CHIMERIC POLYPEPTIDE COMPLEX AND METHODS OF ITS PRODUCTION AND APPLICATION

Abstract

The present invention relates to bispecific chimeric polypeptide complex compositions containing bulky moieties attached to binding domains via cleavable detachable segments which, when cleaved, are capable of simultaneously mediating binding of effector T cells to tumor or cancer target cells and performing cytolysis of tumor cells or cancer cells. The invention also provides compositions and methods for producing and using cleavable chimeric polypeptide complex compositions.

Claims

1. Use of a chimeric polypeptide for the preparation of a medicament for the treatment of cancer in a subject, wherein the chimeric polypeptide comprises a bispecific antibody comprising: (i) an antigen-binding fragment (Fab) having binding specificity for a tumor-specific marker; and (ii) a single-chain variable region fragment (scFv) having binding specificity for CD3, wherein the scFv is attached to a first bulk moiety via a first linker comprising a first peptidyl separable segment capable of being cleaved by at least one mammalian protease; and said first bulk moiety (a) reduces the ability of the scFv to bind to CD3 and (b) comprises an albumin-binding domain, and wherein the Fab is attached to the second bulk moiety via a second linker comprising a second peptidyl detachable segment capable of being cleaved by at least one mammalian protease; and said second bulk moiety reduces the ability of the Fab to bind to a tumor-specific marker.

2. The use according to claim 1, wherein the tumor-specific marker is selected from the group consisting of alpha-4 integrin, Ang2 (angiopoietin 2), B7-H3 (B7 family homolog 3), B7-H6 (B7 family homolog 6), CEACAM5 (carcinoembryonic antigen-related cell adhesion molecule 5), cMET, CTLA4 (cytotoxic T-lymphocyte antigen 4), FOLR1 (folate receptor 1), EpCAM (epithelial cell adhesion molecule), CCR5 (CC family chemokine receptor 5), CD19 (cluster of differentiation 19), HER2 (human epidermal growth factor receptor type 2), HER2 neu (HER2 in rodents), HER3, HER4, HER1 (EGFR (epidermal growth factor receptor)), PD-L1 (programmed death protein ligand 1), PSMA (prostate membrane antigen), CEA (carcinoembryonic antigen), MUC1 (mucin), MUC-2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC16, βhCG (human chorionic gonadotropin-beta), Lewis Y antigen, CD20, CD33, CD38, CD30, CD56 (NCAM (neural cell adhesion molecule)), CD133,ganglioside GD3, 9-O-acetyl-GD3, GM2, Globo H, fucosyl-GM1, GD2, carbonic anhydrase IX, CD44v6, sonic hedgehog gene product (Shh), Wue-1, plasma cell antigen 1, melanoma chondroitin sulfate proteoglycan (MCSP), CCR8, 6-transmembrane prostatic epithelial cell antigen (STEAP), mesothelin, A33 antigen, prostate stem cell antigen (PSCA), Ly-6, desmoglein 4, fetal acetylcholine receptor (fnAChR), CD25, cancer antigen 19-9 (CA19-9), cancer antigen 125 (CA-125), Müllerian inhibitory substance receptor type II (MISIIR), sialylated Tn antigen (sTN), fibroblast activation protein (FAP) antigen, endosialin (CD248), epidermal growth factor receptor variant III (EGFRvIII), tumor-associated antigen L6 (TAL6), SAS (Staphylococcus aureus surface protein), CD63, TAG72 (tumor-associated glycoprotein 72), Thomsen-Friedenreich antigen (TF antigen),insulin-like growth factor I receptor (IGF-IR), Cora antigen, CD7, CD22, CD70, CD79a, CD79b, G250, MT-MMP (membrane-type matrix metalloproteinases), F19 antigen, CA19-9, CA-125, alpha-fetoprotein (AFP), VEGFR1 (vascular endothelial growth factor receptor 1), VEGFR2, DLK1, SP17, ROR1 (retinoic acid receptor-related orphan receptor 1), and EphA2 (ephrin A2 receptor).

3. The use according to claim 2, wherein the tumor-specific marker is PSMA.

4. The use according to claim 2, wherein the tumor-specific marker is EGFR.

5. The use according to any one of claims 1 to 4, wherein cleavage of the first peptidyl separable segment and the second peptidyl separable segment converts the chimeric polypeptide into an activated bispecific antibody, wherein said activated bispecific antibody is capable of causing at least 10 times greater lysis of tumor cells compared to the chimeric polypeptide, when cell lysis is measured in an in vitro assay including tumor cells and a population of peripheral blood mononuclear cells (PBMC) including T cells.

6. Use according to paragraph 5, where in the in vitro analysis: (a) PBMCs are isolated from one or more healthy donors by Ficoll density gradient centrifugation from either whole blood or lymphocyte-rich buffy coat preparations; (b) PBMCs are resuspended and cultured in RPMI-1640 medium containing 10% FCS (fetal calf serum) and HEPES (N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid) at a concentration of 25 mmol / ml at 37°C and 5% CO2 in a humidified incubator until use in the in vitro assay; (c) the tumor cell density is 2.5×105 cells / mL and the PBMC cell density is 1×106 cells / mL in the in vitro assay; (d) tumor cells and PBMCs were co-cultured in assay medium containing PBMCs without phenol red and containing 5% FCS; and / or (e) Cell lysis was measured by lactate dehydrogenase (LDH) release assay, caspase 3 / 7 activity assay, and FACS-based analysis.

7. The use according to claim 6, wherein in the in vitro assay a ratio of effector cell to tumor cell equal to 5:1 is present in the assay medium.

8. The use according to any one of claims 1 to 7, wherein the solid tumor is a metastatic or locally advanced solid tumor.

9. The use according to any one of claims 1 to 8, wherein the cancer is prostate cancer, non-small cell lung cancer, small cell lung cancer, head and neck cancer, colorectal cancer, kidney cancer, triple-negative breast cancer or pancreatic cancer.

10. The use according to any one of claims 1 to 9, wherein CD3 is CD3ε.

11. The use according to any one of claims 1 to 10, wherein at least one protease comprises MMP-2, MMP-7, MMP-9, MMP-13, MMP-14, urokinase-type plasminogen activator (uPA) or matriptase.

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