WO2011026026A1 - Compositions and methods of use of immunotoxins comprising ranpirnase (rap) show potent cytotoxic activity - Google Patents
Compositions and methods of use of immunotoxins comprising ranpirnase (rap) show potent cytotoxic activity Download PDFInfo
- Publication number
- WO2011026026A1 WO2011026026A1 PCT/US2010/047132 US2010047132W WO2011026026A1 WO 2011026026 A1 WO2011026026 A1 WO 2011026026A1 US 2010047132 W US2010047132 W US 2010047132W WO 2011026026 A1 WO2011026026 A1 WO 2011026026A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- seq
- cancer
- antibody
- rap
- toxin
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
- A61K47/6815—Enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
- A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
- A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/06—Antianaemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/77—Internalization into the cell
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- the present invention relates to compositions and methods of use of toxin-antibody constructs (immuno toxins), preferably comprising ranpirnase (Rap) , although the skilled artisan will realize that a wide variety of toxins and other cytotoxic agents are known in the art and any such toxin or cytotoxic agent may be utilized in the claimed compositions and methods.
- the constructs comprise anti -tumor antibodies, such as anti-EGP-1 (anti-Trop-2), anti-CD74, anti-CD22 or anti-CD20.
- compositions and methods are not so limited and the antibody or antibody fragment may bind to an antigen associated with any target tissue, such as a cancer cell, a B cell, a T cell, an autoimmune disease cell, a pathogen, or any other disease-associated target cell for which antibodies are known in the art.
- a target tissue such as a cancer cell, a B cell, a T cell, an autoimmune disease cell, a pathogen, or any other disease-associated target cell for which antibodies are known in the art.
- the immunotoxins are dock-and-lock (DNL) constructs, preferably comprising four copies of ranpirnase attached to an antibody or antibody fragment.
- the toxins or other cytotoxic agents are fusion proteins, each comprising a DDD (dimerization and docking domain) moiety and the antibody or antibody fragment is a fusion protein comprising two AD (anchoring domain) moieties.
- the DDD moieties spontaneously form dimers which bind to an AD moiety, producing a DNL construct comprising four copies of the cyto toxin conjugated to one antibody or antibody fragment.
- the resulting immunotoxins show highly potent cytotoxic activity and may be administered to a subject with a disease to kill disease associated cells.
- the immunotoxins show greater potency against target cells than the parent antibody alone, the cytotoxin alone, a non-conjugated combination of antibody and cytotoxin or cytotoxin conjugated to a control antibody.
- Ribonucleases in particular, Rap (Lee, Exp Opin Biol Ther 2008; 8:813-27) and its more basic variant, amphinase (Ardelt et al., Curr Pharm Biotechnol 2008:9:215-25), are potential anti-tumor agents (Lee and Raines, Biodrugs 2008; 22:53-8).
- Rap is a single-chain ribonuclease of 104 amino acids originally isolated from the oocytes of Rana pipiens . Rap exhibits cytostatic and cytotoxic effects on a variety of tumor cell lines in vitro, as well as antitumor activity in vivo.
- the amphibian ribonuclease enters cells via receptor-mediated endocytosis and once internalized into the cytosol, selectively degrades tRNA, resulting in inhibition of protein synthesis and induction of apoptosis.
- Rap has completed a randomized Phase IHb clinical trial, which compared the effectiveness of Rap plus doxorubicin with that of doxorubicin alone in patients with unresectable malignant mesothelioma, with the interim analysis showing that the MST for the combination was 12 months, while that of the monotherapy was 10 months (Mutti and Gaudino, Oncol Rev 2008;2:61-5). Rap can be administered repeatedly to patients without an untoward immune response, with reversible renal toxicity reported to be dose-limiting (Mikulski et al., J Clin Oncol 2002; 20:274-81; Int J Oncol 1993; 3:57-64).
- Rap and other toxins or cytotoxins may be conjugated to antibodies or antibody fragments for targeted delivery to selected disease-associated cells, such as cancer cells or autoimmune disease cells.
- disease-associated cells such as cancer cells or autoimmune disease cells.
- An exemplary tumor-associated antigen is EGP-I, also known as Trop-2.
- Trop-2 is a type- 1 transmembrane protein and has been cloned from both human (Fornaro et al., Int J Cancer 1995; 62:610-8) and mouse cells (Sewedy et al., Int J Cancer 1998; 75:324-30).
- human Trop-2 In addition to its role as a tumor-associated calcium signal transducer (Ripani et al., Int J Cancer 1998;76:671-6), the expression of human Trop-2 was shown to be necessary for tumorigenesis and invasiveness of colon cancer cells, which could be effectively reduced with a polyclonal antibody against the extracellular domain of Trop-2 (Wang et al., MoI Cancer Ther 2008;7:280-5).
- the murine anti-Trop-2 mAb, mRS7 was generated by hybridoma technology using a crude membrane preparation derived from a surgically removed human primary squamous cell carcinoma of the lung as immunogen (Stein et al., Cancer Res 1990; 50:1330-6).
- Immunoperoxidase staining of frozen tissue sections indicated that the antigen defined by mRS7 is present in tumors of the lung, stomach, bladder, breast, ovary, uterus, and prostate, with most normal human tissues being unreactive (Stein et al., Int J Cancer 1993; 55:938-46).
- the antigen recognized by mRS7 was later shown to be a 46-48 kDa glycoprotein and named epithelial glycoprotein- 1, or EGP-I (Stein et al., Int J Cancer 1994; 8:98-102), which is also referred to in the literature as Trop-2 (Ripani et al., Int J Cancer 1998; 76:671-6).
- Trop-2 Rost al., Int J Cancer 1998; 76:671-6.
- Radiolabeled mRS7 has been shown to effectively target and treat cancer xenografts in nude mice in several earlier studies (Stein et al., Antibody Immunoconj Radiopharm 1991; 4:703-12; Stein et al., Cancer 1994; 73:816-23; Shih et al., Cancer Res 1995; 55:5857s-63s; Stein et al., J Nucl Med 2001; 42:967-74; Stein et al., Crit Rev Oncol Hematol 2001; 39:173- 80).
- immunoconj ugates immunoconj ugates
- RS7 or other disease-targeting antibodies may be attached to Rap or other cytotoxins to provide a more efficacious agent for disease therapy.
- the present invention concerns compositions and methods of use of immunotoxins comprising Ranpirnase (Rap) or other toxins, conjugated to a disease-targeting antibody or antigen-binding antibody fragment.
- the immunotoxin may be of a structure as illustrated in FIG. 1, referred to as 2L-Rap(Q)-hRS7 or 2L-Rap-hRS7, comprising two copies of Rap attached to the N-terminal ends of a humanized anti-Trop-2 antibody (hRS7).
- hRS7 humanized anti-Trop-2 antibody
- the immunotoxins are not so limited and antibodies against other tumor-associated or disease-associated antigens known in the art may be utilized.
- the subject immunotoxin may be made using the dock- and-lock (DNL) technology and may comprise conjugates of antibodies or antigen-binding antibody fragments with Rap or other toxins or cytotoxins.
- DNL dock- and-lock
- the term “immunotoxin” may refer to an immunotoxin made by the DNL technique, or an
- the DNL constructs comprise Rap conjugated to an anti-Trop-2 antibody, such as hRS7.
- an anti-Trop-2 antibody such as hRS7.
- the skilled artisan will be aware that the DNL constructs are not so limited and the subject DNL constructs may comprise an antibody or fragment thereof against any disease-associated antigen, conjugated to ranpirnase or other toxins or cytotoxins known in the art.
- the immunotoxin may comprise a humanized anti-Trop-2 antibody or fragment thereof, such as an hRS7 antibody comprising the heavy chain CDR sequences CDRl (NYGMN, SEQ ID NO:1), CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO:2) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:3) and the light chain CDR sequences CDRl (KASQDVSIAVA, SEQ ID NO:4), CDR2 (SASYRYT, SEQ ID NO:5), and CDR3 (QQHYITPLT, SEQ ID NO:6), attached to human antibody framework (FR) and constant region sequences (see, e.g., U.S. Patent No. 7,238,785, incorporated herein by reference from Col. 34, line 6 to Col. 44, line 37).
- FR human antibody framework
- constant region sequences see, e.g., U.S. Patent No. 7,238,785, incorporated herein by reference from Col. 34, line
- the immunotoxin may comprise a humanized anti- CD20 antibody or fragment thereof, such as veltuzumab, comprising light chain variable region CDRl (RASSSVSYIH, SEQ ID NO:7); CDR2 (ATSNLAS, SEQ ID NO:8); and CDR3 (QQWTSNPPT, SEQ ID NO:9); and heavy chain variable region CDRl (SYNMH, SEQ ID NO:10); CDR2 (AIYPGNGDTSYNQKFKG, SEQ ID NO:11); and CDR3
- veltuzumab comprising light chain variable region CDRl (RASSSVSYIH, SEQ ID NO:7); CDR2 (ATSNLAS, SEQ ID NO:8); and CDR3 (QQWTSNPPT, SEQ ID NO:9); and heavy chain variable region CDRl (SYNMH, SEQ ID NO:10); CDR2 (AIYPGNGDTSYNQKFKG, SEQ ID NO:11); and CDR
- the immunotoxin may comprise a ranpirnase (Rap) amino acid sequence, as is known in the art (see, e.g. NCBI protein database Accession No. 1PU3_A, see also Gorbatyuk et al., J Biol Chem 279:5772-80, 2004).
- Rap ranpirnase
- the immunotoxins may comprise one or more antibodies or fragments thereof which bind to an antigen other than Trop-2 or CD20.
- the antigen(s) may be selected from the group consisting of carbonic anhydrase IX, CCCL 19, CCCL21, CSAp, CDl, CDIa, CD2, CD3, CD4, CD5, CD8, CDI lA, CD14, CD15, CD16, CD18, CD19, IGF-IR, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CDl 33, CD138, CD147, CD154, AFP, PSMA, CEACAM5, CEACAM-6, B7, ED-B of fibronect
- Exemplary antibodies that may be utilized include, but are not limited to, hRl (anti- IGF-IR, U.S. Patent Application Serial No. 12/722,645, filed 3/12/10), hPAM4 (anti-mucin, U.S. Patent No. 7,282,567), hA20 (anti-CD20, U.S. Patent No. 7,251,164), hA19 (anti-CD19, U.S. Patent No. 7,109,304), hIMMU31 (anti-AFP, U.S. Patent No. 7,300,655), hLLl (anti- CD74, U.S. Patent No. 7,312,318), hLL2 (anti-CD22, U.S. Patent No.
- Exemplary toxins that may be incorporated into the immunotoxins include but are not limited to a bacterial toxin, a plant toxin, ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), DNase 1 , Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, Pseudomonas endotoxin, Ranpirnase (Rap) and Rap (N69Q).
- the sequences of each of the recited toxins is known in the art (see for example NCBI database) and clones encoding many of the exemplary toxins are commercially available from Invitrogen, the American Type Culture Collection and other sources known in the art.
- Various embodiments may concern use of the subject immunotoxins to treat or diagnose a disease, including but not limited to non-Hodgkin's lymphomas, B cell acute and chronic lymphoid leukemias, Burkitt lymphoma, Hodgkin's lymphoma, hairy cell leukemia, acute and chronic myeloid leukemias, T cell lymphomas and leukemias, multiple myeloma, glioma, Waldenstrom's macroglobulinemia, carcinomas, melanomas, sarcomas, gliomas, and skin cancers.
- non-Hodgkin's lymphomas B cell acute and chronic lymphoid leukemias, Burkitt lymphoma, Hodgkin's lymphoma, hairy cell leukemia, acute and chronic myeloid leukemias, T cell lymphomas and leukemias, multiple myeloma, glioma, Waldenstrom's macroglobulinemia, carcinomas, melan
- carcinomas may be selected from the group consisting of carcinomas of the oral cavity, gastrointestinal tract, colon, stomach, pulmonary tract, lung, breast, ovary, prostate, uterus, endometrium, cervix, urinary bladder, pancreas, bone, liver, gall bladder, kidney, skin, and testes.
- the subject immunotoxins may be used to treat an autoimmune disease, for example acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonlein purpura, poststreptococcal nephritis, erythema nodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture
- the subject antibodies may be used to treat leukemia, such as chronic lymphocytic leukemia, acute lymphocytic leukemia, chronic myeloid leukemia or acute myeloid leukemia.
- a pharmaceutical composition of the present invention may be use to treat a subject having a metabolic disease, such amyloidosis, or a neurodegenerative disease, such as Alzheimer's disease.
- a pharmaceutical composition of the present invention may be used to treat a subject having an immune-dysregulatory disorder.
- compositions of the present invention also are useful for the therapeutic treatment of infections, where the immunoglobulin component of the immunotoxin specifically binds to a disease-causing microorganism.
- a disease-causing microorganism includes pathogenic bacteria, viruses, fungi and diverse parasites, and the antibody can target these microorganisms, their products or antigens associated with their lesions.
- microorganisms include, but are not limited to: Streptococcus agalactiae, Legionella pneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis, Tetanus toxin, HIV-I, -2, -3, Hepatitis A, B, C, D, Rabies virus, Influenza virus, Cytomegalovirus, Herpes simplex I and II, Human serum parvo-like virus, Papilloma viruses, Polyoma virus, Respiratory syncytial virus, Varicella- Zoster virus, He
- Trypanosoma rhodesiensei Trypanosoma brucei, Schistosoma mansoni, Schistosoma japanicum, Babesia bovis, Elmeria tenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium, and M. pneumoniae. Monoclonal antibodies that bind to these pathogenic microorganisms are well known in the art.
- FIG. 1 Molecular design and size of (Q)-hRS7. Schematic structure of 2L-Rap-X, where X is an IgG and Rap can be Rap(Q)
- FIG. 2 Cell binding curves obtained for PC-3 (A), Calu-3 (B) and 22RvI (C) from
- FIG. 3 Representative data of the IVTT assay (A) showing (Q)-hRS7 and rRap have comparable RNase activity; and (B) plotting the initial rates of rRap (left) and (Q)-hRS7
- FIG. 4 In vitro cytotoxicity of (Q)-hRS7 as evidenced by the MTS assay shown for
- FIG. 5 Therapeutic efficacy of (Q)-hRS7 demonstrated in a Calu-3 human xenograft model to inhibit tumor growth (A) and increase MST (B).
- Nude mice were inoculated subcutaneously with 1 x 10 7 Calu-3 cells. When tumors reached approximately 0.15 cm 3 , mice were treated with either a single intravenous dose of 50 ⁇ g or two injections of 25 ⁇ g administered seven days apart. Control animals received saline.
- FIG. 6 shows RNase Activity by in-vitro transcription translation assay.
- FIG. 7 shows a competition binding assay, demonstrating that hLLl and rap-hLLl fusion protein both have the same affinity for WP, an anti-idiotype antibody of hLLl.
- FIG. 8 shows in vitro cytotoxicity of the fusion protein in Daudi cells:
- FIG. 9 shows in vitro cytotoxicity of the fusion protein in MC/CAR cells by MTS
- FIG. 10 shows blood clearance of 2L-Rap-hLLl - ⁇ 4P in naive SCID mice.
- SCID mice were co-injected intravenously with 88 Y-DTP A-hLLl (O) and 111 In-DTP A-2L-
- FIG. 11 shows treatment of aggressive minimal Daudi lymphoma with 2L-Rap- hLLl- ⁇ 4P or component proteins.
- SCID mice (8-10 mice/group) were inoculated
- mice were treated with a single bolus injection of 2L-Rap-hLLl- ⁇ 4P at the indicated dosages. Control groups were injected with component proteins equivalent to 50 ⁇ g of the immunotoxin or PBS only.
- FIG. 12 shows RNase activity as measured by the in vitro transcription/translation assay. Concentrations of rRap ( ⁇ ), 2L-Rap-hLLl- ⁇ 4P (A), and hLLl- ⁇ 4P ( ⁇ ) were plotted against relative luminescence units (RLU).
- FIG. 13 shows in vitro cytotoxicity of DNL-Rap immunotoxin constructs either treated continuously with immunotoxin or with washing after a 1 hour treatment.
- FIG. 14 shows in vitro cytotoxicity of DNL-Rap immunotoxin constructs in ALL cell lines.
- a “therapeutic agent” is an atom, molecule, or compound that is useful in the treatment of a disease.
- therapeutic agents include antibodies, antibody fragments, peptides, drugs, toxins, enzymes, nucleases, hormones, immunomodulators, antisense oligonucleotides, small interfering RNA (siRNA), chelators, boron compounds, photoactive agents, dyes, and radioisotopes.
- a "diagnostic agent” is an atom, molecule, or compound that is useful in diagnosing a disease.
- useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as with the biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules, and enhancing agents (e.g., paramagnetic ions) for magnetic resonance imaging (MRI).
- an "antibody” as used herein refers to a full-length (i.e., naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes) immunoglobulin molecule (e.g., an IgG antibody) or an immunologically active (i.e., specifically binding) portion of an immunoglobulin molecule, like an antibody fragment.
- An “antibody” includes monoclonal, polyclonal, bispecific, multispecific, murine, chimeric, humanized and human antibodies.
- a "naked antibody” is an antibody or antigen binding fragment thereof that is not attached to a therapeutic or diagnostic agent.
- the Fc portion of an intact naked antibody can provide effector functions, such as complement fixation and ADCC (see, e.g., Markrides, Pharmacol Rev 50:59-87, 1998).
- Other mechanisms by which naked antibodies induce cell death may include apoptosis. (Vaswani and Hamilton, Ann Allergy Asthma Immunol 81 : 105- 119, 1998.)
- an “antibody fragment” is a portion of an intact antibody such as F(ab') 2 , F(ab) 2 , Fab', Fab, Fv, sFv, scFv, dAb and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the full-length antibody.
- antibody fragments include isolated fragments consisting of the variable regions, such as the "Fv” fragments consisting of the variable regions of the heavy and light chains or recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker ("scFv proteins").
- Single-chain antibodies consist of a polypeptide chain that comprises both a V H and a V L domain which interact to form an antigen- binding site.
- the V H and V L domains are usually linked by a peptide of 1 to 25 amino acid residues.
- Antibody fragments also include diabodies, triabodies and single domain antibodies (dAb).
- An antibody or immunotoxin preparation, or a composition described herein is said to be administered in a "therapeutically effective amount” if the amount administered is physiologically significant.
- An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient subject.
- an antibody preparation is physiologically significant if its presence invokes an antitumor response or mitigates the signs and symptoms of an autoimmune disease state.
- physiologically significant effect could also be the evocation of a humoral and/or cellular immune response in the recipient subject leading to growth inhibition or death of target cells.
- monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, removing the spleen to obtain B- lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
- MAbs can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines et al, "Purification of Immunoglobulin G (IgG)," in METHODS IN MOLECULAR
- BIOLOGY, VOL. 10, pages 79-104 The Humana Press, Inc. 1992.
- the antibodies can be sequenced and subsequently prepared by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art. The use of antibody components derived from humanized, chimeric or human antibodies obviates potential problems associated with the immunogenicity of murine constant regions.
- a chimeric antibody is a recombinant protein in which the variable regions of a human antibody have been replaced by the variable regions of, for example, a mouse antibody, including the complementarity-determining regions (CDRs) of the mouse antibody.
- Chimeric antibodies exhibit decreased immunogenicity and increased stability when administered to a subject.
- CDRs complementarity-determining regions
- Leung et ah, Hybridoma 13:469 (1994) produced an LL2 chimera by combining DNA sequences encoding the V ⁇ and V H domains of murine LL2, an anti- CD22 monoclonal antibody, with respective human K and IgGi constant region domains.
- a chimeric or murine monoclonal antibody may be humanized by transferring the mouse CDRs from the heavy and light variable chains of the mouse immunoglobulin into the
- variable domains of a human antibody The mouse framework regions (FR) in the chimeric monoclonal antibody are also replaced with human FR sequences.
- additional modification might be required in order to restore the original affinity of the murine antibody. This can be accomplished by the replacement of one or more human residues in the FR regions with their murine counterparts to obtain an antibody that possesses good binding affinity to its epitope. See, for example, Tempest et ah, Biotechnology 9:266 (1991) and Verhoeyen et ah, Science 239: 1534 (1988).
- those human FR amino acid residues that differ from their murine counterparts and are located close to or touching one or more CDR amino acid residues would be candidates for substitution.
- the phage display technique may be used to generate human antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. MoI. Res. 4:126-40).
- Human antibodies may be generated from normal humans or from humans that exhibit a particular disease state, such as cancer (Dantas-Barbosa et al., 2005).
- the advantage to constructing human antibodies from a diseased individual is that the circulating antibody repertoire may be biased towards antibodies against disease-associated antigens.
- Fab fragment antigen binding protein
- RNAs were converted to cDNAs and used to make Fab cDNA libraries using specific primers against the heavy and light chain immunoglobulin sequences (Marks et al., 1991, J. MoI. Biol. 222:581-97).
- Library construction was performed according to Andris-Widhopf et al. (2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1 st edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY pp. 9.1 to 9.22).
- Fab fragments were digested with restriction endonucleases and inserted into the bacteriophage genome to make the phage display library.
- libraries may be screened by standard phage display methods, as known in the art (see, e.g., Pasqualini and Ruoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J. Nucl. Med. 43:159- 162).
- Phage display can be performed in a variety of formats, for their review, see e.g. Johnson and Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993). Human antibodies may also be generated by in vitro activated B cells. See U.S. Patent Nos.
- transgenic animals that have been genetically engineered to produce human antibodies may be used to generate antibodies against essentially any immunogenic target, using standard immunization protocols.
- Methods for obtaining human antibodies from transgenic mice are disclosed by Green et al, Nature Genet. 7: 13 (1994), Lonberg et al, Nature 5(55:856 (1994), and Taylor et al, Int. Immun. 6:579 (1994).
- a non- limiting example of such a system is the XenoMouse® (e.g., Green et al., 1999, J. Immunol Methods 231 :11 -23) from Abgenix (Fremont, CA).
- the mouse antibody genes have been inactivated and replaced by functional human antibody genes, while the remainder of the mouse immune system remains intact.
- the XenoMouse® was transformed with germline-configured YACs (yeast artificial chromosomes) that contained portions of the human IgH and Igkappa loci, including the majority of the variable region sequences, along accessory genes and regulatory sequences.
- the human variable region repertoire may be used to generate antibody producing B cells, which may be processed into hybridomas by known techniques.
- a XenoMouse® immunized with a target antigen will produce human antibodies by the normal immune response, which may be harvested and/or produced by standard techniques discussed above.
- a variety of strains of XenoMouse® are available, each of which is capable of producing a different class of antibody.
- Transgenically produced human antibodies have been shown to have therapeutic potential, while retaining the pharmacokinetic properties of normal human antibodies (Green et al., 1999).
- the skilled artisan will realize that the claimed compositions and methods are not limited to use of the XenoMouse® system but may utilize any transgenic animal that has been genetically engineered to produce human antibodies.
- Antibody fragments which recognize specific epitopes can be generated by known techniques.
- Antibody fragments are antigen binding portions of an antibody, such as F(ab') 2, Fab', F(ab) 2 , Fab, Fv, sFv and the like.
- F(ab') 2 fragments can be produced by pepsin digestion of the antibody molecule and Fab' fragments can be generated by reducing disulfide bridges of the F(ab') 2 fragments.
- Fab' expression libraries can be constructed (Huse et al, 1989, Science, 246: 1274-1281) to allow rapid and easy identification of monoclonal Fab' fragments with the desired specificity.
- F(ab) 2 fragments may be generated by papain digestion of an antibody.
- a single chain Fv molecule comprises a VL domain and a VH domain.
- the VL and VH domains associate to form a target binding site.
- These two domains are further covalently linked by a peptide linker (L).
- L peptide linker
- An antibody fragment can be prepared by proteolytic hydrolysis of the full length antibody or by expression in E. coli or another host of the DNA coding for the fragment.
- An antibody fragment can be obtained by pepsin or papain digestion of full length antibodies by conventional methods. These methods are described, for example, by Goldenberg, U.S. Patent Nos. 4,036,945 and 4,331,647 and references contained therein. Also, see Nisonoff et al, Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119 (1959), Edelman et al, in METHODS IN ENZYMOLOGY VOL. 1, page 422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
- Antibodies of use may be commercially obtained from a wide variety of known sources.
- a variety of antibody secreting hybridoma lines are available from the American Type Culture Collection (ATCC, Manassas, VA).
- ATCC American Type Culture Collection
- VA Manassas
- a large number of antibodies against various disease targets, including but not limited to tumor-associated antigens, have been deposited at the ATCC and/or have published variable region sequences and are available for use in the claimed methods and compositions. See, e.g., U.S. Patent Nos.
- antibody sequences or antibody-secreting hybridomas against almost any disease-associated antigen may be obtained by a simple search of the ATCC, NCBI and/or USPTO databases for antibodies against a selected disease-associated target of interest.
- the antigen binding domains of the cloned antibodies may be amplified, excised, ligated into an expression vector, transfected into an adapted host cell and used for protein production, using standard techniques well known in the art.
- the antibodies or fragments thereof may be conjugated to one or more therapeutic or diagnostic agents.
- the therapeutic agents do not need to be the same but can be different, e.g. a drug and a radioisotope.
- 131 I can be incorporated into a tyrosine of an antibody or fusion protein and a drug attached to an epsilon amino group of a lysine residue.
- Therapeutic and diagnostic agents also can be attached, for example to reduced SH groups and/or to carbohydrate side chains. Many methods for making covalent or non-covalent conjugates of therapeutic or diagnostic agents with antibodies or fusion proteins are known in the art and any such known method may be utilized.
- a therapeutic or diagnostic agent can be attached at the hinge region of a reduced antibody component via disulfide bond formation.
- such agents can be attached using a heterobifunctional cross-linker, such as iV-succinyl 3-(2-pyridyldithio)propionate (SPDP). Yu et al, Int. J. Cancer 56: 244 (1994). General techniques for such conjugation are well-known in the art. See, for example, Wong, CHEMISTRY OF PROTEIN
- the therapeutic or diagnostic agent can be conjugated via a carbohydrate moiety in the Fc region of the antibody.
- the carbohydrate group can be used to increase the loading of the same agent that is bound to a thiol group, or the carbohydrate moiety can be used to bind a different therapeutic or diagnostic agent.
- the general method involves reacting an antibody component having an oxidized carbohydrate portion with a carrier polymer that has at least one free amine function. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.
- the Fc region may be absent if the antibody used as the antibody component of the immunoconjugate is an antibody fragment. However, it is possible to introduce a
- carbohydrate moiety into the light chain variable region of a full length antibody or antibody fragment. See, for example, Leung et al, J. Immunol 154: 5919 (1995); Hansen et al, U.S. Patent No. 5,443,953 (1995), Leung et al, U.S. patent No. 6,254,868, incorporated herein by reference in their entirety.
- the engineered carbohydrate moiety is used to attach the therapeutic or diagnostic agent.
- a chelating agent may be attached to an antibody, antibody fragment or fusion protein and used to chelate a therapeutic or diagnostic agent, such as a radionuclide.
- exemplary chelators include but are not limited to DTPA (such as Mx-DTPA), DOTA, TETA, NETA or NOTA.
- radioactive metals or paramagnetic ions may be attached to proteins or peptides by reaction with a reagent having a long tail, to which may be attached a multiplicity of chelating groups for binding ions.
- a tail can be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chains having pendant groups to which can be bound chelating groups such as, e.g., ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups known to be useful for this purpose.
- EDTA ethylenediaminetetraacetic acid
- DTPA diethylenetriaminepentaacetic acid
- porphyrins polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups known to be useful for
- Chelates may be directly linked to antibodies or peptides, for example as disclosed in U.S. Patent 4,824,659, incorporated herein in its entirety by reference.
- Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with diagnostic isotopes in the general energy range of 60 to 4,000 keV, such as 125 T 1, 131 ⁇ 1, 123 T 1, 124 T 1, 62 C/-.,u,, 64 ⁇ Cu, 18 ⁇ b?, l l l ⁇ In, 67 ⁇ Ga, 6S n Ga, 99m- I ⁇ c, 94m x I c, U ⁇ C, 13- N ⁇ ⁇ , 15 ⁇ O, 76 D Br , f tor radioimaging.
- chelates when complexed with non-radioactive metals, such as manganese, iron and gadolinium are useful for MRI.
- Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with a variety of metals and radiometals, most particularly with radionuclides of gallium, yttrium and copper, respectively.
- metal-chelate complexes can be made very stable by tailoring the ring size to the metal of interest.
- Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding nuclides, such as 223 Ra for RAIT are encompassed.
- the F-Al conjugate may be complexed with chelating groups, such as DOTA, NOTA or NETA that are attached directly to antibodies or used to label targetable constructs in pre- targeting methods.
- chelating groups such as DOTA, NOTA or NETA
- Such F-18 labeling techniques are disclosed in U.S. Patent No. 7,563,433, the Examples section of which is incorporated herein by reference.
- hRS7 humanized RS7
- cytotoxic RNase moieties suitable for use in the present invention include polypeptides having a native ranpirnase structure and all enzymatically active variants thereof. These molecules advantageously have an N-terminal pyroglutamic acid resides that appears essential for RNase activity and are not substantially inhibited by mammalian RNase inhibitors.
- Nucleic acid that encodes a native cytotoxic RNase may be prepared by cloning and restriction of appropriate sequences, or using DNA amplification with polymerase chain reaction (PCR).
- the amino acid sequence of Rana Pipiens ranpirnase can be obtained from Ardelt et al., J. Biol.
- nucleic acid that encodes a cytotoxic RNase or variant thereof may be synthesized in vitro.
- Chemical synthesis produces a single-stranded oligonucleotide. This may be converted to a double-stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using a short primer and the single strand as a template. While chemical synthesis is most suited to sequences of about 100 bases, longer sequences may be obtained by ligating shorter sequences.
- Example 2, infra provides one illustrative method for obtaining a cytotoxic RNase gene.
- the "dock-and-lock” (DNL) method exploits specific protein/protein interactions that occur between the regulatory (R) subunits of cAMP-dependent protein kinase (PKA) and the anchoring domain (AD) of A-kinase anchoring proteins (AKAPs) (Baillie et al, FEBS Letters. 2005; 579: 3264. Wong and Scott, Nat. Rev. MoI. Cell Biol. 2004; 5: 959).
- PKA which plays a central role in one of the best studied signal transduction pathways triggered by the binding of the second messenger cAMP to the R subunits, was first isolated from rabbit skeletal muscle in 1968 (Walsh et al, J. Biol.
- the structure of the holoenzyme consists of two catalytic subunits held in an inactive form by the R subunits (Taylor, J. Biol. Chem. 1989;264:8443). Isozymes of PKA are found with two types of R subunits (RI and RII), and each type has ⁇ and ⁇ isoforms (Scott, Pharmacol. Ther.
- R subunits have been isolated only as stable dimers and the dimerization domain has been shown to consist of the first 44 amino-terminal residues (Newlon et al, Nat. Struct. Biol. 1999;6:222). Binding of cAMP to the R subunits leads to the release of active catalytic subunits for a broad spectrum of serine/threonine kinase activities, which are oriented toward selected substrates through the compartmentalization of PKA via its docking with AKAPs (Scott et al, J. Biol. Chem. 1990;265;21561)
- AKAP microtubule-associated protein-2
- the AD of AKAPs for PKA is an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem. 1991 ;266: 14188).
- the amino acid sequences of the AD are quite varied among individual AKAPs, with the binding affinities reported for RII dimers ranging from 2 to 90 nM (Alto et al, Proc. Natl. Acad. Sci. USA. 2003;100:4445).
- AKAPs will only bind to dimeric R subunits.
- the AD binds to a hydrophobic surface formed by the 23 amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;
- dimerization domain and AKAP binding domain of human RIIa are both located within the same N-terminal 44 amino acid sequence (Newlon et al, Nat. Struct. Biol. 1999;6:222; Newlon et al, EMBO J. 2001;20:1651), which is termed the DDD herein.
- Entity B is constructed by linking an AD sequence to a precursor of B, resulting in a second component hereafter referred to as b.
- the dimeric motif of DDD contained in a 2 will create a docking site for binding to the AD sequence contained in b, thus facilitating a ready association of a 2 and b to form a trimeric complex composed of a 2 b.
- the a 2 subunit may contain two identical effector moieties, such as an antibody, antibody fragment or cytotoxin, each attached to an identical DDD sequence.
- the trimeric a 2 b complex may comprise two copies of a first effector moieties and one copy of a second effector moiety.
- the effector moiety is a protein or peptide, more preferably an antibody, antibody fragment or toxin, which can be linked to a DDD or AD moiety to form a fusion protein or peptide.
- fusion proteins A variety of methods are known for making fusion proteins, including nucleic acid synthesis, hybridization and/or amplification to produce a synthetic double-stranded nucleic acid encoding a fusion protein of interest. Such double-stranded nucleic acids may be inserted into expression vectors for fusion protein production by standard molecular biology techniques (see, e.g. Sambrook et al., Molecular Cloning, A laboratory manual, 2 nd Ed, 1989).
- the AD and/or DDD moiety may be attached to either the N-terminal or C-terminal end of an effector protein or peptide.
- site of attachment of an AD or DDD moiety to an effector moiety may vary, depending on the chemical nature of the effector moiety and the part(s) of the effector moiety involved in its physiological activity.
- attachment of AD or DDD moieties to an antibody or antibody fragment occurs at the C-terminal end of the heavy chain subunit, at the opposite end of the molecule from the antigen-binding site.
- N-terminal attachment to antibodies or antibody fragments may also be utilized while retaining antigen-binding activity.
- Site-specific attachment of a variety of effector moieties may be performed using techniques known in the art, such as the use of bivalent cross-linking reagents and/or other chemical conjugation techniques.
- the AD and DDD sequences incorporated into the AD and DDD sequences incorporated into the AD and DDD sequences incorporated into the AD and DDD sequences incorporated into the AD and DDD sequences incorporated into the AD and DDD sequences incorporated into the AD and DDD sequences incorporated into the AD and DDD sequences incorporated into the AD and DDD sequences incorporated into the AD and DDD sequences incorporated into the AD and DDD sequences incorporated into the AD and DDD sequences incorporated into the
- immunotoxin DNL construct comprise the amino acid sequences of DDDl and ADl below.
- the AD and DDD sequences comprise the amino acid sequences of DDD2 and AD2, which are designed to promote disulfide bond formation between the DDD and AD moieties.
- DDDl and DDD2 comprise the DDD sequence of the human RIIa form of protein kinase A.
- the DDD and AD moieties may be based on the DDD sequence of the human RIa form of protein kinase A and a corresponding AKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.
- DDD moieties based on the known human Rl ⁇ and Rll ⁇ amino acid sequences may be designed and utilized (see, e.g., NCBI Accession Nos. NP_001158233 and NP_002727, sequences below).
- AD and/or DDD moieties may be utilized in construction of the immunotoxin DNL constructs.
- the structure- function relationships of the AD and DDD domains have been the subject of investigation. (See, e.g., Burns-Hamuro et al., 2005, Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38; Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker et al., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J 400:493-99; Gold et al., 2006, MoI Cell 24:383-95; Kinderman et al., 2006, MoI Cell 24:397-408, the entire text of each of which is incorporated herein by reference.)
- DDD sequence of use for construction of DNL constructs is shown in SEQ ID NO:22, wherein "X" represents a conservative amino acid substitution.
- Conservative amino acid substitutions are discussed in more detail below, but could involve for example substitution of an aspartate residue for a glutamate residue, or a leucine or valine residue for an isoleucine residue, etc. Such conservative amino acid substitutions are well known in the art.
- SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 13) XXIXIXXXLXXLLXXYXVXVLXXXXXLVXFXVXYFXXLXXXXX (SEQ ID NO:22)
- Alto et al. (2003) performed a bioinformatic analysis of the AD sequence of various AKAP proteins to design an RII selective AD sequence called AKAP-IS shown below, with a binding constant for DDD of 0.4 nM.
- the AKAP-IS sequence was designed as a peptide antagonist of AKAP binding to PKA. Residues in the AKAP-IS sequence where
- the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD moiety sequence to prepare cytotoxic DNL constructs.
- Other alternative sequences that might be substituted for the AKAP-IS AD sequence are shown below. Substitutions relative to the AKAP-IS sequence are underlined. It is anticipated that, as with the AKAP-IS sequence, the AD moiety may also include the additional N-terminal residues cysteine and glycine and C- terminal residues glycine and cysteine.
- Figure 2 of Gold et al. disclosed additional DDD-binding sequences from a variety of AKAP proteins, shown below.
- LAWKIAKMIVSDVMQQ (SEQ ID NO:37)
- AKAPIS represents a synthetic RII subunit-binding peptide. All other peptides are derived from the RJI-binding domains of the indicated AKAPs.
- AKAPIS QIEYLAKQIVDNAIQQA (SEQ ID NO : 15)
- AKAP7£-PP-pep PEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO:48)
- AKAP7(5-L314E-pep PEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO:49)
- AKAPl -pep EEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO:50)
- AKAP12-pep NGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO:56)
- Rab32-pep ETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO:58) [091] Residues that were highly conserved among the AD domains of different AKAP proteins are indicated below by underlining with reference to the AKAP IS sequence below. The residues are the same as observed by Alto et al. (2003), with the addition of the C- terminal alanine residue. (See FIG. 4 of Hundsrucker et al. (2006), incorporated herein by reference.) The sequences of peptide antagonists with particularly high affinities for the RII DDD sequence were those of AKAP-IS, AKAP7 ⁇ -wt-pep, AKAP7 ⁇ -L304T-pep and
- Carr et al. examined the degree of sequence homology between different AKAP-binding DDD sequences from human and non-human proteins and identified residues in the DDD sequences that appeared to be the most highly conserved among different DDD moieties. These are indicated below by underlining with reference to the human PKA RIIa DDD sequence. Residues that were particularly conserved are further indicated by italics. The residues overlap with, but are not identical to those suggested by Kinderman et al. (2006) to be important for binding to AKAP proteins. Thus, a potential DDD sequence is indicated in SEQ ID NO:59, wherein "X" represents a conservative amino acid substitution.
- sequence variants of the DDD and/or AD moieties in certain embodiments it may be preferred to introduce sequence variations in the antibody moiety or the linker peptide sequence joining the antibody with the AD sequence.
- sequence variations in the antibody moiety or the linker peptide sequence joining the antibody with the AD sequence in one illustrative example, three possible variants of fusion protein sequences, are shown below.
- the disclosed methods and compositions may involve production and use of proteins or peptides with one or more substituted amino acid residues.
- the structural, physical and/or therapeutic characteristics of native, chimeric, humanized or human antibodies, or AD or DDD sequences may be optimized by replacing one or more amino acid residues.
- the functionalities of native, chimeric, humanized or human antibodies, or AD or DDD sequences may be optimized by replacing one or more amino acid residues.
- FR human framework region
- the therapeutic properties of an antibody such as binding affinity for the target antigen, the dissociation- or off-rate of the antibody from its target antigen, or even the effectiveness of induction of CDC (complement-dependent cytotoxicity) or ADCC (antibody dependent cellular cytotoxicity) by the antibody, may be optimized by a limited number of amino acid substitutions.
- the DDD and/or AD sequences used to make the subject DNL constructs may be further optimized, for example to increase the DDD-AD binding affinity. Potential sequence variations in DDD or AD sequences are discussed above.
- amino acid substitutions typically involve the replacement of an amino acid with another amino acid of relatively similar properties (i.e., conservative amino acid substitutions).
- conservative amino acid substitutions The properties of the various amino acids and effect of amino acid substitution on protein structure and function have been the subject of extensive study and knowledge in the art.
- the hydropathic index of amino acids may be considered (Kyte & Doolittle, 1982, J. MoI. Biol., 157:105-132).
- the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules.
- Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (- 0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
- the use of amino acids whose hydropathic indices are within ⁇ 2 is preferred, within ⁇ 1 are more preferred, and within ⁇ 0.5 are even more preferred.
- Amino acid substitution may also take into account the hydrophilicity of the amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 .+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). Replacement of amino acids with others of similar hydrophilicity is preferred.
- amino acid side chain For example, it would generally not be preferred to replace an amino acid with a compact side chain, such as glycine or serine, with an amino acid with a bulky side chain, e.g., tryptophan or tyrosine.
- a compact side chain such as glycine or serine
- an amino acid with a bulky side chain e.g., tryptophan or tyrosine.
- tryptophan or tyrosine The effect of various amino acid residues on protein secondary structure is also a
- arginine and lysine glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
- amino acid substitutions include whether or not the residue is located in the interior of a protein or is solvent exposed.
- conservative substitutions would include: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala and GIy; He and VaI; VaI and Leu; Leu and He; Leu and Met; Phe and Tyr; Tyr and Trp.
- conservative substitutions would include: Asp and Asn; Asp and GIu; GIu and GIn; GIu and Ala; GIy and Asn; Ala and Pro; Ala and GIy; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; VaI and Leu; Leu and He; He and VaI; Phe and Tyr.
- amino acid substitutions In determining amino acid substitutions, one may also consider the existence of intermolecular or intramolecular bonds, such as formation of ionic bonds (salt bridges) between positively charged residues (e.g., His, Arg, Lys) and negatively charged residues (e.g., Asp, GIu) or disulfide bonds between nearby cysteine residues.
- ionic bonds salt bridges
- positively charged residues e.g., His, Arg, Lys
- negatively charged residues e.g., Asp, GIu
- disulfide bonds between nearby cysteine residues.
- therapeutic agents such as cytotoxic agents, anti- angiogenic agents, pro-apoptotic agents, antibiotics, hormones, hormone antagonists, chemokines, drugs, prodrugs, toxins, enzymes or other agents may be used, either conjugated to the subject immunotoxins or separately administered before, simultaneously with, or after the immuno toxin.
- Drugs of use may possess a pharmaceutical property selected from the group consisting of antimitotic, antikinase, alkylating, antimetabolite, antibiotic, alkaloid, anti- angiogenic, pro-apoptotic agents and combinations thereof.
- Exemplary drugs of use may include 5-fluorouracil, aplidin, azaribine, anastrozole, anthracyclines, bendamustine, bleomycin, bortezomib, bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide,
- mitoxantrone mithramycin, mitomycin, mitotane, navelbine, nitrosourea, plicomycin, procarbazine, paclitaxel, pentostatin, PSI-341, raloxifene, semustine, streptozocin, tamoxifen, taxol, temazolomide (an aqueous form of DTIC), transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vinorelbine, vinblastine, vincristine and vinca alkaloids.
- Toxins of use may include ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), e.g., onconase, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.
- RNase ribonuclease
- Chemokines of use may include RANTES, MCAF, MIPl -alpha, MIPl -Beta and IP-IO.
- anti-angiogenic agents such as angiostatin, baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-PIGF peptides and antibodies, anti-vascular growth factor antibodies, anti-Flk-1 antibodies, anti-Fit- 1 antibodies and peptides, anti-Kras antibodies, anti-cMET antibodies, anti-MIF (macrophage migration-inhibitory factor) antibodies, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin-12, IP-10, Gro- ⁇ , thrombospondin, 2- methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CMlOl, Marimastat, pentosan polysulphate, angiopoietin-2, interferon-alpha, herbimycin A, PNU145156E, 16K
- Immunomodulators of use may be selected from a cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF), an interferon (IFN), erythropoietin, thrombopoietin and a combination thereof. Specifically useful are
- lymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors, such as interleukin (IL), colony stimulating factor, such as granulocyte-colony stimulating factor (G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF), interferon, such as
- interferons- ⁇ , - ⁇ or - ⁇ interferons- ⁇ , - ⁇ or - ⁇ , and stem cell growth factor, such as that designated "Sl factor”.
- cytokines include growth hormones such as human growth hormone, N- methionyl human growth hormone, and bovine growth hormone; parathyroid hormone;
- thyroxine insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor- ⁇ and - ⁇ ; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor;
- FSH follicle stimulating hormone
- TSH thyroid stimulating hormone
- LH luteinizing hormone
- hepatic growth factor prostaglandin, fibroblast growth factor
- prolactin prolactin
- placental lactogen OB protein
- tumor necrosis factor- ⁇ and - ⁇ mullerian-inhibiting substance
- mouse gonadotropin-associated peptide inhibin
- activin vascular endothelial growth factor
- integrin thrombopoietin
- TPO nerve growth factors
- NGF- ⁇ platelet-growth factor
- TGFs transforming growth factors
- TGFs transforming growth factors
- I and -II insulin-like growth factor-I and -II
- EPO erythropoietin
- osteoinductive factors interferons such as interferon- ⁇ , - ⁇ , and - ⁇
- colony stimulating factors CSFs
- CSFs colony stimulating factors
- M-CSF colony stimulating factors
- ILs interleukins
- ILs interleukins
- ILs interleukins
- Radionuclides of use include, but are not limited to- 1 11 In, 177 Lu, 212 Bi, 213 Bi, 21 1 At, 62 Cu, 67 Cu, 90 Y, 125 I, 131 L 32 P, 33 P, 47 Sc, 111 Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au, and 211 Pb.
- the therapeutic radionuclide preferably has a decay-energy in the range of 20 to 6,000 keV, preferably in the ranges 60 to 200 keV for an Auger emitter, 100-2,500 keV for a beta emitter, and 4,000-6,000 keV for an alpha emitter.
- Maximum decay energies of useful beta-particle-emitting nuclides are preferably 20-5,000 keV, more preferably 100-4,000 keV, and most preferably 500-2,500 keV. Also preferred are radionuclides that substantially decay with Auger-emitting particles.
- beta-particle-emitting nuclides are preferably ⁇ l,000 keV, more preferably ⁇ 100 keV, and most preferably ⁇ 70 keV.
- radionuclides that substantially decay with generation of alpha-particles. Such radionuclides include, but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-21 1, Ac-225, Fr- 221, At-217, Bi-213 and Fm-255.
- Decay energies of useful alpha-particle-emitting radionuclides are preferably 2,000-10,000 keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.
- Additional potential radioisotopes of use include 11 C, 13 N, 15 O, 75 Br, 198 Au, 224 Ac, 126 I, 133 I, 77 Br, U3m
- Some useful diagnostic nuclides may include 18 F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, 94 Tc, 94m Tc, 99m Tc, or 111 In.
- Therapeutic agents may include a photoactive agent or dye.
- compositions such as fluorochrome, and other chromogens, or dyes, such as porphyrins sensitive to visible light
- photoradiation phototherapy
- photodynamic therapy this has been termed photoradiation, phototherapy, or photodynamic therapy.
- monoclonal antibodies have been coupled with photoactivated dyes for achieving phototherapy. See Mew et al., J. Immunol. (1983), 130:1473; idem., Cancer Res. (1985), 45:4380; Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83:8744; idem.,
- Other useful therapeutic agents may comprise oligonucleotides, especially antisense oligonucleotides that preferably are directed against oncogenes and oncogene products, such as bcl-2 or p53.
- a preferred form of therapeutic oligonucleotide is siRNA.
- Diagnostic agents are preferably selected from the group consisting of a radionuclide, a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a
- diagnostic agents are well known and any such known diagnostic agent may be used.
- diagnostic agents may include a radionuclide such as 110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 90 Y, 89 Zr, 94m Tc, 94 Tc, 99m Tc, 120 I, 123 I, 124 I, 125 I, 131 I, 154"158 Gd, 32 P, 11 C, 13 N, 15 0, 186 Re, 188 Re, 51 Mn, 52m Mn, 55 Co, 72 As, 75 Br, 76 Br, 82m Rb, 83 Sr, or other gamma-, beta-, or positron-emitters.
- a radionuclide such as 110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 90 Y, 89 Zr,
- Paramagnetic ions of use may include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) or erbium (III).
- Metal contrast agents may include lanthanum (III), gold (III), lead (II) or bismuth (III).
- Ultrasound contrast agents may comprise liposomes, such as gas filled liposomes.
- Radiopaque diagnostic agents may be selected from compounds, barium compounds, gallium compounds, and thallium compounds.
- fluorescent labels are known in the art, including but not limited to fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o- phthaldehyde and fluorescamine.
- Chemiluminescent labels of use may include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt or an oxalate ester.
- Various embodiments concern methods of treating a cancer in a subject, such as a mammal, including humans, domestic or companion pets, such as dogs and cats, comprising administering to the subject a therapeutically effective amount of a cytotoxic immunoconjugate.
- immunological diseases which may be treated with the subject immunotoxins may include, for example, joint diseases such as ankylosing spondylitis, juvenile rheumatoid arthritis, rheumatoid arthritis; neurological disease such as multiple sclerosis and myasthenia gravis; pancreatic disease such as diabetes, especially juvenile onset diabetes; gastrointestinal tract disease such as chronic active hepatitis, celiac disease, ulcerative colitis, Crohn's disease, pernicious anemia; skin diseases such as psoriasis or scleroderma; allergic diseases such as asthma and in transplantation related conditions such as graft versus host disease and allograft rejection.
- joint diseases such as ankylosing spondylitis, juvenile rheumatoid arthritis, rheumatoid arthritis
- neurological disease such as multiple sclerosis and myasthenia gravis
- pancreatic disease such as diabetes, especially juvenile onset diabetes
- gastrointestinal tract disease such as chronic active hepatitis, celi
- the administration of the cytotoxic immunoconjugates can be supplemented by administering concurrently or sequentially a therapeutically effective amount of another antibody that binds to or is reactive with another antigen on the surface of the target cell.
- Preferred additional MAbs comprise at least one humanized, chimeric or human MAb selected from the group consisting of a MAb reactive with CD4, CD5, CD8, CD 14, CD 15, CD 16, CD19, IGF-IR, CD20, CD21, CD22, CD23, CD25, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD70, CD74, CD79a, CD80, CD95, CD126, CD133, CD138, CD154, CEACAM5, CEACAM6, B7, AFP, PSMA, EGP-I, EGP-2, carbonic anhydrase IX, PAM4 antigen, MUCl, MUC2, MUC3, MUC4, MUC5, Ia, MIF, HM1.24, HLA-DR, tenascin, Flt-3, VEGFR, PlGF, ILGF, IL-6, IL-25, tenascin, TRAIL-Rl, TRAIL-R2,
- the immunotoxin therapy can be further supplemented with the administration, either concurrently or sequentially, of at least one therapeutic agent.
- at least one therapeutic agent for example, "CVB" (1.5 g/m 2 cyclophosphamide, 200-400 mg/m 2 etoposide, and 150-200 mg/m 2 carmustine) is a regimen used to treat non-Hodgkin's lymphoma. Patti et ah, Eur. J, Haematol 51: 18 (1993).
- Other suitable combination chemotherapeutic regimens are well-known to those of skill in the art.
- first generation chemotherapeutic regimens for treatment of intermediate- grade non-Hodgkin's lymphoma include C-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone).
- a useful second generation chemotherapeutic regimen is m-BACOD
- a suitable third generation regimen is MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin and leucovorin).
- Additional useful drugs include phenyl butyrate, bendamustine, and bryostatin-1.
- the subject immunotoxins can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the immunotoxin is combined in a mixture with a pharmaceutically suitable excipient.
- a pharmaceutically suitable excipient Sterile phosphate-buffered saline is one example of a pharmaceutically suitable excipient.
- Other suitable excipients are well-known to those in the art. See, for example, Ansel et al, PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990), and revised editions thereof.
- the subject immunotoxins can be formulated for intravenous administration via, for example, bolus injection or continuous infusion.
- the immunotoxin is infused over a period of less than about 4 hours, and more preferably, over a period of less than about 3 hours.
- the first 25-50 mg could be infused within 30 minutes, preferably even 15 min, and the remainder infused over the next 2-3 hrs.
- Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
- the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
- Control release preparations can be prepared through the use of polymers to complex or adsorb the immunotoxins.
- biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebacic acid. Sherwood et al, Bio/Technology 10: 1446 (1992). The rate of release from such a matrix depends upon the molecular weight of the immunotoxin, the amount of immunotoxin within the matrix, and the size of dispersed particles. Saltzman et al., Biophys. J.
- the immunotoxin may also be administered to a mammal subcutaneously or even by other parenteral routes. Moreover, the administration may be by continuous infusion or by single or multiple boluses. Preferably, the immunotoxin is infused over a period of less than about 4 hours, and more preferably, over a period of less than about 3 hours.
- the dosage of an administered immunotoxin for humans will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. It may be desirable to provide the recipient with a dosage of immunotoxin that is in the range of from about 1 mg/kg to 25 mg/kg as a single intravenous infusion, although a lower or higher dosage also may be administered as circumstances dictate.
- the dosage may be repeated as needed, for example, once per week for 4-10 weeks, once per week for 8 weeks, or once per week for 4 weeks. It may also be given less frequently, such as every other week for several months, or monthly or quarterly for many months, as needed in a maintenance therapy.
- an immunotoxin may be administered as one dosage every 2 or 3 weeks, repeated for a total of at least 3 dosages.
- the construct may be administered twice per week for 4-6 weeks. If the dosage is lowered to approximately 200-300 mg/m 2 (340 mg per dosage for a 1.7-m patient, or 4.9 mg/kg for a 70 kg patient), it may be administered once or even twice weekly for 4 to 10 weeks.
- the dosage schedule may be decreased, namely every 2 or 3 weeks for 2-3 months. It has been determined, however, that even higher doses, such as 20 mg/kg once weekly or once every 2-3 weeks can be
- the dosing schedule can optionally be repeated at other intervals and dosage may be given through various parenteral routes, with appropriate adjustment of the dose and schedule.
- the immunotoxins are of use for therapy of cancer.
- cancers include, but are not limited to, carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, or lymphoid malignancies. More particular examples of such cancers are noted below and include: squamous cell cancer (e.g., epithelial squamous cell cancer), Ewing sarcoma, Wilms tumor, astrocytomas, lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrine tumors, medullary thyroid cancer, differentiated thyroid carcinoma, breast cancer, ovarian cancer, colon cancer, rectal
- cancer includes primary malignant cells or tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original malignancy or tumor) and secondary malignant cells or tumors (e.g., those arising from metastasis, the migration of malignant cells or tumor cells to secondary sites that are different from the site of the original tumor).
- primary malignant cells or tumors e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original malignancy or tumor
- secondary malignant cells or tumors e.g., those arising from metastasis, the migration of malignant cells or tumor cells to secondary sites that are different from the site of the original tumor.
- Cancers conducive to treatment methods of the present invention involves cells which express, over-express, or abnormally express IGF-IR.
- cancers or malignancies include, but are not limited to: Acute Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary)
- Lymphoma Central Nervous System Lymphoma, Cerebellar Astrocytoma, Cerebral
- Ependymoma Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,
- Macroglobulinemia Male Breast Cancer, Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's
- compositions described and claimed herein may be used to treat malignant or premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders described above.
- Such uses are indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).
- Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia. It is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplasia characteristically occurs where there exists chronic irritation or inflammation.
- Dysplastic disorders which can be treated include, but are not limited to, anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia, congenital ectodermal dysplasia, craniodiaphysial dysplasia, craniocarpotarsal dysplasia, craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia, ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata, epi
- Additional pre-neoplastic disorders which can be treated include, but are not limited to, benign dysproliferative disorders (e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps or adenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solar keratosis.
- benign dysproliferative disorders e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps or adenomas, and esophageal dysplasia
- leukoplakia keratoses
- Bowen's disease keratoses
- Farmer's Skin Farmer's Skin
- solar cheilitis solar keratosis
- the method of the invention is used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.
- Additional hyperproliferative diseases, disorders, and/or conditions include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, lipos
- lymphangioendotheliosarcoma synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
- Still other embodiments may concern DNA sequences comprising a nucleic acid encoding an antibody, antibody fragment, toxin or constituent fusion protein of an immunotoxin, such as a DNL construct. Fusion proteins may comprise an antibody or fragment or toxin attached to, for example, an AD or DDD moiety.
- Various embodiments relate to expression vectors comprising the coding DNA sequences.
- the vectors may contain sequences encoding the light and heavy chain constant regions and the hinge region of a human immunoglobulin to which may be attached chimeric, humanized or human variable region sequences.
- the vectors may additionally contain promoters that express the encoded protein(s) in a selected host cell, enhancers and signal or leader sequences.
- the light and heavy chain constant regions and hinge region may be from a human EU myeloma immunoglobulin, where optionally at least one of the amino acid in the allotype positions is changed to that found in a different IgGl allotype, and wherein optionally amino acid 253 of the heavy chain of EU based on the EU number system may be replaced with alanine.
- an IgGl sequence may be converted to an IgG4 sequence.
- kits containing components suitable for treating or diagnosing diseased tissue in a patient.
- Exemplary kits may contain one or more
- a device capable of delivering the kit components through some other route may be included.
- One type of device, for applications such as parenteral delivery, is a syringe that is used to inject the composition into the body of a subject.
- Inhalation devices may also be used.
- a therapeutic agent may be provided in the form of a prefilled syringe or autoinjection pen containing a sterile, liquid formulation or lyophilized
- the kit components may be packaged together or separated into two or more containers.
- the containers may be vials that contain sterile, lyophilized formulations of a composition that are suitable for reconstitution.
- a kit may also contain one or more buffers suitable for reconstitution and/or dilution of other reagents.
- Other containers that may be used include, but are not limited to, a pouch, tray, box, tube, or the like. Kit components may be packaged and maintained sterilely within the containers.
- Another component that can be included is instructions to a person using a kit for its use.
- a novel immunotoxin comprising an amphibian ribonuclease recombinantly tethered to a humanized anti-Trop-2 antibody is shown herein to exhibit broad and potent antiproliferative activity against diverse human epithelial cancer cell lines in vitro, such as cervical, breast, colon, pancreatic, ovarian, and prostate cancer, as well as a human lung cancer xenograft in vivo.
- 2L-Rap(Q)-hRS7 also suppressed tumor growth in a prophylactic model of nude mice bearing Calu-3 human non-small cell lung cancer xenografts, with an increase in the median survival time (MST) from 55 to 96 days (P ⁇ 0. OL 1 .
- MST median survival time
- adenocarcinoma line Calu-3
- pancreatic cancer lines Capan-1, BxPC-3, and AsPc-I
- ovarian cancer line SK-OV-3
- FBS heat-inactivated fetal bovine serum
- Antibodies and reagents Milatuzumab fhLLl, anti-CD74), hRS7, recombinant ranpirnase (rRap), and a mouse anti-Rap IgG were from Immunomedics. Fluorescein isothiocynate (FITC)-, phycoerythrin (PE)-, or horseradish peroxidase (HRP)-conjugated goat anti-human (GAH) or goat anti-mouse (GAM) IgG, Fc-specific, antibodies were purchased from Jackson Immuno Research Labs (West Grove, PA).
- FITC Fluorescein isothiocynate
- PE phycoerythrin
- HRP horseradish peroxidase
- GAA goat anti-human
- GAM goat anti-mouse
- GAH IgG conjugated to Alexa Fluor 488, human transferrin conjugated to Alexa Fluor 568, and Hoechst 33258 were acquired from Molecular Probes (Invitrogen, Carlsbad, CA). All restriction enzymes were obtained from New England Biolabs (Beverly, MA).
- an EcoRV restriction site was introduced at the N-terminal (5') side of the hRS7 VL gene using suitable primers by PCR.
- the Xbal-EcoRV fragment of pdHL2-Rap(Q)- L-hLLl-y4P containing Leader peptide-Rap-Linker was ligated with the EcoRV-BamHI fragment generated by PCR containing hRS7 VL gene into an intermediate vector, pBS- Rap(Q)-L-hRS7.
- the Xba-BamHI fragment of pdHL2-hRS7-yl was replaced with Xba- BamHI fragment of pBS-Rap(Q)-L-hRS7.
- IVTT In vitro transcription and translation assay RNase activity was determined in a cell-free system by measuring the activity of de novo synthesized luciferase using the TNT® Quick Coupled Transcription/Translation System (Promega, Madison, WI) per manufacturer's instructions.
- test samples at concentrations ranging from 10 pM to 100 nM in 2 ⁇ L were added to 8 ⁇ L of the TNT® Quick Master Mix containing methionine and luciferase-control DNA and incubated for 2 h at 3O 0 C in a 96-well, round- bottom plate from which 2 ⁇ L were removed for analysis with 50 ⁇ L Bright-Glo substrate in a black 96-well, flat-bottom plate. Plates were read on an Envision chemiluminescence reader. Relative luciferase units (RLU) were plotted against the concentration of test samples.
- RLU Relative luciferase units
- Yeast tRNA degradation assay RNase activity was also determined by measuring the amount of perchloric acid-soluble nucleotides formed using yeast tRNA (Invitrogen) as substrate (Newton et al., Blood 2001 ;97:528-35).
- Each sample was prepared with RNase-free water (Ambion, Austin, TX) in a 1.5-mL RNase-free Eppendorf tube to contain, in a final volume of 100 ⁇ L, 5 nM (Q)-hRS7 or rRap; 10 mM HEPES, pH 6.0; 200 ⁇ g/mL human serum albumin; and a predetermined concentration of tRNA ranging from 100 ⁇ g/mL (3.09 ⁇ M) to 600 ⁇ g/mL (18.54 ⁇ M).
- the enzymatic reaction was performed at 37°C for 2 h and terminated by adding 233 ⁇ L of 3.4% ice-cold perchloric acid to each sample on ice.
- Serial 1 :4 dilutions (200 nM through 1.9xlO "4 nM) of (Q)-hRS7 were made in assay media (RPMI 1640; 10% FBS complete media), and added (50 ⁇ L/well) in triplicates to corresponding wells (final concentrations 100 nM through 0.95 x 10 "4 nM). After incubation for 1.5 h at 4 0 C, the plates were centrifuged at 600 x g for 2 min, blotted dry on paper towels after removal of the media, and washed by adding 150 ⁇ L of ice-cold media into each well followed by centrifugation at 600 x g for 2 min. The media was removed and plates were blotted dry.
- HRP-conjugated GAH antibody was used at a 1 :20,000 dilution and was then added to all the wells (100 ⁇ L/well). For background control, one set of wells received only cells plus the secondary antibody. The plate was incubated for 1 h at 4 0 C. Afterwards, the plate was centrifuged and blotted dry. The cells were then washed twice with ice-cold media followed by a third wash with ice-cold PBS. The procedures of centrifugation, media removal and plate-blotting were repeated following each wash.
- LumiGLO KPL, Gaithersburg, MD
- LumiGLO KPL, Gaithersburg, MD
- hRS7 and hLLl were included as positive and isotype controls, respectively.
- binding of (Q)-hRS7 to human cancer cell lines was determined by flow cytometry on a Guava PCA (Guava Technologies, Inc., Hayward, CA), using the manufacturer's reagents, protocols, and software. Similar studies were performed in parallel for each cell line with hRS7 and hLLl.
- Cells were analyzed by flow cytometry after washing twice with PBS/1% BSA and resuspended in 0.5 mL of PBS/1% BSA. To separate dead from viable cells 1 ⁇ g/mL of propidium iodide was added. For each analysis 10,000 cells were acquired.
- Colony-formation assay Tumor cells were trypsinized and plated in 60-mm dishes (1 x 10 3 cells). Cells were treated with each test article and allowed to form colonies. Fresh media containing the test article were added every 4 days, and after 2 weeks of incubation, colonies were fixed in 4% formaldehyde and stained with Giemsa. Colonies > 50 cells were enumerated under a microscope.
- Alexa Fluor 568-conjugated human transferrin was added with (Q)-hRS7 (10 ⁇ g/mL) or hRS7 (6 ⁇ g/mL) to MDA-MB-468 human breast cancer cells placed (3000 cells in 500 ⁇ L per well) in 8-well chamber slides. After incubation at 37 0 C for 2 h, cells were washed and fixed as described above, then treated with Alexa Fluor 488- conjugated GAH IgG for 15 min at room temperature. After washing twice with PBS, cells were treated with Hoechst 33258 for 15 min at room temperature, washed, and examined under a fluorescence microscope.
- Germantown, NY were injected intravenously with various doses of (Q)-hRS7 ranging from 25 to 400 ⁇ g per mouse and were monitored daily for visible signs of toxicity and body weight change.
- the maximum tolerated dose (MTD) was defined as the highest dose at which no deaths occurred and the body weight loss was 20% or less of pretreatment animal weight (approximately 20 g). Animals that experienced toxic effects were euthanized.
- mice were monitored daily for signs of toxicity and were humanely euthanized and deemed to have succumbed to disease progression if tumors reached greater than 2.0 cm 3 in size or became ulcerated. Additionally, if mice lost more than 20% of initial body weight or otherwise became moribund, they were euthanized. Survival data were analyzed using Kaplan- Meier plots (log-rank analysis) with GraphPad Prism software. Differences were considered statistically significant at P ⁇ 0.05.
- the IVTT assay measures inhibition of protein synthesis due to mRNA degradation by RNase.
- (Q)-hRS7 and rRap have comparable RNase activity in this cell-free assay, whereas no enzymatic activity was observed for hRS7.
- yeast tRNA as substrate, we estimated the kcat/Km (10 9 M “1 s "1 ) of rRap and (Q)-hRS7 to be 4.10 ( ⁇ 0.42) and 1.98, respectively.
- (Q)-hRS7 is most potent against ME-180 (FIG. 4A), T-47D (FIG. 4B), MDA-MB-468, and Calu-3, with EC50 values of 1.5, 2.0, 3.8, and 8.5 nM, respectively.
- FIG. 4A For those cell lines showing less than ⁇ 50% growth inhibition at 100 nM of (Q)-hRS7 with the MTS assay, we also performed colony-formation assays to confirm that (Q)-hRS7 was cytotoxic at 10 or 100 nM to DU- 145, PC-3, MCF7, SK-BR-3, BxPC-3, Capan-1, and SK-OV-3 (not shown).
- (Q)-hRS7 The subcellular location of (Q)-hRS7 was further probed in MDA-MB-468 cells using fluorescence-labeled hTf as a marker for the recycling endosome and Hoechst 33258, which stains the nucleus. It was apparent from the results that (Q)-hRS7 and hTf occupy the same subcellular location in MDA-MB-468 when examined after incubation at 37 0 C for 2 h (not shown). In both cell lines, hRS7 exhibited internalization characteristics similar to (Q)- hRS7, except that it was not visualized by PE-GAM/anti-Rap, as expected (data not shown).
- MTD in mice We determined the MTD of (Q)-hRS7 in normal BALB/c mice given a single intravenous injection to be between 50 ⁇ g and 100 ⁇ g. Other 2L-Rap-X or 2L-Rap(Q)- X fusion proteins made to date have a similar MTD range. In addition, we determined the MTD of (Q)-hRS7 for multiple injections in na ⁇ ve SCID mice to be 80 ⁇ g by giving 20 ⁇ g every five days four times (q5dx4).
- VLS vascular leak syndrome
- HUS hemolytic uremic syndrome
- hRS7 hepatotoxicity
- BL22 a recombinant anti-CD22 immunotoxin composed of the disulfide-stabilized Fv of RFB4 fused to PE38, and similar immunotoxins such as LMB-2 (anti-Tac(Fv)-PE38), also had a very low MTD in mice due to nonspecific liver toxicity, yet BL22 has been reported to be safe and efficacious in clinical trials of patients with hairy-cell leukemia (Kreitman et al., N Engl J Med 2001;345:241-7). Thus, the dose-limiting hepatotoxicity commonly observed in mice may be rarely manifested in humans (Kreitman, BioDrugs 2009;23:l-13).
- Immunogenicity is a more general problem. Most genetically-engineered immunotoxins that have been evaluated in cancer patients induced a strong humoral immune response, which shortens the serum half-life and prevents further administration. Several approaches to reduce the immune response have been tested in experimental animals, with some success reported for deoxyspergualin (Pai et al., Cancer Res 1990;50:7750-3) and CTLA4Ig (Sieall et al., J Immunol 1997; 159:5168-73), and clinical testing of these and other immunosuppressive agents in combination with immunotoxins has been proposed (Frankel, Clin Cancer Res 2004;10:13-5). (Q)- hRS7 will be less
- ImmunoRNases comprising human pancreatic RNase fused to either a human anti-ErbB2 scFv (De Lorenzo et al., Cancer Res 2004;64:4870-4; FEBS Lett 2007;581 :296-300) or a human anti-CD30 scFv-Fc (Menzel et al., Blood 2008; 111 :3830-7), a complete
- (Q)- hRS7 was shown to be effective in inhibiting the growth of Calu-3 human lung cancer xenografts in nude mice, thus validating the antitumor activity and stability of (Q)-hRS7 in vivo, as well as confirming the suitability of adding Trop-2 to the current list of antigens on solid cancers targeted by immunotoxins (Kreitman, AAPS J 2006;8:E532-51; Pastan et al., Nat Rev Cancer 2006; Pastan et al., Annu Rev Med 2007;58:221-37; Schirrmann et al., Exp Opin Biol Ther 2009;9:79-95).
- B 13-24 cells containing an IgG4 gene were purchased from ATCC (ATCC Number CRL-11397) and genomic DNA was isolated. Cells were washed with PBS, resuspended in digestion buffer (100 mM NaCl, 10 mM Tris-HCl pH 8.0, 25 niM EDTA, 0.5% SDS, 0.1 mg/ml proteinase K) and incubated at 50° C. for 18 h. The sample was extracted with an equal volume of phenol/chloroform/isoamyl alcohol and precipitated with 7.5 M
- Genomic DNA was recovered by centrifugation and dissolved in TE buffer. Using genomic DNA as template, the IgG4 gene was amplified by PCR. [0171] Amplified PCR product was cloned into a TOPO-TA sequencing vector (Invitrogen) and confirmed by DNA sequencing. The SacII-Eagl fragment containing the heavy chain constant region of IgGl in pdHL-hLL2 was replaced with SacII-Eagl of the TOPO-TA-IgG4 plasmid to produce the pdHL2-hLL2-IgG4 (pdHL2-hLL2- ⁇ 4) vector.
- a Ser228Pro mutation was introduced in the hinge region of IgG4 to avoid formation of half-molecules.
- a mutated hinge region 56 bp fragment (Pstl-Stul) was synthesized, annealed and replaced with the Pstl-Stul fragment Of IgG 4 . This construction resulted in a final vector pdHL2-hLL2- ⁇ 4P
- a flexible linker comprising three copies of a four glycine-one serine monomer was used to attach the C-terminus of Rap to the N-terminus of Vk of hLLl .
- One rap molecule was attached at the N-terminus of each light chain. Construction of the DNA for this molecule was done by PCR.
- the Xba-BamHI fragment of pdHL2-hLLl- ⁇ 4P was replaced with the Xba-BamHI (Xba-Leader-rap-Linker-Vk-BamHI) fragment of pBS-2L-rap-hLLl to complete the final vector pdHL2-2L-rap-hLLl- ⁇ 4P.
- the vector DNA (30 ⁇ g) was linearized with Sail enzyme and transfected into NSO (4xlO 6 cells/mL) or Sp2/0-Agl4 (5xlO 6 cells/mL) myeloma cells by electroporation (450 V).
- Cells were grown in complete Hybridoma-SFM medium supplemented with low-IgG FBS (10%), penicillin (100 units/mL), streptomycin (100 ⁇ g/mL), L-glutamine (2 mM), sodium pyruvate (1 mM), non-essential amino acids (100 ⁇ M), and methotrexate (0.1 ⁇ M). Positive clones were screened by ELISA.
- plates were coated with 50 ⁇ l of an anti-rap antibody at 5 ug/mL in PBS medium and incubated at 4° C. over night. After washing the plate with PBS and blocking with 2% BSA cell culture supernatants were added. HRP- conjugated goat anti-human IgG.sub.4 antibodies were used for detection and OPD was used as a substrate for color development. Plates were read at 490 run. Positive clones were expanded and frozen for future use. Clone C6 was identified as the best producer and used for further development.
- HPLC Protein purity and concentration were checked on HPLC. A sharp single peak was observed at 7.7 min (not shown), with the retention time indicating the molecule was larger than IgG.
- SDS-PAGE SDS-PAGE was performed under reducing conditions using 4-20% Tris- Glycine gels. A band related to the heavy chain of expected size about 50 kD and two bands of molecular mass about 37 and 39 kD, both larger than the light chain of hLLl (about 25 kD), were observed (not shown). The presence of the two light chains was shown to be due to glycosylation of rap on the fusion protein (see below).
- Mass Spectrometry was performed at The Scripps Research Institute, CA, by the MALDI-TOF method. Two samples were sent for analysis, one in the native state (1.6 mg/mL in 10 mM PBS) and the other in reduced state (1.6 mg/mL in 1 mM HEPES/10 mM DTT, pH 7.5 buffer). The native sample showed one major peak of mass 177150, which is in good agreement with the MW of one IgG plus two raps (not shown). The reduced sample showed three major peaks at 50560 (corresponding to the heavy chain), 38526 and 36700 (corresponding to the two light chains containing rap) (not shown).
- Competition Binding for WP WP is an anti-idiotype antibody of hLLl.
- the affinity of rap-hLLl antibody in comparison with hLLl antibody against WP was evaluated by competition binding assay. Briefly, 96-well plates were coated with 50 ⁇ l of WP at 5 ug/mL and incubated at 4° C. over night. Three types of protein samples, hLLl, rap-hLLl or hA20 were prepared in different 2x dilutions (final concentrations range between 0.49-1000 nM), mixed with an equal volume of 2xHRP-conjugated mLLl antibody (final dilution is
- rap-hLLl was sensitive on both a B-cell lymphoma cell line (Daudi) and a multiple myeloma cell line (MC/CAR).
- rap-hLLl was significantly more potent (cytotoxic) on Daudi cells compared to MC/CAR cells, as reflected by the EC 50 values (FIG. 8 and FIG. 9).
- MC/CAR cells an EC 50 value was not achieved at the concentrations tested. At the highest concentration (56 nM), cell viability was 57%.
- hLLl or free rap, by itself did not demonstrate cytotoxicity in either cell line.
- mice At selected times after dosing (1, 2, 4, 16, 48, 72, 168 h), groups of 5 mice were anesthetized and a blood sample was withdrawn by cardiac puncture. Major tissues were removed, weighed, and placed in containers. Blood samples and tissues were counted in a calibrated gamma counter for 1 11 In (channels 120-480) and 88 Y (channels 600-2000). A crossover curve was generated to correct for the back-scatter of 88 Y energy into the 111 In counting window.
- mice In vivo toxicity Naive SCID or BALB/c mice were injected intravenously with various doses of 2L-Rap-hLLl- ⁇ 4P ranging from 25 to 400 ⁇ g/mouse, and monitored daily for visible signs of toxicity and body weight loss.
- the maximum tolerated dose (MTD) was defined as the highest dose at which no death occurred, and body weight loss was ⁇ 20% of pretreatment animal weight (approximately 20 g). Animals that experienced toxic effects were sacrificed, harvested and subjected to histopathological analysis.
- mice In naive SCID mice, a single intravenous dose of 100, 150, 200, 250, 300 or 400 ⁇ g of 2L-Rap-hLLl- ⁇ 4P resulted in severe weight loss and death of the animals, but all mice survived a dose of 25 or 50 ⁇ g (not shown). In BALB/c mice, all mice survived a single intravenous dose of 30 or 50 ⁇ g of 2L- Rap-hLLl- ⁇ 4P, but not 100 or 200 ⁇ g (not shown). In another experiment, a 75 ⁇ g-dose of 2L-Rap-hLLl- ⁇ 4P was found toxic to SCID mice (data not shown).
- the MTD of 2L-Rap-hLLl- ⁇ 4P given as a single bolus injection is between 50 and 75 ⁇ g in SCID mice and between 50 and 100 ⁇ g in BALB/c mice.
- Gross pathological examination of the dead or sacrificed mice indicated severe liver and spleen toxicity. The liver was pale in color and the spleen was shriveled and smaller than the usual size. Histopathologic examination revealed hepatic and splenic necrosis. Serum samples of the representative mice had elevated levels of alanine aminotransferase (ALT), asparatate aminotransferase (AST) and total bilirubin, suggesting significant liver toxicity at these high doses.
- ALT alanine aminotransferase
- AST asparatate aminotransferase
- total bilirubin suggesting significant liver toxicity at these high doses.
- noncompartmental analysis program WinNonlin, Version 4.1 (Pharsight, Mountain View, Calif).
- the program calculates area under the curve (AUC) using the linear trapezoidal rule with a linear interpolation.
- the elimination rate constant (k ⁇ ) was computed from the terminal half-life (tj/ 2 ⁇ ) assuming first order kinetics. Survival studies were analyzed using Kaplan- Meier plots (log-rank analysis) with GraphPad Prism software. Differences were considered significant at P ⁇ 0.05.
- mice Female SCID mice (8 weeks old, 18-22 g), 8 to 9 per group, were injected intravenously with 1.5x10 7 Daudi cells and received treatments one day later. Mice were examined daily for hind leg paralysis and were weighed weekly. The animals were euthanized when they developed hind leg paralysis or lost 20% of their pretreatment weight. Each set of therapy experiments ended after 180 days.
- mice receiving a single injection of 5, 15, 30, 40 or 50 ⁇ g of 2L-Rap-hLLl- ⁇ 4P were cured. It is noteworthy that the MST of mice receiving a single injection of 1 ⁇ g was 92 days, compared with 28 days of the untreated group (P ⁇ 0.0001), representing a 230% increase.
- the resultant double-stranded PCR product contains cDNA sequence that encodes for 54 amino acid residues of the N-terminal half of the cytotoxic RNase.
- ONNBACK contains the restriction sites HindIII and Ndel to facilitate subcloning into either a staging vector or for in-frame ligation (Ndel site) into the bacterial expression vector. The Nrul site is incorporated in the ONNFOR primer to facilitate in-frame ligation with the cDNA encoding the C- terminal half of the cytotoxic RNase.
- the final double-stranded PCR product contained a cDNA sequence that encoded 51 amino acids of the rest of the C-terminal half of the cytotoxic RNase.
- An Nrul site allowed in-frame ligation with the N-terminal half of the PCR-amplified DNA incorporated in ONCBACK.
- a stop codon and BamHI restriction sites for subcloning into staging or expression vectors were included in the ONCFOR sequence.
- PCR-amplified DNA encoding the N- and C-terminal halves of the cytotoxic RNase after being treated with the appropriate restriction enzymes, were joined at the Nrul sites and subcloned into a staging vector, e.g., pBluescript from Stratagene.
- the ligated sequence encodes a polypeptide of 105 amino acids with an N-terminal Met.
- V-region sequences of hLL2 and hMN-14 have been published. Leung et al., MoI. Immunol., 32: 1413 (1995); U.S. Pat. No. 5,874,540.
- the VK and VH sequences for LL2 and MN- 14 were PCR-amplified using published methods and primers. Sequence analysis of the PCR-amplified DNAs indicated that they encoded proteins typical of antibody VK and VH domains.
- a chimeric antibody constructed based on the PCR-amplified LL2 and MN- 14 sequences exhibited immunoreactivity comparable to their parent antibodies, confirming the authenticity of the sequence obtained.
- VH and VK sequences of hLL2 were used as templates to assemble the hLL2- scFv gene by standard PCR procedures.
- a Met initiation codon at the -1 position was incorporated at the N-terminus of the VL gene, which was linked via a 16 amino acid linker to the VH domain.
- a tail consisting of six histidyl residues was included at the carboxyl end of the VH chain to facilitate the purification of the fusion protein via metal chelate chromatography.
- the immuno toxin fusion protein gene for ranpirnase-hLL2scFv was constructed in a similar fashion by restriction digestion and ligation methods.
- the cDNA sequence when expressed, encoded a fusion protein with ranpirnase attached to the N-terminal end of the LL2 VL sequence via a short linker.
- linkers There are a variety of linkers that can be inserted between the cytotoxic RNase C-terminus and the VL domain N-terminus.
- a preferable linker is the amino acid sequence TRHRQPRGW (SEQ ID NO: 94) from the C-terminal position 273-281 of Pseudomonas exotoxin (PE).
- This sequence has been shown to be a recognition site for intracellular cleavage of PE into active fragments by subtilisins, with cleavage occurring between the G and W residues of the sequence. Chiron et al., J. Biol. Chem., 269:18167 (1994). Incorporation of this sequence facilitates the release of active cytotoxic RNase after internalization of the fusion immunotoxin.
- a 13 -amino acid residue spacer consisting of amino acid residues 48-60 of fragment B of Staphylococcal Protein A, used in the construction of an EDN-scFv fusion, can be used instead to allow for flexible linkage between the cytotoxic RNase and the scFv. Tai et al., Biochemistry, 29:8024 (1990) and Rybak et al., Tumor Targeting, 1 :141 (1995).
- MN- 14 scFv was produced by PCR amplification of cDNA from humanized MN- 14 transfectoma.
- the linker used for MN- 14 scFv was a 15 -amino acid linker and the orientation was VL-linker-VH. After confirmation of the DNA sequences, the single chain construct was subcloned into a eukaryotic expression vector and transfected into an appropriate mammalian host cell for expression.
- Another single chain construct also was made. This was made with the opposite 5'-3' orientation of the heavy and light chains, was assembled in pCANTABE5E (Pharmacia Biotech, Piscataway, NJ.) and expressed in phage. Specific binding of recombinant phage expressing this scFv was demonstrated by ELISA (not shown).
- ranpirnase-MN-14 fusion protein ranpirnase attached via linker to the N-terminus of the VL sequence.
- the DNA fragment encoding ranpirnase was obtained as discussed above.
- a 23 -amino acid linker was used between the ranpirnase sequence and the scFv. Kurucz et al. (1995).
- Fab modules may be produced as fusion proteins containing either a DDD or AD sequence. Independent transgenic cell lines are developed for each fusion protein. Once produced, the modules can be purified if desired or maintained in the cell culture supernatant fluid. Following production, any DDD 2 module can be combined with any AD module to generate a DNL construct.
- the plasmid vector pdHL2 has been used to produce a number of antibodies and antibody-based constructs. See Gillies et al., J Immunol Methods (1989), 125:191-202;
- the di-cistronic mammalian expression vector directs the synthesis of the heavy and light chains of IgG.
- the vector sequences are mostly identical for many different IgG-pdHL2 constructs, with the only differences existing in the variable domain (VH and VL) sequences.
- VH and VL variable domain sequences.
- these IgG expression vectors can be converted into Fab-DDD or Fab- AD expression vectors.
- Fab-DDD expression vectors To generate Fab-DDD expression vectors, the coding sequences for the hinge, CH2 and CH3 domains of the heavy chain are replaced with a sequence encoding the first 4 residues of the hinge, a 14 residue Gly-Ser linker and the first 44 residues of human RIIa (referred to as DDDl).
- ADl AKAP-ZS
- Two shuttle vectors were designed to facilitate the conversion of IgG-pdHL2 vectors to either Fab-DDD 1 or Fab-AD 1 expression vectors, as described below.
- the CHl domain was amplified by PCR using the pdHL2 plasmid vector as a template.
- the left PCR primer consists of the upstream (5') of the CHl domain and a SacII restriction endonuclease site, which is 5' of the CHl coding sequence.
- the right primer consists of the sequence coding for the first 4 residues of the hinge followed by a short linker, with the final two codons comprising a Bam HI restriction site.
- the 410 bp PCR amplimer was cloned into the pGemT PCR cloning vector (Promega, Inc.) and clones were screened for inserts in the T7 (5') orientation.
- the two oligonucleotides designated RIIAl -44 top and RIIAl -44 bottom, that overlap by 30 base pairs on their 3' ends, were synthesized (Sigma Genosys) and combined to comprise the central 154 base pairs of the 174 bp DDDl sequence.
- the oligonucleotides were annealed and subjected to a primer extension reaction with Taq polymerase.
- the duplex was amplified by PCR using the following primers: G4S Bam-Left 5'-GGATCCGGAGGTGGCGGGTCTGGCGGAGGT-S' (SEQ ID NO:68)
- the encoded polypeptide sequence is shown below.
- AKAP-IS Top Two complimentary overlapping oligonucleotides, designated AKAP-IS Top and AKAP-IS Bottom, were synthesized.
- the duplex was amplified by PCR using the following primers:
- This amplimer was cloned into the pGemT vector and screened for inserts in the T7 (5') orientation.
- a 190 bp fragment encoding the DDDl sequence was excised from pGemT with BamHI and Notl restriction enzymes and then ligated into the same sites in CHl-pGemT to generate the shuttle vector CHl-DDDl-pGemT.
- a 110 bp fragment containing the ADl sequence was excised from pGemT with BamHI and Notl and then ligated into the same sites in CHl-pGemT to generate the shuttle vector CHl-ADl-pGemT.
- CHl-DDDl or CHl-ADl can be incorporated into any IgG construct in the pdHL2 vector.
- the entire heavy chain constant domain is replaced with one of the above constructs by removing the SacII/Eagl restriction fragment (CH1-CH3) from pdHL2 and replacing it with the SacII/Eagl fragment of CHl-DDDl or CHl-ADl, which is excised from the respective pGemT shuttle vector.
- DDD or AD The location of the DDD or AD is not restricted to the carboxyl terminal end of CHl.
- a construct was engineered in which the DDDl sequence was attached to the amino terminal end of the VH domain.
- h679-Fd-ADl-pdHL2 is an expression vector for production of h679 Fab with ADl coupled to the carboxyl terminal end of the CHl domain of the Fd via a flexible Gly/Ser peptide spacer composed of 14 amino acid residues.
- a pdHL2-based vector containing the variable domains of h679 was converted to h679-Fd-ADl-pdHL2 by replacement of the SacII/Eagl fragment with the CHl-ADl fragment, which was excised from the CHl-ADl- SV3 shuttle vector with SacII and Eagl.
- C-DDD l-Fd-hMN-14-pdHL2 is an expression vector for production of fusion protein C-DDD 1-Fab-hMN- 14, in which DDDl is linked to hMN-14 Fab at the carboxyl terminus of CHl via a flexible peptide spacer.
- the plasmid vector hMN14(I)-pdHL2 which has been used to produce hMN-14 IgG, was converted to C-DDD l-Fd-hMN-14-pdHL2 by digestion with SacII and Eagl restriction endonucleases to remove the CH1-CH3 domains and insertion of the CHl-DDDl fragment, which was excised from the CH1-DDD1-SV3 shuttle vector with SacII and Eagl.
- N-DDD l-Fd-hMN-14-pdHL2 is an expression vector for production of a stable dimer that comprises two copies of a fusion protein N-DDD 1-Fab-hMN- 14, in which DDDl is linked to hMN-14 Fab at the amino terminus of VH via a flexible peptide spacer.
- the expression vector was engineered as follows. The DDDl domain was amplified by PCR using the two primers shown below.
- hMN-14 Fd sequence was amplified by PCR using the oligonucleotide primers shown below. hMN-14VH left G4S Bam
- CHl-C stop Eag 5'- CGGCCGTCAGCAGCTCTTAGGTTTCTTGTC -3' (SEQ ID NO:78)
- N-DDD 1-hMN-l 4 Fd sequence was excised with Xhol and Eagl restriction enzymes and the 1.28 kb insert fragment was ligated with a vector fragment that was prepared by digestion of C-hMN-14-pdHL2 with those same enzymes.
- the final expression vector is N-DDD l-Fd-hMN-14-pDHL2.
- the 679 antibody binds to an HSG target antigen and may be purified by affinity chromatography.
- the h679-Fd-ADl-pdHL2 vector was linearized by digestion with Sal I restriction endonuclease and transfected into Sp/EEE myeloma cells by electroporation.
- the di-cistronic expression vector directs the synthesis and secretion of both h679 kappa light chain and h679 Fd-ADl, which combine to form h679 Fab-ADl.
- the cells were plated in 96-well tissue culture plates and transfectant clones were selected with 0.05 ⁇ M methotrexate (MTX).
- MTX methotrexate
- Clones were screened for protein expression by ELISA using microtitre plates coated with a BSA- IMP-260 (HSG) conjugate and detection with HRP-conjugated goat anti-human Fab.
- HSG BSA- IMP-260
- HRP-conjugated goat anti-human Fab BIAcore analysis using an HSG (IMP-239) sensorchip was used to determine the productivity by measuring the initial slope obtained from injection of diluted media samples. The highest producing clone had an initial productivity of approximately 30 mg/L.
- a total of 230 mg of h679-Fab-AD 1 was purified from 4.5 liters of roller bottle culture by single-step IMP-291 affinity chromatography. Culture media was concentrated approximately 10-fold by ultrafiltration before loading onto an IMP-291 -affigel column.
- C-DDD l-Fd-hMN-14-pdHL2 and N-DDD l-Fd-hMN-14-pdHL2 vectors were transfected into Sp2/0-derived myeloma cells by electroporation.
- C-DDD 1-Fd-hMN- 14- pdHL2 is a di-cistronic expression vector, which directs the synthesis and secretion of both hMN-14 kappa light chain and hMN-14 Fd-DDDl, which combine to form C-DDD 1-hMN- 14 Fab.
- N-DDD l-hMN-14-pdHL2 is a di-cistronic expression vector, which directs the synthesis and secretion of both hMN-14 kappa light chain and N-DDD 1-Fd-hMN- 14, which combine to form N-DDD 1-Fab-hMN- 14.
- Each fusion protein forms a stable homodimer via the interaction of the DDDl domain.
- Clones were screened for protein expression by ELISA using microtitre plates coated with WI2 (a rat anti-id monoclonal antibody to hMN-14) and detection with HRP-conjugated goat anti-human Fab.
- WI2 a rat anti-id monoclonal antibody to hMN-14
- HRP-conjugated goat anti-human Fab The initial productivity of the highest producing C-DDD 1-Fab-hMN 14 Fab and N-DDDl- Fab-hMN14 Fab clones was 60 mg/L and 6 mg/L, respectively.
- ADl-C is a peptide that was made synthetically consisting of the ADl sequence and a carboxyl terminal cysteine residue, which was used to couple the peptide to Affigel following reaction of the sulfhydryl group with chloroacetic anhydride.
- DDD-containing a 2 structures specifically bind to the ADl-C-Affigel resin at neutral pH and can be eluted at low pH (e.g., pH 2.5).
- C-DDD 1-Fab-hMN- 14 A total of 81 mg of C-DDD 1-Fab-hMN- 14 was purified from 1.2 liters of roller bottle culture by single-step ADl-C affinity chromatography. Culture media was concentrated approximately 10-fold by ultrafiltration before loading onto an ADl-C-affigel column. The column was washed to baseline with PBS and C-DDD 1-Fab-hMN- 14 was eluted with 0.1 M Glycine, pH 2.5. SE-HPLC analysis of the eluate showed a single protein peak with a retention time (8.7 min) consistent with a 107 kDa protein (not shown). The purity was also confirmed by reducing SDS-PAGE, showing only two bands of molecular size expected for the two polypeptide constituents of C-DDD 1-Fab-hMN- 14 (not shown).
- N-DDDl-hMN-14 A total of 10 mg of N-DDDl-hMN-14 was purified from 1.2 liters of roller bottle culture by single-step ADl-C affinity chromatography as described above. SE-HPLC analysis of the eluate showed a single protein peak with a retention time (8.77 min) similar to C-DDD 1-Fab-hMN- 14 and consistent with a 107 kDa protein (not shown). Reducing SDS- PAGE showed only two bands attributed to the polypeptide constituents of N-DDD 1-Fab- hMN- 14 (not shown).
- C-DDD 1-Fab-hMN- 14 The binding activity of C-DDD 1-Fab-hMN- 14 was determined by SE-HPLC analysis of samples in which the test article was mixed with various amounts of WI2.
- a sample prepared by mixing WI2 Fab and C-DDD 1-Fab-hMN- 14 at a molar ratio of 0.75:1 showed three peaks, which were attributed to unbound C-DDD 1-Fab-hMN 14 (8.71 min), C-DDDl- Fab-hMN-14 bound to one WI2 Fab (7.95 min), and C-DDD 1-Fab-hMN 14 bound to two WI2 Fabs (7.37 min) (not shown).
- BIAcore was used to further demonstrate and characterize the specific interaction between the DDl and ADl fusion proteins.
- the experiments were performed by first allowing either h679-Fab-ADl or 679-Fab-NEM to bind to the surface of a high density HSG-coupled (IMP239) sensorchip, followed by a subsequent injection of C- DDD 1-Fab- hMN- 14 or hMN-14 F(ab') 2 .
- IMP239 high density HSG-coupled
- Example 8 Affinity purification of either DDD or AD fusion proteins
- Universal affinity purification systems can be developed by production of DDD or AD proteins, which have lower affinity docking.
- the DDD formed by RIa dimers binds AKAP-IS (ADl) with a 500-fold weaker affinity (225 nM) compared to RIIa.
- ADl AKAP-IS
- 225 nM 500-fold weaker affinity
- RIa dimers formed from the first 44 amino acid resides can be produced and coupled to a resin to make an affinity matrix for purification of any ADl -containing fusion protein.
- Many lower affinity (0.1 ⁇ M) AKAP anchoring domains exist in nature. If necessary, highly predicable amino acid substitutions can be introduced to further lower the binding affinity.
- a low affinity AD can be produced either synthetically or biologically and coupled to resin for use in affinity purification of any DDDl fusion protein.
- N-DDD2-hMN-14-pdHL2 is an expression vector for production of N-DDD2-Fab- hMN-14, which possesses a dimerization and docking domain sequence of DDD2 appended to the amino terminus of the Fd.
- the DDD2 is coupled to the V H domain via a 15 amino acid residue Gly/Ser peptide linker.
- DDD2 has a cysteine residue preceding the dimerization and docking sequences, which are identical to those of DDDl.
- the expression vector was engineered as follows. Two overlapping, complimentary oligonucleotides (DDD2 Top and DDD2 Bottom), which comprise residues 1 - 13 of DDD2, were made synthetically. The oligonucleotides were annealed and phosphorylated with T4 polynucleotide kinase (PNK), resulting in overhangs on the 5' and 3' ends that are compatible for ligation with DNA digested with the restriction endonucleases Ncol and Pstl, respectively.
- PNK polynucleotide kinase
- the duplex DNA was ligated with a vector fragment, DDDl-hMN14 Fd-S V3 that was prepared by digestion with Ncol and Pstl, to generate the intermediate construct DDD2- hMN14 Fd-S V3.
- the final expression vector is N-DDD2-Fd-hMN-14- pdHL2.
- C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production of C-DDD2-Fab- hMN-14, which possesses a dimerization and docking domain sequence of DDD2 appended to the carboxyl terminus of the Fd via a 14 amino acid residue Gly/Ser peptide linker.
- the expression vector was engineered as follows. Two overlapping, complimentary
- oligonucleotides which comprise the coding sequence for part of the linker peptide
- GGGGSGGGCG SEQ ID NO:81
- residues 1 - 13 of DDD2 were made synthetically.
- the oligonucleotides were annealed and phosphorylated with T4 PNK, resulting in overhangs on the 5 ' and 3 ' ends that are compatible for ligation with DNA digested with the restriction endonucleases BamHI and Pstl, respectively.
- the duplex DNA was ligated with the shuttle vector CHl-DDDl-pGemT, which was prepared by digestion with BamHI and Pstl, to generate the shuttle vector CH1-DDD2- pGemT.
- a 507 bp fragment was excised from CHl-DDD2-pGemT with SacII and Eagl and ligated with the IgG expression vector hMN14(I)-pdHL2, which was prepared by digestion with SacII and Eagl.
- the final expression construct is C-DDD2-Fd-hMN-14-pdHL2. h679-Fd-AD2-pdHL2
- h679-Fd-AD2-pdHL2 is an expression vector for the production of h679-Fab-AD2, which possesses an anchor domain sequence of AD2 appended to the carboxyl terminal end of the CHl domain via a 14 amino acid residue Gly/Ser peptide linker.
- AD2 has one cysteine residue preceding and another one following the anchor domain sequence of ADl .
- the expression vector was engineered as follows. Two overlapping, complimentary oligonucleotides (AD2 Top and AD2 Bottom), which comprise the coding sequence for AD2 and part of the linker sequence, were made synthetically. The oligonucleotides were annealed and phosphorylated with T4 PNK, resulting in overhangs on the 5' and 3' ends that are compatible for ligation with DNA digested with the restriction endonucleases BamHI and Spel, respectively.
- duplex DNA was ligated into the shuttle vector CHl-ADl-pGemT, which was prepared by digestion with BamHI and Spel, to generate the shuttle vector CH1-AD2- pGemT.
- a 429 base pair fragment containing CHl and AD2 coding sequences was excised from the shuttle vector with SacII and Eagl restriction enzymes and ligated into h679-pdHL2 vector that prepared by digestion with those same enzymes.
- the final expression vector is h679-Fd-AD2-pdHL2.
- TFl DNL construct
- N-DDD2-Fab-hMN-14 Protein L-purified
- h679-Fab-AD2 IMP-291 -purified
- SE-HPLC did not show any evidence of a 2 b formation (not shown). Instead there were peaks representing 3 4 (7.97 min; 200 kDa), a 2 (8.91 min; 100 kDa) and B (10.01 min; 50 kDa).
- TFl is a highly stable complex.
- HSG IMP- 239
- TFl is a highly stable complex.
- IMP- 239 HSG
- C- DDDl-Fab-hMN-14 and h679-Fab-ADl was tested under similar conditions, the observed increase in response units was accompanied by a detectable drop during and immediately after sample injection, indicating that the initially formed a 2 b structure was unstable.
- TF2 was obtained by reacting C-DDD2-Fab-hMN-14 with h679-Fab-AD2.
- a pilot batch of TF2 was generated with >90% yield as follows.
- Protein L-purified C-DDD2-Fab-hMN-14 200 mg was mixed with h679-Fab-AD2 (60 mg) at a 1.4:1 molar ratio.
- the total protein concentration was 1.5 mg/ml in PBS containing 1 mM EDTA.
- Subsequent steps involving TCEP reduction, HIC chromatography, DMSO oxidation, and IMP-291 affinity chromatography were the same as described for TFl .
- TF2 The functionality of TF2 was determined by BIACORE as described for TFl .
- TF2 C-DDD 1-hMN- 14 + h679-ADl (used as a control sample of noncovalent a 2 b complex), or C- DDD2-hMN-14 + h679-AD2 (used as a control sample of unreduced a 2 and b components) were diluted to 1 ⁇ g/ml (total protein) and pass over a sensorchip immobilized with HSG.
- the response for TF2 was approximately two-fold that of the two control samples, indicating that only the h679-Fab-AD component in the control samples would bind to and remains on the sensorchip.
- WI2 IgG injections demonstrated that only TF2 had a DDD-Fab- hMN-14 component that was tightly associated with h679-Fab-AD as indicated by an additional signal response.
- the additional increase of response units resulting from the binding of WI2 to TF2 immobilized on the sensorchip also corresponds to two fully functional binding sites, each contributed by one subunit of C-DDD2-Fab-hMN-14. This was confirmed by the ability of TF2 to bind two Fab fragments of WI2 (not shown).
- the relative CEA-binding avidity of TF2 was determined by competitive ELISA. Plates were coated (0.5 ⁇ g/well) with a fusion protein containing the A3B3 domain of CEA , which is recognized by hMN-14. Serial dilutions of TFl, TF2 and hMN-14 IgG were made in quadruplicate and incubated in wells containing HRP-conjugated hMN-14 IgG (1 nM). The data indicate that TF2 binds CEA with an avidity that is at least equivalent to that of IgG and two-fold stronger than TFl (not shown).
- TFl and TF2 were designed to be stably tethered structures that could be used in vivo where extensive dilution in blood and tissues would occur.
- the stability of TF2 in human sera was assessed using BIACORE.
- TF2 was diluted to 0.1 mg/ml in fresh human serum, which was pooled from four donors, and incubated at 37 0 C under 5% CO 2 for seven days. Daily samples were diluted 1 :25 and then analyzed by BIACORE using an IMP-239 HSG sensorchip. An injection of WI2 IgG was used to quantify the amount of intact and fully active TF2. Serum samples were compared to control samples that were diluted directly from the stock. TF2 is highly stable in serum, retaining 98% of its bispecific binding activity after 7 days (not shown). Similar results were obtained for TFl in either human or mouse serum (not shown).
- the pdHL2 mammalian expression vector has been used to mediate the expression of many recombinant IgGs (Qu et al., Methods 2005, 36:84-95).
- a plasmid shuttle vector was produced to facilitate the conversion of any IgG-pdHL2 vector into a C-H-AD2-IgG-pdHL2 vector.
- the gene for the Fc was amplified using the pdHL2 vector as a template and the oligonucleotides Fc BgIII Left and Fc Bam-EcoRI Right as primers.
- the amplimer was cloned in the pGemT PCR cloning vector.
- the Fc insert fragment was excised from pGemT with Xbal and BamHI restriction enzymes and ligated with AD2- pdHL2 vector that was prepared by digestion of h679-Fab-AD2-pdHL2 with Xbal and BamHI, to generate the shuttle vector Fc-AD2-pdHL2.
- Epratuzumab, or hLL2 IgG is a humanized anti-human CD22 MAb.
- An expression vector for C-H-AD2-hLL2 IgG was generated from hLL2 IgG-pdHL2, as described above and used to transfect Sp2/0 myeloma cells by electroporation. Following transfection, the cells were plated in 96-well plates and transgenic clones were selected in media containing methotrexate. Clones were screened for C-H-AD2-hLL2 IgG productivity by a sandwich ELISA using 96-well microtitre plates coated with an hLL2-specific anti-idiotype MAb and detection with peroxidase-conjugated anti-human IgG.
- Clones were expanded to roller bottles for protein production and C-H-AD2-hLL2 IgG was purified from the spent culture media in a single step using Protein-A affinity chromatography. SE-HPLC analysis resolved two protein peaks (not shown). The retention time of the slower eluted peak (8.63 min) was similar to hLL2 IgG. The retention time of the faster eluted peak (7.75 min) was consistent with a -300 kDa protein. It was later determined that this peak represents disulfide linked dimers of C-H-AD2-hLL2-IgG. This dimer is reduced to the monomeric form during the DNL reaction.
- hA20 IgG is a humanized anti-human CD20 MAb.
- An expression vector for C-H- AD2-hA20 IgG was generated from hA20 IgG-pDHL2, and used to transfect Sp2/0 myeloma cells by electroporation. Following transfection, the cells were plated in 96-well plates and transgenic clones were selected in media containing methotrexate. Clones were screened for C-H-AD2-hA20 IgG productivity by a sandwich ELISA using 96-well microtitre plates coated with an hA20-specific anti-idiotype MAb and detection with peroxidase-conjugated anti-human IgG.
- Clones were expanded to roller bottles for protein production and C-H- AD2-hA20 IgG was purified from the spent culture media in a single step using Protein-A affinity chromatography. SE-HPLC and SDS-PAGE analyses gave very similar results to those obtained for C-H-AD2-hLL2 IgG (not shown).
- IgG or Fab fusion proteins were constructed and incorporated into DNL constructs.
- the fusion proteins retained the antigen-binding characteristics of the parent antibodies and the DNL constructs exhibited the antigen-binding activities of the incorporated antibodies or antibody fragments.
- a dimer of Rap was covalently tethered to the C-terminus of each heavy chain of the respective IgG.
- a control construct, 14-Rap was made similarly, using labetuzumab (hMN-14), that binds to an antigen (CEAC AM5) not expressed on B-cell lymphomas/leukemias .
- Rap-DDD2 The deduced amino acid sequence of secreted Rap-DDD2 is shown above (S ⁇ Q ID NO:94). Rap, underlined; linker, italics; DDD2, bold; p_Q_, amino-terminal glutamine converted to pyroglutamate. Rap-DDD2 was produced in E. coli as inclusion bodies, which were purified by IMAC under denaturing conditions, refolded and then dialyzed into PBS before purification by Q-Sepharose anion exchange chromatography. SDS-P AGE under reducing conditions resolved a protein band with a Mr appropriate for Rap-DDD2 (18.6 kDa) (not shown). The final yield of purified Rap-DDD2 was 10 mg/L of culture.
- the DNL method was employed to rapidly generate a panel of IgG-Rap conjugates.
- the IgG-AD modules were expressed in myeloma cells and purified from the culture supernatant using Protein A affinity chromatography.
- the Rap-DDD2 module was produced and mixed with IgG-AD2 to form a DNL complex. Since the CH3-AD2-IgG modules possess two AD2 peptides and each can tether a Rap dimer, the resulting IgG-Rap DNL construct comprises four Rap groups and one IgG. IgG-Rap is formed nearly quantitatively from the constituent modules and purified to near homogeneity with Protein A.
- the CH3-AD2-IgG exists as both a monomer, and a disulfide-linked dimer (not shown).
- the IgG-Rap resolves as a cluster of high molecular weight bands of the expected size between those for monomeric and dimeric CH3-AD2-IgG (not shown).
- Reducing conditions, which reduces the conjugates to their constituent polypeptides shows the purity of the IgG-Rap and the consistency of the DNL method, as only bands representing heavy-chain-AD2 (HC- AD2), kappa light chain and Rap-DDD2 are visualized (not shown).
- Rap-DDD2 Reversed phase HPLC analysis of 22-Rap (not shown) resolved a single protein peak at 9.10 min eluting between the two peaks of CH3-AD2-IgG-hLL2, representing the monomeric (7.55 min) and the dimeric (8.00 min) forms.
- the Rap-DDD2 module was isolated as a mixture of dimer and tetramer (reduced to dimer during DNL), which were eluted at 9.30 and 9.55 min, respectively (not shown).
- LC/MS analysis of 22-Rap was accomplished by coupling reversed phase HPLC using a C8 column with ESI-TOF mass spectrometry (not shown).
- the spectrum of unmodified 22-Rap identifies two major species, having either two GOF (GOF/GOF) or one GOF plus one GIF (G0F/G1F) N-linked glycans, in addition to some minor glycoforms (not shown).
- Enzymatic deglycosylation resulted in a single deconvoluted mass consistent with the calculated mass of 22-Rap (not shown).
- the resulting spectrum following reduction with TCEP identified the heavy chain- AD2 polypeptide modified with an N-linked glycan of the GOF or GIF structure as well as additional minor forms (not shown).
- Each of the three subunit polypeptides comprising 22-Rap were identified in the deconvoluted spectrum of the reduced and deglycosylated sample (not shown).
- the results confirm that both the Rap- DDD2 and HC-AD2 polypeptides have an amino terminal glutamine that is converted to pyroglutamate (pQ); therefore, 22-Rap has 6 of its 8 constituent polypeptides modified by pQ.
- IgG-Rap was evaluated with three ALL cell lines (FIG. 14). The relative antigen density was similar among the three lines, with HLA-DR»CD22>CD20. None of the parental MAbs, either alone or combined with rRap, were cytotoxic in these assays.
- the non-targeting 14-Rap showed some activity, similar to the results with NHL lines.
- C2-Rap which targets the most abundant antigen (HLA-DR)
- HLA-DR most abundant antigen
- 20-Rap showed modest cytotoxicity, which was similar to that of the non-targeting 14-Rap. This is in contrast to the results for the NHL lines, which have high CD20 density and were most responsive to 20-Rap.
- the efficacy of IgG-Rap correlates with the relative abundance of the targeted antigen.
- the DNL method provides a modular approach to efficiently tether multiple cytotoxins onto a targeting antibody, resulting in novel immunotoxins that are expected to show higher in vivo potency due to improved pharmacokinetics and targeting specificity.
- LC/MS, RP-HPLC and SDS-PAGE demonstrated the homogeneity and purity of IgG-Rap.
- Targeting Rap with a MAb to a cell surface antigen enhanced its tumor-specific cytotoxicity.
- Antigen density and internalization rate are both critical factors for the observed in vitro potency of IgG-Rap.
- In vitro results show that CD20-, CD22-, or HLA-DR-targeted IgG-Rap have potent biologic activity for therapy of B-cell lymphomas and leukemias.
Abstract
Description
Claims
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2010800386149A CN102596235A (en) | 2009-08-31 | 2010-08-30 | Compositions and methods of use of immunotoxins comprising ranpirnase (RAP) show potent cytotoxic activity |
EP10812707.7A EP2473187A4 (en) | 2009-08-31 | 2010-08-30 | Compositions and methods of use of immunotoxins comprising ranpirnase (rap) show potent cytotoxic activity |
CA2770351A CA2770351C (en) | 2009-08-31 | 2010-08-30 | Compositions and methods of use of immunotoxins comprising ranpirnase (rap) show potent cytotoxic activity |
IN1662DEN2012 IN2012DN01662A (en) | 2009-08-31 | 2010-08-30 | |
BR112012004274A BR112012004274A2 (en) | 2009-08-31 | 2010-08-30 | immunotoxin compositions and methods of use comprising ranpirnase (rap) show potent cytotoxic activity |
AU2010286496A AU2010286496B2 (en) | 2009-08-31 | 2010-08-30 | Compositions and methods of use of immunotoxins comprising ranpirnase (Rap) show potent cytotoxic activity |
JP2012527065A JP6114936B2 (en) | 2009-08-31 | 2010-08-30 | Compositions and methods of use of immunotoxins containing lampyrnase (RAP) exhibiting potent cytotoxic activity |
Applications Claiming Priority (18)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US23847309P | 2009-08-31 | 2009-08-31 | |
US61/238,473 | 2009-08-31 | ||
US26630509P | 2009-12-03 | 2009-12-03 | |
US61/266,305 | 2009-12-03 | ||
US12/644,146 | 2009-12-22 | ||
US12/644,146 US7981398B2 (en) | 2005-04-06 | 2009-12-22 | PEGylation by the dock and lock (DNL) technique |
US31699610P | 2010-03-24 | 2010-03-24 | |
US61/316,996 | 2010-03-24 | ||
US12/731,781 | 2010-03-25 | ||
US12/731,781 US8003111B2 (en) | 2005-04-06 | 2010-03-25 | Dimeric alpha interferon pegylated site-specifically shows enhanced and prolonged efficacy in vivo |
US12/752,649 | 2010-04-01 | ||
US12/752,649 US8034352B2 (en) | 2005-04-06 | 2010-04-01 | Tetrameric cytokines with improved biological activity |
US12/754,140 | 2010-04-05 | ||
US12/754,140 US8722047B2 (en) | 2005-03-03 | 2010-04-05 | Humanized anti-HLA-DR antibodies |
US12/754,740 | 2010-04-06 | ||
US12/754,740 US8562988B2 (en) | 2005-10-19 | 2010-04-06 | Strategies for improved cancer vaccines |
US32396010P | 2010-04-14 | 2010-04-14 | |
US61/323,960 | 2010-04-14 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2011026026A1 true WO2011026026A1 (en) | 2011-03-03 |
Family
ID=43628420
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2010/047132 WO2011026026A1 (en) | 2009-08-31 | 2010-08-30 | Compositions and methods of use of immunotoxins comprising ranpirnase (rap) show potent cytotoxic activity |
Country Status (7)
Country | Link |
---|---|
EP (1) | EP2473187A4 (en) |
JP (1) | JP6114936B2 (en) |
CN (1) | CN102596235A (en) |
BR (1) | BR112012004274A2 (en) |
CA (1) | CA2770351C (en) |
IN (1) | IN2012DN01662A (en) |
WO (1) | WO2011026026A1 (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102559636A (en) * | 2011-12-30 | 2012-07-11 | 百泰生物药业有限公司 | Antibody fusion protein used for leukemia and autoimmune disease and preparation method thereof |
WO2016037985A1 (en) * | 2014-09-08 | 2016-03-17 | Ruprecht-Karls-Universität Heidelberg | Construct for the delivery of a molecule into the cytoplasm of a cell |
WO2016133449A1 (en) * | 2015-02-16 | 2016-08-25 | Xintela Ab | Detection and treatment of malignant tumours in the cns |
WO2016184426A1 (en) * | 2015-05-20 | 2016-11-24 | Tse-Wen Chang | Molecular constructs with targeting and effector elements and their applications |
CN109661400A (en) * | 2016-06-17 | 2019-04-19 | 美真达治疗公司 | For exhausting the composition and method of CD117+ cell |
WO2019222533A1 (en) * | 2018-05-18 | 2019-11-21 | Okogen, Inc. | Methods of treating ocular melanoma |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB201412659D0 (en) * | 2014-07-16 | 2014-08-27 | Ucb Biopharma Sprl | Molecules |
CN108061798B (en) * | 2016-11-07 | 2019-11-08 | 国药中生生物技术研究院有限公司 | Detect the methods and applications of the amount of effective component in HBsAg/anti-HBs complex systems |
CN107217068A (en) * | 2017-07-18 | 2017-09-29 | 哈尔滨紫霞生物科技有限公司 | A kind of method for improving canine recombinant interferon alpha fusion protein antiviral activity |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040001825A1 (en) * | 2002-03-01 | 2004-01-01 | Immunomedics, Inc. | RS7 antibodies |
US20060228300A1 (en) * | 2005-04-06 | 2006-10-12 | Ibc Pharmaceuticals, Inc. | Stably tethered structures of defined compositions with multiple functions or binding specificities |
US20070086942A1 (en) * | 2005-10-19 | 2007-04-19 | Ibc Pharmaceuticals, Inc. | Methods and compositions for generating bioactive assemblies of increased complexity and uses |
US20070140966A1 (en) * | 2005-10-19 | 2007-06-21 | Ibc Pharmaceuticals, Inc. | Multivalent immunoglobulin-based bioactive assemblies |
US20080171067A1 (en) * | 2007-01-17 | 2008-07-17 | Immunomedics, Inc. | Polymeric Carriers of Therapeutic Agents and Recognition Moieties for Antibody-Based Targeting of Disease Sites |
US20090060862A1 (en) * | 2005-04-06 | 2009-03-05 | Immunomedics, Inc. | PEGylation by the Dock and Lock (DNL) Technique |
US20090246214A1 (en) * | 2004-02-13 | 2009-10-01 | Immunomedics, Inc. | Fusion proteins containing recombinant cytotoxic rnases |
US20100196266A1 (en) * | 2005-03-03 | 2010-08-05 | Immunomedics, Inc. | Humanized Anti-HLA-DR Antibodies |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007046893A2 (en) * | 2005-10-19 | 2007-04-26 | Ibc Pharmaceuticals, Inc. | Methods for generating bioactive assemblies and uses thereof |
AU2006232310B9 (en) * | 2005-04-06 | 2011-07-21 | Ibc Pharmaceuticals, Inc. | Improved stably tethered structures of defined compositions with multiple functions or binding specificities |
WO2007075270A2 (en) * | 2005-12-16 | 2007-07-05 | Ibc Pharmaceuticals, Inc. | Multivalent immunoglobulin-based bioactive assemblies |
EP2473186A4 (en) * | 2009-08-31 | 2013-07-10 | Ibc Pharmaceuticals Inc | Bispecific immunocytokine dock-and-lock (dnl) complexes and therapeutic use thereof |
-
2010
- 2010-08-30 CN CN2010800386149A patent/CN102596235A/en active Pending
- 2010-08-30 EP EP10812707.7A patent/EP2473187A4/en not_active Withdrawn
- 2010-08-30 WO PCT/US2010/047132 patent/WO2011026026A1/en active Application Filing
- 2010-08-30 IN IN1662DEN2012 patent/IN2012DN01662A/en unknown
- 2010-08-30 JP JP2012527065A patent/JP6114936B2/en active Active
- 2010-08-30 BR BR112012004274A patent/BR112012004274A2/en not_active IP Right Cessation
- 2010-08-30 CA CA2770351A patent/CA2770351C/en not_active Expired - Fee Related
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040001825A1 (en) * | 2002-03-01 | 2004-01-01 | Immunomedics, Inc. | RS7 antibodies |
US20090246214A1 (en) * | 2004-02-13 | 2009-10-01 | Immunomedics, Inc. | Fusion proteins containing recombinant cytotoxic rnases |
US20100196266A1 (en) * | 2005-03-03 | 2010-08-05 | Immunomedics, Inc. | Humanized Anti-HLA-DR Antibodies |
US20060228300A1 (en) * | 2005-04-06 | 2006-10-12 | Ibc Pharmaceuticals, Inc. | Stably tethered structures of defined compositions with multiple functions or binding specificities |
US20090060862A1 (en) * | 2005-04-06 | 2009-03-05 | Immunomedics, Inc. | PEGylation by the Dock and Lock (DNL) Technique |
US20090191225A1 (en) * | 2005-04-06 | 2009-07-30 | Ibc Pharmaceuticals, Inc. | Stably Tethered Structures of Defined Compositions with Multiple Functions or Binding Specificities |
US20070086942A1 (en) * | 2005-10-19 | 2007-04-19 | Ibc Pharmaceuticals, Inc. | Methods and compositions for generating bioactive assemblies of increased complexity and uses |
US20070140966A1 (en) * | 2005-10-19 | 2007-06-21 | Ibc Pharmaceuticals, Inc. | Multivalent immunoglobulin-based bioactive assemblies |
US20080171067A1 (en) * | 2007-01-17 | 2008-07-17 | Immunomedics, Inc. | Polymeric Carriers of Therapeutic Agents and Recognition Moieties for Antibody-Based Targeting of Disease Sites |
Non-Patent Citations (2)
Title |
---|
See also references of EP2473187A4 * |
VANAMA ET AL.: "Construction, characterization, and mammalian expression of an immunotoxin consisting of ranpimase (Rap) fused to a humanized anti-EGP-1 antibody, hRS7, as a potential therapeutic for prostate cancer", PROC AMER ASSOC CANCER RES, vol. 46, 2005, XP008159980 * |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102559636A (en) * | 2011-12-30 | 2012-07-11 | 百泰生物药业有限公司 | Antibody fusion protein used for leukemia and autoimmune disease and preparation method thereof |
WO2016037985A1 (en) * | 2014-09-08 | 2016-03-17 | Ruprecht-Karls-Universität Heidelberg | Construct for the delivery of a molecule into the cytoplasm of a cell |
US10442863B2 (en) | 2014-09-08 | 2019-10-15 | Lutana Gmbh | Construct for the delivery of a molecule into the cytoplasm of a cell |
JP7080055B2 (en) | 2014-09-08 | 2022-06-03 | ルタナ ゲーエムベーハー | Constructs for delivery of molecules to the cytoplasm of cells |
WO2016133449A1 (en) * | 2015-02-16 | 2016-08-25 | Xintela Ab | Detection and treatment of malignant tumours in the cns |
US10994022B2 (en) | 2015-02-16 | 2021-05-04 | Xintela Ab | Detection and treatment of malignant tumours in the CNS |
WO2016184426A1 (en) * | 2015-05-20 | 2016-11-24 | Tse-Wen Chang | Molecular constructs with targeting and effector elements and their applications |
CN109661400A (en) * | 2016-06-17 | 2019-04-19 | 美真达治疗公司 | For exhausting the composition and method of CD117+ cell |
WO2019222533A1 (en) * | 2018-05-18 | 2019-11-21 | Okogen, Inc. | Methods of treating ocular melanoma |
Also Published As
Publication number | Publication date |
---|---|
JP6114936B2 (en) | 2017-04-19 |
AU2010286496A1 (en) | 2012-02-16 |
IN2012DN01662A (en) | 2015-06-05 |
CN102596235A (en) | 2012-07-18 |
EP2473187A1 (en) | 2012-07-11 |
CA2770351A1 (en) | 2011-03-03 |
JP2013503200A (en) | 2013-01-31 |
CA2770351C (en) | 2019-04-23 |
EP2473187A4 (en) | 2015-09-16 |
BR112012004274A2 (en) | 2016-11-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8551480B2 (en) | Compositions and methods of use of immunotoxins comprising ranpirnase (Rap) show potent cytotoxic activity | |
US10377829B2 (en) | Isolated nucleic acid encoding an anti-IGF-1R antibody | |
US20120276100A1 (en) | Compositions and Methods of Use of Immunotoxins Comprising Ranpirnase (Rap) Show Potent Cytotoxic Activity | |
US9737617B2 (en) | Multiple signaling pathways induced by hexavalent, monospecific and bispecific antibodies for enhanced toxicity to B-cell lymphomas and other diseases | |
US9359443B2 (en) | Combination therapy with anti-CD74 and anti-CD20 antibodies provides enhanced toxicity to B-cell diseases | |
US7901680B2 (en) | Dock-and-lock (DNL) vaccines for cancer therapy | |
US9492561B2 (en) | Dock-and-Lock (DNL) Complexes for delivery of interference RNA | |
AU2010286642B2 (en) | Bispecific immunocytokine dock-and-lock (DNL) complexes and therapeutic use thereof | |
US9446123B2 (en) | Multimeric complexes with improved in vivo stability, pharmacokinetics and efficacy | |
US20110020273A1 (en) | Bispecific Immunocytokine Dock-and-Lock (DNL) Complexes and Therapeutic Use Thereof | |
CA2770351C (en) | Compositions and methods of use of immunotoxins comprising ranpirnase (rap) show potent cytotoxic activity | |
EP2509629A1 (en) | Dock-and-lock (dnl) complexes for delivery of interference rna | |
US20160375108A1 (en) | Compositions and Methods of Use of Immunotoxins Comprising Ranpirnase (Rap) Show Potent Cytotoxic Activity | |
AU2010286496B2 (en) | Compositions and methods of use of immunotoxins comprising ranpirnase (Rap) show potent cytotoxic activity |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 201080038614.9 Country of ref document: CN |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 10812707 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2010286496 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2770351 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 2010286496 Country of ref document: AU Date of ref document: 20100830 Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1662/DELNP/2012 Country of ref document: IN |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2012527065 Country of ref document: JP |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2010812707 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112012004274 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: 112012004274 Country of ref document: BR Kind code of ref document: A2 Effective date: 20120227 |