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BR-112018010537-B1 - A polypeptide that specifically binds to LRP5 and LRP6, its manufacturing method, expression vector, host cell, pharmaceutical composition, and uses.

BR112018010537B1BR 112018010537 B1BR112018010537 B1BR 112018010537B1BR-112018010537-B1

Abstract

BIPARATOPIC POLYPEPTIDES ANTAGONIZING WNT SIGNALING IN TUMOR CELLS. The present invention relates to novel biparatopic LRP5/LRP6 cross-linking polypeptides, and more specifically novel single-domain variable-linking biparatopic LRP5/LRP6 immunoglobulin cross-linking constructs that can inhibit the Wnt signaling pathway. The invention also relates to specific sequences of such polypeptides, methods of their production, and methods of using them, including methods for treating diseases such as cancer.

Inventors

  • Vittoria ZINZALLA
  • Klaus-Peter Kuenkele
  • Marie-Ange Buyse
  • Karen CROMIE
  • Stephanie Staelens
  • Beatrijs STRUBBE

Assignees

  • Ablynx N.V

Dates

Publication Date
20260317
Application Date
20161202
Priority Date
20151204

Claims (18)

  1. 1. Polypeptide that specifically binds LRP5 and LRP6, characterized in that it comprises: a first single variable immunoglobulin domain selected from the group of single variable immunoglobulin domains (i) to (iii) defined by comprising the following CDR sequences: (i): CDR1: TYTVG (= SEQ ID NO:1) CDR2: AIRRRGSSTYYADSVKG (= SEQ ID NO:2) CDR3: DTRTVALLQYRYDY (= SEQ ID NO:3) (ii): CDR1: SYAMG (= SEQ ID NO:4) CDR2: AIRRSGRRTYYADSVKG (= SEQ ID NO:5) CDR3: ARRVRSSTRYNTGTWWWEY (= SEQ ID NO:6) (iii): CDR1: RYTMG (= SEQ ID NO:7) CDR2: AIVRSGGSTYYADSVKG (= SEQ ID NO:8) CDR3: DRRGRGENYILLYSSGRYEY (= SEQ ID NO:9), and a second single variable immunoglobulin domain selected from the group of single variable immunoglobulin domains (iv) and (v) defined by comprising the following CDR sequences: (iv):CDR1: SYAMG (= SEQ ID NO:10)CDR2: AISWSGGSTYYADSVKG (= SEQ ID NO:11)CDR3: SPIPYGSLLRRRNNYDY (= SEQ ID NO:12)(v):CDR1: SYAMG (= SEQ ID NO:13)CDR2: AISWRSGSTYYADSVKG (= SEQ ID NO:14)CDR3: DPRGYGVAYVSAYYEY (= SEQ ID NO:15).
  2. 2. Polypeptide, according to claim 1, characterized in that said first single variable immunoglobulin domain comprises the following CDR sequences: CDR1: TYTVG (= SEQ ID NO:1) CDR2: AIRRRGSSTYYADSVKG (= SEQ ID NO:2) CDR3: DTRTVALLQYRYDY (= SEQ ID NO:3) and in which said second single variable immunoglobulin domain comprises the following CDR sequences: CDR1: SYAMG (= SEQ ID NO:10) CDR2: AISWSGGSTYYADSVKG (= SEQ ID NO:11) CDR3: SPIPYGSLLRRRNNYDY (= SEQ ID NO:12).
  3. 3. Polypeptide according to claim 1, characterized in that said first single variable immunoglobulin domain comprises the following CDR sequences: CDR1: SYAMG (= SEQ ID NO:4)CDR2: AIRRSGRRTYYADSVKG (= SEQ ID NO:5)CDR3: ARRVRSSTRYNTGTWWWEY (= SEQ ID NO:6) and in which said second single variable immunoglobulin domain comprises the following CDR sequences: CDR1: SYAMG (= SEQ ID NO:13)CDR2: AISWRSGSTYYADSVKG (= SEQ ID NO:14)CDR3: DPRGYGVAYVSAYYEY (= SEQ ID NO:15).
  4. 4. Polypeptide, according to claim 1, characterized in that said first single variable immunoglobulin domain comprises the following CDR sequences: CDR1: RYTMG (= SEQ ID NO:7) CDR2: AIVRSGGSTYYADSVKG (= SEQ ID NO:8) CDR3: DRRGRGENYILLYSSGRYEY (= SEQ ID NO:9) and in which said second single variable immunoglobulin domain comprises the following CDR sequences: CDR1: SYAMG (= SEQ ID NO:13) CDR2: AISWRSGSTYYADSVKG (= SEQ ID NO:14) CDR3: DPRGYGVAYVSAYYEY (= SEQ ID NO:15).
  5. 5. Polypeptide according to any one of claims 1 to 4, characterized in that such simple variable immunoglobulin domains are VHH domains, and preferably humanized VHH domains.
  6. 6. Polypeptide according to claim 1 or 2, characterized in that said first single variable immunoglobulin domain has the amino acid sequence as shown in SEQ ID NO:19 and said second single variable immunoglobulin domain has the amino acid sequence as shown in SEQ ID NO:22.
  7. 7. Polypeptide according to claim 1 or 3, characterized in that said first single variable immunoglobulin domain has the amino acid sequence as shown in SEQ ID NO:20 and said second single variable immunoglobulin domain has the amino acid sequence as shown in SEQ ID NO:23.
  8. 8. Polypeptide according to claim 1 or 4, characterized in that said first single variable immunoglobulin domain has the amino acid sequence as shown in SEQ ID NO:21 and said second single variable immunoglobulin domain has the amino acid sequence as shown in SEQ ID NO:23.
  9. 9. Polypeptide according to any one of claims 1 to 8, characterized in that said first and said second single variable immunoglobulin domains are covalently linked by a linker peptide, wherein said linker peptide optionally comprises or consists of a third single variable immunoglobulin domain.
  10. 10. Polypeptide according to claim 9, characterized in that said third immunoglobulin variable domain is a simple albumin-binding immunoglobulin variable domain, and preferably the Alb11 domain, defined by SEQ ID NO:24.
  11. 11. Polypeptide, characterized in that it is selected from the group of polypeptides comprising or consisting of SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.
  12. 12. A bacterial, yeast, or fungal host cell, characterized in that it comprises a nucleic acid molecule encoding a polypeptide, as defined in any one of claims 1 to 11, wherein said host cell is capable of expressing said polypeptide.
  13. 13. Method for manufacturing a polypeptide, as defined in any one of claims 1 to 11, characterized in that it comprises the steps of cultivating a host cell, as defined in claim 12, under conditions that permit the expression of a polypeptide, as defined in any one of claims 1 to 11; and recovering and optionally purifying said polypeptide.
  14. 14. Pharmaceutical composition, characterized in that it comprises (i) as the active ingredient, the polypeptide, as defined in any one of claims 1 to 11, and (ii) a pharmaceutically acceptable vehicle, and optionally (iii) a diluent, excipient, adjuvant and/or stabilizer.
  15. 15. Polypeptide, according to any one of claims 1 to 11, characterized in that it is for use as a medicament in a method for the treatment, prevention or relief of a disease, disorder or condition in a human being or an animal, preferably for use in a method (a) for the treatment of cancer, such as breast cancer, lung cancer, pancreatic cancer, colorectal cancer, sarcomas, ovarian cancer or hepatocellular carcinoma, or (b) for the treatment of idiopathic lung disease, or (c) for the treatment of a retinopathy caused by abnormal Wnt signaling.
  16. 16. Polypeptide, according to any one of claims 1 to 11, characterized in that it is for use in the treatment of cancer in combination with an immune checkpoint inhibitor selected from the group consisting of anti-PD1 antibodies, anti-PDL1 antibodies, anti-CTLA4 antibodies, anti-BTLA antibodies, anti-LAG3 antibodies, and anti-TIM3 antibodies, or in combination with a cancer vaccine.
  17. 17. Use of a polypeptide, as defined in any one of claims 1 to 11, characterized in that it is for manufacturing a pharmaceutical composition and/or a medicament and/or kit for the treatment of cancer or idiopathic lung disease or retinopathy in a patient in need thereof.
  18. 18. Use of a polypeptide, as defined in any one of claims 1 to 11, characterized in that it is for manufacturing a pharmaceutical composition and/or a medicament and/or kits for treating cancer and/or for treating cancer in combination with an immune checkpoint inhibitor selected from the group consisting of anti-PD1 antibodies, anti-PDL1 antibodies, anti-CTLA4 antibodies, anti-BTLA antibodies, anti-LAG3 antibodies, and anti-TIM3 antibodies, or in combination with a cancer vaccine.

Description

FIELD OF THE INVENTION [001] The present invention relates to novel low-density lipoprotein receptor type 5 (LRP5) protein-binding polypeptides and low-density lipoprotein receptor type 6 protein-binding polypeptides. The invention also relates to nucleic acids encoding such polypeptides; to methods for preparing such polypeptides; to host cells expressing or capable of expressing such polypeptides; to compositions comprising such polypeptides; and to uses of such polypeptides or such compositions, in particular for therapeutic purposes in the field of cancer diseases. BACKGROUND OF THE INVENTION [002] Activation of the Wnt signaling pathway requires the binding of extracellular Wnt ligands to the Frizzled receptor and the LRP5 coreceptor (Accessory number: UniProtKB-O75197/LRP5_HUMAN) or its closely related homolog LRP6 (Accessory number: UniProtKB-O75581/LRP6_HUMAN). There are 19 Wnt proteins and 10 Frizzled receptors in mammalian cells. In the absence of Wnt ligand, cytoplasmic beta-catenin is phosphorylated by a protein complex consisting of the Axin and APC structural proteins and the GSK3beta and CK1a kinases. Subsequent recognition by the beta-TrcP ubiquitin ligase induces ubiquitin-mediated degradation of beta-catenin. In the presence of Wnt ligand, Wnt binding to Frizzled and LRP5 or LRP6 induces recruitment of the cytoplasmic effector protein Dvl and phosphorylation of the cytoplasmic tail of LRP5 or LRP6, which provides the binding site for axin. Axin sequestration by LRP5 or LRP6 induces inactivation of the axin-APC-GSK3beta complex and, therefore, stabilization and accumulation of intracellular beta-catenin. Consequently, cytoplasmic beta-catenin levels increase, and beta-catenin migrates to the nucleus and complexes with members of the T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) family of transcription factors. Transcriptional coactivators and basal transcription mechanisms are then recruited, including cAMP response element-binding protein (CREB) or its p300 homolog, inducing the expression of several target genes, including Axin2, cyclin D1, and c-Myc. [003] An additional level of ligand-dependent Wnt pathway regulation is mediated by the E3 ligase RNF43, and its closely related homolog ZNRF3, and by secreted R-spondin proteins (de Lau et al. "The R-spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength". Genes Dev. 2014; 28(4):305-16). RNF43 mediates the ubiquitination of the Frizzled/LRP5 or LRP6 receptor complex on the cell surface, inducing its degradation and thus inhibiting ligand-dependent Wnt pathway activity. RNF43 activity is neutralized by members of the R-spondin family (R-spondin ligands 1 to 4). When the R-spondin ligand is present, it removes RNF43 from the cell surface, allowing enhancement and accumulation of Frizzled/LRP5 or LRP6 Wnt signaling complex in the presence of Wnt ligands. [004] LRP5 and LRP6 function as gatekeepers of ligand-dependent Wnt signaling activation and therefore can be considered as targets for achieving complete blockade of the pathway mediated by all 19 Wnt ligands and 10 Frizzled and R-spondin ligand-enhanced receptors. In particular, Wnt ligands can be divided into a Wnt1 class and a Wnt3a class, each binding to different epitopes/regions of LRP5 and LRP6 for signaling. The ectodomain of LRP5 and LRP6 comprises four repeat units of a beta-propellant connected to an EGF-like domain, followed by three LDLR-like A repeats. Combined structural and functional analyses of LRP5 and LRP6 suggest that Wnt1 (Wnt1 class ligand) binds to a fragment containing beta 1 and 2 thrusters, and Wnt3a binds to a fragment containing beta 3 and 4 thrusters of LRP6. To date, only one low-resolution image of an LRP6 ectodomain containing beta thrusters from regions 1 to 4 has been reported (Ahn et al. "Structural basis of Wnt signaling inhibition by Dickkopf binding to LRP5/6". Dev Cell. 2011; 21(5):862-73). However, the uncertainties of these low-resolution reconstructions (40 Å) and the absence of structural data on the LRP6 ectodomain in the complex with Wnt ligands do not allow for the definition of the exact epitopes in the ligand binding of Wnt1 or Wnt3a. [005] Hyperactivation of Wnt signaling is involved in the pathogenesis of several types of cancer. In some types of cancer, frequent mutations in downstream signaling molecules contribute to the constitutively activated Wnt pathway (e.g., APC mutations in colorectal cancer; beta-carenin activation mutation in hepatocellular carcinoma). Conversely, in Triple-Negative Breast Cancer (TNBC), Non-Small Cell Lung Cancer (NSCLC), pancreatic adenocarcinoma, and in a subgroup of Colorectal Cancer (CRC) and endometrial cancers, Wnt signaling activation is regulated by a ligand-dependent mechanism (i.e., by autocrine/paracrine Wnt activation), as detected by intracellular accumulation of beta-catenin. In NSCLC, TNBC, and pancreatic adenocarcinoma, ligand-dependent Wnt activation is mediated by multiple mech