WO2014158821A1

WO2014158821A1 - Antigen binding constructs to cd70

Info

Publication number: WO2014158821A1
Application number: PCT/US2014/020380
Authority: WO
Inventors: David T. HO; Tove Olafsen; Giti AGAHI; Jean Gudas
Original assignee: Imaginab, Inc.
Priority date: 2013-03-12
Filing date: 2014-03-04
Publication date: 2014-10-02

Abstract

Antigen binding constructs that bind to CD70, for example antibodies, including antibody fragments (such as scFv, mini bodies, and cys-diabodies) that bind to CD70, are described herein. Methods of use are described herein. Embodiments described herein relate generally to antigen binding constructs, such as antibodies, including antibody fragments, that bind to CD70, (such as minibodies, cys-diabodies, and scFv), as well as methods for their use.

Description

ANTIGEN BINDING CONSTRUCTS TO CD70

REFERENCE TO SEQUENCE LISTING, TABLE, OR

COMPUTER PROGRAM LISTING

[0001] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled SeqListIGNAB022WO.txt, created on March 3, 2014, which is 12,809 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD

[0002] Embodiments described herein relate generally to antigen binding constructs, such as antibodies, including antibody fragments, that bind to CD70, (such as minibodies, cys-diabodies, and scFv), as well as methods for their use.

BACKGROUND

[0003] CD70 is a member of the tumor necrosis factor (TNF) superfamily and is the ligand for CD27. Binding of CD70 to CD27 is important for T- and B-cell activation, differentiation and memory cell generation. CD70 is present on activated but not resting B and T lymphocytes and on dendritic cells. CD70 is also expressed on a variety of solid tumors including renal carcinoma, pancreatic, larynx/pharynx, melanoma, ovarian, lung, glioblastoma and colon cancers. In addition, CD70 is expressed on a variety of B cell tumors including non-Hodgkin's lymphoma (NHL), Waldenstrom macroglobulinemia, Hodgkin's lymphoma, B and T cell leukemia and multiple myeloma. Renal cancer tumor cell lines can also express CD70. Background expression of CD70 in normal tissue is low.

SUMMARY [0004] In some embodiments, an antigen binding construct is provided. The antigen binding construct can comprise: a HCDR1 of the HCDR1 in SEQ ID NO: 4, a HCDR2 of the HCDR2 in SEQ ID NO: 4, a HCDR3 of the HCDR3 in SEQID NO: 4, a LCDR1 of the LCDR1 in SEQ ID NO: 2, a LCDR2 of the LCDR2 in SEQ ID NO: 2, and a LCDR3 of the LCDR3 in SEQ ID NO: 2.

[0005] In some embodiments, an antigen binding construct is provided. The antigen binding construct can comprise a LFR1 of the LFR1 in SEQ ID NO: 2, a LFR2 of the LFR2 in SEQ ID NO: 2, a LFR3 of the LFR3 in SEQ ID NO: 2. a LFR4 of the LFR4 in SEQ ID NO: 2, a HFRl of the HFRl in SEQ ID NO: 4, a HFR2 of the HFR2 in SEQ ID NO: 4, a HFR3 of the HFR3 in SEQ ID NO: 4; or a HFR4 of the HFR4 in SEQ ID NO: 4.

[0006] In some embodiments, a humanized cys-diabody that binds to CD70 is provided. The 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 (V_L), and a C-terminal Cysteine.

[0007] In some embodiments, a humanized minibody that binds to CD70 is provided. The minibody can comprise a polypeptide that comprises a single-chain variable fragment (scFv) that binds to CD70, the scFv comprising a variable heavy (V_H) domain linked a variable light (V_L) domain, a hinge-extension domain comprising a human IgGl hinge region, and a human IgG C_H3 sequence.

[0008] In some embodiments, a nucleic acid encoding an antigen binding construct of any of the antigen binding constructs provided herein is provided.

[0009] In some embodiments, a cell line producing an antigen binding construct of any of the antigen binding constructs provided herein is provided.

[0010] In some embodiments, a kit is provided, and can comprise an antigen binding construct as described herein, and a detectable marker.

[0011] In some embodiments, a method of detecting the presence or absence of a CD70 is provided. The method can comprise applying an antigen binding construct to a sample, and detecting a binding or an absence of binding of the antigen binding construct thereof to CD70. [0012] In some embodiments, a method of targeting a therapeutic agent to a CD70 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.

[0013] In some embodiments, a method of neutralizing a B or T lymphocyte cell in a subject in need thereof is provided. The method can comprise administering to the subject an antigen binding construct.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1A is a depiction of the anti-CD70 minibody in the V_HV_L orientation. The minibody forms a covalently bound homodimer that can bind two antigens (for example, CD70).

[0015] FIG. IB is a depiction of the anti-CD70 cys-diabody in the V_LV_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.

[0016] FIG. 1C is a depiction of the assembled cDNA gene expression construct for anti-CD70 minibody in V_LV_H orientation. Abbreviations: SP = signal peptide, V_H = variable heavy domain, V_L = variable light domain, C_H3 = third constant domain, L = linker, H/E = hinge/extension,

[0017] FIG. ID is a depiction of the assembled cDNA gene expression construct for the anti-CD70 cys-diabody in V_LV_H orientation. Abbreviations: 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.

[0018] FIG. IE is a flow chart depicting some embodiments of methods provided herein.

[0019] FIG. 2A depicts variable light chain sequences, and polynucleotide sequences variable light chain sequences. CDRs according to the Chothia definition are boxed. [0020] FIG. 2B depicts variable heavy chain sequences, and polynucleotide sequences encoding variable light chain sequences. CDRs according to the Chothia definition are boxed.

[0021] FIG. 2C depicts some sequences that can be used for various cys-diabodies (for example, within the arrangement of FIG. ID).

[0022] FIG. 2D depicts some sequences that can be used for various minibodies (for example, within the arrangement of FIG. 1C).

[0023] FIG. 2E depicts some sequences of hinge regions that can be employed in various embodiments.

[0024] FIG. 3 depicts a schematic map of pcDNA 3.1 myc-His(-) A, B, C vector.

[0025] FIG. 4 depicts a western blot for various minibodies.

[0026] FIG. 5 depicts a western blot for various cys-diabodies.

[0027] FIG. 6A depicts binding analysis by flow cytometry of V_LV_H minibodies to CD70-expressing human kidney carcinoma 786-0 cells.

[0028] FIG. 6B depicts binding analysis by flow cytometry of V_HV_L minibodies to CD70-expressing human kidney carcinoma 786-0 cells.

[0029] FIG. 6C depicts a binding curve for a V_LV_H minibody to CD70. The concentration of minibody in the transfection supernatant was quantified by ELISA and the binding curve obtained with the minibody provided the EC50 value indicated.

[0030] FIG. 6D depicts a binding curve for a V_HV_L minibody to CD70. The concentration of minibody in the transfection supernatant was quantified by ELISA and the binding curve obtained with the minibody provided the EC50 value indicated.

[0031] FIG. 7A depicts binding analysis by flow cytometry of V_L-5-V_H cys- diabodies to CD70 expressing human kidney carcinoma 786-0 cells.

[0032] FIG. 7B depicts binding analysis by flow cytometry of V_H-5-V_L cys- diabodies to CD70 expressing human kidney carcinoma 786-0 cells.

[0033] FIG. 7C depicts binding analysis by flow cytometry of V_L-8-V_H cys- diabodies to CD70 expressing human kidney carcinoma 786-0 cells.

[0034] FIG. 7D depicts binding analysis by flow cytometry of V_H-8-V_L cys- diabodies to CD70 expressing human kidney carcinoma 786-0 cells. [0035] FIG. 8 depicts an amino acid sequence of the CD70 protein.

DETAILED DESCRIPTION

[0036] Described herein are antigen binding constructs, including antibodies and fragments thereof, such as scFv, cys-diabodies, and minibodies, that bind to a target molecule, CD70. Such antigen binding constructs can be useful for detecting the presence, localization, and/or quantities of the target molecule (CD70 and/or CD70+ cells). Such antigen binding constructs can also be useful for targeting therapeutic agents to cells that express the target molecule. In some embodiments, 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). In some embodiments, methods are provided for using the antigen binding constructs for therapeutic purposes.

[0037] Therapeutic drugs targeting CD70 have been in development. However, apart from the present disclosure, there are no known imaging agents currently in development direct to CD70 expressed on tumor cells, for example in metastatic disease, or on activated inflammatory cells in disease settings.

[0038] Current practice for diagnostic imaging for solid tumors and hematopoietic cancers that express CD70 can have limited specificity and sensitivity. Diagnostic positron emitting tomography (PET) imaging has been performed using 18 F fluorodeoxyglucose ( 18 F- FDG) as tracer relies on the detection of increased glucose metabolism in cancer cells.

However, 18 F-FDG has limitations in sensitivity and specificity particularly for prostate and ovarian cancers that have lower metabolic activity. Additionally, false positives may arise from inflamed or infected tissues when using 18 F-FDG. Hence, there is a need to develop better imaging agents for cancer for improved detection and monitoring of disease. Imaging agents that recognize CD70 may also be important for diagnosing and treating inflammatory, autoimmune diseases and organ transplants where CD70 is expressed on infiltrating activated T and B cells.

[0039] In some embodiments, antigen binding constructs with specific targeting of CD70 that are optimized for diagnostic imaging are provided herein. Since these fragments will bind specifically to cell surface expressed CD70, false readings are less likely in comparison to imaging of cell metabolism. In some embodiments, a CD70-specific antigen binding construct imaging agent provides a substantial improvement in specificity over 18 F-labeled imaging agents such as FDG which are based on detecting changes in metabolism.

[0040] In some embodiments imaging agents that include antigen binding constructs which specifically target CD70 can be useful the development of therapies targeting CD70. For example, the imaging agent can be used to monitor patients during the course of treatment or to select patient populations for therapies that require CD70 expression in the disease setting.

[0041] In some embodiments, the antigen binding constructs provided herein have specific targeting of CD70 that are superior for diagnostic imaging.

[0042] In some embodiments provided herein, the antigen binding construct approaches for targeting the CD70 can harness the binding affinity and specificity advantage of antigen binding constructs compared to other approaches such as small-molecule or peptides. In some embodiments provided herein, the antigen binding constructs have highly specific binding, which can achieve very strong binding affinities (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. For targeting CD70, 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. Some embodiments of the scFv, minibody, and cys-diabody provided herein provide the opportunity for same-day or next-day imaging. 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. [0043] As detailed below, in some embodiments, the CD70 antigen binding constructs are for diagnostics. When labeled with an appropriate radioisotope (e.g., the positron emitter Iodine- 124, Copper-64, and/or Zirconium-89 for PET imaging) or fluorophore (for fluorescent imaging), the antibody fragments can be used for preclinical imaging of CD70 as shown herein. These CD70 antigen binding constructs can also be used as potential SPECT imaging agents by simply changing the radiolabel. For example, the DOTA-conjugated minibody can be radiolabeled with a SPECT radiometal isotope such as Indium-I l l.

[0044] In some embodiments, the antigen binding constructs can be clinical imaging agents (PET/SPECT) in humans. CD70 is overexpressed in multiple cancer types (including renal carcinoma, pancreatic, larynx/pharynx, melanoma, ovarian, lung, glioblastoma, colon cancers. non-Hodgkin's lymphoma (NHL), Waldenstrom macroglobulinemia, Hodgkin's lymphoma, B and T cell leukemia and multiple myeloma. Accordingly, in some embodiments, CD70 antigen binding constructs can be used for targeted diagnostic detection for these cancers. Since CD70 is present on activated but not resting B and T lymphocytes and on dendritic cells, CD70 can also be used for targeted diagnostic detection of inflammatory, autoimmune diseases and organ transplants where CD70 is expressed on infiltrating activated T and/or B cells. In some embodiments, expression of CD70 on infiltrating activated immune cells in the setting of autoimmune and inflammatory disease and in organ transplant rejection is use in diagnosis. In some embodiments, the antigen binding construct can be used as a therapeutic.

Definitions and various embodiments

[0045] The term "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. The term "prevent" does not require the absolute prohibition of the disorder or disease. [0046] 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. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for example by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly, given the present disclosure. For additional guidance, see Remington: The Science and Practice of Pharmacy 21^st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, Pa., 2005.

[0047] 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. In some embodiments, they can be linked via a linking molecule or moiety. In some embodiments, 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. The term "target" or "target molecule" denotes the CD70 protein. Examples of CD70 proteins are known in the art, and include, for example the CD70 protein of SEQ ID NO: 27 (FIG. 8). [0048] The term "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.). The term "antibody" includes cys-diabodies, scFv, and minibodies. Thus, each and every embodiment provided herein in regard to "antibodies" is also envisioned as scFv, cys-diabody, and/or minibody embodiments, unless explicitly denoted otherwise. The term "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. In some embodiments, 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. For full length chains, the light chains are classified as either kappa or lambda. For full length chains, the heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V_L) and variable heavy chain (V_H) refer to these regions of light and heavy chains respectively. As used in this application, an "antibody" encompasses all variations of antibody and fragments thereof. Thus, 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. In some embodiments, the antibody binds specifically to a desired target.

[0049] "Complementarity-determining domains" or "complementarity- determining regions ("CDRs") interchangeably refer to the hypervariable regions of V_L and V_H- The CDRs are the target protein-binding site of the antibody chains that harbors specificity for such target protein. In some embodiments, 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).

[0050] 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. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997)), ImMunoGeneTics database (IMGT) (on the worldwide web at imgt.org/) Giudicelli, V., Duroux, P., Ginestoux, C, Folch, G., Jabado-Michaloud, J., Chaume, D. and Lefranc, M.-P. 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). PMID: 12477501; Brochet, X., Lefranc, M.-P. and Giudicelli, V. IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis Nucl. Acids Res, 36, W503-508 (2008); AbM (Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); the contact definition (MacCallum et al., J. Mol. Biol., 262:732-745 (1996)), and/or the automatic modeling and analysis tool Honegger A, Pliickthun A. (world wide web at bioc dot uzh dot ch/antibody/Numbering/index dot html).

[0051] The term "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. In some embodiments, 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. In some embodiments, CDR3 of the heavy chain variable region is sufficient for the antigen binding construct specificity.

[0052] 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. In this application, 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. In some embodiments, full length heavy and/or light chains are contemplated. In some embodiments, only the variable region of the heavy and/or light chains are contemplated as being present.

[0053] Antibodies can exist as intact immunoglobulins or as a number of fragments produced by digestion with various peptidases. Thus, for example, 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_H1 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. (Paul, Fundamental Immunology 3d ed. (1993). 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. Thus, the term "antibody," as used herein, 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)). [0054] For preparation of monoclonal or polyclonal antibodies, 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. 2006 Jul, 11(13- 14):655-60, Recombinant polyclonal antibodies: the next generation of antibody therapeutics?, Haurum JS). Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express fully human monoclonal antibodies. Alternatively, phage display technology can be used to identify high affinity binders to selected antigens (see, for example, McCafferty et al., supra; Marks et al., Biotechnology, 10:779-783, (1992)).

[0055] Methods for humanizing or primatizing non-human antibodies are well known in the art. Generally, 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. In some embodiments, 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. Accordingly, 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. In practice, 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 in rodent antibodies. [0056] 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.

[0057] 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.

[0058] Other 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.

[0059] The term "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. The term "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.

[0060] 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. For example, 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. The pharmaceutical 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. In some embodiments, the antigen binding construct can be delivered intraoperatively as a local administration during an intervention or resection.

[0061] The term "CD70 dependent disorder" includes any disorder in which CD70 plays a role in the disorder itself. In some embodiments, this denotes over-expression of CD70. Examples of the disorders include, multiple cancer types, such as renal, pancreatic, larynx/pharynx, melanoma, ovarian, lung, glioblastoma and colon cancers as well as non- Hodgkin's lymphoma (NHL), Waldenstrom macroglobulinemia, Hodgkin's lymphoma, B and T cell leukemia and multiple myeloma.

[0062] 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.

[0063] The phrase "specifically (or selectively) bind," when used in the context of describing the interaction between an antigen, for example, a protein, to an antibody or antibody-derived binding agent, refers to a binding reaction that is determinative of the presence of the antigen in a heterogeneous population of proteins and other biologies, for example, in a biological sample, for example, a blood, serum, plasma or tissue sample. Thus, under designated immunoassay conditions, in some embodiments, 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. For example, 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). Typically 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.

[0064] The term "equilibrium dissociation constant (¾, 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.

[0065] The term "isolated, " 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.

[0066] The term "nucleic acid" or "polynucleotide" 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. Specifically, 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)).

[0067] The terms "polypeptide," "peptide," and "protein" are 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.

[0068] The term "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.

[0069] "Conservatively 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. Such 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. One of skill will recognize that 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) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0070] As to 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.

[0071] 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)).

[0072] "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.

[0073] The terms "identical" or percent "identity," in the context of two or more 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. Some embodiments provided herein provide 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 Table 0.1, Table 0.2, and FIGs. 2A-2E, and any one or more of the nucleic acid sequences exemplified in any one of FIGs. 2A-2D. Optionally, 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. With respect to amino acid sequences, 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. With respect to 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.

[0074] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, 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. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0075] 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. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0076] 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). These 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 BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0077] 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. For example, 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. [0078] An indication that two 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. Thus, in some embodiments, 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.

[0079] 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. In some embodiments, the mammal can be an agricultural mammal (for example, equine, ovine, bovine, porcine, camelid) or domestic mammal (for example, canine, feline).

[0080] The term "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.

[0081] The term "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)

[0082] 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. [0083] Anti-CD70 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 Figures 1A-1D.

Table 0.1: Minibodies (N-terminus to C-terminus)

Table 0.2: Cys-Diabodies (N-terminus to C-terminus)

[0084] Depicted in Tables 0.1 and 0.2 are arrangements of sequences for monomers that can be used in minibodies (Table 0.1) and cys-diabodies (Table 0.2). Each row of the table represents the sequence of a monomer construct, with left-to-right representing N-terminus to C-terminus. In some embodiments, the shown sequences of each monomer construct are directly linked to each other. Thus, in some embodiments, 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. In some embodiments, for example, 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. In some embodiments, column 3 and column 6 can be swapped with one another. In some embodiments, 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. Thus, the tables represent all possible combinations, both within a single row and across various rows (and with columns swapped).

[0085] In some embodiments, an antigen binding construct includes a heavy chain CDR1 (HCDR1) of the HCDR 1 in SEQ ID NO: 4; a heavy chain CDR2 (HCDR2) of the HCDR2 in SEQ ID NO: 4; a heavy chain CDR3 (HCDR3) of the HCDR3 in SEQ ID NO: 4; a light chain CDR1 (LCDR1) of the LCDR1 in SEQ ID NO: 2; a light chain CDR2 (LCDR2) of the LCDR2 in SEQ ID NO: 2; and/or a light chain CDR3 (LCDR3) of the LCDR3 in SEQ ID NO: 2. These constructs can be in any of the forms provided herein, including diabodies, cys-diabodies, minibodies, scFv, etc. In some embodiments, the antigen binding construct includes 6, 5, 4, 3, 2, or 1, the above CDRs (some embodiments of the CDRs are indicated in Figures 2 A and 2B). In some embodiments, the antigen binding construct includes HCDR3. In some embodiments, the antigen binding construct binds specifically to the target molecule. In some embodiments, the antigen binding construct competes for binding with one or more of the antibodies having the herein provided CDRs. In some embodiments, 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. [0086] In some embodiments, the antigen binding construct is human or humanized. In some embodiments, the antigen binding construct includes at least one human framework region (for example, those section shown before, between, and after the CDRs shown in FIGs. 2 A and 2B), 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. In some embodiments the antigen binding construct includes a heavy chain FR1 (HFR1) of the HFR1 in SEQ ID NO: 4; a heavy chain FR2 (HFR2) of the HFR2 in SEQ ID NO: 4; a heavy chain FR3 (HFR3) of the HFR3 in SEQ ID NO: 4; a heavy chain FR4 (HFR4) of the HFR4 in SEQ ID NO: 4; a light chain FR1 (LFR1) of the LFR1 in SEQ ID NO: 2; a light chain FR2 (LFR2) of the LFR2 in SEQ ID NO: 2; a light chain FR3 (LFR3) of the LFR3 in SEQ ID NO: 2; and a light chain FR4 (LFR4) of the LFR4 in SEQ ID NO: 2. In some embodiments, the antigen binding construct includes 8, 7, 6, 5, 4, 3, 2, or 1 of the listed FRs.

[0087] In some embodiments, the antigen binding construct includes a detectable marker. In some embodiments, the antigen binding construct includes a therapeutic agent.

[0088] In some embodiments, 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. In some embodiments, 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. In some embodiments, the antigen binding construct is a minibody. In some embodiments, 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 Figures 2 A and 2B). In some embodiments, the antigen binding construct is a monovalent scFv. In some embodiments, a monovalent scFv is provided that includes the HCDRl in the HCDRl of FIG. 2B, the HCDR2 in the HCDR2 of FIG. 2B, the HCDR3 in the HCDR3 of FIG. 2B, the LCDR1 in the LCDR1 of FIG. 2 A, the LCDR2 in the LCDR2 of FIG. 2A, and the LCDR3 in the LCDR3 of FIG. 2A. In some embodiments, the monovalent scFv includes the heavy chain variable region of the heavy chain variable region in FIG. 2B. In some embodiments, the monovalent scFv includes the light chain variable region of the light chain variable region in FIG. 2A. In some embodiments, the monovalent scFv includes the heavy chain variable region of the heavy chain variable region in FIG. 2B, and the light chain variable region of the light chain variable region in FIG. 2A.

[0089] In some embodiments, 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. Thus, bispecific antigen binding constructs can bind to two or more epitopes. In some embodiments, 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. In some embodiments, the first epitope is part of a first antigen, and the second epitope is part of a second antigen, and the bispecific antigen binding construct can thus bind to two different antigens. In some embodiments, the antigen binding construct binds to two epitopes simultaneously.

[0090] In some embodiments, the antigen binding construct has a heavy chain variable region of the heavy chain variable region in SEQ ID NO 4. In some embodiments, 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: 4. In some embodiments, the antigen binding construct has a light chain variable region that includes SEQ ID NO: 2. In some embodiments, 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: 2. In some embodiments, 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.

[0091] 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. In some embodiments, the competing antigen binding construct binds to the same epitope on the target molecule as the reference antigen binding construct. In some embodiments, the reference antigen binding construct binds to a first epitope of the target molecule, and 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. In some embodiments, the first epitope overlaps with the second epitope.

[0092] In some embodiments, 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. In some embodiments, the shorter serum half lives for the minibody and the cys-diabody allow for optimal imaging over a range of times from approximately 6-48 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, which can provide a significant advantage in the clinic with respect to patient care management.

[0093] In addition, the cys-diabody antibody format features the C-terminus cysteine tail. These two sulfhydryl groups (following mild reduction) provide a strategy for site-specific conjugation of functional moieties such as radiolabels that need not interfere with the cys-diabody' s binding activity.

[0094] In some embodiments, the CD70 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-CD70 antibody. For example, 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 murine versions of the CD70 antibody. In some embodiments, an antibody fragment comprises one or more CDR regions from the variable heavy or light regions of a humanized anti-CD70 antibody. Diabodies that bind to the Target Molecule

[0095] In some embodiments, 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. In some embodiments, 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.

[0096] In some embodiments, 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. Thus, in some embodiments, the diabody is bivalent. In some embodiments, 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 Figure IB.

[0097] In some embodiments, the linker can be a peptide. In some embodiments, 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. As described further herein, some cys-diabodies can include a peptide linker that is 5 to 8 amino acids in length. In some embodiments, 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. Naturforsch, 58c, 558- 561). In some embodiments, 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.

[0098] In some embodiments, 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. In some embodiments, the humanized cys-diabody is a homodimer. In some embodiments, the humanized diabody is a heterodimer. In some embodiments, individual monomers are provided that each has a terminal cysteine residue.

[0099] In some embodiments, the scFv of the humanized cys-diabody has a V_HV_L orientation or a V_LV_H orientation. As used herein, a V_HV_L (which may also be referred to herein as "V_HV_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_LV_H orientation means that the V_L domain of the scFv is upstream from the V_H domain. As used herein, "upstream" means toward the N-terminus of an amino acid or toward the 5 ' end of a nucleotide sequence.

[0100] The antibody variable regions can be linked together by a linker as described herein. In some embodiments, the linker is a GlySer linker as described herein (for example, FIG. 2C).

[0101] In some embodiments, the cys-diabody includes a detectable marker.

[0102] In some embodiments, 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).

[0103] In some embodiments, the polypeptide of the monomer includes sequences as shown in Table 0.2. In some embodiments, the polypeptide of the monomer 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 the sequence of the V_L-5-V_H cys-diabody monomer shown in Table 0.2. In some embodiments, the polypeptide of the monomer 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 the sequence of the V_H-5-V_L cys-diabody monomer shown in Table 0.2. In some embodiments, the polypeptide of the monomer 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 the sequence of the V_L-8-V_H cys-diabody monomer shown in Table 0.2. In some embodiments, the polypeptide of the monomer 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 the sequence of the V_H-8-V_L cys-diabody monomer shown in Table 0.2.

[0104] In some embodiments, 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.

[0105] As will be appreciated by those of skill in the art, while the present disclosure generally references "cys-diabodies" alternative arrangements can be employed to achieve the same or similar ends. In some embodiments, any covalently modifiable moiety can be employed in place of one or more of the cysteines. For example, this can include a GlySer linker, a GlyLeu linker, and/or an insert cysteine after a short tag. In some embodiments, the connection can be established via a coiled coil or a leucine zipper. In some embodiments, 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. In some embodiments, rather than the tail providing space between the two polypeptide chains, 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.

[0106] In some embodiments, a chimeric cys-diabody that binds to the target molecule is provided. In some embodiments, the chimeric cys-diabody includes a monomer in the V_LV_H format, and includes the sequence of SEQ ID NO: 2 and/or 4, or of a monomer as shown 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. In some embodiments, the chimeric cys-diabody includes a monomer in the V_HV_L format, and includes the sequence of SEQ ID NO: 2 and/or 4, or of a monomer as shown 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.

[0107] In some embodiments, the cys-diabody includes one or more of the CDRs provided in the CDRs in FIG. 2A and/or 2B .

[0108] In some embodiments, any of the constructs provided herein (including those arrangements noted as cys-diabody embodiments, can be provided as a scFv embodiment. In such embodiments, the construct can still include the cysteine on the tail, but simply not be cross-linked. In other embodiments, the construct need not have the cysteine in a tail or the tail at all.

Linker and/or Tail Options

[0109] In some embodiments, for individual antigen binding constructs, 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.

[0110] In some embodiments, 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. In some embodiments, each linker within a pair for a diabody can be the same length. In some embodiments, each linker within the pair can be a different length. In some embodiments, any combination of linker length pairs can be used, as long as they allow and/or promote the desired combinations. In some embodiments, a modified amino acid can be used.

[0111] Table 0.2 provides Cys-Db variants, V_H-5-V_L, V_H-8-V_L, V_L-5-V_H, and V_L- 8-V_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, V_HV_L and V_LV_H- [0112] In some embodiments, the linker is a GlySer linker. The GlySer linker can be a polypeptide that is rich in Gly and/or Ser residues. In some embodiments, 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%. In some embodiments, 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.

[0113] In some embodiments, 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. In some embodiments, a terminal end of the antibody itself is altered so as to contain a cysteine. In some embodiments, a tail sequence, for example (Gly- Gly-Cys) is added at the C-terminus. In some embodiments, the cysteine tail sequence allows two monomers of a cys-diabody to form disulfide bonds with each other. In some embodiments, 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. In some embodiments, 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. In some embodiments, the tail can include a coiled coil and/or a leucine zipper. As noted above, in some embodiments, 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.

[0114] In some embodiments, the linking option between the two c-terminuses can be achieved by a cysteine, for direct and/or indirect, cross-linking.

[0115] FIGs. 2C-2E depict various optional sequences that can be used in some embodiments of the scFv, cys-diabodies, and/or, minibodies. In some embodiments, the cys- diabody (and or scFv) can be any of those provided herein, but with any of the sequences provided in FIG. 2C. In some embodiments, the minibody can be any of those provided herein, but with any of the sequences provided in FIG. 2D. In some embodiments, any of the antigen binding constructs provided herein can include any of the hinge sequences provided in FIG. 2E.

Minibodies that bind to the Target Molecule

[0116] A "minibody" as described herein includes a homodimer, wherein each monomer is a single-chain variable fragment (scFv) linked to a human IgGl C_H3 domain by a linker, such as a hinge sequence. In some embodiments, the hinge sequence is a human IgGl hinge sequence.

[0117] In some embodiments, the hinge sequence is an artificial hinge sequence. In some embodiments, 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.

[0118] In some embodiments, the artificial hinge sequence includes approximately the first 14 or 15 residues of the human IgGl hinge followed by a linker sequence. In some embodiments, the linker can be any of those provided herein. In some embodiments, the linker can be a GlySer linker sequence that is 6, 7, 8, 9 or 10 amino acids in length. In some embodiments, 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. In some embodiments, association between the C_H3 domains causes the minibody to exist as a stable dimer.

[0119] In some embodiments, 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 Figures 2A and/or 2B, or as shown in Table 0.2). In some embodiments, 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.

[0120] In some embodiments, the polypeptide of the monomer includes sequences as shown in Table 0.1. In some embodiments, the polypeptide of the monomer 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 the sequence of the V_LV_H minibody monomer shown in Table 0.1.

[0121] In some embodiments, the minibody has a variable chain region (heavy, light or heavy and light chain variable region) that is at least about 80% identical to a sequence in SEQ ID NO: 2, and/or 4, for example at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94, 95%, 96%, 97%, 98%, or 99% identity.

[0122] The scFv can have a V_HV_L or a V_LV_H orientation. In some embodiments, 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. In some embodiments, the linker is Gly-Ser-rich and approximately 15-20 amino acids in length. In another embodiment, the linker is GlySer rich and is 18 amino acids in length. In some embodiments, 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. In some embodiments, 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_HV_L or a V_LV_H orientation.

[0123] In some embodiments, 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_H3 sequence. In some embodiments, each monomer of the minibody includes an IgG2, an IgG3, or an IgG4 C_H3. In some embodiments, 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. Thus, 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.

[0124] In some embodiments, a chimeric minibody that binds to the target molecule is provided. In some embodiments, the chimeric minibody includes a monomer in the V_LV_H format, and includes the sequence of SEQ ID NO: 2, 4 or a monomer as shown in Table 0.1, 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. In some embodiments, the chimeric minibody includes a monomer in the V_HV_L format, and includes the sequence of SEQ ID NO: 2, 4 or a monomer as shown in Table 0.1 , 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. In some embodiments, the minibody comprises one or more of the CDRs outlined in FIG. 2A and/or 2B. In some embodiments, the minibody comprises one or more of the variable regions in FIG. 2A and/or 2B.

[0125] In some embodiments, the minibody includes the heavy chain variable region as outlined in Figure 2B. In some embodiments, the minibody includes the light chain variable region as outlined in Figure 2A.

[0126] In some embodiments, the minibody includes one or more of the CDRs provided in the CDRs in FIG. 2A and/or 2B.

Nucleic Acids

[0127] In some embodiments, 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. Thus, in some embodiments, a nucleic acid encoding an antigen binding construct is provided. In some embodiments, the nucleic acid encodes one part or monomer of a cys-diabody or minibody. In some embodiments, 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.

[0128] In some embodiments, an expression vector contains a nucleic acid encoding an antigen binding construct as disclosed herein. In some embodiments, the expression vector includes pcDNA3.1™/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. In some embodiments, the expression vector includes a plasmid. In some embodiments, the vector includes a viral vector, for example a retroviral or adenoviral vector. In embodiments, the vector includes a cosmid, YAC, or BAC.

[0129] In some embodiments, the nucleotide sequence encoding at least one of the minibody monomers comprises at least one of SEQ ID NO: 1, 3, 13, 15, 17, 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.

[0130] In some embodiments, the nucleotide sequence encoding at least one of the cys-diabody monomers includes SEQ ID NO: 1, 3, 5, 7, 9, 11, 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.

Cell Lines

[0131] In some embodiments, a cell line is provided that expresses at least one of the antigen binding constructs described herein. In some embodiments, a mammalian cell line (for example, CHO-K1 cell line) is an expression system to produce the minibodies, cys- diabodies, scFv, or other antibodies as described herein. In some embodiments, 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. Thus, in some embodiments, 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-CD70 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. Other systems can include insect cells and/or plant cells.

Antigen Binding Construct Modifications

[0132] In some embodiments, 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, gormylation and metabolic synthesis of tunicamycin. In some embodiments, the derivative can contain one or more non-natural amino acids.

[0133] In some embodiments, 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. In some embodiments, 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. For example, introduction of 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. Other 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 thiosemicarbazone linkage, a semicarbazone linkage, an oxime linkage and a carbon-carbon linkage. In some embodiments, no cysteine or other linking aspect or tail, need be included in the antigen binding construct.

Detectable markers

[0134] In some embodiments, a modified antigen binding construct is conjugated to a detectable marker. As used herein, 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). In addition, some nanoparticles, for example quantum dots and metal nanoparticles (described below) can be suitable for use as a detection agent. In some embodiments, the detectable marker is IndoCyanine Green (ICG).

[0135] Exemplary 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, ³³P, ⁴⁵Ti, ⁴⁷Sc, ⁵²Fe, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁵Sc, ⁷⁷As, ⁸⁶Y, ⁹⁰Y, ⁸⁹Sr, ⁸⁹Zr, ⁹⁴Tc, ⁹⁴Tc, "mTc, "Mo, ¹⁰⁵Pd, ¹⁰⁵Rh, ^mAg, ^luIn, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm, ^154" ¹⁵⁸Gd, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁹Er, ¹⁷⁵Lu, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹At, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra and ²²⁵ Ac. 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.

[0136] When the detectable marker is a radioactive metal or paramagnetic ion, in some embodiments, 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. Examples of chelating groups that may be used according to the embodiments herein 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. The chelate can be linked to the antigen binding construct by a group which allows formation of a bond to the molecule with minimal loss of immunoreactivity and minimal aggregation and/or internal cross-linking. The same 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 radionuclides, such as Radium-223 for RAIT may be used. In certain embodiments, chelating moieties may be used to attach a PET imaging agent, such as an Aluminum- 18 F complex, to a targeting molecule for use in PET analysis.

[0137] 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 thereof.

[0138] 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 GREEN™, 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.

[0139] 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, β-galactosidase, β-glucoronidase or β-lactamase. Such enzymes may be used in combination with a chromogen, a fluorogenic compound or a luminogenic compound to generate a detectable signal.

[0140] In some embodiments, the antigen binding construct is conjugated to a nanoparticle. The term "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. Metal, dielectric, and semiconductor nanoparticles have been formed, as well as hybrid structures (for example core-shell nanoparticles). 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.

Therapeutic agents

[0141] In some embodiments, 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. Examples of 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.

[0142] 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 immunotherapeutics. In some embodiments 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, bicalutamide, bortezomib, bleomycin, busulfan, calcium leucovorin, citrovorum factor, capecitabine, canertinib, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, cortisone, cyclophosphamide, cytarabine, darbepoetin alfa, dasatinib, daunomycin, decitabine, denileukin diftitox, dexamethasone, dexasone, dexrazoxane, dactinomycin, daunorubicin, decarbazine, docetaxel, doxorubicin, doxifluridine, eniluracil, epirubicin, epoetin alfa, erlotinib, everolimus, exemestane, estramustine, etoposide, filgrastim, fluoxymesterone, fulvestrant, flavopiridol, floxuridine, fludarabine, fluorouracil, flutamide, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin, granulocyte - colony stimulating factor, granulocyte macrophage-colony stimulating factor, hexamethylmelamine, hydrocortisone hydroxyurea, ibritumomab, interferon alpha, interleukin - 2, interleukin-11, isotretinoin, ixabepilone, idarubicin, imatinib mesylate, ifosfamide, irinotecan, lapatinib, lenalidomide, letrozole, leucovorin, leuprolide, liposomal Ara-C, lomustine, mechlorethamine, megestrol, melphalan, mercaptopurine, mesna, methotrexate, methylprednisolone, mitomycin C, mitotane, mitoxantrone, nelarabine, nilutamide, octreotide, oprelvekin, oxaliplatin, paclitaxel, pamidronate, pemetrexed, panitumumab, PEG Interferon, pegaspargase, pegfilgrastim, PEG-L-asparaginase, pentostatin, plicamycin, prednisolone, prednisone, procarbazine, raloxifene, rituximab, romiplostim, ralitrexed, sapacitabine, sargramostim, satraplatin, sorafenib, sunitinib, semustine, streptozocin, tamoxifen, tegafur, tegafur-uracil, temsirolimus, temozolamide, teniposide, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, trimitrexate, alrubicin, vincristine, vinblastine, vindestine, vinorelbine, vorinostat, or zoledronic acid.

[0143] 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.

[0144] In some embodiments 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.

[0145] 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. For example, an antigen binding construct may be radiolabeled with Iodine-131 and conjugated to a lipid carrier, such that the anti-CD70-lipid conjugate forms a micelle. The micelle can incorporate one or more therapeutic or detectable markers. Alternatively, in addition to the carrier, the antigen binding construct may be radiolabeled with Iodine-131 I (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.

[0146] In some embodiments, anti-CD70 antigen binding constructs are conjugated to a therapeutic agent. While these antigen binding constructs can have a shorter half-life compared to a full-length antibody, in some embodiments, 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. CD70 is an internalizing antigen which is very suitable for antibody drug-conjugate approaches. In some embodiments, a therapeutic approach includes radioimmunotherapy by attaching an appropriate radiolabel such as the beta-emitter Yttrium-90 which can deliver cell damage and death to local cancerous tissue or to activated immune cells in the setting of autoimmune and inflammatory disease and in organ transplant rejection. In some embodiments, treatment with these fragments armed with a cytotoxic drug or radionuclide result in less nonspecific toxicity as they will be cleared from the body more rapidly.

[0147] In some embodiments, the antigen binding construct can be connected to a therapeutic agent to treat renal, pancreatic, larynx/pharynx, melanoma, ovarian, lung, glioblastoma or colon cancer, or non-Hodgkin's lymphoma, Waldenstrom macroglobulinemia, Hodgkin's lymphoma, B or T cell leukemia, and/or multiple myeloma.

[0148] In some embodiments, anti-CD70 antigen binding constructs are used as a stand-alone medicament (e.g. antigen binding construct in the absence of therapeutic agent) in the treatment of renal, pancreatic, larynx/pharynx, melanoma, ovarian, lung, glioblastoma or colon cancer, or non-Hodgkin' s lymphoma, Waldenstrom macroglobulinemia, Hodgkin's lymphoma, B or T cell leukemia, or multiple myeloma.

Kits

[0149] In some embodiments, kits are provided. In some embodiments, the kit includes an antigen binding construct as described herein. In some embodiments, the kit includes a nucleic acid that encodes an antigen binding construct as described herein. In some embodiments, the kit includes a cell line that produces an antigen binding construct as described herein. In some embodiments, the kit includes a detectable marker as described herein. In some embodiments, the kit includes a therapeutic agent as described herein. In some embodiments, the kit includes buffers. In some embodiments, the kit includes positive controls, for example CD70, CD70+ cells, or fragments thereof. In some embodiments, the kit includes negative controls, for example a surface or solution that is substantially free of CD70. In some embodiments, the kit includes packaging. In some embodiments, the kit includes instructions.

Methods of detecting the presence or absence of the target molecule

[0150] 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, CD70.

[0151] Figure IE illustrates some embodiments of methods of detecting the presence or absence of CD70. 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. Optionally, 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. [0152] In some embodiments, an antigen binding construct as described herein is applied to a sample in vivo. The antigen binding construct can be administered to a subject. In some embodiments, the subject is a human. In some embodiments, 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. In some embodiments, the antigen binding construct is infused into the subject. In some embodiments, the infusion is intravenous. In some embodiments, the infusion is intraperitoneal. In some embodiments, the antigen binding construct is applied topically or locally (as in the case of an interventional or intraoperative application) to the subject. In some embodiments, a capsule containing the antigen binding construct is applied to the subject, for example orally or intraperitoneally. In some embodiments, the antigen binding construct is selected to reduce the risk of an immunogenic response by subject. For example, for a human subject, the antigen binding construct can be humanized as described herein. In some embodiments, following in vivo application of the antigen binding construct, the sample, or a portion of the sample is removed from the host. In some embodiments, 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.

[0153] In some embodiments, the antigen binding construct is applied to a sample in vitro. In some embodiments, the sample is freshly harvested from a subject, for example a biopsy. In some embodiments, the sample is incubated following harvesting from a subject. In some embodiments, the sample is fixed. In some embodiments the sample includes a whole organ and/or tissue. In some embodiments, the sample includes one or more whole cells. In some embodiments the sample is from cell extracts, for example lysates. In some embodiments, antigen binding construct in solution is added to a solution in the sample. In some embodiments, 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. In some embodiments, lyophilized antigen binding construct is added to a sample that contains solution, thus reconstituting the antigen binding construct. [0154] In some embodiments, 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. In some embodiments, the incubation is within a subject to which the antigen binding construct was administered. In some embodiments, the incubation is within an incubator. In some embodiments, 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.

[0155] In some embodiments, the antigen binding construct that is not bound to the target is optionally removed from the sample. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, following the wash or washes, 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.

[0156] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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%. In some embodiments, 2 days after application of the antigen binding construct, 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%.

[0157] In some embodiments, the presence or absence of the target, CD70, 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.

[0158] In some embodiments, the antigen binding construct includes a detectable marker as described herein. Thus, the presence of the antigen binding construct can be inferred by detecting the detectable marker.

[0159] In some embodiments, 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. For example, 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. In some embodiments, 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.

[0160] In some embodiments, 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. In some embodiments, binding of the absence of binding of the antigen binding construct is detected via two or more forms of imaging. In some embodiments, detection can be via near-infrared (NIR) and/or Cerenkov.

Methods of targeting a therapeutic agent to a cell

[0161] 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 CD70. Thus, some embodiments include methods of targeting a therapeutic agent to a target positive cell. 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. In some embodiments, the antigen binding construct includes at least on therapeutic agent as described herein. In some embodiments, the therapeutic can be directly conjugated to the antigen binding construct via a covalent bond, such as a disulfide bond. In some embodiments, the subject can benefit from the localization of a CD70 positive cell to another cell or agent.

[0162] Optionally, before and/or after administration of the antigen binding construct that includes at least one therapeutic agent, the number and/or localization of the target positive cells of the patient is determined. 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.

[0163] In some embodiments, the CD70 antigen binding construct can be used as a therapeutic agent as stand-alone construct (for example, without a toxin conjugated thereto). 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.

[0164] In some embodiments, another therapeutic approach is radioimmunotherapy via attaching an appropriate radiolabel such as the beta-emitter Yttrium- 90 which can deliver cell damage and death.

[0165] In some embodiments, the antigen binding construct is used to target cells expressing CD70 antigen, including, for example renal, pancreatic, larynx/pharynx, melanoma, ovarian, lung, glioblastoma or colon cancer, or non-Hodgkin's lymphoma, Waldenstrom macroglobulinemia, Hodgkin's lymphoma, B or T cell leukemia, or multiple myeloma cancer cells.

Additional Embodiments

[0166] In some embodiments, 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.

[0167] Additionally, in some embodiments, the cys-diabody fragment component monomers contain C-terminus cysteine residues that form disulfide bonds. These covalently bound cysteine residues can be opened via mild chemical reduction to provide an active thiol group for site specific conjugation. Currently, 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. The location of the C-terminus cysteine residues on the properly folded cys-diabody are opposite the CDRs and prevents steric blocking of CDR-antigen interaction by the conjugated material.

[0168] The ability to image a patient's entire body for the presence of an antibody's target prior to and during treatment provides valuable information for personalized patient management. During the testing of an antibody therapy's safety and efficacy, it is useful to be able to select and test the treatment on patients who express the antibody's target as part of their disease progression.

[0169] In some embodiments, scFv, minibody, and cys-diabody diagnostic fragments matching available antibody therapies allow matching of the patient's disease state with the appropriate antibody therapy.

[0170] In some embodiments, a method of targeting a CD70+ cell to a first antigen is provided. The method can include applying a bispecific antigen binding construct to a sample. The bispecific antigen binding construct can include a CD70 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. In some embodiments, 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. In some embodiments, 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. Without being limited to any one theory, in some embodiments, 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 CD70) on the first cell, and thus brings the target positive cell in proximity to the first cell. For example, a CD70+ cell can be brought into proximity of a cancer cell, and can facilitate an immune response against that cancer cell. In some embodiments, a bispecific antigen binding construct comprised of a CD3 and CD70 fragment can bring a cytoxic T cell in proximity of an activated CD70 expressing immune cell or CD70 expressing tumor cell to result in killing of the target cell.

EXAMPLE 1 : CD70 Antibodies and Antibody Fragments

[0171] The variable (V) regions of the humanized CD70 antibody were reformatted by protein engineering into a minibody and cys-diabody. The minibody format is approximately 80 kDa in size, with each monomer consisting of a single-chain variable fragment (scFv) linked to the human IgGl hinge-C_H3 domain (Figure 1). The variable heavy (V_H) and light (V_L) domains which are involved in the antigen recognition and binding, were connected via a GlySer-rich 18 amino acid linker and that makes up the scFv fragment.

[0172] The scFv, which can be in V_HV_L or V_LV_H orientation, was tethered to the human IgGl C_H3 domain via the human IgGl upper and core hinge regions (15 residues) followed by a 10 amino acid GlySer linker sequence. The minibody in either orientation (V_HV_L-C_H3 or V_LV_H-C_H3) exists as a stable dimer due to the association between the C_H3 domains as well as the formation of disulfide bonds within the hinge regions. To enable secretion of the minibody, a signal sequence was been incorporated to lead the expression construct at the N-terminus (Figure IB, Table 0.1).

[0173] The cys-diabody was a bivalent antibody fragment of -55 kDa in size. It was formed by two identical scFv fragments that end up cross-paired due to a shorter GlySer- rich linker between the V_L and V_H domains in each scFv (Figure IB). A cysteine is preceded by two glycines (GlyGlyCys) at the C-terminus of each scFv which form an inter-chain covalent disulfide bond (Figure ID, Table 0.2). Example 2: Cloning into pcDNA3.1/myc-His

[0174] cDNAs encoding each of the constructs shown in Table 0.1 and 0.2 were cloned into the mammalian expression vector pcDNA3.1/myc-His (-) Version A (Invitrogen Corp; FIG. 3).

Example 3: Expression of Minibodies

[0175] The various pcDNA3.1/myc-His (-) version expression constructs were transiently transfected into CHO-K1 cells to validate expression levels. The transfections were performed using LIPOFECTAMINE™ reagent in a 6-well plate format. Following 72 hours, supernatants were harvested and filtered to remove any cells and debris.

[0176] Western blot analysis was performed on the supernatants to confirm expression of the antigen binding constructs. Supernatants from the transfection of a standard minibody and cys-diabody were used as positive controls. Supernatants were analyzed on SDS-PAGE and transferred to a PVDF membrane. Under non-reducing conditions, the minibodies migrate at the expected molecular weight of 80-90 kDa (FIG. 4). A band representing the monomeric form for the minibody is also detected at approximately 40 kDa (FIG. 4). The cys-diabodies migrate at the expected molecular weight of -55 kDa (FIG. 5). A band representing the monomeric form for the Cys-Db is also detected at -25 kDa (FIG. 5). Minibody variants were detected with horseradish peroxidase (HRP) conjugated anti-IgG (Fc-specific) antibody and cys-diabody variants were detected with HRP conjugated anti-penta-His antibody. Each blot was developed with the HRP substrate 3,3',5,5'-tetramethylbenzidine (TMB). These are representative blots of multiple experiments. The results shown in FIGs. 4-5 are representative blots of multiple experiments.

[0177] Binding analysis of various antigen binding constructs was performed on cells using flow cytometry (FIGs. 6A-6D and FIGs. 7A-7D). Supernatants containing transiently expressed antibody fragments were incubated with CD70-expressing human kidney carcinoma 786-0 cells. Binding of the minibodies (see FIGs. 6A-6B) and the cys- diabodies (see FIGs. 7A-7D) was detected with Allophycocyanin (APC)-conjugated anti- human IgG (Fc-specific) or anti-His-APC antibodies, respectively. Both the minibodies and cys-diabodies showed concentration dependent binding to the 786-0 cells. The concentration of minibody in the transfection supernatant was quantified by ELISA and binding curves were obtained with the minibodies to provide the EC50 values indicated in FIG. 6C and FIG. 6D.

[0178] Supernatants containing transiently expressed minibodies were incubated with CD70 expressing human kidney carcinoma 786-0 cells (see FIGs. 6A-6B). 1X10⁵ 786- 0 cells were incubated with the indicated concentration of minibody and analysis was performed with 10,000 events/point (FIGs. 6A-6B). Binding of minibody variants to 786-0 cells was detected following staining with Allophycocyanin (APC) -conjugated anti-human (Fc specific) IgGantibody. Negative control is the secondary antibody alone (8). All histograms show APC signal vs. cell count. The concentration of minibody in the transfection supernatant was quantified by ELISA and binding curves were obtained with the minibodies to provide the EC50 values indicated in FIG. 6C and FIG. 6D. The graphs in FIGs. 6C and 6D shows the average mean fluorescence intensity (MFI) plotted against the concentration of minibody for respective minibody variants.

[0179] Binding of cys-diabody variants to 786-0 cells following staining with Allophycocyanin (APC) conjugated anti-His antibody. 1X10⁵ 786-0 cells were incubated with indicated dilutions of the cys-diabody supernatant and analysis was performed with 10,000 events/point (see FIGs. 7A-7D). All histograms show APC signal vs. cell count. Staining with the secondary antibody alone was used as a negative control (8).

[0180] In some embodiments, the minibodies, diabodies, and/or scFv constructs provided herein have an EC50 in the pM range, for example, 1 to 1000 pM, including 1-10, 10-100, 10-1000, and 100-1000 pM ranges.

EXAMPLE 4: Therapeutic Treatment Using a Cys-Diabody

[0181] A CD70 cys-diabody that is a homodimer of monomers of Table 0.2 is provided. The cys-diabody is infused intravenously into a subject having renal cancer (or in the alternative, having, pancreatic, larynx/pharynx, melanoma, ovarian, lung, glioblastoma or colon cancer, or non-Hodgkin's lymphoma, Waldenstrom macroglobulinemia, Hodgkin's lymphoma, B or T cell leukemia, or multiple myeloma) in an amount adequate to bind to sufficient levels of CD70 in the subject to provide a lessening of the symptoms of renal cancer (or in the alternative, pancreatic, larynx/pharynx, melanoma, ovarian, lung, glioblastoma or colon cancer, or non-Hodgkin's lymphoma, Waldenstrom macroglobulinemia, Hodgkin's lymphoma, B or T cell leukemia, or multiple myeloma). The cys-diabody is conjugated to Yttrium 90.

EXAMPLE 5: Diagnostics Using a Cys-Diabody

[0182] A CD70 cys-diabody that is a homodimer of monomers of Table 0.2 is provided (or in the alternative, a CD70 minibody of Table 0.1). The cys-diabody (or minibody) is infused intravenously into a subject suspected of having renal cancer. The cys- diabody (or minibody) is radiolabeled with Iodine-124. The subject's kidneys are then examined via PET to determine if the level of Iodine-124 indicates the presence or absence of renal cancer (via displaying the level of CD70).

EXAMPLE 6: In vivo detection of CD70

[0183] 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). Alternatively, the minibody can be radiolabeled by directly radiolabeling with Iodine via Tyrosine residues.

[0184] 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.

[0185] Localization of minibody is used to determine localization of CD70 in the subject.

EXAMPLE 7: In vivo detection of CD70

[0186] 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). Alternatively, the cys-diabody can be radiolabeled by directly radiolabeling with Iodine via Tyrosine residues.

[0187] 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.

[0188] Localization of cys-diabody is used to determine localization of CD70 in the subject.

[0189] In this application, the use of the singular can include the plural unless specifically stated otherwise or unless, as will be understood by one of skill in the art in light of the present disclosure, the singular is the only functional embodiment. Thus, for example, "a" can mean more than one, and "one embodiment" can mean that the description applies to multiple embodiments.

[0190] All references cited herein, including patents, patent applications, papers, text books, and the like, and the references cited therein, to the extent that they are not already, are hereby incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application; including but not limited to defined terms, term usage, described techniques, or the like, this application controls.

[0191] The foregoing description and Examples detail certain embodiments. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the invention may be practiced in many ways and the invention should be construed in accordance with the appended claims and any equivalents thereof.

Claims

We claim:

1. An antigen binding construct that comprises:

a HCDR1 of the HCDR1 in SEQ ID NO: 4;

a HCDR2 of the HCDR2 in SEQ ID NO: 4;

a HCDR3 of the HCDR3 in SEQID NO: 4;

a LCDR1 of the LCDR1 in SEQ ID NO: 2;

a LCDR2 of the LCDR2 in SEQ ID NO: 2; and

a LCDR3 of the LCDR3 in SEQ ID NO: 2.

2. The antigen binding construct thereof of claim 1, further comprising at least one of:

a LFR1 of the LFR1 in SEQ ID NO: 2;

a LFR2 of the LFR2 in SEQ ID NO: 2;

a LFR3 of the LFR3 in SEQ ID NO: 2;

a LFR4 of the LFR4 in SEQ ID NO: 2;

a HFR1 of the HFR1 in SEQ ID NO: 4;

a HFR2 of the HFR2 in SEQ ID NO: 4;

a HFR3 of the HFR3 in SEQ ID NO: 4; or

a HFR4 of the HFR4 in SEQ ID NO: 4.

3. The antigen binding construct of claim 1, wherein the antigen binding construct binds specifically to CD70.

4. The antigen binding construct of claim 1, further comprising a detectable marker selected from the group consisting of at least one of a radioactive substance, a dye, a contrast agent, a fluorescent compound, a bioluminescent compound, an enzyme, an enhancing agent, or a nanoparticle.

5. The antigen binding construct of claim 1, further comprising a therapeutic agent.

6. The antigen binding construct of claim 1, wherein the antigen binding construct is a monovalent scFv.

7. The antigen binding construct of claim 1, wherein the antigen binding construct is bivalent.

8. A humanized cys-diabody that binds to CD70, the cys-diabody comprising a polypeptide that comprises:

a single-chain variable fragment (scFv) comprising a variable heavy (V_H) domain linked to a variable light (V_L); and

a C-terminal cysteine.

9. The humanized cys-diabody of claim 8, wherein the order of the variable domains, from N terminus to C terminus of the polypeptide is V_L, V_H-

10. The humanized cys-diabody of claim 8, wherein the order of the variable domains, from N terminus to C terminus of the polypeptide is V_H, V_L.

11. The humanized cys-diabody of claim 8, further comprising a detectable marker selected from the group consisting of a radioactive substance, a dye, a contrast agent, a fluorescent compound, a bioluminescent compound, an enzyme, an enhancing agent, or a nanoparticle.

12. The humanized cys-diabody of claim 8, wherein the humanized cys-diabody comprises:

a HCDR1 of the HCDR1 in SEQ ID NO: 4;

a HCDR2 of the HCDR2 in SEQ ID NO: 4;

a HCDR3 of the HCDR3 in SEQID NO: 4;

a LCDR1 of the LCDR1 in SEQ ID NO: 2;

a LCDR2 of the LCDR2 in SEQ ID NO: 2; and

a LCDR3 of the LCDR3 in SEQ ID NO: 2.

13. A humanized minibody that binds to CD70, the humanized minibody comprising a polypeptide that comprises:

a single-chain variable fragment (scFv) that binds to CD70, the scFv comprising a variable heavy (V_H) domain linked a variable light (V_L) domain;

a hinge-extension domain comprising a human IgGl hinge region; and a human IgG CH3 sequence.

14. The humanized minibody of claim 13, further comprising a detectable marker selected from the group consisting of a radioactive substance, a dye, a contrast agent, a fluorescent compound, a bioluminescent compound, an enzyme, an enhancing agent, or a nanoparticle.

15. The humanized minibody of claim 13, wherein the humanized minibody comprises:

a HCDR1 of the HCDR1 in SEQ ID NO: 4;

a HCDR2 of the HCDR2 in SEQ ID NO: 4;

a HCDR3 of the HCDR3 in SEQID NO: 4;

a LCDR1 of the LCDR1 in SEQ ID NO: 2;

a LCDR2 of the LCDR2 in SEQ ID NO: 2; and

a LCDR3 of the LCDR3 in SEQ ID NO: 2.

16. A nucleic acid encoding an antibody of any one of claims 1 - 15.

17. A cell line producing an antibody of any of claims 1 - 15.

18. A kit comprising:

an antigen binding construct of any of claims 1 - 15; and

a detectable marker.

19. A method of detecting the presence or absence of a CD70, the method comprising:

applying an antigen binding construct thereof of any one of claims 1 - 15 to a sample; and

detecting a binding or an absence of binding of the antigen binding construct thereof to CD70.

20. The method of claim 19, wherein the antigen binding construct comprises a detectable marker selected from the group consisting of a radioactive substance, a dye, a contrast agent, a fluorescent compound, a bioluminescent compound, an enzyme, an enhancing agent, or a nanoparticle.

21. The method of claim 19, wherein applying the antigen binding construct comprises administering the antibody or fragment thereof to a subject.

23. The method of claim 19, wherein detecting binding or absence of binding of the antigen binding construct thereof to CD70 comprises positron emission tomography.

24. The method of claim 19, the method further comprising applying a secondary antibody or fragment thereof to the sample, wherein the secondary antibody or fragment thereof binds specifically to the antigen binding construct.

25. The method of claim 19, wherein the antibody or fragment thereof is incubated with the sample for no more than 1 hour.

26. A method of targeting a therapeutic agent to a CD70, the method comprising administering to a subject an antigen binding construct of any one of claims 1 - 15, wherein the antigen binding construct is conjugated to a therapeutic agent.

27. A method of neutralizing a B or T lymphocyte cell in a subject in need thereof, the method comprising administering to the subject an antigen binding construct of any one of claims 1 - 15.

28. The method of claim 27, wherein the subject has at least one of renal, pancreatic, larynx/pharynx, melanoma, ovarian, lung, glioblastoma or colon cancer, or non- Hodgkin's lymphoma, Waldenstrom macroglobulinemia, Hodgkin's lymphoma, B or T cell leukemia, or multiple myeloma.