HER2-BINDING TETRAMERIC POLYPEPTIDES

Abstract

The invention relates to a tetrameric polypeptide comprising a first polypeptide chain comprising a first VL antigen binding domain and a first CL constant domain, a second polypeptide chain comprising a first VH antigen binding domain, a first CH1 constant domain, a first CH2 constant domain and a first CH3 constant domain, a first ligand binding to a HER2 D4 epitope linked to the N-terminus of said first VL antigen binding domain or said first VH antigen binding domain by a first interdomain amino acid linker, a third polypeptide chain comprising a second VL antigen binding domain and a second CL constant domain, a fourth polypeptide chain comprising a second VH antigen binding domain, a second CH1 constant domain, a second CH2 constant domain and a second CH3 constant domain and a third ligand binding to a HER2 D4 epitope linked to the N-terminus of said second VL antigen binding domain or said second VH antigen binding domain by a second interdomain amino acid linker, wherein the VL antigen binding domains and the VH antigen binding domains together constitute a second ligand and a fourth ligand binding to a HER2 D1 epitope. The invention further relates to the tetrameric polypeptide for use in a method for the prevention or treatment of a malignant neoplastic disease, an isolated nucleic acid and a host cell for expression of the polypeptide and a method for obtaining the polypeptide.

Claims

1. A tetrameric polypeptide comprising or consisting of a. a first polypeptide chain comprising a first VL antigen binding domain and a first CL constant domain, b. a second polypeptide chain comprising a first VH antigen binding domain, a first CH1 constant domain, a first CH2 constant domain and a first CH3 constant domain, c. a first ligand that specifically binds to a HER2 D4 epitope, wherein said first ligand is comprised in said first polypeptide chain and linked to the N-terminus of said first VL antigen binding domain by a first interdomain amino acid linker, or said first ligand is comprised in said second polypeptide chain and linked to the N-terminus of said first VH antigen binding domain by a first interdomain amino acid linker, d. wherein the first VL antigen binding domain of the first polypeptide chain and the first VH antigen binding domain of the second polypeptide chain together constitute a second ligand, particularly a Fab domain, that specifically binds to a HER2 D1 epitope, e. a third polypeptide chain comprising a second VL antigen binding domain and a second CL constant domain, f. a fourth polypeptide chain comprising a second VH antigen binding domain, a second CH1 constant domain, a second CH2 constant domain and a second CH3 constant domain, g. a third ligand that specifically binds to a HER2 D4 epitope, wherein said third ligand is comprised in said third polypeptide chain and linked to the N-terminus of said second VL antigen binding domain by a second interdomain amino acid linker, or said third ligand is comprised in said fourth polypeptide chain and linked to the N-terminus of said second VH antigen binding domain by a second interdomain amino acid linker, h. wherein the second VL antigen binding domain of the third polypeptide chain and the second VH antigen binding domain of the fourth polypeptide chain together constitute a fourth ligand, particularly an Fab domain, that specifically binds to a HER2 D1 epitope.

2. The polypeptide according to claim 1, wherein said first polypeptide chain and said third polypeptide chain and/or said second polypeptide chain and said fourth polypeptide chain are characterized by a sequence identity with each other of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, wherein most particularly said first polypeptide chain and said third polypeptide chain and/or said second polypeptide chain and said fourth polypeptide chain are identical and/or wherein said first ligand and said third ligand are characterized by a sequence identity with each other of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, wherein most particularly said first ligand and said third ligand are identical.

3. The tetrameric polypeptide according to any one of the preceding claims, wherein said first CH2 constant domain and said first CH3 constant domain of said second polypeptide chain interact with, particularly are covalently linked with, said second CH2 constant domain and said second CH3 constant domain of said fourth polypeptide chain, such that a tetrameric polypeptide is formed.

4. The tetrameric polypeptide according to any one of the preceding claims, wherein said second polypeptide chain comprises a first hinge region between said first CH1 constant domain and said first CH2 constant domain, and said fourth polypeptide chain comprises a second hinge region between said second CH1 constant domain and said second CH2 constant domain, wherein said first hinge region and said second hinge region mediate complex formation between said second polypeptide chain and said fourth polypeptide chain, particularly by at least one disulphide bond, such that a tetrameric polypeptide is formed.

5. The polypeptide according to any one of the preceding claims, wherein said first ligand and/or said third ligand comprises or consists of a single-chain variable fragment polypeptide chain comprising an scFv heavy chain, an scFv linker chain, and an scFv light chain, wherein particularly a. said scFv heavy chain of said first ligand and/or said third ligand comprises a peptide sequence characterized by a sequence identity of 70% or more, more particularly 80% or more, even more particularly 90% or more, even more particularly 95% or more, with a peptide sequence selected from SEQ ID No. 15, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 44, SEQ ID No. 53, SEQ ID No. 54 and SEQ ID No. 80, wherein most particularly said scFv heavy chain of said first ligand and/or said third ligand comprises a peptide sequence identical to a peptide sequence selected from SEQ ID No. 15, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 44, SEQ ID No. 53, SEQ ID No. 54 and SEQ ID No. 80, and b. said scFv light chain of said first ligand and/or said third ligand comprises a peptide sequence characterized by a sequence identity of 70% or more, more particularly 80% or more, even more particularly 90% or more, even more particularly 95% or more, with a peptide sequence selected from SEQ ID No. 14, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 43 and SEQ ID No. 81, wherein most particularly said scFv light chain of said first ligand and/or said third ligand comprises a peptide sequence identical to a peptide sequence selected from SEQ ID No. 14, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 43 and SEQ ID No. 81.

6. The polypeptide according to claim 5, wherein said scFv linker chain comprises a peptide sequence characterized by a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 16, wherein most particularly said scFv linker chain comprises a peptide sequence identical to SEQ ID No. 16.

7. The polypeptide according to any one of the preceding claims, wherein a. said first polypeptide chain and/or said third polypeptide chain comprises a peptide sequence characterized by a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with a peptide sequence selected from SEQ ID No. 18, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 50 and SEQ ID 76, wherein most particularly said first polypeptide chain and/or said third polypeptide chain comprises a peptide sequence identical to a peptide sequence selected from SEQ ID No. 18, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 50 and SEQ ID No. 76, and b. said second polypeptide chain and/or said fourth polypeptide chain comprises a peptide sequence characterized by a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with a peptide sequence selected from SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 40, SEQ ID No. 42, SEQ ID No. 51, SEQ ID No. 52 and SEQ ID 77, wherein most particularly said second polypeptide chain and/or said fourth polypeptide chain comprises a peptide sequence identical to a peptide sequence selected from SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 40, SEQ ID No. 42, SEQ ID No. 51, SEQ ID No. 52 and SEQ ID 77.

8. The polypeptide according to any one of the preceding claims, wherein a. said first polypeptide chain and/or said third polypeptide chain comprises a peptide sequence characterized by a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38 and SEQ ID No. 78, wherein most particularly said first polypeptide chain and/or said third polypeptide chain comprises a peptide sequence identical to SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38 and SEQ ID No. 78, and b. said second polypeptide chain and/or said fourth polypeptide chain comprises a peptide sequence characterized by a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35 and SEQ ID No. 79, wherein most particularly said second polypeptide chain and/or said fourth polypeptide chain comprises a peptide sequence identical to SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35 and SEQ ID No. 79.

9. The polypeptide according to any one of the preceding claims, wherein a. said first polypeptide chain and/or said third polypeptide chain is characterized by a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 1, wherein most particularly said first polypeptide chain and/or said third polypeptide chain is identical to SEQ ID No. 1, and b. said second polypeptide chain and/or said fourth polypeptide chain is characterized by a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 2, wherein most particularly said second polypeptide chain and/or said fourth polypeptide chain is identical to SEQ ID No. 2.

10. The polypeptide according to any one of the preceding claims, wherein a. said first polypeptide chain and/or said third polypeptide chain is characterized by a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 3, wherein most particularly said first polypeptide chain and/or said third polypeptide chain is identical to SEQ ID No. 3, and b. said second polypeptide chain and/or said fourth polypeptide chain is characterized by a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 4, wherein most particularly said second polypeptide chain and/or said fourth polypeptide chain is identical to SEQ ID No. 4.

11. The polypeptide according to any one of the preceding claims, wherein said first interdomain amino acid linker and/or said second interdomain amino acid linker consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, and wherein said first interdomain amino acid linker and/or said second interdomain amino acid linker comprises or consists of amino acids G, A, J, S, T, P, C, V, M and E, particularly wherein said first interdomain amino acid linker and/or said second interdomain amino acid linker comprises or consists of amino acids G, S, A and T, more particularly wherein said first interdomain amino acid linker and/or said second interdomain amino acid linker is characterized by an amino acid sequence (GGGGS)n, with n being 1, 2, 3, 4 or 5 or said first interdomain amino acid linker and/or said second interdomain amino acid linker comprises or consists of a peptide sequence selected from one of SEQ ID No. 17, SEQ ID 55 to SQ ID 69 and SEQ ID 82 to SEQ ID 91 or a functional equivalent peptide sequence characterized by a sequence identity of at least 70%.

12. The polypeptide according to any one of the claims 1 to 11 for use in a method for the prevention or treatment of a malignant neoplastic disease associated with expression of HER2.

13. An isolated nucleic acid encoding at least one of the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain of the polypeptide according to any one of the claims 1 to 11.

14. A host cell which is adapted to produce at least one of the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain of the polypeptide according to any one of the claims 1 to 11, wherein particularly the host cell comprises the isolated nucleic acid according to claim 13, such that the host cell is able to produce at least one of the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain of the polypeptide according to any one of the claims 1 to 11.

15. A method for obtaining the polypeptide according to any one of the claims 1 to 11, wherein the method comprises culturing the host cell according to claim 14, so that at least one of the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain of the polypeptide according to any one of the claims 1 to 11 is produced.

Description

DESCRIPTION OF THE FIGURES

[0326] FIG. 1 shows schemes of biparatopic anti-HER2 binding agents.

[0327] FIG. 2 shows further schemes of biparatopic anti-HER2 binding agents

[0328] FIG. 3 shows a vector map of a plasmid for co-expression of the light and heavy chain of construct 441 in CHO cells (Pymex10 based vector with double expression cassette [CMV GOI polyA]).

[0329] FIG. 4 shows a vector map of a plasmid for co-expression of the light and heavy chain of construct 47C2 in CHO cells (Pymex10 based vector with double expression cassette [CMV GOI polyA]).

[0330] FIG. 5 shows a vector map of a plasmid for expression of construct 841 in CHO cells.

[0331] FIG. 6 shows an elution profile of construct 441 from Protein A affinity chromatography.

[0332] FIG. 7 shows an elution profile of construct 441 from cation exchange chromatography.

[0333] FIG. 8 shows an elution profile of construct 441 from size exclusion chromatography.

[0334] FIG. 9 shows a Coumassie-stained SDS-PAGE gel of fractions from purification of construct 441.

[0335] FIG. 10 shows the viability testing of CHOs during the expression of construct 441 (scFV-IgG). Expression optimization of construct 441 in CHOs cells for indicated time. Cells were cultured in CHOgro medium from Mlrus (MIR 6260) and additionally fed with free cysteine (reduced form) (2), glutathione (3), fetal calf serum (4) or all additives respectively (5). CHO cells were analyzed on CASY cell counter (Scharfe System).

[0336] FIG. 11 shows a Western blot of construct 441 expression, secreted to the medium of CHO cells after indicated times. Cells were cultured in CHOgro medium from Mlrus (MIR 6260) (1) and additionally fed with free cysteine (reduced form) (2), glutathione (3), fetal calf serum (4) or all additives together (5), respectively. Protein was precipitated from medium by acetone precipitation and re-solubilized in SDS PAGE buffer. Proteins were resolved on 4-12% gradient gel and the western blot was analyzed on an Odyssey system (LI-COR). Purified intact full length construct 441 is shown as control (A) and runs above the 170 kDa marker. Molecular weight marker Page ruler from Thermo Scientific is shown in red.

[0337] FIG. 12 shows cell proliferation assays (XTT) with BT474 cells after 4 days of treatment. Trastuzumab (TZB), biparatopic DARPin (6L1G) and different fusion variants of the biparatopic construct. LF IgG HL (murine parent of construct 441), HF IgG HL (murine parent of construct 241) show similar anti-proliferative activity compared to the biparatopic DARPin 6L1G, which is superior to trastuzumab (TZB). HF IgG LH (murine variant, no seq.) and LF IgG LH (murine variant, no seq.) show reduced anti-proliferative activity compared to biparatopic DARPin and higher IC.sub.50 concentrations.

[0338] FIG. 13 shows cell proliferation assays (XTT) with BT474 cells after 4 days of treatment to test the effect of the linker length. Biparatopic DARPin (6L1G) and different fusion linker variants of the biparatopic construct (murine parent construct of 441) are compared. The 2-AA linker (GS) shows highest anti-proliferative activity. The 4-, 7- and 12-AA linkers show similar activity. The 22-AA linker variant shows reduced activity.

[0339] FIG. 14 shows cell proliferation assays (XTT) with BT474 cells after 4 days of treatment. Biparatopic DARPin (6G; 6L1G), biparatopic construct 441 (441), biparatopic construct 411 (humanized kappal VH1) and biparatopic construct 443 (humanized kappa4 VH3). All show similar plateau levels of anti-proliferative activity, except 443, which shows reduced activity.

[0340] FIG. 15 shows cell proliferation assays (XTT) with BT474 cells after 4 days of treatment with different humanized versions of A21 IgG, when fused to TZB scFv. The strategy of humanization is described above. Different variants use humanized kappal VH3 or a humanized kappal VH core graft.

[0341] FIG. 16 shows XTT cell proliferation assay with BT474 cells after 4 day of treatment. Tetravalent IgG (HF IgG HL and LF IgG HL murine) versus bivalent Fab fusions (HF Fab HL and LF Fab HL murine). All constructs show similar plateau and IC50 values.

[0342] FIG. 17 shows XTT cell proliferation assay with SKBR3 cells after 4 day of treatment. Biparatopic DARPin (6G) biparatopic construct (441 tf), trastuzumab (TZB).

[0343] FIG. 18 shows cell proliferation assays (XTT) with CALU-3 cells after 4 days of treatment. Biparatopic DARPin (6G), biparatopic construct (construct 441 (441tf), trastuzumab (TZB).

[0344] FIG. 19 shows cell proliferation assays (XTT) with BT474 cells after 4 days of treatment, testing effect of domain 1 binding unit. Biparatopic construct with A21 (construct 441tf) or 7C2 fusions show different IC50 and plateau level.

[0345] FIG. 20 shows cell proliferation assays (XTT) with BT474 cells after 4 days of treatment, testing the effect of domain 1 binding unit. Biparatopic construct with A21 (construct 441) or with 39S (39s HF IgG H)L

[0346] FIG. 21 shows XTT cell proliferation assays with HCC1419 cells after 4 days of treatment. Biparatopic DARPin (6G; 6L1G), biparatopic construct 441 (441tf) and bivalent LF-oaFabFc (A21-TZB-40a). 441 and 6G show similar inhibition of cell proliferation after 4 days. LF-oaFabFc show slightly reduced inhibition of cell proliferation compared to 441.

[0347] FIG. 22 shows XTT cell proliferation assay with BT474 and HCC1419 cells after 4 day of treatment. All human.

[0348] FIG. 23 shows XTT cell proliferation assay with BT474 and HCC1419 cells after 4 day of treatment. All human.

[0349] FIG. 24 shows a) in the upper panel XTT cell proliferation assays with BT474 (left) and HCC1419 (right) cells after 4 day of treatment; and in the lower panel XTT cell proliferation assays with BT474 (left) and HCC1419 (right) cells after 4 day of treatment (variants with higher affinity (NGS and GGG)); b) repeated experiments with a new expression of NGS.

[0350] FIG. 25 shows XTT cell proliferation assays with BT474 (left) and HCC1419 (right) cells after 4 day of treatment.

[0351] FIG. 26 shows XTT cell proliferation assays with HCC1419 cells grown as 3D spheroids.

[0352] FIG. 27 shows Western Blots 24 hours post treatment (BT474) with indicated agents (murine).

[0353] FIG. 28 shows in the upper panel Induction of apoptosis in BT474 cells after 3 days of treatment. Average number of propidium iodide (PI) positive cells was determined for 4 replicates, counted by cell profiler and was analyzed with Student's t-test. Biparatopic construct (441, 441tf) induced significantly more cell death than trastuzumab (TZB). 441 and biparatopic DARPin (6L1G) show similar level of cell death; and in the lower panel Induction of apoptosis in BT474 cells after 3 days of treatment. Average number of annexin-V positive cells was determined for 3-4 replicates, counted by cell profiler and was analyzed with Student's t-test. Biparatopic construct 441 induced significantly more apoptosis than trastuzumab (TZB). Construct 441 and 6L1G show similar level of apoptosis.

[0354] FIG. 29 shows images of BT474 cells treated with the indicated agents for 3 days.

[0355] FIG. 30 shows Alexa647-labeled trastuzumab (TZB), biparatopic construct 441 and biparatopic one armed constructs oaLF and oaHF were incubated for 1 h at 100 nM concentration with 3 million BT474 cells in 3 ml PBS containing NaN.sub.3 (0.1%) and BSA (1%) at 4° C. Note that BT474 cells were pre-treated with 0.1% NaN.sub.3 in PBS with 1% BSA to block internalization before binding. Cells were analyzed afterwards on CyFlow Space instrument (Partec). All binding agents show specific binding to the surface of HER2-positive BT474 cells.

[0356] FIG. 31 shows the induction of cell death after treatment with 100 nM of indicated agents. BT474, N87, HCC1419 and SKBR3 cells were seeded 24 h before treatment in 96 black clear-well microscopy plates (Nunc), continuously treated for 3 days and stained with HOECHST-33342 (Invitrogen) for total cells and with propidium iodide (Sigma) for membrane-permeable dead cells. Cells were analyzed on a Lionheart FX Automated Microscope (BioTek Instruments) and the number of propidium iodide and HOECHST-33342 positive cells was quantified with Gen5 software (BioTek Instruments). The ratio of propidium iodide and HOECHST-33342 positive cells was calculated for 3 biological replicates and the mean and SD is shown in the corresponding column plots. Biparatopic binding agents (6L1G, 441, 841, LFoa, 241, 641, HFoa, 7C2LF) binding to domain 1 and 4 of HER2 induce continuously more dead cells than trastuzumab (TZB) or the combination of trastuzumab and pertuzumab (TZB+PZB) in HER2-positive cancer cells.

[0357] FIG. 32 shows the half-life of construct 441 in the serum of NSG mice. Drawn sera of mice with previous 441 injections (3 mg/kg) were analyzed by sandwich ELISA. 441 showed an alpha phase of around 4.3 hours, followed by a beta phase of more than 45 hours.

[0358] FIG. 33 shows in-vivo activity of 441 on N87 xenografts in SCID beige mice. After N87 tumors had reached 150 mm.sup.3 in size, mice were treated with eight injections of 441 (10 mg/kg) during four weeks. 441 lead to significant tumor size reduction compared to untreated mice and TZB (10 mg/kg) or huA21G (10 mg/kg) treated mice.

[0359] FIG. 34 shows representative microscopy images of BT-474 cells after treatment for 2 h with trastuzumab (TZB), huA21G (A21), their combination, or 441, and non-treated cells, either without or with addition of an anti-human primary antibody, as controls. Nuclei were stained using 2-(4-amidinophenyl)-1H-indo1-6-carboximidamide (DAPI), antibodies were detected with an anti-human Fc antibody from goat, and lysosomal compartments using an anti-LAMP1 antibody.

[0360] FIG. 35 shows the result of a time-course treatment and subsequent surface protein internalization and degradation assay for the constructs 441 and 841, hA21G, trastuzumab (TZB), the combination of trastuzumab and hA21G (TZB+hA21G), pertuzumab (PZB), the combination of trastuzumab and pertuzumab (TZB+PZB), and the inhibitor of HSP90, geldanamycin (GA).

[0361]

TABLE-US-00001 Sequence listings: SEQ ID No. 1 (“441 polypeptide 1”): EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS GGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIKGSDIVLTQSPDSLAVSLGERATINCRSSQTLLYSNNQKNYLAWYQKKPGQPPKLLI SWAFTRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYSNYPWTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 2 (“441 polypeptide 2”): QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYFINWVREAPGQGLEWMGHISSSYATSTY NQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSK SEQ ID No. 3 (“47C2 polypeptide 1”): EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS GGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIKGSDIVMTQSPDSLAVSLGERATINCRASQSVSGSRFTYMHWYQQKPGQPPKLLI KYASILESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSWEIPPWTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 4 (“47C2 polypeptide 2”): EVQLVQSGAEVKKPGASVKVSCKASGYSFTGYWMNWVRQAPGQGLEWIGMIHPLDAEIRA NQKFRDRVTITVDTSTSTAYLELSSLRSEDTAVYYCARGTYDGGFEYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSK SEQ ID No. 5 (“841 polypeptide 2”): QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYFINWVREAPGQGLEWMGHISSSYATSTY NQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID No. 6 (“87C2 polypeptide 2”): EVQLVQSGAEVKKPGASVKVSCKASGYSFTGYWMNWVRQAPGQGLEWIGMIHPLDAEIRA NQKFRDRVTITVDTSTSTAYLELSSLRSEDTAVYYCARGTYDGGFEYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID No. 7 (“841Fc polypeptide 1”): EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS GGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIKGSDIVLTQSPDSLAVSLGERATINCRSSQTLLYSNNQKNYLAWYQKKPGQPPKLLI SWAFTRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYSNYPWTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 8 (“841Fc polypeptide 2”): QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYFINWVREAPGQGLEWMGHISSSYATSTY NQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 9 (“87C2Fc polypeptide 1”): EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS GGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIKGSDIVMTQSPDSLAVSLGERATINCRASQSVSGSRFTYMHWYQQKPGQPPKLLI KYASILESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSWEIPPWTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 10 (“87C2Fc polypeptide 2”): EVQLVQSGAEVKKPGASVKVSCKASGYSFTGYWMNWVRQAPGQGLEWIGMIHPLDAEIRA NQKFRDRVTITVDTSTSTAYLELSSLRSEDTAVYYCARGTYDGGFEYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 11 (“241 polypeptide 1”): DIVLTQSPDSLAVSLGERATINCRSSQTLLYSNNQKNYLAWYQKKPGQPPKLLISWAFTRKS GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYSNYPWTFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 12 (“241 polypeptide 2”): EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS GGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIKGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYFINWVREAPGQGLEWMGHI SSSYATSTYNQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSK SEQ ID No. 13 (“641 polypeptide 2”): EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS GGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIKGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYFINWVREAPGQGLEWMGHI SSSYATSTYNQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID No. 14 (“scFv TZB L chain”): DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK SEQ ID No. 15 (“scFv TZB H chain”): EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS SEQ ID No. 16 (“scFv TZB linker”): GGGGSGGGGSGGGGSGGGGS SEQ ID No. 17 (“B1 to B2 linker (link1)”): GS SEQ ID No. 18 (“A21 L chain”) DIVLTQSPDSLAVSLGERATINCRSSQTLLYSNNQKNYLAWYQKKPGQPPKLLISWAFTRKS GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYSNYPWTFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 19 (“A21 H chain with Fc”) QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYFINWVREAPGQGLEWMGHISSSYATSTY NQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSK SEQ ID No. 20 (“A21 H chain for Fab”) QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYFINWVREAPGQGLEWMGHISSSYATSTY NQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID No. 21: (“TZB CDR H1”) GFNIKDTYIH SEQ ID No. 22: (“TZB CDR H2”) RIYPTNGYTRYADSVKG SEQ ID No. 23: (“TZB CDR H3”) WGGDGFYAMDY SEQ ID No. 24: (“TZB CDR L1”) RASQDVNTAVA SEQ ID No. 25: (“TZB CDR L2”) SASFLYS SEQ ID No. 26: (“TZB CDR L3”) QQHYTTPPT SEQ ID No. 27: (“A21 CDR H1”) GYSFTGYFIN SEQ ID No. 28: (“A21 CDR H2”) HISSSYATSTYNQKFQG SEQ ID No. 29 (“A21 CDR H3”) SGNYEEYAMDY SEQ ID No. 30: (“A21 CDR L1”) RSSQTLLYSNNQKNYLA SEQ ID No. 31: (“A21 CDR L2”) WAFTRKS SEQ ID No. 32: (“A21 CDR L3”) QQYSNYPWT SEQ ID No. 33: (“7C2 CDR H1”) GYSFTGYWMN SEQ ID No. 34: (“7C2 CDR H2”) MIHPLDAEIRANQKFR SEQ ID No. 35: (“7C2 CDR H3”) GTYDGGFEY SEQ ID No. 36: (“7C2 CDR L1”) RASQSVSGSRFTYMH SEQ ID No. 37: (“7C2 CDR L2”) YASILES SEQ ID No. 38: (“7C2 CDR L3”) QHSWEIPPWT SEQ ID No. 39 (“A21 L chain V1”) DIVLTQSPDSLAVSLGERATINCRSSQTLLYSNNQKNYLAWYQQKPGQPPKLLISWAFTRKS GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYSNYPWTFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 40: (“A21 H chain V1”) QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYFINWVRQAPGQGLEWMGHISSSYATSTY NQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID No. 41: (“A21 L chain V2”) DIVLTQSPDSLAVSLGERATINCRSSQPLEYSNNQWNYLAWYQKKPGQPPKLLISWAFTRK SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCGQYSDYPNTFGQGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 42: (“A21 H chain V2”) QVQLVQSGAEVKKPGASVKVSCKASGYPFTQYFIHWVREAPGQGLEWMGHISSSYATVDY NQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSK SEQ ID No. 43: (“scFv TZB L chain V1”) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK SEQ ID No. 44: (“scFv TZB H chain V1”) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNAYTRYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGTGFYAMDYWGQGTLVTVSS SEQ ID No. 45: (“Alternative Fc part - no mut”) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 46: (“Alternative Fc part - Knob into hole - knob site V1”) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 47: (“Alternative Fc part - Knob into hole - hole site V1”) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 48: (“Alternative Fc part - Knob into hole - knob site V2”) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 49: (“Alternative Fc part - Knob into hole - hole site V2”) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLP PSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 50: (“A21 L chain V3”) DIQLTQSPSSLSASVGDRVTITCRSSQTLLYSNNQKNYLAWYQQKPGKAPKLLISWAFTRKS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSNYPWTFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 51: (“A21 H chain V3”) EVQLVQSGPELVQPGGSVRISCAASGYSFTGYFINWVKQAPGKGLEWISHISSSYATSTYN QSFKGRATFSVDTSSSTAYMQLNSLRAEDTAVYYCVRSGNYEEYAMDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID No. 52: (“A21 H chain V4”) VQLVESGGGLVQPGGSLRLSCAASGYSFTGYFINWVRQAPGKGLEWVSHISSSYATSTYN QSVKGRFTFSVDTSSSTAYLQMNSLRAEDTAVYYCVRSGNYEEYAMDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID No. 53: (“TZB H chain V2”) QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEQMGRIYPTNGYTRY DPKFQGRVTITADTSSNTAYMELSSLRSEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS SEQ ID No. 54: (“TZB H chain V3”) EVQLVQSGPELVQPGGSLRLSCAASGFNIKDTYIHWVKQAPGKGLEWISRIYPTNGYTRYD PSFKGRATISADTSSNTAYLQVNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS SEQ ID No. 55: (“Poly-Gly-linker”) GGGGG SEQ ID No. 56: (“Ova-linker”) GSGSGS SEQ ID No. 57: (“Trans-linker”) GSGGGTGGGSG SEQ ID No. 58: (“Pro-linker”) PPP SEQ ID No. 59: (“PAS-linker 1”) ASPAAPAPASPAAPAPSAPAA SEQ ID No. 60: (“PAS-linker 2”) ASAAAPAAASAAASAPSAAAA SEQ ID No. 61: (“PAS-linker 3”) AASPAAPSAPPAAASPAAPSAPPAA SEQ ID No. 62: (“PAS-linker 4”) ASPASA SEQ ID No. 63: (“PAS-linker 5”) ASPASPASA SEQ ID No. 64: (“XS-linker 1”) PAGSP SEQ ID No. 65: (“XS-linker 2”) STEPS SEQ ID No. 66: (“XS-linker 3”) STEEG SEQ ID No. 67: (“XS-linker 4”) GSAPG SEQ ID No. 68: (“Proper-linker1”) GASTP SEQ ID No. 69: (“Proper-linker2”) GPSAT SEQ ID No. 70: (“39s light chain”) DIVMTQTPLSLSVTPGQPASISCKSSQSVFFRSNNKNILAWYLQKPGQPPQLLIYWASSRES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYFGSPFTFGPGTKVDIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 71: (“39s heavy chain”) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMSWVRQAPGKGLEWVSSISSSSSYIYYA DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGDAYNYYYFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSK SEQ ID No. 72: (“H218 light chain”) QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVS NRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLVFGGGTKLTVLGTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 73: (“H218 heavy chain”) EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGQGLEWMGWISAYNGNTN YAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAREGDGAFDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSK SEQ ID No. 74: (“MF3958 light chain”) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 75: (“MF3958 heavy chain”) QVQLVQSGAEVKKPGASVKLSCKASGYTFTAYYINWVRQAPGQGLEWIGRIYPGSGYTSYA QKFQGRATLTADESTSTAYMELSSLRSEDTAVYFCARPPVYYDSAWFAYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSK SEQ ID No. 76: (“A21 L chain V4”) DIVLTQSPDSLAVSLGERATINCRSSQTLLYSNNQKNYLAWYQKKPGQPPKLLISWAFTRKS GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYSNYPWTFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 77: (“A21 H chain with Fc V2”) QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYFINWVREAPGQGLEWMGHISSSYATSTY NQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSK SEQ ID No. 78: (“7C2 light chain”) DIVMTQSPDSLAVSLGERATINCRASQSVSGSRFTYMHWYQQKPGQPPKLLIKYASILESGV PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSWEIPPWTFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 79: (“7C2 heavy chain”) EVQLVQSGAEVKKPGASVKVSCKASGYSFTGYWMNWVRQAPGQGLEWIGMIHPLDAEIRA NQKFRDRVTITVDTSTSTAYLELSSLRSEDTAVYYCARGTYDGGFEYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSK SEQ ID No. 80: (“TZB-HC”) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSK SEQ ID No. 81: (“TZB-LC”) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID 82 (“GS-linker 1”): GGGGS SEQ ID 83 (“GS-linker 2”): GGGGSGGGGS SEQ ID 84 (“GS-linker 3”): GGGGSGGGGSGGGGS SEQ ID 85 (“GS-linker 4”): GGGGSGGGGSGGGGSGGGGS SEQ ID 86 (“GS-linker 5”): GGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID 87 (“GA-linker”): GAAGAAG SEQ ID 88 (“GT-linker”): GGTGGT SEQ ID 89 (“ST-linker”): STSTS SEQ ID 90 (“PA-linker”): PAPAP SEQ ID 91 (“SA-linker”): SAASAAS SEQ ID 92 (“HF-oaFabFc light chain”/“A21-TZB-2oa light chain”): DIVLTQSPDSLAVSLGERATINCRSSQTLLYSNNQKNYLAWYQKKPGQPPKLLISWAFTRKS GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYSNYPWTFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGESDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK SEQ ID 93 (“HF-oaFabFc heavy chain”/“A21-TZB-2oa heavy chain”): EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS GGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIKGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYFINWVREAPGQGLEWMGHI SSSYATSTYNQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

EXAMPLE 1

Biparatopic Anti-HER2 Binding Agents

[0362] The inventors have generated biparatopic IgG derivatives. In contrast to other available biparatopic HER2-targeting antibodies, e.g. the antibody-drug conjugate (ADC) from Medimmune MED14276 (Li et al., 2016), these IgGs show very strong anti-tumor activity as “naked” binding proteins, i.e., without attached drug (Kast et al., in preparation). Thus, it is believed that these novel biparatopic anti-HER2 IgGs combine the mechanisms of action of trastuzumab plus pertuzumab plus the action of small molecule kinases inhibitors against HER2 in one single molecule. In addition, potential off-target effects of the biparatopic anti-HER2 IgGs are expected to remain far below those of ADC fusions, such as T-DM1 or MED14276, as they can only act on HER2-addicted cells, while ADCs can via their toxin act in many healthy tissue. This opens up the therapeutic windows for new combination therapies. Furthermore, pan-ErbB inhibition by polymerization of HER2 receptors may passively block compensatory activation of other receptor tyrosine kinases (RTKs). The biparatopic anti-HER2 binding agents interfere with the free lateral movement of HER2 receptors on the cell surface of HER2-amplified cancer, yet without inducing signaling competent complexes, which may block the activation of other RTKs. Consequently, biparatopic anti-HER2 binding agents may show strong synergies with small molecule inhibitors, which tend to induce expression of compensatory RTKs that eventually drives escape from therapy. Therefore, biparatopic anti-HER2 IgGs bear a very high potential to elicit strong anti-tumor synergies in combination with small-molecule inhibitors on a broad panel of HER2-amplified cancers. The potential for synergies with small-molecule inhibitors is superior to current single-specificity antibodies or antibody combinations.

[0363] Illustrative schemes of preferred biparatopic IgG constructs are shown in FIGS. 1 and 2

[0364] Data regarding preparation, and biological activity of the biparatopic IgG constructs are shown in FIGS. 3 to 26.

[0365] Protocol for Production of Biparatopic IgGs in CHOs Cells

[0366] Vector Design

[0367] Bicistronic plasmids containing two expression cassettes or two vector systems were constructed for co-transfections. Derivatives of plasmid pYMex10 (Morphosys) were used for the bicistronic strategy. In the resulting plasmid constructs, the coding sequences of the polypeptide chains of the multimeric constructs were each under the control of an individual CMV promotor and terminated by a polyA tail signal as for example taken from bovine growth hormone or simian virus 40 (see FIGS. 3 and 4). pcDNA 3.1 (Thermo) derived vectors were used for co-transfections. Here, the individual polypeptide chains are on a separate plasmid reducing the risk of recombination of homologous elements and further allow to adjust molar ratios of plasmids for the transfections to improve the yield. Exchange of genes of interest is possible by standard cloning techniques. Examples of the resulting constructs are depicted in FIG. 5.

[0368] Expression in CHO-S

[0369] Exponentially growing CHO-S cells (Thermo) were seeded in CHOgro (Mirrus) at a density of four millions per ml in TPP600 bioreactors. Per ml of culture 3 pg of linear polyethylenimine (MW 25,000, PolySciences Inc) and 1.25 μg of highly pure plasmid DNA were added with in-between mixing. Eventually, cultures were supplemented with valproic acid to a final concentration of 1 mM. Proteins were expressed at 31° or 37° C., 8% CO2 and 180 rpm with a 50 mm throw in Kuhner ISF1-X shaker for up to 12 days.

[0370] Purification of Molecules

[0371] Expression cultures were harvested by centrifugation at 1400 rpm and 4° C. for 30 min. Supernatants were furthermore cleared by 0.22 μm filtration and adjusted to pH of 7. All subsequent purification steps were performed at 4° C. Supernatants were applied to PBS equilibrated rProtein A columns (GE) operated on a AEKTA Pure system. After PBS wash, bound protein was eluted by 0.1 M Glycine pH 2.75 (Chromatogram FIG. 6). Fractions of interest were pooled and buffer exchanged to 20 mM Bis/Tris methane 20 mM NaCI pH 6.75. Filtered protein solutions were loaded on Resource S (GE) columns and eluted with a gradient to 100% 20 mM Bis/Tris methane 1000 mM NaCI pH 6.75 (see FIG. 7). Desired fractions were pooled and if required polished on SEC (Superdex200 columns of adequate size; GE) for high purity (FIG. 8). Monomeric fractions were pooled, filtered, and purity analyzed on SDS page (FIG. 9). Eventually, pure protein was used for biochemical and cell assays as well as in-vivo studies.

[0372] HER2-Related Effects of Tetravalent Biparatopic IgGs

[0373] The tetrameric (tetravalent and biparatopic) polypeptide constructs 441 and 47C2 were tested in various assays, which are all well-known to the skilled person, and compared to the tetravalent IgG-fusion MED14276 (without a toxin), the dimeric bivalent biparatopic polypeptide constructs 841, 87C2 and Fc fusions thereof, the bivalent biparatopic DARPin construct 6L1G (see patent application WO 2014/060365 A1), single IgGs (TZB, PZB, A21 and 7C2) and combinations thereof.

[0374] Growth inhibition was tested in an XTT cell viability assay using live-cell high-content microscopy, Hoechst staining and cell count. As shown in Table 1, constructs 441, 47C2, 841, 87C2 and the Fc fusions of 841 and 87C2, 6L1G and the combination of TZB with A21 resulted in full growth inhibition, whereas single antibodies and TZB combined with PZB lead to partial inhibition. Surprisingly, in the absence of a cytotoxic drug, the antibody MED14276 stimulated growth of XTT cells.

[0375] The effect of the constructs on apoptosis/cell death were analyzed by live-cell high-content microscopy with annexin-V and PI staining or by detecting cleaved PARP in cell lysates by Western blot for analysis of PARP cleavage.

[0376] Constructs 441, 47C2, 841, 87C2 and their Fc fusions, 6L1G had an effect on apoptosis, whereas MED14276, single antibodies did not influence apoptosis, and TZB and A21 had a partial effect. The combination TZB+PZB is able to induce apoptosis in a very small fraction of cells or in fragile cell lines.

[0377] HER2 crosslinking on the cell surface, also termed “lockdown”, was measured by fluorescence recovery after photobleaching (FRAP) and single cell localization microscopy. A reduction of the FRAP signal indicates lower mobility of cells and therefore crosslinking in response to the polypeptide constructs.

[0378] 441, 47C2 and 6L1G resulted in lockdown of receptors, and partial crosslinking effect was measured for 841, 87C2 and their Fc fusions as well as the antibody combinations TZB+PZB and TZB+A21. Single antibodies had no effect on crosslinking.

[0379] Furthermore, HER2 internalization into cells was analyzed by a surface protein internalization and degradation assay, confocal microscopy and flow cytometry as described in detail in example 2.

[0380] The tetravalent biparatopic constructs 441, 47C2 and MED14276 displayed a strong effect on HER2 internalization, whereas a recycling inhibition was detected for the combinations TZB+PZB and TZB+A21). The remaining constructs showed no effect.

[0381] HER2 degradation was tested by a surface protein internalization and degradation assay and Western blot detection of total HER2. Here, 441 and 47C2 lead to rapid strong degradation. MED14276 had an effect on degradation, but less strong compared to 441 and 47C2. In contrast, the antibody combinations resulted in slow degradation and the remaining constructs had no effect.

TABLE-US-00002 TABLE 1 Results of assays measuring HER2-related effects of polypeptide constructs “Lockdown″ Growth Apoptosis/ (crosslinking HER2 HER2 Construct inhibition cell death on cells) Internalization Degradation 441/47C2 Yes Yes Yes Strong Rapid, Strong MEDI4276 No No Not Same as 441 Less than (without (stimulating!) determined but 441 toxin) expected 841/87C2 Yes Yes Partial No No and Fc fusions thereof 6L1G Yes Yes Yes No No Single Partial No No No No antibodies TZB + PZB Partial No (yes) Crosslinking Recycling Very slow inhibition (very weak) TZB + A21 Yes Partial Crosslinking Recycling Slow inhibition

[0382] Table 2 shows the results of HER2 binding studies performed with the same constructs as the experiments described above. Binding was determined by flow cytometry and additionally by size-exclusion chromatography/multi angle light scattering (SEC-MALS) in case of complex formation between the biparatopic IgG constructs and HER2.

[0383] All constructs bound the extracellular domains 1, 2 and/or 4 as expected (see Table 2). 441, 47C2 and the combination TZB+A21 displayed an extremely slow off rate which was slower compared to the remaining constructs.

[0384] Interestingly, all biparatopic constructs resulted in a HER2 binding stoichiometry of close to 1:1. Single antibodies and antibody combinations only lead approximately to a 1:2 stoichiometry.

[0385] Serum half-life of construct 441 was further tested by intra venous injection in NSG mice and time resolved detection of biparatopic IgG in blood samples by ELISA. To determine the half-life of 441, 3 mg/kg of purified construct were intravenously injected in NSG mice. At indicated time points mice were blead, whole blood allowed to clot and sera gained by taking supernatants of centrifuged samples. A standard capture ELISA was used to evaluate serum levels of 441 (FIG. 32). Anti-human Fc antibody from mouse was directly coated on maxisorb plates. Bound 441 and sera spiked 441 standards were revealed by anti-kappa chain antibodies from goat conjugated to alkaline phosphatase. Data was fitted with a two-phase decay model and resulting half-life was calculated to be 4.3+45.3 hours (α and β phase).

TABLE-US-00003 TABLE 2 HER2 binding properties and other parameters of polypeptide constructs Bound HER2 Total # Complex molecules Paratope Molecular size on on average per Serum Half weight cells (single Construct Binds to Off Rate in solution molecule life kDa molecule) 441/47C2 HER2 extremely Close to 1:1 4 45 hours 200 Large ECD1/4 slow 2.7 × 10{circumflex over ( )} − 4 MEDI4276 HER2 Not Close to 1:1 4 rapid 200 Not (without toxin) ECD2/4 determined clearance determined But expected (app. 1-3 extremely day) slow 841/87C2 and HER2 Very slow Close to 1:1 2 Not 72(+FC = Not Fc fusions ECD1/4 determined 122) determined thereof 6L1G HER2 Very slow Close to 1:1 2 5 min (12  32 Medium ECD1/4 hours PEGylated) Single HER2 Very slow Close to 1:2 2 12 days 145 Small antibodies ECD1/2/4 TZB + PZB HER2 Very slow Close to 1:2 2 12 days 145 Not ECD2/4 determined TZB + A21 HER2 extremely Close to 1:2 2 12 days 145 Not ECD1/4 slow determined 3.66 × 10{circumflex over ( )} − 4

[0386] Surprisingly, the tetrameric tetravalent biparatopic constructs 441 and 47C2 lead to strong inhibition of cell proliferation, induced cell death by apoptosis and led to crosslinking of HER2 on the cell surface and induced strong HER2 internalization and strong HER2 degradation in addition to their excellent binding properties to HER2. 441 and 47C2 were superior to or scored equally well as all other constructs in all categories.

[0387] TZB+PZB and TZB+A21 resulted in a decrease of total HER2 (very weak in case of TZB+PZB) which was attributed to recycling inhibition, a mechanism by which HER2 is degraded without prior intracellular accumulation (see FIG. 35).

[0388] To determine the effect of construct 441 on tumor growth, SCID beige (Charles River) mice were inoculated on the right flank with five million N87 cells in 50% matrigel (Corning). After tumors had reached around 150 mm.sup.3 mice were treated with 10 mg/kg 441 for eight times with a three to four-day interval. Treated mice responded to 441 with tumor burden reduction. Growth arrest was initially seen for TZB (10 mg/kg) and hA21G (10 mg/kg) treated mice and tumors of control mice (labeled ‘PBS’ in FIG. 33) showed unhindered progression (FIG. 33).

EXAMPLE 2

Enhanced Internalization, Lysosomal Trafficking, and Degradation of HER2 by Revealed Molecule 441

[0389] Microscopy with BT-474 and HCC1419 breast cancer cells For microscopy of fixed samples, cells were seeded at a density of 4.Math.10.sup.4 cm.sup.−2 in p-slides

[0390] (Ibidi, cat. no. 80824) in complete medium. On the next day, cells were treated with the respective molecules. After 2 h, cells were once washed with Dulbecco's phosphate buffered saline (DPBS), and fixed by addition of 4% (w/v) paraformaldehyde dissolved in DPBS and incubation at room temperature for 10 min. Next, cells were washed twice with PBSBA+T (DPBS supplemented with 1% (w/v) bovine serum albumin (BSA), 0.1% (w/v) sodium azide, and 0.5% (w/v) Tween-20). Afterwards, cells were incubated in anti-LAMP antibody (Cell Signaling Technology, cat. no. D401S) dissolved at 1:150 (v/v) in PBSBA+T, further supplemented with 100 ng ml.sup.−1 2-(4-amidinophenyl)-1H-indo1-6-carboximidamide (DAPI) for 30 min at room temperature. Cells were then washed twice with PBSBA+T, and subsequently anti-mouse, conjugated to Alexa Fluor 488 (Thermo Fisher Scientific, cat. no. A11001) and anti-human, conjugated to Alexa Fluor 647 (Thermo Fisher Scientific, cat. no. A-21445) antibodies from goat, dissolved in PBSBA+T, were added and incubated for 30 min at room temperature. Next, cells were washed twice with PBSBA+T, fixed once more by addition of 4% (w/v) paraformaldehyde dissolved in DPBS and incubation at room temperature for 10 min, finally washed once with PBSA, and stored in PBSA (DPBS supplemented with 0.1% (w/v) sodium azide) at 4 ° C. until measurement. Imaging was performed on a SP5 confocal laser scanning microscope (Leica). The images show that for both cell lines, only 441 was able to induce its rapid internalization, and shows strong colocalization with lysosomal (LAMP1-positive) compartments.

[0391] Surface Protein Internalization and Degradation Assay

[0392] To quantify internalization and degradation of HER2 upon treatments, we performed a quantitative surface protein internalization and degradation assay was performed. In brief, a stable Flp-In TREx HEK293 cell line (Thermo Fisher Scientific, cat. no. K650001) was generated according to the instructions of the manufacturer, in which a HaloTag-HER2 receptor fusion can be overexpressed upon induction. For the assay, cells were seeded two days before the first treatment, and one day before treatment, doxycycline was added to induce stable overexpression for 24 h. Treatments (100 nM) were added at indicated time points, referring to the time of cell labeling.

[0393] After completion of the treatment time intervals, cells were labeled in a two-step procedure. A HaloTag ligand containing to Alexa Fluor 660 (HTL-AF660, Promega, cat. no. G8472), which is completely cell-impermeable and therefore stains surface receptors only, was coupled in a first labeling step. A cell-permeable HaloTag ligand containing tetramethyl rhodamine (HTL-TMR, Promega, cat. no. G8252), was, in the second step, applied to stain all receptor fusion, which resides in intracellular compartments. Thus, signals originating from surface and internal receptor are detected in separate channels on a flow cytometer. Therefore, information regarding the localization, and using the rescaling procedure described below, about the quantitative distribution, can be obtained. A commercially available dead-cell stain was used for exclusion of permeabilized (dead) cells from analysis, for which all receptor would appear to be on the surface. Fluorescence intensities in each channel for 2′000-10′000 cells was recorded using a LSR II Fortessa (BD), and single, non-permeabilized cells gated. Mean fluorescence intensities of these populations were obtained using FlowJo 10.4 (FlowJo).

[0394] Data Processing

[0395] To correct for different detection efficiencies of the flow cytometry instrument in the channels for AF660 and TMR, the data were scaled, yielding relative abundances. A control sample (utr.,s.), in which the first (HTL-AF660-labeling) step is omitted, is required to this end. In this sample, all HaloTag molecules will react with HTL-TMR in this “single” (s.) labeling procedure, irrespective of their localization. Using the mean fluorescence intensities (MFI) of the singlet, non-permeabilized cell population, the normalized (feature-scaled) signal S.sub.TMR in the TMR channel for a samples is obtained by normalizing to a single-labeled, untreated control sample (utr.,s.) and background subtraction:

[00003]$\begin{array}{cc}{S}_{\mathrm{TMR}}\left(\mathrm{sample}\right)=\frac{{\mathrm{MFI}}_{\mathrm{TMR}}\left(\mathrm{sample}\right)-{\mathrm{MFI}}_{\mathrm{TMR}}\left(\mathrm{utr}.,\mathrm{unlab}.\right)}{{\mathrm{MFI}}_{\mathrm{TMR}}\left(\mathrm{utr}.,s.\right)-{\mathrm{MFI}}_{\mathrm{TMR}}\left(\mathrm{utr}.,\mathrm{unlab}.\right)},& \#\left(\mathrm{eq}.\mathrm{S1}\right)\end{array}$

where utr.,unlab. represents an untreated, unlabeled control (showing only autofluorescence).

[0396] The first, surface-labeling step with cell-impermeable dye is virtually saturating in the complete two-step (double) labeling procedure. The normalized surface signal S.sub.AF660 can thus be defined in:

[00004]$\begin{array}{cc}{S}_{\mathrm{AF}660}\left(\mathrm{sample}\right)=\frac{{\mathrm{MFI}}_{\mathrm{AF}660}\left(\mathrm{sample}\right)-{\mathrm{MFI}}_{\mathrm{AF}660}\left(\mathrm{utr}.,\mathrm{unlab}.\right)}{{\mathrm{MFI}}_{\mathrm{AF}660}\left(\mathrm{utr}.,s.\right)-{\mathrm{MFI}}_{\mathrm{Af}660}\left(\mathrm{utr}.,\mathrm{unlab}.\right)}.& \#\left(\mathrm{eq}.\mathrm{S2}\right)\end{array}$

[0397] The signal of a sample can be related to the surface signal from a double-labeled control (utr.,d.) and does not require a separate single-stained sample, however, because internal receptor is not accessible to HTL-AF660 and thus cannot be stained.

[0398] ΔS.sub.TMR, the difference from the single to the double labeling procedure in the TMR channel, now exactly corresponds to the number of molecules, which were blocked by the first, surface-specific step. The signal in the AF660 channel in the same double labeling experiment also is a direct correlate of this number of molecules:

ΔS.sub.TMR=S.sub.TMR(utr.,s.)−S.sub.TMR(utr.,d.)=S.sub.AF660,scaled(utr.,d.)#  (eq. S3).

[0399] A correction factor C.sub.A can thus be defined, which relates the measured intensity S.sub.AF660(utr.,d.) (recorded in the AF660 channel) to S.sub.AF660,scaled(utr.,d.) (in the scale of the TMR channel):

S.sub.AF660,scaled(Utr.,d.)=S.sub.AF660(utr.,d.)×C.sub.A#  (eq. S4).

[0400] Using signals from single-labeled and double-labeled, untreated cells, calculation of C.sub.A is possible as follows:

[00005]$\begin{array}{cc}{C}_A=\frac{S_{\mathrm{TMR}}\left(\mathrm{utr}.,s.\right)-S_{\mathrm{TMR}}\left(\mathrm{utr}.,d.\right)}{S_{\mathrm{AF}660}\left(\mathrm{utr}.,d.\right)}=\frac{100\%-S_{\mathrm{TMR}}\left(\mathrm{utr}.,d.\right)}{S_{\mathrm{AF}660}\left(\mathrm{utr}.,d.\right)},& \#\left(\mathrm{eq}.\mathrm{S5}\right)\end{array}$

taking into consideration that the TMR signal of single-labeled, untreated cells was, using eq. S1, scaled to 100% before. Correction of the signals recorded in the AF660 channel, can, for treated samples, now be done according to:

S.sub.AF660,scaled(tre,d.)=C.sub.A×S.sub.AF660(tre.,d.)#  (eq. S6)

[0401] S.sub.TMR(tre.,d.) and S.sub.AF660,scaled(tre.,d.) then truly represent the abundance of internal and surface protein, respectively, and the sum S.sub.AF660,scaled(tre.,d.)+S.sub.TMR(tre.,d.) represents the amount of total protein, for a double-labeled, treated sample, always relative to an untreated control sample.

[0402] Data scaling as described above was performed and the results plotting using MATLAB R2017b (MathWorks), R 3.5.1, Prism 6.07 (GraphPad), and Excel2016 (Microsoft). The results are shown in FIG. 35. In contrast to all other constructs, construct 441 showed almost HER2 internalization after less than 5 minutes.