WO2020082045A1 - Multispecfic anti-hiv antibodies - Google Patents

Multispecfic anti-hiv antibodies Download PDF

Info

Publication number
WO2020082045A1
WO2020082045A1 PCT/US2019/057089 US2019057089W WO2020082045A1 WO 2020082045 A1 WO2020082045 A1 WO 2020082045A1 US 2019057089 W US2019057089 W US 2019057089W WO 2020082045 A1 WO2020082045 A1 WO 2020082045A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
cdr
acid sequence
antibody
region
Prior art date
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.)
Ceased
Application number
PCT/US2019/057089
Other languages
French (fr)
Inventor
Yuxing Li
James STEINHARDT
Javier Guenaga
John R. Mascola
Tae-Wook CHUN
Susan MOIR
Chi-I CHIANG
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Maryland Baltimore
International AIDS Vaccine Initiative Inc
University of Maryland College Park
US Department of Health and Human Services
Original Assignee
University of Maryland Baltimore
International AIDS Vaccine Initiative Inc
University of Maryland College Park
US Department of Health and Human Services
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by University of Maryland Baltimore, International AIDS Vaccine Initiative Inc, University of Maryland College Park, US Department of Health and Human Services filed Critical University of Maryland Baltimore
Priority to US17/285,956 priority Critical patent/US12365722B2/en
Publication of WO2020082045A1 publication Critical patent/WO2020082045A1/en
Anticipated expiration legal-status Critical
Priority to US19/231,954 priority patent/US20250368722A1/en
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—RNA viruses
    • C07K16/112—Retroviridae (F), e.g. leukemia viruses
    • C07K16/114—Lentivirus (G), e.g. human immunodeficiency virus [HIV], feline immunodeficiency virus [FIV] or simian immunodeficiency virus [SIV]
    • C07K16/1145—Env proteins, e.g. gp41, gp110/120, gp160, V3, principal neutralising domain [PND] or CD4-binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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 [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—RNA viruses
    • C07K16/112—Retroviridae (F), e.g. leukemia viruses
    • C07K16/114—Lentivirus (G), e.g. human immunodeficiency virus [HIV], feline immunodeficiency virus [FIV] or simian immunodeficiency virus [SIV]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • ADCC antibody-dependent cell- mediated cytotoxicity
  • the invention provides a multispecific anti-HIV antibody that binds to multiple epitopes on HIV envelope protein, wherein the antibody comprises:
  • the invention provides an engineered cell that expresses an antibody of the invention.
  • the cell is an immune cell.
  • the cell is a B cell.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody of the invention and/or cells of the invention and a pharmaceutically acceptable carrier.
  • the invention provides a method for treating or preventing HIV infection in a subject, comprising administering to the subject an effective amount of a composition comprising an effective amount of a multispecific antibody as described herein.
  • FIG. 11A-B (A) Design of PentaNAb4.0 Forward ScFV (FIG. 11 A) and PentaNAb 4.0 Reverse ScFv (FIG. 11B).
  • FIG. 12A-B Design of a multi-specific antibody HexaNAb 1.0 ScFv which contains PentaNAb4.0 Reverse ScFv with variable domains of dB4C7/UB-42l, an anti-CD4 antibody.
  • the epitopes recognized are the V1/V2 glycan region, the V3-glycan region, the CD4-binding site (CD4bs), the gpl20-gp4l interface, and the gp4l membrane proximal external region (MPER). Furthermore, the present disclosure shows that using an Fc moiety to combine two penta-ScFv molecules that recognize the same series of HIV-l epitopes in either the forward or reverse orientation resulted in a penta-specific bNAb, which displays near-pan neutralization breadth potently. Thus, penta-specific antibodies combining functional moieties of Env bNAbs could achieve exceptional neutralization capacity with profoundly augmented avidity.
  • narrow neutralizing antibody refers to an antibody which inhibits HIV-l infection, as defined by at least about 50% inhibition of infection in vitro, in more than 50%, 60%, 70%, 80%, 90%, 95%, 99% or greater, of a large panel of (greater than 100) HIV-l envelope pseudotyped viruses and/or viral isolates.
  • antibody encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments, dual affinity retargeting antibodies (DART)), single chain Fv (scFv) mutants, multispecific antibodies such as pentaspecific antibodies generated from at least five intact immunoglobulins, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity.
  • antibody fragments such as Fab, Fab', F(ab')2, and Fv fragments, dual affinity retargeting antibodies (DART)
  • scFv single chain Fv mutants
  • multispecific antibodies such as pentaspecific antibodies generated from at least five intact immunoglobulins, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin
  • antigen binding portion or “antigen binding fragment” of an antibody (or simply “antibody portion” or “antibody fragment”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g. , HIV gpl20). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments can be multi-specific formats; specifically binding to two or more different antigens.
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423- 426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" or "antigen binding fragment" of an antibody.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123).
  • Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer- Verlag. New York. 790 pp. (ISBN 3-540-41354-5).
  • vector is intended to refer to a nucleic acid molecule.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g. , replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • an effective amount is used herein to include the amount of an agent (e.g. a multispecific antibody) that, when administered to a patient for treating an infection, is sufficient to effect treatment of the disease (e.g. , by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease or its related comorbidities).
  • the "effective amount” may vary depending on the agent, how it is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, stage of pathological processes, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.
  • An effective amount includes an amount that results in a clinically relevant change or stabilization, as appropriate, of an indicator of a disease or condition.
  • An effective dose of the antibodies or mutants or variants described herein may provide partial or complete biological activity as compared to the biological activity induced by the wild-type or naturally occurring polypeptides upon which the antibodies or mutants or variants are derived.
  • a therapeutically effective dose of the antibodies or mutants or variants described herein may provide a sustained biochemical or biological effect and/or an increased resistance to degradation when placed in solution as compared with the normal affect observed when the naturally occurring and fully processed translated protein is administered to the same subject.
  • an “immunoconjugate” is an antibody or multispecific antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.
  • compositions according to the disclosure can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • the term “subject” is used throughout the specification to describe an animal to which one or more compositions comprising the antibody or antibodies disclosed herein.
  • the subject is a human.
  • the term“subject” and “patient” may be interchangeably used.
  • the term “patient” will refer to human patients suffering from a particular disease or disorder.
  • the subject may be a human suspected of having or being identified as at risk to develop HIV infection.
  • the subject is suspected of having or has been diagnosed with HIV or HIV-l infection or AIDS.
  • the subject may be a human suspected of having or being identified as at risk to develop AIDS or an AIDS-associated disorder.
  • the subject may be a mammal.
  • the multispecific anti-HIV antibody can be present on one or more polypeptide chains. In some embodiments, the amino acid sequences of parts i-v) are present on a single polypeptide chain. In some embodiments, the multispecific anti-HIV antibody comprises two polypeptide chains, with each chain comprising amino acid sequences of parts i-v). In some embodiments, the antibody simultaneously binds the multiple epitopes.
  • Table 1 List of HIV- 1 Pseudovirus Panel: 208 Total Pseudoviruses Included.
  • the amino acid sequences of parts i-v) comprise amino acid sequences of single chain fragment variable (ScFv) moieties, wherein each ScFv moiety comprises an amino acid sequence from a light chain variable region (VL) and an amino acid sequence from a heavy chain variable region (VH) of an antibody.
  • each ScFv moiety comprises an amino acid sequence from a light chain variable region (VL) and an amino acid sequence from a heavy chain variable region (VH) of an antibody.
  • one or more of the ScFv moieties is organized such that the VL is at the amino terminal end of the ScFv moiety and the VH is at the carboxy terminal end of the ScFv moiety.
  • one or more of the ScFv moieties is organized such that the VH is at the amino terminal end of the ScFv moiety and the VL is at the carboxy terminal end of the ScFv moiety.
  • each ScFv moiety is organized such that the VL is at the amino terminal end of the ScFv moiety and the
  • CD4 binding site (CD4bs) epitope iii. an amino acid sequence that binds to the CD4 binding site (CD4bs) epitope
  • the amino acid sequence that binds to the epitope of the Vl/V2-glycan region comprises an amino acid sequence from an antibody selected from the group consisting of VRC26.25 and PGDM1400; the amino acid sequence that binds to the epitope of the V3-glycan region comprises an amino acid sequence from antibody PGT121; the amino acid sequence that binds to the epitope of the CD4-binding site (CD4bs) comprises an amino acid sequence from antibody N6; the amino acid sequence that binds to the epitope of the gpl20/gp4l interface comprises an amino acid sequence from antibody 35022; and the amino acid sequence that binds to the epitope of the membrane proximal external region (MPER) comprises an amino acid sequence from an antibody selected from the group consisting of 10E8n4, l0E8v4_Sl00cF, and 10E8v4_V5R_S 1 OOcF .
  • MPER membrane proximal external region
  • the amino acid sequence from the antibody VRC26.25 comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR Hl comprises QFRFDGYG, CDR H2 comprises ISHDGIKK and CDR H3 comprises
  • the amino acid sequence from the antibody PGDM1400 comprises the VH region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VH region comprises SEQ ID NO:9; and the VL region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VL region comprises amino acids SEQ ID NO: 13.
  • the amino acid sequence from the antibody PGT121 comprises the VH region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VH region comprises SEQ ID NO: 17; and the VL region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VL region comprises amino acids SEQ ID NO:2l .
  • two functional bNAb moieties can be arranged binding to the same protomer (intra-protomer mode) and in some cases to separate protom ers (inter-protom ers) of the HIV-l Env trimer.
  • the shortest distance between the CD4bs targeting bNAb moiety, N6 (with VRC01 as surrogate), and the gpl20/gp4l interface targeting 35022 entity was determined to be an intra-protomer distance.
  • “knob into hole” technology J. B. Ridgway, L. G. Presta, P. Carter, 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng 9, 617-621 (1996)) can be utilized to generate a heterodimer of the “Forward” and “Reverse” PentaNAb arms, with LS mutations (M428L/N434S) in the Fc fragment for elongated antibody in vivo half-lives as well as improved biodistribution into the mucosal compartment.
  • This“knob into hole” technology is not limiting any method of generating PentaNAb dimers known to persons of skill in the art may be used.
  • two Forwards PentaNAbs can be combined through the use of the Fc regions of IgGl.
  • one Forward PentaNAb can be combined with one Reverse PentaNAb through the use of Fc regions of IgGl.
  • the combinations of Forward and Reverse PentaNAbs are advantageous but not limiting.
  • the invention provides a polynucleotide comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:72-80 and SEQ ID NOS: 90, 92, 94, 96, 98, 99 and 107.
  • polynucleotide encoding a polypeptide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any of SEQ ID NOS:72-80 and SEQ ID NOS: 90, 92, 94, 96, 98, 99 and 107.
  • polynucleotide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOS:52-7l, SEQ ID NOS:8l-89 and SEQ ID NOS 91, 93, 95, 97 and 106.
  • the polynucleotides comprise the coding sequence for the mature polypeptide fused in the same reading frame to a polynucleotide which aids, for example, in expression and secretion of a polypeptide from a host cell (e.g.
  • CTGTCC AATCTGGAGTTCTGGGGAC AGGGAACCGC AGTGAC AGT GAGC
  • DNA molecules encoding light chain variable regions and/or heavy chain variable regions can be chemically synthesized using the sequence information provided herein.
  • Synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, including, e.g., expression control sequences, to produce conventional gene expression constructs encoding the desired antibodies. Production of defined gene constructs is within routine skill in the art.
  • the sequences provided herein can be cloned out of hybridomas by conventional hybridization techniques or polymerase chain reaction (PCR) techniques, using synthetic nucleic acid probes whose sequences are based on sequence information provided herein, or prior art sequence information regarding genes encoding the heavy and light chains.
  • Standard techniques of molecular biology may be used to prepare DNA sequences coding for the antibodies or fragments of the antibodies of the present invention. Desired DNA sequences may be synthesized completely or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate.
  • PCR polymerase chain reaction
  • Any suitable host cell/vector system may be used for expression of the DNA sequences encoding the antibody molecules of the present invention or fragments thereof.
  • Bacterial, for example E. coli, and other microbial systems may be used, in part, for expression of antibody fragments such as Fab and F(ab')2 fragments, and especially Fv fragments and single chain antibody fragments, for example, single chain Fvs.
  • Eukaryotic, e.g. mammalian, host cell expression systems may be used for production of larger antibody molecules, including complete antibody molecules.
  • Suitable mammalian host cells include CHO, HEK293T, PER.C6, myeloma or hybridoma cells.
  • the protein coding sequence should be "operably linked" to regulatory or nucleic acid control sequences that direct transcription and translation of the protein.
  • a coding sequence and a nucleic acid control sequence or promoter are said to be “operably linked” when they are covalently linked in such a way as to place the expression or transcription and/or translation of the coding sequence under the influence or control of the nucleic acid control sequence.
  • the "nucleic acid control sequence” can be any nucleic acid element, such as, but not limited to promoters, enhancers, IRES, introns, and other elements described herein that direct the expression of a nucleic acid sequence or coding sequence that is operably linked thereto.
  • promoter will be used herein to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II and that when operationally linked to the protein coding sequences of the invention lead to the expression of the encoded protein.
  • the expression of the antibodies of the present invention can be under the control of a constitutive promoter or of an inducible promoter, which initiates transcription only when exposed to some particular external stimulus, such as, without limitation, antibiotics such as tetracycline, hormones such as ecdysone, or heavy metals.
  • the promoter can also be specific to a particular cell-type, tissue or organ.
  • suitable promoters and enhancers are known in the art, and any such suitable promoter or enhancer may be used for expression of the antibodies of the invention.
  • suitable promoters and/or enhancers can be selected from the Eukaryotic Promoter Database (EPDB).
  • EPDB Eukaryotic Promoter Database
  • Nucleic acids encoding desired antibodies can be incorporated (ligated) into expression vectors, which can be introduced into host cells through conventional transfection or transformation techniques.
  • Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BEK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce IgG protein.
  • Transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the immunoglobulin light and/or heavy chain variable regions. Specific expression and purification conditions will vary depending upon the expression system employed.
  • the antibodies and/or antigens of the invention can be isolated and/or purified or concentrated using any suitable technique known in the art. For example, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, immuno-affmity chromatography, hydroxyapatite chromatography, lectin chromatography, molecular sieve chromatography, isoelectric focusing, gel electrophoresis, or any other suitable method or combination of methods can be used.
  • the antibodies can be made using recombinant DNA methods as described in ET.S. Pat. No. 4,816,567.
  • the polynucleotides encoding a monoclonal antibody can be isolated from mature B-cells or hybridoma cell, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using conventional procedures.
  • the isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which when transfected into host cells such as E.
  • leucine can be replaced with isoleucine or valine, or vice versa; an aspartate with a glutamate or vice versa; a threonine with a serine or vice versa; or a similar conservative replacement of an amino acid with a structurally related amino acid can be made.
  • the polynucleotide(s) encoding a monoclonal antibody can further be modified in a number of different manners using recombinant DNA technology to generate alternative antibodies.
  • the constant domains of the light and heavy chains of, for example, a mouse monoclonal antibody can be substituted 1) for those regions of, for example, a human antibody to generate a chimeric antibody or 2) for a non immunoglobulin polypeptide to generate a fusion antibody.
  • the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody. Site-directed or high-density mutagenesis of the variable region can be used to optimize specificity, affinity, etc. of a monoclonal antibody.
  • modified antibodies can comprise any type of variable region that provides for the association of the antibody with the polypeptides of HIV such as the envelope protein.
  • the constant region mediates several effector functions.
  • binding of the Cl component of complement to antibodies activates the complement system.
  • Activation of complement is important in the opsonisation and lysis of cell pathogens.
  • the activation of complement also stimulates the inflammatory response and can also be involved in autoimmune hypersensitivity.
  • antibodies bind to cells via the Fc region, with a Fc receptor site on the antibody Fc region binding to a Fc receptor (FcR) on a cell.
  • Fc receptors There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (eta receptors), IgA (alpha receptors) and IgM (mu receptors).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the anti-HIV antibodies provide for altered effector functions that, in turn, affect the biological profile of the administered antibody.
  • the deletion or inactivation (through point mutations or other means) of a constant region domain can reduce Fc receptor binding of the circulating modified antibody thereby increasing tumor localization.
  • constant region modifications consistent with this invention, moderate complement binding and thus reduce the serum half-life and nonspecific association of a conjugated cytotoxin.
  • modifications of the constant region can be used to eliminate disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility.
  • modifications to the constant region in accordance with this invention can easily be made using well known biochemical or molecular engineering techniques well within the purview of the skilled artisan.
  • the multispecific anti-HIV antibodies of the invention are useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as the treatment, cure, functional cure, or prevention of HIV infection.
  • therapeutic treatment methods such as the treatment, cure, functional cure, or prevention of HIV infection.
  • the methods of use may be in vitro, ex vivo, or in vivo methods.
  • the multispecific antibodies disclosed herein may be used as neutralizing antibodies, passively administered or given via gene therapies.
  • the anti-HIV antibodies are useful for detecting the presence of HIV in a biological sample.
  • the term "detecting" as used herein encompasses quantitative or qualitative detection.
  • a biological sample comprises a cell or tissue. Certain other methods can be used to detect binding of anti-HIV antibodies to antigens such as envelope protein.
  • Such methods include, but are not limited to, antigen binding assays that are well known in the art, such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays, and immunohistochemistry (IHC).
  • antigen binding assays such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays, and immunohistochemistry (IHC).
  • the antibodies are labeled.
  • Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction.
  • the antibodies are immobilized on an insoluble matrix. Immobilization entails separating the antibody from any antigen that remains free in solution. This conventionally is accomplished by either insolubilizing the antibody before the assay procedure, as by adsorption to a water-insoluble matrix or surface (Bennich et ah, U.S. Pat. No. 3,720,760), or by covalent coupling (for example, using glutaraldehyde cross-linking), or by insolubilizing the antibody after formation of a complex between the antibody and antigen, e.g., by immunoprecipitation.
  • the present invention provides for methods of treating or preventing HIV infection comprising administering a therapeutically effective amount of a multispecific antibody as described herein to a subject (e.g., a subject in need of treatment).
  • a subject e.g., a subject in need of treatment.
  • the subject is a human.
  • a method includes administering to the subject an amount of an anti- HIV antibody effective to prevent an increase in HIV-l titer, virus replication or an amount of an HIV-l protein of one or more HIV strains or isolates in the subject.
  • the antibodies can be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable, parenteral vehicle.
  • a pharmaceutically acceptable, parenteral vehicle examples include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.
  • Nonaqueous vehicles such as fixed oils and ethyl oleate are also used.
  • Liposomes are used as carriers.
  • the vehicle contains minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives.
  • the antibodies are typically formulated in such vehicles at concentrations of about 1 mg/ml to 10 mg/ml.
  • 0.1 mg/kg to about 20 mg/kg body weight (e.g., about 0.1-15 mg/kg/dose) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • the progress of this therapy is readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art.
  • Antibodies of the invention can be coupled to a drug for delivery to a treatment site or coupled to a detectable label to facilitate imaging of a site comprising cells of interest, such as cells infected with HIV.
  • Methods for coupling antibodies to drugs and detectable labels are well known in the art, as are methods for imaging using detectable labels.
  • Labeled antibodies may be employed in a wide variety of assays, employing a wide variety of labels. Detection of the formation of an antibody-antigen complex between an antibody of the invention and an epitope of interest (an HIV epitope) can be facilitated by attaching a detectable substance to the antibody.
  • Suitable detection means include the use of labels such as radionucleotides, enzymes, coenzymes, fluorescers, chemiluminescers, chromogens, enzyme substrates or co-factors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, and the like.
  • labels such as radionucleotides, enzymes, coenzymes, fluorescers, chemiluminescers, chromogens, enzyme substrates or co-factors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, and the like.
  • the antibodies can be tagged with such labels by known methods. For instance, coupling agents such as aldehydes, carbodiimides, dimaleimide, imidates, succinimides, bid-diazotized benzadine and the like are used to tag the antibodies with the above- described fluorescent, chemiluminescent, and enzyme labels.
  • An enzyme is typically combined with an antibody using bridging molecules such as carbodiimides, periodate, diisocyanates, glutaraldehyde and the like.
  • bridging molecules such as carbodiimides, periodate, diisocyanates, glutaraldehyde and the like.
  • the antibodies can be administered as immunoconjugates, conjugated to a second molecule.
  • the second molecule can be a toxin, a label, a radioisotope, a drug, or a chemical compound.
  • An antibody according to the invention may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent, or a radioactive metal ion or radioisotope.
  • a therapeutic moiety such as a cytotoxin, a therapeutic agent, or a radioactive metal ion or radioisotope.
  • radioisotopes include, but are not limited to, 1-131, 1-123, 1-125, Y-90, Re- 188, Re- 186, At-2l l, Cu-67, B ⁇ -212, B ⁇ -213, Pd-l09, Tc-99, In-l l l, and the like.
  • Such antibody conjugates can be used for modifying a given biological response; the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin, TLR agonists (such as TLR7 agonist), or monomethylauristatin E.
  • a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin
  • TLR agonists such as TLR7 agonist
  • monomethylauristatin E monomethylauristatin E.
  • the combined administration includes co administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities.
  • Preferably such combined therapy results in a synergistic therapeutic effect.
  • the antibody, antigen binding fragment, or nucleic acid encoding the antibody or antigen binding fragment can be combined with anti-retroviral therapy.
  • Antiretroviral drugs are broadly classified by the phase of the retrovirus life- cycle that the drug inhibits.
  • compositions including the antibody, antigen binding fragment, or nucleic acid encoding the antibody or antigen binding fragment, that are disclosed herein, are administered depending on the dosage and frequency as required and tolerated by the patient.
  • the composition should provide a sufficient quantity of at least one of the antibodies disclosed herein to effectively treat the patient.
  • the dosage can be administered once, but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy.
  • nucleic acids are direct administration with plasmid DNA, such as with a mammalian expression plasmid.
  • the nucleotide sequence encoding the disclosed antibody, or antibody binding fragments thereof, can be placed under the control of a promoter to increase expression.
  • Another approach is to administer the nucleic acids in the form of mRNA.
  • the subject is administered cells that are engineered to express the anti-HIV antibody.
  • the cells are engineered immune cells, such as B cells.
  • the cells are engineered, autologous cells.
  • an anti-HIV antibody, or antibody binding fragment thereof can also be expressed by attenuated viral hosts or vectors or bacterial vectors.
  • Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus, retrovirus, cytomegalovirus or other viral vectors can be used to express the antibody.
  • vaccinia vectors and methods useful protocols are described in U.S. Pat. No. 4,722,848.
  • BCG Bacllus Calmette Guerin provides another vector for expression of the disclosed antibodies (see Stover, Nature 351:456-460, 1991).
  • compositions comprising one or more antibodies of the invention.
  • the compositions are pharmaceutical compositions.
  • formulations are prepared for storage and use by combining an antibody with a pharmaceutically acceptable vehicle (e.g. carrier, excipient) (. Remington , The Science and Practice of Pharmacy 20th Edition Mack Publishing, 2000).
  • a pharmaceutically acceptable vehicle e.g. carrier, excipient
  • suitable pharmaceutically acceptable vehicles include, but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (e.g.
  • octadecyldimethylbenzyl ammonium chloride hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (e.g.
  • proteins such as serum albumin, gelatin, or immunoglobulins
  • hydrophilic polymers such as polyvinylpyrrolidone
  • amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine
  • carbohydrates such as monosacchandes, disaccharides, glucose, mannose, or dextrins
  • chelating agents such as EDTA
  • sugars such as sucrose, mannitol, trehalose or sorbitol
  • salt-forming counter-ions such as sodium
  • metal complexes e.g. Zn- protein complexes
  • non-ionic surfactants such as TWEEN or polyethylene glycol (PEG).
  • an antibody or combination of antibodies of the present invention can depend on a variety of factors, such as the severity and course of the disease, the responsiveness of the disease, whether the antibody or agent is administered for therapeutic or preventative purposes, previous therapy, patient's clinical history, and so on all at the discretion of the treating physician.
  • the antibody or agent can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved.
  • the administering physician can easily determine optimum dosages, dosing methodologies and repetition rates.
  • dosage is from 0.01 pg to 100 mg per kg of body weight, and can be given once or more daily, weekly, monthly or yearly.
  • the antibody or combination of antibodies is given once every two weeks or once every three weeks.
  • the dosage of the antibody is from about 0.1 mg to about 20 mg per kg of body weight. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.
  • Effective dosages and schedules for administering embodiments of the present invention can be determined empirically.
  • and effective amount of one or more antibodies are administered to neutralize, treat, prevent or eradicate HIV infection.
  • compositions comprising one or more nucleic acid molecules of the invention are administered to the subject.
  • genetic constructs capable of inducing production of antibodies of the present invention may be administered to a patient in need thereof.
  • Controlled-release parenteral formulations can be made as implants, oily injections, or as particulate systems.
  • Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles.
  • Microcapsules contain the therapeutic protein, such as a cytotoxin or a drug, as a central core. In microspheres the therapeutic is dispersed throughout the particle.
  • Polymers can be used for ion-controlled release of the antibody compositions disclosed herein.
  • Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993).
  • the block copolymer, polaxamer 407 exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has been shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990).
  • hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994).
  • liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, Pa. (1993)).
  • Numerous additional systems for controlled delivery of therapeutic proteins are known (see U.S. Pat. No. 5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No.
  • compositions of the invention may be injectable suspensions, solutions, sprays, lyophilized powders, syrups, elixirs and the like. Any suitable form of composition may be used.
  • a nucleic acid or vector of the invention having the desired degree of purity, is mixed with one or more pharmaceutically acceptable carriers and/or excipients.
  • the carriers and excipients must be "acceptable" in the sense of being compatible with the other ingredients of the composition.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, or combinations thereof, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobul
  • compositions can be designed to introduce the antibodies, nucleic acids or expression vectors to a desired site of action and release it at an appropriate and controllable rate.
  • Methods of preparing controlled-release formulations are known in the art.
  • controlled release preparations can be produced by the use of polymers to complex or absorb the immunogen and/or immunogenic composition.
  • a controlled- release formulations can be prepared using appropriate macromolecules (for example, polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) known to provide the desired controlled release characteristics or release profile.
  • Another possible method to control the duration of action by a controlled-release preparation is to incorporate the active ingredients into particles of a polymeric material such as, for example, polyesters, polyamino acids, hydrogels, polylactic acid, polyglycolic acid, copolymers of these acids, or ethylene vinylacetate copolymers.
  • a polymeric material such as, for example, polyesters, polyamino acids, hydrogels, polylactic acid, polyglycolic acid, copolymers of these acids, or ethylene vinylacetate copolymers.
  • compositions can be administered using any suitable delivery method including, but not limited to, intramuscular, intravenous, intradermal, mucosal, and topical delivery. Such techniques are well known to those of skill in the art. More specific examples of delivery methods are intramuscular injection, intradermal injection, and subcutaneous injection. However, delivery need not be limited to injection methods. Further, delivery of DNA to animal tissue has been achieved by cationic liposomes (Watanabe et al., (1994) Mol. Reprod. Dev.
  • delivery routes can be oral, intranasal or by any other suitable route. Delivery also be accomplished via a mucosal surface such as the anal, vaginal or oral mucosa.
  • Dosing schedules can be readily determined for the particular subject and composition.
  • the composition can be administered one or more times to the subject.
  • there is a set time interval between separate administrations of the composition While this interval varies for every subject, typically it can range from 10 days to several weeks, and is often 2, 4, 6 or 8 weeks. In some embodiments, the interval can be typically from 2 to 6 weeks.
  • compositions of the invention can be administered alone, or can be co administered, or sequentially administered, with other HIV immunogens and/or HIV immunogenic compositions, e.g., with "other" immunological, antigenic or vaccine or therapeutic compositions thereby providing multivalent or "cocktail” or combination compositions of the invention and methods of employing them.
  • ingredients and manner (sequential or co-administration) of administration, as well as dosages can be determined taking into consideration such factors as the age, sex, weight, species and condition of the particular subject, and the route of administration.
  • kits of the invention include a suitable container comprising an HIV-l antibody of the invention in either labeled or unlabeled form.
  • the kit further includes reagents for performing the appropriate indirect assay.
  • the kit includes one or more suitable containers including enzyme substrates or derivatizing agents, depending on the nature of the label. Control samples and/or instructions are also included.
  • PentaNAbs broadly neutralizing antibodies
  • Env HIV-l envelope glycoproteins
  • PentaNAbs penta-specific antibodies
  • G4S single chain fragment variable
  • the bNAb functional moieties incorporated in the design target five major HIV neutralization epitopes including the i) V1/V2 apex glycan, ii) V3- base glycan, iii) CD4 binding site, iv) gpl20/gp4l interface, and v) the membrane proximal external region (MPER).
  • the sequence information of the penta-specific antibodies is listed in Table 4.
  • these PentaScFv entities were fused to the fragment crystallizable region (Fc) region of IgGl to accommodate effector functions in a Penta-IgG format.
  • PentaNAb2.0 was initially constructed, which consists of functional moieties of five bNAbs including 10E8n4 SlOOcF, 35022, N6, PGT121, and VRC26.25 to test the rationale.
  • an molecular orientation was used, namely“Forward” orientation with which the MPER bNAb 10E8n4 SlOOcF moiety is at the N terminus of the PentaNAb and the VI V2 glycan bNAb VRC26.25 moiety on the distal side followed by Fc fragment.
  • PentaNAb2.0 in both ScFv and IgG form, displayed improved virus isolate coverage of 99.6%, as compared to individual bNAbs 10E8, VRC01, PGT121 and 35022, which had 97.6%, 90.4%, 63.9%, and 45.2% coverage, respectively.
  • the PentaNAb2.0 in both ScFv and IgG form, displayed significantly improved IC50 geomeans of 0.006 and 0.072 pg/mL, respectively, as compared to 10E8, which has an IC50 mean of 0.299 pg/mL with the best neutralization breadth among all known bNAbs.
  • PentaNAb iterations were assessed with a panel of 26 HIV-l Env-pseudoviruses in TZM-bl target cells. It was found that all of the PentaNAbs in the ScFv format displayed a greater breadth and potency than their respective IgGl counterparts with Fc fragment. Additionally, it was observed that the PentaNAbs in the “reverse” order displayed a greater potency than the“forward” order while maintaining the same breadth.
  • penta-specific antibodies were engineered that are capable of engaging five major neutralization epitopes of HIV-l envelope glycoproteins possess superior antiviral activities to conventional HIV-l bNAbs and previously engineered tri- and tetra-specific HIV-l bNAbs, which may serve as effective agents for HIV-l prevention, treatment and diagnosis.
  • PentaNAb molecules To constitute PentaNAb molecules, five top lead antibodies displaying the best neutralization potency and breadth among bNAbs within each individual epitope cluster were first selected. For example, in one combination VRC26.25 or PGDM1400, PGT121, N6, 35022, and 10E8n4 V5R SlOOcF from the V1/V2, V3-glycan, CD4bs, gpl20/gp4l interface, and MPER epitope cluster, respectively were picked. In order to develop a PentaNAb capable of simultaneously engaging five separate epitopes, the physical distance between the compatible variable heavy (VH) and light (VL) chain region termini (e.g.
  • VH variable heavy
  • VL light chain region termini
  • VH C-terminus followed by N-terminus of VL) of the selected bNAbs was measured to identify the shortest combinations (Fig 2).
  • the PentaNAb was designed such that the VH and VL of each individual bNAb entity was connected between termini with flexible linkers such that the flexible linker would traverse the shortest unimpeded distance possible. It is believed that joining the individual entities of the PentaNAb between the termini with the shortest unimpeded distance in between would avoid excess linker length, which would i) improve protein folding, ii) improve the expression level/yield of the molecule, and iii) improve the coordination of simultaneous engagement of all five epitopes.
  • V2 e.g. VRC26.25
  • V3 e.g. PGT121
  • both VRC26.25 and PGDM1400 are quaternary-dependent antibodies targeting the V1/V2 epitope at the trimer apex (Doria-Rose et al., J Virol 90, 76-91 (2015); Sok et al., Proc Natl Acad Sci El S A 111, 17624-17629 (2014)) (Fig 1), with VH/VL termini close to one another.
  • a linker of sufficient length e.g. a linker with five G4S units is utilized to connect VRC26.25 or PGDM1400 with PGT121.
  • the shortest distance between the CD4bs targeting bNAb moiety, N6 (with VRC01 as surrogate), and the gpl20/gp4l interface targeting 35022 entity was determined to be an intra-protomer distance.
  • the distance between the 35022 and N6 functional variable domains were determined to be 57 A between the C-terminus of 35022 VH domain and the N-terminus of N6 VL domain with the topology of 35022(VL-VH)-N6(VL-VH), and 47 A between the C-terminus of N6 VL and the N- terminus of 35022 VH with the topology of N6(VH-VL)-35022(VH-VL) ( Figure 8B), respectively.
  • five G4S linkers were used to connect N6 and 35022.
  • the shortest distances between the gpl20/gp4l interface targeting 35022 entity and the MPER targeting 10E8n4 entity was determined to be inter-protomer manner.
  • LTsing PDB 5IQ7 superimposed with PDB 5FYK (Stewart- Jones et al ., Cell 165, 813-826 (2016); Kwon et al ., J Virol 90, 5899-5914 (2016)), a model for Env engaged with both 35022 and 10E8 was generated.
  • the distance between the 10E8n4 and 35022 functional variable domains were determined to be 48 A between the C-terminus of 10E8n4 VH domain and the N-terminus of 35022 VL domain with the topology of l0E8v4(VL-VH)-35O22(VL-VH), and 25 A between the C-terminus of 35022 VL and the N-terminus of 10E8n4 VH with the topology of 35022(VH-VL)- lOE8v4(VH-VL) ( Figure 8C), respectively. Therefore, five G4S linkers were used to connect 35022 and 10E8n4.
  • PentaNAb2.0 (Fig. 2, Table 5) was composed consisting of bNAb moieties of VRC26.25, PGT121, N6, 35022, and l0E8v4_Sl00cF from the V1/V2, V3-glycan, CD4bs, gpl20/gp4l interface, and MPER epitope cluster, respectively.
  • the PentaNAb2.0 -ScFvs and full length IgGs were expressed in mammalian 293FreeStyle cells and purified by Ni+ and protein A columns, respectively. All molecules were characterized by SDS-PAGE gels, size exclusion chromatography (SEC), and dynamic light scattering (DLS) (data not shown). Furthermore, the binding specificities of these constructs were validated by ELISA binding assay (not shown), and Bio-Layer Interferometry (BLI), which demonstrated binding to numerous Env ligands including the CD4bs specific RSC3 probe, an MPER peptide, and full length BG505 SOSIP.664 and JR-FL SOSIP.664 Env trimers (data not shown).
  • the PentaNAb2.0 in both ScFv and IgG form, displayed improved virus isolate coverage of 99.6%, as compared to bNAbs 10E8, VRC01 and PGT121, which had 97.6%, 90.4% and 63.9%, respectively.
  • the PentaNAb2.0 in both ScFv and IgG form, displayed significantly improved IC50 geomeans of 0.006 and 0.072 pg/mL, respectively (Figs 3 & 4). Most significantly, with an IC50 mean of 0.006 pg/mL, the PentaNAb2.0 ScFv is the most potent HIV-l multiNAb tested to date by the comprehensive 208-virus panel.
  • Penta-ScFvs and full length IgGs were expressed in mammalian 293FreeStyle cells and purified by Ni+ and protein A columns (Fig 7), respectively. All the proteins have expected molecular weight, as shown in representative SDS-PAGE gels (Fig 7A), size exclusion chromatography (SEC), and dynamic light scattering (DLS) analysis (data not shown). Furthermore, the binding specificities of these antibodies were validated by ELISA binding assay (not shown), and Bio-Layer Interferometry (BLI) (data not shown).
  • the“Forward/Reverse” IgG orientation displayed a potency in between that of the “Forward” and“Reverse” IgG (Fig 7B & 7C).
  • the“Reverse” orientation of Penta- ScFv and Penta-IgG could be applied to generate antiviral agent in the future.
  • HIV human immunodeficiency virus
  • ART antiretroviral therapy
  • our penta-Nabs exhibited superior degrees of HIV inhibition (neutralization of 99.5% of a 208 pseudo-typed HIV panel), exceptional neutralization capacity (IC50 at 0.006 pg/ml) compared to individual bNAbs, and the capacity to neutralize replication-competent HIV isolates from infected individuals whose virus became resistant to various single bNAbs inferring our penta-Nabs the potential to overcome immune-evasion and eliminate persistent HIV reservoirs.
  • PBMCs peripheral blood mononuclear cells
  • VRC01-PGT121 Bi-NAb was combined with 1) the moiety of MPER mAb 10E8 to form Tri-NAb; and 2) another Bi-NAb consisting of gpl20/gp4l interface mAb and 10E8 in two different N-term to C-term orientations to form Tet-NAbl and 2.
  • a scFV was created connecting variable regions of five bNAbs, namely penta-NAb, which engages all of the five conserved epitopes of HIV Env trimer (Fig. 2).
  • Tri-NAb, Tet-NAbs and Penta-NAb demonstrate remarkably improved neutralization breadth and potency compared to the parental mAbs (Fig. 3A).
  • the superior neutralization breadth and potency possessed by the multi-epitope targeting NAbs are likely caused by cooperative and sometimes synergistic effect.
  • the engineered multi-epitope targeting NAbs which target three to five out of the five major conserved HIV Env neutralizing epitopes, could achieve nearly perfect virus coverage with IC50 geometric mean below 0.1 pg/ml.
  • PentaNAb2.0 Reverse ScFv The neutralization profiles of PentaNAb2.0 Reverse ScFv, PentaNAb4.0 Forward ScFv (depicted in FIG. 11 A), and PentaNAb4.0 Reverse ScFv (depicted in FIG. 11B) were tested with 208-virus panel (FIG. 14A & B). It was confirmed that PentaNAb2.0 Reverse ScFv has incremental improvement in potency (IC 50 geometric mean) compared to the initial design, PentaNAb2.0 ScFv (FIG. 14A & B).
  • HexaNAb 1.0 ScFv (FIG. 12), which contains PentaNAb4.0 Reverse ScFv and variable domains of dB4C7/UB-42l, an anti-CD4 antibody (N Engl J Med. 2019 Apr 18; 380(16): 1535-1545. PMID: 30995373; patent US7501494B2) to 1) enhance the antiviral breadth, and 2) potentially inhibit the emergence of resistant virus variants as demonstrated by a recent clinical trial described in this cited reference.
  • the HexaNAb 1.0 ScFv shows 100% virus neutralization breadth and promising potency (IC50 geometric mean -0.005 ug/mL) (FIG. 14 A & B).
  • Penta-specific antibody in full-length IgG form was constructed (Fig. 13), namely PentaNAb4.0 Stem HC LS v2 IgG, expressed as an IgG assembled from a heavy chain and a light chain.
  • the heavy chain consists of V2 bNAb PGDM1400, V3 bNAb PGT121, and the VH domain of CD4bs bNAb N6.
  • the light chain contains the VL domain of N6 and 35022 and 10E8n4 SlOOcF.
  • the PentaNAb4.0 Stem HC LS v2 IgG demonstrates 99.5% virus coverage (207/208 viruses tested) and high potency (IC50 geometric mean -0.02 ug/mL) (FIG. 14A & B), which is comparable to the Sanofi Tri-specific antibodies, 10E8v4xPGDM 1400/VRC01 and l0E8v4xPGDMl400/N6-LS (FIG. 14B).
  • the 5- specificity feature of the PentaNAb4.0 Stem HC LS v2 IgG may inhibit the emergence of neutralization resistant virus better than the other antibodies possessing less specificities (e.g. Tri-specific antibodies).
  • VSS mAb dB4C7/UB-421 s perfect light chain Fv region (SEQ ID NO. 99)
  • PentaNAb2.0 10E8v4_S100cF-5X-35022-5X-N6-5X-PGT121-5X-VRC26.25-IgG3C hinge-IgGl LS (SEQ ID NO.106)

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • AIDS & HIV (AREA)
  • Engineering & Computer Science (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The present invention provides a multispecific anti-HIV antibody that binds to multiple epitopes on HIV envelope protein, wherein the antibody comprises: i. an amino acid sequence that binds to a V1/V2 apex glycan epitope; ii. an amino acid sequence that binds to a V3-base glycan region epitope; iii. an amino acid sequence that binds to a CD4 binding site (CD4bs) epitope; iv. an amino acid sequence that binds to a gp120/gp41 interface epitope; and v. an amino acid sequence that binds to a membrane proximal external region (MPER) epitope.

Description

MULTISPECIFIC ANTI-HIV ANTIBODIES
This application claims priority to U.S. Provisional Application No. 62/749,510, filed 10/23/2018 and U.S. Provisional Application No. 62/748,228 filed October 19, 2018. The entire disclosures of all of the foregoing applications are incorporated by reference herein.
This invention was made with government support under 5R01 AI136756 awarded by NIH. The government has certain rights in the invention.
FIELD OF THE INVENTION
The field of the invention relates to medicine, infectious disease and in particular antibodies which can neutralize HIV-l virus strains.
BACKGROUND OF THE INVENTION
The acquired immunodeficiency syndrome (AIDS) is caused by the human immunodeficiency virus type 1 (HIV-l) (Gallo et al ., Science 224, 500-503. (1984); Barre-Sinoussi et al., Science 220, 868-871. (1983)), and results from infection and depletion of human CD4+ lymphocytes. HIV-l infection of CD4+ host cells is mediated by the viral envelope glycoproteins (Env), which are displayed as trimeric spikes that sparsely coat the surface of the HIV virion. Each trimeric Env complex is composed of three of each exterior envelope glycoprotein, gpl20, and the gp4l transmembrane envelope glycoprotein (Kowalski et al., Science 237, 1351-1355. (1987); Lu et al., Nat Struct Biol 2, 1075-1082 (1995). This trimeric Env complex is the main target of neutralizing antibody responses [reviewed by (Wyatt et al., Science 280, 1884-1888. (1998)].
Over the past two decades, advances in the treatment of human immunodeficiency virus (HIV) infection have led to dramatic improvements in health outcomes of infected individuals receiving antiretroviral therapy (ART). Nonetheless, the vast majority of HIV- infected individuals must remain on continuous, life-long ART in order to maintain suppression of HIV replication and prevent progression to AIDS. While effective at suppressing HIV replication, current ART requires life-long adherence to daily medication regimens and is associated with significant costs, cumulative toxicities, and the potential for emergence of drug-resistant virus. Consequently, there has been considerable interest in strategies that would allow for discontinuation of ART while maintaining suppression of plasma viremia for prolonged periods. In this regard, recent advances in immunogen and antibody cloning technologies have led to the isolation of several highly potent and broadly neutralizing HIV-specific antibodies (bNAb) from B cells of infected individuals. Some bNAbs have exhibited strong activities against HIV and SIV in vitro, in infected animals, and in infected individuals who were not receiving ART.
Recently, broadly neutralizing antibodies (bNAbs) have been explored as prevention and therapy agents for the treatment and management of HIV- 1, as they can i) inhibit virus entry by neutralizing the free infectious virus particles, ii) prevent virus cell- to-cell spread, and iii) eliminate virus-infected cells by binding the Env molecules expressed on the surface of the infected cells and triggering antibody-dependent cell- mediated cytotoxicity (ADCC) through interactions between the IgG Fc region and the Fc receptors on effector cells (primarily NK cells) [reviewed in Burton et al ., Nat Immunol 16, 571-576 (2015)]. Furthermore, the repertoire of isolated bNAbs has drastically increased recently as the process for bNAb isolation and characterization has accelerated with the integration of emerging functional and structural information and new technologies of single B cell sorting and cloning (Burton et al, Proc Natl Acad Sci U S A 88, 10134-10137 (1991); Buchacher et al., AIDS Res Hum Retroviruses 10, 359-369 (1994); Huang et al, Nature 491, 406-412 (2012); Scheid et al, Science 333, 1633-1637 (2011); Wu et al, Science 329, 856-861 (2010); Walker et al, Nature All, 466-470 (2011)). The characterization of HIV- 1 bNAbs and their cognate epitopes on the Env spikes has identified five conserved Env sites of vulnerability including, in the order from the apex to the stem of Env: the Vl/V2-glycan region, the V3- glycan region, the CD4- binding site (CD4bs), the gpl20-gp4l interface (Burton et al, Nat Immunol 16, 571-576 (2015)), and the gp4l membrane proximal external region (MPER) (Burton et al, Nat Immunol 16, 571-576 (2015); Kwong et al, Nat Rev Immunol 13, 693-701 (2013)) (Fig 1).
Administration of a single bNAb as a therapeutic agent has successfully cleared phase I safety clinical trials, demonstrating temporary HIV-l viremia suppression in the majority of patients (Caskey et al, Nature 522, 487-491 (2015); Ledgerwood et al, Clin Exp Immunol 182, 289-301 (2015)). Unfortunately, the HIV virus rapidly develops resistance mutations under the selective pressure of single bNAb, suggesting that passive treatment with a single bNAb is unlikely to result in long-term viremia suppression (Caskey et al, Nature 522, 487-491 (2015); Lynch et al, Sci Transl Med 7, 3 l9ra206 (2015); Bar et al. , N Engl J Med 375, 2037-2050 (2016); Caskey et al. , Nat Med 23, 185- 191 (2017)). Fortunately, some Env mutations associated with bNAb resistance can reduce viral fitness, suggesting that simultaneously targeting different Env epitopes may compromise viral replication (Tebit et al, AIDS Rev 9, 75-87 (2007); Sather et al., J Virol 86, 12676-12685 (20l2);Lynch et al, J Virol 89, 4201-4213 (2015); Pietzsch et al, J Exp Med 207, 1995-2002 (2010)). The quick onset of escaping virus quasi species in these clinical trials with single bNAb agent strongly highlights the need to develop combinational therapy regimens to control virus rebound by preventing emergence of resistant virus. Additionally, in vitro data from previous studies have demonstrated that combinatorial bNAb therapies display a substantial gain of neutralization potency and breadth when two or more bNAbs targeting independent epitopes are combined (Kong et al, J Virol 89, 2659-2671 (2015); Doria-Rose et al, J Virol 86, 3393-3397 (2012)). This in vitro data is further supported by a number of in vivo animal studies in which dual-, triple-, and penta- combinations of bNAbs resulted in improved protective efficacy compared to mono bNAb therapy (Shingai et al, Nature 503, 277-280 (20l3);Klein et al, Nature 492, 118-122 (2012)).
Collectively, these findings suggest that passive immunotherapy with a bNAb(s) could potentially prevent plasma viral rebound in HIV-infected individuals following cessation of ART. For this reason, we recently conducted a clinical trial to investigate whether VRC01 could prevent plasma viral rebound upon discontinuation of ART. While multiple infusions of VRC01 were safe and well-tolerated, the majority of patients experienced plasma viral rebound due to pre-existing and emergent VRC01 -resistant HIV despite adequate levels of antibody in plasma (Lynch, Sci Transl Med 7, 3 l9ra206 (2015); Bar et al, N Engl J Med 375, 2037-2050 (2016)). Therefore, therapeutic strategies involving passive transfer of bNAbs may require a combination of bNAbs that targets multiple regions on HIV Env protein in order to achieve sustained virologic control upon withdrawal of ART.
Of note, the potential significance of the above concept was recently highlighted in an animal experiment in which a trimeric antibody consisting of VRC01, PGDM1400, and 10E8 showed profound potency and breadth against a mixture of SHIV (Xu et al ., Science 358, 85-90 (2017)). Building upon these findings, it is of great importance to explore the effect of multimeric bNAbs (or bNAb-like molecules) that possess broad and potent neutralization capacity against highly heterogeneous infectious viral isolates.
While antibody cocktails demonstrated improved efficacy in preclinical studies, multispecific“single agents” are desirable for manufacturing purposes (Hu et al ., Adv Drug Deliv Rev 98, 19-34 (2016) as well as for improved avidity that may result in enhanced neutralization breadth and potency (Galimidi et al ., Cell 160, 433-446 (2015)). Previous bispecific bNAb designs utilizing CrossMab technology to combine two bNAb Fabs first proved the concept that empirical combinations of bNAb functional moieties in bi-valence format could achieve breadth and coverage (94-97%) superior to individual parental bNAbs (70-90%) (Asokan et al. , J Virol 89, 12501-12512 (2015)). These empirical combinations were recently improved upon with cross-over dual variable (CODV) technology to develop Tri-NAbs with improved potency and breadth (Xu et al ., Science 358, 85-90 (2017)). In addition, using tandem ScFvs format, we engineered a trispecific HIV-l neutralizing antibody and two tetra-specific antibodies, consisting of functional moieties of three and four HIV bNAbs, respectively (Steinhardt et al ., Nat Commun 9, 877 (2018)) (PCT/US2017/057053), which displays elevated neutralization breadth and potency compared to the parental bNAbs.
This background information is provided for informational purposes only. No admission is necessarily intended, nor should it be construed, that any of the preceding information constitutes prior art against the present invention.
SUMMARY OF THE INVENTION
It is to be understood that both the foregoing general description of the embodiments and the following detailed description are exemplary, and thus do not restrict the scope of the embodiments.
Provided herein are multispecific neutralizing antibodies against HIV which in some embodiments utilize a tandem ScFv format to combine five HIV Env bNAb moieties via structure-based rational design. In some embodiments, these antibodies can achieve simultaneous engagement of five separate epitopes by each respective individual bNAb functional moiety to achieve superior binding avidity, profoundly enhanced viral inhibition breadth and potency, and ADCC functions.
By targeting >3 Env epitopes, these multi-NAbs display near pan-isolate neutralization breadth (99.6% coverage), high potency (GMT IC50 = 0 006pg/mL) as assessed by a 208 virus panel and effectively neutralize viral quasi-species isolated from VRC01 clinical trials that are frequently resistant to VRC01. Furthermore, the data herein suggest that these multi-NAbs possess substantially higher antibody-dependent cell- mediated cytotoxicity (ADCC) capacity than their parental bNAbs. Taken together, the data suggest that multi-NAbs may be used as a novel candidate format for the treatment and management of persistent HIV-l infection.
In some embodiments, the present disclosure describes synergistically combined epitope-binding moieties from five bNAbs that target the CD4 binding site, V2 and V3 conserved glycans, gpl20/gp4l interface, as well as the membrane exterior proximal region into a‘single’ penta-specific antibody (penta-NAb). The penta-Nabs exhibited superior degrees of inhibition against pseudo-typed viruses, exceptional neutralization capacity over individual bNAbs, and the capacity to neutralize replication-competent HIV isolates from infected individuals.
In one aspect, the invention provides a multispecific anti-HIV antibody that binds to multiple epitopes on HIV envelope protein, wherein the antibody comprises:
1 an amino acid sequence that binds to a V1/V2 apex glycan epitope;
11 an amino acid sequence that binds to a V3-base glycan region epitope;
111 an amino acid sequence that binds to a CD4 binding site
(CD4bs) epitope;
IV. an amino acid sequence that binds to a gpl20/gp4l interface epitope; and
v. an amino acid sequence that binds to a membrane proximal external region (MPER) epitope. In some embodiments, the amino acid sequences of parts i-v) comprise amino acid sequences of single chain fragment variable (ScFv) moieties, wherein each ScFv moiety comprises an amino acid sequence from a light chain variable region (VL) and an amino acid sequence from a heavy chain variable region (VH) of an antibody. In some embodiments, the VL and VH sequences are separated by one or more linking amino acids. In some embodiments, the linking amino acids comprise one or more tetra-glycine serine (G4S) linkers. In some embodiments, the antibody further comprises an Fc region of an immunoglobulin or a variant thereof. In some embodiments, the antibody comprises a first and second polypeptide chain, wherein the first and second polypeptide chains each comprise five ScFv moieties, wherein each ScFv moiety on a single chain recognizes an individual epitope, wherein each ScFv moiety comprises an amino acid sequence from a light chain variable region (VL) and an amino acid sequence from a heavy chain variable region (VH) of an antibody and an Fc region of an immunoglobulin or a variant thereof.
In some embodiments, the amino acid sequence that binds to the epitope of the Vl/V2-glycan region comprises an amino acid sequence from an antibody selected from the group consisting of VRC26.25 and PGDM1400; the amino acid sequence that binds to the epitope of the V3-glycan region comprises an amino acid sequence from antibody PGT121; the amino acid sequence that binds to the epitope of the CD4-binding site (CD4bs) comprises an amino acid sequence from antibody N6; the amino acid sequence that binds to the epitope of the gpl20/gp4l interface comprises an amino acid sequence from antibody 35022; and the amino acid sequence that binds to the epitope of the membrane proximal external region (MPER) comprises an amino acid sequence from an antibody selected from the group consisting of 10E8n4, l0E8v4_Sl00cF, and 10E8v4_V5R_S 1 OOcF .
In some embodiments, the amino acid sequence from the antibody VRC26.25 comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR Hl comprises QFRFDGYG, CDR H2 comprises ISHDGIKK and CDR H3 comprises
AKDLREDECEEWW SDDF GKQLPC AK SRGGL V GI ADN ; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR Ll comprises TSNIGNNF, CDR L2 comprises ETD and CDR L3 comprises ATWAASLSSARV; the amino acid sequence from the antibody PGDM1400 comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR Hl comprises GNTLKTYD, CDR H2 comprises ISHEGDKK and CDR H3 comprises AKGSKHRLRDYALDDDGALNWAVDVDYLSNLEF; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR Ll comprises HSLIHGDRNNY, CDR L2 comprises LAS and CDR L3 comprises MQGRESPWT; the amino acid sequence from the antibody PGT121 comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR Hl comprises GASISDSY, CDR H2 comprises VHKSGDT and CDR H3 comprises ARTLHGRRIYGIVAFNEWFTYFYMDV; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR Ll comprises SLGSRA, CDR L2 comprises NNQ and CDR L3 comprises HIWDSRVPTKWV; the amino acid sequence from the antibody N6 comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR Hl comprises GYTFTAHI, CDR H2 comprises IKPQYGAV and CDR H3 comprises AR; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR Ll comprises QGVGSD, CDR L2 comprises HTS and CDR L3 comprises QVLQF; the amino acid sequence from the antibody 35022 comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR Hl comprises GYRFNFYH, CDR H2 comprises ISPYSGDK and CDR H3 comprises DDTGTYFCAKGLLRDGSSTWLPYL; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR Ll comprises NSVCCSHKS, CDR L2 comprises EDN and CDR L3 comprises CSYTHNSGCV; the amino acid sequence from the antibody 10E8n4 comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR Hl comprises GFDFDNAW, CDR H2 comprises ITGPGEGWSV and CDR H3 comprises TGYYFCARTGKYYDFWSGYPPGEEYFQD; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR Ll comprises RGDSLRSHYAS, CDR L2 comprises GKNNRPS and CDR L3 comprises SSRDKSGSRLSV; the amino acid sequence from the antibody l0E8v4_Sl00cF comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR Hl comprises GFDFDNAW, CDR H2 comprises ITGPGEGWSV and CDR H3 comprises TGYYFCARTGKYYDFWFGYPPGEEYFQD; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR Ll comprises RGDSLRSHYAS, CDR L2 comprises GKNNRPS and CDR L3 comprises SSRDKSGSRLSV; the amino acid sequence from the antibody l0E8v4_V5R_Sl00cF comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR Hl comprises GFDFDNAW, CDR H2 comprises ITGPGEGWSV and CDR H3 comprises TGYYFCARTGKYYDFWFGYPPGEEYFQD; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR Ll comprises RGDSLRSHYAS, CDR L2 comprises GKNNRPS and CDR L3 comprises SSRDKSGSRLSV.
In another aspect, the invention provides one or more vectors comprising a nucleic acid encoding the antibody or polypeptide of the invention.
In another aspect, the invention provides a cell comprising the one or more vectors of the invention.
In another aspect, the invention provides an engineered cell that expresses an antibody of the invention. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a B cell.
In another aspect, the invention provides a pharmaceutical composition comprising an antibody of the invention and/or cells of the invention and a pharmaceutically acceptable carrier.
In another aspect, the invention provides a method for treating or preventing HIV infection in a subject, comprising administering to the subject an effective amount of a composition comprising an effective amount of a multispecific antibody as described herein.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE FIGURES
The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
FIG. 1. HIV-l Env major epitope clusters and their spatial relationships.
FIG. 2. Schematic of bNAb entities linked together in tandem to form PentaNAb 2.0. (A) Topology of PentaNAb design: from the Env MPER to the Env apex, with the shortest distance between bNAb entities marked. Modeling of Env trimer BG505 SOSIP.664 binding to five bNAbs targeting the five conserved epitopes including the V1V2 glycan, V3 base glycan, CD4 binding site (CD4bs), gpl20/gp4l interface, and membrane proximate exterior region (MPER) simultaneously by superimposing individual bNAb Fab-Env complex structure, only VH-VL regions of Fab are shown. Env trimer in grey, and the bNAb moieties in color; (B) Scheme of the PentaNAb design, with MPER bNAb at the N terminus and V1V2 bNAb connected with CH2-CH3 domains of IgG Fc. Strategies of engineering penta-ScFv by joining the termini of VHs and VLs by linkers in dash lines optimized to accommodate individual VHs and VLs to achieve simultaneous epitope engagement; (C) Linear presentation of PentaNAb 2.0 design.
FIG. 3. Summary of neutralization breadth and potency of PentaNAb2.0 in a comprehensive panel of viruses, in comparison with other multi-specific antibody designs. (A) Comparison of potency-breadth curves and epitope specificities of Tri- and Penta-NAb against a panel of 208 circulating primary HIV-l strains; (B) Heat maps of IC50, breadth and potency are shown as in (A) and Fig. 4. GMT, geometric mean of titer (IC50). The IC50 was adjusted to account for the molarity difference between the antibodies.
FIG. 4. (A) IC50 values (pg/ml) for PentaNAb2.0 and other antibodies tested in a 208 virus panel. The IC50 was adjusted to account for the molarity difference between the antibodies. (B) Summary of IC50 values for PentaNAb2.0 and other antibodies.
FIG. 5. Neutralization capacity of PentaNAbs tested against primary HIV-l viruses isolated from patients involved in VRC01 therapy treatment. Neutralization of virus from patient V07-RD (A), and V09-RD (B), tested with antibodies at 2.5 pg/ml (left) and 10 pg/ml (right), respectively. (C) Summary of IC50 / IC80 (pg/ml) for PentaNAbs tested against primary VRC01 resistant isolates. Neutralization IC50 and IC80 titers (pg/ml) of Penta-scFV and Penta-NAb IgG against HIV isolated from VRC01 clinical trial. The IC50 and IC80 values were adjusted to account for the molarity difference between the antibodies.
FIG. 6. Schemes of various iterations in which bNAb entities can be linked together in tandem to form PentaNAbs in the “Reverse” or “Forward/Reverse” orientation. (A) The“reverse” orientation of the PentaNAb design, in contrast to that in Fig. 2; (B) The PentaNAb“Forward/Reverse” orientation configuration conferred by IgG Fc bearing“knob-into-hole” mutations to form heterodimer.
FIG. 7. Comparison of PentaNAbs in both the Forward, Reverse and Forward/Reverse orders. (A) SDS PAGE gel analysis for PentaNAbs; (B) Summary of neutralization breadth and potency of PentaNAbs against a 26-virus panel of high stringency; (C) Neutralization IC50 values of PentaNAbs against the 26-virus panel. The IC50 was adjusted to account for the molarity difference between the antibodies.
FIG. 8. (A-C) Spatial distance of bNAb functional moieties.
FIG. 9. Bi-ScFv and bi-NAb of VRC01-PGT121 design and EM analysis. (A) Schematic presentation of VRC01-PGT121 ScFv and full length IgG. (B) EM analysis of Env trimer BG505 SOSIP.664 and VRC01-PGT121 ScFv complex suggests bi-specific binding mode. Left, representative EM image of Env trimer BG505 SOSIP.664 and VRC01-PGT121 bi-ScFv complex. Arrows indicate ScFv molecules. Right, EM image of PGV04 (green) and PGT122 (blue) Fab complex with BG505 SOSIP.664 simultaneously. FIG. 10. Levels of HIV replication in NOD-scid IL2Rgnu11 (NSG) mice. PBMCs from an HIV-infected, ART-naive individuals were injected to 8 mice and plasma viremia was monitored over time. The limit of detection was 20 copies/ml as measured by Roche Ampliprep/COBAS HIV-l Test Version 2.
FIG. 11A-B. (A) Design of PentaNAb4.0 Forward ScFV (FIG. 11 A) and PentaNAb 4.0 Reverse ScFv (FIG. 11B). FIG. 12A-B. Design of a multi-specific antibody HexaNAb 1.0 ScFv which contains PentaNAb4.0 Reverse ScFv with variable domains of dB4C7/UB-42l, an anti-CD4 antibody.
FIG. 13A-C. Design of a Penta-specific antibody in full-length IgG form, namely PentaNAb4.0 Stem HC LS v2 IgG (FIG. 13A), expressed as an IgG assembled from a heavy chain (FIG. 13B) and a light chain (FIG 13C). The antibody contains the M428L/N434S (LS) mutation in the Fc domain to elongate antibody in vivo half-life. The heavy chain consists of V2 bNAb PGDM1400, V3 bNAb PGT121, and the VH domain of CD4bs bNAb N6 (FIG. 13B). The light chain contains the VL domain of N6 and 35022 and 10E8n4 SlOOcF (FIG.13C).
FIG. 14A-B depicts the neutralization profiles of PentaNAb2.0 Reverse ScFv, PentaNAb4.0 Forward ScFv, PentaNAb4.0 Reverse ScFv, HexaNAb 1.0 ScFv and PentaNAb4.0 Stem HC LS v2 IgG tested with a 208-virus panel (Fig. 14A & B). The PentaNAb2.0 Reverse ScFv has incremental improvement in potency (IC 50 geometric mean) compared to the initial design, PentaNAb2.0 ScFv. The HexaNAb 1.0 ScFv (FIG. 12) shows 100% virus neutralization breadth and promising potency (IC50 geometric mean -0.005 ug/mL) (Fig. 14 A & B). The PentaNAb4.0 Stem HC LS v2 IgG demonstrates 99.5% virus coverage (207/208 viruses tested) and high potency (IC50 geometric mean -0.02 ug/mL) (Fig. 14A & B).
DETAILED DESCRIPTION OF THE INVENTION
The present disclosure is based on the discovery of highly neutralizing and potent HIV antibodies that are capable of engaging at least five epitopes on HIV envelope protein. In some embodiments, the antibody comprises single-chain variable fragment (ScFv) domains of five bNAbs, specific for the HIV-l envelope epitopes that have been joined to form penta-specific ScFvs (penta-ScFvs). The penta-ScFv crosslinks adjacent HIV-l envelope protomers and demonstrates superior neutralization breadth over its parental bNAbs. The epitopes recognized are the V1/V2 glycan region, the V3-glycan region, the CD4-binding site (CD4bs), the gpl20-gp4l interface, and the gp4l membrane proximal external region (MPER). Furthermore, the present disclosure shows that using an Fc moiety to combine two penta-ScFv molecules that recognize the same series of HIV-l epitopes in either the forward or reverse orientation resulted in a penta-specific bNAb, which displays near-pan neutralization breadth potently. Thus, penta-specific antibodies combining functional moieties of Env bNAbs could achieve exceptional neutralization capacity with profoundly augmented avidity. The penta-specific antibodies described herein can be used in studies aimed at preventing HIV disease progression or mother to child transmission, and curing HIV. Furthermore, the approach described herein, that combines multi-functional moieties of individual bNAbs with profoundly elevated avidity and cooperative effect of multivalence interactions, may be applied to generate superior antibody -based anti-viral therapeutics against other infectious agents.
For the purpose of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is clearly intended (for example in the document where the term is originally used). The use of "or" means "and/or" unless stated otherwise. As used in the specification and claims, the singular form "a," "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof. The use of“comprise,”“comprises,”“comprising,”“include,”“includes,” and “including” are interchangeable and not intended to be limiting. Furthermore, where the description of one or more embodiments uses the term“comprising,” those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language“consisting essentially of’ and/or “consisting of.”
Abbreviations for amino acids are used throughout this disclosure and follow the standard nomenclature known in the art. For example, as would be understood by those of ordinary skill in the art, Alanine is Ala or A; Arginine is Arg or R; Asparagine is Asn or N; Aspartic Acid is Asp or D; Cysteine is Cys or C; Glutamic acid is Glu or E; Glutamine is Gln or Q; Glycine is Gly or G; Histidine is His or H; Isoleucine is Ile or I; Leucine is Leu or L; Lysine is Lys or K; Methionine is Met or M; Phenylalanine is Phe or F; Proline is Pro or P; Serine is Ser or S; Threonine is Thr or T; Tryptophan is Trp or W; Tyrosine is Tyr or Y; and Valine is Val or V.
As used herein, the term "about" means plus or minus 10% of the numerical value of the number with which it is being used.
The term "broad neutralizing antibody" refers to an antibody which inhibits HIV-l infection, as defined by at least about 50% inhibition of infection in vitro, in more than 50%, 60%, 70%, 80%, 90%, 95%, 99% or greater, of a large panel of (greater than 100) HIV-l envelope pseudotyped viruses and/or viral isolates. In some embodiments, the broad neutralizing antibody is an antibody that inhibits HIV- 1 infection as defined by at least about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% inhibition of infection in vitro in more than about 50%, 60%, 70%, 80%, 90%, 95%, 99% or greater, of a large panel of (greater than 100) HIV-l envelope pseudotyped viruses and/or viral isolates. In some embodiments, the disclosure relates to a composition comprising one or a plurality of broad neutralizing antibodies.
As used herein, the term "in combination with," is not intended to imply that the therapy or the therapeutic agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope described herein.
The therapeutic agents can be administered concurrently with, prior to, or subsequent to, one or more other additional therapies or therapeutic agents.
The term "antibody" means a molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within a variable region of the molecule, or any functional fragment, mutant, variant, or derivative thereof which retains the epitope binding features of an immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments, dual affinity retargeting antibodies (DART)), single chain Fv (scFv) mutants, multispecific antibodies such as pentaspecific antibodies generated from at least five intact immunoglobulins, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity.
In some embodiments, the antibody can comprise a sequence from any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.
In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is generally composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
A "conservative amino acid substitution" is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the invention do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence, to the antigen(s), i.e., the envelope protein to which the polypeptide or antibody binds. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well-known in the art (see, e.g., Brummell et al, Biochem. 32: 1180-1187 (1993); Kobayashi et al, Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).
As used herein, the term "CDR" refers to the complementarity determining region within antibody variable sequences. In some embodiments, there are three CDRs in each of the variable regions of the heavy chain and the light chain of an antibody, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The term "CDR set" as used herein refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Rabat (Rabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Rabat CDRs. Chothia and coworkers (Chothia et al., J. Mol. Biol. 196:901-917 (1987) and Chothia et al, Nature 342:877-883 (1989)) found that certain sub-portions within Rabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as LI, L2 and L3 or HI, H2 and H3 where the "L" and the "H" designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Rabat CDRs. Other boundaries defining CDRs overlapping with the Rabat CDRs have been described by Padlan (FASEB J. 9: 133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Rabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments use Rabat or Chothia defined CDRs.
As used herein, the term "fragment" is defined as a physically contiguous portion of the primary structure of a biomolecule. In the case of polypeptides, a fragment may be defined by a contiguous portion of the amino acid sequence of a protein and may be at least 3-5 amino acids, at least 6-10 amino acids, at least 11-15 amino acids, at least 16-24 amino acids, at least 25-30 amino acids, at least 30-45 amino acids and up to the full length of the protein minus a few amino acids. In the case of polynucleotides, a fragment is defined by a contiguous portion of the nucleic acid sequence of a polynucleotide and may be at least 9-15 nucleotides, at least 15-30 nucleotides, at least 31-45 nucleotides, at least 46-74 nucleotides, at least 75-90 nucleotides, and at least 90-130 nucleotides. In some embodiments, fragments of bio molecules are immunogenic fragments.
In some embodiments, the term "functional fragment" means any portion of a polypeptide or amino acid sequence that is of a sufficient length to retain at least partial biological function that is similar to or substantially similar to the parental polypeptide or amino acid sequence upon which the fragment is based. If the fragment is a functional fragment of an antibody or antibody- like molecule, the fragment can possess a binding avidity for one or a plurality of antigens. In some embodiments, a functional fragment is a polypeptide that comprises 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity of any variable region of a polypeptide antibody disclosed herein and has sufficient length to retain at least partial binding affinity to one or a plurality of antigens that bind to the amino acid sequence. In some embodiments, the fragment has a length of at least about 10, about 20, about 30, about 40, about 50 , about 60, about 70, about 80, about 90, or about 100 contiguous amino acids. In some embodiments, the fragment is a fragment of any amino acid sequence disclosed herein and has a length of at least about 50 amino acids. In some embodiments, the fragment has a length of at least about 100, 150, 200, 250, 300, 350, 400, 450, 500, or 550 amino acids.
The term "antigen binding portion" or "antigen binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g. , HIV gpl20). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments can be multi-specific formats; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term "antigen-binding portion" or "antigen binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341 :544-546, Winter et al., PCT publication WO 90/05144 Al herein incorporated by reference), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423- 426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" or "antigen binding fragment" of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer- Verlag. New York. 790 pp. (ISBN 3-540-41354-5).
The term "multispecific antibody" refers to an antibody or antibody-like molecule, or fragment thereof, capable of binding two or more related or unrelated targets, or antigens. Antibody specificity refers to selective recognition of the antibody for a particular epitope, or amino acid sequence, of an antigen. Natural antibodies, for example, are monospecific. Pentaspecific antibodies are antibodies which have five different antigen-binding specificities.
The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three-dimensional structural characteristics, and or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody.
The term "antigen" refers to a polypeptide that can stimulate the production of antibodies or a T cell response in an animal, including polypeptides that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity.
The term "HIV Envelope protein (Env)" refers to the glycoprotein that is found on the surface of HIV. The HIV envelope protein is initially synthesized as a longer precursor protein of 845-870 amino acids in size, designated gpl60. gpl60 forms a homotrimer and undergoes glycosylation within the Golgi apparatus. In vivo, it is then cleaved by a cellular protease into gpl20 and gp4l. gpl20 contains most of the external, surface-exposed, domains of the HIV envelope glycoprotein complex, and it is gpl20 which binds both to cellular CD4 receptors and to cellular chemokine receptors (such as CCR5). gp4l contains a transmembrane domain and remains in a trimeric configuration within the membrane of the virus or the membrane of a host cell; it interacts with gpl20 in a noncovalent manner. The mature gpl20 wildtype polypeptides have about 500 amino acids in the primary sequence. gpl20 is heavily N-glycosylated giving rise to an apparent molecular weight of 120 kD. The polypeptide is comprised of five conserved regions (Cl- 05) and five regions of high variability (VI -V5). Exemplary sequence of wt gpl20 polypeptides are shown on GENBANK, for example accession numbers AAB05604 and AAD12142 (as available on Oct. 16, 2009), incorporated by reference herein. It is understood that there are numerous variation in the sequence of gpl20 from what is given in GENBANK, for example accession numbers AAB05604 and AAD12142, and that these variants are skill recognized in the art as gpl20. The gpl20 core has a molecular structure, which includes two domains: an "inner" domain (which faces gp4l) and an "outer" domain (which is mostly exposed on the surface of the oligomeric envelope glycoprotein complex). The two gpl20 domains are separated by a "bridging sheet" that is not part of either domain. The gpl20 core includes 25 beta strands, 5 alpha helices, and 10 defined loop segments.
The term "CD4 binding site (CD4BS) antibodies” refers to antibodies that bind to the CD4 binding surface of a gpl20 polypeptide. The antibodies interfere with or prevent CD4 from binding to a gpl20 polypeptide.
The term "V3 loops" refers to a loop of about 35 amino acids critical for the binding of the co-receptor and determination of which of the co-receptors will bind. In certain examples the V3 loop includes residues 296-331.
The term "membrane-proximal external region or MPER" refers to a highly conserved region of the gp4l envelope protein. The MPER comprises the last 24 C- terminal amino acids of the gp4l ectodomain,
LLELDKWASLWNWF (N/D)ITNWLWYIK (aa 660 to 683) (Zwick el al. J Virol. 2005 Jan; 79(2): 1252-61).
The term“VI V2 glycan antibody” refers to antibodies that bind to the VI V2 apex and associated conserved glycans N160 and/or N156.
The term“gpl20/gp4l interface antibody” refers to antibodies that bind to a conserved epitope that stretches across gpl20 and gp4l.
The term "vector," as used herein, is intended to refer to a nucleic acid molecule. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g. , replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
"Polynucleotide" or "nucleic acid" as used interchangeably herein, refers to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. A sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may comprise modification(s) made after synthesis, such as conjugation to a label. Other types of modifications include, for example, "caps," substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotides(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-0- methyl-, 2'-0-allyl-, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, .alpha.- anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and basic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S ("thioate"), P(S)S ("dithioate"), (0)NR2 ("amidate"), P(0)R, P(0)OR, CO, or CH2 ("formacetal"), in which each R or R is independently H or substituted or unsubstituted alkyl (1-20C) optionally containing an ether (— 0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
The term "host cell" as used herein is intended to refer to a cell into which exogenous DNA has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but, to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. The host cells can include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life. In some embodiments, eukaryotic cells include protist, fungal, plant and animal cells. In some embodiments, host cells include but are not limited to the prokaryotic cell line E. coir, mammalian cell lines CHO, HEK 293 and COS; the insect cell line Sf9; and the fungal cell Saccharomyces cerevisiae. In some embodiments, the host cell is an immune cell.
Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
The term "inhibit" and its various grammatical forms is used to refer to a restraining, blocking, or limiting of the range or extent of a certain biological event or effect.
The term "effective amount," is used herein to include the amount of an agent (e.g. a multispecific antibody) that, when administered to a patient for treating an infection, is sufficient to effect treatment of the disease (e.g. , by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease or its related comorbidities). The "effective amount" may vary depending on the agent, how it is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, stage of pathological processes, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated. An effective amount includes an amount that results in a clinically relevant change or stabilization, as appropriate, of an indicator of a disease or condition. "Effective amount" refers to an amount of a compound, material, or composition, as described herein effective to achieve a particular biological result such as, but not limited to, biological results disclosed, described, or exemplified herein. Such results may include, but are not limited to, the effective reduction of symptoms associated with any of the disease states mentioned herein, as determined by any means suitable in the art. The effective amount of the composition may be dependent on any number of variables, including without limitation, the species, breed, size, height, weight, age, overall health of the subject, the type of formulation, the mode or manner or administration, the type and/or severity of the particular condition being treated, or the need to modulate the activity of the molecular pathway induced by association of the analog to its receptor. The appropriate effective amount can be routinely determined by those of skill in the art using routine optimization techniques and the skilled and informed judgment of the practitioner and other factors evident to those skilled in the art. An effective dose of the antibodies or mutants or variants described herein may provide partial or complete biological activity as compared to the biological activity induced by the wild-type or naturally occurring polypeptides upon which the antibodies or mutants or variants are derived. A therapeutically effective dose of the antibodies or mutants or variants described herein may provide a sustained biochemical or biological effect and/or an increased resistance to degradation when placed in solution as compared with the normal affect observed when the naturally occurring and fully processed translated protein is administered to the same subject.
An "immunoconjugate" is an antibody or multispecific antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.
The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes; growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
The term "administer" as used herein means to give or to apply. The term "administering" as used herein includes in vivo administration.
The term "linker" refers to a chemical moiety that connects one peptide sequence to another, e.g., one antibody or antigen binding fragment to another. Linkers can also be used to attach antibodies to labels or solid substrates. A linker can include amino acids. Linkers can be straight or branched, saturated or unsaturated carbon chains. They can also include one or more heteroatoms within the chain.
The term "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered. A pharmaceutical composition of the present disclosure can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. To administer an antibody according to the disclosure by certain routes of administration, it may be necessary to coat the antibody with, or coadminister the antibody with, a material to prevent its inactivation. For example, the antibody may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. Pharmaceutically acceptable carriers includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion).
The pharmaceutical compositions according to the disclosure can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
The term "subject" is used throughout the specification to describe an animal to which one or more compositions comprising the antibody or antibodies disclosed herein. In some embodiment, the subject is a human. The term“subject” and "patient" may be interchangeably used. In some instances in the description of the present disclosure, the term "patient" will refer to human patients suffering from a particular disease or disorder. In some embodiments, the subject may be a human suspected of having or being identified as at risk to develop HIV infection. In some embodiments, the subject is suspected of having or has been diagnosed with HIV or HIV-l infection or AIDS. In some embodiments, the subject may be a human suspected of having or being identified as at risk to develop AIDS or an AIDS-associated disorder. In some embodiments, the subject may be a mammal. In some embodiments, the subject may be a non-human animal. In one embodiment, the subject is a human, such as a human being treated or assessed for an HIV infection; or a human having an HIV infection that would benefit from a multispecific antibody as described herein. In some embodiments, the subject is in need of the treatment being administered. In some embodiments, the compositions or pharmaceutical compositions comprise crystalline forms or lyophilized forms of the antibodies, antibody- like molecules or salts thereof.
The term "treat" or "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a disease, condition or disorder, substantially ameliorating clinical or esthetical symptoms of a condition, substantially preventing the appearance of clinical or esthetical symptoms of a disease, condition, or disorder, and protecting from harmful or annoying symptoms. The term "treat" or "treating" as used herein further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting recurrence of symptoms in patients that were previously symptomatic for the disorder(s).
The term "potency" as used herein refers to the neutralization capacity, i.e. the IC50 or IC80 of the antibody, or fragment thereof. The term "IC50" as used herein refers to the concentration of an inhibitor, such as a multispecific antibody, where the response or biological activity is reduced by half. The term "ICxo" as used herein refers to the concentration of an inhibitor (e.g. a multispecific antibody) where the response or biological activity is reduced by eighty percent.
Humanization and primatization refer to in cases where the multi-specific antibodies or the five antibodies forming the multi-specific fusion antibody are non human antibodies, the antibody can be "humanized" to reduce immunogenicity to a human recipient. Methods for humanizing non-human antibodies have been described in the art. See, e.g., Jones et al., Nature 321 :522-525 (1986); Riechmann et al, Nature 332:323-327 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988), and U.S. Pat. No. 4,816,567. Generally, residues from the variable domain of a non-human antibody are "imported" into a human immunoglobulin molecule, resulting in antibodies in which some hypervariable region residues and possibly some FR residues of a human antibody are substituted by residues from analogous sites of non-human antibodies. It is important to humanize a non-human antibody while retaining high affinity for the antigen. To this end, three dimensional immunoglobulin models are commonly available and suitable for use in analyzing proposed humanized sequences in comparison to the parental non-human antibodies. Such analysis permits identification of residues likely involved in recognition and binding of the antigen, and therefore rational design of humanized sequences that retain the specificity and affinity for the antigen.
In some embodiments, multispecific antibodies are formed from anti-HIV human or humanized antibodies. Similarly, a penta-specific fusion antibody or the five antibodies forming the fusion can be "primatized" to reduce immunogenicity to another primate, non-human recipient, e.g., a rhesus recipient. Residues from the variable domain of a donor antibody (such as a non-primate antibody or an antibody of a primate species different from the recipient primate) are "imported" into a nonhuman primate recipient immunoglobulin molecule, resulting in antibodies in which some hypervariable region residues and possibly some FR residues of a nonhuman primate antibody are substituted by residues from analogous sites of donor antibodies. Alternatively, primatized antibodies can be made for use in a desirable primate species by using a recipient immunoglobulin having non-primate sequences or sequences from a different primate species by introducing the Fc fragment, and/or residues, including particularly framework region residues, from the desirable primate, into the recipient immunoglobulin.
By "affinity maturation" is meant when one or more hypervariable region residues of an antibody can be substituted to select for variants that have improved biological properties relative to the parent antibody by employing, e.g., affinity maturation using phage or yeast display. For example, the Fab region of an anti-HIV antibody can be mutated at several sites selected based on available structural information to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from phage particles or on the surface of yeast cells. The displayed variants are then screened for their biological activity (e.g. binding affinity).
Anti-HIV Antibodies
In some embodiments, the invention provides multispecific antibodies that are broadly neutralizing antibodies against HIV. HIV-l is among the most genetically diverse viral pathogens. Of the three main branches of the HIV-l phylogenetic tree, the M (main), N (new), and O (outlier) groups, group M viruses are the most widespread, accounting for over 99% of global infections. This group is presently divided into nine distinct genetic subtypes, or clades (A through K), based on full-length sequences. Env is the most variable HIV-l gene, with up to 35% sequence diversity between clades, 20% sequence diversity within clades, and up to 10% sequence diversity in a single infected person (Shankarappa, R. et al. 1999. ./. Virol. 73: 10489-10502). Clade B is dominant in Europe, the Americas, and Australia. Clade C is common in southern Africa, China, and India and presently infects more people worldwide than any other clade (McCutchan, F E. 2000. Understanding the genetic diversity of HIV-l. AIDS l4(Suppl. 3):S3 l-S44). Clades A and D are prominent in central and eastern Africa.
In some embodiments, the invention provides multispecific antibodies that are broadly neutralizing against HIV. In some embodiments, the antibody has a particularly high potency in neutralizing HIV infection in vitro across multiple clades as shown in the Table 1 and Figure 4A herein. In some embodiments, only low concentrations are required in order to neutralize a given amount of virus. This facilitates higher levels of protection while administering lower amounts of antibody.
In some embodiments, the invention provides a multispecific anti-HIV antibody that binds to multiple epitopes on HIV envelope protein, wherein the antibody comprises:
i. an amino acid sequence that binds to a V1/V2 apex glycan epitope;
ii. an amino acid sequence that binds to a V3-base glycan region epitope;
iii. an amino acid sequence that binds to a CD4 binding site
(CD4bs) epitope;
iv. an amino acid sequence that binds to a gpl20/gp4l interface epitope; and
v. an amino acid sequence that binds to a membrane proximal external region (MPER) epitope. The amino acid sequences of parts i-v) of the multispecific anti-HIV antibody can be present on one or more polypeptide chains. In some embodiments, the amino acid sequences of parts i-v) are present on a single polypeptide chain. In some embodiments, the multispecific anti-HIV antibody comprises two polypeptide chains, with each chain comprising amino acid sequences of parts i-v). In some embodiments, the antibody simultaneously binds the multiple epitopes.
In some embodiments, the multispecific anti-HIV antibody is capable of neutralizing at least 99% of the HIV viruses listed in Table 1 with an IC50 value of less than 50 pg/mL. In some embodiments, the multispecific anti-HIV antibody is capable of neutralizing at least 99.5% of the HIV pseudoviruses listed in Table 1 with an IC50 value of less than 50 pg/mL. In some embodiments, the multispecific anti -HIV antibody has an IC50 geomean of less than or equal to 0.10 pg/ml. In some embodiments, the multispecific anti-HIV antibody has an IC50 geomean of less than or equal to 0.072 pg/ml. In some embodiments, the multispecific anti-HIV antibody has an IC50 geomean of less than or equal to 0.006 pg/ml.
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Table 1. List of HIV- 1 Pseudovirus Panel: 208 Total Pseudoviruses Included.
In some embodiments, the amino acid sequences of parts i-v) comprise amino acid sequences of single chain fragment variable (ScFv) moieties, wherein each ScFv moiety comprises an amino acid sequence from a light chain variable region (VL) and an amino acid sequence from a heavy chain variable region (VH) of an antibody. In some embodiments, one or more of the ScFv moieties is organized such that the VL is at the amino terminal end of the ScFv moiety and the VH is at the carboxy terminal end of the ScFv moiety. In some embodiments, one or more of the ScFv moieties is organized such that the VH is at the amino terminal end of the ScFv moiety and the VL is at the carboxy terminal end of the ScFv moiety. In some embodiments, each ScFv moiety is organized such that the VL is at the amino terminal end of the ScFv moiety and the VH is at the carboxy terminal end of the ScFv moiety.
In some embodiments, the amino acid sequences that bind to the various epitope are separated from each other by one more linking amino acids. In some embodiments, the VL and VH sequences are separated by one or more linking amino acids. The linking amino acid sequences are not limiting. In some embodiments, the linking amino acids comprise one or more tetra-glycine serine (G4S) linkers. In some embodiments, the VL and VH sequences are separated by 1-10 tetra-glycine serine (G4S) linkers. In some embodiments, the VL and VH sequences are separated by 3 tetra-glycine serine (G4S) linkers.
In some embodiments, one or more of the polypeptide chains of the multispecific anti-HIV antibody can further comprises an Fc region of an immunoglobulin or a variant thereof. In some embodiments, the immunoglobulin is IgG. In some embodiments, the immunoglobulin is IgGl. In some embodiments, the antibody comprises an IgGl Fc region variant comprising mutations corresponding to M428L and N434S. In some embodiments, the antibody comprises an immunoglobulin Fc region that has been modified to facilitate heterodimerization.
In some embodiments, the antibody further comprises a sequence tag that facilitates purification of the antibody. In some embodiments, the sequence tag comprises a polyhistidine tag.
In some embodiments, the multispecific anti-HIV antibody comprises a first and second polypeptide chain, wherein the first and second polypeptide chains each comprise a. five ScFv moieties, wherein each ScFv moiety on a single chain recognizes an individual epitope (V1/V2 apex glycan, V3-base glycan region, CD4 binding site (CD4bs), gpl20/gp4l interface, membrane proximal external region (MPER), wherein each ScFv moiety comprises an amino acid sequence from a light chain variable region (VL) and an amino acid sequence from a heavy chain variable region (VH) of an antibody; and
b. an Fc region of an immunoglobulin or a variant thereof.
In some embodiments, the ScFv moieties are separated on the polypeptide chain by one or more linking amino acids. In some embodiments, the linking amino acids comprise one or more tetra-glycine serine (G4S) linkers. In some embodiments, each ScFv moiety is separated by 5 tetra-glycine serine (G4S) linkers.
In some embodiments, the amino acid sequences of parts i-v) are organized on a single polypeptide chain in the following order, from its N-terminus to its C-terminus: i. an amino acid sequence that binds to the V1/V2 apex glycan epitope;
ii. an amino acid sequence that binds to the V3-base glycan region epitope;
iii. an amino acid sequence that binds to the CD4 binding site (CD4bs) epitope;
iv. an amino acid sequence that binds to the gpl20/gp4l
interface epitope; and v. an amino acid sequence that binds to the membrane proximal external region (MPER) epitope.
In some embodiments, the amino acid sequences of parts i-v) are organized on a single polypeptide chain in the following order, from its N-terminus to its C-terminus: i. an amino acid sequence that binds to the membrane proximal external region (MPER) epitope;
ii. an amino acid sequence that binds to the gpl20/gp4l
interface epitope;
iii. an amino acid sequence that binds to the CD4 binding site (CD4bs) epitope;
iv. an amino acid sequence that binds to the V3-base glycan region epitope; and
v. an amino acid sequence that binds to the V1/V2 apex glycan epitope.
In some embodiments, the multispecific anti-HIV antibody comprises a first and second polypeptide chain, wherein the amino acid sequences of parts i-v) are organized in the following order, from its N-terminus to its C-terminus: i. an amino acid sequence that binds to the membrane proximal external region (MPER) epitope;
ii. an amino acid sequence that binds to the gpl20/gp4l
interface epitope;
iii. an amino acid sequence that binds to the CD4 binding site (CD4bs) epitope;
iv. an amino acid sequence that binds to the V3-base glycan region epitope;
v. an amino acid sequence that binds to the V1/V2 apex glycan epitope; and
vi. an Fc region of an immunoglobulin or a variant thereof. In some embodiments, the multispecific anti-HIV antibody comprises a first and second polypeptide chain, wherein the amino acid sequences of parts i-v) are organized in the following order, from its N-terminus to its C-terminus: i. an amino acid sequence that binds to the V1/V2 apex glycan epitope;
ii. an amino acid sequence that binds to the V3-base glycan
region epitope;
iii. an amino acid sequence that binds to the CD4 binding site (CD4bs) epitope;
iv. an amino acid sequence that binds to the gpl20/gp4l
interface epitope;
v. an amino acid sequence that binds to the membrane proximal external region (MPER) epitope; and
vi. an Fc region of an immunoglobulin or a variant thereof.
In some embodiments, the multispecific anti-HIV antibody comprises
1) a first polypeptide chain, wherein the amino acid sequences of parts i-v) are organized in the following order, from its N-terminus to its C-terminus: i. an amino acid sequence that binds to the membrane proximal external region (MPER) epitope;
ii. an amino acid sequence that binds to the gpl20/gp4l
interface epitope;
iii. an amino acid sequence that binds to the CD4 binding site (CD4bs) epitope;
iv. an amino acid sequence that binds to the V3-base glycan
region epitope;
v. an amino acid sequence that binds to the V1/V2 apex glycan epitope; and
vi. an Fc region of an immunoglobulin or a variant thereof; and
2) a second polypeptide chain, wherein the amino acid sequences of parts i-v) are organized in the following order, from its N-terminus to its C-terminus: i. an amino acid sequence that binds to the V1/V2 apex glycan epitope;
ii. an amino acid sequence that binds to the V3-base glycan region epitope;
iii. an amino acid sequence that binds to the CD4 binding site (CD4bs) epitope;
iv. an amino acid sequence that binds to the gpl20/gp4l
interface epitope;
v. an amino acid sequence that binds to the membrane proximal external region (MPER) epitope; and
vi. an Fc region of an immunoglobulin or a variant thereof.
In some embodiments, the amino acid sequence that binds to the epitope of the Vl/V2-glycan region comprises an amino acid sequence from an antibody selected from the group consisting of VRC26.25 and PGDM1400; the amino acid sequence that binds to the epitope of the V3-glycan region comprises an amino acid sequence from antibody PGT121; the amino acid sequence that binds to the epitope of the CD4-binding site (CD4bs) comprises an amino acid sequence from antibody N6; the amino acid sequence that binds to the epitope of the gpl20/gp4l interface comprises an amino acid sequence from antibody 35022; and the amino acid sequence that binds to the epitope of the membrane proximal external region (MPER) comprises an amino acid sequence from an antibody selected from the group consisting of 10E8n4, l0E8v4_Sl00cF, and 10E8v4_V5R_S 1 OOcF .
In some embodiments, the amino acid sequence from the antibody VRC26.25 comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR Hl comprises QFRFDGYG, CDR H2 comprises ISHDGIKK and CDR H3 comprises
AKDLREDECEEWW SDDF GKQLPC AK SRGGL V GI ADN ; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR Ll comprises TSNIGNNF, CDR L2 comprises ETD and CDR L3 comprises ATWAASLSSARV; In some embodiments, the amino acid sequence from the antibody PGDM1400 comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR Hl comprises GNTLKTYD, CDR H2 comprises ISHEGDKK and CDR H3 comprises
AKGSKHRLRD Y ALDDDGALNW A VD VD YL SNLEF ; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR Ll comprises HSLIHGDRNNY, CDR L2 comprises LAS and CDR L3 comprises MQGRESPWT.
In some embodiments, the amino acid sequence from the antibody PGT121 comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR Hl comprises GASISDSY, CDR H2 comprises VHKSGDT and CDR H3 comprises ARTLHGRRI Y GI V AFNEWF T YF YMD V ; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR Ll comprises SLGSRA, CDR L2 comprises NNQ and CDR L3 comprises HIWD SRVPTKW V.
In some embodiments, the amino acid sequence from the antibody N6 comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR Hl comprises GYTFTAHI, CDR H2 comprises IKPQYGAV and CDR H3 comprises AR; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR Ll comprises QGVGSD, CDR L2 comprises HTS and CDR L3 comprises QVLQF.
In some embodiments, the amino acid sequence from the antibody 35022 comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR Hl comprises GYRFNFYH, CDR H2 comprises ISPYSGDK and CDR H3 comprises DDTGTYFCAKGLLRDGSSTWLPYL; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR Ll comprises NSVCCSHKS, CDR L2 comprises EDN and CDR L3 comprises CSYTHNSGCV.
In some embodiments, the amino acid sequence from the antibody 10E8n4 comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR Hl comprises GFDFDNAW, CDR H2 comprises ITGPGEGWSV and CDR H3 comprises TGYYFCARTGKYYDFWSGYPPGEEYFQD; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR Ll comprises RGDSLRSHYAS, CDR L2 comprises GKNNRPS and CDR L3 comprises SSRDKSGSRLSV.
In some embodiments, the amino acid sequence from the antibody l0E8v4_Sl00cF comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR Hl comprises GFDFDNAW, CDR H2 comprises ITGPGEGWSV and CDR H3 comprises
T GY YF CART GK Y YDF WF GYPPGEE YF QD ; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR Ll comprises RGDSLRSHYAS, CDR L2 comprises GKNNRPS and CDR L3 comprises
SSRDKSGSRLSV.
In some embodiments, the amino acid sequence from the antibody l0E8v4_V5R_Sl00cF comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR Hl comprises GFDFDNAW, CDR H2 comprises ITGPGEGWSV and CDR H3 comprises
T GYYF CART GK YYDF WF GYPPGEEYF QD ; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR Ll comprises RGDSLRSHYAS, CDR L2 comprises GKNNRPS and CDR L3 comprises
SSRDKSGSRLSV.
In some embodiments, the amino acid sequence from the antibody VRC26.25 comprises the VH region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VH region comprises SEQ ID NO: l; and the VL region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VL region comprises amino acids SEQ ID NO:5.
In some embodiments, the amino acid sequence from the antibody PGDM1400 comprises the VH region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VH region comprises SEQ ID NO:9; and the VL region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VL region comprises amino acids SEQ ID NO: 13. In some embodiments, the amino acid sequence from the antibody PGT121 comprises the VH region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VH region comprises SEQ ID NO: 17; and the VL region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VL region comprises amino acids SEQ ID NO:2l .
In some embodiments, the amino acid sequence from the antibody N6 comprises the VH region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VH region comprises SEQ ID NO:25; and the VL region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VL region comprises amino acids SEQ ID NO:29.
In some embodiments, the amino acid sequence from the antibody 35022 comprises the VH region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VH region comprises SEQ ID NO:33; and the VL region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VL region comprises amino acids SEQ ID NO:37.
In some embodiments, the amino acid sequence from the antibody 10E8n4 comprises the VH region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VH region comprises SEQ ID NO:4l; and VL region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VL region comprises amino acids SEQ ID NO:45.
In some embodiments, the amino acid sequence from the antibody l0E8v4_Sl00cF comprises the VH region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VH region comprises SEQ ID NO:49; and the VL region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VL region comprises amino acids SEQ ID NO:45.
In some embodiments, the amino acid sequence from the antibody l0E8v4_V5R_Sl00cF comprises the VH region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VH region comprises SEQ ID NO:5l; and the VL region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VL region comprises amino acids SEQ ID NO:45. Variable regions and CDR sequences of the antibodies described herein are shown in Table 2, below.
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0002
Table 2. Amino Acid Sequences of variable domains and CDR sequences.
In some embodiments, the multispecific anti-HIV antibody is capable of binding five different antigen targets. The disclosure features a multispecific antibody that binds five non-overlapping epitopes. In some embodiments, the multispecific anti-HIV antibody comprises an amino acid sequence selected from any of SEQ ID NOS:72-80, as shown below in Table 3.
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Table 3. Penta-Nab names, orientations of variable light and variable heavy chains, and amino acid sequences.
In some embodiments, 3D structure information of broadly neutralizing antibodies (bNAbs) in complex with HIV-l envelope glycoproteins (Env) was used to perform rational design of penta-specific antibodies (PentaNAbs) capable of targeting five major Env neutralizing epitopes. In some embodiments,“PentaNAbs” as referred to herein comprise five functional single chain fragment variable (ScFv) moieties connected in tandem via (G4S)n linkers, such that penta-valence engagement of five individual epitopes on the HIV-l Env by each respective moiety is possible. The bNAb functional moieties incorporated in the design target five major HIV neutralization epitopes including the i) V1/V2 apex glycan, ii) V3-base glycan, iii) CD4 binding site, iv) gpl20/gp4l interface, and v) the membrane proximal external region (MPER). In addition, these PentaScFv entities can be fused to the fragment crystallizable region (Fc) region of IgGl to accommodate effector functions in a Penta-IgG format.
The term“protomer” is the structural unit of an oligomeric protein unit. A protomer may span a single segment of a protein’s primary structure, or may be composed of one or more different protein sequences that spans across a protein’s three- dimensional folded or unfolded protein structure. A protomer may be a repeating structural unit.
In some embodiments, to design the Penta-specific ScFv and bNAbs, the physical distance between the compatible N/C termini of parental bNAb variable heavy (VH) and light chain (VL) domains was estimated to identify the shortest linker combinations allowing optimal VH/VL connection within each bNAb ScFv entity. In some embodiments multiple tandem (G4S)n linkers were used. In some embodiments, at least three G4S linkers were used between the VH and VL domains of a bNAb in the PentaScFv or Penta-bNAb molecule. The number of G4S linkers between VH and VL regions is not limiting and may not be required. In some embodiments there is no linker between the VH and VL regions. The number of G4S linkers between VH and VL domains of a bNab may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen linkers. In some embodiments multiple tandem (G4S)n linkers can be used between individual bNab moieties in the PentaScFv and Penta-bNAb molecules. In some embodiments at least five G4S linkers were used between individual bNAbs. The number of G4S linkers between individual bNabs may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen linkers.
In some embodiments, five G4S linkers were used to connect moieties of bNAbs targeting the V1V2 (e.g. VRC26.25) and V3 (e.g. PGT121) glycans. A linker of sufficient length (e.g. a linker with five G4S units) can be utilized to connect VRC26.25 or PGDM1400 with PGT121. In some embodiments, epitopes located on adjacent protomers of the HIV-l trimer are often more proximate to each other than those on the same protomer. Thus, in some embodiments, two functional bNAb moieties can be arranged binding to the same protomer (intra-protomer mode) and in some cases to separate protom ers (inter-protom ers) of the HIV-l Env trimer. In some embodiments, the shortest distance between the CD4bs targeting bNAb moiety, N6 (with VRC01 as surrogate), and the gpl20/gp4l interface targeting 35022 entity was determined to be an intra-protomer distance. Using the model in PDB 5FYK, the distance between the 35022 and N6 functional variable domains were determined to be 57 A between the C-terminus of 35022 VH domain and the N-terminus of N6 VL domain with the topology of 35022(VL-VH)-N6(VL-VH), and 47 A between the C-terminus of N6 VL and the N- terminus of 35022 VH with the topology of N6(VH-VL)-35022(VH-VL), respectively. Thus, five G4S linkers can be used to connect N6 and 35022. The length of the linkers is not limiting and may be varied.
In some embodiments, five G4S linkers can be used to connect the V3-glycan targeting PGT121 and the CD4bs targeting N6 ScFv entities. The shortest distance between PGT122 (a surrogate for PGT121) and VRC01 (a surrogate for N6) was previously determined between adjacent protomers within an Env trimer (inter-protom er). Using the PDB model 5FYK, the distances between the VRC01 and PGT121 functional variable domains were determined as the follows: 37 A between the C-terminus of VRC01 VH domain and the N-terminus of PGT121 VL domain with VRCOl(VL-VH)- PGTl2l(VL-VH) topology, and 53 A between the C-terminus of PGT121 VL and the N- terminus of VRC01 VH with PGTl2l(VH-VL)-VRC0l(VH-VL) topology. Furthermore, negative stain electron microscopy data demonstrated that one of the Bi-ScFvs previously generated, dVRC0l(VL-VH)-5X-PGTl2l(VL-VH), was able to simultaneously engage both the PGT121 and VRC01 epitopes in an inter-protomer manner as predicted (31), which supports rationale for this linker design.
In some embodiments, the shortest distances between the gpl20/gp4l interface targeting 35022 entity and the MPER targeting 10E8n4 entity was determined to be inter- protomer manner. Using PDB 5IQ7 superimposed with PDB 5FYK, a model for Env engaged with both 35022 and 10E8 was generated. In this model, the distance between the 10E8n4 and 35022 functional variable domains were determined to be 48 A between the C-terminus of 10E8n4 VH domain and the N-terminus of 35022 VL domain with the topology of l0E8v4(VL-VH)-35O22(VL-VH), and 25 A between the C-terminus of 35022 VL and the N-terminus of 10E8n4 VH with the topology of 35022(VH-VL)- lOE8v4(VH-VL) respectively. Therefore, five G4S linkers can be used to connect 35022 and 10E8n4. The linkers used are not limiting and can be optimized.
The type of linker between VH and VL domains of a bNAb is not limited to the use of G4S linkers. The type of linker between individual bNAbs is not limited to the use of G4S linkers. The length, sequence, and/or identity between the linkers separating the VH and VL regions and the linkers separating the individual bNAb moieties may be the same or may be different. The relationship between the types and characteristics of linkers used is not limiting. Such linkers are typically polypeptide sequences of between about 1 and 200 amino acids. The linkers can be any combination of natural or unnatural amino acids, and the sequence identity of the linkers is not limiting. Linkers can be flexible or rigid amino acid subsequences which are synthesized as part of a recombinant fusion protein.
The linkers may be flexible, the linkers may be rigid, or the linkers may be cleavable. Linkers may offer many other advantages for the production of fusion proteins, such as improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles. As such, the linkers in certain embodiments are not limiting and may be optimized for length, flexibility, rigidity, identity, cleavability, chemical composition, or other physical or chemical properties, for reasons that may or may not affect yield, half-life, biological activity, neutralization activity, pharmacokinetic properties, bioavailability, binding affinity, protein size, or stoichiometric binding ratios.
In some embodiments, the linker can be non-peptide based. A chemical linker may be used to connect protein sequences. Such linkers are known to persons of skill in the art. For example, poly(ethylene glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages. In addition to covalent linkage of bNab sequences and linkers, non-covalent methods can be used to produce molecules with bNabs and linkers. Disulfide bonds may be used to form linkers. Chemical conjugation methods are known to persons of skill in the art and may be used to link VH and VL regions, or individual bNab moieties.
In some embodiments, the disclosure relates to an antibody comprising a variable portion and a constant portion, the variable portion comprising any one or plurality of variable heavy and/or light antibody fragments (such as a scFv fragment) capable of binding to the epitopes disclosed herein, and the constant portion comprising an IgG-like domain.
The antibody can be modified to improve certain biological properties of the antibody, e.g., to improve stability, to enhance or reduce effector functions such as antigen-dependent cell-mediated cytoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody, improved or decreased internalization and/or recycling, among others.
In certain embodiments, the multispecific antibody as described herein further includes a modification in the Fc region. In certain embodiments, the multispecific antibodies of the disclosure include mutations that increase binding to the neonatal Fc receptor (FcRn), which recycles IgG in intestinal epithelial cells and increases levels in the serum, extended half-life, enhanced mucosal localization, and conferred more efficient protection against lentivirus infection relative to the wild-type antibody. Enhanced neonatal Fc receptor function improves protection against primate SHIV infection. Such mutations are described, for example in Ko et al. (Nature 514, 642-645 (2014)).
For example, the Fc fragment of some antibodies (derived from human Ig4) can be replaced with human IgGl that increases effector function mediated through FcRs (except FcRn). Such modification may improve the stability of the resulting antibody by about 5 fold. In another example, the IgGl Fc fragment can be modified to improve the recycling of the antibody via the antibody salvage pathway.
Still another type of modification involves alteration of the glycosylation pattern of a parent antibody, including deletions of one or more carbohydrate moieties found in the parent antibody, or addition of one or more carbohydrates (via addition of one or more glycosylation sites) that are not present in the parent antibody.
In some embodiments,“knob into hole” technology (J. B. Ridgway, L. G. Presta, P. Carter, 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng 9, 617-621 (1996)) can be utilized to generate a heterodimer of the “Forward” and “Reverse” PentaNAb arms, with LS mutations (M428L/N434S) in the Fc fragment for elongated antibody in vivo half-lives as well as improved biodistribution into the mucosal compartment. The use of this“knob into hole” technology is not limiting any method of generating PentaNAb dimers known to persons of skill in the art may be used.
PentaNAbs fused to a C-terminal fragment crystallizable region (Fc) region of IgGl, which may or may not contain a Hinge, CH2, and CH3 domains, can accommodate effector functions in a Penta-IgG format. The Fc region may or may not be fused to the C- terminus of each pentaNAb. Such recombinant formats are known to persons of skill in the art. The formats may include, but are not limited to the use of diabodies, minibodies, scFab, or scFv-zippers. For example, the types of recombinant formats may be found in the literature (See Frenzel A, Hust M, and Schirrmann T., Frontiers in Immunology. 2013; 4:217).
In some embodiments, two Forwards PentaNAbs can be combined through the use of the Fc regions of IgGl. In some embodiments, one Forward PentaNAb can be combined with one Reverse PentaNAb through the use of Fc regions of IgGl. The combinations of Forward and Reverse PentaNAbs are advantageous but not limiting.
Polynucleotides
In some embodiments, the invention encompasses polynucleotides comprising polynucleotides that encode a polypeptide as described herein, such as a sequence of an HIV antibody or a fragment of such a polypeptide. For example, in some embodiments, the invention provides a polynucleotide comprising a nucleic acid sequence that encodes a multispecific antibody to HIV Env protein or encodes a fragment of such an antibody. The polynucleotides of the invention can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand.
In some embodiments, the polynucleotides are isolated. In certain embodiments, the polynucleotides are substantially pure.
In some embodiments, the invention provides isolated nucleic acid molecules that encode multispecific anti-HIV antibodies as described herein. In some embodiments, the invention provides isolated nucleic acid molecules that encode isolated variable domains of the antibodies described herein. In some embodiments, the nucleotide sequence is selected from any one of SEQ ID NOS: 52-71 and SEQ ID NOS:8l-89, as shown below in Table 4 and below.
In some embodiments, the invention provides a polynucleotide comprising a polynucleotide encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOS: 1-51 and 72-80.
In some embodiments, the invention provides a polynucleotide comprising a polynucleotide encoding a polypeptide comprising the heavy or light chain variable region selected from the group consisting of SEQ ID NOS: 1, 5, 9, 13, 17 21, 25, 29, 33, 37, 41, 45, 49 and 51.
Also provided is a polynucleotide encoding a polypeptide comprising the heavy or light chain variable region having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any of SEQ ID NOS: 1, 5, 9, 13, 17 21, 25, 29, 33, 37, 41, 45, 49 and 51.
In some embodiments, the invention provides a polynucleotide comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:72-80 and SEQ ID NOS: 90, 92, 94, 96, 98, 99 and 107.
Also provided is a polynucleotide encoding a polypeptide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any of SEQ ID NOS:72-80 and SEQ ID NOS: 90, 92, 94, 96, 98, 99 and 107.
The invention further provides a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NOS: 52-71 and SEQ ID NOS:8l-89.
Also provided is a polynucleotide having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOS:52-7l, SEQ ID NOS:8l-89 and SEQ ID NOS 91, 93, 95, 97 and 106. In some embodiments the polynucleotides comprise the coding sequence for the mature polypeptide fused in the same reading frame to a polynucleotide which aids, for example, in expression and secretion of a polypeptide from a host cell (e.g. a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell). The polypeptide having a leader sequence is a preprotein and can have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides can also encode for a proprotein which is the mature protein plus additional 5' amino acid residues. A mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains.
In certain embodiments the polynucleotides comprise the coding sequence for the mature polypeptide fused in the same reading frame to a marker sequence that allows, for example, for purification of the encoded polypeptide. For example, the marker sequence can be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence can be a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host (e.g. COS-7 cells) is used.
The present invention further relates to variants of the hereinabove described polynucleotides encoding, for example, fragments, analogs, and derivatives.
The polynucleotide variants can contain alterations in the coding regions, non coding regions, or both. In some embodiments the polynucleotide variants contain alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, nucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
Vectors and cells comprising the polynucleotides described herein are also provided. The term "vector" means a construct, which is capable of delivering, and expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells. "Vector" also includes shuttle and expression vectors. In some embodiments, the vector is a plasmid construct and also includes an origin of replication (e.g., the ColEl origin of replication) and a selectable marker (e.g., ampicillin or tetracycline resistance), for replication and selection, respectively. An "expression vector" refers to a vector that contains the necessary control sequences or regulatory elements for expression of the antibodies including antibody fragments of the invention, in bacterial or eukaryotic cells.
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Table 4. Nucleotide Sequences of variable domain anti-HIV antibodies.
Nucleotide sequences of the Penta-Nabs shown in Table 3 are provided below. 1. PentaNAbi.o l0E8v4-5X-35O22-5X-N6-5X-PGTl2l-5X-VRC26.25-His
SEQ ID NO:8l GAATTCGCCGCCACCATGGGATGGAGCTGTATTATTCTGTTTCTGGTCGCTAC
CGCTACCGGAGTGCATTCTTCTGAACTGACCCAGGACCCCGCCGTGAGCGTG
GCCCTGAAGCAGACCGTGACAATCACCTGCAGGGGCGACAGCCTGCGCTCCC
ACTACGCCAGCTGGTATCAGAAGAAGCCTGGCCAGGCCCCAGTGCTGCTGTT
CTACGGCAAGAACAATAGGCCCTCCGGCATCCCTGATCGCTTTTCCGGCTCTG
CCAGCGGAAACAGGGCCAGCCTGACAATCACCGGAGCACAGGCAGAGGACG
AGGCAGATTACTATTGCAGCTCCCGGGACAAGTCCGGCTCTAGACTGAGCGT
GTTCGGCGGCGGCACCAAGCTGACAGTGCTGGGAGGAGGAGGCAGCGGCGG
AGGAGGCTCCGGAGGCGGCGGCTCTGAGGTGCGGCTGGTGGAGTCTGGAGG
AGGCCTGGTGAAGCCAGGAGGCAGCCTGAGACTGAGCTGTTCCGCCTCTGGC
TTCGACTTTGATAATGCCTGGATGACATGGGTGCGGCAGCCACCTGGCAAGG
GCCTGGAGTGGGTGGGAAGAATCACCGGACCAGGAGAGGGATGGTCTGTGG
ACTACGCCGAGAGCGTGAAGGGCCGGTTCACCATCTCCAGAGATAACACCAA
GA AT AC ACTGT AT C T GGAGAT GA AC A AT GT GC GGAC CGAGGAC AC AGGCT AC
TATTTCTGCGCCAGAACCGGCAAGTACTATGATTTTTGGAGCGGCTACCCACC
CGGCGAGGAGTATTTTCAGGACTGGGGCCAGGGCACCCTGGTCATCGTGAGC
AGCGGCGGCGGCGGCAGCGGCGGCGGCGGCTCCGGAGGAGGCGGCTCTGGA
GGAGGAGGCAGCGGGGGAGGGGGCAGCCAGTCCGTGCTGACCCAGTCTGCC
AGCGTGTCCGGCTCTCTGGGACAGAGCGTGACCATCTCCTGTACAGGCCCCA
ACAGCGTGTGCTGTAGCCACAAGAGCATCTCCTGGTACCAGTGGCCTCCAGG
AAGGGCACCTACCCTGATCATCTATGAGGACAATGAGCGGGCCCCAGGCATC
TCCCCCAGATTCTCTGGCTACAAGTCTTATTGGAGCGCCTACCTGACAATCAG
CGACCTGCGCCCCGAGGATGAGACAACATACTATTGCTGTTCCTATACCCACA
ACTCTGGCTGCGT GTTTGGC AC AGGC ACC AAGGT GTCCGT GCTGGGCGGCGG
CGGCAGCGGGGGCGGGGGCTCCGGAGGGGGCGGCTCTCAGGGCCAGCTGGT
GC AGAGCGGAGC AGAGCTGA AGAAGCCTGGAGCC AGCGT GAAGATCTCCTG
TAAGACATCTGGCTACCGGTTCAACTTTTATCACATCAATTGGATCAGGCAGA
CCGCAGGAAGGGGACCAGAGTGGATGGGCTGGATCTCCCCCTACTCTGGCGA
TAAGAACCTGGCCCCAGCCTTCCAGGACAGAGTGATCATGACCACAGATACC
GAGGTGCCAGTGACCAGCTTCACCTCCACCGGAGCCGCCTACATGGAGATCA
GGAATCTGAAGTTCGACGATACAGGCACCTATTTTTGCGCAAAGGGCCTGCT
GAGGGACGGCTCCTCTACCTGGCTGCCTTACCTGTGGGGACAGGGCACCCTG CTGACAGTGAGCTCCGGCGGCGGGGGCAGCGGCGGCGGGGGCTCCGGAGGA
GGAGGCTCTGGAGGAGGGGGCAGCGGAGGAGGCGGCTCCTACATCCACGTG
ACCCAGTCCCCATCTAGCCTGTCTGTGAGCATCGGCGATCGGGTGACCATCAA
CTGTCAGACATCTCAGGGCGTGGGCAGCGACCTGCACTGGTATCAGCACAAG
CCTGGCAGGGCCCCAAAGCTGCTGATCCACCACACATCCTCTGTGGAGGATG
GAGTGCCAAGCCGCTTCTCCGGCTCTGGATTCCACACCTCCTTTAATCTGACA
ATCTCTGACCTGCAGGCCGACGATATCGCCACCTACTATTGCCAGGTGCTGCA
GTTCTTTGGCCGGGGCTCCAGACTGCACATCAAGGGAGGAGGAGGCTCCGGG
GGCGGAGGCTCTGGCGGCGGCGGCAGCCGGGCCCACCTGGTGCAGAGCGGC
ACCGCCATGAAGAAGCCTGGCGCCAGCGTGAGAGTGTCCTGTCAGACATCTG
GCTACACCTTCACCGCCCACATCCTGTTCTGGTTTAGGCAGGCACCAGGAAGA
GGCCTGGAGTGGGTGGGCTGGATCAAGCCCCAGTATGGAGCAGTGAACTTCG
GAGGAGGCTTTCGGGACAGAGTGACACTGACCCGGGACGTGTACAGAGAGA
TCGCCTATATGGATATCAGGGGCCTGAAGCCAGACGATACCGCCGTGTACTA
TTGCGCCAGGGACCGCTCCTACGGCGATAGCTCCTGGGCACTGGACGCATGG
GGACAGGGCACCACAGTGGTGGTGAGCGCCGGCGGCGGAGGCTCCGGCGGC
GGGGGCTCTGGAGGAGGCGGCAGCGGAGGGGGAGGCTCCGGAGGGGGAGGC
TCTAGCGACATCTCCGTGGCCCCTGGCGAGACAGCCAGAATCTCTTGTGGCG
AGAAGTCTCTGGGC AGC AGGGCCGT GC AGT GGT ACC AGC AC AGGGC AGGAC
AGGCACCATCTCTGATCATCTATAACAATCAGGATAGGCCAAGCGGCATCCC
TGAGCGGTTCAGCGGCTCCCCCGACAGCCCTTTTGGCACCACAGCCACACTG
ACCATCACATCCGTGGAGGCAGGCGACGAAGCCGATTACTATTGCCACATCT
GGGATTCCAGAGTGCCAACCAAGTGGGTGTTCGGAGGAGGAACCACACTGAC
AGT GCTGGGAGGGGGGGGCTCTGGCGGCGGGGGC AGCGGGGGAGGAGGCTC
CCAGATGCAGCTGCAGGAGAGCGGACCAGGCCTGGTGAAGCCTAGCGAGAC
ACTGAGCCTGACATGTTCTGTGAGCGGCGCCTCCATCTCTGACAGCTACTGGT
CTTGGATCAGACGGAGCCCCGGCAAGGGCCTGGAATGGATCGGCTACGTGCA
CAAGTCCGGCGATACAAACTATTCCCCATCTCTGAAGTCTCGGGTGAACCTGT
CTCTGGACACCAGCAAGAATCAGGTGAGCCTGTCCCTGGTGGCAGCAACCGC
AGCAGATAGCGGCAAGTACTATTGCGCCAGAACACTGCACGGCAGGCGCATC
TACGGCATCGTGGCCTTTAACGAGTGGTTCACCTACTTTTATATGGACGTGTG
GGGC AAT GGC ACCC AGGT GAC AGTGTCCTCTGGCGGGGGCGGCTCCGGAGGC GGAGGCTCTGGCGGGGGCGGCAGCGGCGGGGGCGGCTCCGGGGGAGGCGGC
TCTCAGAGCGTGCTGACCCAGCCACCTTCCGTGTCTGCCGCACCAGGACAGA
AGGTGACCATCAGCTGTTCCGGCAACACATCCAATATCGGCAACAATTTCGT
GTCTT GGT ACC AGC AGAGGCC AGGAAGGGC ACC AC AGCTGCTGATCT AT GAG
ACAGACAAGCGGCCTTCCGGCATCCCAGATAGATTTTCTGCCAGCAAGTCCG
GCACCAGCGGCACACTGGCAATCACCGGCCTGCAGACAGGCGACGAAGCTG
ATTACTATTGCGCAACCTGGGCAGCCTCCCTGAGCTCCGCCAGGGTGTTCGGA
ACCGGAACAAAAGTGATCGTGCTGGTGGGCGGCGGAGGCTCTGGCGGAGGC
GGCAGCGGCGGGGGGGGCTCCCAGGTGCAGCTGGTGGAGAGCGGCGGCGGC
GTGGTGCAGCCAGGCACCTCCCTGAGGCTGTCTTGTGCAGCAAGCCAGTTCA
GATTTGATGGCTACGGCATGCACTGGGTGCGCCAGGCACCAGGCAAGGGCCT
GGAGTGGGTAGCCTCTATCAGCCACGACGGCATCAAGAAGTACCACGCCGAG
AAAGTGTGGGGCAGGTTCACCATCTCCCGCGATAACTCTAAAAACACACTGT
ATCTGCAGATGAACTCCCTGAGGCCCGAGGACACCGCCCTGTACTATTGCGC
CAAGGACCTGCGCGAGGATGAGTGTGAGGAGTGGTGGTCCGACTACTATGAT
TTTGGC AAGC AGCTGCCTTGCGC A AAGAGC AGGGGAGGCCTGGTGGGAATCG
CCGATAATTGGGGCCAGGGCACCATGGTGACAGTGTCTAGCGGATCCGGACA
CCACCATCACCATCATTAGTGAAAGCTT
2. PentaNAb2.o 10E8v4 _S100cF-5X-35O22-5X-N6-5X-PGT121-5X-VRC26 25-His
SEQ ID NO: 82
GAATTCGCCGCCACCATGGGATGGAGCTGTATTATTCTGTTTCTGGTCGCTAC
CGCTACCGGAGTGCATTCTTCTGAACTGACCCAGGACCCCGCCGTGAGCGTG
GCCCTGAAGCAGACCGTGACAATCACCTGCAGGGGCGACAGCCTGCGCTCCC
ACTACGCCAGCTGGTATCAGAAGAAGCCTGGCCAGGCCCCAGTGCTGCTGTT
CTACGGCAAGAACAATAGGCCCTCCGGCATCCCTGATCGCTTTTCCGGCTCTG
CCAGCGGAAACAGGGCCAGCCTGACAATCACCGGAGCACAGGCAGAGGACG
AGGCAGATTACTATTGCAGCTCCCGGGACAAGTCCGGCTCTAGACTGAGCGT
GTTCGGCGGCGGCACCAAGCTGACAGTGCTGGGAGGAGGAGGCAGCGGCGG
AGGAGGCTCCGGAGGCGGCGGCTCTGAGGTGCGGCTGGTGGAGTCTGGAGG
AGGCCTGGTGAAGCCAGGAGGCAGCCTGAGACTGAGCTGTTCCGCCTCTGGC
TTCGACTTTGATAATGCCTGGATGACATGGGTGCGGCAGCCACCTGGCAAGG
GCCTGGAGTGGGTGGGAAGAATCACCGGACCAGGAGAGGGATGGTCTGTGG ACTACGCCGAGAGCGTGAAGGGCCGGTTCACCATCTCCAGAGATAACACCAA
GA AT AC ACTGT AT C T GGAGAT GA AC A AT GT GC GGAC CGAGGAC AC AGGCT AC
TATTTCTGCGCCAGAACCGGCAAGTACTATGATTTTTGGTTTGGCTACCCACC
CGGCGAGGAGTATTTTCAGGACTGGGGCCAGGGCACCCTGGTCATCGTGAGC
AGCGGCGGCGGCGGCAGCGGCGGCGGCGGCTCCGGAGGAGGCGGCTCTGGA
GG AGG AGGC AGC GGGGG AGGGGGC AGC C AGT C C GT GC T G AC C C AGT C T GC C
AGCGTGTCCGGCTCTCTGGGACAGAGCGTGACCATCTCCTGTACAGGCCCCA
ACAGCGTGTGCTGTAGCCACAAGAGCATCTCCTGGTACCAGTGGCCTCCAGG
AAGGGCACCTACCCTGATCATCTATGAGGACAATGAGCGGGCCCCAGGCATC
TCCCCCAGATTCTCTGGCTACAAGTCTTATTGGAGCGCCTACCTGACAATCAG
CGACCTGCGCCCCGAGGATGAGACAACATACTATTGCTGTTCCTATACCCACA
ACTCTGGCTGCGT GTTTGGC AC AGGC ACC AAGGT GTCCGT GCTGGGCGGCGG
CGGCAGCGGGGGCGGGGGCTCCGGAGGGGGCGGCTCTCAGGGCCAGCTGGT
GC AGAGCGGAGC AGAGCTGA AGAAGCCTGGAGCC AGCGT GAAGATCTCCTG
TAAGACATCTGGCTACCGGTTCAACTTTTATCACATCAATTGGATCAGGCAGA
CCGCAGGAAGGGGACCAGAGTGGATGGGCTGGATCTCCCCCTACTCTGGCGA
TAAGAACCTGGCCCCAGCCTTCCAGGACAGAGTGATCATGACCACAGATACC
GAGGTGCCAGTGACCAGCTTCACCTCCACCGGAGCCGCCTACATGGAGATCA
GGAATCTGAAGTTCGACGATACAGGCACCTATTTTTGCGCAAAGGGCCTGCT
GAGGGACGGCTCCTCTACCTGGCTGCCTTACCTGTGGGGACAGGGCACCCTG
CTGACAGTGAGCTCCGGCGGCGGGGGCAGCGGCGGCGGGGGCTCCGGAGGA
GGAGGCTCTGGAGGAGGGGGCAGCGGAGGAGGCGGCTCCTACATCCACGTG
ACCCAGTCCCCATCTAGCCTGTCTGTGAGCATCGGCGATCGGGTGACCATCAA
CTGTCAGACATCTCAGGGCGTGGGCAGCGACCTGCACTGGTATCAGCACAAG
CCTGGCAGGGCCCCAAAGCTGCTGATCCACCACACATCCTCTGTGGAGGATG
GAGTGCCAAGCCGCTTCTCCGGCTCTGGATTCCACACCTCCTTTAATCTGACA
ATCTCTGACCTGCAGGCCGACGATATCGCCACCTACTATTGCCAGGTGCTGCA
GTTCTTTGGCCGGGGCTCCAGACTGCACATCAAGGGAGGAGGAGGCTCCGGG
GGCGGAGGCTCTGGCGGCGGCGGCAGCCGGGCCCACCTGGTGCAGAGCGGC
ACCGCCATGAAGAAGCCTGGCGCCAGCGTGAGAGTGTCCTGTCAGACATCTG
GCTACACCTTCACCGCCCACATCCTGTTCTGGTTTAGGCAGGCACCAGGAAGA
GGCCTGGAGTGGGTGGGCTGGATCAAGCCCCAGTATGGAGCAGTGAACTTCG GAGGAGGCTTTCGGGACAGAGTGACACTGACCCGGGACGTGTACAGAGAGA
TCGCCTATATGGATATCAGGGGCCTGAAGCCAGACGATACCGCCGTGTACTA
TTGCGCCAGGGACCGCTCCTACGGCGATAGCTCCTGGGCACTGGACGCATGG
GGACAGGGCACCACAGTGGTGGTGAGCGCCGGCGGCGGAGGCTCCGGCGGC
GGGGGCTCTGGAGGAGGCGGCAGCGGAGGGGGAGGCTCCGGAGGGGGAGGC
TCTAGCGACATCTCCGTGGCCCCTGGCGAGACAGCCAGAATCTCTTGTGGCG
AGAAGTCTCTGGGC AGC AGGGCCGT GC AGT GGT ACC AGC AC AGGGC AGGAC
AGGCACCATCTCTGATCATCTATAACAATCAGGATAGGCCAAGCGGCATCCC
TGAGCGGTTCAGCGGCTCCCCCGACAGCCCTTTTGGCACCACAGCCACACTG
ACCATCACATCCGTGGAGGCAGGCGACGAAGCCGATTACTATTGCCACATCT
GGGATTCCAGAGTGCCAACCAAGTGGGTGTTCGGAGGAGGAACCACACTGAC
AGT GCTGGGAGGGGGGGGCTCTGGCGGCGGGGGC AGCGGGGGAGGAGGCTC
CCAGATGCAGCTGCAGGAGAGCGGACCAGGCCTGGTGAAGCCTAGCGAGAC
ACTGAGCCTGACATGTTCTGTGAGCGGCGCCTCCATCTCTGACAGCTACTGGT
CTTGGATCAGACGGAGCCCCGGCAAGGGCCTGGAATGGATCGGCTACGTGCA
CAAGTCCGGCGATACAAACTATTCCCCATCTCTGAAGTCTCGGGTGAACCTGT
CTCTGGACACCAGCAAGAATCAGGTGAGCCTGTCCCTGGTGGCAGCAACCGC
AGCAGATAGCGGCAAGTACTATTGCGCCAGAACACTGCACGGCAGGCGCATC
TACGGCATCGTGGCCTTTAACGAGTGGTTCACCTACTTTTATATGGACGTGTG
GGGC AAT GGC ACCC AGGT GAC AGTGTCCTCTGGCGGGGGCGGCTCCGGAGGC
GGAGGCTCTGGCGGGGGCGGCAGCGGCGGGGGCGGCTCCGGGGGAGGCGGC
TCTCAGAGCGTGCTGACCCAGCCACCTTCCGTGTCTGCCGC ACC AGGAC AGA
AGGTGACCATCAGCTGTTCCGGCAACACATCCAATATCGGCAACAATTTCGT
GTCTT GGT ACC AGC AGAGGCC AGGAAGGGC ACC AC AGCTGCTGATCT AT GAG
ACAGACAAGCGGCCTTCCGGCATCCCAGATAGATTTTCTGCCAGCAAGTCCG
GCACCAGCGGCACACTGGCAATCACCGGCCTGCAGACAGGCGACGAAGCTG
ATTACTATTGCGCAACCTGGGCAGCCTCCCTGAGCTCCGCCAGGGTGTTCGGA
ACCGGAACAAAAGTGATCGTGCTGGTGGGCGGCGGAGGCTCTGGCGGAGGC
GGCAGCGGCGGGGGGGGCTCCCAGGTGCAGCTGGTGGAGAGCGGCGGCGGC
GTGGTGCAGCCAGGCACCTCCCTGAGGCTGTCTTGTGCAGCAAGCCAGTTCA
GATTTGATGGCTACGGCATGCACTGGGTGCGCCAGGCACCAGGCAAGGGCCT
GGAGTGGGTAGCCTCTATCAGCCACGACGGCATCAAGAAGTACCACGCCGAG AAAGTGTGGGGCAGGTTCACCATCTCCCGCGATAACTCTAAAAACACACTGT
ATCTGCAGATGAACTCCCTGAGGCCCGAGGACACCGCCCTGTACTATTGCGC
CAAGGACCTGCGCGAGGATGAGTGTGAGGAGTGGTGGTCCGACTACTATGAT
TTTGGC AAGC AGCTGCCTTGCGC A AAGAGC AGGGGAGGCCTGGTGGGAATCG
CCGAT A ATT GGGGCC AGGGC ACC AT GGT GAC AGT GTCT AGCGGATCCGGAGA
GCTGAAGACCCCTCTGGGCGATACCACACACACATCCCCACGGAGCCCCGAG
CCAAAGTCCTCTGACACCCCACCCCCTAGCCCTAGATCCCCTGAGCCAAAGA
GCTCCGATACACCACCCCCTTCTCCAAGGAGCCCCGAGCCTAAGTCTAGCGA
CACCCCACCCCCTTGCCCCCGCTGTCCAGCACCAGAGCTGCTGGGAGGACCA
AGCGTGTTCCTGTTTCCACCCAAGCCTAAGGATACACTGATGATCTCTCGCAC
CCCCGAGGTGACATGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTG
AAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGC
CCAGGGAGGAGCAGTACAACAGCACCTATCGCGTGGTGTCCGTGCTGACAGT
GCTGC ACC AGGACTGGCTGAACGGC AAGGAGT AC AAGTGC AAGGT GTCC AAT
AAGGCCCTGCCTGCCCCAATCGAGAAGACAATCAGCAAGGCAAAGGGACAG
CCAAGGGAGCCACAGGTGTACACCCTGCCTCCAAGCCGCGAGGAGATGACCA
AGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCTAGCGATATC
GCCGTGGAGTGGGAGTCCAATGGCCAGCCAGAGAACAATTACAAGACCACA
CCCCCTGTGCTGGACTCCGATGGCTCTTTCTTTCTGTATTCTAAGCTGACCGTG
GAC AAGAGCCGGT GGCAGC AGGGC AACGT GTT C AGCTGCTCTGT GCTGC ACG
AAGCCCTGCACAGCCATTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGAAA
ATAGTGAAAGCTT
3. PentaNAbi.o Reverse VRC26.25-5X-PGT121-5X-N6-5X-35022-5X- 10E8v4_S 1 OOcF -Hi s
SEQ ID NO: 83
GAATTCGCCGCCACCATGGGCTGGAGCTGCATCATCCTGTTCCTGGTG
GCAACCGCAACAGGAGTGCACAGCCAGGTGCAGCTGGTGGAGTCCGG
AGGAGGAGTGGTGCAGCCAGGCACCTCTCTGAGGCTGAGCTGTGCAG
CATCCCAGTTCAGATTTGATGGCTACGGAATGCACTGGGTGAGGCAGG
C AC C AGGC A AGGGACTGG AGT GGGT GGC C AGC AT C TCC C AC GACGGC
ATCAAGAAGTACCACGCCGAGAAAGTGTGGGGCAGGTTCACCATCTCT
CGCGATAACAGCAAGAATACACTGTATCTGCAGATGAACAGCCTGAG GCCCGAGGACACCGCCCTGTACTATTGCGCCAAGGACCTGCGCGAGGA T G AGT GT G AGG AGT GGT GGT C C G AC T AC T AT G AT TT T GGC A AGC AGC T GCCTTGCGCAAAGAGCAGGGGAGGACTGGTGGGAATCGCCGACAATT GGGGC C AGGGC AC CAT GGT G AC AGT G AGC AGC GG AGG AGG AGGC TC T GGAGGAGGAGGCAGCGGA
GGCGGCGGCTCTCAGAGCGTGCTGACCCAGCCACCTTCCGTGTCTGCC
GCACCAGGACAGAAGGTGACCATCAGCTGTTCCGGCAACACATCCAAT
ATCGGC AAC AATTTCGT GTCTT GGT ACC AGC AGAGGCCTGGAAGAGC A
CCACAGCTGCTGATCTATGAGACAGACAAGAGGCCCTCCGGCATCCCT
GATCGCTTTTCTGCCAGCAAGTCCGGCACCAGCGGCACACTGGCAATC
ACCGGACTGCAGACAGGCGACGAGGCAGATTACTATTGCGCAACCTG
GGCAGCCTCCCTG
TCTAGCGCCAGGGTGTTCGGCACCGGCACAAAAGTGATCGTGCTGGTG
GGAGGAGGAGGCTCCGGCGGCGGGGGCTCTGGCGGCGGCGGCAGCGG
AGGAGGCGGCTCCGGAGGAGGCGGCTCTC AGAT GC AGCTGC AGGAGA
GCGGACCAGGACTGGTGAAGCCTTCCGAGACCCTGTCTCTGACATGTT
CTGTGAGCGGCGCCTCCATCTCTGATAGCTACTGGAGCTGGATCAGAC
GGAGCCCTGGCAAGGGCCTGGAGTGGATCGGCTACGTGCACAAGTCT
GGCGATACAAACTATTCCCCATCTCTGAAGAGCCGGGTGAACCTGAGC
CTGGACACCTCCAAGAATCAGGTGAGCCTGTCCCTGGTGGCAGCAACC
GC AGC AGAC AGCGGC AAGT ACT ATT GCGCC AGAAC ACTGC ACGGC AG
GCGCATCTACGGCATCGTGGCCTTTAACGAGTGGTTCACCTACTTTTAT
AT GGACGTGTGGGGC AAT GGC ACCC AGGT GAC AGT GTCCTCTGGCGGC
GGCGGCTCTGGCGGAGGAGGCAGCGGAGGAGGAGGCAGCTCCGACAT
CTCTGTGGCACCTGGAGAGACCGCAAGGATCAGCTGTGGAGAGAAGT
CTCTGGGCAGCAGGGCCGTGCAGTGGTACCAGCACAGGGCAGGACAG
GCACCATCCCTGATCATCTATAACAATCAGGACCGGCCATCTGGCATC
CCCGAGAGATTCTCTGGCAGCCCCGATAGCCCTTTTGGCACCACAGCC
ACCCTGACAATCACCTCCGTGGAGGCCGGCGACGAAGCAGATTACTAT
TGCCACATCTGGGACTCCAGAGTGCCAACCAAGTGGGTGTTCGGAGGA
GGAACCACACTGACAGTGCTGGGCGGCGGAGGCTCCGGCGGGGGCGG
CTCTGGAGGCGGCGGCAGCGGAGGGGGCGGCTCCGGCGGCGGCGGCT CTAGGGCACACCTGGTGCAGAGCGGAACCGCAATGAAGAAGCCTGGC
GCCTCTGTGCGCGTGAGCTGTCAGACATCCGGCTACACCTTCACCGCC
C AC ATCCTGTTCTGGTTT AGGC AGGC ACC AGGAAGAGGACTGGAGT GG
GTGGGCTGGATCAAGCCCCAGTATGGAGCAGTGAACTTCGGAGGA
GGCTTTCGGGACAGAGTGACACTGACCCGGGACGTGTACAGAGAGAT
CGCCTATATGGATATCAGGGGCCTGAAGCCCGACGATACCGCCGTGTA
CTATTGCGCCAGGGACCGCTCCTACGGCGATTCTAGCTGGGCACTGGA
CGCATGGGGACAGGGAACCACAGTGGTGGTGAGCGCCGGAGGCGGGG
GCAGCGGCGGCGGGGGCTCCGGAGGCGGAGGCTCTTACATCCACGTG
ACCCAGTCCCCTTCCTCTCTGTCCGTGTCTATCGGCGATCGCGTGACCA
TCAACTGTCAGACAAGCCAGGGAGTGGGCTCCGACCTGCACTGGTATC
AGCACAAGCCTGGCAGGGCCCCAAAGCTGCTGATCCACCACACAAGC
TCCGTGGAGGATGGAGTGCCAAGCCGCTTCAGCGGCTCCGGATTCCAC
ACCAGCTTTAATCTGACAATCTCCGACCTGCAGGCCGACGATATCGCC
ACCTACTATTGCCAGGTGCTGCAGTTCTTTGGCAGGGGCTCCCGCCTGC
ACATCAAGGGCGGCGGCGGCTCTGGGGGCGGGGGCAGCGGCGGGGGG
GGCTCCGGGGGAGGAGGCTCTGGCGGAGGGGGC AGCC AGGGCC AGCT
GGT GC AG AGC GG AGC AG AGC T G A AG A AGC C AGG AGC C TC T GT G A AG A
TCAGCTGTAAGACATCCGGCTACCGGTTCAACTTTTATCACATCAATTG
GAT C AGGC AGACC GC AGGA AGGGGAC C AGAGT GGAT GGGC T GGATCT
CCCCCTACTCTGGCGATAAGAACCTGGCCCCAGCCTTCCAGGACAGAG
TGATCATGACCACAGATACCGAGGTGCCAGTGACCAGCTTCACCTCCA
CCGGAGCCGCCTACATGGAGATCCGGAATCTGAAGTTCGACGATACAG
GCACCTATTTTTGCGCCAAGGGCCTGCTGAGAGACGGCTCTAGCACAT
GGCTGCCATACCTGTGGGGACAGGGCACCCTGCTGACAGTGTCCTCTG
G AGG AGG AGGC T C C GGGGGC GGC GGC TC T GG AGG AGG AGGC TC TC A A
AGCGTGCTGACCCAGTCCGCCTCTGTGAGCGGCTCCCTGGGCCAGTCT
GTGACCATCAGCTGTACAGGCCCCAACTCCGTGTGCTGTTCTCACAAG
TCTATCAGCTGGTACCAGTGGCCACCAGGAAGGGCACCTACCCTGATC
ATCTATGAGGACAATGAGAGGGCACCAGGAATCAGCCCTCGCTTCTCC
GGCTACAAGTCTTATTGGAGCGCCTACCTGACCATTTCCGACCTGCGC
CCCGAGGATGAGACCACATACTATTGCTGTAGCTATACCCACAACTCC GGCTGCGTGTTTGGCACAGGCACCAAGGTGAGCGTGCTGGGAGGAGG
GGGCTCTGGCGGCGGGGGCAGCGGCGGAGGCGGCTCCGGAGGGGGCG
GCTCTGGCGGAGGCGGCAGCGAGGTGCGGCTGGTGGAGAGCGGCGGC
GGCCTGGTGAAGCCAGGCGGCTCTCTGAGACTGTCCTGTTCTGCCAGC
GGCTTCGACTTTGATAATGCCTGGATGACATGGGTGCGGCAGCCTCCT
GGCAAGGGGCTGGAGTGGGTGGGAAGAATCACCGGACCAGGAGAGG
GATGGTCTGTGGACTACGCCGAGAGCGTGAAGGGCCGGTTCACCATCA
GC AGAGAT A AC ACT A A A A AT AC AC T GT ATCTGGAGAT GA AC A AT GT G
CGGACCGAGGACACAGGCTACTATTTCTGCGCCAGAACCGGCAAGTAC
TATGATTTCTGGTTTGGCTACCCCCCTGGCGAGGAGTATTTTCAGGACT
GGGGCCAGGGCACCCTGGTCATCGTGAGCAGCGGCGGGGGAGGCTCC
GGC GGGGGGGGC TC T GG AGG AGGGGGC TC T AGC G AGC T G AC C C AGG A
CCCCGCCGTGTCCGTGGCCCTGAAGCAGACAGTGACCATCACATGCAG
GGGCGACTCCCTGCGCTCTCACTACGCCAGCTGGTATCAGAAGAAGCC
AGGACAGGCACCCGTGCTGCTGTTCTACGGCAAGAACAATCGGCCTTC
CGGCATCCCAGATAGATTTTCCGGCTCTGCCAGCGGAAACAGGGCCAG
CCTGACC AT C AC AGG AGC AC AGGC AG AGG AT GAAGC AGATT ACT ATT
GTTCCTCTCGGGACAAGTCCGGCTCTAGACTGAGCGTGTTCGGCGGCG
GAACCAAGCTGACAGTGCTGGGATCCGGCCACCACCATCACCATCATT
AGTGAAGCTT PentaNAbs.o 10E8v4_S 100cF-5X-35022-5X-N6-5X-PGT 121 -5X-PGDM 1400- His
SEQ ID NO: 84
GAATTCGCCG CCACCATGGG ATGGAGCTGT ATTATTCTGT TTCTGGTCGC TACCGCTACC GGAGTGCATT CTTCTGAACT GACCCAGGAC CCCGCCGTGA GCGTGGCCCT GAAGC AG AC C GTGACAATCA CCTGCAGGGG CGACAGCCTG CGCTCCCACT ACGCCAGCTG GT AT C AG A AG AAGCCTGGCC AGGCCCCAGT GCTGCTGTTC T AC GGC A AG A ACAATAGGCC CTCCGGCATC CCTGATCGCT TTTCCGGCTC TGCCAGCGGA AACAGGGCCA GCCTGACAAT CACCGGAGCA CAGGCAGAGG ACGAGGCAGA TTACTATTGC AGCTCCCGGG ACAAGTCCGG CTCTAGACTG AGCGTGTTCG GCGGCGGCAC CAAGCTGACA GT GCTGGGAG GAGGAGGCAG CGGCGGAGGA GGCTCCGGAG GCGGCGGCTC TGAGGTGCGG CTGGTGGAGT C T GGAGGAGG CCTGGTGAAG CCAGGAGGCA GCCTGAGACT GAGCTGTTCC GCCTCTGGCT TCGACTTTGA TAATGCCTGG AT GAC AT GGG TGCGGCAGCC ACCTGGCAAG GGCCTGGAGT GGGTGGGAAG AATCACCGGA CCAGGAGAGG GATGGTCTGT GGACTACGCC GAGAGCGT GA AGGGCCGGTT CACCATCTCC AGAGATAACA CCAAGAATAC ACTGTATCTG GAGATGAACA ATGTGCGGAC CGAGGACACA GGCTACTATT TCTGCGCCAG AACCGGCAAG TACTATGATT TTTGGTTTGG CTACCCACCC GGCGAGGAGT ATTTTCAGGA CTGGGGCCAG GGCACCCTGG TCATCGTGAG CAGCGGCGGC GGCGGCAGCG GCGGCGGCGG CTCCGGAGGA GGCGGCTCTG GAGGAGGAGG CAGCGGGGGA GGGGGCAGCC AGTCCGTGCT GACCCAGTCT GCCAGCGTGT CCGGCTCTCT GGGACAGAGC GTGACCATCT CCTGTACAGG CCCCAACAGC GTGTGCTGTA GCCACAAGAG CATCTCCTGG TACCAGTGGC CTCCAGGAAG GGCACCTACC CTGATCATCT AT GAGGAC AA TGAGCGGGCC CCAGGCATCT CCCCCAGATT CTCTGGCTAC AAGTCTTATT GGAGCGCCTA CCTGACAATC AGCGACCTGC GCCCCGAGGA T GAGAC A AC A TACTATTGCT GTTCCTATAC CCACAACTCT GGCTGCGTGT TTGGCACAGG CACCAAGGTG TCCGTGCTGG GCGGCGGCGG CAGCGGGGGC GGGGGCTCCG GAGGGGGC GG CTCTCAGGGC CAGCTGGTGC AGAGCGGAGC AGAGCTGAAG AAGCCTGGAG CCAGCGTGAA GATCTCCTGT AAGACATCTG GCTACCGGTT CAACTTTTAT CACATCAATT GGATCAGGCA GACCGCAGGA AGGGGACCAG AGTGGATGGG CTGGATCTCC CCCTACTCTG GCGATAAGAA CCTGGCCCCA GCCTTCCAGG AC AGAGT GAT CATGACCACA GATACCGAGG TGCCAGTGAC CAGCTTCACC TCCACCGGAG CCGCCTACAT GGAGATCAGG AATCTGAAGT TCGACGATAC AGGCACCTAT TTTTGCGCAA AGGGCCTGCT GAGGGACGGC TCCTCTACCT GGCTGCCTTA CCTGTGGGGA CAGGGCACCC TGCTGACAGT GAGCTCCGGC GGC GGGGGC A GCGGCGGCGG GGGCTCCGGA GGAGGAGGCT CTGGAGGAGG GGGCAGCGGA GGAGGCGGCT CCTACATCCA CGTGACCCAG TCCCCATCTA GCCTGTCTGT GAGCATCGGC GATCGGGTGA CCATCAACTG TCAGACATCT CAGGGCGTGG GCAGCGACCT GCACTGGTAT CAGCACAAGC CTGGCAGGGC CCCAAAGCTG CTGATCCACC ACACATCCTC TGTGGAGGAT GGAGTGCCAA GCCGCTTCTC CGGCTCTGGA TTCCACACCT CCTTTAATCT GACAATCTCT GACCTGCAGG CCGACGATAT CGCCACCTAC TATTGCCAGG TGCTGCAGTT CTTTGGCCGG GGCTCCAGAC TGCACATCAA GGGAGGAGGA GGCTCCGGGG GCGGAGGCTC TGGCGGCGGC GGCAGCCGGG CCCACCTGGT GCAGAGCGGC ACCGCCATGA AGAAGCCTGG CGCCAGCGTG AGAGTGTCCT GTCAGACATC TGGCTACACC TTCACCGCCC ACATCCTGTT CTGGTTTAGG CAGGCACCAG GAAGAGGCCT GGAGTGGGTG GGCTGGATCA AGCCCCAGTA TGGAGCAGTG AACTTCGGAG GAGGCTTTCG GGACAGAGTG ACACTGACCC GGGACGTGTA C AGAGAGAT C GCCTATATGG AT AT C AGGGG CCTGAAGCCA GACGATACCG CCGTGTACTA TTGCGCCAGG GACCGCTCCT ACGGCGATAG CTCCTGGGCA CTGGACGCAT GGGGACAGGG CACCACAGTG GTGGTGAGCG CCGGCGGCGG AGGCTCCGGC GGCGGGGGCT CTGGAGGAGG CGGCAGCGGA GGGGGAGGCT CCGGAGGGGG AGGCTCTAGC GACATCTCCG TGGCCCCTGG CGAGACAGCC AGAATCTCTT GT GGC GAG A A GTCTCTGGGC AGCAGGGCCG TGCAGTGGTA CCAGCACAGG GCAGGACAGG CACCATCTCT GATCATCTAT AACAATCAGG ATAGGCCAAG CGGCATCCCT GAGCGGTTCA GCGGCTCCCC CGACAGCCCT TTTGGCACCA CAGCCACACT GACCATCACA TCCGTGGAGG CAGGCGACGA AGCCGATTAC TATTGCCACA TCTGGGATTC CAGAGTGCCA ACCAAGTGGG TGTTCGGAGG AGGAACCACA CTGACAGTGC T GGGAGGGGG GGGCTCTGGC GGC GGGGGC A GCGGGGGAGG AGGCTCCCAG ATGCAGCTGC AGGAGAGCGG ACCAGGCCTG GTGAAGCCTA GCGAGACACT GAGCCTGACA TGTTCTGTGA GCGGCGCCTC CATCTCTGAC AGCTACTGGT CTTGGATCAG ACGGAGCCCC GGCAAGGGCC TGGAATGGAT CGGCTACGTG CACAAGTCCG GCGAT AC AAA CTATTCCCCA TCTCTGAAGT CTCGGGTGAA CCTGTCTCTG GACACCAGCA AGAATCAGGT GAGCCTGTCC CTGGTGGCAG CAACCGCAGC AG AT AGCGGC AAGTACTATT GCGCCAGAAC ACTGCACGGC AGGCGCATCT ACGGCATCGT GGCCTTTAAC GAGTGGTTCA CCTACTTTTA TATGGACGTG TGGGGCAATG GCACCCAGGT GACAGTGTCC TCTGGCGGGG GCGGCTCCGG AGGCGGAGGC TCTGGCGGGG GCGGCAGCGG C GGGGGC GGC TCCGGGGGAG GCGGCTCTGA TTTCGTGCTG ACCCAGTCTC CACATAGTCT GAGCGT GAC A CCCGGCGAAA GCGCATCAAT TTCTTGTAAA TCATCTCATA GTCTGATCCA CGGCGATAGG AACAATTACC TGGCCTGGTA CGTGCAGAAG CCAGGCCGCA GCCCTCAGCT GCTGATCTAC CTGGCAAGCT CCAGGGCATC CGGAGTGCCA GATCGCTTCT CTGGCAGCGG CTCCGATAAG GACTTTACCC TGAAGATCTC CCGGGTGGAG ACAGAGGACG TGGGCACATA CTATTGCATG CAGGGCAGAG AGTCTCCTTG GACCTTCGGC CAGGGCACAA AGGTGGACAT CAAGGGAGGA GGAGGCAGCG GCGGAGGAGG CTCCGGCGGC GGCGGCTCTC AGGCACAGCT GGTGCAGAGC GGACCAGAGG TGAGGAAGCC AGGCACCTCT GTGAAGGTGA GCTGTAAGGC CCCTGGCAAC ACCCTGAAGA CATACGATCT GCACTGGGTG CGGTCTGTGC CAGGACAGGG CCTGCAGTGG AT GGGAT GGA TCAGCCACGA GGGCGACAAG AAAGTGATCG TGGAGCGGTT TAAGGCCAAG GTGACAATCG ATT GGGAC AG AAGCACCAAT ACAGCCTATC TGCAGCTGTC CGGCCTGACC TCTGGCGATA CAGCCGTGTA CTATTGCGCC AAGGGCTCCA AGCACCGGCT GAGAGACTAC GCCCTGTATG ACGATGACGG CGCCCTGAAT TGGGCAGTGG ACGTGGACTA TCTGAGTAAT CTGGAGTTTT GGGGGCAGGG CACCGCAGTG ACAGTGTCTA
GCGGATCCGGACACCACCATCACCATCATTAGTGAAAGCTT
5. PentaNAbs.o Reverse PGDM1400-5X-PGT121-5X-N6-5X-35O22-5X- l0E8v4Sl00cF-His
SEQ ID NO: 85
GAATTCGCCG CCACCATGGG CTGGAGCTGC ATCATCCTGT TCCTGGTGGC AACCGCAACA GGAGTGCACA GCCAGGCACA GCTGGTGCAG AGCGGACCCG A AGT GAGA A A ACCTGGGACT AGCGTCAAAG TGTCATGTAA AGCCCCTGGA AATACCCTGA AGACCTACGA TCTGCACTGG GTGCGGTCCG TGCCTGGACA GGGCCTGCAG TGGATGGGAT GGATCTCTCA CGAGGGCGAC A AG A A AGT G A TCGTGGAGCG GTTCAAGGCC AAGGTGACAA TCGATTGGGA CAGATCCACC AACACAGCCT ACCTGCAGCT GTCTGGCCTG ACCAGCGGCG ATACAGCCGT GTACTACTGT GCCAAGGGCT CTAAGCACCG GCTGAGAGAC TACGCCCTGT ATGACGATGA CGGCGCCCTG AACTGGGCCG TGGATGTGGA CTATCTGTCC AATCTGGAGT TCTGGGGACA GGGAACCGCA GT GAC AGT GA GCTCCGGAGG AGGAGGCTCC GGCGGCGGAG GCTCTGGGGG AGGCGGCAGC GATTTTGTGC TGACCCAGTC TCCACACAGC CTGTCCGTGA CACCCGGCGA GTCTGCCAGC ATCTCCTGCA AGTCTAGCCA CAGCCTGATC CACGGCGACA GGAACAATTA CCTGGCCTGG TACGTGCAGA AGCCAGGCCG CAGCCCTCAG CTGCTGATCT ATCTGGCATC CTCTAGGGCC TCCGGAGTGC CAGATCGCTT CTCTGGCAGC GGCTCCGATA AGGACTTTAC CCTGAAGATC AGCCGGGTGG AGACAGAGGA CGTGGGCACA TACTATTGTA TGCAGGGCCG AGAATCACCT TGGACATTTG GGCAGGGAAC TAAAGTCGAC AT C A A AGGGG GGGGGGGCTC CGGCGGCGGG GGCTCTGGCG GCGGCGGCAG CGGAGGAGGC GGCTCCGGAG GAGGCGGCTC TCAGATGCAG CTGC AGGAGA GCGGACCAGG ACTGGTGAAG CCTTCCGAGA CCCTGTCTCT GACATGTTCT GT GAGCGGCG CCTCCATCTC TGATAGCTAC T GGAGCTGGA TCAGACGGAG CCCTGGCAAG GGCCTGGAGT GGATCGGCTA CGTGCACAAG TCTGGCGATA CAAACTATTC CCCATCTCTG AAGAGCCGGG TGAACCTGAG CCTGGACACC TCCAAGAATC AGGTGAGCCT GTCCCTGGTG GCAGCAACCG CAGCAGACAG CGGCAAGTAC TATTGCGCCA GAACACTGCA CGGCAGGCGC ATCTACGGCA TCGTGGCCTT TAACGAGTGG TTCACCTACT TTTATATGGA CGTGTGGGGC AATGGCACCC AGGTGACAGT GTCCTCTGGC GGCGGCGGCT CTGGCGGAGG AGGCAGCGGA GGAGGAGGCA GCTCCGACAT CTCTGTGGCA CCTGGAGAGA CCGCAAGGAT CAGCTGTGGA GAGAAGTCTC TGGGCAGCAG GGCCGTGCAG TGGTACCAGC ACAGGGCAGG ACAGGCACCA TCCCTGATCA TCTATAACAA TCAGGACCGG CCATCTGGCA TCCCCGAGAG ATTCTCTGGC AGCCCCGATA GCCCTTTTGG CACCACAGCC ACCCTGACAA TCACCTCCGT GGAGGCCGGC GACGAAGCAG ATTACTATTG CCACATCTGG GACTCCAGAG TGCCAACCAA GTGGGTGTTC GGAGGAGGAA CCACACTGAC AGTGCTGGGC GGCGGAGGCT CCGGCGGGGG CGGCTCTGGA GGCGGCGGCA GCGGAGGGGG CGGCTCCGGC GGCGGCGGCT CTAGGGCACA CCTGGTGCAG AGCGGAACCG CAATGAAGAA GCCTGGCGCC TCTGTGCGCG TGAGCTGTCA GACATCCGGC TACACCTTCA CCGCCCACAT CCTGTTCTGG TTTAGGCAGG CACCAGGAAG AGGACTGGAG TGGGTGGGCT GGATCAAGCC CCAGTATGGA GCAGTGAACT TCGGAGGAGG CTTTCGGGAC AGAGTGACAC TGACCCGGGA CGTGTACAGA GAGATCGCCT AT AT GG AT AT CAGGGGCCTG AAGCCCGACG ATACCGCCGT GTACTATTGC GCCAGGGACC GCTCCTACGG CGATTCTAGC TGGGCACTGG ACGCATGGGG ACAGGGAACC ACAGTGGTGG TGAGCGCCGG AGGCGGGGGC AGCGGCGGCG GGGGCTCCGG AGGCGGAGGC TCTTACATCC ACGTGACCCA GTCCCCTTCC TCTCTGTCCG TGTCTATCGG CGATCGCGTG ACCATCAACT GT C AG AC A AG CC AGGGAGT G GGCTCCGACC TGCACTGGTA TCAGCACAAG CCTGGCAGGG CCCCAAAGCT GCTGATCCAC CACACAAGCT CCGTGGAGGA TGGAGTGCCA AGCCGCTTCA GCGGCTCCGG ATTCCACACC AGCTTTAATC TGACAATCTC CGACCTGCAG GCCGACGATA TCGCCACCTA CTATTGCCAG GTGCTGCAGT TCTTTGGCAG GGGCTCCCGC CTGCACATCA AGGGCGGCGG CGGCTCTGGG GGC GGGGGC A GCGGCGGGGG GGGCTCCGGG GGAGGAGGCT CTGGCGGAGG GGGCAGCCAG GGCCAGCTGG TGCAGAGCGG AGC AGAGCTG AAGAAGCCAG GAGCCTCTGT GAAGATCAGC TGTAAGACAT CCGGCTACCG GTTCAACTTT TATCACATCA ATTGGATCAG GCAGACCGCA GGAAGGGGAC CAGAGTGGAT GGGCTGGATC TCCCCCTACT CTGGCGATAA GAACCTGGCC CCAGCCTTCC AGGACAGAGT GATCATGACC ACAGATACCG AGGTGCCAGT GACCAGCTTC ACCTCCACCG GAGCCGCCTA CATGGAGATC CGGAATCTGA AGTTCGACGA TACAGGCACC TATTTTTGCG CCAAGGGCCT GCTGAGAGAC GGCTCTAGCA CATGGCTGCC ATACCTGTGG GGACAGGGCA CCCTGCTGAC AGTGTCCTCT GGAGGAGGAG GCTCCGGGGG CGGCGGCTCT GGAGGAGGAG GCTCTCAAAG CGTGCTGACC CAGTCCGCCT CTGTGAGCGG CTCCCTGGGC CAGTCTGTGA CCATCAGCTG TACAGGCCCC AACTCCGTGT GCTGTTCTCA CAAGTCTATC AGCTGGTACC AGTGGCCACC AGGAAGGGCA CCTACCCTGA TCATCTATGA GGAC A AT GAG AGGGCACCAG GAATCAGCCC TCGCTTCTCC GGCTACAAGT CTTATTGGAG CGCCTACCTG ACCATTTCCG ACCTGCGCCC CGAGGATGAG ACCACATACT ATTGCTGTAG CTATACCCAC AACTCCGGCT GCGTGTTTGG CAC AGGC ACC AAGGT GAGCG TGCTGGGAGG AGGGGGCTCT GGCGGCGGGG GCAGCGGCGG AGGCGGCTCC GGAGGGGGCG GCTCTGGCGG AGGCGGCAGC GAGGTGCGGC TGGTGGAGAG C GGC GGC GGC CTGGTGAAGC CAGGCGGCTC TCTGAGACTG TCCTGTTCTG CCAGCGGCTT CGACTTTGAT AATGCCTGGA TGACATGGGT GCGGCAGCCT CCTGGCAAGG GGCTGGAGT G GGTGGGAAGA ATCACCGGAC CAGGAGAGGG ATGGTCTGTG GACTACGCCG AGAGCGTGAA GGGCCGGTTC ACCATCAGCA GAGATAACAC T A A A A AT AC A CTGTATCTGG AG AT GA AC A A TGTGCGGACC GAGGACACAG GCTACTATTT CTGCGCCAGA ACCGGCAAGT ACTATGATTT CTGGTTTGGC TACCCCCCTG GCGAGGAGT A TTTTCAGGAC TGGGGCCAGG GCACCCTGGT CATCGTGAGC AGCGGCGGGG GAGGCTCCGG CGGGGGGGGC TCTGGAGGAG GGGGCTCTAG CGAGCTGACC CAGGACCCCG CCGTGTCCGT GGCCCTGAAG CAGACAGTGA CCATCACATG CAGGGGCGAC TCCCTGCGCT CTCACTACGC CAGCTGGTAT CAGAAGAAGC CAGGACAGGC ACCCGTGCTG CTGTTCTACG GCAAGAACAA TCGGCCTTCC GGCATCCCAG ATAGATTTTC CGGCTCTGCC AGCGGAAACA GGGCCAGCCT GACCATCACA GGAGCACAGG C AGAGGAT GA AGCAGATTAC TATTGTTCCT CTCGGGACAA GTCCGGCTCT AGACTGAGCG TGTTCGGCGG CGGAACCAAG CTGACAGTGC
TGGGATCCGGACACCACCATCACCATCATTAGTGAAAGCTT
6. PentaNAb4.0 10E8n4 V5R_Sl00cF-5X-35O22-5X-N6-5X-PGTl2l-5X- PGDMl400-His
SEQ ID NO: 86
gaattcgccgccaccatgggatggagctgtattattctgtttctggtcgctaccgctacc
ggagtgcattcttctgaactgacccaggaccccgccgtgagcgtggccctgaagcagacc gtgacaatcacctgcaggggcgacagcctgcgctcccactacgccagctggtatcagaag aagcctggccaggccccagtgctgctgttctacggcaagaacaataggccctccggcatc cctgatcgcttttccggctctgccagcggaaacagggccagcctgacaatcaccggagca caggcagaggacgaggcagattactattgcagctcccgggacaagtccggctctagactg agcgtgttcggcggcggcaccaagctgacagtgctgggaggaggaggcagcggcggagga ggctccggaggcggcggctctgaggtgcggctgCGGgagtctggaggaggcctggtgaag ccaggaggcagcctgagactgagctgttccgcctctggcttcgactttgataatgcctgg atgacatgggtgcggcagccacctggcaagggcctggagtgggtgggaagaatcaccgga ccaggagagggatggtctgtggactacgccgagagcgtgaagggccggttcaccatctcc agagataacaccaagaatacactgtatctggagatgaacaatgtgcggaccgaggacaca ggctactatttctgcgccagaaccggcaagtactatgatttttggtttggctacccaccc ggcgaggagtattttcaggactggggccagggcaccctggtcatcgtgagcagcggcggc ggcggcagcggcggcggcggctccggaggaggcggctctggaggaggaggcagcggggga gggggcagccagtccgtgctgacccagtctgccagcgtgtccggctctctgggacagagc gtgaccatctcctgtacaggccccaacagcgtgtgctgtagccacaagagcatctcctgg taccagtggcctccaggaagggcacctaccctgatcatctatgaggacaatgagcgggcc ccaggcatctcccccagattctctggctacaagtcttattggagcgcctacctgacaatc agcgacctgcgccccgaggatgagacaacatactattgctgttcctatacccacaactct ggctgcgtgtttggcacaggcaccaaggtgtccgtgctgggcggcggcggcagcgggggc gggggctccggagggggcggctctcagggccagctggtgcagagcggagcagagctgaag aagcctggagccagcgtgaagatctcctgtaagacatctggcT AT cggttcaacttttat cacatcaattggatcaggcagaccgcaggaaggggaccagagtggatgggctggatctcc ccctactctggcgataagaacctggccccagccttccaggacagagtgatcatgaccaca gataccgaggtgccagtgaccagcttcacctccaccggagccgcctacatggagatcagg aatctgaagttcgacgatacaggcacctatttttgcgcaaagggcctgctgagggacggc tcctctacctggctgccttacctgtggggacagggcaccctgctgacagtgagctccggc ggcgggggcagcggcggcgggggctccggaggaggaggctctggaggagggggcagcgga ggaggcggctcctacatccacgtgacccagtccccatctagcctgtctgtgagcatcggc gatcgggtgaccatcaactgtcagacatctcagggcgtgggcagcgacctgcactggtat cagcacaagcctggcagggccccaaagctgctgatccaccacacatcctctgtggaggat ggagtgccaagccgcttctccggctctggattccacacctcctttaatctgacaatctct gacctgcaggccgacgatatcgccacctactattgccaggtgctgcagttctttggccgg ggctccagactgcacatcaagggaggaggaggctccgggggcggaggctctggcggcggc ggcagccgggcccacctggtgcagagcggcaccgccatgaagaagcctggcgccagcgtg agagtgtcctgtcagacatctggctacaccttcaccgcccacatcctgttctggtttagg caggcaccaggaagaggcctggagtgggtgggctggatcaagccccagtatggagcagtg aacttcggaggaggctttcgggacagagtgacactgacccgggacgtgtacagagagatc gcctatatggatatcaggggcctgaagccagacgataccgccgtgtactattgcgccagg gaccgctcctacggcgatagctcctgggcactggacgcatggggacagggcaccacagtg gtggtgagcgccggcggcggaggctccggcggcgggggctctggaggaggcggcagcgga gggggaggctccggagggggaggctctagcgacatctccgtggcccctggcgagacagcc
agaatctcttgtggcgagaagtctctgggcagcagggccgtgcagtggtaccagcacagg
gcaggacaggcaccatctctgatcatctataacaatcaggataggccaagcggcatccct
gagcggttcagcggctcccccgacagcccttttggcaccacagccacactgaccatcaca
tccgtggaggcaggcgacgaagccgattactattgccacatctgggattccagagtgcca
accaagtgggtgttcggaggaggaaccacactgacagtgctgggaggggggggctctggc
ggcgggggcagcgggggaggaggctcccagatgcagctgcaggagagcggaccaggcctg
gtgaagcctagcgagacactgagcctgacatgttctgtgagcggcgcctccatctctgac
agctactggtcttggatcagacggagccccggcaagggcctggaatggatcggctacgtg
cacaagtccggcgatacaaactattccccatctctgaagtctcgggtgaacctgtctctg
gacaccagcaagaatcaggtgagcctgtccctggtggcagcaaccgcagcagatagcggc
aagtactattgcgccagaacactgcacggcaggcgcatctacggcatcgtggcctttaac
gagtggttcacctacttttatatggacgtgtggggcaatggcacccaggtgacagtgtcc
tctggcgggggcggctccggaggcggaggctctggcgggggcggcagcggcgggggcggc
tccgggggaggcggctctgatttcgtgctgacccagtctccacatagtctgagcgtgaca
cccggcgaaagcgcatcaatttcttgtaaatcatctcatagtctgatccacggcgatagg
aacaattacctggcctggtacgtgcagaagccaggccgcagccctcagctgctgatctac
ctggcaagctccagggcatccggagtgccagatcgcttctctggcagcggctccgataag
gactttaccctgaagatctcccgggtggagacagaggacgtgggcacatactattgcatg
cagggcagagagtctccttggaccttcggccagggcacaaaggtggacatcaagggagga
ggaggcagcggcggaggaggctccggcggcggcggctctcaggcacagctggtgcagagc
ggaccagaggtgaggaagccaggcacctctgtgaaggtgagctgtaaggcccctggcaac
accctgaagacatacgatctgcactgggtgcggtctgtgccaggacagggcctgcagtgg
atgggatggatcagccacgagggcgacaagaaagtgatcgtggagcggtttaaggccaag
gtgacaatcgattgggacagaagcaccaatacagcctatctgcagctgtccggcctgacc
tctggcgatacagccgtgtactattgcgccaagggctccaagcaccggctgagagactac
gccctgtatgacgatgacggcgccctgaattgggcagtggacgtggactatctgagtaat
ctggagttttgggggcagggcaccgcagtgacagtgtctagcggatccGGACACCACCATCACCAT
CATTAGTGAAAGCTT PentaNAlxo Reverse PGDMl400-5X-PGTl2l-5X-N6-5X-35O22-5X-l0E8v4
V5R SlOOcF-His
SEQ ID NO: 87 gaattcgccgccaccatgggctggagctgcatcatcctgttcctggtggcaaccgcaaca
ggagtgcacagccaggcacagctggtgcagagcggacccgaagtgagaaaacctgggact
agcgtcaaagtgtcatgtaaagcccctggaaataccctgaagacctacgatctgcactgg
gtgcggtccgtgcctggacagggcctgcagtggatgggatggatctctcacgagggcgac
aagaaagtgatcgtggagcggttcaaggccaaggtgacaatcgattgggacagatccacc
aacacagcctacctgcagctgtctggcctgaccagcggcgatacagccgtgtactactgt
gccaagggctctaagcaccggctgagagactacgccctgtatgacgatgacggcgccctg
aactgggccgtggatgtggactatctgtccaatctggagttctggggacagggaaccgca
gtgacagtgagctccggaggaggaggctccggcggcggaggctctgggggaggcggcagc gattttgtgctgacccagtctccacacagcctgtccgtgacacccggcgagtctgccagc
atctcctgcaagtctagccacagcctgatccacggcgacaggaacaattacctggcctgg
tacgtgcagaagccaggccgcagccctcagctgctgatctatctggcatcctctagggcc
tccggagtgccagatcgcttctctggcagcggctccgataaggactttaccctgaagatc
agccgggtggagacagaggacgtgggcacatactattgtatgcagggccgagaatcacct
tggacatttgggcagggaactaaaGTTgacatcaaagggggggggggctccggcggcggg ggctctggcggcggcggcagcggaggaggcggctccggaggaggcggctctcagatgcag ctgcaggagagcggaccaggactggtgaagccttccgagaccctgtctctgacatgttct
gtgagcggcgcctccatctctgatagctactggagctggatcagacggagccctggcaag
ggcctggagtggatcggctacgtgcacaagtctggcgatacaaactattccccatctctg
aagagccgggtgaacctgagcctggacacctccaagaatcaggtgagcctgtccctggtg
gcagcaaccgcagcagacagcggcaagtactattgcgccagaacactgcacggcaggcgc
atctacggcatcgtggcctttaacgagtggttcacctacttttatatggacgtgtggggc
aatggcacccaggtgacagtgtcctctggcggcggcggctctggcggaggaggcagcgga
ggaggaggcagctccgacatctctgtggcacctggagagaccgcaaggatcagctgtgga
gagaagtctctgggcagcagggccgtgcagtggtaccagcacagggcaggacaggcacca
tccctgatcatctataacaatcaggaccggccatctggcatccccgagagattctctggc
agccccgatagcccttttggcaccacagccaccctgacaatcacctccgtggaggccggc
gacgaagcagattactattgccacatctgggactccagagtgccaaccaagtgggtgttc ggaggaggaaccacactgacagtgctgggcggcggaggctccggcgggggcggctctgga ggcggcggcagcggagggggcggctccggcggcggcggctctagggcacacctggtgcag agcggaaccgcaatgaagaagcctggcgcctctgtgcgcgtgagctgtcagacatccggc tacaccttcaccgcccacatcctgttctggtttaggcaggcaccaggaagaggactggag tgggtgggctggatcaagccccagtatggagcagtgaacttcggaggaggctttcgggac agagtgacactgacccgggacgtgtacagagagatcgcctatatggatatcaggggcctg aagcccgacgataccgccgtgtactattgcgccagggaccgctcctacggcgattctagc tgggcactggacgcatggggacagggaaccacagtggtggtgagcgccggaggcgggggc agcggcggcgggggctccggaggcggaggctcttacatccacgtgacccagtccccttcc tctctgtccgtgtctatcggcgatcgcgtgaccatcaactgtcagacaagccagggagtg ggctccgacctgcactggtatcagcacaagcctggcagggccccaaagctgctgatccac cacacaagctccgtggaggatggagtgccaagccgcttcagcggctccggattccacacc agctttaatctgacaatctccgacctgcaggccgacgatatcgccacctactattgccag gtgctgcagttctttggcaggggctcccgcctgcacatcaagggcggcggcggctctggg ggcgggggcagcggcggggggggctccgggggaggaggctctggcggagggggcagccag ggccagctggtgcagagcggagcagagctgaagaagccaggagcctctgtgaagatcagc tgtaagacatccggcT AT cggttcaacttttatcacatcaattggatcaggcagaccgca ggaaggggaccagagtggatgggctggatctccccctactctggcgataagaacctggcc ccagccttccaggacagagtgatcatgaccacagataccgaggtgccagtgaccagcttc acctccaccggagccgcctacatggagatccggaatctgaagttcgacgatacaggcacc tatttttgcgccaagggcctgctgagagacggctctagcacatggctgccatacctgtgg ggacagggcaccctgctgacagtgtcctctggaggaggaggctccgggggcggcggctct ggaggaggaggctctcaaagcgtgctgacccagtccgcctctgtgagcggctccctgggc cagtctgtgaccatcagctgtacaggccccaactccgtgtgctgttctcacaagtctatc agctggtaccagtggccaccaggaagggcacctaccctgatcatctatgaggacaatgag agggcaccaggaatcagccctcgcttctccggctacaagtcttattggagcgcctacctg accatttccgacctgcgccccgaggatgagaccacatactattgctgtagctatacccac aactccggctgcgtgtttggcacaggcaccaaggtgagcgtgctgggaggagggggctct ggcggcgggggcagcggcggaggcggctccggagggggcggctctggcggaggcggcagc gaggtgcggctgCGGgagagcggcggcggcctggtgaagccaggcggctctctgagactg tcctgttctgccagcggcttcgactttgataatgcctggatgacatgggtgcggcagcct cctggcaaggggctggagtgggtgggaagaatcaccggaccaggagagggatggtctgtg gactacgccgagagcgtgaagggccggttcaccatcagcagagataacactaaaaataca
ctgtatctggagatgaacaatgtgcggaccgaggacacaggctactatttctgcgccaga
accggcaagtactatgatttctggtttggctacccccctggcgaggagtattttcaggac
tggggccagggcaccctggtcatcgtgagcagcggcgggggaggctccggcggggggggc
tctggaggagggggctctagcgagctgacccaggaccccgccgtgtccgtggccctgaag
cagacagtgaccatcacatgcaggggcgactccctgcgctctcactacgccagctggtat
cagaagaagccaggacaggcacccgtgctgctgttctacggcaagaacaatcggccttcc
ggcatcccagatagattttccggctctgccagcggaaacagggccagcctgaccatcaca
ggagcacaggcagaggatgaagcagattactattgttcctctcgggacaagtccggctct
agactgagcgtgttcggcggcggaaccaagctgacagtgctgggatccGGACACCACCATCACCA
T C ATT AGT GA A AGC TT PentaNAlxo Reverse PGDMl400-5X-PGTl2l-5X-N6-5X-35O22-5X-l0E8v4 V5R SlOOcFvVH-fused to IgGl LS HC with His Tag
SEQ ID NO: 88
SEQ ID NO: 88
ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCAACCGCAACAGGA
GTGCACAGCCAGGCACAGCTGGTGCAGAGCGGACCCGAAGTGAGAAA
ACCTGGGACTAGCGTCAAAGTGTCATGTAAAGCCCCTGGAAATACCCT
GAAGACCTACGATCTGCACTGGGTGCGGTCCGTGCCTGGACAGGGCCT
GCAGTGGATGGGATGGATCTCTCACGAGGGCGACAAGAAAGTGATCG
T GGAGCGGTTC AAGGCC AAGGT GAC AATCGATTGGGAC AGATCC ACC
AACACAGCCTACCTGCAGCTGTCTGGCCTGACCAGCGGCGATACAGCC
GTGTACTACTGTGCCAAGGGCTCTAAGCACCGGCTGAGAGACTACGCC
CTGTATGACGATGACGGCGCCCTGAACTGGGCCGTGGATGTGGACTAT
CTGTCC AATCTGGAGTTCTGGGGAC AGGGAACCGC AGTGAC AGT GAGC
TCCGGAGGAGGAGGCTCCGGCGGCGGAGGCTCTGGGGGAGGCGGCAG
CGATTTTGTGCTGACCCAGTCTCCACACAGCCTGTCCGTGACACCCGG
CGAGTCTGCCAGCATCTCCTGCAAGTCTAGCCACAGCCTGATCCACGG
CGACAGGAACAATTACCTGGCCTGGTACGTGCAGAAGCCAGGCCGCA GCCCTCAGCTGCTGATCTATCTGGCATCCTCTAGGGCCTCCGGAGTGCC
AGATCGCTTCTCTGGCAGCGGCTCCGATAAGGACTTTACCCTGAAGAT
CAGCCGGGTGGAGACAGAGGACGTGGGCACATACTATTGTATGCAGG
GCCGAGAATCACCTTGGACATTTGGGCAGGGAACTAAAGTTGACATCA
AAGGGGGGGGGGGCTCCGGCGGCGGGGGCTCTGGCGGCGGCGGCAGC
GGAGGAGGCGGCTCCGGAGGAGGCGGCTCTCAGATGCAGCTGCAGGA
GAGCGGACCAGGACTGGTGAAGCCTTCCGAGACCCTGTCTCTGACATG
TTCTGTGAGCGGCGCCTCCATCTCTGATAGCTACTGGAGCTGGATCAG
ACGGAGCCCTGGC AAGGGCCTGGAGT GGATCGGCT ACGT GC AC AAGT
CTGGCGATACAAACTATTCCCCATCTCTGAAGAGCCGGGTGAACCTGA
GCCTGGACACCTCCAAGAATCAGGTGAGCCTGTCCCTGGTGGCAGCAA
CCGCAGCAGACAGCGGCAAGTACTATTGCGCCAGAACACTGCACGGC
AGGCGCATCTACGGCATCGTGGCCTTTAACGAGTGGTTCACCTACTTTT
ATATGGACGTGTGGGGCAATGGCACCCAGGTGACAGTGTCCTCTGGCG
GCGGCGGCTCTGGCGGAGGAGGCAGCGGAGGAGGAGGCAGCTCCGAC
ATCTCTGT GGC ACCTGGAGAGACCGC AAGGAT C AGCTGT GGA
GAGA AGT C TCTGGGC AGC AGGGC CGT GC AGT GGT ACC AGC AC AGGGC
AGGACAGGCACCATCCCTGATCATCTATAACAATCAGGACCGGCCATC
TGGCATCCCCGAGAGATTCTCTGGCAGCCCCGATAGCCCTTTTGGCAC
CACAGCCACCCTGACAATCACCTCCGTGGAGGCCGGCGACGAAGCAG
ATTACTATTGCCACATCTGGGACTCCAGAGTGCCAACCAAGTGGGTGT
TCGGAGGAGGAACCACACTGACAGTGCTGGGCGGCGGAGGCTCCGGC
GGGGGCGGCTCTGGAGGCGGCGGCAGCGGAGGGGGCGGCTCCGGCGG
CGGCGGCTCTAGGGCACACCTGGTGCAGAGCGGAACCGCAATGAAGA
AGCCTGGCGCCTCTGTGCGCGTGAGCTGTCAGACATCCGGCTACACCT
TCACCGCCCACATCCTGTTCTGGTTTAGGCAGGCACCAGGAAGAGGAC
T GGAGT GGGT GGGC T GGAT C A AGCCC C AGT AT GG AGC AGT GA AC TTCG
GAGGAGGCTTTCGGGAC AGAGT GAC ACTGACCCGGGACGT GT AC AGA
GAGATCGCCTATATGGATATCAGGGGCCTGAAGCCCGACGATACCGCC
GTGTACTATTGCGCCAGGGACCGCTCCTACGGCGATTCTAGCTGGGCA
CTGGACGCATGGGGACAGGGAACCACAGTGGTGGTGAGCGCCGGAGG
CGGGGGCAGCGGCGGCGGGGGCTCCGGAGGCGGAGGCTCTTACATCC ACGTGACCCAGTCCCCTTCCTCTCTGTCCGTGTCTATCGGCGATCGCGT GACC AT C AACTGTC AGAC A AGCC AGGGAGT G
GGCTCCGACCTGCACTGGTATCAGCACAAGCCTGGCAGGGCCCCAAAG
CTGCTGATCCACCACACAAGCTCCGTGGAGGATGGAGTGCCAAGCCGC
TTCAGCGGCTCCGGATTCCACACCAGCTTTAATCTGACAATCTCCGACC
TGCAGGCCGACGATATCGCCACCTACTATTGCCAGGTGCTGCAGTTCT
TTGGCAGGGGCTCCCGCCTGCACATCAAGGGCGGCGGCGGCTCTGGGG
GCGGGGGCAGCGGCGGGGGGGGCTCCGGGGGAGGAGGCTCTGGCGGA
GGGGGCAGCCAGGGCCAGCTGGTGCAGAGCGGAGCAGAGCTGAAGAA
GCCAGGAGCCTCTGTGAAGATCAGCTGTAAGACATCCGGCTATCGGTT
CAACTTTTATCACATCAATTGGATCAGGCAGACCGCAGGAAGGGGACC
AGAGTGGATGGGCTGGATCTCCCCCTACTCTGGCGATAAGAACCTGGC
CCCAGCCTTCCAGGACAGAGTGATCATGACCACAGATACCGAGGTGCC
AGTGACCAGCTTCACCTCCACCGGAGCCGCCTACATGGAGATCCGGAA
TCTGAAGTTCGACGATACAGGCACCTATTTTTGCGCCAAGGGCCTGCT
GAGAGACGGCTCTAGCACATGGCTGCCATACCTGTGGGGACAGGGCA
CCCTGCTGACAGTGTCCTCTGGAGGAGGAGGCTCCGGGGGCGGCGGCT
CTGGAGGAGGAGGCTCTCAAAGCGTGCTGACCCAGTCCGCCTCTGTGA
GCGGCTCCCTGGGCCAGTCTGTGACCATCAGCTGTACAGGCCCCAACT
CCGTGTGCTGTTCTCACAAGTCTATCAGCTGGTACCAGTGGCCACCAG
GAAGGGCACCTACCCTGATCATCTATGAGGACAATGAGAGGGCACCA
GGAATCAGCCCTCGCTTCTCCGGCTACAAGTCTTATTGGAGCGCCTAC
CTGACCATTTCCGACCTGCGCCCCGAGGATGAGACCACATACTATTGC
TGTAGCTATACCCACAACTCCGGCTGCGTGTTTGGCACAGGCACCAAG
GT GAGCGTGCTGGGAGGAGGGGGCTCTGGCGGCGGGGGC AGCGGCGG
AGGCGGCTCCGGAGGGGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGC
GGCTGCGGGAGAGCGGCGGCGGCCTGGTGAAGCCAGGCGGCTCTCTG
AGACTGTCCTGTTCTGCCAGCGGCTTCGACTTTGATAATGCCTGGATGA
CATGGGTGCGGCAGCCTCCTGGCAAGGGGCTGGAGTGGGTGGGAAGA
ATCACCGGACCAGGAGAGGGATGGTCTGTGGACTACGCCGAGAGCGT
GAAGGGCCGGTTCACCATCAGCAGAGATAACACTAAAAATACACTGT ATCTGGAGAT GA AC A AT GT GC GGACC GAGGAC AC AGGC T AC T ATTTCT GCGCCAGA
ACCGGCAAGTACTATGATTTCTGGTTTGGCTACCCCCCTGGCGAGGAG
TATTTTCAGGACTGGGGCCAGGGCACCCTGGTCATCGTGAGCAGCGCG
TCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC
ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTC
CCCGAACCTGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC
GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCA
GCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACA
TCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAA
GTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCA
GCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAA
CCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTG
GTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC
GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA
GCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA
CC AGGAC T GGC T GA AT GGC A AGGAGT AC A AGT GC A AGGT C TC C A AC A
AAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGG
CAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGA
GATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA
TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGA
ACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCT
TCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG
AACGTCTTCTCATGCTCCGTGCTGCATGAGGCTCTGCACAGCCACTAC
ACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA PentaNAlxo Reverse PGDMl400-5X-PGTl2l-5X-N6-5X-35O22-5X-l0E8v4 V5R SlOOcF VH-VL-fused to IgGl LS FC with His Tag
SEQ ID NO: 89
gaattcgccgccaccatgggctggagctgcatcatcctgttcctggtggcaaccgcaaca
ggagtgcacagccaggcacagctggtgcagagcggacccgaagtgagaaaacctgggact
agcgtcaaagtgtcatgtaaagcccctggaaataccctgaagacctacgatctgcactgg gtgcggtccgtgcctggacagggcctgcagtggatgggatggatctctcacgagggcgac aagaaagtgatcgtggagcggttcaaggccaaggtgacaatcgattgggacagatccacc aacacagcctacctgcagctgtctggcctgaccagcggcgatacagccgtgtactactgt gccaagggctctaagcaccggctgagagactacgccctgtatgacgatgacggcgccctg aactgggccgtggatgtggactatctgtccaatctggagttctggggacagggaaccgca gtgacagtgagctccggaggaggaggctccggcggcggaggctctgggggaggcggcagc gattttgtgctgacccagtctccacacagcctgtccgtgacacccggcgagtctgccagc atctcctgcaagtctagccacagcctgatccacggcgacaggaacaattacctggcctgg tacgtgcagaagccaggccgcagccctcagctgctgatctatctggcatcctctagggcc tccggagtgccagatcgcttctctggcagcggctccgataaggactttaccctgaagatc agccgggtggagacagaggacgtgggcacatactattgtatgcagggccgagaatcacct tggacatttgggcagggaactaaaGTTgacatcaaagggggggggggctccggcggcggg ggctctggcggcggcggcagcggaggaggcggctccggaggaggcggctctcagatgcag ctgcaggagagcggaccaggactggtgaagccttccgagaccctgtctctgacatgttct gtgagcggcgcctccatctctgatagctactggagctggatcagacggagccctggcaag ggcctggagtggatcggctacgtgcacaagtctggcgatacaaactattccccatctctg aagagccgggtgaacctgagcctggacacctccaagaatcaggtgagcctgtccctggtg gcagcaaccgcagcagacagcggcaagtactattgcgccagaacactgcacggcaggcgc atctacggcatcgtggcctttaacgagtggttcacctacttttatatggacgtgtggggc aatggcacccaggtgacagtgtcctctggcggcggcggctctggcggaggaggcagcgga ggaggaggcagctccgacatctctgtggcacctggagagaccgcaaggatcagctgtgga gagaagtctctgggcagcagggccgtgcagtggtaccagcacagggcaggacaggcacca tccctgatcatctataacaatcaggaccggccatctggcatccccgagagattctctggc agccccgatagcccttttggcaccacagccaccctgacaatcacctccgtggaggccggc gacgaagcagattactattgccacatctgggactccagagtgccaaccaagtgggtgttc ggaggaggaaccacactgacagtgctgggcggcggaggctccggcgggggcggctctgga ggcggcggcagcggagggggcggctccggcggcggcggctctagggcacacctggtgcag agcggaaccgcaatgaagaagcctggcgcctctgtgcgcgtgagctgtcagacatccggc tacaccttcaccgcccacatcctgttctggtttaggcaggcaccaggaagaggactggag tgggtgggctggatcaagccccagtatggagcagtgaacttcggaggaggctttcgggac agagtgacactgacccgggacgtgtacagagagatcgcctatatggatatcaggggcctg aagcccgacgataccgccgtgtactattgcgccagggaccgctcctacggcgattctagc tgggcactggacgcatggggacagggaaccacagtggtggtgagcgccggaggcgggggc agcggcggcgggggctccggaggcggaggctcttacatccacgtgacccagtccccttcc tctctgtccgtgtctatcggcgatcgcgtgaccatcaactgtcagacaagccagggagtg ggctccgacctgcactggtatcagcacaagcctggcagggccccaaagctgctgatccac cacacaagctccgtggaggatggagtgccaagccgcttcagcggctccggattccacacc agctttaatctgacaatctccgacctgcaggccgacgatatcgccacctactattgccag gtgctgcagttctttggcaggggctcccgcctgcacatcaagggcggcggcggctctggg ggcgggggcagcggcggggggggctccgggggaggaggctctggcggagggggcagccag ggccagctggtgcagagcggagcagagctgaagaagccaggagcctctgtgaagatcagc tgtaagacatccggcT AT cggttcaacttttatcacatcaattggatcaggcagaccgca ggaaggggaccagagtggatgggctggatctccccctactctggcgataagaacctggcc ccagccttccaggacagagtgatcatgaccacagataccgaggtgccagtgaccagcttc acctccaccggagccgcctacatggagatccggaatctgaagttcgacgatacaggcacc tatttttgcgccaagggcctgctgagagacggctctagcacatggctgccatacctgtgg ggacagggcaccctgctgacagtgtcctctggaggaggaggctccgggggcggcggctct ggaggaggaggctctcaaagcgtgctgacccagtccgcctctgtgagcggctccctgggc cagtctgtgaccatcagctgtacaggccccaactccgtgtgctgttctcacaagtctatc agctggtaccagtggccaccaggaagggcacctaccctgatcatctatgaggacaatgag agggcaccaggaatcagccctcgcttctccggctacaagtcttattggagcgcctacctg accatttccgacctgcgccccgaggatgagaccacatactattgctgtagctatacccac aactccggctgcgtgtttggcacaggcaccaaggtgagcgtgctgggaggagggggctct ggcggcgggggcagcggcggaggcggctccggagggggcggctctggcggaggcggcagc gaggtgcggctgCGGgagagcggcggcggcctggtgaagccaggcggctctctgagactg tcctgttctgccagcggcttcgactttgataatgcctggatgacatgggtgcggcagcct cctggcaaggggctggagtgggtgggaagaatcaccggaccaggagagggatggtctgtg gactacgccgagagcgtgaagggccggttcaccatcagcagagataacactaaaaataca ctgtatctggagatgaacaatgtgcggaccgaggacacaggctactatttctgcgccaga accggcaagtactatgatttctggtttggctacccccctggcgaggagtattttcaggac tggggccagggcaccctggtcatcgtgagcagcggcgggggaggctccggcggggggggc tctggaggagggggctctagcgagctgacccaggaccccgccgtgtccgtggccctgaag cagacagtgaccatcacatgcaggggcgactccctgcgctctcactacgccagctggtat cagaagaagccaggacaggcacccgtgctgctgttctacggcaagaacaatcggccttcc ggcatcccagatagattttccggctctgccagcggaaacagggccagcctgaccatcaca ggagcacaggcagaggatgaagcagattactattgttcctctcgggacaagtccggctct
agactgagcgtgttcggcggcggaaccaagctgacagtgctgggatccCCCAAATCTTGTGACAA
AACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACC
GTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCC
CGGACCCCTGAGGT C AC AT GCGT GGT GGTGGACGT GAGCC ACGAAGA
CCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAA
TGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTG
TGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGG
AGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGA
AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTAC
ACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCT
GACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTG
GGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCG
TGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGA
CAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGCTGCA
TGAGGCTCTGCACAGCCACTACACGCAGAAGAGCCTCTCCCTGTCTCC
GGGTAAATGA
Methods for producing antibodies, such as those disclosed herein, are known in the art. For example, DNA molecules encoding light chain variable regions and/or heavy chain variable regions can be chemically synthesized using the sequence information provided herein. Synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, including, e.g., expression control sequences, to produce conventional gene expression constructs encoding the desired antibodies. Production of defined gene constructs is within routine skill in the art. Alternatively, the sequences provided herein can be cloned out of hybridomas by conventional hybridization techniques or polymerase chain reaction (PCR) techniques, using synthetic nucleic acid probes whose sequences are based on sequence information provided herein, or prior art sequence information regarding genes encoding the heavy and light chains.
Standard techniques of molecular biology may be used to prepare DNA sequences coding for the antibodies or fragments of the antibodies of the present invention. Desired DNA sequences may be synthesized completely or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate.
Any suitable host cell/vector system may be used for expression of the DNA sequences encoding the antibody molecules of the present invention or fragments thereof. Bacterial, for example E. coli, and other microbial systems may be used, in part, for expression of antibody fragments such as Fab and F(ab')2 fragments, and especially Fv fragments and single chain antibody fragments, for example, single chain Fvs. Eukaryotic, e.g. mammalian, host cell expression systems may be used for production of larger antibody molecules, including complete antibody molecules. Suitable mammalian host cells include CHO, HEK293T, PER.C6, myeloma or hybridoma cells.
In some embodiments, antibodies according to the invention may be produced by i) expressing a nucleic acid sequence according to the invention in a cell, and ii) isolating the expressed antibody product. Additionally, the method may include iii) purifying the antibody.
For the antibodies of the present invention to be expressed, the protein coding sequence should be "operably linked" to regulatory or nucleic acid control sequences that direct transcription and translation of the protein. As used herein, a coding sequence and a nucleic acid control sequence or promoter are said to be "operably linked" when they are covalently linked in such a way as to place the expression or transcription and/or translation of the coding sequence under the influence or control of the nucleic acid control sequence. The "nucleic acid control sequence" can be any nucleic acid element, such as, but not limited to promoters, enhancers, IRES, introns, and other elements described herein that direct the expression of a nucleic acid sequence or coding sequence that is operably linked thereto. The term "promoter" will be used herein to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II and that when operationally linked to the protein coding sequences of the invention lead to the expression of the encoded protein. The expression of the antibodies of the present invention can be under the control of a constitutive promoter or of an inducible promoter, which initiates transcription only when exposed to some particular external stimulus, such as, without limitation, antibiotics such as tetracycline, hormones such as ecdysone, or heavy metals. The promoter can also be specific to a particular cell-type, tissue or organ. Many suitable promoters and enhancers are known in the art, and any such suitable promoter or enhancer may be used for expression of the antibodies of the invention. For example, suitable promoters and/or enhancers can be selected from the Eukaryotic Promoter Database (EPDB).
Nucleic acids encoding desired antibodies can be incorporated (ligated) into expression vectors, which can be introduced into host cells through conventional transfection or transformation techniques. Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BEK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce IgG protein. Transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the immunoglobulin light and/or heavy chain variable regions. Specific expression and purification conditions will vary depending upon the expression system employed.
Following expression, the antibodies and/or antigens of the invention can be isolated and/or purified or concentrated using any suitable technique known in the art. For example, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, immuno-affmity chromatography, hydroxyapatite chromatography, lectin chromatography, molecular sieve chromatography, isoelectric focusing, gel electrophoresis, or any other suitable method or combination of methods can be used.
In some embodiments, the antibodies can be made using recombinant DNA methods as described in ET.S. Pat. No. 4,816,567. The polynucleotides encoding a monoclonal antibody can be isolated from mature B-cells or hybridoma cell, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which when transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, monoclonal antibodies are generated by the host cells. The anti-HIV antibodies can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues. It should be understood that the antibodies of the invention may differ from the exact sequences illustrated and described herein. Thus, the invention contemplates deletions, additions and substitutions to the sequences shown, so long as the sequences function in accordance with the methods of the invention. In this regard, particularly preferred substitutions will generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids. For example, amino acids are generally divided into four families: (1) acidic— aspartate and glutamate; (2) basic— lysine, arginine, histidine; (3) non-polar— alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar— glycine, asparagine, glutamine, cystine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, leucine can be replaced with isoleucine or valine, or vice versa; an aspartate with a glutamate or vice versa; a threonine with a serine or vice versa; or a similar conservative replacement of an amino acid with a structurally related amino acid can be made.
The polynucleotide(s) encoding a monoclonal antibody can further be modified in a number of different manners using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light and heavy chains of, for example, a mouse monoclonal antibody can be substituted 1) for those regions of, for example, a human antibody to generate a chimeric antibody or 2) for a non immunoglobulin polypeptide to generate a fusion antibody. In some embodiments, the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody. Site-directed or high-density mutagenesis of the variable region can be used to optimize specificity, affinity, etc. of a monoclonal antibody.
For the purposes of the present invention, it should be appreciated that modified antibodies can comprise any type of variable region that provides for the association of the antibody with the polypeptides of HIV such as the envelope protein.
Besides their configuration, it is known in the art that the constant region mediates several effector functions. For example, binding of the Cl component of complement to antibodies activates the complement system. Activation of complement is important in the opsonisation and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and can also be involved in autoimmune hypersensitivity. Further, antibodies bind to cells via the Fc region, with a Fc receptor site on the antibody Fc region binding to a Fc receptor (FcR) on a cell. There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (eta receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production.
In certain embodiments, the anti-HIV antibodies provide for altered effector functions that, in turn, affect the biological profile of the administered antibody. For example, the deletion or inactivation (through point mutations or other means) of a constant region domain can reduce Fc receptor binding of the circulating modified antibody thereby increasing tumor localization. In other cases it may be that constant region modifications, consistent with this invention, moderate complement binding and thus reduce the serum half-life and nonspecific association of a conjugated cytotoxin. Yet other modifications of the constant region can be used to eliminate disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility. Similarly, modifications to the constant region in accordance with this invention can easily be made using well known biochemical or molecular engineering techniques well within the purview of the skilled artisan.
Methods
The multispecific anti-HIV antibodies of the invention are useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as the treatment, cure, functional cure, or prevention of HIV infection. The methods of use may be in vitro, ex vivo, or in vivo methods.
In some embodiments, the multispecific antibodies disclosed herein may be used as neutralizing antibodies, passively administered or given via gene therapies. In one aspect, the anti-HIV antibodies are useful for detecting the presence of HIV in a biological sample. The term "detecting" as used herein encompasses quantitative or qualitative detection. In certain embodiments, a biological sample comprises a cell or tissue. Certain other methods can be used to detect binding of anti-HIV antibodies to antigens such as envelope protein. Such methods include, but are not limited to, antigen binding assays that are well known in the art, such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays, and immunohistochemistry (IHC).
In certain embodiments, the antibodies are labeled. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction.
In certain embodiments, the antibodies are immobilized on an insoluble matrix. Immobilization entails separating the antibody from any antigen that remains free in solution. This conventionally is accomplished by either insolubilizing the antibody before the assay procedure, as by adsorption to a water-insoluble matrix or surface (Bennich et ah, U.S. Pat. No. 3,720,760), or by covalent coupling (for example, using glutaraldehyde cross-linking), or by insolubilizing the antibody after formation of a complex between the antibody and antigen, e.g., by immunoprecipitation.
The present invention provides for methods of treating or preventing HIV infection comprising administering a therapeutically effective amount of a multispecific antibody as described herein to a subject (e.g., a subject in need of treatment). In some embodiments, the subject is a human.
Subjects at risk for HIV-related diseases or disorders include patients who have come into contact with an infected person or who have been exposed to HIV-l in some other way. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of HIV-l-related disease or disorder, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
Methods for preventing an increase in HIV-l virus titer, virus replication, virus proliferation or an amount of an HIV-l viral protein in a subject are further provided. In one embodiment, a method includes administering to the subject an amount of an anti- HIV antibody effective to prevent an increase in HIV-l titer, virus replication or an amount of an HIV-l protein of one or more HIV strains or isolates in the subject.
For in vivo treatment of human patients, the patient is usually administered or provided a pharmaceutical formulation including a multispecific anti-HIV antibody of the invention. When used for in vivo therapy, the antibodies of the invention are administered to the patient in therapeutically effective amounts (i.e., amounts that eliminate or reduce the patient's viral burden). The antibodies can be administered to a human patient, in accordance with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The antibodies may be administered parenterally, when possible, at the target cell site, or intravenously. Intravenous or subcutaneous administration of the antibody is preferred in certain embodiments. Therapeutic compositions of the invention are administered to a patient or subject systemically, parenterally, or locally.
For parenteral administration, the antibodies can be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable, parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate are also used. Liposomes are used as carriers. The vehicle contains minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. In some embodiments, the antibodies are typically formulated in such vehicles at concentrations of about 1 mg/ml to 10 mg/ml.
The dose and dosage regimen depends upon a variety of factors readily determined by a physician, such as the nature of the infection and the characteristics of the particular cytotoxic agent or growth inhibitory agent conjugated to the antibody (when used), e.g., its therapeutic index, the patient, and the patient's history. Generally, a therapeutically effective amount of an antibody is administered to a patient. In particular embodiments, the amount of antibody administered is in the range of about 0.1 mg/kg to about 20 mg/kg of patient body weight. Depending on the type and severity of the infection, about 0.1 mg/kg to about 20 mg/kg body weight (e.g., about 0.1-15 mg/kg/dose) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. The progress of this therapy is readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art.
Antibodies of the invention can be coupled to a drug for delivery to a treatment site or coupled to a detectable label to facilitate imaging of a site comprising cells of interest, such as cells infected with HIV. Methods for coupling antibodies to drugs and detectable labels are well known in the art, as are methods for imaging using detectable labels. Labeled antibodies may be employed in a wide variety of assays, employing a wide variety of labels. Detection of the formation of an antibody-antigen complex between an antibody of the invention and an epitope of interest (an HIV epitope) can be facilitated by attaching a detectable substance to the antibody. Suitable detection means include the use of labels such as radionucleotides, enzymes, coenzymes, fluorescers, chemiluminescers, chromogens, enzyme substrates or co-factors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, and the like. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, b-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material is luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, 35S, or sup.3H. Such labeled reagents may be used in a variety of well-known assays, such as radioimmunoassays, enzyme immunoassays, e.g., ELISA, fluorescent immunoassays, and the like.
The antibodies can be tagged with such labels by known methods. For instance, coupling agents such as aldehydes, carbodiimides, dimaleimide, imidates, succinimides, bid-diazotized benzadine and the like are used to tag the antibodies with the above- described fluorescent, chemiluminescent, and enzyme labels. An enzyme is typically combined with an antibody using bridging molecules such as carbodiimides, periodate, diisocyanates, glutaraldehyde and the like. Various labeling techniques are described in Morrison, Methods in Enzymology 32b, 103 (1974), Syvanen et ah, J. Biol. Chem. 284, 3762 (1973) and Bolton and Hunter, Biochem J. 133, 529(1973).
In one embodiment, the antibodies can be administered as immunoconjugates, conjugated to a second molecule. For example, the second molecule can be a toxin, a label, a radioisotope, a drug, or a chemical compound.
An antibody according to the invention may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent, or a radioactive metal ion or radioisotope. Examples of radioisotopes include, but are not limited to, 1-131, 1-123, 1-125, Y-90, Re- 188, Re- 186, At-2l l, Cu-67, Bί-212, Bί-213, Pd-l09, Tc-99, In-l l l, and the like. Such antibody conjugates can be used for modifying a given biological response; the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin, TLR agonists (such as TLR7 agonist), or monomethylauristatin E.
Other therapeutic regimens can be combined with the administration of the anti- HIV antibody of the present invention. The combined administration includes co administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. Preferably such combined therapy results in a synergistic therapeutic effect.
For any application, the antibody, antigen binding fragment, or nucleic acid encoding the antibody or antigen binding fragment can be combined with anti-retroviral therapy. Antiretroviral drugs are broadly classified by the phase of the retrovirus life- cycle that the drug inhibits. The disclosed antibodies can be administered in conjunction with nucleoside analog reverse-transcriptase inhibitors (such as zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, entecavir, and apricitabine), nucleotide reverse transcriptase inhibitors (such as tenofovir and adefovir), non nucleoside reverse transcriptase inhibitors (such as efavirenz, nevirapine, delavirdine, etravirine, and rilpivirine), protease inhibitors (such as saquinavir, ritonavir, indinavir, nelfmavir, amprenavir, lopinavir, fosamprenavir, atazanavir, tipranavir, and darunavir), entry or fusion inhibitors (such as maraviroc and enfuvirtide), maturation inhibitors, (such as bevirimat and vivecon), or a broad spectrum inhibitors, such as natural antivirals. In some examples, a disclosed antibody or active fragment thereof or nucleic acids encoding such is administered in conjunction with IL-15, or conjugated to IL-15.
Single or multiple administrations of the compositions including the antibody, antigen binding fragment, or nucleic acid encoding the antibody or antigen binding fragment, that are disclosed herein, are administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of at least one of the antibodies disclosed herein to effectively treat the patient. The dosage can be administered once, but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy.
One approach to administration of nucleic acids is direct administration with plasmid DNA, such as with a mammalian expression plasmid. The nucleotide sequence encoding the disclosed antibody, or antibody binding fragments thereof, can be placed under the control of a promoter to increase expression. Another approach is to administer the nucleic acids in the form of mRNA.
In some embodiments, the subject is administered cells that are engineered to express the anti-HIV antibody. In some embodiments, the cells are engineered immune cells, such as B cells. In some embodiments, the cells are engineered, autologous cells.
In another approach to using nucleic acids, an anti-HIV antibody, or antibody binding fragment thereof can also be expressed by attenuated viral hosts or vectors or bacterial vectors. Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus, retrovirus, cytomegalovirus or other viral vectors can be used to express the antibody. For example, vaccinia vectors and methods useful protocols are described in U.S. Pat. No. 4,722,848. BCG (Bacillus Calmette Guerin) provides another vector for expression of the disclosed antibodies (see Stover, Nature 351:456-460, 1991). Compositions
The present invention also encompasses compositions comprising one or more antibodies of the invention. In certain embodiments, the compositions are pharmaceutical compositions. In some embodiments, formulations are prepared for storage and use by combining an antibody with a pharmaceutically acceptable vehicle (e.g. carrier, excipient) (. Remington , The Science and Practice of Pharmacy 20th Edition Mack Publishing, 2000). Suitable pharmaceutically acceptable vehicles include, but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (e.g. octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (e.g. less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosacchandes, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn- protein complexes); and non-ionic surfactants such as TWEEN or polyethylene glycol (PEG).
For the treatment or prevention of HIV, the appropriate dosage of an antibody or combination of antibodies of the present invention can depend on a variety of factors, such as the severity and course of the disease, the responsiveness of the disease, whether the antibody or agent is administered for therapeutic or preventative purposes, previous therapy, patient's clinical history, and so on all at the discretion of the treating physician. The antibody or agent can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. In certain embodiments, dosage is from 0.01 pg to 100 mg per kg of body weight, and can be given once or more daily, weekly, monthly or yearly. In certain embodiments, the antibody or combination of antibodies is given once every two weeks or once every three weeks. In certain embodiments, the dosage of the antibody is from about 0.1 mg to about 20 mg per kg of body weight. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.
Effective dosages and schedules for administering embodiments of the present invention can be determined empirically. In some embodiments, and effective amount of one or more antibodies are administered to neutralize, treat, prevent or eradicate HIV infection. In some embodiments, compositions comprising one or more nucleic acid molecules of the invention are administered to the subject. In some embodiments, genetic constructs capable of inducing production of antibodies of the present invention may be administered to a patient in need thereof.
Controlled-release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, Banga, A. J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, Pa., (1995). Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic protein, such as a cytotoxin or a drug, as a central core. In microspheres the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 pm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 pm so that only nanoparticles are administered intravenously. Microparticles are typically around 100 pm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y., pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp. 315-339, (1992).
Polymers can be used for ion-controlled release of the antibody compositions disclosed herein. Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer, polaxamer 407, exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has been shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, Pa. (1993)). Numerous additional systems for controlled delivery of therapeutic proteins are known (see U.S. Pat. No. 5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat. No. 4,957,735; U.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S. Pat. No. 5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268,164; U.S. Pat. No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No. 5,506,206; U.S. Pat. No. 5,271,961; U.S. Pat. No. 5,254,342 and U.S. Pat. No. 5,534,496).
In some embodiments, the compositions of the invention may be injectable suspensions, solutions, sprays, lyophilized powders, syrups, elixirs and the like. Any suitable form of composition may be used. To prepare such a composition, a nucleic acid or vector of the invention, having the desired degree of purity, is mixed with one or more pharmaceutically acceptable carriers and/or excipients. The carriers and excipients must be "acceptable" in the sense of being compatible with the other ingredients of the composition. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, or combinations thereof, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™ PLEIRONICS™ or polyethylene glycol (PEG).
The compositions can be designed to introduce the antibodies, nucleic acids or expression vectors to a desired site of action and release it at an appropriate and controllable rate. Methods of preparing controlled-release formulations are known in the art. For example, controlled release preparations can be produced by the use of polymers to complex or absorb the immunogen and/or immunogenic composition. A controlled- release formulations can be prepared using appropriate macromolecules (for example, polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) known to provide the desired controlled release characteristics or release profile. Another possible method to control the duration of action by a controlled-release preparation is to incorporate the active ingredients into particles of a polymeric material such as, for example, polyesters, polyamino acids, hydrogels, polylactic acid, polyglycolic acid, copolymers of these acids, or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these active ingredients into polymeric particles, it is possible to entrap these materials into microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly methylmethacrylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in New Trends and Developments in Vaccines, Voller et al. (eds.), ETniversity Park Press, Baltimore, Md., 1978 and Remington's Pharmaceutical Sciences, l6th edition.
The compositions can be administered using any suitable delivery method including, but not limited to, intramuscular, intravenous, intradermal, mucosal, and topical delivery. Such techniques are well known to those of skill in the art. More specific examples of delivery methods are intramuscular injection, intradermal injection, and subcutaneous injection. However, delivery need not be limited to injection methods. Further, delivery of DNA to animal tissue has been achieved by cationic liposomes (Watanabe et al., (1994) Mol. Reprod. Dev. 38:268-274; and WO 96/20013), direct injection of naked DNA into animal muscle tissue (Robinson et al., (1993) Vaccine 11 :957-960; Hoffman et al., (1994) Vaccine 12: 1529-1533; Xiang et al., (1994) Virology 199: 132-140; Webster et ah, (1994) Vaccine 12: 1495-1498; Davis et ah, (1994) Vaccine 12: 1503-1509; and Davis et ah, (1993) Hum. Mol. Gen. 2: 1847-1851), or intradermal injection of DNA using "gene gun" technology (Johnston et ah, (1994) Meth. Cell Biol. 43:353-365). Alternatively, delivery routes can be oral, intranasal or by any other suitable route. Delivery also be accomplished via a mucosal surface such as the anal, vaginal or oral mucosa.
Dosing schedules (or regimens) can be readily determined for the particular subject and composition. Hence, the composition can be administered one or more times to the subject. Preferably, there is a set time interval between separate administrations of the composition. While this interval varies for every subject, typically it can range from 10 days to several weeks, and is often 2, 4, 6 or 8 weeks. In some embodiments, the interval can be typically from 2 to 6 weeks.
The compositions of the invention can be administered alone, or can be co administered, or sequentially administered, with other HIV immunogens and/or HIV immunogenic compositions, e.g., with "other" immunological, antigenic or vaccine or therapeutic compositions thereby providing multivalent or "cocktail" or combination compositions of the invention and methods of employing them. Again, the ingredients and manner (sequential or co-administration) of administration, as well as dosages can be determined taking into consideration such factors as the age, sex, weight, species and condition of the particular subject, and the route of administration.
Kits
The present invention also includes kits useful in performing diagnostic and prognostic assays using the antibodies of the present invention. Kits of the invention include a suitable container comprising an HIV-l antibody of the invention in either labeled or unlabeled form. In addition, when the antibody is supplied in a labeled form suitable for an indirect binding assay, the kit further includes reagents for performing the appropriate indirect assay. For example, the kit includes one or more suitable containers including enzyme substrates or derivatizing agents, depending on the nature of the label. Control samples and/or instructions are also included. Application of the teachings of the present invention to a specific problem is within the capabilities of one having ordinary skill in the art in light of the teaching contained herein. Examples of the compositions and methods of the invention appear in the following non-limiting Examples.
EXAMPLES
Example 1. Multi-specific antibodies for HIV-l and their utility
Here, 3D structure information of broadly neutralizing antibodies (bNAbs) in complex with HIV-l envelope glycoproteins (Env) was used to perform rational design of penta-specific antibodies (PentaNAbs) capable of targeting five major Env neutralizing epitopes. These inventions, herein referred to as“PentaNAbs”, consist of five functional single chain fragment variable (ScFv) moieties connected in tandem via (G4S)n linkers, such that penta-valence engagement of five individual epitopes on the HIV-l Env by each respective moiety is possible. The bNAb functional moieties incorporated in the design target five major HIV neutralization epitopes including the i) V1/V2 apex glycan, ii) V3- base glycan, iii) CD4 binding site, iv) gpl20/gp4l interface, and v) the membrane proximal external region (MPER). The sequence information of the penta-specific antibodies is listed in Table 4. In addition, these PentaScFv entities were fused to the fragment crystallizable region (Fc) region of IgGl to accommodate effector functions in a Penta-IgG format.
To design the PentaScFvs, the physical distance between the compatible N/C termini of parental bNAb variable heavy (VH) and light chain (VL) domains was estimated to identify the shortest linker combinations allowing optimal VH/VL connection within each bNAb ScFv entity. Furthermore, to generate molecules capable of simultaneously engaging (“draping” the HIV-l Env) contiguous epitopes on the HIV-l Env surface, the spatial relationship between each neighboring bNAb moiety in the state of cognate epitope binding was assessed. It was found that epitopes located on adjacent protomers of the HIV-l trimer are often more proximate to each other than those on the same protomer. Thus, in some designs, two functional bNAb moieties binding to the same protomer (intra-protomer mode) were arranged and in some cases to separate protomers (inter-protomers) of the HIV-l Env trimer. Finally, PentaNAbs with bNAb moieties connected from N to C terminus in two orientations were designed: from the MPER to V1V2 apex (forward order) or from the V1V2 apex to MPER (reverse order) followed by the Fc fragment to form PentaNAb IgGl molecules. The purpose of these designs is to minimize potential steric hindrance imposed by the addition of Fc fragment to the C terminus of the connected ScFv entities. PentaNAb2.0 was initially constructed, which consists of functional moieties of five bNAbs including 10E8n4 SlOOcF, 35022, N6, PGT121, and VRC26.25 to test the rationale. In this design, an molecular orientation was used, namely“Forward” orientation with which the MPER bNAb 10E8n4 SlOOcF moiety is at the N terminus of the PentaNAb and the VI V2 glycan bNAb VRC26.25 moiety on the distal side followed by Fc fragment.
ETsing a 208-virus panel representing the worldwide diverse circulating HIV-l virus isolates to assess neutralization breath, PentaNAb2.0, in both ScFv and IgG form, displayed improved virus isolate coverage of 99.6%, as compared to individual bNAbs 10E8, VRC01, PGT121 and 35022, which had 97.6%, 90.4%, 63.9%, and 45.2% coverage, respectively. Most significantly, the PentaNAb2.0, in both ScFv and IgG form, displayed significantly improved IC50 geomeans of 0.006 and 0.072 pg/mL, respectively, as compared to 10E8, which has an IC50 mean of 0.299 pg/mL with the best neutralization breadth among all known bNAbs. In addition, PentaNAb2.0 ScFv displays neutralization potency and breadth superior to previously engineered tri- and tetra- specific antibodies to HIV-l Env. Furthermore, PentaNAb2.0, in both ScFv and IgG form effectively neutralizes VRC01 -resistant replication-competent viral isolates from patients participated in the clinical trial involving passive transfer of VRC01. Finally, PentaNAbs were engineered with a“Reverse” orientation with which the VI V2 glycan bNAb VRC26.25 or PGDM1400 moiety is at the N terminus of the PentaNAb and the MPER bNAb 10E8n4 SlOOcF moiety on the distal side followed by Fc fragment. The neutralization capacities of these PentaNAb iterations were assessed with a panel of 26 HIV-l Env-pseudoviruses in TZM-bl target cells. It was found that all of the PentaNAbs in the ScFv format displayed a greater breadth and potency than their respective IgGl counterparts with Fc fragment. Additionally, it was observed that the PentaNAbs in the “reverse” order displayed a greater potency than the“forward” order while maintaining the same breadth. Taken together, penta-specific antibodies were engineered that are capable of engaging five major neutralization epitopes of HIV-l envelope glycoproteins possess superior antiviral activities to conventional HIV-l bNAbs and previously engineered tri- and tetra-specific HIV-l bNAbs, which may serve as effective agents for HIV-l prevention, treatment and diagnosis.
Results
Spatial relationships between HIV-l Env-specifw bNAb epitopes and the design of connecting linker length
To constitute PentaNAb molecules, five top lead antibodies displaying the best neutralization potency and breadth among bNAbs within each individual epitope cluster were first selected. For example, in one combination VRC26.25 or PGDM1400, PGT121, N6, 35022, and 10E8n4 V5R SlOOcF from the V1/V2, V3-glycan, CD4bs, gpl20/gp4l interface, and MPER epitope cluster, respectively were picked. In order to develop a PentaNAb capable of simultaneously engaging five separate epitopes, the physical distance between the compatible variable heavy (VH) and light (VL) chain region termini (e.g. VH C-terminus followed by N-terminus of VL) of the selected bNAbs was measured to identify the shortest combinations (Fig 2). The PentaNAb was designed such that the VH and VL of each individual bNAb entity was connected between termini with flexible linkers such that the flexible linker would traverse the shortest unimpeded distance possible. It is believed that joining the individual entities of the PentaNAb between the termini with the shortest unimpeded distance in between would avoid excess linker length, which would i) improve protein folding, ii) improve the expression level/yield of the molecule, and iii) improve the coordination of simultaneous engagement of all five epitopes.
Five G4S linkers were used to connect moieties of bNAbs targeting the VI V2 (e.g. VRC26.25) and V3 (e.g. PGT121) glycans. The distance between V1/V2 and the V3-glycan targeting antibodies VRC26.25 or PGDM1400 and PGT121, respectively, was not determined as crystallography data of gpl20 bound VRC26.25 or PGDM1400 is currently unavailable. Using the EMD data for VRC26.25 bound to BG505 SOSIP.664, EMD-5856 (Doria-Rose et al ., Nature 509, 55-62 (2014)), the crystal structure of the VRC26.25 Fab (PDB: 5DT1) (Doria-Rose et al. , J Virol 90, 76-91 (2015)), as well as the crystal structure of PGT122 bound BG505 SOSIP.664 (PDB: 5FYK) (Stewart- Jones et al., Cell 165, 813-826 (2016)), a model of VRC26.25 & PGT121 bound Env trimer were generated by manual superposition of BG505 trimer elements. This model was subsequently used to roughly estimate the spatial relationship between VRC26.25 and PGT121 with results similar to other modeling attempts using a combination of molecular dynamics (MD) and molecular dynamics flexible-fitting (MDFF) (Gorman et al ., Nat Struct Mol Biol 23, 81-90 (2016)). Similarly this EM model, EMD-5856 (Doria-Rose et al., Nature 509, 55-62 (2014)), was used to manually fit the crystal structure of the PGDM1400 Fab (PDB: 4RQQ) (Sok et al. , Proc Natl Acad Sci U S A 111, 17624-17629 (2014)), with the crystal structure of PGT122 bound BG505 SOSIP.664 (PDB: 5FYK) (Stewart- Jones et al. , Cell 165, 813-826 (2016)), to briefly estimate the spatial relationship between PGDM1400 and PGT121, since PGDM1400 approaches the gpl20 apex with an angle similar to VRC26.25. According to the EM data, both VRC26.25 and PGDM1400 are quaternary-dependent antibodies targeting the V1/V2 epitope at the trimer apex (Doria-Rose et al., J Virol 90, 76-91 (2015); Sok et al., Proc Natl Acad Sci El S A 111, 17624-17629 (2014)) (Fig 1), with VH/VL termini close to one another. Based on the above stated analysis, a linker of sufficient length (e.g. a linker with five G4S units) is utilized to connect VRC26.25 or PGDM1400 with PGT121.
Five G4S linkers were used to connect the V3-glycan targeting PGT121 and the CD4bs targeting N6 ScFv entities. Previous studies determined that the shortest distance between PGT122 (a surrogate for PGT121) and VRC01 (a surrogate for N6) is between adjacent protomers within an Env trimer (inter-protomer). Elsing the PDB model 5FYK (Stewart- Jones et al., Cell 165, 813-826 (2016)), determined the distances between the VRC01 and PGT121 functional variable domains as the following: 37 A between the C- terminus of VRC01 VH domain and the N-terminus of PGT121 VL domain with VRC0l(VL-VH)-PGTl2l(VL-VH) topology, and 53 A between the C-terminus of PGT121 VL and the N-terminus of VRC01 VH with PGTl2l(VH-VL)-VRC0l(VH-VL) topology (Figure 8A). Furthermore, negative stain electron microscopy data demonstrated that one of the Bi-ScFvs previously generated, dVRC0l(VL-VH)-5X-PGTl2l(VL-VH), was able to simultaneously engage both the PGT121 and VRC01 epitopes in an inter- protomer manner as we had predicted (Steinhardt et al., Nat Commun 9, 877 (2018)), which supports rationale for this linker design.
The shortest distance between the CD4bs targeting bNAb moiety, N6 (with VRC01 as surrogate), and the gpl20/gp4l interface targeting 35022 entity was determined to be an intra-protomer distance. Elsing the model in PDB 5FYK (Stewart- Jones et al ., Cell 165, 813-826 (2016)), the distance between the 35022 and N6 functional variable domains were determined to be 57 A between the C-terminus of 35022 VH domain and the N-terminus of N6 VL domain with the topology of 35022(VL-VH)-N6(VL-VH), and 47 A between the C-terminus of N6 VL and the N- terminus of 35022 VH with the topology of N6(VH-VL)-35022(VH-VL) (Figure 8B), respectively. Thus, five G4S linkers were used to connect N6 and 35022.
Finally, the shortest distances between the gpl20/gp4l interface targeting 35022 entity and the MPER targeting 10E8n4 entity was determined to be inter-protomer manner. LTsing PDB 5IQ7 superimposed with PDB 5FYK (Stewart- Jones et al ., Cell 165, 813-826 (2016); Kwon et al ., J Virol 90, 5899-5914 (2016)), a model for Env engaged with both 35022 and 10E8 was generated. In this model, the distance between the 10E8n4 and 35022 functional variable domains were determined to be 48 A between the C-terminus of 10E8n4 VH domain and the N-terminus of 35022 VL domain with the topology of l0E8v4(VL-VH)-35O22(VL-VH), and 25 A between the C-terminus of 35022 VL and the N-terminus of 10E8n4 VH with the topology of 35022(VH-VL)- lOE8v4(VH-VL) (Figure 8C), respectively. Therefore, five G4S linkers were used to connect 35022 and 10E8n4.
PentaNAbs display exceptional neutralization potency and breadth
Based on the analysis of spatial relationship between the five major bNAb epitopes on HIV-l Env surface, and the design of connecting linker length stated above, PentaNAb2.0 (Fig. 2, Table 5) was composed consisting of bNAb moieties of VRC26.25, PGT121, N6, 35022, and l0E8v4_Sl00cF from the V1/V2, V3-glycan, CD4bs, gpl20/gp4l interface, and MPER epitope cluster, respectively.
Figure imgf000106_0001
Table 5. Summary of PentaNAb names, order of individual antibody entities and orientation of variable light and variable heavy chains.
The PentaNAb2.0 -ScFvs and full length IgGs were expressed in mammalian 293FreeStyle cells and purified by Ni+ and protein A columns, respectively. All molecules were characterized by SDS-PAGE gels, size exclusion chromatography (SEC), and dynamic light scattering (DLS) (data not shown). Furthermore, the binding specificities of these constructs were validated by ELISA binding assay (not shown), and Bio-Layer Interferometry (BLI), which demonstrated binding to numerous Env ligands including the CD4bs specific RSC3 probe, an MPER peptide, and full length BG505 SOSIP.664 and JR-FL SOSIP.664 Env trimers (data not shown).
A comprehensive virus panel consisting of 208 pseudotype virus strains representing the worldwide diversified circulating HIV-l virus isolates was used to analyze and compare the PentaNAb2.0 ScFv and IgG with TetraNAb, TriNAb and BiNAb designs in a previous invention disclosure (PCT/ETS2017/057053), as well as prototypic bNAbs including 10E8, VRC01 and PGT121. Both PentaNAb2.0 ScFv and IgG displayed substantially greater breadth and higher potency than the prototypical bNAbs and BiNAbs, while PentaNAb2.0 ScFv displays potency higher than TetraNAb and TriNAb (Figs. 3A, 3B & 4) by approximately lO-fold. The PentaNAb2.0, in both ScFv and IgG form, displayed improved virus isolate coverage of 99.6%, as compared to bNAbs 10E8, VRC01 and PGT121, which had 97.6%, 90.4% and 63.9%, respectively. The PentaNAb2.0, in both ScFv and IgG form, displayed significantly improved IC50 geomeans of 0.006 and 0.072 pg/mL, respectively (Figs 3 & 4). Most significantly, with an IC50 mean of 0.006 pg/mL, the PentaNAb2.0 ScFv is the most potent HIV-l multiNAb tested to date by the comprehensive 208-virus panel.
Finally, when tested against VRC01 -resistant replication-competent viral isolates from patients participating in the clinical trial involving passive transfer of VRC01, the PentaNAb2.0 ScFv and IgG displayed neutralization capacity superior to previous multiNAb designs as well as the prototypical bNAbs (Fig 5A-5C). Taken together, the data suggest that the PentaNAb could serve as a multifunctional therapeutic agent template for the prevention and treatment of HIV-l infection, outperforming the conventional bNAbs and multi-specific bNAbs engineered previously. Optimized bNAb orientations enhance the potency ofPentaNAbs
PentaNAb2.0 was initially designed and purified in the“Forward” orientation (Fig 2). While PentaNAb2.0 ScFv shows superior potency to Tri- and Tetra-NAb designs, PentaNAb2.0 IgG with Fc fragment fused to the VI V2 glycan antibody moieties at the C terminus of the PentaNAb2.0 ScFv (Fig 2) shows potency virtually identical to Tri- and Tetra-NAb designs. We hypothesized that the steric hindrance imposed by the Fc fragment to the potent VI V2 glycan antibody moieties such as VRC26.25 may result in deleterious effect on neutralization potency.
To test this hypothesis, a series of Penta-ScFvs and full length IgGs (Fig 2, Fig 6) were generated, with combinations and permutations of VH-VL orientations and linker length for optimization (Table 4). Particularly, these designs include either i) the “Forward” orientation: l0E8v4(VL-VH)-35O22(VL-VH)-VRC0l(VL-VH)-5X-
PGTl2l(VL-VH)-VRC26.25/PGDMl400(VL-VH) -Fc whereby the Fc fragment connects with VRC26.25 or PGDM1400 (VI V2 glycan bNAb) and protrudes from the apex of the trimer (Fig 2), or ii) the“Reverse” orientation: VRC26.25/PGDMl400(VH- VL)-PGT 121 (VH-VL)- VRC01 (VH- VL)-35022(VH- VL)- 10E8v4(VH- VL)-Fc whereby the Fc fragment connects with 10E8n4 (MPER bNAb) and protrudes from the trimer MPER region (Fig 6A). Additionally,“knob into hole” technology (Ridgway el al ., Protein Eng 9, 617-621 (1996)) was utilized to generate a heterodimer of the“Forward” and “Reverse” PentaNAb arms for subsequent evaluation, with LS mutations (M428L/N434S) in the Fc fragment for elongated antibody in vivo half-lives as well as improved biodistribution into the mucosal compartment (Ko et al., Nature 514, 642-645 (2014)) (Fig 6B).
Three selected Penta-ScFvs and full length IgGs were expressed in mammalian 293FreeStyle cells and purified by Ni+ and protein A columns (Fig 7), respectively. All the proteins have expected molecular weight, as shown in representative SDS-PAGE gels (Fig 7A), size exclusion chromatography (SEC), and dynamic light scattering (DLS) analysis (data not shown). Furthermore, the binding specificities of these antibodies were validated by ELISA binding assay (not shown), and Bio-Layer Interferometry (BLI) (data not shown). To compare the neutralization potency and breadth of the Penta-ScFvs and IgGs in the“Forward”,“Reverse” and“Forward/Reverse” orientations, a virus panel of high stringency consisting of 26 tier-2 virus strains (Fig 7B & 7C) was used. Of the multiple iterations tested, it was found that the Penta-ScFv and Penta-IgG in the“Reverse” orientation displayed improved potency while maintaining the same breadth (virus coverage) as those in the“Forward” orientation (Fig 7B & 7C). Somewhat expectedly, the“Forward/Reverse” IgG orientation displayed a potency in between that of the “Forward” and“Reverse” IgG (Fig 7B & 7C). Thus, the“Reverse” orientation of Penta- ScFv and Penta-IgG could be applied to generate antiviral agent in the future.
Example 2. Design of Pentaspecific HIV antibodies
Advances in the treatment of human immunodeficiency virus (HIV) infection have led to dramatic improvements in health outcomes of infected individuals receiving antiretroviral therapy (ART) over the course of the last several decades. By suppressing plasma viremia with ART, life expectancy has approached that of uninfected individuals, turning what was once an almost uniformly fatal illness into a manageable chronic disease. Nonetheless, the vast majority of HIV-infected individuals must remain on continuous therapy given that virus inevitably rebounds following discontinuation of ART. Furthermore, current ART requires life-long adherence to daily medication regimens and is associated with significant costs, cumulative toxicities, and the potential for emergence of drug-resistant virus. Consequently, intense efforts have been made in recent years to develop therapeutic strategies aimed at achieving ART-free virol ogic remission in infected individuals. Among those, recent discoveries of a number of highly potent broadly neutralizing HIV-specific antibodies (bNAbs) isolated from B cells of viremic HIV-infected individuals have materialized as a basis for realistic and feasible approaches for HIV prevention and immunotherapy.
Recent clinical trials conducted to assess the safety, tolerability, and virologic efficacy of multiple infusions of several bNAbs have shown to be safe, well-tolerated, and exhibit antiretroviral activities against HIV. However, these human trials have concomitantly shown that therapies involving single bNAbs are unlikely to suppress plasma viremia for prolonged periods of time due to both pre-existing and emergent HIV mutations that are resistant to the antibody-mediated neutralization. Therefore, it is of great interest to develop a combination approach to increase the specificity and potency of bNAbs (or bNAb-like) molecules against a broad spectrum of infectious HIV in patients. In order to achieve this goal, we have developed synergistically combined functional epitope-binding moieties from five bNAbs that target the CD4 binding site, V2 and V3 conserved glycans, gpl20/gp4l interface, as well as the membrane exterior proximal region into a‘single’ penta-specific antibody (penta-NAb).
Thus far, our penta-Nabs exhibited superior degrees of HIV inhibition (neutralization of 99.5% of a 208 pseudo-typed HIV panel), exceptional neutralization capacity (IC50 at 0.006 pg/ml) compared to individual bNAbs, and the capacity to neutralize replication-competent HIV isolates from infected individuals whose virus became resistant to various single bNAbs inferring our penta-Nabs the potential to overcome immune-evasion and eliminate persistent HIV reservoirs.
In this example, it was proposed to:
1) generate multiple penta-Nabs by incorporating five candidate bNAbs exhibiting the highest levels of neutralization against >300 individual infectious viral isolates obtained from the persistent viral reservoir of infected individuals receiving ART.
2) engineer/modify the Fc region to extend the in vivo half-life of each penta-NAb.
3) generate several penta-bNAb/anti-CD3 antibody-based molecules that are capable of binding cell-surface Env and inducing CD8+ T cell-mediated killing of HIV reservoirs in vitro and in vivo.
4) measure the efficacy of penta-NAbs in NSG mice following administration of peripheral blood mononuclear cells (PBMCs) from ART-naive, HIV-infected individuals whose viral reservoirs carry sensitive and/or resistant virus to individual bNAbs.
5) evaluate the capacity of penta-bNAbs and DART molecules to prevent plasma viral rebound in NSG mice injected with from HIV-infected individuals following discontinuation of ART
The overall outcome of this study will provide the first proof of concept that a“5-in-l” antibody can achieve long-lasting broad-spectrum HIV inhibition in vivo, which will substantially advance our basic understanding of protective immunity against persistent virus infection and contribute to the development of safe and effective strategies toward ART-free virologic remission.
Results
A clinical trial was previously conducted to investigate whether a bNAb VRC01 could prevent plasma viral rebound in infected individuals upon discontinuation of ART. While multiple infusions of VRC01 were safe and well-tolerated, the majority of patients experienced plasma viral rebound due to pre-existing and emergent antibody resistant HIV, suggesting therapeutic strategies involving passive transfer of bNAbs may require a combination(s) of bNAbs that targets more than one region on HIV Env protein in order to achieve sustained virologic control in HIV-infected individuals upon withdrawal of ART. Thus, building upon the outcome of the data, it was proposed to test HIV Env- specific antibodies possessing multi-functional moieties with improved antigen binding and/or virus neutralization capacities, which may translate into superior protective efficacy in subsequent studies. The preliminary data, derived from five of the constructs, demonstrated the feasibility of the methodology and linkage to the studies proposed here. Taking the first step toward generating multi -epitope targeting bNAbs, it was sought to test if one could engineer bi-specific single-chain variable fragment (bi-ScFv) consisting of bNAbs VRCOl and PGT121. As shown in Fig. 9A, a series of VRC01-PGT121 bi- ScFvs and full length IgGs were generated with combinations and permutations of VH- VL orientations and linker length. It was shown using single molecule electron microscopy (EM) analysis that the VRC01-PGT121 bi-ScFv binds Env trimer in a bi- specific manner (Fig. 9B). The virus neutralization capacity of the bispecific molecules (Bi-BNAb) against a virus panel consisting of 208 primary virus isolates was evaluated and demonstrated -95% virus neutralization breadth, which is substantially expanded compared to that of the parent mAbs (VRCOl 90% and PGT121 64%).
Using the knob-into-hole technology, the VRC01-PGT121 Bi-NAb was combined with 1) the moiety of MPER mAb 10E8 to form Tri-NAb; and 2) another Bi-NAb consisting of gpl20/gp4l interface mAb and 10E8 in two different N-term to C-term orientations to form Tet-NAbl and 2. Finally, a scFV was created connecting variable regions of five bNAbs, namely penta-NAb, which engages all of the five conserved epitopes of HIV Env trimer (Fig. 2). Interestingly, Tri-NAb, Tet-NAbs and Penta-NAb demonstrate remarkably improved neutralization breadth and potency compared to the parental mAbs (Fig. 3A). The superior neutralization breadth and potency possessed by the multi-epitope targeting NAbs are likely caused by cooperative and sometimes synergistic effect. Thus, the engineered multi-epitope targeting NAbs, which target three to five out of the five major conserved HIV Env neutralizing epitopes, could achieve nearly perfect virus coverage with IC50 geometric mean below 0.1 pg/ml. Furthermore, the neutralization capacity of the Penta-scFV and Penta-Nab was tested, which display neutralization breadth and potency outperforming the parental monoclonal antibodies and combinations (Fig. 5C). These multi-NAbs possessing remarkably improved breadth and potency represent excellent functional modules for next generation candidate therapeutic molecules. In order to generate in vivo efficacy data, NSG mice will be utilized that are injected with PBMCs from HIV-infected viremic and aviremic individuals. As shown in Fig. 10, all 8 mice exhibited robust HIV replication following transfer of 8x106 PBMCs from an viremic individual who was ART -naive. These data demonstrate that the above animal system could be used to measure the efficacy of penta-NAbs and DART molecules against HIV derived from viral reservoirs from infected individuals who may carry sensitive and resistant HIV to certain bNAb.
Example 3
The neutralization profiles of PentaNAb2.0 Reverse ScFv, PentaNAb4.0 Forward ScFv (depicted in FIG. 11 A), and PentaNAb4.0 Reverse ScFv (depicted in FIG. 11B) were tested with 208-virus panel (FIG. 14A & B). It was confirmed that PentaNAb2.0 Reverse ScFv has incremental improvement in potency (IC 50 geometric mean) compared to the initial design, PentaNAb2.0 ScFv (FIG. 14A & B).
A new multi -specific antibody was constructed, namely HexaNAb 1.0 ScFv (FIG. 12), which contains PentaNAb4.0 Reverse ScFv and variable domains of dB4C7/UB-42l, an anti-CD4 antibody (N Engl J Med. 2019 Apr 18; 380(16): 1535-1545. PMID: 30995373; patent US7501494B2) to 1) enhance the antiviral breadth, and 2) potentially inhibit the emergence of resistant virus variants as demonstrated by a recent clinical trial described in this cited reference. The HexaNAb 1.0 ScFv shows 100% virus neutralization breadth and promising potency (IC50 geometric mean -0.005 ug/mL) (FIG. 14 A & B).
A new version of Penta-specific antibody in full-length IgG form was constructed (Fig. 13), namely PentaNAb4.0 Stem HC LS v2 IgG, expressed as an IgG assembled from a heavy chain and a light chain. With theM428L/N434S (LS) mutation in the Fc domain to elongate antibody in vivo half-life, the heavy chain consists of V2 bNAb PGDM1400, V3 bNAb PGT121, and the VH domain of CD4bs bNAb N6. The light chain contains the VL domain of N6 and 35022 and 10E8n4 SlOOcF.
The PentaNAb4.0 Stem HC LS v2 IgG demonstrates 99.5% virus coverage (207/208 viruses tested) and high potency (IC50 geometric mean -0.02 ug/mL) (FIG. 14A & B), which is comparable to the Sanofi Tri-specific antibodies, 10E8v4xPGDM 1400/VRC01 and l0E8v4xPGDMl400/N6-LS (FIG. 14B). The 5- specificity feature of the PentaNAb4.0 Stem HC LS v2 IgG may inhibit the emergence of neutralization resistant virus better than the other antibodies possessing less specificities (e.g. Tri-specific antibodies).
PentaNAb4.0 Reverse ScFv
[PGDM1400 (VH-3X-VL) ] -5X- [PGT121 (VH-3X-VL) ] -5X- [N6 (VH-3X-VL) ] -5X- [35022 (VH-3X-VL) ] -5X- [10E8v4 V5R SlOOcF (VH-3X-VL) ] -His
Amino acid sequence (SEQ ID NO: 90)
MGWSCIILFLVATATGVHSQAQLVQSGPEVRKPGTSVKVSCKAPGNTLKTYDLHWVRSVPGQGLQWMGWISH
EGDKKVIVERFKAKVTIDWDRSTNTAYLQLSGLTSGDTAVYYCAKGSKHRLRDYALYDDDGALNWAVDVDYL
SNLEFWGQGTAVTVSSGGGGSGGGGSGGGGSDFVLTQSPHSLSVTPGESASISCKSSHSLIHGDRNNYLAWY
VQKPGRSPQLLIYLASSRASGVPDRFSGSGSDKDFTLKISRVETEDVGTYYCMQGRESPWTFGQGTKVDIKG
GGGSGGGGSGGGGSGGGGSGGGGSQMQLQESGPGLVKPSETLSLTCSVSGASISDSYWSWIRRSPGKGLEWI
GYVHKSGDTNYSPSLKSRVNLSLDTSKNQVSLSLVAATAADSGKYYCARTLHGRRIYGIVAFNEWFTYFYMD
VWGNGTQVTVSSGGGGSGGGGSGGGGSSDISVAPGETARISCGEKSLGSRAVQWYQHRAGQAPSLIIYNNQD
RPSGIPERFSGSPDSPFGTTATLTITSVEAGDEADYYCHIWDSRVPTKWVFGGGTTLTVLGGGGSGGGGSGG
GGSGGGGSGGGGSRAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVN
FGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVWSAGGGGSGGGG
SGGGGSYIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSG
FHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIKGGGGSGGGGSGGGGSGGGGSGGGGSQGQLVQSGAE
LKKPGASVKISCKTSGYRFNFYHINWIRQTAGRGPEWMGWISPYSGDKNLAPAFQDRVIMTTDTEVPVTSFT
STGAAYMEIRNLKFDDTGTYFCAKGLLRDGSSTWLPYLWGQGTLLTVSSGGGGSGGGGSGGGGSQSVLTQSA
SVSGSLGQSVTISCTGPNSVCCSHKSISWYQWPPGRAPTLIIYEDNERAPGISPRFSGYKSYWSAYLTISDL
RPEDETTYYCCSYTHNSGCVFGTGTKVSVLGGGGSGGGGSGGGGSGGGGSGGGGSEVRLRESGGGLVKPGGS
LRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVR
TEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWGQGTLVIVSSGGGGSGGGGSGGGGSSELTQDPAVSVALKQ
TVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAEDEADYYCS
SRDKSGSRLSVFGGGTKLTVLGSGHHHHHH DNA sequence (SEQ ID NO.91)
ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCAACCGCAACAGGAGTGCACAGCCAGGCACAGCTGGTG CAGAGCGGACCCGAAGTGAGAAAACCTGGGACTAGCGTCAAAGTGTCATGTAAAGCCCCTGGAAATACCCTG AAGACCTACGATCTGCACTGGGTGCGGTCCGTGCCTGGACAGGGCCTGCAGTGGATGGGATGGATCTCTCAC GAGGGCGACAAGAAAGTGATCGTGGAGCGGTTCAAGGCCAAGGTGACAATCGATTGGGACAGATCCACCAAC ACAGCCTACCTGCAGCTGTCTGGCCTGACCAGCGGCGATACAGCCGTGTACTACTGTGCCAAGGGCTCTAAG CACCGGCTGAGAGACTACGCCCTGTATGACGATGACGGCGCCCTGAACTGGGCCGTGGATGTGGACTATCTG TCCAATCTGGAGTTCTGGGGACAGGGAACCGCAGTGACAGTGAGCTCCGGAGGAGGAGGCTCCGGCGGCGGA GGCTCTGGGGGAGGCGGCAGCGATTTTGTGCTGACCCAGTCTCCACACAGCCTGTCCGTGACACCCGGCGAG TCTGCCAGCATCTCCTGCAAGTCTAGCCACAGCCTGATCCACGGCGACAGGAACAATTACCTGGCCTGGTAC GTGCAGAAGCCAGGCCGCAGCCCTCAGCTGCTGATCTATCTGGCATCCTCTAGGGCCTCCGGAGTGCCAGAT CGCTTCTCTGGCAGCGGCTCCGATAAGGACTTTACCCTGAAGATCAGCCGGGTGGAGACAGAGGACGTGGGC ACATACTATTGTATGCAGGGCCGAGAATCACCTTGGACATTTGGGCAGGGAACTAAAGTTGACATCAAAGGG GGGGGGGGCTCCGGCGGCGGGGGCTCTGGCGGCGGCGGCAGCGGAGGAGGCGGCTCCGGAGGAGGCGGCTCT CAGATGCAGCTGCAGGAGAGCGGACCAGGACTGGTGAAGCCTTCCGAGACCCTGTCTCTGACATGTTCTGTG AGCGGCGCCTCCATCTCTGATAGCTACTGGAGCTGGATCAGACGGAGCCCTGGCAAGGGCCTGGAGTGGATC GGCTACGTGCACAAGTCTGGCGATACAAACTATTCCCCATCTCTGAAGAGCCGGGTGAACCTGAGCCTGGAC ACCTCCAAGAATCAGGTGAGCCTGTCCCTGGTGGCAGCAACCGCAGCAGACAGCGGCAAGTACTATTGCGCC AGAACACTGCACGGCAGGCGCATCTACGGCATCGTGGCCTTTAACGAGTGGTTCACCTACTTTTATATGGAC GTGTGGGGCAATGGCACCCAGGTGACAGTGTCCTCTGGCGGCGGCGGCTCTGGCGGAGGAGGCAGCGGAGGA GGAGGCAGCTCCGACATCTCTGTGGCACCTGGAGAGACCGCAAGGATCAGCTGTGGAGAGAAGTCTCTGGGC AGCAGGGCCGTGCAGTGGTACCAGCACAGGGCAGGACAGGCACCATCCCTGATCATCTATAACAATCAGGAC CGGCCATCTGGCATCCCCGAGAGATTCTCTGGCAGCCCCGATAGCCCTTTTGGCACCACAGCCACCCTGACA ATCACCTCCGTGGAGGCCGGCGACGAAGCAGATTACTATTGCCACATCTGGGACTCCAGAGTGCCAACCAAG TGGGTGTTCGGAGGAGGAACCACACTGACAGTGCTGGGCGGCGGAGGCTCCGGCGGGGGCGGCTCTGGAGGC GGCGGCAGCGGAGGGGGCGGCTCCGGCGGCGGCGGCTCTAGGGCACACCTGGTGCAGAGCGGAACCGCAATG AAGAAGCCTGGCGCCTCTGTGCGCGTGAGCTGTCAGACATCCGGCTACACCTTCACCGCCCACATCCTGTTC TGGTTTAGGCAGGCACCAGGAAGAGGACTGGAGTGGGTGGGCTGGATCAAGCCCCAGTATGGAGCAGTGAAC TTCGGAGGAGGCTTTCGGGACAGAGTGACACTGACCCGGGACGTGTACAGAGAGATCGCCTATATGGATATC AGGGGCCTGAAGCCCGACGATACCGCCGTGTACTATTGCGCCAGGGACCGCTCCTACGGCGATTCTAGCTGG GCACTGGACGCATGGGGACAGGGAACCACAGTGGTGGTGAGCGCCGGAGGCGGGGGCAGCGGCGGCGGGGGC TCCGGAGGCGGAGGCTCTTACATCCACGTGACCCAGTCCCCTTCCTCTCTGTCCGTGTCTATCGGCGATCGC GTGACCATCAACTGTCAGACAAGCCAGGGAGTGGGCTCCGACCTGCACTGGTATCAGCACAAGCCTGGCAGG GCCCCAAAGCTGCTGATCCACCACACAAGCTCCGTGGAGGATGGAGTGCCAAGCCGCTTCAGCGGCTCCGGA TTCCACACCAGCTTTAATCTGACAATCTCCGACCTGCAGGCCGACGATATCGCCACCTACTATTGCCAGGTG CTGCAGTTCTTTGGCAGGGGCTCCCGCCTGCACATCAAGGGCGGCGGCGGCTCTGGGGGCGGGGGCAGCGGC GGGGGGGGCTCCGGGGGAGGAGGCTCTGGCGGAGGGGGCAGCCAGGGCCAGCTGGTGCAGAGCGGAGCAGAG CTGAAGAAGCCAGGAGCCTCTGTGAAGATCAGCTGTAAGACATCCGGCTATCGGTTCAACTTTTATCACATC AATTGGATCAGGCAGACCGCAGGAAGGGGACCAGAGTGGATGGGCTGGATCTCCCCCTACTCTGGCGATAAG AACCTGGCCCCAGCCTTCCAGGACAGAGTGATCATGACCACAGATACCGAGGTGCCAGTGACCAGCTTCACC TCCACCGGAGCCGCCTACATGGAGATCCGGAATCTGAAGTTCGACGATACAGGCACCTATTTTTGCGCCAAG GGCCTGCTGAGAGACGGCTCTAGCACATGGCTGCCATACCTGTGGGGACAGGGCACCCTGCTGACAGTGTCC TCTGGAGGAGGAGGCTCCGGGGGCGGCGGCTCTGGAGGAGGAGGCTCTCAAAGCGTGCTGACCCAGTCCGCC TCTGTGAGCGGCTCCCTGGGCCAGTCTGTGACCATCAGCTGTACAGGCCCCAACTCCGTGTGCTGTTCTCAC AAGTCTATCAGCTGGTACCAGTGGCCACCAGGAAGGGCACCTACCCTGATCATCTATGAGGACAATGAGAGG GCACCAGGAATCAGCCCTCGCTTCTCCGGCTACAAGTCTTATTGGAGCGCCTACCTGACCATTTCCGACCTG CGCCCCGAGGATGAGACCACATACTATTGCTGTAGCTATACCCACAACTCCGGCTGCGTGTTTGGCACAGGC ACCAAGGTGAGCGTGCTGGGAGGAGGGGGCTCTGGCGGCGGGGGCAGCGGCGGAGGCGGCTCCGGAGGGGGC GGCTCTGGCGGAGGCGGCAGCGAGGTGCGGCTGCGGGAGAGCGGCGGCGGCCTGGTGAAGCCAGGCGGCTCT CTGAGACTGTCCTGTTCTGCCAGCGGCTTCGACTTTGATAATGCCTGGATGACATGGGTGCGGCAGCCTCCT GGCAAGGGGCTGGAGTGGGTGGGAAGAATCACCGGACCAGGAGAGGGATGGTCTGTGGACTACGCCGAGAGC GTGAAGGGCCGGTTCACCATCAGCAGAGATAACACTAAAAATACACTGTATCTGGAGATGAACAATGTGCGG ACCGAGGACACAGGCTACTATTTCTGCGCCAGAACCGGCAAGTACTATGATTTCTGGTTTGGCTACCCCCCT GGCGAGGAGTATTTTCAGGACTGGGGCCAGGGCACCCTGGTCATCGTGAGCAGCGGCGGGGGAGGCTCCGGC GGGGGGGGCTCTGGAGGAGGGGGCTCTAGCGAGCTGACCCAGGACCCCGCCGTGTCCGTGGCCCTGAAGCAG ACAGTGACCATCACATGCAGGGGCGACTCCCTGCGCTCTCACTACGCCAGCTGGTATCAGAAGAAGCCAGGA CAGGCACCCGTGCTGCTGTTCTACGGCAAGAACAATCGGCCTTCCGGCATCCCAGATAGATTTTCCGGCTCT GCCAGCGGAAACAGGGCCAGCCTGACCATCACAGGAGCACAGGCAGAGGATGAAGCAGATTACTATTGTTCC TCTCGGGACAAGTCCGGCTCTAGACTGAGCGTGTTCGGCGGCGGAACCAAGCTGACAGTGCTGGGATCCGGT CACCACCATCACCACCACTAG
HexaNAbl .0 Reverse ScFv
[dB4C7-UB-421 (VL-3X-VH) ] -5X- [PGDM1400 (VH-3X-VL) ] -5X- [PGT121 (VH-3X-VL) ] - 5X- [N6 (VH-3X-VL) ] -5X- [35022 (VH-3X-VL) ] -5X- [10E8v4 V5R SlOOcF (VH-3X-VL) ] - His
Amino acid sequence (SEQ ID NO: 92)
MGWSCIILFLVATATGVHSDIVLTQSPASLAVSLGQRATITCKAGQSVDYDGDSYMNWYQQKPGQPPKLLIY
VASNLESGIPARFSGSGSGTDFTLNIHPVEENDAATYYCQQSYKDPLTFGQGTKLEIKGGGGSGGGGSGGGG
SQVQLVQSGPELKKPGASVKVSCKASGYTFTDYVIHWVKQATGQGLEWIGEIYPGSGSAYSNAKFKDRVTMT
ADKSSNTAYMELSSLTSDDTAVYFCARRGNGTGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGS
QAQLVQSGPEVRKPGTSVKVSCKAPGNTLKTYDLHWVRSVPGQGLQWMGWISHEGDKKVIVERFKAKVTIDW
DRSTNTAYLQLSGLTSGDTAVYYCAKGSKHRLRDYALYDDDGALNWAVDVDYLSNLEFWGQGTAVTVSSGGG
GSGGGGSGGGGSDFVLTQSPHSLSVTPGESASISCKSSHSLIHGDRNNYLAWYVQKPGRSPQLLIYLASSRA
SGVPDRFSGSGSDKDFTLKISRVETEDVGTYYCMQGRESPWTFGQGTKVDIKGGGGSGGGGSGGGGSGGGGS
GGGGSQMQLQESGPGLVKPSETLSLTCSVSGASISDSYWSWIRRSPGKGLEWIGYVHKSGDTNYSPSLKSRV
NLSLDTSKNQVSLSLVAATAADSGKYYCARTLHGRRIYGIVAFNEWFTYFYMDVWGNGTQVTVSSGGGGSGG
GGSGGGGSSDISVAPGETARISCGEKSLGSRAVQWYQHRAGQAPSLIIYNNQDRPSGIPERFSGSPDSPFGT
TATLTITSVEAGDEADYYCHIWDSRVPTKWVFGGGTTLTVLGGGGSGGGGSGGGGSGGGGSGGGGSRAHLVQ
SGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAWFGGGFRDRVTLTRDVYREI
AYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVWSAGGGGSGGGGSGGGGSYIHVTQSPSSLSV
SIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHTSFNLTISDLQADDIAT
YYCQVLQFFGRGSRLHIKGGGGSGGGGSGGGGSGGGGSGGGGSQGQLVQSGAELKKPGASVKISCKTSGYRF
NFYHINWIRQTAGRGPEWMGWISPYSGDKNLAPAFQDRVIMTTDTEVPVTSFTSTGAAYMEIRNLKFDDTGT
YFCAKGLLRDGSSTWLPYLWGQGTLLTVSSGGGGSGGGGSGGGGSQSVLTQSASVSGSLGQSVTISCTGPNS
VCCSHKSISWYQWPPGRAPTLIIYEDNERAPGISPRFSGYKSYWSAYLTISDLRPEDETTYYCCSYTHNSGC
VFGTGTKVSVLGGGGSGGGGSGGGGSGGGGSGGGGSEVRLRESGGGLVKPGGSLRLSCSASGFDFDNAWMTW
VRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFW
FGYPPGEEYFQDWGQGTLVIVSSGGGGSGGGGSGGGGSSELTQDPAVSVALKQTVTITCRGDSLRSHYASWY
QKKPGQAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLT
VLGSGHHHHHH
DNA sequence (SEQ ID NO: 93)
ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCAACCGCAACAGGAGTGCACAGCGACATCGTGCTGACC CAGTCTCCTGCCAGCCTGGCCGTGAGCCTGGGACAGAGGGCCACCATCACATGCAAGGCCGGCCAGTCTGTG GACTACGATGGCGACAGCTACATGAACTGGTATCAGCAGAAGCCAGGCCAGCCCCCTAAGCTGCTGATCTAT GTGGCCTCCAATCTGGAGTCTGGCATCCCTGCACGCTTCAGCGGCTCCGGCTCTGGAACCGACTTCACCCTG AACATCCACCCAGTGGAGGAGAATGACGCCGCCACCTACTATTGCCAGCAGAGCTACAAGGACCCCCTGACC TTCGGCCAGGGCACAAAGCTGGAGATCAAGGGAGGAGGAGGCAGCGGGGGAGGAGGCTCCGGAGGCGGCGGC TCTCAGGTGCAGCTGGTGCAGTCCGGACCAGAGCTGAAGAAGCCAGGAGCCAGCGTGAAGGTGTCCTGTAAG GCCTCTGGCTACACCTTCACAGATTATGTGATCCACTGGGTGAAGCAGGCAACAGGACAGGGCCTGGAGTGG ATCGGAGAGATCTACCCAGGCAGCGGCTCCGCCTATTCCAACGCCAAGTTTAAGGATCGGGTGACCATGACA GCCGACAAGAGCTCCAATACCGCCTATATGGAGCTGTCTAGCCTGACCTCTGACGATACAGCCGTGTACTTC TGTGCCCGGAGAGGCAACGGCACAGGCTTTGCCTATTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCTGGA GGAGGAGGCAGCGGCGGCGGCGGCTCCGGAGGAGGCGGCTCTGGCGGCGGCGGCAGCGGAGGAGGCGGCTCC CAGGCACAGCTGGTGCAGAGCGGACCCGAAGTGAGAAAACCTGGGACTAGCGTCAAAGTGTCATGTAAAGCC CCTGGAAATACCCTGAAGACCTACGATCTGCACTGGGTGCGGTCCGTGCCTGGACAGGGCCTGCAGTGGATG GGATGGATCTCTCACGAGGGCGACAAGAAAGTGATCGTGGAGCGGTTCAAGGCCAAGGTGACAATCGATTGG GACAGATCCACCAACACAGCCTACCTGCAGCTGTCTGGCCTGACCAGCGGCGATACAGCCGTGTACTACTGT GCCAAGGGCTCTAAGCACCGGCTGAGAGACTACGCCCTGTATGACGATGACGGCGCCCTGAACT GGGCCGTG GATGTGGACTATCTGTCCAATCTGGAGTTCTGGGGACAGGGAACCGCAGTGACAGTGAGCTCCGGAGGAGGA GGCTCCGGCGGCGGAGGCTCTGGGGGAGGCGGCAGCGATTTTGTGCTGACCCAGTCTCCACACAGCCTGTCC GTGACACCCGGCGAGTCTGCCAGCATCTCCTGCAAGTCTAGCCACAGCCTGATCCACGGCGACAGGAACAAT TACCTGGCCTGGTACGTGCAGAAGCCAGGCCGCAGCCCTCAGCTGCTGATCTATCTGGCATCCT CTAGGGCC TCCGGAGTGCCAGATCGCTTCTCTGGCAGCGGCTCCGATAAGGACTTTACCCTGAAGATCAGCCGGGTGGAG AC AGAG GAC GT G G G C AC AT AC TAT T GT AT G C AG G G C C GAGAAT C AC C T T G GAC AT T T G G G C AG G GAAC T AAA GTTGACATCAAAGGGGGGGGGGGCTCCGGCGGCGGGGGCTCTGGCGGCGGCGGCAGCGGAGGAGGCGGCTCC GGAGGAGGCGGCTCTCAGATGCAGCTGCAGGAGAGCGGACCAGGACTGGTGAAGCCTTCCGAGACCCTGTCT CTGACATGTTCTGTGAGCGGCGCCTCCATCTCTGATAGCTACTGGAGCTGGATCAGACGGAGCCCTGGCAAG GGCCTGGAGTGGATCGGCTACGTGCACAAGTCTGGCGATACAAACTATTCCCCATCTCTGAAGAGCCGGGTG AACCTGAGCCTGGACACCTCCAAGAATCAGGTGAGCCTGTCCCTGGTGGCAGCAACCGCAGCAGACAGCGGC AAGTACTATTGCGCCAGAACACTGCACGGCAGGCGCATCTACGGCATCGTGGCCTTTAACGAGT GGTTCACC TACTTTTATATGGACGTGTGGGGCAATGGCACCCAGGTGACAGTGTCCTCTGGCGGCGGCGGCT CTGGCGGA GGAGGCAGCGGAGGAGGAGGCAGCTCCGACATCTCTGTGGCACCTGGAGAGACCGCAAGGATCAGCTGTGGA GAGAAGTCTCTGGGCAGCAGGGCCGTGCAGTGGTACCAGCACAGGGCAGGACAGGCACCATCCCTGATCATC TATAACAATCAGGACCGGCCATCTGGCATCCCCGAGAGATTCTCTGGCAGCCCCGATAGCCCTTTTGGCACC ACAGCCACCCTGACAATCACCTCCGTGGAGGCCGGCGACGAAGCAGATTACTATTGCCACATCT GGGACTCC AGAGTGCCAACCAAGTGGGTGTTCGGAGGAGGAACCACACTGACAGTGCTGGGCGGCGGAGGCT CCGGCGGG GGCGGCTCTGGAGGCGGCGGCAGCGGAGGGGGCGGCTCCGGCGGCGGCGGCTCTAGGGCACACCTGGTGCAG AGCGGAACCGCAATGAAGAAGCCTGGCGCCTCTGTGCGCGTGAGCTGTCAGACATCCGGCTACACCTTCACC GCCCACATCCTGTTCTGGTTTAGGCAGGCACCAGGAAGAGGACTGGAGTGGGTGGGCTGGATCAAGCCCCAG TATGGAGCAGTGAACTTCGGAGGAGGCTTTCGGGACAGAGTGACACTGACCCGGGACGTGTACAGAGAGATC GCCTATATGGATATCAGGGGCCTGAAGCCCGACGATACCGCCGTGTACTATTGCGCCAGGGACCGCTCCTAC GGCGATTCTAGCTGGGCACTGGACGCATGGGGACAGGGAACCACAGTGGTGGTGAGCGCCGGAGGCGGGGGC AGCGGCGGCGGGGGCTCCGGAGGCGGAGGCTCTTACATCCACGTGACCCAGTCCCCTTCCTCTCTGTCCGTG TCTATCGGCGATCGCGTGACCATCAACTGTCAGACAAGCCAGGGAGTGGGCTCCGACCTGCACT GGTATCAG CACAAGCCTGGCAGGGCCCCAAAGCTGCTGATCCACCACACAAGCTCCGTGGAGGATGGAGTGCCAAGCCGC TTCAGCGGCTCCGGATTCCACACCAGCTTTAATCTGACAATCTCCGACCTGCAGGCCGACGATATCGCCACC TACTATTGCCAGGTGCTGCAGTTCTTTGGCAGGGGCTCCCGCCTGCACATCAAGGGCGGCGGCGGCTCTGGG GGCGGGGGCAGCGGCGGGGGGGGCTCCGGGGGAGGAGGCTCTGGCGGAGGGGGCAGCCAGGGCCAGCTGGTG CAGAGCGGAGCAGAGCTGAAGAAGCCAGGAGCCTCTGTGAAGATCAGCTGTAAGACATCCGGCTATCGGTTC AACTTTTATCACATCAATTGGATCAGGCAGACCGCAGGAAGGGGACCAGAGTGGATGGGCTGGATCTCCCCC TACTCTGGC GAT AAGAAC C T G G C C C C AG C C T T C C AG GAC AGAGT GAT CAT GAC C AC AGAT AC C G AG GT G C C A GTGACCAGCTTCACCTCCACCGGAGCCGCCTACATGGAGATCCGGAATCTGAAGTTCGACGATACAGGCACC TATTTTTGCGCCAAGGGCCTGCTGAGAGACGGCTCTAGCACATGGCTGCCATACCTGTGGGGACAGGGCACC CTGCTGACAGTGTCCTCTGGAGGAGGAGGCTCCGGGGGCGGCGGCTCTGGAGGAGGAGGCTCTCAAAGCGTG CTGACCCAGTCCGCCTCTGTGAGCGGCTCCCTGGGCCAGTCTGTGACCATCAGCTGTACAGGCCCCAACTCC GTGTGCTGTTCTCACAAGTCTATCAGCTGGTACCAGTGGCCACCAGGAAGGGCACCTACCCTGATCATCTAT GAGGACAATGAGAGGGCACCAGGAATCAGCCCTCGCTTCTCCGGCTACAAGTCTTATTGGAGCGCCTACCTG ACCATTTCCGACCTGCGCCCCGAGGATGAGACCACATACTATTGCTGTAGCTATACCCACAACT CCGGCTGC GTGTTTGGCACAGGCACCAAGGTGAGCGTGCTGGGAGGAGGGGGCTCTGGCGGCGGGGGCAGCGGCGGAGGC GGCTCCGGAGGGGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGCGGCTGCGGGAGAGCGGCGGCGGCCTGGTG AAGCCAGGCGGCTCTCTGAGACTGTCCTGTTCTGCCAGCGGCTTCGACTTTGATAATGCCTGGATGACATGG GTGCGGCAGCCTCCTGGCAAGGGGCTGGAGTGGGTGGGAAGAATCACCGGACCAGGAGAGGGAT GGTCTGTG GAC T AC G C C GAGAG C GT GAAG GGCCGGTT C AC CAT C AG C AGAGAT AAC AC T AAAAAT AC AC T GT AT C T G GAG ATGAACAATGTGCGGACCGAGGACACAGGCTACTATTTCTGCGCCAGAACCGGCAAGTACTATGATTTCTGG TTTGGCTACCCCCCTGGCGAGGAGTATTTTCAGGACTGGGGCCAGGGCACCCTGGTCATCGTGAGCAGCGGC GGGGGAGGCTCCGGCGGGGGGGGCTCTGGAGGAGGGGGCTCTAGCGAGCTGACCCAGGACCCCGCCGTGTCC GTGGCCCTGAAGCAGACAGTGACCATCACATGCAGGGGCGACTCCCTGCGCTCTCACTACGCCAGCTGGTAT CAGAAGAAGCCAGGACAGGCACCCGTGCTGCTGTTCTACGGCAAGAACAATCGGCCTTCCGGCATCCCAGAT AGAT TTTCCGGCTCTGC C AG C G GAAAC AG G G C C AG C C T GAC CAT C AC AG GAG C AC AG G C AGAG GAT GAAG C A GATTACTATTGTTCCTCTCGGGACAAGTCCGGCTCTAGACTGAGCGTGTTCGGCGGCGGAACCAAGCTGACA GTGCTGGGATCCGGTCACCACCATCACCACCACTAG
PentaNAb4.0 Stem HC LS_v2
HC: PGDM1400-5X-PGT121-5X-N6 HC
[PGDM1400 (VH-3X-VL) ] -5X- [PGT121 (VH-3X-VL) ] -5X- [N6 (VH) ] -CH1-CH2-CH3
Amino acid sequence (SEQ ID NO: 94)
MGWSCIILFLVATATGVHSQAQLVQSGPEVRKPGTSVKVSCKAPGNTLKTYDLHWVRSVPGQGLQWMGWISH
EGDKKVIVERFKAKVTIDWDRSTNTAYLQLSGLTSGDTAVYYCAKGSKHRLRDYALYDDDGALNWAVDVDYL
SNLEFWGQGTAVTVSSGGGGSGGGGSGGGGSDFVLTQSPHSLSVTPGESASISCKSSHSLIHGDRNNYLAWY
VQKPGRSPQLLIYLASSRASGVPDRFSGSGSDKDFTLKISRVETEDVGTYYCMQGRESPWTFGQGTKVDIKG
GGGSGGGGSGGGGSGGGGSGGGGSQMQLQESGPGLVKPSETLSLTCSVSGASISDSYWSWIRRSPGKGLEWI
GYVHKSGDTNYSPSLKSRWLSLDTSKNQVSLSLVAATAADSGKYYCARTLHGRRIYGIVAFNEWFTYFYMD
VWGNGTQVTVSSGGGGSGGGGSGGGGSSDISVAPGETARISCGEKSLGSRAVQWYQHRAGQAPSLIIYNNQD
RPSGIPERFSGSPDSPFGTTATLTITSVEAGDEADYYCHIWDSRVPTKWVFGGGTTLTVLGGGGSGGGGSGG
GGSGGGGSGGGGSRAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAW
FGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYYCARDRSYGDSSWALDAWGQGTTVWSAASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTY
ICNWHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK
DNA sequence (SEQ ID NO: 95)
ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCAACCGCAACCGGTGTGCACAGCCAGGCACAGCTGGTG CAGAGCGGACCCGAAGTGAGAAAACCTGGGACTAGCGTCAAAGTGTCATGTAAAGCCCCTGGAAATACCCTG AAGACCTACGATCTGCACTGGGTGCGGTCCGTGCCTGGACAGGGCCTGCAGTGGATGGGATGGATCTCTCAC GAGGGCGACAAGAAAGTGATCGTGGAGCGGTTCAAGGCCAAGGTGACAATCGATTGGGACAGATCCACCAAC ACAGCCTACCTGCAGCTGTCTGGCCTGACCAGCGGCGATACAGCCGTGTACTACTGTGCCAAGGGCTCTAAG CACCGGCTGAGAGACTACGCCCTGTATGACGATGACGGCGCCCTGAACTGGGCCGTGGATGTGGACTATCTG TCCAATCTGGAGTTCTGGGGACAGGGAACCGCAGTGACAGTGAGCTCCGGAGGAGGAGGCTCCGGCGGCGGA GGCTCTGGGGGAGGCGGCAGCGATTTTGTGCTGACCCAGTCTCCACACAGCCTGTCCGTGACACCCGGCGAG TCTGCCAGCATCTCCTGCAAGTCTAGCCACAGCCTGATCCACGGCGACAGGAACAATTACCTGGCCTGGTAC GTGCAGAAGCCAGGCCGCAGCCCTCAGCTGCTGATCTATCTGGCATCCTCTAGGGCCTCCGGAGTGCCAGAT CGCTTCTCTGGCAGCGGCTCCGATAAGGACTTTACCCTGAAGATCAGCCGGGTGGAGACAGAGGACGTGGGC ACATACTATTGTATGCAGGGCCGAGAATCACCTTGGACATTTGGGCAGGGAACTAAAGTTGACATCAAAGGG GGGGGGGGCTCCGGCGGCGGGGGCTCTGGCGGCGGCGGCAGCGGAGGAGGCGGCTCCGGAGGAGGCGGCTCT CAGATGCAGCTGCAGGAGAGCGGACCAGGACTGGTGAAGCCTTCCGAGACCCTGTCTCTGACATGTTCTGTG AGCGGCGCCTCCATCTCTGATAGCTACTGGAGCTGGATCAGACGGAGCCCTGGCAAGGGCCTGGAGTGGATC GGCTACGTGCACAAGTCTGGCGATACAAACTATTCCCCATCTCTGAAGAGCCGGGTGAACCTGAGCCTGGAC ACCTCCAAGAATCAGGTGAGCCTGTCCCTGGTGGCAGCAACCGCAGCAGACAGCGGCAAGTACTATTGCGCC AGAACACTGCACGGCAGGCGCATCTACGGCATCGTGGCCTTTAACGAGTGGTTCACCTACTTTTATATGGAC GTGTGGGGCAATGGCACCCAGGTGACAGTGTCCTCTGGCGGCGGCGGCTCTGGCGGAGGAGGCAGCGGAGGA GGAGGCAGCTCCGACATCTCTGTGGCACCTGGAGAGACCGCAAGGATCAGCTGTGGAGAGAAGTCTCTGGGC AGCAGGGCCGTGCAGTGGTACCAGCACAGGGCAGGACAGGCACCATCCCTGATCATCTATAACAATCAGGAC CGGCCATCTGGCATCCCCGAGAGATTCTCTGGCAGCCCCGATAGCCCTTTTGGCACCACAGCCACCCTGACA ATCACCTCCGTGGAGGCCGGCGACGAAGCAGATTACTATTGCCACATCTGGGACTCCAGAGTGCCAACCAAG TGGGTGTTCGGAGGAGGAACCACACTGACAGTGCTGGGCGGCGGAGGCTCCGGCGGGGGCGGCTCTGGAGGC GGCGGCAGCGGAGGGGGCGGCTCCGGCGGCGGCGGCTCTAGGGCACACCTGGTGCAGAGCGGAACCGCAATG AAGAAGCCTGGCGCCTCTGTGCGCGTGAGCTGTCAGACATCCGGCTACACCTTCACCGCCCACATCCTGTTC TGGTTTAGGCAGGCACCAGGAAGAGGACTGGAGTGGGTGGGCTGGATCAAGCCCCAGTATGGAGCAGTGAAC TTCGGAGGAGGCTTTCGGGACAGAGTGACACTGACCCGGGACGTGTACAGAGAGATCGCCTATATGGATATC AGGGGCCTGAAGCCCGACGATACCGCCGTGTACTATTGCGCCAGGGACCGCTCCTACGGCGATTCTAGCTGG GCACTGGACGCATGGGGACAGGGAACCACAGTGGTGGTGAGCGCCGCGTCGACCAAGGGCCCATCGGTCTTC CCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTC CCCGAACCTGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTA CAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTAC ATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAA ACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAA CCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGAC CCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAG TACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAG CCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACC TGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAAC TACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAG AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGCTGCATGAGGCTCTGCACAGCCACTACACGCAG AAGAGCCTCTCCCTGTCTCCGGGTAAATGA
LC: N6 KC-CK-35O22-5X-10E8v4 V5R SlOOcF
[N6 (VL) ] -CL-5X- [35022 (VH-3X-VL) ] -5X- [10E8v4 SlOOcF (VH-3X-VL) ]
Amino acid sequence (SEQ ID NO: 96)
MGWSCIILFLVATATGVHSYIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSS
VEDGVPSRFSGSGFHTSFNLTISDLQADDIATYYCQVLQFFGRGSRLHIKRTVAAPSVFIFPPSDEQLKSGT
ASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGSGGGGSQGQLVQSGAELKKPGASVKISCKTSGYRFNFYHI
NWIRQTAGRGPEWMGWISPYSGDKNLAPAFQDRVIMTTDTEVPVTSFTSTGAAYMEIRNLKFDDTGTYFCAK
GLLRDGSSTWLPYLWGQGTLLTVSSGGGGSGGGGSGGGGSQSVLTQSASVSGSLGQSVTISCTGPNSVCCSH
KSISWYQWPPGRAPTLIIYEDNERAPGISPRFSGYKSYWSAYLTISDLRPEDETTYYCCSYTHNSGCVFGTG
TKVSVLGGGGSGGGGSGGGGSGGGGSGGGGSEVRLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPP
GKGLEWVGRITGPGEGWSVDYAESVKGRFTISRDNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPP
GEEYFQDWGQGTLVIVSSGGGGSGGGGSGGGGSSELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPG
QAPVLLFYGKNNRPSGIPDRFSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVL
DNA sequence (SEQ ID NO: 97)
ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCAACCGCAACAGGAGTGCACAGCTACATCCAC GTGACCCAGTCCCCTTCCTCTCTGTCCGTGTCTATCGGCGATCGCGTGACCATCAACTGTCAGACAAGCCAG GGAGTGGGCTCCGACCTGCACTGGTATCAGCACAAGCCTGGCAGGGCCCCAAAGCTGCTGATCCACCACACA AGCTCCGTGGAGGATGGAGTGCCAAGCCGCTTCAGCGGCTCCGGATTCCACACCAGCTTTAATCTGACAATC TCCGACCTGCAGGCCGACGATATCGCCACCTACTATTGCCAGGTGCTGCAGTTCTTTGGCAGGGGCTCCCGC CTGCACATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGAT AACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTC AGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAG
GGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGGAGGAGGGGGCTCTGGCGGCGGGGGC AGCGGCGGAGGCGGCTCCGGAGGGGGCGGCTCTGGCGGAGGCGGCAGCCAGGGCCAGCTGGTGCAGAGCGGA GCAGAGCTGAAGAAGCCAGGAGCCTCTGTGAAGATCAGCTGTAAGACATCCGGCTACCGGTTCAACTTTTAT CACATCAATTGGATCAGGCAGACCGCAGGAAGGGGACCAGAGTGGATGGGCTGGATCTCCCCCTACTCTGGC GAT AAGAAC C T G G C C C C AG C C T T C C AG GAC AGAGT GAT CAT GAC C AC AGAT AC C GAG GT G C C AG T GAC C AG C TTCACCTCCACCGGAGCCGCCTACATGGAGATCCGGAATCTGAAGTTCGACGATACAGGCACCTATTTTTGC GCCAAGGGCCTGCTGAGAGACGGCTCTAGCACATGGCTGCCATACCTGTGGGGACAGGGCACCCTGCTGACA GTGTCCTCTGGAGGAGGAGGCTCCGGGGGCGGCGGCTCTGGAGGAGGAGGCTCTCAAAGCGTGCTGACCCAG TCCGCCTCTGTGAGCGGCTCCCTGGGCCAGTCTGTGACCATCAGCTGTACAGGCCCCAACTCCGTGTGCTGT T C T C AC AAGT C TAT C AG C T G GT AC C AGT G G C C AC C AG GAAG G G C AC C T AC C C T GAT CAT C TAT GAG GAC AAT GAGAGGGCACCAGGAATCAGCCCTCGCTTCTCCGGCTACAAGTCTTATTGGAGCGCCTACCTGACCATTTCC GACCTGCGCCCCGAGGATGAGACCACATACTATTGCTGTAGCTATACCCACAACTCCGGCTGCGTGTTTGGC ACAGGCACCAAGGTGAGCGTGCTGGGAGGAGGGGGCTCTGGCGGCGGGGGCAGCGGCGGAGGCGGCTCCGGA GGGGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGCGGCTGGTGGAGAGCGGCGGCGGCCTGGTGAAGCCAGGC GGCTCTCTGAGACTGTCCTGTTCTGCCAGCGGCTTCGACTTTGATAATGCCTGGATGACATGGGTGCGGCAG CCTCCTGGCAAGGGGCTGGAGTGGGTGGGAAGAATCACCGGACCAGGAGAGGGATGGTCTGTGGACTACGCC GAGAG C GT GAAG GGCCGGTT C AC CAT C AG C AGAGAT AAC AC T AAAAAT AC AC T GT AT C T G GAGAT GAAC AAT GTGCGGACCGAGGACACAGGCTACTATTTCTGCGCCAGAACCGGCAAGTACTATGATTTCTGGTTTGGCTAC CCCCCTGGCGAGGAGTATTTTCAGGACTGGGGCCAGGGCACCCTGGTCATCGTGAGCAGCGGCGGGGGAGGC TCCGGCGGGGGGGGCTCTGGAGGAGGGGGCTCTAGCGAGCTGACCCAGGACCCCGCCGTGTCCGTGGCCCTG AAG C AGAC AGT GAC CAT C AC AT G C AG G G G C GAC TCCCTGCGCTCT C AC T AC G C C AG C T G GT AT C AGAAGAAG CCAGGACAGGCACCCGTGCTGCTGTTCTACGGCAAGAACAATCGGCCTTCCGGCATCCCAGATAGATTTTCC GGCTCTGC C AG C G GAAAC AG G G C C AG C C T GAC CAT C AC AG GAG C AC AG G C AGAG GAT GAAG C AG AT T AC TAT TGTTCCTCTCGGGACAAGTCCGGCTCTAGACTGAGC mAb dB4C7/UB-421’s perfect heavy chain Fv region (SEQ ID NO 98)
QVQLVQSGPELKKPGASVKVSCKASGYTFTDYVIHWVKQATGQGLEWIGEIYPGSGSA
YSNAKFKDRVTMTADKSSNTAYMELSSLTSDDTAVYFCARRGNGTGFAYWGQGTLVT
VSS mAb dB4C7/UB-421’s perfect light chain Fv region (SEQ ID NO. 99)
DIVLTQSPASLAVSLGQRATITCKAGQSVDYDGDSYMNWYQQKPGQPPKLLIYVASNLE
SGIPARFSGSGSGTDFTLNIHPVEENDAATYYCQQSYKDPLTFGQGTKLEIK
CDR1 of Heavy Chain of murine SEQ ID NO. 100 DYVIH
antibody B4
CDR2 of Heavy Chain of murine SEQ I D NO. 101 El YPGSGSAYSN AKFKD antibody B4
CDR3 of Heavy Chain of murine SEQ ID NO. 102 RGNGTGFAY antibody B4
CDR1 of Light Chain of murine SEQ ID NO. 103 KAGQSVDYDGDSYMN antibody B4
CDR2 of Light Chain of murine SEQ ID NO. 104 VASNLES
antibody B4
CDR3 of Light Chain of murine SEQ ID NO. 105 QQSYKDPLT antibody B4
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
PentaNAb2.0 : 10E8v4_S100cF-5X-35022-5X-N6-5X-PGT121-5X-VRC26.25-IgG3C hinge-IgGl LS (SEQ ID NO.106)
ATGGGATGGAGCTGTATTATTCTGTTTCTGGTCGCTACCGCTACCGGAGTGCATTCTTCTGAACTGACCCAG GACCCCGCCGTGAGCGTGGCCCTGAAGCAGACCGTGACAATCACCTGCAGGGGCGACAGCCTGCGCTCCCAC TACGCCAGCTGGTATCAGAAGAAGCCTGGCCAGGCCCCAGTGCTGCTGTTCTACGGCAAGAACAATAGGCCC TCCGGCATCCCTGATCGCTTTTCCGGCTCTGCCAGCGGAAACAGGGCCAGCCTGACAATCACCGGAGCACAG GCAGAGGACGAGGCAGATTACTATTGCAGCTCCCGGGACAAGTCCGGCTCTAGACTGAGCGTGTTCGGCGGC GGCACCAAGCTGACAGTGCTGGGAGGAGGAGGCAGCGGCGGAGGAGGCTCCGGAGGCGGCGGCTCTGAGGTG CGGCTGGTGGAGTCTGGAGGAGGCCTGGTGAAGCCAGGAGGCAGCCTGAGACTGAGCTGTTCCGCCTCTGGC TTCGACTTTGATAATGCCTGGATGACATGGGTGCGGCAGCCACCTGGCAAGGGCCTGGAGTGGGTGGGAAGA ATCACCGGACCAGGAGAGGGATGGTCTGTGGACTACGCCGAGAGCGTGAAGGGCCGGTTCACCATCTCCAGA GATAACACCAAGAATACACTGTATCTGGAGATGAACAATGTGCGGACCGAGGACACAGGCTACTATTTCTGC GCCAGAACCGGCAAGTACTATGATTTTTGGTTTGGCTACCCACCCGGCGAGGAGTATTTTCAGGACTGGGGC CAGGGCACCCTGGTCATCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCTCCGGAGGAGGCGGCTCT GGAGGAGGAGGCAGCGGGGGAGGGGGCAGCCAGTCCGTGCTGACCCAGTCTGCCAGCGTGTCCGGCTCTCTG GGACAGAGCGTGACCATCTCCTGTACAGGCCCCAACAGCGTGTGCTGTAGCCACAAGAGCATCTCCTGGTAC CAGTGGCCTCCAGGAAGGGCACCTACCCTGATCATCTATGAGGACAATGAGCGGGCCCCAGGCATCTCCCCC AGATTCTCTGGCTACAAGTCTTATTGGAGCGCCTACCTGACAATCAGCGACCTGCGCCCCGAGGATGAGACA ACATACTATTGCTGTTCCTATACCCACAACTCTGGCTGCGTGTTTGGCACAGGCACCAAGGTGTCCGTGCTG GGCGGCGGCGGCAGCGGGGGCGGGGGCTCCGGAGGGGGCGGCTCTCAGGGCCAGCTGGTGCAGAGCGGAGCA GAGCTGAAGAAGCCTGGAGCCAGCGTGAAGATCTCCTGTAAGACATCTGGCTACCGGTTCAACTTTTATCAC ATCAATTGGATCAGGCAGACCGCAGGAAGGGGACCAGAGTGGATGGGCTGGATCTCCCCCTACTCTGGCGAT AAGAACCTGGCCCCAGCCTTCCAGGACAGAGTGATCATGACCACAGATACCGAGGTGCCAGTGACCAGCTTC ACCTCCACCGGAGCCGCCTACATGGAGATCAGGAATCTGAAGTTCGACGATACAGGCACCTATTTTTGCGCA AAGGGCCTGCTGAGGGACGGCTCCTCTACCTGGCTGCCTTACCTGTGGGGACAGGGCACCCTGCTGACAGTG AGCTCCGGCGGCGGGGGCAGCGGCGGCGGGGGCTCCGGAGGAGGAGGCTCTGGAGGAGGGGGCAGCGGAGGA GGCGGCTCCTACATCCACGTGACCCAGTCCCCATCTAGCCTGTCTGTGAGCATCGGCGATCGGGTGACCATC AACTGTCAGACATCTCAGGGCGTGGGCAGCGACCTGCACTGGTATCAGCACAAGCCTGGCAGGGCCCCAAAG CTGCTGATCCACCACACATCCTCTGTGGAGGATGGAGTGCCAAGCCGCTTCTCCGGCTCTGGATTCCACACC TCCTTTAATCTGACAATCTCTGACCTGCAGGCCGACGATATCGCCACCTACTATTGCCAGGTGCTGCAGTTC TTTGGCCGGGGCTCCAGACTGCACATCAAGGGAGGAGGAGGCTCCGGGGGCGGAGGCTCTGGCGGCGGCGGC AGCCGGGCCCACCTGGTGCAGAGCGGCACCGCCATGAAGAAGCCTGGCGCCAGCGTGAGAGTGTCCTGTCAG ACATCTGGCTACACCTTCACCGCCCACATCCTGTTCTGGTTTAGGCAGGCACCAGGAAGAGGCCTGGAGTGG GTGGGCTGGATCAAGCCCCAGTATGGAGCAGTGAACTTCGGAGGAGGCTTTCGGGACAGAGTGACACTGACC CGGGACGTGTACAGAGAGATCGCCTATATGGATATCAGGGGCCTGAAGCCAGACGATACCGCCGTGTACTAT TGCGCCAGGGACCGCTCCTACGGCGATAGCTCCTGGGCACTGGACGCATGGGGACAGGGCACCACAGTGGTG GTGAGCGCCGGCGGCGGAGGCTCCGGCGGCGGGGGCTCTGGAGGAGGCGGCAGCGGAGGGGGAGGCTCCGGA GGGGGAGGCTCTAGCGACATCTCCGTGGCCCCTGGCGAGACAGCCAGAATCTCTTGTGGCGAGAAGTCTCTG GGCAGCAGGGCCGTGCAGTGGTACCAGCACAGGGCAGGACAGGCACCATCTCTGATCATCTATAACAATCAG GATAGGCCAAGCGGCATCCCTGAGCGGTTCAGCGGCTCCCCCGACAGCCCTTTTGGCACCACAGCCACACTG ACCATCACATCCGTGGAGGCAGGCGACGAAGCCGATTACTATTGCCACATCTGGGATTCCAGAGTGCCAACC AAGTGGGTGTTCGGAGGAGGAACCACACTGACAGTGCTGGGAGGGGGGGGCTCTGGCGGCGGGGGCAGCGGG GGAGGAGGCTCCCAGATGCAGCTGCAGGAGAGCGGACCAGGCCTGGTGAAGCCTAGCGAGACACTGAGCCTG ACATGTTCTGTGAGCGGCGCCTCCATCTCTGACAGCTACTGGTCTTGGATCAGACGGAGCCCCGGCAAGGGC CTGGAATGGATCGGCTACGTGCACAAGTCCGGCGATACAAACTATTCCCCATCTCTGAAGTCTCGGGTGAAC CTGTCTCTGGACACCAGCAAGAATCAGGTGAGCCTGTCCCTGGTGGCAGCAACCGCAGCAGATAGCGGCAAG TACTATTGCGCCAGAACACTGCACGGCAGGCGCATCTACGGCATCGTGGCCTTTAACGAGTGGTTCACCTAC TTTTATATGGACGTGTGGGGCAATGGCACCCAGGTGACAGTGTCCTCTGGCGGGGGCGGCTCCGGAGGCGGA GGCTCTGGCGGGGGCGGCAGCGGCGGGGGCGGCTCCGGGGGAGGCGGCTCTCAGAGCGTGCTGACCCAGCCA CCTTCCGTGTCTGCCGCACCAGGACAGAAGGTGACCATCAGCTGTTCCGGCAACACATCCAATATCGGCAAC AATTTCGTGTCTTGGTACCAGCAGAGGCCAGGAAGGGCACCACAGCTGCTGATCTATGAGACAGACAAGCGG CCTTCCGGCATCCCAGATAGATTTTCTGCCAGCAAGTCCGGCACCAGCGGCACACTGGCAATCACCGGCCTG CAGACAGGCGACGAAGCTGATTACTATTGCGCAACCTGGGCAGCCTCCCTGAGCTCCGCCAGGGTGTTCGGA ACCGGAACAAAAGTGATCGTGCTGGTGGGCGGCGGAGGCTCTGGCGGAGGCGGCAGCGGCGGGGGGGGCTCC CAGGTGCAGCTGGTGGAGAGCGGCGGCGGCGTGGTGCAGCCAGGCACCTCCCTGAGGCTGTCTTGTGCAGCA AGCCAGTTCAGATTTGATGGCTACGGCATGCACTGGGTGCGCCAGGCACCAGGCAAGGGCCTGGAGTGGGTA GCCTCTATCAGCCACGACGGCATCAAGAAGTACCACGCCGAGAAAGTGTGGGGCAGGTTCACCATCTCCCGC GATAACTCTAAAAACACACTGTATCTGCAGATGAACTCCCTGAGGCCCGAGGACACCGCCCTGTACTATTGC GCCAAGGACCTGCGCGAGGATGAGTGTGAGGAGTGGTGGTCCGACTACTATGATTTTGGCAAGCAGCTGCCT TGCGCAAAGAGCAGGGGAGGCCTGGTGGGAATCGCCGATAATTGGGGCCAGGGCACCATGGTGACAGTGTCT AGCGGATCCGGAGAGCTGAAGACCCCTCTGGGCGATACCACACACACATCCCCACGGAGCCCCGAGCCAAAG TCCTCTGACACCCCACCCCCTAGCCCTAGATCCCCTGAGCCAAAGAGCTCCGATACACCACCCCCTTCTCCA AGGAGCCCCGAGCCTAAGTCTAGCGACACCCCACCCCCTTGCCCCCGCTGTCCAGCACCAGAGCTGCTGGGA GGACCAAGCGTGTTCCTGTTTCCACCCAAGCCTAAGGATACACTGATGATCTCTCGCACCCCCGAGGTGACA TGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTG CACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTATCGCGTGGTGTCCGTGCTGACAGTG CTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAATAAGGCCCTGCCTGCCCCAATC GAGAAGACAATCAGCAAGGCAAAGGGACAGCCAAGGGAGCCACAGGTGTACACCCTGCCTCCAAGCCGCGAG GAGATGACCAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCTAGCGATATCGCCGTGGAG TGGGAGTCCAATGGCCAGCCAGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCCGATGGCTCTTTC TTTCTGTATTCTAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCTCTGTGCTG CACGAAGCCCTGCACAGCCATTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGAAAA
Amino acid: (SEQ ID NO.107)
MGWSCIILFLVATATGVHSSELTQDPAVSVALKQTVTITCRGDSLRSHYASWYQKKPGQAPVLLFYGKNNRP
SGIPDRFSGSASGNRASLTITGAQAEDEADYYCSSRDKSGSRLSVFGGGTKLTVLGGGGSGGGGSGGGGSEV
RLVESGGGLVKPGGSLRLSCSASGFDFDNAWMTWVRQPPGKGLEWVGRITGPGEGWSVDYAESVKGRFTISR
DNTKNTLYLEMNNVRTEDTGYYFCARTGKYYDFWFGYPPGEEYFQDWGQGTLVIVSSGGGGSGGGGSGGGGS
GGGGSGGGGSQSVLTQSASVSGSLGQSVTISCTGPNSVCCSHKSISWYQWPPGRAPTLIIYEDNERAPGISP
RFSGYKSYWSAYLTISDLRPEDETTYYCCSYTHNSGCVFGTGTKVSVLGGGGSGGGGSGGGGSQGQLVQSGA
ELKKPGASVKISCKTSGYRFNFYHINWIRQTAGRGPEWMGWISPYSGDKNLAPAFQDRVIMTTDTEVPVTSF
TSTGAAYMEIRNLKFDDTGTYFCAKGLLRDGSSTWLPYLWGQGTLLTVSSGGGGSGGGGSGGGGSGGGGSGG
GGSYIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGVPSRFSGSGFHT
SFNLTISDLQADDIATYYCQVLQFFGRGSRLHIKGGGGSGGGGSGGGGSRAHLVQSGTAMKKPGASVRVSCQ
TSGYTFTAHILFWFRQAPGRGLEWVGWIKPQYGAVNFGGGFRDRVTLTRDVYREIAYMDIRGLKPDDTAVYY
CARDRSYGDSSWALDAWGQGTTVWSAGGGGSGGGGSGGGGSGGGGSGGGGSSDISVAPGETARISCGEKSL
GSRAVQWYQHRAGQAPSLIIYNNQDRPSGIPERFSGSPDSPFGTTATLTITSVEAGDEADYYCHIWDSRVPT
KWVFGGGTTLTVLGGGGSGGGGSGGGGSQMQLQESGPGLVKPSETLSLTCSVSGASISDSYWSWIRRSPGKG
LEWIGYVHKSGDTNYSPSLKSRVNLSLDTSKNQVSLSLVAATAADSGKYYCARTLHGRRIYGIVAFNEWFTY
FYMDVWGNGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSQSVLTQPPSVSAAPGQKVTISCSGNTSNIGN
NFVSWYQQRPGRAPQLLIYETDKRPSGIPDRFSASKSGTSGTLAITGLQTGDEADYYCATWAASLSSARVFG
TGTKVIVLVGGGGSGGGGSGGGGSQVQLVESGGGWQPGTSLRLSCAASQFRFDGYGMHWVRQAPGKGLEWV
ASISHDGIKKYHAEKVWGRFTISRDNSKNTLYLQMNSLRPEDTALYYCAKDLREDECEEWWSDYYDFGKQLP
CAKSRGGLVGIADNWGQGTMVTVSSGSGELKTPLGDTTHTSPRSPEPKSSDTPPPSPRSPEPKSSDTPPPSP
RSPEPKSSDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTI SKAKGQPREPQVYTLPPSRE
EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVL
HEALHSHYTQKSLSLS PGK
The studies presented above, using prototypic HIV bNAbs to create multispecific Env binding functional moieties with enhanced breadth and/or potency through structure- based rational design and iterative optimization, as well as combinations of the optimized bispecific Abs to form multi-specific Abs will serve as a methodological foundation for the eventual expansion of this line of investigation in regard to the more imaginative anti viral agents described in this disclosure. Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

Claims

What is claimed is:
1. A multispecific anti-HIV antibody that binds to multiple epitopes on HIV
envelope protein, wherein the antibody comprises:
i. an amino acid sequence that binds to a V1/V2 apex glycan epitope;
ii. an amino acid sequence that binds to a V3-base glycan region epitope;
iii. an amino acid sequence that binds to a CD4 binding site (CD4bs) epitope;
iv. an amino acid sequence that binds to a gpl20/gp4l interface epitope; and
v. an amino acid sequence that binds to a membrane proximal external region (MPER) epitope.
2. The multispecific anti -HIV antibody of claim 1, wherein the antibody
simultaneously binds the multiple epitopes.
3. The multispecific anti -HIV antibody of any of claims 1-2, wherein the amino acid sequences of i-v) are present on a single polypeptide chain.
4. The multispecific anti-HIV antibody of any of claims 1-3, wherein the antibody is capable of neutralizing at least 99% of the HIV viruses listed in Table 1 with an IC50 value of less than 50 pg/mL.
5. The multispecific anti -HIV antibody of claim 4, wherein the antibody has an IC50 geomean of less than or equal to 0.10 pg/ml.
6. The multispecific anti-HIV antibody of claim 4, wherein the antibody has an IC50 geomean of less than or equal to 0.072 pg/ml.
7. The multispecific anti-HIV antibody of claim 4, wherein the antibody has an IC50 geomean of less than or equal to 0.006 pg/ml.
8. The multispecific anti-HIV antibody of claim 4, wherein the antibody is capable of neutralizing at least 99.5% of the HIV pseudoviruses listed in Table 1 with an IC50 value of less than 50 pg/mL.
9. The multispecific anti -HIV antibody of any of claims 1-8, wherein the amino acid sequences of parts i-v) comprise amino acid sequences of single chain fragment variable (ScFv) moieties, wherein each ScFv moiety comprises an amino acid sequence from a light chain variable region (VL) and an amino acid sequence from a heavy chain variable region (VH) of an antibody.
10. The multispecific anti -HIV antibody of claim 9, wherein one or more of the ScFv moieties is organized such that the VL is at the amino terminal end of the ScFv moiety and the VH is at the carboxy terminal end of the ScFv moiety.
11. The multispecific anti-HIV antibody of claim 9, wherein one or more of the ScFv moieties is organized such that the VH is at the amino terminal end of the ScFv moiety and the VL is at the carboxy terminal end of the ScFv moiety.
12. The multispecific anti-HIV antibody of claim 9, wherein each ScFv moiety is organized such that the VL is at the amino terminal end of the ScFv moiety and the VH is at the carboxy terminal end of the ScFv moiety.
13. The multispecific anti-HIV antibody of any of claims 9-12, wherein the VL and VH sequences are separated by one or more linking amino acids.
14. The multispecific anti-HIV antibody of claim 13, wherein the linking amino acids comprise one or more tetra-glycine serine (G4S) linkers.
15. The multispecific anti-HIV antibody of claim 14, wherein the VL and VH
sequences are separated by 1-10 tetra-glycine serine (G4S) linkers.
16. The multispecific anti -HIV antibody of claim 15, wherein the VL and VH
sequences are separated by 3 tetra-glycine serine (G4S) linkers.
17. The multispecific anti -HIV antibody of any of claims 1-16, wherein the antibody further comprises an Fc region of an immunoglobulin or a variant thereof.
18. The multispecific anti-HIV antibody of any of claims 1-17, wherein the antibody comprises a first and second polypeptide chain, wherein the first and second polypeptide chains each comprise
a. five ScFv moieties, wherein each ScFv moiety on a single chain
recognizes an individual epitope, wherein each ScFv moiety comprises an amino acid sequence from a light chain variable region (VL) and an amino acid sequence from a heavy chain variable region (VH) of an antibody; and
b. an Fc region of an immunoglobulin or a variant thereof.
19. The multispecific anti -HIV antibody of any of claims 17-18, wherein the immunoglobulin is IgG.
20. The multispecific anti -HIV antibody of any of claims 17-18, wherein the
immunoglobulin is IgGl.
21. The multispecific anti -HIV antibody of any of claims 9-20, wherein the ScFv moieties are separated on the polypeptide chain by one or more linking amino acids.
22. The multispecific anti -HIV antibody of claim 21, wherein the linking amino acids comprise one or more tetra-glycine serine (G4S) linkers.
23. The multispecific anti -HIV antibody of claim 22, wherein each ScFv moiety is separated by 5 tetra-glycine serine (G4S) linkers.
24. The multispecific anti-HIV antibody of any of claims 1-23, wherein the amino acid sequences of parts i-v) of claim 1 are organized on a single polypeptide chain in the following order, from its N-terminus to its C-terminus:
i. an amino acid sequence that binds to the V1/V2 apex glycan epitope;
ii. an amino acid sequence that binds to the V3-base glycan region epitope;
iii. an amino acid sequence that binds to the CD4 binding site (CD4bs) epitope;
iv. an amino acid sequence that binds to the gpl20/gp4l
interface epitope; and
v. an amino acid sequence that binds to the membrane
proximal external region (MPER) epitope.
25. The multispecific anti-HIV antibody of any of claims 1-23, wherein the amino acid sequences of parts i-v) of claim 1 are organized on a single polypeptide chain in the following order, from its N-terminus to its C-terminus:
i. an amino acid sequence that binds to the membrane proximal external region (MPER) epitope;
ii. an amino acid sequence that binds to the gpl20/gp4l
interface epitope; iii. an amino acid sequence that binds to the CD4 binding site (CD4bs) epitope;
iv. an amino acid sequence that binds to the V3-base glycan region epitope; and
v. an amino acid sequence that binds to the V1/V2 apex glycan epitope.
26. The multispecific anti -HIV antibody of any of claims 1-23 and 25, wherein the antibody comprises a first and second polypeptide chain, wherein the amino acid sequences of parts i-v) of claim 1 are organized in the following order, from its N- terminus to its C-terminus:
i. an amino acid sequence that binds to the membrane proximal external region (MPER) epitope;
ii. an amino acid sequence that binds to the gpl20/gp4l
interface epitope;
iii. an amino acid sequence that binds to the CD4 binding site (CD4bs) epitope;
iv. an amino acid sequence that binds to the V3-base glycan region epitope;
v. an amino acid sequence that binds to the V1/V2 apex glycan epitope; and
vi. an Fc region of an immunoglobulin or a variant thereof.
27. The multispecific anti -HIV antibody of any of claims 1-24, wherein the antibody comprises a first and second polypeptide chain, wherein the amino acid sequences of parts i-v) of claim 1 are organized in the following order, from its N-terminus to its C-terminus:
i. an amino acid sequence that binds to the V1/V2 apex glycan epitope;
ii. an amino acid sequence that binds to the V3-base glycan region epitope;
iii. an amino acid sequence that binds to the CD4 binding site (CD4bs) epitope; iv. an amino acid sequence that binds to the gpl20/gp4l
interface epitope;
v. an amino acid sequence that binds to the membrane proximal external region (MPER) epitope; and
vi. an Fc region of an immunoglobulin or a variant thereof.
28. The multispecific anti-HIV antibody of any of claims 1-25, wherein the antibody comprises
1) a first polypeptide chain, wherein the amino acid sequences of parts i-v) of claim 1 are organized in the following order, from its N-terminus to its C-terminus:
i. an amino acid sequence that binds to the membrane proximal external region (MPER) epitope;
ii. an amino acid sequence that binds to the gpl20/gp4l
interface epitope;
iii. an amino acid sequence that binds to the CD4 binding site (CD4bs) epitope;
iv. an amino acid sequence that binds to the V3-base glycan region epitope;
v. an amino acid sequence that binds to the V1/V2 apex glycan epitope; and
vi. an Fc region of an immunoglobulin or a variant thereof; and
2) a second polypeptide chain, wherein the amino acid sequences of parts i-v) of claim 1 are organized in the following order, from its N-terminus to its C-terminus:
i. an amino acid sequence that binds to the V1/V2 apex glycan epitope;
ii. an amino acid sequence that binds to the V3-base glycan region epitope;
iii. an amino acid sequence that binds to the CD4 binding site (CD4bs) epitope;
iv. an amino acid sequence that binds to the gpl20/gp4l
interface epitope; v. an amino acid sequence that binds to the membrane proximal external region (MPER) epitope; and
vi. an Fc region of an immunoglobulin or a variant thereof.
29. The multispecific anti -HIV antibody of any of claims 1-28, 46-47 and 48-49, wherein:
i. the amino acid sequence that binds to the epitope of the Vl/V2-glycan region comprises an amino acid sequence from an antibody selected from the group consisting of VRC26.25 and PGDM1400;
ii. the amino acid sequence that binds to the epitope of the V3-glycan region comprises an amino acid sequence from antibody PGT121;
iii. the amino acid sequence that binds to the epitope of the CD4-binding site (CD4bs) comprises an amino acid sequence from antibody N6; iv. the amino acid sequence that binds to the epitope of the gpl20/gp4l
interface comprises an amino acid sequence from antibody 35022; and v. the amino acid sequence that binds to the epitope of the membrane
proximal external region (MPER) comprises an amino acid sequence from an antibody selected from the group consisting of 10E8n4,
l0E8v4_Sl00cF, and l0E8v4_V5R_Sl00cF.
30. The multispecific anti-HIV antibody of claim 29, wherein
i. the amino acid sequence from the antibody VRC26.25 comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR H1 comprises QFRFDGYG, CDR H2 comprises ISHDGIKK and CDR H3 comprises
AKDLREDECEEWW SDDF GKQLPC AK SRGGL V GI ADN ; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR Ll comprises TSNIGNNF, CDR L2 comprises ETD and CDR L3 comprises ATWAASLSSARV; ii. the amino acid sequence from the antibody PGDM1400 comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR Hl comprises GNTLKTYD, CDR H2 comprises ISHEGDKK and CDR H3 comprises
AKGSKHRLRD Y ALDDD GALNW A VD VD YL SNLEF ; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR L1 comprises HSLIHGDRNNY, CDR L2 comprises LAS and CDR L3 comprises MQGRESPWT;
iii. the amino acid sequence from the antibody PGT121 comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR H1 comprises GASISDSY, CDR H2 comprises VHKSGDT and CDR H3 comprises
ARTLHGRRIYGIVAFNEWFTYFYMDV; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR Ll comprises SLGSRA, CDR L2 comprises NNQ and CDR L3 comprises HIWDSRVPTKWV;
iv. the amino acid sequence from the antibody N6 comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR H1 comprises GYTFTAHI, CDR H2 comprises
IKPQYGAV and CDR H3 comprises AR; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR Ll comprises QGVGSD, CDR L2 comprises HTS and CDR L3 comprises QVLQF;
v. the amino acid sequence from the antibody 35022 comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR H1 comprises GYRFNFYH, CDR H2 comprises ISPYSGDK and CDR H3 comprises
DDTGTYFCAKGLLRDGSSTWLPYL; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR Ll comprises NSVCCSHKS, CDR L2 comprises EDN and CDR L3 comprises CSYTHNSGCV;
vi. the amino acid sequence from the antibody 10E8n4 comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR H1 comprises GFDFDNAW, CDR H2 comprises ITGPGEGWSV and CDR H3 comprises
T GY YF CART GK Y YDF W S GYPPGEE YF QD ; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR L1 comprises RGDSLRSHYAS, CDR L2 comprises GKNNRPS and CDR L3 comprises SSRDKSGSRLSV;
vii. the amino acid sequence from the antibody l0E8v4_Sl00cF comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR Hl comprises GFDFDNAW, CDR H2 comprises ITGPGEGWSV and CDR H3 comprises
T GYYF CART GK YYDF WF GYPPGEEYF QD ; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR L1 comprises RGDSLRSHYAS, CDR L2 comprises GKNNRPS and CDR L3 comprises SSRDKSGSRLSV;
viii. the amino acid sequence from the antibody l0E8v4_V5R_Sl00cF
comprises an amino acid sequence from the VH region comprising CDR Hl, CDR H2 and CDR H3, wherein CDR Hl comprises GFDFDNAW, CDR H2 comprises ITGPGEGWSV and CDR H3 comprises
T GYYF CART GK YYDF WF GYPPGEEYF QD ; and an amino acid sequence from the VL region comprising CDR Ll, CDR L2 and CDR L3, wherein CDR L1 comprises RGDSLRSHYAS, CDR L2 comprises GKNNRPS and CDR L3 comprises SSRDKSGSRLSV.
31. The multispecific anti-HIV antibody of claim 29, wherein
i. the amino acid sequence from the antibody VRC26.25 comprises the VH region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VH region comprises SEQ ID NO: 1; and the VL region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VL region comprises amino acids SEQ ID NO:5;
ii. the amino acid sequence from the antibody PGDM1400 comprises the VH region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VH region comprises SEQ ID NO:9; and the VL region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VL region comprises amino acids SEQ ID NO: l3; iii. the amino acid sequence from the antibody PGT121 comprises the VH region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VH region comprises SEQ ID NO: 17; and the VL region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VL region comprises amino acids SEQ ID NO:2l;
iv. the amino acid sequence from the antibody N6 comprises the VH region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VH region comprises SEQ ID NO:25; and the VL region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VL region comprises amino acids SEQ ID NO:29;
v. the amino acid sequence from the antibody 35022 comprises the VH
region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VH region comprises SEQ ID NO:33; and the VL region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VL region comprises amino acids SEQ ID NO:37;
vi. the amino acid sequence from the antibody 10E8n4 comprises the VH
region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VH region comprises SEQ ID NO:4l; and VL region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VL region comprises amino acids SEQ ID NO:45;
vii. the amino acid sequence from the antibody l0E8v4_Sl00cF comprises the VH region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VH region comprises SEQ ID NO:49; and the VL region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VL region comprises amino acids SEQ ID NO:45; and
viii. the amino acid sequence from the antibody l0E8v4_V5R_Sl00cF
comprises the VH region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VH region comprises SEQ ID NO:5l; and the VL region or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, wherein the VL region comprises amino acids SEQ ID NO:45.
32. The multispecific anti -HIV antibody of any of claims 1-31, wherein the antibody comprises an IgGl Fc region variant comprising mutations corresponding to M428L and N434S.
33. The multispecific anti-HIV antibody of any of claims 1-32, wherein the antibody comprises an immunoglobulin Fc region that has been modified to facilitate heterodimerization.
34. The multispecific anti-HIV antibody of any of claims 1-33, wherein the antibody further comprises a sequence tag that facilitates purification of the antibody.
35. The multispecific anti -HIV antibody of claim 34, wherein the sequence tag
comprises a polyhistidine tag.
36. The multispecific anti-HIV antibody of any of claims 1-35, wherein the antibody comprises any of SEQ ID NOS:72-80.
37. One or more vectors comprising a nucleic acid encoding the antibody or
polypeptide of any of claims 1-36.
38. A cell comprising the one or more vectors of claim 37.
39. An engineered cell that expresses the antibody of any of claims 1-36.
40. The cell of claim 39, wherein the cell is an immune cell.
41. The cell of claim 40, wherein the immune cell is a B cell.
42. A pharmaceutical composition comprising an antibody of any of claims 1-36 and/or cells of any of claims 39-41 and a pharmaceutically acceptable carrier.
43. A method for treating or preventing HIV infection in a subject, comprising
administering to the subject an effective amount of the composition of claim 42.
44. The method of claim 43, wherein the composition is administered in combination with another therapy.
45. The method of claim 44, wherein the therapy is an anti -retroviral therapy.
46. The multispecific anti-HIV antibody of any of claims 1-23, wherein the amino acid sequences of parts i-v) of claim 1 are organized on a single polypeptide chain in the following order, from its N-terminus to its C-terminus: i. an amino acid sequence that binds to a CD4 epitope; ii. an amino acid sequence that binds to a V1/V2 apex glycan epitope; iii. an amino acid sequence that binds to the V3-base glycan region epitope; iv. an amino acid sequence that binds to the CD4 binding site (CD4bs) epitope; v. an amino acid sequence that binds to the gpl20/gp4l interface epitope; and vi. an amino acid sequence that binds to the membrane proximal external region (MPER) epitope.
47. The multispecific anti-HIV antibody of claim 46 wherein the amino acid sequence of (i) is derived from the variable domains of the anti-CD4 antibody dB4C7/UB-42l.
48. The multispecific anti-HIV antibody of any of claims 1-23, wherein the antibody comprises a first and second polypeptide chain, wherein the first polypeptide chain comprises a heavy chain variable region wherein the heavy chain variable region comprises; i. an amino acid sequence that binds to a V1/V2 apex glycan epitope; ii. an amino acid sequence that binds to a V3-base glycan region epitope; and iii. an amino acid sequence that binds to a CD4 binding site (CD4bs) epitope; and wherein the second polypeptide chain comprises a light chain variable region wherein the light chain variable region comprises: i. an amino acid sequence that binds to a CD4 binding site (CD4bs) epitope; ii. an amino acid sequence that binds to a gpl20/gp4l interface epitope; and iii. an amino acid sequence that binds to a membrane proximal external region (MPER) epitope.
49. The multi- specific anti-HIV antibody of claim 48 wherein the Fc domain comprises a M428L/N434S (LS) mutation.
PCT/US2019/057089 2018-10-19 2019-10-18 Multispecfic anti-hiv antibodies Ceased WO2020082045A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US17/285,956 US12365722B2 (en) 2018-10-19 2019-10-18 Multispecific antibodies targeting multiple epitopes on the HIV-1 envelope
US19/231,954 US20250368722A1 (en) 2018-10-19 2025-06-09 Multispecific antibodies targeting multiple epitopes on the hiv-1 envelope

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201862748228P 2018-10-19 2018-10-19
US62/748,228 2018-10-19
US201862749510P 2018-10-23 2018-10-23
US62/749,510 2018-10-23

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US17/285,956 A-371-Of-International US12365722B2 (en) 2018-10-19 2019-10-18 Multispecific antibodies targeting multiple epitopes on the HIV-1 envelope
US19/231,954 Division US20250368722A1 (en) 2018-10-19 2025-06-09 Multispecific antibodies targeting multiple epitopes on the hiv-1 envelope

Publications (1)

Publication Number Publication Date
WO2020082045A1 true WO2020082045A1 (en) 2020-04-23

Family

ID=70284834

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2019/057089 Ceased WO2020082045A1 (en) 2018-10-19 2019-10-18 Multispecfic anti-hiv antibodies

Country Status (2)

Country Link
US (2) US12365722B2 (en)
WO (1) WO2020082045A1 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024094690A1 (en) * 2022-11-02 2024-05-10 VIIV Healthcare UK (No.5) Limited Antigen binding proteins
EP4397748A4 (en) * 2021-08-30 2025-09-17 Kanglin Biotechnology Hangzhou Co Ltd Gene sequence construct for gene therapy against HIV infection
EP4397764A4 (en) * 2021-08-30 2025-09-17 Kanglin Biotechnology Hangzhou Co Ltd Gene sequence construct for gene therapy of human immunodeficiency virus infection
EP4408897A4 (en) * 2021-09-29 2025-10-15 Modex Therapeutics Inc ANTIGEN-BINDING POLYPEPTIDES, ANTIGEN-BINDING POLYPEPTIDE COMPLEXES AND METHODS OF USE THEREOF
EP4408892A4 (en) * 2021-09-29 2025-11-19 Modex Therapeutics Inc Antigen-binding polypeptides, antigen-binding polypeptide complexes and methods for their use in HIV
EP4452317A4 (en) * 2021-12-21 2025-12-10 Modex Therapeutics Inc Conditionally activated antigen-binding polypeptide complexes and methods for using them

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150218257A1 (en) * 2009-03-17 2015-08-06 Theraclone Sciences, Inc. Monoclonal antibodies directed against trimeric forms of the hiv-1 envelope glycoprotein with broad and potent neutralizing activity
WO2017093985A1 (en) * 2015-12-05 2017-06-08 Centre Hospitalier Universitaire Vaudois Hiv binding agents
WO2017133640A1 (en) * 2016-02-03 2017-08-10 National Center For Aids/Std Control And Prevention, Chinese Center For Disease Control And Prevention Broadly neutralizing antibodies against hiv-1 and use thereof
WO2018075564A1 (en) * 2016-10-17 2018-04-26 University Of Maryland, College Park Multispecific antibodies targeting human immunodeficiency virus and methods of using the same
US20180118816A1 (en) * 2015-04-17 2018-05-03 Igm Biosciences, Inc. Multi-valent human immunodeficiency virus antigen binding molecules and uses thereof

Family Cites Families (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3720760A (en) 1968-09-06 1973-03-13 Pharmacia Ab Method for determining the presence of reagin-immunoglobulins(reagin-ig)directed against certain allergens,in aqueous samples
US4235871A (en) 1978-02-24 1980-11-25 Papahadjopoulos Demetrios P Method of encapsulating biologically active materials in lipid vesicles
US4722848A (en) 1982-12-08 1988-02-02 Health Research, Incorporated Method for immunizing animals with synthetically modified vaccinia virus
US4501728A (en) 1983-01-06 1985-02-26 Technology Unlimited, Inc. Masking of liposomes from RES recognition
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4957735A (en) 1984-06-12 1990-09-18 The University Of Tennessee Research Corporation Target-sensitive immunoliposomes- preparation and characterization
US5019369A (en) 1984-10-22 1991-05-28 Vestar, Inc. Method of targeting tumors in humans
US4902505A (en) 1986-07-30 1990-02-20 Alkermes Chimeric peptides for neuropeptide delivery through the blood-brain barrier
US4837028A (en) 1986-12-24 1989-06-06 Liposome Technology, Inc. Liposomes with enhanced circulation time
US5004697A (en) 1987-08-17 1991-04-02 Univ. Of Ca Cationized antibodies for delivery through the blood-brain barrier
EP0368684B2 (en) 1988-11-11 2004-09-29 Medical Research Council Cloning immunoglobulin variable domain sequences.
US5055303A (en) 1989-01-31 1991-10-08 Kv Pharmaceutical Company Solid controlled release bioadherent emulsions
US5271961A (en) 1989-11-06 1993-12-21 Alkermes Controlled Therapeutics, Inc. Method for producing protein microspheres
US5188837A (en) 1989-11-13 1993-02-23 Nova Pharmaceutical Corporation Lipsopheres for controlled delivery of substances
US5268164A (en) 1990-04-23 1993-12-07 Alkermes, Inc. Increasing blood-brain barrier permeability with permeabilizer peptides
US5254342A (en) 1991-09-30 1993-10-19 University Of Southern California Compositions and methods for enhanced transepithelial and transendothelial transport or active agents
DE69311538D1 (en) 1992-03-12 1997-07-17 Alkermes Inc ACTH CONTAINED MICROBALLS WITH CONTROLLED DISCHARGE
US5534496A (en) 1992-07-07 1996-07-09 University Of Southern California Methods and compositions to enhance epithelial drug transport
US5514670A (en) 1993-08-13 1996-05-07 Pharmos Corporation Submicron emulsions for delivery of peptides
DE102004040578B3 (en) 2004-08-21 2006-01-05 Leistritz Ag ECM machine

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150218257A1 (en) * 2009-03-17 2015-08-06 Theraclone Sciences, Inc. Monoclonal antibodies directed against trimeric forms of the hiv-1 envelope glycoprotein with broad and potent neutralizing activity
US20180118816A1 (en) * 2015-04-17 2018-05-03 Igm Biosciences, Inc. Multi-valent human immunodeficiency virus antigen binding molecules and uses thereof
WO2017093985A1 (en) * 2015-12-05 2017-06-08 Centre Hospitalier Universitaire Vaudois Hiv binding agents
WO2017133640A1 (en) * 2016-02-03 2017-08-10 National Center For Aids/Std Control And Prevention, Chinese Center For Disease Control And Prevention Broadly neutralizing antibodies against hiv-1 and use thereof
WO2018075564A1 (en) * 2016-10-17 2018-04-26 University Of Maryland, College Park Multispecific antibodies targeting human immunodeficiency virus and methods of using the same

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4397748A4 (en) * 2021-08-30 2025-09-17 Kanglin Biotechnology Hangzhou Co Ltd Gene sequence construct for gene therapy against HIV infection
EP4397764A4 (en) * 2021-08-30 2025-09-17 Kanglin Biotechnology Hangzhou Co Ltd Gene sequence construct for gene therapy of human immunodeficiency virus infection
EP4408897A4 (en) * 2021-09-29 2025-10-15 Modex Therapeutics Inc ANTIGEN-BINDING POLYPEPTIDES, ANTIGEN-BINDING POLYPEPTIDE COMPLEXES AND METHODS OF USE THEREOF
EP4408882A4 (en) * 2021-09-29 2025-10-15 Modex Therapeutics Inc ANTIGEN-BINDING POLYPEPTIDES, ANTIGEN-BINDING POLYPEPTIDE COMPLEXES AND METHODS OF USE THEREOF
EP4408892A4 (en) * 2021-09-29 2025-11-19 Modex Therapeutics Inc Antigen-binding polypeptides, antigen-binding polypeptide complexes and methods for their use in HIV
EP4408469A4 (en) * 2021-09-29 2025-11-19 Modex Therapeutics Inc Antigen-binding polypeptides, antigen-binding polypeptide complexes and methods for using them
EP4452317A4 (en) * 2021-12-21 2025-12-10 Modex Therapeutics Inc Conditionally activated antigen-binding polypeptide complexes and methods for using them
WO2024094690A1 (en) * 2022-11-02 2024-05-10 VIIV Healthcare UK (No.5) Limited Antigen binding proteins
US12583911B2 (en) 2022-11-02 2026-03-24 VIIV Healthcare UK (No.5) Limited Human immunodeficiency virus GP120 binding proteins

Also Published As

Publication number Publication date
US12365722B2 (en) 2025-07-22
US20250368722A1 (en) 2025-12-04
US20220227845A1 (en) 2022-07-21

Similar Documents

Publication Publication Date Title
US12077571B2 (en) Polyomavirus neutralizing antibodies
US20250368722A1 (en) Multispecific antibodies targeting multiple epitopes on the hiv-1 envelope
US12492241B2 (en) Broadly neutralizing antibodies against HIV
US11149082B2 (en) Multispecific antibodies targeting human immunodeficiency virus and methods of using the same
US12612449B2 (en) HIV-1 antibodies
WO2021108761A1 (en) Broadly neutralizing and potent antibodies against hiv
WO2024123897A1 (en) Broadly neutralizing and potent antibodies against hiv
HK40059983A (en) Polyomavirus neutralizing antibodies
HK40059983B (en) Polyomavirus neutralizing antibodies

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19872895

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19872895

Country of ref document: EP

Kind code of ref document: A1

WWG Wipo information: grant in national office

Ref document number: 17285956

Country of ref document: US