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WO2019173801A1 - Acides nucléiques codant pour des anticorps anti-vih - Google Patents

Acides nucléiques codant pour des anticorps anti-vih Download PDF

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Publication number
WO2019173801A1
WO2019173801A1 PCT/US2019/021493 US2019021493W WO2019173801A1 WO 2019173801 A1 WO2019173801 A1 WO 2019173801A1 US 2019021493 W US2019021493 W US 2019021493W WO 2019173801 A1 WO2019173801 A1 WO 2019173801A1
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Prior art keywords
oligonucleotides
group
seq
acid sequence
fragment
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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PCT/US2019/021493
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English (en)
Inventor
Guy L. Cavet
Sean M. Carroll
Shaun M. Lippow
Wayne Volkmuth
Dongkyoon KIM
Mohammad SAJADI
George K. Lewis
Anthony Devico
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University of Maryland Baltimore
US Department of Veterans Affairs
University of Maryland College Park
Atreca Inc
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University of Maryland Baltimore
US Department of Veterans Affairs
University of Maryland College Park
Atreca Inc
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Publication of WO2019173801A1 publication Critical patent/WO2019173801A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production

Definitions

  • FIG. 1 illustrates an example of a set of overlapping single-stranded
  • oligonucleotides that are used to assemble into a desired polynucleotide disclosed in this application.
  • the arrows represent the 3’ ends of the oligonucleotides and the opposite ends are the 5’ ends.
  • “1”,“2”,“3”,“4”,“5”, and“6” represent the first, second, third, fourth, fifth, and sixth oligonucleotides in the set.
  • FIG. 2 illustrates an example of a set of overlapping double-stranded
  • oligonucleotides that are used to assemble into a desired polynucleotide disclosed in this application.
  • the arrows represent the 3’ ends of the oligonucleotides and the opposite ends are the 5’ ends.
  • “1”,“2”,“3” represent the first, second, third double-stranded
  • FIG. 3 illustrates an example of a ligation-based assembly using a set of non overlapping single-stranded oligonucleotides.
  • the arrows indicate the 3’ ends of the oligonucleotides.
  • the oligonucleotides in the set are indicated by the lines with arrows pointing to the right.
  • HIV antibodies such as L1A1, L1A2, L2A1, and L1A4, in vitro.
  • this disclosure provides an oligonucleotide attached to a solid support, wherein the oligonucleotide encodes a polypeptide comprising: (i) a fragment of the heavy chain of L1A1 having an amino acid sequence selected from the group consisting of SEQ ID NO:65-l42, or a fragment of the light chain of L1A1 having an amino acid sequence selected from SEQ ID NO:340-372, (ii) a fragment of the heavy chain of L1A2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 144-214, or a fragment of the light chain of Ll A2 having an amino acid sequence selected from the group consisting of SEQ ID NO:374-420, (iii) a fragment of the heavy chain of L2A1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 216-276, or a fragment of the light chain of L2A1 having an amino acid sequence selected from the group consisting of SEQ ID NO
  • this disclosure provides an oligonucleotide that encodes a polypeptide comprising (i) a fragment of the heavy chain of L1A1 having an amino acid sequence selected from the group consisting of SEQ ID NO:65-l42, or a fragment of the light chain of L1A1 having an amino acid sequence selected from SEQ ID NO:340-372, (ii) a fragment of the heavy chain of L1A2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 144-214, or a fragment of the light chain of L1A2 having an amino acid sequence selected from the group consisting of SEQ ID NO:374-420, (iii) a fragment of the heavy chain of L2A1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 216-276, or a fragment of the light chain of L2A1 having an amino acid sequence selected from the group consisting of SEQ ID NO:422-476, or (iv) a fragment of the heavy chain of
  • this disclosure provides an oligonucleotide that encodes a polypeptide comprising: (i) a fragment of the heavy chain of L1A1 having an amino acid sequence selected from the group consisting of SEQ ID NO:65-l42, or a fragment of the light chain of L1A1 having an amino acid sequence selected from SEQ ID NO:340-372, (ii) a fragment of the heavy chain of L1A2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 144-214, or a fragment of the light chain of L1A2 having an amino acid sequence selected from the group consisting of SEQ ID NO:374-420, (iii) a fragment of the heavy chain of L2A1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 216-276, or a fragment of the light chain of L2A1 having an amino acid sequence selected from the group consisting of SEQ ID NO:422-476, or (iv) a fragment of the heavy chain
  • this disclosure provides a polynucleotide comprising: (i) a nucleotide sequence encoding a VH sequence selected from the group consisting of SEQ ID NO: l, 3, 5, and 7, wherein the nucleotide sequence comprises at least 1 nucleotide substitution relative to the nucleic acid sequence selected from the group consisting of SEQ ID NO: 33, 35, 37, and 39; (ii) a nucleotide sequence encoding a VL sequence selected from the group consisting of SEQ ID NO:2, 4, 6, and 8, wherein the nucleotide sequence comprises at least 1 nucleotide substitution relative to the nucleic acid sequence selected from the group consisting of SEQ ID NO:34, 36, 38, and 40; (iii) a nucleotide sequence encoding a CDR sequence selected from the group consisting of SEQ ID NO:9-32, wherein the nucleotide sequence comprises at least 1 nucleotide substitution relative to the nu
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NOs:l-32 has at least 80% identity, or at least 90% identity, to any one of SEQ ID NO: 33-64.
  • a reaction mixture comprising an oligonucleotide of any one of embodiments disclosed herein.
  • this disclosure provides a composition comprising a set of oligonucleotides, wherein the set of oligonucleotides collectively encodes a polypeptide comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 consecutive amino acids of a VH or VL of an antibody selected from the group consisting of L2A1, L1A1, L1A2, and L1A4; or collectively encodes the full length sequence of the VH or VL of an antibody selected from the group consisting of L2A1, L1A1, Ll A2, and Ll A4.
  • the polypeptide comprises a CDR3 of a VH or VL of an antibody selected from the group consisting of L2A1, L1A1, L1A2, and L1A4. In some embodiments, the polypeptide further comprises a CDR2 of the VH or VL of the antibody. In some embodiments, the polypeptide further comprises a CDR1 of the VH or VL of the antibody.
  • the set of oligonucleotides are non-overlapping.
  • this disclosure provides a composition
  • a composition comprising a set of oligonucleotides, each oligonucleotide having a sequence region at one end corresponding to a sequence region in one end of an adjacent oligonucleotide, wherein the first oligonucleotide in the set comprises a sequence that aligns to the 5’ end of the target nucleotide sequence and the last oligonucleotide comprises a sequence that aligns to the 3’ end of the target nucleotide sequence, wherein the set of oligonucleotides collectively encodes a polypeptide comprising at least 10, 15, 20, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 consecutive amino acids of a VH or VL of an antibody selected from the group consisting of L2A1, L1A1, L1A2, and L1A4; or collectively encodes the full length sequence of the VH or VL of an antibody selected from the group consist
  • one or more of the oligonucleotides in the set are single- stranded. In some embodiments, one or more of the oligonucleotides in the set are double- stranded having single-stranded overhangs. In some embodiments, one or more of the oligonucleotides in the set of oligonucleotides are phosphorylated at the 5’ end. In some embodiments, the concentrations of the oligonucleotides in the set form a gradient wherein the innermost pair of oligonucleotides being at the lowest concentration and the outermost pair of oligonucleotide being at the highest concentration.
  • this disclosure provides a reaction mixture comprising the composition of any of the embodiments described above, and the reaction mixture may further comprise one or more enzyme selected from the group consisting of a polymerase, an exonuclease, and a ligase.
  • this disclosure provides a method of producing a polynucleotide encoding a desired polypeptide sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 consecutive amino acids of a VH or VL of an antibody selected from the group consisting of L2A1, L1A1, L1A2, and L1A4, or encoding a full length sequence of a VH or VL of an antibody selected from the group consisting of L2A1, L1A1, L1A2, and L1A4; the method comprising: providing a set of oligonucleotides that collectively encodes the desired polypeptide, and linking the set of oligonucleotides to one another to generate a contiguous polynucleotide that encodes the desired polypeptide sequence.
  • this disclosure provides a method of producing a desired polynucleotide encoding a polypeptide comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 consecutive amino acids of a VH or VL of an antibody selected from the group consisting of L2A1, L1A1, L1A2, and L1A4; or the full length sequence of a VH or VL, of an antibody selected from the group consisting of L2A1, L1A1, L1A2, and L1A4, the method comprising: providing a set of oligonucleotides, each having a sequence region at one end corresponding to a sequence region in one end of an adjacent oligonucleotide, wherein the set of oligonucleotides collectively encode the polypeptide, and assembling the set of oligonucleotides into the polynucleotide through the overlapping sequence regions to produce the desired polynu
  • the first oligonucleotide in the set comprises a sequence that aligns to the 5’ end of the target nucleotide sequence and the last oligonucleotide comprises a sequence that aligns to the 3’ end of the target nucleotide sequence.
  • the overlapping sequence regions have a length of 5-50 nucleotides.
  • the assembling step is performed using PCR.
  • the concentrations of the oligonucleotides form a gradient, with the innermost pair of oligonucleotides being at the lowest concentration and the outermost pair of oligonucleotide being at the highest concentration.
  • the overlapping sequences between the oligonucleotides have about the same annealing temperatures across the assembly.
  • the assembling is by ligation.
  • assembling step comprises annealing the oligonucleotides and ligating the oligonucleotides.
  • the method comprises phosphorylating one or more oligonucleotides present in the set of oligonucleotides before ligation.
  • the assembling step comprises further comprises denaturing the oligonucleotides before annealing.
  • the ligation is performed by a ligase that is active at 50 °C or higher.
  • the set of oligonucleotides are double-stranded oligonucleotides with single- stranded overhangs, wherein the overhangs are complementary between adjacent oligonucleotides, wherein the assembling step comprises annealing the overhangs, and contacting the annealed oligonucleotides with a DNA polymerase and/or a DNA ligase to generate the desired polynucleotide.
  • the set of oligonucleotides are double-stranded, overlapping oligonucleotides
  • the assembling step comprises (i) contacting the set of oligonucleotides with an exonuclease to produce single-stranded overhangs, wherein the overhangs are complementary between the adjacent oligonucleotides, (ii) annealing the overhangs, and (iii) contacting the annealed oligonucleotides with a DNA polymerase and a DNA ligase to generate the desired polynucleotide.
  • the method comprises a step of inactivating the exonuclease before step (iii).
  • the exonuclease has 3’ exonuclease activity. In some embodiments, the exonuclease has 5’ exonuclease activity.
  • the assembling step is under constant temperature.
  • the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 9-32. In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 11 or SEQ ID NO: 14. In some embodiments, the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 1-8.
  • the assembling the polynucleotide involves a DNA polymerase and/or a DNA ligase.
  • the DNA polymerase is a thermostable DNA polymerase.
  • the DNA ligase is Taq ligase. DETAILED DESCRIPTION OF THE INVENTION
  • an“antibody” as used herein is any form of antibody or fragment thereof that exhibits the desired biological activity, e.g., binding the specific target antigen.
  • monoclonal antibodies including full- length monoclonal antibodies
  • polyclonal antibodies including full- length monoclonal antibodies
  • human antibodies humanized antibodies
  • chimeric antibodies nanobodies, diabodies
  • multispecific antibodies e.g., bispecific antibodies
  • antibody fragments including but not limited to scFv, Fab, and (Fab’)2, so long as they exhibit the desired biological activity.
  • Antibody fragments comprise a portion of an intact antibody, for example, the antigen-binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (e.g., Zapata et al, Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, a designation reflecting the ability to crystallize readily.
  • Pepsin treatment yields an Fiab'fi fragment that has two antigen combining sites and is still capable of cross-linking antigen.
  • the terms,“HIV antibody” and“anti-HIV antibody” are used synonymously to refer to an antibody that binds to an HIV antigen.
  • variable region or“V-region” refers to an antibody variable region domain comprising the segments of Framework 1, CDR1, Framework 2, CDR2, and Framework 3, including CDR3 and Framework 4, which segments are added to the V- segment as a consequence of rearrangement of the heavy chain and light chain V-region genes during B-cell differentiation.
  • CDR complementarity-determining region
  • HVR hypervariable regions
  • the CDRs are the primary contributors to binding to an epitope of an antigen.
  • the CDRs of each chain are referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also identified by the chain in which the particular CDR is located.
  • an HCDR3 is located in the variable domain of the heavy chain of the antibody in which it is found
  • an LCDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.
  • the term“CDR” may be used interchangeably with“HVR”.
  • amino acid sequences of the CDRs and framework regions can be determined using various well known definitions in the art, e.g., Rabat, Chothia, international
  • ImMunoGeneTics database IMGT
  • AbM ImMunoGeneTics database
  • antigen combining sites are also described in the following: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219-221 (2000); and Lefranc,M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan l;29(l):207-9 (2001); MacCallum et al, Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al, Proc. Natl Acad. Sci.
  • CDRs as determined by Rabat numbering are based, for example, on Rabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, MD (1991)). Chothia CDRs are determined as defined by Chothia (see, e.g., Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
  • HIV antibody and“anti-HIV antibody” are used synonymously to refer to an antibody that binds to an HIV antigen.
  • a "neutralizing anti-HIV antibody” as used herein refers to an antibody that can prevent HIV from initiating and perpetuating an infection in a host and/or in target cells in vitro.
  • the terms“identical” or percent“identity,” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same (e.g., at least 70%, at least 75%, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher) identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region.
  • Alignment for purposes of determining percent amino acid sequence identity can be performed in various methods, including those using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity the BLAST 2.0 algorithms, which are described in Altschul et al, Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al, J. Mol. Biol. 215:403-410 (1990). Thus, BLAST 2.0 can be used with the default parameters described to determine percent sequence.
  • the term“aligns to” or“corresponds to” refers to a sequence that matches, or is complementary to, a region, or the entirety of, a reference sequence.
  • adjacent when referring to amino acids in a reference polypeptide sequence refers to amino acids that occur next to each other, i.e., are contiguous, in the reference polypeptide sequence.
  • “Adjacent” oligonucleotides refer to oligonucleotides that contain regions of sequence that are adjacent to one another (occur next to each other) in a reference polynucleotide sequence that encodes the polypeptide sequence.
  • oligonucleotides that are adjacent to one another may contain overlapping complementary nucleic acid sequences, but when linked, e.g., via an extension reaction, ligation reaction, or chemical synthesis, encode amino acids that are contiguous in the reference polypeptide sequence.
  • “adjacent” oligonucleotides do not contain overlapping sequence, but when linked, encode contiguous amino acids in a reference sequence.
  • the term“overlap”, with reference to the relationship of two oligonucleotides refers to the circumstances where an oligonucleotide contains a sequence at one end, e.g., the 3’ end, that is complementary to a sequence of a second oligonucleotide.
  • oligonucleotides means that these two oligonucleotides do not overlap.
  • the term“native sequence” refers to the nucleic acid or amino acid sequence that exist in nature.
  • the native amino acid sequence of the VH of anti-HIV antibody L1A2 is SEQ ID NO: 1 and the native nucleic acid sequence encoding the VH of anti-HIV antibody L1A2 is SEQ ID NO: 33.
  • nucleic acid and“polynucleotide” and“oligonucleotide” are used interchangeably and as used herein refer to both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above.
  • a nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide, and combinations thereof.
  • the terms also include, but are not limited to, single- and double-stranded forms of DNA.
  • a polynucleotide e.g., a DNA or RNA, may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.
  • the nucleic acid molecules may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analogue, intemucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phospho
  • nucleic acid sequence encompasses its complement unless otherwise specified.
  • a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence.
  • a “nucleic acid”,“polynucleotide” or“oligonucleotide” that is referred to as encoding a particular protein sequence also includes codon-optimized nucleic acids that encode the same polypeptide sequence.
  • oligonucleotides disclosed can be synthesized chemically using the methods well known in the art and/or disclosed herein. Oligonucleotides can also be generated through enzymatic reactions such as ligation or PCR. [0043]
  • substitution denotes the replacement of a nucleotide in a polynucleotide by a different nucleotide or for an amino acid, replacement of an amino acid by a different amino acid in a polypeptide.
  • a polynucleotide “variant,” or a nucleic acid“variant,” as the term is used herein, is a polynucleotide that typically differs from a polynucleotide specifically disclosed herein in one or more substitutions.
  • HIV antibodies such as L1A1, L1A2, L2A1, and L1A4, in vitro, including, but not limited to, oligonucleotides encoding polypeptides comprising sequences of L1A1, L1A2, L2A1, and L1A4; and methods of using such oligonucleotides to produce an L1A1, L1A4, or L2A1 variable region, or desired fragment thereof.
  • this disclosure provides oligonucleotides that are different from the native nucleic acids in the patient in that they are modified in various ways, for example, they are attached to a solid support (e.g., via a cleavable linker), a detectable label, or a blocking group.
  • a solid support e.g., via a cleavable linker
  • detectable label e.g., a detectable label
  • a blocking group e.g., a blocking group.
  • One or more of these modifications can, for example be important for synthesizing the coding sequences of these antibodies in vitro or be useful as a proble.
  • this disclosure provides oligonucleotides that encode the native amino acid sequences of the anti-HIV antibody variable region, or a fragment thereof, such as a CDR, but contain at least one nucleotide substitution relative to the native nucleotide sequence. Furthermore, the present disclosure provides methods of assembling the oligonucleotides to produce a desired polynucleotide that encodes a polypeptide comprising the full length or a fragment of the VH or VL region of an anti-HIV antibody L1A1, L1A2, L1A4, or L2A1.
  • the desired polynucleotide encodes a VH that comprises a HCDR3 of L1A1, L1A2, L1A4, or L2A1; or a VL that comprises a LCDR3 of L1A1, L1A2, L1A4, or L2A1.
  • the desired polynucleotide encodes a VH that comprises a HCDR3 of L1A1, Ll A2, L1A4, or L2A1 and an HCDR1 and/or HCDR2 of L1A1, Ll A2, L1A4, or L2A1.
  • the desired polynucleotide encodes a VH comprising the heavy chain CDRs of any one of antibodies L1A1, L1A2, L1A4., or L2A1. In some embodiments, the desired polynucleotide encodes a VL comprising the heavy chain CDRs of any one of antibodies L1A1, L1A2, L1A4., or L2A1. In some embodiments, a desired polynucleotide encodes a polypeptide comprising a fragment of the full-length VH or VL region in which the fragment is anywhere from 10-128, e.g., 20-120, 30-100 consecutive amino acids of the VH or VL of the anti -HIV antibody.
  • the fragment has at least 15, 20, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 consecutive amino acids of a VH or VL of antibody L1A1, L1A2, L1A4, or L2A1.
  • compositions comprising oligonucleotides that can be used to assemble the desired polynucleotides that encodes a polypeptide comprising the full length or a fragment of the VH or VL region of the anti-HIV antibodies as described herein.
  • nucleic acid sequence by SEQ ID NO encompasses the complement the of sequence provided in the SEQ ID NO and both double- stranded and single-stranded nucleic acids forms.
  • Nucleic acids disclosed herein encode full-length variable regions or fragments of the Ll Al, L1A2, L1A1 and L2A1 anti-HIV antibodies described herein.
  • the amino acid sequences of the VH, VL, and CDR regions are shown in Tables 1 and 2 and the nucleic acid sequences are shown in Tables 3 and 4.
  • Table 3 The nucleic acid sequences encoding the variable regions of the anti-HIV antibodies L1A1, L1A2, L1A4, and L2A1
  • Oligonucleotides provided in this disclosure encode polypeptides comprising the VH or VL region, or fragments thereof, having amino acid sequences of the anti-HIV antibodies L1A1, L1A2, L2A1, or L1A4.
  • the oligonucleotide encodes a polypeptide comprising an amino acid sequence in one of the CDR regions of L1A1, L1A2, L1A41, or L2A1.
  • the oligonucleotide encodes a polypeptide comprising an amino acid sequence in one of the framework regions of L1A1, L1A2, L1A41, or L2A1.
  • the oligonucleotide encodes a peptide having an amino acid sequence that is from at least one CDR and an amino acid sequence that is from at least one framework region of L1A1, L1A2, L1A41, or L2A1.
  • the length of these unique amino acid sequences may vary; in some embodiments, the length of the unique amino acid sequence from the Ll Al, L1A2, L1A41, or L2A1 ranges from 5 to 20 amino acids, e.g., 5, 6, 7, 8, 9,
  • the oligonucleotide encodes a sequence selected from the group consisting of SEQ ID NO:65 -514. In some embodiments, the oligonucleotide encodes a polypeptide that comprises a fragment of the heavy chain of L1A1 having an amino acid sequence selected from the group consisting of SEQ ID NO:65-l42, or a fragment of the light chain of L1A1 having an amino acid sequence selected from SEQ ID NO:340-372.
  • the oligonucleotide encodes a polypeptide that comprises a fragment of the heavy chain of L1A2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 144-214, or a fragment of the light chain of L1A2 having an amino acid sequence selected from the group consisting of SEQ ID NO:374-420.
  • the oligonucleotide encodes a polypeptide that comprises a fragment of the heavy chain of L2A1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 216-276, or a fragment of the light chain of L2A1 having an amino acid sequence selected from the group consisting of SEQ ID NO:422-476.
  • the oligonucleotide encodes a polypeptide that comprises a fragment of the heavy chain of L1A4 having an amino acid sequence selected from the group consisting of SEQ ID NO: 278-338, or a fragment of the light chain of L1A4 having an amino acid sequence selected from the group consisting of SEQ ID NO:478-5l4.
  • an oligonucleotide that encodes a region of a VH or VL of an antibody of L1A2, L1A1, L1A4, and L2A1 is attached to a solid support, e.g., for solid phase nucleic acid synthesis, as described below.
  • Suitable solid supports for use include, but are not limited to, paper,
  • the solid supports may take various forms such as fibers, filters and coated containers.
  • the solid support can have a number of shapes, such as pin, strip, plate, disk, rod, bends, cylindrical structure, particle, including bead, nanoparticles and the like.
  • the support can have variable widths.
  • the support can be hydrophilic or capable of being rendered hydrophilic and includes inorganic powders such as silica, magnesium sulfate, and alumina; natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber containing papers, e.g., filter paper, chromatographic paper, etc.; synthetic or modified naturally occurring polymers, such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly (4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinyl butyrate), polyvinylidene difluoride (PVDF) membrane, glass, controlled pore glass, magnetic controlled pore glass, ceramics, metals, and the like; either used by themselves or in conjunction with other materials.
  • inorganic powders such as silica, magnesium sulfate, and alumina
  • an oligonucleotide that encodes a fragment of a VH or VL of an antibody of L1A2, L1A1, L1A4, and L2A1 is attached to a cleavable linker.
  • the oligonucleotide is attached to a solid support via a cleavable linker.
  • the linker may comprise one or more connecting portions.
  • the cleavable linker portion is an organic group selected from straight chains, branched chains and rings and comprises from 1 to 100 atoms and more preferably from 1 to about 50 atoms.
  • the atoms may be selected from C, H, B, N, O, S, Si, P, halogens and alkali metals.
  • Cleavable linkers include, but are not limited to, hydrolytically cleavable linkers, linkers that undergo reductive cleavage, photochemically cleavable linkers, and enzymatically cleavable linkers .
  • the cleavable linker is a hydrolytically cleavable linker, which is cleaved by hydrolysis.
  • Carboxylic esters and anhydrides, thioesters, carbonate esters, thiocarbonate esters, urethanes, imides, sulfonamides, and sulfonimides are representative as are sulfonate esters.
  • the cleavable linker is one that undergo reductive cleavage.
  • One representative group is an organic group containing a disulfide (S— S) bond which is cleaved by thiols such as ethanethiol, mercaptoethanol, and DTT.
  • Another representative group is an organic group containing a peroxide (O— O) bond.
  • Peroxide bonds can be cleaved by thiols, amines and phosphines.
  • the cleavable linker is a photochemically cleavable linker. Suitable photochemically cleavable linkers include but are not limited to nitro-substituted aromatic ethers and esters of the formula where Rd is H, alkyl or phenyl, and more particularly Ortho-nitrobenzyl esters are cleaved by ultraviolet light according to well-known reactions.
  • the cleavable linker is an enzymatically cleavable linker.
  • Suitable enzymatically cleavable linkers include but are not limited to esters, which are cleaved by esterases and hydrolases, amides and peptides, which are cleaved by proteases and peptidases, and glycoside groups which are cleaved by glycosidases.
  • esters which are cleaved by esterases and hydrolases
  • amides and peptides which are cleaved by proteases and peptidases
  • glycoside groups which are cleaved by glycosidases.
  • Chemical nucleic acid synthesis may be accomplished using biological molecules and protecting groups.
  • Various synthesis methods known in the art can be used, such as H- phosphonate methods, phosphoester method, solid phase phosphoramidite method and the like.
  • H- phosphonate methods such as H- phosphonate methods, phosphoester method, solid phase phosphoramidite method and the like.
  • phosphoester method such as phosphoester method
  • solid phase phosphoramidite method such as phosphoramidite method and the like.
  • the synthesis techniques are based on solid-phase synthesis, in which an oligonucleotide is covalently attached to a solid support via its 3’-terminal hydroxyl group and remain attached to it over the course of the synthesis of the entire oligonucleotide chain.
  • the solid phase synthesis involves
  • step 1 Detritylation, the 5 '-DMT protecting group is removed from the first, solid-support-linked nucleoside.
  • step 2 Coupling, the free 5’-OH of the first, solid- support-linked nucleoside attacks the phosphorus of the incoming second nucleoside, displacing its diisopropylamino group.
  • step 3 Oxidation, the unstable phosphite triester is converted to a stable phosphate triester, which allows the next cycle to proceed to step 1, Detritylation of the second nucleotide.
  • step 4 Solid-support-linked nucleosides with unreacted 5’-OH are acetylated, thereby preventing elongation of sequences with deletion mutations (Capping is performed after Oxidation to drive all water out, which would otherwise inhibit the next cycle of the reaction).
  • Detritylation comprises removal of a 5'-DMT (4,4'-dimethoxytrityl) protecting group of the solid-support-linked nucleoside (contains the terminal 3' base of the
  • the 5'-DMT prevents polymerization of the nucleoside during
  • the 5'-DMT protecting group is removed by TCA (trichloroacetic acid) in the solvent dichloromethane (too concentrated a solution of TCA or too long of a detritylation time leads to depurination and hence, lowers the overall yield of the final oligonucleotide).
  • the products include the 3' terminal nucleoside with a free 5'-OH and a DMT carbocation (resonance structure formed by electron delocalization not shown). The nucleoside proceeds to step 2 in the synthesis while the DMT carbocation absorbs at 495 nm and thereby produces an orange color that can be used to monitor coupling efficiency.
  • Coupling is performed after the DMT has been removed.
  • the free 5'- OH of the solid-support-linked nucleoside is able to react with the next nucleoside, which is added as a phosphoramidite monomer.
  • the diisopropylamino group of the incoming phosphoramidite monomer in the solvent acetonitrile is‘activated’ (protonated) by the acidic catalyst ETT [5-(ethylthio)-lH-tetrazole]
  • the mixing is carried out in the fluid lines of the synthesis instrument as the reagents are delivered to the solid support.
  • the activated phosphoramidite is delivered in a many-fold excess over the solid-support- linked nucleoside to drive the reaction to as close to completion as possible.
  • the products include a dinucleoside with a phosphite triester linkage and a free diisopropylamino group.
  • the phosphite triester formed during the coupling reaction is unnatural and unstable; therefore, it must be converted to a more stable phosphorus species prior to the start of the next cycle through oxidation.
  • Oxidation converts the phosphite triester to the stable phosphate triester. Oxidation of the phosphite triester is achieved with iodine in the presence of water and pyridine.
  • the product is the phosphate triester, which is essentially a standard DNA backbone with a b-cyanoethyl protecting group on the free oxygen.
  • the capping reaction typically produces a the solid-support-linked nucleoside with an acetylated 5 '-OH (pyridine maintains a basic pH thereby preventing detritylation of the phosphoramidite monomer by the free acetate / acetic acid).
  • the 3'-phosphate of the nucleoside is affixed to solid-phase support (typically controlled-pore glass beads, silicon substrates, or glass substrates), and an individual nucleotide of choice is added to a chain growing in the 3 '-5' direction.
  • the second cycle begins by starting with step 1, detritylation, followed by each of the remaining three steps.
  • the number of cycles repeated equals the desired number of bases.
  • Chemical nucleotide synthesis service is also readily available from commercial sources, such as Sigma- Aldrich and ATDBio. Exemplar methods of chemical nucleotide synthesis are described in: www.sigmaaldrich.com/technical-documents/articles/biology/dna- oligonucleotide-synthesis.html and www.atdbio.com/content/l7/Solid-phase-oligonucleotide- synthesis, the content of which are hereby incorporated by reference.
  • the nucleic acid synthesis is on a solid support and product is covalently attached to the solid support.
  • the synthesized sequence is isolated from the solid support by, e.g., alkaline treatment.
  • the synthesized product is covalently attached to the solid support via a cleavable linker and the product is released by e.g., cleaving the cleavable linker.
  • the step of cleaving the cleavable linker involves treatment of the solid phase having nucleic acid bound thereto with a cleaving agent.
  • the treatment usually lasts for a period of time sufficient to break a covalent bond in the cleavable linker portion but not to destroy the nucleic acid.
  • the choice of cleaving agent is determined by the nature of the cleavable linker.
  • the cleaving agent is water or a lower alcohol or a mixture thereof.
  • the cleaving agent preferably contains a base which when added to water raises the pH.
  • Preferred bases are selected from hydroxide salts and alkoxide salts or contains a mineral acid or hydrogen peroxide.
  • Exemplary bases include LiOH, NaOH, KOH, NH40H, NaOCH3, KOCH3, and KOt— Bu.
  • the cleaving agent is a reducing agent selected from thiols, amines and phosphines.
  • exemplary reducing agents include ethanethiol, 2-mercaptoethanol, dithiothreitol, trialkylamine and triphenylphosphine.
  • Photochemically cleavable linker groups require the use of light as the cleaving agent, typically light in the ultraviolet region or the visible region.
  • Enzymatically cleavable linker groups as described above are cleaved by enzymes selected from esterases, hydrolases, proteases, peptidases, peroxidases and glycosidases.
  • Other suitable cleaving agents are described in US20050106576A1, the content of which is hereby incorporated by reference.
  • the oligonucleotide is attached to a blocking group.
  • blocking group or“protective group”, used interchangeably, refers to a part of the nucleotide that inhibits or prevents the nucleotide from forming a covalent linkage to a second nucleotide (e.g., the 3’ OH of a primer nucleotide) during the incorporation step of a nucleic acid polymerization reaction.
  • blocking groups are used in the DNA synthesis to prevent unwanted polymerization during DNA synthesis. The blocking group can be removed from the nucleotide, allowing for nucleotide incorporation during primer extension.
  • Non-limiting examples of blocking groups include 5'-DMT (4,4'-dimethoxytrityl), 2-cyanoethyl groups, and acyl protecting groups.
  • the oligonucleotide is attached to a detectable label, e.g., for use in screening a library of variable region sequences.
  • the detectable label can be any label that allows detection the presence of the oligonucleotide it attached hereto.
  • Detectable labels may be incorporated into a nucleic acid library at a 5' end, at a 3' end, and/or at any nucleotide position within the oligonucleotide.
  • the oligonucleotide may be conjugated to one or more (e.g., two or more) detectable labels.
  • the detectable label is a fluorescent label, such as the Green fluorescent protein, or luciferase.
  • fluorescent labels include but not limited to Alexa 488, Alexa 647, R-phycoerythin, PE-Texas Red, PE-cyanine 5, Peridinin- chlorophyll, PE-cyanine-5.5, PE-cyanine 7, allophycocyanin, APC-cyanine 5.5, APC-cyanine 7, and quantum dots.
  • Other labels known to one skilled in the art can also be used in the nucleic acid probe disclosed herein.
  • the detectable label itself is conjugated to the signal- producing agent.
  • the detectable label is a molecule that can bind a binding partner and the binding partner is linked to a signal producing agent.
  • the detectable label is biotin and the binding partner is streptavidin or vice versa.
  • the signal-producing agent can be any agent that produces quantifiable signal, including but not limited to, chemiluminescence, color, or fluorescence.
  • Non-limiting examples of signal-producing agents include an enzyme, a fluorescent molecule, or the like.
  • Other non-limiting examples of detectable labels and signal-producing agents can be found in US20130209990, hereby incorporated by reference.
  • the signal-producing agent is horseradish peroxidase (HRP).
  • this disclosure provides nucleic acid variants encoding a full length or a fragment of the native VH or VL sequences of antibody Ll Al, Ll A2, L1A4, or L2.
  • nucleic acid variants of the native nucleic acid sequences encode VH or VL sequences of the native anti-HIV antibodies.
  • the nucleic acid variant comprises a nucleotide sequence comprising at least one nucleotide substitution relative to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 33-64, but encodes the same amino acid sequence.
  • the nucleotide sequence comprises at least two, three, four, five, six, or more nucleotide substitutions relative to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 33-64.
  • the nucleic acid variant has a total of 1, 2, 3, 4,
  • the nucleic acid variant is a polynucleotide comprising a nucleotide sequence encoding a fragment or the full length of VH sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, and 7, wherein the nucleotide sequence comprises at least 1 nucleotide substitution relative to the nucleic acid sequence selected from the group consisting of SEQ ID NO: 33, 35, 37, and 39, but the nucleic acid sequence of the variant encodes the same polypeptide sequence as the corresponding native nucleic acid sequence.
  • the nucleic variant is a polynucleotide comprising a nucleotide sequence encoding a fragment or the full length of VL sequence selected from the group consisting of SEQ ID NO:2, 4, 6, and 8, wherein the nucleotide sequence comprises at least 1 nucleotide substitution relative to the nucleic acid sequence selected from the group consisting of SEQ ID NO:34, 36, 38, and 40, but the nucleic acid sequence of the variant encodes the same polypeptide sequence as the corresponding native nucleic acid sequenc.
  • the nucleic variant is a polynucleotide comprising a nucleotide sequence encoding a fragment or the full length of CDR sequence selected from the group consisting of SEQ ID NO: 9-32, wherein the nucleotide sequence comprises at least 1 nucleotide substitution relative to the nucleic acid sequence selected from the group consisting of SEQ ID NO:4l-64, but the nucleic acid sequence of the variant encodes the same polypeptide sequence as the corresponding native nucleic acid sequenc.
  • Oligonucleotide design parameters and assembly methods as described here are for illustrative purposes and are not intended to limit the invention to these particular embodiments.
  • Oligonucleotides may be linked to one another to generate a nucleic acid encoding a desired polypeptide by any method, including enzymatic, e.g., using a polymerase and/or ligase, or chemical linkage.
  • the desired polynucleotide encodes a polypeptide region, e.g., comprising at least 10, 20, 30, 40, 50, 70, 80, 90, or more, amino acids of a VL region of the anti-HIV antibody.
  • the length of the oligonucleotides is not limited to any particular size. In some embodiments, the length is 10- 1000 nucleotides, e.g., 20-600, 20-200, 30-150, or 20 to 70 nucleotides in length. In some embodiments, the length can be more than 1000 nucleotides. Chemical syntheses of longer oligonucleotides are possible, but the intrinsic error rate of each coupling step (typically 0.5%-2%) is such that preparations of longer oligonucleotides are increasingly likely to be riddled with errors, and that the pure desired product will be numerically overwhelmed by sequences containing errors.
  • the molecule is often not chemically synthesized as a single long piece; rather, current methods involve combining many shorter oligonucleotides to build the larger desired sequence, a process often referred to as assembling.
  • Methods for assembling oligonucleotides into a nucleic acid of desired length are well known, for example, as described in Gibson et al, Methods in Enzymology, vol.
  • oligonucleotides are designed by first defining a desired polynucleotide, which encodes a polypeptide comprising a fragment or the full length sequence of the VH or VL of the L1A1, L1A2, L1A4, or L2A1 antibody.
  • the desired polynucleotide encodes a polypeptide comprising 10-128, e.g., 20-120, 30-100 consecutive amino acids of the VH or VL of the anti-HIV antibodies.
  • a set of overlapping oligonucleotides are provided, which collectively encode a polypeptide comprising 10-128, e.g., 20-120, 30-100 consecutive amino acids of the VH or VL of the antibody. Any one of the oligonucleotides in such a set comprises a sequence region complementary to a sequence region in an adjacent oligonucleotide.
  • the nucleic acid sequences and lengths of the oligonucleotides disclosed herein may be determined based on, among other factors, one or more of the following considerations to maximize efficiency: i) the oligonucleotides do not contain codons that are rare for the host expression strain; ii) the oligonucleotides do not contain long strings of the same nucleotide, iii) the oligonucleotides do not form homodimers or unintended heterodimers, iv) the oligonucleotides do not form hairpins, v) the oligonucleotides have a balanced GC content, for example, in the range of 30-70%, e.g., 40-60% total GC content, vi) the melting temperature of the overlapping regions in the oligonucleotides are at least 55°C.
  • Commercial software are readily available for aiding the designing process, such as Primer3, IDT oligoanalyzer
  • the number of the overlapping oligonucleotides in the set may vary.
  • the set comprises at least 2, at least 5, at least 10, at least 15, at least 20, or at least 25 oligonucleotides.
  • the length of each of the oligonucleotides may also vary. In some embodiments the length of each of the oligonucleotides is 10-100 nucleotides, e.g., 20-80 nucleotides, 20-50 nucleotides, or 30-60 nucleotides.
  • the oligonucleotides in the set need not be of the same length and often at least two or more of the oligonucleotides in the set are of different lengths.
  • the oligonucleotides are single-stranded oligonucleotides.
  • the first oligonucleotide comprises a sequence region at its 3’ end that is complementary to a sequence region of a 3’ end of the second oligonucleotide in the set
  • the second oligonucleotide comprises a sequence region at 5’ end that is complementary to the sequence region at the 5’ end of the third oligonucleotide in the set
  • the third oligonucleotide comprises a sequence region at its 3’ end that is complementary to a sequence region of a 3’ end of the second oligonucleotide in the set
  • the second oligonucleotide comprises a sequence region at 5’ end that is complementary to the sequence region at the 5’ end of the third oligonucleotide in the set
  • oligonucleotide comprises a sequence at the 3’ end that is complementary to the sequence region at the 3’ end of the fourth oligo, and so on.
  • the adjacent oligonucleotides in the set are annealed to each other throught the complementary sequences and the set of
  • oligonucleotides collectively comprise the sequence of the desired polynucleotide.
  • One illustrative example of the set of oligonucleotides is shown in FIG. 1.
  • the overlapping sequence region has a length of 5-50 nucleotides, e.g., 10-40, 15-35, or about 20 nucleotides.
  • overlapping single-stranded oligonucleotides when hybridizing to one another through the complementary sequences in the overlapping sequence region comprises a nucleic acid sequence that encodes a polypeptide of the desired length, e.g., having 10-128, e.g., 20-120, 30-100 consecutive amino acids of the VH or VL of the L1A1, Ll A2, Ll A4, or L2A1 antibody .
  • the oligonucleotides in the set are double-stranded oligonucleotides with single-stranded overhangs.
  • the double-stranded oligonucleotides with single-stranded overhangs are generated by annealing single-stranded oligonucleotides designed to be partially complementary such that an overhang (on one or both ends) is present when the two strands are annealed.
  • double- stranded oligonucleotides with single-stranded overhangs are generated by treating double- stranded oligonucleotides with an exonuclease.
  • the overhang may be of either the top or bottom strand (either the 5’ or 3’ end). In some embodiments, the overhang has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or more nucleotides.
  • the 3’ overhang of the top strand of the first oligonucleotide is complementary to the 3’ end of the bottom strand of the second oligonucleotide
  • the 3’ overhang of the top strand of the second oligonucleotide is complementary to the 3’ overhang of bottom strand of the third oligonucleotide, and so on.
  • the overlapping double-stranded oligonucleotides when aligned through the overlapping sequence region, collectively encode a nucleic acid sequence that encode a polypeptide of the desired length, e.g., having 10-128, e.g., 20-120, 30-100 consecutive amino acids of the VH or VL of the antibodies.
  • the oligonucleotides in the set are of the same length.
  • Such oligo sets may be used, for example, in a ligation-based assembly method as described below.
  • FIG. 2 One illustrative example of such oligonucleotide set is shown in FIG. 2.
  • the oligonucleotides in the set are not overlapping and they collectively encode a polypeptide comprising at least 10, 20, 50, 70,
  • oligonucleotides can be single-stranded or double-stranded.
  • this disclosure also provides a composition
  • a composition comprising a set of oligonucleotides, each oligonucleotide having a length of 5 -150 nucleotides, e.g., 5-90, 8-80, 10-75, or 20-60 nucleotides, each having a sequence region at one end corresponding to a sequence region in one end of an adjacent oligonucleotide.
  • the first oligonucleotide in the set comprises a sequence that aligns to the 5’ end of the desired polynucleotide and the last oligonucleotide comprises a sequence that aligns to the 3’ end of a desired polynucleotide, e.g, a polynucleotide encoding a polypeptide comprising a fragment or the full length sequence of the VH or VL of an antibody selected from the group consisting of L2A1, Ll Al, L1A2, and L1A4.
  • the set of the oligonucleotide collectively encode the polypeptide.
  • the oligonucleotides designed as such can be chemically synthesized using methods well known in the art and those described above.
  • oligonucleotides can then be purified by, e.g., polyacrylamide gel electrophoresis (PAGE).
  • PAGE polyacrylamide gel electrophoresis
  • higher coupling efficiency for oligo synthesis (such as IDT’s Ultramer platform) enable longer oligonucleotides of desired complexity to be synthesized, reducing the number of oligonucleotides needed and therefore the complexity of the reaction.
  • This disclosure also provides methods of producing a desired polynucleotide encoding a polypeptide comprising a fragment or the full length of the VH or VL of the anti- HIV antibodies disclosed herein.
  • the method comprises providing a set of oligonucleotides that collectively encode the polypeptide, wherein each of the oligonucleotides has an overlapping sequence region corresponding to a sequence region in an adjacent oligonucleotide and assembling the set of oligonucleotides into the nucleic acid sequence through the overlapping sequence regions to produce the desired polynucleotide.
  • the first oligonucleotide in the set comprises a sequence that aligns to the 5’ end of the target nucleotide sequence and the last oligonucleotide comprises a sequence that aligns to the 3’ end of the target nucleotide sequence.
  • the assembling step is a polymerase chain assembly (PC A).
  • the assembling step is a one-pot PC A, i.e., assembling occurs in a reaction mixture in which all oligonucleotides in the set are pooled together and assembled into the target nucleotide.
  • a single PCR reaction is performed using the oligonucleotides in the set to assemble these oligonucleotides into the desired polynucleotide.
  • oligonucleotides in the reaction form a gradient, with the innermost pair of oligonucleotides being at the lowest concentration and the outermost pair of oligonucleotide (i.e., the first and last oligonucleotides in the set) being at the highest concentration. This promotes amplification of ever longer products and ultimately favors the full-length desired polynucleotide.
  • the overlapping sequences between oligonucleotides are balanced to obtain uniform annealing temperatures across the assembly.
  • the assembling step is performed in a stepwise fashion. This method simplifies the correction of synthetic errors by starting from mini-fragments that are formed by briefly extending oligonucleotide pairs. These pairs then build larger fragments, which are in turn assembled into the final product, the desired polynucleotide.
  • sub-fragments that combine two or more adjacent oligonucleotides are first formed by PCR and the sub-fragments are then amplified using additional oligonucleotides in the set as amplification primers to gradually assemble these oligonucleotides into the desired polynucleotide.
  • additional oligonucleotides in the set as amplification primers to gradually assemble these oligonucleotides into the desired polynucleotide.
  • a first and second oligonucleotides in the set are amplified and assembled into a polynucleotide in a first PCR reaction.
  • oligonucleotide is then added to the mixture containing the polynucleotide in a second PCR reaction using the first and the third oligonucleotides as primers. The steps are reiterated until the last oligonucleotide is added and assembled using the first and last oligonucleotides as amplification primers.
  • Various modifications of the methods are well known, for example, as described in Hughes et al., Methods in Enzymology, Vol (498) 2011, 284-291, hereby incorporated by reference.
  • the assembling step is a ligation-based assembly.
  • a set of oligonucleotides is designed to have sequences spanning one or both strands of the desired polynucleotide.
  • the oligonucleotides have sequences that overlap by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or more nucleotides.
  • one or more of the oligonucleotides are phosphorylated.
  • the oligonucleotides are pooled and annealed through the overlapping sequences and ligated into the desired polynucleotide by a ligase.
  • the oligonucleotides are annealed and assembled into sub-fragments that consist of two or more adjacent oligonucleotides; and the sub-fragments are then annealed and assembled into the desired polynucleotide. This annealing and ligating steps may be reiterated until all the oligonucleotides are assembled into the desired polynucleotide.
  • the adjacent oligonucleotides are ligated through a splint oligo, which anneals to both of the adjacent oligonucleotides through sequences that are complementary to adjacent oligonucleotides, such that the adjacent oligonucleotides can be ligated.
  • the first oligonucleotide can be covalently conjugated to a solid support, such as a bead.
  • the assembly comprises annealing a splint oligo to the adjacement oligonucleotides in the set and ligating the adjacent oligonucleotides.
  • the annealing and ligating steps can be reiterated until the all oligonucleotides in the set are assembled into the desired polynucleotide
  • An illustrative example is shown in FIG. 3.
  • the oligonucleotides in the set are indicated by the lines with arrows pointing to the right.
  • the resulting polynucleotide of any given step may be cloned into a vector, sequenced, and then correct clones can be amplified by PCR to generate new starting double-stranded oligonucleotide material either for further ligation-based assembly, or PCA assembly or multi-enzyme assembly, as disclosed herein.
  • oligonucleotides are first denatured by heat and then cooled to promote annealing and ligation.
  • the ligase is a thermostable ligase. Thermostable ligases are stable and active at much higher temperatures than conventional ligases.
  • the ligase remains active at 40 °C or higher, 50 °C or higher, or 55 °C or higher.
  • the half-life of the ligase is at least 12 hours, at least 24 hours, at least 48 hours at the aforementioned temperatures.
  • Thermostable ligases are well known, such as described in Le et al, DNA Res. 2013 Aug; 20(4): 375-382 and Bhanjadeo et al. 2017 Biochemical and Biophysical Research Communications 485 (2): 492-498, and also commercially available, for example, from Wako Chemical USA, Inc. and New England Biolabs.
  • One illustrative type of suitable thermostable ligases is Ampligase, which has a half-life of 48 hours at 65 °C.
  • no denaturing step is required before the oligonucleotides are annealed and ligated.
  • One of ordinary skill in the art would readily be able to make the determination.
  • the method of assembling further comprises a final PCR step to amplify or assemble the sub-fragments produced by the ligation reaction into the desired polynucleotide.
  • a final PCR step to amplify or assemble the sub-fragments produced by the ligation reaction into the desired polynucleotide.
  • the ligation-based assembly is performed in a stepwise fashion, where the ligated product is tethered to a solid support and oligonucleotides are sequentially ligated to the tethered to the ligated product.
  • An illustrative example of this method is shown in U.S. Pat. No. 9217144, the content of which is hereby incorporated by reference.
  • the set of oligonucleotides used in the ligation-based assembly are non-overlapping and the set of oligonucleotides collectively encode a polypeptide comprising a fragment or the full-length sequence of the VH or VL of an antibody selected from the group consisting of L2A1, Ll Al, L1A2, and Ll A4.
  • adjacent oligonucleotides are ligated into sub-fragments by a ligase that is capable of ligating blunt-end oligonucleotides (e.g., T4 ligase) and sub-fragments are then ligated into the desired polynucleotide that encoding the polypeptide described above.
  • one or more oligonucleotides of the set are phosphorylated before the ligation. In some embodiments, the oligonucleotides are double-stranded. In some embodiments, the oligonucleotides are single-stranded.
  • the assembling step is a multi-enzyme assembly, involving at least an exonuclease, a ligase, and a polymerase.
  • the reaction is an isothermal reaction.
  • An isothermal reaction is one that is performed under constant temperature. In some cases the temperature is between 40-60 °C, in some cases the temperature is about 50 °C.
  • the method may comprise contacting the set of oligonucleotides with an exonuclease so that at least one end of the oligonucleotides is exposed, annealing the overlapping sequences that are complementary between the adjacent oligonucleotides, contacting the annealed oligonucleotides with a DNA polymerase and a DNA ligase to assemble the oligonucleotides.
  • the method further comprises reiterating the annealing, extension and ligation steps to assemble the oligonucleotides into the desired polynucleotide.
  • the DNA exonuclease has a 5’-exonuclease activity (e.g., T5 exonuclease) and the assembly can be performed in one single reaction mixture.
  • the DNA exonuclease has a 3’-exonuclease activity (e.g., T4 DNA polymerase) and the exonuclease is inactivated before contacting the exonuclease-treated oligonucleotides with polymerase and ligases to avoid the exonuclease’s inference on the polymerase and ligase activity.
  • Polymerases suitable for use include but are not limited to, thermostable DNA polymerases such as Phusion® andTaq DNA polymerase.
  • the Taq DNA polymerase is an antibody-bound Taq DNA polymerase.
  • the polymerases are DNA polymerases that do not have strand-displacement activity.
  • Various multi-enzyme assembly methods are well known, for example, as described in Gibson, 2011, Methods in Enzymology, Vol. 498, pp 350-361, the content of which is hereby incorporated by reference.
  • the polynucleotide assembled as above may then be cloned into a vector and sequenced to verify that it contains the correct sequence before being used for antibody production.
  • site-directed mutagenesis is performed to correct errors the clones may have to match the sequence of the desired polynucleotide.
  • the disclosure additionally provides a reaction mixture comprising a set of oligonucleotides as described herein, each oligonucleotide having a desired length, for example, 2 -150 nucleotides, e.g., 5-90, 8-80, 10-75, or 20-60 nucleotides, each having an overlapping sequence region at one end corresponding to a sequence region in one end of an adjacent oligonucleotide.
  • the first oligonucleotide in the set comprises a sequence that aligns to the 5’ end of the target nucleotide sequence and the last oligonucleotide comprises a sequence that aligns to the 3’ end of the target nucleotide sequence.
  • oligonucleotides collectively encode a polypeptide comprising a fragment or the full-length sequence of the VH or VL of an antibody selected from the group consisting of L2A1, L1A1, L1A2, and L1A4.
  • the concentrations of the oligonucleotides in the set form a gradient wherein the innermost pair of oligonucleotides being at the lowest concentration and the outermost pair of oligonucleotide being at the highest concentration.
  • the lowest concentration is 20-60 nM, e.g., about 40 nM, and the highest concentration is 100-300 nM, e.g., about 200 nM.
  • the reaction mixture further comprises one or more enzyme selected from the group consisting of a polymerase, an exonuclease, dNTPs, and a ligase.
  • the reaction mixture further comprises a buffer.
  • the buffer contains one or more of the following: Bis-Tris-propane-HCL, PEG-8000, Tris- HC1, MgCh. and DTT.
  • the reaction mixture further comprises excipients such as carboxymethylcellulose (e.g., 1-10% (v/v)).
  • the reaction mixture further comprises one or more of formamide (e.g., 1-10% (v/v)) and dimethylsulfoxide (e.g., 1-10% (v/v)).
  • the exonuclease has 3’ exonuclease activity.
  • the exonuclease has 5’ exonuclease activity.
  • the ligase is a thermostable DNA ligase.
  • the polymerase is a DNA polymerase that has no strand-displacement activity. In some embodiments, the polymerase is a thermostable DNA polymerase.
  • the target nucleotide produced above can then be cloned into heavy chain or light chain vector and can be transfected into host cells to produce the antibody.
  • Vectors and recombinant methodology for producing recombinant antibodies are well known in the art (see, e.g., Sambrook & Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Ausubel, Current Protocols in Molecular Biology). Reagents, cloning vectors, and kits for genetic manipulation are available from commercial vendors. Suitable vectors include but are not limited to plasmids and viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, and any other vector.
  • Host cells of the present disclosure also include, without limitation, isolated cells, in vitro cultured cells, and ex vivo cultured cells.
  • isolated cells in vitro cultured cells
  • ex vivo cultured cells for a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).
  • Oligonucleotides are designed as described herein. Once designed, oligos ⁇ 40-60nt in length collectively encoding a fragment of the antibody of interest, e.g., a heavy chain variable region, are pooled together and assembled in a one- pot polymerase chain assembly (PCA) reaction. 0.2uM of each oligonucleotides are combined in a 20 pl PCR mixture containing dNTPs, polymerase, and buffer. The mixture is then cycled 55 times according to the manufacturer’s suggested protocol with an extension time capable of extending the whole construct.
  • PCA polymerase chain assembly
  • the reaction is then diluted 1 : 100 into another PCR reaction with the same ingredients except for only the outermost oligonucleotides are used as amplification primers.
  • the PCR material are run on a 1.5% agarose in TAE gel. The correct size band is cut out and the gene construct is then inserted by Gibson assembly or restriction digestion into a cloning vector, transformed into bacteria, and sequence confirmed. The gene construct is then ready for expression in the host vector of choice.
  • oligos are designed with the same criteria as in Example 1, except that both the antisense and sense nucleotide sequences are encoded by the oligos used in the reaction (in PCA assembly the complementary sequences are not needed in their entirety).
  • 5’-phosphorylated oligo- nucleotides with carefully designed overlapping sequences that span both strands of a desired DNA duplex are mixed together, heated to denature the oligonucleotides, and then cooled to promote both annealing of the oligo nucleotides and ligation by the thermostable ligase.
  • reaction mixture contained 2.2 mM of each oligo are incubated with 8 m ⁇ of Pfu DNA ligase (Agilent, 600191 ) and lx reaction buffer. Ligations of the products are conducted as follows: 95 °C 1 min; 55 °C 1.5 min, 70 °C 1.5 min, 95 °C 30 seconds for 15 cycles; 55 °C 2 min; and 70 °C 2 min.

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  • Proteomics, Peptides & Aminoacids (AREA)
  • Communicable Diseases (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Hematology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
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  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Cette invention concerne des compositions d'oligonucléotides, et des procédés d'utilisation de telles compositions pour assembler un polypeptide comprenant des séquences d'anticorps anti-VIH obtenus à partir d'un patient.
PCT/US2019/021493 2018-03-09 2019-03-08 Acides nucléiques codant pour des anticorps anti-vih Ceased WO2019173801A1 (fr)

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Cited By (1)

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US11760789B2 (en) 2017-06-22 2023-09-19 University Of Maryland, Baltimore Broadly neutralizing antibodies against HIV

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WO1994018232A1 (fr) * 1993-02-12 1994-08-18 Repligen Corporation Procedes de generation d'anticorps anti-vih a large spectre de neutralisation et antigenes capables de provoquer la generation de ces anticorps
US5464759A (en) * 1988-12-21 1995-11-07 Bionebraska, Inc. Sequential oligonucleotide syntheses using immunoaffinity techniques
US20150011405A1 (en) * 2013-07-03 2015-01-08 Atreca, Inc. Use of Nanoexpression to Interrogate Antigen Repertoires
US20160289305A1 (en) * 2010-09-24 2016-10-06 International Aids Vaccine Initiative Novel hiv -1 broadly neutralizing antibodies
WO2018237357A1 (fr) * 2017-06-22 2018-12-27 University Of Maryland, Baltimore Anticorps de neutralisation du vih à large spectre contre le vih
WO2019067805A1 (fr) * 2017-09-27 2019-04-04 University Of Southern California Nouvelles plates-formes pour la co-stimulation, nouvelles conceptions de car et autres améliorations pour une thérapie cellulaire adoptive

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Publication number Priority date Publication date Assignee Title
US5464759A (en) * 1988-12-21 1995-11-07 Bionebraska, Inc. Sequential oligonucleotide syntheses using immunoaffinity techniques
WO1994018232A1 (fr) * 1993-02-12 1994-08-18 Repligen Corporation Procedes de generation d'anticorps anti-vih a large spectre de neutralisation et antigenes capables de provoquer la generation de ces anticorps
US20160289305A1 (en) * 2010-09-24 2016-10-06 International Aids Vaccine Initiative Novel hiv -1 broadly neutralizing antibodies
US20150011405A1 (en) * 2013-07-03 2015-01-08 Atreca, Inc. Use of Nanoexpression to Interrogate Antigen Repertoires
WO2018237357A1 (fr) * 2017-06-22 2018-12-27 University Of Maryland, Baltimore Anticorps de neutralisation du vih à large spectre contre le vih
WO2019067805A1 (fr) * 2017-09-27 2019-04-04 University Of Southern California Nouvelles plates-formes pour la co-stimulation, nouvelles conceptions de car et autres améliorations pour une thérapie cellulaire adoptive

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SAJADI ET AL.: "Identification of Near-Pan-neutralizing Antibodies against HIV-1 by Deconvolution of Plasma Humoral Responses", CELL, vol. 173, no. 7, 14 June 2018 (2018-06-14), pages 1783 - 1795, XP055637165 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11760789B2 (en) 2017-06-22 2023-09-19 University Of Maryland, Baltimore Broadly neutralizing antibodies against HIV
US12331105B2 (en) 2017-06-22 2025-06-17 University Of Maryland, Baltimore Broadly neutralizing antibodies against HIV

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