compositions, methods and domain uses of stabilized fibronectin.

专利摘要:
COMPOSITIONS, METHODS AND USES OFDOMAIN OF FIBRONECTIN STABILIZED. The present invention relates to a protein framework based on a consensus protein sequenceof fibronectin type III (FN3) such as the tenth repeat FN3 of thehuman fibronectin (human tenascin), including nucleic acidsisolates encoding a protein framework, vectors, cellshost companies and methods for making and using them. the molecules ofprotein scaffold of the present invention exhibit thermal stability and enhanced chemical stability while having six domains ofModifiable loops that can be engineered to form a partner of binding capable of binding to a target for composite applications,diagnostic and/or therapeutic methods and devices.
公开号:BR112012027863A2
申请号:R112012027863-0
申请日:2011-04-29
公开日:2021-06-29
发明作者:Steven Jacobs
申请人:Janssen Biotech, Inc；
IPC主号:

专利说明:

Descriptive Report of the Patent of Invention for "COMPOSITIONS, METHODS AND USES OF DOMAIN OF FIBRONECTIN STABILIZED".
BACKGROUND 5 Field of the Invention The present invention relates to protein scaffolds with novel properties, including the ability to bind to cellular targets. More particularly, the present invention relates to a protein framework based on a consensus sequence of a fibronectin type III (FN3) repeat. Domain Discussion Monoclonal antibodies are the most widely used class of therapeutic proteins when high affinity and specificity for a target molecule is desired. However, non-antibody proteins that can be engineered to bind to these targets are also of high interest in the biopharmaceutical industry. These "alternative scaffold" proteins may have advantages over traditional antibodies due to their small size, lack of disulfide bonds, high stability, and ability to be expressed in prokaryotic hosts. Innovative purification methods are readily employed; they are easily conjugated to drugs/toxins, efficiently penetrate tissue, and are readily formatted into multispecific ligands (Skerra 2000 J Mol Recognit volume 13 number 4, pages 167 to 187; Binz and Pluckthun 2005 Curr Opin Biotechnol volume 16 number 4 pages 459 to 469). 25 One of these alternative frameworks is the immunoglobulin (Ig) fold. This fold is found in the variable regions of antibodies as well as thousands of non-antibody proteins. It has been shown that an Ig protein of this type, the tenth repeat of fibronectin type III (FN3) obtained from human fibronectin, can tolerate numerous mutations in exposed loops on the surface, while retaining the general structure of the skin fold. IG. In this way, libraries of amino acid variants were integrated with these specific loops and ligands selected for a number of different targets (Koide et al. 1998 J Mol Biol, volume 284 number 4 pages 1141 to 1151; Karatan et al. 2004 Chem Biol, volume 11 number 6 pages 835 to 844). These FN3 domains were also found to bind with high affinity to targets, while retaining important biophysical properties (Parker et al. 2005 Protein Eng Des Sel, volume 18, number 9 pages 435 a 444). Desirable physical properties of potential alternative scaffold molecules include high thermal stability and reversibility of thermal folding and unfolding. Various methods have been applied to increase the apparent thermal stability of proteins and enzymes, including rational design against highly similar thermostable sequences, design of stabilizing disulfide bridges, mutations to increase alpha-helix propensity, manipulation of salt bridges , alteration of protein surface charge, directed evolution, and composition of consensus sequences (Lechmann and Wyss, 2001, Curr Opin Biotechnol, volume 12, number 4 pages 371 to 375). High thermal stability is a desired property of these scaffolds, as it can increase the yield of the recombinant protein obtained, optimize the solubility of the purified molecule, optimize the activity of intracellular scaffolds, decrease immunogenicity, and minimize the need for a cold chain in manufacturing.
SUMMARY OF THE INVENTION The present invention provides a protein framework based on a fibronectin type III repeat protein (FN3), coding or complementary nucleic acids, vectors, host cells, compositions, combinations, formulations, devices and methods for making and using it. In a preferred embodiment, the protein framework is comprised of a consensus sequence of multiple FN3 domains obtained from human Tenascin-C (hereafter in the present document "Tenascin"). In another preferred embodiment, the protein framework of the present invention is a 15 domain FN3 consensus sequence (SEQ ID NO: 1-15) or a variant thereof. In one particular respect
home of the invention, the protein scaffold of the invention has surrogate residues that can cause the scaffold protein to demonstrate increased ability to resist thermal and chemical denaturation.
The protein scaffolds of the invention can be manipulated by methods known in the art, including insertion of residues into loop regions within the scaffold, to form a selective binding domain for a binding partner.
The binding partner can be a soluble molecule or a cellularly anchored molecule, for example, the extracellular domain of a receptor protein. 10 In one embodiment, specific substitutions in the consensus-based sequence of SEQ ID NO: 16 (Tencon) selected for inherent thermal and chemical stability described herein optimize the thermal stability of the Tencon framework by up to 11°C and change the point mean of GdmCl-induced denaturation from 3.4 M to greater than 5 M.
In an embodiment, the substitutions specific to SEQ ID NO: 16 (Tencon) are unitary, such as N46V, E14P, and E86I, and in an alternative embodiment the substitutions are multiple, such as N46V and E86I, all of E14P and N46V and E86I, and all of L17A and N46V and E86I.
Tencon-based polypeptides with optimized stability provide frameworks with improved ease of purification, formulation, and shelf life.
Manipulated binding partners with enhanced overall stability can be produced by introducing randomly selected peptides into loops of the stabilized framework.
The protein scaffolds of the invention can be used as 25 monomeric units or linked to form polymeric structures with the same or different binding partner specificity.
Tencon protein scaffold-based molecules can be further modified to improve one or more in vivo properties related to biodistribution, persistence in the body, or therapeutic efficacy such as association with molecules that alter cellular uptake, particularly from epithelial cell, for example, the Fc region of an antibody, or molecules designed to bind serum proteins such as an albumin binding domain.
In additional embodiments, the protein scaffolds of the invention can be linked to a nucleic acid molecule that can encode the protein scaffold.
The present invention also provides at least one method for expressing at least one protein scaffold polypeptide whose sequence is related to a consensus sequence of multiple FN3 domains in a host cell, which comprises culturing the cell. host as described in the present invention, under conditions in which at least one protein scaffold is expressed in detectable and/or recoverable amounts.
The present invention also features at least one composition comprising: (a) a protein framework based on a consensus sequence of multiple FN3 domains and/or encoding nucleic acid as described in the present invention; and (b) and a suitable and/or pharmaceutically acceptable carrier or diluent.
The present invention further comprises a method for generating a protein scaffold library based on a fibronectin type III repeat protein (FN3), preferably a consensus sequence of multiple FN3 domains, and more preferably a multi-domain consensus sequence. Consensus sequence of multiple FN3 domains of human Tenascin with marked thermal and chemical stability.
Libraries can be generated by changing the amino acid composition of a single loop, or by changing multiple loops or additional positions of the scaffold molecule simultaneously.
Loops that are altered can be lengthened or shortened as needed.
These libraries can be generated to include, at each position, all possible amino acids or a certain subset of amino acids.
Library elements can be used for screening by display, such as by in vitro display (DNA, RNA, ribosome display, etc.) as well as yeast, bacterial and phage display.
The protein scaffolds of the present invention provide enhanced biophysical properties, such as stability under conditions of high osmotic strength and solubility at high concentrations. The domains of scaffold proteins are not disulfide-linked, making them capable of expression and folding in systems lacking the enzymes necessary for disulfide bond formation, including prokaryotic systems, such as E. coli, and in in vitro transcription/translation systems like rabbit reticulocyte lysate system. In a further aspect, the present invention provides a method for generating a scaffold molecule that binds to a specific target by traversing the scaffold library of the invention with the target and 10 detector ligands. In other related aspects, the invention comprises screening methods that can be used to generate or affinity-mature protein scaffolds with the desired activity, for example capable of binding to target proteins with a certain affinity. Affinity maturation can be achieved through iterative cycles of mutagenesis and selection, using systems such as phage display or in vitro display. Mutagenesis during this process may be the result of site-directed mutagenesis to specific framework residues, random mutagenesis due to error-prone PCR, DNA scrambling, and/or a combination of these techniques. The present invention further features any invention described herein.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 SDS-PAGE analysis of purified Tencon performed on a 12% Bis-Tris NuPAGE 4 gel (Invitrogen) and stained with co-omassie blue. N corresponds to native conditions and R to reduced conditions. 25 Figure 2 shows a Tencon circular dichroism analysis in PBS. Figure 3 shows a circular dichroism analysis of the third FN3 domain of tenascin and Tencon in PBS where the melting temperatures of 54°C and 78°C were obtained respectively. Figure 4 shows the design of a phagemid plasmid from pTencon-pIX. Expression is driven by a Lac promoter and secretion via the OmpA signal sequence.
Figure 5 shows myc-Tencon that can be displayed on M13 phage using ELISA demonstrating phage binding to anti-Myc coated, coated CNTO95, and uncoated wells. Figure 6 is a drawing representing the loop structure of the third FN3 domain of human Tenascin. Figure 7 shows the result of Elisa screening of IgG selections by which individual clones were tested for binding to biotinylated IgG or biotinylated HSA as a control. Figures 8A and 8B are graphs showing GdmCl-induced denaturation for single mutants (A) and combinatorial mutants (B) as measured by fluorescence excitation at 280 nm and an emission of 360 nm.
DETAILED DESCRIPTION OF THE INVENTION Abbreviations ADCC = antibody dependent cellular cytotoxicity; CDC = complement dependent cytotoxicity; DSC = differential scanning calorimetry; G = Gibbs Free Energy; IgG = immunoglobulin G; Tm = melting temperature; Definitions & Explanation of Terminology 20 The term "antibody" or "antibody portion" is intended to encompass antibodies, digestion fragments, specified portions and variants thereof, including, without limitation, antibody mimicry, or comprising portions of antibodies that mimic the structure and/or function of an antibody or specified fragment or portion thereof, including, without limitation, single chain antibodies, single domain antibodies, minibodies and fragments thereof. Functional fragments include antigen-binding fragments that bind to the target antigen of interest. For example, antibody fragments capable of binding to a target antigen or portions thereof, including, but not limited to, Fab fragments (for example, by papain digestion), Fab' (for example, by di- pepsin management and partial reduction) and F(ab')2 (eg by pepsin digestion), facb (eg by plasmin digestion),
pFc' (eg by pepsin or plasmin digestion), Fd (eg by pepsin digestion, partial reduction and reaggregation), Fv or scFv (eg by molecular biology techniques) are covered by the term antibody.
The antibody or fragment can be derived from any mammal, such as, but not limited to, human, mouse, rabbit, rat, rodent, primate, goat or any combination thereof, includes antibodies, immunoglobulins, cleavage products and other specified portions and variants of human, primate, rodent, mammalian, chimeric, humanized and/or CDR-grafted. The term "epitope" means a determinant protein capable of specific binding to an antibody or binding domain engineered as one or more loops of a framework-based protein.
Epitopes usually consist of chemically active surface groupings such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.
Conformational and non-conformational epitopes are distinguished such that binding to the former but not the latter is lost in the presence of denaturing solvents.
Conformational epitopes result from the conformational folding of the target molecule that arises when amino acids from different portions of the linear sequence of the target molecule assemble in close proximity in three-dimensional space.
These conformational epitopes are typically distributed on the extracellular side of the plasma membrane.
The terms "Fc", "Fc-containing protein" or "Fc containing molecule", for use in the present invention, refer to a monomeric, dimeric or heterodimeric protein that has at least one domain of immunoglobulin CH2 and CH3. The CH2 and CH3 domains can form at least a part of the dimeric region of the protein/molecule (e.g., antibody). The term "stability" for use herein refers to the ability of a molecule to maintain a folded state under physiological conditions such that it retains at least one of its normal functional activities, e.g. if to a target molecule like a ci-
tocin or serum protein.
Measuring protein stability and protein susceptibility can be viewed as the same or different aspects of protein integrity.
Proteins are sensitive or "unstable" to denaturation caused by heat, ultraviolet or ionizing radiation, changes in ambient osmolarity and pH in liquid solution, mechanical shear force imposed by filtration through small pores, ultraviolet radiation, ionizing radiation, such as gamma irradiation, chemical or heat dehydration, or any other action or force that may cause the disturbance of the protein structure.
The stability of the molecule can be determined using standard methods.
For example, the stability of a molecule can be determined by measuring the thermal melting temperature ("TM"). TM is the temperature in degrees Celsius (°C) at which ½ of the molecules become unfolded.
Typically, the higher the TM, the more stable the molecule.
In addition to heat, the chemical environment also alters the protein's ability to maintain a particular three-dimensional structure.
Chemical denaturation can similarly be measured by a variety of methods.
A chemical denaturant is an agent known to disrupt non-covalent interactions and covalent bonds within a protein, including hydrogen bonds, electrostatic bonds, 20 Van der Waals forces, hydrophobic interactions, or disulfide bonds.
Chemical denaturants include guanidinium hydrochloride, guanidinium thiocyanide, urea, acetone, organic solvents (DMF, benzene, acetonitrile), salts (lithium bromide, ammonium sulfate, lithium chloride, sodium bromide, calcium chloride, sodium chloride ); reducing agents (eg, dithiothreitol, beta-mercaptoethanol, dinitrothiobenzene, and hydrides such as sodium borohydride), nonionic and ionic detergents, acids (eg hydrochloric acid (HCl), acetic acid (CH3COOH) ), halogenated acetic acids), hydrophobic molecules (eg, phospholipids), and targeted denaturants (Jain RK and Hamilton A.
D., Angew.
Chem., volume 114, number 4, 2002). 30 Quantifying the extent of denaturation may depend on the loss of a functional property such as the ability to bind to a target molecule, or on physiochemical properties such as a tendency to aggregation, exposing
site of previously inaccessible solvent residues, or disturbance or formation of disulfide bonds.
In terms of loss of stability, that is, "denature" or "denaturation" of a protein means the process where some or all of the three-dimensional conformation that confers the functional properties of the protein has been lost with a consequent loss of activity and/ or solubility.
Perturbing forces during denaturation include intramolecular bonds, including, but not limited to, electrostatic, hydrophobic, Van der Waals, hydrogen bonding, and disulfide forces.
Protein denaturation can be caused by forces applied to the protein or a solution that comprises the protein as a mechanical (eg compressive or shear) force, thermal, osmotic stress, change in pH, electric or magnetic fields. , ionizing radiation, ultraviolet radiation and dehydration, and by chemical denaturants. A "therapeutically effective" treatment or amount, for use in the present invention, refers to an amount of sufficient volume to bring about a reduction or alleviation of the cause of a disorder or its symptoms. "Attenuating" refers to a reduction in the detrimental effect of the disorder on the patient receiving therapy.
The subject of the invention is preferably a human being, however, it can be considered that any animal in need of treatment for conditions with detrimental effects, disorder, or disease can be treated with a protein based framework designed to that purpose.
Overview The present invention provides an isolated, recombinant and/or synthetic protein scaffold based on a consensus sequence of a fibronectin type III repeat protein (FN3) including, without limitation, mammalian-derived scaffold as well. as compositions and encoded nucleic acid molecules comprising at least one polynucleotide that encodes a protein framework based on the FN3 consensus sequence. The present invention additionally includes, but is not limited to, methods for making and using such nucleic acids and protein scaffolds.
in, including as a discovery platform, and for therapeutic and diagnostic compositions, methods and devices.
The proteinaceous scaffolds of the present invention offer advantages over vast immunoglobulin-based biotherapeutics due to their small and compact size.
In particular, the size and shape of a biological molecule can impact its ability to be administered locally, orally, or to cross the blood-brain barrier; ability to be expressed in low-cost systems such as E. coli; ability to be engineered into bi- or multi-specific molecules that bind to multiple targets or multiple epithets from the same target, suitability for conjugation, ie, to actives, polymers, and probes; ability to be formulated in high concentrations; and the ability of these molecules to effectively penetrate diseased tissue and tumors.
Furthermore, protein scaffolds have many of the properties of antibodies with respect to their folding, which mimics the variable region of an antibody.
This orientation allows the FN3 loops to be exposed similarly to the complementarity-determining regions (CDRs) of the antibody.
They need to be able to bind to cellular targets, and the bonds can be altered, for example affinity matured, to optimize certain binding properties or related properties.
Three of the six loops of the protein framework of the invention correspond topologically to the binding domains of an antibody positioned in the variable domain loops known to be in fact hypervariable (the hypervariable domain loops (HVL) in positions as defined by Kabat with the residues of the complementarity-determining regions (CDRs), that is, antigen-binding regions, of an antibody, while the remaining three loops are exposed to the surface in similar ways. to antibody CDRs.
These loops span or are positioned at or near residues 13-16, 22-28, 38-43, 51-54, 60-64 and 75-81 of SEQ ID NO:16 as shown in Table 3, below, and in Figure 6. Preferably, the loop regions at or around residues 22-28, 51-54 and 75-81 are changed for specificity and binding affinity.
One or more of these loop regions are randomly selected with other loop regions and/or other strands keeping their sequence as main-chain portions to populate a library, and potent linkers can be selected from the library having high affinity for a specific target protein.
One or more of the loop regions can interact with a target protein, similarly to an antibody CDR interaction with the protein.
The frameworks of the present invention can incorporate another 10 subunits, for example via covalent interaction.
All or a portion of an antibody constant region can be linked to the framework to confer antibody-like properties, specifically those properties associated with the Fc region, eg complement activity (ADCC), half-life , etc.
For example, an effector function can be provided and/or controlled, for example, by modifying the C1q binding and/or the Fc R binding and thereby altering the activity of CDC and/or ADCC.
"Effector functions" are responsible for activating or decreasing a biological activity (for example, in an individual). Examples of effector functions include, but are not limited to: C1q binding; complement dependent cytotoxicity (CDC); binding to Fc receptor; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (eg, B cell receptor; BCR), etc.
The functions of this effector may require the Fc region to be combined with a binding domain (eg, protein scaffold loops) and can be evaluated using various assays (eg, Fc binding assays, ADCC assays, CDC assays, etc.). Additional portions can be attached or associated with the scaffold-based or variant-based polypeptide as molecules of a conjugated toxin, albumin or albumin linkers, polyethylene glycol (PEG) can be attached to the scaffold molecule to achieve the desired properties.
These portions can be in-line merges with the scaffold coding sequence and can be generated by standard techniques, by e-
for example, by expressing the fusion protein of a recombinant fusion encoding a vector constructed using publicly available encoding nucleotide sequences.
Alternatively, chemical methods can be used to attach the moieties to a recombinantly produced scaffold-based protein.
The frameworks of the present invention can be used as mono-specific in monomeric form, or as bi- or multi-specific (for different target proteins or different epitopes on the same target protein) in multimeric form.
The attachments between each framework unit can be covalent or non-covalent.
For example, a dimeric bispecific framework has one subunit with specificity for a first target protein or epitope and a second subunit with specificity for a second target protein or epitope.
The scaffold subunits can be joined in a variety of conformations that can increase the valence and therefore the avidity of the antigen binding.
Framework Protein Generation and Production At least one framework protein of the present invention may optionally be produced by a cell line, a mixed cell line, an immortalized cell, or a clonal population of 20 immortalized cells, as is well known in technique.
See, for example, Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, USA (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, NY, USA (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY, 25 USA (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY, USA (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, USA, (1997-2001). Amino acids obtained from a scaffold protein can be altered, added and/or deleted to reduce immunogenicity or reduce, enhance or modify binding, affinity, rate of association, rate of dissociation, avidity, specificity, half-life, stability, solubility or any other suitable characteristic, con-
form is known in the art.
Bioactive scaffold-based proteins can be manipulated with high affinity retention for antigen and other favorable biological properties.
To achieve this goal, scaffold proteins can optionally be prepared through a process of analyzing the parent sequences and various conceptual engineered products using three-dimensional models of the parent and engineered sequences.
Three-dimensional models are commonly available and are familiar to those skilled in the art.
Computer programs are available that illustrate and display likely three-dimensional conformational structures of selected candidate sequences, and can measure possible immunogenicity (eg, the Immunofilter program from Xencor, Inc., of Monrovia, CA, USA ). Inspection of these displays allows analysis of the likely role played by residues in the functioning of the candidate sequence, that is, analysis of residues that influence the ability of the candidate scaffold protein to bind its antigen.
In this way, residues can be selected and combined from the parent and reference sequences so that the desired characteristic, such as affinity for one or more target antigens, is obtained.
Alternatively, or in addition to the above-cited procedures, other suitable engineering methods may be used.
Screening Screening of framework-based protein or of engineered, framework-based protein libraries with varied residues or domains for specific binding to similar proteins or fragments can be conveniently achieved with the use of nucleotide display libraries (display) of DNA or RNA) or peptides, for example, in vitro display.
This method involves screening large collections of peptides for individual members that have the desired function or structure.
The 30 peptides displayed with or without nucleotide sequences can be from 3 to 5,000 or more nucleotides or amino acids in length, often from 5 to 100 amino acids, and often from about 8 to 25 amino acids.
noacids.
In addition to direct chemical synthesis methods for generating peptide libraries, several methods with recombinant DNA have been described.
One type involves the visualization of a peptide sequence on the surface of a bacteriophage or cell.
Each bacteriophage or cell contains the nucleotide sequence that encodes the particular peptide sequence visualized.
The protein scaffolds of the invention can bind to human or other mammalian proteins with a wide range of affinities (KD). In a preferred embodiment, at least one protein scaffold of the present invention may optionally bind a target protein with a very high degree of affinity, for example with a KD equal to or less than about 10 -7 M, including, but not limited to, 0.1 to 9.9 (or any range or value in that range) X 10-8, 10-9, 10-10, 10-11, 10-12, 10-13, 10-14, 10-15 or any range or value within this range, as determined by surface plasmon resonance or the KinExA method, as practiced by those skilled in the art.
The affinity or avidity of a protein scaffold for an antigen can be determined experimentally using any suitable method. (See, for example, Berzofsky, et al., "Antibody-Antigen 20 Interactions", In Fundamental Immunology, Paul, W.
E., Ed., Raven Press: New York, USA (1984); Kuby, Janis Immunology, W. H.
Freeman and Company: New York, USA (1992); and methods described herein). The measured affinity of a specific protein-antigen scaffold interaction may vary if measured under different conditions (eg, osmolarity, 25 pH). Therefore, measurements of affinity and other antigen-binding parameters (eg KD, Kon, Koff) are preferably made with standardized solutions of protein and antigen scaffold, and with a standardized buffer such as the buffer. described here.
Competitive assays can be performed with the protein framework of the present invention, in order to determine which proteins, antibodies and other antagonists compete for binding to a target protein with the protein framework of the present invention, and/or share the epitope region.
Such assays, as is readily known to those of skill in the art, assess competition between antagonists or ligands for a limited number of binding sites on a protein.
The protein and/or antibody is immobilized or rendered insoluble, before or after the competition, and the sample bound to the target protein is separated from the unbound sample, for example by decanting (where has previously rendered the protein or antibody insoluble) or by centrifugation (in cases where the protein or antibody has precipitated after the competitive reaction). Furthermore, competitive binding can be determined by whether the function has been altered by the binding or lack of binding of the protein framework to the target protein, for example, whether the protein framework molecule inhibits or potentiates the enzyme activity of, for example, a bookmark.
ELISA and other functional tests can be used, as is well known in the art. Nucleic acid molecules The nucleic acid molecules of the present invention that encode protein scaffolds may be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA including , but not limited to, cDNA and genomic DNA obtained by cloning or synthetically produced, or any combination thereof.
DNA can be triple-stranded, double-stranded or single-stranded, or any combination thereof.
Any portion of at least one strand of DNA or RNA can be a coding strand, also known as the sense strand, or it can be the non-coding strand, also known as the antisense strand. The isolated nucleic acid molecules of the present invention may include nucleic acid molecules that comprise an open reading frame (ORF), optionally, with one or more introns, for example, but not limited to, at least a specified portion of at least one protein scaffold; nucleic acid molecules that comprise the coding sequence for a protein framework or loop region that binds to the target protein; and nucleic acid molecules which comprise a nucleotide sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the protein scaffold as described herein and/or as known in the art.
Of course, the genetic code is well known in the art.
Therefore, it would be routine for the person skilled in the art to generate such degenerate nucleic acid variants that encode the protein scaffolds of the present invention.
See, for example, Ausubel, et al., above, and such nucleic acid variants are included in the present invention.
As indicated in the present invention, nucleic acid molecules of the present invention which comprise a nucleic acid encoding a protein scaffold may include, but are not limited to, those which encode the amino acid sequence of a protein scaffold fragment. protein by itself; the coding sequence for the entire protein framework or a portion thereof; the coding sequence for a protein framework, fragment or portion, as well as additional sequences, such as the coding sequence of at least one signal leader or fusion peptide, with or without the additional coding sequences above, as at least one intron, along with additional non-coding sequences, including, but not limited to, 5' and 3' non-coding sequences, such as untranslated transcribed sequences that play a role in transcription, processing of mRNA, including splicing and polyadenylation signals (e.g., ribosome binding and mRNA stability); an additional coding sequence that encodes additional amino acids, such as those that provide additional functionalities.
Therefore, the sequence encoding a protein framework can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused protein framework comprising a protein framework fragment or portion.
Nucleic acid molecules The invention also provides nucleic acids encoding the compositions of the invention as isolated polynucleotides or as portions of expression vectors, including vectors compatible with the expression, secretion and/or display of prokaryotic, eukaryotic or filamentous phage of the compositions or the targeted mutagens from them.
The isolated nucleic acids of the present invention can be produced using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and/or (d) combinations thereof, as is well known in the art. .
Polynucleotides useful in the practice of the present invention will encode a functional portion of the protein framework described in the present invention.
Polynucleotides of the present invention encompass nucleic acid sequences that can be used for selective hybridization to a polynucleotide that encodes a protein framework of the present invention.
The present invention features isolated nucleic acids that hybridize under selective hybridization conditions to a polynucleotide shown herein.
Therefore, polynucleotides of this embodiment can be used to isolate, detect and/or quantify nucleic acids that comprise such polynucleotides.
For example, the polynucleotides of the present invention can be used to identify, isolate or amplify partial or full-length clones in a deposited library.
In some embodiments, polynucleotides are isolated genomic or cDNA sequences, or otherwise complementary to a cDNA obtained from a human or mammalian nucleic acid library.
Nucleic acids may conveniently comprise sequences in addition to a polynucleotide of the present invention.
For example, a multicloning site that comprises one or more restriction endonuclease sites can be inserted into the nucleic acid to aid in the isolation of the polynucleotide.
Furthermore, the translatable sequences can be inserted to aid in isolating the translated polynucleotide of the present invention.
For example, a hexahistidine tag sequence provides a convenient means to purify the proteins of the present invention.
The nucleic acid of the present invention, excluding the coding sequence, is optionally a vector, adapter or connector for cloning and/or expression of a polynucleotide of the present invention.
Additional sequences can be added to such sequences.
cloning and/or expression techniques to optimize its function in cloning and/or expression to aid in the isolation of the polynucleotide, or to optimize the introduction of the polynucleotide into a cell.
The use of expression vectors, adapters, and connectors is well known in the art. As indicated in the present invention, nucleic acid molecules of the present invention that comprise a nucleic acid encoding a protein scaffold may include, but are not limited to, those that encode the amino acid sequence of a protein scaffold fragment , by itself; the coding sequence for the entire protein scaffold 10 or a portion thereof; the coding sequence for a protein framework, fragment or portion, as well as additional sequences, such as the coding sequence of at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences, such as at least an intron, along with additional non-coding sequences, including, but not limited to, 5' and 3' non-coding sequences, such as the untranslated, transcribed sequences, which play a role in transcription, mRNA processing, in- including splicing and polyadenylation signals (eg, ribosome binding and mRNA stability); an additional coding sequence that codes for additional amino acids, such as those that provide additional functionality.
Therefore, the sequence encoding a protein framework can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused protein framework, which comprises a protein framework fragment or portion. at.
For bacterial expression including phage-infected bacteria, a preferential secretion signal is a pelB or ompA secretion signal but other secretion signal polypeptide domains can be used as described in US Patent No. 5,658,727. In phage display, a DNA sequence capable of downstream translation encodes a filamentous phage coat protein, e.g. pIII or pIX protein.
Preferred filamentous phage proteins are obtainable from filamentous phage.
mentosus M13, f1, fd, and equivalent filamentous phages.
Thus, a DNA sequence capable of downstream translation encodes an amino acid residue sequence that corresponds to, and is preferably identical to, the coat polypeptide of filamentous phage gene III or gene IX.
The 5 sequences of these coat proteins are known and accessible in public databases such as the NCBI.
A genomic or cDNA library can be screened using a probe based on the sequence of a polynucleotide of the present invention, such as those presented herein.
The probes can be used to hybridize to genomic DNA or cDNA sequences to isolate homologous genes in the same organism or in different organisms.
Those skilled in the art will recognize that varying degrees of stringency of hybridization can be employed in the assay; and the hybridization or wash medium can be severe.
As conditions for hybridization become more stringent, there must be a greater degree of complementarity between probe and target for bidirectional formation to occur.
The degree of stringency can be controlled by one or more of temperature, ionic strength, pH, and the presence of a partially denaturing solvent such as formamide.
For example, the stringency of hybridization is conveniently varied by changing the polarity of the reactant solution through, for example, manipulation of the concentration of formamide within the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding will vary according to the severity of the hybridization medium and/or wash medium.
The degree of complementarity will optimally be 100%, or 70 to 100%, or any range or value in between.
However, it should be understood that minimal sequence variations in probes and primers can be compensated for by reducing the stringency of the hybridization and/or wash medium.
In one aspect of the invention, polynucleotides are constructed using techniques to incorporate randomly selected codons to vary the resulting polypeptide at one or more specific residues, or to add residues at specific locations within the sequence.
sequence.
Various strategies can be used to create libraries of altered polypeptide sequences including rational, semi-rational and random methods.
Rational and semi-rational methods have the advantage over random strategies as they have more control over the consequences of changes introduced in the coding sequence.
Furthermore, by focusing the variation on certain regions of the gene, the universe of all possible amino acid variants can be explored in chosen positions.
A library constructed with the common 10 NNK or NNS diversification scheme introduces a possibility of 32 different codons at each position and all 20 amino acids.
Such a library theoretically grows by 32n for every n number of residues.
In practical terms, however, phage display is limited to sampling libraries of 109 to 1010 variants implying that only 6-7 residues can be varied 15 if full sequence coverage is intended to be achieved in the bi - library.
In this way, semi-rational or "focused" methods can be applied to generate libraries of framework variants by identifying key positions to be varied and choosing the corresponding diversification regime.
A "codon set" refers to a set of different nucleotide triplet sequences used to encode desired variant amino acids.
A standard form of codon designation is the IUB code, which is known in the art and described in the present invention.
A "non-random codon set" refers to a set of codons that encode selected amino acids.
The synthesis of oligonucleotides with selected nucleotide "degeneration" at certain positions is well known in the art, for example, the TRIM approach (Knappek et al., J.
Mol.
Biol. (1999), volume 296, pages 57 to 86); Garrard & Henner, Gene (1993), volume 128, pages 103). Those sets of nucleotides that have certain sets of codons can be synthesized using commercially available nucleotide and nucleoside reagents and apparatus.
A codon set is a set of different nucleotide triplet sequences used to encode the desired variant amino acids.
Codon sets can be represented using symbols to designate particular nucleotides or equimolar mixtures of nucleotides as shown below according to the IUB code. 5 IUB Codes G Guanine A Adenine T Thiamine C Cytosine 10 R (A or G) Y (C or T) M (A or C) K (G or T) S (C or G) 15 W (A or T) H (A or C or T) B (C or G or T) V (A or C or G) D (A or G or T) 20 N (A or C or G or T) For example, in the DVK codon set , D can be the nucleotides A or G or T; V can be A or G or C; and K can be G or T.
This set of codons can have 18 different codons and can encode the amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cis. 25 Focused (eg non-random) libraries can be generated using NNK codons and targeting variety to selected residues or alternatively variants with non-random substitutions can be generated using, for example, DVK codons , which encode 11 amino acids (ACDEGKNRSYW) and a stop codon.
Alternatively, Kunkel mutagenesis can be used to vary desired residues or regions of the polypeptide (Kunkel et al., Methods Enzymol. volume 154, pages 367 to 382, 1987).
Standard cloning techniques are used to clone libraries into an expression vector. The library can be expressed using known systems, for example, expressing the library as fusion proteins. Fusion proteins can be displayed on the surface of any suitable phage. Methods for displaying fusion polypeptides comprising antibody fragments on the surface of a bacteriophage are well known (US 6,969,108 to Griffith; US
6,172,197 granted to McCafferty; US 5,223,409 issued to Ladner; US
6,582,915 granted to Griffiths; US 6,472,147 issued to Janda). Libraries for new polypeptide isolation can be displayed in pIX (WO2009085462A1). Libraries can also be translated in vitro, for example, using ribosome display (Hanes and Pluckthun, Proc. Natl. Acad. Scie. USA, volume 94, page 4937, 1997), mRNA display (Roberts and Szostak , Proc. Natl. Acad. Sci. USA, volume 94, pages 12297, 15 1997), CIS exhibition (Odegrip et. al., Proc. Natl. Acad. Sci. USA, volume 101, pages 2806, 2004) or others cell-free systems (US 5,643,768 granted to Kawasaki). Libraries with diversified regions can be generated using vectors that comprise the polynucleotide encoding the Tencon sequence (SEQ ID NO: 16) or a predetermined mutant thereof. The model construct can have a promoter and signal sequences for the polypeptide chain. To create scaffold libraries, mutagenesis reactions are used with the use of oligonucleotides that encode loop regions (A:B, B:C, C:D, D:E, E:F, and F:G) of the framework. To ensure incorporation of all chosen positions into the random selection scheme, a stop codon (such as TAA) can be incorporated into each desired position that is intended to be diversified. Only clones where the stop codons have been replaced will occur. Modified Framework Polypeptides The modified scaffolds and protein fragments of the invention may comprise one or more moieties that are covalently linked, directly or indirectly, to another protein.
In the case of the addition of peptide residues, the creation of an in-line fusion protein, the addition of these residues can take place by recombinant techniques from a polynucleotide sequence as described above.
In the case of a fixed, attached or conjugated peptide, protein, organic chemical, inorganic chemical or inorganic atom, or any combination thereof, the additional portion that is linked to a protein scaffold or fragment of the invention it is typically linked by other forms in addition to the peptide bond.
The modified protein scaffolds of the invention can be produced by reacting the scaffold or protein fragment with a modifying agent.
For example, organic moieties can be linked to a protein scaffold in a non-site-specific manner through the use of an amine-reactive modifying agent, for example an NHS ester of PEG.
Modified protein scaffolds and fragments comprising an organic portion that is linked to specific sites of a protein scaffold of the present invention can be prepared using suitable methods such as reverse proteolysis (Fisch et al., Bioconjugate Chem., volume 3, pages 147 to 153 (1992); Werlen et al., Bioconjugate Chem., volume 5, pages 411 to 417 (1994); Kumaran et al., Protein Sci. volume 6, number 20 10 , pages 2233 to 2241 (1997); Itoh et al., Bioorg.
Chem., volume 24, number 1 pages 59 to 68 (1996); Capellas et al., Biotechnol.
Bioeng., volume 56, number 4, pages 456 to 463 (1997)), and the methods described in Hermanson, G.
T., Bioconjugate Techniques, Academic Press: San Diego, California, USA (1996). 25 Where a polymer or a chain is attached to the scaffold protein, the polymer or chain may independently be a hydrophilic polymeric group, a fatty acid group or a fatty acid ester group.
For use herein, the term "fatty acid" encompasses mono-carboxylic acids and di-carboxylic acids.
For use in the present invention, the term "hydrophilic polymeric group" refers to an organic polymer that is more soluble in water than in octane.
For example, polylysine is more soluble in water than in octane.
Thus, a protein scaffold modified by the covalent binding of polylysine is encompassed by the invention.
Suitable hydrophilic polymers for modifying the protein scaffolds of the invention may be linear or branched and include, for example, polyalkane glycols (for example PEG, monomethoxy polyethylene glycol (mPEG), 5 PPG and the like), carbohydrates (for example , dextran, cellulose, oligosaccharides, polysaccharides and the like), hydrophilic amino acid polymers (eg, polylysine, polyarginine, polyaspartate and the like), polyalkane oxide (eg, polyethylene oxide, polypropylene oxide and the like). res) and polyvinyl pyrrolidone.
Preferably, the protein scaffold modifying hydrophilic polymer of the invention has a molecular weight of from about 800 to about 150,000 Daltons, as a separate molecular entity.
For example, PEG5,000 and PEG20,000 can be used, where the subscript is the average molecular weight of the polymer in Daltons.
The hydrophilic polymeric group can be substituted with one to about six alkyl, fatty acid or fatty acid ester groups.
Hydrophilic polymers that are substituted by a fatty acid or a fatty acid ester group can be prepared by employing suitable methods.
For example, a polymer comprising an amine group can be coupled to a fatty acid carboxylate or fatty acid ester, and an activated carboxylate (eg, activated with 20 N,N-carbonyl diimidazole) to a fatty acid or fatty acid ester can be coupled to a hydroxyl group in a polymer.
Fatty acids and fatty acid esters suitable for modifying the protein scaffolds of the invention may be saturated or may contain one or more units of unsaturation.
Fatty acids that are suitable for modifying the protein scaffolds of the invention include, for example, n-dodecanoate (C12, laurate), n-tetradecanoate (C14, myristate), n-octadecanoate (C18, stearate), n-eicosanoate (C20, arachidate), n-docosanoate (C22, behenate), n-triacontanoate (C30), n-tetracontanoate (C40), cis-9-octadecanoate (C18, oleate), all cis- 5,8,11, 14-30 eicosatetraenoate (C20, arachidonate), octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like.
Suitable fatty acid esters include dicarboxylic acid mono-esters.
cos which comprise a linear or branched lower alkyl group.
The lower alkyl group may comprise from one to about twelve, preferably from one to about six, carbon atoms.
Fc-containing proteins can be compared for functionality by several well-known in vitro assays.
In particular, affinity for members of the Fc RI, Fc RII and Fc RIII family of Fc receptors is of interest.
These measurements could be made using recombinant soluble forms of the receptors from cell-associated forms of the receptors.
Furthermore, affinity for FcRn, the receptor responsible for the prolonged circulating half-life of IgGs, can be measured, for example, by BIAcore using soluble recombinant FcRn.
Cell-based functional assays, such as ADCC assays and CDC assays, provide criteria on the likelihood of functional consequences of particular variant structures.
In one embodiment, the ADCC assay is configured to have NK cells as the primary effector cell, thus reflecting the functional effects of the Fc receptor RIIIA.
Phagocytosis assays can also be used to compare immune effector functions of different variants, such as assays that measure cellular responses, such as superoxide or inflammatory mediator release.
In vivo models can be used in the same way as, for example, in the case of using anti-CD3 antibody variants to measure T cell activation in mice, an activity that is dependent on Fc domains that interconnect with specific ligands such as Fc receptors. Host Cell Selection or Host Cell Manipulation 25 As described here, the host cell chosen for scaffold-based protein expression is an important contributor to the final composition, including, without limitation, variation in the composition of the oligosaccharide moieties that decorate the protein, if desired, for example in the CH2 domain of the immunoglobulin when present.
Thus, one aspect of the invention involves the selection of appropriate hose cells for use and/or development of a production cell expressing the desired therapeutic protein.
Additionally, the host cell can be of mammalian origin or can be selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, lymphoma, yeast, insect or plant cells, or any cells derived, immortalized or transformed therefrom. Alternatively, the host cell can be selected from a species or organism incapable of glycosylation of polypeptides, for example, a prokaryotic cell or organism, such as and from E. coli spp, Klebsiella spp. or natural or engineered Pseudomonas spp. 10 Selection of Binding Domains Polypeptides or fusion proteins or components and domains thereof may also be obtained by selection from libraries of such domains or components, e.g., a phage library. A phage library can be created by inserting a random oligonucleotide library or a polynucleotide library containing sequences of interest such as antibody domains from B cells from an immunized animal or human (Smith, G.P.
1985. Science, volume 228, pages 1315 to 1317). Antibody phage libraries contain heavy (H) and light (L) chain variable region pairs in a phage that allows expression of Fv fragments or single chain Fab fragments ( Hoogenboom, et al. 2000, Immunol. Today, volume 21, number 8, pages 371 to 378. The diversity of a phagemid library can be manipulated to increase and/or alter the specificities of the library's polypeptides to produce and subsequently identify additional desirable molecular properties and polynucleotides that encode them. Other libraries of target binding components that may include in addition to antibody variable regions are ribosome display, CIS display, yeast display, bacterial display, and mammalian cell display. method of translating mRNAs into their cognate proteins while keeping the protein fixed to the RNA. The nucleic acid coding sequence is retrieved by RT-
PCR (Mattheakis, L.C. et al. 1994. Proc.
Natl.
Academic
Sci.
USA, volume 91, page 9022). The CIS display is an alternative in vitro display method in which the library is constructed as a fusion protein with RepA.
During in vitro translation, RepA binds in cis to the DNA from which it was produced, providing a direct link between genotype and phenotype. (Odegrip et. al., Proc.
Natl.
Academic
Sci.
USA, volume 101, page 2806, 2004). Yeast display is based on the construction of membrane-associated alpha-agglutinin yeast adhesion receptor fusion proteins, aga1 and aga2, a part of the pairing-type system (Broder, et al. 1997. Nature 10 Biotechnology , volume 15, pages 553 to 557). A bacterial display is based on target fusion to exported bacterial proteins that associate with the cell membrane or cell wall (Chen and Georgiou 2002. Biotechnol Bioeng, volume 79, pages 496 to 503). Similarly, mammalian display systems are based on the creation of a fusion protein between a polypeptide containing randomly selected sequences and a secreted membrane anchor protein.
Uses of Framework-Based Molecules The compositions of the framework-based molecules described herein and generated by any of the methods described above can be used to diagnose, monitor, modulate, treat, alleviate, help prevent the incidence of, or reduce the symptoms of human diseases or specific pathologies in cells, tissues, organs, fluid, or in general, a host.
A framework-based molecule engineered for a specific purpose can be used to treat an immune-mediated disease or immune deficiency, metabolic disease, a cardiovascular disorder or disease; a malignant disease; neurological disorder or disease; an infection such as a bacterial, viral or parasitic infection; or other known or specified related condition including swelling, pain, and tissue necrosis or fibrosis. Such a method may comprise administering an effective amount of a composition or a pharmaceutical composition comprising at least one scaffold protein to a cell, tissue.
of the organ, animal or patient in need of such modulation, treatment, relief, prevention or reduction in symptoms, effects or mechanisms.
The effective amount may comprise from about 0.001 to 500 mg/kg by single (eg bolus), multiple or continuous administration, or enough to achieve a serum concentration of 0.01 to 5,000 ug/ml of serum concentration by single, multiple or continuous administration, or any effective range or value within that range, as performed and determined using known methods as described in the present invention or known in the relevant art. 10 Structure-Based Protein Compositions Target-binding framework proteins that are modified or unmodified, monovalent, bi- or multivalent, and mono-, bi- or multi-target, can be isolated using procedures separations well known in the art by capture, immobilization, partitioning, or sedimentation and purified to the extent necessary for commercial applicability.
For therapeutic use, scaffold-based proteins can be formulated in an appropriate manner of administration that includes, but is not limited to, parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intra- abdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraostial, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intraretal, intrarenal, intraretinal, intraspinal, intrasynovial, 25 intrathoracic, intrauterine, intravesical, intralesional, bolus, vaginal, rectal, buccal, subligual, intranasal or transdermal.
At least one protein scaffold composition can be prepared for use in the form of tablets or capsules; nasal powders, drops or aerosols; a gel, ointment, lotion, suspension, or incorporated into a therapeutic bandage or "patch" delivery system as known in the art.
The invention provides stable formulations of a protein based scaffold, which is preferably a phosphate-buffered aqueous saline solution or solution.

权利要求:
Claims (1)
[1]
of mixed salt, as well as preserved solutions and formulations, as well as multi-purpose preserved formulations, suitable for pharmaceutical or veterinary use, which comprise at least one protein based scaffold in a pharmaceutically acceptable formulation.
Suitable vehicles and their formulation, including other human proteins, e.g., human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, D.B. ed., Lipincott Williams and Wilkins, Philadelphia, PA, USA 2006, Part 5, Pharmaceutical Manufacturing, pages 691 to 1092, See specifically pages 958 to 989. The compositions can be used with, or incorporated into a formulation single, other actives known to be beneficial in treating the disorder, condition, or disease or it can be an active tested by preparing framework-based protein combinations with innovative compositions and actives. Although the invention has been described in general terms, embodiments of the invention will be further described in the following examples, which are not to be understood as limiting the scope of the claims.
Example 1. Construction of Fc 20 Glycosylation Variants Tencon Design The third FN3 domain of human Tenascin (SEQ ID NO: 3) can be used as an alternative framework capable of being engineered to bind to specific target molecules via surface exposed loops, structurally analogous to the complementarity determining regions (CDR) of the antibody.
The melting temperature of this domain is 54°C in PBS, in its native form.
To produce a scaffold molecule with similar structure and improved physical properties, such as optimized thermal stability, a consensus sequence was designed based on an alignment of 15 FN3 domains obtained from human Tenascin 30 (SEQ ID NOs: 1 to 15) . Analysis of the multiple sequence alignment in Table 1 shows that these 15 domains have sequence identities to each other in the range.
xa from 13 to 80%, with a mean sequence identity between pairs of 29%. A consensus sequence (SEQ ID NO: 16) was designed by incorporating the most conserved (frequent) amino acid at each position of the alignment shown in Table 1. In paired alignments, the consensus sequence of the present invention (SEQ ID NO :16), designated as Tencon, is identical to the FN3 domains of Tenascin at 34 to 59% of positions, with an average sequence identity of 43%. Protein expression and purification The Tencon amino acid sequence (SEQ ID NO: 16) was back-translated, resulting in the DNA sequence shown in SEQ ID NO: 17. This sequence was assembled by PCR overlay, subcloned into a vector modified pET15, transformed into E. coli BL21 Star(DE3) (Invitrogen) and applied to LB agar plates containing 75 µg/ml carbenicillin.
A single colony was selected and cultured overnight at 37°C in 50 ml of TB medium containing 2% glucose and 100 µg/ml carbenicillin.
This culture was used to seed 500 ml of self-induction medium (Overnight Express Instant TB medium, Novagen) in a 2.5 L Ultra Yield flask (Thomson Instrument Company). Proliferation and expression were done using a dual program (3 hours at 37°C, 300 rpm, followed by 16 hours at 30°C, 250 rpm) in an ATR Multitron shaking incubator.
The culture was harvested and centrifuged at 7,000 rpm for 15 minutes in a JL8.1 rotor to form pellets with the cells.
Cells were resuspended in 30 ml of buffer containing 20 mM sodium phosphate, pH 7.5, 500 mM NaCl, 10% glycerol, 20 mM imidazole, 0.37 mg/ml lysozyme, Complete Protease Inhibitor 1X (EDTA-free; Roche) and Benzonase (Sigma-Aldrich, 0.25 µl/ml final) and lysed with a Misonix XL2020 sonicator for 5 minutes on ice in pulse mode (5 seconds on, 30 seconds off). Insoluble material was removed by centrifugation at 17,000 rpm for 30 minutes in a JA-17 rotor. Tencon protein was purified from the soluble lysate in a two-step chromatographic process.
First, the protein was captured by immobilized metal affinity chromatography, adding 2 ml of Ni-NTA agarose microspheres (Qiagen) to the lysate and placing it on a rocking platform for 1 hour at 4°C.
The resin was then packed into a Poly-Prep column (Bio-Rad) and washed with 20 mM sodium phosphate, pH 7.5, 500 mM NaCl, 10% glycerol and 20 mM imidazole to remove the unbound material.
Proteins were eluted from the resin with 20 mM sodium phosphate, pH 7.5, 500 mM NaCl, 10% glycerol and 500 mM imidazole.
The fractions were analyzed by SDS-PAGE, either with Coomassie staining or with Western blot, using anti-His conjugated antibody HRP-10 (Immunology Consultants Laboratory). The desired fractions were pooled and dialyzed in PBS at pH 7.4. As a second purification step, the protein was loaded onto a Superdex-75 HiLoad 16/60 column (GE Healthcare) equilibrated in PBS.
Fractions were analyzed by SDS-PAGE, and those containing Tencon were pooled and 15 concentrated using a Centriprep UltraCel YM-3 concentrator (A-micon). Protein concentration was determined using a BioTek plate reader to measure sample absorbance at 280 nm.
The final preparation was analyzed by Coomassie staining (figure 1), Western blot 20 with anti-His antibody, and by HPLC-SEC using a G3000SW-XL column (TOSOH Biosciences) equilibrated in PBS.
Analysis by SDS-PAGE shows that Tencon migrates between 6 and 14 kDa, according to the expected mass of 10.7 kDa for the monomeric protein.
A yield of >50 mg of pure Tencon protein per liter of culture was obtained. 25 Biophysical characterization Tencon's structure and stability were characterized by circular dichroism spectroscopy and differential scanning calorimetry, respectively.
Circular dichroism measurements were made on an AVIV spectrometer at 20°C in PBS, and with a concentration of 0.2 mg/mL. 30 The spectrum in Figure 8 shows a minimum at 218 nm, which suggests a beta-sheet structure, as expected for a protein belonging to the FN3 family, as designed.
Scanning calorimetry data differs.
were obtained by heating solutions to 0.5 mg/ml of the 3rd FN3 domain of Tenascin or Tencon in PBS, from 35°C to 95°C, at a speed of 1°C/minute in an N- calorimeter DSCII (Applied Thermodynamics). First, the curve for the bulk buffer block was subtracted to yield the profiles shown in Figure 3. From these data, melting temperatures of 54°C and 78°C were calculated for the 3rd domain FN3 and for Tencon, respectively, using the CpCalc (Applied Thermodynamics) software. The folding and unfolding of both domains is reversible at these temperatures. 10 Immunogenicity analysis A computer program that models the immunogenicity of amino acid sequences for humans was used to compare the predicted immunogenicity of amino acid sequences representing the 3rd FN3 domain of human Tenascin, Tencon, and various therapeutic antibodies ( shown in Table 2). Chimeric mAbs and a human mAb (adalimumab) analyzed with the program were followed by applying a limit of tolerance (remove 9 mer peptides with 100% identity to sequence encoded by human germline). The tolerance limit has not been applied to Tenascin or Tencon.
Tolerance limit 20 assumes broad tolerance of T cells to germline-encoded mAb sequences, and focuses analysis on novel sequences, primarily on CDRs and flanking domains.
These analyzes predict a low immunogenic risk for both Tenascin and Tencon based on the probability that a 9 mer peptide, derived from the analyzed sequence, will bind to one or more HLA molecules.
The score is weighted against the prevalence of each HLA allele.
The scores for the models were summed for each sequence to obtain a single number describing the overall PIR for each sequence (sum of scores). The results of this analysis are summarized in Table 2. Tenascin had the lowest total score (11.9). Tencon, like Tenascin, scored mainly in non-binding and predicted low immunogenic risk aggretopes (score = 13.2). Tenascin and Tencon sequences scored favorably compared to therapeutic antibodies.
Display of Tencon in M13 phage by pIX fusion The gene encoding the Tencon amino acid sequence was subcloned by PCR into the phagemid expression vector pPep9 and by restriction digestion cloning, resulting in the vector pTencon-pIX.
This system expresses Myc-tagged Tencon at the amino-terminus as a carboxy-terminal to amino-terminal fusion of the M13 pIX protein (figure 4). The Lac promoter allows for lower expression levels without IPTG and increased expression after addition of IPTG.
The OmpA signal sequence was attached to the amino terminus of Tencon to promote efficient translocation to the periplasm.
A short TSGGGGS connector (SEQ ID NO: 141 was constructed between Tencon and pIX to prevent steric interactions between these proteins.
To confirm the surface display of 15 M13 phage particles, pTencon-pIX was transformed into E. coli XL1-Blue, and a single colony was used to inoculate a 5 mL culture of LB supplemented with ampicillin.
This culture was grown at 37°C until reaching the mid-log phase, at which point 610 PFU of VCSM13 helper phage were added, and the culture was incubated at 37°C for 10 minutes without shaking, followed by 50 20 minutes with shaking.
The recovered helper phage culture was then diluted in 50 mL of 2YT medium supplemented with ampicillin and kanamycin, and grown at 37°C under agitation until the OD600 reached 0.7, at which point IPTG was added to a final concentration of 1 mM, the temperature being reduced to 30°C.
After 16 hours, the culture was centrifuged at 4,000 X g for 25-20 minutes, and the supernatant was collected and stored at 4°C for analysis.
Binding of the phage particles to an anti-Myc antibody (Invitrogen) was used to confirm the display of the Myc-Tencon construct on the surface of M13 phage. A Maxisorp plate was coated overnight at a concentration of 2.5 ug/ml with -Myc or an anti-v 30 antibody (negative control) and blocked with SuperBlock T20 (Pierce). Serial twofold dilutions of the phagemid culture supernatant described above were made in PBS and added to the wells of the coated plate.
After 1 hour,
the plate was washed with TBST and an anti-M13 HRP antibody was added to each well and washed with TBST following a 1 hour incubation.
Roche BD ELISA POD substrate was added, and luminescence was detected in a plate reader (Tecan). Figure 5 shows that Myc-Tencon 5 phage particles bind to anti-myc coated wells, but not to those with antiv or to uncoated control wells on the plate, in a concentration-dependent manner, confirming the specific display of Myc-Tencon on M13 phage particles. An additional phagemid vector can be constructed to display 10 Tencon and library members (see Example 2) on M13 phage as fusions to coat the pIII protein.
For this system, the gene for pIX is replaced by a gene encoding a truncated version of pIII (Bass et al., 1990). Additional changes compared to the system shown in Figure 4 include the replacement of the OmpA signal sequence by the 15 signal sequence for DsbA, as secretion using this sequence has been shown to be beneficial for the display of alternative scaffold molecules stable (Steiner et al., 2006). Example 2: Generation of Tencon Libraries Tencon variant libraries can be made using many different methods, depending on the desired complexity and the relative location of the mutations in the molecule.
DNA synthesis methods are preferred to create mutations spread throughout the Tencon gene.
Restriction enzyme cloning can also be used to recombine DNA fragments containing mutations in different regions of the gene.
Saturation mutagenesis in a small defined region, such as a single Tencon loop, can be introduced through the use of a degenerate oligonucleotide and direct oligonucleotide mutagenesis (Kunkel et al., 1987). A Tencon library, the FG7 library, was constructed, designed to replace the FG loop with 7 random amino acids using oligonucleotide-directed mutagenesis.
An oligonucleotide (TconFG7-For-5'pho) was synthesized to have a 21 base pair (bp) degenerate sequence of NNS at the positions encoding the FG loop and two flanking nucleotide sequences with 20 to 27 bp of complementarity with the Tencon encoding sequence. In this design, all twenty amino acids are capable of representation in the FG loop. The calculated diversity at the 5 nucleotide level is 1.3 x 109. TconFG7-For5’pho: (SEQ ID NO: 18)
GAATACACCGTTTCTATCTACGGTGTTNNSNNSNNSNNSNNSNNSNNSC
CGCTGTCTGCGGAATTCAC The template for oligonucleotide-directed mutagenesis, pDsbA-10 Tencon-Asc-loop-Myc-pIII, was constructed by replacing the Tencon F:G loop encoding the sequence with a stem-loop sequence containing an AscI restriction site . This system allows for the elimination of initial template DNA after mutagenesis by digesting the resulting DNA with AscI prior to transformation. To purify a single-stranded DNA template 15 for mutagenesis, a single colony of E. coli CJ236 bearing pDsbA-Tencon-Asc-loop-Myc-pIII was placed in 5 ml of 2YT growth medium with carbenicillin (50 ug/ ml final concentration) and chloramphenicol (10 ug/ml). After 6 hours, VCSM13 helper phage was added to a final concentration of 1010 PFU/ml, and incubated without shaking for 10 minutes, before being transferred to 150 ml of 2YT with carbenicillin (10 ug/ml) and uridine (0 .25 µg/ml) and incubated at 37°C with shaking at 200 rpm, overnight. Cells were pelleted by centrifugation, supernatant was collected, and phage were pelleted with PEG NaCl. Single-stranded DNA was purified from this pellet using a QIAprep 25 Spin M13 kit (Qiagen) according to the manufacturer's instructions. For annealing ligation of the degenerate oligonucleotide to the template, 5 µg of the template DNA was combined with oligo TconFG7-For-5-pho at a 10:1 molar ratio in Tris-HCl (50 mM, pH 7.5) and MgCl 2 (10 mM), with incubation at 90°C for 2 minutes, 60°C for 3 minutes, 30 and 20°C for 5 minutes. After the annealing reaction, ATP (10 mM), dNTPs (25 mM each), DTT (100 mM), T4 ligase (7 units) and T7 DNA polymerase (10 units) were added to the reaction mixture, with incubation -
at 14°C for 6 hours, followed by 20°C for 12 hours.
The resulting DNA was purified using a PCR purification kit (Qiagen), and recovered in 100 uL of water.
Library DNA was digested with 10 units AscI for 4 hours and then purified again with Qiagen's PCR purification kit 5 .
The final DNA from the library was recovered in 50 µl of water.
The resulting double-stranded DNA product was then transformed into E. coli MC1061F’ by electroporation.
Transformants were collected in 20 ml of SOC medium, and allowed to recover for 1 hour at 37°C.
At the end of recovery, a 10 aliquot of the transformation was serially diluted and applied to plates with carbenicillin (100 ug/ml) containing 1% glucose to assess the total number of transformants.
The remainder of the SOC culture was then used to inoculate 1 L of 2xYT medium with carbenicillin and 1% glucose, being grown until OD600 reached 0.6. 100 ml of this culture was inoculated with 15 M13 helper phage at 1010/ml and incubated at 37°C before centrifugation.
The resulting cell pellet was resuspended in 500 ml of unused 2xYT medium containing carbenicillin (100 ug/ml) and kanamycin (35 ug/ml) and cultured at 30°C overnight prior to centrifugation.
Phage particles were precipitated by the addition of PEG/NaCl and stored at -80°C.
A second library, BC6/FG7, was designed to simultaneously introduce diversity into Tencon's B:C and F:G loops.
For that, two oligonucleotides were synthesized: Tc-BC6-For-5’phos and POP149. The forward oligo was phosphorylated and contained 18 NNS codon bases at each position encoding the B:C loop, while the reverse oligo was biotinylated at the 5' end and contained 21 NNS codon bases at each encoding position the F:G loop.
Both oligonucleotides are flanked by two 18 bp nucleotide sequences identical to the region preceding and following the region to be mutagenized (see below for primer details). Tc-BC6-For-5’phos: (SEQ ID NO: 19) gactctctgcgtctgtcttggNNSNNSNNSNNSNNSNNSTTCGACTCTTTCCTGATC
CAGTACC POP 2149: (SEQ ID NO: 20)
GTGAATTCCGCAGACAGCGGSNNSNNSNNSNNSNNSNNSNNAACACCG
TAGATAGAAACGGTG 5 To build the library, sixteen 100 uL PCR reactions were performed using oligos Tc-CB6-For5'phos and POP2149 to amplify the DNA template for Tencon, introducing NNS codons into the B:C and F: loops. G simultaneously in the process. The double-stranded PCR product was mixed with streptavidin magnetic beads (Dynal) in 10 B&W buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 M NaCl, 0.1% Tween-20) and incubated for 20 minutes, pulled down with a magnet and washed twice with B&W buffer. The direct strand was eluted from the microspheres with 300 uL of 150 mM NaOH. This "megaprimer", a mixture of long primers of more than 8 x 1016 in theoretical diversity, was used for annealing binding to a single-stranded library template. The construction of the library was carried out as described above for the FG7 library. Example 3: IgG Ligand Selection To perform selections of Tencon 20 library members that bind IgG, a recombinant IgG (human IgG1 subtype) was biotinylated using sulfo-NHS-LC-biotin (Pierce) beforehand. of dialysis in PBS. For selections, 200 µl of phages displaying the FG7 or BC6/FG7 libraries were blocked with 200 µl of chemical blocker before addition of biotinylated IgG at concentrations of 500 nM (cycle 1) or 100 nM (cycles 2 and 3). 25 Bound phage were recovered by magnetic microspheres with neutrovidin (Seradyne) in cycle 1, or with magnetic microspheres with streptavidin (Promega) in cycles 2 and 3. Unbound phage were washed from the microspheres using 5 to 10 washes with 1 ml of Tris-buffered saline with Tween (TBST), followed by two washes of 1 ml with 30 ml of Tris-buffered saline (TBS). Bound phage were eluted from microspheres by the addition of mid-log phase of E. coli MC1061F’. Infected cells were plated on LB agar plates supplemented with carbohydrate.
benicilline and glucose.
The next day, cells were scraped from the plate and grown to mid-log phase, before recovery with VCSM13 helper phage and overnight cultivation.
The phage particles were isolated by precipitation in PEG/NaCl and used for the next round of selections.
After 3 cycles of panning against IgG, the result was subcloned into a pET27 vector modified to include a ligase-independent cloning site, by PCR amplification of the Tencon gene.
This PCR product was annealed to the vector and transformed into 10 BL21-GOLD(DE3) cells (Stratagene). Individual colonies were collected in 1 mL cultures in 96-deep well plates (Corning) and grown to saturation overnight at 37°C.
The next day, 50 microL of the overnight culture was used to inoculate a 1 mL culture without prior use.
Cultures were grown at 37°C for 15 ± 2 hours before adding 1 mM IPTG and lowering the temperature to 30°C.
Cells were collected by centrifugation 16 hours after induction, and lysed with 100 microL of BugBuster (Novagen). The resulting lysates were clarified by centrifugation and used to test for IgG binding by ELISA. 20 Maxisorp plates (Nunc) were coated with 0.1 µg of anti-HIS antibodies (Qiagen), left overnight, washed with TBST and blocked with Starting Block T20 (Thermo Scientific). Clarified lysates diluted 1:4 in Starting Block were added to the plates and allowed to bind for 1 hour before washing with TBST.
Biotinylated IgG or biotinylated HSA was added at a concentration of 1 µg/ml, washing with TBST after 1 hour of incubation.
Detection of bound IgG or HSA was achieved by addition of streptavidin-HRP (Jackson Immunoresearch), followed by detection with POD substrate for chemiluminescence.
The results of the ELISA test are shown in Figure 7. The constructs that bound biotinylated IgG more than 10 times compared to biotinylated HSA, as judged by the signal from the ELISA assay, were sequenced.
After completion of several selection experiments, 60 unique binding sequences of bi-
FG7 library and 10 unique sequences from BC6FG7 library were obtained; Table 4 shows representative sequences of IgG binders in which the B:C and/or F:G loops are shown to the extent that they become different from those of SEQ ID NO:16. well, numerous mutations in other regions of the framework. The Tencon protein designed, expressed and purified in the present invention has improved thermal stability at 26°C over that of the 3rd FN3 domain of human Tenascin, which was used as an alternative scaffold molecule. Based on this increased stability, this scaffold molecule is likely to be more compatible with amino acid substitution, and easier to manufacture. Mutations that decrease protein stability tend to be better tolerated in the context of a more stable framework, and therefore a framework with optimized stability is likely to produce more functional and well-folded ligands from a library of strain variants. framework.
Table 1 (1) 1 10 20 30 40 50 60 70 80 90 100 1 (1) ---SPPKDLVVTEVTEETVNLAWDN-EMRVTEYLVVYTPTH--EGGLEMQFRVPGDQTSTIIQELEPGVEYFIRVFAILENKKSIPVSARVAT------- 2 (1) TYLPAPEGLKFKSIKETSVTEYLVVYTPTH--EGGLEMQFRVPGDQTSTIIQELEPGVEYFIRVFAILENKKSIPVSARVAT------- 2 (1) TYLPAPEGLKFKSIKETSVTEYNVEGFRLHQRGNT ) --- DAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKD - 4 VPGDRTTIDLTEDENQYSIGNLKPDTEYEVSLISRRGDMSSNPAKETFTT ------- (1) TGLDAPRNLRRVSQTDNSITLEWRNGKAAIDSYRIKYAPISGGDHAEVDVPKSQQATTKTTLTGLRPGTEYGIGVSAVKEDKESNPATINAATELDTPKD 5 (1) --- ---- DTPKDLQVSETAETSLTLLWKTPLAKFDRYRLNYSLPT GQWVGVQLPRNTTSYVLRGLEPGQEYNVLLTAEKGRHKSKPAKSKPARVK ----- 6 (1) -QAPELENLTVTEVGWDGLRLNWTAADQAYEHFIIQVQEAN - KVEAARNLTVPGSLRAVDIPGLKAATPYTVSIYGVIQGYRTPVLSAEASTGE --- - 7 (1) -ETPNLGEVVVAEVGWDALKLNWTAPEGAYEYFFIQVQEAD - TVEAAQNLTVPGGLRSTDLPGLKAATHYTITIRGVTQDFSTTPLSVEVLTE ------ 8 (1) -EVPDMGNLTVTEVSWDALRLNWTTPDGTYDQFTIQVQEAD - QVEEAHNLTVPGSLRSMEIPGLRAGTPYTVTLHGEVRGHSTRPLAVEVVTE ------ 9 (1) -DLPQLGDLAVSEVGWDGLRLNWTAADNAYEHFVIQVQEVN - KVEAAQNLTLPGSLRAVDIPGLEAATPYRVSIYGVIRGYRTPVLSAEAS TAKEPE-- 10 (1) -KEPEIGNLNVSDITPESFNLSWMATDGIFETFTIEIIDSN - RLLETVEYNISGAERTAHISGLPPSTDFIVYLSGLAPSIRTKTISATATTE ------ 11 (1) -ALPLLENLTISDINPYGFTVSWMASENAFDSFLVTVVDSG - KLLDPQEFTLSGTQRKLELRGLITGIGYEVMVSGFTQGHQTKPLRAEIVTE ------ 12 (1) -AEPEVDNLLVSDATPDGFRLSWTADEGVFDNFVLKIRDTK - KQSEPLEITLLAPERTRDLTGLREATEYEIELYGISKGRRSQTVSAIATTAM ----- 13 (1 ) --- --- GSPKEVIFSDITENSATVSWRAPTAQVESFRITYVPITG GTPSMVTVDGTKTQTRLVKLIPGVEYLVSIIAMKGFEESEPVSGSFTTAL ----- 14 (1) --- ---- DGPSGLVTANITDSEALARWQPAIATVDSYVISYTGEK VPEITRTVSGNTVEYALTDLEPATEYTLRIFAEKGPQKSSTITAKFTTDL ----- 15 (1) DSPRDLTATEVQSETALLTWRPPRASVTGYLLVYESVD --- ---- ---- Table GTVKEVIVGPDTTSYSLADLSPSTHYTAKIQALNGPLRSNMIQTIFTTIGL 2. Sequence Description Sum of 1st Sum of 2nd Sum of scores Sum of scores score scores (chain) ions (molecule) Tenascin Alternate framework 6.01 5.85 11.86 11.86 Tencon Alternate framework 5.83 7 .37 13.20 13.20 adalimumab Vh humanized mAb 9.45 8.06 17.50 45.42 Vl 15.29 12.63 27.92 cetuximab Vh chimeric mAb 17, 63 16.89 34.52 64.44 Vl 14.45 15.47 29.92 Rituximab Chimeric Vh mAb 16.57 14.38 30.96 61.65 Vl 16.63 14.06 30.69 basiliximab Chimeric Vh mAb 16.48 13.40 29.89 58.98 Value 16.05 13.05 29.09
Table 3. Loops Loop Residues of SEQ ID NO: 16 Amino acid sequence A-B 13-16 TEDS B-C 22-28 TAPDAAF C-D 38-43 SEKVGE D-E 51-54 GSER E-F 60-64 GLKPG F-G 75-81 KGGHRSN
Table 4. IgG Linked Frameworks Clone No. Loop B:C Loop Residues F:G Mutation Residues 22 to 28 (SEQ ID NO) 75 to 81 (SEQ ID NO) framework 1 SYGFNN (21) QIGPIIP (46) 2 TYEGES (22) QIGPIIP (46) 3 TYESES (23) QIGPIIP (46) 4 TNWMDS (24) SIRTIDS (47) 5 KSVFIM (25) PKFHSPL (48) 6 YSSYAT (26) WKTTIWF (49) 7 RFHPFP (27) RKNWKTR (50) 8 MMCMPL (28) RLFRIYQ (51) 9 YCRVRD (29) WLSRSYD (52) 10 SYGFNN (21) WLSRSYD (52) 11 MDCFMG (30) WLSRSCD (53) 12 TYRFNS (31) WMGPYCD (54) 13 ASRRSL (32) RRRRYSF (55) 14 TIESES (33) HIVPMVP (56) 15 TL*MQS (34) QIEPIIR (57) 16 IYDSES (35) PSAANNP (58) 17 VRLRYVQ (59) 18 QVGPLIP (60) 19 RIGPILP ( 61) 20 QIGPLLP (62) 21 RIGPLLP (63) 22 QVGPLLP (64) 23 RIGPMLP (65) 24 QIGPVLP (66) 25 RIGPVLP (67) 26 QIGPMMP (68) 27 QVGPLVP (69) 28 QIGPMLP (70) R18P 29 QVGPILP (71) 30 QVGPLLP (64) 31 QVGPMLP (72) 32 QIGPIVP (73) I33V 33 MIGPLLP (74) 34 QIGPLFP (75) 35 QIGPVLP (66) T59A 36 QIGPMVP (76) 37 QIGPIVP (77)
38 RIEPILP (78) V74G 39 VAGSVWP (79) 40 REGATLY (80) 41 KQIPPIL (81) S38G 42 LSLSSVL (82) 43 HMLLPLP (83) V74A 44 MIGPLIP (84) 45 TIGPHIP (85) 46 EIGPCLP (86) 47 EIGPVLP (87) 48 KIGPILP (88) Y35H 49 MIGPVLP (89) 50 QIGPILP (90) S52P 51 QIGPILP (90) Q36R 52 QIGPILP (90) 53 EVGPILP (91) 54 QVGPLLP (92) A23T 55 QIGPILP (93) 56 QIGPCVP ( 94) 57 QIGPLVP (95) 58 RGLVMPM (96) V74A 59 MIGPILP (97) 60 QIGPILP (90) E37G 61 QIGPILP (90) T68A 62 QIGPILP (90) T22I 63 QIGPILP (90) S52F 64 QIGPILP (90) Y56H 65 QIGPILP (90) A44V 66 QIGPILP (90) P24S 67 RIGPILP (61) 68 CIGPMVP (98) 69 FIGPVLP (99) 70 HIGPILP (100) 71 HIGPIMP (101) 72 HIGPYLP (102) 73 HVGPILP (103) 74 IIGPLLP (104) 75 LIGPLLP (105) 76 MVGPLLP (106) 77 NIGPYLP (107) 78 NIGPYLP (108) 79 QIGPHLP (109) 80 QIGPIIP (46) 82 QIGPILG (110) 83 QIGPILS (111) 83 QIGPILT (112) 84 QIGPIMP (113) 85 QIGPIPI (114) 86 QIGPLN (115) 87 QIGPLLP (62) 88 QIGPVFP (116) 89 QIGPVLS (117)
90 QIGPWLP (118) 92 QVGPILP (71) 93 QVGPILR (118) 94 QVGPIMN (119) 95 QVGPIMP (120) 96 QVGPIMP (121) 97 QVGPLLS (122) 98 QVGPVLP (123) 99 QVGPVLT (124) 100 RIGPIMP (125) 101 RIGPIVP (126) 102 RIGPMFP (127) 103 RIGPMIP (128) 104 RIGPMVP (129) 105 RIGPVIP (130) 106 RVGPILP (131) 107 RVGPLLP (132) 108 TVGPHIP (133) 109 DRKRFI (36) PSWRSNW (134) 110 EFWRGS (37) QIGPLLP (62) 111 GLLDPL (38) ALRATLE (135) 112 GLLVPE (39) KYGYLTP (136) 113 MASDGL (40) RIGPMLP (137) 114 NKTETN (41) NPFCSRF (138) 115 QAERKV (42) QIGPLLP (62) 116 QAERKV (42) RIGPLLP (63) 117 SQVCTL (43) YYLHQWC (139) 118 YFDKDS (44) QIGPLLP (62) 119 YFECEP (45) HIVPLLR (140)
Sequences: SEQ ID NO. 1: sppkdlvvtevteetvnlawdnemrvteylvvytpthegglemqfrvpgdqtstiiqelepgveyfirvfaile nkksipvsarvat 5 SEQ ID NO. 2: tylpapeglkfksiketsvevewdpldiafetweiifrnmnkedegeitkslrrpetsyrqtglapgqeyeislh ivknntrgpglkrvtttrld SEQ ID NO. 3: dapsqievkdvtdttalitwfkplaeidgieltygikdvpgdrttidltedenqysignlkpdteyevslisrrgd 10 mssnpaketftt SEQ ID NO. 4 tgldaprnlrrvsqtdnsitlewrngkaaidsyrikyapisggdhaevdvpksqqattkttltglrpgteygigv savkedkesnpatinaateldtpkd
SEQ ID NO. 5 dtpkdlqvsetaetsltllwktplakfdryrlnyslptgqwvgvqlprnttsyvlrglepgqeynvlltaekgrhks kpakskparvk SEQ ID NO. 6 5 qapelenltvtevgwdglrlnwtaadqayehfiiqvqeankveaarnltvpgslravdipglkaatpytvsiy gviqgyrtpvlsaeastge SEQ ID NO. 7 etpnlgevvvaevgwdalklnwtapegayeyffiqvqeadtveaaqnltvpgglrstdlpglkaathytitirg vtqdfsttplsvevlte 10 SEQ ID NO. 8 evpdmgnltvtevswdalrlnwttpdgtydqftiqvqeadqveeahnltvpgslrsmeipglragtpytvtlh gevrghstrplavevvte SEQ ID NO. 9 dlpqlgdlavsevgwdglrlnwtaadnayehfviqvqevnkveaaqnltlpgslravdipgleaatpyrvsi 15 ygvirgyrtpvlsaeastakepe SEQ ID NO. 10 kepeignlnvsditpesfnlswmatdgifetftieiidsnrlletveynisgaertahisglppstdfivylsglaps irtktisatatte SEQ ID NO. 11 20 alpllenltisdinpygftvswmasenafdsflvtvvdsgklldpqeftlsgtqrklelrglitgigyevmvsgftq ghqtkplraeivte SEQ ID NO. 12 aepevdnllvsdatpdgfrlswtadegvfdnfvlkirdtkkqsepleitllapertrdltglreateyeielygiskg rrsqtvsaiattam 25 SEQ ID NO. 13 gspkevifsditensatvswraptaqvesfrityvpitggtpsmvtvdgtktqtrlvklipgveylvsiiamkgfe esepvsgsfttal SEQ ID NO. 14 dgpsglvtanitdsealarwqpaiatvdsyvisytgekvpeitrtvsgntveyaltdlepateytlrifaekgpq 30 ksstitakfttdl SEQ ID NO. 15 dsprdltatevqsetalltwrpprasvtgyllvyesvdgtvkevivgpdttsysladlspsthytakiqalngplr snmiqtifttigl SEQ ID NO. 16
LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGS
ERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT 5 SEQ ID NO. 17 ctgccggcgccgaaaaacctggttgtttctgaagttaccgaagactctctgcgtctgtcttggaccgcgccg gacgcggcgttcgactctttcctgatccagtaccaggaatctgaaaaagttggtgaagcgatcaacctgac cgttccgggttctgaacgttcttacgacctgaccggtctgaaaccgggtaccgaatacaccgtttctatctac ggtgttaaaggtggtcaccgttctaacccgctgtctgcggaattcaccacc 10 Tencon sequence showing loops (SEQ ID NO: 16) AB BC CD 1-LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEA-44 EF FG 45-INLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT-89 Example 4: Tencon mutations Stabilization mutants were engineered to optimize stability Tencon framework folding tools previously described in this document (SEQ ID NO: 16). Several point mutations were made to produce the individual residue substitution of SEQ ID NO: 16, such as N46V (Ten-con17 - SEQ ID NO:142), E14P (Tencon18 - SEQ ID NO:143), E11N (Ten-con19 – SEQ ID NO:144), E37P (Tencon20 – SEQ ID NO:145), and G73Y (Tencon21 – SEQ ID NO: 146) which were predicted to improve stability by the PoPMuSiC v2.0 program (Dehouck, Grosfils et al. 2009). It was previously discovered that the E86I mutant (Tencon22 – SEQ ID NO: 147) stabilizes a homologous protein, the 3rd FN3 domain of human Tenascin (WO2009/086116A2). Finally, the L17A mutation was found to significantly stabilize Tencon during alanine scanning experiments in which all Tencon loop residues were independently replaced with alanine (data not shown). Following an initial round of stability testing (see below), the mutants with-
binatorials N46V/E86I (Tencon 23 - SEQ ID NO: 148), E14P/N46V/E86I (Tencon24 - SEQ ID NO: 149), and L17A/N46V/E86I (Tencon25 - SEQ ID NO: 150) were produced to further increase stability.
Expression and purification 5 Mutations in the Tencon coding sequence were made using a QuikChange mutagenesis kit (Stratagene). The resulting plasmids were transformed into BL21-GOLD (DE3) E. coli (Stratagene) for expression.
A single colony was selected and grown overnight at 37°C in 2 ml of TB medium containing 100 µg/ml ampicillin. This culture was used to seed 100 ml of self-inducing medium (Overnight Express medium Instant TB, Novagen) in a 500 mL vial with flow stoppers for mixing contents and cultured at 37°C for 16 hours.
The culture was harvested by centrifugation at 4000xg for 20 minutes and the pelleted cells resuspended in 5 ml of BugBuster HT (Novagen) per gram of wet cell pellet.
After 30 minutes of incubation at room temperature, the lysates were clarified by centrifugation at 30,000xg for 20 minutes and loaded onto a 3 mL Ni-NTA superflux column (Novagen) by gravity.
After loading, each column was washed with 15 mL of a buffer containing 50 mM sodium phosphate at pH 7.4, 500 mM NaCl, and 10 mM imidazole.
Bound protein was then eluted from the column using 10 ml of a buffer containing 50 mM sodium phosphate pH 7.4, 500 mM NaCl, and 250 mM imidazole.
Protein purity was assessed by SDS-PAGE.
Before biophysical analysis, each mutant was dialyzed completely in PBS at pH 7.4. 28 to 33 mg of purified protein were obtained for each mutant from 100 ml of culture.
Characterization of Thermal Stability The thermal stabilities of the source Tencon and of each mutant were measured by differential scanning capillary calorimetry (CVD). Each sample was extensively dialyzed in contact with PBS at pH 7.4 and diluted to a concentration of 2-3 mg/mL.
Melting temperatures were measured for these samples using an instrument
VP-DSC equipped with an autosampler (MicroCal, LLC). The samples were heated from 10°C to 95°C or 100°C at a rate of 1°C per minute.
A buffer scan was just completed between each sample scan to calculate a baseline for data integration. 5 Data were fitted to a two-state unfolding model followed by subtraction of the only buffer sign.
Reversibility of thermal denaturation was determined by repeating the scan for each sample without removing it from the cell.
Reversibility was calculated by comparing the area under the curve of the 1st scan with the 2nd scan.
The results of the DSC experiments are shown in Table 5 as the values derived from complete melting curves.
The simple mutants Tencon17, Tencon18, Tencon19, and Tencon22 optimized thermal stability compared to the source tencon sequence.
Only Tencon21 was significantly destabilizing.
The Tencon23, Tencon24, and Tencon25 combinatorial mutant samples all had a significantly greater improvement in stability, indicating that the projected mutations are additive with respect to the optimization of thermal stability.
Guanidine Hydrochloride Denaturation The abilities of Tencon and each mutant to remain doubled in treatment with increasing concentrations of guanidine hydrochloride (GdmCl) as measured by tryptophan fluorescence were used to assess stability.
Tencon contains only one tryptophan residue.
The tryptophan residue is buried in the hydrophobic core and thus the fluorescence emission at 360 nm is a sensitive measure of the folded state of this protein. 200 uL of a solution containing 50 mM sodium phosphate in 7.0, 150 mM NaCl, and varying concentrations of GdmCl from 0.48 to 6.63 M were pipetted into black 96-well plates, without ligation ( Greiner) to produce a 17-point titer. 10 l of a solution containing the tencon mutants was added to each well across the entire plate to produce a final protein concentration of 23 M, and mixed by pipetting gently up and down.
After incubation at room temperature for 24 hours, fluorescence was read using a Spectramax M5 plate reader (Molecular Devices) with excitation at 280 nm and emission at 360 nm.
The data generated from these curves are shown in Figure 8. The fluorescence signal was converted to a split fraction using the equation (Pace 1986 Methods Enzymol, volume 131, pages 266 to 280):
Where yF is the fluorescence signal from the folded sample and yu from the unfolded sample.
The midpoints of the split transition and the transition slope were determined by fitting with the equation below 10 (Clarke, Hamill et al. 1997):
Where F is the fluorescence at a given denaturant concentration, N and D are the y-intercepts of the native and denatured state, N and D are the baseline slopes for the native and denatured state, [D ] is the concentration of GdmCl, [D]50% the concentration of GdmCl at the point where 50% of the sample is denatured, m the angular coefficient of transition, R the gas constant, and T the temperature.
The free bending energy for each sample was estimated using the equation (Pace 1986 supra; Clarke, Hamill et al. 1997 J Mol Biol, volume 270, number 5 pages 771 to 778):
It is often difficult to measure the exact slope of the transition, m, for such curves.
Additionally, the mutations described here are not expected to alter the folding mechanism of tencon.
In this way, the m value for each mutant was measured and the values averaged (Pace 1986 supra) to produce an m = 14.83 kJ/mol/M (3544 cal/mol/M) used for all free energy calculations.
The results of these calculations are shown in Table 5. The results for the GdmCl unfolding experiments demonstrated that the same 20 mutants that stabilize Tencon with respect to thermal stability also stabilize the protein against GdmCl-induced denaturation.
Size Exclusion Chromatography Size exclusion chromatography (CET) was used to assess the aggregation status of WT tencon and each mutant. 5 μl of 5 each sample was injected onto a Superdex 75 5/150 column (GE Healthcare) at a flow rate of 0.3 mL/min with a mobile PBS phase.
Elution from the column was monitored by absorbance at 280 nm.
To assess the aggregation status, the column was previously calibrated with globular molecular weight standards (Sigma). All samples were tested, with the exception of Tencon21, eluting in a peak at an elution volume consistent with that of a monomeric sample.
Tencon21 eluted with 2 peaks, indicating the presence of aggregates.
Table 5. Construct Mutations Tm [D]50% G(H2O) (kJ/mol (kJ (Kcal)) (M) (kcal/mol)) Tencon 16 326.52 (78.04) 3.4 50.21 (12.0) (SEQ ID NO: 16) Tencon17 (SEQ ID NO: 142) N46V 342.59 (81.88) 3.6 53.56 (12.8) Tencon18 (SEQ ID NO: 143) E14P 346 .31 (82.77) 3.5 51.89 (12.4) Tencon19 (SEQ ID NO: 144) E11N 330.54 (79.00) 3.4 50.21 (12.0) Tencon20 (SEQ ID NO: 145) E37P 323.84 (77.40) 3.4 50.21 (12.0) Tencon21 (SEQ ID NO: 146) G73Y 282.67 (67.56) 2.4 35.6 (8, 5) Tencon22 (SEQ ID NO: 147) E86I 346.35 (82.78) 3.7 54.81 (13.1) Tencon23 (SEQ ID NO: 148) N46V/E86I 362.54 (86.65) 4 .1 60.67 (14.5) Tencon24 (SEQ ID NO:149) E14P/N46V/E86I 365.97 (87.47) 4.0 59.41 (14.2) Tencon25 (SEQ ID NO:150) L17A/N46V/E86I 387.98 (92.73) 5.1 75.73 (18.1) Tencon26 (SEQ ID NO: 151) L17A 355.22 (84.9) 4.6 67.78 (16, two)
It will be clear that the invention may be practiced differently from that particularly described in the above-mentioned description and examples.
Numerous modifications and variations of the present invention are possible in light of the above teachings and are, therefore, within the scope of the appended claims.

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法律状态:
2021-07-20| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2021-07-20| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI N? 10196/2001, QUE MODIFICOU A LEI N? 9279/96, A CONCESS?O DA PATENTE EST? CONDICIONADA ? ANU?NCIA PR?VIA DA ANVISA. CONSIDERANDO A APROVA??O DOS TERMOS DO PARECER N? 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL N? 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVID?NCIAS CAB?VEIS. |

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2021-09-08| B07G| Grant request does not fulfill article 229-c lpi (prior consent of anvisa) [chapter 7.7 patent gazette]|Free format text: NOTIFICACAO DE DEVOLUCAO DO PEDIDO EM FUNCAO DA REVOGACAO DO ART. 229-C DA LEI NO 9.279, DE 1996, POR FORCA DA LEI NO 14.195, DE 2021 |

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2021-09-21| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US32998010P| true| 2010-04-30|2010-04-30|

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US61/329,980|2010-04-30|

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PCT/US2011/034512|WO2011137319A2|2010-04-30|2011-04-29|Stabilized fibronectin domain compositions, methods and uses|

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