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WO2009018438A1 - Découverte de protéine utilisant un affichage de ribosome intracellulaire - Google Patents

Découverte de protéine utilisant un affichage de ribosome intracellulaire Download PDF

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Publication number
WO2009018438A1
WO2009018438A1 PCT/US2008/071747 US2008071747W WO2009018438A1 WO 2009018438 A1 WO2009018438 A1 WO 2009018438A1 US 2008071747 W US2008071747 W US 2008071747W WO 2009018438 A1 WO2009018438 A1 WO 2009018438A1
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protein
construct
acid molecule
deoxyribonucleic acid
cell
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PCT/US2008/071747
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English (en)
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Matthew P. Delisa
Lydia Contreras-Martinez
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Cornell Research Foundation, Inc.
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Priority to US12/671,446 priority Critical patent/US9879252B2/en
Publication of WO2009018438A1 publication Critical patent/WO2009018438A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1041—Ribosome/Polysome display, e.g. SPERT, ARM
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to protein discovery using intracellular ribosome display.
  • Ribosome display is a powerful approach for affinity and stability maturation of recombinant antibodies.
  • ribosome display is performed entirely in vitro, there are several limitations to this approach including technical challenges associated with: (i) efficiently expressing and stalling antibodies on ribosomes using cell-free translation mixtures; and (ii) folding of antibodies in buffers where the concentration and composition of factors varies from that found in the intracellular milieu.
  • scFv single-chain variable fragment
  • V H and V L covalently linked variable domains
  • One aspect of the present invention relates to a method of identifying a protein that binds to a target molecule and has intracellular functionality.
  • This method includes providing a construct comprising a deoxyribonucleic acid molecule encoding the protein which binds to the target molecule, with the deoxyribonucleic acid molecule being coupled to a stall sequence.
  • a host cell is transformed with the construct and then cultured under conditions effective to form, within the host cell, a complex of the protein whose translation has been stalled, the mRNA encoding the protein, and ribosomes.
  • the protein in the complex is in a properly folded, active form and the complex is recovered from the cell.
  • Another aspect of the present invention relates to a construct which includes a deoxyribonucleic acid molecule encoding a protein that binds to a target molecule and an SecM stalling sequence coupled to the deoxyribonucleic acid molecule.
  • the deoxyribonucleic acid molecule and the SecM stalling sequence are coupled with sufficient distance between them to permit expression of their encoded protein, within the cell, in a properly folded, active form.
  • Another aspect of the present invention relates to a method of identifying a protein that binds to a target molecule and has intracellular functionality.
  • This method includes providing a construct comprising a deoxyribonucleic acid molecule encoding the protein which binds to the target molecule, said deoxyribonucleic acid molecule being coupled to a stall sequence.
  • a cell-free extract preparation containing ribosomes is also provided.
  • the method further involves contacting the construct with the cell-free extract preparation containing ribosomes under conditions effective for ribosome translation and the formation of a complex of the protein whose translation has been stalled, the mRNA encoding the protein, and the ribosomes.
  • the protein in the complex is in a properly folded, active form and the complex is recovered.
  • Applicants have developed a novel method for intracellular ribosome display that takes advantage of the recently discovered Escherichia coli SecM translation arrest mechanism. This is the first evidence that the encoding mRNA of SecM-stalled heterologous proteins remains stably attached to ribosomes, thereby enabling creation of stalled antibody-ribosome-mRNA (ARM) complexes entirely inside of living cells.
  • ARM antibody-ribosome-mRNA
  • intracellular ribosome display naturally selects for proteins that are correctly folded and soluble under reducing conditions, in the face of macromolecular crowding and in the presence of all cellular factors (e.g., chaperones, isomerases, proteases, etc.) that impact protein solubility.
  • cytoplasmic stability, and thus intracellular function, of an scFv can be enhanced 2-3 fold after only a single round of mutagenesis and selection using intracellular ribosome display.
  • Figure IA-B illustrate the principle of intracellular ribosome display for affinity and stability maturation of protein (scFv) libraries in accordance with the present invention.
  • a plasmid-encoded scFv library is first amplified by PCR, whereby a flexible linker sequence and SecM17 fusion partner are introduced.
  • scFv-SecM17 fusions is induced; mRNA is transcribed and translated entirely in vivo.
  • RNA is isolated from the dissociated ARM complexes and reverse transcribed to cDNA.
  • Figure IB shows a schematic drawing of unfused and SecM17-fused scFv constructs which include: a FLAG epitope tag (F); the anti- ⁇ -gal scFvl3 sequence; a c-Myc epitope tag (M); a 6x-his tag (H); a thrombin cleavage site (T); a flexible Gly-Ser linker (GS); a SecM stall sequence, FSTPVWISQAQGIRAGP (SEQ ID NO: 1); and a stop codon (star).
  • F FLAG epitope tag
  • M c-Myc epitope tag
  • H 6x-his tag
  • T a thrombin cleavage site
  • GS flexible Gly-Ser linker
  • SecM stall sequence FSTPVWISQAQGIRAGP
  • Figures 2A-B show the in vivo expression of scFv-SecMl 7 fusion proteins, in accordance with the present invention.
  • Figure 2A is a Western blot analysis of soluble fractions isolated from BL21(DE3) E. coli cells, expressing unfused (upper panel) or SecM17-fused (lower panel) wt scFvl3 (wt) or solubility- enhanced scFvl 3-R4 (R4) using anti-FLAG IgG. Induced (+) and uninduced (-) samples are shown for scFvl3-SecM17 fusions. An equivalent amount of total protein was loaded in each lane.
  • Figure 2B shows a normalized ELISA signal from soluble fractions prepared from cells expressing the unfused or SecM17-fused scFv constructs as indicated on ⁇ -gal-coated plates. An equivalent amount of total protein was assayed in each well. Absorbance values for each sample were normalized to the absorbance measured for the value for scFvl3-R4-SecM17. Data is the average of three replicate experiments and error bars represent the standard error of the mean. [0016] Figures 3A-D show that antibody fragments and mRNA are ribosome- associated.
  • FIG. 4D shows the solubility and binding activity of selected scFvl3 fragments. Western blot analysis was conducted for soluble fractions recovered from BL2I (DE3) cells expressing unfused versions of scFvl3 and related variants from plasmid pET28a ( Figure 4A) and pTrc99A ( Figure 4B).
  • Clones scFvl3-Rl, scFvl3-R2, and scFvl 3-R4 were isolated previously after 1, 2, and 4 rounds of evolution, respectively (Martineau, et. al., "Expression of an Antibody Fragment at High Levels in the Bacterial Cytoplasm,” J MoI Biol. 280: 1 17-27 (1998), which is hereby incorporated by reference in its entirety).
  • Clones S20 and S23 represent the two best clones isolated in this study after a single round of mutagenesis.
  • Figure 4C show the results of whole cell ⁇ -gal assays of intact X-gal treated AMEF 959 cells expressing wt scFvl 3 or related variants from plasmid pTrc99A. An equivalent number of cells were analyzed in each well.
  • AMEF ⁇ -gal activation is reported as the change in X-gal hydrolysis for 959 cells expressing an scFvl3 variant normalized to the change in X-gal hydrolysis for 959 cells expressing scFvl 3-R4.
  • Data is the average of six replicate experiments and the error bars represent the standard error of the mean.
  • Absorbance values for each sample were normalized to the absorbance measured for scFvl 3-R4 -expressing cells.
  • Figures 5A-B show the mutations in scFvl 3 fragments selected by intracellular ribosome display.
  • Figure 5A shows the amino acid sequence alignment of wt scFvl3 (SEQ ID NO: 2) and related variants, S20 (SEQ ID NO: 3) and S23 (SEQ ID NO: 4). The sequence of wt scFvl3 is written in single letter amino acid code. Numbering of amino acid residues in V H and V L and the labeling of CDRs is according to Kabat numbering scheme. Immunodetection epitopes are italicized.
  • Figure 5B shows the location of mutations for clones S20 and S23 in scFv!3 structure.
  • the structure was previously modeled by homology (Martineau, et. al., "Expression of an Antibody Fragment at High Levels in the Bacterial Cytoplasm," J. MoI. Biol. 280: 1 17-27 (1998), which is hereby incorporated by reference in its entirety).
  • the drawing was generated with MacPyMOL.
  • the V H is shown in olive, the VL in green, the disulfide bonds in black, and the mutations for clones S20 and S23 in red and purple, respectively.
  • Asterisks indicate mutations that are shared between selected variants and those isolated previously by Martineau, et, al., "Expression of an Antibody Fragment at High Levels in the Bacterial Cytoplasm," J. MoI. Biol 280: 1 17-27 (1998), which is hereby incorporated by reference in its entirety).
  • Figures 6A-D show the isolation of 70S ribosome fractions by sucrose density gradient centrifugation.
  • the absorbance (254 nm) profile of gradient fractions shows accumulation of 70S ribosomes in fractions 23-28 (peak 70S- containing fraction indicated by asterisk).
  • These figures show fractions generated from cells expressing: scFvl3 ( Figure ⁇ A); scFvl3-R4 ( Figure 6B); scFvl 3-SecM17 ( Figure 6C); and scFvl3-R4-SecM17 ( Figure 6D).
  • One aspect of the present invention relates to a method of identifying a protein that binds to a target molecule and has intracellular functionality.
  • This method includes providing a construct comprising a deoxyribonucleic acid molecule encoding the protein which binds to the target molecule, with the deoxyribonucleic acid molecule being coupled to a stall sequence.
  • a host cell is transformed with the construct and then cultured under conditions effective to form, within the host cell, a complex of the protein whose translation has been stalled, the mRNA encoding the protein, and ribosomes.
  • the protein in the complex is in a properly folded, active form and the complex is recovered from the cell.
  • the protein that binds to the target molecule having intracellular functionality of the present invention can include any ligand binding protein. Suitable ligand binding proteins, include high-affinity antibody fragments (e.g., Fab, Fab' and F(ab') 2 ), single-chain Fv antibody fragments, nanobodies or nanobody fragments, fluorobodies, or aptamers.
  • the protein is a single-chain variable fragment antibody, an antibody in which the heavy chain and the light chain of a traditional two chain antibody have been joined to form one chain.
  • a linker peptide is inserted between the two chains to allow for proper folding and creation of an active binding site.
  • the methods of the present invention can be used to generate libraries of single-chain antibodies that are cytoplasmically stable and intracellularly functional. The libraries of single-chain antibodies are useful for screening and selection of antibodies having the desired high-affinity binding properties.
  • ligand binding proteins suitable for use in the methods of the present invention include biotin-binding proteins, lipid-binding proteins, periplasmic binding proteins, lectins, serum albumins, enzymes, phosphate and sulfate binding proteins, immunophilins, metal lothionein, or various other receptor proteins.
  • the method of the present invention can be used to generate peptide libraries that are useful for screening high-affinity ligand binding partners.
  • a deoxyribonucleic acid molecule encoding the protein of interest is coupled to a stall sequence.
  • a stall sequence is any sequence that interacts with residues in the ribosomal exit tunnel to stall translation, resulting in the display of the protein of interest on the ribosome surface.
  • the SecM stall sequence is encoded by a nucleic acid sequence set forth in SEQ ID NO: 25: ttc age acg ccc gtc tgg ata age cag gcg caa ggc ate cgt get ggc cct. This corresponds to DNA bases 448-498 of the secMgene. See Schmidt, et al., "Nucleotide Sequence of the sec A Gene and secA (Ts) Mutations Preventing Protein Export in Escherichia Coli," J. Bacteriol. 170:3404-14 (1988), which is hereby incorporated by reference in its entirety.
  • a consensus for the SecM protein is FXXXXWIXXXXGIRAGP; where X can be any amino acid (SEQ ID NO: 26).
  • Suitable stall sequences includes: (1) cat leader 5-mer peptide from Gram-positive bacteria; and (2) cmlA leader 8 ⁇ mer peptide from Gram-negative bacteria (see Lovett, et al., "Nascent Peptide Regulation of Translation,” J Bacteriol 176(21):6415-7 (1994), which is hereby incorporated by reference in its entirety).
  • Generating proteins of interest according to the methods of the present invention can be carried out using the techniques described herein or using any other standard technique known in the art.
  • the fusion protein i.e.
  • the protein of interested coupled to a stall sequence can be prepared by translation of an in-frame fusion of the deoxyribonucleic acid molecule encoding the protein of interest and the polynucleotide stall sequences, i.e., a hybrid gene.
  • the hybrid gene encoding the fusion polypeptide is inserted into an expression vector which is used to transform or transfect a host cell.
  • the deoxyribonucleic acid molecule encoding the protein or polypeptide of interest is inserted into an expression vector in which the polynucleotide encoding the stall polypeptide is already present.
  • the stall polypeptide or protein of the fusion protein is preferably fused to the C -terminal, end of the protein or polypeptide of interest.
  • Fusions between the deoxyribonucleic acid molecule encoding the protein or polypeptide of interest and a stall polynucleotide sequence may be such that the nucleic acid sequence encoding the protein or polypeptide of interest is directly contiguous with the nucleic acid sequence encoding the stall polypeptide or protein of the present invention.
  • the deoxyribonucleic acid molecule encoding the protein of interest may be coupled to the stall polynucleotide sequence by way of a linker sequence such as the flexible 8-residue Gly-Ser linker described herein having the sequence, AGSAAGSG (SEQ ID NO:27),
  • the Gly-Ser linker may comprise between 10-50 Gly/Ser units depending on the optimal separation needed between the ribosome and the target protein.
  • other suitable linkers include a GIy linker or the flexible linkers from an immunoglobulin disclosed in U.S. Patent No.
  • the linker may also contain a protease-specific cleavage site so that the protein of interest may be controllably released from the stall polypeptide or protein.
  • protease sites include those specific to cleavage by factor Xa, enterokinase, collagenase, Igase (from Neisseria gonorrhoeae), thrombin, and TEV (Tobacco Etch Virus) protease.
  • one or more polynucleotide encoding marker proteins can also be positioned between the deoxyribonucleic acid molecule encoding the protein of interest and the stall polynucleotide sequence.
  • Marker proteins are well known in the art and include affinity protein markers, such as chitin binding protein, maltose binding protein, glutathione-s-transferase, and the poly(His) tag; epitope markers, such as the V5-tag, c-myc-tag, HA-tag, or FLAG-tag.
  • a c-Myc epitope tag, a 6x-His tag, a thrombin cleavage site, and a linker are all positioned within the construct between the deoxyribonucleic acid molecule encoding the protein of interest and stalling sequence.
  • the nucleic acid construct containing the deoxyribonucleic acid molecule encoding the protein of interest and the stall polynucleotide with the optional c-Myc epitope tag, 6x-His tag, thrombin cleavage site, and linker positioned in between preferably also contains a polynucleotide sequence encoding a marker sequence upstream (5') of the deoxyribonucleic acid molecule encoding the protein of interest. Any of the marker proteins mentioned above (i.e.
  • chitin binding protein maltose binding protein, glutathione-s-transferase, and the poly(His) tag; epitope markers, such as the V5-tag, c-myc-tag or the HA-tag) are suitable.
  • a polynucleotide encoding a FLAG-tag is inserted upstream of the deoxyribonucleic acid molecule encoding the protein of interest.
  • a stop codon is inserted at the 3 'end of the polynucleotide encoding the stall sequence.
  • the nucleic acid construct encoding the fusion protein is inserted into an expression system to which the molecule is heterologous.
  • the heterologous nucleic acid molecule is inserted into the expression system or vector in proper sense (5'— »3') orientation relative to the promoter and any other 5' regulatory molecules, and correct reading frame.
  • the preparation of the nucleic acid constructs can be carried out using standard cloning methods well known in the art as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory Press, Cold Springs Harbor, New York (1989), which is hereby incorporated by reference in its entirety.
  • Suitable expression vectors include those which contain replicon and control sequences that are derived from species compatible with the host cell. For example, if E. coli is used as a host cell, plasmids such as pUC19, pUC18 or pBR322 may be used. Other suitable expression vectors are described in Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrook and Russell, 2001, Cold Spring Harbor Laboratory Press, which is hereby incorporated by reference in its entirety.
  • RNA transcription and messenger RNA e.g., DNA transcription and messenger RNA (“mRNA”) translation
  • mRNA messenger RNA
  • Transcription of DNA is dependent upon the presence of a promoter, which is a DNA sequence that directs the binding of RNA polymerase, and thereby promotes mRNA synthesis. Promoters vary in their "strength" (i.e., their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters to obtain a high level of transcription and, hence, expression and surface display.
  • any one of a number of suitable promoters may also be incorporated into the expression vector carrying the deoxyribonucleic acid molecule encoding the protein of interest coupled to a stall sequence.
  • promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the P R and P L promoters of coliphage lambda and others, including but not limited, to / ⁇ cUV5, ompF, bla, Ipp, and the like, may be used to direct high levels of transcription of adjacent DNA segments.
  • a hybrid tr ⁇ -lac ⁇ ]V5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
  • SD Shine- Dalgarno
  • Host cells suitable for expressing and displaying the fusion protein on the ribosome surface include any one of the more commonly available gram negative bacteria. Suitable microorganisms include Pseudomonas aeruginosa, Escherichia coli, Salmonella gastroenteritis (typhimirium), S. typhi, S, enteriditis, Shigella flexneri, S, sonnie, S dysente ⁇ ae, Neisseria gonorrhoeae, N. meningitides, Haemophilus influenzae H.
  • pleuropneumoniae Pasteurella haemolytica, P. multilocida, Legionella pneumophila, Treponema pallidum, T. denticola, T. orales, Borrelia burgdorferi, Borrelia spp. Leptospira interrogans, Klebsiella pneumoniae, Proteus vulgaris, P. morganii, P. mirabilis, Rickettsia prowazeki, R, typhi, R. richettsii, Porphyromonas (Bacteriodes) gingivalis, Chlamydia psittaci, C. pneumoniae, C.
  • B. can is, Spirillum minus, Pseudomonas mallei, Aeromonas hydrophila, A salmonicida, and Yersinia pestis,
  • the host cell is E. coli.
  • An additional preferred embodiment includes the utilization of an E. coli host strain carrying mutations in the both thioredoxin reductase (trxB) and glutathione reductase (gor) genes (e.g., Origami ) wherein disulfide bond formation in the cytoplasm is significantly enhanced.
  • trxB gor mutant strain can be used to affinity- and/or stability-mature scFvs that are stalled and folded under oxidizing conditions.
  • eukaryotic cells such as mammalian and yeast, and baculovirus systems are also suitable host cells that can be used in accordance with the methods of the present invention.
  • Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells and many others. Methods for transforming/transfecting host cells with expression vectors are well-known in the art and depend on the host system selected, as described in
  • suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus.
  • suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophage.
  • cellular ribosome fractions are prepared and incubated with an immobilized antigen, protein binding partner, or marker protein binding partner that specifically recognizes and selectively binds to the protein of interest or marker protein that is displayed on the surface of the ribosome.
  • the antigen, protein binding partner, or marker binding protein can be immobilized on any solid surface or support, such as a polystyrene microtiter plate, column, or a magnetic bead (e.g. Dynabeads ® ).
  • the antigen or protein binding partner can be co-expressed in vivo, in the cell, along with the protein of interest displayed on the ribosome.
  • the marker binding protein can be an antibody recognizing the epitope tag immobilized on the solid support.
  • the complex can be recovered using affinity purification media such as, NTA- agarose, HisPur resin or Talon resin.
  • Dissociating the bound protein-mRNA-ribosome complex from the solid support can be carried out using any appropriate chelating or elution buffer readily used in the art.
  • the protein-mRNA-ribosome complex is dissociated using EDTA.
  • the method of the present invention additionally includes isolating the mRNA from the recovered complex.
  • the isolated mRNA is reverse transcribed to form a cDNA encoding the protein, and a construct, comprising the cDNA coupled to the stall sequence, is formed.
  • the steps of transforming, culturing, and recovering, as described above, are repeated to enrich the protein recovered.
  • Methods for isolating and reverse transcribing RNA are well known in the art (See Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization with Nucleic Acid Probes. Part I. Theory and Nucleic Acid Preparation, P.
  • RNA can be isolated by using the guanidinium isothiocyanate-uitracentrifugation method, the guanidinium and phenol-chloroform method, the lithium chloride-SDS-urea method or poly A+ / mRNA from tissue lysates using oligo(dT) cellulose method. It is important when isolating the RNA that enough high quality RNA is isolated.
  • the mRNA can be reverse transcribed to form cDNA using any commercially available kit following manufacturer's instructions. Typically, the reaction is carried out using either oligo- dT or random decamer primers and a the reverse transcriptase enzyme.
  • the steps of transforming, culturing, and recovering, as described above, are repeated to enrich the protein recovered.
  • the enriched protein can be characterized by direct amino acid sequencing. Generally, protein sequencing is carried out using mass spectrometry or Edman degradation reaction.
  • the protein can further be characterized based on affinity screening (i.e. is ability to bind to a ligand binding partner) using an panning, chromatography, or an ELISA based assay.
  • the protein can also be characterized by its activity in an enzyme based assay.
  • the stability of the identified protein of the present invention can be enhanced by altering or optimizing cellular conditions. Stability can generally be defined as the propensity of a molecule to exist in its folded and active state. Since stalled proteins, i.e. proteins that are displayed on the surface of the ribosome due to stalled translation, undergo folding in the cytoplasm, potent molecular chaperones and/or isomerases can be co-expressed in the host cell to enhance the stability, solubility and/or native folding capacity.
  • Preferred molecular chaperones include DnaK, DnaH, GrpE, GroEL, or GroES and a preferred isomerase is the protein disulfide isomerase.
  • the stability of the identified protein can also be enhanced via the addition of oxidized and reduced glutathione.
  • the stability of the identified protein can further be enhanced by mutating the deoxyribonucleic acid molecule encoding the protein to produce variant nucleic acid sequences encoding variants with amino acid sequences. Methods of site directed or random mutagenesis are well known in the art and are suitable for use in the method of the present invention (See Current Protocols in Molecular Biology, Ausubel et al. eds., (1992), which is hereby incorporated by reference in its entirety).
  • Another aspect of the present invention relates to a construct which includes a deoxyribonucleic acid molecule encoding a protein that binds to a target molecule and an SecM stalling sequence coupled to the deoxyribonucleic acid molecule.
  • the deoxyribonucleic acid molecule and the SecM stalling sequence are coupled with sufficient distance between them to permit expression of their encoded protein, within the cell, in a properly folded, active form.
  • This construct can be incorporated into an expression vector or a host cell as described above.
  • Another aspect of the present invention relates to a method of identifying a protein that binds to a target molecule and has intracellular functionality. This method includes providing a construct comprising a deoxyribonucleic acid molecule encoding the protein which binds to the target molecule, said deoxyribonucleic acid molecule being coupled to a stall sequence. A cell-free extract preparation containing ribosomes is also provided.
  • the method further involves contacting the construct with the cell-free extract preparation containing ribosomes under conditions effective for ribosome translation and the formation of a complex of the protein whose translation has been stalled, the mRNA encoding the protein, and the ribosomes.
  • the protein in the complex is in a properly folded, active form and the complex is recovered.
  • the protein to be identified can be any ligand binding protein and the stall sequence is any sequence that interacts with residues in the ribosomal exit tunnel to stall translation.
  • the ligand binding protein is a single chain antibody.
  • a primary advantage of using a cell-free translation system to achieve ribosome display is the ability to easily manipulate selection biases to enhance stability of the identified protein.
  • stability can generally be defined as the propensity of a molecule to exist in its folded and active state.
  • a stability selection pressure may disrupt or prevent a polypeptide folding correctly such that it does not attain an active or fully active state.
  • a stability selection pressure may affect the ability of a polypeptide to remain in its folded and active state.
  • a stability selection pressure may differentiate in some way between polypeptides that are in a folded and active state and those that are not.
  • a stability selection pressure may be a chemical denaturant, such as urea, guanidine HCl (GuHCl) or thiocyanate, for example, sodium thiocyanate.
  • a stability selection pressure may be a reducing agent, such as dithiothreitol (DTT), Tris[2-carboxyethyl] phosphine hydrochloride (TCEP), mercaptoethanol or glutathione.
  • a stability selection pressure may be a physical denaturant, such as pH or temperature, in particular increased temperature.
  • a selection pressure may be a protease or enzyme capable of degrading protein.
  • a selection pressure may be depletion of chaperons or small molecule protein folding inhibitors.
  • a stability selection pressure may be the use of hydrophobic interaction chromatography (HlC).
  • HlC Hydrophobic interaction chromatography
  • HlC techniques have been used as a part of protein purification strategies as well as an analytical tool for the detection of protein conformational changes (reviewed in Queiroz et al., "Hydrophobic Interaction Chromatography of Proteins, " J Biotech. 87: 143-159 (2001 ), which is hereby incorporated by reference in its entirety.
  • HIC is based on hydrophobic attraction between the HIC matrix and the protein molecules.
  • the HIC matrix consists of small non-polar groups (butyl, octyl or phenyl) attached to a hydrophilic polymer backbone (e.g.
  • HIC cross-linked dextran or agarose
  • Many proteins generally considered to be hydrophilic, also have sufficient numbers of hydrophobic groups allowing interaction with the H ⁇ C matrix, HIC is sensitive enough to interact with non-polar groups normally buried within the tertiary structure of the protein but exposed due to incorrect folding. The strength of the interaction is dependent upon the type of matrix, type and concentration of salt, pH, additives, and temperature.
  • the present invention is suitable for a number of uses.
  • scFv stability-enhanced single-chain variable fragment
  • intracellular ribosome display method of the present invention the cytoplasmic stability, and thus intracellular function, of an scFc can be enhanced 2-3 fold after only a single round of mutagenesis and selection.
  • Another use of the present invention involves isolation of functionally enhanced disulfide-bond containing antibody fragments.
  • a trxB gor host mutant strain in which the redox potential of the cytoplasm favors the formation of disulfide bonds in proteins is used to affinity- and/or stability-mature scFvs that are stalled and folded under oxidizing conditions.
  • the present invention can also be employed in the screening of naive libraries for cytoplasmically functional proteins with specific binding affinity to a given target molecule:
  • the technology of the present invention is suited for the selection of intracellularly functional antibodies that bind a specific antigen target from na ⁇ ve (i.e., not stemming from preimmunized cells) libraries.
  • Stability-enhancement/evolution of proteins under optimized cellular conditions can also be carried out with the present invention. Since stalled proteins undergo folding in the cytoplasm, it is relatively straightforward and inexpensive to optimize in vivo folding conditions by co-expressing potent molecular chaperones and/or isomerases.
  • the present invention can be used for protein engineering experiments (i.e. by random mutagenesis) under conditions where the cellular environment is tuned to better suit the specific folding requirements of a particular target protein.
  • the present invention can also be combined with in vitro/in vivo selection strategies for protein engineering via ribosome display.
  • Example 1 Bacterial Strains and Plasmids.
  • E. colt strain BL21(DE3) was used throughout except for in vivo ⁇ -gal activation experiments where the E, coli 959 strain was used which carries the AMEF ⁇ -gal gene (Martineau et al., "Expression of an Antibody Fragment at High Levels in the Bacterial Cytoplasm," JMoI Biol 280: 1 17-27 (1998), which is hereby incorporated by reference in its entirety). Plasmids encoding the SecM stall sequence fusions were constructed as follows.
  • a 285-nucleotide segment of the secM gene containing the 17-amino acid stall sequence (FSTPVWISQAQGIRAGP (SEQ ID NO: I)), plus additional downstream regions, was amplified from plasmid pNH21 by PCR using primers (S'-CTCATGGTCGACTTCAGCACGCCCGTCTGG-S' (SEQ ID NO: 5)) and (5'- CTCATGCTCGAGTTAAAGCTTCTGCGCAACTGTTGGGAAGC-3' (SEQ ID NO: 6)) to introduce a SaIi restriction site at the 5' end and an Xhol-Hin ⁇ lM restriction site at the 3 'end.
  • This PCR product was Sall-Xhol digested and Iigated into the same sites of pET28a (Novagen). Second, removal of the additional SecM downstream regions performed by introducing a Hin ⁇ lll restriction site immediately after the 17-residue stall sequence using site-directed mutagenesis (Stratagene QuikChange ® Kit) and primers (5'- GGCATCCGTGCTGGCCCTAAGCTTCAACGCCTCACCTAACAA-S' (SEQ ID NO: 7) and 5'-
  • Each PCR product was digested with TVcoI and Sail and Iigated into jVcoI-S ⁇ /I-digested pET- SecM to yield the intermediate constructs pET-scFvl B-SecMiy and pET-scFvl3-R4- SecMl 7'.
  • a Sad restriction site was inserted immediately before the Sail restriction site using QuikChange ® and primers (5'-
  • This PCR product was Sacl-S all-digested and ligated into similarly digested pET-scFvl3-SecM 17' or ⁇ ET-scFvl3-R4-SecM17'.
  • the additional NDPK sequence was excised by first inserting an EcoRl restriction site before the NDPK sequence using QuikChange ® and primers (5'- GTGCCGCGCGGCAGCCATGAATTCATGCATGCTATAAATATTGC-S ' (SEQ ID NO: 16) and 5'- GCAATATTTATAGCATGCATGAATTCATGGCTGCCGCGCGGCAC-S' (SEQ ID NO: 17)).
  • Plasmids encoding the unfused scFv sequences were constructed by amplifying the scFvB (or the scFvl 3 variants Rl, R2 and R4) sequence by PCR from plasmids pPM163, pPM163-Rl , pPM163-R2, and pPM163-R4 (Martineau et al., "Expression of an Antibody Fragment at High Levels in the Bacterial Cytoplasm," J MoI Biol 280: 117-27 (1998), which is hereby incorporated by reference in its entirety) with primers (5'- GCGATGCCATGGCCGACTACAAGGACGATGACGACAAGGGAGCCGAGGT GCAGCTG -3' (SEQ ID NO: 20) and 5'-
  • GCGATGGAGCTCTTATGCGGCCCCATTCAG-3' (SEQ ID NO: 21 )) that introduce a FLAG epitope tag and an Ncol restriction site at the 5 'end and a Sad restriction site at the 3 'end.
  • This PCR product was digested with Ncol and Sad and ligated into similarly digested pET28a.
  • a library of random mutants was constructed by error-prone PCR of the scFvl 3 gene sequence using pET-scFvl3-SecM17 as template and skewing the nucleotide and magnesium concentrations as described (DeLisa et al., "Genetic Analysis of the Twin Arginine Translocator Secretion Pathway in Bacteria," J Biol Chem 277: 29825-31 (2002) and Fromant et al., "Direct Random Mutagenesis of Gene-Sized DNA Fragments Using Polymerase Chain Reaction," Anal Biochem 224:347-53 (1995), which are hereby incorporated by reference in their entirety) to generate a 1.5% error-rate library. Error-prone PCR products were amplified with using primers
  • cloni ExpressTM BL21(DE3) cells (Lucigen) and serial dilutions of these cells were plated on kanamycin (50 ⁇ g/ml) to determine the number of independent transformants. Transformed ceils were selected on LB plates containing kanamycin (50 ⁇ g/ml). Library cells were pooled, cultured, and induced for scFv expression prior to isolation of ribosomes for panning and selection experiments.
  • Example 3 Cell Fractionation.
  • Cells transformed with the pET28a-derived scFv constructs were grown in 10-ml cultures at 37 0 C in Luria-Bertani (LB) supplemented with kanamycin (50 ⁇ g/ml). Protein synthesis was induced by adding 1 mM isopropyl- ⁇ -D- thiogalactopyranoside (IPTG) when cells reached to mid log phase (OD($ ⁇ r-0.5). Cells were harvested after 1 hour of induction and pelleted by centrifugation for 15 min at 4 0 C and 3,500 rpm.
  • IPTG isopropyl- ⁇ -D- thiogalactopyranoside
  • the soluble fraction was prepared by resuspending the pellet in 300 ml of phosphate buffered saline (PBS) solution followed by sonication (Branson Sonifier). The sonicant was spun for 15 min at 4 0 C and 1,300 rpm and the resulting supernatant was collected as the soluble fraction.
  • PBS phosphate buffered saline
  • Ribosomes were isolated according to a procedure modified from Evans et al., "Homogeneous Stalled Ribosome Nascent Chain Complexes Produced in vivo or in vitro," Nat Methods 2:757-62 (2005), which is hereby incorporated by reference in its entirety. Specifically, 100-ml cultures grown as above were induced with 1 niM IPTG at an OD600-0.5 and grown at 30 0 C for an additional 30 min.
  • Buffer C (20 mM Tris-HCl pH 7.5, 50 mM NH 4 Cl, 25 mM MgCh) ice cubes were added to each culture flask, rapidly shaken for 1 min on ice, and incubated on ice for an additional 30 min. Next, cells were pelleted by centrifugation as above and resuspended in 600 ⁇ l of cold Buffer C.
  • ribosomes were isolated by ultracentrifugation for 35 h at 24,000 rpm and 4 0 C using a Beckman LS 8 ultracentrifuge with an SW28 rotor.
  • the crude ribosome pellet was resuspended in 200 ⁇ l Buffer C and ultracentrifuged in a 10 to 40% (w/v) sucrose gradient in Buffer A (20 nM Tris-HCl pH 7.5, 100 mM NH 4 Cl, 25 mM MgCl 2 ) for 17 h at 22,000 rpm and 4°C in a SW41 rotor. Gradient fractionation was performed manually by pipetting 250 ⁇ l at a time from the top part of the gradient. All collected samples were stored at 4°C for further analysis.
  • a 96-well BD FALCON plate was coated with 65 ⁇ l of 1 mg/ml ⁇ -gal
  • GCGATGGAGCTCTTATGCGGCCCCATTCAG-3' (SEQ ID NO: 24), which binds the 3' end of scFvl3 and introduces a Sad restriction site and a stop codon.
  • PCR amplification was performed in a second step with the same reverse primer and forward primer 5'-
  • the pellet was dried of all remaining TCA and directly resuspended in 45 ⁇ l SDS- PAGE loading buffer.
  • the following primary antibodies were used with the corresponding dilution in parenthesis: mouse anti-GroEL (1 : 10,000; Sigma); mouse anti-FLAG (1 : 1,500; Stratagene).
  • the secondary antibodies were goat anti-mouse and goat anti-rabbit horseradish peroxidase conjugates (Promega) each diluted 1 : 10,000.
  • a Bradford protein assay was performed on all samples to verify that an equal amount of total protein was loaded to each lane.
  • fractions were normalized by rRNA content as measured by (see Figure 6).
  • membranes were first probed with primary antibodies and, following development, stripped in Tris-buffered saline supplemented with 2% SDS and 0.7 M ⁇ -mercaptoethanol. Stripped membranes were reblocked and probed with anti-GroEL antibody.
  • Example 7 - ELISA.
  • Cell iysates and ribosome samples were analyzed by ELISA according to the same steps described above for ribosome panning with the following modifications: (1 ) plates were coated with 100 ⁇ l of 10 ⁇ g/ml ⁇ -ga! (Sigma) in PBS; and (2) 0.5% BSA was used instead of non-fat milk in the blocking solution. Following the washes after the samples were applied, instead of sample elution, 50 ⁇ l of anti-FLAG antibody (Stratagene) at a 1 :5,000 dilution in blocking solution was added to each well and incubated for 1 h at 4 0 C.
  • anti-FLAG antibody (Stratagene)
  • Strain AMEF 959 (Martineau et al., "Expression of an Antibody Fragment at High Levels in the Bacterial Cytoplasm,” J MoI Biol 280:1 17-27 ⁇ 1998), which is hereby incorporated by reference in its entirety) was transformed with each scFvl3 variant encoded in plasmid pTrc99A.
  • ⁇ - gal activity was measured using a whole cell assay modified from Arnold et a!., "Influences of Transporter Associated with Antigen Processing (TAP) on the Repertoire of Peptides Associated With the Endoplasmic Reticulum-Resident Stress Protein gp96," J Exp Med 186:461 -6 ( 1997), which is hereby incorporated by reference in its entirety. Specifically, 5-bromo ⁇ 4-chloro-3-indolyl- ⁇ D- galactopyranoside (X-gal) was added in each well to a final concentration of 1 mM and absorbance at 620 nm was recorded over 4-10 h at 37°C to measure active ⁇ -gal.
  • TEP Antigen Processing
  • human scFv! 3 was chosen as a model. This was originally isolated by Winter and coworkers in a two-step procedure: first, in vitro phage display was used to isolate scFv binders to native E.
  • 70S ribosomes were isolated (see Figure 6) from soluble proteins and other cell lysate components by sucrose cushion centrif ⁇ gation.
  • ribosome preparations were probed for the presence of trigger factor (TF) which is known to dock on the ribosome near the exit tunnel (Kramer et al., "L23 Protein Functions as a Chaperone Docking Site on the Ribosome, ' ' Nature 419: 171-4 (2002), which is hereby incorporated by reference in its entirety).
  • TF trigger factor
  • Ribosome-associated RNA was isolated from dissociated complexes and the mRNA encoding the scFvl3 sequence was amplified using RT-PCR using general primers that annealed to both scFvl 3 and scFvl3-R4, Remarkably, ribosome fractions corresponding to unfused scFvs yielded no distinct PCR bands whereas both the scFvl 3-SecM17 and the solubility-enhanced scFvl 3-R4- SecMl 7 constructs gave rise to a substantial PCR product corresponding in size to the full-length scFvl3 sequence (Figure 3C); sequencing of each PCR product identified these as the wt scFvl3 and scFvl 3-R4 mRNA sequences, respectively.
  • PCR products were ligated into an expression plasmid that was transformed into E. coli and 10 randomly selected single clones were analyzed for each mixture to determine the identity of the plasmid-encoded scFv.
  • a complete cycle of mutagenesis and screening was performed to improve the solubility of wt scFvl3.
  • the resulting error- prone PCR product was ligated in-frame with the SecM stall sequence and, following transformation, a cell library containing ⁇ 5xl O 6 transformants was obtained.
  • Members of this cell library were induced and ribosome fractions were prepared and screened by panning on immobilized ⁇ -gal to isolate clones exhibiting enhanced solubility relative to wt scFvl3.
  • a total of twelve unique clones were obtained after only a single round of selection and each was evaluated in an unfused format (i.e., lacking the SecM stall sequence in pET28a) for soluble expression and antigen binding.
  • the present intracellular display strategy is suitable to isolate any stability-enhanced antibody with binding affinity for any antigen (not limited to ⁇ -gal).
  • the only requirements, which are the same for traditional in vitro ribosome display, are that the antibody fragment must be amenable to display in the context of ARM complexes and that the binding target is known and available in a purified form to allow for selection.
  • intracellular ribosome display offers a number of advantages. For instance, expression and stalling of proteins on ribosomes is less technically challenging as these steps are performed entirely inside cells, requiring only an inducer (e.g., IPTG) to initiate the entire process from start to finish.
  • an inducer e.g., IPTG
  • bacterial cell culture but not cell-free translation, can be easily scaled to produce large quantities and high concentrations of stalled ribosome complexes that might be necessary for various applications such as making biophysical measurements using NMR.
  • stalled scFvs undergo folding in the cytoplasm, it is relatively straightforward and inexpensive to optimize in vivo folding conditions by co-expressing potent molecular chaperones and/or isomerases (Jurado et al, "Production of Functional Single-Chain Fv Antibodies in the Cytoplasm of Escherichia coli " JMoI Biol 320:1-10 (2002) and Levy et al., "Production of
  • Such a strategy would also reduce the likelihood of false positives that may arise due to undesired antibody folding upon removal of ARM complexes from the cytoplasmic environment /yr ⁇ r to the panning procedure.
  • this is regulated by instantaneous cooling of the ARM complexes to 4 0 C (where the kinetics of folding are extremely slow) and by performing the biopanning step immediately after complexes are isolated.
  • the ability to display a functional binding protein on ribosomes and simultaneously express its interacting partner could potentially be used to engineer and even block protein-protein interactions inside cells.
  • intracellular ribosome display is a powerful complementary method to in vitro ribosome display for the directed evolution of proteins and should find use in the engineering of potent binding proteins that are soluble inside host cells for applications in functional genomics and proteomics as well as molecular medicine.

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Abstract

La présente invention concerne un procédé d'identification d'une protéine qui se lie à une molécule cible et qui possède une fonctionnalité intracellulaire. Ce procédé comprend la fourniture d'une construction comportant une molécule d'acide désoxyribonucléique codant la protéine qui se lie à la molécule cible, la molécule d'acide désoxyribonucléique étant couplée à une séquence d'arrêt. Une cellule hôte est transformée avec la construction puis mise en culture dans des conditions efficaces pour former, dans la cellule hôte, un complexe de la protéine dont la traduction a été arrêtée, l'ARNm codant la protéine, et des ribosomes. La protéine dans le complexe est sous une forme active correctement pliée et le complexe est récupéré de la cellule. Ce procédé peut être effectué avec une préparation d'extrait dépourvue de cellule contenant des ribosomes au lieu d'une cellule hôte. La présente invention concerne également une construction qui comprend une molécule d'acide désoxyribonucléique codant une protéine qui se lie à une molécule cible et une séquence d'arrêt SecM couplée à une molécule d'acide désoxyribonucléique. La molécule d'acide désoxyribonucléique et la séquence d'arrêt SecM sont couplées avec une distance suffisante entre elles pour permettre une expression de leur protéine codée, à l'intérieur de la cellule, sous une forme active correctement pliée.
PCT/US2008/071747 2007-06-13 2008-07-31 Découverte de protéine utilisant un affichage de ribosome intracellulaire WO2009018438A1 (fr)

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WO2016077526A1 (fr) 2014-11-12 2016-05-19 Siamab Therapeutics, Inc. Composés interagissant avec le glycane et procédés d'utilisation
US9399676B2 (en) 2013-05-06 2016-07-26 Scholar Rock, Inc. Compositions and methods for growth factor modulation
WO2017083582A1 (fr) 2015-11-12 2017-05-18 Siamab Therapeutics, Inc. Composés interagissant avec le glycane et méthodes d'utilisation
US9879087B2 (en) 2014-11-12 2018-01-30 Siamab Therapeutics, Inc. Glycan-interacting compounds and methods of use
US11253609B2 (en) 2017-03-03 2022-02-22 Seagen Inc. Glycan-interacting compounds and methods of use
US11401330B2 (en) 2016-11-17 2022-08-02 Seagen Inc. Glycan-interacting compounds and methods of use

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US20030165945A1 (en) * 2000-04-28 2003-09-04 Bird Timothy A. Human Pellino polypeptides
US20040265984A1 (en) * 2001-09-24 2004-12-30 Ada Yonath Methods of growing crystals of free, antibiotic-complexed, and substrate-complexed large ribosomal subunits, and methods of rationally designing or identifying antibiotics using structure coordinate data derived from such crystals
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US20060177862A1 (en) * 2000-03-31 2006-08-10 Cambridge Antibody Technology Limited Ribosome display
US20030165945A1 (en) * 2000-04-28 2003-09-04 Bird Timothy A. Human Pellino polypeptides
US20040265984A1 (en) * 2001-09-24 2004-12-30 Ada Yonath Methods of growing crystals of free, antibiotic-complexed, and substrate-complexed large ribosomal subunits, and methods of rationally designing or identifying antibiotics using structure coordinate data derived from such crystals

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9399676B2 (en) 2013-05-06 2016-07-26 Scholar Rock, Inc. Compositions and methods for growth factor modulation
US9573995B2 (en) 2013-05-06 2017-02-21 Scholar Rock, Inc. Compositions and methods for growth factor modulation
US9758576B2 (en) 2013-05-06 2017-09-12 Scholar Rock, Inc. Compositions and methods for growth factor modulation
US10597443B2 (en) 2013-05-06 2020-03-24 Scholar Rock, Inc. Compositions and methods for growth factor modulation
US10981981B2 (en) 2013-05-06 2021-04-20 Scholar Rock, Inc. Compositions and methods for growth factor modulation
US11827698B2 (en) 2013-05-06 2023-11-28 Scholar Rock, Inc. Compositions and methods for growth factor modulation
USRE49435E1 (en) 2014-11-12 2023-02-28 Seagen Inc. Glycan-interacting compounds and methods of use
WO2016077526A1 (fr) 2014-11-12 2016-05-19 Siamab Therapeutics, Inc. Composés interagissant avec le glycane et procédés d'utilisation
US9879087B2 (en) 2014-11-12 2018-01-30 Siamab Therapeutics, Inc. Glycan-interacting compounds and methods of use
EP4183806A2 (fr) 2014-11-12 2023-05-24 Seagen Inc. Composés interagissant avec le glycane et procédés d'utilisation
WO2017083582A1 (fr) 2015-11-12 2017-05-18 Siamab Therapeutics, Inc. Composés interagissant avec le glycane et méthodes d'utilisation
US11028181B2 (en) 2015-11-12 2021-06-08 Seagen Inc. Glycan-interacting compounds and methods of use
US11401330B2 (en) 2016-11-17 2022-08-02 Seagen Inc. Glycan-interacting compounds and methods of use
US11253609B2 (en) 2017-03-03 2022-02-22 Seagen Inc. Glycan-interacting compounds and methods of use

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