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WO1997030089A1 - Nouvelle proteine de fusion anticorps-cytokine et methodes d'elaboration et d'utilisation de cette proteine - Google Patents

Nouvelle proteine de fusion anticorps-cytokine et methodes d'elaboration et d'utilisation de cette proteine Download PDF

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Publication number
WO1997030089A1
WO1997030089A1 PCT/US1997/001420 US9701420W WO9730089A1 WO 1997030089 A1 WO1997030089 A1 WO 1997030089A1 US 9701420 W US9701420 W US 9701420W WO 9730089 A1 WO9730089 A1 WO 9730089A1
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WIPO (PCT)
Prior art keywords
igg3
fusion protein
antibody
antigen
cytokine
Prior art date
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PCT/US1997/001420
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English (en)
Inventor
Eric T. Harvill
Sherie L. Morrison
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Regents Of The University Of California
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Priority to AU19524/97A priority Critical patent/AU1952497A/en
Publication of WO1997030089A1 publication Critical patent/WO1997030089A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/05—Immunological preparations stimulating the reticulo-endothelial system, e.g. against cancer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • 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
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention was developed at least in part by grants CA-16858 and AI-29470 (from the National Institutes of Health) and DHHS PHS National Institutional Research Service Award T 32 CA09056. Thus, the U.S. government may have certain rights with regard to the present invention.
  • the present invention concerns an anti-hapten/IL-2 fusion protein and methods of producing and using the same, and more particularly to a method for targeting either (a) the antibody or hapten thereof (i.e., antigenic binding partner) to a cell bearing a cytokine receptor on its surface (e.g., T cells) or (b) the cytokine to a cell bearing the hapten of the antibody on its surface.
  • a cytokine receptor e.g., T cells
  • Interleukin 2 The therapeutic value of Interleukin 2 (IL-2) is limited by its short half life and systemic toxicity.
  • One approach to overcoming these problems is to fuse this protein to an antibody, a protein with a long half life and the ability to target a unique antigen within the body.
  • Interleukin-2 is an important cytokine involved in the generation of an effective cell-mediated immune response.
  • the activities of IL-2 include stimulation of T cells to proliferate and become cytotoxic. 1,2,3 IL-2 also stimulates cytotoxicity in natural killers (NK), macrophages and a variety of other cell types, giving rise to LAK cells. 4
  • NK natural killers
  • 4 These promising in vitro activities have lead to the in vivo use of IL-2 to treat cancer and Acquired Immune Deficiency Syndrome (AIDS) patients.
  • AIDS Acquired Immune Deficiency Syndrome
  • IL-2 usefulness might be achieved by extending its in vivo half-life to allow constant, subtoxic levels to be
  • MAbs Monoclonal Antibodies
  • IgG1-IL2 fusion was reported to be 30- to 100-fold less active than IL-2.
  • antibodies with greater specificity and affinity for tumor-associated antigens provide increasingly useful reagents in the diagnosis and therapy of human cancer, their ability to kill tumor cells is suboptimal. Conjugation with toxin or radionuclide increases the ability of antibodies to kill tumors but the inaccessibility of many tumor cells within a dense tumor mass and the loss of antigen expression by rapidly mutating tumor cells make it difficult for these therapeutics alone to kill every tumor cell and effect a cure.
  • An alternative strategy is to use anti-tumor antibodies to activate an immune response against the tumor.
  • An ongoing immune response should be able to access the entire tumor and should be directed against many different tumor associated antigens so that antigen modulation should not render tumor cells resistant. Indeed, the lysis of tumor cells by
  • IL-2 is a potent stimulator of the immune system.
  • One approach to improving the usefulness of IL-2 is to target it to the site of the tumor, thereby increasing its effective local concentration without causing systemic
  • Antibody-IL-2 fusion proteins have been constructed with the expectation that the antibody binding specificity may target IL-2 to the site of the tumor, reducing systemic toxicity while improving the local immune response. These fusion proteins have been shown to bind antigen 12,14 and retain antibody effector functions including the ability to stimulate antibody dependent cell-mediated cytotoxicity and bind to Fc ⁇ RIII. 16 The IL-2 moiety retains the ability to stimulate the proliferation of an IL-2 dependent cell line with differences in specific activity that may relate to the location of the fusion. 14
  • Ab-IL-2 fusion proteins were shown to stimulate the increased cytotoxicity of a tumor specific T cell line. 12.14 In severe combined immune deficient (scid) mice reconstituted with human LAK cells, an Ab-IL-2 fusion protein prevented tumor metastases from growing in the lungs following
  • one object of the present invention is to provide a novel antibody-cytokine fusion protein which retains at least some of the half-life characteristics of the antibody (e.g., which lengthens the effective therapeutic duration of the cytokine) and at least some of the biological properties and/or activities of the cytokine.
  • a further object of the present invention is to provide a novel method of producing the present antibody-cytokine fusion protein.
  • a further object of the present invention is to provide a novel method for delivering a cytokine or moiety or fragment exhibiting at least some of the biological activity of the cytokine to a cell bearing an antigen on its surface which binds (preferably specifically). to the antibody moiety of the present fusion protein.
  • a further object of the present invention is to deliver an antigen which binds specifically to the antibody moiety of the present fusion protein to a cell having a cytokine
  • receptor on its surface e.g., B cells, T cells, etc.
  • a further object of the present invention is to provide a novel method of stimulating an immune response to an antigen, in which the antigen is dansylated, the antibody moiety is an anti-dansyl monoclonal antibody and the dansylated antigen and anti-dansyl monoclonal antibody-IL2 fusion protein are
  • an animal capable of producing an immune response e.g., a bird such as a chicken, duck, goose or ostrich or a mammal such as a rabbit, mouse, rat, goat, horse or primate
  • an immune response e.g., a bird such as a chicken, duck, goose or ostrich or a mammal such as a rabbit, mouse, rat, goat, horse or primate
  • a further object of the present invention is to provide a novel method for increasing the immune response of a patient having a suppressed immune system (e.g., patients being administered immunosuppressants such as those having recently undergone tissue or organ transplants, patients suffering from acquired immune deficiency syndrome, etc.) using the present method, but in which the antigen is the dansylated B cell or T cell which is uninfected by a retrovirus (e.g., human
  • a further object of the present invention is to provide a novel composition for use in the present methods of
  • Ab is an antibody (preferably a monoclonal antibody, more preferably an anti-dansyl monoclonal antibody), L is a covalent bond or linking group (preferably of from 1 to 10 naturally-occurring amino acids, more preferably of from l to 5 naturally-occurring amino acids, and most preferably a cysteine residue), and Ck is a cytokine, lymphokine
  • interleukin interleukin, macrophage arming factor, lymphocyte inhibition factor, monocyte chemotactic and activating factor and
  • Figs. 1A and 1B show the exemplary IgG3-IL2 assembled and secreted as an H 2 L 2 heterotetramer in TAAG cells incubated with [ 35 S]methionine as the sole source of methionine in the
  • Fig. 2 is a graph depicting Fc ⁇ RI binding by IgG3 and IgG3-IL2, in which U937 cells stimulated with gamma Interferon were incubated with 125 I-labeled IgG3 and varying
  • Fig. 3 is a graph showing complement mediated hemolysis by IgG3 and IgG3-IL2, in which IgG3-IL2 and IgG3 were
  • Fig. 4 is a graph showing the stimulation of CTLL-2 cell proliferation by hrIL-2 and IgG3-IL2, in which the abilities of hrIL-2 and two separate preparations of IgG3-IL2 to
  • Fig. 5 is a bar graph showing the generation of LAK activity by various concentrations of hrIL-2 or IgG3-IL2, in which PBL cultured for 3 days with hrIL-2 or IgG3-IL2 at the indicated doses were tested for their cytotoxicity against 5lCr-loaded Raji target cells; results are expressed as the additional percentage of total 51 Cr specifically released by LAK cells compared to that of unstimulated PBL (average of three samples with the indicated standard deviation);
  • Figs. 6A, 6B and 6C are graphs showing IL-2R binding of hrIL-2 and IgG3-IL2, in which YT-1 cells were incubated at 4oC for 3 h with 125 I-labeled IL-2 and varying concentrations of IgG3-IL2 as unlabeled competitor to assess binding to the IL- 2R; untreated cells were used to evaluate binding to the intermediate affinity IL-2R ⁇ (Fig. 6A), and YT-1 cells pretreated with forskolin (YT-l*) were used to assess binding to the high affinity IL-2R ⁇ (Fig.
  • YT-1 cells were incubated with 125I-labeled IgG3-IL2 and varying concentrations of IgG3-IL2 or hrIL-2 as unlabeled competitor (Fig. 6C); the percent inhibition of binding is plotted against the concentration of the unlabeled competitor and the amount required for 50% inhibition of binding is indicated;
  • Figure 7 shows the half-life of 125 I-labeled IgG3 and 125 I-labeled lgG3-IL2 injected intraperitoneally into BALB/c mice, in which iodinated proteins were injected and the residual radioactivity determined over time using a whole body gamma counter (data are expressed as the percent of the initial injected radioactivity present at each time point);
  • Figure 8 shows the tissue distribution of 125 I-labeled IgG3 and 125 I-labeled lgG3-IL2 injected intraperitoneally into BALB/c mice, in which mice were sacrificed 4 hours after injection and the radioactivity present in selected organs determined (values are expressed as the radiolocalization index, defined as [(cpm/gram of tissue) / (cpm/gram of the entire animal)] ⁇ 100);
  • Figure 10 shows the IgG3-IL2 stimulated antibody response to DNS-BSA-Sepharose, in which five mice per group were injected with DNS-BSA-Sepharose alone (open triangles), DNS- BSA-Sepharose bound by antidansyl IgG3 (open squares) or DNS- BSA-Sepharose bound by antidansyl IgG3-IL2 (open circles); identical booster injections were delivered on day 70
  • serum was collected at the intervals shown, pooled within each group and analyzed by ELISA on DNS-BSA coated plates with isotype-specific secondary antibodies.
  • the present invention concerns an anti-hapten/IL-2 fusion protein.
  • the hapten is dansyl
  • the isotype of the antibody is human IgG3
  • the IL-2 is human.
  • virtually any hapten specificity can be used and the isotype and source of the antibody and the specific IL-2 can be changed without adversely impacting the general usefulness of this invention.
  • IL-2 has been used in vaccines to increase the immune response to attached antigens. 19,20.22.22,23 Although the mechanism is not well
  • IL-2 may target the molecule to particular cells for more effective antigen presentation and/or may deliver a stimulatory signal that directly or indirectly enhances the immune response to the attached antigen. Physically linking the antigen and IL-2 has been shown to be critical to
  • the present fusion protein (e.g., IgG3-IL2) may be capable of binding dansyl, a hapten that can be easily linked to primary amine groups.
  • the present fusion protein can therefore be used to potentiate the immune response to any substance that can be conjugated to dansyl. Therefore, the present fusion protein should allow a panel of antigens to be rapidly tested in order to both (a) determine the mechanism by which a cytokine (e.g., IL-2) affects the immune response, and (b) identify the protein (s) or peptide (s) that will be most useful in a therapeutic application.
  • a cytokine e.g., IL-2
  • IL-2 interleukin 2
  • IL-2R interleukin 2 receptor
  • NK natural killer
  • LAK lymphokine activated killer
  • MAb monoclonal antibody
  • Ig immunoglobulin
  • C complement
  • DNS N,N dimethyl-1-aminonaphthalene-5-sulfonyl chloride
  • BSA bovine serum albumin
  • SRBC sheep red blood cell
  • IMDM Iscove's modified Dulbecco's medium
  • PCR polymerase chain reaction
  • PAGE polyacrylamide gel electrophoresis
  • CM complete media
  • V immunoglobulin variable region
  • c immunoglobulin constant region
  • H immunoglobulin heavy chain
  • L immunoglobulin light chain
  • PBS phosphate buffered saline
  • ELISA enzyme linked immunosorbent assay
  • Fc ⁇ R Fc gamma receptor
  • ADCC antibody dependent cell-mediated cytotoxicity
  • scid severe combined
  • the antibody combining site retains its ability to bind antigen, and the IL-2 retains its ability to activate the cellular immune system.
  • IgG3-IL2 The exemplary fusion protein of the present invention, termed IgG3-IL2, is appropriately N-glycosylated within the IgG3 C H 2 domain, binds the human high affinity Fc receptor (Fc ⁇ RI) with an affinity slightly lower than that of IgG3, and is able to activate complement via the classical pathway to lyse antigen coated sheep red blood cells (SRBC).
  • Fc ⁇ RI human high affinity Fc receptor
  • the extremely short half-life of IL-2 in vivo poses a major obstacle to its effective use as a therapeutic.17
  • the need for stable blood levels has led to difficult regimens of continuous infusion or multiple daily injections of IL-2 and has prompted efforts to extend its half-life in vivo by conjugating it to PEG.
  • the short half-life of IL-2 is attributed in part to its small size (15 kDa), which is below the molecular weight cut off of the kidney and is presumed to cause it to be rapidly eliminated from the blood.9
  • Fusing IL-2 to an antibody may increase its in vivo half- life both by increasing its size and by incorporating the half-life characteristics of the antibody.
  • a previous fusion of IL-2 to IgG1 resulted in a molecule with a half-life of only 0.3 hours, leading to the conclusion that continuous infusion would be necessary in therapeutic
  • lgG3-IL2 demonstrate that this molecule has a half-life of at least 1 hour, preferably at least 2 hours, more preferably at least 4 hours and most preferably at least 7 hours, greatly extended compared to that of IL-2 or prior IgG1-IL2 fusion proteins.
  • IgG3 has other characteristics that make it a particularly advantageous isotype for fusion.
  • IgG3 is unique among all of the IgGs in having an extended hinge region of 62 amino acids. This hinge serves as a spacer,26 separating the Fab from the Fc with its attached IL-2. This spacing may allow the fusion protein to retain a more Ig-like conformation with its associated extended half-life.
  • the present IgG3-IL2 fusion protein contains a single cysteine between the last amino acid of C H 3 and the first amino acid of the mature IL-2 in each heavy chain. It is possible that these free cysteines interact with each other or with other cysteine residues to stabilize the molecule. Alternatively, the orientation of the molecule may partially protect IL-2 so that it is less accessible to proteases.
  • IL-2 is quickly eliminated before it can reach dense and poorly vascularized tumor tissue.
  • tissue distribution of the present fusion protein shows that this molecule, with its longer half-life, is able to travel throughout the body and infiltrate distant organs.
  • the ability of the present IgG3-IL2 to permeate organs suc h as the heart and lung suggests that it will reach tumor tissue in cancer patients.
  • Anti-tumor forms e.g., those in which the Ab moiety binds specifically to an antigen on the surface of a tumor cell or to a dansylated form thereof
  • Anti-tumor forms should, therefore, be able to bind and localize to tumor tissue in quantities sufficient to achieve immune activation at the site of the tumor.
  • IgG3-IL2 When used to stimulate the proliferation of the IL-2 dependent cell line CTLL-2, IgG3-IL2 has a specific activity slightly lower than that of human recombinant IL-2 (hrIL-2). In marked contrast, when comparable unit concentrations (as defined by the standard CTLL-2 proliferation assay) are used to stimulate human peripheral blood lymphocytes (PBL), IgG3-IL2 generates significantly greater lymphokine activated killer (LAK) cell cytotoxicity than does hrIL-2.
  • LAK lymphokine activated killer
  • IgG3-IL2 shows a greater affinity than hrIL-2 for the high affinity IL-2R, consisting of ⁇ , ⁇ and ⁇ subunits.
  • the exemplary IgG3-IL2 fusion protein possesses a combination of the biological properties of IgG3 and IL-2, including antigen binding, complement activation, Fc-yRI
  • IgG3-IL2 to target (a) humoral and cell-mediated immune activation to the site of an antigen of interest and/or (b) an antigen to
  • IL-2R bearing cells or organs bearing cells or organs.
  • Antibody-IL-2 fusion proteins may be of greater
  • the presence of the antibody variable region may target the protein, allowing for higher doses of IL-2 at the location of the antigen of interest.- The presence of IL-2 at that site should generate a cell-mediated immune response to a broad range of antigenic epitopes not limited to the antibody's target ligand.
  • IL-2 is unstable in vivo and is quickly eliminated by the kidney due to its small size (15 kDa).
  • IL-2 The resulting short half-life of IL-2 may be extended by MAb-conjugation due to both the size and stability of the MAb.25 Third, by increasing the permeability of nearby blood vessels, an antibody-IL2 fusion protein may increase immune access to the antibody's target ligand. This has been
  • MAb-IL2 may cross-link IL-2R-bearing effector cells with antigen-bearing target cells or, alternatively, target some bound soluble antigen to IL-2R bearing cells.
  • the present invention also concerns a method of stimulating an immune response to an antigen, comprising administering the present fusion protein (i) to a patient in need thereof (i.e., of a stimulated immune response to the antigen) or (ii) to an animal which exhibits an immune
  • the cytokine moiety is IL-2
  • the receptor is an IL-2 receptor
  • the antigen or hapten is a dansylated polypeptide, protein, or amino sugar (e.g., such as those located on a cell surface)
  • the antibody is an anti-dansyl monoclonal antibody.
  • the present method of stimulating an immune response to an antigen is useful for increasing the immune response to one or more antigens associated with one or more particular
  • the amino group-containing hapten is an HIV or other retroviral coat protein, a viral coat protein, an enterotoxin or other bacterial toxin, a fungal protein, an antigenic region of such a protein or toxin, an amino sugar of a bacterial or fungal cell wall, etc.
  • the present method is also useful for treating a viral, bacterial, fungal or retroviral infection.
  • a fusion protein was created linking the N-terminus of human IL-2 to the C-terminus of human IgG3.
  • an IgG1-IL2 fusion protein binds antigen, generates ADCC, stimulates T cell proliferation and increases the cytotoxicity of various cell types. 12,13,14,29
  • the exemplary IgG3-IL2 is shown to be similar to IgG3 in its N-glycosylation and its ability to activate complement to lyse antigen coated cells. Further, IgG3-lL2 binds Fc ⁇ RI with an affinity only slightly lower than that of IgG3. lgG3-IL-2, like IL-2, is able to sustain CTLL-2 proliferation and generate LAK cells from human PBL.
  • the exemplary IgG3-IL2 binds the intermediate affinity IL-2 receptor (IL-2R/ ⁇ ) with an affinity slightly lower than that of hrIL-2, but shows significantly higher affinity than hrIL-2 for the high affinity IL-2 receptor (IL-2R ⁇ ).
  • the discrepancy between proliferative activity and LAK generation of IgG3-IL2 may be viewed in light of the affinity for the receptor forms and known functions of the receptor subunits.
  • IgG3-IL2 an exemplary fusion protein designed to combine the antigen specificity and effector functions of human IgG3 with the immune stimulatory activities of IL-2, has been engineered and expressed. By fusing these two molecules, a therapeutic agent with an improved repertoire of properties and activities has been provided.
  • IgG3-IL2 is W-glycosylated, activates complement, binds Fc ⁇ RI, binds both intermediate and high affinity IL-2Rs, and generates LAK cell cytotoxicity.
  • IgG3-IL2 has significantly improved properties compared to IL-2.
  • IgG3-IL2 may require that the heavy chain gene fusion be transcribed, translated, assembled with its light chain and secreted by transfected cells.
  • Fig. 1 demonstrates that the exemplary IgG3-IL2 is properly assembled and secreted as the expected 200-kDa H 2 L 2 , disulfide-linked heterotetramer and that it retains its ability to bind
  • the present invention further concerns a method of producing a fusion protein, comprising:
  • the antibody comprises one or more a light chains and/or heavy chains (more preferably two of each), which may be expressed from the same or different polynucleotide strands; the linking group L is a cysteine residue; and the cytokine is IL-2 (preferably human IL-2).
  • the linking group may join the C-terminus of the C H 3 chain of the antibody to the N-terminus of the cytokine.
  • the cell line is preferably one capable of expressing the polynucleotide(s) and permitting assembly of antibody chains, subunits and/or fragments (where necessary) without
  • the present fusion protein may be recovered by
  • Cultured cells may be lysed or ruptured prior to recovering the fusion protein.
  • the Fc region of IgG3, and its N-glycosylation site, are known to be involved in both Fc receptor binding and
  • IgG3-IL2 transcribed, translated, post-translationally modified, assembled and secreted as desired.
  • IgG3-IL2 binds to the high affinity Fc ⁇ RI with an affinity only slightly less than that of IgG3. It has been further shown that IgG3-IL2 is able to activate complement to lyse antigen coated cells (Fig. 3).
  • Activation of complement leads to lysis of antigen-bearing cells and release of chemotactic factors.
  • Figs. 2 and 3 demonstrate that the Fc region of IgG3-IL2 molecules, retains its appropriate structure and effector functions.
  • the exemplary IgG3- IL2 differs in two significant ways from the previously described fusion proteins: (1) human IgG3 is used instead of IgG1 for the fusion, and (2) the present IgG3-IL2 fusion protein does not have a linker separating IL-2 from the end of CH3, but instead, has a cysteine residue at the joint.
  • the present IgG3-IL2 fusion protein is of intermediate cytokine activity, being 30-60% as potent as IL-2 in
  • the therapeutic value of IL-2 lies in its ability to stimulate an immune response, including generating increased cytotoxic activity from a variety of immune cells.
  • the generation of LAK cells from PBL is a property of IL-2 that has been well studied and applied to the treatment of human cancer. 1,4,32
  • the ability of the IgG1-IL2 fusion protein to direct the killing of LAK cells generated with IL-2 was previously reported. 13 However, the ability of this fusion protein itself to generate LAK cells was not examined.
  • IgG3-IL2 was found to stimulate more cytotoxicity than hrIL-2 at every effector to target ratio, even when hrIL-2 was added at a 10-fold higher unit/ml concentration (Fig. 5).
  • hrIL-2 and IgG3-IL2 differ significantly in their relative ability to generate (l) proliferative activity in the CTLL-2 assay and (2)
  • cytotoxicity in the LAK assay This difference may reflect the stability of the antibody molecule, which may enable the fusion protein to remain active for a longer period of time during the 3-to 5-day culture involved in the LAK assay.
  • the bivalency of lgG3-IL2 may affect the signaling of the molecule or the internalization and
  • IgG3-IL2 may also lead to an increased overall affinity for its membrane bound receptor, leading to greater receptor occupancy and greater signaling.
  • IgG3-IL2 exhibits a slightly lower affinity than hrIL-2 for the intermediate affinity IL-2R( ⁇ ) (Fig. 6A). In marked
  • IgG3-IL2 shows a considerably higher affinity than hrIL-2 for the high affinity IL-2R( ⁇ ) (Fig. 6B).
  • IgG3-IL2 exhibited higher affinity than hrIL2 for the high affinity IL-2R( ⁇ ) (Fig. 6C).
  • IgG3 an antibody moiety
  • IL-2 may alter the structure of IL-2 or sterically hinder it such that selected portions of IL-2 are less accessible for binding. Additionally, the size and structure of IgG3-IL2 may impair its binding to multiple, large IL-2R ( ⁇ ) dimers, making it effectively monovalent for this intermediate
  • the fast association time of the alpha subunit may speed the initial contact between the cell surface and IgG3-IL2,
  • the dansyl specificity of this protein could potentially be used to deliver any dansylated molecule to IL- 2R bearing cells or organs.
  • Candidate molecules include toxins which may be delivered specifically to IL-2R bearing cells for the therapy of virally infected T-cells, T-cell leukemias or autoimmune diseases.
  • response to an antigen may further comprise the step of dansylating a target antigen prior to administering the same (either separately from or in combination with an anti-dansyl Ab-L-Ck fusion protein) to the animal or patient.
  • the improved binding to the high affinity form of the IL- 2R may increase the specificity of IgG3-IL2 for various T cell types.
  • targeting of dansylated antigens to IL- 2R bearing cells may affect the overall immune response to that antigen by altering the cells that encounter and
  • the fusion protein may be labeled, for example with a radioactive isotope (e.g., 3 H, 35 S, 32 P, 125 I, 51 Cr, etc.), a fluorescent label (e.g., dansyl,
  • target e.g., tissue, organ or cell
  • IL-2 activity can be changed by changing the antigen binding specificity of the IgG3-IL2 fusion proteins.
  • Tumor specific fusion proteins have been shown to suppress the growth of metastases in a human LAK cell
  • IL-2 has a very short half-life in vivo
  • Antibodies with their in vivo half- lives of days, could be expected to contribute to an extended half-life for an antibody-IL2 fusion protein by increasing the size and stability of the molecule.
  • an Ab-cytokine (preferably IgG3-IL-2) fusion protein (IgG3-IL2) injected into the peritoneum of mice demonstrates a half-life of at least 1 hour, preferably at least 2 hours, more preferably at least 4 hours and most preferably at least 7 hours, much longer than that of IL-2' or of the previously described fusion between
  • the present Ab-cytokine fusion protein (preferably IgG3- IL2) escapes the peritoneal cavity and permeates organs
  • IL-2R cytokine receptors
  • the present inventors show herein that the mouse antibody response to an injected antigen i ⁇ enhanced when that antigen is bound by lgG3-IL2, but not IgC3. increases were noted, especially at early time points, in the production of antigen-specific antibodies of all measured isotypes: IgA, IgM, IgG1, IgG2 and IgG3. The IgG2 response was particularly dramatic, in agreement with previous reports that IL-2 increases secretion of this isotype. 37
  • the antibody is a monoclonal antibody of the IgG2, IgG3, IgA or IgM isotype.
  • the antibody moiety is a monoclonal antibody that specifically binds to a dansyl hapten.
  • the hapten group dansyl is very reactive and can be easily attached by known methods to virtually any protein.
  • an anti-dansyl/IL-2 fusion protein When an anti-dansyl/IL-2 fusion protein is injected along with a dansylated antigen, there is an greatly increased antibody response to the antigen. Presumably, the anti- dansyl/IL-2 fusion protein remains localized at the site of the antigen, triggering a more potent immune response. A similar approach can be used to potentiate the immune response to a large number of different antigens. Potential uses of the present fusion protein include:
  • mice models are shown hereinbelow to tolerate doses of 100 ⁇ g of the present IgG3-IL2, although with some weight gain. This dose of over 7,000 units/g is 70 times greater than the dose of hrIL-2 that causes similar weight gain in humans, indicating that IgG3-IL2, with its extended half-life in vivo, is not proportionally increased in toxicity. 17
  • a major objective of the present invention is to
  • a cytokine e.g., IL-2
  • One application of the present fusion protein is thus to target a cytokine (e.g., IL-2) to the site of a solid tumor, where it may stimulate an anti-tumor immune response.
  • cytokine e.g., IL-2
  • antibody-IL-2 fusion proteins have utility in generating an anti-tumor imune response (see, e.g., the present disclosure and ref. 9 hereinbelow).
  • a further application of the present IgG3-IL2 fusion protein is in the production of vaccines against molecules attached to IL-2 via the antigenic specificity of the
  • HSV herpes simplex virus
  • the present anti-dansyl lgG3-IL2 fusion protein provides a tool which can be used to 3oin bioactive IL-2 to any protein of interest to potentiate the immune response against the associated protein.
  • the hapten dansyl can be easily linked to any primary amine present on a potential antigen, and the resulting dansylated antigen is then recognized by the high affinity IgG3-IL2.
  • the antigen-antibody complexes can then be inoculated into animals, and the resulting immune response may provide protection against challenge with antigen bearing virus, bacteria or tumor cells.
  • the results indicate that the antibody response is greatly enhanced by the attached fusion protein IgG3-IL2, but not by attached Ab (IgG3) alone.
  • Antigen coated beads generate an antibody response that is changed only slightly when the beads are bound by human IgG3 before injection. However, when bound by the present fusion protein (IgG3-IL2), the increase in antibody response is dramatic. All isotypes are increased starting early after injection and remain elevated compared to controls for the length of the study. This early response may reflect the ability of the present fusion protein (IgG3-IL2) to directly stimulate antibody producing cells, relieving the need for T helper cells for activation. Alternatively, the local
  • concentration of IL-2 may stimulate other nearby cells to produce cytokines that then act on the B cells to stimulate antibody production.
  • IgG2 The particularly dramatic increase in IgG2 caused by the present fusion protein (e.g., IgG3-IL2) is consistent with the earlier observation that IL-2 stimulates increased IgG2a production in vitro . 36 Thus, present in vivo results confirm earlier predictions based on in vitro results. Ab (IgG3) alone had only minimal effect on the antibody response, with notable increases in IgG1 response and the IgM response to boost.
  • the effects of IgG3 do not resemble those of IgG3-IL2 in either scale or pattern.
  • immunized with a complex of the present fusion protein e.g., IgG3-IL2
  • a complex of the present fusion protein e.g., IgG3-IL2
  • IgG3-IL2 the present fusion protein
  • IL-2 may improve the immune status of such patients. 39
  • the extended half-life of the present fusion protein e.g., IgG3-lL2
  • its ability to attach to an antigen of interest may greatly improve upon the ability of IL-2 to bolster the immunity of such patients.
  • the extended half-life and versatility of the present fusion protein provides a novel tool for both scientists and clinicians to use targeted IL-2 as an immune stimulator.
  • the properties of the present fusion protein e.g., IgG3-IL2 suggest it may be useful in other areas, such as in treatment of AIDS and of secondary opportunistic infections associated therewith.
  • the long half-life of this protein may allow immune compromised patients to be treated with relatively constant blood concentrations of IL-2, effectively boosting their immune system.
  • anti-dansyl IgG3-IL2 may be any suitable protein capable of recognition a panel of dansylated antigens.
  • the increased half-life and immune stimulatory activities of the present fusion protein preferably IgG3-IL2
  • cytokine- e.g., IL-2
  • RNA from PMA and PHA stimulated Jurkat cells was used to generate cDNA by reverse transcription using an oligo dT primer.
  • This cDNA was used as template for a polymerase chain reaction (PCR) using oligonucleotide primers flanking the mature IL-2 coding sequence: primer 1, 5"-ACAACACGTG
  • the PCR product was cloned into pBluescript and sequenced. Using the Pmll and EcoRI sites introduced during the PCR reaction, the IL-2 cDNA was subcloned into a vector created to allow fusion of various genes to the human IgG3 heavy chain at the 3' end of the IgG3 constant domain ((C k 3).” This vector contains a PvuII site which yields a blunt end two base pairs after the end of C H 3. The IL-2 cDNA fragment digested with PmlI results in a single base pair before the first codon of mature IL-2.
  • the resulting fused genes encode a fusion protein linking the last amino acid of C H 3 to the first amino acid of mature IL-2 with a single codon, generated as a consequence of the cloning strategy, encoding a cysteine between them.
  • the fused gene was then joined to a mouse anti-dansyl (dansyl: 5- dimethylaminonaphthalene 1-sulfonyl chloride) variable region in the expression vector pSV2 ⁇ Hgpt. 38
  • the expression vector encoding the chimeric mouse/human kappa ( ⁇ ) light chain specific for DNS (pSV 184 ⁇ HneoV DNS Huk) is available from the present inventors.
  • IgG3-IL2 by an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • 35 S-labeled IgG3-IL was prepared by growing TAAG cells, in the presence or absence of tunicamycin, in DMEM minus methionine (Gibco, Grand Island, NY) supplemented with [ 3S S]methionine (ICN, Irvine, CA) and precipitating the protein from the supernatant with dansyl-BSA coated Sepharose.
  • Unlabeled IgG3-IL2 was purified from culture supernatants using an affinity column with the dansyl isomer 2-dimethylamino-naphthalene-5-sulfonyl chloride
  • hrlL-2 was kindly provided by Cetus Corporation (Emeryville, CA). The specific activity of the hrIL-2 was 1.8 ⁇ 10 7 International Units (IU)/mg of protein. Protein concentration was
  • YT-1 a human natural killer-like cell line that
  • CTLL-2 an IL-2 dependent murine T cell line
  • Raji an NK-resistant human Burkitt's lymphoma
  • UCLA William Clark
  • YT-1, U937 and Raji were grown in RPMI 1640 medium supplemented with 10% (vol/vol) calf serum (HyClone, Logan, UT) .
  • YT-l cells were induced to express the high affinity IL-2R by stimulation with 10 ⁇ M forskolin (Sigma Chemical Co., St. Louis, MO) for 24 h at 3-5 ⁇ 10 5 cells/ml (designated YT-1 + ) .
  • U937 cells were stimulated with interferon- ⁇ (100 units/ml) for 2 days before binding assays to increase Fc ⁇ RI expression.
  • P3X63Ag8.653 cells were cultured in Iscove's modified Dulbecco's medium (XHDM; Gibco, Grand Island, NY) with 5% calf serum (HyClone, Logan, UT).
  • CTLL-2 cells were cultured in RPMI 1640 medium supplemented with 10% bovine calf serum and IL-2.
  • IgG3 and IgG3-IL2 were radiolabeled with Na- 125 l
  • 125 I-labeled IL-2 was purchased from New England Nuclear (Wilmington, DE).
  • RPMI 1640 supplemented with 10% (vol/vol) fetal calf serum (CM) and resuspended at 2-4 ⁇ 10 7 cells/ml in CM. Aliquots of this cell suspension (90 ⁇ l) were then added to 10 ⁇ l of CM containing radiolabeled ligand and cold competitor at varying concentrations in 0.5-ml tubes.
  • SRBC Complement-mediated hemolysis Sheep red blood cells
  • DNS-BSA Complement-mediated hemolysis Sheep red blood cells
  • 0.2 ml packed, antigen coated SRBC were incubated for 1 h at 37°C with [ 51 Cr] sodium chromate (Amersham Corp., Arlington Heights, IL) in 2 ml of fresh Gel-HBS buffer (0.01 M Hepes, 0.15 M NaCl, 0.5 mM MgCl 2 , 0.15 mM CaCl 2 , 0.1% gelatin, pH 7.4).
  • a 96-well plate containing serial dilutions of hrIL-2 or IgG3-IL2 in 50 ⁇ l CM was seeded with 50 ⁇ l CM containing 2-4 ⁇ 10 4 CTLL-2 cells previously depleted of IL-2 for 4 h. After incubation for 18 h, 1 ⁇ Ci [methyl- 3 H] thymidine in 24 ⁇ l CM was added to each well and incubated for 6 h longer. The cells were then harvested from the wells with dH 2 O and washed through glass microfiber filters (Whatman Ltd., Maidstone, UK). The filters were then immersed in EcoLume (Amersham, Arlington Heights, IL) and quantitated in a Beckman LS3150T liquid scintillation counter (Beckman Instruments, Fullerton, CA).
  • LAK cell activity was determined as previously described. 4 Briefly, PBMC were isolated from human blood using a ficoll gradient and depleted of monocytes and macrophages by
  • IgG3-IL2 is assembled and secreted as H 2 L 2
  • IgG3 is composed of two heavy chains (H) and two light chains (L) assembled and secreted as a disulfide-linked heterotetramer (H 2 L 2 ).
  • H heavy chains
  • L light chains
  • the intact protein migrates slightly faster than a 200- kDa molecular weight standard.
  • ⁇ - mercaptoethanol Upon treatment with ⁇ - mercaptoethanol, it is reduced to subunits of 28 kDa,
  • IgG3-lL2 binds FcyRI and activates complement
  • IgG3-IL2 contains the Fc region of IgG3 and should
  • IgG3 -IL2 stimulates both proliferation and cytotoxicity in order to determine if the IL-2 portion of the protein is functional, IgG3-IL2 was tested for its ability to
  • hrIL-2 stimulated some proliferation at
  • hrIL-2 and IgG3-IL2 increased killing in a dose-dependent manner (Fig. 5). Maximal killing (defined as greater than 95% 51 Cr release) was generated at 1000 IU/ml for hrIL-2 and an effector to target (E/T) ratio of 50. In contrast, lgG3-IL-2 required only 100 units/ml for maximal killing at an E/T of 50. It is noteworthy that IgG3-IL2 consistently generated more efficient killing than a 10-fold higher concentration of hrIL-2 at all E/T ratios. Furthermore, significant activity is generated at IgG3-IL2 concentrations of 10 IU/ml, a concentration at which very little activity was observed with IL-2 at any E/T ratio. The data is representative of numerous similar experiments.
  • IgG3-IL2 binds both intermediate and high affinity IL-2 receptors
  • IL-2R ⁇ high affinity IL-2 receptor
  • IgG3-IL2 has a
  • 125 I-labeled IgG3-IL2 binding to YT-1 + cells in the presence of varying concentrations of unlabeled hrlL-2 or lgG3-IL2 was examined (Fig. 6C). Both hrIL-2 and IgG3-IL2 inhibited binding of 125 I-labeled IgG3-IL2 to these cells in a dose-dependent manner and to similar maximal values. Half maximal inhibition occurred for hrIL-2 at 10-15 nM and for IgG3-IL2 at 0.9-1 nM, again indicating that IgG3-IL2 has a higher affinity than hrIL-2 for this form of the IL-2 receptor. Unlabeled IgG3 did not affect binding to IL-2R in these assays (data not shown).
  • Chimeric IgG3 and IgG3-IL2 contain mouse variable regions specific for dansyl (5-dimethylaminonaphthalene-1-sulfonyl chloride) and human constant regions of IgG3, kappa light chain and human IL-2. Expression and affinity purification of these proteins was performed according to Experiment 1 above. Purity was assessed by Coomasie blue staining of SDS-polyacrylamide gels. Protein concentration was determined by a bicinchoninic acid-based protein assay (BCA Protein Assay, Pierce Chemical Co., Rockford, IL).
  • lgG3 and IgG3-IL2 were radiolabeled with Na- 125 I (Amersham, Arlington Heights, IL) using Iodobeads (Pierce Chemical Co., Rockford, IL) according to the manufacturer's protocol. Proteins analyzed by SDS-PAGE were visualized by exposure on a phosphor screen and analyzed with a phosphorimager (Molecular Dynamics, Sunnyvale, CA).
  • 125 I-labeled IgG3 and 125 I-labeled IgG3-IL2 were injected intraperitoneally into adult female BALB/c mice (Taconic Farm, Germantown, NY).
  • a whole body gamma counter (Wm. B. Johnson & Assoc. Inc., Montville, NJ) was used to determine the residual radioactivity. The percentages expressed in Figure 1 were determined by comparing the residual counts per minute to the counts per minute immediately after injection. Biodistribution and toxicity
  • mice Female BALB/c mice were treated with KI and injected with 125 I-labeled protein as described above. Four hours after injection, mice were sacrificed and dissected to determine the tissue distribution of the injected protein. Organs were weighed on a Mettler H30 analytical balance (Mettler,
  • the samples were analyzed by SDS-PAGE under non-reducing and reducing conditions and were visualized by exposure on a phosphor screen with subsequent analysis on a phosphorimager (Molecular Dynamics, Sunnyvale, CA).
  • Dansyl was conjugated to bovine serum albumin (BSA) at a ratio of 40 to 1 to make dansyl-BSA (DNS-BSA), which was used to coat Sepharose as previously described.15
  • BSA bovine serum albumin
  • DNS-BSA dansyl-BSA
  • mice five mice per group were inoculated by intraperitoneal injection with 0.5 ml of PBS containing 0.2 ml of packed DNS- BSA-Sepharose, alone or prebound to 10 ⁇ g of IgG3 or 10 ⁇ g of IgG3-IL2. 31ood was collected in microfuge tubes by nicking the tail on the indicated days, and cells removed from serum by brief centrifugation in a microfuge.
  • Blood was pooled from the five animals of each group and diluted 1 to 1000 (IgA, IgM, IgG2, IgG3, Kappa light chain) or 1 to 5000 (IgG1) in PBS with 1% gelatin for analysis.
  • ELISAs were performed in 96-well plates (Corning Glass Works, Corning, NY) coated with DNS-BSA at a concentration of 100 ⁇ g/ml and blocked with 1% BSA in PBS. Serum diluted in PBS was allowed to bind to the plate at 4°C overnight. The plate was washed with PBS six times and incubated with the indicated isotype-specific, alkaline phosphatase-linked antibody for 1 hour at 37°C, then washed again.
  • Substrate P-nitrophenyl phosphate, Sigma
  • IgG3 is a larger molecule with a long serum half-life. 45 The half-life of IgG3- IL2 was examined in vivo to determine if fusing IL-2 to IgG3 would result in a molecule with improved pharmacokinetics.
  • IgG3 or IgG3-IL2 were injected into the peritoneal cavity of mice and the residual radioactivity measured over time (Fig. 7).
  • the fusion protein shows a half- life of 7 hours, reduced compared to IgG3 but greatly
  • the in vivo fate of an injected protein determines in part its utility as a therapeutic agent.
  • the 125 I content of various organs were measured four hours after injection (Fig. 8). The relative concentration within each organ was
  • IgG3 and IgG3-IL2 permeated organs within and beyond the peritoneal cavity, the site of injection.
  • IgG3 was present at the highest concentration in blood.
  • IgG3-IL2 was present at the highest concentration in the thymus and, to a lesser degree, in other organs containing IL- 2R bearing cells such as the spleen and lymph nodes. Although present at five times the average concentration of the rest of the animal, the small size of the thymus of adult mice
  • mice tolerated large doses of IgG3-IL2 without serious toxicity. Intraperitoneal injection of 100 ⁇ g did not result in any noticeable morbidity, but did cause some weight gain. Approximately 48 hours after injection, mice had gained an average (n 3) of 1 gram, or 6% of their total body weight. This increase was transient, and weights returned to normal after 96 hours.
  • mice When following the fate of iodinated proteins, there is a concern that the residual label remains associated with the injected protein. To address this concern, four hours after intraperitoneal injection of 125 I-IgG3 or IgG3-IL2, mice were sacrificed, the serum collected and the labeled protein recovered as described above. Greater than 50% of the
  • Sepharose beads coated with DNS-BSA were mixed with PBS, anti-dansyl lgG3 or IgG3-IL2 for 1 hour at 4°C. The beads were then injected into the abdomen of mice, and blood was collected periodically to determine the antibody response to DNS-BSA. A second, identical booster injection was delivered on day 70 to all mice.
  • DNS-BSA coated plates were used with isotype specific detecting antibodies in ELISAs to quantitate the response (Fig. 10).
  • IgM increases over time and responds to the boost with a further increase.
  • IgA shows a similar pattern, although levels appear to stabilize at 60 days and respond more dramatically to the boost.
  • IgGl increases rapidly to a
  • IgG2 maximal , level. 25 days after the initial inoculation and begins to decline after 40 days, but responds to the boost with an increase.
  • IgG3 shows a very large increase compared to the almost undetectable response of the controls at 20 days, a decline after 40 days and no response to the boost.
  • IgG3 increases to 50 days and declines thereafter with only a small response to the boost.
  • the two control groups showed similar antibody response to antigen with only small differences noted in most isotypes.
  • the IgG3 coated beads did, however, stimulate higher levels of IgG1 overall and generate an increased IgM response to boost.

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Abstract

Par fusion d'une cytokine (par exemple IL-2) à un anticorps (par exemple IgG3), on obtient une molécule présentant les caractéristiques fonctionnelles des deux protéines. Les caractéristiques pharmacocinétiques d'une telle protéine de fusion peuvent être considérablement améliorées par rapport à celles de la cytokine seule (telle IL-2) et les fusions anticorps-IL-2 précédemment décrites. La molécule est intacte; elle peut être extraite du sang d'une souris quelques heures après une injection intrapéritonéale. La présente protéine de fusion atteint également des organes éloignés les uns des autres dans tout l'organisme de l'animal. La demi-vie de 7 heures in vivo d'une molécule IL-2 IgG3 typique est bien supérieure à celle d'une molécule IL-2, ce qui peut la rendre plus utile qu'une molécule IL-2 pour de multiples applications in vivo. Il est apparu que d'autres protéines de fusion IL-2 utilisées comme vaccin déclenchent une réponse immunitaire plus forte par rapport à la protéine fusionnée et ont été étudiées pour la prévention et pour le traitement des tumeurs et des virus exprimant ces antigènes. La protéine de fusion typique IgG3-IL2 se lie à un haptène pouvant être conjugé à la plupart des antigènes visés. Des antigènes peuvent donc être liés à l'IL-2 bioactive sans les complexités et les incertitudes des fusions d'IL-2 avec chaque antigène individuellement. Cette démarche a été testée en utilisant la sérum-albumine bovine (BSA) comme antigène modèle. La réponse des anticorps à cette BSA liée à IgG3-IL2 est plus forte que celle de la BSA, seule, ou liée à IgG3. Ce système devrait être utile pour potentialiser la réponse immunitaire à l'antigène et sélectionner les antigènes destinés à des vaccins.
PCT/US1997/001420 1996-02-13 1997-02-11 Nouvelle proteine de fusion anticorps-cytokine et methodes d'elaboration et d'utilisation de cette proteine WO1997030089A1 (fr)

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EP1442750A1 (fr) * 1997-11-20 2004-08-04 Vical Incorporated Traitement de cancer à l'aide de polynucléotides exprimant des cytokines et préparations correspondantes
US6838260B2 (en) 1997-12-08 2005-01-04 Emd Lexigen Research Center Corp. Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
WO2005021592A2 (fr) * 2003-08-28 2005-03-10 Merck Patent Gmbh Augmentation de la demi-vie circulante de proteines de type interleukine 2
US6969517B2 (en) 2001-05-03 2005-11-29 Emd Lexigen Research Center Corp. Recombinant tumor specific antibody and use thereof
US6992174B2 (en) 2001-03-30 2006-01-31 Emd Lexigen Research Center Corp. Reducing the immunogenicity of fusion proteins
US7067110B1 (en) 1999-07-21 2006-06-27 Emd Lexigen Research Center Corp. Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens
US7091321B2 (en) 2000-02-11 2006-08-15 Emd Lexigen Research Center Corp. Enhancing the circulating half-life of antibody-based fusion proteins
US7141651B2 (en) 1999-08-09 2006-11-28 Emd Lexigen Research Center Corp. Multiple cytokine protein complexes
US7148321B2 (en) 2001-03-07 2006-12-12 Emd Lexigen Research Center Corp. Expression technology for proteins containing a hybrid isotype antibody moiety
US7169904B2 (en) 2002-12-17 2007-01-30 Emd Lexigen Research Center Corp. Immunocytokine sequences and uses thereof
US7186804B2 (en) 2001-12-04 2007-03-06 Emd Lexigen Research Center Corp. IL-2 fusion proteins with modulated selectivity
US7211253B1 (en) 1999-11-12 2007-05-01 Merck Patentgesellschaft Mit Beschrankter Haftung Erythropoietin forms with improved properties
US7268120B1 (en) 1997-11-20 2007-09-11 Vical Incorporated Methods for treating cancer using cytokine-expressing polynucleotides
US7323549B2 (en) 2003-12-30 2008-01-29 Emd Lexigen Research Center Corp. IL-7 fusion proteins
US7432357B2 (en) 2004-01-22 2008-10-07 Merck Patent Gmbh Anti-cancer antibodies with reduced complement fixation
US7465447B2 (en) 2003-12-31 2008-12-16 Merck Patent Gmbh Fc-erythropoietin fusion protein with improved pharmacokinetics
US7517526B2 (en) 2000-06-29 2009-04-14 Merck Patent Gmbh Enhancement of antibody-cytokine fusion protein mediated immune responses by combined treatment with immunocytokine uptake enhancing agents
US7589179B2 (en) 2004-12-09 2009-09-15 Merck Patent Gmbh IL-7 variants with reduced immunogenicity
US7736652B2 (en) * 2002-03-21 2010-06-15 The Regents Of The University Of California Antibody fusion proteins: effective adjuvants of protein vaccination
WO2011112935A2 (fr) * 2010-03-12 2011-09-15 The Regents Of The University Of California Protéines de fusion de type anticorps ayant une activité de liaison à l'héparine perturbée
US8691952B2 (en) 2005-12-30 2014-04-08 Merck Patent Gmbh Anti-CD19 antibodies with reduced immunogenicity
US8907066B2 (en) 2009-04-22 2014-12-09 Merck Patent Gmbh Antibody fusion proteins with a modified FcRn binding site
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US7470675B2 (en) 1997-11-20 2008-12-30 Vical Incorporated Methods for treating cancer using interferon-ω-expressing polynucleotides
EP1442750A1 (fr) * 1997-11-20 2004-08-04 Vical Incorporated Traitement de cancer à l'aide de polynucléotides exprimant des cytokines et préparations correspondantes
US6838260B2 (en) 1997-12-08 2005-01-04 Emd Lexigen Research Center Corp. Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
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US7067110B1 (en) 1999-07-21 2006-06-27 Emd Lexigen Research Center Corp. Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens
US7141651B2 (en) 1999-08-09 2006-11-28 Emd Lexigen Research Center Corp. Multiple cytokine protein complexes
US7582288B2 (en) 1999-08-09 2009-09-01 Merck Patent Gmbh Methods of targeting multiple cytokines
US7211253B1 (en) 1999-11-12 2007-05-01 Merck Patentgesellschaft Mit Beschrankter Haftung Erythropoietin forms with improved properties
US7091321B2 (en) 2000-02-11 2006-08-15 Emd Lexigen Research Center Corp. Enhancing the circulating half-life of antibody-based fusion proteins
US7507406B2 (en) 2000-02-11 2009-03-24 Emd Serono Research Center, Inc. Enhancing the circulating half-life of antibody-based fusion proteins
US7517526B2 (en) 2000-06-29 2009-04-14 Merck Patent Gmbh Enhancement of antibody-cytokine fusion protein mediated immune responses by combined treatment with immunocytokine uptake enhancing agents
US7148321B2 (en) 2001-03-07 2006-12-12 Emd Lexigen Research Center Corp. Expression technology for proteins containing a hybrid isotype antibody moiety
US6992174B2 (en) 2001-03-30 2006-01-31 Emd Lexigen Research Center Corp. Reducing the immunogenicity of fusion proteins
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US6969517B2 (en) 2001-05-03 2005-11-29 Emd Lexigen Research Center Corp. Recombinant tumor specific antibody and use thereof
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US11208496B2 (en) 2005-12-30 2021-12-28 Cancer Research Technology Ltd. Anti-CD19 antibodies with reduced immunogenicity
US8907066B2 (en) 2009-04-22 2014-12-09 Merck Patent Gmbh Antibody fusion proteins with a modified FcRn binding site
US8617557B2 (en) 2010-03-12 2013-12-31 The Regents Of The University Of California Antibody fusion with IL-12 proteins with disrupted heparin-binding activity
WO2011112935A3 (fr) * 2010-03-12 2012-03-29 The Regents Of The University Of California Protéines de fusion de type anticorps ayant une activité de liaison à l'héparine perturbée
WO2011112935A2 (fr) * 2010-03-12 2011-09-15 The Regents Of The University Of California Protéines de fusion de type anticorps ayant une activité de liaison à l'héparine perturbée

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