Methods of screening, selecting, and identifying cytotoxic recombinant polypeptides based on an interim diminution of ribotoxicity

Patent metadata

Field	Value
Publication number	US-2016355803-A1
Application number	US-201515117174-A
Country	US
Kind code	A1
Filing date	Feb 4, 2015
Priority date	Feb 5, 2014
Publication date	Dec 8, 2016
Grant date	—

Abstract

The present invention relates to methods of screening libraries of chimeric molecules comprising ribotoxic polypeptides, where screening is based on the interim reduction or elimination of ribotoxicity and the methods can identify cytotoxic molecules, each comprising a binding region and a ribotoxic region which jointly possess a desired assay-selectable characteristic, such as, e.g., binding to a target biomolecule, binding to a target cell, and/or cellular internalization.

First claim

1 - 53 . (canceled)

54 . A method for identifying an amino acid sequence for use in constructing one or more chimeric cytotoxic protein,
wherein the one or more chimeric cytotoxic protein comprises:
i) a ribotoxic region comprising a polypeptide and capable of inactivating a ribosome, and
ii) a binding region comprising a polypeptide and capable of binding at least one target biomolecule;
wherein the method comprises the steps of:
a) providing a plurality of chimeric proteins, each protein comprising:
i) a modified ribotoxic region having at least one amino acid substitution, deletion, insertion, or addition as compared to a corresponding unmodified ribotoxin region, so as to reduce or eliminate ribotoxicity of the modified ribotoxic region thereby minimizing unwanted selection bias caused by the presence of ribotoxicity, and
ii) a binding region comprising a polypeptide and capable of binding at least one target biomolecule;

b) screening the plurality of chimeric proteins to identify a protein with at least one assay-screenable characteristic selected from:
target biomolecule binding affinity, target biomolecule binding selectivity, target cell binding affinity, target cell binding selectivity, target cell internalization, and improved expression; and

c) identifying the amino acid sequences of the polypeptide regions of the protein identified in step b) in order to construct one or more chimeric cytotoxic protein derived from the identified protein or comprising a binding region in the identified protein associated with a more ribotoxic form of a modified ribotoxic region present in the identified protein.

55 . The method of claim 54 , further comprising the step of:
d) producing the one or more chimeric cytotoxic protein, wherein the producing step comprises:
i) providing a polynucleotide encoding the one or more chimeric cytotoxic protein, and
ii) expressing the polynucleotide using a host cell or cell-free translation system.

56 . The method of claim 54 , wherein the one or more chimeric cytotoxic protein is a fusion polypeptide comprising the ribotoxin region fused to the binding region.

57 . The method of claim 56 , wherein the plurality of chimeric proteins is a plurality of fusion polypeptides, each fusion polypeptide comprising the modified ribotoxin region fused to a binding region comprising a polypeptide and capable of binding at least one target biomolecule.

58 . The method of claim 57 , wherein the modified ribotoxic region is derived from a toxin selected from the group consisting of:
abrin, agrostin, amarandin, amaranthin, Amaranthus antiviral/RIP, angiogenin, A. patens RIP, Articulatin D, asparin, aspergillin, Aspfl, balsamin, B. hispida RIP, bouganin, Bougainvillea×buttiana antiviral protein 1, benincasin, bouganin, B. rubra RIP, bryodin, B. spectabilis RIP, B. vulgaris RIP, C. album RIP, camphorin, C. aculeatum -systemic resistance inducing protein, C. cristata RIP, C. figarei RIP, charantin, charybdin, cinnamomin, clavin, C. moschata RIP, cochinin B, colocins, crotin, cucurmosin, curcin, Dianthus spp. RIP, Corynebacterium spp. diphtheria toxin, dodecandrin, ebulin, ebulitin, E. hyemalis RIP, euserratin, eutirucallin, flammin, flammulin, foetidissimin, gelonin, gigantin, gypsophilin, H. crepitans RIP, Heterotepalin, hispin, hirsutellin A, H. orientalis RIP, H. vulgare RIP, hypsin, insularin, I. hollandica RIP, lagenin, lamjapin, lanceolin, L. cylindrical RIP, luffacylin, luffaculin, luffagulin, luffin, L. usitatissimum RIP, lychnin, lyophyllin, manutin, marmorin, mapalmin, M. charantia lectin, M. crystallinum RIP, melonin, mexin, Mirabilis spp. RIP, mitogillin, modeccin, MOR, Mormordica spp. RIP, momorsgrovin, moschatin, musarmin, N. tabacum RIP, nigrin, nigritin, ocymoidin, pachyerosin, P. californicum lectin, pepocin, petroglaucin, petrograndin, Phytolacca spp. RIP, pisavin, pleuturegin, Pluturegin, A. thaliana pectin methyl transferase, P. multiforum RIP, pokeweed antiviral protein, porrectin, Aeromonas spp. Pseudomonas toxin ( A. hydrophila pseudomonas -like toxin), pulchellin, quinqueginsin, R. communis agglutinin, restrictocin, ricin, riproximin, saporin, sarcin, sativin, S. cereale RIP, sechiumin, Shiga toxin, Shiga-like toxin, sieboldin b, S. nigra RIP, S. ocymoides RIP, Spinacia oleracea protein, stellarin, stenodactylin, texanin, tricholin, Trichosanthes spp. RIP, Triticum spp. RIP, V. album RIP, velin, velutin, verotoxin, V. hispanica RIP, vircumin, volkensin, V. volvacea RIP, Volvarin, Yucca leaf protein, Z. diploperennis RIP, Z. mays RIP, and any ribotoxic fragment of any of the foregoing.

59 . The method of claim 58 , further comprising before step a), the steps of:
a′) providing an expression library of diverse nucleic acids constructed from a plurality of polynucleotides encoding a plurality of fusion polypeptides; and b′) expressing the expression library of diverse nucleic acids such that a plurality of fusion polypeptides is produced.

60 . The method of claim 59 , further comprising before step a′), the steps of:
a″) providing a library comprising a plurality of diverse polynucleotides encoding a plurality of binding regions, wherein at least two subsets of polynucleotides encode polypeptides with different binding regions; and
b″) joining the polynucleotides of the library to a toxin template polynucleotide encoding a modified ribotoxic region in an operable combination to construct an expression library of diverse nucleic acids that encode a plurality of fusion polypeptides, each fusion polypeptide comprising a binding region fused with the modified ribotoxic region; and
optionally,
c″) recombining the polynucleotides of the library of joined polynucleotides to an expression polynucleotide template to construct an expression library of diverse nucleic acids capable of expressing a plurality of fusion polypeptides, each fusion polypeptide comprising a binding region fused to the modified ribotoxic region.

61 . The method of claim 59 , wherein the binding region is selected from the group consisting of:
complementary determining region 3 fragment, constrained FR3-CDR3-FR4 polypeptide, single-domain antibody fragment, single-chain variable fragment, antibody variable fragment, antigen-binding fragment, Fd fragment, fibronectin-derived 10 th fibronectin type III domain, tenascin type III domain, ankyrin repeat motif domain, low-density-lipoprotein-receptor-derived A-domain, lipocalin, Kunitz domain, Protein-A-derived Z domain, gamma-B crystalline-derived domain, ubiquitin-derived domain, Sac7d-derived polypeptide, Fyn-derived SH2 domain, engineered antibody mimic, and any genetically manipulated counterparts of any of the foregoing that retain binding functionality.

62 . The method of claim 61 , wherein the expression library is operable using a protein display method selected from the group consisting of:
bacteriophage display, RNA display, ribosome display, DNA display, bead surface display, virus display, microorganism display, and mammalian cell display.

63 . The method of claim 62 , wherein at least one binding region is capable of binding to a target biomolecule found in physical association with at least one type of malignant cell.

64 . The method of claim 63 , wherein the modified ribotoxic region comprises or consists of an amino acid sequence that has at least 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.7% sequence identity to a sequence selected from any one of SEQ ID NOs: 1-39 or a ribotoxic fragment thereof.

65 . A method for producing a nucleic acid encoding a chimeric cytotoxic protein, where

Assignees

• Molecular Templates Inc

Inventors

• Poma Eric
• Willert Erin
• Kim Jason
• Higgins Jack

Classifications

• G01N2500/20

  cell-free systems · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C12N15/1037

  Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display · CPC title

• C12N15/1062 Primary

  mRNA-Display, e.g. polypeptide and encoding template are connected covalently · CPC title

• C12N15/1041

  Ribosome/Polysome display, e.g. SPERT, ARM · CPC title