Abstract:
An immunotoxin for use in the treatment of leishmaniasis A wherein the immunotoxin comprises a portion which is specifically binding to the cellular surface receptor CD64 as a component A and a cell killing portion as a component B, wherein the cell killing portion alters the function, gene expression, or viability of a cell thereby killing  Leishmania -infected macrophages and by this eliminates  Leishmania.

Description:
[0001]    The present invention pertains to an immunotoxin for use in the treatment of leishmaniasis. 
         [0002]    Leishmaniasis is a neglected infectious disease, despite the 1.5-2.0 million new cases every year and a population of 350 million at risk [1]-[3]. The causative agent is a protozoan parasite from the genus  Leishmania , transmitted by bites from infected phlebotomine sand flies. Cutaneous leishmaniasis (CL) is an emerging infectious disease in several countries, due to behavioural and environmental changes, as well as malnutrition and HIV co-infection [2]. Due to worldwide increasing therapeutic failure and drug resistance, as well as serious side effects of current chemotherapy, new drugs are urgently needed for CL [4]. In addition, no surrogate markers are available to monitor clinical response during often lengthy periods of treatment or to predict therapeutic failure. 
         [0003]    J. Van Weyenbergh et al. discloses in Abstract/Cytokine 63 (2013) 243-314 that CD64-targeted immunotoxins selectively induce apoptosis and decrease parasite survival in  Leishmania -infected human macrophages in vitro. CD64-immunotoxin treatment in vivo decreases lesion size, parasite load and inflammation in infected HuCD64-transgenic mice, but not control WT mice. The authors conclude that the results reveal the therapeutic potential of CD64-immunotoxin treatment in cutaneous leishmaniasis, but also challenge the clinical relevance of Thl boosting, as suggested in established murine models, for therapeutic and vaccination strategies in human leishmaniasis. 
         [0004]    WO 2005/052007 A1 discloses a heterologous, recombinantly prepared complex which comprises at least one cytotoxic domain and at least one CD64-specific binding domain, especially of human origin, and nucleic acids and vectors coding for such a complex. It further reports about methods for influencing cell growth and the physiology of CD64-positive cells with the complex according to the invention or with vectors containing the nucleic acid coding therefore. Further vectors and hosts for producing the complex are disclosed. The disclosure further relates to the preparation and distribution of medicaments based on the complex or vectors coding therefore, for the treatment of diseases based on a pathological proliferation and/or increased activity of structurally defined cell populations. This applies, in particular, to tumour diseases, allergies, autoimmune diseases, infectious diseases, chronic inflammation or transplantations (immune suppression). 
         [0005]    An object of the present invention is to provide a new therapeutic approach for treating leishmaniasis. 
         [0006]    The present invention is based on the observation that monocyte CD64 (FcγRI) ex vivo expression is elevated in two independent cohorts of cutaneous leishmaniasis patients and significantly predicts therapeutic failure. CD64-targeted immunotoxins composed of a receptor-specific antibody combined with a cell killing portion selectively induce apoptosis and decrease parasite survival in  Leishmania -infected human macrophages. In vivo immunotoxin treatment of infected HuCD64-transgenic mice decreases lesion size, parasite load and inflammation, providing proof-of-concept for targeting the pathogen by eliminating the host cell in protozoan infection. The invention was verified by means of the CD64-targeting immunotoxin H22xRA composed of the H22 full length antibody chemically conjugated to the plant ribosome-inactivating protein ricin A and H22-ETA a recombinant fusion protein of H22 single-chain antibody fragment genetically fused to a mutant of  Pseudomonas  exotoxin A. However to avoid any undue limitation, the present invention as described herein is of course not limited to the use of that specific molecule. It must be clear that the skilled person understands, that once the mechanism underlying the therapeutic approach provided by the present invention is elucidated, a plethora of potential molecules can be designed which are able to treat successfully leichmaniasis. 
         [0007]    Previously, it was shown in a Tuberculosis infection model in mice, that specific in vitro elimination of mycobacteria infected Alveolar Macrophages with H22xRA immunotoxin did not lead to pathogen elimination, but rather to exacerbation of the disease, followed by death of the mice. This indicates that for  Leishmania  infection, the H22 based immunotoxin provides additional selectivity resulting in bringing the parasite laden monocytic cells into apoptosis, concurrently killing the intracellular parasites, without disturbing the abiding immune response, fighting the  Leishmania  parasite [5] incorporated by reference. 
         [0008]    The object underlying the invention is accomplished by an immunotoxin for use in the treatment of leishmaniasis wherein the immunotoxin comprises a portion which is specifically binding to the cellular surface receptor CD64 as a component A and a cell killing portion as a component B, wherein the cell killing portion alters the function, gene expression, or viability of a cell thereby killing  Leishmania -infected macrophages and by this eliminates  Leishmania.    
         [0009]    Necessary is the component A which binds to the CD64 receptor molecule which expression in macrophages is elevated, which are infected with  Leishmania . This component must have an affinity to the CD64 molecule. 
         [0010]    Furthermore a component B must be present which is able to interact after uptake by the infected cell, i.e a macrophage, with that cell and kill that cell either non specifically or by an induced cell-death. The present invention therefore provides an immunotoxin for use in the treatment of leishmaniasis wherein the immunotoxin comprises a portion, which is specifically binding to the cellular surface receptor CD64, and a cell killing portion. 
         [0011]    In particular the immunotoxin of the invention comprises a cell killing portion which is covalently bonded to the portion specifically binding to the cellular surface receptor CD64. 
         [0012]    In one embodiment of the invention the immunotoxin is a recombinant protein or the portion specifically binding to the cellular surface receptor CD64 is linked directly to the cell-killing portion or linked via a linking group. 
         [0013]    In a further embodiment of the invention the portion of the immunotoxin which is specifically binding to the cellular surface receptor CD64 is selected from the group consisting of antibodies or their derivatives or fragments, synthetic peptides or molecules, ligands, receptor binding molecules, and their structural analogs, mutants or combinations thereof. 
         [0014]    The term “mutant” as used herein is well understood by the person skilled in the art. Mutants are basically proteins which are the product of mutated genes, i. e. proteins which have an altered sequence of amino acids in comparison with the wild type. The result of an amino acid exchange in the mutant may be substantially neutral i.e. without effects on the function, for example if a conservative exchange of amino acids occurs. Mutant in the sense of the present invention comprises also activating mutations, change the gene product such that its effect gets stronger (enhanced activation). Methods for obtaining and identifying mutants are well known in the art, such as site directed mutagenesis in particular in combination with high-trough-put screening (HTS). 
         [0015]    The skilled person knows the term “structural analog” as chemical compound that can be imagined to arise from another compound, if one atom or group of atoms is replaced with another atom or group of atoms. According to the invention the structural analog shall have substantially the same activity as the compound from which it has been derived. 
         [0016]    The term “small molecule” is understood as a low molecular weight organic compound, typically involved in a biological process as a substrate or product typically in within a mass range of 50-1500 daltons (Da). Some examples of small molecules include: sugars, lipids, amino acids, fatty acids, phenolic compounds, alkaloids etc. 
         [0017]    In still another embodiment of the invention the portion, which is specifically binding to the cellular surface receptor CD64, is a recombinant molecule. 
         [0018]    In yet another embodiment of the invention the cell killing portion alters the function, gene expression, or viability of a cell in particular by inactivating molecules responsible for protein biosynthesis or activating components of cell-inherent apoptosis pathways. 
         [0019]    In a further embodiment of the invention the cell killing portion is cytotoxic in particular a molecule selected from the group consisting of a member of ADP-ribosylating enzymes, such as the  Pseudomonas  Exotoxin A, Diphtheria-, Cholera- or the Pertussis-, Botulinumtoxin; or a member of the ribosome-inactivating proteins such as Dianthin, Saporin, Bryodin, Gelonin, Ricin, Abrin, Pokeweed Antiviral Protein (PAP) or Restrictocin; or is a member of the RNases (Phosphodiesterases) such as the Bovine seminal RNase, BovineRNase A, Bovine pancreatic RNase, Angiogenin, Eosinophil-derived Neurotoxin (EDN), Eosinophilic Cationic Protein (ECP), Onconase, or Bullfrog Lectin; or is a member of the prodrug-activating enzymes such as Calicheamicin, Glucose Oxidase, Carboxypeptidase, Alkaline Phosphatase, Cytosindeaminase, β-Glucosidase, β-Glucuronidase, β-Lactamase, Nitroreductase, Thymidinkinase or Purin Nukleosid Phosphorylase; or is a member of the cathepsin protease family; or a member of the calpains; or a member of the granzymes; or a member of the microtubule-binding proteins including tau; or any derivative of the above mentioned proteins; or a combination thereof. The term “derivative” in the context of proteins means covalently modified proteins, in particular amidated, alkylated, phosphorylated, acylated, glycosylated, etc. N- or C-terminally or side chain modified proteins. 
         [0020]    In particular the cell killing portion of the immunotoxin of the invention include all kinds of substances that can display proven efficacy e.g. as chemotherapeutical agents and may be selected from the group of alkylating agents (e.g. cyclophosphamide, cholrambucil), anthracyclins (doxorubicin, daunomycin), maytansinoids (maytansinoid DM1), anti-metabolites, plant alkaloids and terpenoids as the  Vinca  alkaloids (vinblastine, vincristine vinorebline, vindesin) Podophyllotoxin and structural analogs hereof and taxanes (paclitaxel, docetaxel, taxotere) or topoisomerase inhibitors (camptothecins), synthetic toxins as ellipticine analogs or synthetic analogs of tumor antibiotics as duocarmycin or CC1065, other tubulin binding agents as halichondrin B, hemiasterlins and dolastatins or analogs as monomethyl-auristatin E; component B may also be selected from the group of small molecules having cytotoxic/cytostatic activities like alkylating agents (like Cyclophosphamide, Mechlorethamine, Chlorambucil, Melphalan) or anthracyclines (like Danorubicin, Doxorubicin, Epirubicin, Idarubicin, Mitoxantrone, Valrubicin) or cytoskeletal disruptors (like Paclitaxel, Docetaxel) or Epothilones (like) or Inhibitors of topoisomerase II (like Etoposide, Teniposide, Tafluposide) or nucleotide analogs and precursor analogs (like azacididine, azathioprine, capecitabine, cytarabine, doxofluridine, fluorouracil, gemcitabine, mercaptopurine, methotrexate, tioguanine) or peptide antibiotics (like bleomycin) or platinum-based agents (like carboplatin, cisplatin, oxaliplatin) or retinoids (like all-trans retinoic acid) or  vinca  alkaloids and structural analogs (like vinblastine, vincristine, vindestine, vinorelbine), beta ray emitting nuclides like Iodine-131, Yttrium-90, Lutetium-177, from the group of Aromatase Inhibitors (like Aminoglutethimide, Anastrozole, Letrozole, Vorozole, Exemestane, 4-androstene-3,6,17-trione, 1,4,6-androstatrien-3,17-dione, Formestane, Testolactone), Carbonic Anhydrase Inhibitors (like Acetazolamide, Methazolamide, Dorzolamide, Topiramate), Cholinesterase Inhibitors (Organophosphates like Metrifonate, Carbamates like Physostigmine, Neostigmine, Pyridostigmine, Ambenonium, Demarcarium, Rivastigmine, Phananthrine like Galantamine, Piperidine like Donepezil, Tacrine, Edophonium, or Phenothiazines), Cyclooxygenase Inhibitors (like Celecoxib, Rofecoxib, Etoricoxib, Acetaminophen, Diclofenac, Ibuprofen), Folic Acid Antagonists (like Methotrexate), Hydroxymethylglutaryl-CoA Reductase Inhibitors (like Atorvastatin, Cerivastatin, Fluvastatin, Lovastatin, Mevastatin, Pitavastatin, Pravastatin, Rosuvastatin, Simvastatin, Vytorin, Advicor, Caduet), Integrase Inhibitors (like Raltegravir, Elvitegravir), Lipoxygenase Inhibitors (like Zileutron), Monoamine Oxidase Inhibitors (like Isocarboxazid, Moclobemide, Phenelzine, Tranylcypromine, Selegiline, Rasagiline, Nialamide, Iproniazid, Iproclozide, Toloxatone, Linezolid, Tryptamines, Dienolide, Detxtroamphetamine), Nucleic Acid Synthesis Inhibitors, Phosphodiesterase Inhibitors (like Caffeine, Theopyline, 3-isobutyl-1-methylxanthine, Vinpocetine, EHNA, Enoximone, Lirinone, PDE3, Mesembrine, Rolipram, Ibudilast, Sildenafil, Tadalafil, Vardenafil, Udenafil, Avanafil), Protease Inhibitors (like Saquinavir, Ritonavir, Idinavir, Nelfinavir, Amprenavir, Lopinavir, Atazanavir, Fosamprenavir, Tipranavir, Darunavir), Protein Kinase Inhibitors (like Imatinib, Geftinib, Pegaptanib, Sorafenib, Dasatinib, Sunitinib, Erlotinib, Nilotinib, Lapatinib), Protein Synthesis Inhibitors (like Anisomycin, Cycloheximide, Chloramphenicol, Tetracycline, Streptomycin, Erythromycin, Puromycin, etc.), Proton Pump Inhibitors (like Omeprazole, Lansoprazole, Esomeprazole, Pantoprazole, Rabeprazole), from the group of oligonucleotides nucleic acids like small interfering RNAs (siRNAs) or a short hairpin RNA (shRNA), an antisense DNA or RNA, a double stranded RNA (dsRNA) or a micro RNA (miRNA) might be used to down-regulate specific key elements of regulative pathways within a cell. 
         [0021]    In a particular embodiment of the immunotoxin of the invention at least one supplementary component C is additionally present. Preferably the component C regulates expression of a gene encoding the complex. It may also be used for purification of the recombinant complex or its individual components alone as A or B, or of the components together as AB. It may also contain structural subcomponents which stimulate internalization of the immunotoxin or its individual components, in particular of the cell killing portion, into a macrophage as target cell. The component C may also trigger translocation of the cell killing portion into a subcellular compartment or stimulate proteolytic removal of the portion which is specifically binding to the cellular surface receptor CD64 from the cell killing portion or trigger intracellular activation of the cell killing portion. The skilled person readily understands that component C may possess only a single one of the features as described hereinabove or combinations thereof. 
         [0022]    In a further embodiment of the invention the cell killing portion is cytotoxic in particular a molecule selected from the group consisting of a member of ADP-ribosylating enzymes, such as the  Pseudomonas  Exotoxin A, Diphtheria-, Cholera- or the Pertussis-, Botulinumtoxin; or a member of the ribosome-inactivating proteins such as Dianthin, Saporin, Bryodin, Gelonin, Ricin, Abrin, Pokeweed Antiviral Protein (PAP) or Restrictocin; or is a member of the RNases (Phosphodiesterases) such as the Bovine seminal RNase, BovineRNase A, Bovine pancreatic RNase, Angiogenin, Eosinophil-derived Neurotoxin (EDN), Eosinophilic Cationic Protein (ECP), Onconase, or Bullfrog Lectin; or is a member of the prodrug-activating enzymes such as Calicheamicin, Glucose Oxidase, Carboxypeptidase, Alkaline Phosphatase, Cytosindeaminase, β-Glucosidase, β-Glucuronidase, β-Lactamase, Nitroreductase, Thymidinkinase or Purin Nukleosid Phosphorylase; or is a member of the cathepsin protease family; or a member of the calpains; or a member of the granzymes; or a member of the microtubule-binding proteins including tau; or any derivative of the above mentioned proteins; or a combination thereof. 
         [0023]    A single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (V H ) and light chains (V L ) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the V H  with the C-terminus of the V L , or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. These molecules were created to facilitate phage display, where it is highly convenient to express the antigen-binding domain as a single peptide. As an alternative, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma. ScFvs have many uses, e.g., flow cytometry, immunohistochemistry, and as antigen-binding domains of artificial T cell receptors. Unlike monoclonal antibodies, which are often produced in mammalian cell cultures, scFvs are more often produced in bacteria cell cultures such as  E. coli. [ 6] 
     
    
     
         [0024]      FIG. 1 a - e    show CD64-directed immunotoxins induce apoptosis in a CD64-selective manner and exert leishmanicidal activity in vitro. 
           [0025]      FIG. 2 a - c    show CD64-directed immunotoxin demonstrates therapeutic potential in vivo. 
           [0026]      FIG. 3 a - d    show CD64 is a biomarker for therapeutic response in cutaneous leishmaniasis. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0027]    Bacterial Strains, Mammalian Cells, and Plasmids 
         [0028]      E. coli  BL21 Derivatives including (DE3) (F −  ompT hsdS B (r B   − m B   − ) gal dcm rne131 DE3) were as host for bacterial synthesis of ETA′-, Ang-, and tau-based fusion proteins. The bacterial expression vector pBM1.1 is derived from the pET27b plasmid (Novagen, Madison, USA), and is used for N-terminal fusion of Sfi I/Not I-ligands to the modified deletion mutant of  Pseudomonas aeruginosa  Exotoxin A plasmids were prepared by the alkaline lysis method and purified using plasmid preparation kits from Qiagen (Hilden, Germany) [7], incorporated by reference. All standard cloning procedures were carried out as described by HETK293T cells were used as host for expression of GB, GM, and Ang-based fusion proteins [8], incorporated by reference. The construction of pMS plasmids encoding GB-H22 has already been described [9], incorporated by reference. 
         [0029]    Construction and Expression of CD64-Specific Recombinant Immunotoxins 
         [0030]    After transformation into BL21 (DE3) strains, H22(scFv)-ETA′ (SEQ ID NO: 1), H22(scFv)-Ang (SEQ ID NO: 2), H22(scFv)- Cat AD-Ang (SEQ ID NO: 3), H22(scFv)-Ang GGRR  (SEQ ID NO: 4), H22(scFv)- Cat AD-Ang GGRR  (SEQ ID NO: 5), H22(scFv)- Cat AD-GB (SEQ ID NO: 6), H22(scFv)- Cat AD-GB R201K  (SEQ ID NO: 7), H22(scFv)-MAP (SEQ ID NO: 8), fusion proteins were periplasmically expressed under osmotic stress in the presence of compatible solutes as described by Barth et al. 2000. Briefly, transformed bacteria were harvested 15 h after IPTG induction. The bacterial pellet was resuspended in sonication-buffer (75 mM Tris/HCl (pH 8), 300 mM NaCl, 1 capsule of protease inhibitors/50 ml (Complete™, Roche Diagnostics, Mannheim, Germany), 5 mM DTT, 10 mM EDTA, 10% (v/v) glycerol) at 4° and sonicated 6 times for 30 s at 200 W. The m22(scFv)-ETA′ fusion proteins were enriched by IMAC (immobilized metal-ion affinity chromatography) using nickel-nitriloacetic chelating Sepharose (Qiagen) and SEC (size exclusion chromatography) with Bio-Prep SE-100/17 (Biorad, München, Germany) columns according to the manufacturer&#39;s instructions. Recombinant Protein was eluted with PBS (pH 7.4) and 1 M NaCl, analyzed by Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE), quantified by densitometry (GS-700 Imaging Densitometer; Biorad) after Coomassie staining in comparison with BSA standards and verified by Bradford assays (Biorad). 
         [0031]    HEK293T cells were used as expression cell line. The cells were transfected with 1 μg DNA, GB-H22(scFv) (SEQ ID NO: 9), GB R201K -H22(scFv) (SEQ ID NO: 10), GM-H22(scFv) (SEQ ID NO: 11), H22(scFv)- Cat AD-GB (SEQ ID NO: 6), H22(scFv)- Cat AD-GB R201K  (SEQ ID NO: 7), H22(scFv)-Ang (SEQ ID NO: 2), H22- Cat AD-Ang (SEQ ID NO: 3), H22(scFv)-Ang GGRR  (SEQ ID NO: 4), and H22(scFv)- Cat AD-Ang GGRR  (SEQ ID NO: 5), according to the manufacturer&#39;s instructions using RotiFect (Roth). The used pMS plasmid contains the EGFP reporter gene so that expression of the corresponding protein could be verified by its green fluorescence via fluorescence microscopy. 
         [0032]    The secreted protein was purified from the supernatant of the cells via Immobilized Metal-ion Affinity Chromatography (IMAC) and Fast Performance Liquid Chromatography (FPLC). The cleared supernatant was supplemented with 10 mM imidazole and loaded to an XK16/20 column (Amersham/GE Healthcare) containing 8 ml Sepharose 6 Fast Flow resin (Clontech/Takara). The used buffers such as incubation, washing and elution buffer were described before [10], incorporated by reference. The eluted protein was re-buffered into 20 mM Tris, pH 7.4, 50 mM NaCl, concentrated, aliquoted and stored at −80° C. For activation prior to use Enterokinase was added to the protein (0.02 U/μg) with 2 mM CaCl2 for 16 h incubation at 23° C. The protein concentration was calculated after SDS-PAGE analysis and Coomassie staining using AIDA Image Analyzer Software (Raytest Isotopenmessgeräte GmbH). 
         [0033]    In Vitro Cytotoxic Activity 
         [0034]    To characterize the cytotoxic activity of the recombinant anti-CD64 immunotoxins in vitro, growth inhibition of AML-derived cell lines HL-60, U937 or in vitro differentiated macrophages was documented by XTT-based colorimetric assay (see Table 1). 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                   
                 Expression 
                   
                 Target 
                   
               
               
                 Lead 
                 system 
                 Yield 
                 cells 
                 IC50/EC50 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 ETA′ 
                 
                   E. coli 
                 
                 1 
                 mg/l 
                 U937 
                 186 
                 pM 
               
               
                   
                 
                   E. coli 
                 
                 1-1.5 
                 mg/l 
                 U937 
                 140 
                 pM 
               
               
                   
                 
                   E. coli 
                 
                 1 
                 mg/l 
                 HL-60 
                 157 
                 pM 
               
             
          
           
               
                   
                 
                   E. coli 
                 
                 not mentioned 
                 HL-60 
                 100-900 
                 pM 
               
             
          
           
               
                   
                 
                   E. coli 
                 
                 * 
                 HL-60 
                 22 pM-2.5 nM 
               
             
          
           
               
                   
                 
                   E. coli 
                 
                 * 
                 hM1φ 
                 0.689-15.08 
                 nM 
               
               
                   
                 
                   E. coli 
                 
                 not mentioned 
                 hM1φ 
                 214 
                 pM 
               
             
          
           
               
                 GB 
                 HEK293T 
                 ~1 
                 mg/l 
                 U937 
                 1.7-17 
                 nM 
               
             
          
           
               
                   
                 HEK293T 
                 1-2 mg/l 
                 HL-60 
                 4-7 
                 nM 
               
               
                   
                 HEK293T 
                 not mentioned 
                 HL-60 
                 0.2-2 
                 nM 
               
             
          
           
               
                   
                 HEK293T 
                 1.5 
                 mg/l 
                 hM1φ 
                 414 
                 pM 
               
               
                   
                 HEK293T 
                 1-2 
                 mg/l 
                 hM1φ 
                 140 
                 pM 
               
             
          
           
               
                 CatAd-Gb 
                 HEK293T 
                 not mentioned 
                 HL-60 
                 60-330 
                 pM 
               
               
                 GBmut 
                 HEK293T 
                 not mentioned 
                 HL-60 
                 4-7 
                 nM 
               
             
          
           
               
                   
                 HEK293T 
                 1-2 
                 mg/l 
                 HL-60 
                 2.4 
                 nM 
               
               
                   
                 HEK293T 
                 1.5 
                 mg/l 
                 HL-60 
                 5.7 
                 nM 
               
               
                   
                 HEK293T 
                 4.9 
                 mg/l 
                 HL-60 
                 0.34-1.13 
                 nM 
               
               
                   
                 HEK293T 
                 4.9 
                 mg/l 
                 hM1φ 
                 0.18-0.43 
                 nM 
               
               
                 CatAd- 
                 HEK293T 
                 ~0.4 
                 mg/l ** 
                 HL-60 
                 12-96.6 
                 pM 
               
               
                 Gb R201K   
                 HEK293T 
                 ~0.4 
                 mg/l ** 
                 hM1φ 
                 56.9-64 
                 pM 
               
               
                 GM 
                 HEK293T 
                 1 
                 mg/l 
                 HL-60 
                 1.2-6.4 
                 nM 
               
               
                 Ang 
                 
                   E. coli 
                 
                 ~1 
                 mg/l 
                 U937 
                 200 
                 pM 
               
             
          
           
               
                   
                 HEK293T 
                 not mentioned 
                 HL-59 
                 2.8-5 
                 nM 
               
             
          
           
               
                   
                 HEK293T 
                 1.7 
                 mg/l 
                 HL-60 
                 10 
                 nM 
               
               
                   
                 HEK293T 
                 1.7 
                 mg/l 
                 hM1φ 
                 290 
                 pM 
               
             
          
           
               
                   
                 HEK293T 
                 1-2 mg/l 
                 hM1φ 
                 108 
                 pM 
               
             
          
           
               
                 Ang GGRR   
                 HEK293T 
                 0.4-2.7 
                 mg/l 
                 HL-60 
                 6.7-0.57 
                 nM 
               
               
                   
                 HEK293T 
                 0.4-2.7 
                 mg/l 
                 hM1φ 
                 153-43 
                 pM 
               
               
                 CatAD- 
                 
                   E. coli 
                 
                 ~1 
                 mg/l 
                 U937 
                 10 
                 pM 
               
             
          
           
               
                 Ang 
                 HEK293T 
                 not mentioned 
                 HL-60 
                 0.56-1.9 
                 nM 
               
             
          
           
               
                 CatAD- 
                 HEK293T 
                 0.5 
                 mg/l 
                 HL-60 
                 0.64 
                 nM 
               
               
                 Ang GGRR   
                 HEK293T 
                 0.5 
                 mg/l 
                 hM1φ 
                 0.79 
                 nM 
               
               
                 MAP 
                 
                   E. coli 
                 
                 ~1 
                 mg/l 
                 HL-60 
                 40 
                 pM 
               
             
          
           
               
                   
                 
                   E. coli 
                 
                 ~1 
                 mg/l 
                 hM1φ 
                 unaffected 
               
               
                   
                   
               
             
          
         
       
     
         [0035]    Parasite Culture, Immunotoxin Preparation, and In Vitro Assays 
         [0036]      Leishmania amazonensis  (MHOM/BR/87/BA125) cultures were maintained in vitro as proliferating promastigotes in Schneider&#39;s insect medium (Sigma Chemical Co., St. Louis, Mo.), supplemented with 10% FCS at 25° C. 
         [0037]    The construction and purification of CD64-directed immunotoxins H22xRA and H22-ETA single chain Fv have been described previously [10], [11], incorporated by reference. PBMCs were isolated by Ficoll-Hypaque gradient centrifugation. Monocytes were separated by adherence for 30 min, differentiated into macrophages for 7 days in RPMI+10% FCS (Gibco-BRL) and then infected with  Leishmania amazonensis  (5:1 ratio) before immunotoxin treatment (24-48 h). Intracellular parasite survival was quantified by transformation of amastigotes into motile promastigotes, which were allowed to proliferate in Schneider&#39;s medium for 8 days at 23° C. Apoptosis was assessed by nuclear fragmentation (Hoechst 33258 or hematoxyline/eosine staining, quantified by microscopy) and annexin V-staining (quantified by flow cytometry, FACSort, BD Biosciences). 
         [0038]    Both immunotoxins were tested using  Leishmania amazonensis -infected human macrophages from healthy donors, an established preclinical in vitro model. A significant time- and dose-dependent decrease in parasite survival was observed following treatment of infected macrophages with both H22xRA and H22-ETA ( FIG. 1 c - d   ). This immunotoxin-induced leishmanicidal activity was associated with host cell apoptosis, as shown in  FIG. 1 e   . Control cultures exhibit characteristic large pleomorphic macrophage nuclei and small elongated  Leishmania  amastigote nuclei in infected cells ( FIG. 1 e   ). Upon immunotoxin treatment, both macrophage and  Leishmania  amastigote nuclei display nuclear fragmentation, structural degradation and loss of DNA content typical of apoptosis (arrows in  FIG. 1 e   ), indicating that host cell-targeting results in concurrent intracellular pathogen elimination. 
         [0039]    Legend to  FIG. 1 : 
         [0040]    CD64-directed immunotoxins induce apoptosis in a CD64-selective manner and exert leishmanicidal activity in vitro. a, In vitro apoptosis (quantified as % of nuclear fragmentation) of CD64 “high” (MFI&gt;50) and “low” (MFI&lt;50) monocytes from healthy donors cultured for 48 h in the presence or absence of 1, 10 or 100 ng of H22-ETA. b, In vitro apoptosis (quantified as % of annexin V-positive cells by flow cytometry) of untreated CD64 “low” and IFNγ-treated CD64 “high” monocytes from two healthy donors (mean±SEM) cultured for 48 h in the presence or absence of 1, 10 or 100 ng/ml of H22xRA. In vitro survival assay of  Leishmania  promastigotes recovered from  Leishmania amazonensis -infected macrophages (duplicate cultures from four normal donors, mean±SEM) cultured for 24, 48 and 72 h in the presence or absence of c 100 ng/ml of H22-RA and d 10 and 50 ng/ml of H22-ETA. e, In vitro apoptosis visualized by Hoechst 33258 staining of uninfected and  Leishmania amazonensis -infected macrophages from a representative healthy donor cultured for 48 h in the presence or absence of 50 ng/ml of H22-ETA immunotoxin (arrows indicate nuclear fragmentation and/or DNA degradation). 
         [0041]    In Vivo Infection and Immunotoxin Treatment 
         [0042]    Animal husbandry, experimentation and welfare complied with the International Guiding Principles for Biomedical Research Involving Animals and was approved by the Animal Care Ethics Committee from Uniklinikum Aachen. Human CD64-transgenic (described by Heijnen et al. [12], incorporated by reference) and WT C57BL6 mice were used at 8-12 weeks of age. Stationary-phase promastigotes (10 5  parasites in 10 μl of saline) of  Leishmania amazonensis  were inoculated into the right ear dermis using a 27.5-gauge needle. At 6 weeks post-infection, both groups of mice were treated with 10 intralesional injections of 70 ng of H22-ETA (10 ul 1×10 −7 M in saline) on alternate days. Littermate WT mice were used as controls for non-specific effects of the immunotoxin, since murine CD64 is not recognized by the human mAb or immunotoxin. Lesion size was monitored every other day from 6 to 9 weeks post-infection using a digital micrometer (series 227/201 Mitutoyo Japan). Infected ears were aseptically excised at 9 weeks post-infection, photographed, scored for inflammation in a blinded manner by a trained pathologist and homogenized in Schneider&#39;s medium. Parasite load was determined using a quantitative limiting-dilution assay. Homogenates were serially diluted in Schneider&#39;s medium with 10% FCS and seeded into 96-well plates containing biphasic blood agar (Novy-Nicolle-McNeal) medium. The number of viable parasites was determined from the highest dilution at which promastigotes could be grown after up to 2 weeks of incubation at 25° C. 
         [0043]    Following intradermal  Leishmania amazonensis  infection in the ear and lesion development, short-term intralesional treatment with H22-ETA halted disease progression in huCD64-transgenic mice but not in WT control littermates, used as controls for possible non-specific effects of the immunotoxin. As shown in  FIG. 2   a, b, c , H22-ETA treatment caused a four-fold decrease in lesion size (p=0.0017), a three-fold decrease in inflammation score (p=0.0052), as well as a five-fold decrease in parasite load (p=0.030). Notably, only 10 intradermal injections were sufficient to achieve a significant therapeutic effect in infected mice, which ascertains the in vivo applicability of the anti-CD64 IT, since patients typically receive 1 to 3 cycles of pentavalent antimonials, i.e. 20 to 60 intravenous injections to achieve clinical cure. 
         [0044]    Legend to  FIG. 2   
         [0045]    CD64-directed immunotoxin demonstrates therapeutic potential in vivo. WT and HuCD64-Tg C57BL6 mice were infected intradermally in the right ear with 10 5    Leishmania amazonensis  stationary phase promastigotes. Following lesion development at 6 weeks post infection, both groups received 10 doses of 100 ng H22-ETA, intralesionally on alternate days. a, Lesion size was measured as right ear thickness, corrected for left ear values of each individual mouse (*p&lt;0.05, **p&lt;0.01, unpaired t test). b, Right ear inflammation was scored on a scale from 0 (absent) to 3 (severe), (**p=0.0052, Mann-Whitney test). c, Parasite load was determined by quantifying  Leishmania  promastigotes in serial dilutions of ear homogenates in Schneider&#39;s Insect Medium (*p=0.030, unpaired t test following log transformation). 
         [0046]    Staining of Blood Cells, Recruitment and Follow Up of Patients 
         [0047]    Peripheral blood samples (10 ml) were collected from patients and healthy controls (Salvador-Bahia urban area, no history of residence in endemic areas) by venipuncture using heparin as an anticoagulant. For whole-blood staining (cohort I), 50 μl of whole blood was diluted with an equal volume of PBS containing 1% BSA and 0.1% sodium azide, followed by staining for 30 min on ice with fluorescein-conjugated anti-CD64 (clone 22, Immunotech-Coulter, Marseille, France; 10.1, Pharmingen, BD Biosciences, US) and lineage markers CD14 (monocytes), CD3 (T cells), CD19 (B cells), CD16b (neutrophils), CD49d (eosinophils) and CD56 (NK cells) or isotype-matched control antibodies (all from Immunotech-Coulter, Marseille, France). Staining was followed by fixation and erythrocyte lysis (whole blood lysing solution, Becton-Dickinson, San Jose, Calif.). For PBMC staining (cohort II), 200,000 mononuclear cells (purified by Ficoll/Hypaque gradient centrifugation) were stained with the same antibody cocktails as cohort I. Since the drastic lysis and fixation step for whole blood was omitted, MFIs in cohort II were higher in both controls and patients. For each sample, 10,000-20,000 events were acquired in a flow cytometer (FACSort, Becton-Dickinson) and analyzed using CellQuest software. Monocytes were gated according to their characteristic forward-scatter and side-scatter as previously described 9  and were confirmed to be CD14 + , CD3 − , CD19 − , CD16b −  and CD56 − . 
         [0048]    Patients with cutaneous leishmaniasis from two consecutive cohorts were recruited and treated in two outpatient clinics (Jequié and Jiquiriçá, Bahia State, North-East Brazil) covering the same rural area, which has a low socio-economic status and a high incidence of infection with  Leishmania braziliensis . This study was approved by the Ethics Committee of the University Hospital Professor Edgard Santos (first cohort, recruitment 2000-2001, follow-up until 2005) and of the Gonçalo Moniz Research Center (second cohort, recruitment 2002-2004, follow-up until 2006). Healthy controls (n=40) were analyzed in parallel within the same time frame and with the same staining protocol. Informed consent was obtained from all patients and healthy controls. A total of 53 patients provided blood samples of sufficient quality for flow cytometry analysis at the time of diagnosis with cutaneous leishmaniasis (as described 9 , according to characteristic lesion morphology, positive skin test, seropositivity towards  Leishmania  antigen and/or the presence of parasites in the lesion). Clinical and demographic data from both cohorts are listed in Table 2. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Clinical characteristics of cutaneous leishmaniasis patients 
               
             
          
           
               
                   
                   
                   
                 Disease 
                 Lesion 
                 Healing 
                   
               
               
                   
                   
                 Age 
                 duration 
                 diameter 
                 time 
                 Treatment  
               
               
                   
                 Gender 
                 (years) 
                 (days) 
                 (cm) 
                 (days) 
                 cycles 
               
               
                   
               
               
                 Cohort  
                 16M/5F 
                 31.3 ± 4.3 
                 60 ± 7 
                 2.5 ± 0.3 
                  99 ± 22 
                 1.04 ± 0.04 
               
               
                 I 
                   
                   
                   
                   
                   
                   
               
               
                 Cohort  
                 16M/16F 
                 29.9 ± 2.8 
                 37 ± 3 
                 2.0 ± 0.2 
                 142 ± 18 
                  1.9 ± 0.2 
               
               
                 II 
                   
                   
                   
                   
                   
                   
               
               
                   
               
             
          
         
       
     
         [0049]    To take both healing time (complete cicatrisation of lesions) and drug dosage (one cycle of standard treatment equals 20 days of intravenous pentavalent antimony) into account, therapeutic response was scored on a scale of 1-3 where 1 is fast (1 cycle and &lt;60 d), 2 is intermediate (1-3 cycles and &lt;360 d) and 3 is slow or non-healing (&gt;3 cycles or &gt;360 d). One patient form cohort I and five patients from cohort II did not have a complete two-year follow-up and were excluded from therapeutic response analysis. 
         [0050]    Ex vivo expression of CD64 (FcγRI) in monocytes was significantly elevated in two independent CL cohorts. In the first cohort, CD64 mean fluorescence intensity (MFI) increased 2.4-fold in patients compared to healthy donors (p=0.0039;  FIG. 3 a   ), In the second cohort, a strikingly similar 2.7-fold increase in MFI was observed in patients vs. controls (p&lt;0.0001;  FIG. 3 b   ). In addition, the percentage of CD64-positive cells was significantly increased in both cohorts (Supplementary  FIG. 3 a - b   ). CD64 expression was also analyzed in an additional group of 17 patients (second cohort) following standard antimonial therapy. As shown in  FIG. 3 c   , the MFI of monocyte CD64 was reduced significantly following treatment, compared to patients before treatment (p=0.014). CD64 expression did not differ significantly between treated patients and controls (p=0.13), indicating that increased CD64 expression at diagnosis reflects disease status and is not an intrinsic feature of leishmaniasis patients. Moreover, monocyte surface levels of CD32 (FcγRII) were not significantly different before or after treatment (p=0.84, results not shown), indicating a selective up-regulation of CD64 during active disease in human CL. CD64 MFI positively correlated to therapeutic failure in the first cohort (Supplementary  FIG. 3 c   , r=0.65, p=0.0018). This selective association between CD64 and therapeutic response was validated in a second larger cohort (Supplementary  FIG. 3 d   , r=0.51, p=0.006). Since the fast, intermediate and slow/non-healing phenotypes were similarly distributed in both cohorts, a joint analysis was possible following normalization of CD64 MFI. As shown in  FIG. 3 d   , normalized CD64 expression was strongly associated with therapeutic response (p&lt;0.0001). Both fast vs. slow healing patients (area under the ROC curve 0.97, p=0.0004, sensitivity 95.8% (95% CI [78.9-99.9%]), specificity 83.3% (95% CI [35.9-99.6%], likelihood ratio 5.8) and fast vs. intermediate and slow healing patients (area under the ROC curve 0.81, p=0.0003, sensitivity 66.7% (95% CI [44.7-84.4%], specificity 90.5% (95% CI [69.6-98.8%], likelihood ratio 7.0) could be significantly discriminated through their CD64 level at diagnosis, thus authenticating its clinical value as a biomarker. 
         [0051]    Legend to  FIG. 3   
         [0052]    CD64 is a biomarker for therapeutic response in cutaneous leishmaniasis. a, b, Flow cytometric analysis of ex vivo monocyte CD64 expression (as mean fluorescence intensity, MFI), in 53 cutaneous leishmaniasis (CL) patients (cohort I and II, **p=0.0039, ***p&lt;0.0001, respectively, t test with Welch&#39;s correction) and 40 normal donors. c, Flow cytometric analysis of ex vivo monocyte CD64 expression (MFI) in CL patients before and after treatment (cohort II, *p=0.014, t test with Welch&#39;s correction). d, Normalized CD64 levels for both cohorts of CL patients classified according to therapeutic response (fast, intermediate or slow healing as described in Supplementary Methods, ANOVA p&lt;0.0001, post-test for linear trend p&lt;0.0001). This study was approved by the Ethics Committee of the University Hospital Professor Edgard Santos (first cohort, recruitment 2000-2001, follow-up until 2005) and of the Gonçalo Moniz Research Center (second cohort, recruitment 2002-2004, follow-up until 2006). 
         [0053]    List of Amino Acid Sequences (Single Letter Code of Amino Acids) 
         [0000]    
       
         
               
               
             
           
               
                 Sequence 1: H22(scFv)-ETA′ 
                   
               
               
                 MKYLLPTAAAGLLLLAAQPAMAMGHHHHHHHHHHSSGHIDDDDKHMKLMAQPAMAQVQLVESGGGVVQ 
               
               
                   
               
               
                 PGRSLRLSCSSSGFIFSDNYMYWVRQAPGKGLEWVATISDGGSYTYYPDSVKGRFTISRDNSKNTLFL 
               
               
                   
               
               
                 QMDSLRPEDTGVYFCARGYYRYEGAMDYWGQGTPVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSAS 
               
               
                   
               
               
                 VGDRVTITCKSSQSVLYSSNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTIS 
               
               
                   
               
               
                 SLQPEDIATYYCHQYLSSWTFGQGTKLEIKAAAELASGGPEGGSLAALTAHQACHLPLETFTRHRQPR 
               
               
                   
               
               
                 GWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAES 
               
               
                   
               
               
                 ERFVRQGTGNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQ 
               
               
                   
               
               
                 NWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQ 
               
               
                   
               
               
                 DQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGR 
               
               
                   
               
               
                 LETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK 
               
               
                   
               
               
                 Sequence 2: H22(scFv)-Ang 
               
               
                 METDTLLLWVLLLWVPGSTGDAAQPAMAQVQLVESGGGVVQPGRSLRLSCSSSGFIFSDNYMYWVRQA 
               
               
                   
               
               
                 PGKGLEWVATISDGGSYTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGYYRYEGAMD 
               
               
                   
               
               
                 YWGQGTPVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCKSSQSVLYSSNQKNYLAW 
               
               
                   
               
               
                 YQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYLSSWTFGQGTKL 
               
               
                   
               
               
                 EIKAAALESRQDNSRYTHFLTQHYDAKPQGRDDRYCESIMRRRGLTSPCKDINTFIHGNKRSIKAICE 
               
               
                   
               
               
                 NKNGNPHRENLRISKSSFQVTTCKLHGGSPWPPCQYRATAGFRNVVVACENGLPVHLDQSIFRRPAEH 
               
               
                   
               
               
                 EFRGGPEQKLISEEDLNSAVDHHHHHH 
               
               
                   
               
               
                 Sequence 3: H22(scFv)- cat AD-Ang 
               
               
                 METDTLLLWVLLLWVPGSTGDAAQPAMAQVQLVESGGGVVQPGRSLRLSCSSSGFIFSDNYMYWVRQA 
               
               
                   
               
               
                 PGKGLEWVATISDGGSYTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGYYRYEGAMD 
               
               
                   
               
               
                 YWGQGTPVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCKSSQSVLYSSNQKNYLAW 
               
               
                   
               
               
                 YQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYLSSWTFGQGTKL 
               
               
                   
               
               
                 EIKAAAGGGGSALALPLSSIFSRIGDPGGPYVHDEVDRGPPGSRQDNSRYTHFLTQHYDAKPQGRDDR 
               
               
                   
               
               
                 YCESIMRRRGLTSPCKDINTFIHGNKRSIKAICENKNGNPHRENLRISKSSFQVTTCKLHGGSPWPPC 
               
               
                   
               
               
                 QYRATAGFRNVVVACENGLPVHLDQSIFRRPAEHEFRGGPEQKLISEEDLNSAVDHHHHHH 
               
               
                   
               
               
                 Sequence 4: H22(scFv)-Ann GGRR   
               
               
                 METDTLLLWVLLLWVPGSTGDAAQPAMAQVQLVESGGGVVQPGRSLRLSCSSSGFIFSDNYMYWVRQA 
               
               
                   
               
               
                 PGKGLEWVATISDGGSYTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGYYRYEGAMD 
               
               
                   
               
               
                 YWGQGTPVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCKSSQSVLYSSNQKNYLAW 
               
               
                   
               
               
                 YQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYLSSWTFGQGTKL 
               
               
                   
               
               
                 EIKAAALESRQDNSRYTHFLTQHYDAKPGGRRDRYCESIMRRRGLTSPCKDINTFIHGNKRSIKAICE 
               
               
                   
               
               
                 NKNGNPHRENLRISKSSFQVTTCKLHGGSPWPPCQYRATAGFRNVVVACENGLPVHLDQSIFRRPAEH 
               
               
                   
               
               
                 EFRGGPEQKLISEEDLNSAVDHHHHHH 
               
               
                   
               
               
                 Sequence 5: H22(scFv)- cat AD-Ann GGRR   
               
               
                 METDTLLLWVLLLWVPGSTGDAAQPAMAQVQLVESGGGVVQPGRSLRLSCSSSGFIFSDNYMYWVRQA 
               
               
                   
               
               
                 PGKGLEWVATISDGGSYTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGYYRYEGAMD 
               
               
                   
               
               
                 YWGQGTPVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCKSSQSVLYSSNQKNYLAW 
               
               
                   
               
               
                 YQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYLSSWTFGQGTKL 
               
               
                   
               
               
                 EIKAAAGGGGSALALPLSSIFSRIGDPGGPYVHDEVDRGPPGSRQDNSRYTHFLTQHYDAKPGGRRDR 
               
               
                   
               
               
                 YCESIMRRRGLTSPCKDINTFIHGNKRSIKAICENKNGNPHRENLRISKSSFQVTTCKLHGGSPWPPC 
               
               
                   
               
               
                 QYRATAGFRNVVVACENGLPVHLDQSIFRRPAEHEFRGGPEQKLISEEDLNSAVDHHHHHH 
               
               
                   
               
               
                 Sequence 6: H22(scFv)- cat AD-GB 
               
               
                 METDTLLLWVLLLWVPGSTGDAAQPAMAQVQLVESGGGVVQPGRSLRLSCSSSGFIFSDNYMYWVRQA 
               
               
                   
               
               
                 PGKGLEWVATISDGGSYTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGYYRYEGAMD 
               
               
                   
               
               
                 YWGQGTPVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCKSSQSVLYSSNQKNYLAW 
               
               
                   
               
               
                 YQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYLSSWTFGQGTKL 
               
               
                   
               
               
                 EIKAAAGGGGSALALPLSSIFSRIGDPGGPYVHDEVDRGPIIGGHEAKPHSRPYMAFLMIWDQKSLKR 
               
               
                   
               
               
                 CGGFLIRDDFVLTAAHCWGSSINVTLGAHNIKEQEPTQQFIPVKRAIPHPAYNPKNFSNDIMLLQLER 
               
               
                   
               
               
                 KAKRTRAVQPLRLPSNKAQVKPGQTCSVAGWGQTAPLGKHSHTLQEVKMTVQEDRKCESDLRHYYDST 
               
               
                   
               
               
                 IELCVGDPEIKKTSFKGDSGGPLVCNKVAQGIVSYGRNNGMPPRACTKVSSFVHWIKKTMKRYAEHHH 
               
               
                   
               
               
                 HHH 
               
               
                   
               
               
                 Sequence 7: H22(scFv)- cat AD-GB R201K   
               
               
                 METDTLLLWVLLLWVPGSTGDAAQPAMAQVQLVESGGGVVQPGRSLRLSCSSSGFIFSDNYMYWVRQA 
               
               
                   
               
               
                 PGKGLEWVATISDGGSYTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGYYRYEGAMD 
               
               
                   
               
               
                 YWGQGTPVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCKSSQSVLYSSNQKNYLAW 
               
               
                   
               
               
                 YQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYLSSWTFGQGTKL 
               
               
                   
               
               
                 EIKAAAGGGGSALALPLSSIFSRIGDPGGPYVHDEVDRGPIIGGHEAKPHSRPYMAFLMIWDQKSLKR 
               
               
                   
               
               
                 CGGFLIRDDFVLTAAHCWGSSINVTLGAHNIKEQEPTQQFIPVKRAIPHPAYNPKNFSNDIMLLQLER 
               
               
                   
               
               
                 KAKRTRAVQPLRLPSNKAQVKPGQTCSVAGWGQTAPLGKHSHTLQEVKMTVQEDRKCESDLRHYYDST 
               
               
                   
               
               
                 IELCVGDPEIKKTSFKGDSGGPLVCNKVAQGIVSYGKNNGMPPRACTKVSSFVHWIKKTMKRYAEHHH 
               
               
                   
               
               
                 HHH 
               
               
                   
               
               
                 Sequence 8: H22(scFv)-MAP 
               
               
                 HHHHHHHHHHSSGHIDDDDKHMKLMAQPAMAQVQLVESGGGVVQPGRSLRLSCSSSGFIFSDNYMYWV 
               
               
                   
               
               
                 RQAPGKGLEWVATISDGGSYTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGYYRYEG 
               
               
                   
               
               
                 AMDYWGQGTPVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCKSSQSVLYSSNQKNY 
               
               
                   
               
               
                 LAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYLSSWTFGQG 
               
               
                   
               
               
                 TKLEIKAAAMAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDTPSLEDE 
               
               
                   
               
               
                 AAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSS 
               
               
                   
               
               
                 GEPPKSGDRSGYSSPGSPGTPGSRSRTPALPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDL 
               
               
                   
               
               
                 KNVKSKIGATENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSK 
               
               
                   
               
               
                 CGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHG 
               
               
                   
               
               
                 AEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGLPKKKRKV 
               
               
                   
               
               
                 Sequence 9: GB-H22(scFv) 
               
               
                 HIDDDDKIIGGHEAKPHSRPYMAFLMIWDQKSLKRCGGFLIRDDFVLTAAHCWGSSINVTLGAHNIKE 
               
               
                   
               
               
                 QEPTQQFIPVKRAIPHPAYNPKNFSNDIMLLQLERKAKRTRAVQPLRLPSNKAQVKPGQTCSVAGWGQ 
               
               
                   
               
               
                 TAPLGKHSHTLQEVKMTVQEDRKCESDLRHYYDSTIELCVGDPEIKKTSFKGDSGGPLVCNKVAQGIV 
               
               
                   
               
               
                 SYGRNNGMPPRACTKVSSFVHWIKKTMKRYGSKLAEHEGDAAQPAMAQVQLVESGGGVVQPGRSLRLS 
               
               
                   
               
               
                 CSSSGFIFSDNYMYWVRQAPGKGLEWVATISDGGSYTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPE 
               
               
                   
               
               
                 DTGVYFCARGYYRYEGAMDYWGQGTPVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTIT 
               
               
                   
               
               
                 CKSSQSVLYSSNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIA 
               
               
                   
               
               
                 TYYCHQYLSSWTFGQGTKLEIKAAAGPHHHHHH 
               
               
                   
               
               
                 Sequence 10: GB R201K -H22(scFv) 
               
               
                 HIDDDDKIIGGHEAKPHSRPYMAFLMIWDQKSLKRCGGFLIRDDFVLTAAHCWGSSINVTLGAHNIKE 
               
               
                   
               
               
                 QEPTQQFIPVKRAIPHPAYNPKNFSNDIMLLQLERKAKRTRAVQPLRLPSNKAQVKPGQTCSVAGWGQ 
               
               
                   
               
               
                 TAPLGKHSHTLQEVKMTVQEDRKCESDLRHYYDSTIELCVGDPEIKKTSFKGDSGGPLVCNKVAQGIV 
               
               
                   
               
               
                 SYGKNNGMPPRACTKVSSFVHWIKKTMKRYAEHEGDAAQPAMAQVQLVESGGGVVQPGRSLRLSCSSS 
               
               
                   
               
               
                 GFIFSDNYMYWVRQAPGKGLEWVATISDGGSYTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGV 
               
               
                   
               
               
                 YFCARGYYRYEGAMDYWGQGTPVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCKSS 
               
               
                   
               
               
                 QSVLYSSNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC 
               
               
                   
               
               
                 HQYLSSWTFGQGTKLEIKAAAGPHHHHHH 
               
               
                   
               
               
                 Sequence 11: GM-H22(scFv) 
               
               
                 HIDDDDKIIGGREVIPHSRPYMASLQRNGSHLCGGVLVHPKWVLTAAHCLAQRMAQLRLVLGLHTLDS 
               
               
                   
               
               
                 PGLTFHIKAAIQHPRYKPVPALENDLALLQLDGKVKPSRTIRPLALPSKRQVVAAGTRCSMAGWGLTH 
               
               
                   
               
               
                 QGGRLSRVLRELDLQVLDTRMCNNSRFWNGSLSPSMVCLAADSKDQAPCKGDSGGPLVCGKGRVLAGV 
               
               
                   
               
               
                 LSFSSRVCTDIFKPPVATAVAPYVSWIRKVTGRSAAEHEGDAAQPAMAQVQLVESGGGVVQPGRSLRL 
               
               
                   
               
               
                 SCSSSGFIFSDNYMYWVRQAPGKGLEWVATISDGGSYTYYPDSVKGRFTISRDNSKNTLFLQMDSLRP 
               
               
                   
               
               
                 EDTGVYFCARGYYRYEGAMDYWGQGTPVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTI 
               
               
                   
               
               
                 TCKSSQSVLYSSNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDI 
               
               
                   
               
               
                 ATYYCHQYLSSWTFGQGTKLEIKAAAGPHHHHHH 
               
             
          
         
       
     
       REFERENCES 
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