OPTIMIZED CROSS-SPECIES SPECIFIC BISPECIFIC SINGLE CHAIN ANTIBODY CONTRUCTS

Abstract

The present invention provides to a bispecific single chain antibody construct binding to a target cell surface antigen via a first binding domain and to the T cell surface antigen CD3 via a second binding domain, wherein serum albumin is fused to the C-terminus of the antibody construct. Moreover, the invention provides a polynucleotide encoding the antibody construct, a vector comprising said polynucleotide and a host cell transformed or transfected with said vector. Furthermore, the invention provides a process for the production of the antibody construct of the invention, a medical use of said antibody construct and a kit comprising said antibody construct.

Claims

1. A bispecific single chain antibody construct comprising a first binding domain which binds to a target cell surface antigen and a second binding domain which binds to a T cell surface antigen CD3, wherein: the second binding domain binds to an epitope of the human and Callithrix jacchus, Saguinus oedipus or Saimiri sciureus CD3ε chain, wherein the epitope is part of the amino acid sequence of SEQ ID NO: 7, 8, 9, or 10 and comprises at least the amino acid sequence Gln-Asp-Gly-Asn-Glu (SEQ ID NO: 1), wherein a serum albumin is fused to the C-terminus of the construct; and wherein the bispecific single chain antibody construct does not have [[an]]the amino acid sequence of SEQ ID NO: 2 or 3.

2. The antibody construct according to claim 1, wherein the serum albumin is a human serum albumin or an FcRn binding optimized variant thereof.

3. The antibody construct according to claim 1, wherein the serum albumin comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 12 and 608 to 628.

4. The antibody construct according to claim 1, wherein the serum albumin is linked to the antibody construct via a peptide linker.

5. The antibody construct according to claim 4, wherein the peptide linker has the amino acid sequence (GGGGS).sub.n (SEQ ID NO: 13).sub.n wherein n is an integer in the range of 1 to 5.

6. The antibody construct according to claim 1, wherein the second binding domain comprises a VL region having CDR-L1-L3 and a VH region having CDR-H1-H3 selected from the group consisting of: (a) CDR-L1-L3 as depicted in SEQ ID NOs: 14-16 and CDR-H1-H3 as depicted in SEQ ID NOs: 17-19; (b) CDR-L1-L3 as depicted in SEQ ID NOs: 26-28 and CDR-H1-H3 as depicted in SEQ ID NOs: 29-31; (c) CDR-L1-L3 as depicted in SEQ ID NOs: 38-40 and CDR-H1-H3 as depicted in SEQ ID NOs: 41-43; (d) CDR-L1-L3 as depicted in SEQ ID NOs: 50-52 and CDR-H1-H3 as depicted in SEQ ID NOs: 53-55; (e) CDR-L1-L3 as depicted in SEQ ID NOs: 62-64 and CDR-H1-H3 as depicted in SEQ ID NOs: 65-67; (f) CDR-L1-L3 as depicted in SEQ ID NOs: 74-76 and CDR-H1-H3 as depicted in SEQ ID NOs: 77-79; (g) CDR-L1-L3 as depicted in SEQ ID NOs: 86-88 and CDR-H1-H3 as depicted in SEQ ID NOs: 89-91; (h) CDR-L1-L3 as depicted in SEQ ID NOs: 98-100 and CDR-H1-H3 as depicted in SEQ ID NOs: 101-103; (i) CDR-L1-L3 as depicted in SEQ ID NOs: 110-112 and CDR-H1-H3 as depicted in SEQ ID NOs: 113-115; and (j) CDR-L1-L3 as depicted in SEQ ID NOs: 122-124 and CDR-H1-H3 as depicted in SEQ ID NOs: 125-127.

7. The antibody construct according to claim 1, wherein the second binding domain comprises pairs of VH and VL chains selected from the group consisting of: (a) a VH-chain comprising the amino acid sequence set forth in SEQ ID NO: 20 and a VL-chain comprising the amino acid sequence set forth in SEQ ID NO: 22; (b) a VH-chain comprising the amino acid sequence set forth in SEQ ID NO: 32 and a VL-chain comprising the amino acid sequence set forth in SEQ ID NO: 34; (c) a VH-chain comprising the amino acid sequence set forth in SEQ ID NO: 44 and a VL-chain comprising the amino acid sequence set forth in SEQ ID NO: 46; (d) a VH-chain comprising the amino acid sequence set forth in SEQ ID NO: 56 and a VL-chain comprising the amino acid sequence set forth in SEQ ID NO: 58; (e) a VH-chain comprising the amino acid sequence set forth in SEQ ID NO: 68 and a VL-chain comprising the amino acid sequence set forth in SEQ ID NO: 70; (f) a VH-chain comprising the amino acid sequence set forth in SEQ ID NO: 80 and a VL-chain comprising the amino acid sequence set forth in SEQ ID NO: 82; (g) a VH-chain comprising the amino acid sequence set forth in SEQ ID NO: 92 and a VL-chain comprising the amino acid sequence set forth in SEQ ID NO: 94; (h) a VH-chain comprising the amino acid sequence set forth in SEQ ID NO: 104 and a VL-chain comprising the amino acid sequence set forth in SEQ ID NO: 106; (i) a VH-chain comprising the amino acid sequence set forth in SEQ ID NO: 116 and a VL-chain comprising the amino acid sequence set forth in SEQ ID NO: 118; and (j) a VH-chain comprising the amino acid sequence set forth in SEQ ID NO: 128 and a VL-chain comprising the amino acid sequence set forth in SEQ ID NO: 130.

8. The antibody construct according to claim 1, wherein the second binding domain comprises the amino acid sequence set forth in SEQ ID NO: 24, SEQ ID NO: 36, SEQ ID NO: 48, SEQ ID NO: 60, SEQ ID NO: 72, SEQ ID NO: 84, SEQ ID NO: 96, SEQ ID NO: 108, SEQ ID NO: 120 or SEQ ID NO: 132.

9. The antibody construct according to claim 1, wherein the target cell surface antigen is a tumor antigen or a viral antigen on the surface of an infected host cell.

10. The antibody construct according to claim 9, wherein the tumor antigen is selected from the group consisting of CDH19, MSLN, DLL3, FLT3, EGFRvIII, CD33, CD19, CD20, and CD70.

11. The antibody construct according to claim 1, wherein the antibody construct comprises the following elements starting from the N-terminus: (a) an scFv binding to the target cell surface antigen comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 629-675, 684, 694, 704, 714, 724, 734, 744, 754, 764, 774, 784, 794, 804, 814, 824, 834, 844, 854, 864, 874, 884, 894, 904, 914, 924, 934, 944, 954, 964, 974, 984, 994, 1004, 1014, 1024, 1034, 1044, 1054, 1064, 1074, 1084, 1094, 1104, 1114, 1124, 1134, 1144, 1154, 1164, 1174, 1184, 1194, 1204, 1214, 1224, 1234, 1244, 1254, 1264, 1274, 1284, 1294, 1304, 1314, 1324, 1334, 1344, 1354, 1364, 1374, 1384, 1394, 1404, 1414, 1424, 1434, 1444, 1454, 1464, 1474, 1484, 1494, 1504, 1514, 1524, 1534, 1544, 1554, 1564, 1574, 1584, 1594, 1604, 1614, 1624, 1634, 1644, 1654, 1664, 1674, 1684, 1694, 1704, 1714, 1724, 1734, 1744, 1754, 1764, 1774, 1784, 1794, 1804, 1814, 1824, 1834, 1844, 1854, 1864, 1874, 1884, 1894, 1904, 1914, 1924, 1934, 1944, 1954, 1964, 1974, 2004, 2014, 2024, 2034, 2044, 2054, 2064, 2074, 2084, 2094, 2104, 2114, 2124, 2134, 2144, 2154, 2164, 2174, 2184, 2194, 2204, 2214, 2224, 2234, 2244, 2254, 2264, 2274, 2284, 2294, 2304, 2314, 2324, 2334, 2344, 2354, 2364, 2374, 2384, 2394, 2404, 2414, 2424, 2434, 2444, 2454, 2464, 2474, 2484, 2494, 2504, 2514, 2524, 2534, 2544, 2554, 2564, 2574, 2584, 2594, 2604, 2614, 2624, 2634, 2644, 2654, 2664, 2674, 2684, 2694, and 2704; (b) a peptide linker comprising the amino acid sequence selected from the group consisting of: SEQ ID NOs: 13 and 2707-2709; (c) an scFv binding to the T cell surface antigen CD3 comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 36, 48, 60, 72, 84, 96, 108, 120, 132 and 2706; (d) a peptide linker comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 13 and 2707-2709; (e) a serum albumin comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 12 and 608 to 628; and (f) optionally a His-tag.

12. A polynucleotide encoding the antibody construct according to claim 1.

13. A vector comprising the polynucleotide according to in claim 12.

14. A host cell transformed or transfected with the polynucleotide according to claim 12.

15. A process for producing the antibody construct according to claim 1, said process comprising culturing a host cell comprising a polynucleotide encoding a bispecific single chain antibody construct comprising a first binding domain which binds to a target cell surface antigen and a second binding domain which binds to a T cell surface antigen CD3, wherein the second binding domain binds to an epitope of the human and Callithrix jacchus, Saquinus oedipus or Saimiri sciureus CD3ε chain, wherein the epitope is part of the amino acid sequence of SEQ ID NO: 7, 8, 9, or 10 and comprises at least the amino acid sequence Gln-Asp-Gly-Asn-Glu (SEQ ID NO: 1), wherein a serum albumin is fused to the C-terminus of the construct; and wherein the bispecific single chain antibody construct does not have the amino acid sequence of SEQ ID NO: 2 or 3 under conditions allowing the expression of the antibody construct, and, optionally, recovering the produced antibody construct from the culture.

16. A pharmaceutical composition comprising the antibody construct according to claim 1.

17. (canceled)

18. A method for treating or ameliorating a proliferative disease, a tumorous disease, a viral disease or an immunological disorder comprising the step of administering to a subject in need thereof an effective amount of the antibody construct according to claim 1.

19. A kit comprising the antibody construct according to claim 1 and a recipient and, optionally, directions for use.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0246] FIG. 1:

[0247] T activation of BiTE® antibody constructs in the absence of an albumin fusion at the C-terminus of the molecule. Expression of CD25 and CD69 on CD4.sup.+ (panel A and C) and CD8.sup.+ (panel B and D) T cells was determined by flow cytometry.

[0248] FIG. 2:

[0249] T activation of BiTE® antibody constructs in the presence (lower panel) of an albumin fusion at the C-terminus of the molecule. Expression of CD25 and CD69 on CD4.sup.+ (panel A and C) and CD8.sup.+ (panel B and D) T cells was determined by flow cytometry.

[0250] FIG. 3:

[0251] FACS analysis of CDH19/CD3 bispecific antibodies on indicated cell lines: 1) CHO cells stably transfected with human CDH19, 2) human CD3 positive human T cell line HBP-ALL, 3) CHO cells stably transfected with cynomolgus CDH19, 4) macaque T cell line 4119 LnPx, 5) human melanoma cell line CHL-1 expressing native human CDH19, 6) untransfected CHO cells. Negative controls [1) to 6)]: detection antibodies without prior CDH19/CD3 bispecific antibody.

[0252] FIG. 4:

[0253] Cytotoxic activity of CDH19/CD3 bispecific antibodies as measured in a 48-hour FACS-based cytotoxicity assay. Effector cells: CD3-expressing macaque T cell line 4119LnPx. Target cells: cynomolgus CDH19-transfected CHO cells. Effector to target cell (E:T)-ratio: 10:1. The figure shows the results for CDH19 2G6 302×12C HALB and for a negative control.

[0254] FIG. 5:

[0255] Pharmacokinetics of BiTE® antibody constructs

[0256] Four molecules named 1) 2G6-156; 2) 2G6-LFcBy; 3) 2G6-LFcBy-156; 4) 2G6-D3HSA were tested in the cynomolgus monkey in the context of a pharmacokinetic (PK) study. The figure shows the results in connection with Example 4.

[0257] FIG. 6:

[0258] Absence of “unspecific” T cell activation for bispecific single chain antibody constructs comprising a human/chimpanzee specific CD3 binding domain using AMG 110.

[0259] FIG. 7:

[0260] T activation in the by bispecific antibody constructs binding to different cell surface target structures in absence and presence of an albumin fusion at the C-terminus of the molecule. Expression of CD25 and CD69 on T cells was determined by flow cytometry.

[0261] 7a FACS-based activation assay (48 h) with human PBMC (donors #669, #729, #736) and Influenza specific BITE® antibody constructs; BiTE® start concentration=1 μg/mL (1:10 dilution);

[0262] 7b FACS-based activation assay (48 h) with human PBMC (donors #453, #458, #551) and DLL3 specific BITE® antibody constructs; BiTE® start concentration =1 μg/mL (1:10 dilution);

[0263] 7c Human PBMC were cultured in the absence of target cells with Mesothelin BiTE® or the HSA-variant of the Mesothelin BiTE® antibody construct for 48 h. The expression of the activation marker CD69 on T cells was determined by immunostaining and flow cytometry;

[0264] FIG. 8:

[0265] T activation in the by bispecific antibody constructs binding to different cell surface target structures in absence and presence of target cells. Expression of CD25 and CD69 on T cells was determined by flow cytometry.

[0266] 8a: Isolated T cells from healthy human donors were co-cultured in the presence or absence of target cells (Raji) (E:T cell ratio 10:1) and Full length HSA-CD19 BITE® for 48 h. The expression of the activation markers CD69 or CD25 on T cells was determined by immunostaining and flow cytometry;

[0267] 8b: Isolated T cells from healthy human donors were co-cultured in the presence or absence of target cells (Raji) (E:T cell ratio 10:1) and Full length HSA-CD20 BITE® for 48 h. The expression of the activation markers CD69 or CD25 on T cells was determined by immunostaining and flow cytometry;

[0268] 8c: Human PBMC were co-cultured in the presence or absence of target cells (U251vIII) (E:T cell ratio 10:1) and EGFRvIII-BITE® or the HSA-variant of the EGFRvIII-BITE® for 48 h. The expression of the activation markers CD69 on T cells was determined by immunostaining and flow cytometry.

[0269] FIG. 9:

[0270] Tumor-accumulation of CDH19-HALB BiTE® antibody construct.

[0271] FIG. 10:

[0272] Mean PK profiles of six BiTE®-HALB fusion proteins after single dose administration in cynomolgus monkeys.

[0273] FIG. 11:

[0274] Pharmacokinetic profiles of CD33-1×I2C-HALB and CD33-2×I2C6-HALB constructs administered in repeated dose schedules at different doses and different consecutive schedules. For reasons of comparability, described repeated dose profiles are dose-normalized to 120 μg/kg. The PK profile of the reference compound 8 administered at 30 μg/kg/day, cIV for 7 days, is shown in orange.

[0275] FIG. 12:

[0276] Pharmacokinetic profiles of modified HALB HLE modalities based on the EGFRvIII-BiTE antibody construct. Seven different EGFRvIII-HLE BiTE antibody constructs generated by mutations within the HALB wild-type sequence were tested in the cynomolgus monkey in the context of a pharmacokinetic (PK) study. Differences in the hALB variants are based on different, increased FcRn-binding affinities.

[0277] It should be understood that the inventions herein are not limited to particular methodology, protocols, or reagents, as such can vary. The discussion and examples provided herein are presented for the purpose of describing particular embodiments only and are not intended to limit the scope of the present invention, which is defined solely by the claims.

[0278] All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

Examples

[0279] The following examples are provided for the purpose of illustrating specific embodiments or features of the present invention. These examples should not be construed as to limit the scope of this invention. The examples are included for purposes of illustration, and the present invention is limited only by the claims.

Example 1

[0280] T cell activation of BiTE® molecules in the presence and the absence of an albumin fusion at the C-terminus of the molecule [0281] BiTE® constructs (serial dilutions: 0.1 pg/mL-1 μg/mL): [0282] 1. CDH19 2G6 302×I2C-H6; 258.7 μg/mL (SoW0416; Aliquot Lot: 140514_APu01); Untagged BiTE® [0283] 2. CDH19 2G6 302×I2C-HALB-DY-H6; 260 μg/mL (SoW0445; Aliquot Lot: 140514_APu03); Half-life extended full length [0284] Human PBMC effector cells (4 donors; #875, #889, #891, #895) [0285] 48 h incubation time [0286] Determination of CD25 and CD69 expression on CD4+and CD8+T cells with flow cytometer and antigen-specific conjugates mAb

[0287] In a similar setting the capacity of different BiTE® antibody constructs to activate T cell was analyzed. Those BiTE® antibody constructs do not comprise the CD3 specific binding domain of bispecific single chain antibody constructs of the invention but merely a human/chimpanzee specific CD3 binding domain. The corresponding BiTE® antibody construct is AMG 110, which is an EpCAM and CD3 specific BiTE® antibody construct. PBMC were incubated with increasing AMG 110 concentrations for 24 or 48 hrs, T cell activation was determined by staining for CD25 or CD69 surface expression by by flow cytometry on a BD FACSCanto II instrument. The result of this analysis is shown in FIG. 6. Moreover, the generic modality of eliminating the target independent T cell activation in the defined extreme high BiTE® antibody construct concentrations was demonstrated for a greater number of additional bispecific antibody constructs with specificity for different target structures as shown in FIG. 7 (7a: Influenza A antigen; 7b: DLL3; 7c: mesothelin (MSLN)).

Example 2

Bispecific Binding and Interspecies Cross-Reactivity

[0288] For confirmation of binding to human and cyno CDH19 and to human and macaque CD3, bispecific antibodies were tested by flow cytometry using indicated cell lines. CHO cells transfected with human CDH19, with cyno CDH19, the human melanoma cell line CHL-1 expressing native human CDH19, CD3-expressing human T cell leukemia cell line HPB-ALL (DSMZ, Braunschweig, ACC483) and the CD3-expressing macaque T cell line 4119LnPx (Knappe A, et al., Blood, 2000, 95, 3256-3261) were used as antigen positive cell lines. Moreover, untransfected CHO cells were used as negative control.

[0289] For flow cytometry 200,000 cells of the respective cell lines were incubated for 30 min on ice with 50 μl of purified bispecific antibody at a concentration of ≦5 μg/ml. The cells were washed twice in PBS/10% FCS and binding of the constructs was detected with a murine anti-His antibody (AbD Serotec; diluted 1:1000 in 50 μl PBS/10% FCS). After washing, bound anti-His antibodies were detected with an Fc gamma-specific antibody (Dianova) conjugated to phycoerythrin, diluted 1:100 in PBS/10% FCS. Samples were measured by flow cytometry on a FACSCanto II instrument and analyzed by FACSDiva software (both from Becton Dickinson).

[0290] The CDH19/CD3 bispecific antibodies stained CHO cells transfected with human CDH19, cyno CDH19, the human CDH19-expressing melanoma cell lines CHL-1 as well as human and macaque T cells. Moreover, there was no staining of untransfected CHO cells (see FIG. 3).

Example 3

Cytotoxic Activity

Target Cell Labeling

[0291] For the analysis of cell lysis in flow cytometry assays, the fluorescent membrane dye DiOC.sub.18 (DiO) (Molecular Probes, #V22886) was used to label cynomolgus CDH19 positive CHO cells—as target cells and distinguish them from effector cells. Briefly, cells were harvested, washed once with PBS and adjusted to 10.sup.6 cell/mL in PBS containing 2% (v/v) FBS and the membrane dye DiO (5 μL/10.sup.6 cells). After incubation for 3 min at 37° C., cells were washed twice in complete RPMI medium and the cell number adjusted to 1.25×10.sup.5 cells/mL. The vitality of cells was determined using 0.5% (v/v) isotonic EosinG solution (Roth, #45380).

Flow Cytometry Based Analysis

[0292] This assay was designed to quantify the lysis of cynomolgus CDH19-transfected CHO cells in the presence of serial dilutions of CDH19 bispecific antibodies.

[0293] Equal volumes of DiO-labeled target cells and effector cells (i.e. CD3-expressing macaque T cell line 4119LnPx) were mixed, resulting in an E:T cell ratio of 10:1. 160 μL of this suspension were transferred to each well of a 96-well plate. 40 μL of serial dilutions of the CDH19 bispecific antibodies and a negative control bispecific (an CD3-based bispecific antibody recognizing an irrelevant target antigen) or RPMI complete medium as an additional negative control were added. The bispecific antibody-mediated cytotoxic reaction proceeded for 48 hours in a 7% CO.sub.2 humidified incubator. Then cells were transferred to a new 96-well plate and loss of target cell membrane integrity was monitored by adding propidium iodide (PI) at a final concentration of 1 μg/mL. PI is a membrane impermeable dye that normally is excluded from viable cells, whereas dead cells take it up and become identifiable by fluorescent emission.

[0294] Samples were measured by flow cytometry on a FACSCanto II instrument and analyzed by FACSDiva software (both from Becton Dickinson).

[0295] Target cells were identified as DiO-positive cells. PI-negative target cells were classified as living target cells. Percentage of cytotoxicity was calculated according to the following formula:

[00001]$.Math.\mathrm{Cytotoxicity}.Math.\left[\%\right]=\frac{n_{\mathrm{dead}.Math..Math.\mathrm{target}.Math..Math.\mathrm{cells}}}{n_{\mathrm{target}.Math..Math.\mathrm{cells}}}\times 100$$n=\mathrm{number}.Math..Math.\mathrm{of}.Math..Math.\mathrm{events}$

[0296] Using GraphPad Prism 6 software (Graph Pad Software, San Diego), the percentage of cytotoxicity was plotted against the corresponding bispecific antibody concentrations. Dose response curves were analyzed with the four parametric logistic regression models for evaluation of sigmoid dose response curves with fixed hill slope and EC.sub.50 values were calculated.

[0297] The cytotox results using the above described system for CDH19 2G6 302×12C HALB and for a negative control are shown in FIG. 4

Example 4

Pharmacokinetics of BITE® Antibody Constructs

[0298] Four molecules named 1) 2G6-156; 2) 2G6-LFcBy; 3) 2G6-LFcBy-156; 4) 2G6-D3HSA were tested in the cynomolgus monkey in the context of a pharmacokinetic (PK) study. In this PK study a dose of 6 μg/kg was administered as a single intravenous bolus injection. For each of the above compounds, a group of 2 animals were used. Blood samples were collected and serum was prepared for determination of serum concentration for each drug in both animals. Serum drug levels were measured using an immunoassay. The serum concentration-time data were used to determine PK parameters. Blood sample was collected at the following time points: predose, 0.05, 0.25, 0.5, 1, 4, 8, 24, 48, 72, 168, 240, and 336 hours post dose. The PK parameters were determined using standard non-compartmental analysis (NCA) methods.

[0299] For all drugs tested, serum levels were quantifiable for the vast majority of time points in all animals after drug administration. The PK profiles showed a biphasic exponential decline for all drugs tested. Using NCA methods, the following parameters were estimated: AUC.sub.inf (Area under the serum concentration-time curve), V.sub.ss (volume of distribution at steady state), CL (systemic clearance), MRT (mean residence time), and Terminal t.sub.1/2 (half-life estimated from terminal phase). The PK parameters (mean of n=2) of each compound tested are summarized below:

[0300] The AUC.sub.inf was 568 hr*ng/mL, 366 hr*ng/mL, 1796 hr*ng/mL, and 1383 hr*ng/mL respectively for compounds 1, 2, 3 and 4. The V.sub.Ss was 446 mL/kg, 594 mL/kg, 193 mL/kg, and 80.7 mL/kg respectively for compounds 1, 2, 3 and 4. Systemic clearance was 11.3 mL/hr/kg, 16.1 mL/hr/kg, 4.3 mL/hr/kg, and 4.9 mL/hr/kg respectively for compounds 1, 2, 3 and 4. The MRT value for compounds 1, 2, 3 and 4 were 42.3 hr, 39.8 hr, 48.7 hr, and 18.1 hr respectively and that for terminal half-life was 44.3 hr, 31.2 hr, 40.3 hr, and 13.5 hr respectively for compounds 1, 2, 3 and 4.

[0301] The terminal half-life and MRT of each of compounds 1, 2 and 3 were much higher (>2-folds) than those for compound 4. Compounds 1, 2 and 3 present a longer half-life version of BiTE® antibody constructs.

[0302] Results see FIG. 5.

Example 5

BiTE® Induced CD69 or CD25 Expression on T Cells in Presence and Absence of Target Cells

[0303] Isolated T cells from healthy human donors were co-cultured in the presence or absence of target cells

[0304] For CD19/CD3 and CD20/CD3 bispecific antibody constructs Raji cells were used as target cells. An E:T cell ratio of 10:1 was used and CD19-HALB-BITE (FIG. 8a) and CD20-HALB-BiTE (FIG. 8b) were incubated for 48 h. The expression of the activation markers CD69 or CD25 on T cells was determined by immunostaining and flow cytometry and antigen-specific conjugates mAb.

[0305] For EGFRvIII/CD3 bispecific antibody constructs U251vIII cells were used as target cells. At an E:T of 10:1 EGFRvIII-BiTE or the HSA-variant of the EGFRvIII-BITE were incubated for 48 h. The expression of the activation markers CD69 on T cells was determined by immunostaining and flow cytometry (FIG. 8c).

Example 6

Tumor-Accumulation of CDH19-HALB BITE

[0306] CDH19-HALB BiTE, labeled with CW800 (nearinfrared dye), injected i.v. into the lateral tail vein of tumor-bearing mice (CDH19-positive tumor), in vivo analysis using “In Vivo Xtreme Imager” (Bruker) at the indicated time post injection (FIG. 9).

Example 7

Pharmacokinetics of BiTE® Antibody Constructs after Single Administration

[0307] Different target binding BiTE® antibodies were fused to a single wild type human Albumin (HALB) moiety and were tested in the cynomolgus monkey in the context of pharmacokinetic (PK) studies. The pharmacokinetics of six BiTE®-HALB antibodies are shown exemplarily. The corresponding nomenclature of these molecules is briefly summarized in Table 2, below.

TABLE-US-00002 TABLE 2 Compound nomenclature of five single dosed BiTE ®-HALB antibodies compound synonym test compound name Compound 1 MS-3 x I2C6-HALB Compound 2 MS-4 x I2C6-HALB Compound 3 MS-5 x I2C6-HALB Compound 4 CD33-1 x I2C6-HALB Compound 5 CDH19cc x I2C6-HALB Compound 6 CDH19 x I2C6-HALB

[0308] The BiTE®-HALB antibodies were administered as intravenous short (30 min) infusions at 12 μg/kg (compounds 1-3 and 5) and 15 μg/kg (compounds 4 and 6), respectively. For each of the above named compounds a group of three animals was used. Blood samples were collected and serum was prepared for determination of serum concentrations. Serum BiTE® antibody construct levels were measured using an immunoassay. The assay is performed by capturing the BiTE® via its target moiety, while an antibody directed against the CD3-binding part of the construct was used for detection. The serum concentration-time profiles were used to determine PK parameters. Blood sampling time points are listed in Table 3 below.

TABLE-US-00003 TABLE 3 Blood sampling time points during the PK study blood blood blood sampling time sampling time sampling time points cmpd. 1-3 points cmpd. 4-5 points cmpd. 6 [h] [h] [h] 0 0 0 0.05 0.08 0.08 0.25 0.25 0.25 0.5 1 1 1 4 2 4 8 4 8 16 8 24 24 24 48 48 48 72 72 72 168 120 96 240 168 168 240 240

[0309] The pharmacokinetic parameters were determined using standard non-compartmental analysis (NCA) methods.

[0310] For all tested proteins serum levels were quantifiable for all time points in all animals after BiTE® antibody construct administration. The PK profiles describe a biphasic, exponential decline after each of the single test item administrations (FIG. 10).

[0311] Using non compartmental analysis, the following PK parameters were estimated: AUCinf (Area under the serum concentration—time curve), Vss (volume of distribution at steady state), CL (systemic clearance) and terminal t½ (terminal half-life).

[0312] The PK parameter as mean of n=3 for each tested compound are summarized in Table 4 below.

TABLE-US-00004 TABLE 4 Pharmacokinetic parameters after single dose administration of six various BiTE ®-HALB proteins in cynomolgus monkeys. terminal t1/2 AUCinf Cl Vss test item [h] [h*ng/mL] [mL/h/kg] [mL/kg] MS-3 x I2C6-HALB 75 11419 1.1 66 MS-4 x I2C6-HALB 77 11593 1.0 65 MS-5 x I2C6-HALB 70 10262 1.2 64 CD33-1 x I2C6-HALB 48 4498 3.3 161 CDH19cc x I2C6-HALB 53 2226 6.7 303 CDH19 x I2C6-HALB 52 3199 3.8 349

[0313] The AUCinf for the different BiTE®-HALB variants ranged between 2226 h*ng/mL and 11593 h*ng/mL, depending on the BiTE® target context. All analyzed HALB fusions achieved terminal half-lives of at least 48 hours after single low dose administration at 12 and 15 μg/kg. Systemic clearance values were 1.1 mUh/kg, 1.0 mL/h/kg, 1.2 mL/h/kg, 3.3 mL/h/kg, 6.7 mL/h/kg and 3.8 mUh/kg respectively for compounds 1-6. The corresponding volume of distribution was 66 mL/kg, 65 mL/kg, 64 mL/kg, 161 mL/kg, 303 mL/kg and 349 mL/kg, respectively.

[0314] The differences in pharmacokinetic behavior of the various BiTE®-HALB antibodies was clearly related to the different BiTE® moieties and their targets, respectively. The HALB-moiety of each of the analyzed was represented by the wild type human Albumin and was not changed between the constructs.

Example 8

Pharmacokinetics of BITE® Antibody Constructs after Repeated Administration

[0315] Three molecules, CD33-1×I2C6-HALB (compound 4) and CD33-2×I2C6-HALB (compound 7) were tested in the cynomolgus monkey in the context of repeated dose pharmacokinetic (PK) studies. Each molecule represents a BiTE® moiety directed against one common target protein fused one copy of the wild type human serum albumin (HALB) moiety. Compound 8 represents a naked, non HALB-fused version of compound 7 and is named CD33-2×I2C6

[0316] The compounds were administered at different concentrations and in different dose schedules as described in Table 5 below.

TABLE-US-00005 TABLE 5 Administered doses with corresponding dose schedules analyzed in repeated dose set up of CD33-1 x I2C6 BiTE ® variants Administered dose Dose Consecutive test item [μg/kg] schedule doses CD33-1 x I2C6- 60 Q2D 4 HALB 80 (every 180 other day) CD33-2 x I2C6- 120 Q3D 2 HALB (every third day) 120 Q5D (every fifth day) CD33-2 x I2C6 30 μg/kg/day cIV 1

[0317] In these studies two or four consecutive doses of 60 μg/kg, 80 μg/kg, 180 μg/kg and 120 μg/kg were administered as intravenous short (30 min) infusions for compounds 4 and 7. Compound 8 was administered as continuous infusion (cIV) at 30 μg/kg/day for 7 days. For each of the above compounds a group of three animals were used. Blood samples were collected and serum was prepared for determination of serum concentrations. Serum BiTE® antibody construct levels were measured using an immunoassay. The assay is performed by capturing the BiTE® via its target moiety, while an antibody directed against the CD3-binding part of the construct was used for detection. The serum concentration-time profiles are described in FIG. 11.

[0318] Repeated administration of BiTE®-HALB antibodies leads to accumulation of serum trough levels over dosing, whereas the maximum achieved serum concentrations (cmax) stays nearly stable over repeated dosing (Table 6Fehler! Verweisquelle konnte nicht gefunden werden.). For all described administered doses and dose schedules the exposures over 7 days of the CD33-1×I2C6-HALB compounds were clearly increased compared to the exposure of a single cIV administered CD33-1×I2C6 naked, BiTE® antibody construct within the same time frame (FIG. 11).

TABLE-US-00006 TABLE 6 Maximum and trough serum concentrations of CD33 x I2C6 BiTE ® antibody construct variations administered at different dose schedules and various doses C.sub.max after dosing Dose Dose [ng/mL] test item [μg/kg] schedule 1.sup.st 2.sup.nd 3.sup.rd 4.sup.th CD33-1 x I2C6- 60 Q2D 1447 1092 1230 1214 HALB 80 (every 1961 1701 1691 1333 180 other day) 5053 3871 4163 2171 CD33-2 x I2C6- 120 Q3D 3631 2891 — — HALB (every third day) Q5D 3133 2630 — — (every fifth day) CD33-1 x I2C6- 60 Q2D 84 147 190 395 HALB 80 (every 132 229 231 412 180 other day) 394 735 676 1052 CD33-2 x I2C6- 120 Q3D 243 80 — — HALB (every third day) Q5D 100 22 — — (every fifth day)

[0319] Every other day administration of compound 4 at different dose levels shows dose linear behavior with stable AUC/Dose values (Table 7Table) of the BiTE®-HALB antibody construct in a dose range of 60 μg/kg to 180 μg/kg.

TABLE-US-00007 TABLE 7 Exposure over dose of CD33-1 x I2C6-HALB BiTE ® antibodies administered at 60, 80 and 180 μg/kg compared to canonical CD33-2 x I2C6 BiTE ® Administered dose test item [μg/kg] AUC.sub.0-168h/Dose CD33-1 x I2C6-HALB 60 0.341 80 0.341 180 0.416 CD33-2 x I2C6 30 μg/kg/day 0.027

[0320] The mean AUC/Dose of the CD33-1×I2C6-HALB BITE® antibody construct is 10-fold increased, compared to the AUC/Dose of the corresponding canonical BiTE® antibody construct.

Example 9

Pharmacokinetics of BiTE® Antibody Constructs with Different Albumin-Based Half-Life Extensions

[0321] Eight molecules were tested in the cynomolgus monkey in the context of a pharmacokinetic (PK) study. Each molecule represents a fusion of the same BiTE® protein to wild type or FcRn-affinity modified human serum albumin (HALB). The names of the test compounds are briefly summarized in Table 8, below.

TABLE-US-00008 TABLE 8 Compound nomenclature compound synonym test compound name Compound 9  EvIII-1 x I2C6-HALB   Compound 10 EvIII-1 x I2C6-HALB098 Compound 11 EvIII-1 x I2C6-HALB114 Compound 12 EvIII-1 x I2C6-HALB131 Compound 13 EvIII-1 x I2C6-HALB133 Compound 14 EvIII-1 x I2C6-HALB135 Compound 15 EvIII-1 x I2C6-HALB253 Compound 16 EvIII-1 x I2C6-HALB254

[0322] In this study two consecutive doses of 80 μg/kg were administered as intravenous short (30 min) infusions. For each of the above compounds a group of three animals were used. Blood samples were collected and serum was prepared for determination of serum concentrations. Serum BiTE® antibody levels were measured using an immunoassay. The assay is performed by capturing the BiTE® via its target moiety, while an antibody directed against the CD3-binding part of the construct was used for detection. The serum concentration-time profiles were used to determine PK parameters. Blood sampling time points are listed in Table 9, below.

TABLE-US-00009 TABLE 9 Blood sampling time points during the PK study blood sampling time points [h] 0.08 1 2 4 8 16 24 48 72 96 96 97 100 120 144 168 192 216 240

[0323] The pharmacokinetic parameters were determined using standard non-compartmental analysis (NCA) methods. The pharmacokinetic terminal phase of the single drugs was described using the decay period of the second test item administration. For all tested proteins serum levels were quantifiable for all time points in all animals after BiTE® antibody administration. The PK profiles describe a biphasic, exponential decline after each of the single test item administrations (FIG. 12).

[0324] Using non compartmental analysis, the following PK parameters were estimated: AUCinf (Area under the serum concentration—time curve), Vss (volume of distribution at steady state), CL (systemic clearance) and terminal t½ (terminal half-life). The PK parameter as mean of n=3 for each tested compound are summarized in the table below.

TABLE-US-00010 x-fold terminal increase in t1/2 t1/2 AUCinf Cl Vss compared to test item [h] [h*ng/mL] [mL/h/kg] [mL/kg] EGFRvIII-HALB EvIII-1 x I2C6-HALB 51.2 169109 0.95 110.21 1 EvIII-1 x I2C6-HALB098 67.6 208504 0.77 109.03 1.32 EvIII-1 x I2C6-HALB114 79.7 248438 0.64 99.87 1.56 EvIII-1 x I2C6-HALB131 69.8 194542 0.82 118.34 1.36 EvIII-1 x I2C6-HALB133 54.2 203041 0.79 98.35 1.06 EvIII-1 x I2C6-HALB135 71.1 246928 0.65 95.05 1.39 EvIII-1 x I2C6-HALB253 71.2 216000 0.74 106.44 1.39 EvIII-1 x I2C6-HALB254 66.8 189131 0.85 117.72 1.30

[0325] In respect to terminal half-life, all tested affinity-increased EGFRvIII-HALB variants achieve a factor of up to 1.56 fold (compound 11) longer half-life then the wild-type HALB fusion protein in the cyno repeated dose setting. They thereby all represent a half-life extended (HLE) version of the corresponding wild-type albumin.

TABLE-US-00011 Lengthy table referenced here US20170349668A1-20171207-T00001 Please refer to the end of the specification for access instructions.

TABLE-US-LTS-00001 LENGTHY TABLES The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).