WO2014164067A1 - Constructions de liaison à l'antigène se liant à cd30 - Google Patents

Constructions de liaison à l'antigène se liant à cd30 Download PDF

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WO2014164067A1
WO2014164067A1 PCT/US2014/020318 US2014020318W WO2014164067A1 WO 2014164067 A1 WO2014164067 A1 WO 2014164067A1 US 2014020318 W US2014020318 W US 2014020318W WO 2014164067 A1 WO2014164067 A1 WO 2014164067A1
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antigen binding
binding construct
seq
diabody
cys
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PCT/US2014/020318
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English (en)
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David T. HO
Tove Olafsen
Giti AGAHI
Jean Gudas
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Imaginab, Inc.
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Publication of WO2014164067A1 publication Critical patent/WO2014164067A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/53—Hinge
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components

Definitions

  • Embodiments described herein relate generally to antigen binding constructs, such as antibodies, including antibody fragments, that bind to CD30, (such as minibodies, cys-diabodies, and scFv), as well as methods for their use.
  • antigen binding constructs such as antibodies, including antibody fragments, that bind to CD30, (such as minibodies, cys-diabodies, and scFv), as well as methods for their use.
  • CD30 also known as tumor necrosis factor receptor superfamily, member 8 or TNFRSF8 is a member of the TNF superfamily and the receptor for the CD30 ligand. CD30 is found on activated but not resting B and T lymphocytes and other immune cells. CD30 is also expressed on a variety of hematopoietic cell tumors including Hodgkins lymphoma (HL), Anaplastic Large Cell Lymphoma (ALCL) a subset of Cutaneous T Cell Lymphomas (CTCL) and on rare solid tumors such as embryonal carcinomas CD30 is overexpressed in some carcinomas and lymphomas (including HL, ALCL, CTCL and other T lymphoproliferative disorders).
  • HL Hodgkins lymphoma
  • ALCL Anaplastic Large Cell Lymphoma
  • CCL Cutaneous T Cell Lymphomas
  • CD30 is overexpressed in some carcinomas and lymphomas (including HL, ALCL, CTCL and other T lymphoproliferative disorders).
  • an antigen binding construct is provided.
  • the antigen binding construct can comprise a HCDR1 of the HCDR1 in SEQ ID NO: 9 or 17, a HCDR2 of the HCDR2 in SEQ ID NO: 9 or 17, a HCDR3 of the HCDR3 in SEQID NO: 9 or 17, a LCDR1 of the LCDR1 in SEQ ID NO: 3, 6, or 13, a LCDR2 of the LCDR2 in SEQ ID NO: 3, 6, or 13; and a LCDR3 of the LCDR3 in SEQ ID NO: 3, 6, or 13.
  • an antigen binding construct can comprise a LFR1 of the LFR1 in SEQ ID NO: 3, 6, or 13, a LFR2 of the LFR2 in SEQ ID NO: 3, 6, or 13, a LFR3 of the LFR3 in SEQ ID NO: 3, 6, or 13, a LFR4 of the LFR4 in SEQ ID NO: 3, 6, or 13, a HFRl of the HFRl in SEQ ID NO: 9 or 17, a HFR2 of the HFR2 in SEQ ID NO: 9 or 17, a HFR3 of the HFR3 in SEQ ID NO: 9 or 17; or a HFR4 of the HFR4 in SEQ ID NO: 9 or 17.
  • a humanized cys-diabody that binds to CD30 is provided.
  • the humanized cys-diabody can comprise a polypeptide that comprises a single- chain variable fragment (scFv) comprising a variable heavy (V H ) domain linked to a variable light (Vj_), and C-terminal Cysteine.
  • scFv single- chain variable fragment
  • V H variable heavy
  • Vj_ variable light
  • a humanized minibody that binds to CD30 can comprise a polypeptide that comprises a single- chain variable fragment (scFv) that binds to CD30, the scFv comprising a variable heavy (V H ) domain linked a variable light (Vj_) domain, a hinge-extension domain comprising a human IgGl hinge region; and a human IgG CH3 sequence.
  • scFv single-chain variable fragment
  • nucleic acid encoding an antigen binding construct of any one of the antigen binding constructs is provided.
  • a cell line producing an antigen binding construct of any one of the antigen binding constructs is provided.
  • kits can comprise an antigen binding construct as described herein and a detectable marker.
  • a method of detecting the presence or absence of a CD30 is provided.
  • the method can comprise applying an antigen binding construct thereof to a sample, and detecting a binding or an absence of binding of the antigen binding construct thereof to CD30.
  • a method of targeting a therapeutic agent to CD30 is provided. The method can comprise administering to a subject an antigen binding construct, wherein the antigen binding construct is conjugated to a therapeutic agent.
  • a method of neutralizing a T cell or B cell in a subject in need thereof is provided.
  • the method can comprise administering to the subject an antigen binding construct.
  • FIG. 1A is a depiction of the anti-CD30 minibody in the V H V L orientation.
  • the minibody forms a covalently bound homodimer that can bind two antigens (for example, CD30).
  • FIG. IB is a depiction of the anti-CD30 cys-diabody in the V L V H orientation.
  • the shortened linker forces cross-pairing of two scFv enabling binding to two antigens and the formation of a covalent bond between the two terminal cysteines.
  • FIG. 1C is a depiction of the assembled cDNA gene expression construct for anti-CD30 minibody in V L V H orientation.
  • SP signal peptide
  • V H variable heavy domain
  • V L variable light domain
  • C H 3 third constant domain
  • L linker
  • H/E hinge/extension
  • FIG. ID is a depiction of the assembled cDNA gene expression construct for the anti-CD30 cys-diabody in V L V H orientation.
  • SP signal peptide
  • V H variable heavy domain
  • V L variable light domain
  • L linker
  • GGC glycine, glycine, cysteine. Any of the sequences provided herein can be used in any combination provided in FIGs. 1C and/or ID.
  • FIG. IE is a flow chart depicting some embodiments of methods provided herein.
  • FIGs. 2A, 2B and 2C depict sequences showing the humanization of antigen binding constructs to the target molecule.
  • FIGs. 3A, 3B, and 3C, and 3D depict some variable light chain sequences that can be used for antigen binding constructs against the target molecule.
  • FIGs. 3E, 3F, and 3G depict some variable heavy chain sequences that can be used for antigen binding constructs to the target molecule.
  • FIG. 4A depicts some sequences that can be used for various cys-diabodies (for example, within the arrangement of FIG. ID).
  • FIG. 4B depicts some sequences that can be used for various minibodies (for example, within the arrangement of FIG. 1C).
  • FIG. 4C depicts some sequences of hinge regions that can be employed in various embodiments.
  • FIG. 5 depicts a map of pcDNA 3.1/wyc-His(-) A, B, C vector.
  • FIG. 6A depicts a western blot of minibodies. Supernatants were run on SDS-PAGE and transferred to PVDF membrane. Chimeric and Version A humanized minibody variants were detected with horse radish peroxidase (HRP) conjugated anti-human IgG (Fc-specific). Shown are representative blots of multiple experiments.
  • HRP horse radish peroxidase
  • FIG. 6B depicts a western blot of cys-diabodies. Supernatants were run on SDS-PAGE and transferred to PVDF membrane. Version A humanized cys-diabody variants were detected with anti-penta-His HRP antibody. Each blot was developed after incubation with the HRP substrate 3,3',5,5'-tetramethylbenzidine (TMB). Shown are representative blots of multiple experiments.
  • TMB 3,3',5,5'-tetramethylbenzidine
  • FIG. 7A depicts flow cytometry data detecting binding of antigen binding constructs to CD30 expressed on the surface of HH cells.
  • FIG. 7B depicts binding curves were generated to provide EC50 values.
  • the amount of minibody in the transfection supernatant was quantified by ELISA.
  • the EC50 values for the minibodies were between 0.158 nM to 0.375 nM.
  • FIG. 8 depicts flow cytometry analysis of cys-diabody variants. Binding of cys-diabody variants to HH cells was detected with allophycocyanin (APC)-conjugated anti- His antibody. 1X10 3 HH cells were incubated with indicated dilutions of the cys-diabody supernatant and flow cytometric analysis was performed with 10,000 events/point. All histograms show APC signal vs. cell count. Staining with the secondary antibody alone was used as a negative control (red).
  • FIG. 9 A is an image of a gel for version Bl humanized minibody variants detected with horse radish peroxidase (HRP) conjugated anti-human IgG (Fc-specific).
  • HRP horse radish peroxidase
  • FIG. 9B is an image of a gel for version Bl humanized cys-diabody variants detected with horse radish peroxidase (HRP) conjugated anti-penta-His antibody.
  • HRP horse radish peroxidase
  • FIG. 10 provides a sequence of a CD30 protein.
  • antigen binding constructs including antibodies and fragments thereof, such as scFv, cys-diabodies, and minibodies, that bind to a target molecule, CD30.
  • Such antigen binding constructs can be useful for detecting the presence, localization, and/or quantities of the target molecule (CD30 and/or CD30+ cells).
  • Such antigen binding constructs can also be useful for targeting therapeutic agents to cells that express the target molecule.
  • methods are provided for detecting the presence or absence of the target molecule (or "target") using antigen binding constructs (including antibodies, and constructs such as scFv, cys-diabodies, and/or minibodies).
  • methods are provided for using the antigen binding constructs for therapeutic purposes.
  • the antigen binding constructs provided herein have specific targeting of CD30 that are superior for diagnostic imaging. Since background expression of CD30 in normal tissue is low, CD30 can be a target for therapy. Therapeutic drugs are in development against CD30. However, there are no known imaging agents currently in development which specifically target CD30.
  • Methods of diagnostic imaging include using positron emission tomography (PET) and 18 F fluorodeoxyglucose ( 18 F-FDG) as tracers, can rely on the detection of increased glucose metabolism in cancer cells.
  • 18 F-FDG can have limitations in sensitivity and specificity for some cancers, for example prostate and ovarian cancers that have lower metabolic activity. Additionally, false positives may arise from inflamed or infected tissues when using 18 F-FDG.
  • Antigen binding constructs according so some embodiments herein bind specifically to CD30 expressed on the cell surface, making false readings less likely in comparison to tracers that target cell metabolism. In some embodiments, antigen binding constructs can be used for imaging hematopoietic cancers and/or for metastatic disease.
  • the antigen binding construct approaches for targeting CD30 can harness the binding affinity and specificity advantage of antigen binding constructs compared to other approaches such as small-molecule or peptides.
  • the antigen binding constructs have highly specific binding, which can achieve very strong binding affinities (greater than picomolar binding affinities).
  • Antigen binding constructs can exhibit higher binding specificity and affinity than other formats for binding targets such as small molecules or peptides.
  • antibody fragments allow for a distinct advantage over a full-length antibody for imaging.
  • the scFv, minibody, and cys-diabody antibody fragments can have superior pharmacokinetic properties for faster diagnostic imaging while maintaining the binding specificity and affinity of the parental antibody.
  • Current technology utilizes imaging with the full length antibodies which often requires significantly longer times (-7-8 days post-injection) to produce high contrast images due to the slow serum clearance of the intact antibody.
  • Same-day or next-day imaging also provides a logistical solution to the problem facing many patients who travel great distances to receive treatment/diagnosis since the duration of travel stays or the need to return one week later would be eliminated when imaging with minibody or cys- diabody fragments versus intact antibodies.
  • the CD30 antigen binding constructs are for diagnostics.
  • an appropriate radionuclide e.g., the positron emitter Iodine-124, Copper-64, and Zirconium-89 for PET imaging
  • fluorophore for fluorescent imaging
  • the antibody fragments can be used for preclinical imaging of CD30 as shown herein.
  • These CD30 antigen binding constructs can also be used as potential SPECT imaging agents by simply changing the radiolabel.
  • the DOTA- conjugated minibody can be radiometal labeled with a SPECT radionuclide such as Indium- 111.
  • the antigen binding constructs can be clinical imaging agents (PET/SPECT) in humans. Since CD30 is expressed in multiple cancer types (including a variety of hematopoietic cell tumors including Hodgkins lymphoma (HL), Anaplastic Large Cell Lymphoma (ALCL), and a subset of Cutaneous T Cell Lymphomas (CTCL), other T cell lymphoproliferative disorders, and on rare solid tumors such as embryonal carcinomas, and on the surface of activated immune cells in diabetes, asthma, colitis, graft versus host disease (GVHD), atherosclerosis and other inflammatory conditions.
  • HL Hodgkins lymphoma
  • ACL Anaplastic Large Cell Lymphoma
  • CTCL Cutaneous T Cell Lymphomas
  • rare solid tumors such as embryonal carcinomas
  • GVHD graft versus host disease
  • these CD30 antigen binding constructs can be used for targeted diagnostic detection for these cancers, and/or for determining the status of inflammatory diseases that may influence treatments. Since CD30 is expressed on activated T and B cells, CD30 antigen binding constructs can be used for diagnoses of inflammatory and autoimmune diseases.
  • treating or “treatment” of a condition may refer to preventing the condition, slowing the onset and/or rate of development of the condition, reducing the risk of developing the condition, preventing and/or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, generating a complete or partial regression of the condition, or some combination thereof.
  • prevent does not require the absolute prohibition of the disorder or disease.
  • a “therapeutically effective amount” or a “therapeutically effective dose” is an amount that produces a desired therapeutic effect in a subject, such as preventing, treating a target condition, delaying the onset of the disorder and/or symptoms, and/or alleviating symptoms associated with the condition. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and/or the route of administration.
  • the term "antigen binding construct” includes all varieties of antibodies, including binding fragments thereof. Further included are constructs that include 1, 2, 3, 4, 5, and/or 6 CDRs. In some embodiments, these CDRs can be distributed between their appropriate framework regions in a traditional antibody. In some embodiments, the CDRs can be contained within a heavy and/or light chain variable region. In some embodiments, the CDRs can be within a heavy chain and/or a light chain. In some embodiments, the CDRs can be within a single peptide chain. In some embodiments, the CDRs can be within two or more peptides that are covalently linked together. In some embodiments, they can be covalently linked together by a disulfide bond.
  • the antigen binding proteins are non- covalent, such as a diabody and a monovalent scFv. Unless otherwise denoted herein, the antigen binding constructs described herein bind to the noted target molecule.
  • target or “target molecule” denotes the CD30 protein. Examples of CD30 proteins are known in the art, and include, for example the CD30 protein of SEQ ID NO: 41, in FIG. 10.
  • antibody includes, but is not limited to, genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, chimeric antibodies, fully human antibodies, humanized antibodies, antibody fragments, scFv, and heteroconjugate antibodies (for example, bispecific antibodies, diabodies, triabodies, tetrabodies, etc.).
  • antibody includes cys-diabodies, scFv, and minibodies.
  • scFv cys-diabody
  • minibody embodiments unless explicitly denoted otherwise.
  • antibody includes a polypeptide 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.
  • a full length antibody can be composed of two identical pairs of polypeptide chains, each pair having one "light” and one "heavy” chain (connected through a disulfide bond.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • variable light chain (V L ) and variable heavy chain (V H ) refer to these regions of light and heavy chains respectively.
  • an "antibody” encompasses all variations of antibody and fragments thereof.
  • antibodies within the scope of this concept are full length antibodies, chimeric antibodies, humanized antibodies, single chain antibodies (scFv), Fab, Fab', and multimeric versions of these fragments (for example, F(ab') 2 ) with the same binding specificity.
  • the antibody binds specifically to a desired target.
  • CDRs complementarity-determining domains
  • V L and V H The CDRs are the target protein-binding site of the antibody chains that harbors specificity for such target protein.
  • CDRl-3 there are three CDRs (CDRl-3, numbered sequentially from the N-terminus) in each V L and/or V H , constituting about 15- 20% of the variable domains.
  • the CDRs are structurally complementary to the epitope of the target protein and are thus directly responsible for the binding specificity.
  • the remaining stretches of the V L or V H the so-called framework regions (FRs), exhibit less variation in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4. W.H. Freeman & Co., New York, 2000).
  • the positions of the CDRs and framework regions can be determined using various well known definitions in the art, for example, Kabat (Wu, T. T., E. A. Kabat. 1970. An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity. J. Exp. Med. 132; 211-250; Kabat, E. A., Wu, T. T., Perry, H., Gottesman, K., and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242, Bethesda, MD), Chothia Chothia and Lesk, J. Mol.
  • IMGT/LIGM-DB the IMGT® comprehensive database of immunoglobulin and T cell receptor nucleotide sequences Nucl. Acids Res., 34, D781-D784 (2006), PMID: 16381979; Lefranc, M.-P., Pommie, C, Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, G., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains Dev. Comp. Immunol., 27, 55-77 (2003).
  • binding specificity determinant or “BSD” interchangeably refer to the minimum contiguous or non-contiguous amino acid sequence within a complementarity determining region necessary for determining the binding specificity of an antibody.
  • a minimum binding specificity determinant can be within one or more CDR sequences.
  • the minimum binding specificity determinants reside within (i.e., are determined solely by) a portion or the full-length of the CDR3 sequences of the heavy and light chains of the antibody.
  • CDR3 of the heavy chain variable region is sufficient for the antigen binding construct specificity.
  • an "antibody variable light chain” or an “antibody variable heavy chain” as used herein refers to a polypeptide comprising the V L or V H , respectively.
  • the endogenous V L is encoded by the gene segments V (variable) and J (junctional), and the endogenous V H by V, D (diversity), and J.
  • Each of V L or V H includes the CDRs as well as the framework regions.
  • antibody variable light chains and/or antibody variable 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 V L or V H , as one skilled in the art will readily recognize.
  • full length heavy and/or light chains are contemplated.
  • only the variable regions of the heavy and/or light chains are contemplated as being present.
  • Antibodies can exist as intact immunoglobulins or as a number of fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2 , a dimer of Fab' which itself is a light chain (V L -C L ) joined to V H -C H 1 by a disulfide bond.
  • the F(ab)' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)' 2 dimer into an Fab' monomer.
  • the Fab' monomer is a Fab with part of the hinge region.
  • antibody While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology.
  • antibody also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (for example, single chain Fv) or those identified using phage display libraries (see, for example, McCafferty et al., Nature 348:552-554 (1990)).
  • any technique known in the art can be used (see, for example, Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96. Alan R. Liss, Inc. 1985; Advances in the production of human monoclonal antibodies Shixia Wang, Antibody Technology Journal 2011:1 1-4; J Cell Biochem. 2005 Oct 1;96(2):305-13; Recombinant polyclonal antibodies for cancer therapy; Sharon J, Liebman MA, Williams BR; and Drug Discov Today.
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain.
  • import residues which are typically taken from an import variable domain.
  • the terms "donor” and "acceptor” sequences can be employed.
  • humanization can be essentially performed following the method of Winter and co-workers (see, for example, Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non- human species.
  • humanized antibodies are typically human antibodies in which some complementarity determining region ("CDR") residues and possibly some framework (“FR”) residues are substituted by residues from analogous sites
  • a "chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, for example, an enzyme, toxin, hormone, growth factor, and drug; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • Antibodies further include one or more immunoglobulin chains that are chemically conjugated to, or expressed as, fusion proteins with other proteins. It also includes bispecific antibodies.
  • a bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.
  • antigen-binding fragments or antibody portions of the invention include bivalent scFv (diabody), bispecific scFv antibodies where the antibody molecule recognizes two different epitopes, single binding domains (sdAb or nanobodies), and minibodies.
  • antibody fragment includes, but is not limited to one or more antigen binding fragments of antibodies alone or in combination with other molecules, including, but not limited to Fab', F(ab') 2 , Fab, Fv, rlgG (reduced IgG), scFv fragments, single domain fragments (nanobodies), peptibodies, minibodies, diabodies, and cys-diabodies.
  • scFv refers to a single chain Fv ("fragment variable”) antibody in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form one chain.
  • a pharmaceutically acceptable carrier may be a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body.
  • the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof.
  • Each component of the carrier is "pharmaceutically acceptable” in that it is be compatible with the other ingredients of the formulation. It also must be suitable for contact with any tissue, organ, or portion of the body that it may encounter, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.
  • compositions described herein may be administered by any suitable route of administration.
  • a route of administration may refer to any administration pathway known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, transdermal (for example, topical cream or ointment, patch), or vaginal.
  • Transdermal administration may be accomplished using a topical cream or ointment or by means of a transdermal patch.
  • “Parenteral” refers to a route of administration that is generally associated with injection, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.
  • the antigen binding construct can be delivered intraoperatively as a local administration during an intervention or resection.
  • the term "CD30 dependent disorder” includes any disorder in which CD30 plays a role in the disorder itself. In some embodiments, this denotes over-expression of CD30. Examples of the disorders include, multiple cancer types, such as different carcinomas and lymphomas (including HL, ALCL, CTCL and other T lymphoproliferative disorders).
  • a minibody is an antibody format that has a smaller molecular weight than the full-length antibody while maintaining the bivalent binding property against an antigen. Because of its smaller size, the minibody has a faster clearance from the system and enhanced penetration when targeting tumor tissue. With the ability for strong targeting combined with rapid clearance, the minibody is advantageous for diagnostic imaging and delivery of cytotoxic/radioactive payloads for which prolonged circulation times may result in adverse patient dosing or dosimetry.
  • a biological sample for example, a blood, serum, plasma or tissue sample.
  • the antibodies or binding agents with a particular binding specificity bind to a particular antigen at least two times the background and do not substantially bind in a significant amount to other antigens present in the sample.
  • Specific binding to an antibody or binding agent under such conditions may require the antibody or agent to have been selected for its specificity for a particular protein.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, for example, Harlow & Lane, Using Antibodies, A Laboratory Manual (1998), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • a specific or selective binding reaction will produce a signal at least twice over the background signal and more typically at least than 10 to 100 times over the background.
  • Equilibrium dissociation constant (3 ⁇ 4, M) refers to the dissociation rate constant (k d , time “1 ) divided by the association rate constant (k a , time "1 , M “1 ). Equilibrium dissociation constants can be measured using any known method in the art.
  • the antibodies of the present invention generally will have an equilibrium dissociation constant of less than about 10 - " 7 or 10 - " 8 M, for example, less than about 10 - " 9 M or 10 - " 10 M, in some embodiments, less than about 10 "11 M, 10 "12 M, or 10 "13 M.
  • nucleic acid or protein when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. In some embodiments, it can be in either a dry or aqueous solution. Purity and homogeneity can be determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames that flank the gene and encode a protein other than the gene of interest. The term "purified” denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. In some embodiments, this can denote that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure of molecules that are present under in vivo conditions.
  • nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double- stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (for example, degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • polypeptide As used interchangeably herein 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 polymer.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar 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, for example, hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, for example, an alpha-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, for example, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (for example, 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 in a manner similar to a naturally occurring amino acid.
  • Constantly modified variants applies to both amino acid and nucleic 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 sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • 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 every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • 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" 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. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: 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, for example, Creighton, Proteins (1984)).
  • Percentage of sequence identity can be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (for example, a polypeptide of the invention), which does not comprise additions or deletions, for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same sequences.
  • Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (for example, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over a specified region, or, when not specified, over the entire sequence of a reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides, respectively, exemplified herein (for example, any one or more of the variable regions exemplified in any one of FIGs. 2A, 2B 2C, 3A, 3B, 3C, 3D, 3E, 3F, and 3G; any one or more of the CDRs exemplified in any one of FIGs.
  • the identity exists over a region that is at least about 15, 25 or 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length, or over the full length of the reference sequence.
  • identity or substantial identity can exist over a region that is at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, optionally at least about 150, 200 or 250 amino acids in length, or over the full length of the reference sequence.
  • shorter amino acid sequences for example, amino acid sequences of 20 or fewer amino acids, in some embodiments, substantial identity exists when one or two amino acid residues are conservatively substituted, according to the conservative substitutions defined herein.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol.
  • BLAST and BLAST 2.0 algorithms Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra).
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
  • the terms "subject,” “patient,” and “individual” interchangeably refer to an entity that is being examined and/or treated. This can include, for example, a mammal, for example, a human or a non-human primate mammal.
  • the mammal can also be a laboratory mammal, for example, mouse, rat, rabbit, hamster.
  • the mammal can be an agricultural mammal (for example, equine, ovine, bovine, porcine, camelid) or domestic mammal (for example, canine, feline).
  • therapeutically acceptable amount or “therapeutically effective dose” interchangeably refer to an amount sufficient to effect the desired result. In some embodiments, a therapeutically acceptable amount does not induce or cause undesirable side effects. A therapeutically acceptable amount can be determined by first administering a low dose, and then incrementally increasing that dose until the desired effect is achieved.
  • co-administer refers to the administration of two active agents in the blood of an individual or in a sample to be tested. Active agents that are coadministered can be concurrently or sequentially delivered.
  • Antigen binding constructs including antibodies and binding fragments
  • Antigen binding constructs that bind to the target are described herein.
  • An antigen binding construct is a molecule that includes one or more portions of an immunoglobulin or immunoglobulin-related molecule that specifically binds to, or is immunologically reactive with the target molecule.
  • Anti-CD30 antibody fragments such as minibodies and cys-diabody fragments are provided in some embodiments.
  • the antibody fragments can be used, for example, for imaging. Schematic representations of exemplary minibody and cys-diabody fragments are illustrated in FIGs. 1A-1D.
  • an antigen binding construct includes a heavy chain CDRl (HCDRl) of the HCDR 1 in SEQ ID NOs: 7, 9, or 17; a heavy chain CDR2 (HCDR2) of the HCDR2 in SEQ ID NOs: 7, 9, or 17; a heavy chain CDR3 (HCDR3) of the HCDR3 in SEQ ID NOs: 7, 9, or 17; a light chain CDRl (LCDR1) of the LCDR1 in SEQ ID NOs: 1, 3, 4, 6, or 13; a light chain CDR2 (LCDR2) of the LCDR2 in SEQ ID NOs: 1, 3, 4, 6, or 13; and/or a light chain CDR3 (LCDR3) of the LCDR3 in SEQ ID NOs: 1, 3, 4, 6, or 13.
  • HCDRl heavy chain CDRl
  • HCDR2 heavy chain CDR2
  • HCDR3 HCDR3
  • the antigen binding construct includes 6, 5, 4, 3, 2, or 1, the above CDRs (some embodiments of the CDRs are indicated in FIGs. 2A, 2B, 2C, 3A, 3B, 3C, 3D, 3E, 3F, and 3G).
  • the antigen binding construct includes HCDR3.
  • the antigen binding construct binds specifically to the target molecule.
  • the antigen binding construct competes for binding with one or more of the antibodies having the herein provided CDRs.
  • the antigen binding construct includes at least the 3 heavy chain CDRs noted herein. In some embodiments, the antigen binding construct includes heavy chain CDR3. In some embodiments, the antigen binding construct further includes any one of the heavy chain CDR2 sequences provided herein.
  • the antigen binding construct is human or humanized.
  • the antigen binding construct includes at least one human framework region, or a framework region with at least about 80% sequence identity, for example at least about 80%, 85%, 90%, 93%, 95%, 97%, or 99% identity to a human framework region.
  • the antigen binding construct includes a heavy chain FRl (HFRl) of the HFRl in SEQ ID NO: 7, 8, 9, or 17; a heavy chain FR2 (HFR2) of the HFR2 in SEQ ID NO: 7, 8, 9, or 17; a heavy chain FR3 (HFR3) of the HFR3 in SEQ ID NO: 7, 8, 9, or 17; a heavy chain FR4 (HFR4) of the HFR4 in SEQ ID NO: 7, 8, 9, or 17; a light chain FRl (LFR1) of the LFR1 in SEQ ID NO: 1, 3, 4, 6, or 13; a light chain FR2 (LFR2) of the LFR2 in SEQ ID NO: 1, 3, 4, 6, or 13; a light chain FR3 (LFR3) of the LFR3 in SEQ ID NO: 1, 3, 4, 6, or 13; and a light chain FR4 (LFR4) of the LFR4 in SEQ ID NO: 1, 3, 4, 6, or 13.
  • the antigen binding construct includes 8, 7, 6, 5, 4,
  • the antigen binding construct includes a detectable marker. In some embodiments, the antigen binding construct includes a therapeutic agent.
  • the antigen binding construct is bivalent.
  • Bivalent antigen binding construct can include at least a first antigen binding domain, for example a first scFv, and at least a second antigen binding domain, for example a second scFv.
  • a bivalent antigen binding construct is a multimer that includes at least two monomers, for example at least 2, 3, 4, 5, 6, 7, or 8 monomers, each of which has an antigen binding domain.
  • the antigen binding construct is a minibody.
  • the antigen binding construct is a diabody, including, for example, a cys- diabody.
  • the minibody and/or the cys-diabody can include any of the CDR and heavy chain variable region and/or light chain variable region embodiments provided herein (for example, the CDR sequences provided in FIGs. 2A, 2B, 2C, 3A, 3B, 3C, 3D, 3E, 3F, and 3G.
  • the antigen binding construct is a monovalent scFv.
  • a monovalent scFv is provided that includes the HCDRl in the HCDRl of FIG. 2C, 3E, 3F, or 3G; the HCDR2 in the HCDR2 of FIG. 2C, 3E, 3F, or 3G, the HCDR3 in the HCDR3 of FIG.
  • the monovalent scFv includes the heavy chain variable region of the heavy chain variable region in 2C, 3E, 3F, or 3G. In some embodiments, the monovalent scFv includes the light chain variable region of the light chain variable region in 2A, 2B, 3A, 3B, 3C, or 3D.
  • the monovalent scFv includes the heavy chain variable region of the heavy chain variable region in FIG. 2C, 3E, 3F, or 3G and the light chain variable region of the light chain variable region in FIG. 2A, 2B, 3A, 3B, 3C, or 3D.
  • the antigen binding construct is arranged as outlined in tables 0.1 and 0.2 below:
  • the construct can include any of the constructs on a single row in Table 0.1 or Table 0.2. In some embodiments, the constructs can include any combination in Table 0.1 or Table 0.2.
  • the first item in the first row, column 2 can be combined with the first row, column 3 to the first row column 4, to the first row column 5, to the first row, column 6.
  • column 3 and column 6 can be swapped with one another.
  • the first item in the first row, column 2 can be combined with the first row, column 3 to the second row column 4, to the second row column 5, to the second row, column 6.
  • the tables represent all possible combinations, both within a single row and across various rows (and with columns swapped).
  • the antigen binding construct is bispecific.
  • Bispecific constructs can include at least a first binding domain, for example an scFv that binds specifically to a first epitope, and at least a second binding domain, for example an scFv that binds specifically to a second epitope.
  • bispecific antigen binding constructs can bind to two or more epitopes.
  • the first epitope and the second epitope are part of the same antigen, and the bispecific antigen binding construct can thus bind to two epitopes of the same antigen.
  • the first epitope is part of a first antigen
  • the second epitope is part of a second antigen
  • the bispecific antigen binding construct can thus bind to two different antigens.
  • the antigen binding construct binds to two epitopes simultaneously.
  • the antigen binding construct has a heavy chain variable region of the heavy chain variable region in SEQ ID NO: 9.
  • the antigen binding construct has a heavy chain variable region that includes a sequence with at least about 80% identity, for example at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9.
  • the antigen binding construct has a heavy chain variable region of the heavy chain variable region in SEQ ID NO 17.
  • the antigen binding construct has a heavy chain variable region that includes a sequence with at least about 80% identity, for example at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 17.
  • the antigen binding construct has a heavy chain variable region of the heavy chain variable region in SEQ ID NO 8.
  • the antigen binding construct has a heavy chain variable region that includes a sequence with at least about 80% identity, for example at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8.
  • the antigen binding construct has a light chain variable region that includes SEQ ID NO: 3.
  • the antigen binding construct has a light chain variable region that includes a sequence with least about 80% identity, for example at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 3.
  • the antigen binding construct has a light chain variable region that includes SEQ ID NO: 6.
  • the antigen binding construct has a light chain variable region that includes a sequence with least about 80% identity, for example at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 6.
  • the antigen binding construct has a light chain variable region that includes SEQ ID NO: 13.
  • the antigen binding construct has a light chain variable region that includes a sequence with least about 80% identity, for example at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 13.
  • the antigen binding construct is a human antigen binding construct and has a heavy chain variable region, a light chain variable region, or a heavy and light chain that is at least as identical as at least the heavy and/or light chain variable sequences noted above.
  • Some embodiments provided herein include an antigen binding construct that competes for binding to the target molecule with one or more antigen binding constructs provided herein.
  • the competing antigen binding construct binds to the same epitope on the target molecule as the reference antigen binding construct.
  • the reference antigen binding construct binds to a first epitope of the target molecule
  • the competing antigen binding construct binds to a second epitope of the target molecule, but interferes with binding of the reference antigen binding construct to the target molecule, for example by sterically blocking binding of the reference antigen binding construct, or by inducing a conformational change in the target molecule.
  • the first epitope overlaps with the second epitope.
  • the scFv, minibody, and cys-diabody formats have advantageous pharmacokinetic characteristics for diagnostic imaging and certain therapeutic applications while maintaining the high binding affinity and specificity of a parental antibody. Compared to imaging with the full-length parental antibody, the pharmacokinetics are more desirable for these fragments in that they are able to target the antigen and then rapidly clear the system for rapid high-contrast imaging.
  • the shorter serum half-lives for the minibody and the cys-diabody allow for optimal imaging over a range of times, approximately 8- 48 hours post injection for the minibody and 2-24 hours post- injection for the cys-diabody. The rapid serum clearance together with better tissue penetration can allow for same day imaging, providing a significant advantage in the clinic with respect to patient care management.
  • the cys-diabody antibody format features the C-terminus cysteine tail.
  • These two sulfhydryl groups provide a strategy for site-specific conjugation of functional moieties such as radiolabels that need not interfere with the cys-diabody' s binding activity.
  • the CD30 antibody fragments can comprise one, two, or three of the variable light region CDRs and/or one, two, or three of the variable heavy region CDRs from an anti-CD30 antibody.
  • an antibody fragment may contain one, two or three of the variable region CDRs and/or one, two, or three of the variable heavy region CDRs of mu antigen binding construct.
  • an antibody fragment comprises one or more CDR regions from the variable heavy or light regions of a humanized anti-CD30 antibody.
  • the antigen binding construct can be a diabody.
  • the diabody can include a first polypeptide chain which includes a heavy (V H ) chain variable domain connected to a light chain variable domain (V L ) on the first polypeptide chain.
  • the light and heavy variable chain domains can be connected by a linker.
  • the linker can be of the appropriate length to reduce the likelihood of pairing between the two domains on the first polypeptide chain and a second polypeptide chain comprising a light chain variable domain (V L ) linked to a heavy chain variable domain V H on the second polypeptide chain connected by a linker that is too short to allow significant pairing between the two domains on the second polypeptide chain.
  • the appropriate length of the linker encourages chain pairing between the complementary domains of the first and the second polypeptide chains and can promote the assembly of a dimeric molecule with two functional antigen binding sites.
  • the diabody is bivalent.
  • the diabody can be a cysteine linked diabody (a cys-Db). A schematic of a cys-Db binding to two antigen sites is illustrated in FIG. IB.
  • the linker can be a peptide.
  • the linker can be any suitable length that promotes such assembly, for example, between 1 and 20 amino acids, such as 5 and 10 amino acids in length.
  • some cys-diabodies can include a peptide linker that is 5 to 8 amino acids in length.
  • the linker need not be made from, or exclusively from amino acids, and can include, for example, modified amino acids (see, for example, Increased Resistance of Peptides to Serum Proteases by Modification of their Amino Groups, Rossella Galati, Alessandra Verdina, Giuliana Falasca, and Alberto Chersi, (2003) Z.
  • the linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the linker can be from 2 to 30 angstroms in length, for example 2.5 to 27 angstroms.
  • the antigen binding construct includes a humanized cys-diabody.
  • the humanized cys-diabody can include a single-chain variable fragment (scFv) that includes a variable heavy (V H ) domain linked to a variable light (V L ) domain, and a C-terminal Cysteine.
  • the humanized cys-diabody is a homodimer.
  • the humanized diabody is a heterodimer.
  • individual monomers are provided that each have a cysteine terminal residue.
  • the scFv of the humanized cys-diabody has a V H - V L orientation or a V L -V H orientation.
  • a V H -V L (which may also be referred to herein as "V H V L ") orientation means that the variable heavy domain (V H ) of the scFv is upstream from the variable light domain (V L ) and a V L V H orientation means that the V L domain of the scFv is upstream from the V H domain.
  • upstream means toward the N-terminus of an amino acid or toward the 5 ' end of a nucleotide sequence.
  • the antibody variable regions can be linked together by a linker as described herein.
  • the linker is a GlySer linker as described herein (See FIG. 4A).
  • the cys-diabody includes a detectable marker.
  • the cys-diabody includes a pair of monomers. Each monomer can include a polypeptide. In some embodiments, the polypeptides of the monomers are identical (for example, cys-diabody can be a homodimer). In some embodiments, the polypeptides of the monomers are different (for example, the cys-diabody can be a heterodimer).
  • the cysteines are cross-linked with one another. In some embodiments, the cysteines are reduced, and thus, these tail forming cysteines do not form a disulfide bond with one another. In some embodiments, one or more of the "tail forming" cysteines form a covalent bond with one or more detectable marker, such as a fluorescent probe.
  • any covalently modifiable moiety can be employed in place of one or more of the cysteines.
  • this can include a GlySer linker, a GlyLeu linker, and/or an insert cysteine after a short tag.
  • the connection can be established via a coiled coil or a leucine zipper.
  • the "tail" itself can include functional groups on its end so that it can selectively bind to a desired residue and/or location at the ends of each of the polypetides, in place of the disulfide bond itself.
  • the covalently modifiable moieties can be attached directly to the end of the heavy or light chain polypeptide, but the two covalently modifiable moieties can be connected by a linker.
  • a chimeric cys-diabody that binds to the target molecule is provided.
  • the chimeric cys-diabody includes a monomer in the V L -V H format, and includes a sequence as outlined in Table 0.2 or a sequence having at least about 80% identity thereto, for example at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%% identity thereto.
  • the chimeric cys-diabody includes a monomer in the V H -V L format, and includes the sequence as outlined in Table 0.2 or a sequence having at least about 80% identity thereto, for example at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%% identity thereto.
  • the cys-diabody includes one or more of the CDRs provided in the CDRs in 2A, 2B 2C, 3A, 3B, 3C, 3D, 3E, 3F, and/or 3G.
  • any of the constructs provided herein can be provided as a scFv embodiment.
  • the construct can still include the cysteine on the tail, but simply not be cross-linked.
  • the construct need not have the cysteine in a tail or the tail at all.
  • the heavy and light chain variable domains can associate in different ways. For this reason, the use of different linker lengths allows for conformational flexibility and range-of-motion to ensure formation of the disulfide bonds.
  • the two linker lengths can be somewhere between (and including) about 1 to 50 amino acids, for example, 2 to 15, 2 to 14, 3 to 13, 4 to 10, or 5 amino acids to 8 amino acids.
  • each linker within a pair for a diabody can be the same length.
  • each linker within the pair can be a different length.
  • any combination of linker length pairs can be used, as long as they allow and/or promote the desired combinations.
  • a modified amino acid can be used.
  • Table 0.2 provides Cys-Db variants, V H 5V L , V H 8V L , V L 5V H , and V L 8V H .
  • Producing and testing the expression and binding of all four variants allows for identification of a desired format for protein production for each new Cys-Db. Evaluating the set of variants can help to make certain that a high-quality, stable protein is produced where the disulfide bridge is available. Therefore, engineering a Cys-Db can involve using two distinct linker lengths, not one - as in the minibody, as well as both orientations of the variable regions, VH VL and VI/VH.
  • the linker is a GlySer linker.
  • the GlySer linker can be a polypeptide that is rich in Gly and/or Ser residues.
  • at least about 40% of the amino acid residues of the GlySer linker are Gly, Ser, or a combination of Gly and Ser, for example at least about 40%, 50%, 60%, 70%, 80%, or 90%.
  • the GlySer linker is at least about 2 amino acids long, for example at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 amino acids long.
  • a cysteine is added at the C-terminus of the diabody.
  • This cysteine can allow the diabody complex to form covalent cysteine bonds and provides the option for available sulfur residues for site-specific conjugation of functional moieties such as radiolabels.
  • a terminal end of the antibody itself is altered so as to contain a cysteine.
  • a tail sequence for example (Gly- Gly-Cys) is added at the C-terminus.
  • the cysteine tail sequence allows two monomers of a cys-diabody to form disulfide bonds with each other.
  • the cysteine tail sequence allows a cys-diabody to form disulfide linkages with a detectable moiety such as a detectable marker and/or therapeutic agent.
  • the sulfhydryl groups of the cysteine tail can undergo mild reduction prior to site-specific conjugation of a desired functional moiety, for example a detectable marker and/or therapeutic agent.
  • the tail is at least about 1 amino acid long, for example at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 amino acids long. In some embodiments, the tail is 3 to 8 amino acids in length.
  • the tail can and/or include a coiled coil and/or a leucine zipper.
  • the cysteine is located at the c-terminus; however, this does not require that the cysteine be located as the last c-terminal amino acid. Instead, this denotes that the cysteine can be part of any of the residues that are located in the c-terminus of the protein.
  • the linking option between the two C-termini can be achieved by a cysteine, for direct and/or indirect, cross-linking.
  • FIGs. 4A-4C depict various optional sequences that can be used in some embodiments of the scFv, cys-diabodies, and/or, minibodies.
  • the cys- diabody (and or scFv) can be any of those provided herein, but with any of the sequences provided in FIG. 4A.
  • the minibody can be any of those provided herein, but with any of the sequences provided in FIG. 4B.
  • any of the antigen binding constructs provided herein can include any of the hinge sequences provided in FIG. 4C.
  • a "minibody” as described herein includes a homodimer, wherein each monomer is a single-chain variable fragment (scFv) linked to a human IgGl C H 3 domain by a linker, such as a hinge sequence.
  • a linker such as a hinge sequence.
  • the hinge sequence is a human IgGl hinge sequence.
  • the hinge sequence is an artificial hinge sequence.
  • the hinge sequence can be an IgG hinge from any one or more of the four classes.
  • the artificial hinge sequence may include a portion of a human IgGl hinge and a GlySer linker sequence.
  • the artificial hinge sequence includes approximately the first 14 or 15 residues of the human IgGl hinge followed by a linker sequence.
  • the linker can be any of those provided herein.
  • the linker can be a GlySer linker sequence that is 6, 7, 8, 9 or 10 amino acids in length.
  • the artificial hinge sequence includes approximately the first 15 residues of the IgGl hinge followed by a GlySer linker sequence that is about 10 amino acids in length.
  • association between the C H 3 domains causes the minibody to exist as a stable dimer.
  • the minibody scFv sequence can include CDR and/or FR, and or variable region sequences that are similar and/or the same to a diabody sequence described herein (for Example, as found in FIGs. 2A, 2B 2C, 3A, 3B, 3C, 3D, 3E, 3F, and/or 3G).
  • the minibody scFv has a sequence (CDR, CDRs, full set of 6 CDRS, heavy chain variable region, light chain variable region, heavy and light chain variable regions, etc) that is at identical to a scFv of a cys-diabody described herein.
  • the minibody has a sequence that is at least about 80% identical to a sequence as outlined in Table 0.1, for example at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94, 95%, 96%, 97%, 98%, or 99% identity.
  • the scFv can have a V H V L or a V L V H orientation.
  • the V H and V L are linked to each other by an amino acid linker sequence.
  • the amino acid linker can be a linker as described herein.
  • the linker is Gly-Ser-rich and approximately 15-20 amino acids in length.
  • the linker is Gly-Ser rich and is 18 amino acids in length.
  • the linker length varies between (and including) about 1 to 50 amino acids, for example, 2 to 30, 3 to 20, 4 to 15, or 5 amino acids to 8 amino acids.
  • the minibody scFv has a sequence that is at least about 80% identical to a scFv of a cys-diabody described herein, for example at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94, 95%, 96%, 97%, 98%, or 99% identity.
  • the scFv can have a V H V L or a V L V H orientation.
  • each monomer of the minibody includes the following elements, from N-terminus to C-terminus: (a) an scFv sequence that includes a V H domain linked to a V L domain and that binds to the target molecule, (b) a hinge-extension domain comprising a human IgGl hinge region, and (c) a human IgG C H 3 sequence.
  • each monomer of the minibody includes an IgG2, an IgG3, or an IgG4 C H 3.
  • the minibody is encoded by a nucleic acid can be expressed by a cell, a cell line or other suitable expression system as described herein.
  • a signal sequence can be fused to the N-terminus of the scFv to enable secretion of the minibody when expressed in the cell or cell line.
  • the minibody comprises one or more of the CDRs outlined in FIG. 2A, 2B 2C, 3A, 3B, 3C, 3D, 3E, 3F, and/or 3G. In some embodiments, the minibody comprises one or more of the variable regions in FIG. 2A, 2B 2C, 3A, 3B, 3C, 3D, 3E, 3F, and/or 3G. [0125] In some embodiments, the minibody includes the heavy chain variable region as outlined in FIG. 2C, 3E, 3F, and/or 3G . In some embodiments, the minibody includes the light chain variable region as outlined in FIG. 2A, 2B, 3A, 3B, 3C, and/or 3D.
  • the minibody includes one or more of the CDRs provided in the CDRs in FIG. 2A, 2B 2C, 3A, 3B, 3C, 3D, 3E, 3F, and/or 3G.
  • the minibody and/or cys-diabody and/or antibody and/or scFv includes one or more of the residues in the humanized sequence shown in FIG. 2A and/or 2B and/or 2C that is denoted with an asterisk.
  • the remaining sequence can be varied.
  • the sequence can have 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent or greater identity to the remaining sections of the sequence.
  • the human and/or humanized antigen binding construct will include one or more of the asterisked residues in FIG.
  • the antigen binding construct includes one or more of the underlined residues in FIG.
  • the antigen binding construct includes one or more of the underlined residues in FIG. 2A and/or 2B and/or 2C as well as the boxed CDR sections, whereas other residues are allowed to vary.
  • the antigen binding construct includes a LCDR1 region that includes at least one of a Lys at position 1, Gin at position 4, Asp at position 7, Asp at position 9, Asp at position 11, Ser at position 12, Tyr at position 13, Met at position 14, and/or Asn at position 15, as shown in FIG. 2A.
  • the antigen binding construct includes a LCDR2 region that includes at least one of a Ala at position 1 , Leu at position 5, Glu at position 6, and/or Ser at position 7, as shown in FIG. 2A.
  • the antigen binding construct includes a LCDR3 region that includes at least one of a Gin at position 1, Glu at position 5, and/or Asp at position 6, as shown in FIG. 2A.
  • the antigen binding construct includes a LCDR1 region that includes at least one of a Arg at position 1, Asp at position 7, Phe at position 8, Asp at position 9, Gly at position 10, Asp at position 11, Ser at position 12, Tyr at position 13, Met at position 14, and/or Asn at position 15, as shown in FIG. 2B.
  • the antigen binding construct includes a LCDR2 region that includes at least one of a Ala at position 1, Leu at position 5, Glu at position 6, and/or Ser at position 7, as shown in FIG. 2B.
  • the antigen binding construct includes a LCDR3 region that includes at least one of a Gin at position 1, Glu at position 5, and/or Asp at position 6, as shown in FIG. 2B.
  • the antigen binding construct includes a LFR1 region that includes at least one of a Pro at position 15, and/or Thr at position 22, as shown in FIG. 2A.
  • the antigen binding construct includes a LFR2 region that includes at a Val at position 12, as shown in FIG. 2A.
  • the antigen binding construct includes a LFR3 region that includes at least one of a Val at position 2, Thr at position 18, Asn at position 20, Ala at position 24, Asn at position 25, Thr at position 27, and/or Asn at position 29 as shown in FIG. 2A.
  • the antigen binding construct includes a LFR4 region that includes at least one of a Gin at position 3, and/or Val at position 7, as shown in FIG. 2A.
  • the antigen binding construct includes a LFR1 region that includes at least one of a Glu at position 1, Thr at position 10, Ser at position 12, Leu at position 13, Pro at position 15, Glu at position 17, and/or Leu at position 21 as shown in FIG. 2B.
  • the antigen binding construct includes a LFR2 region that includes at least one of a Ala at position 9, Arg at position 11, and/or Leu at position 12, as shown in FIG. 2B.
  • the antigen binding construct includes a LFR3 region that includes at least one of a Ala at position 4, Phe at position 6, Ser at position 20, Leu at position 22, G at position 23, Pro at position 24, Asp at position 26, Val at position 29, and/or Tyr at position 31 as shown in FIG. 2B.
  • the antigen binding construct includes a LFR4 region that includes at least one of a Thr at position 5, and/or He at position 9, as shown in FIG. 2B.
  • the antigen binding construct includes a HFR1 region that includes at least one of a Val at position 2, Leu at position 4, Val at position 5, Ala at position 9, Lys at position 12, and/or Val at position 20, as shown in FIG. 2C.
  • the antigen binding construct includes a HFR2 region that includes at least one of a Tyr at position 2, He at position 3, Thr at position 4, Arg at position 7, Ala at position 9, Gly at position 13, and/or He at position 17 as shown in FIG. 2C.
  • the antigen binding construct includes a HFR3 region that includes at least one of a Asn at position 3, Glu at position 4, Lys at position 6, Lys at position 8, Ala at position 9, He at position 11, Arg at position 13, Ala at position 17, Tyr at position 21, Glu at position 23, Arg at position 28, Tyr at position 36, and/or Asn at position 39 as shown in FIG. 2C.
  • the antigen binding construct includes a HFR4 region that includes at least one of a Leu at position 6, and/or Ser at position 11, as shown in FIG. 2C.
  • the antigen binding construct can include one or more of the asterisked residues in FIG. 2A and/or 2B and/or 2C, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, or 79.
  • the CDR residues are maintained and the residues with the asterisk are maintained, but one or more of the other residues are allowed to vary.
  • the polypeptides of the antigen binding constructs can be encoded by nucleic acids and expressed in vivo or in vitro, or these peptide can be synthesized chemically.
  • a nucleic acid encoding an antigen binding construct is provided.
  • the nucleic acid encodes one part or monomer of a cys-diabody or minibody.
  • the nucleic acid encodes two or more monomers, for example, at least 2 monomers.
  • Nucleic acids encoding multiple monomers can include nucleic acid cleavage sites between at least two monomers, can encode transcription or translation start site between two or more monomers, and/or can encode proteolytic target sites between two or more monomers.
  • an expression vector contains a nucleic acid encoding an antigen binding construct as disclosed herein.
  • the expression vector includes pcDNA3.1TM/myc-His (-) Version A vector for mammalian expression (Invitrogen, Inc.) or a variant thereof.
  • the pcDNA3.1 expression vector features a CMV promoter for mammalian expression and both mammalian (Neomycin) and bacterial (Ampicillin) selection markers.
  • the expression vector includes a plasmid.
  • the vector includes a viral vector, for example a retroviral or adenoviral vector.
  • the vector includes a cosmid, YAC, or BAC.
  • the nucleotide sequence encoding at least one of the minibody monomers comprises at least one of SEQ ID NO: 10, 11, 12, 14, 15, 16, 18, 27, 29, 31,, or a sequence having at least about 80% identity, for example about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94, 95%, 96%, 97%, 98%, 99%, or greater identity thereto.
  • the nucleotide sequence encoding at least one of the cys-diabody monomers includes SEQ ID NO: 10, 12, 14, 16, 18, 19, 21, 23, 25, or a sequence having at least about 80% identity, for example about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94, 95%, 96%, 97%, 98%, 99% or greater identity thereto.
  • a cell line that expresses at least one of the antigen binding constructs described herein.
  • a mammalian cell line for example, CHO-K1 cell line
  • a mammalian cell line is an expression system to produce the minibodies, cys- diabodies, scFv, or other antibodies as described herein.
  • the minibodies, cys-diabodies, scFv, and other antibodies or antibody fragments described herein are non-glycosylated, and a mammalian expression system is not required, as such post- translational modifications are not needed.
  • one or more of a wide variety of mammalian or non-mammalian expression systems are used to produce the antigen binding constructs disclosed herein (for example, anti-CD30 minibodies and cys- diabodies) including, but not limited to mammalian expression systems (for example, CHO- Kl cells), bacterial expression systems (for example, E. Coli, B. subtilis) yeast expression systems (for example, Pichia, S. cerevisiae) or any other known expression system.
  • mammalian expression systems for example, CHO- Kl cells
  • bacterial expression systems for example, E. Coli, B. subtilis
  • yeast expression systems for example, Pichia, S. cerevisiae
  • Other systems can include insect cells and/or plant cells.
  • the antigen binding construct includes at least one modification.
  • exemplary modifications include, but are not limited to, antigen binding constructs that have been modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, and linkage to a cellular ligand or other protein. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation and metabolic synthesis of tunicamycin.
  • the derivative can contain one or more non-natural amino acids.
  • the antigen binding construct is conjugated to another substance to form an anti-target conjugate.
  • the conjugates described herein can be prepared by known methods of linking antigen binding constructs with lipids, carbohydrates, protein or other atoms and molecules.
  • the conjugate is formed by site- specific conjugation using a suitable linkage or bond. Site-specific conjugation is more likely to preserve the binding activity of an antigen binding construct.
  • the substance may be conjugated or attached at the hinge region of a reduced antigen binding construct via disulfide bond formation.
  • cysteine residues at the C-terminus of a scFv fragment such as those that can be introduced in the cys-diabodies described herein, allows site-specific thiol-reactive coupling at a site away from the antigen binding site to a wide variety of agents.
  • linkages or bonds used to form the conjugate can include, but are not limited to, a covalent bond, a non-covalent bond, a sulfide linkage, a hydrazone linkage, a hydrazine linkage, an ester linkage, an amido linkage, and amino linkage, an imino linkage, a thiosemicabazone linkage, a semicarbazone linkage, an oxime linkage and a carbon-carbon linkage.
  • no cysteine or other linking aspect or tail need be included in the antigen binding construct.
  • a modified antigen binding construct is conjugated to a detectable marker.
  • a detectable marker includes an atom, molecule, or compound that is useful in diagnosing, detecting or visualizing a location and/or quantity of a target molecule, cell, tissue, organ and the like.
  • Detectable markers that can be used in accordance with the embodiments herein include, but are not limited to, radioactive substances (for example, radioisotopes, radionuclides, radiolabels or radiotracers), dyes, contrast agents, fluorescent compounds or molecules, bioluminescent compounds or molecules, enzymes and enhancing agents (for example, paramagnetic ions).
  • some nanoparticles for example quantum dots and metal nanoparticles (described below) can be suitable for use as a detection agent.
  • the detectable marker is IndoCyanine Green (ICG).
  • radioactive substances that can be used as detectable markers in accordance with the embodiments herein include, but are not limited to, 18 F, 18 F-FAC, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, 99 mTc, "Mo, 105 Pd, 105 Rh, m Ag, m In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154"
  • Exemplary paramagnetic ions substances that can be used as detectable markers include, but are not limited to ions of transition and lanthanide metals (for example metals having atomic numbers of 6 to 9, 21-29, 42, 43, 44, or 57-71). These metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.
  • the detectable marker is a radioactive metal or paramagnetic ion
  • the marker can be reacted with a reagent having a long tail with one or more chelating groups attached to the long tail for binding these ions.
  • the long tail can be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which may be bound to a chelating group for binding the ions.
  • chelating groups examples include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTP A), DOTA, NOTA, NOGADA, NETA, deferoxamine (DfO), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups.
  • EDTA ethylenediaminetetraacetic acid
  • DTP A diethylenetriaminepentaacetic acid
  • DOTA DOTA
  • NOTA NOGADA
  • NETA deferoxamine
  • porphyrins porphyrins
  • polyamines crown ethers
  • bis-thiosemicarbazones polyoximes, and like groups.
  • chelates when complexed with non-radioactive metals, such as manganese, iron and gadolinium are useful for MRI, when used along with the antigen binding constructs and carriers described herein.
  • Macrocyclic chelates such as NOTA, NOGADA, DOTA, and TETA are of use with a variety of metals and radiometals including, but not limited to, radionuclides of gallium, yttrium and copper, respectively.
  • Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding nuclides, such as 223 Ra for RAIT may be used.
  • chelating moieties may be used to attach a PET imaging agent, such as an Al- 18 F complex, to a targeting molecule for use in PET analysis.
  • Exemplary contrast agents that can be used as detectable markers in accordance with the embodiments of the disclosure include, but are not limited to, barium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid, iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide, iohexyl, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetric acid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid, ipodate, meglumine, metrizamide, metrizoate, propyliodone, thallous chloride, or combinations
  • Bioluminescent and fluorescent compounds or molecules and dyes that can be used as detectable markers in accordance with the embodiments of the disclosure include, but are not limited to, fluorescein, fluorescein isothiocyanate (FITC), OREGON GREENTM, rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC), Cy3, Cy5, and the like), fluorescent markers (for example, green fluorescent protein (GFP), phycoerythrin, and the like), autoquenched fluorescent compounds that are activated by tumor-associated proteases, enzymes (for example, luciferase, horseradish peroxidase, alkaline phosphatase, and the like), nanoparticles, biotin, digoxigenin or combination thereof.
  • fluorescent markers for example, green fluorescent protein (GFP), phycoerythrin, and the like
  • enzymes for example, luciferase, horseradish peroxidase, alkaline phosphata
  • Enzymes that can be used as detectable markers in accordance with the embodiments of the disclosure include, but are not limited to, horseradish peroxidase, alkaline phosphatase, acid phoshatase, glucose oxidase, ⁇ -galactosidase, ⁇ -glucoronidase or ⁇ -lactamase. Such enaymes may be used in combination with a chromogen, a fluorogenic compound or a luminogenic compound to generate a detectable signal.
  • the antigen binding construct is conjugated to a nanoparticle.
  • nanoparticle refers to a microscopic particle whose size is measured in nanometers, for example, a particle with at least one dimension less than about 100 nm. Nanoparticles can be used as detectable substances because they are small enough to scatter visible light rather than absorb it. For example, gold nanoparticles possess significant visible light extinction properties and appear deep red to black in solution. As a result, compositions comprising antigen binding constructs conjugated to nanoparticles can be used for the in vivo imaging of T-cells in a subject. At the small end of the size range, nanoparticles are often referred to as clusters.
  • Nanospheres, nanorods, and nanocups are just a few of the shapes that have been grown.
  • Semiconductor quantum dots and nanocrystals are examples of additional types of nanoparticles.
  • Such nanoscale particles when conjugated to an antigen binding construct, can be used as imaging agents for the in vivo detection of T-cells as described herein.
  • an antigen binding construct is conjugated to a therapeutic agent.
  • a "therapeutic agent” as used herein is an atom, molecule, or compound that is useful in the treatment of cancer, inflammation, other disease conditions, or to otherwise suppress an immune response, for example immunosuppression in organ transplants.
  • therapeutic agents include, but are not limited to, drugs, chemotherapeutic agents, therapeutic antibodies and antibody fragments, toxins, radioisotopes, enzymes (for example, enzymes to cleave prodrugs to a cytotoxic agent at the site of the antigen binding construct binding), nucleases, hormones, immunomodulators, antisense oligonucleotides, chelators, boron compounds, photoactive agents and dyes, and nanoparticles.
  • drugs chemotherapeutic agents, therapeutic antibodies and antibody fragments, toxins, radioisotopes, enzymes (for example, enzymes to cleave prodrugs to a cytotoxic agent at the site of the antigen binding construct binding), nucleases, hormones, immunomodulators, antisense oligonucleotides, chelators, boron compounds, photoactive agents and dyes, and nanoparticles.
  • chemotherapeutic agents for example, enzymes to cleave prodrugs to a cytotoxic agent at the site
  • Chemotherapeutic agents are often cytotoxic or cytostatic in nature and may include alkylating agents, antimetabolites, anti-tumor antibiotics, topoisomerase inhibitors, mitotic inhibitors hormone therapy, targeted therapeutics and immuno therapeutics.
  • the chemotherapeutic agents that may be used as detectable markers in accordance with the embodiments of the disclosure include, but are not limited to, 13-cis- Retinoic Acid, 2-Chlorodeoxyadenosine, 5-Azacitidine, 5-Fluorouracil, 6-Mercaptopurine, 6- Thioguanine, actinomycin-D, adriamycin, aldesleukin, alemtuzumab, alitretinoin, all- transretinoic acid, alpha interferon, altretamine, amethopterin, amifostine, anagrelide, anastrozole, arabinosylcytosine, arsenic trioxide, amsacrine, aminocamptothecin, aminoglutethimide, asparaginase, azacytidine, bacillus calmette-guerin (BCG), bendamustine, bevacizumab, bexarotene, bicalu
  • Toxins that may be used in accordance with the embodiments of the disclosure include, but are not limited to, ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.
  • nanoparticles are used in therapeutic applications as drug carriers that, when conjugated to an antigen binding construct, deliver chemotherapeutic agents, hormonal therapeutic agents, radio therapeutic agents, toxins, or any other cytotoxic or anti-cancer agent known in the art to cancerous cells that overexpress the target on the cell surface.
  • any of the antigen binding constructs described herein may be further conjugated with one or more additional therapeutic agents, detectable markers, nanoparticles, carriers or a combination thereof.
  • an antigen binding construct may be radiolabeled with Iodine-131 and conjugated to a lipid carrier, such that the anti-CD30-lipid conjugate forms a micelle.
  • the micelle can incorporate one or more therapeutic or detectable markers.
  • the antigen binding construct may be radiolabeled with Iodine-131 (for example, at a tyrosine residue) and conjugated to a drug (for example, at the epsilon amino group of a lysine residue), and the carrier may incorporate an additional therapeutic or detectable marker.
  • the antigen binding construct can be connected to a therapeutic to treat a form of cancer, including, but not limited to: a variety of hematopoietic cell tumors including Hodgkins lymphoma (HL), Anaplastic Large Cell Lymphoma (ALCL), and a subset of Cutaneous T Cell Lymphomas (CTCL), other T cell lymphoproliferative disorders, and rare solid tumors such as embryonal carcinomas
  • a form of cancer including, but not limited to: a variety of hematopoietic cell tumors including Hodgkins lymphoma (HL), Anaplastic Large Cell Lymphoma (ALCL), and a subset of Cutaneous T Cell Lymphomas (CTCL), other T cell lymphoproliferative disorders, and rare solid tumors such as embryonal carcinomas
  • the antigen binding construct can be used as a tool for the development of therapies targeting CD30.
  • the antigen binding construct is used to monitor patients following treatment of therapy targeting CD30, or to select patient populations for therapies that require CD30 expression in the disease setting.
  • CD30 is expressed on immune cells infiltrates in diseases such as diabetes, asthma, colitis, graft versus host disease (GVHD), atherosclerosis and other inflammatory conditions.
  • antigen binding constructs that bind to CD30 can aid in identifying patients with increased disease severity and/or those who are at increased risk and in need of additional or tailored therapy.
  • the antigen binding construct can be connected to a therapeutic to treat diabetes, asthma, colitis, graft versus host disease (GVHD), atherosclerosis and other inflammatory conditions
  • kits are provided.
  • the kit includes an antigen binding construct as described herein.
  • the kit includes a nucleic acid that encodes an antigen binding construct as described herein.
  • the kit includes a cell line that produces an antigen binding construct as described herein.
  • the kit includes a detectable marker as described herein.
  • the kit includes a therapeutic agent as described herein.
  • the kit includes buffers.
  • the kit includes positive controls, for example CD30, CD30+ cells, or fragments thereof.
  • the kit includes negative controls, for example a surface or solution that is substantially free of CD30.
  • the kit includes packaging.
  • the kit includes instructions.
  • Antigen binding constructs can be used to detect the presence or absence of the target molecule in vivo and/or in vitro. Accordingly, some embodiments include methods of detecting the presence or absence of the target. The method can include applying an antigen binding construct to a sample. The method can include detecting a binding or an absence of binding of the antigen binding construct to the target molecule, CD30.
  • FIG. IE illustrates some embodiments of methods of detecting the presence or absence of CD30. It will be appreciated that the steps shown in FIG. IE can be performed in any sequence, and/or can be optionally repeated and/or eliminated, and that additional steps can optionally be added to the method.
  • An antigen binding construct as described herein can be applied to a sample 100.
  • An optional wash 110 can be performed.
  • a secondary antigen binding construct can be applied to the sample 120.
  • An optional wash can be performed 130.
  • a binding or absence of binding of the antigen binding construct to the target molecule can be detected 140.
  • an antigen binding construct as described herein is applied to a sample in vivo.
  • the antigen binding construct can be administered to a subject.
  • the subject is a human.
  • the subject is a non- human mammal, for example a rat, mouse, guinea pig, hamster, rabbit, dog, cat, cow, horse, goat, sheep, donkey, pig, monkey, or ape.
  • the antigen binding construct is infused into the subject.
  • the infusion is intravenous.
  • the infusion is intraperitoneal.
  • the antigen binding construct is applied topically or locally (as in the case of an interventional or intraoperative application) to the subject.
  • a capsule containing the antigen binding construct is applied to the subject, for example orally or intraperitoneally.
  • the antigen binding construct is selected to reduce the risk of an immunogenic response by subject.
  • the antigen binding construct can be humanized as described herein.
  • following in vivo application of the antigen binding construct the sample, or a portion of the sample is removed from the host.
  • the antigen binding construct is applied in vivo, is incubated in vivo for a period of time as described herein, and a sample is removed for analysis in vitro, for example in vitro detection of antigen binding construct bound to the target molecule or the absence thereof as described herein.
  • the antigen binding construct is applied to a sample in vitro.
  • the sample is freshly harvested from a subject, for example a biopsy.
  • the sample is incubated following harvesting from a subject.
  • the sample is fixed.
  • the sample includes a whole organ and/or tissue.
  • the sample includes one or more whole cells.
  • the sample is from cell extracts, for example lysates.
  • antigen binding construct in solution is added to a solution in the sample.
  • antigen binding construct in solution is added to a sample that does not contain a solution, for example a lyophilized sample, thus reconstituting the sample.
  • lyophilized antigen binding construct is added to a sample that contains solution, thus reconstituting the antigen binding construct.
  • the antigen binding construct is optionally incubated with the sample.
  • the antigen binding construct can be incubated for a period of no more than about 14 days, for example no more than about 14 days, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or no more than about 23 hours, for example no more than about 23 hours, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, 0.25, or 0.1 hour, including ranges between any two of the listed values.
  • the incubation is within a subject to which the antigen binding construct was administered.
  • the incubation is within an incubator.
  • the incubator is maintained at a fixed temperature, for example about 21°C, room temperature, 25°C, 29°C, 34°C, 37°C, or 40°C.
  • the antigen binding construct that is not bound to the target is optionally removed from the sample.
  • the sample is washed. Washing a sample can include removing solution that contains unbound antigen binding construct, and adding solution that does not contain antigen binding construct, for example buffer solution.
  • an in vitro sample is washed, for example by aspirating, pipetting, pumping, or draining solution that contains unbound antigen binding construct, and adding solution that does not contain antigen binding construct.
  • an in vivo sample is washed, for example by administering to the subject solution that does not contain antigen binding construct, or by washing a site of topical antigen binding construct administration.
  • the wash is performed at least two times, for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times.
  • at least about 50% of unbound antibody is removed from the sample, for example at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or greater.
  • unbound antigen binding construct is eliminated from the sample. Following application of the antigen binding construct to the sample, antigen binding construct bound to the target reaches an equilibrium with antigen binding construct unbound to the target, so that at some time after application of the antigen binding construct, the amount of antigen binding construct bound to the target does not substantially increase. After this time, at least part of the quantity of the antigen binding construct that is unbound to the target can be eliminated. In some embodiments, unbound antigen binding construct is eliminated by metabolic or other bodily processes of the subject to whom the antibody or fragment was delivered.
  • unbound antigen binding construct is eliminated by the addition of an agent that destroys or destabilized the unbound antigen binding construct, for example a protease or a neutralizing antibody.
  • an agent that destroys or destabilized the unbound antigen binding construct for example a protease or a neutralizing antibody.
  • 1 day after application of the antigen binding construct at least about 30% of the antigen binding construct that was applied has been eliminated, for example at least about 30%, 40%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.9%.
  • At least about 40% of the antigen binding construct that was applied has been eliminated, for example at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.9%.
  • the presence or absence of the target, CD30 is detected.
  • the presence or absence of the target can be detected based on the presence or absence of the antigen binding construct in the sample. After removal and/or elimination of the antigen binding construct from the sample, for example by washing and/or metabolic elimination, remaining antigen binding construct in the sample can indicate the presence of the target, while an absence of the antigen binding construct in the sample can indicate the absence of the target.
  • the antigen binding construct includes a detectable marker as described herein.
  • the presence of the antigen binding construct can be inferred by detecting the detectable marker.
  • a secondary antigen binding construct is used to detect the antigen binding construct.
  • the secondary antigen binding construct can bind specifically to the antigen binding construct.
  • the secondary antigen binding construct can include a polyclonal or monoclonal antibody, diabody, minibody, etc. against the host type of the antibody, or against the antigen binding construct itself.
  • the secondary antigen binding construct can be conjugated to a detectable marker as described herein.
  • the secondary antigen binding construct can be applied to the sample.
  • the secondary antigen binding construct is applied to the sample in substantially the same manner as the antigen binding construct. For example, if the antigen binding construct was infused into a subject, the secondary antigen binding construct can also be infused into the subject.
  • binding or the absence of binding of the antigen binding construct is detected via at least one of: positron emission tomography (PET), single- photon emission computed tomography (SPECT), magnetic resonance imaging (NMR), or detection of fluorescence emissions.
  • PET can include, but is not limited to microPET imaging.
  • binding of the absence of binding of the antigen binding construct is detected via two or more forms of imaging.
  • detection can be via near-infrared (NIR) and/or Cerenkov.
  • Antigen binding constructs can be used to target a therapeutic molecule, for example a cytotoxin to a target positive cell, such as a cell expressing CD30.
  • a therapeutic agent for example a cytotoxin to a target positive cell, such as a cell expressing CD30.
  • the method can include administering an antigen binding construct as described herein to a subject.
  • the subject can be a subject in need, for example a subject in need of elimination or neutralization of at least some target positive cells.
  • the antigen binding construct includes at least on therapeutic agent as described herein.
  • the therapeutic can be directly conjugated to the antigen binding construct via a covalent bond, such as a disulfide bond.
  • the subject can benefit from the localization of a CD30 positive cell to another cell or agent.
  • the number and/or localization of the target positive cells of the patient is determined before and/or after administration of the antigen binding construct that includes at least one therapeutic agent. For example, determining the number and/or localization of target positive cells prior to administration can indicate whether the patient is likely to benefit from neutralization and/or elimination of the target positive cells. Determining the number and/or localization of the target positive cells after administration can indicate whether the target positive cells were eliminated in the patient.
  • the CD30 antigen binding construct can be used as a therapeutic agent as stand-alone construct (for example, without a toxin conjugated thereto). CD30 is an internalizing antigen which is very suitable for antibody drug-conjugate approaches. While the fragments have a shorter half-life compared to the intact antibody which would be less optimal for therapy, these formats can exhibit improved tumor penetration based on their smaller size and be therapeutically effective when appropriately armed with a cytotoxic drug or radioisotope.
  • another therapeutic approach is radioimmunotherapy via attaching an appropriate radiolabel such as the beta-emitter Yttrium- 90 which can deliver cell damage and death.
  • an appropriate radiolabel such as the beta-emitter Yttrium- 90 which can deliver cell damage and death.
  • treatment with antigen binding constructs linked to a cytotoxic drug or radioisotope can result in less nonspecific toxicity as they will be cleared from the body more rapidly than antibodies or fragments with longer half-lives.
  • the antigen binding construct is used to target cells expressing CD30 antigen, including, for example, activated but not resting B and T lymphocytes and other immune cells.
  • CD30 antigen including, for example, activated but not resting B and T lymphocytes and other immune cells.
  • hematopoietic cell tumors including Hodgkins lymphoma (HL), Anaplastic Large Cell Lymphoma (ALCL) a subset of Cutaneous T Cell Lymphomas (CTCL), and on rare solid tumors such as embryonal carcinomas.
  • the scFv, minibody and/or cys-diabody antibody fragments have superior pharmacokinetic properties for diagnostic imaging.
  • Current technology utilizes imaging with the intact antibody which requires significantly longer times (-7-8 days post-injection) to produce high contrast images due to the slow serum clearance of full length antibodies.
  • the minibody and cys-diabody provide the opportunity for same-day or next-day imaging. Each day is vital for patients with an aggressively progressing disease, and the ability to identify the proper therapeutic approach at an earlier time-point has the potential to improve patient survival.
  • Same-day or next-day imaging also provides a logistical solution to the problem facing many patients who travel great distances to receive treatment/diagnosis since the duration of travel stays or the need to return one week later would be eliminated when imaging with minibody or cys-diabody fragments versus full length antibodies.
  • the cys-diabody fragment component monomers contain c-terminus cysteine residues that form disulfide bonds. These covalently bound cys-diabody cysteine residues can be opened via mild chemical reduction to provide an active thiol group for site specific conjugation.
  • conjugation of antibodies relies on non-specific targeting of tyrosine or lysine residues which are commonly located in the functionally important complementary determining regions (CDRs) of antibodies whereas cysteine residues are rarely located in the CDRs.
  • CDRs complementary determining regions
  • scFv, minibody, and cys-diabody diagnostic fragments matching available antibody therapies allow matching of the patient's disease state with the appropriate antibody therapy.
  • a method of targeting a CD30+ cell to a first antigen can include applying a bispecific antigen binding construct to a sample.
  • the bispecific antigen binding construct can include a CD30 antigen binding construct as described herein.
  • the bispecific antibody can include an antigen binding construct that binds to the first antigen, for example 1 , 2, 3, 4, 5, or 6 CDR's, an scFv, or a monomer of a minibody or cys-diabody.
  • the bispecific antibody includes 1 , 2, or 3 HCDR's of an antigen binding construct as described herein, and/or 1, 2, or 3 LCDR's of an antigen binding construct as described herein.
  • the bispecific antigen binding construct includes a scFv of an antigen binding construct as described herein. In some embodiment, the bispecific antigen binding construct includes a V H or V L sequence as described herein. In some embodiments, the bispecific antigen binding construct includes a minibody or cys-diabody monomer as described herein. In some embodiments, the bispecific antigen binding construct is applied to a sample in vivo, for example an organ or tissue of a subject. In some embodiments, the bispecific antigen binding construct is applied to an in vitro sample.
  • the bispecific antigen binding construct binds to the target on the target positive cell, and binds to the first antigen (which can be different from CD30) on the first cell, and thus brings the target positive cell in proximity to the first cell.
  • a CD30+ cell can be brought into proximity of a cancer cell, and can facilitate an immune response against that cancer cell.
  • EXAMPLE 1 CD30 Antibodies and Antibody Fragments
  • the murine variable regions of the murine anti-human CD30 antibody were humanized by grafting the murine Complimentary Determining Region (CDR) onto a human framework.
  • the murine V genes were run against the human V germline database. The human V gene with highest sequence homology was examined for critical residues and similarity to antigen binding loop structures.
  • V L and V H CDRS of the murine anti-human CD30 antibody were then incorporated into the human acceptor variable region framework, replacing the human CDRs (FIG. 2A-C). Selected mouse residues were kept in the human framework.
  • the humanized variable regions from this humanization are named version B. Since two human acceptor sequences were identified for the variable light domain, there are two versions of the humanized V L (FIG. 2A-B, version Bl and B2).
  • the resulting humanized variable regions (huCD30) were then formatted into two minibody variants (Table 0.1) and four cys-diabody variants (Table 0.2).
  • variable light sequences named 1IUV L _VA and variable heavy sequences named 1IUV L _VA were also humanized using a different approach (see FIGs. 3C and 3G, variable light sequences named 1IUV L _VA and variable heavy sequences named 1IUV L _VA).
  • chimeric anti-human CD30 minibody variants were also engineered (FIGs. 9, 10). These were composed of the murine anti-human CD30 variable domains fused to the human IgGl hinge-C H 3 and served as a reference and/or positive control in binding studies.
  • the fragments were initially evaluated for their expression levels and binding to recombinant and cellular hCD30 following cloning of their cDNAs into the pcDNA3.1/myc-His (-) Version A vector for mammalian expression (Invitrogen Corp; FIG. 5).
  • variable gene sequences from the version A humanization two minibody variants were engineered based on the orientation of the variable genes (V L -V H and V H -V L ) ⁇ the scFv; and two chimeric minibodies were also made based on the murine variable gene sequences.
  • An additional four Cys-Db variants were generated based on version A humanized sequences.
  • V L -V H and V H -V Two variants differed in the orientation (V L -V H and V H -V and two in linker lengths (5 and 8 amino acids).
  • the cDNAs for these fragments were cloned into the mammalian expression vector pcDNA3.1/myc-His (-) Version A (Invitrogen Corp; FIG. 5).
  • Binding of these antibody fragments to cells that express cell surface CD30 was determined using flow cytometry. Supernatants containing transiently expressed antibody fragments were incubated with CD30 expressing HH cells. Binding of the minibodies and the cys-diabodies was detected with R-Phycoerythrin (PE)-conjugated anti- human IgG (Fc-specific) or anti-His-PE antibodies, respectively. The minibody and cys- diabody variants showed concentration-dependent binding to cell surface CD30 expressed on the surface of HH cells (FIG. 7A). The amount of minibody in the transfection supernatant was quantified by ELISA and binding curves were generated to provide EC50 values (FIG. 7B). The EC50 values for the minibodies were between 0.158 nM to 0.375 nM.
  • Flow cytometry analysis was also performed to detect binding of cys- diabody variants to HH cells following staining with allophycocyanin (APC) conjugated anti- His antibody (FIG. 8).
  • APC allophycocyanin
  • 1X10 3 HH cells were incubated with indicated dilutions of the cys- diabody supernatant and flow cytometric analysis was performed with 10,000 events/point. All histograms show APC signal vs. cell count. Staining with the secondary antibody alone was used as a negative control (red).
  • a humanized CD30 cys-diabody from Table 0.2 is provided.
  • the cys- diabody is infused intravenously into a subject having Hodgkins lymphoma (HL), Anaplastic Large Cell Lymphoma (ALCL), and a subset of Cutaneous T Cell Lymphomas (CTCL), or embryonal carcinoma, or activated immune cells in diabetes, asthma, colitis, graft versus host disease (GVHD), or atherosclerosis in an amount adequate to bind to sufficient levels of CD30 in the subject to provide a lessening of the symptoms of Hodgkins lymphoma (HL), Anaplastic Large Cell Lymphoma (ALCL), and a subset of Cutaneous T Cell Lymphomas (CTCL), embryonal carcinoma, diabetes, asthma, colitis, graft versus host disease (GVHD), or atherosclerosis.
  • HL Hodgkins lymphoma
  • ACL Anaplastic Large Cell Lymphoma
  • CTCL Cutaneous T Cell
  • a minibody from Table 0.1 is conjugated with a relevant chelator via Lysine residues on the minibody and subsequenty radiolabeled with an isotope of Inl 11 (or in the alternative, Zr89 or Cu64).
  • the minibody can be radiolabeled by directly radiolabeling with Iodine via Tyrosine residues.
  • the minibody is infused intravenously into a healthy human subject.
  • the minibody is incubated in the human subject for 10 minutes post-infusion. On the same day as the incubation, the localization of the minibody is detected via a PET scan or external scintillation system.
  • a cys-diabody from Table 0.2 is conjugated with a relevant chelator via its cysteine residue on the cys-diabody and subsequenty radiolabeled with an isotope of Inl 11 (or in the alternative, Zr89 or Cu64).
  • the cys-diabody can be radiolabeled by directly radiolabeling with Iodine via Tyrosine residues.
  • the cys-diabody is infused intravenously into a healthy human subject.
  • the cys-diabody is incubated in the human subject for 10 minutes post-infusion. On the same day as the incubation, the localization of the cys-diabody is detected via a PET scan or external scintillation system.

Abstract

L'invention concerne des constructions de liaison à l'antigène qui se lient à CD30, par exemple des anticorps, comprenant des fragments d'anticorps (tels que scFv, des mini-anticorps, et cys-diabodies) qui se lient à CD30. L'invention concerne aussi des procédés d'utilisation.
PCT/US2014/020318 2013-03-12 2014-03-04 Constructions de liaison à l'antigène se liant à cd30 WO2014164067A1 (fr)

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WO2022074206A1 (fr) 2020-10-08 2022-04-14 Affimed Gmbh Lieurs trispécifiques
WO2023007023A1 (fr) 2021-07-30 2023-02-02 Affimed Gmbh Corps duplex
WO2023078968A1 (fr) 2021-11-03 2023-05-11 Affimed Gmbh Liants de cd16a bispécifiques

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CN108610420A (zh) * 2016-12-13 2018-10-02 科济生物医药（上海）有限公司 抗cd19的人源化抗体以及靶向cd19的免疫效应细胞
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CN108610420B (zh) * 2016-12-13 2023-09-26 科济生物医药（上海）有限公司 抗cd19的人源化抗体以及靶向cd19的免疫效应细胞
WO2022074206A1 (fr) 2020-10-08 2022-04-14 Affimed Gmbh Lieurs trispécifiques
WO2023007023A1 (fr) 2021-07-30 2023-02-02 Affimed Gmbh Corps duplex
WO2023078968A1 (fr) 2021-11-03 2023-05-11 Affimed Gmbh Liants de cd16a bispécifiques

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