[go: up one dir, main page]

WO2018049188A1 - Agoniste puissant à large spectre permettant d'améliorer l'efficacité d'un vaccin chez les animaux d'élevage - Google Patents

Agoniste puissant à large spectre permettant d'améliorer l'efficacité d'un vaccin chez les animaux d'élevage Download PDF

Info

Publication number
WO2018049188A1
WO2018049188A1 PCT/US2017/050722 US2017050722W WO2018049188A1 WO 2018049188 A1 WO2018049188 A1 WO 2018049188A1 US 2017050722 W US2017050722 W US 2017050722W WO 2018049188 A1 WO2018049188 A1 WO 2018049188A1
Authority
WO
WIPO (PCT)
Prior art keywords
fragment
swine
antibody
antigen
mab
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2017/050722
Other languages
English (en)
Inventor
Waithaka MWANGI
Surya WAGHELA
Cameron Martin
Jocelyn BRAY
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Texas A&M University System
Texas A&M University
Original Assignee
Texas A&M University System
Texas A&M University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Texas A&M University System, Texas A&M University filed Critical Texas A&M University System
Publication of WO2018049188A1 publication Critical patent/WO2018049188A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen

Definitions

  • the present invention relates generally to vaccine adjuvants. More specifically, the invention relates to methods and compositions for improving vaccine efficacy in livestock and companion species, including, but not limited to, cattle, swine, sheep, goats, horses and dogs.
  • APCs antigen presenting cells, B cells, and macrophages
  • Activation of these APCs is an innate response that adjuvants as well as live vaccines stimulate through Pathogen- Associated Molecular Patterns (PAMPs), chemokines and cytokines.
  • PAMPs Pathogen- Associated Molecular Patterns
  • activated helper T cells provide APC activation by expressing CD40 ligand (CD40L) and thereby stimulate production of inflammatory cytokines by the APCs.
  • CD40L CD40 ligand
  • Signaling via the CD40 receptor is also critical for antibody isotype switching and for effective priming of helper CD4 T cells as well as cytotoxic T lymphocytes (CTLs).
  • CD40 Cluster of differentiation 40
  • B cells B cells, macrophages, dendritic cells (DCs), endothelial cells and fibroblasts
  • DCs dendritic cells
  • CD40 is also expressed on several types of human cancer cells including bladder, breast, and ovarian (Paulie, et al., Cancer Immunol. Immunother. 20:23-28, 1985; Noelle, et al, Immunol. Today 13:431-433, 1992; Foy, et al, J. Exp. Med.
  • CD40L (CD154), a natural ligand for CD40, is a cytokine expressed either on the surface or secreted by activated CD4 + T cells (Clark and Ledbetter, Proc. Natl. Acad. Sci. USA 83:4494-4498, 1986; Hill, et al., J. Immunol. 174:41-50, 2005).
  • the CD40L interacts with CD40 by crosslinking multiple CD40 molecules and thereby provides a critical signal for APC activation (Noelle, et al., 1992, supra; Foy, et al., 1993, supra).
  • CD40-CD40L interaction stimulates B cells to undergo somatic hypermutation, class switch recombination, clonal expansion, and upregulation of major histocompatibility complex II (MHCII) and secretion of proinflammatory cytokines.
  • MHCII major histocompatibility complex II
  • proinflammatory cytokines for example, humans suffering from X-linked hyper-IgM syndrome are deficient in either CD40 or CD40L, and thus do not undergo class switch recombination or somatic hypermutation.
  • the X-linked hyper IgM syndrome leads to high proportions of IgMs and low levels of IgA, IgE, and IgG present in the serum, absence of germinal centers, and the inability to mount a T-cell- dependent humoral response (Aruffo, et al, Cell 72:291-300, 1993).
  • CD40L interacts with CD40 on macrophages, it stimulates the cells to synthesize and release nitric oxide, and upregulate MHCII and proinflammatory cytokines (Grewal and Flavell, Immunol. Today 17:410- 414, 1996; Noelle, Immunity 4:415-419, 1996).
  • Naive T cells require two distinct signals from APCs for proper activation and induction of differentiation: signal 1 is provided by peptide antigens in the context of MHC molecules, while signal 2 is delivered by costimulatory molecules such as CD80 or CD86 present on DCs (Haase, et al., Scand. J. Immunol. 59:237-245, 2004).
  • costimulatory molecules such as CD80 or CD86 present on DCs
  • antigen-loaded DCs to provide these signals effectively, they require activation to up-regulate surface expression of MHC -peptide complexes and costimulatory molecules, and to secrete pro-inflammatory molecules such as IL-12 (Fujii, et al., J. Exp. Med. 199, 1607-1618, 2004).
  • DC activation is an innate response that adjuvants as well as live vaccines stimulate through pattern recognition receptor (PRR)-ligand signaling, chemokine and cytokine secretion (Gallucci, et al., Nat. Med. 5: 1249-1255, 1999).
  • PRR pattern recognition receptor
  • chemokine and cytokine secretion Gallucci, et al., Nat. Med. 5: 1249-1255, 1999.
  • Expression of CD80/CD86 is upregulated by PRR ligands, TNF-a and IFN- ⁇ , as well as interaction between CD40 on APCs and CD40L (Gallucci, et al., 1999, supra; Fujii, et al., 2004, supra; Haase, et ah, 2004, supra).
  • Agonistic mAbs against CD40 directly mimic CD4 + T-cell help in vivo in response to T-cell dependent antigens (Banchereau, et al., Annu. Rev. Immunol. 12:881-922, 1994; Banchereau, et al., Adv. Exp. Med. Biol. 378:79-83, 1995; Barr, et al., Immunology 109:87-92, 2003; Bishop, In: Seminars in Immunology, Vol. 21, p. 255, 2009).
  • CD40-targeted antigen delivery up to 1000-fold increased antibody responses was reported (Barr and Heath, Immunology 102:39-43, 2001; Barr et al., 2003, supra).
  • CD40 agonists like membrane-associated CD40L, soluble CD40L (sCD40L), or anti-CD40 antibodies evokes distinct functional responses
  • sCD40L soluble CD40L
  • Antibody class switching is also attributed to such agonistic anti-CD40 antibodies following interaction with CD40 on B cells.
  • the present invention overcomes one or more deficiencies in the art by providing an anti-swine CD40 mAb that has agonistic activity in a variety of livestock species.
  • the present invention provides an anti-swine CD40 monoclonal antibody, or a fragment thereof.
  • the anti-swine CD40 monoclonal antibody or fragment thereof increases immunogenicity of the antigen in a plurality of species.
  • the plurality of species are selected from the group consisting of pigs, cattle, sheep and goats.
  • the anti- swine CD40 antibody or fragment thereof is defined as a recombinant diabody, an scFv, an Fab fragment, an F(ab')2 fragment, a disulfide linked Fv or a whole immunoglobulin molecule.
  • the anti-swine CD40 antibody or fragment thereof is from clone 2E4E4.
  • the anti-swine CD40 antibody or fragment thereof is comprised in a composition comprising an antigen.
  • the antigen is a whole killed virus, inactivated virus or an attenuated virus.
  • the antigen is conjugated to the anti-swine CD40 monoclonal antibody, or a fragment thereof.
  • the anti-swine CD40 antibody or fragment thereof comprises a pharmaceutically acceptable carrier.
  • the anti- swine CD40 antibody or fragment thereof comprises an additional adjuvant that is distinct from said anti-swine CD40 monoclonal antibody, or a fragment thereof.
  • the anti- swine CD40 antibody or fragment thereof has a first light-chain variable domain having an amino acid sequence comprising SEQ ID NO:2, a second light-chain variable domain having an amino acid sequence comprising SEQ ID NO:3, a third light-chain variable domain having an amino acid sequence comprising SEQ ID NO:4, a first heavy-chain variable domain having an amino acid sequence comprising SEQ ID NO:5, a second heavy-chain variable domain having an amino acid sequence comprising SEQ ID NO:6, and a heavy-chain variable domain having an amino acid sequence comprising SEQ ID NO:7, or any combination of combinations thereof.
  • the present invention additionally provides a pharmaceutical composition, comprising an antigen, a pharmaceutically acceptable carrier, and an anti-swine CD40 monoclonal antibody, or a fragment thereof.
  • the composition increases immunogenicity of the antigen in a plurality of species.
  • the composition is administered intravenously, intra-arterially, intra-peritoneally, intramuscularly, intradermally, orally, dermally, nasally, buccally, rectally, vaginally, by inhalation, or by topical administration.
  • the present invention also provides a composition comprising an anti-swine CD40 monoclonal antibody, or a fragment thereof.
  • the anti-swine CD40 monoclonal antibody or fragment thereof increases immunogenicity of an antigen in a plurality of species.
  • the anti-swine CD40 monoclonal antibody is from clone 2E4E4.
  • the present invention further provides a method of increasing the immunogenicity of an antigen, comprising combining the antigen with an anti- swine CD40 monoclonal antibody, or a fragment thereof.
  • the anti- swine CD40 monoclonal antibody or fragment thereof increases immunogenicity of the antigen in a plurality of species.
  • the antigen is conjugated to the anti-swine CD40 monoclonal antibody, or a fragment thereof.
  • FIG. 1 Alignment of swine, bovine, caprine, and ovine CD40 amino acid sequences. The signal sequence is shown where the consensus sequence is highlighted in green color (amino acid 1-19), whereas the consensus sequence of the transmembrane domain is highlighted in red color (amino acid 192-215). The percentage identity of the extracellular domains of bovine, ovine, and caprine CD40 protein sequences to that of swine is 74%, 75%, and 75% respectively.
  • FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E and FIG. 2F Reactivity of the mAb 2E4E4 against HEK 293A cells expressing swine or bovine CD40. Evaluation of the mAb 2E4E4 specificity against swine and bovine CD40 was performed by immunocytometric and flow cytometric analysis.
  • FIG. 2 A Analysis of purified mAb 2E4E4 and a defined IgGl isotype control mAb by PAGE.
  • FIG. 2B HEK 293A cells transfected with a construct expressing full length swine CD40 and probed with mAb 2E4E4.
  • FIG. 2C Reactivity of the mAb 2E4E4 against HEK 293A cells expressing swine or bovine CD40. Evaluation of the mAb 2E4E4 specificity against swine and bovine CD40 was performed by immunocytometric and flow cytometric analysis.
  • FIG. 2D Sham treated HEK 293 A cells probed with the mAb 2E4E4.
  • FIG. 2E Flow cytometric analysis of HEK 293A cells transfected with a construct encoding full length swine CD40 probed with either the mAb2E4E4 (red) or IgGl isotype control (blue).
  • FIG. 2F Flow cytometric analysis of HEK 293A cells transfected with a construct a encoding full length bovine CD40 probed with either the mAb2E4E4 (red) or IgGl isotype control (blue).
  • FIG. 3A and FIG. 3B The mAb 2E4E4 recognized CD40 on stimulated swine and bovine PBMCs.
  • FIG. 3A Flow cytometry performed on non-stimulated (red) or LPS-stimulated (orange) swine PBMCs and then probed with the mAb 2E4E4. IgGl isotype control (blue) was also used to probe LPS-stimulated swine PBMCs.
  • FIG. 3B Flow cytometry performed on non-stimulated (red) or LPS-stimulated (orange) bovine PBMCs and probed with the mAb 2E4E4. IgGl isotype control (blue) was also used to probe LPS-stimulated bovine PBMCs.
  • FIG. 5A, FIG. 5B, FIG. 5C and FIG. 5D Proliferation of swine and bovine PBMC by mAb 2E4E4 stimulation.
  • FIG. 5A Swine PBMC response after incubation with 2E4E4 FIG. 5B.
  • FIG. 6 Nitric oxide production by macrophages following stimulation by mAb 2E4E4. Agonistic effect of 2E4E4 was verified by nitric oxide assay using bovine macrophages incubated with graded amount of the mAb 2E4E4 (black) or IgGl isotype control (grey). Each column represents mean ⁇ of N0 2 ⁇ of triplicate wells stimulated with the mAb 2E4E4 at each concentration + SD; * P ⁇ 0.0001.
  • FIG. 7A, FIG. 7B, FIG. 7C and FIG. 7D Pro-inflammatory cytokine response upregulation by mAb 2E4E4.
  • Intracellular cytokine staining was used to evaluate the ability of mAb 2E4E4 to upregulate pro-inflammatory cytokines in PBMCs.
  • Swine PBMCs were incubated with the mAb 2E4E4, LPS, or media alone, harvested at 12 hours (grey) and 24 hours (black), and then probed with mAbs against IL-la (FIG. 7A), TNF-a (FIG. 7B), IL-6 (FIG. 7C) or IL-8 (FIG. 7D).
  • Each column represents the mean florescent intensity of two wells + SD.
  • FIG. 8 Amino acid sequence of 2E4E4 light and heavy chain variable domains (SEQ ID NO: l).
  • FIG. 9 Amino acid (SEQ ID NO: l) sequence of 2E4E4 diabody. Shown are light chain V K CDR1 (SEQ ID NO:2), light chain V K CDR2 (SEQ ID NO:3), light chain V K CDR3 (SEQ ID NO:4), heavy chain V H CDR1 (SEQ ID NO:5), heavy chain V H CDR2 (SEQ ID NO:6), and heavy chain V H CDR3 (SEQ ID NO:7).
  • the present invention provides agonistic anti-CD40 monoclonal antibodies (mAbs) that are potent at activating bovine and porcine antigen presenting cells.
  • mAbs agonistic anti-CD40 monoclonal antibodies
  • One of the agonistic anti-CD40 mAb is termed 2E4E4, and the amino acid sequence of the light and heavy chain variable domains is shown in FIG. 8.
  • a recombinant version of the agonist (diabody) has also been generated (nucleotide and amino acid sequence of the 2E4E4 diabody is shown in FIG. 9).
  • This mAb is used to generate an adjuvant for enhancing or increasing the immune response to a wide variety of antigens in these two food animal species, as well as other livestock species, including, but not limited to, sheep and goats.
  • the mAb or its recombinant versions can be used as a standalone stimulant in a vaccine formulation or conjugated to an antigen for targeting to APCs as well as adjuvant activity.
  • the anti-CD40 monoclonal antibody, or a fragment thereof is present in an immunologically effective amount.
  • the agonist can be used to enhance efficacy of whole killed virus vaccines, live attenuated vaccines, inactivated virus vaccines, purified subunit antigen or engineered chimeric polypeptide containing defined protective B cell, CD4 T cell, and cytotoxic T lymphocyte (CTL) epitopes from bovine, porcine, ovine and/or caprine pathogens.
  • CTL cytotoxic T lymphocyte
  • the mAb is modified to replace the constant regions of the mouse IgG heavy and light chains with cognate bovine or swine IgG components.
  • the term "vaccine composition” includes at least one immunogenic antigen or immunogen, along with one or more of the presently described agonistic anti-CD40 monoclonal antibodies, or fragments thereof, in a pharmaceutically acceptable carrier useful for inducing an immune response in a host.
  • Vaccine compositions can be administered in dosages and by techniques well known to those skilled in the medical or veterinary arts, taking into consideration such factors as the age, sex, weight, species and condition of the recipient animal, and the route of administration.
  • the term "host cell” refers to any mammalian cell, whether located in vitro or in vivo. For example, host cells may be located in a transgenic animal.
  • a peptide, polypeptide, protein or polynucleotide coding for such a molecule is "immunogenic” (and thus an "antigen” or “immunogen” within the present invention), if it is capable of inducing an immune response.
  • immunogenicity is more specifically defined as the ability to induce a T-cell response.
  • an "antigen” or an “immunogen” would be a molecule that is capable of inducing an immune response, and in the case of the present invention, a molecule capable of inducing a T-cell response.
  • peptide is used herein to generally designate a series of amino acid residues, connected one to the other typically by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acids.
  • the peptides are generally about 8 or about 9 amino acids in length, but can be as long as about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29 or about 30 amino acids in length.
  • polypeptide is used herein to generally designate a series of amino acid residues, connected one to the other typically by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acids.
  • polypeptide length is not critical to the invention as long as the correct epitopes are maintained.
  • polypeptide is meant to refer to molecules containing more than about 30 amino acid residues, for example about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 200, about 250, about 300, about 400, about 500 amino acids or greater.
  • protein is used herein to generally designate a full length amino acid sequence.
  • the antigen in the vaccine may be an antigenic determinant.
  • An "antigenic determinant” or “epitope” as used herein refers to a portion of an antigen that contacts a particular antibody.
  • numerous regions of the protein may induce the production of antibodies that bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to as antigenic determinants.
  • isolated means that the material is removed from its original environment (e.g. , the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • the invention is directed to a vaccine that is composed of an isolated viral antigen.
  • a viral "antigen” or “immunogen” as used herein refers to a non-infectious virus or immunogenic portion, fragment or derivative thereof.
  • the antigen may be a nucleic acid antigen and/or a peptide antigen and optionally may include lipids, such as those found in viral lipid envelopes.
  • an antigen or immunogen may comprise a viral like particle (VLP), whole organism, killed, attenuated, inactivated or live; a subunit or portion of an organism; a recombinant vector containing an insert with immunogenic properties; a piece or fragment of DNA capable of inducing an immune response upon presentation to a host animal; a protein, a glycoprotein, a lipoprotein, a polypeptide, a peptide, an epitope, a hapten, or any combination thereof.
  • VLP viral like particle
  • the antigen, and thus the vaccine is composed of an inactivated or attenuated virus.
  • the virus may be inactivated by, for instance, heat, formalin, or a detergent.
  • the virus is attenuated by virtue of one or more mutation in the viral genome.
  • a viral antigen may also be a nucleic acid encoding a viral peptide.
  • the nucleic acid may be delivered in a vector and/or operably linked to a heterologous promoter and transcription terminator.
  • a "vector" may be any of a number of nucleic acid molecules into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell.
  • Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to, plasmids, phagemids, and virus genomes.
  • a cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell.
  • An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably linked to regulatory sequences and may be expressed as an RNA transcript.
  • a coding sequence and regulatory sequences are said to be "operably linked” when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame- shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
  • a promoter region is operably linked to a coding sequence if the promoter region is capable of effecting transcription of that DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide.
  • reference to a DNA sequence includes both single stranded and double stranded DNA.
  • the specific sequence unless the context indicates otherwise, refers to the single strand DNA of such sequence, the duplex of such sequence with its complement (double stranded DNA) and the complement of such sequence.
  • the term "coding region" refers to that portion of a gene which either naturally or normally codes for the expression product of that gene in its natural genomic environment, i.e., the region coding in vivo for the native expression product of the gene.
  • the coding region can be from a normal, mutated or altered gene, or can even be from a DNA sequence, or gene, wholly synthesized in the laboratory using methods well known to those of skill in the art of DNA synthesis.
  • Pharmaceutically acceptable carriers are well known and are usually liquids, in which an active therapeutic agent is formulated.
  • the carrier generally does not provide any pharmacological activity to the formulation, though it may provide chemical and/or biological stability, release characteristics, and the like.
  • Exemplary formulations can be found, for example, in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2000, and include, but are not limited to, saline, water, buffered water, 0.3% glycine, hyaluronic acid, dextrose and the like.
  • compositions can be used for parenteral administration, such as subcutaneous, intradermal, intramuscular, or intraperitoneal, or intravenous, intra-arterial, oral, dermal, nasal, buccal, rectal, vaginal administration, by inhalation, or by topical administration.
  • parenteral administration such as subcutaneous, intradermal, intramuscular, or intraperitoneal, or intravenous, intra-arterial, oral, dermal, nasal, buccal, rectal, vaginal administration, by inhalation, or by topical administration.
  • the peptides and optionally other molecules are dissolved or suspended in a pharmaceutically acceptable, preferably aqueous carrier.
  • the peptides can also be administered together with immune stimulating substances, such as cytokines.
  • the composition can contain excipients, such as buffers, binding agents, blasting agents, diluents, flavors, lubricants, etc.
  • excipients such as buffers, binding agents, blasting agents, diluents, flavors, lubricants, etc.
  • An extensive listing of excipients that can be used in such a composition can be, for example, taken from Handbook of Pharmaceutical Excipients, 5th ed., edited by Raymond Rowe, Paul Sheskey and Sian Owen, Pharmaceutical Press, 2006.
  • Suitable excipients include, but are not limited to, antioxidants like ascorbic acid or glutathione, preserving agents such as phenol, m-cresol, methyl- or propylparaben, chlorobutanol, thiomersal (thimerosal) or benzalkoniumchloride, stabilizer, framework former such as saccharose, lactose, maltose, trehalose, mannitose, mannitol and/or sorbitol, mannitol and/or lactose and solubilizer such as polyethyleneglycols (PEG), i.e., PEG 3000, 3350, 4000 or 6000, or cyclodextrins, i.e., hydroxypropyl-P-cyclodextrin, sulfobutylethyl-P-cyclodextrin or ⁇ -cyclodextrin, or dextrans or poloxamers, i.e.,
  • a pharmaceutically acceptable salt refers to a derivative of antigenic or immunogenic agent modified by making acid or base salts of the agent.
  • acid salts are prepared from the free base (typically wherein the neutral form of the drug has a neutral — NH 2 group) involving reaction with a suitable acid.
  • Suitable acids for preparing acid salts include both organic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, as well as inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid phosphoric acid and the like.
  • preparation of basic salts of acid moieties which may be present on a peptide are prepared using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or the like.
  • the pharmaceutical compositions of the present invention may also include sugars, sugar alcohols, amino acids such as glycine, arginine, glutamic acid and others as framework former.
  • the sugars may be mono-, di- or trisaccharide. These sugars may be used alone, as well as in combination with sugar alcohols. Examples of sugars include glucose, mannose, galactose, fructose or sorbose as monosaccharides, saccharose, lactose, maltose or trehalose as disaccharides and raffinose as a trisaccharide.
  • a sugar alcohol may be, for example, mannitose.
  • Preferred ingredients are saccharose, lactose, maltose, trehalose, mannitol and/or sorbitol.
  • Known vaccine adjuvants for use in addition to the presently described agonistic anti-CD40 monoclonal antibodies include, but are not limited to, oil and water emulsions, oil-in-water emulsions, water-in-oil emulsions, water-in-oil-in-water emulsions, saponin, aluminum hydroxide, dextran sulfate, carbomer, sodium alginate, (N,N-dioctadecyl-N',Nt-bis(2- hydroxyethyl)-propanediamine), paraffin oil, muramyl dipeptide, cationic lipids, DMRIE, DOPE, and TLR ligands such as CpG oligonucleotides.
  • Additional adjuvants for use with the presently described agonistic anti-CD40 monoclonal antibodies include, but are not limited to, 1018 ISS, aluminium salts, Amplivax, AS 15, BCG, CP-870,893, CpG7909, CyaA, Mologen's dSLIM, GM CSF, IC30, IC31, Imiquimod, ImuFact IMP321, interferon-a or - ⁇ , IS Patch, ISS, ISCOMs, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, and other non-toxic LPS derivatives, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM- 174, OM- 197-MP-EC, ONTAK, PepTel® vector system, PLG microparticles, resiquimod, SRL172, Virosomes and other Virus-like particles, YF-17D
  • Kits of the present invention preferably comprise a formulation of one or more of the presently described agonistic anti-CD40 monoclonal antibodies in a suitable container and instructions for its use.
  • suitable containers include, for example, bottles, vials (e.g. , dual chamber vials), syringes (such as dual chamber syringes) and test tubes.
  • the container may be formed from a variety of materials such as glass or plastic.
  • Example 1 Generation and characterization of a cross-reactive anti-CD40 agonistic monoclonal antibody
  • CD40 Cluster of Differentiation 40
  • APCs Antigen Presenting Cells
  • CD40L The CD40 ligand (CD40L) is expressed on activated CD4+ T cells, and upon interaction with CD40 leads to enhanced antigen presentation, nitric oxide (NO) expression, proinflammatory cytokine expression by APCs, and stimulation of B cells to undergo somatic hypermutation, immunoglobulin class switching, and proliferation.
  • Anti-CD40 monoclonal antibodies (mAbs) capable of crosslinking CD40 molecules act as agonists and mimic native CD40-CD40L interactions.
  • An anti-swine CD40 mAb was developed, designated 2E4E4, and its agonistic activity on cells from livestock species was characterized.
  • Immunocytometric analysis of HEK 293A cells transfected with plasmids expressing swine or bovine CD40 genes demonstrated the specificity of the mAb 2E4E4.
  • Recognition of CD40 by the mAb 2E4E4 was validated by flow cytometry on primary swine and bovine peripheral blood mononuclear cells (PBMCs). Further validation was provided by immunohistochemistry on swine, bovine, caprine, and ovine spleen sections.
  • PBMCs peripheral blood mononuclear cells
  • Swine PBMCs stimulated with the mAb 2E4E4 revealed a significant increase in expression of MHC-II, TNF-a, IL-6, and IL-8 by intracellular flow cytometry.
  • the mAb 2E4E4 stimulated robust swine, bovine, caprine, and ovine PBMC proliferation, and activated bovine macrophages to upregulate significant NO synthesis.
  • HEK human embryonic kidney
  • PBMCs peripheral blood mononuclear cells
  • HEK 293A cells were grown in DMEM supplemented with 10% FBS, GlutamaxTM, HEPES (0.01 M), non-essential amino acids (0.1 M), and 2-mercaptoethanol (0.1 M).
  • Swine, bovine, caprine, and ovine PBMCs were cultured in RPMI 1640 supplemented with 10% FBS, GlutamaxTM, non-essential amino acids (0.1 M), HEPES (0.01 M), 2-mercaptoethanol (0.1 M), and Penicillin- Streptomycin (100 U/mL).
  • FreeStyleTM 293-F cells were grown in FreestyleTM Expression Media (Thermoscientific) in an atmosphere of 8% C0 2 at 37°C.
  • the PCR product encoding the swCD40ED was cloned into PCR-TOPO vector (Invitrogen), and following colony screening and DNA sequencing of positive clones, one construct encoding the authentic swCD40ED was modified by overlap extension PCR to incorporate a secretory signal sequence at the 5' terminus and the FLAG-tag at the 3'-terminus.
  • the resultant gene and the PCR product encoding full length swCD40 were sub-cloned into the eukaryotic expression vector pcDNA2.2-TOPO (Invitrogen) and verified by sequencing.
  • a construct encoding full length bovine CD40 (boCD40) was similarly generated.
  • Recombinant swCD40ED was expressed as a FLAG-tagged protein by transfecting HEK 293 Free-Style cells (Invitrogen) and affinity purified using anti-FLAG M2-agarose affinity chromatography (Sigma) as previously described (Hope, et al., J. Immunol. Methods 301: 114-123, 2005; Mwangi, et al., J. Leukoc. Biol. 78:401-411, 2005).
  • Monoclonal antibody production Monoclonal antibodies (mAbs) against swCD40ED were produced as previously described (Waghela, et al., Vet. Parasitol. 94: 133-139, 2000). Briefly, three female BALB/c mice were inoculated subcutaneously three times every 2 weeks with 50 ⁇ g of recombinant swCD40ED in RIBI adjuvant. Seroconversion was monitored on a weekly basis by ELISA using plates coated with recombinant swCD40ED.
  • the mouse with the best anti-swCD40ED antibody response was stimulated by intraorbital injection of 50 ⁇ g of the recombinant swCD40ED without adjuvant.
  • the spleen was harvested for preparing single cell suspension for electrofusion with Sp2/0 myeloma cells (ATCC, Manassas, VA).
  • Hybridomas were plated in 96-well cell culture plates (Nunc) and grown in hypoxanthine- aminopterin-thymidine (HAT) medium.
  • Primary screening was performed by ELISA on day 14 post-fusion using ELISA plates coated with recombinant swCD40ED (100 ng/well).
  • Proliferation assay was used to test ELISA positive hybridomas for agonistic effect on swine PBMCs. Positive hybridoma clones identified by proliferation assay were subcloned by limiting dilution and retested by ELISA and proliferation assays. The leading candidate, clone 2E4E4, was selected for further analysis.
  • HEK 293 A cell monolayers were transfected with constructs encoding either swCD40 (pcDNAswCD40) or boCD40 (pcDNAboCD40) using Polyethylenimine (Polyscience) as previously described (Hopkins, et ah, In: L.J. Hartley (Ed.) Protein Expression in Mammalian Cells: Methods and Protocols. Humana Press, Totowa, NJ, p. 251-268, 2012).
  • the monolayers were fixed with cold methanol, rinsed with PBS, blocked with 10% FBS/TBS solution, and incubated for 1 hour at room temperature with 5 ⁇ g of the mAb 2E4E4 or 5 ⁇ g of an IgGl isotype control (Biolegends).
  • the cell monolayers were washed 3X with blocking buffer and then incubated for 1 hour with Alkaline Phosphatase AffiniPure F(ab') 2 Fragment Donkey Anti-Mouse IgG (H+L) (Jackson Immuno Research Laboratories, Inc.).
  • Flow cytometry Transfection of HEK 293A cells.
  • the pcDNAswCD40 and pcDNAboCD40 constructs were used to transfect HEK 293A cell monolayers using Polyethylenimine (Polyscience) as previously described (Hopkins, et ah, 2012, supra). Following 48 hour incubation, one million transfected cells were added to each well of a 96 well V-bottom plate and stained with Zombie RedTM Fixable Viability Kit (Biolegend) following the manufacturer's protocol.
  • the pcDNAswCD40 and pcDNAboCD40 transfected HEK 293A cells were incubated with 5 ⁇ g of the mAb 2E4E4 or 5 ⁇ g of IgGl isotype control for 30 minutes and washed 3X with blocking buffer (cDMEM with 0.05% sodium azide/20% bovine serum). The cells were incubated for 30 minutes with AffiniPure F(ab') 2 Fragment Donkey Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc.), washed 3X with block buffer, and stored in FACS fixer (12.5% formaldehyde/PBS). Data was collected using BDfacscalibur (Becton Dickinson) and data analysis was done using FlowJo 10 software (FlowJo).
  • the swine and bovine PBMCs were incubated for 30 minutes on ice with either 5 ⁇ g of the mAb 2E4E4-FITC or 5 ⁇ g of IgGl isotype control (Biolegends) conjugated to FITC, washed 3X with blocking buffer, and fixed using FACS Fixer. FACS data was collected and analyzed as above.
  • Swine PBMCs were seeded in a 12 well plate at 4 million PBMCS per well in 1 mL of cRPMI media alone, media containing LPS (10 ⁇ g/ml), or media containing graded amounts of the mAb 2E4E4 (0.5, 1.0, 2.5, or 5.0 ⁇ g/ml).
  • PBMCs PBMCs were added to each well of a 96 well v-bottom plate (Axygen), blocked (20% swine serum/FACS media) for 30 minutes on ice, and then incubated for 30 minutes with 5 ⁇ g of mouse anti-swine MHCII-FITC (Monoclonal Antibody Center Washington State University, clone MSA3).
  • the PBMCs were washed 3X with blocking buffer, and then fixed and stored in FACS fixer. FACS data was collected and analyzed as above.
  • the cells were labeled for 12 hours with 0.3 ⁇ of H-thymidine and incorporation of the isotope by the cells was determined using liquid scintillation counter (Becton-Dickinson).
  • the stimulation index (SI) was calculated for both 2E4E4 and IgGl by dividing the treatment (2E4E4 or IgGl control) by the media control.
  • Nitric oxide assay The level of Nitrite (N0 2 ⁇ ) released by activated bovine macrophages was measured by a Griess assay using monocyte-derived macrophages generated as previously described (Shoda, et ah, J. Interferon Cytokine Res. 19: 1169-1177, 1999).
  • the macrophages (200,000 cells/well) were added in triplicate wells of a 96 well flat bottom plate containing graded amounts of the mAb 2E4E4 (0.5, 1.0, 2.5, 5.0, or 10 ⁇ g/ml), IgGl isotype control (0.5, 1.0, 2.5, 5.0, or 10 ⁇ g/ml), LPS (10 ⁇ g/ml), or media alone.
  • macrophage supernatants were tested for nitrite concentration using Nitric Oxide Assay kit (ThermoFisher) following manufacturer's protocol. Nitrite released was presented as ⁇ N0 2 " .
  • Intracellular cytokine staining For intracellular cell cytokine staining, swine PBMCS (4 million cells) were added to each well of a 12 well plate containing graded amounts of the mAb 2E4E4 (1.0, or 2.5 ⁇ g/ml), LPS (1 ⁇ g/ml) or media alone. The cells were incubated for 12 or 24 hours and twelve hours before the PBMCs were harvested, Brefeldin A was added to each well at a final concentration of ⁇ g/ml.
  • the PBMCs were plated in 96 well v-bottom plate (5 x 10 5 cells/well), incubated for 15 minutes in PERM/WASHTM, blocked (20% porcine serum in IX PERM/WASHTM buffer), and further incubated with 5 ⁇ g of mouse anti-swine TNF-a clone 103314 (R&D Systems), mouse anti-swine IL-la clone 85733.11 (R&D Systems), mouse anti- swine IL-6 clone 77830 (R&D Systems), or mouse anti-swine IL-8 Clone 105115 (R&D Systems) for 1 hour. After 3 washes with blocking buffer, the cells were fixed and stored in FACS fixer. Flow data was collected, and analyzed as above.
  • a mouse anti-swine CD40 mAb designated 2E4E4 was generated by immunizing mice using the extracellular domain of swine CD40 (FIG. 1).
  • the mAb 2E4E4 was shown to be an IgGl with a kappa light chain (FIG. 2A).
  • Immunocytometric analysis of HEK 293 A cells transfected with a construct expressing full length swine CD40 confirmed that the mAb 2E4E4 recognized CD40, whereas sham treated cells were negative (FIG. 2B and FIG. 2D, respectively). This outcome was confirmed by performing flow cytometry on similarly transfected cells.
  • the mAb 2E4E4 but not IgGl isotype control, strongly recognized surface- expressed swine CD40 (FIG. 2E). Since swine and bovine CD40 protein sequences are highly conserved (FIG. 1), mAb 2E4E4 was tested for binding to bovine CD40. Indeed, immunocytometric and flow cytometric analysis of HEK 293A cells transfected with a construct expressing full length bovine CD40 yielded similar results (FIG. 2C and FIG. 2F). These outcomes showed that the mAb 2E4E4 can bind to both swine and bovine CD40, and hence all further experiments were designed to evaluate the interaction of the mAb 2E4E4 with bovine CD40 as well. mAb 2E4E4 recognized native swine and bovine CD40
  • Flow cytometric analysis showed that the mAb 2E4E4 bound to the CD40 expressed on LPS-stimulated swine (FIG. 3A) and bovine (FIG. 3B) PBMCs.
  • the mAb 2E4E4 showed significant fluorescence on stimulated PBMCs (FIG. 3A and FIG. 3B, orange) as compared to non-stimulated controls (FIG. 3A and FIG. 3B, red).
  • a specific signal was also detected on non-stimulated PBMCs probed with the mAb 2E4E4, but not on cells probed with an IgGl isotype control (FIG. 3A and FIG. 3B, blue).
  • IHC data also showed cross-reactivity of the mAb 2E4E4 to caprine and ovine CD40, but not an IgGl isotype control (FIG. 4C and FIG. 4D, respectively, compared to FIG. 4G and FIG. 4H).
  • FIG. 4C and FIG. 4D respectively, compared to FIG. 4G and FIG. 4H.
  • the mAb 2E4E4 showed significant agonistic effects on swine, bovine, caprine, and ovine PBMCs.
  • the mAb 2E4E4 stimulated significant (P ⁇ 0.001) proliferation of swine and bovine PBMCs compared to the IgGl isotype control (FIG. 5A and FIG. 5B).
  • the mAb 2E4E4 also had significant stimulatory effect on ovine and caprine PBMCs in a dosage dependent manner (FIG. 5C and FIG. 5D).
  • Flow cytometry confirmed that the mAb 2E4E4 upregulated MHCII expression on swine PBMCs.
  • a significant increase in MHCII expression was observed when swine PBMCs were stimulated with the mAb 2E4E4 and the response was dose dependent (Table 1).
  • Swine PBMCs were incubated with the mAb 2E4E4 or LPS for 24 hours and MHCII upregulation was determined by flow cytometric analysis. Data is presented as percentage of MHCII positive cells compared to IgGl isotype control.
  • the MHCII upregulation is expected to result in enhanced antigen presentation by APCs. Further confirmation of the mAb 2E4E4 agonistic effect was validated by intracellular cytokine staining. Upregulation of TNF-a, IL-la, and IL-8 was observed at 12 hours post-stimulation (FIG. 7A, FIG. 7B and FIG. 7C). Unlike TNF-a, IL-la, and IL-8, no significant IL-6 expression was observed until 24 hours post-stimulation (FIG. 7D). These results showed that the mAb 2E4E4 stimulates the upregulation of MHCII and release of proinflammatory cytokines, indicating that the mAb 2E4E4 is capable of being used as vaccine adjuvant.
  • an anti-swine CD40 mAb designated 2E4E4
  • the mAb 2E4E4 was shown to be specific to swine CD40, but it also had robust cross-reactivity against bovine, caprine, and ovine cells and spleen tissues.
  • the mAb 2E4E4 had agonistic effects on swine cells and in addition, it showed broad agonistic effects against bovine, caprine, and ovine cells.
  • numbers expressing quantities used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term "about.”
  • the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value.
  • the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
  • the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise.
  • the term “or” as used herein, including the claims, is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Virology (AREA)
  • Biophysics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Genetics & Genomics (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne des anticorps monoclonaux, et des fragments et des dérivés de ceux-ci, qui augmentent l'immunogénicité d'un antigène chez diverses espèces. L'invention concerne également des compositions et des compositions pharmaceutiques comprenant les anticorps monoclonaux, ainsi que des méthodes d'augmentation de l'immunogénicité d'un antigène chez diverses espèces.
PCT/US2017/050722 2016-09-09 2017-09-08 Agoniste puissant à large spectre permettant d'améliorer l'efficacité d'un vaccin chez les animaux d'élevage Ceased WO2018049188A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201662385631P 2016-09-09 2016-09-09
US62/385,631 2016-09-09

Publications (1)

Publication Number Publication Date
WO2018049188A1 true WO2018049188A1 (fr) 2018-03-15

Family

ID=61562245

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2017/050722 Ceased WO2018049188A1 (fr) 2016-09-09 2017-09-08 Agoniste puissant à large spectre permettant d'améliorer l'efficacité d'un vaccin chez les animaux d'élevage

Country Status (1)

Country Link
WO (1) WO2018049188A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140050732A1 (en) * 2010-09-29 2014-02-20 Universite De Liege Combination for use in the treatment and/or prevention of mastitis

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140050732A1 (en) * 2010-09-29 2014-02-20 Universite De Liege Combination for use in the treatment and/or prevention of mastitis

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LEE, WET ET AL.: "In Vitro Testing of an Anti- CD 40 Monoclonal Antibody, Clone 2C10, in Primates and Pigs", TRANSPL IMMUNOL., vol. 33, no. 3, November 2015 (2015-11-01), pages 185 - 191, XP055465048 *
MARTIN, C ET AL.: "Characterization of a Broadly Reactive Anti- CD 40 Agonistic Monoclonal Antibody for Potential Use as an Adjuvant", PLOS ONE, vol. 12, no. 1, 20 January 2017 (2017-01-20), XP055473581 *

Similar Documents

Publication Publication Date Title
ES2432357T3 (es) Anticuerpos enteramente humanos contra 4-1BB humano (CD137)
TWI571264B (zh) 抗ｃｄ４０抗體及其用途
ES2807232T3 (es) Anticuerpos anti-CD40 y usos de los mismos
JP2022527506A (ja) Ｃｄ４０活性化特性を有する組換えタンパク質
JP2023528017A (ja) 重症急性呼吸器症候群コロナウイルス２（ｓａｒｓ－ｃｏｖ－２）ポリペプチドおよびワクチン目的でのその使用
ZA200602834B (en) Fully human antibodies against human 4-1BB (CD137)
Melgoza-González et al. Recent advances in antigen targeting to antigen-presenting cells in veterinary medicine
JP2023502712A (ja) 抗ｐｄ－１／ｉｌ－１５免疫サイトカインによる新しいｐｄ－１標的免疫療法
WO2014039840A1 (fr) Compositions de vaccin contre le vih et procédés associés
JP2023554587A (ja) Ｓａｒｓ－ｃｏｖ－２ スパイクタンパク質の受容体結合ドメインにコンジュゲートまたは融合している抗体およびワクチン目的でのそれらの使用
WO2018049188A1 (fr) Agoniste puissant à large spectre permettant d'améliorer l'efficacité d'un vaccin chez les animaux d'élevage
JP2024540505A (ja) ユニバーサルサルベコウイルスワクチン
CN110603053A (zh) 重组衣原体活化的b细胞平台及其使用方法
JP2025533079A (ja) ニパウイルス感染に対するｄｃ標的化ワクチン
AU2023390817A1 (en) Optimized tag moiety
CN118742321A (zh) 用于预防性和治疗性治疗由SARS-CoV-2引起的疾病的免疫原性构建体和疫苗
WO2022161598A1 (fr) Anticorps ciblant largement des coronavirus et leurs utilisations
JP2023527916A (ja) アゴニスト抗ｃｄ４０抗体
EP1861123A2 (fr) Effet synergique d'agent bloquant le tgf-beta et d'agent immunogène sur les tumeurs
HK1231897B (zh) 抗-cd40抗体及其用途
HK1085226B (en) Fully human antibodies against human 4-1bb (cd137)
NZ546017A (en) Fully human antibodies against human 4-1BB (cd137)

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17849630

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17849630

Country of ref document: EP

Kind code of ref document: A1