US20140050720A1 - Ultralong complementarity determining regions and uses thereof - Google Patents

Ultralong complementarity determining regions and uses thereof Download PDF

Info

Publication number
US20140050720A1
US20140050720A1 US13/737,913 US201313737913A US2014050720A1 US 20140050720 A1 US20140050720 A1 US 20140050720A1 US 201313737913 A US201313737913 A US 201313737913A US 2014050720 A1 US2014050720 A1 US 2014050720A1
Authority
US
United States
Prior art keywords
antibody
sequence
ultralong cdr3
antibodies
derived
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.)
Abandoned
Application number
US13/737,913
Other languages
English (en)
Inventor
Vaughn Smider
Omar A. Bazirgan
Dan Ekiert
Hongyuan Helen Mao
Peter Schultz
Feng Wang
Ian Wilson
Yong Zhang
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.)
Scripps Research Institute
Fabrus Inc
Original Assignee
Scripps Research Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Scripps Research Institute filed Critical Scripps Research Institute
Priority to US13/737,913 priority Critical patent/US20140050720A1/en
Publication of US20140050720A1 publication Critical patent/US20140050720A1/en
Assigned to THE CALIFORNIA INSTITUTE FOR BIOMEDICAL RESEARCH reassignment THE CALIFORNIA INSTITUTE FOR BIOMEDICAL RESEARCH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SCHULTZ, PETER, ZHANG, YONG, WANG, FENG
Assigned to THE SCRIPPS RESEARCH INSTITUTE reassignment THE SCRIPPS RESEARCH INSTITUTE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SMIDER, VAUGHN, SCHULTZ, PETER, ZHANG, YONG, WANG, FENG, EKIERT, DAMIAN, WILSON, IAN
Assigned to FABRUS, INC. reassignment FABRUS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BAZIRGAN, Omar A., MAO, Hongyuan Helen, SMIDER, VAUGHN
Assigned to THE SCRIPPS RESEARCH INSTITUTE reassignment THE SCRIPPS RESEARCH INSTITUTE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: THE CALIFORNIA INSTITUTE FOR BIOMEDICAL RESEARCH
Assigned to THE SCRIPPS RESEARCH INSTITUTE reassignment THE SCRIPPS RESEARCH INSTITUTE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FABRUS, INC.
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor

Definitions

  • immunoglobulin constructs comprising at least a portion of an ultralong CDR3, methods of making such constructs, pharmaceutical compositions and medicaments comprising such constructs, and methods of using such constructs and compositions to prevent, inhibit, and/or treat a disease or condition in a subject.
  • Antibodies are natural proteins that the vertebrate immune system forms in response to foreign substances (antigens), primarily for defense against infection.
  • antibodies have been induced in animals under artificial conditions and harvested for use in therapy or diagnosis of disease conditions, or for biological research.
  • Each individual antibody producing cell produces a single type of antibody with a chemically defined composition, however, antibodies obtained directly from animal serum in response to antigen inoculation actually comprise an ensemble of non-identical molecules (e.g., polyclonal antibodies) made from an ensemble of individual antibody producing cells.
  • Some bovine antibodies have unusually long VH CDR3 sequences compared to other vertebrates. For example, about 10% of IgM contains “ultralong” CDR3 sequences, which can be up to 61 amino acids long. These unusual CDR3s often have multiple cysteines. Functional VH genes form through a process called V(D)J recombination, wherein the D-region encodes a significant proportion of CDR3. A unique D-region encoding an ultralong sequence has been identified in cattle. Ultralong CDR3s are partially encoded in the cattle genome, and provide a unique characteristic of their antibody repertoire in comparison to humans. Kaushik et al. (U.S. Pat. Nos. 6,740,747 and 7,196,185) disclose several bovine germline D-gene sequences unique to cattle stated to be useful as probes and a bovine VDJ cassette stated to be useful as a vaccine vector.
  • the present disclosure provides antibodies that comprise an utralong CDR3 including, libraries that comprise an ultralong CDR3, and uses thereof.
  • the present disclosure also provides a library of antibodies or binding fragments thereof, wherein the antibodies or binding fragments thereof comprise an ultralong CDR3.
  • the ultralong CDR3 is 35 amino acids in length or longer, 40 amino acids in length or longer, 45 amino acids in length or longer, 50 amino acids in length or longer, 55 amino acids in length or longer, or 60 amino acids in length or longer.
  • the ultralong CDR3 is 35 amino acids in length or longer.
  • the ultralong CDR3 comprises 3 or more cysteine residues, 4 or more cysteine residues, 5 or more cysteine residues, 6 or more cysteine residues, 7 or more cysteine residues, 8 or more cysteine residues, 9 or more cysteine residues, 10 or more cysteine residues, 11 or more cysteine residues, or 12 or more cysteine residues.
  • the ultralong CDR3 comprises 3 or more cysteine residues.
  • the antibodies or binding fragments thereof comprise a cysteine motif.
  • the cysteine motif is selected from the group consisting of SEQ ID NOS: 45-156. In some embodiments of each or any of the above or below mentioned embodiments, the cysteine motif is selected from the group consisting of SEQ ID NOS: 45-99. In some embodiments of each or any of the above or below mentioned embodiments, the cysteine motif is selected from the group consisting of SEQ ID NOS: 45-135. In some embodiments of each or any of the above or below mentioned embodiments, the cysteine motif is selected from the group consisting of SEQ ID NOS: 100-135. In some embodiments of each or any of the above or below mentioned embodiments, the cysteine motif is selected from the group consisting of SEQ ID NOS: 136-156.
  • the ultralong CDR3 comprises a non-human DH or a derivative thereof.
  • the ultralong CDR3 comprises a JH sequence or a derivative thereof.
  • the ultralong CDR3 comprises: a non-human VH sequence or a derivative thereof, a non-human DH sequence or a derivative thereof and/or a JH sequence or derivative thereof.
  • the ultralong CDR3 comprises an additional amino acid sequence comprising two to six amino acid residues or more positioned between the VH sequence and the DH sequence.
  • the ultralong CDR3 comprises a non-bovine sequence.
  • the non-bovine sequence is a non-antibody or a human sequence.
  • the non-bovine sequence replaces at least a portion of the ultralong CDR3.
  • the non-bovine sequence is a hormone, lymphokine, interleukin, chemokine, cytokine, toxin, or combination thereof.
  • the non-bovine sequence is a cytokine.
  • the cytokine is granulocyte colony-stimulating factor (G-CSF).
  • the ultralong CDR3 comprises an additional sequence that is a linker.
  • the linker is linked to a C-terminus, a N-terminus, or both C-terminus and N-terminus of the non-antibody sequence.
  • the linker is (GGGGS) n (SEQ ID NO: 339), where n is an integer between 0 and 5.
  • the linker is (GSG) n (SEQ ID NO: 342), GGGSGGGGS (SEQ ID NO: 337) or GGGGSGGGS (SEQ ID NO: 338)
  • the ultralong CDR3 comprises a X 1 X 2 X 3 X 4 X 5 X n motif, wherein X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V), wherein X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein X 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), wherein X 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), wherein X 5 is glutamine (Q), and wherein n is 27-54.
  • X 1 is threonine (T), glycine (G), alanine (A
  • the X 1 X 2 X 3 X 4 X 5 motif is TTVHQ (SEQ ID NO: 159) or TSVHQ (SEQ ID NO: 160).
  • the ultralong CDR3 further comprises a (X a X b ) z motif, wherein X a is any amino acid residue, X b is an aromatic amino acid selected from the group consisting of: tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and wherein z is 1-4.
  • the (X a X b ) z motif is YXYXYX.
  • the ultralong CDR3 comprises a X 1 X 2 X 3 X 4 X 5 X n (X a X b ) z motif, wherein X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V), wherein X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein X 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), wherein X 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and wherein X 5 is glutamine (Q), wherein X a is any
  • the X 1 X 2 X 3 X 4 X 5 motif is TTVHQ (SEQ ID NO: 159) or TSVHQ (SEQ ID NO: 160), and wherein the (X a X b ) z motif is YXYXYX.
  • the ultralong CDR3 comprises TSVHQETKKYQ (SEQ ID NO. 157).
  • the ultralong CDR3 comprises VHQETKKYQ (SEQ ID NO: 158).
  • the ultralong CDR3 comprises CTTVHQX n (SEQ ID NO. 223).
  • the ultralong CDR3 comprises CTSVHQX n (SEQ ID NO. 223), wherein n is 1-8.
  • the ultralong CDR3 comprises TTVHQ (SEQ ID NO. 159).
  • the ultralong CDR3 comprises TSVHQ (SEQ ID NO. 160).
  • the ultralong CDR3 comprises VHQ.
  • the ultralong CDR3 comprises KKQ.
  • the ultralong CDR3 comprises VYQ.
  • the ultralong CDR3 comprises CX 1 X 2 X 3 X 4 Q (SEQ ID NO: 228), wherein X 1 is T, S, A, or G, wherein X 2 is T, S, A, P, or I, wherein X 3 is V or K, and wherein X 4 is H, K, or Y.
  • the ultralong CDR3 comprises CX 1 X 2 VHQ (SEQ ID NO: 230), wherein X 1 is T, S, A, or G, and wherein X 2 is T, S, A, P, or I.
  • the ultralong CDR3 comprises CX 1 X 2 VX 3 Q (SEQ ID NO: 232), wherein X 1 is T, S, A, or G, wherein X 2 is T, S, A, P, or I, and wherein X 3 is H, Y, or K.
  • the ultralong CDR3 comprises CX 1 X 2 KKQ (SEQ ID NO: 234), wherein X 1 is T, S, A, or G, and wherein X 2 is T, S, A, P, or I.
  • the ultralong CDR3 comprises YTYNYEW (SEQ ID NO: 235).
  • the ultralong CDR3 comprises YX 1 YX 2 (SEQ ID NO: 296), wherein X 2 is E or D.
  • the ultralong CDR3 comprises YX 1 YX 2 Y (SEQ ID NO: 297).
  • the ultralong CDR3 comprises YEX, wherein X is H, W, N, F, I or Y.
  • the ultralong CDR3 comprises YDX, wherein X is H, W, N, F, I or Y.
  • the ultralong CDR3 comprises XYE, wherein X is T, S, N or I.
  • the ultralong CDR3 comprises XYD, wherein X is T, S, N or I.
  • the ultralong CDR3 comprises Y(E/D)X 1 X n W (SEQ ID NOS: 304-305), wherein X 1 is H, W, N, F, I or Y, and wherein n is 1-4.
  • the antibodies or binding fragments thereof are chimeric, human engineered, or humanized.
  • the ultralong CDR3 is a ruminant CDR3.
  • the ruminant is a cow.
  • the present disclosure also provides a library of polynucleotides encoding for antibodies or binding fragments thereof, wherein the encoded antibodies or binding fragments thereof comprise an ultralong CDR3.
  • the ultralong CDR3 is 35 amino acids in length or longer, 40 amino acids in length or longer, 45 amino acids in length or longer, 50 amino acids in length or longer, 55 amino acids in length or longer, or 60 amino acids in length or longer.
  • the ultralong CDR3 is 35 amino acids in length or longer.
  • the ultralong CDR3 comprises 3 or more cysteine residues, 4 or more cysteine residues, 5 or more cysteine residues, 6 or more cysteine residues, 7 or more cysteine residues, 8 or more cysteine residues, 9 or more cysteine residues, 10 or more cysteine residues, 11 or more cysteine residues, or 12 or more cysteine residues.
  • the ultralong CDR3 comprises 3 or more cysteine residues.
  • the antibodies or binding fragments thereof comprise a cysteine motif.
  • the cysteine motif is selected from the group consisting of SEQ ID NOS: 45-156. In some embodiments of each or any of the above or below mentioned embodiments, the cysteine motif is selected from the group consisting of SEQ ID NOS: 45-99. In some embodiments of each or any of the above or below mentioned embodiments, the cysteine motif is selected from the group consisting of SEQ ID NOS: 45-135. In some embodiments of each or any of the above or below mentioned embodiments, the cysteine motif is selected from the group consisting of SEQ ID NOS: 100-135. In some embodiments of each or any of the above or below mentioned embodiments, the cysteine motif is selected from the group consisting of SEQ ID NOS: 136-156.
  • the ultralong CDR3 comprises a non-human DH or a derivative thereof.
  • the ultralong CDR3 comprises a JH sequence or a derivative thereof.
  • the ultralong CDR3 comprises: a non-human VH sequence or a derivative thereof, a non-human DH sequence or a derivative thereof and/or a JH sequence or derivative thereof.
  • the ultralong CDR3 comprises an additional amino acid sequence comprising two to six amino acid residues or more positioned between the VH sequence and the DH sequence.
  • the ultralong CDR3 comprises a non-bovine sequence.
  • the non-bovine sequence is a non-antibody or a human sequence.
  • the non-bovine sequence replaces at least a portion of the ultralong CDR3.
  • the non-antibody sequence is a hormone, lymphokine, interleukin, chemokine, cytokine, toxin, or combination thereof.
  • the non-bovine sequence is a cytokine.
  • the cytokine is granulocyte colony-stimulating factor (G-CSF).
  • the ultralong CDR3 comprises an additional sequence that is a linker.
  • the linker is linked to a C-terminus, a N-terminus, or both C-terminus and N-terminus of the non-antibody sequence.
  • the linker is (GGGGS) n (SEQ ID NO: 339), where n is an integer between 0 and 5.
  • the linker is (GSG) n (SEQ ID NO: 342), GGGSGGGGS (SEQ ID NO: 337) or GGGGSGGGS (SEQ ID NO: 338).
  • the ultralong CDR3 comprises a X 1 -X 2 -X 3 -X 4 -X 5 motif, wherein X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V), wherein X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein X 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), wherein X 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and wherein X 5 is glutamine (Q).
  • X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine
  • the X 1 X 2 X 3 X 4 X 5 motif is TTVHQ (SEQ ID NO:159) or TSVHQ (SEQ ID NO: 160).
  • the ultralong CDR3 further comprises a (X a X b ) z motif, wherein X a is an aromatic amino acid selected from the group consisting of: tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), wherein X b is any amino acid residue, and wherein z is 1-4.
  • X a is an aromatic amino acid selected from the group consisting of: tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), wherein X b is any amino acid residue, and wherein z is 1-4.
  • the (X a X b ) z motif is YXYXYX.
  • the ultralong CDR3 comprises a X 1 X 2 X 3 X 4 X 5 X n (X 6 X 7 ) z motif, wherein X 1 is threonine (T), glycine (G), alanine (A), serine (S), or valine (V), wherein X 2 is serine (S), threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein X 3 is valine (V), alanine (A), threonine (T), or aspartic acid (D), wherein X 4 is histidine (H), threonine (T), arginine (R), tyrosine (Y), phenylalanine (F), or leucine (L), and wherein X 5 is glutamine (Q), wherein X a is any amino acid
  • the X 1 X 2 X 3 X 4 X 5 motif is TTVHQ (SEQ ID NO: 159) or TSVHQ (SEQ ID NO: 160), and wherein the (X a -X b ) z motif is YXYXYX.
  • the ultralong CDR3 comprises TSVHQETKKYQ (SEQ ID NO. 157).
  • the ultralong CDR3 comprises VHQETKKYQ (SEQ ID NO: 158).
  • the ultralong CDR3 comprises CTTVHQXn (SEQ ID NO. 223).
  • the ultralong CDR3 comprises CTSVHQXn (SEQ ID NO. 224), wherein n is 1-8.
  • the ultralong CDR3 comprises TTVHQ (SEQ ID NO. 159).
  • the ultralong CDR3 comprises TSVHQ (SEQ ID NO. 160).
  • the ultralong CDR3 comprises VHQ.
  • the ultralong CDR3 comprises KKQ.
  • the ultralong CDR3 comprises VYQ.
  • the ultralong CDR3 comprises CX 1 X 2 X 3 X 4 Q, wherein X 1 is T, S, A, or G, wherein X 2 is T, S, A, P, or I, wherein X 3 is V or K, and wherein X 4 is H, K, or Y.
  • the ultralong CDR3 comprises CX 1 X 2 VHQ, wherein X 1 is T, S, A, or G, and wherein X 2 is T, S, A, P, or I.
  • the ultralong CDR3 comprises CX 1 X 2 VX 3 Q, wherein X 1 is T, S, A, or G, wherein X 2 is T, S, A, P, or I, and wherein X 3 is H, Y, or K.
  • the ultralong CDR3 comprises CX 1 X 2 KKQ, wherein X 1 is T, S, A, or G, and wherein X 2 is T, S, A, P, or I.
  • the ultralong CDR3 comprises YTYNYEW (SEQ ID NO: 235).
  • the ultralong CDR3 comprises YX 1 YX 2 , wherein X 2 is E or D.
  • the ultralong CDR3 comprises YX 1 YX 2 Y.
  • the ultralong CDR3 comprises YEX, wherein X is H, W, N, F, I or Y.
  • the ultralong CDR3 comprises YDX, wherein X is H, W, N, F, I or Y.
  • the ultralong CDR3 comprises XYE, wherein X is T, S, N or I.
  • the ultralong CDR3 comprises XYD, wherein X is T, S, N or I.
  • the ultralong CDR3 comprises Y(E/D)X 1 X n W, wherein X 1 is H, W, N, F, I or Y, and wherein n is 1-4.
  • the antibodies or binding fragments thereof are chimeric, human engineered, or humanized.
  • the ultralong CDR3 is a ruminant CDR3.
  • the ruminant is a cow.
  • the antibodies or binding fragments thereof are present in a spatially addressed format.
  • the polynucleotides coding for the antibodies or binding fragments thereof are present in a spatially addressed format.
  • the present disclosure also provides a library of vectors comprising any of the library of polynucleotides disclosed herein.
  • the present disclosure also provides a library of host cells comprising the any library of vectors disclosed herein.
  • the cell is a bacteria, virus, or bacteriophage.
  • the antibodies or binding fragments thereof are displayed on the cell surface.
  • the antibodies or binding fragments thereof are secreted from the cell.
  • the antibodies or binding fragments thereof are present in a spatially addressed format.
  • the present disclosure also provides an antibody or binding fragment thereof comprising an ultralong CDR3, wherein the ultralong CDR3 comprises a non-bovine sequence.
  • the ultralong CDR3 is 35 amino acids in length or longer, 40 amino acids in length or longer, 45 amino acids in length or longer, 50 amino acids in length or longer, 55 amino acids in length or longer, or 60 amino acids in length or longer.
  • the ultralong CDR3 is 35 amino acids in length or longer.
  • the ultralong CDR3 comprises 3 or more cysteine residues, 4 or more cysteine residues, 5 or more cysteine residues, 6 or more cysteine residues, 7 or more cysteine residues, 8 or more cysteine residues, 9 or more cysteine residues, 10 or more cysteine residues, 11 or more cysteine residues, or 12 or more cysteine residues.
  • the ultralong CDR3 comprises 3 or more cysteine residues.
  • the antibodies or binding fragments thereof comprise a cysteine motif.
  • the cysteine motif is selected from the group consisting of SEQ ID NOS: 45-156. In some embodiments of each or any of the above or below mentioned embodiments, the cysteine motif is selected from the group consisting of SEQ ID NOS: 45-99. In some embodiments of each or any of the above or below mentioned embodiments, the cysteine motif is selected from the group consisting of SEQ ID NOS: 45-135. In some embodiments of each or any of the above or below mentioned embodiments, the cysteine motif is selected from the group consisting of SEQ ID NOS: 100-135. In some embodiments of each or any of the above or below mentioned embodiments, the cysteine motif is selected from the group consisting of SEQ ID NOS: 136-156.
  • the non-bovine sequence is a non-antibody sequence or a human sequence.
  • the ultralong CDR3 comprises a non-human DH or a derivative thereof.
  • the ultralong CDR3 comprises a JH sequence or a derivative thereof.
  • the ultralong CDR3 comprises: a non-human VH sequence or a derivative thereof, a non-human DH sequence or a derivative thereof and/or a JH sequence or derivative thereof.
  • the ultralong CDR3 comprises an additional amino acid sequence comprising two to six amino acid residues or more positioned between the VH sequence and the DH sequence.
  • the non-bovine sequence replaces at least a portion of the ultralong CDR3.
  • the non-bovine sequence is a hormone, lymphokine, interleukin, chemokine, cytokine, toxin, or combination thereof.
  • the non-bovine sequence is a cytokine.
  • the cytokine is granulocyte colony-stimulating factor (G-CSF).
  • the antibodies or binding fragments thereof are chimeric, human engineered, or humanized.
  • the ultralong CDR3 is a ruminant CDR3.
  • the ruminant is a cow.
  • the present disclosure also provides a polynucleotide encoding for any antibody or binding fragment thereof disclosed herein.
  • the present disclosure also provides a vector comprising any polynucleotide encoding for the antibody or binding fragment thereof disclosed herein.
  • the present disclosure also provides a host cell comprising any vector disclosed herein.
  • the cell is a bacteria, virus, or bacteriophage.
  • the antibody or binding fragment thereof is displayed on the surface of the cell.
  • the antibody or binding fragment thereof is secreted from the cell.
  • the present disclosure also provides a method of producing an antibody or binding fragment thereof comprising an ultralong CDR3 or fragment thereof comprising culturing a host cell comprising a polynucleotide encoding any of the antibodies disclosed herein under conditions wherein the polynucleotide sequence is expressed and the antibody or binding fragment thereof comprising an ultralong CDR3 or fragment thereof is produced.
  • the methods further comprise recovering the antibody or binding fragment thereof comprising an ultralong CDR3 or fragment thereof from the host cell culture.
  • the present disclosure also provides a pharmaceutical composition comprising any antibody or binding fragment thereof disclosed herein.
  • the present disclosure also provides a method of treating a mammal in need thereof comprising administering to the mammal an amount of any antibody disclosed herein.
  • the mammal is a human.
  • a recombinant antibody or fragment thereof wherein at least a portion of the recombinant antibody or fragment thereof is based on or derived from at least a portion of an ultralong CDR3.
  • an antibody or fragment thereof comprising at least a portion of an ultralong CDR3 sequence and at least a portion of a non-bovine sequence.
  • an antibody or fragment thereof comprising (a) a first antibody sequence, wherein at least a portion of the first antibody sequence is derived from at least a portion of an ultralong CDR3; (b) a non-antibody sequence; and (c) optionally, a second antibody sequence, wherein at least a portion of the second antibody sequence is derived from at least a portion of an ultralong CDR3.
  • the antibodies disclosed herein may be a chimeric, human engineered, or humanized antibody.
  • the antibodies disclosed herein may be a bovine engineered, bovinized, or fully bovine antibody.
  • the antibodies disclosed herein may comprise a Fab, a scFv, dsFv, diabody, (dsFv) 2 , minibody, flex minibody or bi-specific fragment.
  • the antibodies disclosed herein may be an isolated antibody.
  • the antibodies disclosed herein may further comprise a non-antibody sequence.
  • the non-antibody sequence may be derived from a mammal.
  • the mammal may be a bovine, human, or non-bovine mammal.
  • the antibodies disclosed herein may comprise a non-antibody sequence derived from a non-bovine animal.
  • the non-bovine animal may be a scorpion.
  • the non-bovine animal may be a lizard.
  • the lizard may be a gila monster.
  • the non-antibody sequence may be a derived from a growth factor.
  • the growth factor may be a GCSF, GMCSF or FGF21.
  • the GCSF may be a bovine GCSF.
  • the GCSF may be a human GCSF.
  • the GMCSF and/or the FGF21 may be from a human.
  • the non-antibody sequence may be a derived from a cytokine.
  • the cytokine may be a beta-interferon.
  • the non-antibody sequence may be a derived from a hormone.
  • the hormone may be an exendin-4, GLP-1, somatostatin, or erythropoietin.
  • the GLP-1 and/or erythropoietin may be from a human.
  • the non-antibody sequence may be a derived from a toxin.
  • the toxin may be a Moka1, VM-24, ziconotide, chlorotoxin, or protoxin2 (ProTxII).
  • the non-antibody sequence may be IL8, ziconotide, somatostatin, chlorotoxin, SDF1(alpha), or IL21.
  • the non-antibody sequence may comprise an amino acid sequence based on or derived from any of SEQ ID NOS: 317-332.
  • the non-antibody sequence may replace at least a portion of the ultralong CDR3.
  • the non-antibody sequence may be inserted into the sequence of the ultralong CDR3.
  • the non-antibody sequence may be conjugated to at least a portion of the antibody (e.g., ultralong CDR3, variable region, heavy chain, light chain).
  • the non-antibody sequence may be attached to the ultralong CDR3, linker, cleavage site, non-bovine sequence, non-ultralong CDR3 antibody sequence, or combination thereof.
  • the non-antibody sequence may be adjacent to the ultralong CDR3, linker, cleavage site, non-bovine sequence, non-ultralong CDR3 antibody sequence, or combination thereof.
  • the antibodies disclosed herein may comprise an ultralong CDR3 may be based on or derived from a cow ultralong CDR3. At least a portion of the antibodies disclosed herein may be from a mammal. At least a portion of the first antibody sequence and/or at least a portion of the second antibody sequence of the antibodies disclosed herein may be from a mammal. The mammal may be a bovine, human or non-bovine mammal.
  • the antibodies disclosed herein may comprise 3 or more amino acids in length.
  • the antibodies disclosed herein may comprise a sequence that may be based on or derived from an ultralong CDR3 disclosed herein.
  • the antibodies disclosed herein may comprise 1 or more amino acid residues based on or derived from a stalk domain of the ultralong CDR3.
  • the antibodies disclosed herein may comprise 1 or more amino acid residues based on or derived from a knob domain of the ultralong CDR3.
  • At least a portion of the antibodies disclosed herein may be based on or derived from at least a portion of an ultralong CDR3 disclosed herein.
  • the portion of the antibody based on or derived from at least a portion of the ultralong CDR3 may be 20 or fewer amino acids in length.
  • the portion of the antibody based on or derived from at least a portion of the ultralong CDR3 may be 3 or more amino acids in length
  • the antibodies disclosed herein may comprise 1 or more conserved motifs derived from a stalk domain of the ultralong CDR3.
  • the 1 or more conserved motifs derived from the stalk domain of the ultralong CDR3 may comprise any of the stalk domain conserved motifs disclosed herein.
  • the portion of the ultralong CDR3s disclosed herein may comprise at least a portion of a stalk domain of the ultralong CDR3, at least a portion of the knob domain of the ultralong CDR3, or a combination thereof.
  • the antibodies disclosed herein may comprise a sequence selected from any one of SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336.
  • the antibodies disclosed herein may comprise a sequence that may be 50% or more homologous to a sequence selected from any one of SEQ ID NOS: 157-224 and 235-295.
  • a portion of any of the antibodies disclosed herein may be based on or derived from at least a portion of a single ultralong CDR3 sequence.
  • a portion of the antibodies disclosed herein may be based on or derived from at least a portion of two or more different ultralong CDR3 sequences.
  • the portion of the ultralong CDR3 is based on or derived from a BLV1H12 ultralong CDR3 sequence.
  • the portion of the ultralong CDR3 may be based on or derived from a sequence that may be 50% or more homologous to a BLV1H12 ultralong CDR3 sequence.
  • the portion of the ultralong CDR3 may be based on or derived from a BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, or F18 ultralong CDR3 sequence.
  • the portion of the ultralong CDR3 may be based on or derived from a sequence that may be 50% or more homologous to a BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, or F18 ultralong CDR3 sequence.
  • the antibodies disclosed herein may comprise a first and/or second antibody sequence that comprises 3 or more amino acids in length.
  • a portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 and/or the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 may be 20 or fewer amino acids in length.
  • a portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 and/or the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 may be 3 or more amino acids in length.
  • the first and/or second antibody sequences comprise one or more amino acid residues based on or derived from a stalk domain of the ultralong CDR3.
  • the first and/or second antibody sequences may comprise one or more amino acid residues based on or derived from a knob domain of the ultralong CDR3.
  • the one or more amino acid residues derived from the knob domain of the ultralong CDR3 may be a serine and/or cysteine residue.
  • the first and/or second antibody sequences may comprise one or more conserved motifs derived from a stalk domain of the ultralong CDR3.
  • the one or more conserved motifs derived from the stalk domain of the ultralong CDR3 may comprise a sequence selected from any one of SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 and/or the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 comprises a sequence selected from any one of SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 and/or the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 may comprise a sequence that may be 50% or more homologous to a sequence selected from any one of SEQ ID NOS: 157-224 and 235-295.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 may comprise a sequence selected from any one of SEQ ID NOS: 157-234.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 may comprise a sequence that may be 50% or more homologous to a sequence selected from any one of SEQ ID NOS: 157-224.
  • the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 comprises a sequence selected from any one of SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336.
  • the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 may comprise a sequence that may be 50% or more homologous to a sequence selected from any one of SEQ ID NOS: 235-295.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 and the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 may be derived from the same ultralong CDR3 sequence.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 and the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 is derived from two or more different ultralong CDR3 sequences.
  • the portions of the ultralong CDR3 of the first and/or second antibody sequences may be based on or derived from a BLV1H12 ultralong CDR3 sequence.
  • the portions of the ultralong CDR3 of the first and/or second antibody sequences may be based on or derived from a sequence that may be 50% or more homologous to a BLV1H12 ultralong CDR3 sequence.
  • the portions of the ultralong CDR3 of the first and/or second antibody sequences may be based on or derived from a BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, or F18 ultralong CDR3 sequence.
  • the portions of the ultralong CDR3 of the first and/or second antibody sequences may be based on or derived from a sequence that may be 50% or more homologous to a BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, or F18 ultralong CDR3 sequence.
  • the ultralong CDR3 is based on or derived from an ultralong CDR3 that may be 35 or more amino acids in length.
  • the ultralong CDR3 may be based on or derived from an ultralong CDR3 comprising 3 or more cysteine residues.
  • the ultralong CDR3 is based on or derived from an ultralong CDR3 may comprise one or more cysteine motifs.
  • the one or more cysteine motifs may be selected from the group consisting of SEQ ID NOS: 45-156.
  • the cysteine motif is selected from the group consisting of SEQ ID NOS: 45-99.
  • the cysteine motif is selected from the group consisting of SEQ ID NOS: 45-135.
  • the cysteine motif is selected from the group consisting of SEQ ID NOS: 100-135.
  • the cysteine motif is selected from the group consisting of SEQ ID NOS: 136-156.
  • the antibodies disclosed herein may be based on or derived from an ultralong CDR3 that may be 35 or more amino acids in length.
  • the antibodies disclosed herein may be based on or derived from an ultralong CDR3 comprising 3 or more cysteine residues.
  • the antibodies disclosed herein may be based on or derived from an ultralong CDR3 may comprise 1 or more cysteine motifs.
  • the antibodies disclosed herein may comprise an ultralong CDR3 that is 35 or more amino acids in length.
  • the antibodies disclosed herein may comprise an ultralong CDR3 comprising 3 or more cysteine residues.
  • the antibodies disclosed herein may comprise an ultralong CDR3 comprising one or more cysteine motifs.
  • the ultralong CDR3 may be a heavy chain CDR3.
  • the ultralong CDR3 may comprise an amino acid sequence derived from or based on a non-human DH sequence.
  • the ultralong CDR3 may comprise an amino acid sequence derived from or based on a JH sequence.
  • the ultralong CDR3 may comprise an amino acid sequence derived from or based on a non-human VH sequence; an amino acid sequence derived from or based on a non-human DH sequence; and/or an amino acid sequence derived from or based on a JH sequence.
  • the ultralong CDR3 may comprise an additional amino acid sequence comprising at least about two amino acid residues positioned between the VH derived amino acid sequence and the DH derived amino acid sequence.
  • any of the antibodies disclosed herein may comprise a sequence based on or derived from a sequence selected from SEQ ID NOS: 24-44, the antibody or binding fragment thereof encoded by a DNA sequence based on or derived from the DNA of SEQ ID NOS: 2-22.
  • Any of the antibodies disclosed herein may comprise a sequence based on or derived from a sequence selected from SEQ ID NO: 23, the antibody or binding fragment thereof encoded by a DNA sequence based on or derived from the DNA of SEQ ID NO: 1.
  • any of the ultralong CDR3s disclosed herein may comprise a sequence based on or derived from a sequence selected from SEQ ID NOS: 24-44. Any of the antibodies disclosed herein may comprise a sequence based on or derived from a sequence selected from SEQ ID NO: 23. Any of the ultralong CDR3s disclosed herein may be encoded by a DNA sequence that may be derived from or based on SEQ ID NOS: 2-22. Any of the antibodies disclosed herein may comprise a portion encoded by a DNA sequence that may be derived from or based on SEQ ID NO: 1.
  • any of the antibodies disclosed herein may comprise one or more linkers. Any of the antibodies disclosed herein may comprise a first linker sequence. Any of the antibodies disclosed herein may comprise a second linker sequence. The first and second linker sequences may comprise the same sequence. The first and second linker sequences may comprise different sequences. The first and/or second linker sequences may be the same length. The first and/or second linker sequences may be different lengths. The first and/or second linker sequences may be 3 or more amino acids in length.
  • the first and/or second linker sequence may attach the non-antibody sequence to the portion based on or derived from the portion of the ultralong CDR3.
  • the first and/or second linker sequences may attach the non-antibody sequence to the first antibody sequence.
  • the first and/or second linker sequences may attach the non-antibody sequence to the second antibody sequence.
  • the first and/or second linker sequences may be adjacent to a non-antibody sequence, a portion of an ultralong CDR3 sequence, a cleavage site sequence, a non-bovine sequence, an antibody sequence, or a combination thereof.
  • the first and/or second linker sequences may comprise one or more glycine residues.
  • the first and/or second linker sequences may comprise two or more consecutive glycine residues.
  • the first and/or second linker sequences may comprise one or more serine residues.
  • the first and/or second linker sequence may comprise one or more polar amino acid residues.
  • the one or more polar amino acid residues may be selected from serine, threonine, asparagine, or glutamine.
  • the polar amino acid residues may comprise uncharged side chains.
  • any of the antibodies disclosed herein may comprise one or more cleavage sites.
  • the one or more cleavage sites may comprise a recognition site for a protease.
  • the protease may be a Factor Xa or thrombin.
  • the one or more cleavage sites may comprise an amino acid sequence of IEGR.
  • the one or more cleavage site may be between a first antibody sequence and the non-antibody sequence.
  • the one or more cleavage sites may be between a second antibody sequence and the non-antibody sequence.
  • the one or more cleavage sites may be between the one or more linkers and the non-antibody sequence.
  • the one or more cleavage sites may be between a first antibody sequence and the one or more linkers.
  • the one or more cleavage sites may be between a second antibody sequence and the one or more linkers.
  • the one or more cleavage sites may be adjacent to a non-antibody sequence, a portion of an ultralong CDR3 sequence, a linker sequence, an antibody sequence, or a combination thereof.
  • antibodies or binding fragments thereof wherein the antibodies or binding fragments thereof may comprise an ultralong CDR3.
  • nucleic acid library comprising a plurality of polynucleotides comprising sequences coding for antibodies or binding fragments thereof, wherein the antibodies or binding fragments thereof may comprise an ultralong CDR3.
  • nucleic acid library comprising a plurality of polynucleotides comprising sequences coding for antibodies or binding fragments thereof, wherein the antibodies or binding fragments thereof may comprise any of the antibodies disclosed herein.
  • polynucleotide comprising a nucleic acid sequence that encodes a variable region, wherein the variable region may comprise an ultralong CDR3.
  • vector comprising a polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence that encodes a variable region, wherein the variable region may comprise an ultralong CDR3.
  • host cell comprising a polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence that encodes a variable region, wherein the variable region may comprise an ultralong CDR3.
  • polynucleotide comprising a nucleic acid sequence that encodes the antibody or binding fragment thereof of any of the antibodies disclosed herein.
  • vector comprising a polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence that encodes the antibody or binding fragment thereof of any of the antibodies disclosed herein.
  • host cell comprising a polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence that encodes the antibody or binding fragment thereof of any of the antibodies disclosed herein.
  • an antibody or binding fragment thereof comprising an ultralong CDR3 or fragment thereof comprising culturing a host cell comprising a polynucleotide, wherein the polynucleotide may comprise a nucleic acid sequence that encodes the antibody or binding fragment thereof of any of the antibodies disclosed herein under conditions wherein the polynucleotide sequence may be expressed and the antibody or binding fragment thereof comprising an ultralong CDR3 or fragment thereof may be produced.
  • the method may comprise recovering the antibody or binding fragment thereof comprising the ultralong CDR3 or fragment thereof from the host cell culture.
  • composition comprising any of the antibodies disclosed herein.
  • the pharmaceutical compostion may comprise two or more antibodies, wherein at least one of the two or more antibodies comprises at least a portion of an ultralong CDR3.
  • composition comprising (a) an antibody or fragment thereof comprising sequence based on or derived from at least a portion of an ultralong CDR3; and (b) a pharmaceutically acceptable excipient.
  • the antibodies disclosed herein comprise an ultralong CDR3 sequence and a non-antibody sequence.
  • the non-antibody sequence is selected from the group comprising Moka1, Vm24, human GLP-1, Exendin-4, beta-interferon, human EPO, human FGF21, human GMCSF, human interferon-beta, bovine GCSF, human GCSF, be IL8, ziconotide, somatostatin, chlorotoxin, SDF1(alpha), IL21 and a derivative or variant thereof.
  • the non-antibody sequence may comprise an amino acid sequence based on or derived from any of SEQ ID NOS: 317-332.
  • the disease or condition may be selected from the group comprising autoimmune disease, heteroimmune disease or condition, inflammatory disease, pathogenic infection, thromboembolic disorder, respiratory disease or condition, metabolic disease, central nervous system (CNS) disorder, bone disease, cancer, blood disorder, obesity, diabetes, osteoporosis, anemia, or pain.
  • autoimmune disease heteroimmune disease or condition
  • inflammatory disease pathogenic infection
  • thromboembolic disorder respiratory disease or condition
  • metabolic disease central nervous system (CNS) disorder
  • bone disease cancer
  • blood disorder obesity, diabetes, osteoporosis, anemia, or pain.
  • the disease or condition would benefit from the modulation of an ion channel.
  • the ion channel may be selected from the group comprising a potassium ion channel, sodium ion channel, or acid sensing ion channel.
  • the ion channel may be selected from the group comprising Kv1.3 ion channel, Nav1.7 ion channel and acid sensing ion channel (ASIC).
  • the disease or condition would benefit from the modulation of a receptor.
  • the receptor may be selected from the group comprising GLP1R, GCGR, EPO receptor, FGFR, FGF21R, CSFR, GMCSFR, IL8R, IL21R and GCSFR.
  • the disease or condition may be mastitis.
  • the subject may be a mammal.
  • the mammal may be a bovine or human.
  • the crystals may have a space group P2 1 2 1 2 1 .
  • the crystal has the unit cell dimensions of “a” between about 40 to 80 angstroms, between 45 to about 75 angstroms, or between about 50 to about 75 angstroms; “b” between about 40 to 140 angstroms, between about 50 to about 130 angstroms, between about 55 to about 130 angstroms; and “c” between 100 to about 350 angstroms, between 120 to about 340 angstroms, or between about 125 to about 330 angstroms.
  • the crystal may comprise a bovine antibody or portion thereof.
  • the crystal may comprise a Fab fragment based on or derived from a bovine antibody.
  • the crystal may be an isolated crystal.
  • any antibody comprising an ultralong CDR3 may be used including, for example, any of the above mentioned antibodies comprising an ultralong CDR3.
  • FIG. 1A-C present the identification of a new structural domain in bovine antibodies.
  • FIG. 1A shows a comparison of CDR H3 length amongst murine, human, and bovine repertoires. An ultralong subset of over 60 amino acids is uniquely found in bovine heavy chains.
  • FIG. 1B shows sequences of representative CDR H3 from murine (mu), human (hu), or bovine sequences from the literature along with six bovine sequences (B-S1 to B-S4, and B-L1 and B-L2) from our deep sequencing results. The conserved cysteine of framework 3 and tryptophan of framework 4 that define CDR H3 boundaries in all antibody variable regions are shaded in grey for reference. The lengths of the CDR H3s are indicated at the right.
  • the murine antibodies include D44.1, an anti-HEL antibody, 93F3, an aldolase, and OKT3, a therapeutic antibody targeting human CD3. This antibody is unusual in having a free cysteine in CDR H3.
  • the human antibodies include Yvo, a cryoglobulin, CR6261, an anti-influenza A hemaglutinin, and PG9, an anti-HIV antibody which has one of the longest human CDR H3s.
  • the bovine antibodies represent the ultralong sequences in the literature, and short sequences for comparison. BLV5B8 and BLV1H12 (indicated in bold) were used in our structure determinations. Relatively conserved TTVHQ and CPDG motifs are in bold.
  • FIG. 1C shows crystal structures of BLV1H12 (left) and BLV5B8 (middle) Fabs compared to the 93F3 Fab with a “normal” CDR H3 (right).
  • a superlong, two ⁇ -stranded stalk protrudes from each bovine V H immunoglobulin domain and terminates in an unusual three disulfide-linked knob domain.
  • FIG. 2A-C present the structural diversity in ultralong bovine antibodies.
  • FIG. 2A shows a comparison of the structure of the two knobs showing differences in disulfide patterns. Close up views of the knobs of BLV1H12 (left) and BLV5H8 (right) are shown, in addition to a two-dimensional representation of the knob and its disulfide pattern. Disulfides are in numbered and circled. The sequences of the knob regions are shown below, with cysteines conserved with the D H 2 germline gene segment underlined. The disulfide pattern is indicated above each sequence.
  • FIG. 2B shows an overlay of the variable regions of BLV1H12 and BLV5B8 shows structural homology in the variable regions except the upper part of the stalk and knob, which are significantly divergent.
  • FIG. 2C shows surface and charge density representation of BLV1H12 (left) and BLV5B8 (right) showing different shapes and charge in the knob region.
  • FIG. 3A-C present the genetic basis for ultralong antibody formation.
  • FIG. 3A shows the identification of V H BUL, a germline variable region used in ultralong antibodies.
  • the leader sequence is in light grey, coding sequence is indicated with the amino acid translation above, the intron is in italics, and the unique TTVHQ extension, which forms a portion of the ascending strand of the stalk is in bold.
  • the recombination signal sequence heptamer and nonamer are underlined.
  • FIG. 3B shows the V H BUL region is found on chromosome 21.
  • FIG. 3C shows a schematic of the bovine immunoglobulin loci depicting V H BUL, D H 2, and V ⁇ 1x, which are preferentially used in ultralong antibodies.
  • the process of V(D)J recombination assembles the gene segments to form functional ultralong heavy and light chain genes. (bottom left) The V-D-J regions mapped onto the BLV1H12 Fab structure.
  • V H BUL is unique in encoding CDR H1 and CDR5H2 residues that interact with the stalk, as well as a TTVHQ motif that initiates the ascending ⁇ -strand.
  • V ⁇ 1x light chain encodes CDR L1 and CDR L2 residues that interact with the stalk.
  • Arrows indicate areas of potential junctional diversity. Relatively long V-D insertions are indicated in purple. It is unclear whether this sequence results from N-additions, gene conversion, or another mechanism.
  • Bottom right A detailed depiction of the interactions of CDR H1, H2, L1, and L2 with the stalk of BLV1H12, as well as the location of the YxYxY motif of the descending strand.
  • FIG. 4A-C depict deep sequence diversity of bovine ultralong V H CDR H3s.
  • FIG. 4A shows the distribution of the number of cysteines in bovine ultralong CDR H3s of IgM and IgG.
  • FIG. 4B shows the length distribution of ultralong CDR H3s. Note that clonal sequences selected during an immune response can bias the proportion at any given length.
  • FIG. 4C shows representative sequences of ultralong bovine V H CDR H3s. The terminal portion of the V H BUL region is shown, along with junctional diversity at the V-D joint, D H 2 and J H (top). The sequences of BLVH12 and BLV5B8 are shown for comparison, followed by 20 ultralong CDR H3 sequences (bottom).
  • Cysteines conserved with D H 2 are underlined.
  • the conserved cysteine and tryptophan that define the CDR H3 boundaries in all antibody variable regions are highlighted in grey for reference.
  • the CPDG motif is underlined in grey and the region of the descending strand encoding a YxYxY motif is underlined in grey.
  • FIG. 5A-C show that cysteine mutations contribute to ultralong CDR H3 diversity.
  • FIG. 5A shows that the consensus of ultralong CDR H3 deep sequences aligns with D H 2. A consensus sequence for three deep sequencing runs (from two cows) were determined, and aligned with one another and with D H 2. The consensus aligns well except for some areas of insertions/deletions. Thus, either a single D H gene, or highly related genes, produce the diversity of sequences in ultralong CDR H3 antibodies.
  • FIG. 5B shows that the D H 2 gene region analysis showing residues that can readily mutate to cysteine, including SH hotspots. The nucleotide sequence is above and translated amino-acid sequence below.
  • RGYW hotspots which are recognized by AID for SH and/or gene conversion, are boxed. Nucleotides at positions 3, 15, 19, 21, 25, 27, 31, 33, 39, 43, 45, 49, 51, 57, 60, 64, 73, 75, 79, 81, 84, 88, 90, 93, 97, 102, 106, 112, 117, 121, 123, 127, 129, 133, 139, and 145 in (B) can be altered in a single mutation to a cysteine-encoding codon.
  • FIG. 5C shows affinity maturation groups show mutation to and from cysteine. Several groups of clonally related sequences were identified and analyzed for somatic hypermutation. Three groups are shown as examples (labeled 1 to 3 on the left). Sequence differences from cysteine are highlighted in grey. The number of times each sequence is represented in the cluster is shown at the right.
  • FIG. 6A-D show bovine antibodies with ultralong CDR H3s bind antigen.
  • FIG. 6A shows immunization experimental scheme for identifying antigen-specific, ultralong CDR H3 antibodies. Heavy chain variable region mRNA was isolated, amplified by RT PCR, and paired with the invariant light chain to produce a small library of IgG produced in HEK293 cells.
  • FIG. 6B shows ELISA of 132 ultralong CDR H3 antibodies against BVDV (left), and binding activity of the “hits” B8, B9, and H12 in a titration assay (right).
  • FIG. 6C shows the sequences of B8, H9, and H12 in comparison to BLV1H12 and the germline D H 2 region.
  • FIG. 6D shows that H12 binds NS2-3 on cells.
  • a flag-tagged BVDV NS2-3 protein construct was transfected into HEK293A cells and stained with anti-Flag as a positive control (left), the H12 antibody (middle), and B8 (right). Binding assays with untransfected cells are shown on the bottom.
  • FIG. 7A-B depict a model for ultralong CDR H3 diversification into novel minifolds.
  • FIG. 7A shows a schematic of the D H 2 knob with 4 cysteines is shown on the left, with SH and/or gene conversion leading to a multitude of new cysteine patterns and new loops on the right.
  • FIG. 7B shows mechanisms for generating antibody diversity. In humans and mice (left), combinatorial diversity through V(D)J recombination and V H -V L pairing creates a multitude of different binding sites, which are further optimized following antigen exposure by somatic hypermutation.
  • FIG. 8A-J depict schemes showing attachment of bovine G-CSF onto the knob domain of a heavy chain region of bovine BLV1H12 antibody to design an immunoglobulin construct described herein.
  • FIG. 8A shows a ribbon diagram of a heavy chain region and light chain region of bovine BLV1H12 antibody. The boxed region highlights the extended region of the ultralong CDR3 comprising the stalk and knob domain.
  • FIG. 8B shows a ribbon diagram of a growth factor (e.g., bovine G-CSF) inserted into or replacing a portion of the knob domain of a heavy chain region of a bovine BLV1H12 antibody.
  • FIG. 8A shows a ribbon diagram of a growth factor (e.g., bovine G-CSF) inserted into or replacing a portion of the knob domain of a heavy chain region of a bovine BLV1H12 antibody.
  • a growth factor e.g., bovine G-CSF
  • FIG. 8C shows a ribbon diagram of a toxin (e.g., Moka1, VM-24) inserted into or replacing a portion of the knob domain of a heavy chain region of a bovine BLV1H12 antibody.
  • FIG. 8D shows a ribbon diagram of a receptor ligand (e.g., GLP-1, exendin-4) inserted into or replacing a portion of the knob domain of a heavy chain region of a bovine BLV1H12 antibody.
  • FIG. 8E shows a ribbon diagram of a hormone (e.g., erythropoietin) inserted into or replacing a portion of the knob domain of a heavy chain region of a bovine BLV1H12 antibody.
  • FIG. 8F shows a cartoon depicting a heavy chain region and light chain region of bovine BLV1H12 antibody.
  • FIG. 8G shows a cartoon of a growth factor (e.g., bovine G-CSF) inserted into or replacing a portion of the knob domain of a heavy chain region of a bovine BLV1H12 antibody.
  • FIG. 8H shows a cartoon of a toxin (e.g., Moka1, VM-24) inserted into or replacing a portion of the knob domain of a heavy chain region of a bovine BLV1H12 antibody.
  • FIG. 8G shows a cartoon of a growth factor (e.g., bovine G-CSF) inserted into or replacing a portion of the knob domain of a heavy chain region of a bovine BLV1H12 antibody.
  • FIG. 8H shows a cartoon of a toxin (e.g., Moka1, VM-24) inserted into or replacing a portion of the knob domain of a heavy chain region of a
  • FIG. 8I shows a cartoon of a receptor ligand (e.g., GLP-1, exendin-4) inserted into or replacing a portion of the knob domain of a heavy chain region of a bovine BLV1H12 antibody.
  • the left panel shows both ends of the receptor ligand inserted into or replacing a portion of the knob domain; the right panel shows the N-terminus of the receptor ligand released from the knob domain after treatment with a protease.
  • FIG. 8J shows a cartoon of a hormone (e.g., erythropoietin) inserted into or replacing a portion of the knob domain of a heavy chain region of a bovine BLV1H12 antibody.
  • a hormone e.g., erythropoietin
  • Said attachment of the growth factor, toxin, receptor ligand, and hormone can be by means of a polypeptide linker of sequence GGGGS (Ab-peptide L1) or GGGSGGGGS and GGGGSGGGS (Ab-peptide L2).
  • Another construct (Ab-peptide L0) in which attachment is not by means of a linker is not shown in this cartoon.
  • FIG. 9A-9E present illustrative mouse NFS-60 cell proliferative activity for the Ab-bGCSF fusion proteins.
  • FIG. 9A-9B depict proliferative activity of bovine G-CSF and human G-CSF respectively.
  • FIG. 9C depicts the lack of proliferative activity for bovine BLV1H12 antibody in the absence of G-CSF.
  • FIG. 9D depicts proliferative activity of bovine G-CSF inserted into or replacing a portion of the knob domain in the absence of a linker (Ab-bGCSF L0).
  • FIG. 9E depicts proliferative activity of bovine G-CSF inserted into or replacing a portion of the knob domain by means of a polypeptide linker of sequence GGGGS (Ab-bGCSF L1).
  • FIG. 10A-10E present human granulocyte progenitor cell proliferative activities of the Ab-GCSF fusion proteins.
  • FIG. 10A-10B depict proliferative activity of bovine G-CSF and human G-CSF respectively.
  • FIG. 10C depicts the lack of proliferative activity for bovine BLV1H12 antibody (Ab) in the absence of G-CSF.
  • FIG. 10D depicts proliferative activity of bovine G-CSF inserted into or replacing a portion of the knob domain in the absence of a linker (Ab-bGCSF L0).
  • FIG. 10E depicts proliferative activity of bovine G-CSF inserted into or replacing a portion of the knob domain, said attachment by means of a polypeptide linker of sequence GGGGS (Ab-bGCSF L1).
  • FIG. 11A-11B depict pharmacokinetics of Ab-bGCSF fusion proteins in mice.
  • FIG. 12A-12B provide Proliferative activities of Ab-bGCSF fusion proteins on mice neutrophils that are blood stained and counted at the 10th day post-injection.
  • FIG. 13A-13C display expression and purification of Ab-bGCSF fusion proteins in Pichia pastoris .
  • FIG. 13A shows a map of Pichia expression vector for an immunoglobulin construct provided herein.
  • FIG. 13B provides a western blot post-induction of expression of the immunoglobulin constructs Ab-bGCSF L0 and Ab-bGCSF L1.
  • FIG. 13C provides SDS-PAGE gel of purified Ab-bGCSF L0 and Ab-bGCSF L1 expressed in Pichia at a yield of ⁇ 70 ⁇ g per 100 mL culture.
  • FIG. 14A-14C show vectors for expression of the immunoglobulin constructs described herein in free style HEK 293 cells.
  • FIG. 14A provides a vector of Ab-bGCSF L0 heavy chain for expression in free style HEK 293 cells.
  • FIG. 14B provides a vector of Ab-bGCSF L1 heavy chain for expression in free style HEK 293 cells.
  • FIG. 14C provides a vector of Ab-bGCSF light chain for expression in free style HEK 293 cells.
  • FIG. 15A shows SDS-PAGE gel of purified antibody fusions.
  • FIG. 15A SDS-PAGE gel of purified Ab-bGCSF L0 and Ab-bGCSF L1 proteins from HEK 293 cells.
  • FIG. 15B provides a SDS PAGE of the immunoglobulin constructs Ab-Protoxin2 comprising a GGGGS linker attached to both ends of protoxin2.
  • FIG. 16A-16B provide SDS PAGE and activities of the immunoglobulin constructs Ab-Moka1 L0 (no linker) and Ab-Moka1 L1 (linker).
  • FIG. 16A provides a SDS PAGE of the immunoglobulin fusion proteins Ab-Moka1 L0 and Ab-Moka1 L1.
  • FIG. 16B provides BLV1H12-Moka1 fusion proteins inhibitory activities on T cells activation
  • FIG. 17 provides BLV1H12-Moka1 fusion proteins inhibitory activities on human peripheral blood mononuclear cells (PBMCs)
  • PBMCs peripheral blood mononuclear cells
  • FIG. 18A-18C provide SDS PAGE and activities of the immunoglobulin constructs Ab-VM24 L1 (GGGGS linker) and Ab-VM24 L2 (GGGSGGGGS and GGGGSGGGS linkers).
  • FIG. 18A provides a SDS PAGE of the immunoglobulin fusion proteins Ab-VM24 L1 and Ab-VM24 L2.
  • FIG. 18B provides BLV1H12-VM24 L1 fusion protein inhibitory activities on T cells activation.
  • FIG. 18C provides BLV1H12-VM24 L2 fusion protein inhibitory activities on T cells activation.
  • FIG. 19 provides a SDS PAGE of the immunoglobulin constructs Ab-GLP-1 and Ab-Exendin-4.
  • FIG. 20 provides activity of Ab-GLP-1 and Ab-Ex4 on HEK293 cells expressing GLP-1 receptor.
  • FIG. 21 provides proliferative activity of Ab-hEPO fusion proteins on TF1 cells.
  • FIG. 22A-C depicts ultralong CDR3 sequences.
  • the 5 full length ultralong CDR H3s reported in the literature contain between four and eight cysteines and are not highly homologous to one another; however, some conservation of cysteine residues with D H 2 could be found when the first cysteine of these CDR H3s was “fixed” prior to alignment.
  • Four of the seven sequences (BLV1H12, BLV5D3, BLV8C11, and BF4E9) contain four cysteines in the same positions as D H 2, but also have additional cysteines.
  • BLV5B8 has two cysteines in common with the germline D H 2.
  • B-L1 and B-L2 are from initial sequences from bovine spleen, and the remaining are selected ultralong CDR H3 sequences from deep sequencing data.
  • the first group contains the longest CDR H3s identified, and appear clonally related.
  • the * indicates a sequence represented 167 times, suggesting it was strongly selected for function.
  • Several of the eight-cysteine sequences appear selected for function as they were represented multiple times, indicated in parentheses. Other representative sequences of various lengths are indicated in the last group.
  • the framework cysteine and tryptophan residues that define the CDR H3 boundaries are double-underlined.
  • sequences BLV1H12 through UL-77 (left-most column) presented in Tables 2A-C are depicted broken apart into four segments to identify the segments of amino acid residues that are derived from certain germline sequences. Moving from left to right, the first segment is derived from the V H germline and is represented throughout the disclosure as a X 1 X 2 X 3 X 4 X 5 motif. The second segment is a string of spacer amino acid residues designated throughout the disclosure as X n residues. The third segment is a string of amino acid residues derived from the germline D H 2 region and the fourth segment is a string of amino acid residues derived from the germline J H 1 region.
  • the antibodies and fragments thereof comprise at least a portion of an ultralong CDR3.
  • the portion of the ultralong CDR3 may be derived from or based on an ultralong CDR3 sequence.
  • the portion of the ultralong CDR3 may be derived from or based on a stalk domain of an ultralong CDR3 sequence.
  • the portion of the ultralong CDR3 may be derived from or based on a knob domain of an ultralong CDR3 sequence.
  • the antibodies and fragments thereof may further comprise one or more therapeutic polypeptides. The therapeutic polypeptides may be inserted into the portion of the ultralong CDR3.
  • the therapeutic polypeptides may replace one or more amino acid residues in the amino acid sequence of the portion of the ultralong CDR3.
  • the therapeutic polypeptides may replace one or more nucleotides in the nucleic acid sequence of the portion of the ultralong CDR3.
  • the therapeutic polypeptides may be conjugated or attached to the portion of the ultralong CDR3.
  • the antibodies and fragments disclosed herein may further comprise one or more linkers. Additionally, the antibodies and fragments disclosed herein further comprise a cleavage site. A portion of the antibodies and fragments disclosed herien may be based on or derived from an antibody sequence from a different animal or specie from with the ultralong CDR3 is derived.
  • the ultralong CDR3 may be derived from or based on a bovine antibody sequence and the additional and another portion of the antibody sequence may be derived from or based on a non-bovine antibody sequence. Further details of the antibodies and fragments thereof are described herein.
  • the present disclosure provides antibodies or immunoglobulin constructs comprising ultralong CDR3 sequences or portions thereof.
  • the present disclosure provides an antibody comprising an ultralong CDR3.
  • the ultralong CDR3 may be 35 amino acids in length or more (e.g., 40 or more, 45 or more, 50 or more, 55 or more, 60 or more).
  • the ultralong CDR3 may comprise at least a portion of a knob domain of a CDR3, at least a portion of a stalk domain of a CDR3, or a combination thereof.
  • the portion of the knob domain of the CDR3 may comprise one or more conserved motifs derived from the knob domain of the ultralong CDR3.
  • the portion of the stalk domain of the CDR3 may comprise one or more conserved motis derived from the stalk domain of the ultralong CDR3.
  • Such an antibody may comprise at least 3 cysteine residues or more (e.g., 4 or more, 6 or more, 8 or more) within the ultralong CDR3.
  • the antibody may comprise one or more cysteine motifs.
  • the antibody may comprise a non-antibody sequence within the ultralong CDR3.
  • the antibody comprises a non-bovine sequence.
  • the non-bovine sequence can be linked to the ultralong CDR3 sequence.
  • the antibody may further comprise a linker.
  • the linker comprises an amino acids sequence of (GSG) n , GGGSGGGGS or GGGGSGGGS.
  • the antibody may comprise a non-bovine sequence within the ultralong CDR3.
  • the antibody may further comprise an antibody sequence, wherein the antibody sequence does not comprise an ultralong CDR3 sequence.
  • the antibody may further comprise an antibody sequence, wherein the amino acid sequence identitity of the antibody peptide sequence to the ultralong CDR3 peptide sequence is about 40% or less (e.g., about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, 10% or less, about 5% or less, about 3% or less, about 1% or less).
  • the antibody may comprise a cytotoxic agent or therapeutic polypeptide.
  • the cytotoxic agent or therapeutic polypeptide may be conjugated to the ultralong CDR3.
  • the antibody may bind to a target.
  • the target may be a protein target.
  • the protein target may be a transmembrane protein target.
  • the antibody may comprise at least a portion of a BLV1H12 and/or BLVCV1 antibody. Alternatively, or additionally, the antibody comprises at least a portion of a BLV5D3, BLV8C11, BF1H1, BLV5B8 and/or F18 antibody.
  • the antibody may comprise at least a portion of a human antibody.
  • the antibody may be a chimeric, recombinant, engineered, synthetic, humanized, fully human, or human engineered antibody.
  • the antibody may comprise antibody sequences from two or more different antibodies. The two or more different antibodies may be from the same species.
  • the specie may be a bovine specie, human specie, or murine specie.
  • the two or more different antibodies may be from the same type of animal.
  • the two or more different antibodies may be from a cow.
  • the two or more different antibodies may be from a human.
  • the two or more different antibodies are from different species.
  • the two or more different antibodies are from a human specie and bovine specie.
  • the two or more diffent antibodies are from a bovine specie and a non-bovine specie.
  • the two or more different antibodies are from a human specie and a non-human specie.
  • the present disclosure provides an antibody comprising an ultralong CDR3, wherein the CDR3 is 35 amino acids in length or more and is derived from or based on a non-human sequence.
  • the ultralong CDR3 sequence may be derived from any species that naturally produces ultralong CDR3 antibodies, including ruminants such as cattle ( Bos taurus ).
  • the antibody may comprise at least a portion of a BLV1H12 and/or BLVCV1 antibody.
  • the antibody comprises at least a portion of a BLV5D3, BLV8C11, BF1H1, BLV5B8 and/or F18 antibody.
  • the ultralong CDR3 sequence may be derived from a camelid or shark CDR3 sequence.
  • the present disclosure provides an antibody comprising an ultralong CDR3, wherein the CDR3 comprises a non-antibody sequence.
  • the non-antibody sequence may be derived from any protein family including, but not limited to, chemokines, growth factors, peptides, cytokines, cell surface proteins, serum proteins, toxins, extracellular matrix proteins, clotting factors, secreted proteins, etc.
  • the non-antibody sequence may be derived from a therapeutic polypeptide.
  • the non-antibody sequence may be of human or non-human origin.
  • the non-antibody sequence may comprise a synthetic sequence.
  • the non-antibody sequence may comprise a portion of a non-antibody protein such as a peptide or domain.
  • the non-antibody sequence of an ultralong CDR3 may contain mutations from its natural sequence, including amino acid changes (e.g., substitutions), insertions or deletions. Engineering additional amino acids at the junction between the non-antibody sequence may be done to facilitate or enhance proper folding of the non-antibody sequence within the antibody.
  • the CDR3 may be 35 amino acids in length or more.
  • the ultralong CDR3 may comprise at least a portion of a knob domain of a CDR3, at least a portion of a stalk domain of a CDR3, or a combination thereof.
  • the portion of the knob domain of the CDR3 may comprise one or more conserved motifs derived from the knob domain of the ultralong CDR3.
  • the portion of the stalk domain of the CDR3 may comprise one or more conserved motis derived from the stalk domain of the ultralong CDR3.
  • the antibody comprises at least 3 cysteine residues or more.
  • the antibody can comprise one or more cysteine motifs.
  • the antibody may comprise at least a portion of a BLV1H12 and/or BLVCV1 antibody.
  • the antibody comprises at least a portion of a BLV5D3, BLV8C11, BF1H1, BLV5B8 and/or F18 antibody.
  • the present disclosure provides an antibody comprising an ultralong CDR3 and a non-bovine sequence.
  • the ultralong CDR3 can be derived from a ruminant.
  • the ruminant can be a bovine.
  • the non-bovine sequence can be derived from or based on a non-bovine mammal sequence.
  • the non-bovine sequence can be derived from or based on a human, mouse, rat, sheep, dog, and/or goat sequence.
  • the ultralong CDR3 sequence can comprise the non-bovine sequence.
  • the non-bovine sequence is linked to the ultralong CDR3 sequence.
  • the non-bovine sequence can be derived from or based on at least a portion of an antibody sequence.
  • the antibody sequence can encode a variable region, constant region or a combination thereof.
  • the CDR3 may be 35 amino acids in length or more.
  • the ultralong CDR3 may comprise at least a portion of a knob domain of a CDR3, at least a portion of a stalk domain of a CDR3, or a combination thereof.
  • the portion of the knob domain of the CDR3 may comprise one or more conserved motifs derived from the knob domain of the ultralong CDR3.
  • the portion of the stalk domain of the CDR3 may comprise one or more conserved motis derived from the stalk domain of the ultralong CDR3.
  • the antibody comprises at least 3 cysteine residues or more.
  • the antibody can comprise one or more cysteine motifs.
  • the present disclosure provides an antibody comprising an ultralong CDR3, wherein the CDR3 is 35 amino acids in length or more and comprises at least 3 cysteine residues or more, including, for example, 4 or more, 6 or more, and 8 or more.
  • the present disclosure provides for an antibody comprising an ultralong CDR3 wherein the CDR3 is 35 amino acids in length or more and comprises at least 3 cysteine residues or more and wherein the ultralong CDR3 is a component of a multispecific antibody.
  • the multispecific antibody may be bispecific or comprise greater valencies.
  • the present disclosure provides an antibody comprising an ultralong CDR3, wherein the CDR3 is 35 amino acids in length or more and comprises at least 3 cysteine residues or more, wherein the ultralong CDR3 is a component of an immunoconjugate.
  • the present disclosure provides an antibody comprising an ultralong CDR3, wherein the CDR3 is 35 amino acids in length or more and comprises at least 3 cysteine residues or more, wherein the antibody comprising an ultralong CDR3 binds to a transmembrane protein target.
  • transmembrane targets may include, but are not limited to, GPCRs, ion channels, transporters, and cell surface receptors.
  • the present disclosure provides an antibody comprising an ultralong CDR3, wherein the antibody comprising an ultralong CDR3 binds to a transmembrane protein target.
  • transmembrane targets may include, but are not limited to, GPCRs, ion channels, transporters, and cell surface receptors.
  • the CDR3 may be 35 amino acids in length or more.
  • the ultralong CDR3 may comprise at least a portion of a knob domain of a CDR3, at least a portion of a stalk domain of a CDR3, or a combination thereof.
  • the portion of the knob domain of the CDR3 may comprise one or more conserved motifs derived from the knob domain of the ultralong CDR3.
  • the portion of the stalk domain of the CDR3 may comprise one or more conserved motis derived from the stalk domain of the ultralong CDR3.
  • the antibody comprises at least 3 cysteine residues or more.
  • the antibody can comprise one or more cysteine motifs.
  • an immunoglobulin construct comprising a mammalian immunoglobulin heavy chain comprising at least a portion of complementarity-determining region 3 (CDR3H); and a therapeutic polypeptide, wherein the therapeutic polypeptide is inserted into or replaces at least a portion of the CDR3H.
  • the immunoglobulin construct may comprise one or more linkers.
  • the one or more linkers can connect the therapeutic polypeptide to the heavy chain.
  • the linker comprises an amino acid acid sequence of (GSG)n (SEQ ID NO: 342), GGGSGGGGS (SEQ ID NO: 337) or GGGGSGGGS.
  • the therapeutic polypeptide is selected from a hormone, a lymphokine, an interleukin, a chemokines, a cytokine and combinations thereof.
  • the therapeutic polypeptide is a cytokine.
  • the therapeutic polypeptide is a colony stimulating factor polypeptide.
  • the colony stimulating factor is macrophage colony-stimulating factor (M-C SF), Granulocyte-macrophage colony-stimulating factor (GM-CSF), Granulocyte colony-stimulating factor (G-CSF) or fragment, or variant thereof.
  • the colony stimulating factor is mammalian G-CSF or derivative or variant thereof.
  • the colony stimulating factor is bovine G-CSF or derivative or variant thereof.
  • the therapeutic polypeptide is Moka1, Vm24, human GLP-1, Exendin-4, human EPO, human FGF21, human GMCSF, or human interferon-beta.
  • immunoglobulin constructs comprising a mammalian immunoglobulin heavy chain comprising a knob domain in the complementarity-determining region 3 (CDR3H) or fragment thereof; and a therapeutic polypeptide attached to said knob domain of the CDR3H, wherein said mammalian immunoglobulin is a bovine immunoglobulin.
  • the bovine immunoglobulin is a BLV1H12 antibody.
  • at least a portion of the knob domain is replaced by the therapeutic polypeptide.
  • the knob domain of the CDR3H may comprise one or more conserved motifs derived from the knob domain of the ultralong CDR3H.
  • the immunoglobulin construct may further comprise at least a portion of a stalk domain in the CDR3H.
  • the portion of the stalk domain of the CDRH3 may comprise one or more conserved motis derived from the stalk domain of the ultralong CDR33H.
  • antibodies or fragments thereof comprising a stalk domain in the complementarity-determining region 3 (CDR3H) or fragment thereof; and a therapeutic polypeptide.
  • the complementarity-determining region 3 (CDR3H) is derived from a bovine ultralong CDR3H.
  • the therapeutic polypeptide can be any of the therapeutic polypeptides disclosed herein.
  • the therapeutic polypeptide is Moka1, Vm24, GLP-1, Exendin-4, human EPO, human FGF21, human GMCSF, or human interferon-beta.
  • the therapeutic polypeptide can be attached to the stalk domain.
  • the antibody or fragment thereof further comprises a linker. The linker can attach the therapeutic polypeptide to the stalk domain.
  • the antibody or fragment thereof further comprises at least a portion of a knob domain in the CDR3H.
  • the linker attaches the therapeutic polypeptide to the knob domain.
  • the knob domain is attached to the stalk domain.
  • the portion of the knob domain of the CDR3 may comprise one or more conserved motifs derived from the knob domain of the ultralong CDR3.
  • the stalk domain of the CDR3 may comprise one or more conserved motis derived from the stalk domain of the ultralong CDR3.
  • an antibody or fragment thereof is provided herein.
  • the antibody or fragment thereof can comprise at least one immunoglobulin domain or fragment thereof; and a therapeutic polypeptide or derivative or variant thereof.
  • the therapeutic polypeptide, or derivative or variant thereof can be attached to the immunoglobulin domain.
  • the therapeutic polypeptide is Moka1, Vm24, GLP-1, Exendin-4, human EPO, human FGF21, human GMCSF, human interferon-beta, or derivative or variant thereof.
  • the immunoglobulin domain is an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M.
  • the immunoglobulin domain can be an immunoglobulin heavy chain region or fragment thereof.
  • the immunoglobulin domain is from a mammalian antibody.
  • the immunoglobulin domain is from a chimeric antibody.
  • the immunoglobulin domain is from an engineered antibody or recombinant antibody.
  • the immunoglobulin domain is from a humanized, human engineered or fully human antibody.
  • the mammalian antibody is a bovine antibody.
  • the mammalin antibody is a human antibody.
  • the mammalian antibody is a murine antibody.
  • the immunoglobulin domain is a heavy chain region comprising a knob domain in the complementarity-determining region 3 (CDR3H) or fragment thereof. The therapeutic polypeptide can be attached to the knob domain.
  • the immunoglobulin domain is a heavy chain region comprising a stalk domain in the complementarity-determining region 3 (CDR3H) or fragment thereof.
  • the therapeutic polypeptide is attached to the stalk domain.
  • the antibody or fragment thereof further comprises a linker.
  • the linker can attach the therapeutic polypeptide to the immunoglobulin domain or fragment thereof.
  • the knob domain of the CDR3 may comprise one or more conserved motifs derived from the knob domain of the ultralong CDR3.
  • the stalk domain of the CDR3 may comprise one or more conserved motis derived from the stalk domain of the ultralong CDR3.
  • an immunoglobulin construct comprising at least one immunoglobulin domain or fragment thereof; and a G-CSF polypeptide or derivative or variant thereof attached to said immunoglobulin domain.
  • the immunoglobulin domain is an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M.
  • the immunoglobulin domain is an immunoglobulin heavy chain region or fragment thereof.
  • the immunoglobulin domain is from a mammalian or chimeric antibody.
  • the immunoglobulin domain is from a humanized, human engineered or fully human antibody.
  • the mammalian antibody is a bovine antibody.
  • the mammalian antibody is a human antibody. In other instances, the mammalian antibody is a murine antibody.
  • the immunoglobulin domain is a heavy chain region comprising a knob domain in the complementarity-determining region 3 (CDR3H) or fragment thereof.
  • the G-CSF polypeptide is attached to the knob domain.
  • the immunoglobulin domain may be a heavy chain region comprising a stalk domain in the complementarity-determining region 3 (CDR3H) or fragment thereof.
  • the G-CSF polypeptide may be attached to the stalk domain.
  • the knob domain of the CDR3 may comprise one or more conserved motifs derived from the knob domain of the ultralong CDR3.
  • the stalk domain of the CDR3 may comprise one or more conserved motis derived from the stalk domain of the ultralong CDR3.
  • an immunoglobulin construct comprising at least one immunoglobulin domain or fragment thereof; and a G-CSF polypeptide or derivative or variant thereof attached to said immunoglobulin domain, wherein said G-CSF polypeptide is a bovine G-CSF polypeptide or derivative or variant thereof.
  • a pharmaceutical composition comprising an immunoglobulin construct provided herein, and a pharmaceutically acceptable carrier.
  • a method of preventing or treating a disease in a mammal in need thereof comprising administering a pharmaceutical composition described herein to said mammal.
  • the immunoglobulin domain may be a heavy chain region comprising a knob domain in the complementarity-determining region 3 (CDR3H) or fragment thereof.
  • the G-CSF polypeptide may be attached to the knob domain.
  • the immunoglobulin domain may be a heavy chain region comprising a stalk domain in the complementarity-determining region 3 (CDR3H) or fragment thereof.
  • the G-CSF polypeptide may be attached to the stalk domain.
  • the knob domain of the CDR3 may comprise one or more conserved motifs derived from the knob domain of the ultralong CDR3.
  • the stalk domain of the CDR3 may comprise one or more conserved motis derived from the stalk domain of the ultralong CDR3.
  • an antibody or fragment thereof comprising: (a) a first antibody sequence, wherein at least a portion of the first antibody sequence is derived from at least a portion of an ultralong CDR3; and (b) a non-antibody sequence.
  • the antibody or fragment thereof may further comprise a second antibody sequence, wherein at least a portion of the second antibody sequence is derived from at least a portion of an ultralong CDR3.
  • the ultralong CDR3 from which the first antibody sequence and/or second antibody sequence may be derived from a ruminant.
  • the ruminant can be a cow.
  • At least a portion of the first antibody sequence and/or at least a portion of the second antibody sequence can be derived from a mammal.
  • the mammal may be a bovine.
  • the mammal is a non-bovine mammal, such as a human.
  • the first and/or second antibody sequences may be 3 or more amino acids in length.
  • the amino acids may be consecutive amino acids.
  • the amino acids are non-consecutive amino acids.
  • the first and/or second antibody sequences may comprise a bovine antibody sequence comprising 3 or more amino acids in length.
  • the bovine antibody may be a BLVH12, BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, or F18 antibody.
  • the first and/or second antibody sequences may comprise a human antibody sequence comprising 3 or moreore amino acids in length.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 and/or the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 can be 20 or fewer amino acids in length.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 and/or the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 may be 3 or more amino acids in length.
  • the first and/or second antibody sequences can comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 30 or more, or 40 or more amino acid residues derived from a knob domain of the ultralong CDR3.
  • the 1 or more amino acid residues derived from the knob domain of the ultralong CDR3 may be a serine and/or cysteine residue.
  • the first and/or second antibody sequences may comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more amino acid residues derived from a stalk domain of the ultralong CDR3.
  • the first and/or second antibody sequences may comprise 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more conserved motifs derived from a stalk domain of the ultralong CDR3.
  • the one or more conserved motifs derived from the stalk domain of the ultralong CDR3 may comprise a sequence selected from any one of SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 and/or the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 may comprise a sequence selected from any one of SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 and/or the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 may comprise a sequence that is 50% or more homologous to a sequence selected from any one of SEQ ID NOS: 157-224 and 235-295.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 may comprise a sequence selected from any one of SEQ ID NOS: 157-234.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 may comprise a sequence that is 50% or more homologous to a sequence selected from any one of SEQ ID NOS: 157-224.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 may comprise a sequence that is 50% or more homologous to a sequence selected from any one of SEQ ID NOS: 225-227.
  • the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 may comprise a sequence selected from any one of SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336.
  • the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 may comprise a sequence that may be 50% or more homologous to a sequence selected from any one of SEQ ID NOS: 235-295.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 and the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 may be derived from the same ultralong CDR3 sequence.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 and the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 may be derived from two or more different ultralong CDR3 sequences.
  • the portions of the ultralong CDR3 of the first and/or second antibody sequences may be based on or derived from a BLV1H12 ultralong CDR3 sequence.
  • the portions of the ultralong CDR3 of the first and/or second antibody sequences may be based on or derived from a BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, or F18 ultralong CDR3 sequence.
  • the antibody may further comprise one or more linker sequences.
  • the present disclosure also provides antibodies that comprise a heavy chain polypeptide, wherein the heavy chain polypeptide comprises at least a portion of an ultralong CDR3 sequence.
  • the heavy chain polypeptide may comprise a polypeptide sequence based on or derived from a polypeptide sequence of any one of SEQ ID NOS: 24-44.
  • the heavy chain polypeptide may comprise a polypeptide sequence encoded by a DNA sequence based on or derived from the DNA sequence of any one of SEQ ID NOS: 2-22.
  • antibodies comprising a heavy chain polypeptide wherein the heavy chain polypeptide comprises an ultralong CDR3 sequence and the heavy chain polypeptide sequences are substantially similar to those polypeptide sequences provided by any one of SEQ ID NOS: 24-44.
  • a heavy chain polypeptide sequence may be considered substantially similar to a polypeptide sequence provided by any one of SEQ ID NOS: 24-44 where the heavy chain polypeptide sequence shares 60%, 70%, 80%, 90%, 95%, 99%, or more nucleic acid identity to a nucleotide sequence provided by any one of SEQ ID NOS: 24-44.
  • the antibodies may further comprise a light chain polypeptide.
  • the light chain polypeptide may comprise a polypeptide sequence based on or derived from a polypeptide sequence of SEQ ID NO: 23.
  • the light chain polypeptide may comprise a polypeptide sequence encoded by a DNA sequence based on or derived from the DNA sequence of SEQ ID NO: 1.
  • antibodies further comprising a light chain polypeptide, wherein the light chain polypeptide comprises an ultralong CDR3 sequence and the light chain polypeptide sequences are substantially similar to those polypeptide sequences provided by SEQ ID NO: 23.
  • a light chain polypeptide sequence may be considered substantially similar to a polypeptide sequence provided by SEQ ID NO: 1 where the light chain polypeptide sequence shares 60%, 70%, 80%, 90%, 95%, 99%, or more nucleic acid identity to a nucleotide sequence provided by any one of SEQ ID NO: 1.
  • the antibody may have therapeutic activity in an animal.
  • the antibody can have therapeutic activity in infectious disease in a subject.
  • the antibody may comprise a monoclonal antibody, polyclonal antibody, chimeric antibody, recombinant antibody, engineered antibody, or synthetic antibody.
  • the antibody may comprise a mammalian antibody.
  • the antibody may comprise a bovine antibody.
  • the antibody may comprise a G-CSF polypeptide, or derivative or variant thereof.
  • the antibody may comprise a mammalian G-CSF polypeptide, or derivative or variant thereof.
  • the antibody may comprise a bovine G-CSF, or derivative or variant thereof.
  • a pharmaceutical composition of therapeutic formulation comprises an antibody described herein and a pharmaceutically acceptable carrier.
  • the antibody is used in a method of treating a subject in need thereof, with a therapeutically effective amount of the antibody or a pharmaceutical composition described herein.
  • a nucleic acid molecule or a complement thereof encodes a therapeutic immunoglobulin described herein.
  • the present disclosure provides genetic sequences (e.g., genes, nucleic acids, polynucleotides) encoding antibodies comprising ultralong CDR3 sequences or portions thereof.
  • the present disclosure provides genetic sequences (e.g., genes, nucleic acids, polynucleotides) encoding antibodies comprising the knob domain and/or knob domain of ultralong CDR3 sequences.
  • the present disclosure provides genetic sequences encoding an antibody or immunoglobulin construct described herein.
  • the present disclosure also provides genetic sequences (e.g., genes, nucleic acids, polynucleotides) encoding an ultralong CDR3 or portion thereof.
  • the present disclosure also provides genetic sequences (e.g., genes, nucleic acids, polynucleotides) encoding the knob domain and/or knob domain of an ultralong CDR3.
  • the present disclosure provides genetic sequences encoding an antibody comprising an ultralong CDR3.
  • the ultralong CDR3 may be 35 amino acids in length or more (e.g., 40 or more, 45 or more, 50 or more, 55 or more, 60 or more).
  • the ultralong CDR3 may comprise at least a portion of a knob domain of a CDR3, at least a portion of a stalk domain of a CDR3, or a combination thereof.
  • Such an antibody may comprise at least 3 cysteine residues or more (e.g., 4 or more, 6 or more, 8 or more) within the ultralong CDR3.
  • the antibody may comprise one or more cysteine motifs.
  • the antibody may comprise a non-antibody sequence within the ultralong CDR3.
  • the antibody comprises a non-bovine sequence.
  • the antibody may further comprise an antibody sequence.
  • the antibody may comprise a cytotoxic agent or therapeutic polypeptide.
  • the cytotoxic agent or therapeutic polypeptide may be conjugated to the ultralong CDR3.
  • the antibody may bind to a target.
  • the target may be a protein target, such as a transmembrane protein target.
  • the present disclosure provides genetic sequences encoding an antibody comprising an ultralong CDR3, wherein the CDR3 is 35 amino acids in length or more and is derived from or based on a non-human sequence.
  • the genetic sequences encoding the ultralong CDR3 may be derived from any species that naturally produces ultralong CDR3 antibodies, including ruminants such as cattle ( Bos taurus ).
  • the ultralong CDR3 sequence may be derived from a camelid or shark CDR3 sequence.
  • the present disclosure provides genetic sequences encoding an antibody comprising an ultralong CDR3, wherein the CDR3 comprises a non-antibody protein sequence.
  • the genetic sequences encoding the non-antibody protein sequences may be derived from any protein family including, but not limited to, chemokines, growth factors, peptides, cytokines, cell surface proteins, serum proteins, toxins, extracellular matrix proteins, clotting factors, secreted proteins, etc.
  • the non-antibody sequence may be derived from a therapeutic polypeptide.
  • the non-antibody protein sequence may be of human or non-human origin.
  • the non-antibody sequence may comprise a synthetic sequence.
  • the non-antibody sequence may comprise a portion of a non-antibody protein such as a peptide or domain.
  • the non-antibody protein sequence of an ultralong CDR3 may contain mutations from its natural sequence, including amino acid changes (e.g., substitutions), insertions or deletions. Engineering additional amino acids at the junction between the non-antibody sequence may be done to facilitate or enhance proper folding of the non-antibody sequence within the antibody.
  • the CDR3 may be 35 amino acids in length or more.
  • the ultralong CDR3 may comprise at least a portion of a knob domain of a CDR3, at least a portion of a stalk domain of a CDR3, or a combination thereof.
  • the antibody comprises at least 3 cysteine residues or more.
  • the antibody can comprise one or more cysteine motifs.
  • the present disclosure provides genetic sequences encoding an antibody comprising an ultralong CDR3 and a non-bovine sequence.
  • the ultralong CDR3 can be derived from a ruminant.
  • the ruminant can be a bovine.
  • the non-bovine sequence can be derived from or based on a non-bovine mammal sequence.
  • the non-bovine sequence can be derived from or based on a human, mouse, rat, sheep, dog, and/or goat sequence.
  • the non-bovine sequence can be within the ultralong CDR3.
  • the non-bovine sequence is linked or attached to the ultralong CDR3 sequence.
  • the non-bovine sequence can be derived from or based on at least a portion of an antibody sequence.
  • the antibody sequence can encode a variable region, constant region or a combination thereof.
  • the CDR3 may be 35 amino acids in length or more.
  • the ultralong CDR3 may comprise at least a portion of a knob domain of a CDR3, at least a portion of a stalk domain of a CDR3, or a combination thereof.
  • the antibody comprises at least 3 cysteine residues or more.
  • the antibody can comprise one or more cysteine motifs.
  • the present disclosure provides genetic sequences encoding an antibody comprising an ultralong CDR3, wherein the CDR3 is 35 amino acids in length or more and comprises at least 3 cysteine residues or more, including, for example, 4 or more, 6 or more, and 8 or more.
  • the present disclosure provides genetic sequences encoding an antibody comprising an ultralong CDR3 wherein the CDR3 is 35 amino acids in length or more and comprises at least 3 cysteine residues or more and wherein the ultralong CDR3 is a component of a multispecific antibody.
  • the multispecific antibody may be bispecific or comprise greater valencies.
  • the present disclosure provides genetic sequences encoding an antibody comprising an ultralong CDR3, wherein the CDR3 is 35 amino acids in length or more and comprises at least 3 cysteine residues or more, wherein the ultralong CDR3 is a component of an immunoconjugate.
  • the present disclosure provides genetic sequences encoding an antibody comprising an ultralong CDR3 wherein the CDR3 is 35 amino acids in length or more and comprises at least 3 cysteine residues or more and wherein the antibody comprising an ultralong CDR3 binds to a transmembrane protein target.
  • transmembrane targets may include, but are not limited to, GPCRs, ion channels, transporters, and cell surface receptors.
  • the present disclosure provides genetic sequences encoding an antibody comprising an ultralong CDR3, wherein the antibody comprising an ultralong CDR3 binds to a transmembrane protein target.
  • transmembrane targets may include, but are not limited to, GPCRs, ion channels, transporters, and cell surface receptors.
  • the CDR3 may be 35 amino acids in length or more.
  • the ultralong CDR3 may comprise at least a portion of a knob domain of a CDR3, at least a portion of a stalk domain of a CDR3, or a combination thereof.
  • the antibody comprises at least 3 cysteine residues or more.
  • the antibody can comprise one or more cysteine motifs.
  • the present disclosure provides genetic sequences encoding an antibody or fragment thereof comprising: (a) a first antibody sequence, wherein at least a portion of the first antibody sequence is derived from at least a portion of an ultralong CDR3; and (b) a non-antibody sequence.
  • the antibody or fragment thereof may further comprise a second antibody sequence, wherein at least a portion of the second antibody sequence is derived from at least a portion of an ultralong CDR3.
  • the ultralong CDR3 from which the first antibody sequence and/or second antibody sequence may be derived from a ruminant.
  • the ruminant can be a cow.
  • At least a portion of the first antibody sequence and/or at least a portion of the second antibody sequence can be derived from a mammal.
  • the mammal may be a bovine.
  • the mammal is a non-bovine mammal, such as a human.
  • the first and/or second antibody sequences may be 3 or more amino acids in length.
  • the amino acids may be consecutive amino acids.
  • the amino acids are non-consecutive amino acids.
  • the first and/or second antibody sequences may comprise a bovine antibody sequence comprising 3 or more amino acids in length.
  • the bovine antibody may be a BLVH12, BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, or F18 antibody.
  • the first and/or second antibody sequences may comprise a human antibody sequence comprising 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, or 70 or more amino acids in length.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 and/or the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 can be 20 or fewer amino acids in length.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 and/or the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 may be 3 or more amino, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, or 20 or more acids in length.
  • the first and/or second antibody sequences can comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 30 or more, or 40 or more amino acid residues derived from a knob domain of the ultralong CDR3.
  • the 1 or more amino acid residues derived from the knob domain of the ultralong CDR3 may be a serine and/or cysteine residue.
  • the first and/or second antibody sequences may comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more amino acid residues derived from a stalk domain of the ultralong CDR3.
  • the first and/or second antibody sequences may comprise 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more conserved motifs derived from a stalk domain of the ultralong CDR3.
  • the one or more conserved motifs derived from the stalk domain of the ultralong CDR3 may comprise a sequence selected from any one of SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 and/or the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 may comprise a sequence selected from any one of SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 and/or the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 may comprise a sequence that is 50% or more homologous to a sequence selected from any one of SEQ ID NOS: 157-224 and 235-295.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 may comprise a sequence selected from any one of SEQ ID NOS: 157-234.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 may comprise a sequence that is 50% or more homologous to a sequence selected from any one of SEQ ID NOS: 157-224.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 may comprise a sequence that is 50% or more homologous to a sequence selected from any one of SEQ ID NOS: 225-227.
  • the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 may comprise a sequence selected from any one of SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336.
  • the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 may comprise a sequence that may be 50% or more homologous to a sequence selected from any one of SEQ ID NOS: 235-295.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 and the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 may be derived from the same ultralong CDR3 sequence.
  • the portion of the first antibody sequence derived from at least a portion of the ultralong CDR3 and the portion of the second antibody sequence derived from at least a portion of the ultralong CDR3 may be derived from two or more different ultralong CDR3 sequences.
  • the portions of the ultralong CDR3 of the first and/or second antibody sequences may be based on or derived from a BLV1H12 ultralong CDR3 sequence.
  • the portions of the ultralong CDR3 of the first and/or second antibody sequences may be based on or derived from a BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, or F18 ultralong CDR3 sequence.
  • the antibody may further comprise one or more linker sequences.
  • the present disclosure also provides isolated genetic sequences (e.g., genes, nucleic acids, polynucleotides, oligonucleotides) such as genetic sequences encoding antibodies that comprise an ultralong CDR sequence including, for example, a CDR3 sequence as provided by any one of SEQ ID NOS: 2-22. Also provided are ultralong CDR3 nucleic acid sequences that are substantially similar to those CDR3 sequences provided by any one of SEQ ID NOS: 2-22.
  • a CDR3 sequence may be considered substantially similar to a CDR3 sequence provided by any one of SEQ ID NOS: 2-22 where the CDR3 sequence shares 80%, 85%, 90%, 95%, 99%, or more, nucleic acid sequence identity to a CDR3 sequence provided by any one of SEQ ID NOS: 2-22 or hybridizes to any one of SEQ ID NOS: 2-22 under stringent hybridization conditions.
  • the present disclosure also provides isolated genetic sequences (e.g., genes, nucleic acids, polynucleotides, oligonucleotides) such as genetic sequences encoding antibodies that comprise a heavy chain polypeptide, wherein the heavy chain polypeptide comprises at least a portion of an ultralong CDR3 sequence.
  • the heavy chain polypeptide may comprise a polypeptide sequence of any one of SEQ ID NOS: 24-44.
  • the heavy chain polypeptide may comprise a polypeptide sequence encoded by the DNA of any one of SEQ ID NOS: 2-22.
  • isolated genetic sequences e.g., genes, nucleic acids, polynucleotides, oligonucleotides
  • genetic sequences encoding antibodies comprising a heavy chain polypeptide, wherein the heavy chain polypeptide comprises an ultralong CDR3 sequence and the heavy chain polypeptide sequences are substantially similar to those polypeptide sequences provided by any one of SEQ ID NOS: 24-44.
  • a heavy chain polypeptide sequence may be considered substantially similar to a polypeptide sequence provided by any one of SEQ ID NOS: 24-44 where the heavy chain polypeptide sequence shares 60%, 70%, 80%, 90%, 95%, 99%, or more nucleic acid identity to a nucleotide sequence provided by any one of SEQ ID NOS: 24-44 or hybridizes to any one of SEQ ID NOS: 24-44 under stringent hybridization conditions.
  • the isolated genetic sequences e.g., genes, nucleic acids, polynucleotides, oligonucleotides
  • genetic sequences encoding antibodies may further comprise a light chain polypeptide.
  • the light chain polypeptide may comprise a polypeptide sequence of SEQ ID NO: 23.
  • the light chain polypeptide may comprise a polypeptide sequence encoded by the DNA of SEQ ID NO: 1.
  • isolated genetic sequences e.g., genes, nucleic acids, polynucleotides, oligonucleotides
  • genetic sequences encoding antibodies further comprising a light chain polypeptide wherein the light chain polypeptide comprises an ultralong CDR3 sequence and the light chain polypeptide sequences are substantially similar to those polypeptide sequences provided by SEQ ID NO: 23.
  • a light chain polypeptide sequence may be considered substantially similar to a polypeptide sequence provided by SEQ ID NO: 1 where the light chain polypeptide sequence shares 60%, 70%, 80%, 90%, 95%, 99%, or more nucleic acid identity to a nucleotide sequence provided by any one of SEQ ID NO: 1 or hybridizes to SEQ ID NOS: 1 under stringent hybridization conditions.
  • the present disclosure provides collections, libraries and arrays of antibodies comprising ultralong CDR3 sequences.
  • members of the collections, libraries, or arrays may exhibit sequence diversity.
  • the present disclosure provides a library or an array of antibodies comprising ultralong CDR3 sequences wherein at least two members of the library or array differ in the positions of at least one of the cysteines in the ultralong CDR3 sequence.
  • Structural diversity may be enhanced through different numbers of cysteines in the ultralong CDR3 sequence (e.g., at least 3 or more cysteine residues such as 4 or more, 6 or more and 8 or more) and/or through different disulfide bond formation, and hence different loop structures.
  • the present disclosure provides for a library or an array of antibodies comprising ultralong CDR3 sequences wherein at least two members of the library or the array differ in at least one amino acid located between cysteines in the ultralong CDR3.
  • members of the library or the array can contain cysteines in the same positions of CDR3, resulting in similar overall structural folds, but with fine differences brought about through different amino acid side chains.
  • Such libraries or arrays may be useful for affinity maturation.
  • the present disclosure provides libraries or arrays of antibodies comprising ultralong CDR3 sequences wherein at least two of the ultralong CDR3 sequences differ in length (e.g., 35 amino acids in length or more such as 40 or more, 45 or more, 50 or more, 55 or more and 60 or more).
  • the amino acid and cysteine content may or may not be altered between the members of the library or the array.
  • Different lengths of ultralong CDR3 sequences may provide for unique binding sites, including, for example, due to steric differences, as a result of altered length.
  • the present disclosure provides libraries or arrays of antibodies comprising ultralong CDR3 sequences wherein at least two members of the library differ in the human framework used to construct the antibody comprising an ultralong CDR3.
  • the present disclosure provides libraries or arrays of antibodies comprising ultralong CDR3 sequences wherein at least two members of the library or the array differ in having a non-antibody protein sequence that comprises a portion of the ultralong CDR3.
  • Such libraries or arrays may contain multiple non-antibody protein sequences, including for chemokines, growth factors, peptides, cytokines, cell surface proteins, serum proteins, toxins, extracellular matrix proteins, clotting factors, secreted proteins, viral or bacterial proteins, etc.
  • the non-antibody protein sequence may be of human or non-human origin and may be comprised of a portion of a non-antibody protein such as a peptide or domain.
  • the non-antibody protein sequence of the ultralong CDR3 may contain mutations from its natural sequence, including amino acid changes (e.g., substitutions), or insertions or deletions. Engineering additional amino acids at the junction between the non-antibody sequence within the ultralong CDR3 may be done to facilitate or enhance proper folding of the non-antibody sequence within the antibody.
  • the present disclosure provides libraries or arrays of antibodies comprising ultralong CDR3 sequences wherein at least two members of the library or the array differ in having a non-bovine sequence.
  • the non-bovine sequence can be derived from or based on a non-bovine mammal sequence.
  • the non-bovine sequence can be derived from or based on a human, mouse, rat, sheep, dog, and/or goat sequence.
  • the non-bovine sequence can be within the ultralong CDR3.
  • the non-bovine sequence is linked or attached to the ultralong CDR3 sequence.
  • the non-bovine sequence can be derived from or based on at least a portion of an antibody sequence.
  • the antibody sequence can encode a variable region, constant region or a combination thereof.
  • the present disclosure provides libraries or arrays of antibodies comprising ultralong CDR3 sequences wherein at least two members of the library or array differ in having a cytoxic agent or therapeutic polypeptide that is conjugated to the ultralong CDR3.
  • the cytoxic agent or therapeutic polypeptide may include, but is not limited to, a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (e.g., a radioconjugate).
  • the cytotoxic agent or therapeutic polypeptide can be encoded by a non-antibody sequence.
  • the present disclosure provides libraries or arrays of antibodies comprising ultralong CDR3 sequences wherein at least two members of the library or array differ in binding to targets.
  • the target can be a protein target.
  • the protein target can be a transmembrane protein target.
  • transmembrane targets may include, but are not limited to, GPCRs, ion channels, transporters, and cell surface receptors.
  • the libraries or the arrays of the present disclosure may be in several formats well known in the art.
  • the library or the array may be an addressable library or an addressable array.
  • the library or array may be in display format, for example, the antibody sequences may be expressed on phage, ribosomes, mRNA, yeast, or mammalian cells.
  • the present disclosure provides cells comprising genetic sequences encoding antibodies comprising ultralong CDR3 sequences or portions thereof.
  • the present disclosure provides cells comprising genetic sequences encoding antibodies comprising at least a portion of a knob domain or at least a portion of a knob domain of an ultralong CDR3 sequence.
  • the present disclosure provides cells comprising genetic sequences (e.g., genes, nucleic acids, polynucleotides) encoding an ultralong CDR3 or portion thereof.
  • the present disclosure also provides cells comprising genetic sequences (e.g., genes, nucleic acids, polynucleotides) encoding the knob domain and/or knob domain of an ultralong CDR3.
  • the present disclosure provides cells expressing an antibody comprising an ultralong CDR3.
  • the cells may be prokaryotic or eukaryotic, and an antibody comprising an ultralong CDR3 may be expressed on the cell surface or secreted into the media.
  • an antibody preferentially contains a motif for insertion into the plasmid membrane such as a membrane spanning domain at the C-terminus or a lipid attachment site.
  • an antibody comprising an ultralong CDR3 may be secreted into the periplasm.
  • the cells are eukaryotic, they may be transiently transfected with genetic sequences encoding an antibody comprising an ultralong CDR3.
  • a stable cell line or stable pools may be created by transfecting or transducing genetic sequences encoding an antibody comprising an ultralong CDR3 by methods well known to those of skill in the art.
  • Cells can be selected by fluorescence activated cell sorting (FACS) or through selection for a gene encoding drug resistance.
  • FACS fluorescence activated cell sorting
  • Cells useful for producing antibodies comprising ultralong CDR3 sequences include prokaryotic cells like E. coli , eukaryotic cells like the yeasts Saccharomyces cerevisiae and Pichia pastoris , insect cells (e.g., Sf9, Hi5), chinese hamster ovary (CHO) cells, monkey cells like COS-1, or human cells like HEK-293, HeLa, SP-1.
  • the present disclosure provides methods for making libraries comprising antibodies comprising ultralong CDR3 sequences. Methods for making libraries of spatially addressed libraries are described in WO 2010/054007. Methods of making libraries in yeast, phage, E. coli , or mammalian cells are well known in the art.
  • the present disclosure also provides methods of screening libraries of antibodies comprising ultralong CDR3 sequences.
  • An “ultralong CDR3” or an “ultralong CDR3 sequence”, used interchangeably herein, comprises a CDR3 or CDR3 sequence that is not derived from a human antibody sequence.
  • An ultralong CDR3 may be 35 amino acids in length or longer, for example, 40 amino acids in length or longer, 45 amino acids in length or longer, 50 amino acids in length or longer, 55 amino acids in length or longer, or 60 amino acids in length or longer.
  • the length of the ultralong CDR3 may include a non-antibody sequence.
  • An ultralong CDR3 may comprise at least a portion of a knob domain and/or knob domain.
  • An ultralong CDR3 may comprise a non-antibody sequence, including, for example, a cytokine, chemokine, growth factor or hormone sequence.
  • the ultralong CDR3 is a heavy chain CDR3 (CDR-H3 or CDRH3).
  • the ultralong CDR3 is a sequence derived from or based on a ruminant (e.g., bovine) sequence.
  • An ultralong CDR3 may comprise at least 3 or more cysteine residues, for example, 4 or more cysteine residues, 6 or more cysteine residues, or 8 or more cysteine residues.
  • An ultralong CDR3 may comprise one or more cysteine motifs.
  • An ultralong CDR3 may comprise an amino acid sequence that is derived from or based on SEQ ID NOS: 23-44 or is encoded by a DNA sequence that is derived from or based on SEQ ID NOS: 2-22.
  • a variable region that comprises an ultralong CDR3 may include an amino acid sequence that is derived from or based on SEQ ID NOS: 23-44 or is encoded by a DNA sequence that is derived from or based on SEQ ID NOS: 2-22. Such a sequence may be derived from or based on a bovine germline VH gene sequence.
  • An ultralong CDR3 may comprise a sequence derived from or based on a non-human DH gene sequence (see, e.g., Koti, et al. (2010) Mol. Immunol. 47: 2119-2128).
  • An ultralong CDR3 may comprise a sequence derived from or based on a JH sequence, (see e.g., Hosseini, et al. (2004) Int.
  • an ultralong CDR3 may comprise a sequence derived from or based on a non-human VH sequence and/or a sequence derived from or based on a non-human DH sequence and/or a sequence derived from or based on a JH sequence, and optionally an additional sequence comprising two to six amino acids or more such as, for example, between the VH derived sequence and the DH derived sequence.
  • an ultralong CDR3 may comprise a sequence that is about 50% or more homologous to a sequence derived from or based on SEQ ID NOS: 23-44.
  • the ultralong CDR3 may comprise a sequence that is about 60%, 70%, 80%, 85%, 90%, 95%, 97% or more homologous to a sequence derived from or based on SEQ ID NOS: 23-44.
  • an ultralong CDR3 may comprise a sequence that aligns to 5 or more amino acids to a sequence derived from or based SEQ ID NOS: 23-44.
  • the ultralong CDR3 may comprise a sequence that aligns to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more amino acids to a sequence derived from or based SEQ ID NOS: 23-44.
  • an ultralong CDR3 may comprise a sequence that comprises 5 or more consecutive amino acids to a sequence derived from or based SEQ ID NOS: 23-44.
  • the ultralong CDR3 may comprise a sequence that comprises 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more consecutive amino acids to a sequence derived from or based SEQ ID NOS: 23-44.
  • an ultralong CDR3 may comprise a sequence that is about 50% or more homologous to a DNA sequence that is derived from or based on a SEQ ID NOS: 2-22.
  • the ultralong CDR3 may comprise a sequence that is about 60%, 70%, 80%, 85%, 90%, 95%, 97% or more homologous to a DNA sequence that is derived from or based on SEQ ID NOS: 2-22.
  • an ultralong CDR3 may comprise a sequence that aligns to 5 or more nucleic acids to a DNA sequence that is derived from or based SEQ ID NOS: 2-22.
  • the ultralong CDR3 may comprise a sequence that aligns to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 80, 100, 120, 140, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600 or more nucleic acids to a DNA sequence that is derived from or based SEQ ID NOS: 2-22.
  • an ultralong CDR3 may comprise a sequence that comprises 5 or more consecutive nucleic acids to a DNA sequence that is derived from or based SEQ ID NOS: 2-22.
  • the ultralong CDR3 may comprise a sequence that comprises 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 80, 100, 120, 140, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600 or more consecutive amino acids to a DNA sequence that is derived from or based SEQ ID NOS: 2-22.
  • an ultralong CDR3 may comprise a sequence that is about 50% or more homologous to a sequence derived from or based on a knob domain sequence.
  • the ultralong CDR3 may comprise a sequence that is about 60%, 70%, 80%, 85%, 90%, 95%, 97% or more homologous to a sequence derived from or based on a knob domain sequence.
  • an ultralong CDR3 may comprise a sequence that aligns to 5 or more amino acids to a sequence derived from or based a knob domain sequence.
  • the ultralong CDR3 may comprise a sequence that aligns to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more amino acids to a sequence derived from or based a knob domain sequence
  • an ultralong CDR3 may comprise a sequence that comprises 5 or more consecutive amino acids to a sequence derived from or based a knob domain sequence
  • the ultralong CDR3 may comprise a sequence that comprises 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more consecutive amino acids to a sequence derived from or based a knob domain sequence.
  • an ultralong CDR3 may comprise a sequence that is about 50% or more homologous to a sequence derived from or based on a knob domain sequence.
  • the ultralong CDR3 may comprise a sequence that is about 60%, 70%, 80%, 85%, 90%, 95%, 97% or more homologous to a sequence derived from or based on a knob domain sequence.
  • an ultralong CDR3 may comprise a sequence that aligns to 5 or more amino acids to a sequence derived from or based a knob domain sequence
  • the ultralong CDR3 may comprise a sequence that aligns to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more amino acids to a sequence derived from or based a knob domain sequence
  • an ultralong CDR3 may comprise a sequence that comprises 5 or more consecutive amino acids to a sequence derived from or based a stalk domain sequence
  • the ultralong CDR3 may comprise a sequence that comprises 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more consecutive amino acids to a sequence derived from or based a stalk domain sequence.
  • the antibodies disclosed herein may comprise at least a portion of an ultralong CDR3 derived from or based on a sequence of any of the ultralong CDR3s disclosed herein.
  • the sequence of the ultralong CDR3 or a portion thereof may be modified or altered to contain one or more non-bovine antibody-based nucleotides and/or amino acids.
  • the modifications and/or alterations in the sequence of the ultralong CDR3 or portion thereof may improve one or more features of the expressed antibody. For example, the modifications and/or alterations may improve expression, folding, half-life, activity and/or solubility of the antibody.
  • an “isolated” biological molecule refers to a biological molecule that has been identified and separated and/or recovered from at least one component of its natural environment.
  • Antagonist refers to any molecule that partially or fully blocks, inhibits, or neutralizes an activity (e.g., biological activity) of a polypeptide. Also encompassed by “antagonist” are molecules that fully or partially inhibit the transcription or translation of mRNA encoding the polypeptide. Suitable antagonist molecules include, e.g., antagonist antibodies or antibody fragments; fragments or amino acid sequence variants of a native polypeptide; peptides; antisense oligonucleotides; small organic molecules; and nucleic acids that encode polypeptide antagonists or antagonist antibodies. Reference to “an” antagonist encompasses a single antagonist or a combination of two or more different antagonists.
  • “Agonist” refers to any molecule that partially or fully mimics a biological activity of a polypeptide.
  • agonist molecules that stimulate the transcription or translation of mRNA encoding the polypeptide.
  • Suitable agonist molecules include, e.g., agonist antibodies or antibody fragments; a native polypeptide; fragments or amino acid sequence variants of a native polypeptide; peptides; antisense oligonucleotides; small organic molecules; and nucleic acids that encode polypeptides agonists or antibodies.
  • Reference to “an” agonist encompasses a single agonist or a combination of two or more different agonists.
  • an “isolated” antibody refers to one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody will be purified (1) to greater than 95% by weight of antibody (e.g., as determined by the Lowry method), and preferably to more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence (e.g., by use of a spinning cup sequenator), or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions (e.g., using CoomassieTM blue or, preferably, silver stain).
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Similarly, isolated antibody includes the antibody in medium around recombinant cells. An isolated antibody may be prepared by at least one purification step.
  • an “isolated” nucleic acid molecule refers to a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid.
  • An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells.
  • an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that express an antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
  • Variable domain residue numbering as in Kabat or amino acid position numbering as in Kabat refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain.
  • a heavy chain variable domain may include a single amino acid insert (e.g., residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc according to Kabat) after heavy chain FR residue 82.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.
  • “Substantially similar,” or “substantially the same”, refers to a sufficiently high degree of similarity between two numeric values (generally one associated with an antibody disclosed herein and the other associated with a reference/comparator antibody) such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values).
  • the difference between said two values is preferably less than about 50%, preferably less than about 40%, preferably less than about 30%, preferably less than about 20%, preferably less than about 10% as a function of the value for the reference/comparator antibody.
  • Binding affinity generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure.
  • an “on-rate” or “rate of association” or “association rate” or “k on ” can be determined with a surface plasmon resonance technique such as Biacore (e.g., Biacore A100, BiacoreTM-2000, BiacoreTM-3000, Biacore, Inc., Piscataway, N.J.) carboxymethylated dextran biosensor chips (CM5, Biacore Inc.) and according to the supplier's instructions.
  • Biacore e.g., Biacore A100, BiacoreTM-2000, BiacoreTM-3000, Biacore, Inc., Piscataway, N.J.
  • CM5, Biacore Inc. carboxymethylated dextran biosensor chips
  • Vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • phage vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • 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, “recombinant vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. Accordingly, “plasmid” and “vector” may, at times, be used interchangeably as the plasmid is a commonly used form of vector.
  • Gene refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, precursor, or RNA (e.g., mRNA, rRNA, tRNA).
  • the polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) of the full-length or fragment are retained.
  • the term also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5′ and 3′ ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full-length mRNA. Sequences located 5′ of the coding region and present on the mRNA are referred to as 5′ non-translated sequences. Sequences located 3′ or downstream of the coding region and present on the mRNA are referred to as 3′ non-translated sequences.
  • the term “gene” encompasses both cDNA and genomic forms of a gene.
  • a genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.”
  • Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns can contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript.
  • mRNA messenger RNA
  • the mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.
  • genomic forms of a gene can also include sequences located on both the 5′ and 3′ end of the sequences that are present on the RNA transcript.
  • flanking sequences or regions are located 5′ or 3′ to the non-translated sequences present on the mRNA transcript.
  • the 5′ flanking region can contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene.
  • the 3′ flanking region can contain sequences that direct the termination of transcription, post transcriptional cleavage and polyadenylation.
  • Polynucleotide refers to polymers of nucleotides of any length, and include DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after synthesis, such as by conjugation with a label.
  • Other types of modifications include, for example, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radio
  • any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports.
  • the 5′ and 3′ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms.
  • Other hydroxyls may also be derivatized to standard protecting groups.
  • Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and a basic nucleoside analogs such as methyl riboside.
  • One or more phosphodiester linkages may be replaced by alternative linking groups.
  • linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S(“thioate”), P(S)S (“dithioate”), “(O)NR 2 (“amidate”), P(O)R, P(O)OR′, CO or CH 2 (“formacetal”), in which each R or R′ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
  • Oligonucleotide refers to short, generally single stranded, generally synthetic polynucleotides that are generally, but not necessarily, less than about 200 nucleotides in length.
  • oligonucleotide and polynucleotide are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.
  • Stringent hybridization conditions refer to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH.
  • Tm thermal melting point
  • the Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5 ⁇ SSC, and 1% SDS, incubating at 42° C., or, 5 ⁇ SSC, 1% SDS, incubating at 65° C., with wash in 0.2 ⁇ SSC, and 0.1% SDS at 65° C.
  • Recombinant when used with reference to a cell, nucleic acid, protein, antibody or vector indicates that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein, the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are overexpressed or otherwise abnormally expressed such as, for example, expressed as non-naturally occurring fragments or splice variants.
  • nucleic acid By the term “recombinant nucleic acid” herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases and endonucleases, in a form not normally found in nature. In this manner, operably linkage of different sequences is achieved.
  • an isolated nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined are both considered recombinant for the purposes of this disclosure.
  • a recombinant nucleic acid is made and introduced into a host cell or organism, it will replicate non-recombinantly, e.g., using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes disclosed herein.
  • a “recombinant protein” is a protein made using recombinant techniques, e.g., through the expression of a recombinant nucleic acid as depicted herein.
  • Percent (%) amino acid sequence identity refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MegAlign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • Polypeptide,” “peptide,” “protein,” and “protein fragment” may be used interchangeably to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.
  • “Conservatively modified variants” applies to both amino acid and nucleic acid sequences.
  • “Amino acid variants” refers to amino acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical or associated (e.g., naturally contiguous) sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
  • nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes silent variations of the nucleic acid.
  • AUG which is ordinarily the only codon for methionine
  • TGG which is ordinarily the only codon for tryptophan
  • nucleic acid which encodes a polypeptide is implicit in a described sequence with respect to the expression product, but not with respect to actual probe sequences.
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” including where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles disclosed herein.
  • conservative substitutions include: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
  • Antibodies are glycoproteins having similar structural characteristics. While antibodies may exhibit binding specificity to a specific antigen, immunoglobulins may include both antibodies and other antibody-like molecules which generally lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.
  • antibody “immunoglobulin” and “immunoglobulin construct” are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity) and may also include certain antibody fragments (as described in greater detail herein).
  • the term “antibody” can refer to a full length antibody or a portion thereof.
  • An antibody can refer to a peptide comprising at least one antibody sequence.
  • the antibody sequence can comprise 5 or more amino acids of an antibody sequence.
  • the antibody sequence can comprise 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids of an antibody sequence.
  • the 5 or more amino acids may be consecutive amino acids of an antibody sequence.
  • the 5 or more amino acids are non-consecutive amino acids of an antibody sequence.
  • the 5 or more amino acids may comprise a conserved motif within the antibody sequence.
  • the 5 or more amino acids may comprise a conserved motif within an ultralong CDR3 sequence.
  • An antibody can be human, humanized, fully human and/or affinity matured.
  • An antibody can be a chimeric antibody.
  • An antibody can be a recombinant, engineered, or synthetic antibody.
  • An antibody may be a bovine, bovine engineered, fully bovine and/or affinity matured.
  • the bovine engineered antibody may comprise one or more nucleotides or peptides derived from a bovine antibody sequence.
  • a fully bovine antibody may comprise replacing one or more nucleotides or peptides from a non-bovine antibody sequence with one or more nucleotides or peptides based on a bovine antibody sequence.
  • An antibody may refer to immunoglobulins and immunoglobulin portions, whether natural or partially or wholly synthetic, such as recombinantly produced, including any portion thereof containing at least a portion of the variable region of the immunoglobulin molecule that is sufficient to form an antigen binding site.
  • an antibody or portion thereof includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen binding site.
  • an antibody may refer to an antibody that contains two heavy chains (which can be denoted H and H′) and two light chains (which can be denoted L and L′), where each heavy chain can be a full-length immunoglobulin heavy chain or a portion thereof sufficient to form an antigen binding site (e.g. heavy chains include, but are not limited to, VH, chains VH-CH1 chains and VH-CH1-CH2-CH3 chains), and each light chain can be a full-length light chain or a thereof sufficient to form an antigen binding site (e.g. light chains include, but are not limited to, VL chains and VL-CL chains). Each heavy chain (H and H′) pairs with one light chain (L and L′, respectively).
  • antibodies minimally include all or at least a portion of the variable heavy (VH) chain and/or the variable light (VL) chain.
  • the antibody also can include all or a portion of the constant region.
  • a full-length antibody is an antibody having two full-length heavy chains (e.g. VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and hinge regions, such as antibodies produced by antibody secreting B cells and antibodies with the same domains that are produced synthetically.
  • an “antibody” refers to a protein of the immunoglobulin family or a polypeptide comprising fragments of an immunoglobulin that is capable of noncovalently, reversibly, and in a specific manner binding a corresponding antigen.
  • An exemplary antibody structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD), connected through a disulfide bond.
  • the recognized immunoglobulin genes include the ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either ⁇ or ⁇ .
  • Heavy chains are classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (VL) and variable heavy chain (VH) refer to these regions of light and heavy chains respectively.
  • the antibodies provided herein comprise at least one immunoglobulin domain from an avian antibody, reptilian antibody, amphibian antibody, insect antibody, or chimeric combinations thereof.
  • the antibodies can comprise at least one immunoglobulin domain from a chimeric antibody.
  • the chimeric antibody can be derived from two or more different species (e.g., mouse and human, bovine and human).
  • the antibodies can comprise at least one immunoglobulin domain from an engineered, recombinant or synthetic antibody.
  • engineered, recombinant or synthetic antibodies are created using antibody genes made in a laboratory or taken from cells.
  • the antibody genes can be derived from one or more mammals.
  • the antibody genes are derived from a human.
  • the antibody genes may be derived from a bovine.
  • the antibodies disclosed herein comprise at least one immunoglobulin domain from a humanized, human engineered or fully human antibody.
  • the antibody may comprise antibody sequences from two or more different antibodies.
  • the two or more different antibodies may be from the same species.
  • the specie may be a bovine specie, human specie, or murine specie.
  • the two or more different antibodies may be from the same type of animal.
  • the two or more different antibodies may be from a cow.
  • the two or more different antibodies may be from a human.
  • the two or more different antibodies are from different species.
  • the two or more different antibodies are from a human specie and bovine specie.
  • the two or more diffent antibodies are from a bovine specie and a non-bovine specie.
  • the two or more different antibodies are from a human specie and a non-human specie.
  • the two or more different antibodies may be from different animals.
  • the two different animals are a human and a cow.
  • the different animals may be from the same specie.
  • the different animals may be a cow and a water buffalo.
  • variable refers to the fact that certain portions of the variable domains (also referred to as variable regions) differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions (HVRs) both in the light-chain and the heavy-chain variable domains. CDRs include those specified as Kabat, Chothia, and IMGT as shown herein within the variable region sequences. The more highly conserved portions of variable domains are called the framework (FR).
  • CDRs complementarity-determining regions
  • HVRs hypervariable regions
  • variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest , Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab′) 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
  • Fv refers to an antibody fragment which contains an antigen-recognition and antigen-binding site.
  • this region consists of a dimer of one heavy and one light chain variable domain in non-covalent association.
  • scFv single chain Fv
  • one heavy chain and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv (scFv) species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer.
  • the six CDRs confer antigen-binding specificity to the antibody.
  • a single variable domain or half of an Fv comprising only three CDRs specific for an antigen
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
  • Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.
  • Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab′) 2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • the “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 .
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • Antibody fragments comprise only a portion of an intact antibody, wherein the portion preferably retains at least one, preferably most or all, of the functions normally associated with that portion when present in an intact antibody.
  • antibody fragments include Fab, Fab′, F(ab′)2, single-chain Fvs (scFv), Fv, dsFv, diabody (e.g., (ds Fv) 2 ), Fd and Fd′ fragments Fab fragments, Fd fragments, scFv fragments, linear antibodies, single-chain antibody molecules, minibodies, flex minibodies, bispecific fragments, and multispecific antibodies formed from antibody fragments (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov).
  • an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen.
  • an antibody fragment for example one that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half life modulation, ADCC function and complement binding.
  • an antibody fragment is a monovalent antibody that has an in vivo half life substantially similar to an intact antibody.
  • such an antibody fragment may comprise on antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.
  • an antibody fragment or antibody portion refers to any portion of a full-length antibody that is less than full length but contains at least a portion of the variable region of the antibody sufficient to form an antigen binding site (e.g. one or more CDRs) and thus retains the a binding specificity and/or an activity of the full-length antibody;
  • antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically, e.g. recombinantly produced derivatives.
  • a “dsFv” refers to an Fv with an engineered intermolecular disulfide bond, which stabilizes the VH-VL pair.
  • a “Fd fragment” refers to a fragment of an antibody containing a variable domain (VH) and one constant region domain (CH1) of an antibody heavy chain.
  • a “Fab fragment” refers to an antibody fragment that contains the portion of the full-length antibody that would results from digestion of a full-length immunoglobulin with papain, or a fragment having the same structure that is produced synthetically, e.g. recombinantly.
  • a Fab fragment contains a light chain (containing a VL and CL portion) and another chain containing a variable domain of a heavy chain (VH) and one constant region domain portion of the heavy chain (CH1); it can be recombinantly produced.
  • a “F(ab′)2 fragment” refers to an antibody fragment that results from digestion of an immunoglobulin with pepsin at pH 4.0-4.5, or a synthetically, e.g. recombinantly, produced antibody having the same structure.
  • the F(ab′)2 fragment contains two Fab fragments but where each heavy chain portion contains an additional few amino acids, including cysteine residues that form disulfide linkages joining the two fragments; it can be recombinantly produced.
  • a “Fab′ fragment” refers to a fragment containing one half (one heavy chain and one light chain) of the F(ab′)2 fragment.
  • Fd′ fragment refers to a fragment of an antibody containing one heavy chain portion of a F(ab′)2 fragment.
  • a “Fv′ fragment” refers to a fragment containing only the VH and VL domains of an antibody molecule.
  • a “scFv fragment” refers to an antibody fragment that contains a variable light chain (VL) and variable heavy chain (VH), covalently connected by a polypeptide linker in any order.
  • the linker is of a length such that the two variable domains are bridged without substantial interference.
  • Exemplary linkers are (Gly-Ser)n residues with some Glu or Lys residues dispersed throughout to increase solubility.
  • Diabodies are dimeric scFv; diabodies typically have shorter peptide linkers than scFvs, and they preferentially dimerize.
  • HsFv refers to antibody fragments in which the constant domains normally present in a Fab fragment have been substituted with a heterodimeric coiled-coil domain (see, e.g., Arndt et al. (2001) J Mol. Biol. 7:312:221-228).
  • “Hypervariable region”, “HVR”, or “HV”, as well as “complementary determining region” or “CDR”, may refer to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops.
  • antibodies comprise six hypervariable or CDR regions; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3).
  • a number of hypervariable region or CDR delineations are in use and are encompassed herein.
  • the Kabat Complementarity Determining Regions are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.
  • Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)).
  • the AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, (Chothia “CDRs”) and are used by Oxford Molecular's AbM antibody modeling software.
  • the “contact” hypervariable regions are based on an analysis of the available complex crystal structures. The residues from each of these hypervariable regions are noted below. (See also, for example, FIG. 1 and bold, italicized text for Kabat CDRs and underlined text for Chothia CDRs for 12.3 ICI antibody).
  • IMGT referes to the international ImMunoGeneTics Information System, as described by Lefrace et al., Nucl. Acids, Res. 37; D1006-D1012 (2009), including for example, IMGT designated CDRs for antibodies (see also, for example, FIG. 1 and bracketed text for 12.3 1C1 antibody).
  • Hypervariable regions may comprise “extended hypervariable regions” as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in the VL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in the VH.
  • the variable domain residues are numbered according to Kabat et al., Supra for each of these definitions.
  • “Framework” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined. “Framework regions” (FRs) are the domains within the antibody variable region domains comprising framework residues that are located within the beta sheets; the FR regions are comparatively more conserved, in terms of their amino acid sequences, than the hypervariable regions.
  • “Monoclonal antibody” refers to an antibody from a population of substantially homogeneous antibodies, that is, for example, the individual antibodies comprising the population are identical and/or bind the same epitope(s), except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts.
  • Such monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences.
  • the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones or recombinant DNA clones.
  • the selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this disclosure.
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present disclosure may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler et al., Nature, 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681, (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No.
  • phage display technologies see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al. J. Immunol.
  • “Humanized” or “Human engineered” forms of non-human (e.g., murine, bovine) antibodies are chimeric antibodies that contain amino acids represented in human immunoglobulin sequences, including, for example, wherein minimal sequence is derived from non-human immunoglobulin.
  • humanized antibodies may be human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in non-human (e.g., rodent) antibodies.
  • humanized or human engineered antibodies may be non-human (e.g., rodent) antibodies in which some residues are substituted by residues from analogious sites in human antibodies (see, e.g., U.S. Pat. No. 5,766,886).
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody, including, for example non-antibody sequences such as a chemokine, growth factor, peptide, cytokine, cell surface protein, serum protein, toxin, extracellular matrix protein, clotting factor, or secreted protein sequence.
  • Humanized antibodies include human engineered antibodies, for example, as described by U.S. Pat. No. 5,766,886, including methods for preparing modified antibody variable domains.
  • a humanized antibody may comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • a humanized antibody optionally may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Hybrid antibodies refer to immunoglobulin molecules in which pairs of heavy and light chains from antibodies with different antigenic determinant regions are assembled together so that two different epitopes or two different antigens can be recognized and bound by the resulting tetramer.
  • “Chimeric” antibodies have a portion of the heavy and/or light chain identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see e.g., Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
  • Humanized antibody refers to a subset of chimeric antibodies.
  • Single-chain Fv or “scFv” antibody fragments may comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • an “antigen” refers to a predetermined antigen to which an antibody can selectively bind.
  • the target antigen may be polypeptide, carbohydrate, nucleic acid, lipid, hapten or other naturally occurring or synthetic compound.
  • the target antigen is a polypeptide.
  • Epitopes or “antigenic determinant”, used interchangeably herein, refer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody.
  • the antigen is a polypeptide
  • epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding are typically lost upon protein denaturing.
  • An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
  • Antibodies may bind to the same or a different epitope on an antigen.
  • Antibodies may be characterized in different epitope bins. Whether an antibody binds to the same or different epitope as another antibody (e.g., a reference antibody or benchmark antibody) may be determined by competition between antibodies in assays (e.g., competitive binding assays).
  • Competition between antibodies may be determined by an assay in which the immunoglobulin under test inhibits specific binding of a reference antibody to a common antigen.
  • Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay or enzyme-linked immunosorbent assay (EIA or ELISA), sandwich competition assay including an ELISA assay (see Stahli et al., Methods in Enzymology 9:242-253 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol.
  • solid phase direct labeled assay solid phase direct labeled sandwich assay (see Harlow and Lane, “Antibodies, A Laboratory Manual,” Cold Spring Harbor Press (1988)); solid phase direct label RIA using 1-125 label (see Morel et al., Molec. Immunol. 25(1):7-15 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552 (1990)); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol., 32:77-82 (1990)).
  • Competition binding assays may be performed using Surface Plasmon Resonance (SPR), for example, with a Biacore® instrument for kinetic analysis of binding interactions.
  • SPR Surface Plasmon Resonance
  • an antibody comprising an ultralong CDR3 of unknown epitope specificity may be evaluated for its ability to compete for binding against a comparator antibody (e.g., a BA1 or BA2 antibody as described herein).
  • An assay may involve the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test immunoglobulin and a labeled reference immunoglobulin.
  • Competitive inhibition may be measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess.
  • An assay may include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur.
  • a competing antibody when present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 50%, or at least about 70%, or at least about 80%, or least about 90%, or at least about 95%, or at least about 99% or about 100% for a competitor antibody.
  • an antibody “selectively binds” or “specifically binds” means that the antibody reacts or associates more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to an antigen or an epitope than with alternative substances, including unrelated proteins. “Selectively binds” or “specifically binds” may mean, for example, that an antibody binds to a protein with a K D of at least about 0.1 mM, or at least about 1 ⁇ M or at least about 0.1 ⁇ M or better, or at least about 0.01 ⁇ M or better. Because of the sequence identity between homologous proteins in different species, specific binding can include an antibody that recognizes a given antigen in more than one species.
  • Non-specific binding and “background binding” when used in reference to the interaction of an antibody and a protein or peptide refer to an interaction that is not dependent on the presence of a particular structure (e.g., the antibody is binding to proteins in general rather that a particular structure such as an epitope).
  • Diabodies refer to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et. al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
  • a “human antibody” refers to one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • affinity matured antibody refers to one with one or more alterations in one or more CDRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s).
  • Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen.
  • Affinity matured antibodies are produced by procedures known in the art. Marks et al., Bio/Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described by: Barbas et al., Proc Nat. Acad. Sci.
  • Antibody effector functions refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • cytotoxic cells e.g., Natural Killer (NK) cells, neutrophils, and macrophages
  • NK cells Natural Killer cells
  • neutrophils neutrophils
  • macrophages cytotoxic cells
  • the antibodies “arm” the cytotoxic cells and are absolutely required for such killing.
  • the primary cells for mediating ADCC, NK cells express Fc ⁇ RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII.
  • ADCC activity of a molecule of interest is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu Rev. Immunol 9:457-92 (1991).
  • an in vitro ADCC assay may be performed.
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. Proc. Natl. Acad. Sci. USA 95:652-656 (1998).
  • “Effector cells” are leukocytes which express one or more FcRs and perform effector functions. Preferably, the cells express at least Fc ⁇ RIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred.
  • the effector cells may be isolated from a native source, e.g., from blood.
  • Fc receptor or “FcR” describes a receptor that binds to the Fc region of an antibody.
  • the preferred FcR is a native sequence human FcR.
  • a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • Fc ⁇ RII receptors include Fc ⁇ RIIA (an “activating receptor”) and Fc ⁇ RIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain
  • Inhibiting receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • FcR FcR
  • the term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulates homeostasis of immunoglobulins.
  • FcRn neonatal receptor
  • antibody variants with improved or diminished binding to FcRs have been described (see, e.g., Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001)).
  • Binding to human FcRn in vivo and serum half life of human FcRn high affinity binding polypeptides can be assayed, e.g., in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with the Fc variant polypeptides.
  • “Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) which are bound to their cognate antigen.
  • a CDC assay for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.
  • Fc region-comprising polypeptide refers to a polypeptide, such as an antibody or immunoadhesin (see definitions below), which comprises an Fc region.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during purification of the polypeptide or by recombinant engineering the nucleic acid encoding the polypeptide.
  • blocking antibody or an “antagonist” antibody refers to one which inhibits or reduces biological activity of the antigen it binds.
  • Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.
  • Antist antibody refers to an antibody which mimics (e.g., partially or fully) at least one of the functional activities of a polypeptide of interest.
  • acceptor human framework refers to a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework, or from a human consensus framework.
  • An acceptor human framework “derived from” a human immunoglobulin framework or human consensus framework may comprise the same amino acid sequence thereof, or may contain pre-existing amino acid sequence changes. Where pre-existing amino acid changes are present, preferably no more than 5 and preferably 4 or less, or 3 or less, pre-existing amino acid changes are present.
  • a “human consensus framework” refers to a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.
  • the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
  • the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.
  • the subgroup is subgroup kappa I as in Kabat et al., supra.
  • the subgroup is subgroup III as in Kabat et al., supra.
  • “Disorder” or “disease” refers to any condition that would benefit from treatment with a substance/molecule (e.g., an antibody comprising an ultralong CDR3 as disclosed herein) or method disclosed herein. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.
  • Treatment refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • antibodies disclosed herein are used to delay development of a disease or disorder.
  • “Individual” refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs and horses), primates, mice and rats.
  • “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, rodents (e.g., mice and rats), and monkeys; domestic and farm animals; and zoo, sports, laboratory, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.
  • the mammal is selected from a human, rodent, or monkey.
  • “Pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.
  • “Pharmaceutically acceptable salt” refers to a salt of a compound that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound.
  • “Pharmaceutically acceptable excipient, carrier or adjuvant” refers to an excipient, carrier or adjuvant that can be administered to a subject, together with at least one antibody of the present disclosure, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.
  • “Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant, excipient, or carrier with which at least one antibody of the present disclosure is administered.
  • Providing a prognosis”, “prognostic information”, or “predictive information” refer to providing information, including for example the presence of cancer cells in a subject's tumor, regarding the impact of the presence of cancer (e.g., as determined by the diagnostic methods of the present disclosure) on a subject's future health (e.g., expected morbidity or mortality, the likelihood of getting cancer, and the risk of metastasis).
  • Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder.
  • those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented.
  • Providing a diagnosis” or “diagnostic information” refers to any information, including for example the presence of cancer cells, that is useful in determining whether a patient has a disease or condition and/or in classifying the disease or condition into a phenotypic category or any category having significance with regards to the prognosis of or likely response to treatment (either treatment in general or any particular treatment) of the disease or condition.
  • diagnosis refers to providing any type of diagnostic information, including, but not limited to, whether a subject is likely to have a condition (such as a tumor), whether a subject's tumor comprises cancer stem cells, information related to the nature or classification of a tumor as for example a high risk tumor or a low risk tumor, information related to prognosis and/or information useful in selecting an appropriate treatment.
  • Selection of treatment can include the choice of a particular chemotherapeutic agent or other treatment modality such as surgery or radiation or a choice about whether to withhold or deliver therapy.
  • a “human consensus framework” refers to a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.
  • the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
  • the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.
  • the subgroup is subgroup kappa I as in Kabat et al., supra.
  • the subgroup is subgroup III as in Kabat et al., supra.
  • acceptor human framework refers to a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below.
  • An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
  • the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.
  • Antigen-binding site refers to the interface formed by one or more complementary determining regions.
  • An antibody molecule has two antigen combining sites, each containing portions of a heavy chain variable region and portions of a light chain variable region.
  • the antigen combining sites can contain other portions of the variable region domains in addition to the CDRs.
  • an “antibody light chain” or an “antibody heavy chain” refers to a polypeptide comprising the VL or VH, respectively.
  • the VL is encoded by the minigenes V (variable) and J (junctional), and the VH by minigenes V, D (diversity), and J.
  • Each of VL or VH includes the CDRs as well as the framework regions.
  • antibody light chains and/or antibody heavy chains may, from time to time, be collectively referred to as “antibody chains.” These terms encompass antibody chains containing mutations that do not disrupt the basic structure of VL or VH, as one skilled in the art will readily recognize.
  • “Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures.
  • native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide bonded. From N- to C-terminus, each heavy chain has a variable region (V H), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (V L), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain.
  • V H variable heavy domain
  • V L variable region
  • CL constant light domain
  • the light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (K), based on the amino acid sequence of its constant domain.
  • “Combinatorial library” refers to collections of compounds formed by reacting different combinations of interchangeable chemical “building blocks” to produce a collection of compounds based on permutations of the building blocks.
  • the building blocks are the component V, D and J regions (or modified forms thereof) from which antibodies are formed.
  • the terms “library” or “collection” are used interchangeably.
  • a “combinatorial antibody library” refers to a collection of antibodies (or portions thereof, such as Fabs), where the antibodies are encoded by nucleic acid molecules produced by the combination of V, D and J gene segments, particularly human V, D and J germline segments.
  • the combinatorial libraries herein typically contain at least 50 different antibody (or antibody portions or fragment) members, typically at or about 50, 100, 500, 103, 1 ⁇ 103, 2 ⁇ 103, 3 ⁇ 103, 4 ⁇ 103, 5 ⁇ 103, 6 ⁇ 103, 7 ⁇ 103, 8 ⁇ 103, 9 ⁇ 103, 1 ⁇ 104, 2 ⁇ 104, 3 ⁇ 104, 4 ⁇ 104, 5 ⁇ 104, 6 ⁇ 104, 7 ⁇ 104, 8 ⁇ 104, 9 ⁇ 104, 1 ⁇ 105, 2 ⁇ 105, 3 ⁇ 105, 4 ⁇ 105, 5 ⁇ 105, 6 ⁇ 105, 7 ⁇ 105, 8 ⁇ 105, 9 ⁇ 105, 106, 107, 108, 109, 1010, or more different members.
  • the resulting libraries or collections of antibodies or portions thereof can be screened for binding to a target protein or modulation of a functional activity.
  • a “human combinatorial antibody library” refers to a collection of antibodies or portions thereof, whereby each member contains a VL and VH chains or a sufficient portion thereof to form an antigen binding site encoded by nucleic acid containing human germline segments produced as described herein.
  • a “variable germline segment” refers to V, D and J groups, subgroups, genes or alleles thereof. Gene segment sequences are accessible from known database (e.g., National Center for Biotechnology Information (NCBI), the international ImMunoGeneTics Information System® (IMGT), the Kabat database and the Tomlinson's VBase database (Lefranc (2003) Nucleic Acids Res., 31:307-310; Martin et al., Bioinformatics Tools for Antibody Engineering in Handbook of Therapeutic Antibodies, Wiley-VCH (2007), pp. 104-107). Tables 3-5 list exemplary human variable germline segments.
  • germline segments are set forth in SEQ ID NOS: 10-451 and 868.
  • a germline segment includes modified sequences thereof, that are modified in accord with the rules of sequence compilation provided herein to permit practice of the method.
  • germline gene segments include those that contain one amino acid deletion or insertion at the 5′ or 3′ end compared to any of the sequences of nucleotides set forth in SEQ ID NOS:10-451, 868.
  • variable heavy chain germline segments are assembled such that the VH segment is 5′ to the DH segment which is 5′ to the JH segment, thereby resulting in a nucleic acid sequence encoding a VH chain.
  • Variable light chain germline segments are assembled such that the VL segment is 5′ to the JL segment, thereby resulting in a nucleic acid sequence encoding a VL chain.
  • a constant gene segment or segments also can be assembled onto the 3′ end of a nucleic acid encoding a VH or VL chain.
  • Linkage refers to the joining of germline segments. Linkage can be direct or indirect. Germline segments can be linked directly without additional nucleotides between segments, or additional nucleotides can be added to render the entire segment in-frame, or nucleotides can be deleted to render the resulting segment in-frame. It is understood that the choice of linker nucleotides is made such that the resulting nucleic acid molecule is in-frame and encodes a functional and productive antibody.
  • “In-frame” or “linked in-frame” with reference to linkage of human germline segments means that there are insertions and/or deletions in the nucleotide germline segments at the joined junctions to render the resulting nucleic acid molecule in-frame with the 5′ start codon (ATG), thereby producing a “productive” or functional full-length polypeptide.
  • the choice of nucleotides inserted or deleted from germline segments, particularly at joints joining various VD, DJ and VJ segments, is in accord with the rules provided in the method herein for V(D)J joint generation. For example, germline segments are assembled such that the VH segment is 5′ to the DH segment which is 5′ to the JH segment.
  • nucleotides can be inserted or deleted from the individual VH, DH or JH segments, such that the resulting nucleic acid molecule containing the joined VDJ segments are in-frame with the 5′ start codon (ATG).
  • a portion of an antibody includes sufficient amino acids to form an antigen binding site.
  • a “reading frame” refers to a contiguous and non-overlapping set of three-nucleotide codons in DNA or RNA. Because three codons encode one amino acid, there exist three possible reading frames for given nucleotide sequence, reading frames 1, 2 or 3. For example, the sequence ACTGGTCA will be ACT GGT CA for reading frame 1, A CTG GTC A for reading frame 2 and AC TGG TCA for reading frame 3. Generally for practice of the method described herein, nucleic acid sequences are combined so that the V sequence has reading frame 1.
  • stop codon refers to a three-nucleotide sequence that signals a halt in protein synthesis during translation, or any sequence encoding that sequence (e.g. a DNA sequence encoding an RNA stop codon sequence), including the amber stop codon (UAG or TAG)), the ochre stop codon (UAA or TAA)) and the opal stop codon (UGA or TGA)). It is not necessary that the stop codon signal termination of translation in every cell or in every organism. For example, in suppressor strain host cells, such as amber suppressor strains and partial amber suppressor strains, translation proceeds through one or more stop codon (e.g. the amber stop codon for an amber suppressor strain), at least some of the time.
  • stop codon e.g. the amber stop codon for an amber suppressor strain
  • VH variable heavy chain or a “variable light” (VL) chain
  • VH domain VL domain
  • VH domain VL domain
  • heavy chain germline segments are designated as VH, DH and JH, and compilation thereof results in a nucleic acid encoding a VH chain.
  • Light chain germline segments are designated as VL or JL, and include kappa and lambda light chains (V ⁇ and J ⁇ ; V ⁇ and J ⁇ ) and compilation thereof results in a nucleic acid encoding a VL chain. It is understood that a light chain is either a kappa or lambda light chain, but does not include a kappa/lambda combination by virtue of compilation of a V ⁇ and J ⁇ .
  • a “degenerate codon” refers to three-nucleotide codon that specifies the same amino acid as a codon in a parent nucleotide sequence.
  • One of skill in the art is familiar with degeneracy of the genetic code and can identify degenerate codons.
  • “Diversity” with respect to members in a collection refers to the number of unique members in a collection. Hence, diversity refers to the number of different amino acid sequences or nucleic acid sequences, respectively, among the analogous polypeptide members of that collection.
  • a collection of polynucleotides having a diversity of 104 contains 104 different nucleic acid sequences among the analogous polynucleotide members.
  • the provided collections of polynucleotides and/or polypeptides have diversities of at least at or about 102, 103, 104, 105, 106, 107, 108, 109, 1010 or more.
  • Sequence diversity refers to a representation of nucleic acid sequence similarity and is determined using sequence alignments, diversity scores, and/or sequence clustering. Any two sequences can be aligned by laying the sequences side-by-side and analyzing differences within nucleotides at every position along the length of the sequences. Sequence alignment can be assessed in silico using Basic Local Alignment Search Tool (BLAST), an NCBI tool for comparing nucleic acid and/or protein sequences. The use of BLAST for sequence alignment is well known to one of skill in the art. The Blast search algorithm compares two sequences and calculates the statistical significance of each match (a Blast score). Sequences that are most similar to each other will have a high Blast score, whereas sequences that are most varied will have a low Blast score.
  • BLAST Basic Local Alignment Search Tool
  • polypeptide domain refers to a part of a polypeptide (a sequence of three or more, generally 5 or 7 or more amino acids) that is a structurally and/or functionally distinguishable or definable.
  • exemplary of a polypeptide domain is a part of the polypeptide that can form an independently folded structure within a polypeptide made up of one or more structural motifs (e.g. combinations of alpha helices and/or beta strands connected by loop regions) and/or that is recognized by a particular functional activity, such as enzymatic activity or antigen binding.
  • a polypeptide can have one, typically more than one, distinct domains.
  • the polypeptide can have one or more structural domains and one or more functional domains.
  • a single polypeptide domain can be distinguished based on structure and function.
  • a domain can encompass a contiguous linear sequence of amino acids.
  • a domain can encompass a plurality of non-contiguous amino acid portions, which are non-contiguous along the linear sequence of amino acids of the polypeptide.
  • a polypeptide contains a plurality of domains.
  • each heavy chain and each light chain of an antibody molecule contains a plurality of immunoglobulin (Ig) domains, each about 110 amino acids in length.
  • Ig domain refers to a domain, recognized as such by those in the art, that is distinguished by a structure, called the Immunoglobulin (Ig) fold, which contains two beta-pleated sheets, each containing anti-parallel beta strands of amino acids connected by loops. The two beta sheets in the Ig fold are sandwiched together by hydrophobic interactions and a conserved intra-chain disulfide bond. Individual immunoglobulin domains within an antibody chain further can be distinguished based on function. For example, a light chain contains one variable region domain (VL) and one constant region domain (CL), while a heavy chain contains one variable region domain (VH) and three or four constant region domains (CH). Each VL, CL, VH, and CH domain is an example of an immunoglobulin domain.
  • VL variable region domain
  • CL constant region domain
  • CH constant region domain
  • variable domain refers to a specific Ig domain of an antibody heavy or light chain that contains a sequence of amino acids that varies among different antibodies.
  • Each light chain and each heavy chain has one variable region domain (VL, and, VH).
  • VL, and, VH variable region domain
  • the variable domains provide antigen specificity, and thus are responsible for antigen recognition.
  • Each variable region contains CDRs that are part of the antigen binding site domain and framework regions (FRs).
  • a “constant region domain” refers to a domain in an antibody heavy or light chain that contains a sequence of amino acids that is comparatively more conserved among antibodies than the variable region domain.
  • Each light chain has a single light chain constant region (CL) domain and each heavy chain contains one or more heavy chain constant region (CH) domains, which include, CH1, CH2, CH3 and CH4.
  • CH1 and CL domains extend the Fab arm of the antibody molecule, thus contributing to the interaction with antigen and rotation of the antibody arms.
  • Antibody constant regions can serve effector functions, such as, but not limited to, clearance of antigens, pathogens and toxins to which the antibody specifically binds, e.g. through interactions with various cells, biomolecules and tissues.
  • an “antibody or portion thereof that is sufficient to form an antigen binding site” means that the antibody or portion thereof contains at least 1 or 2, typically 3, 4, 5 or all 6 CDRs of the VH and VL sufficient to retain at least a portion of the binding specificity of the corresponding full-length antibody containing all 6 CDRs.
  • a sufficient antigen binding site at least requires CDR3 of the heavy chain (CDRH3). It typically further requires the CDR3 of the light chain (CDRL3).
  • CDRH3 heavy chain
  • CDRL3 CDR3 of the light chain
  • CDR-L1 corresponds to residues L24-L34
  • CDR-L2 corresponds to residues L50-L56
  • CDR-L3 corresponds to residues L89-L97
  • CDR-H1 corresponds to residues H31-H35, 35a or 35b depending on the length
  • CDR-H2 corresponds to residues H50-H65
  • CDR-H3 corresponds to residues H95-H102.
  • a “peptide mimetic” refers to a peptide that mimics the activity of a polypeptide.
  • an erythropoietin (EPO) peptide mimetic is a peptide that mimics the activity of Epo, such as for binding and activation of the EPO receptor.
  • An “address” refers to a unique identifier for each locus in a collection whereby an addressed member (e.g. an antibody) can be identified.
  • An addressed moiety is one that can be identified by virtue of its locus or location. Addressing can be effected by position on a surface, such as a well of a microplate. For example, an address for a protein in a microwell plate that is F9 means that the protein is located in row F, column 9 of the microwell plate. Addressing also can be effected by other identifiers, such as a tag encoded with a bar code or other symbology, a chemical tag, an electronic, such RF tag, a color-coded tag or other such identifier.
  • An “array” refers to a collection of elements, such as antibodies, containing three or more members.
  • spatial array refers to an array where members are separated or occupy a distinct space in an array.
  • spatial arrays are a type of addressable array.
  • Examples of spatial arrays include microtiter plates where each well of a plate is an address in the array.
  • Spacial arrays include any arrangement wherein a plurality of different molecules, e.g., polypeptides, are held, presented, positioned, situated, or supported.
  • arrays can include microtiter plates, such as 48-well, 96-well, 144-well, 192-well, 240-well, 288-well, 336-well, 384-well, 432-well, 480-well, 576-well, 672-well, 768-well, 864-well, 960-well, 1056-well, 1152-well, 1248-well, 1344-well, 1440-well, or 1536-well plates, tubes, slides, chips, flasks, or any other suitable laboratory apparatus.
  • arrays can also include a plurality of sub-arrays.
  • a plurality of sub-arrays encompasses an array where more than one arrangement is used to position the polypeptides.
  • multiple 96-well plates could constitute a plurality of sub-arrays and a single array.
  • an “addressable library” or “spatially addressed library” refers to a collection of molecules such as nucleic acid molecules or protein agents, such as antibodies, in which each member of the collection is identifiable by virtue of its address.
  • an “addressable array” refers to one in which the members of the array are identifiable by their address, the position in a spatial array, such as a well of a microtiter plate, or on a solid phase support, or by virtue of an identifiable or detectable label, such as by color, fluorescence, electronic signal (i.e. RF, microwave or other frequency that does not substantially alter the interaction of the molecules of interest), bar code or other symbology, chemical or other such label.
  • a spatial array such as a well of a microtiter plate, or on a solid phase support
  • an identifiable or detectable label such as by color, fluorescence, electronic signal (i.e. RF, microwave or other frequency that does not substantially alter the interaction of the molecules of interest), bar code or other symbology, chemical or other such label.
  • the members of the array are located at identifiable loci on the surface of a solid phase or directly or indirectly linked to or otherwise associated with the identifiable label, such as affixed to a microsphere or other particulate support (herein referred to as beads) and suspended in solution or spread out on a surface.
  • a microsphere or other particulate support herein referred to as beads
  • An addressable combinatorial antibody library refers to a collection of antibodies in which member antibodies are identifiable and all antibodies with the same identifier, such as position in a spatial array or on a solid support, or a chemical or RF tag, bind to the same antigen, and generally are substantially the same in amino acid sequence.
  • identifier such as position in a spatial array or on a solid support, or a chemical or RF tag
  • reference to an “addressable arrayed combinatorial antibody library” means that the antibody members are addressed in an array.
  • “In silico” refers to research and experiments performed using a computer. In silico methods include, but are not limited to, molecular modeling studies, biomolecular docking experiments, and virtual representations of molecular structures and/or processes, such as molecular interactions.
  • the antibody members of a library can be designed using a computer program that selects component V, D and J germline segments from among those input into the computer and joins them in-frame to output a list of nucleic acid molecules for synthesis.
  • the recombination of the components of the antibodies in the collections or libraries provided herein can be performed in silico by combining the nucleotide sequences of each building block in accord with software that contains rules for doing so. The process could be performed manually without a computer, but the computer provides the convenience of speed.
  • a “database” refers to a collection of data items.
  • reference to a database is typically with reference to antibody databases, which provide a collection of sequence and structure information for antibody genes and sequences.
  • Exemplary antibody databases include, but are not limited to, IMGT®, the international ImMunoGeneTics information system (imgt.cines.fr; see e.g., Lefranc et al.
  • a database also can be created by a user to include any desired sequences.
  • the database can be created such that the sequences are inputted in a desired format (e.g., in a particular reading frame; lacking stop codons; lacking signal sequences).
  • the database also can be created to include sequences in addition to antibody sequences.
  • Screening refers to identification or selection of an antibody or portion thereof from a collection or library of antibodies and/or portions thereof, based on determination of the activity or property of an antibody or portion thereof. Screening can be performed in any of a variety of ways, including, for example, by assays assessing direct binding (e.g. binding affinity) of the antibody to a target protein or by functional assays assessing modulation of an activity of a target protein.
  • “Activity towards a target protein” refers to binding specificity and/or modulation of a functional activity of a target protein, or other measurements that reflects the activity of an antibody or portion thereof towards a target protein.
  • a “target protein” or “protein target” refers to candidate proteins or peptides that are specifically recognized by an antibody or portion thereof and/or whose activity is modulated by an antibody or portion thereof. Modulating the activity can comprise increasing, decreasing, stimulating, or preventing the activity or expression of the target protein.
  • a target protein includes any peptide or protein that contains an epitope for antibody recognition. Target proteins include proteins involved in the etiology of a disease or disorder by virtue of expression or activity. Exemplary target proteins are described herein. In some instances, the target protein is a transmembrane protein target. Transmembrane protein targets include, but are not limited to, GPCRs, ion channels, transporters, and cell surface receptors.
  • Ion channels may be potassium ion channels, sodium ion channels, calcium ion channels, and voltage gated channels.
  • the antibodies disclosed herein modulate a Kv1.3 ion channel, Nav1.7 ion channel, or acid sensing ion channel (ASIC).
  • the antibodies disclosed herein may modulate cell surface receptors such as GLP1R, GCGR, EPO receptor, FGFR, FGF21R, CSFR, GMCSFR, and GCSFR.
  • Additional target proteins include, but are not limited to, cytokines, kinases, interferons, hormones, and growth factors.
  • the target proteins can be from a mammal or non-mammal.
  • the target proteins can be from a human. Alternatively, the target proteins are from a bovine.
  • “Hit” refers to an antibody or portion thereof identified, recognized or selected as having an activity in a screening assay.
  • “Iterative” with respect to screening means that the screening is repeated a plurality of times, such as 2, 3, 4, 5 or more times, until a “Hit” is identified whose activity is optimized or improved compared to prior iterations.
  • High-throughput refers to a large-scale method or process that permits manipulation of large numbers of molecules or compounds, generally tens to hundreds to thousands of compounds. For example, methods of purification and screening can be rendered high-throughput. High-throughput methods can be performed manually. Generally, however, high-throughput methods involve automation, robotics or software.
  • BLAST Basic Local Alignment Search Tool
  • blastn is a program that compares a nucleotide query sequence against a nucleotide sequence database (e.g. GenBank).
  • BlastP is a program that compares an amino acid query sequence against a protein sequence database.
  • a BLAST bit score is a value calculated from the number of gaps and substitutions associated with each aligned sequence. The higher the score, the more significant the alignment.
  • a “human protein” refers to a protein encoded by a nucleic acid molecule, such as DNA, present in the genome of a human, including all allelic variants and conservative variations thereof.
  • a variant or modification of a protein is a human protein if the modification is based on the wildtype or prominent sequence of a human protein.
  • “Naturally occurring amino acids” refer to the 20 L-amino acids that occur in polypeptides. The residues are those 20 ⁇ -amino acids found in nature which are incorporated into protein by the specific recognition of the charged tRNA molecule with its cognate mRNA codon in humans.
  • Non-naturally occurring amino acids refer to amino acids that are not genetically encoded.
  • a non-natural amino acid is an organic compound that has a structure similar to a natural amino acid but has been modified structurally to mimic the structure and reactivity of a natural amino acid.
  • Non-naturally occurring amino acids thus include, for example, amino acids or analogs of amino acids other than the 20 naturally-occurring amino acids and include, but are not limited to, the D-isostereomers of amino acids.
  • Exemplary non-natural amino acids are known to those of skill in the art.
  • Nucleic acids include DNA, RNA and analogs thereof, including peptide nucleic acids (PNA) and mixtures thereof. Nucleic acids can be single or double-stranded.
  • probes or primers which are optionally labeled, such as with a detectable label, such as a fluorescent or radiolabel, single-stranded molecules are contemplated. Such molecules are typically of a length such that their target is statistically unique or of low copy number (typically less than 5, generally less than 3) for probing or priming a library.
  • a probe or primer contains at least 14, 16 or 30 contiguous nucleotides of sequence complementary to or identical to a gene of interest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleic acids long.
  • a “peptide” refers to a polypeptide that is from 2 to 40 amino acids in length.
  • amino acids which occur in the various sequences of amino acids provided herein are identified according to their known, three-letter or one-letter abbreviations (Table 1).
  • the nucleotides which occur in the various nucleic acid fragments are designated with the standard single-letter designations used routinely in the art.
  • amino acid is an organic compound containing an amino group and a carboxylic acid group.
  • a polypeptide contains two or more amino acids.
  • amino acids include the twenty naturally-occurring amino acids, non-natural amino acids and amino acid analogs (i.e., amino acids wherein the ⁇ -carbon has a side chain).
  • amino acid residue refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages.
  • the amino acid residues described herein are presumed to be in the “L” isomeric form. Residues in the “D” isomeric form, which are so designated, can be substituted for any L-amino acid residue as long as the desired functional property is retained by the polypeptide.
  • NH2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide.
  • amino acid residue sequences represented herein by formulae have a left to right orientation in the conventional direction of amino-terminus to carboxyl-terminus.
  • amino acid residue is broadly defined to include the amino acids listed in the Table of Correspondence (Table 1) and modified and unusual amino acids, such as those referred to in 37 C.F.R. ⁇ 1.821-1.822, and incorporated herein by reference.
  • a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues, to an amino-terminal group such as NH2 or to a carboxyl-terminal group such as COOH.
  • L-amino acid is identified by the standard three letter code (or single letter code) or the standard three letter code (or single letter code) with the prefix “L-”; the prefix “D-” indicates that the stereoisomeric form of the amino acid is D.
  • an “immunoconjugate” refers to an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent, non-antibody peptide or therapeutic polypeptide.
  • An immunoconjugate may include non-antibody sequences.
  • the non-antibody sequence can be conjugated to the antibody.
  • the non-antibody sequence can be within the antibody sequence.
  • non-antibody peptide refers to a peptide encoded by a non-antibody antibody sequence.
  • a non-antibody peptide may be a hormone, a lymphokine, an interleukin, a chemokines, a cytokine or a peptide toxin.
  • therapeutic polypeptide As used herein, the terms “therapeutic polypeptide,” “therapeutic peptides,” and therapeutic immunoglobulin construct” mean one or more peptides having demonstrated or potential use in treating, preventing, or ameliorating one or more diseases, disorders, or conditions in a subject in need thereof, as well as related peptides.
  • Therapeutic peptides include peptides found to have use in treating, preventing, or ameliorating one or more diseases, disorders, or conditions after the time of filing of this application.
  • Related peptides include fragments of therapeutic peptides, therapeutic peptide variants, and therapeutic peptide derivatives that retain some or all of the therapeutic activities of the therapeutic peptide.
  • therapeutic polypeptide or “therapeutic peptides” encompass modifications to the therapeutic peptides defined and/or disclosed herein.
  • the therapeutic polypeptide is selected from a hormone, a lymphokine, an interleukin, a chemokines, a cytokine, a peptide toxin, and combinations thereof.
  • Therapeutic polypeptides can be peptides encoded by non-antibody sequences.
  • a derivative or a variant of a polypeptide is said to share “homology” or be “homologous” with the peptide if the amino acid sequences of the derivative or variant has at least 50% identity with the original peptide.
  • the derivative or variant is at least 75% the same as that of either the peptide or a fragment of the peptide having the same number of amino acid residues as the derivative.
  • the derivative or variant is at least 85% the same as that of either the peptide or a fragment of the peptide having the same number of amino acid residues as the derivative.
  • the amino acid sequence of the derivative is at least 90% the same as the peptide or a fragment of the peptide having the same number of amino acid residues as the derivative. In some embodiments, the amino acid sequence of the derivative is at least 95% the same as the peptide or a fragment of the peptide having the same number of amino acid residues as the derivative. In certain embodiments, the derivative or variant is at least 99% the same as that of either the peptide or a fragment of the peptide having the same number of amino acid residues as the derivative.
  • Kb kilobases
  • bp base pairs
  • Kb kilobases
  • bp base pairs
  • kD kilo-Daltons
  • Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.
  • Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letters, 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et al., Nucleic Acids Res., 12:6159-6168 (1984). Purification of oligonucleotides is by either native polyacrylamide gel electrophoresis or by anion-exchange chromatography as described in Pearson & Reanier, J. Chrom., 255:137-149 (1983).
  • sequence of the cloned genes and synthetic oligonucleotides can be verified after cloning using, e.g., the chain termination method for sequencing double-stranded templates of Wallace et al., Gene, 16:21-26 (1981).
  • the nucleic acids encoding recombinant polypeptides of the present disclosure may be cloned into an intermediate vector before transformation into prokaryotic or eukaryotic cells for replication and/or expression.
  • the intermediate vector may be a prokaryote vector such as a plasmid or shuttle vector.
  • cattle are the only species where ultralong CDR3 sequences have been identified.
  • other species for example other ruminants, may also possess antibodies with ultralong CDR3 sequences.
  • Exemplary antibody variable region sequences comprising an ultralong CDR3 sequence identified in cattle include those designated as: BLV1H12 (see, SEQ ID NO: 22), BLV5B8 (see, SEQ ID NO: 23), BLV5D3 (see, SEQ ID NO: 24) and BLV8C11 (see, SEQ ID NO: 25) (see, e.g., Saini, et al. (1999) Eur. J. Immunol. 29: 2420-2426; and Saini and Kaushik (2002) Scand. J. Immunol.
  • BF4E9 see, SEQ ID NO: 26
  • BF1H1 see, SEQ ID NO: 27
  • F18 see, SEQ ID NO: 28
  • bovine antibodies are identified and/or produced. Multiple techniques exist to identify and/or produce antibodies.
  • Antibodies of the present disclosure may be isolated by screening including, high-throughput screening, of combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al.
  • Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas.
  • Phage display libraries of bovine antibodies may be a source of bovine antibody gene sequences, including ultralong CDR3 sequences.
  • a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
  • a humanized antibody comprises one or more variable domains in which CDRs (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences.
  • a humanized antibody optionally will also comprise at least a portion of a human constant region.
  • some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.
  • Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci.
  • Antibodies with ultralong CDR3 sequences may also include non-antibody sequences, such as cytokines, therapeutic polypeptides or growth factors, into the CDR3 region.
  • the resultant antibody can be effective in treating or preventing a disease or condition.
  • an antibody comprising an ultralong CDR3 inhibits tumor metastasis.
  • the cytokine, therapeutic polypeptide or growth factor may be shown to have an antiproliferative effect on at least one cell population.
  • the resultant antibody modulates the expression or activity of a target (e.g., protein target, transmembrane protein target).
  • a target e.g., protein target, transmembrane protein target.
  • an antibody comprising an ultralong CDR3 inhibits or blocks an ion channel.
  • the non-antibody sequence may be a hormone, a lymphokine, an interleukin, a chemokines, a cytokine, a peptide toxin, and combinations thereof.
  • cytokines, therapeutic polypeptides, toxins, lymphokines, growth factors, or other hematopoietic factors include Granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), Granulocyte-macrophage colony-stimulating factor (GM-CSF), Meg-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN (e.g., ⁇ -interferon, ⁇ -interferon, a ⁇ -interferon), TNF-
  • Additional growth factors for use in the antibodies and/or pharmaceutical compositions of the present disclosure include: angiogenin, bone morphogenic protein-1, bone morphogenic protein-2, bone morphogenic protein-3, bone morphogenic protein-4, bone morphogenic protein-5, bone morphogenic protein-6, bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein-10, bone morphogenic protein-11, bone morphogenic protein-12, bone morphogenic protein-13, bone morphogenic protein-14, bone morphogenic protein-15, bone morphogenic protein receptor IA, bone morphogenic protein receptor IB, brain derived neurotrophic factor, ciliary neutrophic factor, ciliary neutrophic factor receptor, cytokine-induced neutrophil chemotactic factor 1, cytokine-induced neutrophil, chemotactic factor 2, cytokine-induced neutrophil chemotactic factor 2, endothelial cell growth factor, endothelin 1, epidermal growth factor, epithelial-derived neutrophil attractant, fibroblast
  • the therapeutic polypeptide is a mammalian G-CSF, a growth hormone, a leptin, a ⁇ -interferon, a ⁇ -interferon, a ⁇ -interferon, a GM-CSF, a IL-11, a IL-10, a mokal (e.g., Moka, mokatoxin-1), or a VM-24.
  • the therapeutic polypeptide is a glucagon-like peptide 1 (GLP-1), exendin-4 (Ex-4), erythropoietin (EPO), fibroblast growth factor (FGF21), IL8, ziconotide, somatostatin, chlorotoxin, SDF1(alpha), IL21, or derivative or variant thereof.
  • GLP-1 glucagon-like peptide 1
  • Ex-4 exendin-4
  • EPO erythropoietin
  • FGF21 fibroblast growth factor
  • IL8 ziconotide
  • somatostatin chlorotoxin
  • SDF1(alpha) IL21
  • derivative or variant thereof glucagon-like peptide 1
  • the G-CSF may be a bovine G-CSF.
  • the G-CSF, GM-CSF, EPO, FGF21, ⁇ -interferon and GLP-1 may be from a human.
  • the non-antibody sequence may comprise an amino acid sequence based on or derived from any of SEQ ID NOS: 317-332.
  • the non-antibody sequence may comprise an amino acid sequence that is 50%, 60%, 70% 80%, 90%, 95%, 97%, 99% identical to any of SEQ ID NOS: 317-332.
  • the non-antibody sequence may comprise an amino acid sequence that comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues of SEQ ID NOS: 317-332.
  • the amino acid residues may be consecutive. Alternatively, the amino acid residues are non-consecutive.
  • the non-antibody sequence may comprise at least a portion of any of SEQ ID NOS: 317-332.
  • the antibodies disclosed herein may comprise one or more sequences based on or derived from a mammalian, avian, reptilian, amphibian, fish, insect, bug, or arachnid sequence.
  • Mammals include, but are not limited to, cows, bison, buffalo, humans, mice, dogs, cats, sheep, goats, or rabbits.
  • Avians include, but are not limited to, chicken, geese, doves, eagles, sparrows, and pidgeons.
  • Reptiles include, but are not limited to, lizards, gators, snakes, and turtles.
  • Amphibians include, but are not limited to, frogs, salamanders, toads, and newts.
  • Fish include, but are not limited to, tuna, salmon, whales, and sharks.
  • Insects, bugs, and arachnids include, but are not limited to, flies, mosquitos, spiders, and scorpions.
  • the non-antibody sequence may be based on or derived from a bovine or human sequence.
  • the non-antibody sequence is based on or derived from a lizard, snail, snake or scorpion sequence.
  • the lizard may be a gila monster.
  • the snail may be a cone snail.
  • the non-antibody sequence is linked to an end of an ultralong CDR3 sequence.
  • the non-antibody sequence can be linked to the 5′ end or 3′ end of the ultralong CDR3 nucleotide sequence.
  • the non-antibody sequence can be linked to the N-terminus or C-terminus of the ultralong CDR3 peptide sequence.
  • the non-antibody sequence is inserted within an ultralong CDR3 sequence.
  • the non-antibody sequence is inserted between the stalk domain of an ultralong CDR3 sequence.
  • the non-antibody sequence can be inserted within the stalk domain of an ultralong CDR3 sequence.
  • the non-antibody sequence is inserted between the stalk domain and the knob domain of an ultralong CDR3 sequence.
  • the non-antibody sequence is inserted within the knob domain of an ultralong CDR3 sequence.
  • the non-antibody sequence replaces at least a portion of an ultralong CDR3 sequence.
  • the non-antibody sequence can replace about 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more amino acids of the ultralong CDR3 peptide sequence.
  • the non-antibody sequence can replace about 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more of the ultralong CDR3 peptide sequence.
  • the non-antibody sequence can replace at least a portion of a knob domain of an ultralong CDR3.
  • the non-antibody sequence can replace about 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more amino acids of the knob domain of an ultralong CDR3 peptide sequence.
  • the non-antibody sequence can replace about 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more of the knob domain of the ultralong CDR3 peptide sequence.
  • the non-antibody sequence can replace at least a portion of a stalk domain of an ultralong CDR3.
  • the non-antibody sequence can replace about 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 20 or more amino acids of the stalk domain of an ultralong CDR3 peptide sequence.
  • the non-antibody sequence can replace about 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more of the stalk domain of the ultralong CDR3 peptide sequence.
  • the amino acids may be consecutive amino acids. Alternatively, the amino acids are non-consecutive amino acids.
  • the ultralong CDR3 may comprise one or more conserved motifs.
  • the conserved motifs may be stalk domain conserved motifs as disclosed herein. Alternatively, the conserved motifs may be knob domain conserved motifs as disclosed herein.
  • the non-antibody sequence replaces at least a portion of an ultralong CDR3 sequence.
  • the non-antibody sequence can replace about 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more, 55 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more nucleotides of the ultralong CDR3 nucleotide sequence.
  • the non-antibody sequence can replace about 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more of the ultralong CDR3 nucleotide sequence.
  • the non-antibody sequence can replace at least a portion of a knob domain of an ultralong CDR3.
  • the non-antibody sequence can replace about 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more, 55 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more nucleotides of the knob domain of an ultralong CDR3 nucleotide sequence.
  • the non-antibody sequence can replace about 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more of the knob domain of the ultralong CDR3 nucleotide sequence.
  • the non-antibody sequence can replace at least a portion of a stalk domain of an ultralong CDR3.
  • the non-antibody sequence can replace about 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more nucleotides of the stalk domain of an ultralong CDR3 nucleotide sequence.
  • the non-antibody sequence can replace about 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more of the stalk domain of the ultralong CDR3 nucleotide sequence.
  • the nucleotides may be consecutive nucleotides. Alternatively, the nucleotides are non-consecutive nucleotides.
  • the ultralong CDR3 may comprise one or more conserved motifs.
  • the conserved motifs may be stalk domain conserved motifs as disclosed herein. Alternatively, the conserved motifs may be knob domain conserved motifs as disclosed herein.
  • An antibody comprising an ultralong CDR3 sequence and a non-antibody sequence may further comprise one or more cleavage sites between the ultralong CDR3 sequence and the non-antibody sequence.
  • the one or more cleavage sites may be in front of the N-terminus of the non-antibody peptide sequence.
  • a cleavage site is inserted at the N-terminus of the non-antibody peptide sequence and at the C-terminus of the ultralong CDR3 peptide sequence.
  • the one or more cleave sites are behind the C-terminus of the non-antibody peptide sequence.
  • the cleavage site is inserted at the C-terminus of the non-antibody peptide sequence and at the N-terminus of the ultralong CDR3 peptide sequence.
  • the one or more cleavage sites may flank both the N-terminus and the C-terminus of the non-antibody peptide sequence.
  • the one or more cleavage sites may be upstream of the non-antibody nucleotide sequence.
  • the one or more cleavage sites may be at the 5′ end of the non-antibody nucleotide sequence and at the 3′ end of the ultralong CDR3 nucleotide sequence.
  • the one or more cleavage sites may be downstream of the non-antibody nucleotide sequence.
  • the one or more cleavage sites may be at the 3′ end of the non-antibody nucleotide sequence and at the 5′ end of the ultralong CDR3 nucleotide sequence.
  • the one or more cleavage sites may flank both the 5′ end and the 3′ end of the non-antibody nucleotide sequence.
  • the one or more cleavage sites may directly flank the non-antibody sequence. For example, there are zero nucleotides or amino acids between the cleavage site sequence and the non-antibody sequence.
  • the one or more cleavage sites may indirectly flank the non-antibody sequence.
  • nucleotides between the cleavage site nucleotide sequence and the non-antibody nucleotide sequence there are one or more nucleotides between the cleavage site nucleotide sequence and the non-antibody nucleotide sequence. There may be 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 12 or more, 15 or more, 20 or more nucleotides between the cleavage site nucleotide sequence and the non-antibody nucleotide sequence. In another example, there are one or more amino acids between the cleavage site peptide sequence and the non-antibody peptide sequence.
  • the cleavage site may be adjacent to the sequence based on or derived from the ultralong CDR3 sequence, linker sequence, non-antibody sequence, non-bovine sequence, or a combination thereof.
  • the cleavage site may be between the sequence based on or derived from the ultralong CDR3 sequence and the linker sequence.
  • the cleavage site may be between the sequence based on or derived from the ultralong CDR3 sequence and the non-antibody sequence.
  • the cleavage site may be between the linker sequence and the non-antibody sequence.
  • the cleavage site may be for a protease.
  • the protease may be a serine protease, threonine protease, cysteine protease, aspartate protease, or metalloprotease.
  • the protease may include, but is not limited to, Factor Xa protease, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidain, bromelain, calpains, caspases, cathepsins, Mir1-CP, papain, HIV-1 protease, chymosin, renin, cathepsin D, pepsin, plasmepsin, nepenthesin, metalloexopeptidases, and metalloendopeptidases.
  • the cleavage site may be a cleavage site for Factor Xa or thrombin.
  • the cleavage site may comprise the amino acid sequence of IEGR.
  • the cleavage site is for a nuclease.
  • the antibody comprising the ultralong CDR3 sequence and non-antibody sequence may be cleaved by one or more proteases. Cleavage of the antibody by the one or more protease can result in release of one or more ends of the non-antibody peptide from the ultralong CDR3 region of the antibody.
  • cleavage of the antibody results in release of the N-terminus of the non-antibody peptide from the ultralong CDR3 region.
  • cleavage of the antibody results in release of the C-terminus of the non-antibody peptide from the ultralong CDR3 region.
  • the non-antibody sequence may be linked to the ultralong CDR3 sequence via one or more linkers.
  • the non-antibody sequence may be inserted with an ultralong CDR3 sequence.
  • two or more linkers are used to link the non-antibody sequence to the ultralong CDR3 sequence.
  • the two or more linkers may comprise the same sequence.
  • the two or more linkers comprise different sequences.
  • the one or more linker sequences may be the same length.
  • the one or more linker sequences may be different lengths.
  • the one or more linker sequences may be 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more amino acids in length.
  • the one or more linker sequences may comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more glycine residues.
  • the one or more linker sequences may comprise 2 or more, 3 or more, 4 or more, or 5 or more consecutive glycine residues.
  • the one or more linker sequences may comprise 1 or more serine residues.
  • the one or more linker sequences may comprise 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more polar amino acid residues.
  • the polar amino acid residues may be selected from serine, threonine, asparagine, or glutamine.
  • the polar amino acid residues may comprise uncharged side chains.
  • the linkers may be attached to the N-terminal, C-terminal, or both N- and C-termini of the non-antibody peptide sequence.
  • the linkers may be attached to the 5′-end, 3′-end, or both the 5′- and 3′ ends of the non-antibody nucleotide sequence.
  • the linker may comprise amino acid residues.
  • Exemplary amino acid linker components include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
  • Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe).
  • Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly).
  • the linker may comprise an amino acid sequence of GGGSGGGGS (SEQ ID NO: 337) or GGGGSGGGS (SEQ ID NO: 338).
  • Amino acid residues which comprise an amino acid linker component include those occurring naturally, as well as minor amino acids and non-naturally occurring amino acids including analogs, such as citrulline.
  • Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzymes, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.
  • the ultralong CDR3 may be based on or derived from a single ultralong CDR3 sequence. Alternatively, the ultralong CDR3 is based on or derived from two or more ultralong CDR3 sequences. The two or more ultralong CDR3 sequences may be from the same animal. Alternatively, the two or more ultralong CDR3 sequences are from two or more different animals.
  • the ultralong CDR3 may comprise at least a portion of a stalk domain of an ultralong CDR3.
  • the antibodies disclosed herein may comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more 7 or more, 8 or more, 9 or more, or 10 or more amino acids derived from or based on the stalk domain of the ultralong CDR3.
  • the antibodies disclosed herien may comprise 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer amino acids derived from or based the stalk domain of the ultralong CDR3.
  • the amino acids may be consecutive amino acids. Alternatively, the amino acids are non-consecutive amino acids.
  • the antibodies disclosed herein may comprise a sequence that is 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, or 100% homologous the sequence of the stalk domain of the ultralong CDR3.
  • the ultralong CDR3 may comprise one or more conserved motifs derived from or based on a stalk domain of the ultralong CDR3.
  • the antibodies disclosed herein may comprise 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more conserved motifs derived from or based on the stalk domain of the ultralong CDR3.
  • the one or more conserved motifs derived from or based on the stalk domain of the ultralong CDR3 may comprise a sequence selected from any one of SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336.
  • the antibodies disclosed herein may comprise a sequence that is 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, or 100% homologous to a sequence selected from any one of SEQ ID NOS: 157-224 and 235-295.
  • the antibodies disclosed herein may comprise a sequence that is 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, or 100% homologous to a sequence selected from any one of SEQ ID NOS: 225-227.
  • the one or more conserved motifs derived from or based on the stalk domain of the ultralong CDR3 may comprise a CT(T/S)VHQ motif.
  • the one or more conserved motifs derived from or based on the stalk domain of the ultralong CDR3 comprise a CT(T/S)VHQX n motif.
  • n is between 1 to 8, between 1 to 7, between 1 to 6, between 1 to 5, between 1 to 4, or between 1 to 3.
  • the one or more conserved motifs derived from or based on the stalk domain of the ultralong CDR3 may comprise a CX 1 X 2 X 3 X 4 Q motif.
  • X 1 may be a T, S, A, or G residue.
  • X 2 may be a T, S, A, P, or I residue.
  • X 3 may be a V or K residue.
  • X 4 may be an H, K, or Y residue.
  • the one or more conserved motifs derived from or based on the stalk domain of the ultralong CDR3 may comprise an X 1 X 2 VHQ motif.
  • X 1 may be a T, S, A, or G residue.
  • X 2 may be a T, S, A, P or I residue.
  • the one or more conserved motifs derived from or based on the stalk domain of the ultralong CDR3 may comprise a CX 1 X 2 VHQ motif.
  • X 1 may be a T, S, A, or G residue.
  • X 2 may be a T, S, A, P or I residue.
  • the one or more conserved motifs derived from or based on the stalk domain of the ultralong CDR3 may comprise an X 1 X 2 VX 3 Q motif.
  • X 1 may be a T, S, A, or G residue.
  • X 2 may be a T, S, A, P or I residue.
  • X 3 may be an H, Y or K residue.
  • the one or more conserved motifs derived from or based on the stalk domain of the ultralong CDR3 may comprise a CX 1 X 2 VX 3 Q motif.
  • X 1 may be a T, S, A, or G residue.
  • X 2 may be a T, S, A, P or I residue.
  • X 3 may be an H, Y or K residue.
  • the one or more conserved motifs derived from or based on the stalk domain of the ultralong CDR3 may comprise an X 1 X 2 KKQ motif.
  • X 1 may be a T, S, A, or G residue.
  • X 2 may be a T, S, A, P or I residue.
  • the one or more conserved motifs derived from or based on the stalk domain of the ultralong CDR3 may comprise a CX 1 X 2 KKQ motif.
  • X 1 may be a T, S, A, or G residue.
  • X 2 may be a T, S, A, P or I residue.
  • the one or more conserved motifs derived from or based on the stalk domain of the ultralong CDR3 may comprise an YX 1 YX 2 motif.
  • X 1 may be a T, S, N, or I residue.
  • X 2 may be an E or D residue.
  • the one or more conserved motifs derived from or based on the stalk domain of the ultralong CDR3 may comprise an YX 1 YX 2 Y motif.
  • X 1 may be an L, S, T, or R residue.
  • X 2 may be a T, S, N or I residue.
  • the one or more conserved motifs derived from or based on the stalk domain of the ultralong CDR3 may comprise an YX 1 YX 2 YX 3 motif.
  • X 1 may be an L, S, T, or R residue.
  • X 2 may be a T, S, N or I residue.
  • X 3 may be an E or D residue.
  • the one or more conserved motifs derived from or based on the stalk domain of the ultralong CDR3 may comprise an YX 1 YX 2 YX 3 X 4 motif
  • X 1 may be an L, S, T, or R residue.
  • X 2 may be a T, S, N or I residue.
  • X 3 may be an E or D residue.
  • X 4 may be an H, W, N, F, I or Y residue.
  • the one or more conserved motifs derived from or based on the stalk domain of the ultralong CDR3 may comprise an Y(E/D)X motif X may be an H, W, N, F, I or Y residue.
  • the one or more conserved motifs derived from or based on the stalk domain of the ultralong CDR3 may comprise an XY(E/D) motif X may be a T, S, N or I residue.
  • the one or more conserved motifs derived from or based on the stalk domain of the ultralong CDR3 may comprise an Y(E/D)X 1 X n W motif X 1 may be an H, W, N, F, I or Y residue. In some instances, n is between 1 to 4, between 1 to 3, or between 1 to 2.
  • the one or more conserved motifs derived from or based on the stalk domain of the ultralong CDR3 may comprise an Y(E/D)X 1 X 2 X 3 X 4 X 5 W motif X 1 may be an H, W, N, F, I or Y residue.
  • X 2 may be an Y, H, G, or N residue.
  • X3 may be a V, I, or A residue.
  • X 4 may be a D, N, T, or E residue.
  • X 5 may be an A, V, S, or T residue.
  • the antibodies disclosed herein may comprise a first conserved motif derived from or based on the stalk domain of the ultralong CDR3 selected from any of SEQ ID NOS: 157-234 and a second conserved motif derived from or based on the stalk domain of the ultralong CDR3 selected from any of SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336.
  • the antibodies disclosed herein may comprise a first conserved motif derived from or based on the stalk domain of the ultralong CDR3 selected from a group comprising CT(T/S)VHQX n , CX 1 X 2 X 3 X 4 Q, X 1 X 2 VHQ, CX 1 X 2 VHQ, X 1 X 2 VX 3 Q, CX 1 X 2 VX 3 Q, X 1 X 2 KKQ, and CX 1 X 2 KKQ and a second conserved motif derived from or based on the stalk domain of the ultralong CDR3 selected from the group comprising YX 1 YX 2 , YX 1 YX 2 Y, YX 1 YX 2 YX 3 , YX 1 YX 2 YX 3 X 4 , Y(E/D)X, XY(E/D), Y(E/D)X 1 X n W, and Y(E/D)X 1 X 2 X 3
  • the ultralong CDR3 may comprise at least a portion of a knob domain of an ultralong CDR3.
  • the antibodies disclosed herein may comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more 7 or more, 8 or more, 9 or more, or 10 or more amino acids derived from or based on the knob domain of the ultralong CDR3.
  • the antibodies disclosed herien may comprise 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer amino acids derived from or based the knob domain of the ultralong CDR3.
  • the amino acids may be consecutive amino acids. Alternatively, the amino acids are non-consecutive amino acids.
  • the antibodies disclosed herein may comprise a sequence that is 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, or 100% homologous the sequence of the knob domain of the ultralong CDR3.
  • the ultralong CDR3 may comprise one or more conserved motifs derived from or based on a knob domain of the ultralong CDR3.
  • the antibodies disclosed herein may comprise 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more conserved motifs derived from or based on the knob domain of the ultralong CDR3.
  • the one or more conserved motifs derived from or based on the knob domain may comprise a cysteine motif as disclosed herein.
  • one or more conserved motifs derived from or based on the knob domain comprises a C(P/S)DG motif.
  • the antibodies disclosed herein may comprise a sequence based on or derived from a mammal.
  • the mammal may be a bovine.
  • the mammal is a non-bovine mammal, such as a human.
  • the antibody sequences may be 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more 45 or more, 50 or more, 55 or more, 60 or more, 65 or more 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more 190 or more, 200 or more, 220 or more, 230 or more, 240 or more 250 or more 260 or more, 270 or more, 280 or more, 290 or more or 300 or more amino acids in length.
  • the antibody sequences may comprise a bovine antibody sequence comprising 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more 45 or more, 50 or more, 55 or more, 60 or more, 65 or more 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more 190 or more, 200 or more, 220 or more, 230 or more, 240 or more 250 or more 260 or more, 270 or more, 280 or more, 290 or more or 300 or more amino acids in length.
  • the bovine antibody may be a BLVH12, BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, or F18 antibody.
  • the antibody sequences may comprise a human antibody sequence comprising 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more 45 or more, 50 or more, 55 or more, 60 or more, 65 or more 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more 190 or more, 200 or more, 220 or more, 230 or more, 240 or more 250 or more 260 or more, 270 or more, 280 or more, 290 or more or 300 or more amino acids in length.
  • the amino acids may be consecutive amino acids.
  • the antibody sequence based on or derived from at least a portion of the ultralong CDR3 can be 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, or 5 or fewer amino acids in length.
  • the antibody sequence based on or derived from at least a portion of the ultralong CDR3 may be 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more amino acids in length.
  • the amino acids may be consecutive amino acids. Alternatively, the amino acids are non-consecutive amino acids.
  • the antibody sequence based on or derived from at least a portion of the ultralong CDR3 can contain 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more nucleic acid modifications or alterations in the nucleotide sequence of the ultralong CDR3 from which it is based on or derived from.
  • the modifications and/or alterations may comprise substitutions, deletions, and/or insertions. Substitutions may comprise replacing one nucleic acid with another nucleic acid.
  • the nucleic acid may be a natural nucleic acid or a non-natural nucleic acid.
  • the antibody sequence based on or derived from at least a portion of the ultralong CDR3 can contain 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 50 or more, or 60 or more amino acid modifications or alterations in the peptide sequence of the ultralong CDR3 from which it is based on or derived from.
  • the modifications and/or alterations may comprise substitutions, deletions, and/or insertions.
  • substitutions may comprise replacing one amino acid with another amino acid.
  • the amino acids to be substituted may contain one or more similar features to the amino acid by which it is replaced. The features may include, but are not limited to, size, polarity, hydrophobicity, acidity, side chain, and bond formations.
  • the amino acid may be a natural amino acid or a non-natural amino acid.
  • the half-life of an antibody described herein is greater than the half-life of the un-conjugated therapeutic peptide or un-conjugated non-antibody peptide that is incorporated in the antibody. In some embodiments, the half-life of an antibody provided herein is greater than 4 hours when administered to a subject.
  • the half-life of an antibody provided herein is greater than 4 hours, greater than 6 hours, greater than 12 hours, greater than 24 hours, greater than 36 hours, greater than 2 days, greater than 3 days, greater than 4 days, greater than 5 days, greater than 6 days, greater than 7 days, greater than 8 days, greater than 9 days, greater than 10 days, greater than 11 days, greater than 12 days, greater than 13 days, or greater than 14 days when administered to a subject.
  • the subject is a mammal.
  • the subject is a mouse or a bovine.
  • the subject is a human.
  • a pharmaceutical composition comprising the antibody is administered to the subject once a day, every two days, every three days, every 4 days, every 7 days, every 10 days, every 14 days, every 21 days, every 28 days, every 2 months, or every three months.
  • the antibodies may be modified or altered to reduce immunogenicity.
  • the sequence of a partially bovine or non-bovine antibody may be modified or altered to reduce immunogenicity to humans.
  • a non-human antibody may be humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
  • a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences.
  • a humanized antibody optionally will also comprise at least a portion of a human constant region.
  • some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.
  • a non-human antibody e.g., the antibody from which the HVR residues are derived
  • the antibodies comprising an ultralong CDR3 as disclosed herein are preferably monoclonal. Also encompassed within the scope of the disclosure are Fab, Fab′, Fab′-SH and F(ab′) 2 fragments of the antibodies comprising an ultralong CDR3 as provided herein. These antibody fragments can be created by traditional means, such as enzymatic digestion, or may be generated by recombinant techniques. Such antibody fragments may be chimeric or humanized. These fragments are useful for the diagnostic and therapeutic purposes set forth below.
  • Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
  • the antibodies comprising an ultralong CDR3 as disclosed herein can be made using a hybridoma cell-based method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods.
  • Hybridoma cells can be generated by fusing B cells producing a desired antibody with an immortalized cell line, usually a myeloma cell line, so that the resulting fusion cells will be an immortalized cell line that secrets a particular antibody.
  • myeloma cells can be first transfected with a nucleic acid encoding a germline antibody V region and can be screened for the expression of the germline V region. Those myeloma cells with highest level of proteolytic light chain expression can be subsequently fused with B cells that produce an antibody with desired target protein specificity.
  • the fusion cells will produce two types of antibodies: one is a heterologous antibody containing an endogenous antibody chain (either heavy or light) operably joined to the recombinant germline V region (either heavy or light), and the other is the same antibody that the parental B cells would secrete (e.g. both endogenous heavy and light chains).
  • the operably joined heterologous heavy and light chains can be isolated by conventional methods such as chromatography and identification can be confirmed by target protein binding assays, assays identifying a unique tag of the germline polypeptide, or endopeptidase activity assays described in other sections of this disclosure. In some cases, where the heterologous antibody is the predominant type in quantity among the two types of antibodies, such isolation may not be needed.
  • the hybridoma cells may be seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • myeloma cell lines may be murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X 63 -Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA.
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
  • Culture medium in which hybridoma cells are growing is assayed for production of antibodies comprising an ultralong CDR3.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells may be determined by immunoprecipitation or by an in vitro binding assay, such as an enzyme-linked immunoadsorbent assay (ELISA).
  • ELISA enzyme-linked immunoadsorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the antibodies comprising an ultralong CDR3 as disclosed herein may be made by using combinatorial libraries to screen for synthetic antibody clones with the desired activity or activities.
  • synthetic antibody clones are selected by screening phage libraries containing phage that display various fragments of antibody variable regions (e.g., scFv or Fab) fused to phage coat protein.
  • phage libraries may be panned, for example, by affinity chromatography against the desired antigen.
  • Clones expressing antibody fragments capable of binding to the desired antigen may be adsorbed to the antigen and thus separated from the non-binding clones in the library.
  • the binding clones may then be eluted from the antigen, and can be further enriched by additional cycles of antigen adsorption/elution.
  • Any of the antibodies comprising an ultralong CDR3 as disclosed herein may be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length antibody comprising an ultralong CDR3 clone using the VH and VL (e.g., from scFv or Fab) sequences from the phage clone of interest and suitable constant region (Fc) sequences described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.
  • the antigen-binding domain of an antibody is formed from two variable (V) regions, one each from the light (VL) and heavy (VH) chains, that both present three hypervariable loops or complementarity-determining regions (CDRs).
  • Variable domains may be displayed functionally on phage, either as single-chain Fv (scFv, also referred to as single-chain antibody (SCA)) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).
  • scFv or SCA encoding phage clones and Fab encoding phage clones may be separately or collectively referred to as “Fv phage clones” or “Fv clones”.
  • Repertoires of VH and VL genes may be separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be searched for antigen-binding clones as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).
  • Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas.
  • the naive repertoire may be cloned to provide a single source of human antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J. 12: 725-734 (1993).
  • naive libraries can also be made synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
  • Filamentous phage is used to display antibody fragments by fusion to the minor coat protein pIII.
  • Protein pIII may include truncated forms of pIII.
  • the antibody fragments can be displayed as single chain Fv fragments, in which VH and VL domains are connected on the same polypeptide chain by a flexible polypeptide spacer, (e.g., as described by Marks et al., J. Mol.
  • Fab fragments in which one chain is fused to pIII (e.g., a truncated pIII) and the other is secreted into the bacterial host cell periplasm where assembly of a Fab-coat protein structure which becomes displayed on the phage surface by displacing some of the wild type coat proteins, (e.g., as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991)).
  • pIII e.g., a truncated pIII
  • Nucleic acid encoding antibody variable gene segments are recovered from the cells of interest and may be amplified or copies made by recombinant DNA techniques (e.g., Kunkel mutagenesis).
  • the desired DNA may be obtained by isolating genomic DNA or mRNA from lymphocytes followed by polymerase chain reaction (PCR) with primers matching the 5′ and 3′ ends of rearranged VH and VL genes as described in Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse V gene repertoires for expression.
  • the V genes may be amplified from cDNA and genomic DNA, with back primers at the 5′ end of the exon encoding the mature V-domain and forward primers based within the J-segment as described in Orlandi et al. (1989) and in Ward et al., Nature, 341: 544-546 (1989).
  • back primers can also be based in the leader exon as described in Jones et al., Biotechnol., 9: 88-89 (1991), and forward primers within the constant region as described in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732 (1989).
  • degeneracy may be incorporated in the primers as described in Orlandi et al. (1989) or Sastry et al. (1989).
  • Library diversity may be enhanced or maximized by using PCR primers targeted to each V-gene family in order to amplify available VH and VL arrangements present in the immune cell nucleic acid sample, for example, as described in the method of Marks et al., J. Mol. Biol., 222: 581-597 (1991) or as described in the method of Orum et al., Nucleic Acids Res., 21: 4491-4498 (1993).
  • rare restriction may be introduced within the PCR primer as a tag at one end as described in Orlandi et al. (1989), or by further PCR amplification with a tagged primer as described in Clackson et al., Nature, 352: 624-628 (1991).
  • Repertoires of synthetically rearranged V genes may be derived in vitro from V gene segments.
  • Most of the human VH-gene segments have been cloned and sequenced (e.g., reported in Tomlinson et al., J. Mol. Biol., 227: 776-798 (1992)), and mapped (e.g., reported in Matsuda et al., Nature Genet., 3: 88-94 (1993); these cloned segments (including all the major conformations of the H1 and H2 loop) may be used to generate diverse VH gene repertoires with PCR primers encoding H3 loops of diverse sequence and length as described in Hoogenboom and Winter, J. Mol.
  • VH repertoires may also be made with all the sequence diversity focused in a long H3 loop of a single length as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992).
  • Human V ⁇ and V ⁇ . segments have been cloned and sequenced (reported in Williams and Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used to make synthetic light chain repertoires.
  • Synthetic V gene repertoires based on a range of VH and VL folds, and L3 and H3 lengths, will encode antibodies of considerable structural diversity.
  • germline V-gene segments can be rearranged in vitro according to the methods of Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
  • Repertoires of antibody fragments may be constructed by combining VH and VL gene repertoires together in several ways. Each repertoire may be created in different vectors, and the vectors recombined in vitro, for example, as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo by combinatorial infection, for example, the loxP system described in Waterhouse et al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivo recombination approach exploits the two-chain nature of Fab fragments to overcome the limit on library size imposed by E. coli transformation efficiency.
  • Naive VH and VL repertoires are cloned separately, one into a phagemid and the other into a phage vector.
  • the two libraries are then combined by phage infection of phagemid-containing bacteria so that each cell contains a different combination and the library size is limited only by the number of cells present (about 10 12 clones).
  • Both vectors contain in vivo recombination signals so that the VH and VL genes are recombined onto a single replicon and are co-packaged into phage virions.
  • These large libraries may provide large numbers of diverse antibodies of good affinity (K d ⁇ 1 of about 10 ⁇ 8 M).
  • the repertoires may be cloned sequentially into the same vector, for example, as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or assembled together by PCR and then cloned, for example, as described in Clackson et al., Nature, 352: 624-628 (1991).
  • PCR assembly may also be used to join VH and VL DNAs with DNA encoding a flexible peptide spacer to form single chain Fv (scFv) repertoires.
  • in cell PCR assembly may be used to combine VH and VL genes within lymphocytes by PCR and then clone repertoires of linked genes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837 (1992).
  • the antibodies produced by naive libraries can be of moderate affinity (K d ⁇ 1 of about 10 6 to 10 7 M ⁇ 1 ), but affinity maturation may also be mimicked in vitro by constructing and reselecting from secondary libraries as described in Winter et al. (1994), supra.
  • mutation can be introduced at random in vitro by using error-prone polymerase (reported in Leung et al., Technique, 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol. Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl. Acad. Sci. USA, 89: 3576-3580 (1992).
  • affinity maturation may be performed by randomly mutating one or more CDRs, for example, using PCR with primers carrying random sequence spanning the CDR of interest, in selected individual Fv clones and screening for higher affinity clones.
  • WO 9607754 described a method for inducing mutagenesis in a complementarity determining region of an immunoglobulin light chain to create a library of light chain genes.
  • Another effective approach is to recombine the VH or VL domains selected by phage display with repertoires of naturally occurring V domain variants obtained from unimmunized donors and screen for higher affinity in several rounds of chain reshuffling as described in Marks et al., Biotechnol., 10: 779-783 (1992). This technique allows the production of antibodies and antibody fragments with affinities in the 10 ⁇ 9 M range.
  • the phage library samples are contacted with an immobilized protein under conditions suitable for binding of at least a portion of the phage particles with the adsorbent. Normally, the conditions, including pH, ionic strength, temperature and the like are selected to mimic physiological conditions.
  • the phages bound to the solid phase are washed and then eluted by acid, e.g., as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or by alkali, (e.g., as described in Marks et al., J. Mol.
  • Phages may be enriched 20-1,000-fold in a single round of selection. Moreover, the enriched phages may be grown in bacterial culture and subjected to further rounds of selection.
  • the efficiency of selection depends on many factors, including the kinetics of dissociation during washing, and whether multiple antibody fragments on a single phage can simultaneously engage with antigen.
  • Antibodies with fast dissociation kinetics (and weak binding affinities) may be retained by use of short washes, multivalent phage display and high coating density of antigen in solid phase. The high density not only stabilizes the phage through multivalent interactions, but favors rebinding of phage that has dissociated.
  • DNA encoding the hybridoma-derived monoclonal antibodies or phage display Fv clones disclosed herein is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions of interest from hybridoma or phage DNA template).
  • the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of the desired monoclonal antibodies in the recombinant host cells.
  • DNA encoding Fv clones as disclosed herein may be combined with known DNA sequences encoding heavy chain and/or light chain constant regions (e.g., the appropriate DNA sequences can be obtained from Kabat et al., supra) to form clones encoding full or partial length heavy and/or light chains.
  • constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions may be obtained from any human or animal species.
  • a Fv clone derived from the variable domain DNA of one animal (such as human) species and then fused to constant region DNA of another animal species to form coding sequence(s) for “hybrid”, full length heavy chain and/or light chain is included in the definition of “chimeric” and “hybrid” antibody as used herein.
  • a Fv clone derived from human variable DNA is fused to human constant region DNA to form coding sequence(s) for all human, full or partial length heavy and/or light chains.
  • DNA encoding an antibody comprising an ultralong CDR3 derived from a hybridoma disclosed herein may also be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of homologous murine sequences derived from the hybridoma clone (e.g., as in the method of Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).
  • DNA encoding a hybridoma or Fv clone-derived antibody or fragment can be further modified by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In this manner, “chimeric” or “hybrid” antibodies are prepared that have the binding specificity of the Fv clone or hybridoma clone-derived antibodies disclosed herein.
  • the present disclosure provides antibody genes and proteins including, for example, chimeric, recombinant, engineered, synthetic, hybrid, bovine, fully bovine, bovinized, human, fully human or humanized antibody genes or proteins that comprise an ultralong CDR3 sequence.
  • the antibodies disclosed herein may selectively or specifically bind to an epitope of a target protein.
  • the antibody may be an antagonist (e.g., blocking) antibody or an agonist antibody.
  • variable region of the heavy and light chains are encoded by multiple germline gene segments separated by non-coding regions, or introns, and often are present on different chromosomes.
  • the genes for the human immunoglobulin heavy chain region contains approximately 65 variable (VH) genes, 27 Diversity (DH) genes, and 6 Joining (JH) genes.
  • the human kappa ( ⁇ ) and lambda ( ⁇ ) light chains are also each encoded by a similar number of VL and JL gene segments, but do not include any D gene segments.
  • Exemplary VH, DH, JH and VL (V ⁇ or V ⁇ ) and JL (J ⁇ or J ⁇ ) germline gene segments are set forth in WO 2010/054007.
  • germline DNA is rearranged whereby one DH and one JH gene segment of the heavy chain locus are recombined, which is followed by the joining of one VH gene segment forming a rearranged VDJ gene that encodes a VH chain.
  • allelic exclusion is regulated by in-frame or “productive” recombination of the VDJ segments, which occurs in only about one-third of VDJ recombinations of the variable heavy chain.
  • ⁇ heavy chain that gets expressed on the surface of a pre-B cell and transmits a signal to shut off further heavy chain recombination, thereby preventing expression of the allelic heavy chain locus.
  • the surface-expressed ⁇ heavy chain also acts to activate the kappa ( ⁇ ) locus for rearrangement.
  • the lambda ( ⁇ ) locus is only activated for rearrangement if the K recombination is unproductive on both loci.
  • the light chain rearrangement events are similar to the heavy chain, except that only the VL and JL segments are recombined. Before primary transcription of each, the corresponding constant chain gene is added. Subsequent transcription and RNA splicing leads to mRNA that is translated into an intact light chain or heavy chain.
  • variable regions of antibodies confer antigen binding and specificity due to recombination events of individual germline V, D and J segments, whereby the resulting recombined nucleic acid sequences encoding the variable region domains differ among antibodies and confer antigen-specificity to a particular antibody.
  • the variation is limited to three complementarity determining regions (CDR1, CDR2, and CDR3) found within the N-terminal domain of the heavy (H) and (L) chain variable regions.
  • the CDRs are interspersed with regions that are more conserved, termed “framework regions” (FR).
  • FR framework regions
  • Each VH and VL is typically composed of three CDRs and four FRs arranged from the amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Sequence variability among VL and VH domains is generally limited to the CDRs, which are the regions that form the antigen binding site.
  • VH genes encode the N-terminal three framework regions, the first two complete CDRs and the first part of the third CDR), the DH gene encodes the central portion of the third CDR, and the JH gene encodes the last part of the third CDR and the fourth framework region.
  • VL genes encode the first CDR and second CDR.
  • the third CDR (CDRL3) is formed by the joining of the VL and JL gene segments. Hence, CDRs 1 and 2 are exclusively encoded by germline V gene segment sequences.
  • VH and VL chain CDR3s form the center of the Ag-binding site, with CDRs 1 and 2 form the outside boundaries; the FRs support the scaffold by orienting the H and L CDRs.
  • an antigen binding site typically requires at least four of the CDRs make contact with the antigen's epitope, with CDR3 of both the heavy and light chain being the most variable and contributing the most specificity to antigen binding (see, e.g., Janis Kuby, Immunology, Third Edition, New York, W.H. Freeman and Company, 1998, pp. 115-118).
  • CDRH3 which includes all of the D gene segment, is the most diverse component of the Ab-binding site, and typically plays a critical role in defining the specificity of the Ab. In addition to sequence variation, there is variation in the length of the CDRs between the heavy and light chains.
  • the constant regions are encoded by sequences that are more conserved among antibodies. These domains confer functional properties to antibodies, for example, the ability to interact with cells of the immune system and serum proteins in order to cause clearance of infectious agents. Different classes of antibodies, for example IgM, IgD, IgG, IgE and IgA, have different constant regions, allowing them to serve distinct effector functions.
  • V, D, and J can provide nearly 2 ⁇ 10 7 different antibodies with both high affinity and specificity. Additional diversity is introduced by nucleotide insertions and deletions in the joining segments and also by somatic hypermutation of V regions. The result is that there are approximately 10 10 antibodies present in an individual with differing antigen specificities.
  • the present disclosure encompasses antibody fragments.
  • antibody fragments include, but are not limited to, Fab, Fab′, Fab′-SH, F(ab′)2, Fv, Fv′, Fd, Fd′, scFv, hsFv fragments, and diabodies, and other fragments described below.
  • Diabodies are antibody fragments with two antigen binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129134 (2003).
  • Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
  • a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516).
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.
  • Fab′-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab′) 2 fragments (see, e.g., Carter et al., Bio/Technology 10: 163-167 (1992)).
  • F(ab′) 2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab′) 2 fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues (see, e.g., in U.S. Pat. No. 5,869,046).
  • Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.
  • the antibody of choice is a single chain Fv fragment (scFv or single chain antibody (SCA)).
  • scFv or single chain antibody (SCA) single chain Fv fragment
  • Fv and sFv are the only species with intact combining sites that are devoid of constant regions; thus, they are suitable for reduced nonspecific binding during in vivo use.
  • sFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an sFv. See Antibody Engineering, ed. Borrebaeck, Supra.
  • the antibody fragment may also be a “linear antibody”, for example, as described in U.S. Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.
  • Humanized antibodies comprising an ultralong CDR3.
  • Humanized antibodies may include human engineered antibodies (see, e.g., Studnicka et al. (1994) Protein Eng. 7(6) 805-814; and U.S. Pat. No. 5,766,886).
  • Various methods for humanizing non-human antibodies are known in the art.
  • a humanized antibody can have one or more amino acid residues introduced into it from a source which is human or non-human. Humanization may be performed following the method of Studnicka (see, e.g., Studnicka et al. (1994) Protein Eng. 7(6) 805-814; and U.S. Pat. No. 5,766,886), including the preparation of modified antibody variable domains.
  • Humanization may alternatively be performed following the method of Winter and co-workers (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), by substituting hypervariable region sequences for the corresponding sequences of a human antibody.
  • such “humanized” or “human engineered” antibodies are chimeric antibodies, including wherein substantially less than an intact human variable domain has been substituted by or incorporated into the corresponding sequence from a non-human species.
  • humanized antibodies may be human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • humanized or human engineered antibodies may be non-human (e.g, rodent) antibodies in which some residues are substituted by residues from analogious sites in human antibodies (see, e.g., Studnicka et al. (1994) Protein Eng. 7(6) 805-814; and U.S. Pat. No. 5,766,886).
  • variable domains both light and heavy
  • the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is important to reduce antigenicity.
  • sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences.
  • the human sequence which is closest to that of the rodent is then accepted as the human framework for the humanized antibody (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901).
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.
  • the same framework may be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623).
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
  • Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
  • FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
  • the hypervariable region residues are directly and most substantially involved in influencing antigen binding.
  • the humanized antibodies comprising an ultralong CDR3 may be deimmunized.
  • Methods of deimmunizing an antibody or protein are well known in the art.
  • the immunogenicity of therapeutic proteins such as antibodies is thought to result from the presence of T-cell epitopes which can bind MHC class II molecules and generate a proliferative and cytokine response in CD4+ helper T-cells. These CD4+ helper cells then collaborate with B-cells to generate an antibody response against the therapeutic protein. Removal of the T-cell epitopes are thought to be key steps in deimmunizing a recombinant protein. T-cell epitopes can be predicted by in silico algorithms that identify residues required for binding MHC.
  • epitopes can be identified directly by utilizing peripheral blood mononuclear cells from panels of human donors and measuring their response against the therapeutic protein when incubated with antigen presenting cells.
  • peripheral blood mononuclear cells from panels of human donors and measuring their response against the therapeutic protein when incubated with antigen presenting cells.
  • Such in silico and in vitro systems are well known in the art [Jones T D, Crompton L J, Can F J, Baker M P. Methods Mol Biol. 2009; 525:405-23, Deimmunization of monoclonal antibodies; and Baker M, and Jones T D. The identification and removal of immunogenicity in therapeutic proteins. Curr. Opin. Drug Discovery Dev. 2007; (2007); 10(2): 219-227].
  • the residues of the peptide can be mutated one by one and tested for T-cell activation until a mutation is found which disrupts MHC II binding and T-cell activation.
  • Such mutations when found in an individual peptide, can be encoded directly in the recombinant therapeutic protein. Incubation of the whole protein with antigen presenting cells will not induce a significant CD4+ response, indicating successful deimmunization.
  • bovine antibodies comprising an ultralong CDR3.
  • the bovine antibodies may be recombinant antibodies, engineered antibodies, synthetic antibodies, bovinized antibodies, or fully bovine antibodies.
  • Bovinized antibodies may include bovine engineered antibodies.
  • Methods for producing a bovinized antibody may comprise introducing one or more amino acid residues into it from a source which is a bovine.
  • methods for producing a bovinized antibody may comprise introducing one or more amino acid residues into it from a source which is a non-bovine.
  • Bovinization may be performed by preparing a modified antibody variable domains. Alternatively, bovinization may be performed by substituting hypervariable region sequences for the corresponding sequences of a bovine antibody.
  • Bovinized or bovine engineered antibodies are chimeric antibodies.
  • Chimeric antibodies may include antibodies wherein substantially less than an intact bovine variable domain has been substituted by or incorporated into the corresponding sequence from a non-bovine species.
  • Bovinized or bovine engineered antibodies may be bovine antibodies in which some hypervariable region residues and constant region residues are substituted by residues from analogous sites in non-bovine antibodies.
  • bovinized, bovine engineered or fully bovine antibodies may be non-bovine (e.g, human) antibodies in which some residues are substituted by residues from analogious sites in bovine antibodies.
  • a bovine immunoglobuline region can be used to replace a non-bovine (e.g., human, rodent) immunoglobulin region to produce a fully bovine, bovinized or bovine engineered antibody.
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. For example, one of the binding specificities may be for a first antigen and the other may be for any other antigen. Exemplary bispecific antibodies may bind to two different epitopes of the same protein. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular protein. These antibodies possess a binding arm specific for the particular protein and an arm which binds the cytotoxic agent (e.g., saporin, anti-interferon- ⁇ , vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies may be prepared as full length antibodies or antibody fragments (e.g., F(ab′) 2 bispecific antibodies).
  • cytotoxic agent e.g., saporin, anti-interferon- ⁇ , vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten
  • bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305: 537 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J., 10: 3655 (1991).
  • antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1), containing the site necessary for light chain binding, present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
  • the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm.
  • This asymmetric structure may facilitate the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation.
  • This approach is disclosed in WO 94/04690.
  • For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).
  • the interface between a pair of antibody molecules may can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the C H3 domain of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan).
  • Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies include cross-linked or “heteroconjugate” antibodies.
  • one of the antibodies in the heteroconjugate may be coupled to avidin, the other to biotin.
  • Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/00373, and EP 03089).
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
  • bispecific antibodies may be prepared using chemical linkage.
  • Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′) 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
  • the Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced may be used as agents for the selective immobilization of enzymes.
  • bispecific antibodies have been produced using leucine zippers. See, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
  • VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
  • sFv single-chain Fv
  • Antibodies with more than two valencies are contemplated.
  • trispecific antibodies can be prepared. See, e.g., Tutt et al. J. Immunol. 147: 60 (1991).
  • a multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind.
  • the antibodies of the present disclosure may be multivalent antibodies (which are other than of the IgM class) with three or more antigen binding sites (e.g., tetravalent antibodies), which may be produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody.
  • the multivalent antibody may comprise a dimerization domain and three or more antigen binding sites.
  • a preferred dimerization domain may comprise (or consist of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fe region.
  • a preferred multivalent antibody may comprise (or consist of) three to about eight, but preferably four, antigen binding sites.
  • the multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains.
  • the polypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1.
  • the polypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain.
  • a multivalent antibody may preferably further comprises at least two (and preferably four) light chain variable domain polypeptides.
  • a multivalent antibody may, for instance, comprise from about two to about eight light chain variable domain polypeptides.
  • the light chain variable domain polypeptides may comprise a light chain variable domain and, optionally, further comprise a CL domain.
  • amino acid sequence modification(s) of the antibodies comprising an ultralong CDR3 as described herein are contemplated.
  • Amino acid sequence variants including, for example, conservatively modified variants, of the antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, or by peptide synthesis.
  • Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics.
  • the amino acid alterations may be introduced in the subject antibody amino acid sequence at the time that sequence is made.
  • a useful method for identification of certain residues or regions of the antibody that are preferred locations for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085.
  • a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with antigen.
  • Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed immunoglobulins are screened for the desired activity.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide.
  • Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
  • N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
  • glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites).
  • the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).
  • the carbohydrate attached thereto may be altered.
  • antibodies with a mature carbohydrate structure that lacks fucose attached to an Fc region of the antibody have been described (see, e.g., US 2003/0157108, US 2004/0093621.
  • Antibodies with a bisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached to an Fc region of the antibody have been described (see, e.g., WO 2003/011878, and U.S. Pat. No. 6,602,684).
  • Antigen-binding molecules with modified glycosylation have been described (see, e.g., WO 99/54342, U.S. Pat. Nos. 6,602,684 and 7,517,670, and US 2004/0072290; see also, e.g., U.S. Pat. Nos. 7,214,775 and 7,682,610).
  • the preferred glycosylation variant herein comprises an Fc region, wherein a carbohydrate structure attached to the Fc region lacks fucose.
  • Such variants have improved ADCC function.
  • the Fc region further comprises one or more amino acid substitutions therein which further improve ADCC, for example, substitutions at positions 298, 333, and/or 334 of the Fc region (Eu numbering of residues).
  • Examples of publications related to “defucosylated” or “fucose-deficient” antibodies include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614 (now U.S. Pat. No. 6,946,292) US 2002/0164328 (now U.S. Pat. No.
  • variants are an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue.
  • the sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 2 under the heading of “preferred substitutions”. If such substitutions result in a change in biological activity, then more substantial changes, denominated “exemplary substitutions”, or as further described below in reference to amino acid classes, may be introduced and the products screened.
  • Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring amino acids are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: asp, glu; (4) basic: his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody).
  • a parent antibody e.g., a humanized or human antibody
  • the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated.
  • a convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site.
  • the antibodies thus generated are displayed from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed.
  • alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding.
  • Nucleic acid molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the antibody.
  • the Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions including that of a hinge cysteine.
  • a human Fc region sequence e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region
  • an amino acid modification e.g., a substitution
  • an antibody used in methods disclosed herein may comprise one or more alterations as compared to the wild type counterpart antibody, e.g., in the Fc region. These antibodies would nonetheless retain substantially the same characteristics required for therapeutic utility as compared to their wild type counterpart. For example, it is thought that certain alterations can be made in the Fc region that would result in altered (e.g., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in WO99/51642. See also Duncan & Winter Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No.
  • WO00/42072 and WO 2004/056312 describe antibody variants with improved or diminished binding to FcRs. See, also, Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001). Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005/0014934 (Hinton et al.).
  • These antibodies comprise an Fc reg on with one or more substitutions therein which improve binding of the Fc region to FcRn.
  • Polypeptide variants with altered Fc region amino acid sequences and increased or decreased Clq binding capability are described in U.S. Pat. No. 6,194,551, WO99/51642. See, also, Idusogie et al. J. Immunol. 164:4178-4184 (2000).
  • the present disclosure contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious.
  • In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks Fc ⁇ R binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • NK cells express Fc ⁇ RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu Rev. Immunol. 9:457-492 (1991).
  • Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'! Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc.
  • non-radioactive assays methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTecllrlology, Inc. Mountain View, Calif.; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).
  • Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.
  • a CDC assay may be performed (see, for example, Gazzano-Santoro et al., Immunol. Methods 202:163 (1996); Cragg, M. S.
  • FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int'l Immunol. 18(12):1759-1769 (2006)).
  • Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056).
  • Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).
  • an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).
  • alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. Immunol. 164: 41784184 (2000).
  • CDC Complement Dependent Cytotoxicity
  • Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus are described in US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn.
  • Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).
  • the antibodies comprising an ultralong CDR3 as disclosed herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available.
  • the moieties suitable for derivatization of the antibody are water soluble polymers.
  • water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g.
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody may vary, and if more than one polymers are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
  • nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression.
  • DNA encoding the antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • nucleic acid encoding an antibody comprising an ultralong CDR3, a variable region comprising an ultralong CDR3, or an ultralong CDR3, is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. Many vectors are available.
  • vector depends in part on the host cell to be used. Generally, preferred host cells are of either prokaryotic or eukaryotic (generally mammalian) origin. It will be appreciated that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species.
  • Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus.
  • eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the CMV promoter, SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase.
  • markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase.
  • high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a nucleic acid sequence encoding a partially human ultralong CDR3 antibody chain under the direction of the polyhedrin promoter or other strong baculovirus promoters.
  • Polynucleotide sequences encoding polypeptide components of the antibodies disclosed herein can be obtained using standard recombinant techniques. Desired polynucleotide sequences may be isolated and sequenced from antibody producing cells such as hybridoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. Many vectors that are available and known in the art can be used for the purpose of the present disclosure.
  • Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector.
  • Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides.
  • the vector components generally include, but are not limited to: an origin of replication, a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert and a transcription termination sequence.
  • V regions comprising an ultralong CDR3 may optionally be fused to a C-region to produce an antibody comprising constant regions.
  • plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts.
  • the vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells.
  • E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species.
  • pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides easy means for identifying transformed cells.
  • pBR322 its derivatives, or other microbial plasmids or bacteriophage may also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of endogenous proteins.
  • promoters which can be used by the microbial organism for expression of endogenous proteins. Examples of pBR322 derivatives used for expression of particular antibodies have been described (see, e.g., U.S. Pat. No. 5,648,237).
  • phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts.
  • bacteriophage such as ⁇ GEMTM-11 may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.
  • the expression vectors disclosed herein may comprise two or more promoter-cistron pairs, encoding each of the polypeptide components.
  • a promoter is an untranslated regulatory sequence located upstream (5′) to a cistron that modulates its expression.
  • Prokaryotic promoters typically fall into two classes, inducible and constitutive. Inducible promoter is a promoter that initiates increased levels of transcription of the cistron under its control in response to changes in the culture condition, e.g., the presence or absence of a nutrient or a change in temperature.
  • the selected promoter can be operably linked to cistron DNA encoding the light or heavy chain by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector disclosed herein.
  • Both the native promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of the target genes.
  • heterologous promoters are utilized, as they generally permit greater transcription and higher yields of expressed target gene as compared to the native target polypeptide promoter.
  • Promoters suitable for use with prokaryotic hosts include: an ara B promoter, a PhoA promoter, ⁇ -galactamase and lactose promoter systems, a tryptophan (trp) promoter system and hybrid promoters such as the tac or the trc promoter.
  • trp tryptophan
  • Other promoters that are functional in bacteria are suitable as well.
  • Their nucleotide sequences have been published, thereby enabling a skilled worker operably to ligate them to cistrons encoding the target light and heavy chains (e.g., Siebenlist et al. (1980) Cell 20: 269) using linkers or adaptors to supply any required restriction sites.
  • Suitable bacterial promoters are well known in the art and fully described in scientific literature such as Sambrook and Russell, supra, and Ausubel et al, supra.
  • Bacterial expression systems for expressing antibody chains of the recombinant catalytic polypeptide are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene, 22:229-235 (1983); Mosbach et al., Nature, 302:543-545 (1983)).
  • each cistron within the recombinant vector comprises a secretion signal sequence component that directs translocation of the expressed polypeptides across a membrane.
  • the signal sequence may be a component of the vector, or it may be a part of the target polypeptide DNA that is inserted into the vector.
  • the signal sequence should be one that is recognized and processed (e.g., cleaved by a signal peptidase) by the host cell.
  • the signal sequence is substituted by a prokaryotic signal sequence selected, for example PelB, OmpA, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, and MBP.
  • a prokaryotic signal sequence selected, for example PelB, OmpA, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, and MBP.
  • the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.
  • the production of the immunoglobulins according to the disclosure can occur in the cytoplasm of the host cell, and therefore does not require the presence of secretion signal sequences within each cistron.
  • immunoglobulin light and heavy chains are expressed, folded and assembled to form functional immunoglobulins within the cytoplasm.
  • Certain host strains e.g., the E. coli trxB-strains
  • Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.
  • the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell, Human Embryonic Kidney (HEK) cell or lymphoid cell (e.g., YO, NSO, Sp20 cell).
  • CHO Chinese Hamster Ovary
  • HEK Human Embryonic Kidney
  • lymphoid cell e.g., YO, NSO, Sp20 cell.
  • antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed.
  • For expression of antibody fragments and polypeptides in bacteria see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol.
  • the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gemgross, Nat. Biotech.
  • Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the above-mentioned bacteria having defined genotypes are known in the art and described in, for example, Bass et al., Proteins, 8:309-314 (1990).
  • E. coli, Serratia , or Salmonella species can be suitably used as the host when well known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon.
  • plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon.
  • the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may desirably be incorporated in the cell culture.
  • Plant cell cultures can also be utilized as hosts. See, e.g. U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125, 978, and 6,417,429 (describing PLANTIBODIESTM technology for producing antibodies in transgenic plants). Vertebrate cells may also be used as hosts.
  • mammalian cell lines that are adapted to grow in suspension may be useful.
  • Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., Gen VII′01.
  • TM4 cells baby hamster kidney cells
  • CV1 African green monkey kidney cells
  • HELA human cervical carcinoma cells
  • canine kidney cells MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TR1 cells, as described, e.g., in Mather et al., Annals NI'. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells.
  • CHO Chinese hamster ovary
  • DHFR′ CHO cells Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)
  • myeloma cell lines such as YO, NSO and Sp2/0.
  • Yazaki and Wu Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.].), pp. 255-268 (2003).
  • a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody.
  • nucleic acid encoding an antibody comprising an ultralong CDR3 is inserted into one or more expression vectors for further cloning and/or expression in a host cell.
  • Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • Host cells are transformed with such expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • Transformation means introducing DNA into the prokaryotic host so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant.
  • transformation is done using standard techniques appropriate to such cells.
  • the calcium treatment employing calcium chloride is generally used for bacterial cells that contain substantial cell-wall barriers.
  • Another method for transformation employs polyethylene glycol/DMSO.
  • Yet another technique used is electroporation.
  • Prokaryotic cells used to produce the polypeptides disclosed herein are grown in media known in the art and suitable for culture of the selected host cells.
  • suitable media include luria broth (LB) plus necessary nutrient supplements.
  • the media also contains a selection agent, chosen based on the construction of the expression vector, to selectively permit growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to media for growth of cells expressing ampicillin resistant gene.
  • any necessary supplements besides carbon, nitrogen, and inorganic phosphate sources may also be included at appropriate concentrations introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source.
  • the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, dithioerythritol and dithiothreitol.
  • the prokaryotic host cells are cultured at suitable temperatures.
  • the preferred temperature ranges from about 20° C. to about 39° C., more preferably from about 25° C. to about 37° C., even more preferably at about 30° C.
  • the pH of the medium may be any pH ranging from about 5 to about 9, depending mainly on the host organism.
  • the pH is preferably from about 6.8 to about 7.4, and more preferably about 7.0.
  • an inducible promoter is used in the expression vector disclosed herein, protein expression is induced under conditions suitable for the activation of the promoter.
  • an ara B or phoA promoter may be used for controlling transcription of the polypeptides.
  • a variety of inducers may be used, according to the vector construct employed, as is known in the art.
  • the expressed polypeptides of the present disclosure are secreted into and recovered from the periplasm of the host cells or transported into the culture media. Protein recovery from the periplasm typically involves disrupting the microorganism, generally by such means as osmotic shock, sonication or lysis. Once cells are disrupted, cell debris or whole cells may be removed by centrifugation or filtration. The proteins may be further purified, for example, by affinity resin chromatography. Alternatively, proteins that are transported into the culture media may be isolated therein. Cells may be removed from the culture and the culture supernatant being filtered and concentrated for further purification of the proteins produced. The expressed polypeptides can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.
  • PAGE polyacrylamide gel electrophoresis
  • Antibody production may be conducted in large quantity by a fermentation process.
  • Various large-scale fed-batch fermentation procedures are available for production of recombinant proteins.
  • Large-scale fermentations have at least 1000 liters of capacity, preferably about 1,000 to 100,000 liters of capacity. These fermentors use agitator impellers to distribute oxygen and nutrients, especially glucose (a preferred carbon/energy source).
  • Small scale fermentation refers generally to fermentation in a fermentor that is no more than approximately 100 liters in volumetric capacity, and can range from about 1 liter to about 100 liters.
  • induction of protein expression is typically initiated after the cells have been grown under suitable conditions to a desired density, e.g., an OD550 of about 180-220, at which stage the cells are in the early stationary phase.
  • a desired density e.g., an OD550 of about 180-220
  • inducers may be used, according to the vector construct employed, as is known in the art and described above. Cells may be grown for shorter periods prior to induction. Cells are usually induced for about 12-50 hours, although longer or shorter induction time may be used.
  • various fermentation conditions can be modified.
  • additional vectors overexpressing chaperone proteins such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl cis, trans-isomerase with chaperone activity) may be used to co-transform the host prokaryotic cells.
  • the chaperone proteins have been demonstrated to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host cells. (see e.g., Chen et al.
  • host strains deficient for proteolytic enzymes can be used for the present disclosure.
  • host cell strains may be modified to effect genetic mutation(s) in the genes encoding known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations thereof.
  • E. coli protease-deficient strains are available (see, e.g., Joly et al. (1998), supra; U.S. Pat. No. 5,264,365; U.S. Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996)).
  • E. coli strains deficient for proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins may be used as host cells in the expression systems disclosed herein.
  • Standard protein purification methods known in the art can be employed.
  • the following procedures are exemplary of suitable purification procedures: fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.
  • Protein A immobilized on a solid phase is used for immunoaffinity purification of the full length antibody products disclosed herein.
  • Protein A is a 41 kD cell wall protein from Staphylococcus aureas which binds with a high affinity to the Fc region of antibodies (see, e.g., Lindmark et al (1983) J. Immunol. Meth. 62:1-13).
  • the solid phase to which Protein A is immobilized is preferably a column comprising a glass or silica surface, more preferably a controlled pore glass column or a silicic acid column. In some applications, the column has been coated with a reagent, such as glycerol, in an attempt to prevent nonspecific adherence of contaminants.
  • the preparation derived from the cell culture as described above is applied onto the Protein A immobilized solid phase to allow specific binding of the antibody of interest to Protein A.
  • the solid phase is then washed to remove contaminants non-specifically bound to the solid phase.
  • the antibody of interest is recovered from the solid phase by elution.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • a vector for use in a eukaryotic host cell may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest.
  • the heterologous signal sequence selected preferably is one that is recognized and processed (e.g., cleaved by a signal peptidase) by the host cell.
  • mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal are available.
  • the DNA for such precursor region is ligated in reading frame to DNA encoding the antibody.
  • an origin of replication component is not needed for mammalian expression vectors.
  • the SV40 origin may be used only because it contains the early promoter.
  • Selection genes may contain a selection gene, also termed a selectable marker.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, where relevant, or (c) supply critical nutrients not available from complex media.
  • One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.
  • Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.
  • cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR.
  • Mtx methotrexate
  • An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).
  • host cells transformed or co-transformed with DNA sequences encoding an antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3′-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.
  • APH aminoglycoside 3′-phosphotransferase
  • Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the antibody polypeptide nucleic acid.
  • Promoter sequences are known for eukaryotes.
  • Virtually alleukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated.
  • Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide.
  • N may be any nucleotide.
  • AATAAA sequence At the 3′ end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3′ end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.
  • Antibody polypeptide transcription from vectors in mammalian host cells is controlled, for example, by 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 and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from hetero
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment.
  • a system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978.
  • the Rous Sarcoma Virus long terminal repeat can be used as the promoter.
  • Enhancer sequences are now known from mammalian genes (globin, elastase, albumin, ⁇ -fetoprotein, and insulin).
  • An enhancer from a eukaryotic cell virus may also be used. Examples include the 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, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elements for activation of eukaryotic promoters.
  • the enhancer may be spliced into the vector at a position 5′ or 3′ to the antibody polypeptide-encoding sequence, but is preferably located at a site 5′ from the promoter.
  • Expression vectors used in eukaryotic host cells will typically also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5′ and, occasionally 3′, untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding an antibody.
  • One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and the expression vector disclosed therein.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein include higher eukaryote cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/ ⁇ DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.
  • COS-7 monkey kidney CV1 line transformed by SV40
  • human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)
  • baby hamster kidney cells
  • mice sertoli cells TM4, Mather, Biol. Reprod. 23:243-251 (1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into a host cell (see, e.g., Sambrook and Russell, supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least both genes into the host cell capable of expressing germline antibody polypeptide.
  • Host cells are transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the host cells used to produce an antibody of this disclosure may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCINTM drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the antibody can be produced intracellularly, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon® ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • the antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique.
  • affinity chromatography is the preferred purification technique.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.
  • Protein A can be used to purify antibodies that are based on human ⁇ 1, ⁇ 2, or ⁇ 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)).
  • Protein G is recommended for all mouse isotypes and for human ⁇ 3 (Guss et al., EMBO J. 5:15671575 (1986)).
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABXTM resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
  • Soluble forms of antibody or fragment present either in the cytoplasm or released from the periplasmic space may be further purified using methods known in the art, for example Fab fragments are released from the bacterial periplasmic space by osmotic shock techniques.
  • inclusion bodies comprising an antibody or fragment have formed, they can often bind to the inner and/or outer cellular membranes and thus will be found primarily in the pellet material after centrifugation.
  • the pellet material can then be treated at pH extremes or with chaotropic agent such as a detergent, guanidine, guanidine derivatives, urea, or urea derivatives in the presence of a reducing agent such as dithiothreitol at alkaline pH or tris carboxyethyl phosphine at acid pH to release, break apart, and solubilize the inclusion bodies.
  • a reducing agent such as dithiothreitol at alkaline pH or tris carboxyethyl phosphine at acid pH to release, break apart, and solubilize the inclusion bodies.
  • the soluble antibody or fragment can then be analyzed using gel electrophoresis, immunoprecipitation or the like. If it is desired to isolate a solublized antibody or antigen binding fragment isolation may be accomplished
  • the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25 M salt).
  • an antibody or fragment may not be biologically active upon isolation.
  • Various methods for “refolding” or converting a polypeptide to its tertiary structure and generating disulfide linkages can be used to restore biological activity. Such methods include exposing the solubilized polypeptide to a pH usually above 7 and in the presence of a particular concentration of a chaotrope. The selection of chaotrope is very similar to the choices used for inclusion body solubilization, but usually the chaotrope is used at a lower concentration and is not necessarily the same as chaotropes used for the solubilization.
  • the refolding/oxidation solution will also contain a reducing agent or the reducing agent plus its oxidized form in a specific ratio to generate a particular redox potential allowing for disulfide shuffling to occur in the formation of the protein's cysteine bridge(s).
  • Some of the commonly used redox couples include cysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride, dithiothreitol(DTT)/dithiane DTT, and 2-mercaptoethanol(bME)/di-thio-b(ME).
  • GSH glutathione
  • DTT dithiothreitol
  • bME 2-mercaptoethanol
  • a cosolvent may be used to increase the efficiency of the refolding, and common reagents used for this purpose include glycerol, polyethylene glycol of various molecular weights, arginine and the like.
  • immunoconjugates comprising any of the antibodies comprising an ultralong CDR3 as described herein conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (e.g., a radioconjugate).
  • the immunoconjugate comprises any of the antibodies comprising an ultralong CDR3 as described herein conjugated to a peptide.
  • the peptide may be a non-antibody peptide, therapeutic polypeptide, cytokine, hormone or growth factor.
  • the peptide may be encoded by a non-antibody sequence.
  • the antibody-drug conjugates may be used for the local delivery of cytotoxic or cytostatic agents.
  • drugs to kill or inhibit tumor cells in the treatment of cancer Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278) allows targeted delivery of the drug moiety to tumors, and intracellular accumulation therein, where systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (Baldwin et al., (1986) Lancet pp. (Mar.
  • Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem. Letters 10: 1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res.
  • bacterial toxins such as diphtheria toxin
  • plant toxins such as ricin
  • small molecule toxins such as geldanamycin
  • maytansinoids EP 1391213; Liu et al., (1996)
  • the toxins may effect their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.
  • ZEVALIN® is an antibody-radioisotope conjugate composed of a murine IgG1 kappa monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes and 111 In or 90 Y radioisotope bound by a thiourea linker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al (2002) J. Clin. Oncol.
  • ZEVALIN has activity against B-cell non-Hodgkin's Lymphoma (NHL), administration results in severe and prolonged cytopenias in most patients.
  • MYLOTARGTM (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate composed of a hu CD33 antibody linked to calicheamicin, was approved in 2000 for the treatment of acute myeloid leukemia by injection (Drugs of the Future (2000) 25(7):686; U.S. Pat. Nos.
  • Cantuzumab mertansine an antibody drug conjugate composed of the huC242 antibody linked via the disulfide linker SPP to the maytansinoid drug moiety, DM1
  • CanAg such as colon, pancreatic, gastric, and others.
  • MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), an antibody drug conjugate composed of the anti-prostate specific membrane antigen (PSMA) monoclonal antibody linked to the maytansinoid drug moiety, DM1, is under development for the potential treatment of prostate tumors.
  • PSMA anti-prostate specific membrane antigen
  • auristatin peptides auristatin E (AE) and monomethylauristatin (MMAE), synthetic analogs of dolastatin, were conjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Y on carcinomas) and cAC10 (specific to CD30 on hematological malignancies) (Doronina et al (2003) Nature Biotechnology 21(7):778-784) and are under therapeutic development.
  • AE auristatin E
  • MMAE monomethylauristatin
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa ), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 131 I, 131 In, 90 Y, and 186 Re.
  • Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-succinimidyl-3
  • a ricin immunotoxin may be prepared as described in Vitetta et al., Science, 238: 1098 (1987).
  • Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
  • Conjugates of an antibody and one or more small molecule toxins such as a calicheamicin, maytansinoids, dolastatins, aurostatins, a trichothecene, and CC 1065, and the derivatives of these toxins that have toxin activity, are also contemplated herein.
  • the immunoconjugate comprises an antibody (full length or fragments) comprising an ultralong CDR3 as disclosed herein conjugated to one or more maytansinoid molecules.
  • Maytansinoids are mitototic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in U.S. Pat. Nos.
  • Maytansinoid drug moieties are attractive drug moieties in antibody drug conjugates because they are: (i) relatively accessible to prepare by fermentation or chemical modification, derivatization of fermentation products, (ii) amenable to derivatization with functional groups suitable for conjugation through the non-disulfide linkers to antibodies, (iii) stable in plasma, and (iv) effective against a variety of tumor cell lines.
  • Immunoconjugates containing maytansinoids, methods of making same, and their therapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020, 5,416,064 and EP 0 425 235. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprising a maytansinoid designated DM1 linked to the monoclonal antibody C242 directed against human colorectal cancer. The conjugate was found to be highly cytotoxic towards cultured colon cancer cells, and showed antitumor activity in an in vivo tumor growth assay.
  • the drug conjugate achieved a degree of cytotoxicity similar to the free maytansinoid drug, which could be increased by increasing the number of maytansinoid molecules per antibody molecule.
  • the A7-maytansinoid conjugate showed low systemic cytotoxicity in mice.
  • Antibody-maytansinoid conjugates are prepared by chemically linking an antibody to a maytansinoid molecule without significantly diminishing the biological activity of either the antibody or the maytansinoid molecule. See, e.g., U.S. Pat. No. 5,208,020. An average of 3-4 maytansinoid molecules conjugated per antibody molecule has shown efficacy in enhancing cytotoxicity of target cells without negatively affecting the function or solubility of the antibody, although even one molecule of toxin/antibody would be expected to enhance cytotoxicity over the use of naked antibody. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in U.S.
  • Preferred maytansinoids are maytansinol and maytansinol analogues modified in the aromatic ring or at other positions of the maytansinol molecule, such as various maytansinol esters.
  • linking groups known in the art for making antibody-maytansinoid conjugates, including, for example, those disclosed in U.S. Pat. Nos. 5,208,020, 6,441,163, or EP Patent 0 425 235, Chari et al., Cancer Research 52:127-131 (1992).
  • Antibody-maytansinoid conjugates comprising the linker component SMCC may be prepared.
  • the linking groups include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents, disulfide and thioether groups being preferred. Additional linking groups are described and exemplified herein.
  • Conjugates of the antibody and maytansinoid may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such
  • Particularly preferred coupling agents include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 (1978)) and N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a disulfide linkage.
  • SPDP N-succinimidyl-3-(2-pyridyldithio)propionate
  • SPP N-succinimidyl-4-(2-pyridylthio)pentanoate
  • the linker may be attached to the maytansinoid molecule at various positions, depending on the type of the link.
  • an ester linkage may be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction may occur at the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group.
  • the linkage is formed at the C-3 position of maytansinol or a maytansinol analogue.
  • the immunoconjugate comprises an antibody disclosed herein conjugated to dolastatins or dolostatin peptidic analogs and derivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).
  • Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965).
  • the dolastatin or auristatin drug moiety may be attached to the antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO 02/088172).
  • Exemplary auristatin embodiments include the N-terminus linked monomethylauristatin drug moieties DE and DF, (see, e.g., U.S. Pat. No. 7,498,298).
  • peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments.
  • Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method (see, e.g., E. Schroder and K. Lubke, “The Peptides”, volume 1, pp 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry.
  • the auristatin/dolastatin drug moieties may be prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc.
  • the immunoconjugate comprises an antibody disclosed herein conjugated to one or more calicheamicin molecules.
  • the calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations.
  • For the preparation of conjugates of the calicheamicin family see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296.
  • Structural analogues of calicheamicin which may be used include, but are not limited to, ⁇ 1 I , ⁇ 2 I , ⁇ 3 I , N-acetyl- ⁇ 1 I , PSAG and ⁇ I 1 (see, e.g., Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patents).
  • Another anti-tumor drug that the antibody can be conjugated is QFA which is an antifolate.
  • QFA is an antifolate.
  • Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody mediated internalization greatly enhances their cytotoxic effects.
  • antitumor agents that can be conjugated to the antibodies disclosed herein include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LL-E33288 complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296).
  • Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa ), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232 published Oct. 28, 1993.
  • the present disclosure further contemplates an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).
  • a compound with nucleolytic activity e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase.
  • the antibody may comprise a highly radioactive atom.
  • radioactive isotopes are available for the production of radioconjugated antibodies. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu.
  • the conjugate When used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc 99m or I 123 , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
  • NMR nuclear magnetic resonance
  • the radiolabels or other labels may be incorporated in the conjugate in known ways.
  • the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen.
  • Labels such as tc 99m or I 123 , Re 186 , Re 188 and In 111 can be attached via a cysteine residue in the peptide.
  • Yttrium-90 can be attached via a lysine residue.
  • the IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989) describes other methods.
  • Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987).
  • Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
  • the linker may be a “cleavable linker” facilitating release of the cytotoxic drug in the cell.
  • an acid-labile linker for example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020) may be used.
  • ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A). See pages 467-498, 2003-2004 Applications Handbook and Catalog.
  • an antibody (Ab) is conjugated to one or more drug moieties (D), e.g., about 1 to about 20 drug moieties per antibody, through a linker (L).
  • An ADC of Formula I [Ab-(L-D) p ] may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent, to form Ab-L, via a covalent bond, followed by reaction with a drug moiety D; and (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form D-L, via a covalent bond, followed by reaction with the nucleophilic group of an antibody. Additional methods for preparing ADC are described herein.
  • the linker may be composed of one or more linker components.
  • exemplary linker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl 4-(2-pyridylthio)pentanoate (“SPP”), N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”), and N-Succinimidyl (4-iodo-acetyl)aminobenzoate (“SIAB”). Additional linker components are known in the art and some are disclosed herein (see, e.g., U.S. Pat. No. 7,498,298).
  • the linker may comprise amino acid residues.
  • Exemplary amino acid linker components include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
  • Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe).
  • Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly).
  • Amino acid residues which comprise an amino acid linker component include those occurring naturally, as well as minor amino acids and non-naturally occurring amino acids including analogs, such as citrulline.
  • Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzymes, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.
  • Nucleophilic groups on antibodies include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups, e.g., lysine, (iii) side chain thiol groups, e.g., cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated.
  • Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, e.g., cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol).
  • a reducing agent such as DTT (dithiothreitol).
  • Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles.
  • Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol.
  • Reactive thiol groups may be introduced into the antibody (or fragment thereof) by introducing one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or more non-native cysteine amino acid residues).
  • Antibody drug conjugates disclosed herein may also be produced by modification of the antibody to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug.
  • the sugars of glycosylated antibodies may be oxidized, e.g., with periodate oxidizing reagents, to form aldehyde or ketone groups which may react with the amine group of linker reagents or drug moieties.
  • the resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g., by borohydride reagents to form stable amine linkages.
  • reaction of the carbohydrate portion of a glycosylated antibody with either glactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques).
  • proteins containing N-terminal serine or threonine residues can react with sodium meta-periodate, resulting in production of an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852).
  • Such aldehyde can be reacted with a drug moiety or linker nucleophile.
  • nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.
  • a fusion protein comprising the antibody and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis.
  • the length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.
  • the antibody may be conjugated to a “receptor” (such streptavidin) for utilization in tumor pre-targeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a radionucleotide).
  • a receptor such streptavidin
  • a ligand e.g., avidin
  • cytotoxic agent e.g., a radionucleotide
  • Hybridoma cells can be generated by fusing B cells producing a desired antibody with an immortalized cell line, usually a myeloma cell line, so that the resulting fusion cells will be an immortalized cell line that secrets a particular antibody.
  • myeloma cells can be first transfected with a nucleic acid encoding a germline antibody V region and can be screened for the expression of the germline V region. Those myeloma cells with highest level of proteolytic light chain expression can be subsequently fused with B cells that produce an antibody with desired target protein specificity.
  • the fusion cells will produce two types of antibodies: one is a heterologous antibody containing an endogenous antibody chain (either heavy or light) operably joined to the recombinant germline V region (either heavy or light), and the other is the same antibody that the parental B cells would secrete (e.g. both endogenous heavy and light chains).
  • the operably joined heterologous heavy and light chains can be isolated by conventional methods such as chromatography and identification can be confirmed by target protein binding assays, assays identifying a unique tag of the germline polypeptide, or endopeptidase activity assays described in other sections of this disclosure. In some cases, where the heterologous antibody is the predominant type in quantity among the two types of antibodies, such isolation may not be needed.
  • Hybridomas. Including bovine hybridomas may be a source of bovine antibody gene sequences, including ultralong CDR3 sequences.
  • a nucleic acid sequence encoding a germline antibody polypeptide of the present disclosure can be introduced into a non-human mammal to generate a transgenic animal that expresses the germline antibody polypeptide.
  • the transgene expressed by the transgenic mammals of the present disclosure need not replace at least one allele of the endogenous coding sequence responsible for the variable regions of antibody chains following somatic recombination. Due to allelic exclusion, the presence of an exogenous, post-somatic rearrangement version of the germline V region DNA will inhibit the endogenous alleles of pre-somatic rearrangement V minigenes from undergoing somatic rearrangement and contributing to the makeup of antibody chains this mammal may produce.
  • heterologous antibodies comprising one endogenously rearranged antibody chain, and one transgenic gene which was rearranged a priori.
  • heterologous antibodies are invaluable in research and in treating certain conditions in live subjects.
  • a method that directs the integration of the transgene to the locus of an endogenous allele will fully serve the purpose of practicing the present disclosure as well.
  • transgenic animal of the present disclosure is mouse, whereas a number of other transgenic animals can also be produced using the same general method. These animals include, but are not limited to: rabbits, sheep, cattle, and pigs (Jaenisch Science 240:1468-1474 (1988); Hammer et al., J. Animal. Sci. 63:269 (1986); Hammer et al. Nature 315:680 (1985); Wagner et al., Theriogenology 21:29 (1984)).
  • Antibodies comprising an ultralong CDR3, antibody fragments, nucleic acids, or vectors disclosed herein can be formulated in compositions, especially pharmaceutical compositions.
  • Such compositions with antibodies comprising an ultralong CDR3 comprise a therapeutically or prophylactically effective amount of antibodies comprising an ultralong CDR3, antibody fragment, nucleic acid, or vector disclosed herein in admixture with a suitable carrier, e.g., a pharmaceutically acceptable agent.
  • a suitable carrier e.g., a pharmaceutically acceptable agent.
  • antibodies comprising an ultralong CDR3, antibody fragments, nucleic acids, or vectors disclosed herein are sufficiently purified for administration before formulation in a pharmaceutical composition.
  • compositions for use in the present pharmaceutical compositions include carriers, excipients, diluents, antioxidants, preservatives, coloring, flavoring and diluting agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, and surfactants.
  • Neutral buffered saline or saline mixed with serum albumin are exemplary appropriate carriers.
  • the pharmaceutical compositions may include antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, pluronics, or polyethylene glycol (PEG).
  • antioxidants such as ascorbic acid
  • low molecular weight polypeptides such as serum albumin, gelatin, or immunoglobulins
  • hydrophilic polymers such as polyviny
  • suitable tonicity enhancing agents include alkali metal halides (preferably sodium or potassium chloride), mannitol, sorbitol, and the like.
  • Suitable preservatives include benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid and the like. Hydrogen peroxide also may be used as preservative.
  • Suitable cosolvents include glycerin, propylene glycol, and PEG.
  • Suitable complexing agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxy-propyl-beta-cyclodextrin.
  • Suitable surfactants or wetting agents include sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapal, and the like.
  • the buffers may be conventional buffers such as acetate, borate, citrate, phosphate, bicarbonate, or Tris-HCl.
  • Acetate buffer may be about pH 4-5.5, and Tris buffer can be about pH 7-8.5. Additional pharmaceutical agents are set forth in Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company, 1990.
  • the composition may be in liquid form or in a lyophilized or freeze-dried form and may include one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and/or bulking agents (see, for example, U.S. Pat. Nos. 6,685,940, 6,566,329, and 6,372,716).
  • a lyoprotectant is included, which is a non-reducing sugar such as sucrose, lactose or trehalose.
  • the amount of lyoprotectant generally included is such that, upon reconstitution, the resulting formulation will be isotonic, although hypertonic or slightly hypotonic formulations also may be suitable.
  • lyoprotectant concentrations for sugars e.g., sucrose, lactose, trehalose
  • sugars e.g., sucrose, lactose, trehalose
  • concentrations for sugars in the pre-lyophilized formulation are from about 10 mM to about 400 mM.
  • a surfactant is included, such as for example, nonionic surfactants and ionic surfactants such as polysorbates (e.g., polysorbate 20, polysorbate 80); poloxamers (e.g., poloxamer 188); poly(ethylene glycol) phenyl ethers (e.g., Triton); sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, and surfact
  • surfactant that may be present in the pre-lyophilized formulation are from about 0.001-0.5%.
  • High molecular weight structural additives may include for example, acacia, albumin, alginic acid, calcium phosphate (dibasic), cellulose, carboxymethylcellulose, carboxymethylcellulose sodium, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, dextran, dextrin, dextrates, sucrose, tylose, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose, disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressible sugar, magnesium aluminum silicate, maltodextrin, polyethylene oxide, polymethacrylates, povidone, sodium alginate, tragacanth microcrystalline cellulose, starch, and
  • compositions may be suitable for parenteral administration.
  • Exemplary compositions are suitable for injection or infusion into an animal by any route available to the skilled worker, such as intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, or intralesional routes.
  • a parenteral formulation typically will be a sterile, pyrogen-free, isotonic aqueous solution, optionally containing pharmaceutically acceptable preservatives.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringers' dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like.
  • Preservatives and other additives may also be present, such as, for example, anti-microbials, anti-oxidants, chelating agents, inert gases and the like. See generally, Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980.
  • compositions described herein may be formulated for controlled or sustained delivery in a manner that provides local concentration of the product (e.g., bolus, depot effect) and/or increased stability or half-life in a particular local environment.
  • the compositions can include the formulation of antibodies comprising an ultralong CDR3, antibody fragments, nucleic acids, or vectors disclosed herein with particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., as well as agents such as a biodegradable matrix, injectable microspheres, microcapsular particles, microcapsules, bioerodible particles beads, liposomes, and implantable delivery devices that provide for the controlled or sustained release of the active agent which then can be delivered as a depot injection.
  • Such sustained- or controlled-delivery means are known and a variety of polymers have been developed and used for the controlled release and delivery of drugs.
  • Such polymers are typically biodegradable and biocompatible.
  • Polymer hydrogels including those formed by complexation of enantiomeric polymer or polypeptide segments, and hydrogels with temperature or pH sensitive properties, may be desirable for providing drug depot effect because of the mild and aqueous conditions involved in trapping bioactive protein agents (e.g., antibodies comprising an ultralong CDR3). See, for example, the description of controlled release porous polymeric microparticles for the delivery of pharmaceutical compositions in WO 93/15722.
  • Suitable materials for this purpose include polylactides (see, e.g., U.S. Pat. No. 3,773,919), polymers of poly-(a-hydroxycarboxylic acids), such as poly-D-( ⁇ )-3-hydroxybutyric acid (EP 133,988A), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers, 22: 547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981), and Langer, Chem.
  • polylactides see, e.g., U.S. Pat. No. 3,773,919
  • polymers of poly-(a-hydroxycarboxylic acids) such as poly-D-( ⁇ )-3-hydroxybutyric acid (EP 133,988A)
  • biodegradable polymers include poly(lactones), poly(acetals), poly(orthoesters), and poly(orthocarbonates).
  • Sustained-release compositions also may include liposomes, which can be prepared by any of several methods known in the art (see, e.g., Eppstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-92 (1985)).
  • the carrier itself, or its degradation products, should be nontoxic in the target tissue and should not further aggravate the condition. This can be determined by routine screening in animal models of the target disorder or, if such models are unavailable, in normal animals.
  • Microencapsulation of recombinant proteins for sustained release has been performed successfully with human growth hormone (rhGH), interferon-(rhIFN-), interleukin-2, and MN rgp120.
  • rhGH human growth hormone
  • interferon-(rhIFN-) interferon-(rhIFN-)
  • interleukin-2 interleukin-2
  • MN rgp120 MN rgp120.
  • the sustained-release formulations of these proteins were developed using poly-lactic-coglycolic acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties.
  • PLGA poly-lactic-coglycolic acid
  • the degradation products of PLGA, lactic and glycolic acids can be cleared quickly within the human body.
  • the degradability of this polymer can be depending on its molecular weight and composition. Lewis, “Controlled release of bioactive agents from lactide/glycolide polymer,” in: M. Chasin and R.
  • sustained release compositions include, for example, EP 58,481A, U.S. Pat. No. 3,887,699, EP 158,277A, Canadian Patent No. 1176565, U. Sidman et al., Biopolymers 22, 547 [1983], R. Langer et al., Chem. Tech. 12, 98 [1982], Sinha et al., J. Control. Release 90, 261 [2003], Zhu et al., Nat. Biotechnol. 18, 24 [2000], and Dai et al., Colloids Surf B Biointerfaces 41, 117 [2005].
  • Bioadhesive polymers are also contemplated for use in or with compositions of the present disclosure.
  • Bioadhesives are synthetic and naturally occurring materials able to adhere to biological substrates for extended time periods.
  • Carbopol and polycarbophil are both synthetic cross-linked derivatives of poly(acrylic acid).
  • Bioadhesive delivery systems based on naturally occurring substances include for example hyaluronic acid, also known as hyaluronan.
  • Hyaluronic acid is a naturally occurring mucopolysaccharide consisting of residues of D-glucuronic and N-acetyl-D-glucosamine.
  • Hyaluronic acid is found in the extracellular tissue matrix of vertebrates, including in connective tissues, as well as in synovial fluid and in the vitreous and aqueous humor of the eye. Esterified derivatives of hyaluronic acid have been used to produce microspheres for use in delivery that are biocompatible and biodegradable (see, for example, Cortivo et al., Biomaterials (1991) 12:727-730; EP 517,565; WO 96/29998; Illum et al., J. Controlled Rel. (1994) 29:133-141).
  • Exemplary hyaluronic acid containing compositions of the present disclosure comprise a hyaluronic acid ester polymer in an amount of approximately 0.1% to about 40% (w/w) of an antibody comprising an ultralong CDR3 to hyaluronic acid polymer.
  • Both biodegradable and non-biodegradable polymeric matrices may be used to deliver compositions of the present disclosure, and such polymeric matrices may comprise natural or synthetic polymers. Biodegradable matrices are preferred. The period of time over which release occurs is based on selection of the polymer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable.
  • Exemplary synthetic polymers which may be used to form the biodegradable delivery system include: polymers of lactic acid and glycolic acid, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyanhydrides, polyurethanes and co-polymers thereof, poly(butic acid), poly(valeric acid), alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose
  • Exemplary natural polymers include alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.
  • the polymer optionally is in the form of a hydrogel (see, for example, WO 04/009664, WO 05/087201, Sawhney, et al., Macromolecules, 1993, 26, 581-587) that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multi-valent ions or other polymers.
  • Delivery systems also include non-polymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di- and tri-glycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di- and tri-glycerides
  • hydrogel release systems silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • Specific examples include, but are not limited to: (a) erosional systems in which the product is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189 and 5,736,152 and (b) diffusional systems in which a product permeates at a
  • Liposomes containing the product may be prepared by methods known methods, such as for example (DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; JP 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324).
  • compositions may be administered locally via implantation into the affected area of a membrane, sponge, or other appropriate material on to which an antibody comprising an ultralong CDR3, antibody fragment, nucleic acid, or vector disclosed herein has been absorbed or encapsulated.
  • the device may be implanted into any suitable tissue or organ, and delivery of an antibody comprising an ultralong CDR3 antibody fragment, nucleic acid, or vector disclosed herein can be directly through the device via bolus, or via continuous administration, or via catheter using continuous infusion.
  • a pharmaceutical composition comprising an antibody comprising an ultralong CDR3, antibody fragment, nucleic acid, or vector disclosed herein may be formulated for inhalation, such as for example, as a dry powder Inhalation solutions also may be formulated in a liquefied propellant for aerosol delivery. In yet another formulation, solutions may be nebulized. Additional pharmaceutical composition for pulmonary administration include, those described, for example, in WO 94/20069, which discloses pulmonary delivery of chemically modified proteins.
  • the particle size should be suitable for delivery to the distal lung. For example, the particle size may be from 1 ⁇ m to 5 ⁇ m; however, larger particles may be used, for example, if each particle is fairly porous.
  • formulations containing antibodies comprising an ultralong CDR3, antibody fragments, nucleic acids, or vectors disclosed herein may be administered orally.
  • Formulations administered in this fashion may 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 when bioavailability is maximized and pre-systemic degradation is minimized.
  • Additional agents may be included to facilitate absorption of a selective binding agent. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders also can be employed.
  • Another preparation may involve an effective quantity of an antibody comprising an ultralong CDR3, antibody fragment, nucleic acid, or vector disclosed herein in a mixture with non-toxic excipients which are suitable for the manufacture of tablets.
  • 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.
  • Suitable and/or preferred pharmaceutical formulations may be determined in view of the present disclosure and general knowledge of formulation technology, depending upon the intended route of administration, delivery format, and desired dosage. Regardless of the manner of administration, an effective dose may be calculated according to patient body weight, body surface area, or organ size. Further refinement of the calculations for determining the appropriate dosage for treatment involving each of the formulations described herein are routinely made in the art and is within the ambit of tasks routinely performed in the art. Appropriate dosages may be ascertained through use of appropriate dose-response data.
  • antibodies comprising an ultralong CDR3 or fragments thereof are provided with a modified Fc region where a naturally-occurring Fc region is modified to increase the half-life of the antibody or fragment in a biological environment, for example, the serum half-life or a half-life measured by an in vitro assay.
  • a modified Fc region where a naturally-occurring Fc region is modified to increase the half-life of the antibody or fragment in a biological environment, for example, the serum half-life or a half-life measured by an in vitro assay.
  • molecules such as PEG or other water soluble polymers, including polysaccharide polymers
  • This may also be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment (e.g., by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle, e.g., by DNA or peptide synthesis) (see, International Publication No. WO96/32478).
  • Salvage receptor binding epitope refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
  • a salvage receptor binding epitope may include a region wherein any one or more amino acid residues from one or two loops of a Fc domain are transferred to an analogous position of the antibody fragment. Even more preferably, three or more residues from one or two loops of the Fc domain are transferred. Still more preferred, the epitope is taken from the CH2 domain of the Fc region (e.g., of an IgG) and transferred to the CH1, CH3, or VH region, or more than one such region, of the antibody. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the C L region or V L region, or both, of the antibody fragment. See also WO 97/34631 and WO 96/32478 which describe Fc variants and their interaction with the salvage receptor.
  • Mutation of residues within Fc receptor binding sites may result in altered effector function, such as altered ADCC or CDC activity, or altered half-life.
  • Potential mutations include insertion, deletion or substitution of one or more residues, including substitution with alanine, a conservative substitution, a non-conservative substitution, or replacement with a corresponding amino acid residue at the same position from a different IgG subclass (e.g., replacing an IgG1 residue with a corresponding IgG2 residue at that position).
  • a pharmaceutical composition comprising an antibody comprising an ultralong CDR3; and a pharmaceutically acceptable carrier.
  • the antibody may comprise a therapeutic polypeptide, or derivative or variant thereof.
  • the therapeutic polypeptide may be a non-antibody sequence.
  • the therapeutic polypeptide, or derivative or variant thereof can be within the ultralong CDR3.
  • the therapeutic polypeptide is Moka1, Vm24, GLP-1, Exendin-4, human EPO, human FGF21, human GMCSF, human interferon-beta, human GCSF, bovine GCSF or derivative or variant thereof.
  • the antibody is an immunoconjugate as described herein.
  • the antibody can comprise one or more immunoglobulin domains.
  • the immunoglobulin domain is an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M.
  • the immunoglobulin domain can be an immunoglobulin heavy chain region or fragment thereof.
  • the immunoglobulin domain is from a mammalian antibody.
  • the immunoglobulin domain is from a chimeric antibody.
  • the immunoglobulin domain is from an engineered antibody or recombinant antibody.
  • the immunoglobulin domain is from a humanized, human engineered or fully human antibody.
  • the mammalian antibody may be a bovine antibody.
  • the mammalin antibody may be a human antibody.
  • the mammalian antibody is a murine antibody.
  • the ultralong CDR3 can comprise at least a portion of a knob domain in the CDR3.
  • the therapeutic polypeptide can be attached to the knob domain.
  • the ultralong CDR3 comprises at least a portion of a stalk domain in the CDR3.
  • the therapeutic polypeptide may be attached to the stalk domain.
  • the antibody further comprises a linker.
  • the linker can be within the ultralong CDR3.
  • the linker can attach the therapeutic polypeptide to the immunoglobulin domain or fragment thereof. In other instances, the linker attaches the therapeutic polypeptide to the knob domain or stalk domain.
  • the pharmaceutical composition comprising an immunoglobulin construct comprising a heavy chain polypeptide comprising a sequence based on or derived from a sequence selected from any one of SEQ ID NOS: 24-44 and the polypeptide sequence encoded by the DNA any one of SEQ ID NOS: 2-22; and a light chain polypeptide comprising a sequence selected from SEQ ID NO: 23 and a polypeptide sequence encoded by the DNA of SEQ ID NO: 1; and a pharmaceutically acceptable carrier.
  • the disease is an infectious disease such as mastitis.
  • the mammal in need is a dairy animal selected from a list comprising cow, camel, donkey, goat, horse, reindeer, sheep, water buffalo, moose and yak. In some embodiments, the mammal in need is bovine.
  • compositions disclosed herein may be useful for providing prognostic or providing diagnostic information.
  • kits which comprise one or more compounds or compositions packaged in a manner which facilitates their use to practice methods of the present disclosure.
  • a kit includes a compound or composition described herein (e.g., a composition comprising an antibody comprising an ultralong CDR3 alone or in combination with a second agent), packaged in a container with a label affixed to the container or a package insert that describes use of the compound or composition in practicing the method.
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody comprising an ultralong CDR3 as disclosed herein; and (b) a second container with a composition contained therein, wherein the composition comprises a further therapeutic agent.
  • the article of manufacture in this embodiment disclosed herein may further comprise a package insert indicating that the first and second compositions can be used to treat a particular condition.
  • the article of manufacture 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.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • the composition comprising the antibody is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to mammals, such as humans, bovines, felines, canines, and murines.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the amount of the composition described herein which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a Therapeutic protein can be determined by standard clinical techniques.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses are extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • compositions comprising one or more antibodies comprising an ultralong CDR3 as disclosed herein to said subject.
  • the composition can further comprise a pharmaceutically acceptable carrier.
  • the subject may be a mammal.
  • the mammal may be a human.
  • the mammal is a bovine.
  • the antibody may comprise a therapeutic polypeptide, or derivative or variant thereof.
  • the therapeutic polypeptide can be encoded by a non-antibody sequence.
  • the therapeutic polypeptide, or derivative or variant thereof can be attached to the immunoglobulin domain.
  • the therapeutic polypeptide, or derivative or variant thereof may be within the ultralong CDR3.
  • the therapeutic polypeptide, or derivative or variant thereof is conjugated to the ultralong CDR3.
  • the therapeutic polypeptide is Moka1, Vm24, human GLP-1, Exendin-4, human EPO, human FGF21, human GMCSF, human interferon-beta, or derivative or variant thereof.
  • the antibody may be an immunoconjugate as described herein.
  • the antibody can comprise one or more immunoglobulin domains.
  • the immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M.
  • the immunoglobulin domain can be an immunoglobulin heavy chain region or fragment thereof. In some instances, the immunoglobulin domain is from a mammalian antibody.
  • the immunoglobulin domain is from a chimeric antibody.
  • the immunoglobulin domain may be from an engineered antibody or recombinant antibody.
  • the immunoglobulin domain may be from a humanized, human engineered or fully human antibody.
  • the mammalian antibody can be a bovine antibody.
  • the mammalin antibody may be a human antibody.
  • the mammalian antibody is a murine antibody.
  • the ultralong CDR3 may be 35 amino acids in length or more.
  • the ultralong CDR3 may comprise at least 3 cysteine residues or more.
  • the ultralong CDR3 may comprise one or more cysteine motifs.
  • the one or more cysteine motifs may be based on or derived from SEQ ID NOS: 45-156.
  • the one or more cysteine motifs may be based on or derived from SEQ ID NOS: 45-99.
  • the one or more cysteine motifs may be based on or derived from SEQ ID NOS: 100-135.
  • the one or more cysteine motifs may be based on or derived from SEQ ID NOS: 136-156.
  • the ultralong CDR3 can comprise at least a portion of a knob domain.
  • the knob domain may comprise a conserved motif within the knob domain of an ultralong CDR3.
  • the knob domain may comprise a cysteine motif disclosed herein.
  • the therapeutic polypeptide can be attached to the knob domain.
  • the ultralong CDR3 comprises at least a portion of a stalk domain.
  • the stalk domain may comprise a conserved motif within the stalk domain of an ultralong CDR3.
  • the conserved motif within the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 157-224.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 157-161.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 223-234.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 235-239.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 296-299.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 300-303.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from a first sequence selected from the derived from SEQ ID NOS: 300-303.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence basegroup comprising SEQ ID NOS: 157-234 and a second sequence selected from the group comprising SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336.
  • the antibodies disclosed herein may comprise 2 or more, 3 or more, 4 or more, 5 or more sequences based on or derived from SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336.
  • the stalk domain may comprise a T(S/T)VHQ motif.
  • the therapeutic polypeptide can be attached to the stalk domain.
  • the antibody, antibody fragment or immunoglobulin construct further comprises a linker. The linker can attach the therapeutic polypeptide to the immunoglobulin domain or fragment thereof. In other instances, the linker attaches the therapeutic polypeptide to the knob domain or stalk domain.
  • the disease or condition is an autoimmune disease, heteroimmune disease or condition, inflammatory disease, pathogenic infection, thromboembolic disorder, respiratory disease or condition, metabolic disease, central nervous system (CNS) disorder, bone disease or cancer.
  • the disease or condition is a blood disorder.
  • the disease or condition is obesity, diabetes, osteoporosis, anemia, or pain.
  • a method of preventing or treating a disease or condition in a subject in need thereof comprising administering to the subject a composition comprising: an immunoglobulin construct comprising a heavy chain polypeptide comprising a sequence that is substantially similar to a sequence selected from SEQ ID NOS: 24-44; and a light chain polypeptide comprising the sequence that is substantially similar to a sequence of SEQ ID NO: 23.
  • the heavy chain polypeptide sequence may share 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or more amino acid sequence identity to a heavy chain sequence provided by any one of SEQ ID NOS: 24-44.
  • the light chain polypeptide sequence may share 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or more amino acid sequence identity to a light chain sequence provided by SEQ ID NO: 23.
  • the disease or condition is an autoimmune disease, heteroimmune disease or condition, inflammatory disease, pathogenic infection, thromboembolic disorder, respiratory disease or condition, metabolic disease, central nervous system (CNS) disorder, bone disease or cancer.
  • the disease or condition is a blood disorder.
  • the disease or condition is obesity, diabetes, osteoporosis, anemia, or pain.
  • a method of preventing or treating a disease or condition in a subject in need thereof comprising administering to the subject a composition comprising: an immunoglobulin construct comprising a heavy chain polypeptide comprising a polypeptide sequence encoded by a DNA sequence that is substantially similar to a sequence selected from SEQ ID NOS: 2-22; and a light chain polypeptide comprising a polypeptide sequence encoded by a DNA sequence that is substantially similar to a sequence of SEQ ID NO: 1.
  • the heavy chain nucleotide sequence may share 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or more homology to a heavy chain sequence provided by any one of SEQ ID NOS: 2-22.
  • the light chain nucleotide sequence may share 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or more homology to a light chain sequence provided by SEQ ID NO: 1.
  • the disease or condition is an autoimmune disease, heteroimmune disease or condition, inflammatory disease, pathogenic infection, thromboembolic disorder, respiratory disease or condition, metabolic disease, central nervous system (CNS) disorder, bone disease or cancer.
  • the disease or condition is a blood disorder.
  • the disease or condition is obesity, diabetes, osteoporosis, anemia, or pain.
  • Disclosed herein in some embodiments is a method of preventing or treating an autoimmune disease in a subject in need thereof comprising administering a composition comprising one or more antibodies comprising an ultralong CDR3 as disclosed herein to said subject.
  • the composition can further comprise a pharmaceutically acceptable carrier.
  • the subject may be a mammal.
  • the mammal may be a human.
  • the mammal is a bovine.
  • the antibody may comprise a therapeutic polypeptide, or derivative or variant thereof.
  • the therapeutic polypeptide can be encoded by a non-antibody sequence.
  • the therapeutic polypeptide, or derivative or variant thereof can be attached to the immunoglobulin domain.
  • the therapeutic polypeptide, or derivative or variant thereof may be within the ultralong CDR3.
  • the therapeutic polypeptide, or derivative or variant thereof is conjugated to the ultralong CDR3.
  • the therapeutic polypeptide is Moka1, VM-24 or beta-interferon or derivative or variant thereof.
  • the antibody may be an immunoconjugate as described herein.
  • the antibody can comprise one or more immunoglobulin domains.
  • the immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M.
  • the immunoglobulin domain can be an immunoglobulin heavy chain region or fragment thereof.
  • the immunoglobulin domain is from a mammalian antibody.
  • the immunoglobulin domain is from a chimeric antibody.
  • the immunoglobulin domain may be from an engineered antibody or recombinant antibody.
  • the immunoglobulin domain may be from a humanized, human engineered or fully human antibody.
  • the mammalian antibody can be a bovine antibody.
  • the mammalin antibody may be a human antibody. In other instances, the mammalian antibody is a murine antibody.
  • the ultralong CDR3 may be 35 amino acids in length or more.
  • the ultralong CDR3 may comprise at least 3 cysteine residues or more.
  • the ultralong CDR3 may comprise one or more cysteine motifs.
  • the one or more cysteine motifs may be based on or derived from SEQ ID NOS: 45-156.
  • the one or more cysteine motifs may be based on or derived from SEQ ID NOS: 45-99.
  • the one or more cysteine motifs may be based on or derived from SEQ ID NOS: 100-135.
  • the one or more cysteine motifs may be based on or derived from SEQ ID NOS: 136-156.
  • the ultralong CDR3 comprises at least a portion of a knob domain.
  • the knob domain may comprise a conserved motif within the knob domain of an ultralong CDR3.
  • the knob domain may comprise a cysteine motif disclosed herein.
  • the Moka1, VM-24, beta-interferon, or a derivative or variant thereof can be attached to the knob domain.
  • the ultralong CDR3 comprises at least a portion of a stalk domain.
  • the stalk domain may comprise a conserved motif within the stalk domain of an ultralong CDR3.
  • the conserved motif within the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 157-224.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 157-161.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 223-234.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 235-239.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 296-299.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 300-303.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from a first sequence selected from the derived from SEQ ID NOS: 300-303.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence basegroup comprising SEQ ID NOS: 157-234 and a second sequence selected from the group comprising SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336.
  • the antibodies disclosed herein may comprise 2 or more, 3 or more, 4 or more, 5 or more sequences based on or derived from SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336.
  • the stalk domain may comprise a T(S/T)VHQ motif.
  • the Moka1, VM-24, beta-interferon, or a derivative or variant thereof can be attached to the stalk domain.
  • the antibody, antibody fragment or immunoglobulin construct further comprises a linker.
  • the linker can attach Moka1, VM-24, beta-interferon, or a derivative or variant thereof to the immunoglobulin domain or fragment thereof.
  • the linker attaches Moka1, VM-24, beta-interferon, or a derivative or variant thereof to the knob domain or stalk domain.
  • the autoimmune disease is a T-cell mediated autoimmune disease.
  • T-cell mediated autoimmune diseases include, but are not limited to, multiple sclerosis, type-1 diabetes, and psoriasis.
  • Lupus can include, but is not limited to, acute cutaneous lupus erythematosus, subacute cutaneous lupus erythematosus, chronic cutaneous lupus erythematosus, discoid lupus erythematosus, childhood discoid lupus erythematosus, generalized discoid lupus erythematosus, localized discoid lupus erythematosus, chilblain lupus erythematosus (hutchinson), lupus erythematosus-lichen planus overlap syndrome, lupus erythematosus panniculitis (lupus erythematosus profundus), tumid lupus erythematosus, verrucous lupus erythematosus (hypertrophic lupus erythematosus), complement deficiency syndromes, drug-induced
  • a method of preventing or treating a disease or condition which would benefit from the modulation of a potassium voltage-gated channel in a subject in need thereof comprising administering a composition comprising one or more antibodies comprising an ultralong CDR3 as disclosed herein to said subject.
  • the composition can further comprise a pharmaceutically acceptable carrier.
  • the potassium voltage-gated channel is a KCNA3 or K v 1.3 channel.
  • the subject may be a mammal.
  • the mammal may be a human.
  • the mammal is a bovine.
  • the antibody may comprise a therapeutic polypeptide, or derivative or variant thereof.
  • the therapeutic polypeptide can be encoded by a non-antibody sequence.
  • the therapeutic polypeptide, or derivative or variant thereof can be attached to the immunoglobulin domain.
  • the therapeutic polypeptide, or derivative or variant thereof may be within the ultralong CDR3.
  • the therapeutic polypeptide, or derivative or variant thereof is conjugated to the ultralong CDR3.
  • the therapeutic polypeptide is Moka1, VM-24, or derivative or variant thereof.
  • the antibody may be an immunoconjugate as described herein.
  • the antibody can comprise one or more immunoglobulin domains.
  • the immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M.
  • the immunoglobulin domain can be an immunoglobulin heavy chain region or fragment thereof.
  • the immunoglobulin domain is from a mammalian antibody.
  • the immunoglobulin domain is from a chimeric antibody.
  • the immunoglobulin domain may be from an engineered antibody or recombinant antibody.
  • the immunoglobulin domain may be from a humanized, human engineered or fully human antibody.
  • the mammalian antibody can be a bovine antibody.
  • the mammalin antibody may be a human antibody.
  • the mammalian antibody is a murine antibody.
  • the ultralong CDR3 may be 35 amino acids in length or more.
  • the ultralong CDR3 may comprise at least 3 cysteine residues or more.
  • the ultralong CDR3 may comprise one or more cysteine motifs.
  • the one or more cysteine motifs may be based on or derived from SEQ ID NOS: 45-156.
  • the one or more cysteine motifs may be based on or derived from SEQ ID NOS: 45-99.
  • the one or more cysteine motifs may be based on or derived from SEQ ID NOS: 100-135.
  • the one or more cysteine motifs may be based on or derived from SEQ ID NOS: 136-156.
  • the ultralong CDR3 comprises at least a portion of a knob domain.
  • the knob domain may comprise a conserved motif within the knob domain of an ultralong CDR3.
  • the knob domain may comprise a cysteine motif disclosed herein.
  • the Moka1, VM-24, or a derivative or variant thereof can be attached to the knob domain.
  • the ultralong CDR3 comprises at least a portion of a stalk domain.
  • the stalk domain may comprise a conserved motif within the stalk domain of an ultralong CDR3.
  • the conserved motif within the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 157-224.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 157-161.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 223-234.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 235-239.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 296-299.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 300-303.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from a first sequence selected from the derived from SEQ ID NOS: 300-303.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence basegroup comprising SEQ ID NOS: 157-234 and a second sequence selected from the group comprising SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336.
  • the antibodies disclosed herein may comprise 2 or more, 3 or more, 4 or more, 5 or more sequences based on or derived from SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336.
  • the stalk domain may comprise a T(S/T)VHQ motif.
  • the Moka1, VM-24, or a derivative or variant thereof can be attached to the stalk domain.
  • the antibody, antibody fragment or immunoglobulin construct further comprises a linker.
  • the linker can attach Moka1, VM-24, or a derivative or variant thereof to the immunoglobulin domain or fragment thereof.
  • the linker attaches Moka1, VM-24, beta-interferon, or a derivative or variant thereof to the knob domain or stalk domain.
  • the disease or condition is an autoimmune disease.
  • the autoimmune disease can be a T-cell mediated autoimmune disease.
  • modulating a potassium voltage-gated channel comprises inhibiting or blocking a potassium voltage-gated channel.
  • the disease or condition is episodic ataxia, seizure, or neuromyotonia.
  • a method of preventing or treating a metabolic disease or condition in a subject in need thereof comprising administering a composition comprising one or more antibodies comprising an ultralong CDR3 as disclosed herein to said subject.
  • the composition can further comprise a pharmaceutically acceptable carrier.
  • the subject may be a mammal.
  • the mammal may be a human.
  • the mammal is a bovine.
  • the antibody may comprise a therapeutic polypeptide, or derivative or variant thereof.
  • the therapeutic polypeptide can be encoded by a non-antibody sequence.
  • the therapeutic polypeptide, or derivative or variant thereof can be attached to the immunoglobulin domain.
  • the therapeutic polypeptide, or derivative or variant thereof may be within the ultralong CDR3.
  • the therapeutic polypeptide, or derivative or variant thereof is conjugated to the ultralong CDR3.
  • the therapeutic polypeptide is GLP-1, Exendin-4, FGF21, or derivative or variant thereof.
  • the GLP-1 may be a human GLP-1.
  • the FGF21 is a human FGF21.
  • the antibody may be an immunoconjugate as described herein.
  • the antibody can comprise one or more immunoglobulin domains.
  • the immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M.
  • the immunoglobulin domain can be an immunoglobulin heavy chain region or fragment thereof.
  • the immunoglobulin domain is from a mammalian antibody.
  • the immunoglobulin domain is from a chimeric antibody.
  • the immunoglobulin domain may be from an engineered antibody or recombinant antibody.
  • the immunoglobulin domain may be from a humanized, human engineered or fully human antibody.
  • the mammalian antibody can be a bovine antibody.
  • the mammalin antibody may be a human antibody.
  • the mammalian antibody is a murine antibody.
  • the ultralong CDR3 may be 35 amino acids in length or more.
  • the ultralong CDR3 may comprise at least 3 cysteine residues or more.
  • the ultralong CDR3 may comprise one or more cysteine motifs.
  • the one or more cysteine motifs may be based on or derived from SEQ ID NOS: 45-156.
  • the one or more cysteine motifs may be based on or derived from SEQ ID NOS: 45-99.
  • the one or more cysteine motifs may be based on or derived from SEQ ID NOS: 100-135.
  • the one or more cysteine motifs may be based on or derived from SEQ ID NOS: 136-156.
  • the ultralong CDR3 comprises at least a portion of a knob domain.
  • the knob domain may comprise a conserved motif within the knob domain of an ultralong CDR3.
  • the knob domain may comprise a cysteine motif disclosed herein.
  • the GLP-1, Exendin-4, FGF21, or a derivative or variant thereof can be attached to the knob domain.
  • the ultralong CDR3 comprises at least a portion of a stalk domain.
  • the stalk domain may comprise a conserved motif within the stalk domain of an ultralong CDR3.
  • the conserved motif within the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 157-224.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 157-161.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 223-234.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 235-239.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 296-299.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 300-303.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from a first sequence selected from the derived from SEQ ID NOS: 300-303.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence basegroup comprising SEQ ID NOS: 157-234 and a second sequence selected from the group comprising SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336.
  • the antibodies disclosed herein may comprise 2 or more, 3 or more, 4 or more, 5 or more sequences based on or derived from SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336.
  • the conserved motif with the stalk domain of the ultralong CDR3 may comprise a polypeptide sequence based on or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336.
  • the stalk domain may comprise a T(S/T)VHQ motif
  • the GLP-1, Exendin-4, FGF21, or a derivative or variant thereof can be attached to the stalk domain.
  • the antibody, antibody fragment or immunoglobulin construct further comprises a linker.
  • the linker can attach GLP-1, Exendin-4, FGF21, or a derivative or variant thereof to the immunoglobulin domain or fragment thereof. In other instances, the linker attaches GLP-1, Exendin-4, FGF21, or a derivative or variant thereof to the knob domain or stalk domain.
  • Metabolic diseases and/or conditions can include disorders of carbohydrate metabolism, amino acid metabolism, organic acid metabolism (organic acidurias), fatty acid oxidation and mitochondrial metabolism, porphyrin metabolism, purine or pyrimidine metabolism, steroid metabolism, mitochondrial function, peroxisomal function, urea cycle disorder, urea cycle defects or lysosomal storage disorders. In some instances, the metabolic disease or condition is diabetes.
  • the metabolic disese or condition is glycogen storage disease, phenylketonuria, maple syrup urine disease, glutaric acidemia type 1, Carbamoyl phosphate synthetase I deficiency, alcaptonuria, Medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD), acute intermittent porphyria, Lesch-Nyhan syndrome, lipoid congenital adrenal hyperplasia, congenital adrenal hyperplasia, Kearns-Sayre syndrome, Zellweger syndrome, Gaucher's disease, or Niemann Pick disease.
  • MCADD Medium-chain acyl-coenzyme A dehydrogenase deficiency
  • a method of preventing or treating a central nervous system (CNS) disorder in a subject in need thereof comprising administering a composition comprising one or more antibodies comprising an ultralong CDR3 as disclosed herein to said subject.
  • the composition can further comprise a pharmaceutically acceptable carrier.
  • the subject may be a mammal.
  • the mammal may be a human.
  • the mammal is a bovine.
  • the antibody may comprise a therapeutic polypeptide, or derivative or variant thereof.
  • the therapeutic polypeptide can be encoded by a non-antibody sequence.
  • the therapeutic polypeptide, or derivative or variant thereof can be attached to the immunoglobulin domain.
  • the therapeutic polypeptide, or derivative or variant thereof may be within the ultralong CDR3.
  • the therapeutic polypeptide, or derivative or variant thereof is conjugated to the ultralong CDR3.
  • the therapeutic polypeptide is GLP-1, Exendin-4, or derivative or variant thereof.
  • the GLP-1 may be a human GLP-1.
  • the antibody may be an immunoconjugate as described herein.
  • the antibody can comprise one or more immunoglobulin domains.
  • the immunoglobulin domain may be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M.
  • the immunoglobulin domain can be an immunoglobulin heavy chain region or fragment thereof.
  • the immunoglobulin domain is from a mammalian antibody.
  • the immunoglobulin domain is from a chimeric antibody.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)
  • Virology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
US13/737,913 2012-01-09 2013-01-09 Ultralong complementarity determining regions and uses thereof Abandoned US20140050720A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US13/737,913 US20140050720A1 (en) 2012-01-09 2013-01-09 Ultralong complementarity determining regions and uses thereof

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201261584680P 2012-01-09 2012-01-09
US201261671629P 2012-07-13 2012-07-13
US13/737,913 US20140050720A1 (en) 2012-01-09 2013-01-09 Ultralong complementarity determining regions and uses thereof

Publications (1)

Publication Number Publication Date
US20140050720A1 true US20140050720A1 (en) 2014-02-20

Family

ID=48782074

Family Applications (3)

Application Number Title Priority Date Filing Date
US13/737,913 Abandoned US20140050720A1 (en) 2012-01-09 2013-01-09 Ultralong complementarity determining regions and uses thereof
US13/737,910 Active 2034-01-27 US10774132B2 (en) 2012-01-09 2013-01-09 Ultralong complementarity determining regions and uses thereof
US16/742,759 Active US11390665B2 (en) 2012-01-09 2020-01-14 Ultralong complementarity determining regions and uses thereof

Family Applications After (2)

Application Number Title Priority Date Filing Date
US13/737,910 Active 2034-01-27 US10774132B2 (en) 2012-01-09 2013-01-09 Ultralong complementarity determining regions and uses thereof
US16/742,759 Active US11390665B2 (en) 2012-01-09 2020-01-14 Ultralong complementarity determining regions and uses thereof

Country Status (7)

Country Link
US (3) US20140050720A1 (enExample)
EP (1) EP2802603A4 (enExample)
JP (2) JP6684490B2 (enExample)
CN (1) CN104520321A (enExample)
AU (2) AU2013208003B2 (enExample)
CA (1) CA2863224A1 (enExample)
WO (1) WO2013106485A2 (enExample)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150191517A1 (en) * 2012-07-27 2015-07-09 Purdue Pharma L.P. Novel sodium channel blocking peptides and the use thereof
WO2016115218A1 (en) * 2015-01-14 2016-07-21 The California Institute For Biomedical Research Antibody drug conjugates for the treatment of immune conditions
US9644021B2 (en) 2013-01-11 2017-05-09 The California Institute For Biomedical Research Bovine fusion antibodies
US9663499B2 (en) 2013-06-07 2017-05-30 The California Institute For Biomedical Research Small molecule inhibitors of fibrosis
EP3155018A4 (en) * 2014-06-06 2018-01-10 The California Institute for Biomedical Research Constant region antibody fusion proteins and compositions thereof
US10336735B2 (en) 2014-12-10 2019-07-02 The Scripps Research Institute Small molecule inhibitors of fibrosis
US10562980B2 (en) 2012-01-09 2020-02-18 The Scripps Research Institute Humanized antibodies
US10640574B2 (en) 2013-07-18 2020-05-05 Taurus Biosciences, Llc Humanized antibodies with ultralong complementary determining regions
US10683353B2 (en) * 2013-07-11 2020-06-16 The Scripps Research Institute Coiled coil immunoglobulin fusion proteins and compositions thereof
US10774132B2 (en) 2012-01-09 2020-09-15 The Scripps Research Instittue Ultralong complementarity determining regions and uses thereof

Families Citing this family (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EA201790946A8 (ru) * 2014-12-01 2018-10-31 Зе Скриппс Рисёрч Инститьют Способы и композиции, связанные с функциональными полипептидами, встроенными в гетерологичные белковые каркасы
EP3288972A1 (en) * 2015-05-01 2018-03-07 Medimmune Limited Novel phage display library, members thereof and uses of the same
BR112018010937A2 (pt) * 2015-12-04 2018-12-04 Novartis Ag composições de anticorpos enxertados com citocina e métodos de uso para imunorregulação
DK3387019T3 (da) 2015-12-09 2022-01-10 Scripps Research Inst Relaxin-immunoglobulinfusionsproteiner og anvendelsesfremgangsmåder
EP3786292A1 (en) * 2016-01-08 2021-03-03 Maxion Therapeutics Limited Binding members with altered diversity scaffold domains
WO2018067198A1 (en) 2016-10-03 2018-04-12 The Regents Of The University Of California Inhibitory antibodies and methods of use thereof
WO2018075857A1 (en) 2016-10-20 2018-04-26 I-Mab Novel cd47 monoclonal antibodies and uses thereof
CN112717135A (zh) * 2016-11-22 2021-04-30 上海交通大学医学院 酸敏感离子通道调控剂的用途
US20200362058A1 (en) * 2017-05-24 2020-11-19 Novartis Ag Antibody-cytokine engrafted proteins and methods of use
JOP20190271A1 (ar) * 2017-05-24 2019-11-21 Novartis Ag بروتينات مطعّمة بسيتوكين- الجسم المضاد وطرق الاستخدام للاضطرابات المتعلقة بالمناعة
WO2018215937A1 (en) * 2017-05-24 2018-11-29 Novartis Ag Interleukin-7 antibody cytokine engrafted proteins and methods of use in the treatment of cancer
IL322309A (en) * 2017-05-24 2025-09-01 Novartis Ag IL2 antibody grafted proteins and methods of use in cancer treatment
GB201711208D0 (en) 2017-07-12 2017-08-23 Iontas Ltd Ion channel inhibitors
US11572405B2 (en) 2018-01-12 2023-02-07 Bristol-Myers Squibb Company Combination therapy with anti-IL-8 antibodies and anti-PD-1 antibodies for treating cancer
JP7515410B2 (ja) * 2018-06-13 2024-07-12 クリスタル バイオサイエンス インコーポレイテッド 複数のジスルフィド架橋によって安定化され、遺伝子変換によって多様化された長いｃｄｒ－ｈ３ｓで抗体を作製するトランスジェニックニワトリ
CA3097443A1 (en) * 2018-11-12 2020-05-22 I-Mab Biopharma Us Limited Fusion proteins containing cd47 antibodies and cytokines
US11332546B2 (en) 2019-05-21 2022-05-17 The Regents Of The University Of California Protease inhibitory antibodies and methods of use thereof
WO2021081440A2 (en) * 2019-10-24 2021-04-29 Minotaur Therapeutics, Inc. Chimeric cytokine modified antibodies and methods of use thereof
IL296732A (en) * 2020-03-27 2022-11-01 UCB Biopharma SRL Autonomous knob domain peptides
CN112034169A (zh) * 2020-08-27 2020-12-04 华威特（江苏）生物制药有限公司 用于检测牛病毒性腹泻病毒1型的直接免疫荧光试剂及其试剂盒
WO2022232321A1 (en) * 2021-04-28 2022-11-03 Minotaur Therapeutics, Inc. Humanized chimeric bovine antibodies and methods of use
IL308087A (en) * 2021-05-12 2023-12-01 Applied Biomedical Science Inst Methods for the screening and expression of disulfide-bonded polypeptides
EP4337689A1 (en) * 2021-05-12 2024-03-20 Applied Biomedical Science Institute Binding polypeptides against sars cov-2 and uses thereof
WO2023192478A1 (en) 2022-04-01 2023-10-05 Bristol-Myers Squibb Company Combination therapy with anti-il-8 antibodies and anti-pd-1 antibodies for treating cancer
TWI897189B (zh) 2023-01-18 2025-09-11 美商基利科學股份有限公司 具有經改變重鏈基因座之嵌合基因轉殖免疫球蛋白小鼠及其製造及使用方法

Family Cites Families (235)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US30985A (en) 1860-12-18 Thomas l
US3887699A (en) 1969-03-24 1975-06-03 Seymour Yolles Biodegradable polymeric article for dispensing drugs
US3854480A (en) 1969-04-01 1974-12-17 Alza Corp Drug-delivery system
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
US3896111A (en) 1973-02-20 1975-07-22 Research Corp Ansa macrolides
US4151042A (en) 1977-03-31 1979-04-24 Takeda Chemical Industries, Ltd. Method for producing maytansinol and its derivatives
US4137230A (en) 1977-11-14 1979-01-30 Takeda Chemical Industries, Ltd. Method for the production of maytansinoids
FR2413974A1 (fr) 1978-01-06 1979-08-03 David Bernard Sechoir pour feuilles imprimees par serigraphie
US4263428A (en) 1978-03-24 1981-04-21 The Regents Of The University Of California Bis-anthracycline nucleic acid function inhibitors and improved method for administering the same
US4307016A (en) 1978-03-24 1981-12-22 Takeda Chemical Industries, Ltd. Demethyl maytansinoids
US4265814A (en) 1978-03-24 1981-05-05 Takeda Chemical Industries Matansinol 3-n-hexadecanoate
JPS5562090A (en) 1978-10-27 1980-05-10 Takeda Chem Ind Ltd Novel maytansinoid compound and its preparation
JPS5566585A (en) 1978-11-14 1980-05-20 Takeda Chem Ind Ltd Novel maytansinoid compound and its preparation
US4256746A (en) 1978-11-14 1981-03-17 Takeda Chemical Industries Dechloromaytansinoids, their pharmaceutical compositions and method of use
JPS55164687A (en) 1979-06-11 1980-12-22 Takeda Chem Ind Ltd Novel maytansinoid compound and its preparation
JPS55102583A (en) 1979-01-31 1980-08-05 Takeda Chem Ind Ltd 20-acyloxy-20-demethylmaytansinoid compound
JPS55162791A (en) 1979-06-05 1980-12-18 Takeda Chem Ind Ltd Antibiotic c-15003pnd and its preparation
JPS55164685A (en) 1979-06-08 1980-12-22 Takeda Chem Ind Ltd Novel maytansinoid compound and its preparation
JPS55164686A (en) 1979-06-11 1980-12-22 Takeda Chem Ind Ltd Novel maytansinoid compound and its preparation
US4309428A (en) 1979-07-30 1982-01-05 Takeda Chemical Industries, Ltd. Maytansinoids
JPS5645483A (en) 1979-09-19 1981-04-25 Takeda Chem Ind Ltd C-15003phm and its preparation
JPS5645485A (en) 1979-09-21 1981-04-25 Takeda Chem Ind Ltd Production of c-15003pnd
EP0028683A1 (en) 1979-09-21 1981-05-20 Takeda Chemical Industries, Ltd. Antibiotic C-15003 PHO and production thereof
WO1982001188A1 (en) 1980-10-08 1982-04-15 Takeda Chemical Industries Ltd 4,5-deoxymaytansinoide compounds and process for preparing same
US4450254A (en) 1980-11-03 1984-05-22 Standard Oil Company Impact improvement of high nitrile resins
EP0052322B1 (de) 1980-11-10 1985-03-27 Gersonde, Klaus, Prof. Dr. Verfahren zur Herstellung von Lipid-Vesikeln durch Ultraschallbehandlung, Anwendung des Verfahrens und Vorrichtung zur Durchführung des Verfahrens
US4675189A (en) 1980-11-18 1987-06-23 Syntex (U.S.A.) Inc. Microencapsulation of water soluble active polypeptides
PH19942A (en) 1980-11-18 1986-08-14 Sintex Inc Microencapsulation of water soluble polypeptides
US4419446A (en) 1980-12-31 1983-12-06 The United States Of America As Represented By The Department Of Health And Human Services Recombinant DNA process utilizing a papilloma virus DNA as a vector
US4315929A (en) 1981-01-27 1982-02-16 The United States Of America As Represented By The Secretary Of Agriculture Method of controlling the European corn borer with trewiasine
US4313946A (en) 1981-01-27 1982-02-02 The United States Of America As Represented By The Secretary Of Agriculture Chemotherapeutically active maytansinoids from Trewia nudiflora
IE52535B1 (en) 1981-02-16 1987-12-09 Ici Plc Continuous release pharmaceutical compositions
JPS57192389A (en) 1981-05-20 1982-11-26 Takeda Chem Ind Ltd Novel maytansinoid
US4485045A (en) 1981-07-06 1984-11-27 Research Corporation Synthetic phosphatidyl cholines useful in forming liposomes
DE3374837D1 (en) 1982-02-17 1988-01-21 Ciba Geigy Ag Lipids in the aqueous phase
DE3218121A1 (de) 1982-05-14 1983-11-17 Leskovar, Peter, Dr.-Ing., 8000 München Arzneimittel zur tumorbehandlung
EP0102324A3 (de) 1982-07-29 1984-11-07 Ciba-Geigy Ag Lipide und Tenside in wässriger Phase
US4601978A (en) 1982-11-24 1986-07-22 The Regents Of The University Of California Mammalian metallothionein promoter system
US4452775A (en) 1982-12-03 1984-06-05 Syntex (U.S.A.) Inc. Cholesterol matrix delivery system for sustained release of macromolecules
US4560655A (en) 1982-12-16 1985-12-24 Immunex Corporation Serum-free cell culture medium and process for making same
US4657866A (en) 1982-12-21 1987-04-14 Sudhir Kumar Serum-free, synthetic, completely chemically defined tissue culture media
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4544545A (en) 1983-06-20 1985-10-01 Trustees University Of Massachusetts Liposomes containing modified cholesterol for organ targeting
HUT35524A (en) 1983-08-02 1985-07-29 Hoechst Ag Process for preparing pharmaceutical compositions containing regulatory /regulative/ peptides providing for the retarded release of the active substance
DE3483949D1 (de) 1983-09-26 1991-02-21 Udo Dr Med Ehrenfeld Mittel und erzeugnis fuer die diagnose und therapie von tumoren sowie zur behandlung von schwaechen der zelligen und humoralen immunabwehr.
US4767704A (en) 1983-10-07 1988-08-30 Columbia University In The City Of New York Protein-free culture medium
EP0143949B1 (en) 1983-11-01 1988-10-12 TERUMO KABUSHIKI KAISHA trading as TERUMO CORPORATION Pharmaceutical composition containing urokinase
DE3413608A1 (de) 1984-04-11 1985-10-24 Hoechst Ag, 6230 Frankfurt Implantierbare zubereitungen von regulatorischen peptiden mit gesteuerter freisetzung und verfahren zu ihrer herstellung
US4965199A (en) 1984-04-20 1990-10-23 Genentech, Inc. Preparation of functional human factor VIII in mammalian cells using methotrexate based selection
US4970198A (en) 1985-10-17 1990-11-13 American Cyanamid Company Antitumor antibiotics (LL-E33288 complex)
GB8516415D0 (en) 1985-06-28 1985-07-31 Celltech Ltd Culture of animal cells
US4980286A (en) 1985-07-05 1990-12-25 Whitehead Institute For Biomedical Research In vivo introduction and expression of foreign genetic material in epithelial cells
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
US5618920A (en) 1985-11-01 1997-04-08 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
US6548640B1 (en) 1986-03-27 2003-04-15 Btg International Limited Altered antibodies
US4927762A (en) 1986-04-01 1990-05-22 Cell Enterprises, Inc. Cell culture medium with antioxidant
IL85035A0 (en) 1987-01-08 1988-06-30 Int Genetic Eng Polynucleotide molecule,a chimeric antibody with specificity for human b cell surface antigen,a process for the preparation and methods utilizing the same
US5079233A (en) 1987-01-30 1992-01-07 American Cyanamid Company N-acyl derivatives of the LL-E33288 antitumor antibiotics, composition and methods for using the same
AU600575B2 (en) 1987-03-18 1990-08-16 Sb2, Inc. Altered antibodies
US4975278A (en) 1988-02-26 1990-12-04 Bristol-Myers Company Antibody-enzyme conjugates in combination with prodrugs for the delivery of cytotoxic agents to tumor cells
US5053394A (en) 1988-09-21 1991-10-01 American Cyanamid Company Targeted forms of methyltrithio antitumor agents
US5606040A (en) 1987-10-30 1997-02-25 American Cyanamid Company Antitumor and antibacterial substituted disulfide derivatives prepared from compounds possessing a methyl-trithio group
US5770701A (en) 1987-10-30 1998-06-23 American Cyanamid Company Process for preparing targeted forms of methyltrithio antitumor agents
WO1989007142A1 (en) 1988-02-05 1989-08-10 Morrison Sherie L Domain-modified constant region antibodies
EP0435911B1 (en) 1988-09-23 1996-03-13 Cetus Oncology Corporation Cell culture medium for enhanced cell growth, culture longevity and product expression
GB8823869D0 (en) 1988-10-12 1988-11-16 Medical Res Council Production of antibodies
WO1990005144A1 (en) 1988-11-11 1990-05-17 Medical Research Council Single domain ligands, receptors comprising said ligands, methods for their production, and use of said ligands and receptors
US5175384A (en) 1988-12-05 1992-12-29 Genpharm International Transgenic mice depleted in mature t-cells and methods for making transgenic mice
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5133974A (en) 1989-05-05 1992-07-28 Kv Pharmaceutical Company Extended release pharmaceutical formulations
DE3920358A1 (de) 1989-06-22 1991-01-17 Behringwerke Ag Bispezifische und oligospezifische, mono- und oligovalente antikoerperkonstrukte, ihre herstellung und verwendung
ATE144793T1 (de) 1989-06-29 1996-11-15 Medarex Inc Bispezifische reagenzien für die aids-therapie
US5208020A (en) 1989-10-25 1993-05-04 Immunogen Inc. Cytotoxic agents comprising maytansinoids and their therapeutic use
CA2026147C (en) 1989-10-25 2006-02-07 Ravi J. Chari Cytotoxic agents comprising maytansinoids and their therapeutic use
US5959177A (en) 1989-10-27 1999-09-28 The Scripps Research Institute Transgenic plants expressing assembled secretory antibodies
DE69133566T2 (de) 1990-01-12 2007-12-06 Amgen Fremont Inc. Bildung von xenogenen Antikörpern
JPH06500011A (ja) 1990-06-29 1994-01-06 ラージ　スケール　バイオロジー　コーポレイション 形質転換された微生物によるメラニンの製造
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
KR100272077B1 (ko) 1990-08-29 2000-11-15 젠팜인터내셔날,인코포레이티드 이종 항체를 생산할 수 있는 전이유전자를 가진 인간이외의 동물
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5122469A (en) 1990-10-03 1992-06-16 Genentech, Inc. Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins
US5264365A (en) 1990-11-09 1993-11-23 Board Of Regents, The University Of Texas System Protease-deficient bacterial strains for production of proteolytically sensitive polypeptides
US5508192A (en) 1990-11-09 1996-04-16 Board Of Regents, The University Of Texas System Bacterial host strains for producing proteolytically sensitive polypeptides
WO1992009690A2 (en) 1990-12-03 1992-06-11 Genentech, Inc. Enrichment method for variant proteins with altered binding properties
US5571894A (en) 1991-02-05 1996-11-05 Ciba-Geigy Corporation Recombinant antibodies specific for a growth factor receptor
US6565841B1 (en) 1991-03-15 2003-05-20 Amgen, Inc. Pulmonary administration of granulocyte colony stimulating factor
IT1247472B (it) 1991-05-31 1994-12-17 Fidia Spa Processo per la preparazione di microsfere contenenti componenti biologicamente attivi.
EP0590058B1 (en) 1991-06-14 2003-11-26 Genentech, Inc. HUMANIZED Heregulin ANTIBODy
GB9114948D0 (en) 1991-07-11 1991-08-28 Pfizer Ltd Process for preparing sertraline intermediates
WO1993006217A1 (en) 1991-09-19 1993-04-01 Genentech, Inc. EXPRESSION IN E. COLI OF ANTIBODY FRAGMENTS HAVING AT LEAST A CYSTEINE PRESENT AS A FREE THIOL, USE FOR THE PRODUCTION OF BIFUNCTIONAL F(ab')2 ANTIBODIES
US5362852A (en) 1991-09-27 1994-11-08 Pfizer Inc. Modified peptide derivatives conjugated at 2-hydroxyethylamine moieties
FI941572L (fi) 1991-10-07 1994-05-27 Oncologix Inc Anti-erbB-2-monoklonaalisten vasta-aineiden yhdistelmä ja käyttömenetelmä
WO1993008829A1 (en) 1991-11-04 1993-05-13 The Regents Of The University Of California Compositions that mediate killing of hiv-infected cells
US6146850A (en) * 1991-11-04 2000-11-14 Xoma Corporation Proteins encoding gelonin sequences
AU3178993A (en) 1991-11-25 1993-06-28 Enzon, Inc. Multivalent antigen-binding proteins
US5407686A (en) 1991-11-27 1995-04-18 Sidmak Laboratories, Inc. Sustained release composition for oral administration of active ingredient
DE69233204T2 (de) 1991-12-13 2004-07-15 Xoma Corp., Berkeley Verfahren und materialien zur herstellung von modifizierten variablen antikörperdomänen und ihre therapeutische verwendung
US5667988A (en) 1992-01-27 1997-09-16 The Scripps Research Institute Methods for producing antibody libraries using universal or randomized immunoglobulin light chains
ATE419355T1 (de) 1992-02-06 2009-01-15 Novartis Vaccines & Diagnostic Marker für krebs und biosynthetisches bindeprotein dafür
US5470582A (en) 1992-02-07 1995-11-28 Syntex (U.S.A.) Inc. Controlled delivery of pharmaceuticals from preformed porous polymeric microparticles
GB9207479D0 (en) * 1992-04-06 1992-05-20 Scotgen Ltd Novel antibodies for treatment and prevention of respiratory syncytial virus infection in animals and man
ZA932522B (en) 1992-04-10 1993-12-20 Res Dev Foundation Immunotoxins directed against c-erbB-2(HER/neu) related surface antigens
ATE149570T1 (de) 1992-08-17 1997-03-15 Genentech Inc Bispezifische immunoadhesine
US20080152586A1 (en) 1992-09-25 2008-06-26 Avipep Pty Limited High avidity polyvalent and polyspecific reagents
ES2091684T3 (es) 1992-11-13 1996-11-01 Idec Pharma Corp Aplicacion terapeutica de anticuerpos quimericos y radiomarcados contra el antigeno de diferenciacion restringida de los linfocitos b humanos para el tratamiento del linfoma de las celulas b.
ES2151541T3 (es) 1992-12-02 2001-01-01 Alkermes Inc Microesferas que contienen hormona del crecimiento de liberacion prolongada.
US5635483A (en) 1992-12-03 1997-06-03 Arizona Board Of Regents Acting On Behalf Of Arizona State University Tumor inhibiting tetrapeptide bearing modified phenethyl amides
US5780588A (en) 1993-01-26 1998-07-14 Arizona Board Of Regents Elucidation and synthesis of selected pentapeptides
AU6235294A (en) 1993-02-02 1994-08-29 Scripps Research Institute, The Methods for producing polypeptide binding sites
WO1994029351A2 (en) 1993-06-16 1994-12-22 Celltech Limited Antibodies
US6372716B1 (en) 1994-04-26 2002-04-16 Genetics Institute, Inc. Formulations for factor IX
US5773001A (en) 1994-06-03 1998-06-30 American Cyanamid Company Conjugates of methyltrithio antitumor agents and intermediates for their synthesis
BR9509201A (pt) 1994-09-09 1997-12-30 Takeda Chemical Industries Ltd Preparação de liberação sustemtada uso de um sal de metal polivalente insolúvel em água ou ligeiramente solúvel em água e processo para produzir uma preparação de liberação sustentada
US5789199A (en) 1994-11-03 1998-08-04 Genentech, Inc. Process for bacterial production of polypeptides
US5663149A (en) 1994-12-13 1997-09-02 Arizona Board Of Regents Acting On Behalf Of Arizona State University Human cancer inhibitory pentapeptide heterocyclic and halophenyl amides
GB9501079D0 (en) 1995-01-19 1995-03-08 Bioinvent Int Ab Activation of T-cells
US5840523A (en) 1995-03-01 1998-11-24 Genetech, Inc. Methods and compositions for secretion of heterologous polypeptides
CA2216919C (en) 1995-03-28 2007-09-18 Fidia Advanced Biopolymers Srl Nanospheres comprising a biocompatible polysaccharide
US5641870A (en) 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
US5869046A (en) 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
US6121022A (en) 1995-04-14 2000-09-19 Genentech, Inc. Altered polypeptides with increased half-life
US6096871A (en) 1995-04-14 2000-08-01 Genentech, Inc. Polypeptides altered to contain an epitope from the Fc region of an IgG molecule for increased half-life
EP0739981A1 (en) * 1995-04-25 1996-10-30 Vrije Universiteit Brussel Variable fragments of immunoglobulins - use for therapeutic or veterinary purposes
JP4312259B2 (ja) 1995-04-27 2009-08-12 アムジェン フレモント インク． 免疫したゼノマウス（ＸｅｎｏＭｏｕｓｅ）に由来するヒト抗体
EP0823941A4 (en) 1995-04-28 2001-09-19 Abgenix Inc HUMAN ANTIBODIES DERIVED FROM IMMUNIZED XENO MOUSES
US5714586A (en) 1995-06-07 1998-02-03 American Cyanamid Company Methods for the preparation of monomeric calicheamicin derivative/carrier conjugates
EP0831787B1 (en) 1995-06-07 2001-08-22 Alkermes Controlled Therapeutics, Inc. Composition for sustained release of human growth hormone
US5712374A (en) 1995-06-07 1998-01-27 American Cyanamid Company Method for the preparation of substantiallly monomeric calicheamicin derivative/carrier conjugates
ZA965368B (en) 1995-07-14 1997-01-14 Novo Nordisk As A pharmaceutical formulation
US6685940B2 (en) 1995-07-27 2004-02-03 Genentech, Inc. Protein formulation
US5736152A (en) 1995-10-27 1998-04-07 Atrix Laboratories, Inc. Non-polymeric sustained release delivery system
DE19544393A1 (de) 1995-11-15 1997-05-22 Hoechst Schering Agrevo Gmbh Synergistische herbizide Mischungen
GB9603256D0 (en) 1996-02-16 1996-04-17 Wellcome Found Antibodies
WO1997034631A1 (en) 1996-03-18 1997-09-25 Board Of Regents, The University Of Texas System Immunoglobin-like domains with increased half lives
AU2660397A (en) 1996-04-05 1997-10-29 Board Of Regents, The University Of Texas System Methods for producing soluble, biologically-active disulfide bond-containing eukaryotic proteins in bacterial cells
DE69738539T2 (de) 1996-12-03 2009-03-26 Amgen Fremont Inc. Vollkommen humane Antikörper die EGFR binden
US6083715A (en) 1997-06-09 2000-07-04 Board Of Regents, The University Of Texas System Methods for producing heterologous disulfide bond-containing polypeptides in bacterial cells
EP0994903B1 (en) 1997-06-24 2005-05-25 Genentech, Inc. Methods and compositions for galactosylated glycoproteins
GB9714680D0 (en) 1997-07-11 1997-09-17 Medical Res Council Recombinant production of proteins
US6040498A (en) 1998-08-11 2000-03-21 North Caroline State University Genetically engineered duckweed
AU759779B2 (en) 1997-10-31 2003-05-01 Genentech Inc. Methods and compositions comprising glycoprotein glycoforms
JP4460155B2 (ja) 1997-12-05 2010-05-12 ザ・スクリプス・リサーチ・インステイチユート マウス抗体のヒト化
AUPP221098A0 (en) 1998-03-06 1998-04-02 Diatech Pty Ltd V-like domain binding molecules
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
DE69937291T2 (de) 1998-04-02 2008-07-10 Genentech, Inc., South San Francisco Antikörpervarianten und fragmente davon
JP4334141B2 (ja) 1998-04-20 2009-09-30 グリカート バイオテクノロジー アクチェンゲゼルシャフト 抗体依存性細胞傷害性を改善するための抗体のグリコシル化操作
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
BR0008758A (pt) 1999-01-15 2001-12-04 Genentech Inc Variantes de polipeptìdeos parentais com funçãoefetora alterada, polipeptìdeos, composição ácidonucleico isolado, vetor, célula hospedeira,método para produzir uma variante depolipeptìdeo, método para o tratamento de umadesordem em mamìferos e método para produziruma região fc variante
CA2363035C (en) 1999-02-22 2007-09-04 Symyx Technologies, Inc. Compositions comprising nickel and their use as catalyst in oxidative dehydrogenation of alkanes
CA2369292C (en) 1999-04-09 2010-09-21 Kyowa Hakko Kogyo Co. Ltd. Method of modulating the activity of functional immune molecules
GB9912350D0 (en) * 1999-05-26 1999-07-28 European Molecular Biology Lab Embl Modified cytokine
US6566329B1 (en) 1999-06-28 2003-05-20 Novo Nordisk A/S Freeze-dried preparation of human growth hormone
JP4944324B2 (ja) 1999-07-13 2012-05-30 ボルダー バイオテクノロジー， インコーポレイテッド 免疫グロブリン融合タンパク質
JP2003512821A (ja) 1999-10-04 2003-04-08 メディカゴ インコーポレイテッド 外来性遺伝子の転写調節方法
US7125978B1 (en) 1999-10-04 2006-10-24 Medicago Inc. Promoter for regulating expression of foreign genes
WO2001029246A1 (fr) 1999-10-19 2001-04-26 Kyowa Hakko Kogyo Co., Ltd. Procede de production d'un polypeptide
US20030144474A1 (en) 2000-06-05 2003-07-31 Sunol Molecular Corporation T cell receptor fusions and conjugates and methods of use thereof
CA2419894A1 (en) 2000-09-15 2002-03-21 Coley Pharmaceutical Gmbh Process for high throughput screening of cpg-based immuno-agonist/antagonist
US6946292B2 (en) 2000-10-06 2005-09-20 Kyowa Hakko Kogyo Co., Ltd. Cells producing antibody compositions with increased antibody dependent cytotoxic activity
US7064191B2 (en) 2000-10-06 2006-06-20 Kyowa Hakko Kogyo Co., Ltd. Process for purifying antibody
US7196185B2 (en) 2001-04-20 2007-03-27 Azad Kumar Kaushik Bovine germline D-genes and their application
US6740747B2 (en) 2001-04-20 2004-05-25 Azad Kaushik Bovine VDJ cassette, BF1H1, suitable for antigenization
US6884869B2 (en) 2001-04-30 2005-04-26 Seattle Genetics, Inc. Pentapeptide compounds and uses related thereto
US6441163B1 (en) 2001-05-31 2002-08-27 Immunogen, Inc. Methods for preparation of cytotoxic conjugates of maytansinoids and cell binding agents
FI117667B (fi) * 2001-07-05 2007-01-15 Univ Zuerich Farmaseuttinen koostumus, joka soveltuu käytettäväksi ortopediassa ja hammaslääketieteessä
CA2455365C (en) 2001-08-03 2014-07-29 Glycart Biotechnology Ag Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity
WO2003030821A2 (en) 2001-10-05 2003-04-17 Human Genome Sciences, Inc. Albumin fusion proteins
ATE430580T1 (de) 2001-10-25 2009-05-15 Genentech Inc Glycoprotein-zusammensetzungen
AU2002364586A1 (en) 2001-12-21 2003-07-30 Delta Biotechnology Limited Albumin fusion proteins
US20040093621A1 (en) 2001-12-25 2004-05-13 Kyowa Hakko Kogyo Co., Ltd Antibody composition which specifically binds to CD20
US8491896B2 (en) 2002-06-14 2013-07-23 Immunomedics, Inc. Anti-pancreatic cancer antibodies
US7736652B2 (en) 2002-03-21 2010-06-15 The Regents Of The University Of California Antibody fusion proteins: effective adjuvants of protein vaccination
WO2003084570A1 (en) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. DRUG CONTAINING ANTIBODY COMPOSITION APPROPRIATE FOR PATIENT SUFFERING FROM FcϜRIIIa POLYMORPHISM
JPWO2003085118A1 (ja) 2002-04-09 2005-08-11 協和醗酵工業株式会社 抗体組成物の製造方法
WO2003085119A1 (en) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. METHOD OF ENHANCING ACTIVITY OF ANTIBODY COMPOSITION OF BINDING TO FcϜ RECEPTOR IIIa
CN1930288B (zh) 2002-04-09 2012-08-08 协和发酵麒麟株式会社 基因组被修饰的细胞
CA2481920A1 (en) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Antibody composition-containing medicament
ES2362419T3 (es) 2002-04-09 2011-07-05 Kyowa Hakko Kirin Co., Ltd. Células con depresión o deleción de la actividad de la proteína que participa en el transporte de gdp-fucosa.
EP2301959A3 (en) * 2002-04-09 2013-03-13 The Scripps Research Institute Motif-grafted hybrid polypeptides and uses thereof
ATE386810T1 (de) 2002-06-14 2008-03-15 Dyax Corp Rekombination von nukleinsäurebibliotheksmitgliedern
WO2004009664A2 (en) 2002-07-19 2004-01-29 Omeros Corporation Biodegradable triblock copolymers, synthesis methods therefor, and hydrogels and biomaterials made there from
EP1391213A1 (en) 2002-08-21 2004-02-25 Boehringer Ingelheim International GmbH Compositions and methods for treating cancer using maytansinoid CD44 antibody immunoconjugates and chemotherapeutic agents
US7361740B2 (en) 2002-10-15 2008-04-22 Pdl Biopharma, Inc. Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis
PT1572744E (pt) 2002-12-16 2010-09-07 Genentech Inc Variantes de imunoglobulina e utilizações destas
AU2004205684A1 (en) 2003-01-23 2004-08-05 Genentech, Inc. Methods for producing humanized antibodies and improving yield of antibodies or antigen binding fragments in cell culture
WO2005007809A2 (en) 2003-05-30 2005-01-27 Alexion Pharmaceuticals, Inc. Antibodies and fusion proteins that include engineered constant regions
EP1636261B1 (en) 2003-06-26 2007-08-01 MERCK PATENT GmbH Thrombopoietin proteins with improved properties
AU2004279742A1 (en) 2003-10-08 2005-04-21 Kyowa Hakko Kirin Co., Ltd. Fused protein composition
EP1705251A4 (en) 2003-10-09 2009-10-28 Kyowa Hakko Kirin Co Ltd PROCESS FOR PRODUCING ANTIBODY COMPOSITION BY RNA INHIBITION OF FUNCTION OF $ G (A) 1,6-FUCOSYLTRANSFERASE
NZ583292A (en) 2003-11-06 2012-03-30 Seattle Genetics Inc Monomethylvaline compounds capable of conjugation to ligands
BRPI0406606A (pt) 2003-11-13 2005-12-06 Hanmi Pharm Ind Co Ltd Composição farmacêutica compreendendo uma região fc de imunoglobulina como um veìculo
JPWO2005053742A1 (ja) 2003-12-04 2007-06-28 協和醗酵工業株式会社 抗体組成物を含有する医薬
CA2549956C (en) * 2003-12-22 2016-04-12 Glaxo Group Limited Immunoglobulins
WO2005082353A2 (en) 2004-02-20 2005-09-09 Arqule, Inc. Use of beta-lapachone for treating or preventing cancer
EP1576952A1 (en) 2004-03-18 2005-09-21 OctoPlus Technologies B.V. Hydrogel microspheres with improved release profile
CA2561686C (en) 2004-03-31 2014-12-02 Genentech, Inc. Humanized anti-tgf-beta antibodies
BR122019012028B1 (pt) 2004-04-13 2023-09-26 F. Hoffmann-La Roche Ag Anticorpos anti-p-selectina, molécula de ácido nucléico, vetor, e composição
CA2567655C (en) 2004-06-02 2015-06-30 Diatech Pty Ltd Binding moieties based on shark ignar domains
TWI380996B (zh) 2004-09-17 2013-01-01 Hoffmann La Roche 抗ｏｘ４０ｌ抗體
WO2006036834A2 (en) 2004-09-24 2006-04-06 Amgen Inc. MODIFIED Fc MOLECULES
US7947839B2 (en) 2004-12-01 2011-05-24 Genentech, Inc. Heterocyclic-substituted bis-1,8 naphthalimide compounds, antibody drug conjugates, and methods of use
EP2650020B1 (en) 2005-05-06 2016-08-24 Providence Health & Services - Oregon Trimeric OX40-immunoglobulin fusion protein and methods of use
US20070160617A1 (en) 2005-06-20 2007-07-12 Psma Development Company, Llc PSMA antibody-drug conjugates
JP4616237B2 (ja) 2006-11-07 2011-01-19 日本電信電話株式会社 シリコン化合物薄膜の形成方法
AU2008248598B2 (en) 2007-05-07 2011-11-17 Novartis Ag Organic compounds
EP2170960B1 (en) * 2007-07-13 2015-07-29 Bac Ip B.V. Single-domain antigen-binding proteins that bind mammalian igg
EP2185719B1 (en) 2007-08-02 2013-11-13 NovImmune SA Anti-rantes antibodies and methods of use thereof
ES2687808T3 (es) 2007-09-26 2018-10-29 Chugai Seiyaku Kabushiki Kaisha Región constante de anticuerpo modificado
CA3049612C (en) 2008-04-24 2023-01-10 Dyax Corp. Libraries of genetic packages comprising novel hc cdr1, cdr2, and cdr3 and novel lc cdr1, cdr2, and cdr3 designs
EP2113255A1 (en) 2008-05-02 2009-11-04 f-star Biotechnologische Forschungs- und Entwicklungsges.m.b.H. Cytotoxic immunoglobulin
JP5346507B2 (ja) * 2008-06-30 2013-11-20 公益財団法人東京都医学総合研究所 抗体複合体、抗原検出方法、及び抗体複合体製造方法
US8586543B2 (en) * 2008-08-19 2013-11-19 Merck Sharp & Dohme Corp. IL-8 biomarker for monitoring cancer treatment with certain ERK inhibitors
EP2331570B1 (en) 2008-09-10 2014-08-27 Philochem AG Display library for antibody selection
EP2344545A4 (en) 2008-10-06 2012-10-03 Commw Scient Ind Res Org CRYSTALLINE STRUCTURE OF BETA-AMYLOID PEPTIDE
EP2356270B1 (en) 2008-11-07 2016-08-24 Fabrus Llc Combinatorial antibody libraries and uses thereof
US20100184684A1 (en) * 2009-01-06 2010-07-22 C3 Jian, Inc. Antibacterial and antifungal peptides
HRP20230487T1 (hr) 2009-02-13 2023-07-21 Immunomedics, Inc. Imunokonjugati s unutarstaničnom vezom koja se može rascijepiti
EP2408474B1 (en) 2009-03-18 2019-06-26 Armagen, Inc. Compositions and methods for blood-brain barrier delivery of igg-decoy receptor fusion proteins
JP2012523244A (ja) * 2009-04-14 2012-10-04 トロジャン テクノロジーズ リミテッド 治療用アンテナペディア−抗体分子およびその使用法
CA2781519A1 (en) 2009-09-16 2011-03-24 Genentech, Inc. Coiled coil and/or tether containing protein complexes and uses thereof
HUE044865T2 (hu) 2009-10-09 2019-11-28 Armagen Inc Eljárások és készítmények a központi idegrendszerben iduronát-2-szulfatáz-aktivitás növelésére
US9023996B2 (en) * 2009-12-23 2015-05-05 Synimmune Gmbh Anti-FLT3 antibodies
EP2407487A1 (en) 2010-07-14 2012-01-18 F-Star Biotechnologische Forschungs - und Entwicklungsges. M.B.H. Multispecific modular antibody
US8481038B2 (en) 2010-11-15 2013-07-09 Five Prime Therapeutics, Inc. Treatment of cancer with elevated dosages of soluble FGFR1 fusion proteins
WO2012169822A2 (ko) 2011-06-10 2012-12-13 강원대학교산학협력단 암세포 증식 억제와 혈관신생 억제를 위한 융합 단백질 및 이를 포함한 항암 조성물
EP2717924A4 (en) 2011-06-10 2015-04-22 Harvard College MODULATION OF BETA CELL PROLIFERATION IN THE PANCREATIC GLAND
CA2863224A1 (en) 2012-01-09 2013-07-18 The Scripps Research Institute Ultralong complementarity determining regions and uses thereof
JP2015509091A (ja) 2012-01-09 2015-03-26 ザ スクリプス リサーチ インスティテュート ヒト化抗体
US8956000B2 (en) 2012-07-20 2015-02-17 Stephane Martinez System and method for illumination of a rain gutter
WO2014110368A1 (en) * 2013-01-11 2014-07-17 The California Institute For Biomedical Research Bovine fusion antibodies
US20160237156A1 (en) 2013-07-11 2016-08-18 The California Institute For Biomedical Research Immunoglobulin fusion proteins and compositions thereof
US20160168231A1 (en) 2013-07-18 2016-06-16 Fabrus, Inc. Antibodies with ultralong complementarity determining regions
CN106031321A (zh) 2014-01-08 2016-10-12 探戈科技公司 高功率便携装置和对接系统

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10562980B2 (en) 2012-01-09 2020-02-18 The Scripps Research Institute Humanized antibodies
US11390665B2 (en) 2012-01-09 2022-07-19 The Scripps Research Institute Ultralong complementarity determining regions and uses thereof
US10774132B2 (en) 2012-01-09 2020-09-15 The Scripps Research Instittue Ultralong complementarity determining regions and uses thereof
US9718865B2 (en) * 2012-07-27 2017-08-01 Purdue Pharma L.P. Sodium channel blocking peptides and the use thereof
US20150191517A1 (en) * 2012-07-27 2015-07-09 Purdue Pharma L.P. Novel sodium channel blocking peptides and the use thereof
US10259863B2 (en) 2013-01-11 2019-04-16 The California Institute For Biomedical Research Bovine fusion antibodies
US9644021B2 (en) 2013-01-11 2017-05-09 The California Institute For Biomedical Research Bovine fusion antibodies
US9981956B2 (en) 2013-06-07 2018-05-29 The Scripps Research Institute Small molecule inhibitors of fibrosis
US9663499B2 (en) 2013-06-07 2017-05-30 The California Institute For Biomedical Research Small molecule inhibitors of fibrosis
US10683353B2 (en) * 2013-07-11 2020-06-16 The Scripps Research Institute Coiled coil immunoglobulin fusion proteins and compositions thereof
US11673959B2 (en) 2013-07-11 2023-06-13 The Scripps Research Institute Coiled coil immunoglobulin fusion proteins and compositions thereof
US10640574B2 (en) 2013-07-18 2020-05-05 Taurus Biosciences, Llc Humanized antibodies with ultralong complementary determining regions
US11530493B2 (en) 2013-07-18 2022-12-20 Taurus Biosciences, Llc Humanized antibodies with ultralong complementary determining regions
EP3155018A4 (en) * 2014-06-06 2018-01-10 The California Institute for Biomedical Research Constant region antibody fusion proteins and compositions thereof
US10336735B2 (en) 2014-12-10 2019-07-02 The Scripps Research Institute Small molecule inhibitors of fibrosis
WO2016115218A1 (en) * 2015-01-14 2016-07-21 The California Institute For Biomedical Research Antibody drug conjugates for the treatment of immune conditions

Also Published As

Publication number Publication date
WO2013106485A3 (en) 2014-03-06
JP6684490B2 (ja) 2020-04-22
CA2863224A1 (en) 2013-07-18
US11390665B2 (en) 2022-07-19
US20140086871A1 (en) 2014-03-27
EP2802603A4 (en) 2015-11-04
WO2013106485A2 (en) 2013-07-18
JP2018140992A (ja) 2018-09-13
AU2013208003A1 (en) 2014-08-07
CN104520321A (zh) 2015-04-15
EP2802603A2 (en) 2014-11-19
US10774132B2 (en) 2020-09-15
JP2015504896A (ja) 2015-02-16
AU2018201832A1 (en) 2018-04-12
AU2013208003B2 (en) 2017-12-14
US20200181241A1 (en) 2020-06-11

Similar Documents

Publication Publication Date Title
US11390665B2 (en) Ultralong complementarity determining regions and uses thereof
US20230287598A1 (en) Humanized antibodies with ultralong complementary determining regions
US10562980B2 (en) Humanized antibodies
US9644021B2 (en) Bovine fusion antibodies
US20160168231A1 (en) Antibodies with ultralong complementarity determining regions

Legal Events

Date Code Title Description
AS Assignment

Owner name: THE CALIFORNIA INSTITUTE FOR BIOMEDICAL RESEARCH,

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SCHULTZ, PETER;WANG, FENG;ZHANG, YONG;SIGNING DATES FROM 20140828 TO 20140905;REEL/FRAME:033790/0495

AS Assignment

Owner name: THE SCRIPPS RESEARCH INSTITUTE, CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SMIDER, VAUGHN;EKIERT, DAMIAN;SCHULTZ, PETER;AND OTHERS;SIGNING DATES FROM 20140324 TO 20141212;REEL/FRAME:034520/0989

Owner name: FABRUS, INC., CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SMIDER, VAUGHN;BAZIRGAN, OMAR A.;MAO, HONGYUAN HELEN;SIGNING DATES FROM 20140705 TO 20140709;REEL/FRAME:034521/0133

AS Assignment

Owner name: THE SCRIPPS RESEARCH INSTITUTE, CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:FABRUS, INC.;REEL/FRAME:034533/0961

Effective date: 20140709

Owner name: THE SCRIPPS RESEARCH INSTITUTE, CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:THE CALIFORNIA INSTITUTE FOR BIOMEDICAL RESEARCH;REEL/FRAME:034533/0858

Effective date: 20141215

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION