard dissociation binding entropy (|ΔS°‡diss|) of 100 J/mol*K or more, and (iii) the absolute standard binding entropy (|ΔS°|) of 100 J/mol*K or more.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 Illustration of the Binding Late (BL) and Stability Late (SL) data of exemplary anti-PTH antibodies.

(2) FIG. 2 Binding Late/Complex Stability plot of 549 hybridoma primary cultures: the encircled data spot shows sufficient antigen response signal and 100% complex stability, whereas the enframed data spot shows no sufficient antigen response.

(3) FIG. 3 Secondary antibody response of the <IgGFCγM>R antibody capture system versus the analyte monoclonal anti-TSH antibody at 25 nM, 50 nM, 75 nM and 100 nM and at increasing temperatures.

(4) FIG. 4 a) Exemplary concentration-dependent sensograms of the temperature-dependent antibody-PTH interaction of antibody M D1.1. The kinetics were measured in HBS-EP pH 7.4 at 25° C., 3 min. association time, 5 min. dissociation time, fitting according to Langmuir; b) Exemplary concentration-dependent sensograms of the temperature-dependent antibody-PTH interaction of antibody M 9.3.1. The kinetics were measured in HBS-EP pH 7.4 at 25 C, 3 min. association time, 15 min. dissociation time, fitting according to a Langmuir 1.1. model.

(5) FIG. 5 Calculation of thermodynamic parameters according to the linear equations of van't Hoff, Eyring and Arrhenius. Exemplary plots shown for antibody M D1.1.

(6) FIG. 6 a) Exemplary concentration-dependent sensograms of the temperature-dependent antibody-PTH interaction of antibody M 9.10.20. The kinetics were measured in HBS-EP pH 7.4 at 25° C., 3 min. association time, 15 min. dissociation time, fitting according to Langmuir; b) Exemplary concentration-dependent sensograms of the temperature-dependent antibody-PTH interaction of antibody M 1F8. The kinetics were measured in HBS-EP pH 7.4 at 25° C., 3 min. association time, 5 min. dissociation time, fitting according to a Langmuir 1.1. model.

(7) FIG. 7 a) Three dimensional rate map of the data of antibody M D1.1. b) Three dimensional rate map of the data of antibody M 9.3.1.

(8) FIG. 8 a) Three dimensional rate map of the data of antibody M 9.10.20; b) Three dimensional rate map of the data of antibody M 1F8.

(9) FIG. 9 a) Double logarithmic plot of the temperature-dependent characteristics of 34 exemplary antibodies; b) Double logarithmic plot of the temperature-dependent characteristics of three exemplary antibodies: filled circles—antibody with increasing affinity with increasing temperature, open circles—antibody with constant affinity with increasing temperature, squares—antibody with decreasing affinity with increasing temperature.

(10) FIG. 10 a) Affinity plot of 13 anti-PTH antibodies from the PTH screening indicating affinities at 25° C. (X-axis) and 37° C. (Y-axis). b) Dissociation rate constant plot of the same antibodies as in a) at 25° C. (X-axis) and 37° C. (Y-axis).

(11) FIG. 11 Equilibrium thermodynamics plot of 12 exemplified anti-PTH antibodies calculated according to van't Hoff.

(12) FIG. 12 Transition state thermodynamic plot of the activation enthalpies ΔH°‡ass of 12 exemplified anti-PTH antibodies, calculated according to the Eyring equation.

(13) FIG. 13 Transition state thermodynamic plot of the activation entropy ΔS°‡ass (entropic burden), calculated according to the Eyring equation.

(14) FIG. 14 Transition state thermodynamic plot of the dissociation entropy ΔS°‡diss of 13 exemplary anti-PTH antibodies, calculated according to the Eyring equation.

(15) FIG. 15 Temperature- and concentration-dependent measurements of interactions: a) Due to inhomogeneous capture kinetics in the temperature gradient, the R.sub.MAX value varies and no equilibrium was achieved at any temperature step during the association phases of the different sensograms; b) Due to an adapted mAb concentration and prolonged association phases the R.sub.MAX values are homogeneous, the equilibrium was achieved at temperatures >35° C.

EXAMPLE 1

(16) Immunization of Mice

(17) Balb/c mice 8-12 weeks old were subjected to intraperitoneal immunization with 100 human recombinant PTH (Parathyroid hormone) derivatives formulated as a KLH (keyhole limpet haemocyanin) fusion in complete Freud's adjuvant. Recombinant N-terminal and C-terminal PTH fragments as well as full length (1-84) PTH were used as antigens. PTH derivatives were produced synthetically by peptide synthesis.

(18) The immunization was performed 4 times: initial boost, 6 weeks, 10 weeks and 14 weeks after the initial boost. The second and third immunization was done using incomplete Freud's adjuvant. The final boost was done i.v. using 100 μg antigen three days before the hybridoma fusion took place. The production of hybridoma primary cultures was done according to Köhler and Milstein (Kohler, G., et al., Nature 256(5517) (1975) 495-497). The hybridomas were isolated in 96-well micro titer plates (MTPs) by limited dilution and were screened for antigen binding by ELISA methods according to the manufacturer's manual. Primary hybridoma cell cultures, which showed a positive color formation upon antigen binding in ELISA, were transferred into the kinetic screening process.

EXAMPLE 2

(19) Preparation of the CM5 Sensor Chip

(20) The BIAcore A100 system under the control of the Software V.1.1 was prepared like follows: A BIAcore CM5 sensor (series S) was mounted into the system and was hydrodynamically addressed according to the manufacturer's recommendations. A polyclonal rabbit IgG antibody (<IgGFCγM>R, Jackson ImmunoResearch Laboratories Inc., USA) at 30 μg/ml was immobilized at 10,000 RU on spots 1, 2, 4 and 5 in the flow cells 1, 2, 3 and 4 via EDC/NHS chemistry according to the manufacturer's instructions using 10 mM sodium acetate buffer pH 4.5 as pre-concentration buffer. The sensor surface was finally blocked with ethanolamine.

EXAMPLE 3

(21) Kinetic Screening of Primary Hybridoma Culture Supernatants

(22) Hybridoma culture supernatants from different PTH immunization campaigns conducted according to Example 1 were processed as outlined below.

(23) The spots 2 and 4 of a sensor chip obtained according to Example 2 were used as a reference (1-2, 5-4). In order to capture antibody on the sensor surface hybridoma culture supernatants were diluted 1:5 with running buffer HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% P20, BIAcore) and were injected at 30 μl/min for 1 min. Subsequently, the respective antigen was injected at 30 μl/min for 2 min. association time. The dissociation phase was monitored for 5 min. Finally the surface was regenerated with a 2 min. injection of 100 mM phosphoric acid.

(24) The sensor was preconditioned by repeated cycles of antibody capturing and regeneration. The monoclonal mouse antibody mAb<TSH>M-1.20 IgG1k (Roche Diagnostics GmbH, Mannheim, Germany) was repeatedly injected for 2 min. at 30 μl/min at 50 nM in HBS-EP and the chip was regenerated using 100 mM H.sub.3PO.sub.4 by a 2 min. injection at 30 μl/min.

(25) For the selection of primary hybridomas the following procedure was used: A Binding Late (BL) reference point was set shortly before the antigen's injection ended. A

(26) Stability Late (SL) reference point was set shortly before the end of the complex dissociation phase. The BL and SL data were graphically visualized (FIG. 1). The data was used to calculate the antigen complex stability using formula (I):
antigen-complex-stability=(1−[BL(RU)−SL(RU)/BL(RU)])  (I)

(27) (see FIG. 2). E.g. the encircled data spots show sufficient antigen response signal and 100% complex stability, whereas the enframed data spot shows no sufficient antigen response.

(28) Thus, the top 10% hybridomas according to antigen response signal and complex stability have been selected.

EXAMPLE 4

(29) Hybridoma Cloning and Antibody Production

(30) Anti-PTH antibody producing hybridoma primary cultures, which were selected in Example 3, were subcloned using the cell sorter FACSAria (Becton Dickinson) under the control software V4.1.2. The deposited single clones were incubated under suitable conditions for further proliferation in 24 well plates and were subsequently transferred to the thermodynamic screening process according to Example 5 after having determined the antibody concentration in solution using ELISA methods according to the instruction of the manufacturer.

EXAMPLE 5

(31) Thermodynamic Screening of Secondary Hybridoma Culture Supernatants

(32) Subsequent to the kinetic screening, in which hybridoma cells secreting antibodies with high antibody-antigen-complex stability have been identified, the secreted antibodies were further characterized by a thermodynamic screening employing the determination of the temperature-dependent kinetics in order to determine the antigen-antibody complex thermostability and in order to calculate the thermodynamic properties.

(33) A CM5 sensor series S was mounted into the BIAcore T100 System driven under the control software V1.1.1 and preconditioned by 1 min. injection at 100 μl/min of a mixture comprising 0.1% SDS, 50 mM NaOH, 10 mM HCl and 100 mM H.sub.3PO.sub.4.

(34) In case of screening antibodies of murine origin, a polyclonal rabbit anti-murine-IgG antibody (<IgGFCγM>R, Jackson ImmunoResearch Laboratories Inc., USA) at 30 μg/ml was immobilized at 6,000 RU on flow cells 1, 2, 3, 4 with EDC/NHS chemistry according to the manufacturer's instructions using 10 mM sodium acetate buffer pH 4.5 as pre-concentration buffer. Finally, the sensor surface was blocked with ethanolamine.

(35) As reference antibody the monoclonal murine anti-TSH antibody 1.20 (IgG1k, mouse) was temperature-dependently titrated at different concentrations on the above prepared capture system to determine the capture capability of the system (see FIG. 3).

(36) TABLE-US-00003 TABLE 3 Response levels of monoclonal murine anti-TSH antibody 1.20 at different concentrations under increasing temperatures on the <IgGFCγM>R antibody capture system. RU at ° C.: AK_nM 17° C. 21° C. 25° C. 29° C. 33° C. 37° C. TSH_25 91 106 122 137 153 170 TSH_50 187 206 226 248 273 295 TSH_75 225 248 270 293 318 340 TSH_100 270 292 315 342 369 394

(37) These concentration values of the monoclonal murine anti-TSH antibody 1.20 were used as a reference for the calculation of the antibody capturing from the hybridoma cultures in order to achieve similar secondary antibody response levels at different temperatures.

(38) Kinetic measurements at different temperatures were performed at 20 μl/min., the flow rate was 30 μl/min., 50 μl/min., 100 μl/min., respectively. The sample injection of recombinant synthetic full length PTH 1-84 (9.4 kDa) was done for 30 sec., 90 sec., 180 sec., respectively, or other suitable injection times in order to achieve ligand saturation or entry into the binding equilibrium during the complex association phase (see FIG. 4 a)). The dissociation rate was monitored first for up to 300 sec. and further for 15 min (see FIG. 4 b)). The PTH injections were repeated in different concentration steps of at least five concentrations. As control one concentration step was analyzed twice to control the reproducibility of the assay. Flow cell 1 served as a reference. A buffer injection was used instead of an antigen injection to double reference the data by buffer signal subtraction. The capture system was regenerated using 100 mM H.sub.3PO.sub.4 by a 2 min. injection at 100 μl/min. The regeneration procedure was optimized to guarantee quantitative surface regeneration also at 13° C., 17° C. and 21° C. At these temperatures the regeneration solution was injected three times whereas at 25° C., 29° C., 33° C. and 37° C. the regeneration solution was injected one time.

(39) The data obtained was evaluated according to a 1:1 binary Langmuir interaction model in order to calculate the association rate constant ka [1/Ms], the dissociation rate constant kd [1/s] and the resulting affinity constant K.sub.D [M] at different temperatures. Thermodynamic equilibrium data was calculated according to the linear form of the Van′t Hoff equation. Transition State thermodynamics were calculated according to the Eyring and Arrhenius equations using e.g. the BIAcore T100 evaluation software V.1.1.1 or the program Origin 7sri v. 7.0300.

EXAMPLE 6

(40) Example for the Necessity to Adjust Homogeneous R.sub.MAX Values

(41) A BIAcore T100 device was mounted with a CM5 series-S BIAcore sensor, and was immobilized with 6000 RU <IgGFCyM>R (Jackson ImmunoResearch Laboratories Inc., USA) on each flow cell according to the manufacturer's instructions. The non-optimized experiment used 40 nM capture antibody at 20 μl/min., in HBS-EP buffer (0.05% P20). The sample buffer was the system buffer, supplemented with 1 mg/ml CMD.

(42) The antigen was injected after the capturing of the secondary antibody in 6 concentration steps of 1.2 nM, 4 nM, 11 nM, 33 nM, 100 nM and 300 nM, whereby 11 nM were used as a double control and 0 nM were used as reference. The antigen was injected at 100 μl/min for 2 min. association and 5 min. dissociation, followed by a HBS-EP wash of 15 min. at 30 μl/min. and a regeneration with 10 mM glycine pH 1.7 at 3 μl/min for 3 min. Concentration-dependent measurements were done at 4° C., 11° C., 18° C., 25° C., 32° C., and 40° C.

(43) The optimized system was used like described above, but with the exceptions that the antibody to be captured was injected for 3 min. association time at different concentration steps of 100 nM at 15° C., 80 nM at 20° C., 60 nM at 25° C., 50 nM at 30° C., 40 nM at 35° C. and 40 nM at 40° C.

(44) Finally kinetics and thermodynamics were determined using the BIAcore evaluation software.