ANTIBODY QUANTIFICATION IN BIOLOGICAL SAMPLES

Abstract

The invention relates to a method for quantifying bispecific antibodies, in particular bispecific antibody therapeutics, in biological samples by quantifying a unique signature peptide of said antibody by mass spectrometry. The invention relates also to a kit comprising the unique signature peptide.

Claims

1. A method for quantifying a bispecific antibody in a biological sample, wherein the antibody comprises an engineered human IgG CH3 heterodimer comprising several substitutions in the CH3 domains, including at least two substitutions at positions 80 to 88 of a first CH3 domain, wherein the method comprises quantifying a signature peptide of said antibody by mass spectrometry, wherein the signature peptide is a tryptic peptide corresponding to positions 80 to 88 of said first CH3 domain, and wherein the amino acid positions are indicated according to IGMT® numbering.

2. The method according to claim 1, wherein the signature peptide consists of a sequence: TABLE-US-00011 (SEQ ID NO: 1) TX.sub.1PPX.sub.2LX.sub.3SX.sub.4GSFX.sub.5LX.sub.6SX.sub.7 wherein X.sub.1 represents T or D, X.sub.2 represents V, L, P or M, X.sub.3 represents D, Q or E, X.sub.4 represents D or Q, X.sub.5 represents F, A or W, X.sub.6 represents S, W or H, and X.sub.7 represents K or R, with the proviso that when X.sub.1 is T, then at least one of X.sub.2, X.sub.3, X.sub.4, X.sub.5, and X.sub.7 is such that X.sub.2 is L, P or M; X.sub.3 is Q or E; X.sub.4 is Q; X.sub.5 is A or W; and X.sub.7 is R.

3. The method according to claim 2, wherein the signature peptide is selected from the group consisting of: TTPPVLDSDGSFALSSK (SEQ ID NO: 3), TDPPLLESDGSFALSSR (SEQ ID NO: 4), TDPPLLESQGSFALSSR (SEQ ID NO: 5), TTPPPLQSDGSFWLWSK (SEQ ID NO: 6) and TTPPMLESDGSFFLHSK (SEQ ID NO: 7), preferably SEQ ID NO: 4 or SEQ ID NO: 5.

4. The method according to any one of the preceding claims, wherein the bispecific antibody comprises a human IgG CH3 domain heterodimer engineered using T-cell receptor-based immunoglobulin domain interface, wherein the first CH3 domain is from human IgG1 and comprises at least the substitutions F85.1A and Y86S and the second CH3 domain is from human IgG1 or IgG3 and comprises at least the substitutions S20K, T22V, K26T, K79Y, K88W and T90N, and wherein said positions are indicated according to IGMT® numbering.

5. The method according to claim 4, wherein: the first CH3 domain further comprises one or more of the following substitutions: Q3E, Y5A, L7F, S20T, T22V, K26T, T81D, V84L, D84.2E, K88R and T90R; preferably Q3E, Y5A, L7F, S20T, T22V, K26T, T81D, V84L, D84.2E, K88R and T90R or Q3E, Y5A, L7F, S20T, T22V, K26T, T81D, V84L, D84.2E and K88R; and the second CH3 domain further comprises one or more of the following substitutions: Q3A, D12E, L14M, N44S, V84M, F85.1S, Y86V, V101I, H115R and Y116F; preferably F85.1S and Y86V; F85.1S, Y86V and Q3A for a CH3 domain from human IgG1, and D12E, L14M, N44S, V84M, F85.1S, Y86V, V101I, H115R and Y116F fora CH3 domain from human IgG3.

6. The method according to any one of the preceding claims, wherein the antibody comprises a human IgG CH3 domain heterodimer engineered using immunoglobulin domain interface exchange between human IgG and IgD CH3 domains, wherein the first CH3 domain comprises the substitutions: Q3V, Y5L, K26S, V84P, D84.2Q, F85.1W and Y86W and the second CH3 domain comprises the substitutions: S20W, K79A, T81A, K88V and T90R.

7. The method according to any one of the preceding claims, wherein the antibody comprises a human IgG CH3 domain heterodimer engineered using immunoglobulin domain interface exchange between human IgG and IgM CH3 domains, wherein the first CH3 domain comprises the substitutions: Q3D, K26T, V84M, D84.2E and Y86H and the second CH3 domain comprises the substitutions: S20T, K79V, T81S and K88I.

8. The method according to any one of the preceding claims, wherein the bispecific antibody comprises a Fc, a Fab and a scFv from human immunoglobulin.

9. The method according to any one of the preceding claims, wherein the bispecific antibody is a therapeutic antibody.

10. The method according to any one of the preceding claims, wherein the bispecific antibody is a bispecific anti-CD3 antibody, preferably anti-CD3 and anti-Her2, anti-CD3 and anti-CD38 or anti-CD3 and anti-EGFR bispecific antibody.

11. The method according to any one of the preceding claims, which comprises the steps of: a) purifying the bispecific antibody from the biological sample by immunocapture, b) digesting the bispecific antibody obtained in step a) with trypsin or trypsin/Lys C to generate peptides comprising the signature peptide, and c) subjecting the peptides obtained in step b) to mass spectrometry to determine the amount of signature peptide in the biological sample by comparison with an internal standard or with the use of calibration standards.

12. The method according to claim 11, wherein the immunocapture is performed with an antibody specific for the bispecific antibody; preferably an anti-idiotype antibody, an antibody against the signature peptide, or a combination thereof; preferably the immunocapture is performed on a solid support or using immunomagnetic separation; more preferably using biotinylated antibody and streptavidin coated magnetic beads.

13. The method according to any one of the preceding claims, wherein the mass spectrometry comprises two-dimensional nano-liquid chromatography coupled to electrospray-ionization Orbitrap mass spectrometry.

14. The method according to any one of the preceding claims, wherein the biological sample is a human biological sample, preferably a human body fluid, more preferably human serum.

15. The method according to any one of the preceding claims, wherein the lower limit of quantification of the bispecific antibody is 50 pg/ml and the detection range of the bispecific antibody is from 50 pg/ml to 5000 pg/ml in human serum.

16. A kit for quantifying a bispecific antibody in a biological sample using the method of any one of the preceding claims, comprising at least a signature peptide as defined in any one of claims 1 to 3; preferably further comprising an antibody specific for the bispecific antibody as defined in claim 12.

Description

FIGURE LEGENDS

[0096] FIG. 1 represents LC-HRMS/MS profile of GBR 1302 tryptic digest.

A. GBR 1302 tryptic digest showing peptide TDPPLLESDGSFALSSR (SEQ ID NO: 4) with m/z 896.44 (M+2H).sup.2+ B. GBR 1302 tryptic digest showing peptide EPEVATFPPSR (SEQ ID NO: 10) with m/z 615.31 (M+2H).sup.2+. C. Human serum blank.

[0097] FIG. 2 represents GBR 1302 quantification in human serum by LC-HRMS/MS.

[0098] FIG. 3 represents GBR 1342 quantification in monkey serum by LC-HRMS/MS.

[0099] FIG. 4: Geometric mean serum profile for GBR1302.

EXAMPLES

Materials and Methods

1. Materials

[0100] Bispecific Antibodies [0101] GBR 1302: anti-CD3/anti-Her2 human IgG1 BEAT® bispecific antibody [0102] GBR 1342: anti-CD3/anti-CD38 human IgG1 BEAT® bispecific antibody [0103] GBR 1372: anti-CD3/anti-EGFR human IgG1 BEAT® bispecific antibody

[0104] Internal Standard [0105] GBR 1302 IS: Stable Isotope labelled (SIL) signature peptide SEQ ID NO: 4 (m/z 901.44 (M+2H).sup.2+). Working solution 100 pg/mL in 30:70 ACN:Water [0106] GBR 1342 IS: Stable Isotope labelled (SIL) signature peptide SEQ ID NO: 5 (m/z 907.96 (M+2H).sup.21; Working solution 100 pg/mL in 30:70 ACN:Water

[0107] Reagents [0108] Biological matrix: human or monkey serum [0109] Streptavidin Beads (DynaBeads Streptavidin T1, p/n 650-02, Invitrogen) [0110] Biotinylated OKT3 antibody (Abpro and inhouse) [0111] Biotinylated 9G7 antibody (Abpro and inhouse) [0112] Biotinylated SP34 antibody (Bio-rad and inhouse) [0113] TCEP Reducing Solution (75 mM TCEP in water, freshly prepared) [0114] Iodoacetamide Alkylation (IAA) Solution (150 mM IAA in water, freshly prepared) [0115] Trypsin Solution (Trypsin Gold (PROMEGA) 50 μg/mL in water; freshly prepared) [0116] Acetonitrile (ACN) solution: 30:70 ACN:Water [0117] PBS/BSA: 0.1% BSA in 10 mM PBS [0118] TrisHC1, 1M, pH 8.3 [0119] HCl 30 mM

[0120] Calibration Standards and Quality Control (QC) Samples [0121] QC samples: 50, 150, 300, 750, 4000 pg/mL of bispecific antibody in biological matrix [0122] Standards: STD1 to STD8: 50, 75, 100, 250, 500, 1000, 3000 and 5000 pg/mL of bispecific antibody in biological matrix

2. Methods

2.1 Sample Preparation

[0123] Samples, quality controls, standards, zero samples and blanks in PBS-BSA are distributed in wells of Plate A, biotinylated antibody (2 μg) is then added, and Plate A is incubated overnight at 4° C. with shaking. Streptavidin T1 beads are then added in the wells and Plate A is incubated for 1 h at room temperature to bind up biotinylated-antibody captured analyte. Beads are then transferred to a collection plate (Plate B) placed on a magnetic stand and washed twice with CHAPs buffer and twice with PBS buffer. Antibody elution is performed by adding 30 mM HCl into Plate B, mixing for 3 min and transferring the eluate into Plate C containing 1 M Tris pH 8.3; and repeating the elution step.

[0124] Internal standard is added to samples, quality controls, standards and zero samples of Plate C. 30:70 ACN:Water is added to control blanks of Plate C. 75 mM TCEP is added to each well, after mixing for 1 min, the plate is incubated at 56° C. for 45 min. The plate is cooled down at RT for 10 min. 150 mM IAA is added to each well, and after mixing for 1 min, the plate is incubated at RT for 35 min in the dark. Trypsin (50 μg/mL) is added to each well, and after mixing for 1 min, the plate is incubated at 37° C., overnight with shaking.

2.3 Chromatography

[0125]

TABLE-US-00005 Autosampler Dionex ultimate 3000 AS Injection vol 100 μL Loading and micro pump Dionex. Flow rate: Loading pump: 50 to 400 ul/min (flow gradient) Micro pump: 300 to 400 μL/min (flow gradient) Nano LC pump Dionex ultimate 3000 (NCS-3500RS) nano pump Flow rate: 600 nL/min Trap Column μ-Precolumn Cartridge P/N 160454 fitted with an Thermo Acclaim PepMap100, C18 300 μm × 5 mm, 5 μm Analytical Column Thermo Acclaim PepMap C18 75 μm × 15 cm, 5 μm Column Temperature 60° C. Mobile Phase: Nano A: 98% H.sub.2O, 2% ACN with  0.1% formic acid B: 90% ACN, 10% water with  0.1% formic acid Mobile Phase: (1) C: 60% IPA, 30% ACN, 10% water with (1) Loading  1% formic acid (2) Micro pump (2) A: 60% IPA, 30% ACN, 10% water with  1% formic acid B: 1% formic acid in water [0126] Retention time: GBR 1302 4.40±1.0 minutes [0127] GBR 1302-IS 4.40±1.0 minutes [0128] GBR 1342 4.10±1.0 minutes [0129] GBR 1342-IS 4.10±1.0 minutes

[0130] The method is performed using 2D Trap-Nano LC Configuration in which Thermo QE and Dionex ultimate 3000 RSLC nano LC are coupled with Thermo Easy-Spray source. The samples are first loaded by loading pump onto a trap column followed by switching to a nanoLC analytical column operated at a flow rate of 600 nL/min by a nano pump. The analytical column coiled into a loop is intimately coupled with a linear restrictor emitter. The trap column and analytical column are both washed with high organic solvent to elute highly retained endogenous components by nano pump and micropump.

2.4 Mass Spectrometry

[0131]

TABLE-US-00006 Mass Spectrometry Thermo Q-Exactive Series mass spec Ion mode Positive ion mode Experiment Ionization PRM (Target MS2) Thermo Easy Spray Compounds for inclusion list: GBR1302: m/z 896.441 z = +2 GBR1302 IS: m/z 901.445 z = +2 GBR1342: m/z 902.96 z = +2 GBR1302 IS: m/z 907.96 z = +2 Collision energy 21 Spray Voltage 2300 V Orbitrap Resolution 17,500 AGC Target 5 e5 Max Injection Time 50 ms S-lens RF amplitude 90 Capillary Tem. 275° C. Fragment (charge state z = +2) GRB1302: m/z 788.404 GRB1302 IS: m/z 793.408 GRB1342: m/z 794.919 GRB1342 IS: m/z 799.923 [0132] MS Acquisition time: GRB1302 and GRB1302 IS: 4.1 min to 5.1 min [0133] GRB1342 and GRB1342 IS: 4 min to 5 min

Example 1: Identification of a Unique Signature Peptide for Bispecific Antibody Quantification in Human or Non-Human Primate Serum

[0134] In a first approach, potential signature peptides for quantifying bispecific antibodies in human serum were selected using standard rules for selection: 6-15 aa; no chemical chemical reactive residues (Tryptophan (W), Methionine (M), Cysteine (C)); no inclusion of 2R, 2K and RK; no potential PTM (Tyrosine (Y), Threonine (T), Serine(S), Lysine (K)); preferably containing Proline (P); R in P proximity (potential missed tryptic cleavage). Based on these rules, 15 signature peptides (SPs) were selected in human serum spiked with GBR 1302. Two peptides LYSGVPSR (SEQ ID NO: 8) and FTISADTSK (SEQ ID NO: 9) were selected for optimization based on their mass response intensities. It was observed that the selected signature peptides were present in the blank human serum as well as in GBR 1302 spiked human serum sample suggesting that they were not specific for GBR 1302.

[0135] In a second approach, the software Expasy peptide cutter (http://web.expasy.org/peptide_cutter/) was used to predict the peptides generated by Trypsin digestion of GBR 1302, GBR 1342 and GBR 1372 bispecific antibodies. The resulting tryptic peptide sequences were then compared to the human plasma proteome (NCBI BLAST) to exclude peptides which were not unique to the bispecific antibodies (present in the plasma proteome).

[0136] Two unique signature peptides were found in LC-HRMS/MS profile of GBR 1302 tryptic digest: TDPPLLESDGSFALSSR (SEQ ID NO: 4) with m/z 896.44 (M+2H).sup.2+ and EPEVATFPPSR (SEQ ID NO: 10) with m/z 615.31 (M+2H).sup.2+ (FIGS. 1A and 1B). It was observed that they were unique for GBR 1302 and not present in blank human serum and Trastuzumab (FIG. 1C).

[0137] It was also observed that both peptides were situated in the engineered CH3 heterodimer which is present in all the bispecific antibodies generated by immunoglobulin domain interface exchange technology. The SP of SEQ ID NO: 4 is situated from positions 80 to 88 of IgG CH3 domain according to IGMT numbering. The SP of SEQ ID NO: 10 is situated from positions 1 to 11 of IgG CH3 domain according to IGMT numbering.

[0138] Based on mass response intensity, signature peptide TDPPLLESDGSFALSSR (SEQ ID NO: 4) was selected for bispecific antibody quantification.

[0139] GBR 1372 comprises the same Fc heterodimer as GBR 1302. The same unique signature peptide (SEQ ID NO: 4) was found in LC-HRMS/MS profile of GBR 1372 tryptic digest.

[0140] GBR 1342 comprises a Fc heterodimer which differs from that of GBR 1302 and GBR 1372 by a D84.4Q substitution. The corresponding signature peptide TDPPLLESQGSFALSSR (SEQ ID NO: 5) with m/z 902.96 (M+2H).sup.2+ was found in LC-HRMS/MS profile of GBR 1342 tryptic digest.

[0141] These results show that the signature peptide from positions 80 to 88 of one CH3 domain of an engineered human IgG CH3 heterodimer is a unique signature peptide which can be used for the quantification of all the bispecific antibodies having an engineered human IgG CH3 heterodimer in human serum.

Example 2: High Sensitivity LC-HRMS/MS Assay for Bispecific Antibody Quantification in Human or Monkey Serum

[0142] A LC-HRMS/MS assay based on the detection of the unique signature peptide identified in example 1 was developed for bispecific antibody quantification in human or non-human primate serum. GBR 1302 was quantified in human serum based on MS analysis of the signature peptide SEQ ID NO: 4. GBR 1342 was quantified in monkey serum based on MS analysis of the signature peptide SEQ ID NO: 5. The steps of the assay are disclosed in details the materials and methods section. Briefly, bispecific antibody spiked in human or monkey serum was immunopurified using biotinylated anti-idiotype antibody and streptavidin coated immunomagnetic beads. Bispecific antibody internal standard (IS; stable-isotope-labeled (SIL) signature peptide) was added to immunopurified bispecific antibody before pretreatment with TCEP and iodoacetamide and trypsin digestion. Trypsin digest was then subjected to 2D Trap-Nano LC-Nano ESI MS/MS using Thermo Q-Exactive Orbitrap Mass Spectrometer.

GBR 1302 Quantification in Human Serum

[0143] A linear calibration curve was established with a mean correlation coefficient of R.sup.2=(0.9978) using 8 standards (STD1 to STD8: 50, 75, 100, 250, 500, 1000, 3000 and 5000 pg/mL). Calibration curve was linear for 2 orders of magnitude and gave a LLOQ of 50 pg/mL (FIG. 2). GBR 1302 concentrations derived using the linear regression curves generated in these experiments displayed an accuracy of (97.8-100.9%) and a % CV (imprecision)<9.4, FIG. 2 and Table 1.

TABLE-US-00007 TABLE 1 GBR 1302 quantification in human serum: accuracy and precision STD 1 STD 2 STD 3 STD 4 STD 5 STD 6 STD 7 STD 8 Theor. Conc. 50 75 100 250 500 1000 3000 5000 Found Conc. pg/mL #1 48.46 76.14 98.42 269.95 480.97 986.58 3108.91 4890.80 #2 51.89 74.45 99.29 236.42 494.30 970.26 3060.55 5202.19 Mean 50.18 75.30 98.86 253.19 487.63 978.42 3084.73 5046.50 S.D. 2.42 1.19 0.62 23.71 9.43 11.54 34.20 220.19 % CV 4.8 1.6 0.6 9.4 1.9 1.2 1.1 4.4 % Theoretical 100.4 100.4 98.9 101.3 97.5 97.8 102.8 100.9 % Dev 0.4 0.4 −1.1 1.3 −2.5 −2.2 2.8 0.9 n 2 2 2 2 2 2 2 2

GBR 1342 Quantification in Monkey Serum

[0144] A linear calibration curve was established with a mean correlation coefficient of R.sup.2=(0.9966) using 8 standards (STD1 to STD8: 50, 75, 100, 250, 500, 1000, 3000 and 5000 pg/mL). Calibration curve was linear for 2 orders of magnitude and gave a LLOQ of 50 pg/mL (FIG. 3). GBR 1342 concentrations derived using the linear regression curves generated in these experiments displayed an accuracy of (94.3-101.8%) and a % CV (imprecision)<5.7, FIG. 3 and Table 2.

TABLE-US-00008 TABLE 2 GBR 1342 quantification in monkey serum: accuracy and precision Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Std 7 Std 8 Theor. Conc. 50 75 100 250 500 1000 3000 5000 Found Conc. #1 53.66 75.20 97.11 255.77 529.56 915.63 3103.21 5144.84 #2 46.56 75.18 102.49 238.59 486.42 969.49 3087.14 5038.80 Mean 50.11 75.19 99.80 247.18 507.99 942.56 3095.18 5091.82 S.D. 5.02 0.02 3.81 12.15 30.50 38.08 11.36 74.98 % CV 10 0 3.8 4.9 6 4 0.4 1.5 % Theoretical 100.2 100.3 99.8 98.9 101.6 94.3 103.2 101.8 % Dev 0.2 0.3 −0.2 −1.1 1.6 −5.7 3.2 1.8 n 2 2 2 2 2 2 2 2

[0145] These results show that the LC-HRMS/MS assay according to the present invention can achieve bispecific antibody quantification in human and non-human primate serum with a high sensitivity (LLOQ of 100 pg/mL), a wide range of detection (two orders of magnitude, 50 pg/mL to 5000 pg/mL) and good precision and accuracy. Consequently, the LC-HRMS/MS assay according to the present invention is a very performant assay for preclinical and clinical studies of bispecific antibody therapeutics.

Example 3: Pharmacokinetics Studies of GBR1302 in Patients with Progressive HER2-Positive Solid Tumors

Material and Methods

[0146] To evaluate the pharmacokinetic of GBR1302 in adults with progressive HER2-positive solid tumors for which no standard or curative treatment is available, a phase 1, first-in-human, open-label, multicenter, dose-escalation study was carried. Subjects received intravenous GBR 1302 on Day 1 and Day 15 in 28-day treatment cycles at escalating dose levels, starting at 1 ng/kg. The first 4 cohorts consisted of a single subject; subsequent cohorts are being enrolled using a 3+3 design. Blood samples were collected for pharmacokinetic (PK) and antidrug antibody (ADA) analyses (secondary endpoints). Quantification of GBR 1302 serum concentrations (for PK) and detection/confirmation of anti GBR 1302 antibodies (for immunogenicity) were performed using validated LC/MS/MS and ELISA methods, respectively. PK parameters were evaluated using standard non-compartmental methods.

[0147] The following PK parameters were estimated: [0148] Maximum observed serum concentration (C.sub.max) [0149] Area under the serum concentration-time curve from time 0 to time of the last measurable concentration (AUC.sub.last) [0150] Time of maximum observed serum concentration (T.sub.max) [0151] Time of last observed serum concentration (T.sub.last) [0152] Serum elimination half-life (t.sub.1/2) [0153] Last measurable plasma concentration (C.sub.last)

[0154] To assess immunogenicity antidrug antibody (ADA) response was measured.

Results:

[0155] Pharmacokinetic of GBR1302 was studied in 31 subjects over a dose range of 1 ng/kg to 750 ng/kg, as shown in Tables 3 and 4, and FIG. 4.

TABLE-US-00009 TABLE 3 Individual subject PK. NCA analysis using Phoenix WinNonlin version 8.0 linear trapezoidal method with IV infusion dosing. For PK analysis, actual infusion start and end times were used along with actual elapsed PK sampling time points upto cohort 8. For cohort 9, actual PK sampling times were not available, hence scheduled sampling time were used for PK calculations. CHRT SUBJ Dosing_ Dose C.sub.max AUC.sub.last T.sub.max T.sub.last t.sub.1/2 C.sub.last ID ID Occasion (ng/kg) (ng/mL) (hr*ng/mL) (hr) (hr) (hr) (ng/mL) 1 102- Dose 1 1 NC NC NC NC NC NC 001 1 102- Dose 2 3 0.145 NC 4 4 NC NC 001 2 101- Dose 1 3 NC NC NC NC NC NC 001 2 101- Dose 2 10 0.0911 NC 4 4 NC NC 001 2 101- Dose 3 10 NC NC NC NC NC NC 001 3 102- Dose 1 10 0.109 NC 4.01 4.01 NC NC 002 3 102- Dose 2 30 0.433 24.4 4.01 144 NC 0.062 002 3 102- Dose 3 30 0.269 21.1 4.00 146.17 NC 0.0623 002 3 102- Dose 4 30 0.395 24.0 2.00 146.67  .sup.b67.2 0.069 002 4 101- Dose 1 30 0.32 10.0 4.07 48.00 NC 0.117 002 4 101- Dose 2 60 0.559 31.8 4.07 143.97 NC 0.0689 002 4 101- Dose 3 60 0.606 54.4 2.22 311.72 .sup. 127 0.0627 002 4 101- Dose 4 60 0.528 69 2.00 335.02 .sup. 114 0.0558 002 5 101- Dose 1 60 0.327 10.7 4.02 48.08 NC 0.129 003 5 101- Dose 1 60 0.362 11.9 4.05 49.22 NC 0.134 004 5 101- Dose 1 60 0.602 18.6 4.28 46.92 NC 0.229 005 5 101- Dose 1 60 0.403 30.7 4.03 143.67 NC 0.0786 006 5 102- Dose 1 60 0.545 54.5 4.00 312.08 .sup. 123 0.053 004 5 103- Dose 1 60 0.63 18.9 4.08 48 NC 0.169 001 5 103- Dose 1 60 0.468 27.1 4.02 144.03 NC 0.0659 002 5 104- Dose 1 60 0.884 87.6 4.08 316.25  .sup. 92.8 0.0673 002 5 101- Dose 2 100 0.575 32.4 4.08 143.53 NC 0.0663 003 5 101- Dose 2 100 0.726 60 4.07 313.2 .sup. 114 0.0585 004 5 101- Dose 2 100 0.789 53.5 4.08 144 NC 0.123 006 5 102- Dose 2 100 1.08 105 4 359.17 .sup.b150 0.0803 004 5 103- Dose 2 100 0.821 64.5 4.27 335.23 .sup. 115 0.0586 002 5 104- Dose 2 100 1.78 186 4.15 335.5  .sup. 98.1 0.0931 002 5 101- Dose 3 100 0.669 64 2 335.37 .sup. 119 0.0512 004 5 101- Dose 3 100 0.867 73.3 2.08 315.17 .sup.  85.8 0.0584 006 5 102- Dose 3 100 0.899 98.2 2 335.67 .sup.b150 0.0875 004 5 103- Dose 3 100 1.04 77.3 4 335.73 .sup.b177 0.0624 002 5 104- Dose 3 100 1.98 204 2 360.08 .sup. 116 0.121 002 5 101- Dose 4 100 0.832 63.5 1.03 312.6  .sup. 92.7 0.0624 004 5 102- Dose 4 100 1.02 79.5 2 335.67 .sup. 117 0.0618 004 5 103- Dose 4 100 1.33 83.4 1.03 335.27  .sup. 84.6 0.072 002 5 104- Dose 4 100 2.92 191 1 287.42  .sup. 95.4 0.181 002 6 101- Dose 1 100 0.666 38 4.12 142.67 NC 0.103 007 6 103- Dose 1 100 0.533 28.1 4.07 119.65 NC 0.0751 005 6 201- Dose 1 100 0.733 58.7 4.2 313.08 .sup. 125 0.0611 001 6 101- Dose 2 200 1.19 107 4.08 314.87 .sup. 119 0.101 007 6 103- Dose 2 200 0.929 79.2 6.8 292.8 NC 0.0598 005 6 201- Dose 2 200 1.05 83.4 4.1 337.4  .sup. 94.5 0.0695 001 6 101- Dose 3 200 1.15 94.9 2.03 335.08 .sup.b154 0.0754 007 6 103- Dose 3 200 1.02 67.9 4 335.6 .sup. 123 0.065 005 6 201- Dose 3 200 1.94 146 3.92 336.85 .sup. 104 0.111 001 6 101- Dose 4 200 1.39 78.8 2 141.33  .sup.b67.5 0.261 007 6 201- Dose 4 200 1.68 136 4.27 315.78 .sup.  86.9 0.0864 001 7 101- Dose 1 200 2.17 126 4.67 333.93 .sup. 126 0.0675 009 7 101- Dose 1 200 1.39 102 5.75 333.67 .sup.b117 0.12 010 7 103- Dose 1 200 2.17 224 4.07 286.43 .sup.b138 0.246 006 7 101- Dose 2 300 2.54 184 4.65 315  .sup. 95.1 0.13 009 7 101- Dose 2 300 2.44 368 4.03 530.63 .sup. 113 0.0876 010 7 103- Dose 2 300 3.05 322 4 382.97 .sup. 109 0.156 006 7 101- Dose 3 300 1.93 325 2.03 334.83 NC 0.141 010 7 103- Dose 3 300 2.42 143 2 95.72 NC 0.765 006 7 101- Dose 4 300 3.62 NC 2 4.07 NC NC 010 8 101- Dose 1 300 3.21 273 6.22 334.92 .sup.b127 0.198 011 8 101- Dose 1 300 2.45 182 4.02 333.17 .sup. 108 0.123 012 8 201- Dose 1 300 2.02 224 4.18 335.53 .sup.b139 0.154 002 8 101- Dose 2 500 6.59 495 6.37 336.95 .sup. 126 0.302 011 8 101- Dose 2 500 4.45 332 4.03 336.35 .sup.b130 0.205 012 8 201- Dose 2 500 2.54 216 4.18 335.52 .sup.b165 0.194 002 8 101- Dose 3 500 5.68 436 2.5 335.22 .sup.b153 0.377 011 8 101- Dose 3 500 6.23 299 2.03 335.88 .sup.b142 0.215 012 8 201- Dose 3 500 4.71 265 2 311.93  .sup.b85.5 0.268 002 8 101- Dose 4 500 6.92 402 2 338.37 .sup.b146 0.334 011 8 101- Dose 4 500 5.43 278 1.03 336.58 .sup.b165 0.217 012 9 101- Dose 1 500 7.05 626 4.02 334.08 .sup.b197 0.661 013 9 101- Dose 1 500 4.46 377 11.28 360.2 .sup. 145 0.226 014 9 106- Dose 1 500 3.14 199 4 143.83 NC 0.6 001 9 106- Dose 1 500 5.51 321 6 334.83 .sup. 117 0.161 002 9 107- Dose 1 500 6.13 287 5.17 168.92 NC 0.181 001 9 201- Dose 1 500 6.24 346 4.02 143.87 NC 0.877 004 9 203- Dose 1 500 5.01 359 9.55 335.27 .sup. 105 0.216 001 9 204- Dose 1 500 5.24 170 8.4 49.45 NC 1.87 001 9 204- Dose 1 500 7.88 784 3.93 335.5 .sup. 155 0.813 002 9 204- Dose 1 500 8.02 727 4.02 336.58  .sup. 94.3 0.321 003 9 209- Dose 1 500 7.94 423 4.33 146.17 NC 0.802 001 9 209- Dose 1 500 4.04 132 2.05 48.22 NC 1.49 002 9 209- Dose 1 500 13.2 898 5.15 260.23  .sup. 74.8 0.648 003 9 209- Dose 1 500 4.55 253 4 336.73 .sup.b208 0.143 006 9 209- Dose 1 500 5.39 266 7.92 145.92 NC 0.642 007 9 101- Dose 2 750 16.2 861 4.03 146.08 NC 2.24 013 9 101- Dose 2 750 9.14 576 4.02 289.33 .sup. 115 0.441 014 9 106- Dose 2 750 7.56 242 5.92 48 NC 2.87 002 9 203- Dose 2 750 8.51 577 4.17 360.68 .sup. 138 0.373 001 9 204- Dose 2 750 12.4 954 3.98 337.02 .sup.b160 0.895 002 9 204- Dose 2 750 13.3 1290 4.02 309.98 .sup. 111 1.26 003 9 209- Dose 2 750 20.8 1490 4.05 526.05 .sup. 104 0.137 003 9 209- Dose 2 750 4.28 NC 4.02 4.02 NC NC 006 9 203- Dose 3 750 10.7 318 3.17 46.27 NC 3.34 001 9 204- Dose 3 750 14.4 824 2 335.6 .sup.b141 0.672 002 9 209- Dose 3 750 13.3 884 4.28 312.75  .sup. 81 0.44 003 9 204- Dose 4 750 10.6 590 1.02 360.52 .sup. 149 0.522 002 .sup.bflagged because the either the R2 adjusted is <0.8, and/or AUC % extrapolated is >20%, and/or duration of Kel estimation is <1.5-fold of the resultant t.sub.1/2. .sup.bflagged because the either the R2 adjusted is <0.8, and/or AUC % extrapolated is >20%, and/or duration of Kel estimation is <1.5-fold of the resultant t1/2

TABLE-US-00010 TABLE 4 Summary PK parameters. [Mean (SD)] of GBR 1302. Summary PK parameters [Mean (SD)] of GBR 1302 Cohort Dosing_ Dose C.sub.max AUC.sub.last #T.sub.max T.sub.last t.sub.1/2 C.sub.last ID Occasion (ng/kg) (ng/mL) (hr*ng/mL) (hr) (hr) (hr) (ng/mL) Cohort 1 Dose 1 1 NC NC NC NC NC NC (N = 1) Cohort 1 Dose 2 3 0.145 NC 4.00 4.00 NC NC (N = 1) Cohort 2 Dose 1 3 NC NC NC NC NC NC (N = 1) Cohort 2 Dose 2 10 0.091 NC 4.00 4.00 NC NC (N = 1) Cohort 2 Dose 3 10 NC NC NC NC NC NC (N = 1) Cohort 3 Dose 1 10 0.109 NC 4.01 4.01 NC NC (N = 1) Cohort 3 Dose 2 30 0.433 24.4 4.01 144.00 NC 0.062 (N = 1) Cohort 3 Dose 3 30 0.269 21.1 4.00 146.17 NC 0.062 (N = 1) Cohort 3 Dose 4 30 0.395 24.0 2.00 146.67 NC 0.069 (N = 1) Cohort 4 Dose 1 30 0.32 10.0 4.07 48.00 NC 0.117 (N = 1) Cohort 4 Dose 2 60 0.559 31.8 4.07 143.97 NC 0.069 (N = 1) Cohort 4 Dose 3 60 0.606 54.4 2.22 311.72 127 0.063 (N = 1) Cohort 4 Dose 4 60 0.528 69.0 2.00 335.02 114 0.056 (N = 1) Cohort 5 Dose 1 60 0.528 32.5 4.04 96.45 .sup.a108 0.116 (N = 8) (0.181) (26.3) (4.00-4.28)  (46.92-316.25) (21.4) (0.061) Cohort 5 Dose 2 100 0.962 83.6 4.08 324.22 .sup.b109 0.080 (N = 6) (0.433) (55.5) (4.00-4.27) (143.53-359.17) (9.48) (0.025) Cohort 5 Dose 3 100 1.09 103 2.00 335.67 .sup.b107 0.076 (N = 5) (0.514) (57.6) (2.00-4.00) (315.17-360.08) (18.4) (0.029) Cohort 5 Dose 4 100 1.53 104 1.03 323.94  .sup. 97.4 0.094 (N = 4) (0.952) (58.4) (1.00-2.00) (287.42-335.67) (13.8) (0.058) Cohort 6 Dose 1 100 0.644 41.6 4.12 142.67 .sup.c125 0.080 (N = 3) (0.102) (15.6) (4.07-4.20) (119.65-313.08) (0.021) Cohort 6 Dose 2 200 1.06 89.9 4.10 314.87 .sup.a107 0.077 (N = 3) (0.131) (15.0) (4.08-6.8)  (292.8-337.4) (17.3) (0.022) Cohort 6 Dose 3 200 1.37 103 3.92 335.60 .sup.a114 0.084 (N = 3) (0.498) (39.7) (2.03-4.00) (335.08-336.85) (13.4) (0.024) Cohort 6 Dose 4 200 1.54 107 3.14 228.56 .sub.  .sup.c86.9 0.174 (N = 2) (0.205) (40.4) (2.00-4.27) (141.33-315.78) (0.123) Cohort 7 Dose 1 200 1.91 151 4.67 333.67 .sup.c126 0.145 (N = 3) (0.45) (64.6) (4.07-5.75) (286.43-333.93) (0.092) Cohort 7 Dose 2 300 2.68 291 4.03 382.97 .sup. 106 0.125 (N = 3) (0.327) (95.8) (4.00-4.65)   (315-530.63) (9.39) (0.035) Cohort 7 Dose 3 300 2.18 234 2.02 215.28 NC 0.453 (N = 2) (0.346) (129) (2.00-2.03)  (95.72-334.83) (0.441) Cohort 7 Dose 4 300 3.62 NC 2.00 4.07 NC 2.32 (N = 1) Cohort 8 Dose 1 300 2.56 226 4.18 334.92 .sup.a108 0.158 (N = 3) (0.603) (45.6) (4.02-6.22) (333.17-335.53) (0.038) Cohort 8 Dose 2 500 4.53 348 4.18 336.35 .sup.a126 0.234 (N = 3) (2.03) (140) (4.03-6.37) (335.52-336.95) (0.059) Cohort 8 Dose 3 500 5.54 333 2.03 335.22 NC 0.287 (N = 3) (0.770) (90.5) (2.00-2.50) (311.93-335.88) (0.083) Cohort 8 Dose 4 500 6.18 340 1.52 337.48 NC 0.276 (N = 2) (1.05) (87.7) (1.03-2.00) (336.58-338.37) (0.083) Cohort 9 Dose 1 500 6.25 411 4.33 260.23 .sup.d115 0.643 (N = 15) (2.42) (236)  (2.05-11.28) (48.22-360.2) (30.5) (0.499) Cohort 9 Dose 2 750 11.5 .sup.e856  4.03 299.66 .sup.f117 .sup.e1.56 (N = 8) (5.28) (435) (3.98-5.92)  (4.02-526.05) (14.7) (1.45) Cohort 9 Dose 3 750 12.8 675 3.17 312.75  .sup.c81 1.48 (N = 3) (1.90) (311) (2.00-4.28) (46.27-335.6) (1.61) Cohort 9 Dose 4 750 10.6 590 1.02 360.52 .sup. 149 0.522 (N = 1) NC: Not Calculable; #Median (Min-Max); .sup.aN = 2; .sup.bN = 3; .sup.cN = 1; .sup.dN = 6; .sup.eN = 7; .sup.fN = 4.

[0156] Serum concentrations were less than the lower limit of quantification of 50 pg/mL at the first dose (1 ng/kg), and only transient concentrations were observed at 3 and 10 ng/kg dose levels. Evaluable PK profiles were observed from 30 ng/kg onwards. GBR 1302 showed maximum plasma concentration (C.sub.max) around the end of infusion, after which serum concentrations declined bi-exponentially with a mean terminal half-life of around 4 to 7 days. Both C.sub.max and area under the curve (AUC.sub.0-4) showed a near dose-proportional increase up to 750 ng/kg (maximum evaluated dose). None of the samples collected from subjects up to cohort 5 showed positive ADA response. These results show a favorable, linear PK, and none of the subjects evaluated so far showed positive ADA response.