JP2022504472A - HER2-binding tetrameric polypeptide - Google Patents

Abstract

The present invention relates to a tetramer polypeptide, wherein the tetramer polypeptide comprises a first VL antigen binding domain and a first CL constant domain, a first polypeptide chain, and a first VH antigen binding domain. The first VL antigen-binding domain or the first by means of a second polypeptide chain containing a first CH1 constant domain, a first CH2 constant domain and a first CH3 constant domain, and a first interdomain amino acid linker. A first ligand that binds to the HER2 D4 epitope linked to the N-terminal of the VH antigen-binding domain, a third polypeptide chain containing a second VL antigen-binding domain and a second CL constant domain, and a second. The second VL antigen by a fourth polypeptide chain containing a VH antigen-binding domain, a second CH1 constant domain, a second CH2 constant domain and a second CH3 constant domain, and a second interdomain amino acid linker. It comprises a binding domain or a third ligand that binds to the HER2 D4 epitope linked to the N-terminal of the second VH antigen binding domain, and both the VL antigen binding domain and the VH antigen binding domain bind to the HER2 D1 epitope. It constitutes a second ligand and a fourth ligand. The present invention further relates to applications of tetrameric polypeptides in methods of preventing or treating malignant neoplastic diseases, host cells for expressing isolated nucleic acids and polypeptides, and methods of obtaining polypeptides.

Description

The present invention relates to a tetrameric polypeptide having a binding site for two HER2 epitopes D1 and a binding site for two HER2 epitopes D4.

Members of the HER family of receptor tyrosine kinases are important mediators for cell growth, differentiation, migration and survival. The receptor family includes four different members, including the epidermal growth factor receptor (EGFR, ErbB1 or HER1), HER2 (ErbB2 or p185 <neu>), HER3 (ErbB3) and HER4 (ErbB4). Members of the EGFR family are closely related single-stranded modular glycoproteins with extracellular ligand binding regions, single transmembrane domains and intracellular tyrosine kinases, followed by key identification for downstream signal protein docking. Followed by the phosphorylation site of.

The extracellular region of the HER receptor family contains two homologous ligand binding domains (domains 1 and 3) and two cysteine rich domains (domains 2 and 4) that are important for receptor dimerization. In the absence of ligand, the HER receptor normally exists as an inert monomer known as a "tether" structure characterized by a close interaction between domains 2 and 4. Binding of the ligand to the extracellular domain causes conformational rearrangement, exposing dimerized domains 2 and 4. Therefore, binding of growth factors to the HER receptor induces a change in conformation that allows dimerization of the receptor. After extracellular receptor dimerization, the transmembrane helix switches to an active conformation to allow the intracellular kinase domains to trans-autophosphorylate with each other. This phosphorylation allows the recruitment of specific downstream signaling proteins.

Epidermal growth factor receptor 1 (EGFR) is involved in human malignancies as a cause. In particular, increased expression of EGFR was observed in breast cancer, bladder cancer, lung cancer, head cancer, cervical cancer, gastric cancer and glioblastoma.

Human epidermal growth factor receptor 2 (also known as HER2, ErbB2 or Neu, UniProtKB / Swiss-Prot No. P04626) consists of 1233 amino acids, structurally similar to EGFR and 4 subdomains 1. It has an extracellular domain consisting of ~ 4, a transmembrane domain, a near-membrane domain, an intracellular cytoplasmic tyrosine kinase, and a regulatory C-terminal region. However, the structure of the extracellular space of HER2 differs from other EGF receptors in important respects. In other EGF receptors, domain 2 binds to domain 4 in the non-activated state. Upon binding to domains 1 and 3, the activating growth factor (ligand) selects a conformation that allows the dimerization arm to extend from domain 2 to interact with the ErbB dimer partner. And make it stable. On the other hand, HER2 has a fixed conformation that resembles the ligand activation state of other receptor members. That is, there is no interaction between domains 2-4 and the dimerization loop in domain 2 is persistently exposed. HER2 is activated by forming a heterodimer complex with other ErbB family members and is indirectly regulated by EGFR and HER3 ligands. HER2 is the preferred heterodimerization partner for the other three ErbB receptors, enhancing the affinity of other ErbB receptors for ligands by slowing the dissociation rate of the ligand-receptor complex. Therefore, HER2 reinforces and prolongs signal transduction.

Excessive HER2 on the cell surface causes transformation of epithelial cells from multiple tissues. Amplification of the human homologue (also known as HER2) of the neu gene has been observed in breast and ovarian cancers and is associated with a poor prognosis (US 4,968,603). Overexpression of HER2 was also observed in other cancers, including gastric cancer, endometrial cancer, salivary adenocarcinoma, lung cancer, kidney cancer, colon cancer, thyroid cancer, pancreatic cancer and bladder cancer.

HER2-targeting antibody Drebin and colleagues have proposed an antibody against the rat neu gene product p185 <neu> disclosed in US 6,733,752.

Hudziak et al. (1989, Mol. Cell. Biol. 9 (3), 1165-1172) described the generation of the HER2 antibody group characterized by the human breast tumor cell line SkBr-3. Using a cell proliferation assay, maximal inhibition was obtained with an antibody called 4D5. In addition, antibody 4D5 has been found to sensitize HER2-overexpressing breast tumor cell lines to the cytotoxic effects of TNF- [α], see also US5,677,171. Recombinant humanized forms of mouse HER2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2, trastuzumab or Herceptin; US 5,821,337) are used for patients with HER2 overexpressing metastatic breast cancer who have previously received extensive anticancer therapy. It is clinically effective. Herceptin has been approved for use in patients with HER2-positive metastatic gastric (gastric) cancer in combination with chemotherapy.

Herceptin is widely used in the treatment of patients with early-stage and metastatic breast cancer in which the tumor overexpresses the HER2 protein and / or the HER2 gene is amplified. Treatment of breast cancer patients with Herceptin / trastuzumab is recommended, for example, for patients with HER2-positive disease and is now routinely practiced, see US2002 / 0064785, US2003 / 0170234A1, US2003 / 0134344 and US2003 / 0147884. .. Therefore, prior art has focused on the eligibility of breast cancer patients for trastuzumab / Herceptin therapy based on high HER2 protein expression levels (eg, defined as HER2 (3+) by immunohistochemistry (IHC)). High HER2 (protein) expression detected in samples obtained from patients such as breast tissue biopsies and breast tissue sections by immunohistochemical methods, or in tissues derived from metastatic sites (eg, HER2 (+++)). Or HER2 gene amplification (eg, the number of HER2 gene copies is higher than 4 copies of the HER2 gene per tumor cell) or both), defined as the presence of HER2-positive disease in breast cancer. One of the methods frequently used to detect overexpression and amplification of HER2 at the genetic level is fluorescence in situ hybridization (FISH), which is also described in US2003 / 0152987.

The humanized antibody pertuzumab is the first new class of agents known as HER dimerization inhibitors (HDIs). Pertuzumab inhibits the ability to form an active heterodimeric receptor complex by binding to HER2 in its dimerization domain, ultimately blocking the downstream signal cascade that causes cell growth and division. .. Pertuzumab is for the extracellular domain 2 of HER2. Compared to trastuzumab, which acts by binding to domain 4 of HER2, pertuzumab inhibits dimerization of HER2 with HER3 and other members of the EGFR receptor family in the presence of the corresponding activating ligand. It is a HER dimerization inhibitor. Pertuzumab blocks the growth-stimulating effects and cell survival signals activated by the ligands of HER1, HER3 and HER4 by blocking complex formation. Pertuzumab, under the name Perjeta, is used to treat patients with HER2-positive metastatic breast cancer who have not previously received anti-HER2 therapy or chemotherapy for metastatic disease in combination with trastuzumab and docetaxel. Approved by the FDA. Pertuzumab is a fully humanized recombinant monoclonal antibody based on the human IgG1 ([κ]) framework sequence. Patent publications relating to the selection of patients subject to pertuzumab and its therapy include US20060073143A1, US20030086924, US20040013667A1 and US20040106161A1.

Trastuzumab was found to be clinically useful, for example, in terms of prolonging survival when used in combination with chemotherapy compared to chemotherapy alone, but most HER2-positive breast cancer patients did not respond. (The overall response rate was 45% when Herceptin was used in combination with chemotherapy, and 29% when chemotherapy alone was used).

Therefore, although it has been shown that monoclonal antibody therapy against HER2 can provide improved treatment for metastatic breast cancer that overexpresses HER2, for example, there is still considerable room for improvement.

Non-antibody scaffolds targeting HER2 Recently, alternative target proteins have been proposed that are more diverse in molecular structure, heterodimer and abundant compared to antibody fragments derived from human immunoglobulins and constructs and formats derived from antibodies. By making body assemblies, more molecular formats are possible and new biological functions are introduced. Many such target proteins have been described (reviewed in (Binz et al., 2005, Nat. Biotech, 23, 1257-1268)). Non-limiting examples of such target proteins include camel antibodies, protein scaffolds derived from the protein A domain (called "alphabodies", Affibody AB), tendamistats (α-amylase inhibitors, streptomyces. 74 amino acid β-sheet protein from Tendere), fibronectin, lipocalin (“Antibody”, Pieris), T cell receptor, ankyrin (designed ankyrin repeat protein called “DARPins”, Univ. Zurich and Molecular Partners; US12014; US12014 (See), A domain of several receptors (“Avivers”, Avidia) and PDZ domain, fibronectin domain (FN3) (“Adectins”, Adnexus), consensus fibronectin domain (“Centyrins”, Centyrex / Johnson & Johnson), "Affiliins", SCIL protein) and Notchone (Moore and Cochrane, 2012, Methods in Energy 503, 223-251 and references cited therein).

Multimers and multispecific assemblies can be constructed from these proteins (Caravella and Lugovskoy, 2010, Current Opinions in Chemical Biology, 14, 520-528; Vanlandschoot et al., 2011, Antirevi). 407; Lofblom et al., 2011, Chemical Opinion in Biotechnology, 22, 843-848, Boersma et al., 2011, Curr. Opin. Biotechnol., 22, 849-857). It is also possible to fuse these and other peptide domains with an antibody to make so-called Zybodies (Zyngenia Inc., Gaithersburg, MD).

A common feature of scaffolds with these different intrinsic properties is that they can be made to bind to specific epitopes by using selection techniques known to those of skill in the art (Binz et al., Ibid.).

For example, each of the different domains of HER2 can be expressed individually in insect cells utilizing the baculovirus expression system, as demonstrated for Domain 1 and Domain 4 (Free et al., 2012, Nat). Biotechnol., 30, 997-1001). This ensures that the selected binder is directed to the target domain. The HER2 domain is then biotinylated as described above (Zahnd et al., 2006, J. Biol. Chem. 281 (46), 35167-75), streptavidin-coated magnetic beads, or streptavidin. Alternatively, it can be immobilized on a microtiter plate coated with neutral avidin (Steiner et al., 2008, J. Mol. Biol. 382,121-11227; Zahnd et al., 2007, J. Mol. Biol. 369,1015-1028.). The HER2 domain so immobilized can serve as a target for a diverse protein library of phage display format or ribosome display format. A wide variety of different antibody libraries have already been published (Mondon P. et al., 2008, Frontiers in Bioscience. 13, 1117-1129) and techniques for selecting binding antibodies are known to those of skill in the art. Phage displays are suitable formats for antibody fragments (Fab fragments, scFv fragments, or single domain antibodies) (Hoogenboom, 2005, Nature Biotechnology., 23 (9), 1105-1116) and other scaffolds containing disulfide bonds. Is also applicable to scaffolds that do not contain disulfide bonds (eg, Steiner et al., 2008, J. Mol. Biol., 382,121-11227) (Rentero et al., 2011, Chimia., 65 (eg). 11), 843-5, Skera A., 2007, Current Opinion in Biotechnology., 18 (4), 295-304). Similarly, ribosome displays are applicable to antibody fragments (Hane's et al., 2000, Nat. Biotechnol., 18, 1287-1292) and other scaffolds (Zahnd et al., 2007, Nat. Methods, 4). , 269-279; Zahnd et al., 2007, J. Mol. Biol., 369, 1015-28.). A third powerful technique is yeast display (Pepper et al., 2008, Combinatorial Chemistry & High Throughput Screening, 11 (2), 127-134). In this case, the library of target binding proteins is displayed on the surface of the yeast and each of the HER2 domains is either directly labeled with a fluorescent dye or the His tag is detected with an anti-His tag antibody and the anti-His tag antibody. Is also detected with secondary antibodies. Such methods are known to those of skill in the art (Boder et al., 2000, Methods in Enzymeology, 328, 430-44).

Another possibility of engineering is to connect these binders to create bispecific or higher multivalent binding molecules. Such connections can be made by fusing two or more of these binding molecules genetically, by cross-linking molecules that are chemically expressed separately, or by adding a dimerization domain. It is feasible (including independent dependent claims of each or any combination thereof) (eg, Stephan et al., 2011, J. Mol Biol., 413, 826-843; Boersma et al., 2011). , J. Biol. Chem., 286, 41273-41285).

Bispecific anti-HER2 camelid antibody constructs (bispecific Nanobodies) are shown in US2011059090. This document relates to bispecific molecules that simultaneously target HER2 in extracellular domain 2 defined in competition with pertuzumab and domain 4 defined in competition with trastuzumab. This molecule is stated to exhibit stronger antiproliferative activity than trastuzumab (Herceptin) as a result of direct comparison in an in vitro cell culture model using the SkBr3 cell line.

Since there are no known HER2-specific ligands, current HER2-targeting strategies aim to bind to the interaction surface and block receptor dimerization. Current findings on HER2 receptor dimerization are largely based on the ligand-bound crystal structure of the EGFR homodimer, which is widely recognized as the active mode of all EGF receptor family members. (Garret et al., 2002, Cell, 110, 763-773). The two EGFR molecules show back-to-back interactions. Extending this finding to possible interactions with HER2 and other members of the EGFR family, one interaction interface resides in domain 2 of the extracellular component of HER2. Pertuzumab is known to bind to domain 2 and reliably block receptor interactions at this interface. Other known interactions are located in domain 4 of the extracellular component of HER2. It is believed that this interaction interface is blocked by trastuzumab. However, the two antibodies, pertuzumab and trastuzumab, cannot completely block all HER2 interactions, even when used simultaneously. The interaction between the extracellular portion and the kinase domain of HER2 is thought to be linked so that some residual interactions are possible even when blocked by pertuzumab or trastuzumab, which is consistent with crystal structure data. (Lu et al., 2010, Mol. Cell. Biol., 22, 5432-5443). According to the bispecific ligand (US2011 / 0059090) that simultaneously binds to two epitopes (pertuzumab and trastuzumab), cell growth in the cell culture model is reduced by about 50%, but the effect of trastuzumab is about 40. %Decrease. However, the same effect can be achieved by treatment with a mixture of trastuzumab and pertuzumab.

Patent application WO2014 / 060365 comprises a first polypeptide ligand that binds to HER2 extracellular domain 1 (D1 epitope) and a second polypeptide ligand that binds to HER2 extracellular domain 4 (D4 epitope). A bispecific HER2 targeting agent in which the polypeptide ligand of No. 1 and the second polypeptide ligand are separated by a linker is described. The most active of these biparatopic binders binds to two separate HER2 molecules primarily in an intermolecular binding mode. Thereby, these biparatopic bivalent binding agents crosslink HER2_ECD1 of one HER2 and HER2_ECD4 of another HER2 molecule with these paratopes and preferably short peptide linkers, resulting in intermolecular binding rather than intramolecular binding. Further contribute. The bivalent binding mode of the biparatopic binding agent induces polymerization of the HER2 receptor on the cell surface, thus forming a productive HER2 / HER3 or HER2 / EGFR heterodimer or HER2 / HER2 homodimer. Cannot (Tamaskovic et al., 2016, Jost et al., 2013). Therefore, HER2 and HER3 phosphorylation is inhibited in HER2 overexpressing cancer cells, followed by arrest of proliferation and anti-apoptotic signaling from the HER2 / HER3 receptor, and finally cell death is induced by apoptosis. However, these biparatopic binders do not affect the total expression of the HER2 receptor (Tamaskovic et al., 2016). Total expression of the HER2 receptor is still relatively high and can cause incomplete inactivation of the HER2 receptor. Moreover, these constructs do not exhibit the desired pharmacokinetic properties required for systemic application. Desired functions such as long serum half-life in the bloodstream and protein recycling mechanism by binding to FcRn receptors have not been realized in these constructs. Moreover, these biparatopic constructs do not utilize antibody effector functions such as complement-dependent cellular cytotoxicity (CDC) or antibody-dependent-cell-mediated cytotoxicity (ADCC). These effector functions can contribute to further enhancing the antitumor activity of the biparatopic binder in vivo. Finally, these biparatopic binders are not further modified to evade T cell epitopes and therefore tend to potentially induce an immune response. This can significantly reduce tolerability and serum levels after repeated doses in immunocompromised patients.

In addition, tetravalent biparatopic HER2 target antibody drug conjugates comprising a fusion of trastuzumab and 39S antibody variable sequences that bind to D4 and D2 epitopes of HER2 have been described (Li et al., 2016, Cancer Cell, 29). , 117-129). This complex was commercialized by Medimmune under the name MEDI4276 and tested in a Phase 1/2 clinical trial (NCT025765448). However, the armless form of biparatopic IgG fusion constructs not fused with the tubricin toxic payload is not sufficient to induce inhibition of cancer cell proliferation. Conversely, the non-complex form of this IgG fusion molecule induces cancer cell proliferation at high concentrations in the HER2-overexpressing cancer cell model. This can be caused by binding to HER2_ECD2 and HER2_ECD4 to increase the formation of HER2 homodimers and heterodimers capable of signaling. On the other hand, the non-complex IgG fusion protein form of this tetravalent biparatopic HER2 target antibody can induce downregulation of HER2 receptor expression (Li et al., 2016, Cancer Cell, 29, 117-129). .. Taken together, the tetravalent scFv_4D5-IgG_39S fusion protein downregulates HER2 expression but does not show inhibition of HER2 signaling and instead activates cancer cell proliferation in specific HER2 overexpression models. Invite. This also indicates that downregulation of HER2 is not easily associated with inhibition of cancer cell growth. Obviously, inhibition of HER2-targeted agents on HER2-dependent signaling pathways is essential for clinical application. For example, trastuzumab (Herceptin) does block HER3 signaling and does significantly reduce cell proliferation in HER2-overexpressing cancers without PI3K pathway activation mutations.

Based on the prior art described above, an object of the present invention provides means and methods for providing an improved HER2 targeting agent in view of the above drawbacks of the prior art, in particular other small molecules that bind to the HER2 targeting agent. In the absence of molecular toxins is to provide HER2 targeting agents that exhibit improved HER2 signaling inhibition, expression downregulation, polymerization and clustering, inhibition of receptor diffusion, degradation and / or inhibition of recycling. This object is achieved by the subject matter of the independent claims herein.

The present invention relates to tetrameric polypeptides.
A first polypeptide chain containing a first VL antigen binding domain and a first CL constant domain in the direction N to C,
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 in the direction N to C.
A first ligand that specifically binds to the HER2 D4 epitope, is contained in the first polypeptide chain, and is linked to the N-terminus of the first VL antigen-binding domain by a first interdomain amino acid linker. Alternatively, the first ligand contained in the second polypeptide chain and linked to the N-terminus of the first VH antigen binding domain by the first interdomain amino acid linker.
A second ligand that specifically binds to the HER2 D1 epitope, which is composed of the first VL antigen-binding domain of the first polypeptide chain and the first VH antigen-binding domain of the second polypeptide chain. Especially with the Fab domain,
A third polypeptide chain containing a second VL antigen binding domain and a second CL constant domain in the direction N to C,
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 in the direction N to C.
A third ligand that specifically binds to the HER2 D4 epitope, is contained in the third polypeptide chain, and is linked to the N-terminus of the second VL antigen-binding domain by a second interdomain amino acid linker. Alternatively, a third ligand contained in the fourth polypeptide chain and linked to the N-terminus of the second VH antigen-binding domain by a second interdomain amino acid linker.
A fourth ligand that specifically binds to the HER2 D1 epitope, which is composed of the second VL antigen-binding domain of the third polypeptide chain and the second VH antigen-binding domain of the fourth polypeptide chain. , In particular with, or consists of Fab domains.

Thus, the tetrameric polypeptide according to the invention contains twice as many different HER2-binding paratopes, i.e., tetravalent. The polypeptide is a biparatopic because it contains binding sites for two different HER2 epitopes, D1 and D4, in a single molecule.

Surprisingly, the polypeptide exhibits superior HER2 inactivation compared to conventional antibodies and bivalent biparatopic polypeptides (combined with two binding paratopes), cell growth and proliferation, It is also effective in apoptosis and other forms of cell death, HER2 internalization and inhibition of HER2 recycling, HER2 expression downregulation and HER2 degradation, HER2 cross-linking, inhibition of HER2 dimerization and reduction of HER2 receptor surface mobility. In addition, increased molecular size excludes renal filtration and FcRn-mediated recycling enhances pharmacokinetic properties. Finally, due to the presence of the Fc moiety, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDC) may occur. Finally, the construct consists of non-immunogenic sequences, as all sequences are human antibody sequences. In summary, the tetrameric polypeptide according to the invention is a promising candidate for systemic therapy for HER2-expressing cancer.

In another embodiment, the invention comprises a compound of the invention or a pharmaceutically acceptable salt thereof, and at least one of at least one pharmaceutically acceptable carrier, diluent or excipient. Regarding pharmaceutical compositions.

The present invention further relates to applications of tetrameric polypeptides in methods of preventing or treating malignant neoplastic diseases, isolated nucleic acids encoding the polypeptides, host cells for producing the polypeptides, and acquisition of the polypeptides. Regarding the method.

The scheme of the biparatopic anti-HER2 binder is shown. A further scheme of biparatopic anti-HER2 binders is shown. A vector map of a plasmid for co-expressing the light and heavy chains of construct 441 in CHO cells (Pymex10 based vector with dual expression cassette [CMV GOI polyA]) is shown. A vector map of a plasmid for co-expressing the light and heavy chains of construct 47C2 in CHO cells (Pymex10 based vector with dual expression cassette [CMV GOI polyA]) is shown. A vector map of the plasmid for expressing construct 841 in CHO cells is shown. The elution profile of construct 441 from Protein A affinity chromatography is shown. The elution profile of construct 441 from cation exchange chromatography is shown. The elution profile of construct 441 from size exclusion chromatography is shown. The Coomassie stained SDS-PAGE gel of the fraction from the purification of construct 441 is shown. The life-and-death discrimination test of CHO in the expression of construct 441 (scFV-IgG) is shown. Expression optimization of construct 441 in CHO cells was performed at the indicated time. Cells were cultured in CHOgro medium (MIR 6260) from MIrus, and free cysteine (reduced form) (2), glutathione (3), fetal bovine serum (4), or all additives (5) were added, respectively. .. CHO cells were analyzed on a CASY cell counter (Schalfe System). Western blots of construct 441 expression secreted into the medium of CHO cells after the indicated time are shown. Cells are cultured in CHOgro medium (MIR6260) from MIrus (1), with free cysteine (reduced) (2), glutathione (3), fetal bovine serum (4), or all additives (5), respectively. added. Protein was precipitated from the medium by acetone precipitation and redissolved in SDS PAGE buffer. Proteins were separated on a 4-12% gradient gel and Western blots were analyzed on the Odyssey system (LI-COR). The purified intact full-length construct 441 is shown as control (A) and exceeds the 170 kDa marker. Page ruler, a molecular weight marker manufactured by Thermo Scientific, is shown in red. A cell proliferation assay (XTT) using BT474 cells after treatment for 4 days is shown. Different fusion variants of trastuzumab (TZB), biparatopic DARPin (6L1G), and biparatopic constructs are shown. LF IgG HL (rat parent of construct 441) and HF IgG HL (rat parent of construct 241) show similar antiproliferative activity compared to biparatopic DARPin 6L1G and are superior to trastuzumab (TZB). HF IgG LH (rat variant, no sequence) and LF IgG LH (rat sequence, no sequence) show reduced antiproliferative activity and higher IC50 concentrations compared to biparatopic DARPin. A cell proliferation assay (XTT) using BT474 cells after treatment for 4 days to test the effect of linker length is shown. Biparatopic DARPin (6L1G) and different fusion linker variants of the biparatopic construct (441 murine parent construct) are compared. 2-AA linker (GS) exhibits the highest antiproliferative activity. The 4-AA, 7-AA and 12-AA linkers show similar activity. The 22-AA linker variant exhibits reduced activity. A cell proliferation assay (XTT) using BT474 cells after treatment for 4 days is shown. Biparatopic DARPin (6G; 6L1G), biparatopic construct 441 (441), biparatopic construct 411 (humanized κ1 VH1) and biparatopic construct 443 (humanized κ4 VH3) are shown. Everything else shows similar plateau-level antiproliferative activity, except that 443 shows reduced activity. A cell proliferation assay (XTT) using BT474 cells after treatment with a different humanized form of A21 IgG for 4 days when fused to TZB scFv is shown. The humanization strategy is as described above. Different variants use humanized κ1 VH3 or humanized κ1 VH core transplanted tissue. The XTT cell proliferation assay using BT474 cells after treatment for 4 days is shown. The tetravalent IgG (HF IgG HL and LF IgG HL mice) and the divalent Fab fusion (HF Fab HL and LF Fab HL mice) are compared. All constructs show similar plateau and IC50 values. The XTT cell proliferation assay using SKBR3 cells after treatment for 4 days is shown. Byparatopic DARPin (6G), byparatopic construct (441 ft), trastuzumab (TZB) are shown. A cell proliferation assay (XTT) using CARU-3 cells after treatment for 4 days is shown. Biparatopic DARPin (6G), biparatopic construct (construction 441 (441tf)), trastuzumab (TZB) are shown. A cell proliferation assay (XTT) using BT474 cells after treatment for 4 days is shown to test the effect of domain 1 binding units. Biparatopic constructs with A21 (construct 441tf) or 7C2 fusion show different IC50s and plateau levels. A cell proliferation assay (XTT) using BT474 cells after treatment for 4 days is shown to test the effect of domain 1 binding units. Biparatopic constructs comprising A21 (construct 441) or 39S (39s HF IgG HL) are shown. The XTT cell proliferation assay using HCC1419 cells after treatment for 4 days is shown. Biparatopic DARPin (6G; 6L1G), biparatopic construct 441 (441tf) and divalent LF-oaFabFc (A21-TZB-4oa) are shown. 441 and 6G show similar inhibition of cell proliferation after 4 days. Compared to 441, LF-oaFabFc has slightly reduced inhibition of cell proliferation. The XTT cell proliferation assay using BT474 and HCC1419 cells after treatment for 4 days is shown. All are of human origin. The XTT cell proliferation assay using BT474 and HCC1419 cells after treatment for 4 days is shown. All are of human origin. a) Top panel of XTT cell proliferation assay with BT474 (left) and HCC1419 (right) cells treated for 4 days, and XTT with BT474 (left) and HCC1419 (right) cells treated for 4 days. The lower panel of the cell proliferation assay (higher affinity variants (NGS and GGG)), b) shows repeated experiments with new expression of NGS. The XTT cell proliferation assay using BT474 (left) and HCC1419 (right) cells after treatment for 4 days is shown. The XTT cell proliferation assay in which HCC1419 cells are grown into 3D spheroids is shown. Western blots after 24-hour post-treatment (BT474) with the indicated agent (rat) are shown. The top panel of induction of apoptosis in BT474 cells after treatment for 3 days is shown. The average number of propidium iodide (PI) -positive cells was determined after repeating 4 times, counted by Cell Profiler, and analyzed by Student's t-test. Compared to trastuzumab (TZB), the biparatopic construct (441,441tf) clearly induced more cell death. 441 and biparatopic DARPin (6L1G) show similar levels of cell death. Also shown is a lower panel of induction of apoptosis in BT474 cells after treatment for 3 days. The average number of annexin V-positive cells was determined after repeating 3-4 times, counted by Cell Profiler, and analyzed by Student's t-test. Compared to trastuzumab (TZB), the biparatopic construct 441 clearly induced more apoptosis. Constructs 441 and 6L1G show similar levels of apoptosis. Images of BT474 cells after treatment with the indicated agents for 3 days are shown. Alexa647 labeled trastuzumab (TZB), biparatopic construct 441 and biparatopic one-arm constructs oaLF and oaHF in 3 ml containing NaN3 (0.1%) and BSA (1%) with 3 million BT474 cells at a concentration of 100 nM. The results of incubation in PBS at 4 ° C. for 1 hour are shown. It is not noted that prior to binding, BT474 cells were pretreated with PBS containing 1% BSA with 0.1% NaN3 to block internalization. The cells were then analyzed by a CyFlow Space instrument (Partec). All binders exhibit specific binding to the surface of HER2-positive BT474 cells. The induction of cell death after treatment with the indicated agent of 100 nM is shown. Twenty-four hours prior to treatment, BT474, N87, HCC1419 and SKBR3 cells were inoculated into 96 black clearwell microscope plates (Nunc), treated continuously for 3 days and all cells stained with HOECHT-33342 (Invitrogen). , Membrane-permeable dead cells were stained with propidium iodide (Sigma). Cells were analyzed with a Lionheart FX automated microscope (BioTek Instruments) and the number of propidium iodide and HOECHT-33342 positive cells was quantified with Gen5 software (BioTek Instruments). The ratio of propidium iodide to HOECHST-3342 positive cells is calculated after 3 biological iterations and the mean and SD are shown in the corresponding bar graph. Compared to trastuzumab (TZB) or a combination of trastuzumab and pertuzumab (TZB + PZB), biparatopic binding agents (6L1G, 441, 841, LFoa, 241, 641, HFoa, 7C2LF) that bind to domains 1 and 4 of HER2 Persistently induce more dead cells in HER2-positive cancer cells. The half-life of construct 441 in the serum of NSG mice is shown. The collected sera of the mice previously injected with 441 (3 mg / kg) by the sandwich ELISA method were analyzed. 441 showed an alpha phase of about 4.3 hours, followed by a beta phase of 45 hours or more. Shows 441 in vivo activity against N87 xenografts in SCID beige mice. After the size of the N87 tumor reaches 150 mm3, mice are treated with 441 (10 mg / kg) injected 8 times every 4 weeks. Compared to untreated mice and mice treated with TZB (10 mg / kg) or huA21G (10 mg / kg), 441 significantly reduces tumor size. Trastuzumab (TZB), huA21G (A21), BT-474 cells after treatment with their combination or 441 for 2 hours, and untreated (with or without anti-human primary antibody) cells to be controlled. A typical microscope image is shown. Nuclei were stained with 2- (4-amidinophenyl) -1H-indole-6-carboxymidamide (DAPI), the antibody was detected with an anti-human Fc antibody derived from goat, and the lysosomal compartment was detected with an anti-LAMP1 antibody. Constructs 441 and 841, hA21G, trastuzumab (TZB), trastuzumab plus hA21G combination (TZB + hA21G), pertuzumab (PZB), trastuzumab plus pertuzumab combination (TZB + PZB), and HSP90 inhibitor geldanamycin (GA). The results of the treatment and subsequent surface protein internalization and degradation assays are shown.

Specific description of the invention

Terms and Definitions Unless otherwise defined, all technical and scientific terms used herein are in the art (eg, cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). It has the same meaning as what is normally recognized by those skilled in the art. Standard techniques include molecular, genetic, and biochemical methods (generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, ColdS. Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc.) and chemical methods.

The term "a subject includes an object" in the context of the present specification includes individual embodiments in which the subject comprises the subject, that is, the term "contains" is synonymous with "consists". In other individual embodiments, the subject is one of several different things contained in the subject.

The term ligand in the context of the present specification relates to a region of a target, particularly a polypeptide that binds to HER2.

The term interdomain amino acid linker in the context of the present specification relates to a polypeptide linker that covalently binds the C-terminus of the first polypeptide domain to the N-terminus of the second polypeptide domain.

The term epitope in the context of the present specification relates to the region of the antigen molecule to which the antibody binds.

The term Fab domain in the context of the present specification refers to a first strand consisting of a VL domain that is (covalently) linked to the CL domain at the C end and a VH domain that is (covalently) linked to the CH1 domain at the C end. It relates to an antibody molecule comprising a second chain consisting of a CL domain and a CH1 domain linked by a disulfide bond.

The term VL antigen binding domain in the context of the present specification relates to the variable domain of the light chain of an antibody, particularly the immunoglobulin G light chain.

The term VH antigen-binding domain in the context of the present specification relates to the variable domain of an antibody heavy chain, particularly an immunoglobulin G heavy chain.

The term CL constant domain in the context of the present specification relates to the constant domain of the light chain of an antibody, particularly the immunoglobulin G light chain.

The term CH1 constant domain in the context of the present specification relates to the first constant domain of antibody heavy chains, particularly immunoglobulin G heavy chains.

The term CH2 constant domain in the context of the present specification relates to a second constant domain of an antibody heavy chain, particularly an immunoglobulin G heavy chain.

The term CH3 constant domain in the context of the present specification relates to a third constant domain of antibody heavy chains, particularly immunoglobulin G heavy chains.

The term single-chain variable fragment in the context of the present specification is a VH antigen-binding domain (also referred to as scFv heavy chain) that covalently binds to a VL antigen-binding domain (also referred to as scFv light chain) by a polypeptide linker (scFv linker chain). Will be).

As used herein, the term positive, when used in the context of marker expression, refers to the expression of an antigen as measured by a fluorescently labeled antibody, which refers to fluorescence on a structure (eg, a cell) referred to as "positive" for the label. Have at least 30% higher median fluorescence intensity (≧ 30%), especially ≧ 50% or ≧ 80%, compared to staining with an isotype-matched fluorescently labeled antibody that is not specifically bound to the same target. Is. Such a representation of a marker is indicated by the superscript "plus" ( ⁺ ) following the marker's name, for example CD4 ⁺ .

As used herein, the term negative refers to the expression of an antigen as measured by a fluorescently labeled antibody when used in the context of marker expression, with the median fluorescence intensity being specifically bound to the same target. Not less than 30% higher than the median fluorescence intensity of isotype-matched antibodies, especially less than 15% higher. Such a representation of a marker is indicated by the superscript minus ( ^- ) following the marker's name, for example ^CD127- .

For high expression of markers, for example, high expression of CD25 is the expression level of the marker in a clearly distinguishable cell population detected by FACS compared to other populations characterized by lower fluorescence intensity per cell. , Refers to exhibiting the highest fluorescence intensity per cell. The high representation is indicated by the superscript "high" or "hi" following the name of the marker, eg CD25 ^high . The term "highly expressed" refers to the same characteristics.

Regarding the low expression of the marker, for example, the low expression of CD25 is the expression level of the marker in a clearly distinguishable cell population detected by FACS compared to other populations characterized by higher fluorescence intensity per cell. , Refers to exhibiting the lowest fluorescence intensity per cell. Low expression is indicated by the superscript "low" or "lo" following the name of the marker, eg CD25 ^low . The term "lowly expressed" refers to the same characteristics.

Marker expression can be measured by techniques such as fluorescence microscopy, flow cytometry, ELISA, ELISA or multiplex analysis.

The term polypeptide in the context of the present specification relates to a molecule consisting of 50 or more amino acids forming a linear chain in which amino acids are connected by peptide bonds. The amino acid sequence of a polypeptide can represent the amino acid sequence of an entire protein (physiologically discovered) or a fragment thereof. As used herein, the terms "polypeptide" and "protein" are used interchangeably and include proteins and fragments thereof. As used herein, polypeptides are disclosed as amino acid residue sequences.

The term peptide in the context of the present specification is a molecule consisting of up to 50 amino acids, particularly 8-30 amino acids, more particularly 8-15 amino acids, forming a linear chain in which the amino acids are connected by peptide bonds. Regarding.

The amino acid residue sequence is provided from the amino terminus to the carboxyl terminus. Uppercase letters in the sequence position refer to the L-amino acid of the one-letter code (Stryer, Biochemistry, 3rd ed. ^P.21 ). Lowercase letters in the amino acid sequence position refer to the corresponding D- or (2R) -amino acid. The sequence is written from left to right in the direction from the amino terminus to the carboxy terminus. According to the standard nomenclature, the amino acid residue sequences are alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine ( Gln, Q), glutamine (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met) , M), Phenylalanine (Phe, F), Proline (Pro, P), Serin (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Val. , V), named with a three-letter or one-letter code. J is leucine or isoleucine.

The term gene refers to a polynucleotide containing at least one open reading frame (ORF) capable of encoding a particular polypeptide or protein after being transcribed and translated. Polynucleotide sequences can be used to identify larger fragments or full-length coding sequences of related genes. Methods of isolating larger fragment sequences are known to those of skill in the art.

The term gene expression or gene product refers to the process and its products that produce nucleic acids (RNAs) or amino acids (eg, peptides or polypeptides) when a gene is transcribed and translated.

As used herein, expression refers to the process by which DNA is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells. Expression can be measured at the level of transcription and translation, ie in mRNA and / or protein products.

Sequences that are similar or homologous to or homologous to the sequences disclosed herein (eg, at least about 70% sequence identity) are also part of the invention. In some embodiments, at the amino acid level, sequence identity is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, It can be 99% or more. At the nucleic acid level, sequence identity is about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99. % Or more. Substantial identity also exists when the nucleic acid segment hybridizes to the complement of the strand under selective hybridization conditions (eg, highly stringent hybridization conditions). Nucleic acid may be present in whole cell, cytolytic, or partially purified or substantially pure form.

The calculation of "homology" or "sequence identity" or "similarity" between two sequences (these terms are used interchangeably herein) is performed as follows. Align sequences for optimal comparison (eg, gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment and are non-homologous. You can ignore the array). In a specific embodiment, the length of the reference sequence aligned for comparison is at least 30%, particularly at least 40%, more particularly at least 50%, even more particularly at least 60%, and even more of the length of the reference sequence. In particular, at least 70%, 80%, 90% and 100%. Subsequently, the amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If the position of the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecule is assumed to be identical at that position (as used herein). In addition, the "homogeneity" of an amino acid or nucleic acid corresponds to the "identity" of an amino acid or nucleic acid). The percent identity between the two sequences is the number of identical positions shared in the sequence, taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap. Is a function of. For circulating and related proteins, the sequence of one of the partners is appropriately divided into 2 to achieve the optimal alignment of functionally equivalent residues required to calculate the percent identity. Need to be aligned in one section.

In the context of the present specification, the terms sequence identity and percent of sequence identity refer to values determined by comparing two aligned sequences. Sequence alignment methods for comparison are known in the art. Sequence alignment for comparison is described in Smith and Waterman, 1981, Adv. Apple. Math. , 2,482 Local Homology Algorithm, Needleman and Wunsch, 1970, J. Mol. Mol. Biol. , 48, 443 Global Alignment Algorithm, Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. , 85, 2444, or computerized implementations of these algorithms, including, but not limited to, Clustal, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analysis is published, for example, by the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/).

One example for comparing amino acid sequences is the BLASTP algorithm using the following default settings: Expect threshold: 10, Word size: 3, Maximum match within query range (Max). matrix in a questionnaire: 0, Matrix: BLASTUM62, Gap Costs: Existence 11, Extension 1, Compositional adaptation: Conditional composition score matrix correction. compositional score matrix algorithm). One example for comparing nucleic acid sequences is the BLASTN algorithm with the following default settings: Expect threshold: 10, Word size: 28, Maximum match within query range (Max). Matches in a nucleic acid): 0, Match / Mismatch Scores: 1. -2, Gap Costs: Linear. Unless otherwise stated, the sequence identity values provided herein are a set of BLAST programs (Altschul et al., 1990, J. Mol. Biol, respectively) using the above default parameters for comparison of proteins and nucleic acids. ., 215, 403-410) refers to the value obtained.

In the context of this specification, the term antibody refers to all antibodies, immunoglobulin type G (IgG), type A (IgA), type D (IgD), type E (IgE) or type M (IgM), optional. Including, but not limited to, antigen-binding fragments thereof or single strands thereof, and related or derived constructs thereof. All antibodies are glycoproteins containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region ( _VH ) and a heavy chain constant region ( _CH ). The heavy chain constant region is composed of three domains, _CH 1, _CH 2 and _CH 3. Each light chain is composed of a light chain variable region (abbreviated herein as _VL ) and a light chain constant region ( _CL ). The light chain constant region is composed of one domain _CL . The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component of the classical complement system. Similarly, the term includes so-called Nanobodies or single domain antibodies, which are antibody fragments consisting of a single monomeric variable antibody domain.

In the context of the present specification, the term humanized antibody is first produced by a non-human species of immune cell and its protein sequence is modified to enhance similarity to naturally produced antibody variants in humans. Refers to an antibody. As used herein, the term humanized antibody includes an antibody in which a CDR sequence derived from a germline of another mammalian species, such as a mouse, is transplanted into a human framework sequence. The framework region may be separately modified within the human framework sequence and within the CDR sequence derived from the germline of another mammalian species.

The term antibody-like molecule in the context of the present specification refers to a molecule that has a high affinity (Kd ≤ 10E-8 mol / l) and can specifically bind to another molecule or target. The antibody-like molecule binds to its target in the same manner as the specific binding of the antibody. The term antibody-like molecule includes repeat proteins such as designed ankyrin repeat proteins (Molecular Partners, Zurich), modified antibody mimetic proteins that exhibit high specificity and high affinity target protein binding (US2012 / 142611, US2016 / 2503411,). US 2016/075767 and US 2015/368302, all of which are incorporated herein by reference). The term antibody-like molecule further includes, but is not limited to, polypeptides derived from armadillo repeat proteins, polypeptides derived from leucine-rich repeat proteins, and polypeptides derived from tetratricopeptide repeat proteins.

The term antibody-like molecule further includes specific binding polypeptides derived from:
・ Protein A domain,
・ Fibronectin domain FN3,
・ Consensus fibronectin domain,
Lipocalin (see Skera, 2000, Biochim. Biophyss. Acta, 1482 (1-2), 337-50),
Polypeptides derived from zinc finger proteins (see Kwan et al., 2003, Structure, 11 (7), 803-813),
Src homology domain 2 (SH2) or Src homology domain 3 (SH3),
・ PDZ domain,
・ Gamma crystallin,
・ Ubiquitin,
・ Cysteine knot polypeptide or notchton,
・ Cystatin,
・ Sac7d,
-Triple Helix Coiled Coil (also known as Alpha Body),
-Knitz domain or Knitz-type protease inhibitor, and-Carbohydrate binding module 32-2.

The term polypeptide derived from the protein A domain refers to a molecule that is a derivative of protein A and can specifically bind to the Fc and Fab regions of immunoglobulins.

The term armadillo repeat protein refers to a polypeptide containing at least one armadillo repeat characterized by a pair of alpha helices that form a hairpin structure.

The term humanized camel antibody in the context of the present specification consists only of heavy or heavy chain variable domains (VHH domains) so that its amino acid sequence enhances similarity to naturally produced antibodies in humans. A modified antibody that, when administered to humans, exhibits reduced immunogenicity. A common strategy for humanizing camel antibodies is described in Vincke et al. , 2009, J Biol Chem. , 284 (5), 3273-3284 and US2011 / 165621.

In the context of the present specification, the term fragment crystallizable (Fc) region is used in a known sense in the fields of cell biology and immunology, and the _CH 2 domain and _CH 3 are covalently linked by disulfide bonds. Refers to an antibody fraction containing two identical heavy chain fragments composed of domains.

The term specific binding in the context of the present invention refers to the properties of a ligand that binds to those targets with a given affinity and target specificity. The affinity of such a ligand is indicated by the dissociation constant of the ligand. A specifically reacting ligand has a dissociation constant of ^≤10-7 mol / L when bound to its target, but the dissociation constant is at least three orders of magnitude higher in interaction with the molecule and the molecule has a chemical composition. Overall similar to the target, but with a different three-dimensional structure.

In the context of the present specification, the term dissociation constant (KD) is used in a known sense in the fields of chemistry and physics, where a complex composed of [almost two] different components is reversible to its constituents. Refers to the equilibrium constant that measures the tendency to dissociate. The complex may be, for example, an antibody-antigen complex AbAg composed of antibody Ab and antigen Ag. _KD is expressed as a molar concentration [mol / l] and corresponds to the concentration of [Ab] when half of the binding site of [Ag] is occupied. That is, the concentration of unbound [Ab] is equal to the concentration of the [AbAg] complex. The dissociation constant can be calculated based on the following equation.

［Ａｂ］は抗体の濃度であり、［Ａｇ］は抗原の濃度であり、［ＡｂＡｇ］は、抗体抗原複合体の濃度である。

[Ab] is the concentration of the antibody, [Ag] is the concentration of the antigen, and [AbAg] is the concentration of the antibody-antigen complex.

In the context of the present specification, the terms dissociation rate (Koff, [1 / sec]) and binding rate (Kon, [1 / sec * M]) are used in a known sense in the fields of chemistry and physics. Refers to the rate constant that measures the dissociation (Koff) or association (Kon) between an antibody and its target antigen. Koff and Kon can be determined experimentally using methods already established in the art. Surface plasmon resonance is employed as a method for determining Koff and Kon of an antibody. This is a principle in biosensor systems such as the Biacore ™ or ProteOn ™ system. They are also used to determine the dissociation constant KD using the following equation.

本明細書で使用されるように、医薬組成物という用語は、本発明の化合物又はその医薬的に許容される塩、及び少なくとも１つの医薬的に許容される担体を指す。いくつかの実施形態において、本発明による医薬組成物は、局所、非経口又は注射可能な投与に適する形態で提供される。

As used herein, the term pharmaceutical composition refers to a compound of the invention or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition according to the invention is provided in a form suitable for topical, parenteral or injectable administration.

As used herein, the term pharmaceutically acceptable carrier is known to those of skill in the art, such as any solvent, dispersion medium, coating, surfactant, antioxidant, preservative (eg,). , Antibacterial agents, antifungal agents), isotonic agents, absorption retarders, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, etc. (See, for example, Remington: the Science and Practice of Pharmacy, ISBN 0857110624).

As used herein, the term treatment of any disease or disorder (eg, cancer), in one embodiment, ameliorate the disease or disorder (eg, at least one of the disease or its clinical manifestations). To delay, prevent, or reduce). In another embodiment, "treatment" refers to reducing or ameliorating at least one physical parameter, including one that may not be identifiable by the patient. In yet another embodiment, "treatment" is the regulation of physical (eg, identifiable symptom stabilization), physiological (eg, physical parameter stabilization) or both disorders or disorders. Point to. Unless otherwise specified below, methods for assessing the treatment and / or prevention of a disease are known in the art.

In the context of this specification, the term dimer refers to a unit consisting of two subunits.

In the context of the present specification, the term homodimer refers to a dimer composed of two subunits that are the same or are highly similar members of similar subunits.

In the context of the present specification, the term amino acid linker refers to a variable length polypeptide for connecting two polypeptides to produce a single-stranded polypeptide. Exemplary embodiments of linkers for carrying out the inventions detailed herein are oligopeptides consisting of 1, 2, 3, 4, 5, 10, 20, 30, 40 or 50 amino acids. It is a chain. A non-limiting example of an amino acid linker is the polypeptide GGGGSGGGGS (SEQ ID NO: 83).

A first aspect of the invention relates to tetrameric polypeptides.

According to the first alternative of the first aspect, the tetrameric polypeptide is:
A first polypeptide chain containing a first VL antigen binding domain and a first CL constant domain in the direction N to C,
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 in the direction from N to C.
A first ligand that specifically binds to the HER2 D4 epitope, is contained in the first polypeptide chain, and is linked to the N-terminus of the first VL antigen-binding domain by a first interdomain amino acid linker. Or with the first ligand, which is contained in the second polypeptide chain and is linked to the N-terminus of the first VH antigen binding domain by the first interdomain amino acid linker.
A second that specifically binds to the HER2 D1 epitope, which is composed of the first VL antigen-binding domain of the first polypeptide chain and the first VH antigen-binding domain of the second polypeptide chain together. With ligands, especially Fab domains,
A third polypeptide chain containing a second VL antigen binding domain and a second CL constant domain in the direction N to C,
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 in the direction from N to C.
A third ligand that specifically binds to the HER2 D4 epitope, is contained in the third polypeptide chain, and is linked to the N-terminus of the second VL antigen-binding domain by a second interdomain amino acid linker. Or with a third ligand that is contained in the fourth polypeptide chain and is linked to the N-terminus of the second VH antigen binding domain by a second interdomain amino acid linker.
A fourth that specifically binds to the HER2 D1 epitope, which is composed of the second VL antigen-binding domain of the third polypeptide chain and the second VH antigen-binding domain of the fourth polypeptide chain together. Contains or consists of ligands, particularly Fab domains.

According to the second alternative of the first aspect, the tetrameric polypeptide is:
A first polypeptide chain containing a first VL antigen binding domain and a first CL constant domain in the direction N to C,
-Contains a first VH antigen binding domain and a first CH1 constant domain in the direction from N to C, particularly further contains a first CH2 constant domain, and more particularly a first CH2 constant domain is deleted at its C-terminus. The second polypeptide chain and
A first ligand that specifically binds to the HER2 D4 epitope, is contained in the first polypeptide chain, and is linked to the N-terminus of the first VL antigen-binding domain by a first interdomain amino acid linker. Or with the first ligand, which is contained in the second polypeptide chain and is linked to the N-terminus of the first VH antigen binding domain by the first interdomain amino acid linker.
A second that specifically binds to the HER2 D1 epitope, which is composed of the first VL antigen-binding domain of the first polypeptide chain and the first VH antigen-binding domain of the second polypeptide chain together. With ligands, especially Fab domains,
A third polypeptide chain containing a second VL antigen binding domain and a second CL constant domain in the direction N to C,
-Contains a second VH antigen binding domain and a second CH1 constant domain in the direction from N to C, particularly further contains a second CH2 constant domain, and more particularly a second CH2 constant domain is deleted at its C-terminus. With the fourth polypeptide chain,
A third ligand that specifically binds to the HER2 D4 epitope, is contained in the third polypeptide chain, and is linked to the N-terminus of the second VL antigen-binding domain by a second interdomain amino acid linker. Or with a third ligand that is contained in the fourth polypeptide chain and is linked to the N-terminus of the second VH antigen binding domain by a second interdomain amino acid linker.
A fourth that specifically binds to the HER2 D1 epitope, which is composed of the second VL antigen-binding domain of the third polypeptide chain and the second VH antigen-binding domain of the fourth polypeptide chain together. Contains or consists of ligands, particularly Fab domains.

All of the following embodiments can be combined with both the first and second alternatives of the first aspect of the invention.

The VL antigen-binding domain and CL constant domain of the first and third polypeptide chains are the domains of the immunoglobulin G light chain, and the VH antigen-binding domain and CH1, CH2, CH3 constants of the second and fourth polypeptides. The domain is a domain of immunoglobulin G heavy chain. In other words, the first and third polypeptide chains each contain an immunoglobulin G light chain, and the second and fourth polypeptide chains each contain an immunoglobulin G heavy chain. The immunoglobulin light and heavy chains of the tetrameric polypeptide according to the invention form second and fourth ligands that specifically bind to the HER2 D1 epitope.

In addition, the polypeptide containing the first and third ligands that specifically bind to the HER2 D4 epitope is fused to the N-terminal of the immunoglobulin G heavy chain or immunoglobulin G light chain by an interdomain amino acid linker, thus 4 A tetramer polypeptide with one HER2 binding site is formed, of which two of the four HER2 binding sites bind to the D4 epitope and two bind to the D1 epitope.

Surprisingly, the tetrameric polypeptide according to the invention is superior to conventional antibodies and other antibody-like molecules such as divalent biparatopic polypeptides (which have two binding regions in total), HER2. It exhibits inactivation and is also effective for cell proliferation, apoptosis, HER2 internalization and HER2 degradation. Therefore, the tetrameric polypeptide according to the present invention is a promising candidate for the therapy of HER2-expressing cancer.

Without being bound by theory, the CH1, CH2, CH3 and CL domains, especially the CH2 and CH3 domains, are the further effects of the tetramer polypeptide of the invention, especially the inhibition of cell growth and proliferation, apoptosis and other forms. Induction of cell death, HER2 internalization, HER2 recycling inhibition and HER2 degradation, HER2 cross-linking, reduced HER2 surface mobility, HER2 expression downregulation, and HER2 dimer by positioning variable domains at specific angles and distances. It has been shown to contribute to the inhibition of cell formation and signal transduction.

In some embodiments, the first polypeptide chain and the third polypeptide chain have 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with each other. Most particularly, the first polypeptide chain and the third polypeptide chain are the same.

In some embodiments, the first polypeptide chain is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79 with the third polypeptide chain. %, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, It has 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, the second polypeptide chain and the fourth polypeptide chain have 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with each other. Most particularly, the second polypeptide chain and the fourth polypeptide chain are the same.

In some embodiments, the second polypeptide chain is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79 with the fourth polypeptide chain. %, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, It has 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, the immunoglobulin light chain of the first polypeptide chain and the immunoglobulin light chain of the third polypeptide chain are 70% or more, particularly 80% or more, more particularly 90% or more, more particularly particularly, of each other. It has 95% or more sequence identity, and most particularly, the immunoglobulin light chain of the first polypeptide chain and the immunoglobulin light chain of the third polypeptide chain are the same.

In some embodiments, the immunoglobulin light chain of the first polypeptide chain is 70%, 71%, 72%, 73%, 74%, 75%, with the immunoglobulin light chain of the third polypeptide chain. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, the immunoglobulin heavy chain of the second polypeptide chain and the immunoglobulin heavy chain of the fourth polypeptide chain are 70% or more, particularly 80% or more, more particularly 90% or more, more particularly particularly, of each other. It has 95% or more sequence identity, and most particularly, the immunoglobulin heavy chain of the second polypeptide chain and the immunoglobulin heavy chain of the fourth polypeptide chain are the same.

In some embodiments, the immunoglobulin heavy chain of the second polypeptide chain is 70%, 71%, 72%, 73%, 74%, 75%, with the immunoglobulin heavy chain of the fourth polypeptide chain. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, the first and third ligands have 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with each other, most particularly particularly. The first ligand and the third ligand are the same.

In some embodiments, the first ligand is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% with the third ligand. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 Has%, 98%, 99% or 100% sequence identity.

In other words, the first ligan is substantially the same as the third ligand.

In some embodiments, the second and fourth ligands have 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with each other, most particularly particularly. The second ligand and the fourth ligand are the same.

In some embodiments, the second ligand is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% with the fourth ligand. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 Has%, 98%, 99% or 100% sequence identity.

In other words, the second ligand is substantially the same as the fourth ligand.

In some embodiments, the first CH2 constant domain and the first CH3 constant domain of the second polypeptide chain are the second CH2 constant domain and the second CH3 constant domain of the fourth polypeptide chain. It interacts to form a tetrameric polypeptide. In particular, the CH2 and CH3 constant domains of the second and fourth polypeptide chains are dimerized. In particular, in combination with the interaction of the first and second polypeptide chains with the third and fourth polypeptide chains, especially by dimerization of the corresponding VL and VH antigen binding domains and CL and CH1 constant domains. , First, second, third and fourth polypeptides tetramers are formed. Therefore, in terms of multimer formation, the tetrameric polypeptide of the present invention resembles an antibody.

In some embodiments, the second polypeptide chain comprises a first hinge region between the first CH1 constant domain and the first CH2 constant domain, and the fourth polypeptide chain is a second. A second hinge region is included between the CH1 constant domain and the second CH2 constant domain, and the first and second hinge regions are particularly composed of at least one disulfide bond, more particularly the first disulfide bond and The second disulfide bond mediates complex formation between the second polypeptide chain and the fourth polypeptide chain to form a tetramer polypeptide. Complex formation of CH1 and CH2 constant domains can occur by the hinge region, i.e., by the formation of disulfide bonds between cysteine residues, as in antibodies.

In some embodiments, the CH2 constant domain and / or the CH3 constant domain of the second polypeptide chain is deleted at its C-terminus.

In some embodiments, the CH2 constant domain and / or the CH3 constant domain of the fourth polypeptide chain is deleted at its C-terminus.

In some embodiments, the first ligand comprises or consists of a single chain variable fragment polypeptide chain comprising a scFv heavy chain, a scFv linker chain and a scFv light chain. In some embodiments, the scFv heavy chain is the VH domain of 4D5 (particularly SEQ ID NO: 80) and the scFv light chain is the VL domain of 4D5 (particularly SEQ ID NO: 81).

In some embodiments, the single chain variable fragment polypeptide chain comprises a scFv heavy chain, a scFv linker chain and a scFv light chain in the direction N to C.

In some embodiments, the single chain variable fragment polypeptide chain comprises a scFv light chain, a scFv linker chain and a scFv heavy chain in the direction N to C.

In other words, the scFv heavy chain and the scFv light chain may be provided in any direction in the single chain variable fragment polypeptide chain.

In some embodiments, the third ligand comprises or consists of a single chain variable fragment polypeptide chain comprising a scFv heavy chain, a scFv linker chain and a scFv light chain in the direction N to C. In some embodiments, the scFv heavy chain is the VH domain of 4D5 (particularly SEQ ID NO: 80) and the scFv light chain is the VL domain of 4D5 (particularly SEQ ID NO: 81).

In some embodiments
The scFv heavy chain of the first ligand is a peptide sequence and 70 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. % More, especially 80% or more, more particularly 90% or more, and even more particularly 95% or more of peptide sequences having or consisting of sequence identity, most particularly the scFv heavy chain of the first ligand is a sequence. Contains or consists of the same peptide sequence as the peptide sequence selected from 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.
The scFv light chain of the first ligand is 70% or more, particularly 80% or more, with the 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. More particularly, the scFv light chain of the first ligand contains or consists of a peptide sequence having a sequence identity of 90% or more, even more particularly 95% or more, and the scFv light chain of the first ligand is SEQ ID NO: 14, SEQ ID NO: 24, sequence. Contains or consists of the same peptide sequence as the peptide sequence selected from No. 25, SEQ ID NO: 26, SEQ ID NO: 43 and SEQ ID NO: 81.

In some embodiments
The scFv heavy chain of the first ligand is a peptide sequence and 70 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. %, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, Peptide sequences with 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. Including or consisting of them
The scFv light chain of the first ligand is 70%, 71%, 72%, with the 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. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% containing or consisting of peptide sequences having sequence identity.

In some embodiments
The scFv heavy chain of the third ligand is a peptide sequence and 70 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. % More, especially 80% or more, more particularly 90% or more, and even more particularly 95% or more of peptide sequences having or consisting of sequence identity, most particularly the scFv heavy chain of the third ligand is a sequence. Contains or consists of the same peptide sequence as the peptide sequence selected from 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.
The scFv light chain of the third ligand is 70% or more, particularly 80% or more, with the 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. More particularly, the scFv light chain of the third ligand contains or consists of a peptide sequence having a sequence identity of 90% or more, more particularly 95% or more, and the scFv light chain of the third ligand is SEQ ID NO: 14, SEQ ID NO: 24, sequence. Contains or consists of the same peptide sequence as the peptide sequence selected from No. 25, SEQ ID NO: 26, SEQ ID NO: 43 and SEQ ID NO: 81.

In some embodiments, the scFv heavy chain of the third ligand is 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. 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% with selected peptide sequences , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Contains or consists of peptide sequences with identity
The scFv light chain of the third ligand is 70%, 71%, 72%, with the 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. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% containing or consisting of peptide sequences having sequence identity.

In some embodiments
The scFv heavy chain of the first ligand and the third ligand is 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, respectively. Contains or consists of a peptide sequence having 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with the peptide sequence to be expressed, most particularly the first ligand and The scFv heavy chain of the third ligand is identical to the 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, respectively. Contains or consists of the peptide sequences of
The scFv light chain of the first ligand and the third ligand is 70% or more of the 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, respectively. , In particular 80% or more, more particularly 90% or more, and even more particularly 95% or more, containing or consisting of peptide sequences having sequence identity, most particularly the scFv light chain of the first and third ligands. Contains or consists of the same peptide sequence as the 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, respectively.

In some embodiments
The scFv heavy chain of the first ligand and the third ligand is 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, respectively. 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identical Contains or consists of sex peptide sequences
The scFv light chain of the first ligand and the third ligand is 70% with the 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, respectively. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% containing peptide sequences with sequence identity? Or consists of them.

The scFv light chain and heavy chain may consist entirely of the peptide sequence defined above, or the scFv light chain and heavy chain may comprise the peptide sequence defined above, where the scFv light chain. And heavy chains may contain other peptide sequences.

In some embodiments, the scFv light chain of the first ligand comprises or consists of the VL antigen binding domain of antibody 4D5, and the scFv heavy chain of the first ligand contains the VH antigen binding domain of antibody 4D5. Includes or consists of it.

In some embodiments, the scFv light chain of the third ligand comprises or consists of the VL antigen binding domain of antibody 4D5, and the scFv heavy chain of the third ligand comprises the VH antigen binding domain of antibody 4D5. Includes or consists of it.

In the context of the present specification, the term 4D5 refers to the humanized monoclonal antibody trastuzumab, also known as Herceptin, and is also referred to herein as "TZB" for the membrane proximal domain IV of HER2 (Cho et al.,. 2003).

In some embodiments, the scFv linker chain comprises a peptide sequence having a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more with SEQ ID NO: 16, and most particularly particularly. The scFv linker chain comprises the same SEQ ID NO: 16 as the peptide sequence.

In some embodiments, the scFv linker chains are SEQ ID NO: 16 and 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81. %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Contains peptide sequences with 98%, 99% or 100% sequence identity.

In some embodiments
The first polypeptide chain is 70% or more of the 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. , Especially 80% or more, more particularly 90% or more, and even more particularly 95% or more, and the first polypeptide chain contains SEQ ID NO: 18, SEQ ID NO: 30, SEQ ID NO: 31. , The same peptide sequence as the peptide sequence selected from SEQ ID NO: 32, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 50 and SEQ ID NO: 76.
The second polypeptide chain is 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 NO: 77. The second polypeptide chain comprises a peptide sequence having a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more, and most particularly, the second polypeptide chain is 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 NO: 77.

In some embodiments
The first polypeptide chain is 70% with the 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. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% containing peptide sequences with sequence identity.
The second polypeptide chain is 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 NO: 77. 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identical Includes a sex peptide sequence.

In some embodiments
The third polypeptide chain is 70% or more of the 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. , Especially 80% or more, more particularly 90% or more, and even more particularly 95% or more, and the third polypeptide chain contains SEQ ID NO: 18, SEQ ID NO: 30, SEQ ID NO: 31. , The same peptide sequence as the peptide sequence selected from SEQ ID NO: 32, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 50 and SEQ ID NO: 76.
The fourth polypeptide chain is 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 NO: 77. The fourth polypeptide chain comprises a peptide sequence having a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more, and most particularly, the fourth polypeptide chain is 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 NO: 77.

In some embodiments
The third polypeptide chain is 70% with the 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. 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% containing peptide sequences with sequence identity.
The fourth polypeptide chain is 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 NO: 77. 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identical Includes a sex peptide sequence.

In some embodiments
The first polypeptide chain and the third polypeptide chain are 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, respectively. Contains a peptide sequence having 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with the peptide sequence to be expressed, most particularly the first polypeptide chain and the third poly. The peptide chain comprises the same peptide sequence as the 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, respectively.
The second polypeptide chain and the fourth polypeptide chain are 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, and sequences, respectively. It comprises a peptide sequence having 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with the peptide sequence selected from No. 52 and SEQ ID NO: 77, most particularly the second. The polypeptide chain and the fourth polypeptide chain are 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 NO, respectively. Contains the same peptide sequence as the peptide sequence selected from number 77.

In some embodiments
The first polypeptide chain and the third polypeptide chain are 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, respectively. 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identical Contains sexual peptide sequences
The second polypeptide and the fourth polypeptide chain are 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:, respectively. 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% with the peptide sequence selected from 52 and SEQ ID NO: 77. , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99. Includes peptide sequences with% or 100% sequence identity.

Therefore, the VL and VH antigen binding domains of the first, second, third and fourth polypeptide chains specifically bind to domain I of HER2 A21 (Hu S. et al., 2008, Proteins, 70). , 938-949) may be substantially identical to the VL and VH antigen binding domains of the scFv fragment.

In some embodiments
The first polypeptide chain has 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 78. The first polypeptide chain comprises the same peptide sequences as SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 78.
The second polypeptide chain has 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 79. The second polypeptide chain comprises the same peptide sequences as SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 79.

In some embodiments
The first polypeptide chain is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78 with SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 78. %, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, Contains peptide sequences with 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
The second polypeptide chain is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78 with SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 79. %, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, Includes peptide sequences with 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments
The third polypeptide chain has 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 78. The third polypeptide chain comprises the same peptide sequences as SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 78.
The fourth polypeptide chain has 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 79. The fourth polypeptide chain comprises the same peptide sequences as SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 79.

In some embodiments
The third polypeptide chain is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78 with SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 78. %, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, Contains peptide sequences with 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
The fourth polypeptide chain is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78 with SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 79. %, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, Includes peptide sequences with 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments
The first polypeptide chain and the third polypeptide chain are 70% or more, particularly 80% or more, more particularly 90% or more, and more particularly particularly with SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 78, respectively. It contains a peptide sequence having 95% or more sequence identity, and most particularly, the first polypeptide chain and the third polypeptide chain are identical to SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 78, respectively. Contains the peptide sequence of
The second polypeptide chain and the fourth polypeptide chain are 70% or more, particularly 80% or more, more particularly 90% or more, and more particularly particularly with SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 79, respectively. It contains a peptide sequence having 95% or more sequence identity, and most particularly, the second polypeptide chain and the fourth polypeptide chain are identical to SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 79, respectively. Contains the peptide sequence of.

In some embodiments
The first polypeptide chain and the third polypeptide chain are 70%, 71%, 72%, 73%, 74%, 75% with SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 78, respectively. , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% containing peptide sequences with sequence identity.
The second polypeptide chain and the fourth polypeptide chain are 70%, 71%, 72%, 73%, 74%, 75% with SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 79, respectively. , 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 Includes peptide sequences with %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

Thus, the immunoglobulin light chains and heavy chains of the first, second, third and fourth polypeptide chains are ErbB2 antibodies called 7C2 (US 7,371,376) that specifically bind to domain I of HER2. May include an IgG domain (VL, CL, VH, CH1, CH2 and / or CH3) that is substantially identical to the corresponding domain of.

In some specific embodiments, the tetramer polypeptide according to the invention is an immunoglobulin G-type antibody (particularly human or humanized) having essentially two identical heavy chains and two identical light chains. It is a monoclonal IgG antibody), and both the antigen-specific variable heavy chain and the light chain form ligands (second and fourth ligands) that specifically react with the D1 domain of Her2, and each light chain or each heavy chain is , N-terminally linked polypeptide, the polypeptide comprising a scFv polypeptide chain composed of a heavy chain linked by an scFv linker chain and a light chain variable region, and the scFv polypeptide chain from 1 to 1. It is linked to the N-terminal of an immunoglobulin heavy chain or light chain by an interdomain amino acid linker consisting of 20 amino acids.

In some embodiments
The first polypeptide chain has 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with SEQ ID NO: 1, most particularly the first polypeptide chain. Is the same as SEQ ID NO: 1
The second polypeptide chain has 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with SEQ ID NO: 2, most particularly the second polypeptide chain. Is the same as SEQ ID NO: 2.

In some embodiments
The first polypeptide chain is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82 with SEQ ID NO: 1. %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Has 99% or 100% sequence identity,
The second polypeptide chain is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82 with SEQ ID NO: 2. %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Has 99% or 100% sequence identity.

In some embodiments
The third polypeptide chain has 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with SEQ ID NO: 1, and most particularly, the third polypeptide chain. Is the same as SEQ ID NO: 1
The fourth polypeptide chain has 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with SEQ ID NO: 2, and most particularly, the fourth polypeptide chain. Is the same as SEQ ID NO: 2.

In some embodiments
The third polypeptide chain is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82 with SEQ ID NO: 1. %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Has 99% or 100% sequence identity,
The fourth polypeptide chain is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82 with SEQ ID NO: 2. %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Has 99% or 100% sequence identity.

In some embodiments
The first polypeptide chain and the third polypeptide chain have sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, and particularly 95% or more, respectively, with SEQ ID NO: 1 and most. In particular, the first polypeptide chain and the third polypeptide chain are identical to SEQ ID NO: 1.
The second polypeptide chain and the fourth polypeptide chain have sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, and particularly 95% or more, respectively, with SEQ ID NO: 2, and most of them. In particular, the second polypeptide chain and the fourth polypeptide chain are identical to SEQ ID NO: 2.

In some embodiments
The first and third polypeptide chains are SEQ ID NO: 1 and 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, respectively. , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96. %, 97%, 98%, 99% or 100% sequence identity,
The second and fourth polypeptide chains are SEQ ID NO: 2 and 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, respectively. , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96. Has%, 97%, 98%, 99% or 100% sequence identity.

The resulting tetrameric polypeptide is called "441" (due to identity). In this construct, the IgG light chain (of the first and third polypeptide chains) containing the VL antigen binding domain of antibody A21 is a scFv containing the VL and VH antigen binding domains of 4D5 (trastuzumab or Herceptin, HER2 D4 binder). It is fused to the fragment at the N-terminal and combined with an IgG heavy chain (second and fourth polypeptide chains) containing the VH antigen binding domain of antibody A21. The VL and VH antigen binding domains of A21 both constitute the HER2 D1 binder.

In some embodiments
The first polypeptide chain has 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with SEQ ID NO: 3, and most particularly, the first polypeptide chain. Is the same as SEQ ID NO: 3
The second polypeptide chain has 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with SEQ ID NO: 4, most particularly the second polypeptide chain. Is the same as SEQ ID NO: 4.

In some embodiments
The first polypeptide chain is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82 with SEQ ID NO: 3. %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Has 99% or 100% sequence identity,
The second polypeptide chain is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82 with SEQ ID NO: 4. %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Has 99% or 100% sequence identity.

In some embodiments
The third polypeptide chain has 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with SEQ ID NO: 3, and most particularly, the third polypeptide chain. Is the same as SEQ ID NO: 3
The fourth polypeptide chain has 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with SEQ ID NO: 4, and most particularly, the fourth polypeptide chain. Is the same as SEQ ID NO: 4.

In some embodiments
The third polypeptide chain is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82 with SEQ ID NO: 3. %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Has 99% or 100% sequence identity,
The fourth polypeptide chain is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82 with SEQ ID NO: 4. %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Has 99% or 100% sequence identity.

In some embodiments
The first polypeptide chain and the third polypeptide chain have sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, and particularly 95% or more, respectively, with SEQ ID NO: 3, respectively, and the most. In particular, the first polypeptide chain and the third polypeptide chain are identical to SEQ ID NO: 3.
The second polypeptide chain and the fourth polypeptide chain have sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, and particularly 95% or more, respectively, with SEQ ID NO: 4, and most In particular, the second polypeptide chain and the fourth polypeptide chain are identical to SEQ ID NO: 4.

In some embodiments
The first polypeptide chain and the third polypeptide chain are SEQ ID NO: 3 and 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, respectively. , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96. %, 97%, 98%, 99% or 100% sequence identity,
The second and fourth polypeptide chains are SEQ ID NO: 4 and 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, respectively. , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96. Has%, 97%, 98%, 99% or 100% sequence identity.

The resulting tetrameric polypeptide is called "47C2" (due to identity). In this construct, the IgG light chain (of the first and third polypeptide chains) containing the VL antigen binding domain of antibody 7C2 is scFv containing the VL and VH antigen binding domains of 4D5 (trastuzumab or Herceptin, HER2 D4 binder). It is fused to the fragment at the N-terminal and combined with an IgG heavy chain (second and fourth polypeptide chains) containing the VH antigen binding domain of antibody 7C2. The VL and VH antigen binding domains of 7C2 both constitute the HER2 D1 binder.

In some embodiments
The first polypeptide chain has 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with SEQ ID NO: 11, and most particularly, the first polypeptide chain. Is the same as SEQ ID NO: 11
The second polypeptide chain has 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with SEQ ID NO: 12, most particularly the second polypeptide chain. Is the same as SEQ ID NO: 12.

In some embodiments
The first polypeptide chain is SEQ ID NO: 11 and 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82. %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Has 99% or 100% sequence identity,
The second polypeptide chain is SEQ ID NO: 12 and 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82. %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Has 99% or 100% sequence identity.

In some embodiments
The third polypeptide chain has 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with SEQ ID NO: 11, and most particularly, the third polypeptide chain. Is the same as SEQ ID NO: 11
The fourth polypeptide chain has 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with SEQ ID NO: 12, and most particularly, the fourth polypeptide chain. Is the same as SEQ ID NO: 12.

In some embodiments
The third polypeptide chain is SEQ ID NO: 11 and 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82. %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Has 99% or 100% sequence identity,
The fourth polypeptide chain is SEQ ID NO: 12 and 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82. %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Has 99% or 100% sequence identity.

In some embodiments
The first polypeptide chain and the third polypeptide chain have sequence identities of 70% or more, particularly 80% or more, more particularly 90% or more, and particularly 95% or more, respectively, with SEQ ID NO: 11 and most. In particular, the first polypeptide chain and the third polypeptide chain are identical to SEQ ID NO: 11.
The second polypeptide chain and the fourth polypeptide chain have sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, and particularly 95% or more, respectively, with SEQ ID NO: 12, and most of all. In particular, the second polypeptide chain and the fourth polypeptide chain are identical to SEQ ID NO: 12.

In some embodiments
The first polypeptide chain and the third polypeptide chain are SEQ ID NO: 11 and 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, respectively. , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96. %, 97%, 98%, 99% or 100% sequence identity,
The second and fourth polypeptide chains are SEQ ID NO: 12 and 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, respectively. , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96. Has%, 97%, 98%, 99% or 100% sequence identity.

The resulting tetrameric polypeptide is called "241" (due to identity). In this construct, the IgG heavy chain (of the second and fourth polypeptide chains) containing the VH antigen binding domain of antibody A21 is a scFv containing the VL and VH antigen binding domains of 4D5 (trastuzumab or Herceptin, HER2 D4 binder). It is fused to the fragment at the N-terminal and combined with an IgG light chain (first and third polypeptide chains) containing the VL antigen binding domain of antibody A21. The VL and VH antigen binding domains of A21 both constitute the HER2 D1 binder.

In some embodiments, the VH antigen-binding domain is selected from the VH antigen-binding domain of A21 (particularly SEQ ID NO: 40, 42, 51, 52 or 77) and the VH antigen-binding domain of 7C2 (particularly SEQ ID NO: 79). , The VL antigen-binding domain is selected from the VL antigen-binding domain of A21 (particularly SEQ ID NO: 39, 41, 50 or 76) and the VL antigen-binding domain of 7C2 (particularly SEQ ID NO: 78).

In some embodiments, the first and third polypeptide chains are SEQ ID NO: 1, or at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%. Or identical to a functionally equivalent peptide sequence with 99% sequence identity, the second and fourth polypeptide chains are SEQ ID NO: 2, or at least 70%, 75%, 80%, 85%, It is identical to a functionally equivalent peptide sequence having 90%, 95%, 96%, 97%, 98% or 99% sequence identity (in the case of sequence identity, it is construct 441).

In some embodiments, the first and third polypeptide chains are SEQ ID NO: 3, or at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%. Or identical to a functionally equivalent peptide sequence with 99% sequence identity, the second and fourth polypeptide chains are SEQ ID NO: 4, or at least 70%, 75%, 80%, 85%, It is identical to a functionally equivalent peptide sequence having 90%, 95%, 96%, 97%, 98% or 99% sequence identity (in the case of sequence identity, it is construct 47C2).

In some embodiments, the first and third polypeptide chains are SEQ ID NO: 11, or at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%. Or identical to a functionally equivalent peptide sequence with 99% sequence identity, the second and fourth polypeptide chains are SEQ ID NO: 12, or at least 70%, 75%, 80%, 85%, It is identical to a functionally equivalent peptide sequence having 90%, 95%, 96%, 97%, 98% or 99% sequence identity (in the case of sequence identity, it is construct 241).

The interdomain amino acid linker is not limited in terms of amino acid composition, but in the specific embodiments envisioned herein, amino acids that are shown to contribute to the flexibility of the linker are selected. We have already shown that a linker consisting of G, S and / or T residues, such as repeats of (GG _m S) and (GG _m T), works, where m is selected from 1 to 3. The total length of the linker does not exceed 20, 25, and thus 30. It has been shown that a short interdomain linker of one or two amino acids works. The first and third polypeptides may contain the same interdomain amino acid linker or may contain different interdomain amino acid linkers.

In some embodiments, the interdomain amino acid linkers are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24 or 25 amino acids.

As can be seen from the results by the present inventors regarding the length and sequence composition of the interdomain amino acid linker, the equivalent length of up to 25 amino acids, which is not expected to interfere with the solubility of the obtained protein for structure prediction. It is expected that a linker with a protein will work.

In the polypeptide according to any one of the above embodiments, the interdomain amino acid linker comprises or comprises the amino acids G, A, J, S, T, P, C, V, M and E. In particular, the interdomain amino acid linker comprises or consists of the amino acids G, S, A and T.

In a specific embodiment, the interdomain amino acid linker is (GGΣ) _n , where n is an integer, n ≧ 4 (particularly n is 4, 5, 6, 7 or 8), where Σ is. It is selected from S and T.

In a specific embodiment, the interdomain amino acid linker is (GGS) _n , where n is an integer and n ≧ 4 (particularly n is 4, 5, 6, 7 or 8).

In a specific embodiment, the interdomain amino acid linker is (GGT) _n , where n is an integer and n ≧ 4 (particularly n is 4, 5, 6, 7 or 8).

In a specific embodiment, the interdomain amino acid linker is (GΣG) _n , where n is an integer, n ≧ 4 (particularly n is 4, 5, 6, 7 or 8), where Σ is. It is selected from S and T.

In a specific embodiment, the interdomain amino acid linker is (ΓΓΣ) _n , where n is an integer, n ≧ 4 (particularly n is 4, 5, 6, 7 or 8) and each Γ. Is selected from A, G and V independently of the other Γ, and Σ is selected from S and T.

Solubility and flexibility are important considerations when choosing a linker sequence. Those skilled in the art can be described by Chen et al. , 2013, Advanced Drag Delivery Reviews, 65, 1357-1369 and Evers et al. , 2006, Biochemistry, 45, 13183-13192, the composition and length of this sequence can be readily altered based on the teachings herein and the knowledge available for linker design.

In some embodiments, the interdomain amino acid linker is characterized by the amino acid sequence (GGGGS) _n , where n is 1, 2, 3, 4 or 5.

In some embodiments, the interdomain amino acid linker comprises or comprises a sequence characterized by one of SEQ ID NO: 17, SEQ ID NO: 55-SEQ ID NO: 69 and SEQ ID NO: 82-SEQ ID NO: 91. It is an array.

In some embodiments, the interdomain amino acid linker is a peptide sequence selected from one of SEQ ID NO: 17, SEQ ID NO: 55-SEQ ID NO: 69 and SEQ ID NO: 82-SEQ ID NO: 91, or at least 70% of the sequences. Contains or consists of functionally equivalent peptide sequences with identity.

A second aspect of the invention relates to an application (HER2-positive cancer) in a method of preventing or treating a malignant neoplastic disease associated with the expression of HER2 in a polypeptide according to the first aspect.

Similarly, a dosage form for preventing or treating a malignant neoplastic disease associated with HER2 expression, comprising the tetrameric polypeptide of the invention, is provided.

Those skilled in the art will appreciate that any drug specifically mentioned can be present as a pharmaceutically acceptable salt of said drug. Pharmaceutically acceptable salts include ionic drugs and counter-charged counterions. Non-limiting examples of pharmaceutically acceptable anionic salt forms are acetate, benzoate, besilate, hydrogen tartrate, bromide, carbonate, chloride, citrate, edetate, edicyl. Acids, embonates, estrates, fumarates, gluceptates, gluconates, hydrobromide, hydrochlorides, iodides, emulsions, lactobionates, malate, maleate , Mandelate, Mesilate, Methyl Bromide, Methyl Sulfate, Mucate, Napsylate, Nitrate, Pamoate, Phosphate, Diphosphate, Salicylate, Disalicylate, Includes stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminum, benzathine, calcium, ethylenediamine, lysine, magnesium, meglumine, potassium, prokine, sodium, tromethamine and zinc.

The dosage form may be used for enteral administration such as nasal, buccal, rectal, transdermal or oral administration, or may be inhaled or suppository. Alternatively, parenteral administration such as subcutaneous, intravenous, intrahepatic or intramuscular injection may be used. If necessary, pharmaceutically acceptable carriers and / or excipients may be present.

Topical administration is also within the advantageous use of the present invention. Benson and Watkinson, Topical and Transdermal Drug Delivery (Eds.): Principles and Practice (1st Edition, Wiley 2011, ISBN-13: 978-0470450291), and Guy and Handcraft: Transdermal Drug Delivery Systems : Revised and Expanded (2 nd Ed , CRC Press 2002, ISBN-13: ^{978-0824708610} ), Osborne and Amann (Eds.): Topical Drug Delivery Formations (1st Ed. CRC Press 789: 198; As such, a wide range of possible formulations for providing topical formulations are known to those of skill in the art.

The third aspect of the present invention is the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain of the tetrameric polypeptide according to the first aspect of the present invention. With respect to isolated nucleic acids encoding at least one of. In particular, the isolated nucleic acid may be included in the plasmid for expression in a bacterial or eukaryotic host cell. Nucleic acid sequences encoding the first, second, third and fourth polypeptides may be provided on the same plasmid or separate plasmids for co-expression on the same host.

The fourth aspect of the present invention is the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain of the tetrameric polypeptide according to the first aspect of the present invention. With respect to host cells compatible with at least one production of. In particular, the host cell is a bacterial cell or eukaryotic cell. More particularly, the host cell is a Chinese hamster ovary (CHO) cell.

In some embodiments, at least one of a first polypeptide chain, a second polypeptide chain, a third polypeptide chain and a fourth polypeptide chain of a polypeptide according to the first aspect of the invention. Includes the isolated nucleic acid according to the third aspect of the invention so that it can be produced. In particular, the first, second, third and fourth polypeptides may be co-produced in the same cell or may be produced separately and combined in vitro.

A fifth aspect of the present invention relates to a method for obtaining a polypeptide according to the first aspect of the present invention, the first polypeptide chain, the second polypeptide chain, the second polypeptide chain according to the first aspect of the present invention. Includes culturing host cells according to a fourth aspect of the invention to produce at least one of the 3 and 4 polypeptide chains.

Pharmaceutical Compositions and Administration Another aspect of the invention relates to pharmaceutical compositions comprising the tetrameric polypeptide of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In a further embodiment, the composition comprises at least two pharmaceutically acceptable carriers described herein.

In some embodiments of the invention, the tetrameric polypeptides of the invention are typically in pharmaceutical dosage form to provide a product that is easy to control the dosage of the drug and is classy and easy to handle to the patient. It is formulated.

In embodiments of the invention relating to topical use of the tetramer polypeptide of the invention, the pharmaceutical composition is topically administered, such as a sprayable formulation delivered by aqueous solution, suspension, ointment, cream, gel or aerosol. It is formulated in a suitable form and contains an active ingredient and one or more of a solubilizer, a stabilizer, a tonicity enhancing agent, a buffer and an antiseptic known to those skilled in the art.

The pharmaceutical composition can be formulated for oral, parenteral or rectal administration. Further, the pharmaceutical compositions of the present invention include, but are not limited to, solid forms (including, but not limited to, capsules, tablets, pills, granules, powders or suppositories) or liquid forms (solutions, suspensions or emulsions). (Not limited to these).

The dosing regimen of the compounds of the invention comprises the pharmacodynamic properties of a particular drug and its mode of administration and route, recipient species, age, gender, health status, physical illness and weight, nature and extent of symptoms, combination therapy. It depends on known factors such as type, frequency of treatment, route of administration, patient's renal and hepatic function, and desired effects. In some embodiments, the compounds of the invention may be administered in once-daily doses, or the total daily dose may be administered in divided doses of 2, 3, or 4 times daily. May be done.

The pharmaceutical compositions of the present invention can be subjected to conventional pharmaceutical processes such as sterilization and / or conventional inert diluents, smoothing agents, or buffers, and preservatives, stabilizers, wetting agents, emulsifying agents. And an adjuvant such as a buffer can be included. They may be manufactured by standard processes such as conventional mixing, granulation, thawing or lyophilization processes. Many procedures and methods for preparing pharmaceutical compositions are known in the art, eg, L. Lachman et al. See The Theory and Practice of Internal Practice, 4th Ed, 2013 (ISBN 8123922892).

The present invention relates to the following items, which are further clearly stated as claims.
Item 1: A first polypeptide chain comprising a first ligand, an interdomain amino acid linker and a first immunoglobulin domain that binds to the HER2 D4 epitope.
A second polypeptide chain comprising a second immunoglobulin domain, wherein the first immunoglobulin domain and the second immunoglobulin domain both constitute a second ligand, particularly the Fab domain, which binds to the HER2 D1 epitope. The second polypeptide chain and
A third polypeptide chain comprising a third ligand, an interdomain amino acid linker and a third immunoglobulin domain that binds to the HER2 D4 epitope.
A fourth polypeptide chain comprising a fourth immunoglobulin domain, wherein the third immunoglobulin domain and the fourth immunoglobulin domain both constitute a fourth ligand, particularly the Fab domain, which binds to the HER2 D1 epitope. A tetrameric polypeptide comprising or consisting of a fourth polypeptide chain.
Item 2: In the polypeptide according to item 1, the first immunoglobulin domain is substantially the same as the third immunoglobulin domain, and / or the second immunoglobulin domain is the second immunoglobulin domain. It is substantially the same as the immunoglobulin domain of 4.
Item 3: In the polypeptide according to item 1 or 2, the first ligand is substantially the same as the third ligand.
Item 4: In the polypeptide according to any one of the above items, the first ligand and / or the third ligand is a single chain variable fragment containing a scFv heavy chain, a scFv linker chain and a scFv light chain. Contains or consists of polypeptide chains.
Item 5: In the polypeptide according to item 4, the scFv heavy chain is the VH domain of 4D5, and the scFv light chain is the VL domain of 4D5.
Item 6: In the polypeptide according to any one of items 1 to 5,
-The first immunoglobulin domain is the VL domain, the second immunoglobulin domain is the VH domain, and / or the third immunoglobulin domain is the VL domain, the fourth immunoglobulin domain. Is a VH domain, or-the first immunoglobulin domain is a VH domain, the second immunoglobulin domain is a VL domain, and / or the third immunoglobulin domain is a VH domain. The fourth immunoglobulin domain is the VL domain.
Item 7: In the polypeptide according to item 6, the VH domain is linked to the CH1 domain at the C-terminus.
Item 8: In the polypeptide according to item 7, the CH1 domain is linked to the CH2 domain or the CH2 domain and the CH3 domain at the C-terminus.
Item 9: In the polypeptide according to any one of items 6 to 8, the VL domain is linked to the CL domain at the C-terminus.
Item 10: In the polypeptide according to any one of items 6 to 9, the VH domain is selected from the VH domain of A21 and the VH domain of 7C2, and the VL domain is the VL domain of A21 and the VL of 7C2. Selected from the domain.
Item 11: In the polypeptide according to any one of the above items, the interdomain amino acid linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 13. It consists of 14, 15, 16, 17, 18, 19 or 20 amino acids.
Item 12: In the polypeptide according to any one of the above items, the interdomain amino acid linker comprises or contains amino acids G, A, J, S, T, P, C, V, M and E. In particular, the interdomain amino acid linker comprises or consists of the amino acids G, S, A and T.
Item 13: In the polypeptide according to any one of the above items, the interdomain amino acid linker is characterized by the amino acid sequence (GGGGS) _n , where n is 1, 2, 3, 4 or 5.
Item 14: In the bispecific HER2 polypeptide according to any one of the above items, the interdomain amino acid linker is any of SEQ ID NO: 17, SEQ ID NO: 55 to SEQ ID NO: 69 and SEQ ID NO: 82 to SEQ ID NO: 91. Contains or is such a sequence characterized by one or the other.
Item 15: In the polypeptide according to any one of the above items, the interdomain amino acid linker is selected from one of SEQ ID NO: 17, SEQ ID NO: 55 to SEQ ID NO: 69 and SEQ ID NO: 82 to SEQ ID NO: 91. Contains or consists of functionally equivalent peptide sequences having at least 70% sequence identity.
Item 16: Application in a method for preventing or treating a malignant neoplastic disease related to the expression of Her2 of the polypeptide according to any one of the above items.

Alternatives to a single separable feature, such as an isotype protein or coding sequence, ligand type or medical indication, are laid out herein as "embodiments", but are disclosed herein. It is understood that such alternatives can be freely combined to form separate embodiments of the invention. Accordingly, alternative embodiments of any detectable label can be combined with alternative embodiments of any ligand, and these combinations are combined with any medical indication or diagnostic method described herein. be able to.

The present invention will be further described through the following examples and drawings to provide further embodiments and advantages. These examples are intended to illustrate the invention, but do not limit its scope.

Array list:

Example 1: Biparatopic Anti-HER2 Binder We have produced a biparatopic IgG derivative. Compared to other available biparatopic HER2 target antibodies such as antibody drug conjugates (ADCs) (Li et al., 2016) from Medimmune MEDI4276, these IgGs are drug-free (drug-free). Kast et al., Under preparation) As a "naked" binding protein, it exhibits very strong antitumor activity. Therefore, it is recognized that these novel biparatopic anti-HER2 IgGs combine the mechanism of action of trastuzumab and pertuzumab and the mechanism of action of small molecule kinase inhibitors on HER2 in a single molecule. Furthermore, while ADC can act on many healthy tissues with its toxin, biparatopic anti-HER2 IgG can only act on HER2-poisoned cells, so its potential off-target effects include T-DM1 and MEDI4276. It is expected to be much lower than the effect of the ADC fusion of. Therefore, a new therapeutic concentration range of combination therapy is pioneered. In addition, pan-ErbB inhibition by polymerization of the HER2 receptor may passively block compensatory activation of other receptor tyrosine kinases (RTKs). Byparatopic anti-HER2 binders are free lateral of HER2 receptors on the cell surface of HER2-amplified cancers without inducing complexes with signaling capabilities that may block the activation of other RTKs. Interfere with movement. As a result, the biparatopic anti-HER2 binder can exhibit a strong synergistic effect with the small molecule inhibitor, which ultimately induces the expression of compensatory RTKs that act to escape treatment. Tend to do. Therefore, in a wide range of HER2-amplified cancers, biparatopic anti-HER2 IgG has a very high potential to elicit a strong antitumor synergistic effect in combination with small molecule inhibitors. The potential for synergistic effects with small molecule inhibitors is superior to current monospecific antibodies or combinations of antibodies.

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

Data on the preparation and biological activity of biparatopic IgG constructs are shown in FIGS. 3-26.

Design of Experiments for Producing Biparatopic IgG in CHO Cells Vector Design A bicistronic plasmid containing two expression cassettes or two vector systems is constructed for co-transfection. Derivatives of the plasmid pYMex10 (Morphosys) are used in the bicistronic strategy. In the resulting plasmid construct, the coding sequence of the polypeptide chain of the multimer construct is regulated by the respective CMV promoter, respectively, and ends with a poly A tail signal obtained from, for example, bovine growth hormone or Simian virus 40 (FIG. 3 and FIG. See Figure 4). A vector derived from pcDNA3.1 (Thermo) is used for co-transfection. Here, each polypeptide chain is in a separate plasmid, allowing the molar ratio of the transfection plasmid to be adjusted to reduce the risk of recombination of homologous elements and further improve yield. The target gene can be exchanged by standard cloning technology. An example of the resulting structure is shown in FIG.

Expression in CHO-S In a TPP600 bioreactor, CHO-S cells (Thermo) that grow exponentially are inoculated into CHOgro (Mirrus) at a density of 4 million per ml. To 1 ml of culture, add 3 μg of linear polyethyleneimine (MW25,000, Polysciences Inc) and 1.25 μg of high-purity plasmid DNA while mixing. Finally, add valproic acid to the culture to a final concentration of 1 mM. In the Kuhner ISF1-X shaker, the protein is expressed at 31 ° C. or 37 ° C., 8% CO2, 180 rpm, and 50 mm amplitude for as long as 12 days.

Molecule Purification Expression cultures are obtained by centrifugation at 1400 rpm and 4 ° C. for 30 minutes. The supernatant is further clarified by filtration to 0.22 μm and the pH is adjusted to 7. All subsequent purification steps are performed at 4 ° C. The supernatant is poured into a PBS-equilibrium rProtein A column (GE) operated by the AEKTA Pure system. After washing with PBS, the bound protein is eluted with 0.1 M glycine (pH 2.75) (chromogram in FIG. 6). Combine the target fractions and replace the buffer with 20 mM Bis / Tris methane, 20 mM NaCl (pH 6.75). The filtered protein solution is loaded onto a Resource S (GE) column and eluted with a gradient to 100% 20 mM Bis / Tris methane, 1000 mM NaCl (pH 6.75) (see Figure 7). The desired fractions are combined and, if necessary, finished with SEC (Appropriately sized Superdex 200 column, GE) to obtain high purity (FIG. 8). The monomeric fractions are combined, filtered and analyzed for purity by SDSPAGE (FIG. 9). Finally, pure proteins are used in biochemistry and cell assays, as well as in vivo studies.

HER2-related effects of tetravalent biparatopic IgG Tested with tetrameric (tetravalent and biparatopic) polypeptide constructs 441 and 47C2 in various assays known to those of skill in the art, and tetravalent IgG fusion MEDI4276 ( Dimeric divalent biparatopic polypeptide construct 841, 87C2 and their Fc fusion, dimeric biparatopic DARPin construct 6L1G (see patent application WO2014 / 060365A1), single IgG (TZB, Compare with PZB, A21 and 7C2) and combinations thereof.

Growth inhibition is tested with an XTT cell viability assay using live cell high-content microscopy, Hoechst staining and cell counting. As shown in Table 1, the Fc fusions of constructs 441, 47C2, 841, 87C2, and 841 and 87C2, 6L1G, and the combination of TZB and A21 completely inhibit growth, but with a single antibody and TZB and PZB. Partially inhibits by the combination of. Surprisingly, in the absence of cytotoxic agents, the antibody MEDI4276 stimulates the growth of XTT cells.

The effect of constructs on apoptosis / cell death by detecting live cell high-content microscopy with Annexin V and PI staining, or by detecting divided PARPs in cell lysates by Western blot to analyze PARP division. analyse.

Constructs 441, 47C2, 841, 87C2 and their Fc fusion, 6L1G, affect apoptosis, MEDI4276, a single antibody, do not affect apoptosis, and TZB and A21 partially. The combination of TZB + PZB can induce apoptosis in very few cells or fragile cell lines.

HER2 cross-linking on the cell surface, also called "lockdown", is measured by fluorescence recovery (FRAP) after photobleaching and single cell localized microscopy. A decrease in FRAP signal indicates low cell mobility. Therefore, it crosslinks in response to the polypeptide construct.

441, 47C2 and 6L1G lead to receptor lockdown and measure some cross-linking effects on 841, 87C2 and their Fc fusions, as well as the TZB + PZB and TZB + A21 antibody combinations. Single antibodies have no effect on cross-linking.

Further, as described in detail in Example 2, the internalization of HER2 into cells is analyzed by surface protein internalization and degradation assays, confocal microscopy and flow cytometry.

The tetravalent biparatopic constructs 441, 47C2 and MEDI4276 showed a strong effect on HER2 internalization, but the combination of TZB + PZB and TZB + A21 detected recycling inhibition. Other constructs had no effect.

HER2 degradation is tested by total HER2 surface protein internalization and degradation assay and Western blot detection. Here, 441 and 47C2 cause rapid and strong decomposition. MEDI4276 is effective against decomposition, but not as strong as 441 and 47C2. In contrast, antibody combinations lead to slow degradation and other constructs are ineffective.

Table 2 shows the results of HER2 binding studies performed using the same constructs as the above experiments. If flow cytometry further forms a complex of biparatopic IgG constructs with HER2, binding is determined by size exclusion chromatography / multiangle light scattering (SEC-MALS).

All constructs bound to extracellular domains 1, 2, and / or 4 as expected (see Table 2). The combination of 441, 47C2 and TZB + A21 exhibits extremely slow dissociation rates and is slower than other constructs.

Interestingly, all biparatopic constructs result in near 1: 1 HER2-bonded stoichiometry. The combination of a single antibody and an antibody results in only approximately 1: 2 stoichiometry.

In addition, the serum half-life of construct 441 is tested by intravenous injection into NSG mice and time-resolved detection of biparatopic IgG in blood samples by ELISA. To determine the half-life of 441, 3 mg / kg purified constructs are injected intravenously into NSG mice. At the indicated time points, serum is obtained by drawing blood from mice, coagulating whole blood, and collecting the supernatant of the centrifuged sample. Serum levels of 441 are assessed using standard capture ELISA (FIG. 32). Anti-human Fc antibody from mice is applied directly to a maxisorb plate. Standards for binding 441 and serum spike 441 are revealed by anti-κ chain antibody from goats conjugated to alkaline phosphatase. As a result of fitting the data by the two-phase decay model, the obtained half-life was calculated to be 4.3 + 45.3 hours (α phase and β phase).

Surprisingly, the tetravalent tetravalent biparatopic constructs 441 and 47C2 have excellent binding properties to HER2, also strongly inhibit cell proliferation, cause cell death by apoptosis, and HER2 on the cell surface. It causes cross-linking, causing strong HER2 internalization and strong HER2 degradation. 441 and 47C2 are superior to any of the other constructs in all categories or have comparable scores.

TZB + PZB and TZB + A21 lead to a decrease in total HER2 (very weak in the case of TZB + PZB) due to recycling inhibition, a mechanism by which HER2 degrades without prior intracellular accumulation (FIG. 35). reference).

To determine the effect of construct 441 on tumor growth, the right flank of SCID Beige (Charles River) mice is inoculated with 5 million N87 cells in 50% Corning. After the tumor reaches about 150 mm ³ , the mice are treated 8 times with 10 mg / kg 441 at intervals of 3-4 days. The treated mice have a reduced tumor mass in response to 441. Growth arrest was first seen in mice treated with TZB (10 mg / kg) and hA21G (10 mg / kg), and tumors in control mice (labeled "PBS" in FIG. 33) were undisturbed. It was shown that it will continue to develop into (Fig. 33).

Example 2: Internalization, lysosome transport and degradation of HER2 reinforced by disclosed molecule 441 Microscopic examination with BT-474 and ^HCC1419 breast cancer cells To microscopically examine fixed samples, 4.10. Inoculate μslides (Ibidi, cat.no. 80824) in complete medium at a density of cm ^-2 . The next day, treat the cells with the corresponding molecule. After 2 hours, cells are washed once with Dulbeccoline phosphate buffered saline (DPBS), 4% (w / v) paraformaldehyde lysed in DPBS is added, and the cells are fixed by incubating at room temperature for 10 minutes. .. Next, PBSBA + T (1% (w / v) bovine serum albumin (BSA), 0.1% (w / v) sodium azide, and 0.5% (w / v) Tween-20 were added. Wash the cells twice with DPBS). Then, the anti-LAMP antibody was dissolved in PBSBA + T at 1: 150 (v / v), and 100 ng ml ^-1 2- (4-amidinophenyl) -1H-indole-6-carboxymidamide (DAPI) was added. In Cell Signaling Technology, cat.no. D401S), cells are incubated for 30 minutes at room temperature. The cells were then washed twice with PBSBA + T, followed by an anti-mouse antibody conjugated to Alexa Fluor 488 (Thermo Fisher Scientific, cat.no. A11001), and Alexa Fluor 647 (Thermo Fisher Scientific, cat. Goat-derived anti-human antibody conjugated to 21445) and dissolved in PBSBA + T is added and incubated at room temperature for 30 minutes. The cells were then washed twice with PBSBA + T, re-fixed with 4% (w / v) paraformaldehyde lysed in DPBS, incubated for 10 minutes at room temperature, and finally washed once with PBSA. Store in PBSA (DPBS with 0.1% (w / v) sodium azide added) at 4 ° C. before measurement. Images are formed on the SP5 confocal laser scanning microscope (Leica). The images show that for the two cell lines, only 441 can induce its rapid internalization and exhibits strong co-localization with the lysosomal (LAMP1-positive) compartment.

Surface Protein Internalization and Degradation Assays Quantitative surface protein internalization and degradation assays were performed to quantify HER2 internalization and degradation during treatment. Briefly, based on the manufacturer's instructions, a stable Flp-In TREX HEK293 cell line (Thermo Fisher Scientific, cat.no. K650001) is generated and the HaloTag-HER2 receptor fusion is overexpressed at the time of induction. be able to. For the assay, cells are inoculated 2 days prior to the initial treatment and doxycycline is added 1 day prior to the initial treatment to induce stable overexpression for 24 hours. The treated product (100 nM) is added at the indicated time point with reference to the time of cell labeling.

After a treatment time interval, the cells are labeled in a two-step manner. In the first labeling step, a HaloTag ligand containing Alexa Fluor 660 (HTL-AF660, Promega, cat.no. G8472) was bound, and since the ligand is completely cell impermeable, only the surface receptor was used. Stain. In the second step, a cell-permeable HaloTag ligand containing tetramethylrhodamine (HTL-TMR, Promega, cat.no. G8252) is used to stain all receptor fusions present in the intracellular compartment. Thereby, signals from the surface and internal receptors are detected in separate channels of the flow cytometer. Therefore, information on localization can be obtained and information on quantitative distribution can be obtained by the rescaling procedure described below. Since all receptors appear on the surface, commercially available dead cell stains are used to exclude permeable (dead) cells from the analysis. LSR II Fortessa (BD) is used to record the fluorescence intensity of each channel of 2,000 to 10,000 cells and gate a single impermeable cell. The average fluorescence intensity of these populations is obtained using FlowJo 10.4 (FlowJo).

Data processing To compensate for the different detection efficiencies in the AF660 and TMR channels of the flow cytometry instrument, the data is scaled to calculate the relative abundance. Therefore, a control sample (utr., S.) Is required in which the first (HTL-AF660 label) step is omitted. In this sample, all HaloTag molecules, regardless of their localization, react with HTL-TMR in this "single" (s.) Labeling procedure. Normalization of the TMR channel of the sample by normalization of a single-labeled untreated control sample (utr., S.) and background removal using the average fluorescence intensity (MFI) of the singlet impermeable cell population. (Characteristic scaling) Obtain the signal _STMR .

However, at tr. , Unlab. Represents an untreated, unlabeled control (showing only autofluorescence).

The first surface labeling step with the cell impermeable dye is substantially sufficient with a complete two-step (double) labeling procedure. Therefore, the normalized surface signal S _AF660 can be defined as follows.

Sample signals could be associated with surface signals from double-labeled controls (utr., D.) and could not be stained because HTL-AF660 does not have access to internal receptors, and separate single stained samples Not needed.

ΔS _TMR is the difference between a single labeling procedure and a double labeling procedure on the TMR channel and corresponds exactly to the number of molecules blocked in the first surface-specific step. The signal on the AF660 channel of the same double labeling experiment is also directly related to this number of molecules.

Therefore, a correction factor _{CA can be defined, and the intensity S AF660 (} utr., D.) (recorded on the AF660 channel) measured by the correction factor is S _{AF660, scaling} (utr., D.) ( (At the scale of the TMR channel).

Using signals from single-labeled and double _- labeled untreated cells, CA can be calculated as follows.

Given that the TMR signal of the single-labeled untreated cells was previously scaled to 100% by equation S1, for the treated sample, the correction of the signal recorded on the AF660 channel is performed as follows: be able to.

Subsequently, _STMR (tre., D.) And S _{AF660, scaling} (tre., D.) Represent the abundance of internal protein and surface protein, respectively, and S _{AF660, scaling} (tre., D.) + S. The sum of _TMRs (tre., D.) Represents the total amount of protein in the double-labeled treated sample and is always relative to the untreated control sample.

The data scaling described above is performed and the results are plotted by MATLAB R2017b (MathWorks), R3.5.1, Prism6.07 (GraphPad) and Excel2016 (Microsoft). The results are shown in FIG. Compared to all other constructs, construct 441 showed almost internalization of HER2 within 5 minutes.

Claims (15)

(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 the HER2 D4 epitope, which is contained in the first polypeptide chain and is contained in the first interdomain amino acid linker to form the first VL antigen-binding domain. With a first ligand linked to the N-terminus or contained in the second polypeptide chain and linked to the N-terminus of the first VH antigen binding domain by a first interdomain amino acid linker. ,
(D) A third that specifically binds to the HER2 D1 epitope, which is composed of the first VL antigen-binding domain of the first polypeptide chain and the first VH antigen-binding domain of the second polypeptide chain together. Two ligands, especially the Fab domain,
(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 the HER2 D4 epitope, which is contained in the third polypeptide chain and is contained in the second interdomain amino acid linker to the second VL antigen-binding domain. With a third ligand linked to the N-terminus or contained in the fourth polypeptide chain and linked to the N-terminus of the second VH antigen binding domain by a second interdomain amino acid linker. ,
(H) A second HER2 D1 epitope that specifically binds to the HER2 D1 epitope, which is composed of the second VL antigen-binding domain of the third polypeptide chain and the second VH antigen-binding domain of the fourth polypeptide chain together. With 4 ligands, especially the Fab domain,
Tetrameric polypeptides comprising or consisting of. The first polypeptide chain and the third polypeptide chain, and / or the second polypeptide chain and the fourth polypeptide chain are 70% or more, particularly 80% or more, more particularly 90% of each other. These are further characterized by 95% or more sequence identity, most particularly the first polypeptide chain and the third polypeptide chain, and / or the second polypeptide chain and the fourth poly. The peptide chains are identical and / or the first and third ligands are 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more sequence identity with each other. The polypeptide of claim 1, characterized, most particularly the said first ligand and said third ligand are identical. The first CH2 constant domain and the first CH3 constant domain of the second polypeptide chain interact with the second CH2 constant domain and the second CH3 constant domain of the fourth polypeptide chain. The tetrameric polypeptide according to claim 1 or 2, wherein the tetrameric polypeptide is formed by acting and particularly covalently binding. The second polypeptide chain comprises a first hinge region between the first CH1 constant domain and the first CH2 constant domain, and the fourth polypeptide chain is the second CH1 constant. A second hinge region is included between the domain and the second CH2 constant domain, and the first hinge region and the second hinge region are particularly formed by at least one disulfide bond to form the second polypeptide chain. The tetrameric polypeptide according to any one of claims 1 to 3, wherein a tetrameric polypeptide is formed by mediating complex formation between the above-mentioned fourth polypeptide chain and the above-mentioned fourth polypeptide chain. The first ligand and / or the third ligand comprises or consists of a single chain variable fragment polypeptide chain comprising a scFv heavy chain, a scFv linker chain and a scFv light chain, in particular.
(A) The scFv heavy chain of the first ligand and / or the third ligand is 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 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more of the peptide sequence characterized by sequence identity with the peptide sequence selected from SEQ ID NO: 80, most particularly the first. And / or the scFv heavy chain of the third ligand is 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. Contains the same peptide sequence as the peptide sequence to be
(B) The scFv light chain of the first ligand and / or the third ligand is 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. It comprises a peptide sequence characterized by sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more with the peptide sequence, most particularly the first ligand and / or the third. The scFv light chain of the ligand of the above comprises the same peptide sequence as the 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.
The polypeptide according to any one of claims 1 to 4. The scFv linker chain comprises a peptide sequence characterized by sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, and even more particularly 95% or more with SEQ ID NO: 16, most particularly the scFv linker chain. The polypeptide according to claim 5, wherein the polypeptide comprises the same peptide sequence as SEQ ID NO: 16. (A) The first polypeptide chain and / or the third polypeptide chain comprises 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 It comprises 70% or more, in particular 80% or more, more particularly 90% or more, and even more particularly 95% or more, a peptide sequence characterized by sequence identity with the peptide sequence selected from SEQ ID NO: 76, most particularly the first. The polypeptide chain and / or the third polypeptide chain is 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. Contains the same peptide sequence as the peptide sequence
(B) The second polypeptide chain and / or the fourth polypeptide chain is 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, Includes a peptide sequence selected from SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 77 and a peptide sequence characterized by sequence identity of 70% or more, especially 80% or more, more particularly 90% or more, and even more particularly 95% or more. Most particularly, the second polypeptide chain and / or the fourth polypeptide chain is 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. , Containing the same peptide sequence as the peptide sequence selected from SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 77.
The polypeptide according to any one of claims 1 to 6. (A) The first polypeptide chain and / or the third polypeptide chain is 70% or more, particularly 80% or more, more particularly 90 with SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 78. % And more, and particularly more than 95%, comprising a peptide sequence characterized by sequence identity, most particularly the first polypeptide chain and / or the third polypeptide chain being SEQ ID NO: 36, SEQ ID NO: 37. , Containing the same peptide sequences as SEQ ID NO: 38 and SEQ ID NO: 78.
(B) The second polypeptide chain and / or the fourth polypeptide chain is 70% or more, particularly 80% or more, more particularly 90 with SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 79. % And more, and particularly more than 95%, comprising a peptide sequence characterized by sequence identity, most particularly the second polypeptide chain and / or the fourth polypeptide chain being SEQ ID NO: 33, SEQ ID NO: 34. , Containing the same peptide sequences as SEQ ID NO: 35 and SEQ ID NO: 79,
The polypeptide according to any one of claims 1 to 7. (A) The sequence identity of the first polypeptide chain and / or the third polypeptide chain is 70% or more, particularly 80% or more, more particularly 90% or more, and further particularly 95% or more with SEQ ID NO: 1. Characterized by, most particularly, said first polypeptide chain and / or said third polypeptide chain is identical to SEQ ID NO: 1.
(B) The second polypeptide chain and / or the fourth polypeptide chain has a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, and further particularly 95% or more with SEQ ID NO: 2. Characterized by, most particularly, said second polypeptide chain and / or said fourth polypeptide chain is identical to SEQ ID NO: 2.
The polypeptide according to any one of claims 1 to 8. (A) The sequence identity of the first polypeptide chain and / or the third polypeptide chain is 70% or more, particularly 80% or more, more particularly 90% or more, and further particularly 95% or more with SEQ ID NO: 3. Characterized by, most particularly, said first polypeptide chain and / or said third polypeptide chain is identical to SEQ ID NO: 3.
(B) The second polypeptide chain and / or the fourth polypeptide chain has a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, and further particularly 95% or more with SEQ ID NO: 4. Characterized by, most particularly, said second polypeptide chain and / or said fourth polypeptide chain is identical to SEQ ID NO: 4.
The polypeptide according to any one of claims 1 to 9. The first interdomain amino acid linker and / or the second interdomain amino acid linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19 or 20 amino acids, the first interdomain amino acid linker and / or the second interdomain amino acid linker comprises amino acids G, A, J, S, T, P. Whether the first interdomain amino acid linker and / or the second interdomain amino acid linker comprises or comprises C, V, M and E, in particular the amino acids G, S, A and T. , Or, in particular, the first interdomain amino acid linker and / or the second interdomain amino acid linker is characterized by the amino acid sequence (GGGGS) n, where n is 1, 2, 3, 4 Or 5, or the first interdomain amino acid linker and / or the second interdomain amino acid linker is any one of SEQ ID NO: 17, SEQ ID NO: 55 to SEQ ID NO: 69 and SEQ ID NO: 82 to SEQ ID NO: 91. The polypeptide according to any one of claims 1 to 10, comprising or consisting of a peptide sequence selected from one or a functionally equivalent peptide sequence characterized by at least 70% sequence identity. .. The polypeptide according to any one of claims 1 to 11, for use in a method for preventing or treating a malignant neoplastic disease associated with the expression of HER2. It encodes at least one of a first polypeptide chain, a second polypeptide chain, a third polypeptide chain and a fourth polypeptide chain of the polypeptide according to any one of claims 1 to 11. , Isolated nucleic acid. Suitable for the production of 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 claims 1 to 11. 1st polypeptide chain, 2nd polypeptide chain, 3rd polypeptide chain and 4th polypeptide of the polypeptide according to any one of claims 1 to 11. A host cell comprising the isolated nucleic acid of claim 13 so that at least one of the peptide chains can be produced. The method for obtaining the polypeptide according to any one of claims 1 to 11, wherein the first polypeptide chain and the second poly of the polypeptide according to any one of claims 1 to 11 are obtained. A method comprising culturing a host cell according to claim 14 to produce at least one of a peptide chain, a third polypeptide chain and a fourth polypeptide chain.

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