EP3953384A1 - Trispecific and/or trivalent binding proteins using the cross-over-dual-variable domain (codv) format for treatment of hiv infection - Google Patents

Trispecific and/or trivalent binding proteins using the cross-over-dual-variable domain (codv) format for treatment of hiv infection

Info

Publication number
EP3953384A1
EP3953384A1 EP20722421.3A EP20722421A EP3953384A1 EP 3953384 A1 EP3953384 A1 EP 3953384A1 EP 20722421 A EP20722421 A EP 20722421A EP 3953384 A1 EP3953384 A1 EP 3953384A1
Authority
EP
European Patent Office
Prior art keywords
seq
amino acid
acid sequence
polynucleotide
sequence
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.)
Pending
Application number
EP20722421.3A
Other languages
German (de)
French (fr)
Inventor
Mangaiarkarasi ASOKAN
Christian Beil
Jochen Beninga
Joerg Birkenfeld
Mark Connors
Richard A. Koup
Young Do Kwon
Peter D. Kwong
Qingbo LIU
Paolo Lusso
John R. Mascola
Gary J. Nabel
Amarendra PEGU
Ercole Rao
Ronnie WEI
Ling Xu
Zhi-Yong Yang
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.)
Sanofi SA
United States Department of Health and Human Services
Original Assignee
Sanofi SA
United States 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 Sanofi SA, United States Department of Health and Human Services filed Critical Sanofi SA
Publication of EP3953384A1 publication Critical patent/EP3953384A1/en
Pending legal-status Critical Current

Links

Classifications

    • 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]
    • C07K16/1145—Env proteins, e.g. gp41, gp110/120, gp160, V3, principal neutralising domain [PND] or CD4-binding site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C07K2317/30—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
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C07K2317/53—Hinge
    • 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
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the disclosure relates to trispecific and/or trivalent binding proteins comprising four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein a first pair of polypeptides forming the binding protein possess dual variable domains having a cross-over orientation.
  • the disclosure also relates to methods for making trispecific and/or trivalent binding proteins and uses of such binding proteins for treating and/or preventing HIV/AIDS.
  • ART Anti-retroviral therapy
  • ART transcriptase
  • integrase integrase
  • PI viral protease
  • Treatment using ART or combination of different classes of ART results in inhibition of HIV- 1 replication and subsequent reduction of viremia, often to undetectable level (aviremic status).
  • ART greatly helps HIV patients in controlling their disease progression, and containing the global HIV epidemic, it does require patients taking daily medicines often following a strict regimen. About 10% patients fail therapy each year due to drug toxicity, suboptimal adherence and emerging drug resistance. As more HIV patients can live a normal life span (over 80 years), chronic complications are of particular concern, such as aging and drug-drug interaction, and cardiovascular/renal/bone toxicities. The economic burden treating HIV/AIDS has not subsided thus far.
  • the HIV-1 reservoirs are neither affected by ART nor the host immune system as they do not express viral proteins. Yet, a small proportion of cells among the reservoirs are randomly reactivated by unknown mechanism(s), which are responsible for recurrence of viremia once ART is stopped.
  • multispecific binding proteins e.g ., antibodies
  • the binding proteins bind one or more HIV target proteins and a CD3 polypeptide.
  • the binding proteins bind an HIV target protein, a CD28 polypeptide, and a CD3 polypeptide.
  • TCE trispecific anti-HIV/CD28xCD3 T cell engager
  • binding proteins comprising four polypeptide chains that form the three antigen binding sites that specifically bind one or more HIV target proteins, wherein a first polypeptide chain comprises a structure represented by the formula:
  • a second polypeptide chain comprises a structure represented by the formula:
  • a third polypeptide chain comprises a structure represented by the formula:
  • polypeptide chain comprises a structure represented by the formula:
  • V L1 is a first immunoglobulin light chain variable domain
  • V L2 is a second immunoglobulin light chain variable domain
  • V L3 is a third immunoglobulin light chain variable domain
  • V H1 is a first immunoglobulin heavy chain variable domain
  • V H2 is a second immunoglobulin heavy chain variable domain
  • V H3 is a third immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H1 is an immunoglobulin C H1 heavy chain constant domain
  • C H2 is an immunoglobulin C H2 heavy chain constant domain
  • C H3 is an immunoglobulin C H3 heavy chain constant domain
  • hinge is an immunoglobulin hinge region connecting the C H1 and C H2 domains; and L 1 , L2, L 3 and L 4 are amino acid linkers;
  • polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair
  • V H1 and V L1 form a first antigen binding site
  • V H2 and V L2 form a second antigen binding site that binds a CDS polypeptide
  • the V H2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22)
  • the V L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of
  • QSLVHX 1 NX 2 X 3 TY wherein X 1 is E or Q, X 2 is A or L, and X 3 is Q, R, or F (SEQ ID NO:293), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30); and wherein V H3 and V L3 form a third antigen binding site that binds an HIV target protein.
  • the first binding site binds a CD28 polypeptide (e.g ., a human CD28 polypeptide).
  • the V H1 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:31), a CDR- H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:33), and the V L1 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO: 36).
  • the V H1 domain comprises the amino acid sequence of
  • V L1 domain comprises the amino acid sequence of
  • the CDR-L1 sequence of the V L2 domain comprises an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO:24), QSLVHENLQTY (SEQ ID NO:25), QSLVHENLFTY (SEQ ID NO:26), and QSLVHENLRTY (SEQ ID NO:27).
  • the V H2 domain comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and the V L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:24), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).
  • VH antibody heavy chain variable
  • the V H2 domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and the V L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO:25), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).
  • the V H2 domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and the V L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO:26), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).
  • the V H2 domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and the V L2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO:27), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).
  • the V H2 domain comprises the amino acid sequence of
  • V L2 domain comprises an amino acid sequence selected from the group consisting of
  • DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHQNAQT YL S W YLQKPGQ SPQ SLI YK V S NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQ YPFTFGSGTKVEIK
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:54.
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:55.
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:56.
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:57.
  • the third antigen binding site binds an HIV target protein selected from the group consisting of glycoprotein 120, glycoprotein 41 and glycoprotein 160.
  • the V H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of NCPIN (SEQ ID NO: l) a CDR-H2 sequence comprising the amino acid sequence of WMKPRHGAV S YARQLQG (SEQ ID NO:2), and a CDR-H3 sequence comprising the amino acid sequence of GKYCTARDYYNWDFEH (SEQ ID NO:3)
  • the V L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of RTSQYGSLA (SEQ ID NO:4), a CDR-L2 sequence comprising the amino acid sequence of SGSTRAA (SEQ ID NO:5), and a CDR-L3 sequence comprising the amino acid sequence of QQYEF (SEQ ID NO:6).
  • the Vro domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7) a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAV (SEQ ID NO:8) or IKPQYGAT (SEQ ID NO:9), and a CDR-H3 sequence comprising the amino acid sequence of DRSYGDSSWALDA (SEQ ID NO: 10), and the V L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO: 11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO: 12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO: 13).
  • the V H3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of DCTLN (SEQ ID NO: 14) a CDR-H2 sequence comprising the amino acid sequence of WLKPRW GAVNY ARPLQG (SEQ ID NO: 15), and a CDR-H3 sequence comprising the amino acid sequence of GKNCDYNWDFEH (SEQ ID NO: 16), and the V L3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of
  • V H3 domain comprises the amino acid sequence of
  • V L3 domain comprises the amino acid sequence of
  • the V H3 domain comprises the amino acid sequence of
  • V S S (SEQ ID NO:44), and/or the V L3 domain comprises the amino acid sequence of
  • the V H3 domain comprises the amino acid sequence of
  • V L3 domain comprises the amino acid sequence of
  • V H3 domain comprises the amino acid sequence of
  • V L3 domain comprises the amino acid sequence of
  • V H3 domain comprises the amino acid sequence of
  • V L3 domain comprises the amino acid sequence of
  • V H3 domain comprises the amino acid sequence of
  • V S S (SEQ ID NO:50), and/or the V L3 domain comprises the amino acid sequence of
  • At least one of L 1 , L 2 , L 3 or L 4 is independently 0 amino acids in length.
  • L 1 , L 2 , L 3 and L 4 each independently are zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:40), GGGGSGGGGSGGGGS (SEQ ID NO: 41), S, RT, TKGPS (SEQ ID NO: 39), GQPKAAP (SEQ ID NO: 38), and GGSGSSGSGG (SEQ ID NO: 42).
  • L 1 , L 2 , L 3 and L 4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:40), GGGGSGGGGSGGGGS (SEQ ID NO:41), S, RT, TKGPS (SEQ ID NO:39), GQPKAAP (SEQ ID NO: 38), and
  • L 1 comprises the sequence GQPKAAP (SEQ ID NO: 38)
  • L 2 comprises the sequence TKGPS (SEQ ID NO:39)
  • L 3 comprises the sequence S
  • L 4 comprises the sequence RT.
  • at least one of L 1 , L 2 , L 3 or L 4 comprises the sequence DKTHT (SEQ ID NO:37).
  • L 1 , L 2 , L 3 and L 4 comprise the sequence DKTHT (SEQ ID NO:37).
  • the hinge-C H2 -C H3 domains of the second and the third polypeptide chains are human IgG4 hinge-C H2 -C H3 domains, and wherein the hinge-C H2 -C H3 domains each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L235A.
  • the hinge-C H2 -C H3 domains of the second and the third polypeptide chains are human IgG4 hinge-C H2 -C H3 domains, and wherein the hinge-C H2 - C H3 domains each comprise amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236.
  • the hinge-C H2 -C H3 domains of the second and the third polypeptide chains are human IgG4 hinge-C H2 -C H3 domains, and wherein the hinge-C H2 -C H3 domains each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K.
  • the hinge-C H2 -C H3 domains of the second and the third polypeptide chains are human IgG1 hinge-C H2 -C H3 domains, and wherein the hinge-C H2 -C H3 domains each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A.
  • the hinge-C H2 -C H3 domains of the second and the third polypeptide chains are human IgG1 hinge-C H2 -C H3 domains, and wherein the hinge-C H2 - C H3 domains each comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgG1 according to EU Index, wherein the amino acid substitutions are S298N, T299A, and Y300S.
  • the hinge-C H2 -C H3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the hinge-C H2 -C H3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W.
  • the hinge-C H2 -C H3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W; and wherein the hinge-C H2 -C H3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:61;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:62 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:62;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:63;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:64 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:64.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:65 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:65;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:66 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:66;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:67 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:67;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:68 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:68.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:69 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:69;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:70 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:70;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:71 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:71;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:72 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:72.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:73 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 73;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:74 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:74;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:75 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:75;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:76 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:76.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:77 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:77;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:78 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:78;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:79 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:79;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:80 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:80.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:81 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:81;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:82 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 82;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:83 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 83;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 84 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:84.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:85 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:85;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:86 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:86;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:87 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:87;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:88 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:88.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:89 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:89;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:90 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:90;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:91 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:91;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:92 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:92.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:93 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:93;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:94 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 94;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:95 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 95;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 96 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:96.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 97 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:97;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:98 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:98;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:99 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:99;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 100.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 101 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 101;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 102;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 103 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 104.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 105;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 107 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 107;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 108 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 109 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 109;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 110 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 110;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 111 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 111;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 112.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: l 13 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 113;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 114;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 115;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 116 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: l 16.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: l 17;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 119;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 121;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 122 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 123 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 123;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 129;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 130;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 131 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 131;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 132.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 133;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 135;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 136.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 137;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 138;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 139;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 140.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 141;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 142;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 143;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 144.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 145;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 146;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 147;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 148.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 149;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 150;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 151;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 152 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 152.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 153;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 154;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 155;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 156.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 157;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 158;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 159;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 160.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 161;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 162 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 162;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 163 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 163;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 164 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 164.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 165 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 165;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 166 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 166;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 167 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 167;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 168 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 168.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 169;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 170 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 170;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 171 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 171;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 172.
  • the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 173;
  • the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 174;
  • the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 175 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 175;
  • the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 176.
  • nucleic acid molecules comprising a nucleotide sequence encoding the binding protein of any one of the above embodiments.
  • expression vectors comprising the nucleic acid molecule of any one of the above embodiments.
  • isolated host cells comprising the nucleic acid molecule of any one of the above embodiments or the expression vector of any one of the above embodiments.
  • the host cell is a mammalian or insect cell.
  • compositions comprising the binding protein of any one of the above embodiments and a
  • kits for preventing and/or treating HIV infection in a patient comprising administering to the patient a therapeutically effective amount of at least one binding protein of any one of the above embodiments or the pharmaceutical composition of any one of the above embodiments.
  • the binding protein is co-administered with standard anti-retroviral therapy.
  • administration of the at least one binding protein results in the elimination of one or more latently and/or chronically HIV-infected cells in the patient.
  • the patient is a human.
  • the binding protein or pharmaceutical composition of any one of the above embodiments is provided for the prevention and/or treatment of HIV infection in a patient.
  • the binding protein is to be co-administered with standard anti-retroviral therapy. In some embodiments, the binding protein causes the elimination of one or more latently and/or chronically HIV-infected cells in the patient. In some embodiments, the patient is a human.
  • the binding protein or pharmaceutical composition of any one of the above embodiments is provided for use in the manufacture of a medicament for the prevention and/or treatment of HIV infection in a patient.
  • the binding protein is to be co-administered with standard anti-retroviral therapy.
  • the binding protein causes the elimination of one or more latently and/or chronically HIV-infected cells in the patient.
  • the patient is a human.
  • a vector system comprising one or more vectors encoding a first, second, third, and fourth polypeptide chain of a binding protein of any one of the above embodiments.
  • the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein.
  • kits comprising one, two, three, or four polypeptide chains of a binding protein according to any one of the above
  • kits further comprise instructions for using the polypeptide chain or binding protein according to any of the methods or uses described herein, e.g. , supra.
  • kits comprising one, two, three, or four polynucleotides according to any one of the above embodiments.
  • kits of polynucleotides comprising one, two, three, or four polynucleotides of a kit of polynucleotides comprising: (a) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 177, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 178, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 179, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 180; (b) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 181, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 182, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 180;
  • FIG. 1 provides a schematic representation of a trispecific binding protein comprising four polypeptide chains that form three antigen binding sites that binds three target proteins: CD28, CD3, and HIV Env.
  • a first pair of polypeptides possess dual variable domains having a cross-over orientation (VH1-VH2 and VL2-VL1) forming two antigen binding sites (VH1 and VL1; VH2 and VL2) that recognize CD28 and CD3, resepectively, and a second pair of polypeptides possess a single variable domain (VH3 and VL3) forming a single antigen binding site that recognizes HIV Env.
  • the trispecific binding protein shown in FIG. 1 uses a constant region with a“knobs-into-holes” mutation, where the knob is on the second pair of polypeptides with a single variable domain.
  • FIG. 2 shows a schematic representation of a trispecific T cell Engager (TCE) strategy for using the anti-HIV trispecific binding protein shown in FIG. 1 to target and eliminate the HIV reservoir.
  • TCE T cell Engager
  • the present disclosure provides trispecific and/or trivalent binding proteins comprising four polypeptide chains that form three antigen binding sites that specifically bind to one or more human immunodeficiency virus (HIV) target proteins and/or one or more T-cell receptor target proteins, wherein a first pair of polypeptides forming the binding protein possess dual variable domains having a cross-over orientation, and wherein a second pair of polypeptides possess a single variable domain.
  • HIV human immunodeficiency virus
  • polynucleotide refers to single-stranded or double- stranded nucleic acid polymers of at least 10 nucleotides in length.
  • the nucleotides comprising the polynucleotide can be ribonucleotides or
  • deoxyribonucleotides or a modified form of either type of nucleotide.
  • modifications include base modifications such as bromuridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as
  • polynucleotide specifically includes single-stranded and double-stranded forms of DNA.
  • An "isolated polynucleotide” is a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which: (1) is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.
  • An "isolated polypeptide” is one that: (1) is free of at least some other polypeptides with which it would normally be found, (2) is essentially free of other polypeptides from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a polypeptide with which the "isolated polypeptide" is associated in nature, (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature.
  • Such an isolated polypeptide can be encoded by genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof.
  • the isolated polypeptide is substantially free from polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).
  • Naturally occurring antibodies typically comprise a tetramer.
  • Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full-length "light” chain (typically having a molecular weight of about 25 kDa) and one full-length "heavy” chain (typically having a molecular weight of about 50-70 kDa).
  • the terms "heavy chain” and “light chain” as used herein refer to any combination of polypeptide chains
  • each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids that typically is responsible for antigen recognition.
  • the carboxy-terminal portion of each chain typically defines a constant domain responsible for effector function.
  • a full-length heavy chain immunoglobulin polypeptide includes a variable domain (VH) and three constant domains (C H1 , C H2 , and C H3 ), wherein the VH domain is at the amino-terminus of the polypeptide and the C H3 domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide includes a variable domain (VL) and a constant domain (CL), wherein the VL domain is at the amino-terminus of the polypeptide and the CL domain is at the carboxyl-terminus.
  • Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4.
  • IgM has subclasses including, but not limited to, IgM1 and IgM2.
  • IgA is similarly subdivided into subclasses including, but not limited to, IgA1 and IgA2.
  • variable and constant domains typically are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids.
  • the variable regions of each light/heavy chain pair typically form an antigen binding site.
  • the variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs.
  • both light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • CDR set 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 and Lesk, 1987, J. Mol. Biol.
  • 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, 1995, FASEB J. 9: 133- 39; MacCallum, 1996, J. Mol. Biol. 262(5): 732-45; and Lefranc, 2003, Dev. Comp.
  • CDR boundary definitions may not strictly follow one of the herein 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 certain embodiments use Rabat or Chothia defined CDRs. Identification of predicted CDRs using the amino acid sequence is well known in the field, such as in Martin, A.C. "Protein sequence and structure analysis of antibody variable domains," In Antibody Engineering, Vol. 2. Kontermann R., Diibel S., eds. Springer-Verlag, Berlin, p.
  • the amino acid sequence of the heavy and/or light chain variable domain may be also inspected to identify the sequences of the CDRs by other conventional methods, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability.
  • the numbered sequences may be aligned by eye, or by employing an alignment program such as one of the CLUSTAL suite of programs, as described in Thompson, 1994, Nucleic Acids Res. 22: 4673-80.
  • Molecular models are conventionally used to correctly delineate framework and CDR regions and thus correct the sequence-based assignments.
  • Fc refers to a molecule comprising the sequence of a non-antigen-binding fragment resulting from digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and can contain the hinge region.
  • the original immunoglobulin source of the native Fc is preferably of human origin and can be any of the immunoglobulins, although IgG1 and IgG2 are preferred.
  • Fc molecules are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent ( i.e ., disulfide bonds) and non-covalent association.
  • the number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgAl, and IgGA2).
  • class e.g., IgG, IgA, and IgE
  • subclass e.g., IgG1, IgG2, IgG3, IgAl, and IgGA2
  • Fc disulfide-bonded dimer resulting from papain digestion of an IgG.
  • Fc as used herein is generic to the monomeric, dimeric, and multimeric forms.
  • a F(ab) fragment typically includes one light chain and the VH and C H1 domains of one heavy chain, wherein the V H -C H1 heavy chain portion of the F(ab) fragment cannot form a disulfide bond with another heavy chain polypeptide.
  • a F(ab) fragment can also include one light chain containing two variable domains separated by an amino acid linker and one heavy chain containing two variable domains separated by an amino acid linker and a C H1 domain.
  • a F(ab') fragment typically includes one light chain and a portion of one heavy chain that contains more of the constant region (between the C H1 and C H2 domains), such that an interchain disulfide bond can be formed between two heavy chains to form a F(ab')2 molecule.
  • binding protein refers to a non-naturally occurring (or recombinant or engineered) molecule that specifically binds to at least one target antigen.
  • a trispecific binding protein of the present disclosure typically comprises four polypeptide chains that form at least three antigen binding sites, wherein a first polypeptide chain has a structure represented by the formula:
  • a third polypeptide chain has a structure represented by the formula:
  • polypeptide chain has a structure represented by the formula:
  • V L1 is a first immunoglobulin light chain variable domain
  • V L2 is a second immunoglobulin light chain variable domain
  • V L3 is a third immunoglobulin light chain variable domain
  • V H1 is a first immunoglobulin heavy chain variable domain
  • V H2 is a second immunoglobulin heavy chain variable domain
  • V H3 is a third immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H1 is the immunoglobulin C H1 heavy chain constant domain
  • hinge is an immunoglobulin hinge region connecting the C H1 and C H2 domains
  • L 1 , L 2 , L 3 and L 4 are amino acid linkers
  • polypeptide of formula I and the polypeptide of formula II form a cross over light chain-heavy chain pair.
  • a "recombinant" molecule is one that has been prepared, expressed, created, or isolated by recombinant means.
  • binding proteins having biological and immunological specificity to between one and three target antigens.
  • nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins.
  • expression vectors comprising nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins.
  • host cells that express such binding proteins (i.e., comprising nucleic acid molecules or vectors encoding polypeptide chains that form such binding proteins).
  • swapability refers to the interchangeability of variable domains within the binding protein format and with retention of folding and ultimate binding affinity.
  • Full swapability refers to the ability to swap the order of both V H1 and V H2 domains, and therefore the order of V L1 and V L2 domains, in the polypeptide chain of formula I or the polypeptide chain of formula II (i.e., to reverse the order) while
  • VH and VL refer only to the domain's location on a particular protein chain in the final format.
  • V H1 and V H2 could be derived from V L1 and V L2 domains in parent antibodies and placed into the V H1 and V H2 positions in the binding protein.
  • V L1 and V L2 could be derived from V H1 and V H2 domains in parent antibodies and placed in the V H1 and V H2 positions in the binding protein.
  • the VH and VL designations refer to the present location and not the original location in a parent antibody.
  • V H and VL domains are therefore "swappable.”
  • the term "antigen” or “target antigen” or “antigen target” as used herein refers to a molecule or a portion of a molecule that is capable of being bound by a binding protein, and additionally is capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen.
  • a target antigen may have one or more epitopes.
  • the binding protein is capable of competing with an intact antibody that recognizes the target antigen.
  • HIV Human Immunodeficiency Virus
  • HIV infection generally encompasses infection of a host, particularly a human host, by the human immunodeficiency virus (HIV) family of retroviruses including, but not limited to, HIV I, HIV II, HIV III (also known as HTLV-II, LAV-1, LAV-2). HIV can be used herein to refer to any strains, forms, subtypes, clades and variations in the HIV family.
  • HIV infection will encompass the treatment of a person who is a carrier of any of the HIV family of retroviruses or a person who is diagnosed with active AIDS, as well as the treatment or prophylaxis of the AIDS-related conditions in such persons.
  • AIDS as used herein means Acquired Immunodeficiency Syndrome. AIDS is caused by HIV.
  • CD4bs or “CD4 binding site” refer to the binding site for CD4 (cluster of differentiation 4), which is a glycoprotein found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells.
  • CD3 is cluster of differentiation factor 3 polypeptide and is a T-cell surface protein that is typically part of the T cell receptor (TCR) complex.
  • CD28 is cluster of differentiation 28 polypeptide and is a T-cell surface protein that provides co-stimulatory signals for T-cell activation and survival.
  • glycoprotein 160 or "gpl60 protein” refers to the envelope glycoprotein complex of HIV and which is a homotrimer that is cleaved into gpl20 and gp41 subunits.
  • MPER refers to the membrane-proximal external region of glycoprotein 41 (gp41), which is a subunit of the envelope protein complex of retroviruses, including HIV.
  • glycocan refers to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan.
  • glycan refers to the HIV-1 envelope glycoprotein gpl20.
  • T-cell engager refers to binding proteins directed to a host's immune system, more specifically the T cells' cytotoxic activity as well as directed to a HIV target protein.
  • trimer apex refers to apex of HIV-1 envelope glycoprotein gpl20.
  • the term "monospecific binding protein” refers to a binding protein that specifically binds to one antigen target.
  • the term "monovalent binding protein” refers to a binding protein that has one antigen binding site.
  • binding protein refers to a binding protein that specifically binds to two different antigen targets.
  • binding protein refers to a binding protein that has two binding sites.
  • trimer binding protein refers to a binding protein that specifically binds to three different antigen targets.
  • trivalent binding protein refers to a binding protein that has three binding sites.
  • the trivalent binding protein can bind to one antigen target.
  • the trivalent binding protein can bind to two antigen targets.
  • the trivalent binding protein can bind to three antigen targets.
  • An "isolated" binding protein is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the binding protein, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the binding protein will be purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated binding proteins include the binding protein in situ within recombinant cells since at least one component of the binding protein's natural environment will not be present.
  • substantially pure or “substantially purified” as used herein refer to a compound or species that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition). In some
  • a substantially purified fraction is a composition wherein the species comprises at least about 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will comprise more than about 80%, 85%, 90%, 95%, or 99% of all macromolar species present in the composition. In still other embodiments, the species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
  • a "neutralizing" binding protein as used herein refers to a molecule that is able to block or substantially reduce an effector function of a target antigen to which it binds.
  • substantially reduce means at least about 60%, preferably at least about 70%, more preferably at least about 75%, even more preferably at least about 80%, still more preferably at least about 85%, most preferably at least about 90% reduction of an effector function of the target antigen.
  • epitope includes any determinant, preferably a polypeptide determinant, capable of specifically binding to an immunoglobulin or T-cell receptor.
  • epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, 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 or binding protein.
  • a binding protein is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
  • a binding protein is said to specifically bind an antigen when the equilibrium dissociation constant is £ 10 -8 M, more preferably when the equilibrium dissociation constant is £ 10 -9 M, and most preferably when the dissociation constant is £ 10 -10 M.
  • the dissociation constant (KD) of a binding protein can be determined, for example, by surface plasmon resonance.
  • surface plasmon resonance analysis measures real-time binding interactions between ligand (a target antigen on a biosensor matrix) and analyte (a binding protein in solution) by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor; Piscataway, NJ).
  • SPR surface plasmon resonance
  • Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen).
  • KD as used herein refers to the dissociation constant of the interaction between a particular binding protein and a target antigen.
  • the term "specifically binds" as used herein refers to the ability of a binding protein or an antigen-binding fragment thereof to bind to an antigen containing an epitope with an Kd of at least about 1 x 10 -6 M, 1 x 10 -7 M, 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M, 1 x 10 -12 M, or more, and/or to bind to an epitope with an affinity that is at least two-fold greater than its affinity for a nonspecific antigen.
  • linker refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient mobility for the domains of the light and heavy chains to fold into cross over dual variable region immunoglobulins.
  • a linker is inserted at the transition between variable domains or between variable and constant domains, respectively, at the sequence level.
  • the transition between domains can be identified because the approximate size of the immunoglobulin domains are well understood.
  • the precise location of a domain transition can be determined by locating peptide stretches that do not form secondary structural elements such as beta-sheets or alpha-helices as demonstrated by experimental data or as can be assumed by techniques of modeling or secondary structure prediction.
  • the linkers described herein are referred to as L 1 , which is located on the light chain between the C-terminus of the V L2 and the N- terminus of the V L1 domain; and L 2 , which is located on the light chain between the C- terminus of the V L1 and the N-terminus of the CL domain.
  • the heavy chain linkers are known as L 3 , which is located between the C-terminus of the V H1 and the N-terminus of the V H2 domain; and L 4 , which is located between the C-terminus of the V H2 and the N- terminus of the C H1 domain.
  • vector refers to any molecule (e.g ., nucleic acid, plasmid, or virus) that is used to transfer coding information to a host cell.
  • vector includes a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double-stranded DNA molecule into which additional DNA segments may be inserted.
  • viral vector Another type of vector, wherein additional DNA segments may be inserted into the viral genome.
  • 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 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.
  • 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").
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector may be used interchangeably herein, as a plasmid is the most commonly used form of vector.
  • viral vectors e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses
  • viral vectors e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses
  • recombinant host cell refers to a cell into which a recombinant expression vector has been introduced.
  • a recombinant host cell or host cell is intended to refer not only to the particular subject cell, but also 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 such cells are still included within the scope of the term "host cell” as used herein.
  • host cell expression systems can be used to express the binding proteins, including bacterial, yeast, baculoviral, and mammalian expression systems (as well as phage display expression systems).
  • a suitable bacterial expression vector is pUC19.
  • a host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chains of the binding protein such that the polypeptide chains are expressed in the host cell and, preferably, secreted into the medium in which the host cells are cultured, from which medium the binding protein can be recovered.
  • transformation refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain a new DNA.
  • a cell is transformed where it is genetically modified from its native state.
  • the transforming DNA may recombine with that of the cell by physically integrating into a chromosome of the cell, or may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid.
  • a cell is considered to have been stably transformed when the DNA is replicated with the division of the cell.
  • transfection refers to the uptake of foreign or exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA has been introduced inside the cell membrane.
  • transfection techniques are well known in the art. Such techniques can be used to introduce one or more exogenous DNA molecules into suitable host cells.
  • non-naturally occurring refers to the fact that the object can be found in nature and has not been manipulated by man.
  • a polynucleotide or polypeptide that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man is naturally-occurring.
  • non-naturally occurring refers to an object that is not found in nature or that has been structurally modified or synthesized by man.
  • the twenty conventional amino acids and their abbreviations follow conventional usage.
  • Stereoisomers e.g ., D-amino acids
  • unnatural amino acids and analogs such as a-, a-di substituted amino acids, N- alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for the polypeptide chains of the binding proteins. Examples of
  • unconventional amino acids include: 4-hydroxyproline, g-carboxyglutamate, e-N,N,N- trimethyllysine, e-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, s-N-methylarginine, and other similar amino acids and imino acids (e.g ., 4-hydroxyproline).
  • the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl- terminal direction, in accordance with standard usage and convention.
  • Naturally occurring residues may be divided into classes based on common side chain properties:
  • Conservative amino acid substitutions may involve exchange of a member of one of these classes with another member of the same class.
  • Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class.
  • a skilled artisan will be able to determine suitable variants of the polypeptide chains of the binding proteins using well-known techniques. For example, one skilled in the art may identify suitable areas of a polypeptide chain that may be changed without destroying activity by targeting regions not believed to be important for activity.
  • patient as used herein includes human and animal subjects.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures.
  • Those in need of treatment include those having the disorder as well as those prone to have a disorder or those in which the disorder is to be prevented.
  • binding proteins can be used to treat humans infected with HIV, or humans susceptible to HIV infection, or ameliorate HIV infection in a human subject infected with HIV.
  • the binding proteins can also be used to prevent HIV in a human patient.
  • treating humans infected with HIV include those subjects who are at any one of the several stages of HIV infection progression, which, for example, include acute primary infection syndrome (which can be asymptomatic or associated with an influenza-like illness with fevers, malaise, diarrhea and neurologic symptoms such as headache), asymptomatic infection (which is the long latent period with a gradual decline in the number of circulating CD4 + T cells), and AIDS (which is defined by more serious AIDS-defming illnesses and/or a decline in the circulating CD4 cell count to below a level that is compatible with effective immune function).
  • acute primary infection syndrome which can be asymptomatic or associated with an influenza-like illness with fevers, malaise, diarrhea and neurologic symptoms such as headache
  • asymptomatic infection which is the long latent period with a gradual decline in the number of circulating CD4 + T cells
  • AIDS which is defined by more serious AIDS-defming illnesses and/or a decline in the circulating CD4 cell count to below a level that is compatible with effective immune function).
  • treating or preventing HIV infection will also encompass treating suspected infection by HIV after suspected past exposure to HIV by e.g., contact with HIV-contaminated blood, blood transfusion, exchange of body fluids,“unsafe” sex with an infected person, accidental needle stick, receiving a tattoo or acupuncture with contaminated instruments, or transmission of the virus from a mother to a baby during pregnancy, delivery or shortly thereafter.
  • composition or “therapeutic composition” as used herein refer to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.
  • pharmaceutically acceptable carrier or “physiologically acceptable carrier” as used herein refers to one or more formulation materials suitable for
  • a therapeutically effective amount when used in reference to a pharmaceutical composition comprising one or more binding proteins refer to an amount or dosage sufficient to produce a desired therapeutic result. More specifically, a therapeutically effective amount is an amount of a binding protein sufficient to inhibit, for some period of time, one or more of the clinically defined pathological processes associated with the condition being treated. The effective amount may vary depending on the specific binding protein that is being used, and also depends on a variety of factors and conditions related to the patient being treated and the severity of the disorder. For example, if the binding protein is to be administered in vivo , factors such as the age, weight, and health of the patient as well as dose response curves and toxicity data obtained in preclinical animal work would be among those factors considered. The determination of an effective amount or therapeutically effective amount of a given pharmaceutical composition is well within the ability of those skilled in the art.
  • One embodiment of the disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a binding protein.
  • the binding protein of the disclosure is a trispecific and/or trivalent binding protein comprising four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein a first polypeptide chain comprises a structure represented by the formula:
  • a second polypeptide chain comprises a structure represented by the formula:
  • a third polypeptide chain comprises a structure represented by the formula:
  • polypeptide chain comprises a structure represented by the formula:
  • V L1 is a first immunoglobulin light chain variable domain
  • V L2 is a second immunoglobulin light chain variable domain
  • V L3 is a third immunoglobulin light chain variable domain
  • V H1 is a first immunoglobulin heavy chain variable domain
  • V H2 is a second immunoglobulin heavy chain variable domain
  • V H3 is a third immunoglobulin heavy chain variable domain
  • CL is an immunoglobulin light chain constant domain
  • C H1 is an immunoglobulin C H1 heavy chain constant domain
  • C H2 is an immunoglobulin C H2 heavy chain constant domain
  • C H3 is an immunoglobulin C H3 heavy chain constant domain
  • hinge is an immunoglobulin hinge region connecting the C H1 and C H2 domains.
  • L 1 , L 2 , L 3 and L 4 are amino acid linkers
  • polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair.
  • the first polypeptide chain and the second polypeptide chain have a cross-over orientation that forms two distinct antigen binding sites.
  • the V H1 and V L1 form a binding pair and form the first antigen binding site.
  • the V H2 and V L2 form a binding pair and form the second antigen binding site.
  • the third polypeptide and the fourth polypeptide form a third antigen binding site.
  • the V H3 and V L3 form a binding pair and form the third antigen binding site.
  • one or more of the antigen binding sites binds an HIV target protein.
  • V H3 and V L3 form a third antigen binding site that binds an HIV target protein.
  • V H1 and V L1 form a first antigen binding site that binds a T cell target protein
  • V H2 and V L2 form a second antigen binding site that binds a T cell target protein
  • V H3 and V L3 form a third antigen binding site that binds an HIV target protein.
  • V H1 and V L1 form a first antigen binding site that binds a T cell target protein
  • V H2 and V L2 form a second antigen binding site that binds a CD3 polypeptide
  • V H3 and V L3 form a third antigen binding site that binds an HIV target protein.
  • V H1 and V L1 form a first antigen binding site that binds a CD28 polypeptide
  • V H2 and V L2 form a second antigen binding site that binds a CD3 polypeptide
  • V H3 and V L3 form a third antigen binding site that binds an HIV target protein.
  • the binding proteins specifically bind to one or more HIV target proteins (e.g ., as described infra ) and one or more target proteins on a T-cell including T cell receptor complex.
  • These T-cell engager binding proteins are capable of recruiting T cells transiently to target cells and, at the same time, activating the cytolytic activity of the T cells.
  • the T-cell engager trispecific antibodies can be used to activate HIV- 1 reservoirs and redirect/activate T cells to lyse latently infected HIV-1 + T cells.
  • target proteins on T cells include but are not limited to CD3 and CD28, among others.
  • the trispecific binding proteins may be generated by combining the antigen binding domains of two or more monospecific antibodies (parent antibodies) into one antibody. See International Publication Nos. WO 2011/038290 A2, WO 2013/086533 Al, WO 2013/070776 Al, WO 2012/154312 Al, and WO 2013/163427 Al.
  • the binding proteins of the disclosure may be prepared using domains or sequences obtained or derived from any human or non-human antibody, including, for example, human, murine, or humanized antibodies.
  • the trivalent binding protein is capable of binding three different antigen targets.
  • the binding protein is trispecific and one light chain-heavy chain pair is capable of binding two different antigen targets or epitopes and one light chain-heavy chain pair is capable of binding one antigen target or epitope.
  • a binding protein of the present disclosure binds one or more HIV target proteins and one or more T cell target proteins.
  • the binding protein is capable of specifically binding one HIV target protein and two different epitopes on a single T cell target protein.
  • the binding protein is capable of specifically binding one HIV target protein and two different T cell target proteins (e.g., CD28 and CD3).
  • the first and second polypeptide chains of the binding protein form two antigen binding sites that specifically target two T cell target proteins, and the third and fourth polypeptide chains of the binding protein form an antigen binding site that specifically binds an HIV target protein.
  • the one or more HIV target proteins are one or more of glycoprotein 120, glycoprotein 41, and glycoprotein 160.
  • the one or more T cell target proteins are one or more of CD3 and CD28.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:61; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:62 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:62; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:63 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:63; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 64 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:64.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:65 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:65; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:66 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:66; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:67 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:67; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 68.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:69 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:69; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:70 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:70; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:71 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:71; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 72 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:72.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:73 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:73; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:74 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:74; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:75 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:75; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:76 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:76.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:77 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:77; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:78 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:78; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:79 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:79; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 80 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:80.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:81 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:81; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:82 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:82; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:83 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:83; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 84 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:84.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:85 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:85; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:86 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:86; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:87 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:87; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:88 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:88.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:89 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:89; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:90 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:90; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:91 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:91; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 92 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:92.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:93 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:93; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:94 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:94; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:95 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:95; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 96 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:96.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:97 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:97; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:98 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:98; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:99 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:99; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 100.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 101 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 101; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 102; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 103 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 104.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 105; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 107 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 107; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 108 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 109 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 109; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 110 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 110; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 111 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 111; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 112.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 113; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 114; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 115; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 116 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 116.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 117; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 119; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 121; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 122 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 123 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 123; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 129; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 130; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 131 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 131; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 132.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 133; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 135; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 136.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 137; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 138; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 139; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 140.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 141; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 142; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 143; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 144.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 145; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 146; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 147; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 148.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 149; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 150; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 151; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 152 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 152.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 153; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 154; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 155; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 156.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 157; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 158; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 159; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 160.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 161; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 162 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 162; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 163 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 163; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 164 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 164.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 165 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 165; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 166 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 166; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 167 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 167; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 168 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 168.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 169; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 170 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 170; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 171 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 171; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 172.
  • a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 173; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 174; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 175 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 175; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 176.
  • Certain aspects of the present disclosure relate to binding proteins that comprise an antigen binding site that binds an HIV target protein or polypeptide.
  • the HIV target protein is glycoprotein 120, glycoprotein 41, or glycoprotein 160.
  • a binding protein binds one or more of: glycoprotein 120, glycoprotein 41, and glycoprotein 160.
  • Exemplary HIV target proteins include, without limitation, MPER of the HIV-1 gp41 protein, a CD4 binding site of the HIV- 1 gpl20 protein, a glycan in the V3 loop of the HIV-1 gpl20 protein, or a trimer apex of the HIV-1 gpl20 protein or gpl60.
  • a binding protein of the present disclosure comprises an antigen binding site that binds a CD4 binding site of the HIV-1 gpl20 protein.
  • Exemplary antigen binding sites that bind HIV target proteins contemplated for use herein include, without limitation, those described in International Publication No. WO2017/074878, such as those from antibodies CD4BS“a”, CD4BS“b”, MPER, MPER IOOW, V1/V2“a”, V1/V2“b”, or V3.
  • a binding protein comprising an antigen binding site that binds an HIV target protein is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent, or multispecific and/or multivalent.
  • a binding protein that comprises an antigen binding site that binds an HIV target protein is a trispecific binding protein comprising four polypeptides that form three antigen binding sites.
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of NCPIN (SEQ ID NO: l) a CDR- H2 sequence comprising the amino acid sequence of WMKPRHGAVSYARQLQG (SEQ ID NO:2), and a CDR-H3 sequence comprising the amino acid sequence of
  • VH antibody heavy chain variable
  • GKYCTARDYYNWDFEH SEQ ID NO:3
  • VL antibody light chain variable domain comprising a CDR-L1 sequence comprising the amino acid sequence of
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of NCPIN (SEQ ID NO: l) a CDR-H2 sequence comprising the amino acid sequence of
  • WMKPRHGAV S YARQLQG (SEQ ID NO:2), and a CDR-H3 sequence comprising the amino acid sequence of GKYCTARDYYNWDFEH (SEQ ID NO:3); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of RTSQYGSLA (SEQ ID NO:4), a CDR-L2 sequence comprising the amino acid sequence of SGSTRAA (SEQ ID NO:5), and a CDR-L3 sequence comprising the amino acid sequence of QQYEF (SEQ ID NO:6).
  • VL antibody light chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7) a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAV (SEQ ID NO:8) or IKPQYGAT (SEQ ID NO:9); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO: 11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO: 12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO: 13).
  • VH antibody heavy chain variable
  • VL antibody light chain variable domain comprising a CDR-L1 sequence comprising the amino acid sequence of GYTFTAHI
  • IKPQYGAV SEQ ID NO:8 or I
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR- H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7) a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAV (SEQ ID NO:8) or
  • IKPQYGAT SEQ ID NO:9; and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO: 11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO: 12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO: 13).
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7) a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAV (SEQ ID NO:8); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO: 11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO: 12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO: 13).
  • VH antibody heavy chain variable
  • VL antibody light chain variable domain comprising a CDR-L1 sequence comprising the amino acid sequence of GYTFTAHI
  • IKPQYGAV SEQ ID NO:8
  • VL antibody light chain variable domain comprising a CDR-L
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7) a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAV (SEQ ID NO: 8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO: 11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO: 12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO: 13).
  • VH antibody heavy chain variable
  • VL antibody light chain variable domain comprising a CDR-L1 sequence comprising the amino acid sequence of GYTFTAHI
  • IKPQYGAV SEQ ID NO: 8
  • VL antibody light chain variable domain comprising a CDR-L1 sequence
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7) a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAT (SEQ ID NO:9); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO: 11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO: 12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO: 13).
  • VH antibody heavy chain variable
  • VL antibody light chain variable domain comprising a CDR-L1 sequence comprising the amino acid sequence of GYTFTAHI
  • IKPQYGAT SEQ ID NO:9
  • VL antibody light chain variable domain comprising a CDR-L
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7) a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAT (SEQ ID NO:9); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO: 11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO: 12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO: 13).
  • VH antibody heavy chain variable
  • VL antibody light chain variable domain comprising a CDR-L1 sequence comprising the amino acid sequence of GYTFTAHI
  • IKPQYGAT SEQ ID NO:9
  • VL antibody light chain variable domain comprising a CDR-L1 sequence
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of DCTLN (SEQ ID NO: 14) a CDR- H2 sequence comprising the amino acid sequence of WLKPRWGAVNYARPLQG (SEQ ID NO: 15), and a CDR-H3 sequence comprising the amino acid sequence of
  • VH antibody heavy chain variable
  • GKNCDYNWDFEH SEQ ID NO: 16
  • an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of RTSQYGSLA (SEQ ID NO: 17), a CDR-L2 sequence comprising the amino acid sequence of SGSTRAA (SEQ ID NO: 18), and a CDR-L3 sequence comprising the amino acid sequence of QQYEF (SEQ ID NO: 19).
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of DCTLN (SEQ ID NO: 14) a CDR- H2 sequence comprising the amino acid sequence of WLKPRWGAVNYARPLQG (SEQ ID NO: 15), and a CDR-H3 sequence comprising the amino acid sequence of
  • VH antibody heavy chain variable
  • GKNCDYNWDFEH SEQ ID NO: 16
  • an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of RTSQYGSLA (SEQ ID NO: 17), a CDR-L2 sequence comprising the amino acid sequence of SGSTRAA (SEQ ID NO: 18), and a CDR-L3 sequence comprising the amino acid sequence of QQYEF (SEQ ID NO: 19).
  • a binding protein of the present disclosure comprises an antigen binding site with a VH domain comprising an extended heavy chain FR3 loop of antibody VRC03, e.g., as described in Liu, Q. et al. (2019) Nat. Commun. 10:721.
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:43, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:45.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:43, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:45.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • V S S (SEQ ID NO:44), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:44, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:45.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:44, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:45.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQY GAVNF GGGFRDRVTLTRD VYREIAYMDIRGLKPDDTAVYY C ARDRS Y GDS SWALD AWGQGTTVVVSA (SEQ ID NO:46), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:46, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:49.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:46, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:49.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • VL antibody light chain variable domain
  • VL antibody light chain variable domain
  • an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:47, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:49.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:47, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:49.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least
  • VH antibody heavy chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:48, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:49.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:48, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:49.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:50, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:51.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:50, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:51.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • the binding protein is a trispecific binding protein.
  • the trispecific binding protein comprising an antigen binding site that binds an HIV target protein, an antigen binding site that binds a CD28 polypeptide, and an antigen binding site that binds a CD3 polypeptide.
  • the binding protein is a trispecific binding protein comprising four
  • polypeptides comprising three antigen binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair ( e.g ., as described herein).
  • the VH and VL domains of any of the anti-CD38 antigen binding sites described above represent V H3 and V L3 and form a third antigen binding site that binds an HIV target protein.
  • V H1 and V L1 form a first antigen binding site that binds a CD28 polypeptide
  • V H2 and V L2 form a second antigen binding site that binds a CD3 polypeptide
  • the VH and VL domains of any of the anti-HIV antigen binding sites described above and/or in Table 1 represent V H3 and V L3 and form a third antigen binding site that binds an HIV target protein.
  • a binding protein comprising an anti-HIV antigen binding site of the present disclosure comprises 1, 2, 3, 4, 5, or all 6 CDR sequences of an anti-HIV antibody described in Table 1.
  • a binding protein comprising an anti-HIV antigen binding site of the present disclosure comprises a VH domain sequence and/or VL domain sequence of an anti-HIV antibody described in Table 1.
  • CD28 polypeptide is a human CD28 polypeptide, also known as Tp44.
  • Human CD28 polypeptides are known in the art and include, without limitation, the polypeptides represented by NCBI Accession Numbers XP_011510499.1 , XP_011510497.1 , XP_011510496.1 ,
  • a binding protein comprising an antigen binding site that binds a CD28 polypeptide is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent, or multispecific and/or multivalent.
  • a binding protein that comprises an antigen binding site that binds a CD28 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites.
  • a binding protein that comprises an antigen binding site that binds a CD28 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, and one of which binds a CD3 polypeptide.
  • a binding protein that comprises an antigen binding site that binds a CD28 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, one of which binds a CD3 polypeptide, and one of which binds an HIV target protein.
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:33); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO:36).
  • VH antibody heavy chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:33); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO:36).
  • VH antibody heavy chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNV NTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWG KGTTVTVSS (SEQ ID NO:59), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:59, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:60.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:59, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:60.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • the binding protein is a trispecific binding protein.
  • the trispecific binding protein comprising an antigen binding site that binds an HIV target protein, an antigen binding site that binds a CD28 polypeptide, and an antigen binding site that binds a CD3 polypeptide.
  • the binding protein is a trispecific binding protein comprising four
  • polypeptides comprising three antigen binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair ( e.g ., as described herein).
  • the VH and VL domains of any of the anti-CD28 antigen binding sites described above represent V H1 and V L1 and form a first antigen binding site that binds a CD28 polypeptide.
  • the VH and VL domains of any of the anti-CD28 antigen binding sites described above represent V H1 and V L1 and form a first antigen binding site that binds a CD28 polypeptide, V H2 and V L2 form a second antigen binding site that binds a CD3 polypeptide, and V H3 and V L3 and form a third antigen binding site that binds an HIV target protein.
  • a binding protein comprising an anti-CD28 antigen binding site of the present disclosure comprises 1, 2, 3, 4, 5, or all 6 CDR sequences of an anti-CD28 antibody described in Table 2.
  • a binding protein comprising an anti-CD28 antigen binding site of the present disclosure comprises a VH domain sequence and/or VL domain sequence of an anti-CD28 antibody described in Table 2.
  • CD3 polypeptide is a human CD3 polypeptide, including CD3-delta (also known as T3D, IMD19, and CD3-DELTA), CD3-epsilon (also known as T3E, IMD18, and TCRE), and CD3-gamma (also known as T3G, IMD17, and CD3-GAMMA).
  • CD3-delta also known as T3D, IMD19, and CD3-DELTA
  • CD3-epsilon also known as T3E, IMD18, and TCRE
  • CD3-gamma also known as T3G, IMD17, and CD3-GAMMA
  • Human CD3 polypeptides are known in the art and include, without limitation, the polypeptides represented by NCBI Accession Numbers XP 006510029.1 or NP_031674.1, or a polypeptide produced from NCBI Gene ID Numbers 915, 916, or 917.
  • a binding protein comprising an antigen binding site that binds a CD3 polypeptide is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent, or multispecific and/or multivalent.
  • a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites.
  • a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, and one of which binds a CD3 polypeptide.
  • a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, one of which binds a CD3 polypeptide, and one of which binds an HIV target protein.
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein Xi is E or Q, X2 is A or L, and X3 is Q, R, or F (SEQ ID NO:293), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYP
  • the CDR- L1 sequence of the V L2 domain comprises an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO:24), QSLVHENLQTY (SEQ ID NO:25), QSLVHENLFTY (SEQ ID NO:26), and Q SL VHENLRT Y (SEQ ID NO:27).
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:24), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).
  • VH antibody heavy chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR- H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR- H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:24), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).
  • VH antibody heavy chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO:25), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).
  • VH antibody heavy chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR- H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR- H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO:25), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).
  • VH antibody heavy chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO:26), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).
  • VH antibody heavy chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR- H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR- H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO:26), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).
  • VH antibody heavy chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO:27), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).
  • VH antibody heavy chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR- H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR- H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO:27), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).
  • VH antibody heavy chain variable
  • DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHQNAQT YL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 54),
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VET) domain comprising the amino acid sequence of SEQ ID NO:52, and/or an antibody light chain variable (VL) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57.
  • VT antibody heavy chain variable
  • VL antibody light chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VET) domain comprising the amino acid sequence of SEQ ID NO:52, and an antibody light chain variable (VL) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57.
  • VET antibody heavy chain variable
  • VL antibody light chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:54.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 54.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:55.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 55.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least
  • VH antibody heavy chain variable
  • VL antibody light chain variable domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:56.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 56.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:57.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 57.
  • VH antibody heavy chain variable
  • VL antibody light chain variable
  • anti-CD3 binding sites are described herein with high affinity binding to human CD3 polypeptides and potential manufacturing liabilities (e.g ., deamidation sites) removed.
  • the binding protein is a trispecific binding protein.
  • the trispecific binding protein comprising an antigen binding site that binds an HIV target protein, an antigen binding site that binds a CD28 polypeptide, and an antigen binding site that binds a CD3 polypeptide.
  • the binding protein is a trispecific binding protein comprising four
  • polypeptides comprising three antigen binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair (e.g., as described herein).
  • the VH and VL domains of any of the anti-CD3 antigen binding sites described above represent V H2 and V L2 and form a second antigen binding site that binds a CD3 polypeptide.
  • V H1 and V L1 form a first antigen binding site that binds a CD28 polypeptide
  • the VH and VL domains of any of the anti-CD3 antigen binding sites described above and/or in Table 3 represent V H2 and V L2 and form a second antigen binding site that binds a CD3 polypeptide
  • V H3 and V L3 form a third antigen binding site that binds an HIV target protein.
  • a binding protein comprising an anti-CD3 antigen binding site of the present disclosure comprises 1, 2, 3, 4, 5, or all 6 CDR sequences of an anti-CD3 antibody described in Table 3.
  • a binding protein comprising an anti-CD3 antigen binding site of the present disclosure comprises a VH domain sequence and/or VL domain sequence of an anti-CD3 antibody described in Table 3.
  • the linkers can also be 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids long.
  • L 1 , L 2 , L 3 , and L 4 in one binding protein may all have the same amino acid sequence or may all have different amino acid sequences.
  • linkers include, for example, GGGGSGGGGS (SEQ ID NO:40), GGGGSGGGGSGGGGS (SEQ ID NO: 41), S, RT, TKGPS (SEQ ID NO: 39), GQPKAAP (SEQ ID NO: 38), GGSGSSGSGG (SEQ ID NO: 42), and DKTHT (SEQ ID NO:37), as well as those disclosed in International Publication Nos. WO2017/074878 and W02017/180913.
  • linkers comprising randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine, and proline have been shown to be suitable in the binding proteins.
  • the identity and sequence of amino acid residues in the linker may vary depending on the type of secondary structural element necessary to achieve in the linker. For example, glycine, serine, and alanine are best for linkers having maximum flexibility.
  • glycine, proline, threonine, and serine are useful if a more rigid and extended linker is necessary. Any amino acid residue may be considered as a linker in combination with other amino acid residues to construct larger peptide linkers as necessary depending on the desired properties.
  • the length of L 1 is at least twice the length of L 3 .
  • the length of L 2 is at least twice the length of L 4 .
  • the length of L 1 is at least twice the length of L 3
  • the length of L 2 is at least twice the length of L 4 .
  • L 1 is 3 to 12 amino acid residues in length
  • L 2 is 3 to 14 amino acid residues in length
  • L 3 is 1 to 8 amino acid residues in length
  • L 4 is 1 to 3 amino acid residues in length.
  • L 1 is 5 to 10 amino acid residues in length
  • L 2 is 5 to 8 amino acid residues in length
  • L 3 is 1 to 5 amino acid residues in length
  • L 4 is 1 to 2 amino acid residues in length.
  • L 1 is 7 amino acid residues in length
  • L 2 is 5 amino acid residues in length
  • L 3 is 1 amino acid residue in length
  • L 4 is 2 amino acid residues in length.
  • L 1 , L 2 , L 3 and L 4 each independently are zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:40), GGGGS GGGGSGGGGS (SEQ ID NO: 41), S, RT, TKGPS (SEQ ID NO: 39), GQPKAAP (SEQ ID NO: 38), and GGSGSSGSGG (SEQ ID NO: 42).
  • L 1 , L 2 , L 3 and L 4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:40), GGGGS GGGGS GGGGS (SEQ ID NO:41), S, RT, TKGPS (SEQ ID NO:39), GQPKAAP (SEQ ID NO: 38), and
  • L 1 comprises the sequence GQPKAAP (SEQ ID NO: 38)
  • L 2 comprises the sequence TKGPS (SEQ ID NO:39)
  • L 3 comprises the sequence S
  • L 4 comprises the sequence RT.
  • At least one of L 1 , L 2 , L 3 or L 4 comprises the sequence
  • L 1 , L 2 , L 3 and L 4 comprise the sequence DKTHT (SEQ ID NO:37). Fc regions and constant domains
  • a binding protein of the present disclosure comprises a second polypeptide chain further comprising an Fc region linked to C H1 , the Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains.
  • a binding protein of the present disclosure comprises a third polypeptide chain further comprising an Fc region linked to C H1 , the Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains.
  • a binding protein of the present disclosure comprises a second polypeptide chain further comprising an Fc region linked to C H1 , the Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising an Fc region linked to C H1 , the Fc region comprising an immunoglobulin hinge region and C H2 and Cm immunoglobulin heavy chain constant domains.
  • a binding protein of the present disclosure comprises a full-length antibody heavy chain or a polypeptide chain comprising an Fc region.
  • the Fc region is a human Fc region, e.g ., a human IgG1, IgG2, IgG3, or IgG4 Fc region.
  • the Fc region includes an antibody hinge, C H1 , C H2 , C H3 , and optionally Cm domains.
  • the Fc region is a human IgG1 Fc region.
  • the Fc region is a human IgG4 Fc region.
  • the Fc region includes one or more of the mutations described infra.
  • the Fc region is an Fc region of one of the heavy chain polypeptides (e.g, polypeptide 2 or 3) of a binding protein shown in Table 4.
  • the heavy chain constant region is a constant region of one of the heavy chain polypeptides (e.g, polypeptide 2 or 3) of a binding protein shown in Table 4.
  • the light chain constant region is a constant region of one of the light chain polypeptides (e.g, polypeptide 1 or 4) of a binding protein shown in Table 4.
  • a binding protein of the present disclosure includes one or two Fc variants.
  • Fc variant refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fc variants, and their interaction with the salvage receptor, are known in the art.
  • Fc variant can comprise a molecule or sequence that is humanized from a non-human native Fc.
  • a native Fc comprises regions that can be removed because they provide structural features or biological activity that are not required for the antibody-like binding proteins of the invention.
  • Fc variant comprises a molecule or sequence that lacks one or more native Fc sites or residues, or in which one or more Fc sites or residues has be modified, that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody- dependent cellular cytotoxicity (ADCC).
  • ADCC antibody- dependent cellular cytotoxicity
  • a binding protein of the present disclosure (e.g ., a trispecific binding protein) comprises a“knob” mutation on the second polypeptide chain and a“hole” mutation on the third polypeptide chain.
  • a binding protein of the present disclosure comprises a“knob” mutation on the third polypeptide chain and a “hole” mutation on the second polypeptide chain.
  • the“knob” mutation comprises substitution(s) at positions corresponding to positions 354 and/or 366 of human IgG1 or IgG4 according to EU Index.
  • the amino acid substitutions are S354C, T366W, T366Y, S354C and T366W, or S354C and T366Y.
  • the“knob” mutation comprises substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to EU Index.
  • the amino acid substitutions are S354C and T366W.
  • the“hole” mutation comprises substitution(s) at positions corresponding to positions 407 and, optionally, 349, 366, and/or 368 and of human IgG1 or IgG4 according to EU Index.
  • the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S, and/or L368A.
  • the“hole” mutation comprises substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to EU Index.
  • the amino acid substitutions are Y349C, T366S, L368A, and Y407V.
  • the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitution(s) at positions corresponding to positions 366 and optionally 354 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are T366W or T366Y and optionally S354C; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 407 and optionally 349, 366, and/or 368 and of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y407V or Y407T and optionally Y
  • the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitution(s) at positions corresponding to positions 407 and optionally 349, 366, and/or 368 and of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S, and/or L368A; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 366 and optionally 354 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are
  • the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitution at position corresponding to position 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitution is T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 366, 368, and/or 407 and of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are T366S,
  • the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitution(s) at positions corresponding to positions 366, 368, and/or 407 and of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are T366S, L368A, and/or Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitution at position corresponding to position 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitution is T366W.
  • the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V.
  • the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3
  • the second Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W.
  • the first and/or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.
  • the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, S354C, T366W, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366,
  • the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366,
  • substitutions are S228P, Y349C, T366S, L368A, Y407V, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, S354C, T366W, and R409K.
  • the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A, L235A, S354C, and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349,
  • the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349,
  • substitutions are F234A, L235A, Y349C, T366S, L368A, and Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A, L235A, S354C, and T366W.
  • a binding protein of the present disclosure comprises one or more mutations to reduce effector function, e.g ., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/or antibody- dependent cellular cytotoxicity (ADCC).
  • ADCP Fc receptor-mediated antibody-dependent cellular phagocytosis
  • CDC complement-dependent cytotoxicity
  • ADCC antibody- dependent cellular cytotoxicity
  • the second polypeptide chain further comprises a first Fc region linked to C H1 , the first Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains; wherein the third polypeptide chain further comprises a second Fc region linked to C H1 , the second Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains; wherein the first and second Fc regions are human IgG1 Fc regions; and wherein the first and the second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to EU Index, wherein the amino acid substitutions are L234A and L235A.
  • the Fc regions of the second and the third polypeptide chains are human IgG1 Fc regions, and wherein the Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to EU Index, wherein the amino acid substitutions are L234A and L235A.
  • the second polypeptide chain further comprises a first Fc region linked to C H1 , the first Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains; wherein the third polypeptide chain further comprises a second Fc region linked to C H1 , the second Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains; wherein the first and second Fc regions are human IgG1 Fc regions; and wherein the first and the second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A.
  • the Fc regions of the second and the third polypeptide chains are human IgG1 Fc regions, and wherein the Fc regions each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A.
  • the Fc regions of the second and the third polypeptide chains are human IgG4 Fc regions, and the Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L235A.
  • the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to C H1 , the first Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to C H1 , the second Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to C H1 , the second Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to C H1 , the second Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to C H1 , the second F
  • immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains wherein the first and the second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L235A.
  • the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, F234A, L235A, S354C, T366W, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an
  • immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K.
  • the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234,
  • substitutions are S228P, F234A, L235A, S354C, T366W, and R409K.
  • the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate Fey I and/or Fcyll binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate Fcyl and/or Fcyll binding but do not affect FcRn binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 228 and/or 409 of human IgG4 according to EU Index. In some embodiments, the amino acid substitutions are S228P and /or R409K.
  • the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 234 and/or 235 of human IgG4 according to EU Index.
  • the amino acid substitutions are F234A and/or L235A.
  • the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 228, 234, 235, and/or 409 of human IgG4 according to EU Index.
  • the amino acid substitutions are S228P, F234A, L235A, and /or R409K.
  • the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index.
  • the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236.
  • the Fc region is a human IgG4 Fc region comprising amino acid mutations at substitutions corresponding to positions 228, 233-236, and/or 409 of human IgG4 according to EU Index.
  • the amino acid mutations are S228P; E233P, F234V, L235A, and a deletion at 236; and /or R409K.
  • the Fc region comprises one or more mutations that reduce or eliminate Fc receptor binding and/or effector function of the Fc region (e.g ., Fc receptor- mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/or antibody-dependent cellular cytotoxicity (ADCC)).
  • the Fc region is a human IgG1 Fc region comprising one or more amino acid substitutions at positions corresponding to positions 234, 235, and/or 329 of human IgG1 according to EU Index.
  • the amino acid substitutions are L234A, L235A, and/or P329A.
  • the Fc region is a human IgG1 Fc region comprising amino acid substitutions at positions corresponding to positions 298, 299, and/or 300 of human IgG1 according to EU Index.
  • the amino acid substitutions are S298N, T299A, and/or Y300S.
  • a binding protein of the present disclosure comprises one or more mutations to improve stability, e.g ., of the hinge region and/or dimer interface of IgG4 ( See e.g., Spiess, C. et al. (2013) J. Biol. Chem. 288:26583-26593).
  • the mutation comprises substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K.
  • the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to C H1 , the first Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to C H1 , the second Fc region comprising an immunoglobulin hinge region and C H2 and C H3 immunoglobulin heavy chain constant domains; wherein the first and second Fc regions are human IgG4 Fc regions; and wherein the first and the second Fc regions each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K.
  • a binding protein of the present disclosure comprises knob and hole mutations and one or more mutations to improve stability.
  • the first and/or second Fc regions are human IgG4 Fc regions.
  • the Fc region is a human IgG1 Fc region comprising one or more amino acid substitutions at positions corresponding to positions 234, 235, and/or 329 of human IgG1 according to EU Index.
  • the amino acid substitutions are L234A, L235A, and/or P329A.
  • the Fc region is a human IgG1 Fc region comprising amino acid substitutions at positions corresponding to positions 298, 299, and/or 300 of human IgG1 according to EU Index.
  • the amino acid substitutions are S298N, T299A, and/or Y300S. Nucleic acids
  • nucleic acid molecules comprising a nucleotide sequence encoding any of the binding proteins described herein.
  • Exemplary and non-limiting nucleic acid sequences are provided in Table 5.
  • kits of polynucleotides e.g. , that encode one or more polypeptides of a binding protein as described herein.
  • a kit of polynucleotides of the present disclosure comprises one, two, three, or four polynucleotides of a kit of polynucleotides comprising: (a) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 177, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 178, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 179, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 180; (b) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 181,
  • the vector system comprises one or more vectors encoding a first, second, third, and fourth polypeptide chain of any of the binding proteins described herein.
  • the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein, e.g ., as shown in the polynucleotides of Table 5.
  • the vector system comprises a first vector encoding the first and second polypeptide chains of the binding protein, and a second vector encoding the third and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and third polypeptide chains of the binding protein, and a second vector encoding the second and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and fourth polypeptide chains of the binding protein, and a second vector encoding the second and third polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first, second, third, and fourth polypeptide chains of the binding protein.
  • the one or more vectors of the vector system may be any of the vectors described herein.
  • the one or more vectors are expression vectors.
  • the first, second, third, and fourth polynucleotides are present on one or more expression vectors, e.g ., one, two, three, or four expression vectors.
  • polynucleotides that encode the polypeptides which form the binding proteins, incorporate these polynucleotides into recombinant expression vectors, and introduce such vectors into host cells.
  • Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Similarly, conventional techniques may be used for chemical syntheses, chemical analyses,
  • the isolated nucleic acid is operably linked to a
  • a promoter may refer to nucleic acid control sequences which direct transcription of a nucleic acid.
  • a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence of a binding protein if the promoter affects the transcription or expression of the coding sequence.
  • promoters may include, but are not limited to, promoters obtained from the genomes of viruses (such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40 (SV40), and the like), from heterologous eukaryotic promoters (such as the actin promoter, an immunoglobulin promoter, from heat- shock promoters, and the like), the CAG-promoter (Niwa et al., Gene 108(2): 193-9, 1991), the phosphoglycerate kinase (PGK)-promoter, a tetracycline-inducible promoter (Masui et al., Nucleic Acids Res.
  • viruses such as polyoma virus, fowlpox virus, aden
  • polynucleotides encoding binding proteins of the present disclosure may be under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein or other suitable promoter that will be readily recognized by one skilled in the art.
  • the isolated nucleic acid is incorporated into a vector.
  • the vector is an expression vector.
  • Expression vectors may include one or more regulatory sequences operatively linked to the polynucleotide to be expressed.
  • regulatory sequence includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals).
  • Suitable enhancers may include, but are not limited to, enhancer sequences from mammalian genes (such as globin, elastase, albumin, a-fetoprotein, insulin and the like), and enhancer sequences from a eukaryotic cell virus (such as SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, adenovirus enhancers, and the like).
  • mammalian genes such as globin, elastase, albumin, a-fetoprotein, insulin and the like
  • enhancer sequences from a eukaryotic cell virus such as SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, adenovirus enhancers, and the like).
  • suitable vectors may include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenoviral, vaccinia viral, Sindbis-viral, measles, herpes viral, lentiviral, retroviral, adeno-associated viral vectors, etc.).
  • Expression vectors can be used to transfect host cells, such as, for example, bacterial cells, yeast cells, insect cells, and mammalian cells.
  • Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art, and can be used to transfect any cell of interest.
  • a host cell comprising one or more isolated polynucleotides, vectors, and/or vector systems described herein.
  • an isolated host cell of the present disclosure is cultured in vitro.
  • the host cell is a bacterial cell (e.g, an E. coli cell).
  • the host cell is a yeast cell (e.g, an S. cerevisiae cell).
  • the host cell is an insect cell.
  • insect host cells may include, for example, Drosophila cells (e.g., S2 cells), Trichoplusia ni cells (e.g, High Five TM cells), and Spodoptera frugiperda cells (e.g, Sf21 or Sf9 cells).
  • the host cell is a mammalian cell.
  • mammalian host cells may include, for example, human embryonic kidney cells (e.g, 293 or 293 cells subcloned for growth in suspension culture), Expi293TM cells, CHO cells, baby hamster kidney cells (e.g, BHK, ATCC CCL 10), mouse sertoli cells (e.g, TM4 cells), monkey kidney cells (e.g, CV1 ATCC CCL 70), African green monkey kidney cells (e.g, VERO-76, ATCC CRL-1587), human cervical carcinoma cells (e.g, HELA, ATCC CCL 2), canine kidney cells (e.g, MDCK, ATCC CCL 34), buffalo rat liver cells (e.g, BRL 3A, ATCC CRL 1442), human lung cells (e.g, W138, ATCC CCL 75), human liver cells (e.g, Hep G2, HB 8065), mouse mammary tumor cells (e.g, MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4
  • the method includes a) culturing a host cell (e.g, any of the host cells described herein) comprising an isolated nucleic acid, vector, and/or vector system (e.g, any of the isolated nucleic acids, vectors, and/or vector systems described herein) under conditions such that the host cell expresses the binding protein; and b) isolating the binding protein from the host cell.
  • a host cell e.g, any of the host cells described herein
  • an isolated nucleic acid, vector, and/or vector system e.g, any of the isolated nucleic acids, vectors, and/or vector systems described herein
  • Methods of isolating proteins from cultured host cells are well known to one of ordinary skill in the art, including, for example, by affinity chromatography (e.g, two step affinity chromatography comprising protein A affinity chromatography followed by size exclusion chromatography) .
  • affinity chromatography e.g, two step affinity chromatography comprising protein A affinity chromatography followed by size exclusion chromatography
  • Therapeutic or pharmaceutical compositions comprising binding proteins are within the scope of the disclosure.
  • Such therapeutic or pharmaceutical compositions can comprise a therapeutically effective amount of a binding protein, or binding protein-drug conjugate, in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.
  • Acceptable formulation materials are nontoxic to recipients at the dosages and concentrations employed.
  • the pharmaceutical composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition.
  • Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or
  • hydroxypropyl-beta-cyclodextrin fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorb
  • the optimal pharmaceutical composition will be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, and desired dosage. Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the binding protein.
  • the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature.
  • a suitable vehicle or carrier for injection can be water, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration.
  • Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute.
  • binding protein compositions can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or an aqueous solution. Further, the binding protein can be formulated as a lyophilizate using appropriate excipients such as sucrose.
  • compositions of the disclosure can be selected for parenteral delivery or subcutaneous. Alternatively, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally.
  • compositions is within the skill of the art.
  • the formulation components are present in concentrations that are acceptable to the site of administration.
  • buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
  • the therapeutic compositions for use can be in the form of a pyrogen-free, parenterally acceptable, aqueous solution comprising the desired binding protein in a pharmaceutically acceptable vehicle.
  • a particularly suitable vehicle for parenteral injection is sterile distilled water in which a binding protein is formulated as a sterile, isotonic solution, properly preserved.
  • Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which can then be delivered via a depot injection.
  • Hyaluronic acid can also be used, and this can have the effect of promoting sustained duration in the circulation.
  • Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.
  • a pharmaceutical composition can be formulated for inhalation.
  • a binding protein can be formulated as a dry powder for inhalation.
  • Binding protein inhalation solutions can also be formulated with a propellant for aerosol delivery.
  • solutions can be nebulized.
  • binding proteins that are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules.
  • a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized.
  • Additional agents can be included to facilitate absorption of the binding protein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.
  • Another pharmaceutical composition can involve an effective quantity of binding proteins in a mixture with non-toxic excipients that are suitable for the manufacture of tablets.
  • Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
  • compositions of the disclosure will be evident to those skilled in the art, including formulations involving binding proteins in sustained- or controlled-delivery formulations.
  • Techniques for formulating a variety of other sustained- or controlled-delivery means such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art.
  • Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules.
  • Sustained release matrices can include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid.
  • Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art.
  • compositions to be used for in vivo administration typically must be sterile. This can be accomplished by filtration through sterile filtration membranes.
  • compositions are lyophilized, sterilization using this method can be conducted either prior to, or following, lyophilization and reconstitution.
  • the composition for parenteral administration can be stored in lyophilized form or in a solution.
  • parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the pharmaceutical composition can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.
  • Such formulations can be stored either in a ready -to-use form or in a form ( e.g ., lyophilized) requiring reconstitution prior to administration.
  • kits for producing a single-dose administration unit can each contain both a first container having a dried protein and a second container having an aqueous formulation. Also included within the scope of this disclosure are kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes).
  • the effective amount of a binding protein pharmaceutical composition to be employed therapeutically will depend, for example, upon the therapeutic context and objectives.
  • One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the binding protein is being used, the route of administration, and the size (body weight, body surface, or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
  • Dosing frequency will depend upon the pharmacokinetic parameters of the binding protein in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect.
  • the composition can therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages can be ascertained through use of appropriate dose-response data.
  • the route of administration of the pharmaceutical composition is in accord with known methods, e.g, orally; through injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release systems; or by implantation devices.
  • the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.
  • composition can also be administered locally via implantation of a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated.
  • a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated.
  • the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed- release bolus, or continuous administration.
  • the pharmaceutical compositions can be used to prevent and/or treat HIV infection.
  • the pharmaceutical compositions can be used as a standalone therapy or in combination with standard anti-retroviral therapy.
  • the disclosure also relates to a kit comprising a binding protein and other reagents useful for detecting target antigen levels in biological samples.
  • reagents can include a detectable label, blocking serum, positive and negative control samples, and detection reagents.
  • the kit comprises a composition comprising any binding protein, polynucleotide, vector, vector system, and/or host cell described herein.
  • the kit comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing a condition (e.g HIV infection) and may have a sterile access port (for example the container may be an
  • the label or package insert indicates that the composition is used for preventing, diagnosing, and/or treating the condition of choice.
  • the article of manufacture or kit may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • Certain aspects of the present disclosure relate to methods of preventing HIV infection in a patient, treating HIV infection in a patient, preventing AIDS in a patient, and treating AIDS in a patient, using any of the binding proteins or pharmaceutical compositions disclosed herein.
  • Any of the binding proteins or pharmaceutical compositions disclosed herein may find use in a method of the present disclosure, e.g ., methods for preventing HIV infection in a patient, treating HIV infection in a patient, preventing AIDS in a patient, and treating AIDS in a patient.
  • FIG. 1 illustrates an exemplary and non-limiting format for a trispecific binding protein that may be employed in the methods and uses described herein.
  • a proposed mechanism by which the binding protein shown in FIG. 1 may result in elimination of the HIV reservoir cells in a patient involves: (1) activation of latently infected CD4+ T cells via the anti-CD28 and anti-CD3 arms of the trispecific binding protein; (2) recruitment of CD8+ T cells to activated, latently infected CD4+ T cells via anti-Env and anti-CD3 arms; (3) activation of engaged CD8+ T cells via the anti-CD28 and anti-CD3 arms; and (4) killing of latently infected CD4+ T cells through a Perforin/Granzyme mechanism.
  • this mechanism is thought to activate and subsequently kill HIV-1 reservoir cells, providing a novel strategy for attacking the HIV-1 reservoir in a patient.
  • the methods of the present disclosure comprise
  • the at least one binding protein is administered in combination with an anti-retroviral therapy (e.g, an anti-HIV therepy).
  • an anti-retroviral therapy e.g, an anti-HIV therepy.
  • the at least one binding protein is administered before the anti-retroviral therapy. In some embodiments, the at least one binding protein is administered concurrently with the anti-retroviral therapy. In some embodiments, the at least one binding protein is administered after the anti-retroviral therapy. In some embodiments, the at least one binding protein is co-administered with any standard anti-retroviral therapy known in the art.
  • administration of the at least one binding protein or pharmaceutical composition results in elimination of one or more latently and/or chronically HIV-infected cells in the patient.
  • administration of the at least one binding protein results in neutralization of one or more HIV virions and results in elimination of one or more latently and/or chronically HIV-infected cells in the patient.
  • the patient is a human. Table 4. Trispecific binding protein polypeptide sequences

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • AIDS & HIV (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Communicable Diseases (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Using the Cross-Over-Dual-Variable Domain (CODv) format, the present disclosure relates to compositions comprising trispecific and/ or trivalent binding proteins comprising four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein a first pair of polypeptides forming the binding protein possess dual variable domains having a cross-over orientation, and wherein a second pair of polypeptides possess a single variable domain. Also provided herein are methods for making trispecific and/or trivalent binding proteins and uses of such binding proteins for the treatment and/or prevention of HIV/AIDS.

Description

TRISPECIFIC AND/OR TRIVALENT BINDING PROTEINS USING THE CROSS-OVER-DUAL-VARIABLE DOMAIN (CODV) FORMAT FOR TREATMENT OF HIV INFECTION
[0001] This application claims priority to U.S. Provisional Application No. 62/831,415, filed April 9, 2019, and EP Application No. EP19306312.0, filed October 8, 2019, the disclosures of each of which are incorporated herein by reference in their entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 183952031940SEQLIST.TXT, date recorded: March 25, 2020, size: 580 KB).
FIELD
[0003] The disclosure relates to trispecific and/or trivalent binding proteins comprising four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein a first pair of polypeptides forming the binding protein possess dual variable domains having a cross-over orientation. The disclosure also relates to methods for making trispecific and/or trivalent binding proteins and uses of such binding proteins for treating and/or preventing HIV/AIDS.
BACKGROUND
[0004] Anti-retroviral therapy (ART) has been the standard of care for HIV/AIDS patients in the past decades. ART drugs target internal proteins such as reverse
transcriptase (RT), integrase (IN), and viral protease (PI) by inhibiting reverse transcription of HIV-1 genome, integration of HIV-1 genome, and proteolytic cleavage of protein precursors that are necessary for the production of infectious viral particles. Treatment using ART or combination of different classes of ART results in inhibition of HIV- 1 replication and subsequent reduction of viremia, often to undetectable level (aviremic status). Although ART greatly helps HIV patients in controlling their disease progression, and containing the global HIV epidemic, it does require patients taking daily medicines often following a strict regimen. About 10% patients fail therapy each year due to drug toxicity, suboptimal adherence and emerging drug resistance. As more HIV patients can live a normal life span (over 80 years), chronic complications are of particular concern, such as aging and drug-drug interaction, and cardiovascular/renal/bone toxicities. The economic burden treating HIV/AIDS has not subsided thus far.
[0005] HIV latently infects long-lived resting memory CD4+ T cells and others as a form of proviral DNA integrated into the host genome. The latently infected cells survive for decades and self-renew like stem cells via homeostatic proliferation, which is regarded as an HIV-1 reservoir. The HIV-1 reservoirs are neither affected by ART nor the host immune system as they do not express viral proteins. Yet, a small proportion of cells among the reservoirs are randomly reactivated by unknown mechanism(s), which are responsible for recurrence of viremia once ART is stopped.
[0006] Therefore, a need exists for developing HIV/AIDS treatments to target the HIV- 1 reservoir(s), and ultimately eliminate them completely, achieving a cure, or long term remission of HIV without any further treatment. Any therapeutic strategy to eliminate the HIV-1 reservoir needs to activate the reservoir first, followed by elimination of the activated HIV-1 reservoir cells.
[0007] All references cited herein, including patent applications, patent publications, and UniProtKB/Swiss-Prot Accession numbers are herein incorporated by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference.
BRIEF SUMMARY
[0008] To meet these and other needs, provided herein are multispecific binding proteins ( e.g ., antibodies) that form three antigen binding sites. In some embodiments, the binding proteins bind one or more HIV target proteins and a CD3 polypeptide. In some embodiments, the binding proteins bind an HIV target protein, a CD28 polypeptide, and a CD3 polypeptide. The trispecific anti-HIV/CD28xCD3 T cell engager (TCE) concept disclosed herein is thought to be an effective eliminator of the HIV-1 reservoir through activation by anti-CD3, co-activation by anti-CD28, and subsequent killing of activated HIV-1 reservoir cells through anti-HIV/anti-CD28 by engaging activated CD8 T cells, providing a potential strategy for attacking the HIV-1 reservoir. In addition, anti-CD3 binding sites are described with high affinity binding to human CD3 polypeptides and potential manufacturing liabilities (e.g., deamidation sites) removed.
[0009] In some embodiments, provided herein are binding proteins comprising four polypeptide chains that form the three antigen binding sites that specifically bind one or more HIV target proteins, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2-L1-VL1-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VH1-L3-VH2-L4-CH1-hinge-CH2-CH3 [P]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-CH1-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV]
wherein:
VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VH1 is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is an immunoglobulin CH1 heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;
wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair;
wherein VH1 and VL1 form a first antigen binding site;
wherein VH2 and VL2 form a second antigen binding site that binds a CDS polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of
QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (SEQ ID NO:293), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30); and wherein VH3 and VL3 form a third antigen binding site that binds an HIV target protein.
[0010] In some embodiments, the first binding site binds a CD28 polypeptide ( e.g ., a human CD28 polypeptide). In some embodiments, the VH1 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:31), a CDR- H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:33), and the VL1 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO: 36). In some embodiments, the VH1 domain comprises the amino acid sequence of
QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGN VNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDV WGKGTTVTVSS (SEQ ID NO:59), and/or the VL1 domain comprises the amino acid sequence of
DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHT GVPSRF SGSGSGTDFTLTIS SLQPEDIAT YYCQQGQTYP YTF GQGTKLEIK (SEQ ID NO: 60).
[0011] In some embodiments, the CDR-L1 sequence of the VL2 domain comprises an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO:24), QSLVHENLQTY (SEQ ID NO:25), QSLVHENLFTY (SEQ ID NO:26), and QSLVHENLRTY (SEQ ID NO:27). In some embodiments, the VH2 domain comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:24), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30). In some embodiments, the VH2 domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO:25), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30). In some embodiments, the VH2 domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO:26), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30). In some embodiments, the VH2 domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO:27), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30). In some embodiments, the VH2 domain comprises the amino acid sequence of
QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKD K SN S Y AT Y Y AD S VKGRFTI SRDD SKNTL YLQMN SLR AEDT A V Y Y CRGV Y Y AL SPF DYWGQGTLVTVSS (SEQ ID NO:52), and/or the VL2 domain comprises an amino acid sequence selected from the group consisting of
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHQNAQT YL S W YLQKPGQ SPQ SLI YK V S NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQ YPFTFGSGTKVEIK
(SEQ ID NO: 54),
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLQTYL S W YLQKPGQ SPQ SLI YK V S NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK
(SEQ ID NO: 55),
DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVS NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK
(SEQ ID NO:56), and DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLRTYL S WYLQKPGQ SPQ SLIYK V S NRF S GVPDRF S GS GS GTDF TLKI SRVE AED V GV Y Y CGQGT Q YPF TF GSGTK VEIK
(SEQ ID NO:57). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:54. In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:55. In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:56. In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:57.
[0012] In some embodiments, the third antigen binding site binds an HIV target protein selected from the group consisting of glycoprotein 120, glycoprotein 41 and glycoprotein 160. In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of NCPIN (SEQ ID NO: l) a CDR-H2 sequence comprising the amino acid sequence of WMKPRHGAV S YARQLQG (SEQ ID NO:2), and a CDR-H3 sequence comprising the amino acid sequence of GKYCTARDYYNWDFEH (SEQ ID NO:3), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of RTSQYGSLA (SEQ ID NO:4), a CDR-L2 sequence comprising the amino acid sequence of SGSTRAA (SEQ ID NO:5), and a CDR-L3 sequence comprising the amino acid sequence of QQYEF (SEQ ID NO:6). In some embodiments, the Vro domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7) a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAV (SEQ ID NO:8) or IKPQYGAT (SEQ ID NO:9), and a CDR-H3 sequence comprising the amino acid sequence of DRSYGDSSWALDA (SEQ ID NO: 10), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO: 11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO: 12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO: 13). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of DCTLN (SEQ ID NO: 14) a CDR-H2 sequence comprising the amino acid sequence of WLKPRW GAVNY ARPLQG (SEQ ID NO: 15), and a CDR-H3 sequence comprising the amino acid sequence of GKNCDYNWDFEH (SEQ ID NO: 16), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of
RTSQYGSLA (SEQ ID NO: 17), a CDR-L2 sequence comprising the amino acid sequence of SGSTRAA (SEQ ID NO: 18), and a CDR-L3 sequence comprising the amino acid sequence of QQYEF (SEQ ID NO: 19). In some embodiments, the VH3 domain comprises the amino acid sequence of
QVRLSQSGGQMKKPGDSMRISCRASGYEFINCPINWIRLAPGKRPEWMGWMKPRH GAVSYARQLQGRVTMTRDMYSETAFLELRSLTSDDTAVYFCTRGKYCTARDYYN WDFEHWGQGTPVTVSS (SEQ ID NO:43), and/or the VL3 domain comprises the amino acid sequence of
SLTQ SPGTL SL SPGET All S CRT S Q Y GSL AW Y QQRPGQ APRL VI Y S GSTRA AGIPDRF SGSRWGPDYNLTISNLESGDF GVYY CQQYEFF GQGTKVQVDIK (SEQ ID NO:45).
In some embodiments, the VH3 domain comprises the amino acid sequence of
QVRLSQSGGQMKKPGDSMRISCRASGYEFINCPINWIRLAPGKRPEWMGWMKPRH GAVSYARQLQGRVTMTRQLSQDPDDPDWGTAFLELRSLTSDDTAVYFCTRGKYC T ARD YYNWDFEHW GQGTP VT V S S (SEQ ID NO:44), and/or the VL3 domain comprises the amino acid sequence of
SLTQ SPGTL SL SPGET All S CRT S Q Y GSL AW Y QQRPGQ APRL VI Y S GSTRA AGIPDRF SGSRWGPDYNLTISNLESGDF GVYY CQQYEFF GQGTKVQVDIK (SEQ ID NO:45).
In some embodiments, the VH3 domain comprises the amino acid sequence of
RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQ
Y GAVNF GGGFRDRVTLTRD VYREIAYMDIRGLKPDDT AV YY C ARDRS Y GD S SW A LDAWGQGTTVVVSA (SEQ ID NO:46), and/or the VL3 domain comprises the amino acid sequence of
YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDG VP SRF SGSGFHT SFNLTISDLQ ADDIAT YY CQ VLQFF GRGSRLHIK (SEQ ID NO:49). In some embodiments, the VH3 domain comprises the amino acid sequence of
RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQ
Y GATNF GGGFRDRVTLTRD VYREI AYMDIRGLKPDDT AVYY C ARDRS Y GD S SWA LDAWGQGTTVVVSA (SEQ ID NO:47), and/or the VL3 domain comprises the amino acid sequence of
YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDG VP SRF SGSGFHT SFNLTISDLQ ADDIAT YY CQ VLQFF GRGSRLHIK (SEQ ID NO:49). In some embodiments, the VH3 domain comprises the amino acid sequence of
RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQ
Y GA VNF GGGFRDR VTLTRQL S QDPDDPD W GI A YMDIRGLKPDDT A V Y Y C ARDRS
Y GD S S WALD AW GQGTT VVV S A (SEQ ID NO:48), and/or the VL3 domain comprises the amino acid sequence of
YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDG VP SRF SGSGFHT SFNLTISDLQ ADDIAT YY CQ VLQFF GRGSRLHIK (SEQ ID NO:49). In some embodiments, the VH3 domain comprises the amino acid sequence of
Q VQL VQ S GGQMKKP GE SMRI S CRA S GYEFID C TLNWIRL AP GKRPEWMGWLKPR WGAVNYARPLQGRVTMTRQLSQDPDDPDWGTAFLELRSLTVDDTAVYFCTRGKN CD YNWDFEHW GRGTP VI V S S (SEQ ID NO:50), and/or the VL3 domain comprises the amino acid sequence of
LTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFS GSRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIK (SEQ ID NO:51).
[0013] In some embodiments that may be combined with any other embodiments described herein, at least one of L1, L2, L3 or L4 is independently 0 amino acids in length.
In some embodiments, L1, L2, L3 and L4 each independently are zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:40), GGGGSGGGGSGGGGS (SEQ ID NO: 41), S, RT, TKGPS (SEQ ID NO: 39), GQPKAAP (SEQ ID NO: 38), and GGSGSSGSGG (SEQ ID NO: 42). In some embodiments, L1, L2, L3 and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:40), GGGGSGGGGSGGGGS (SEQ ID NO:41), S, RT, TKGPS (SEQ ID NO:39), GQPKAAP (SEQ ID NO: 38), and
GGSGSSGSGG (SEQ ID NO:42). In some embodiments, L1 comprises the sequence GQPKAAP (SEQ ID NO: 38), L2 comprises the sequence TKGPS (SEQ ID NO:39), L3 comprises the sequence S, and L4 comprises the sequence RT. In some embodiments, at least one of L1, L2, L3 or L4 comprises the sequence DKTHT (SEQ ID NO:37). In some embodiments, L1, L2, L3 and L4 comprise the sequence DKTHT (SEQ ID NO:37). [0014] In some embodiments that may be combined with any other embodiments described herein, the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgG4 hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L235A. In some embodiments, the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgG4 hinge-CH2-CH3 domains, and wherein the hinge-CH2- CH3 domains each comprise amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgG4 hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgG1 hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgG1 hinge-CH2-CH3 domains, and wherein the hinge-CH2- CH3 domains each comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgG1 according to EU Index, wherein the amino acid substitutions are S298N, T299A, and Y300S. In some embodiments, the hinge-CH2-CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the hinge-CH2-CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W. In some embodiments, the hinge-CH2-CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W; and wherein the hinge-CH2-CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V.
[0015] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:61; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:62 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:62; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:63; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:64 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:64. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:65 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:65; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:66 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:66; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:67 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:67; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:68 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:68. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:69 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:69; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:70 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:70; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:71 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:71; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:72 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:72. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:73 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 73; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:74 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:74; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:75 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:75; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:76 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:76. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:77 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:77; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:78 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:78; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:79 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:79; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:80 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:80. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:81 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:81; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:82 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 82; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:83 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 83; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 84 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:84. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:85 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:85; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:86 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:86; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:87 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:87; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:88 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:88. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:89 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:89; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:90 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:90; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:91 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:91; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:92 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:92. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:93 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:93; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:94 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 94; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:95 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 95; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 96 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:96. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 97 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:97; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:98 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:98; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:99 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:99; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 100. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 101 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 101; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 102; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 103 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 104. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 105; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 107 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 107; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 108 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 109 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 109; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 110 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 110; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 111 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 111; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: l 13 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 113; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 114; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 115; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 116 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: l 16. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: l 17; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 119; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 121; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 122 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 123 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 123; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 129; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 130; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 131 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 131; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 132. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 133; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 135; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 136. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 137; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 138; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 139; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 141; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 142; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 143; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 144. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 145; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 146; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 147; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 148. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 149; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 150; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 151; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 152 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 152. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 153; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 154; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 155; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 156. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 157; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 158; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 159; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 160. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 161; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 162 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 162; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 163 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 163; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 164 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 164. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 165 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 165; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 166 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 166; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 167 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 167; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 168 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 168. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 169; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 170 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 170; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 171 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 171; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 172. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 173; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 174; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 175 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 175; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 176.
[0016] In some embodiments, provided herein are isolated nucleic acid molecules comprising a nucleotide sequence encoding the binding protein of any one of the above embodiments. In some embodiments, provided herein are expression vectors comprising the nucleic acid molecule of any one of the above embodiments. In some embodiments, provided herein are isolated host cells comprising the nucleic acid molecule of any one of the above embodiments or the expression vector of any one of the above embodiments. In some embodiments, the host cell is a mammalian or insect cell.
[0017] In some embodiments, provided herein are pharmaceutical compositions comprising the binding protein of any one of the above embodiments and a
pharmaceutically acceptable carrier.
[0018] In some embodiments, provided herein are methods of preventing and/or treating HIV infection in a patient comprising administering to the patient a therapeutically effective amount of at least one binding protein of any one of the above embodiments or the pharmaceutical composition of any one of the above embodiments. In some embodiments, the binding protein is co-administered with standard anti-retroviral therapy. In some embodiments, administration of the at least one binding protein results in the elimination of one or more latently and/or chronically HIV-infected cells in the patient. In some embodiments, the patient is a human. [0019] In some embodiments, the binding protein or pharmaceutical composition of any one of the above embodiments is provided for the prevention and/or treatment of HIV infection in a patient. In some embodiments, the binding protein is to be co-administered with standard anti-retroviral therapy. In some embodiments, the binding protein causes the elimination of one or more latently and/or chronically HIV-infected cells in the patient. In some embodiments, the patient is a human.
[0020] In some embodiments, the binding protein or pharmaceutical composition of any one of the above embodiments is provided for use in the manufacture of a medicament for the prevention and/or treatment of HIV infection in a patient. In some embodiments, the binding protein is to be co-administered with standard anti-retroviral therapy. In some embodiments, the binding protein causes the elimination of one or more latently and/or chronically HIV-infected cells in the patient. In some embodiments, the patient is a human.
[0021] In some embodiments, provided herein is a vector system comprising one or more vectors encoding a first, second, third, and fourth polypeptide chain of a binding protein of any one of the above embodiments. In some embodiments, the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein.
[0022] In some embodiments, provided herein are kits comprising one, two, three, or four polypeptide chains of a binding protein according to any one of the above
embodiments. In some embodiments, the kits further comprise instructions for using the polypeptide chain or binding protein according to any of the methods or uses described herein, e.g. , supra.
[0023] In some embodiments, provided herein are kits comprising one, two, three, or four polynucleotides according to any one of the above embodiments. In some
embodiments, provided herein are kits of polynucleotides comprising one, two, three, or four polynucleotides of a kit of polynucleotides comprising: (a) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 177, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 178, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 179, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 180; (b) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 181, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 182, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 183, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 184; (c) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 185, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 186, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 187, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 188; (d) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 189, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 190, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 191, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 192; (e) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 193, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 194, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 195, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 196; (f) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 197, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 198, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 199, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:200; (g) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:201, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:202, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:203, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:204; (h) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:205, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:206, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:207, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:208; (i) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:209, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:210, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:211, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:212; (j) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:213, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:214, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:215, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:216; (k) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:217, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:218, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:219, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:220; (1) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:221, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:222, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:223, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:224; (m) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:225, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:226, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:227, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:228; (n) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:229, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:230, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:231, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:232; (o) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:233, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:234, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:235, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:236; (p) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:237, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:238, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:239, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:240; (q) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:241, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:242, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:243, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:244; (r) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:245, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:246, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:247, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:248; (s) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:249, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:250, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:251, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:252; (t) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:253, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:254, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:255, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:256; (u) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:257, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:258, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:259, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:260; (v) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:261, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:262, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:263, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:264; (w) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:265, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:266, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:267, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:268; (x) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:269, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:270, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:271, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:272; (y) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:273, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:274, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:275, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:276; (z) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:277, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:278, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:279, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:280; (aa) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:281, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:282, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:283, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:284; (bb) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:285, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:286, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:287, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:288; or (cc) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:289, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:290, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:291, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:292. In some embodiments, the first, second, third, and fourth polynucleotides are present on one or more expression vectors, e.g ., one, two, three, or four expression vectors.
[0024] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 provides a schematic representation of a trispecific binding protein comprising four polypeptide chains that form three antigen binding sites that binds three target proteins: CD28, CD3, and HIV Env. A first pair of polypeptides possess dual variable domains having a cross-over orientation (VH1-VH2 and VL2-VL1) forming two antigen binding sites (VH1 and VL1; VH2 and VL2) that recognize CD28 and CD3, resepectively, and a second pair of polypeptides possess a single variable domain (VH3 and VL3) forming a single antigen binding site that recognizes HIV Env. The trispecific binding protein shown in FIG. 1 uses a constant region with a“knobs-into-holes” mutation, where the knob is on the second pair of polypeptides with a single variable domain.
[0026] FIG. 2 shows a schematic representation of a trispecific T cell Engager (TCE) strategy for using the anti-HIV trispecific binding protein shown in FIG. 1 to target and eliminate the HIV reservoir. DETAILED DESCRIPTION
[0027] The present disclosure provides trispecific and/or trivalent binding proteins comprising four polypeptide chains that form three antigen binding sites that specifically bind to one or more human immunodeficiency virus (HIV) target proteins and/or one or more T-cell receptor target proteins, wherein a first pair of polypeptides forming the binding protein possess dual variable domains having a cross-over orientation, and wherein a second pair of polypeptides possess a single variable domain.
[0028] The following description sets forth exemplary methods, parameters, and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.
Definitions
[0029] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
[0030] It is understood that aspects and embodiments of the disclosure described herein include“comprising,”“consisting,” and“consisting essentially of’ aspects and
embodiments.
[0031] The term "polynucleotide" as used herein refers to single-stranded or double- stranded nucleic acid polymers of at least 10 nucleotides in length. In certain embodiments, the nucleotides comprising the polynucleotide can be ribonucleotides or
deoxyribonucleotides or a modified form of either type of nucleotide. Such modifications include base modifications such as bromuridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as
phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate. The term
"polynucleotide" specifically includes single-stranded and double-stranded forms of DNA.
[0032] An "isolated polynucleotide" is a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which: (1) is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence. [0033] An "isolated polypeptide" is one that: (1) is free of at least some other polypeptides with which it would normally be found, (2) is essentially free of other polypeptides from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a polypeptide with which the "isolated polypeptide" is associated in nature, (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such an isolated polypeptide can be encoded by genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof. Preferably, the isolated polypeptide is substantially free from polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).
[0034] Naturally occurring antibodies typically comprise a tetramer. Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full-length "light" chain (typically having a molecular weight of about 25 kDa) and one full-length "heavy" chain (typically having a molecular weight of about 50-70 kDa). The terms "heavy chain" and "light chain" as used herein refer to any
immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids that typically is responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant domain responsible for effector function. Thus, in a naturally occurring antibody, a full-length heavy chain immunoglobulin polypeptide includes a variable domain (VH) and three constant domains (CH1, CH2, and CH3 ), wherein the VH domain is at the amino-terminus of the polypeptide and the CH3 domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide includes a variable domain (VL) and a constant domain (CL), wherein the VL domain is at the amino-terminus of the polypeptide and the CL domain is at the carboxyl-terminus.
[0035] Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgA1 and IgA2. Within full-length light and heavy chains, the variable and constant domains typically are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See , e.g., FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., Raven Press, 2nd ed., 1989), which is incorporated by reference in its entirety for all purposes. The variable regions of each light/heavy chain pair typically form an antigen binding site. The variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope. From the amino-terminus to the carboxyl-terminus, both light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
[0036] The term "CDR set" 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 and Lesk, 1987, J. Mol. Biol. 196: 901-17; Chothia et al ., 1989, Nature 342: 877-83) 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 chain 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, 1995, FASEB J. 9: 133- 39; MacCallum, 1996, J. Mol. Biol. 262(5): 732-45; and Lefranc, 2003, Dev. Comp.
Immunol. 27: 55-77. Still other CDR boundary definitions may not strictly follow one of the herein 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 certain embodiments use Rabat or Chothia defined CDRs. Identification of predicted CDRs using the amino acid sequence is well known in the field, such as in Martin, A.C. "Protein sequence and structure analysis of antibody variable domains," In Antibody Engineering, Vol. 2. Kontermann R., Diibel S., eds. Springer-Verlag, Berlin, p. 33-51 (2010). The amino acid sequence of the heavy and/or light chain variable domain may be also inspected to identify the sequences of the CDRs by other conventional methods, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. The numbered sequences may be aligned by eye, or by employing an alignment program such as one of the CLUSTAL suite of programs, as described in Thompson, 1994, Nucleic Acids Res. 22: 4673-80. Molecular models are conventionally used to correctly delineate framework and CDR regions and thus correct the sequence-based assignments.
[0037] The term "Fc" as used herein refers to a molecule comprising the sequence of a non-antigen-binding fragment resulting from digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and can contain the hinge region. The original immunoglobulin source of the native Fc is preferably of human origin and can be any of the immunoglobulins, although IgG1 and IgG2 are preferred. Fc molecules are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent ( i.e ., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgAl, and IgGA2). One example of a Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG. The term " Fc" as used herein is generic to the monomeric, dimeric, and multimeric forms.
[0038] A F(ab) fragment typically includes one light chain and the VH and CH1 domains of one heavy chain, wherein the VH-CH1 heavy chain portion of the F(ab) fragment cannot form a disulfide bond with another heavy chain polypeptide. As used herein, a F(ab) fragment can also include one light chain containing two variable domains separated by an amino acid linker and one heavy chain containing two variable domains separated by an amino acid linker and a CH1 domain. [0039] A F(ab') fragment typically includes one light chain and a portion of one heavy chain that contains more of the constant region (between the CH1 and CH2 domains), such that an interchain disulfide bond can be formed between two heavy chains to form a F(ab')2 molecule.
[0040] The term "binding protein" as used herein refers to a non-naturally occurring (or recombinant or engineered) molecule that specifically binds to at least one target antigen.
A trispecific binding protein of the present disclosure, unless otherwise specified, typically comprises four polypeptide chains that form at least three antigen binding sites, wherein a first polypeptide chain has a structure represented by the formula:
VL2- L1-VL1- L2-CL [I]
and a second polypeptide chain has a structure represented by the formula:
VH1-L3- VH2-L4-CH1-hinge-CH2-CH3 [II]
and a third polypeptide chain has a structure represented by the formula:
VH3-CH1 [III]
and a fourth polypeptide chain has a structure represented by the formula:
VL3-CL [IV]
wherein:
VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VH1 is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is the immunoglobulin CH1 heavy chain constant domain; and
hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains;
L1, L2, L3 and L4 are amino acid linkers;
and wherein the polypeptide of formula I and the polypeptide of formula II form a cross over light chain-heavy chain pair.
[0041] A "recombinant" molecule is one that has been prepared, expressed, created, or isolated by recombinant means.
[0042] One embodiment of the disclosure provides binding proteins having biological and immunological specificity to between one and three target antigens. Another embodiment of the disclosure provides nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins. Another embodiment of the disclosure provides expression vectors comprising nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins. Yet another embodiment of the disclosure provides host cells that express such binding proteins (i.e., comprising nucleic acid molecules or vectors encoding polypeptide chains that form such binding proteins).
[0043] The term "swapability" as used herein refers to the interchangeability of variable domains within the binding protein format and with retention of folding and ultimate binding affinity. "Full swapability" refers to the ability to swap the order of both VH1 and VH2 domains, and therefore the order of VL1 and VL2 domains, in the polypeptide chain of formula I or the polypeptide chain of formula II (i.e., to reverse the order) while
maintaining full functionality of the binding protein as evidenced by the retention of binding affinity. Furthermore, it should be noted that the designations VH and VL refer only to the domain's location on a particular protein chain in the final format. For example, VH1 and VH2 could be derived from VL1 and VL2 domains in parent antibodies and placed into the VH1 and VH2 positions in the binding protein. Likewise, VL1 and VL2 could be derived from VH1 and VH2 domains in parent antibodies and placed in the VH1 and VH2 positions in the binding protein. Thus, the VH and VL designations refer to the present location and not the original location in a parent antibody. VH and VL domains are therefore "swappable." [0044] The term "antigen" or "target antigen" or "antigen target" as used herein refers to a molecule or a portion of a molecule that is capable of being bound by a binding protein, and additionally is capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. A target antigen may have one or more epitopes.
With respect to each target antigen recognized by a binding protein, the binding protein is capable of competing with an intact antibody that recognizes the target antigen.
[0045] The term "HIV" as used herein means Human Immunodeficiency Virus. As used herein, the term“HIV infection” generally encompasses infection of a host, particularly a human host, by the human immunodeficiency virus (HIV) family of retroviruses including, but not limited to, HIV I, HIV II, HIV III (also known as HTLV-II, LAV-1, LAV-2). HIV can be used herein to refer to any strains, forms, subtypes, clades and variations in the HIV family. Thus, treating HIV infection will encompass the treatment of a person who is a carrier of any of the HIV family of retroviruses or a person who is diagnosed with active AIDS, as well as the treatment or prophylaxis of the AIDS-related conditions in such persons.
[0046] The term "AIDS" as used herein means Acquired Immunodeficiency Syndrome. AIDS is caused by HIV.
[0047] The terms "CD4bs" or "CD4 binding site" refer to the binding site for CD4 (cluster of differentiation 4), which is a glycoprotein found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells.
[0048] The term "CD3" is cluster of differentiation factor 3 polypeptide and is a T-cell surface protein that is typically part of the T cell receptor (TCR) complex.
[0049] "CD28" is cluster of differentiation 28 polypeptide and is a T-cell surface protein that provides co-stimulatory signals for T-cell activation and survival.
[0050] The term "glycoprotein 160" or "gpl60 protein" refers to the envelope glycoprotein complex of HIV and which is a homotrimer that is cleaved into gpl20 and gp41 subunits.
[0051] The term "MPER" refers to the membrane-proximal external region of glycoprotein 41 (gp41), which is a subunit of the envelope protein complex of retroviruses, including HIV.
[0052] The term "glycan" refers to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan. In the disclosed binding proteins, glycan refers to the HIV-1 envelope glycoprotein gpl20.
[0053] The term "T-cell engager" refers to binding proteins directed to a host's immune system, more specifically the T cells' cytotoxic activity as well as directed to a HIV target protein.
[0054] The term "trimer apex" refers to apex of HIV-1 envelope glycoprotein gpl20.
[0055] The term "monospecific binding protein" refers to a binding protein that specifically binds to one antigen target.
[0056] The term "monovalent binding protein" refers to a binding protein that has one antigen binding site.
[0057] The term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigen targets.
[0058] The term "bivalent binding protein" refers to a binding protein that has two binding sites. [0059] The term "trispecific binding protein" refers to a binding protein that specifically binds to three different antigen targets.
[0060] The term "trivalent binding protein" refers to a binding protein that has three binding sites. In particular embodiments the trivalent binding protein can bind to one antigen target. In other embodiments, the trivalent binding protein can bind to two antigen targets. In other embodiments, the trivalent binding protein can bind to three antigen targets.
[0061] An "isolated" binding protein is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the binding protein, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the binding protein will be purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated binding proteins include the binding protein in situ within recombinant cells since at least one component of the binding protein's natural environment will not be present.
[0062] The terms "substantially pure" or "substantially purified" as used herein refer to a compound or species that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition). In some
embodiments, a substantially purified fraction is a composition wherein the species comprises at least about 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will comprise more than about 80%, 85%, 90%, 95%, or 99% of all macromolar species present in the composition. In still other embodiments, the species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
[0063] A "neutralizing" binding protein as used herein refers to a molecule that is able to block or substantially reduce an effector function of a target antigen to which it binds.
As used herein, "substantially reduce" means at least about 60%, preferably at least about 70%, more preferably at least about 75%, even more preferably at least about 80%, still more preferably at least about 85%, most preferably at least about 90% reduction of an effector function of the target antigen.
[0064] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specifically binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, 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 or binding protein. In certain embodiments, a binding protein is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. In some embodiments, a binding protein is said to specifically bind an antigen when the equilibrium dissociation constant is £ 10-8 M, more preferably when the equilibrium dissociation constant is £ 10-9 M, and most preferably when the dissociation constant is £ 10-10 M.
[0065] The dissociation constant (KD) of a binding protein can be determined, for example, by surface plasmon resonance. Generally, surface plasmon resonance analysis measures real-time binding interactions between ligand (a target antigen on a biosensor matrix) and analyte (a binding protein in solution) by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor; Piscataway, NJ). Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen). The term "KD, " as used herein refers to the dissociation constant of the interaction between a particular binding protein and a target antigen.
[0066] The term "specifically binds" as used herein refers to the ability of a binding protein or an antigen-binding fragment thereof to bind to an antigen containing an epitope with an Kd of at least about 1 x 10-6 M, 1 x 10-7 M, 1 x 10-8 M, 1 x 10-9 M, 1 x 10-10 M, 1 x 10-11 M, 1 x 10-12 M, or more, and/or to bind to an epitope with an affinity that is at least two-fold greater than its affinity for a nonspecific antigen.
[0067] The term "linker" as used herein refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient mobility for the domains of the light and heavy chains to fold into cross over dual variable region immunoglobulins. A linker is inserted at the transition between variable domains or between variable and constant domains, respectively, at the sequence level. The transition between domains can be identified because the approximate size of the immunoglobulin domains are well understood. The precise location of a domain transition can be determined by locating peptide stretches that do not form secondary structural elements such as beta-sheets or alpha-helices as demonstrated by experimental data or as can be assumed by techniques of modeling or secondary structure prediction. The linkers described herein are referred to as L1, which is located on the light chain between the C-terminus of the VL2 and the N- terminus of the VL1 domain; and L2, which is located on the light chain between the C- terminus of the VL1 and the N-terminus of the CL domain. The heavy chain linkers are known as L3, which is located between the C-terminus of the VH1 and the N-terminus of the VH2 domain; and L4, which is located between the C-terminus of the VH2 and the N- terminus of the CH1 domain.
[0068] The term "vector" as used herein refers to any molecule ( e.g ., nucleic acid, plasmid, or virus) that is used to transfer coding information to a host cell. The term "vector" includes a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA molecule into which additional DNA segments may be inserted. Another type of vector is a viral vector, wherein additional DNA segments may be inserted 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. In addition, 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. The terms "plasmid" and "vector" may be used interchangeably herein, as a plasmid is the most commonly used form of vector.
However, the disclosure is intended to include other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.
[0069] The phrase "recombinant host cell" (or "host cell") as used herein refers to a cell into which a recombinant expression vector has been introduced. A recombinant host cell or host cell is intended to refer not only to the particular subject cell, but also 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 such cells are still included within the scope of the term "host cell" as used herein. A wide variety of host cell expression systems can be used to express the binding proteins, including bacterial, yeast, baculoviral, and mammalian expression systems (as well as phage display expression systems). An example of a suitable bacterial expression vector is pUC19. To express a binding protein recombinantly, a host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chains of the binding protein such that the polypeptide chains are expressed in the host cell and, preferably, secreted into the medium in which the host cells are cultured, from which medium the binding protein can be recovered.
[0070] The term "transformation" as used herein refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain a new DNA. For example, a cell is transformed where it is genetically modified from its native state. Following transformation, the transforming DNA may recombine with that of the cell by physically integrating into a chromosome of the cell, or may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid. A cell is considered to have been stably transformed when the DNA is replicated with the division of the cell. The term "transfection" as used herein refers to the uptake of foreign or exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are well known in the art. Such techniques can be used to introduce one or more exogenous DNA molecules into suitable host cells.
[0071] The term "naturally occurring" as used herein and applied to an object refers to the fact that the object can be found in nature and has not been manipulated by man. For example, a polynucleotide or polypeptide that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man is naturally-occurring. Similarly, "non-naturally occurring" as used herein refers to an object that is not found in nature or that has been structurally modified or synthesized by man.
[0072] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Stereoisomers ( e.g ., D-amino acids) of the twenty conventional amino acids; unnatural amino acids and analogs such as a-, a-di substituted amino acids, N- alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for the polypeptide chains of the binding proteins. Examples of
unconventional amino acids include: 4-hydroxyproline, g-carboxyglutamate, e-N,N,N- trimethyllysine, e-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, s-N-methylarginine, and other similar amino acids and imino acids ( e.g ., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl- terminal direction, in accordance with standard usage and convention.
[0073] Naturally occurring residues may be divided into classes based on common side chain properties:
(1) hydrophobic: Met, Ala, Val, Leu, lie, Phe, Trp, Tyr, Pro;
(2) polar hydrophilic: Arg, Asn, Asp, Gin, Glu, His, Lys, Ser, Thr ;
(3) aliphatic: Ala, Gly, lie, Leu, Val, Pro;
(4) aliphatic hydrophobic: Ala, Ile, Leu, Val, Pro;
(5) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;
(6) acidic: Asp, Glu;
(7) basic: His, Lys, Arg;
(8) residues that influence chain orientation: Gly, Pro;
(9) aromatic: His, Trp, Tyr, Phe; and
(10) aromatic hydrophobic: Phe, Trp, Tyr.
[0074] Conservative amino acid substitutions may involve exchange of a member of one of these classes with another member of the same class. Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class.
[0075] A skilled artisan will be able to determine suitable variants of the polypeptide chains of the binding proteins using well-known techniques. For example, one skilled in the art may identify suitable areas of a polypeptide chain that may be changed without destroying activity by targeting regions not believed to be important for activity.
Alternatively, one skilled in the art can identify residues and portions of the molecules that are conserved among similar polypeptides. In addition, even areas that may be important for biological activity or for structure may be subject to conservative amino acid
substitutions without destroying the biological activity or without adversely affecting the polypeptide structure. [0076] The term "patient" as used herein includes human and animal subjects.
[0077] The terms "treatment" or "treat" as used herein refer to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those having the disorder as well as those prone to have a disorder or those in which the disorder is to be prevented. In particular embodiments, binding proteins can be used to treat humans infected with HIV, or humans susceptible to HIV infection, or ameliorate HIV infection in a human subject infected with HIV. The binding proteins can also be used to prevent HIV in a human patient.
[0078] It should be understood as that treating humans infected with HIV include those subjects who are at any one of the several stages of HIV infection progression, which, for example, include acute primary infection syndrome (which can be asymptomatic or associated with an influenza-like illness with fevers, malaise, diarrhea and neurologic symptoms such as headache), asymptomatic infection (which is the long latent period with a gradual decline in the number of circulating CD4+T cells), and AIDS (which is defined by more serious AIDS-defming illnesses and/or a decline in the circulating CD4 cell count to below a level that is compatible with effective immune function). In addition, treating or preventing HIV infection will also encompass treating suspected infection by HIV after suspected past exposure to HIV by e.g., contact with HIV-contaminated blood, blood transfusion, exchange of body fluids,“unsafe” sex with an infected person, accidental needle stick, receiving a tattoo or acupuncture with contaminated instruments, or transmission of the virus from a mother to a baby during pregnancy, delivery or shortly thereafter.
[0079] The terms "pharmaceutical composition" or "therapeutic composition" as used herein refer to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.
[0080] The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" as used herein refers to one or more formulation materials suitable for
accomplishing or enhancing the delivery of a binding protein.
[0081] The terms "effective amount" and "therapeutically effective amount" when used in reference to a pharmaceutical composition comprising one or more binding proteins refer to an amount or dosage sufficient to produce a desired therapeutic result. More specifically, a therapeutically effective amount is an amount of a binding protein sufficient to inhibit, for some period of time, one or more of the clinically defined pathological processes associated with the condition being treated. The effective amount may vary depending on the specific binding protein that is being used, and also depends on a variety of factors and conditions related to the patient being treated and the severity of the disorder. For example, if the binding protein is to be administered in vivo , factors such as the age, weight, and health of the patient as well as dose response curves and toxicity data obtained in preclinical animal work would be among those factors considered. The determination of an effective amount or therapeutically effective amount of a given pharmaceutical composition is well within the ability of those skilled in the art.
[0082] One embodiment of the disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a binding protein.
Trispecific and/or Trivalent Binding Proteins
[0083] Certain aspects of the present disclosure relate to trispecific and/or trivalent binding proteins. Any of the CDRs or variable domains of any of the antigen binding proteins described herein may find use in a trispecific binding protein of the present disclosure. Trispecific binding proteins of various formats are contemplated. In some embodiments, the binding protein of the disclosure is a trispecific and/or trivalent binding protein comprising four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2- LI-VL1- L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VH1- L3-VH2- L4-CH1-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-CH1-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV]
wherein:
VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VH1 is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain; VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is an immunoglobulin CH1 heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin CH3 heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and
L1, L2, L3 and L4 are amino acid linkers;
and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair.
[0084] In some embodiments, the first polypeptide chain and the second polypeptide chain have a cross-over orientation that forms two distinct antigen binding sites. In some embodiments, the VH1 and VL1 form a binding pair and form the first antigen binding site.
In some embodiments, the VH2 and VL2 form a binding pair and form the second antigen binding site. In some embodiments, the third polypeptide and the fourth polypeptide form a third antigen binding site. In some embodiments, the VH3 and VL3 form a binding pair and form the third antigen binding site.
[0085] In some embodiments, one or more of the antigen binding sites binds an HIV target protein. In some embodiments, VH3 and VL3 form a third antigen binding site that binds an HIV target protein. In some embodiments, VH1 and VL1 form a first antigen binding site that binds a T cell target protein, VH2 and VL2 form a second antigen binding site that binds a T cell target protein, and VH3 and VL3 form a third antigen binding site that binds an HIV target protein. In some embodiments, VH1 and VL1 form a first antigen binding site that binds a T cell target protein, VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, and VH3 and VL3 form a third antigen binding site that binds an HIV target protein. In some embodiments, VH1 and VL1 form a first antigen binding site that binds a CD28 polypeptide, VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, and VH3 and VL3 form a third antigen binding site that binds an HIV target protein.
[0086] In some embodiments, the binding proteins specifically bind to one or more HIV target proteins ( e.g ., as described infra ) and one or more target proteins on a T-cell including T cell receptor complex. These T-cell engager binding proteins are capable of recruiting T cells transiently to target cells and, at the same time, activating the cytolytic activity of the T cells. The T-cell engager trispecific antibodies can be used to activate HIV- 1 reservoirs and redirect/activate T cells to lyse latently infected HIV-1+ T cells. Examples of target proteins on T cells include but are not limited to CD3 and CD28, among others. In some embodiments, the trispecific binding proteins may be generated by combining the antigen binding domains of two or more monospecific antibodies (parent antibodies) into one antibody. See International Publication Nos. WO 2011/038290 A2, WO 2013/086533 Al, WO 2013/070776 Al, WO 2012/154312 Al, and WO 2013/163427 Al. The binding proteins of the disclosure may be prepared using domains or sequences obtained or derived from any human or non-human antibody, including, for example, human, murine, or humanized antibodies.
[0087] In some embodiments of the disclosure, the trivalent binding protein is capable of binding three different antigen targets. In one embodiment, the binding protein is trispecific and one light chain-heavy chain pair is capable of binding two different antigen targets or epitopes and one light chain-heavy chain pair is capable of binding one antigen target or epitope.
[0088] In some embodiments, a binding protein of the present disclosure binds one or more HIV target proteins and one or more T cell target proteins. In some embodiments, the binding protein is capable of specifically binding one HIV target protein and two different epitopes on a single T cell target protein. In some embodiments, the binding protein is capable of specifically binding one HIV target protein and two different T cell target proteins (e.g., CD28 and CD3). In some embodiments, the first and second polypeptide chains of the binding protein form two antigen binding sites that specifically target two T cell target proteins, and the third and fourth polypeptide chains of the binding protein form an antigen binding site that specifically binds an HIV target protein. In some embodiments, the one or more HIV target proteins are one or more of glycoprotein 120, glycoprotein 41, and glycoprotein 160. In some embodiments, the one or more T cell target proteins are one or more of CD3 and CD28.
[0089] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:61; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:62 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:62; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:63 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:63; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 64 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:64.
[0090] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:65 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:65; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:66 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:66; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:67 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:67; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 68.
[0091] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:69 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:69; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:70 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:70; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:71 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:71; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 72 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:72.
[0092] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:73 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:73; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:74 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:74; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:75 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:75; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:76 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:76.
[0093] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:77 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:77; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:78 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:78; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:79 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:79; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 80 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:80.
[0094] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:81 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:81; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:82 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:82; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:83 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:83; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 84 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:84.
[0095] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:85 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:85; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:86 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:86; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:87 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:87; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:88 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:88.
[0096] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:89 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:89; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:90 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:90; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:91 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:91; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 92 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:92.
[0097] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:93 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:93; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:94 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:94; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:95 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:95; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 96 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:96.
[0098] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:97 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:97; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:98 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:98; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:99 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:99; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 100.
[0099] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 101 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 101; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 102; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 103 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 104.
[0100] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 105; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 107 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 107; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 108 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108.
[0101] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 109 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 109; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 110 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 110; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 111 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 111; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 112.
[0102] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 113; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 114; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 115; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 116 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 116.
[0103] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 117; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 119; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120.
[0104] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 121; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 122 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 123 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 123; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124.
[0105] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 129; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 130; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 131 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 131; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 132.
[0106] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 133; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 135; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 136.
[0107] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 137; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 138; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 139; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 140.
[0108] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 141; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 142; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 143; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 144.
[0109] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 145; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 146; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 147; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 148.
[0110] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 149; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 150; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 151; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 152 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 152.
[0111] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 153; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 154; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 155; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 156.
[0112] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 157; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 158; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 159; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 160.
[0113] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 161; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 162 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 162; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 163 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 163; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 164 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 164.
[0114] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 165 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 165; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 166 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 166; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 167 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 167; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 168 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 168.
[0115] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 169; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 170 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 170; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 171 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 171; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 172.
[0116] In some embodiments, a binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 173; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 174; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 175 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 175; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 176.
Exemplary and non-limiting polypeptides that may find use in any of the trispecific binding proteins described herein are provided in Table 4.
Anti-HIV Binding Sites
[0117] Certain aspects of the present disclosure relate to binding proteins that comprise an antigen binding site that binds an HIV target protein or polypeptide.
[0118] In some embodiments, the HIV target protein is glycoprotein 120, glycoprotein 41, or glycoprotein 160. In some embodiments, a binding protein binds one or more of: glycoprotein 120, glycoprotein 41, and glycoprotein 160. Exemplary HIV target proteins include, without limitation, MPER of the HIV-1 gp41 protein, a CD4 binding site of the HIV- 1 gpl20 protein, a glycan in the V3 loop of the HIV-1 gpl20 protein, or a trimer apex of the HIV-1 gpl20 protein or gpl60. For example, in some embodiments, a binding protein of the present disclosure comprises an antigen binding site that binds a CD4 binding site of the HIV-1 gpl20 protein. Exemplary antigen binding sites that bind HIV target proteins contemplated for use herein include, without limitation, those described in International Publication No. WO2017/074878, such as those from antibodies CD4BS“a”, CD4BS“b”, MPER, MPER IOOW, V1/V2“a”, V1/V2“b”, or V3.
[0119] In some embodiments, a binding protein comprising an antigen binding site that binds an HIV target protein is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent, or multispecific and/or multivalent. In some embodiments, a binding protein that comprises an antigen binding site that binds an HIV target protein is a trispecific binding protein comprising four polypeptides that form three antigen binding sites.
[0120] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of NCPIN (SEQ ID NO: l) a CDR- H2 sequence comprising the amino acid sequence of WMKPRHGAVSYARQLQG (SEQ ID NO:2), and a CDR-H3 sequence comprising the amino acid sequence of
GKYCTARDYYNWDFEH (SEQ ID NO:3); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of
RTSQYGSLA (SEQ ID NO:4), a CDR-L2 sequence comprising the amino acid sequence of SGSTRAA (SEQ ID NO:5), and a CDR-L3 sequence comprising the amino acid sequence of QQYEF (SEQ ID NO:6). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of NCPIN (SEQ ID NO: l) a CDR-H2 sequence comprising the amino acid sequence of
WMKPRHGAV S YARQLQG (SEQ ID NO:2), and a CDR-H3 sequence comprising the amino acid sequence of GKYCTARDYYNWDFEH (SEQ ID NO:3); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of RTSQYGSLA (SEQ ID NO:4), a CDR-L2 sequence comprising the amino acid sequence of SGSTRAA (SEQ ID NO:5), and a CDR-L3 sequence comprising the amino acid sequence of QQYEF (SEQ ID NO:6).
[0121] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7) a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAV (SEQ ID NO:8) or IKPQYGAT (SEQ ID NO:9); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO: 11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO: 12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO: 13). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR- H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7) a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAV (SEQ ID NO:8) or
IKPQYGAT (SEQ ID NO:9); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO: 11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO: 12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO: 13). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7) a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAV (SEQ ID NO:8); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO: 11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO: 12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO: 13). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7) a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAV (SEQ ID NO: 8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO: 11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO: 12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO: 13). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7) a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAT (SEQ ID NO:9); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO: 11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO: 12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO: 13). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7) a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAT (SEQ ID NO:9); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO: 11), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO: 12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO: 13).
[0122] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of DCTLN (SEQ ID NO: 14) a CDR- H2 sequence comprising the amino acid sequence of WLKPRWGAVNYARPLQG (SEQ ID NO: 15), and a CDR-H3 sequence comprising the amino acid sequence of
GKNCDYNWDFEH (SEQ ID NO: 16); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of RTSQYGSLA (SEQ ID NO: 17), a CDR-L2 sequence comprising the amino acid sequence of SGSTRAA (SEQ ID NO: 18), and a CDR-L3 sequence comprising the amino acid sequence of QQYEF (SEQ ID NO: 19). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of DCTLN (SEQ ID NO: 14) a CDR- H2 sequence comprising the amino acid sequence of WLKPRWGAVNYARPLQG (SEQ ID NO: 15), and a CDR-H3 sequence comprising the amino acid sequence of
GKNCDYNWDFEH (SEQ ID NO: 16); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of RTSQYGSLA (SEQ ID NO: 17), a CDR-L2 sequence comprising the amino acid sequence of SGSTRAA (SEQ ID NO: 18), and a CDR-L3 sequence comprising the amino acid sequence of QQYEF (SEQ ID NO: 19).
[0123] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site with a VH domain comprising an extended heavy chain FR3 loop of antibody VRC03, e.g., as described in Liu, Q. et al. (2019) Nat. Commun. 10:721.
[0124] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
QVRLSQSGGQMKKPGDSMRISCRASGYEFINCPINWIRLAPGKRPEWMGWMKPRHG AVSYARQLQGRVTMTRDMYSETAFLELRSLTSDDTAVYFCTRGKYCTARDYYNWD FEHWGQGTPVTVSS (SEQ ID NO:43), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
SLTQ SPGTL SL SPGET All S CRT S Q Y GSL AW Y QQRPGQ APRL VI Y S GSTRA AGIPDRF S GSRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIK (SEQ ID NO:45). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:43, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:45. In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:43, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:45.
[0125] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
QVRLSQSGGQMKKPGDSMRISCRASGYEFINCPINWIRLAPGKRPEWMGWMKPRHG AV S YARQLQGRVTMTRQL SQDPDDPDW GTAFLELRSLT SDDT AVYF CTRGK Y CT A RD YYNWDFEHW GQGTP VT V S S (SEQ ID NO:44), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
SLTQ SPGTL SL SPGET All S CRT S Q Y GSL AW Y QQRPGQ APRL VI Y S GSTRA AGIPDRF S GSRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIK (SEQ ID NO:45). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:44, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:45. In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:44, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:45.
[0126] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQY GAVNF GGGFRDRVTLTRD VYREIAYMDIRGLKPDDTAVYY C ARDRS Y GDS SWALD AWGQGTTVVVSA (SEQ ID NO:46), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
YfflVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGV P SRF S GS GFHT SFNLTISDLQ ADDI AT Y Y C Q VLQFF GRGSRLHIK (SEQ ID NO:49). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:46, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:49. In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:46, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:49.
[0127] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQY GATNF GGGFRDRVTLTRD VYREI AYMDIRGLKPDDT AVYY C ARDRS Y GD S SWALD AWGQGTTVVVSA (SEQ ID NO:47), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGV P SRF S GS GFHT SFNLTISDLQ ADDI AT YY C Q VLQFF GRGSRLHIK (SEQ ID NO:49). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:47, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:49. In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:47, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:49.
[0128] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQY GAVNFGGGFRDRVTLTRQLSQDPDDPDWGIAYMDIRGLKPDDTAVYYCARDRSYG DSSWALDAWGQGTTVVVSA (SEQ ID NO:48), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGV P SRF S GS GFHT SFNLTISDLQ ADDI AT Y Y C Q VLQFF GRGSRLHIK (SEQ ID NO:49). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:48, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:49. In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:48, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:49.
[0129] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
Q VQL VQ S GGQMKKP GE SMRI S CRA S GYEFID C TLNWIRL AP GKRPE WMGWLKPRW GAVNYARPLQGRVTMTRQLSQDPDDPDWGTAFLELRSLTVDDTAVYFCTRGKNCD YNWDFEHW GRGTP VI V S S (SEQ ID NO:50), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
LTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSG SRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIK (SEQ ID NO:51). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:50, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:51. In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:50, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:51.
[0130] In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprising an antigen binding site that binds an HIV target protein, an antigen binding site that binds a CD28 polypeptide, and an antigen binding site that binds a CD3 polypeptide. In some embodiments, the binding protein is a trispecific binding protein comprising four
polypeptides comprising three antigen binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair ( e.g ., as described herein). In some embodiments, the VH and VL domains of any of the anti-CD38 antigen binding sites described above represent VH3 and VL3 and form a third antigen binding site that binds an HIV target protein. In some embodiments, VH1 and VL1 form a first antigen binding site that binds a CD28 polypeptide, VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, and the VH and VL domains of any of the anti-HIV antigen binding sites described above and/or in Table 1 represent VH3 and VL3 and form a third antigen binding site that binds an HIV target protein.
[0131] Sequences of exemplary anti-HIV antigen binding sites are provided in Table 1. In some embodiments, a binding protein comprising an anti-HIV antigen binding site of the present disclosure comprises 1, 2, 3, 4, 5, or all 6 CDR sequences of an anti-HIV antibody described in Table 1. In some embodiments, a binding protein comprising an anti-HIV antigen binding site of the present disclosure comprises a VH domain sequence and/or VL domain sequence of an anti-HIV antibody described in Table 1.
Table 1. Anti-HIV binding protein sequences.
Anti-CD28 Binding Sites
[0132] Certain aspects of the present disclosure relate to binding proteins that comprise an antigen binding site that binds a CD28 polypeptide. In some embodiments, the CD28 polypeptide is a human CD28 polypeptide, also known as Tp44. Human CD28 polypeptides are known in the art and include, without limitation, the polypeptides represented by NCBI Accession Numbers XP_011510499.1 , XP_011510497.1 , XP_011510496.1 ,
NP_001230007.1, NP_001230006.1, or NP_006130.1, or a polypeptide produced from NCBI Gene ID Number 940. In some embodiments, a binding protein comprising an antigen binding site that binds a CD28 polypeptide is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent, or multispecific and/or multivalent. In some embodiments, a binding protein that comprises an antigen binding site that binds a CD28 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites. In some embodiments, a binding protein that comprises an antigen binding site that binds a CD28 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, and one of which binds a CD3 polypeptide. In some embodiments, a binding protein that comprises an antigen binding site that binds a CD28 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, one of which binds a CD3 polypeptide, and one of which binds an HIV target protein.
[0133] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:33); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO:36). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:33); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO:36).
[0134] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNV NTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWG KGTTVTVSS (SEQ ID NO:59), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTG VPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO:60). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:59, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:60. In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:59, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:60.
[0135] In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprising an antigen binding site that binds an HIV target protein, an antigen binding site that binds a CD28 polypeptide, and an antigen binding site that binds a CD3 polypeptide. In some embodiments, the binding protein is a trispecific binding protein comprising four
polypeptides comprising three antigen binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair ( e.g ., as described herein). In some embodiments, the VH and VL domains of any of the anti-CD28 antigen binding sites described above represent VH1 and VL1 and form a first antigen binding site that binds a CD28 polypeptide. In some embodiments, the VH and VL domains of any of the anti-CD28 antigen binding sites described above represent VH1 and VL1 and form a first antigen binding site that binds a CD28 polypeptide, VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, and VH3 and VL3 and form a third antigen binding site that binds an HIV target protein.
[0136] Sequences of exemplary anti-CD28 antigen binding sites are provided in Table 2. In some embodiments, a binding protein comprising an anti-CD28 antigen binding site of the present disclosure comprises 1, 2, 3, 4, 5, or all 6 CDR sequences of an anti-CD28 antibody described in Table 2. In some embodiments, a binding protein comprising an anti-CD28 antigen binding site of the present disclosure comprises a VH domain sequence and/or VL domain sequence of an anti-CD28 antibody described in Table 2.
Table 2. Anti-CD28 binding protein sequences.
Anti-CD3 Binding Sites
[0137] Certain aspects of the present disclosure relate to binding proteins that comprise an antigen binding site that binds a CD3 polypeptide. In some embodiments, the CD3 polypeptide is a human CD3 polypeptide, including CD3-delta (also known as T3D, IMD19, and CD3-DELTA), CD3-epsilon (also known as T3E, IMD18, and TCRE), and CD3-gamma (also known as T3G, IMD17, and CD3-GAMMA). Human CD3 polypeptides are known in the art and include, without limitation, the polypeptides represented by NCBI Accession Numbers XP 006510029.1 or NP_031674.1, or a polypeptide produced from NCBI Gene ID Numbers 915, 916, or 917. In some embodiments, a binding protein comprising an antigen binding site that binds a CD3 polypeptide is monospecific and/or monovalent, bispecific and/or bivalent, trispecific and/or trivalent, or multispecific and/or multivalent. In some embodiments, a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites. In some embodiments, a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, and one of which binds a CD3 polypeptide. In some embodiments, a binding protein that comprises an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides that form three antigen binding sites, one of which binds a CD28 polypeptide, one of which binds a CD3 polypeptide, and one of which binds an HIV target protein.
[0138] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein Xi is E or Q, X2 is A or L, and X3 is Q, R, or F (SEQ ID NO:293), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30). In some embodiments, the CDR- L1 sequence of the VL2 domain comprises an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO:24), QSLVHENLQTY (SEQ ID NO:25), QSLVHENLFTY (SEQ ID NO:26), and Q SL VHENLRT Y (SEQ ID NO:27).
[0139] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:24), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).
In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR- H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR- H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:24), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).
[0140] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO:25), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR- H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR- H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO:25), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).
[0141] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO:26), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR- H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR- H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO:26), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).
[0142] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO:27), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR- H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR- H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO:27), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30).
[0143] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKS N S YAT YY AD S VKGRFTISRDD SKNTLYLQMN SLRAEDT AVYY CRGVYY AL SPFD Y WGQGTLVTVSS (SEQ ID NO:52), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHQNAQT YL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 54),
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLQTYL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:55),
DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNR F SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:56), and
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLRTYL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:57). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VET) domain comprising the amino acid sequence of SEQ ID NO:52, and/or an antibody light chain variable (VL) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57. In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VET) domain comprising the amino acid sequence of SEQ ID NO:52, and an antibody light chain variable (VL) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57.
[0144] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VET) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKS N S YAT YY AD S VKGRFTISRDD SKNTLYLQMN SLRAEDT AVYY CRGVYY AL SPFD Y WGQGTLVTVSS (SEQ ID NO:52), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHQNAQT YL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:54). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:54. In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 54.
[0145] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKS N S YAT YY AD S VKGRFTISRDD SKNTL YLQMN SLRAEDT AVYY CRGVYY AL SPFD Y WGQGTLVTVSS (SEQ ID NO:52), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLQTYL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:55). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:55. In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 55.
[0146] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKS N S YAT YY AD S VKGRFTISRDD SKNTLYLQMN SLRAEDT AVYY CRGVYY AL SPFD Y WGQGTLVTVSS (SEQ ID NO:52), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNR F SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:56). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:56. In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 56.
[0147] In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKS N S YAT YY AD S VKGRFTISRDD SKNTLYLQMN SLRAEDT AVYY CRGVYY AL SPFD Y WGQGTLVTVSS (SEQ ID NO:52), and/or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLRTYL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:57). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:57. In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:52, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 57.
[0148] Advantageously, anti-CD3 binding sites are described herein with high affinity binding to human CD3 polypeptides and potential manufacturing liabilities ( e.g ., deamidation sites) removed.
[0149] In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprising an antigen binding site that binds an HIV target protein, an antigen binding site that binds a CD28 polypeptide, and an antigen binding site that binds a CD3 polypeptide. In some embodiments, the binding protein is a trispecific binding protein comprising four
polypeptides comprising three antigen binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair (e.g., as described herein). In some embodiments, the VH and VL domains of any of the anti-CD3 antigen binding sites described above represent VH2 and VL2and form a second antigen binding site that binds a CD3 polypeptide. In some embodiments, VH1 and VL1 form a first antigen binding site that binds a CD28 polypeptide, the VH and VL domains of any of the anti-CD3 antigen binding sites described above and/or in Table 3 represent VH2 and VL2 and form a second antigen binding site that binds a CD3 polypeptide, and VH3 and VL3 form a third antigen binding site that binds an HIV target protein.
[0150] Sequences of exemplary anti-CD3 antigen binding sites are provided in Table 3. In some embodiments, a binding protein comprising an anti-CD3 antigen binding site of the present disclosure comprises 1, 2, 3, 4, 5, or all 6 CDR sequences of an anti-CD3 antibody described in Table 3. In some embodiments, a binding protein comprising an anti-CD3 antigen binding site of the present disclosure comprises a VH domain sequence and/or VL domain sequence of an anti-CD3 antibody described in Table 3.
Table 3. Anti-CD3 binding protein sequences.
Linkers
[0151] In some embodiments, the linkers L1, L2, L3, and L4 range from no amino acids (length=0) to about 100 amino acids long, or less than 100, 50, 40, 30, 20, or 15 amino acids or less. The linkers can also be 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids long. L1, L2, L3, and L4 in one binding protein may all have the same amino acid sequence or may all have different amino acid sequences.
[0152] Examples of suitable linkers include, for example, GGGGSGGGGS (SEQ ID NO:40), GGGGSGGGGSGGGGS (SEQ ID NO: 41), S, RT, TKGPS (SEQ ID NO: 39), GQPKAAP (SEQ ID NO: 38), GGSGSSGSGG (SEQ ID NO: 42), and DKTHT (SEQ ID NO:37), as well as those disclosed in International Publication Nos. WO2017/074878 and W02017/180913. The examples listed above are not intended to limit the scope of the disclosure in any way, and linkers comprising randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine, and proline have been shown to be suitable in the binding proteins. [0153] The identity and sequence of amino acid residues in the linker may vary depending on the type of secondary structural element necessary to achieve in the linker. For example, glycine, serine, and alanine are best for linkers having maximum flexibility. Some combination of glycine, proline, threonine, and serine are useful if a more rigid and extended linker is necessary. Any amino acid residue may be considered as a linker in combination with other amino acid residues to construct larger peptide linkers as necessary depending on the desired properties.
[0154] In some embodiments, the length of L1 is at least twice the length of L3. In some embodiments, the length of L2 is at least twice the length of L4. In some embodiments, the length of L1 is at least twice the length of L3, and the length of L2 is at least twice the length of L4. In some embodiments, L1 is 3 to 12 amino acid residues in length, L2 is 3 to 14 amino acid residues in length, L3 is 1 to 8 amino acid residues in length, and L4 is 1 to 3 amino acid residues in length. In some embodiments, L1 is 5 to 10 amino acid residues in length, L2 is 5 to 8 amino acid residues in length, L3 is 1 to 5 amino acid residues in length, and L4 is 1 to 2 amino acid residues in length. In some embodiments, L1 is 7 amino acid residues in length, L2 is 5 amino acid residues in length, L3 is 1 amino acid residue in length, and L4 is 2 amino acid residues in length.
[0155] In some embodiments, L1, L2, L3 and L4 each independently are zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:40), GGGGS GGGGSGGGGS (SEQ ID NO: 41), S, RT, TKGPS (SEQ ID NO: 39), GQPKAAP (SEQ ID NO: 38), and GGSGSSGSGG (SEQ ID NO: 42). In some embodiments, L1, L2, L3 and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:40), GGGGS GGGGS GGGGS (SEQ ID NO:41), S, RT, TKGPS (SEQ ID NO:39), GQPKAAP (SEQ ID NO: 38), and
GGSGSSGSGG (SEQ ID NO:42). In some embodiments, L1 comprises the sequence GQPKAAP (SEQ ID NO: 38), L2 comprises the sequence TKGPS (SEQ ID NO:39), L3 comprises the sequence S, and L4 comprises the sequence RT.
[0156] In some embodiments, at least one of L1, L2, L3 or L4 comprises the sequence
DKTHT (SEQ ID NO:37). In some embodiments, L1, L2, L3 and L4 comprise the sequence DKTHT (SEQ ID NO:37). Fc regions and constant domains
[0157] In some embodiments, a binding protein of the present disclosure comprises a second polypeptide chain further comprising an Fc region linked to CH1, the Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains. In some embodiments, a binding protein of the present disclosure comprises a third polypeptide chain further comprising an Fc region linked to CH1, the Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains. In some embodiments, a binding protein of the present disclosure comprises a second polypeptide chain further comprising an Fc region linked to CH1, the Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising an Fc region linked to CH1, the Fc region comprising an immunoglobulin hinge region and CH2 and Cm immunoglobulin heavy chain constant domains.
[0158] In some embodiments, a binding protein of the present disclosure comprises a full-length antibody heavy chain or a polypeptide chain comprising an Fc region. In some embodiments, the Fc region is a human Fc region, e.g ., a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region includes an antibody hinge, CH1, CH2, CH3 , and optionally Cm domains. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the Fc region is a human IgG4 Fc region. In some
embodiments, the Fc region includes one or more of the mutations described infra. In some embodiments, the Fc region is an Fc region of one of the heavy chain polypeptides (e.g, polypeptide 2 or 3) of a binding protein shown in Table 4. In some embodiments, the heavy chain constant region is a constant region of one of the heavy chain polypeptides (e.g, polypeptide 2 or 3) of a binding protein shown in Table 4. In some embodiments, the light chain constant region is a constant region of one of the light chain polypeptides (e.g, polypeptide 1 or 4) of a binding protein shown in Table 4.
[0159] In some embodiments, a binding protein of the present disclosure includes one or two Fc variants. The term "Fc variant" as used herein refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fc variants, and their interaction with the salvage receptor, are known in the art. Thus, the term "Fc variant" can comprise a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises regions that can be removed because they provide structural features or biological activity that are not required for the antibody-like binding proteins of the invention. Thus, the term "Fc variant" comprises a molecule or sequence that lacks one or more native Fc sites or residues, or in which one or more Fc sites or residues has be modified, that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody- dependent cellular cytotoxicity (ADCC).
[0160] In some embodiments, a binding protein of the present disclosure ( e.g ., a trispecific binding protein) comprises a“knob” mutation on the second polypeptide chain and a“hole” mutation on the third polypeptide chain. In some embodiments, a binding protein of the present disclosure comprises a“knob” mutation on the third polypeptide chain and a “hole” mutation on the second polypeptide chain. In some embodiments, the“knob” mutation comprises substitution(s) at positions corresponding to positions 354 and/or 366 of human IgG1 or IgG4 according to EU Index. In some embodiments, the amino acid substitutions are S354C, T366W, T366Y, S354C and T366W, or S354C and T366Y. In some embodiments, the“knob” mutation comprises substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to EU Index. In some embodiments, the amino acid substitutions are S354C and T366W. In some embodiments, the“hole” mutation comprises substitution(s) at positions corresponding to positions 407 and, optionally, 349, 366, and/or 368 and of human IgG1 or IgG4 according to EU Index. In some embodiments, the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S, and/or L368A. In some embodiments, the“hole” mutation comprises substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to EU Index. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A, and Y407V.
[0161] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitution(s) at positions corresponding to positions 366 and optionally 354 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are T366W or T366Y and optionally S354C; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 407 and optionally 349, 366, and/or 368 and of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S, and/or L368A.
[0162] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitution(s) at positions corresponding to positions 407 and optionally 349, 366, and/or 368 and of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S, and/or L368A; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 366 and optionally 354 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are T366W or T366Y and optionally S354C.
[0163] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitution at position corresponding to position 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitution is T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 366, 368, and/or 407 and of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are T366S,
L368A, and/or Y407V.
[0164] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitution(s) at positions corresponding to positions 366, 368, and/or 407 and of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are T366S, L368A, and/or Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitution at position corresponding to position 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitution is T366W.
[0165] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3
immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W. In some embodiments, the first and/or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.
[0166] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, S354C, T366W, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366,
368, 407, and 409 of human IgG4 according to EU Index, wherein the amino acid
substitutions are S228P, Y349C, T366S, L368A, Y407V, and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366,
368, 407, and 409 of human IgG4 according to EU Index, wherein the amino acid
substitutions are S228P, Y349C, T366S, L368A, Y407V, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, S354C, T366W, and R409K.
[0167] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A, L235A, S354C, and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349,
366, 368, and 407 of human IgG4 according to EU Index, wherein the amino acid
substitutions are F234A, L235A, Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349,
366, 368, and 407 of human IgG4 according to EU Index, wherein the amino acid
substitutions are F234A, L235A, Y349C, T366S, L368A, and Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A, L235A, S354C, and T366W.
[0168] In some embodiments, a binding protein of the present disclosure comprises one or more mutations to reduce effector function, e.g ., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/or antibody- dependent cellular cytotoxicity (ADCC). In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; wherein the first and second Fc regions are human IgG1 Fc regions; and wherein the first and the second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to EU Index, wherein the amino acid substitutions are L234A and L235A. In some embodiments, the Fc regions of the second and the third polypeptide chains are human IgG1 Fc regions, and wherein the Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to EU Index, wherein the amino acid substitutions are L234A and L235A. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; wherein the first and second Fc regions are human IgG1 Fc regions; and wherein the first and the second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and the third polypeptide chains are human IgG1 Fc regions, and wherein the Fc regions each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and the third polypeptide chains are human IgG4 Fc regions, and the Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L235A. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to CH1, the second Fc region comprising an
immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and wherein the first and the second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L235A.
[0169] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, F234A, L235A, S354C, T366W, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an
immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235,
354, 366, and 409 of human IgG4 according to EU Index, wherein the amino acid
substitutions are S228P, F234A, L235A, S354C, T366W, and R409K.
[0170] In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate Fey I and/or Fcyll binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate Fcyl and/or Fcyll binding but do not affect FcRn binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 228 and/or 409 of human IgG4 according to EU Index. In some embodiments, the amino acid substitutions are S228P and /or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 234 and/or 235 of human IgG4 according to EU Index. In some embodiments, the amino acid substitutions are F234A and/or L235A. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 228, 234, 235, and/or 409 of human IgG4 according to EU Index. In some embodiments, the amino acid substitutions are S228P, F234A, L235A, and /or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid mutations at substitutions corresponding to positions 228, 233-236, and/or 409 of human IgG4 according to EU Index. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and a deletion at 236; and /or R409K.
[0171] In some embodiments, the Fc region comprises one or more mutations that reduce or eliminate Fc receptor binding and/or effector function of the Fc region ( e.g ., Fc receptor- mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/or antibody-dependent cellular cytotoxicity (ADCC)). [0172] In some embodiments, the Fc region is a human IgG1 Fc region comprising one or more amino acid substitutions at positions corresponding to positions 234, 235, and/or 329 of human IgG1 according to EU Index. In some embodiments, the amino acid substitutions are L234A, L235A, and/or P329A. In some embodiments, the Fc region is a human IgG1 Fc region comprising amino acid substitutions at positions corresponding to positions 298, 299, and/or 300 of human IgG1 according to EU Index. In some embodiments, the amino acid substitutions are S298N, T299A, and/or Y300S.
[0173] In some embodiments, a binding protein of the present disclosure comprises one or more mutations to improve stability, e.g ., of the hinge region and/or dimer interface of IgG4 ( See e.g., Spiess, C. et al. (2013) J. Biol. Chem. 288:26583-26593). In some embodiments, the mutation comprises substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and a third polypeptide chain further comprising a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; wherein the first and second Fc regions are human IgG4 Fc regions; and wherein the first and the second Fc regions each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K. In some embodiments, a binding protein of the present disclosure comprises knob and hole mutations and one or more mutations to improve stability. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.
[0174] In some embodiments, the Fc region is a human IgG1 Fc region comprising one or more amino acid substitutions at positions corresponding to positions 234, 235, and/or 329 of human IgG1 according to EU Index. In some embodiments, the amino acid substitutions are L234A, L235A, and/or P329A. In some embodiments, the Fc region is a human IgG1 Fc region comprising amino acid substitutions at positions corresponding to positions 298, 299, and/or 300 of human IgG1 according to EU Index. In some embodiments, the amino acid substitutions are S298N, T299A, and/or Y300S. Nucleic acids
[0175] Other aspects of the present disclosure relate to isolated nucleic acid molecules comprising a nucleotide sequence encoding any of the binding proteins described herein. Exemplary and non-limiting nucleic acid sequences are provided in Table 5.
[0176] Other aspects of the present disclosure relate to kits of polynucleotides, e.g. , that encode one or more polypeptides of a binding protein as described herein. In some embodiments, a kit of polynucleotides of the present disclosure comprises one, two, three, or four polynucleotides of a kit of polynucleotides comprising: (a) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 177, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 178, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 179, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 180; (b) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 181, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 182, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 183, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 184; (c) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 185, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 186, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 187, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 188; (d) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 189, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 190, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 191, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 192; (e) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 193, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 194, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 195, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 196; (f) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 197, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 198, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 199, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:200; (g) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:201, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:202, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:203, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:204; (h) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:205, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:206, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:207, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:208; (i) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:209, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:210, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:211, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:212; (j) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:213, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:214, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:215, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:216; (k) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:217, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:218, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:219, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:220; (1) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:221, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:222, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:223, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:224; (m) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:225, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:226, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:227, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:228; (n) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:229, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:230, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:231, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:232; (o) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:233, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:234, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:235, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:236; (p) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:237, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:238, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:239, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:240; (q) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:241, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:242, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:243, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:244; (r) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:245, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:246, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:247, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:248; (s) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:249, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:250, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:251, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:252; (t) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:253, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:254, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:255, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:256; (u) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:257, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:258, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:259, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:260; (v) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:261, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:262, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:263, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:264; (w) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:265, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:266, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:267, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:268; (x) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:269, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:270, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:271, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:272; (y) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:273, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:274, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:275, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:276; (z) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:277, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:278, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:279, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:280; (aa) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:281, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:282, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:283, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:284; (bb) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:285, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:286, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:287, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:288; or (cc) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:289, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:290, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:291, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:292.
[0177] Other aspects of the present disclosure relate to a vector system comprising one or more vectors encoding a first, second, third, and fourth polypeptide chain of any of the binding proteins described herein. In some embodiments, the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein, e.g ., as shown in the polynucleotides of Table 5. In some embodiments, the vector system comprises a first vector encoding the first and second polypeptide chains of the binding protein, and a second vector encoding the third and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and third polypeptide chains of the binding protein, and a second vector encoding the second and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and fourth polypeptide chains of the binding protein, and a second vector encoding the second and third polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first, second, third, and fourth polypeptide chains of the binding protein. The one or more vectors of the vector system may be any of the vectors described herein. In some embodiments, the one or more vectors are expression vectors. In some embodiments, the first, second, third, and fourth polynucleotides are present on one or more expression vectors, e.g ., one, two, three, or four expression vectors.
[0178] Standard recombinant DNA methodologies are used to construct the
polynucleotides that encode the polypeptides which form the binding proteins, incorporate these polynucleotides into recombinant expression vectors, and introduce such vectors into host cells. See e.g., Sambrook et al, 2001 , MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 3rd ed.). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Similarly, conventional techniques may be used for chemical syntheses, chemical analyses,
pharmaceutical preparation, formulation, delivery, and treatment of patients.
[0179] In some embodiments, the isolated nucleic acid is operably linked to a
heterologous promoter to direct transcription of the binding protein-coding nucleic acid sequence. A promoter may refer to nucleic acid control sequences which direct transcription of a nucleic acid. A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence of a binding protein if the promoter affects the transcription or expression of the coding sequence. Examples of promoters may include, but are not limited to, promoters obtained from the genomes of viruses (such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40 (SV40), and the like), from heterologous eukaryotic promoters (such as the actin promoter, an immunoglobulin promoter, from heat- shock promoters, and the like), the CAG-promoter (Niwa et al., Gene 108(2): 193-9, 1991), the phosphoglycerate kinase (PGK)-promoter, a tetracycline-inducible promoter (Masui et al., Nucleic Acids Res. 33:e43, 2005), the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the promoter for 3- phosphoglycerate kinase, the promoters of yeast acid phosphatase, and the promoter of the yeast alpha-mating factors. Polynucleotides encoding binding proteins of the present disclosure may be under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein or other suitable promoter that will be readily recognized by one skilled in the art.
[0180] In some embodiments, the isolated nucleic acid is incorporated into a vector. In some embodiments, the vector is an expression vector. Expression vectors may include one or more regulatory sequences operatively linked to the polynucleotide to be expressed. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Examples of suitable enhancers may include, but are not limited to, enhancer sequences from mammalian genes (such as globin, elastase, albumin, a-fetoprotein, insulin and the like), and enhancer sequences from a eukaryotic cell virus (such as SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, adenovirus enhancers, and the like). Examples of suitable vectors may include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenoviral, vaccinia viral, Sindbis-viral, measles, herpes viral, lentiviral, retroviral, adeno-associated viral vectors, etc.). Expression vectors can be used to transfect host cells, such as, for example, bacterial cells, yeast cells, insect cells, and mammalian cells. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art, and can be used to transfect any cell of interest.
Host cells
[0181] Other aspects of the present disclosure relate to a host cell (e.g., an isolated host cell) comprising one or more isolated polynucleotides, vectors, and/or vector systems described herein. In some embodiments, an isolated host cell of the present disclosure is cultured in vitro. In some embodiments, the host cell is a bacterial cell (e.g, an E. coli cell). In some embodiments, the host cell is a yeast cell (e.g, an S. cerevisiae cell). In some embodiments, the host cell is an insect cell. Examples of insect host cells may include, for example, Drosophila cells (e.g., S2 cells), Trichoplusia ni cells (e.g, High Five™ cells), and Spodoptera frugiperda cells (e.g, Sf21 or Sf9 cells). In some embodiments, the host cell is a mammalian cell. Examples of mammalian host cells may include, for example, human embryonic kidney cells (e.g, 293 or 293 cells subcloned for growth in suspension culture), Expi293™ cells, CHO cells, baby hamster kidney cells (e.g, BHK, ATCC CCL 10), mouse sertoli cells (e.g, TM4 cells), monkey kidney cells (e.g, CV1 ATCC CCL 70), African green monkey kidney cells (e.g, VERO-76, ATCC CRL-1587), human cervical carcinoma cells (e.g, HELA, ATCC CCL 2), canine kidney cells (e.g, MDCK, ATCC CCL 34), buffalo rat liver cells (e.g, BRL 3A, ATCC CRL 1442), human lung cells (e.g, W138, ATCC CCL 75), human liver cells (e.g, Hep G2, HB 8065), mouse mammary tumor cells (e.g, MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, a human hepatoma line (e.g, Hep G2), and myeloma cells (e.g, NS0 and Sp2/0 cells).
[0182] Other aspects of the present disclosure relate to a method of producing any of the binding proteins described herein. In some embodiments, the method includes a) culturing a host cell (e.g, any of the host cells described herein) comprising an isolated nucleic acid, vector, and/or vector system (e.g, any of the isolated nucleic acids, vectors, and/or vector systems described herein) under conditions such that the host cell expresses the binding protein; and b) isolating the binding protein from the host cell. Methods of culturing host cells under conditions to express a protein are well known to one of ordinary skill in the art. Methods of isolating proteins from cultured host cells are well known to one of ordinary skill in the art, including, for example, by affinity chromatography (e.g, two step affinity chromatography comprising protein A affinity chromatography followed by size exclusion chromatography) .
Pharmaceutical Compositions
[0183] Therapeutic or pharmaceutical compositions comprising binding proteins are within the scope of the disclosure. Such therapeutic or pharmaceutical compositions can comprise a therapeutically effective amount of a binding protein, or binding protein-drug conjugate, in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration. [0184] Acceptable formulation materials are nontoxic to recipients at the dosages and concentrations employed.
[0185] The pharmaceutical composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or
hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides - e.g ., sodium or potassium chloride - or mannitol sorbitol), delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants (see, e.g,
REMINGTON'S PHARMACEUTICAL SCIENCES (18th Ed., A.R. Gennaro, ed., Mack Publishing Company 1990), and subsequent editions of the same, incorporated herein by reference for any purpose).
[0186] The optimal pharmaceutical composition will be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, and desired dosage. Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the binding protein.
[0187] The primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier for injection can be water, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute. In one embodiment of the disclosure, binding protein compositions can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or an aqueous solution. Further, the binding protein can be formulated as a lyophilizate using appropriate excipients such as sucrose.
[0188] The pharmaceutical compositions of the disclosure can be selected for parenteral delivery or subcutaneous. Alternatively, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such
pharmaceutically acceptable compositions is within the skill of the art.
[0189] The formulation components are present in concentrations that are acceptable to the site of administration. For example, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
[0190] When parenteral administration is contemplated, the therapeutic compositions for use can be in the form of a pyrogen-free, parenterally acceptable, aqueous solution comprising the desired binding protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which a binding protein is formulated as a sterile, isotonic solution, properly preserved. Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which can then be delivered via a depot injection. Hyaluronic acid can also be used, and this can have the effect of promoting sustained duration in the circulation. Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.
[0191] In one embodiment, a pharmaceutical composition can be formulated for inhalation. For example, a binding protein can be formulated as a dry powder for inhalation. Binding protein inhalation solutions can also be formulated with a propellant for aerosol delivery. In yet another embodiment, solutions can be nebulized.
[0192] It is also contemplated that certain formulations can be administered orally. In one embodiment of the disclosure, binding proteins that are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. For example, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption of the binding protein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.
[0193] Another pharmaceutical composition can involve an effective quantity of binding proteins in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions can be prepared in unit-dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
[0194] Additional pharmaceutical compositions of the disclosure will be evident to those skilled in the art, including formulations involving binding proteins in sustained- or controlled-delivery formulations. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art.
[0195] Pharmaceutical compositions to be used for in vivo administration typically must be sterile. This can be accomplished by filtration through sterile filtration membranes.
Where the composition is lyophilized, sterilization using this method can be conducted either prior to, or following, lyophilization and reconstitution. The composition for parenteral administration can be stored in lyophilized form or in a solution. In addition, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
[0196] Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations can be stored either in a ready -to-use form or in a form ( e.g ., lyophilized) requiring reconstitution prior to administration.
[0197] The disclosure also encompasses kits for producing a single-dose administration unit. The kits can each contain both a first container having a dried protein and a second container having an aqueous formulation. Also included within the scope of this disclosure are kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes).
[0198] The effective amount of a binding protein pharmaceutical composition to be employed therapeutically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the binding protein is being used, the route of administration, and the size (body weight, body surface, or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
[0199] Dosing frequency will depend upon the pharmacokinetic parameters of the binding protein in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition can therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages can be ascertained through use of appropriate dose-response data.
[0200] The route of administration of the pharmaceutical composition is in accord with known methods, e.g, orally; through injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release systems; or by implantation devices. Where desired, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.
[0201] The composition can also be administered locally via implantation of a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed- release bolus, or continuous administration.
[0202] The pharmaceutical compositions can be used to prevent and/or treat HIV infection. The pharmaceutical compositions can be used as a standalone therapy or in combination with standard anti-retroviral therapy.
[0203] The disclosure also relates to a kit comprising a binding protein and other reagents useful for detecting target antigen levels in biological samples. Such reagents can include a detectable label, blocking serum, positive and negative control samples, and detection reagents. In some embodiments, the kit comprises a composition comprising any binding protein, polynucleotide, vector, vector system, and/or host cell described herein. In some embodiments, the kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing a condition ( e.g HIV infection) and may have a sterile access port (for example the container may be an
intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). In some embodiments, the label or package insert indicates that the composition is used for preventing, diagnosing, and/or treating the condition of choice. Alternatively, or additionally, the article of manufacture or kit may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
Methods and Uses for Binding Proteins
[0204] Certain aspects of the present disclosure relate to methods of preventing HIV infection in a patient, treating HIV infection in a patient, preventing AIDS in a patient, and treating AIDS in a patient, using any of the binding proteins or pharmaceutical compositions disclosed herein. Any of the binding proteins or pharmaceutical compositions disclosed herein may find use in a method of the present disclosure, e.g ., methods for preventing HIV infection in a patient, treating HIV infection in a patient, preventing AIDS in a patient, and treating AIDS in a patient.
[0205] FIG. 1 illustrates an exemplary and non-limiting format for a trispecific binding protein that may be employed in the methods and uses described herein. As shown in FIG. 2, a proposed mechanism by which the binding protein shown in FIG. 1 may result in elimination of the HIV reservoir cells in a patient involves: (1) activation of latently infected CD4+ T cells via the anti-CD28 and anti-CD3 arms of the trispecific binding protein; (2) recruitment of CD8+ T cells to activated, latently infected CD4+ T cells via anti-Env and anti-CD3 arms; (3) activation of engaged CD8+ T cells via the anti-CD28 and anti-CD3 arms; and (4) killing of latently infected CD4+ T cells through a Perforin/Granzyme mechanism. Advantageously, this mechanism is thought to activate and subsequently kill HIV-1 reservoir cells, providing a novel strategy for attacking the HIV-1 reservoir in a patient.
[0206] In some embodiments, the methods of the present disclosure comprise
administering to the patient a therapeutically effective amount of at least one of the binding proteins or pharmaceutical compositions described herein.
[0207] In some embodiments, the at least one binding protein is administered in combination with an anti-retroviral therapy (e.g, an anti-HIV therepy). In some
embodiments, the at least one binding protein is administered before the anti-retroviral therapy. In some embodiments, the at least one binding protein is administered concurrently with the anti-retroviral therapy. In some embodiments, the at least one binding protein is administered after the anti-retroviral therapy. In some embodiments, the at least one binding protein is co-administered with any standard anti-retroviral therapy known in the art.
[0208] In some embodiments, administration of the at least one binding protein or pharmaceutical composition results in elimination of one or more latently and/or chronically HIV-infected cells in the patient. In some embodiments, administration of the at least one binding protein results in neutralization of one or more HIV virions and results in elimination of one or more latently and/or chronically HIV-infected cells in the patient. In some embodiments, the patient is a human. Table 4. Trispecific binding protein polypeptide sequences
Table 5. Trispecific binding protein polynucleotide sequences

Claims

CLAIMS What is claimed is:
1. A binding protein comprising four polypeptide chains that form three antigen binding sites that specifically bind one or more HIV target proteins, wherein a first polypeptide chain comprises a structure represented by the formula:
VL2- L1-VL1-L2-CL [I]
and a second polypeptide chain comprises a structure represented by the formula:
VH1-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]
and a third polypeptide chain comprises a structure represented by the formula:
VH3-CH1-hinge-CH2-CH3 [III]
and a fourth polypeptide chain comprises a structure represented by the formula:
VL3-CL [IV]
wherein:
VL1 is a first immunoglobulin light chain variable domain;
VL2 is a second immunoglobulin light chain variable domain;
VL3 is a third immunoglobulin light chain variable domain;
VH1 is a first immunoglobulin heavy chain variable domain;
VH2 is a second immunoglobulin heavy chain variable domain;
VH3 is a third immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is an immunoglobulin CH1 heavy chain constant domain;
CH2 is an immunoglobulin CH2 heavy chain constant domain;
CH3 is an immunoglobulin Cm heavy chain constant domain;
hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;
wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and wherein VH1 and VL1 form a first antigen binding site; wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:20), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:21), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:22), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein Xi is E or Q, X2 is A or L, and X3 is Q, R, or F (SEQ ID NO:293), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:30); and wherein VH3 and VL3 form a third antigen binding site that binds an HIV target protein.
2. The binding protein of claim 1, wherein the first binding site binds a CD28 polypeptide.
3. The binding protein of claim 2, wherein the VH1 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:33), and the VL1 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO:36).
4. The binding protein of claim 3, wherein the VH1 domain comprises the amino acid sequence of
QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNV NTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWG KGTTVTVSS (SEQ ID NO:59), and/or the VL1 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTG VPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO: 60).
5. The binding protein of any one of claims 1-4, wherein the CDR-L1 sequence of the VL2 domain comprises an amino acid sequence selected from the group consisting of Q SLVHQNAQT Y (SEQ ID NO:24), QSLVHENLQTY (SEQ ID NO:25), QSLVHENLFTY (SEQ ID NO:26), and QSLVHENLRTY (SEQ ID NO:27).
6. The binding protein of claim 5, wherein the VH2 domain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKS N S YAT YY AD S VKGRFTISRDD SKNTLYLQMN SLRAEDT AVYY CRGVYY AL SPFD Y WGQGTLVTVSS (SEQ ID NO:52), and/or the VL2 domain comprises an amino acid sequence selected from the group consisting of
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHQNAQT YL S WYLQKPGQ SPQ SLIYK V SN RFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 54),
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLQTYL S WYLQKPGQ SPQ SLIYK V SN RFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:55),
DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNR F SGVPDRF SGS GS GTDFTLKI SRVE AED V GV Y Y C GQGT Q YPF TFGS GTK VEIK (SEQ ID NO:56), and
DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLRTYL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVE AED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:57).
7. The binding protein of any one of claims 1-6, wherein the third antigen binding site binds an HIV target protein selected from the group consisting of glycoprotein 120, glycoprotein 41 and glycoprotein 160.
8. The binding protein of claim 7, wherein:
(a) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of NCPIN (SEQ ID NO: l) a CDR-H2 sequence comprising the amino acid sequence of WMKPRHGAVSYARQLQG (SEQ ID NO:2), and a CDR-H3 sequence comprising the amino acid sequence of GKYCTARDYYNWDFEH (SEQ ID NO:3), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of RTSQYGSLA (SEQ ID NO:4), a CDR-L2 sequence comprising the amino acid sequence of SGSTRAA (SEQ ID NO:5), and a CDR-L3 sequence comprising the amino acid sequence of QQYEF (SEQ ID NO: 6);
(b) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTAHI (SEQ ID NO:7) a CDR-H2 sequence comprising the amino acid sequence of IKPQYGAV (SEQ ID NO: 8) or IKPQYGAT (SEQ ID NO: 9), and a CDR-H3 sequence comprising the amino acid sequence of DRSYGDSSWALDA (SEQ ID NO: 10), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGVGSD (SEQ ID NO: 1 1), a CDR-L2 sequence comprising the amino acid sequence of HTS (SEQ ID NO: 12), and a CDR-L3 sequence comprising the amino acid sequence of CQVLQF (SEQ ID NO: 13); or
(c) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of DCTLN (SEQ ID NO: 14) a CDR-H2 sequence comprising the amino acid sequence of WLKPRW GAVNY ARPLQG (SEQ ID NO: 15), and a CDR-H3 sequence comprising the amino acid sequence of GKNCDYNWDFEH (SEQ ID NO: 16), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of RTSQYGSLA (SEQ ID NO: 17), a CDR-L2 sequence comprising the amino acid sequence of SGSTRAA (SEQ ID NO: 18), and a CDR-L3 sequence comprising the amino acid sequence of QQYEF (SEQ ID NO: 19).
9. The binding protein of claim 8, wherein:
(a) the VH3 domain comprises the amino acid sequence of
QVRLSQSGGQMKKPGDSMRISCRASGYEFINCPINWIRLAPGKRPEWMGWMKPRHG AVSYARQLQGRVTMTRDMYSETAFLELRSLTSDDTAVYFCTRGKYCTARDYYNWD FEHWGQGTPVTVSS (SEQ ID NO:43), and/or the VL3 domain comprises the amino acid sequence of
SLTQ SPGTL SL SPGET All S CRT S Q Y GSL AW Y QQRPGQ APRL VI Y S GSTRA AGIPDRF S GSRWGPDYNLTISNLESGDF GVYY CQQYEFF GQGTKVQVDIK (SEQ ID NO:45);
(b) the VH3 domain comprises the amino acid sequence of
QVRLSQSGGQMKKPGDSMRISCRASGYEFINCPINWIRLAPGKRPEWMGWMKPRHG AV S YARQLQGRVTMTRQL SQDPDDPDW GT AFLELRSLT SDDT AVYF CTRGK Y CT A RD YYNWDFEHW GQGTP VT V S S (SEQ ID NO:44), and/or the VL3 domain comprises the amino acid sequence of
SLTQ SPGTL SL SPGET All S CRT S Q Y GSL AW Y QQRPGQ APRL VI Y S GSTRA AGIPDRF S GSRWGPDYNLTISNLESGDF GVYY CQQYEFF GQGTKVQVDIK (SEQ ID NO:45);
(c) the VH3 domain comprises the amino acid sequence of RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQY GAVNF GGGFRDRVTLTRD VYREIAYMDIRGLKPDDTAVYY C ARDRS Y GDS SWALD AWGQGTTVVVSA (SEQ ID NO:46), and/or the VL3 domain comprises the amino acid sequence of
YfflVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGV P SRF S GS GFHT SFNLTISDLQ ADDI AT Y Y C Q VLQFF GRGSRLHIK (SEQ ID NO:49);
(d) the VH3 domain comprises the amino acid sequence of
RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQY GATNF GGGFRDRVTLTRD VYREI AYMDIRGLKPDDT AVYY C ARDRS Y GD S S WALD AWGQGTTVVVSA (SEQ ID NO:47), and/or the VL3 domain comprises the amino acid sequence of
YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGV P SRF S GS GFHT SFNLTISDLQ ADDI AT YY C Q VLQFF GRGSRLHIK (SEQ ID NO:49);
(e) the VH3 domain comprises the amino acid sequence of
RAHLVQSGTAMKKPGASVRVSCQTSGYTFTAHILFWFRQAPGRGLEWVGWIKPQY GAVNFGGGFRDRVTLTRQLSQDPDDPDWGIAYMDIRGLKPDDTAVYYCARDRSYG D S S WALD AW GQGTT VVV S A (SEQ ID NO:48), and/or the VL3 domain comprises the amino acid sequence of
YIHVTQSPSSLSVSIGDRVTINCQTSQGVGSDLHWYQHKPGRAPKLLIHHTSSVEDGV P SRF S GS GFHT SFNLTISDLQ ADDI AT YY C Q VLQFF GRGSRLHIK (SEQ ID NO:49); or
(f) the VH3 domain comprises the amino acid sequence of
Q VQL VQ S GGQMKKP GE SMRI S CRA S GYEFID C TLNWIRL AP GKRPEWMGWLKPRW GAVNYARPLQGRVTMTRQLSQDPDDPDWGTAFLELRSLTVDDTAVYFCTRGKNCD YNWDFEHWGRGTPVIVSS (SEQ ID NO:50), and/or the VL3 domain comprises the amino acid sequence of
LTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSG SRWGPD YNLTISNLESGDF GVYY CQQYEFF GQGTKVQVDIK (SEQ ID NO:51).
10. The binding protein of any one of claims 1-9, wherein at least one of L1, L2, L3 or L4 is independently 0 amino acids in length.
11. The binding protein of any one of claims 1-9, wherein (a) L1, L2, L3 and L4 each independently are zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:40), GGGGS GGGGS GGGGS (SEQ ID NO: 41), S, RT, TKGPS (SEQ ID NO: 39), GQPKAAP (SEQ ID NO: 38), and GGSGSSGSGG (SEQ ID NO: 42); or (b) L1, L2, L3 and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:40), GGGGS GGGGS GGGGS (SEQ ID NO:41), S, RT, TKGPS (SEQ ID NO:39), GQPKAAP (SEQ ID NO: 38), and GGSGSSGSGG (SEQ ID NO:42).
12. The binding protein of any one of claims 1-9, wherein L1 comprises the sequence GQPKAAP (SEQ ID NO: 38), L2 comprises the sequence TKGPS (SEQ ID NO:39), L3 comprises the sequence S, and L4 comprises the sequence RT.
13. The binding protein of any one of claims 1-9, wherein at least one of L1, L2, L3 or L4 comprises the sequence DKTHT (SEQ ID NO:37).
14. The binding protein of claim 13, wherein L1, L2, L3 and L4 comprise the sequence DKTHT (SEQ ID NO:37).
15. The binding protein of any one of claims 1-14, wherein the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgG4 hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L235A.
16. The binding protein of any one of claims 1-14, wherein the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgG4 hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236.
17. The binding protein of any one of claims 1-16, wherein the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgG4 hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K.
18. The binding protein of any one of claims 1-14, wherein the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgG1 hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A.
19. The binding protein of any one of claims 1-14, wherein the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgG1 hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgG1 according to EU Index, wherein the amino acid substitutions are S298N, T299A, and Y300S.
20. The binding protein of any one of claims 1-19, wherein the hinge-CH2-CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the hinge-CH2- CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W.
21. The binding protein of any one of claims 1-19, wherein the hinge-CH2-CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W; and wherein the hinge-CH2-CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V.
22. The binding protein of claim 1, wherein:
(a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:61 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:61; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:62 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:62; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:63 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:63; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:64 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 64;
(b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:65 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:65; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:66 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:66; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:67 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:67; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:68 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:68;
(c) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:69; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:70 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:70; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:71 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:71; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:72 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 72;
(d) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:73 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:73; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:74 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:74; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:75 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:75; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:76 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:76;
(e) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:77 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:77; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:78 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:78; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:79 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:79; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:80 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:80;
(f) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:81 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:81; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 82 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:82; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:83 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:83; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:84 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:84;
(g) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:85 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:85; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:86 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:86; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:87 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:87; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:88 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:88;
(h) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:89 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:89; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:90 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:90; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:91 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:91; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:92 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 92;
(i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:93 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:93; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:94 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:94; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:95 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:95; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:96 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:96;
(j) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:97 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:97; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:98 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:98; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:99 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:99; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 100;
(k) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 101 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 101; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 102; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 103 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 104; (l) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 105; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 107 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 107; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 108 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108;
(m) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 109 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 109; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 110 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 110; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 111 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 111; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 112;
(n) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 113; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 114; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 115; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 116 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 116;
(o) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 117; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 119; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120;
(p) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 121; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 122 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 123 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 123; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124;
(q) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 129; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 130; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 131 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 131; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 132;
(r) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 133; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 135; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 136;
(s) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 137; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 138; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 139; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 140;
(t) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 141; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 142; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 143; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 144;
(u) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 145; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 146; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 147; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 148;
(v) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 149; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 150; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 151; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 152 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 152;
(x) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 153; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 154; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 155; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 156;
(y) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 157; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 158; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 159; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 160;
(z) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 161; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 162 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 162; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 163 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 163; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 164 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 164;
(aa) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 165 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 165; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 166 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 166; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 167 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 167; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 168 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 168;
(bb) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 169; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 170 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 170; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 171 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 171; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 172; or
(cc) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 173; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 174; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 175 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 175; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 176.
23. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the binding protein of any one of claims 1-22.
24. An expression vector comprising the nucleic acid molecule of claim 23.
25. An isolated host cell comprising the nucleic acid molecule of claim 23 or the expression vector of claim 24.
26. The isolated host cell of claim 25, wherein the host cell is a mammalian or insect cell.
27. A pharmaceutical composition comprising the binding protein of any one of claims 1- 22 and a pharmaceutically acceptable carrier.
28. A method of preventing and/or treating HIV infection in a patient comprising administering to the patient a therapeutically effective amount of at least one binding protein of any one of claims 1-22 or the pharmaceutical composition of claim 27.
29. The method of claim 28, wherein the binding protein is co-administered with standard anti-retroviral therapy.
30. The method of claim 28 or claim 29, wherein administration of the at least one binding protein results in the elimination of one or more latently and/or chronically HIV-infected cells in the patient.
31. The method of any one of claims 28-30, wherein the patient is a human.
32. The binding protein of any one of claims 1-22 or the pharmaceutical composition of claim 27 for the prevention and/or treatment of HIV infection in a patient.
33. The binding protein for use of claim 32, wherein the binding protein is to be co- administered with standard anti -retroviral therapy.
34. The binding protein for use of claim 32 or claim 33, wherein the binding protein causes the elimination of one or more latently and/or chronically HIV-infected cells in the patient.
35. The binding protein for use of any one of claims 32-34, wherein the patient is a human.
36. A vector system comprising one or more vectors encoding a first, second, third, and fourth polypeptide chain of a binding protein of any one of claims 1-22.
37. The vector system of claim 36, wherein the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein.
38. A kit of polynucleotides, comprising:
(a) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 177, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 178, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 179, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 180;
(b) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 181, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 182, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 183, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 184;
(c) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 185, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 186, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 187, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 188;
(d) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 189, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 190, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 191, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 192;
(e) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 193, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 194, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 195, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 196;
(f) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 197, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 198, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 199, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:200;
(g) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:201, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:202, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:203, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:204;
(h) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:205, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:206, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:207, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:208;
(i) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:209, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:210, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:211, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:212;
(j) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:213, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:214, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:215, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:216;
(k) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:217, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:218, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:219, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:220;
(l) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:221, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:222, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:223, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:224;
(m) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:225, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:226, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:227, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:228;
(n) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:229, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:230, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:231, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:232;
(o) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:233, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:234, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:235, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:236;
(p) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:237, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:238, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:239, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:240;
(q) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:241, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:242, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:243, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:244;
(r) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:245, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:246, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:247, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:248;
(s) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:249, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:250, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:251, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:252;
(t) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:253, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:254, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:255, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:256;
(u) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:257, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:258, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:259, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:260;
(v) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:261, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:262, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:263, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:264;
(w) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:265, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:266, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:267, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:268;
(x) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:269, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:270, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:271, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:272;
(y) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:273, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:274, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:275, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:276;
(z) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:277, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:278, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:279, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:280;
(aa) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:281, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:282, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:283, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:284;
(bb) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:285, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:286, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:287, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:288; or
(cc) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:289, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:290, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:291, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:292.
39. The kit of claim 38, wherein the first, second, third, and fourth polynucleotides are present on one or more expression vectors.
EP20722421.3A 2019-04-09 2020-04-08 Trispecific and/or trivalent binding proteins using the cross-over-dual-variable domain (codv) format for treatment of hiv infection Pending EP3953384A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201962831415P 2019-04-09 2019-04-09
EP19306312 2019-10-08
PCT/US2020/027313 WO2020210386A1 (en) 2019-04-09 2020-04-08 Trispecific and/or trivalent binding proteins using the cross-over-dual-variable domain (codv) format for treatment of hiv infection

Publications (1)

Publication Number Publication Date
EP3953384A1 true EP3953384A1 (en) 2022-02-16

Family

ID=70471109

Family Applications (1)

Application Number Title Priority Date Filing Date
EP20722421.3A Pending EP3953384A1 (en) 2019-04-09 2020-04-08 Trispecific and/or trivalent binding proteins using the cross-over-dual-variable domain (codv) format for treatment of hiv infection

Country Status (15)

Country Link
EP (1) EP3953384A1 (en)
JP (1) JP7676316B2 (en)
KR (1) KR20210149796A (en)
CN (1) CN114096561A (en)
AU (1) AU2020273156A1 (en)
BR (1) BR112021020346A2 (en)
CA (1) CA3136147A1 (en)
CO (1) CO2021014988A2 (en)
IL (1) IL286932A (en)
MA (1) MA55609A (en)
MX (1) MX2021012384A (en)
PH (1) PH12021500042A1 (en)
SG (1) SG11202111011SA (en)
TW (1) TW202104274A (en)
WO (1) WO2020210386A1 (en)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MX2022004881A (en) 2019-10-25 2022-05-16 Sanofi Sa METHODS TO ANALYZE INCORRECT PAIRING OF CHAINS IN MULTI-SPECIFIC BINDING PROTEINS.
TW202330594A (en) * 2021-09-29 2023-08-01 美商莫德斯醫療公司 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
CN118317979A (en) * 2021-09-29 2024-07-09 摩德斯医疗股份有限公司 Antigen-binding polypeptides, antigen-binding polypeptide complexes, and methods of using the same
WO2026047100A1 (en) * 2024-08-29 2026-03-05 Fundació Privada Institut De Recerca Sobre Immunopatologies-Caixa Irsicaixa Bispecific antibody for t cell activation for the killing of hiv infected cells

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9175070B2 (en) 2009-09-25 2015-11-03 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Neutralizing antibodies to HIV-1 and their use
WO2012154312A1 (en) 2011-05-09 2012-11-15 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Neutralizing antibodies to hiv-1 and their use
CN104080805B (en) 2011-11-07 2017-02-22 美国政府健康及人类服务部 Neutralizing gp41 antibodies and their use
CN104271597B (en) 2011-12-08 2018-05-25 美国政府(由卫生和人类服务部的部长所代表) HIV-1 neutralizing antibody and use thereof
WO2013163427A1 (en) 2012-04-25 2013-10-31 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Antibodies to treat hiv-1 infection
US20140213772A1 (en) * 2012-12-28 2014-07-31 Abbvie, Inc. Cross-over dual variable domain immunoglobulin constructs
WO2017011414A1 (en) * 2015-07-10 2017-01-19 Duke University Bispecific molecules comprising an hiv-1 envelope targeting arm
ES2894304T3 (en) * 2015-10-25 2022-02-14 Sanofi Sa Trispecific and/or trivalent binding proteins for the prevention or treatment of HIV infection
EP3443006B1 (en) 2016-04-13 2023-08-02 Sanofi Trispecific and/or trivalent binding proteins
WO2018002902A1 (en) 2016-07-01 2018-01-04 Glaxosmithkline Intellectual Property (No.2) Limited Antibody-drug conjugates and therapeutic methods using the same
CN111971299A (en) * 2018-02-21 2020-11-20 美国政府(由卫生和人类服务部的部长所代表) Neutralizing antibodies to HIV-1Env and uses thereof
JP7664175B2 (en) * 2019-04-09 2025-04-17 サノフイ Trispecific binding proteins, methods and uses thereof
JP7518286B2 (en) * 2020-08-25 2024-07-17 ギリアード サイエンシーズ， インコーポレイテッド Multispecific antigen-binding molecules targeting HIV and methods of use

Also Published As

Publication number Publication date
MX2021012384A (en) 2022-02-21
SG11202111011SA (en) 2021-11-29
JP2022526826A (en) 2022-05-26
CN114096561A (en) 2022-02-25
JP7676316B2 (en) 2025-05-14
IL286932A (en) 2021-10-31
MA55609A (en) 2022-02-16
CO2021014988A2 (en) 2022-01-17
TW202104274A (en) 2021-02-01
WO2020210386A1 (en) 2020-10-15
AU2020273156A1 (en) 2021-12-02
PH12021500042A1 (en) 2022-06-06
CA3136147A1 (en) 2020-10-15
BR112021020346A2 (en) 2021-12-07
KR20210149796A (en) 2021-12-09

Similar Documents

Publication Publication Date Title
JP7724814B2 (en) Trispecific and/or trivalent binding proteins for the prevention or treatment of HIV infection
US20230357401A1 (en) Trispecific anti-cd38, anti-cd28, and anti-cd3 binding proteins and methods of use for treating viral infection
US12227573B2 (en) Trispecific binding proteins, methods, and uses thereof
JP7676316B2 (en) Trispecific and/or trivalent binding proteins using crossover dual variable domain (CODV) formats for the treatment of HIV infection - Patents.com
CA3136821A1 (en) Trispecific binding proteins, methods, and uses thereof
HK40098941A (en) Trispecific and/or trivalent binding proteins for prevention or treatment of hiv infection
HK40100590A (en) Trispecific and/or trivalent binding proteins for prevention or treatment of hiv
HK40052791A (en) Trispecific and/or trivalent binding proteins for prevention or treatment of hiv infection
HK1253385B (en) Trispecific and/or trivalent binding proteins for prevention or treatment of hiv infection

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20211109

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAX Request for extension of the european patent (deleted)
RAV Requested validation state of the european patent: fee paid

Extension state: TN

Effective date: 20211109

Extension state: MA

Effective date: 20211109

REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 40068710

Country of ref document: HK

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20241206