EP3953384A1 - Trispezifische und/oder trivalente bindeproteine unter verwendung eines cross-over-dual-variable-domain(codv)-formats zur behandlung einer hiv-infektion - Google Patents

Trispezifische und/oder trivalente bindeproteine unter verwendung eines cross-over-dual-variable-domain(codv)-formats zur behandlung einer hiv-infektion

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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
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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
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English (en)
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
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Application filed by Sanofi SA, United States Department of Health and Human Services filed Critical Sanofi SA
Publication of EP3953384A1 publication Critical patent/EP3953384A1/de
Pending legal-status Critical Current

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    • 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

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EP20722421.3A 2019-04-09 2020-04-08 Trispezifische und/oder trivalente bindeproteine unter verwendung eines cross-over-dual-variable-domain(codv)-formats zur behandlung einer hiv-infektion Pending EP3953384A1 (de)

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US201962831415P 2019-04-09 2019-04-09
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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

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MX2022004881A (es) 2019-10-25 2022-05-16 Sanofi Sa Metodos para analizar el emparejamiento incorrecto de cadenas en proteinas de union multiespecificas.
TW202330594A (zh) * 2021-09-29 2023-08-01 美商莫德斯醫療公司 抗原結合多肽、抗原結合多肽複合物及其使用方法
EP4408892A4 (de) * 2021-09-29 2025-11-19 Modex Therapeutics Inc Antigenbindende polypeptide, antigenbindende polypeptidkomplexe und verfahren zur verwendung davon in hiv
CN118317979A (zh) * 2021-09-29 2024-07-09 摩德斯医疗股份有限公司 抗原结合多肽、抗原结合多肽复合物及其使用方法
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

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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 (zh) 2011-11-07 2017-02-22 美国政府健康及人类服务部 gp41中和性抗体及其用途
CN104271597B (zh) 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的中和抗体及其用途
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
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WO2017011414A1 (en) * 2015-07-10 2017-01-19 Duke University Bispecific molecules comprising an hiv-1 envelope targeting arm
ES2894304T3 (es) * 2015-10-25 2022-02-14 Sanofi Sa Proteínas de unión triespecíficas y/o trivalentes para la prevención o tratamiento de infección por VIH
EP3443006B1 (de) 2016-04-13 2023-08-02 Sanofi Trispezifische und/oder dreiwertige bindungsproteine
WO2018002902A1 (en) 2016-07-01 2018-01-04 Glaxosmithkline Intellectual Property (No.2) Limited Antibody-drug conjugates and therapeutic methods using the same
CN111971299A (zh) * 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) HIV-1 Env的中和抗体及其用途
JP7664175B2 (ja) * 2019-04-09 2025-04-17 サノフイ 三重特異性結合性タンパク質、方法、およびその使用
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SG11202111011SA (en) 2021-11-29
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JP7676316B2 (ja) 2025-05-14
IL286932A (en) 2021-10-31
MA55609A (fr) 2022-02-16
CO2021014988A2 (es) 2022-01-17
TW202104274A (zh) 2021-02-01
WO2020210386A1 (en) 2020-10-15
AU2020273156A1 (en) 2021-12-02
PH12021500042A1 (en) 2022-06-06
CA3136147A1 (en) 2020-10-15
BR112021020346A2 (pt) 2021-12-07
KR20210149796A (ko) 2021-12-09

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