EP4204448A2 - Anti-golph2 antibodies for macrophage and dendritic cell differentiation - Google Patents

Anti-golph2 antibodies for macrophage and dendritic cell differentiation

Info

Publication number
EP4204448A2
EP4204448A2 EP21769136.9A EP21769136A EP4204448A2 EP 4204448 A2 EP4204448 A2 EP 4204448A2 EP 21769136 A EP21769136 A EP 21769136A EP 4204448 A2 EP4204448 A2 EP 4204448A2
Authority
EP
European Patent Office
Prior art keywords
antibody
seq
antigenic peptide
antigen
sequence
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.)
Withdrawn
Application number
EP21769136.9A
Other languages
German (de)
French (fr)
Inventor
Frank Stenner-Liewen
Norbert MARKULY
Heike LIEWEN
Yang Liu
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.)
Cureab GmbH
Universitaet Basel
Original Assignee
Cureab GmbH
Universitaet Basel
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 Cureab GmbH, Universitaet Basel filed Critical Cureab GmbH
Publication of EP4204448A2 publication Critical patent/EP4204448A2/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6056—Antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention is in the field of immunotherapy.
  • the invention relates to targetbinding molecules, preferably antibodies or target-binding fragments thereof, that specifically bind to the extracellular part of the protein GOLPH2, or to an antigenic portion thereof. It was surprisingly found by the inventors that binding of the target-binding molecule to the extracellular part of GOLPH2, when expressed on the cell surface of a monocyte or a monocyte-derived cell, induces the maturation of said monocyte or monocyte-derived cell. Furthermore, the inventors unexpectedly found that antibodies directed against GOLPH2, if conjugated or complexed with an antigen, elicit specific and reproducible presentation of this antigen on the surface of myeloid immune cells, which in turn results in effective and lasting T-cell stimulation.
  • the invention further relates to immunoconjugates comprising the target-binding molecules, complexes or constructs, in particular the anti- GOLPH2 antibodies, or target-binding fragments thereof.
  • the target-binding molecules and immunoconjugates of the invention may be administered alone, as a therapeutic conjugate or in combination with other therapeutic agents, such as chemotherapeutic agents, antigens, vaccines or checkpoint inhibitors.
  • the present invention also relates to nucleotide sequences encoding the targetbinding molecule of the invention, as well as vectors and host cells containing the nucleotide sequences.
  • the target-binding molecules, antibodies and compositions of the invention are useful in initiating and/or enhancing an immune response in a subject.
  • the target-binding molecule may be used in methods for generating antigen-presenting cells and activated T cell. This versatile approach can be exploited in a variety of vaccine therapies, such that an antigen of choice, be it against an infectious disease or a tumor may be utilized for raising an immune response in the vaccinated individual.
  • Golgi protein-73 (GP73), alternatively named Golgi Membrane Protein 1 (G0LM1), or Golgi- associated-Phosphoprotein 2 (GOLPH2) is a single pass transmembrane type II protein. Its genuine function is unknown.
  • the genomic sequence of GOLPH2 predicts 11 exons and two splicing variants.
  • the transcript variant 1 (NM_016548.3) is 3100nt in length and contains exons 2 to 11, while transcript variant 2 (NM_177937.2) is 3092nt in length and contains exons 1, and 3 to 11. Both variants encode the same open reading frame. The biological significance of these variants is currently not clear; see Kim et al., Golgi phosphoprotein 2 in physiology and in diseases.
  • GOLPH2 is an integral membrane protein of the cis- and medial-Golgi apparatus.
  • GOLPH2 can cycle out of the cis Golgi to endosomes and the cell surface; see Puri S et al. Cycling of early Golgi proteins via the cell surface and endosomes upon lumenal pH disruption Traffic 2002, 3:641-653.
  • GOLPH2 endosomal trafficking allows for proprotein convertase furin mediated cleavage, resulting in its release into the extracellular space, and provides a molecular explanation for its presence as a serum biomarker for hepatocellular carcinoma (HCC); see Bachert C et al., Endosomal trafficking and proprotein convertase cleavage of cis Golgi protein GOLPH2 produces marker for hepatocellular carcinoma Traffic 2007, 8: 1415-1423; Marrero JA et al., GOLPH2, a resident Golgi glycoprotein, is a novel serum marker for hepatocellular carcinoma J Hepatol 2005, 43: 1007-1012; Mao Y et al., Golgi protein 73 (GOLPH2) is a valuable serum marker for hepatocellular carcinoma Gut 2010, 59: 1687-1693); Li X et al., Serum golgi phosphoprotein 2 level: a better marker
  • GOLPH2 has been shown to be highly expressed in several malignancies including hepatocellular, cholangiocellular, esophageal, renal, prostate and various other carcinomas but not in adjacent non-tumor tissue. Patients with GOLPH2- positive HCC have a higher tumor grade than patients with GOLPH2 -negative HCCs. In bile duct carcinomas (BDC) GOLPH2 expression correlates with better overall survival whereas in HCC GOLPH2 overexpression has been found to be associated with increased risk of metastasis, a higher probability of recurrence and a worse survival; see Riener et al.
  • BDC bile duct carcinomas
  • Antibodies for targeting GOLPH2 have been described in WO 2014/144355 A2, CN105699653 A and CN105734059 A.
  • the use of antibodies to inhibit GOLPH2 to enhance cell-mediated immunity in cancer patients has been described in WO2012/112798 Al.
  • GOLPH2 has been proposed as a biomarker for diagnosis of lung cancer WO 2011/093675 A2 or as a test in systemic inflammatory conditions e.g. sepsis WO2013/083781 A2.
  • GOLPH2 is expressed on normal epithelial cells and other tissues to varying degrees.
  • GOLPH2 has been described in the context of melanoma (Donizy P, et al., Golgi- related proteins GOLPH2 (GP73/GOLM1) and GOLPH3 (GOPP1/MIDAS) in cutaneous melanoma: Patterns of expression and prognostic significance. Int J Mol Sci. 2016; 17).
  • GOLPH2 positive macrophages have been identified as tumor-associated macrophages (TAMs).
  • TAMs have been recognized and novel drugs to alter their phenotype from a so-called M2 macrophage towards less pro-tumorigenic macrophage are in development (Hu, Guorong et al. “Nanoparticles Targeting Macrophages as Potential Clinical Therapeutic Agents against Cancer and Inflammation.” Frontiers in immunology Vol. 10 1998. 21 Aug. 2019).
  • Monocytes are a type of leukocyte, or white blood cell. Monocytes represent the largest type of leukocytes and can differentiate into macrophages and myeloid lineage dendritic cells. As a part of the vertebrate innate immune system, monocytes also influence the process of adaptive immunity. There are at least three subclasses of monocytes in human blood based on their phenotypic receptors.
  • Monocytes are produced by the bone marrow from precursors called monoblasts, bipotent cells that differentiated from hematopoietic stem cells. Monocytes circulate in the bloodstream for about one to three days and then typically move into tissues throughout the body where they differentiate into macrophages and dendritic cells. They constitute between three and eight percent of the leukocytes in the blood. About half of the body's monocytes are stored as a reserve in the spleen in clusters in the red pulp's Cords of Billroth. Moreover, monocytes are the largest corpuscle in blood.
  • Macrophages are responsible for protecting tissues from foreign substances, but are also suspected to be important in the formation of important organs like the heart and brain. They are cells that possess a large smooth nucleus, a large area of cytoplasm, and many internal vesicles for processing foreign material.
  • Monocytes and their macrophage and dendritic cell progeny serve three main functions in the immune system. These are phagocytosis, antigen presentation, and cytokine production. Phagocytosis is the process of uptake of microbes and particles followed by digestion and destruction of this material. Monocytes can perform phagocytosis using intermediary (opsonising) proteins such as antibodies or complement that coat the pathogen, as well as by binding to the microbe directly via patternrecognition receptors that recognize pathogens. Monocytes are also capable of killing infected host cells via antibody-dependent cell-mediated cytotoxicity. Vacuolization may be present in a cell that has recently phagocytized foreign matter.
  • Microbial fragments that remain after such digestion can serve as antigens.
  • the fragments can be incorporated into MHC molecules and then trafficked to the cell surface of monocytes (and macrophages and dendritic cells). This process is called antigen presentation and it can lead to activation of T lymphocytes, which then mount a specific immune response against the antigen.
  • Macrophages are a type of white blood cell of the immune system, that engulfs and digests cellular debris, foreign substances, microbes, cancer cells, and anything else that does not have the type of proteins specific to healthy body cells on its surface in a process called phagocytosis.
  • phagocytes are found in essentially all tissues, where they patrol for potential pathogens by amoeboid movement. They take various forms (with various names) throughout the body (e.g., histiocytes, Kupffer cells, alveolar macrophages, microglia, and others), but all are part of the mononuclear phagocyte system. Besides phagocytosis, they play a critical role in nonspecific defense (innate immunity) and also help initiate specific defense mechanisms (adaptive immunity) by recruiting other immune cells such as lymphocytes. For example, they are important as antigen presenters to T cells. In humans, dysfunctional macrophages cause severe diseases such as chronic granulomatous disease that result in frequent infections.
  • innate immunity nonspecific defense
  • adaptive immunity adaptive immunity
  • macrophages Beyond increasing inflammation and stimulating the immune system, macrophages also play an important anti-inflammatory role and can decrease immune reactions through the release of cytokines. Macrophages that encourage inflammation are called Ml macrophages, whereas those that decrease inflammation and encourage tissue repair are called M2 macrophages.
  • Ml and M2 There are several activated forms of macrophages. In spite of a spectrum of ways to activate macrophages, there are two main groups designated Ml and M2. Ml "killer" macrophages are activated by LPS and IFN-y, and secrete high levels of IL-12 and low levels of IL-10. Ml macrophages have pro-inflammatory, bactericidal, and phagocytic functions. In contrast, the M2 "repair” designation (also referred to as alternatively activated macrophages) broadly refers to macrophages that function in constructive processes like wound healing and tissue repair, and those that turn off damaging immune system activation by producing anti-inflammatory cytokines like IL- 10.
  • M2 is the phenotype of resident tissue macrophages, and can be further elevated by IL-4. M2 macrophages produce high levels of IL-10, TGF-beta and low levels of IL-12. Tumor-associated macrophages are mainly of the M2 phenotype, and seem to promote tumor growth.
  • DCs Dendritic cells
  • Their main function is to process antigen material and present it on the cell surface to the T cells of the immune system. They act as messengers between the innate and the adaptive immune systems.
  • Dendritic cells are present in those tissues that are in contact with the external environment, such as the skin (where there is a specialized dendritic cell type called the Langerhans cell) and the inner lining of the nose, lungs, stomach and intestines. They can also be found in an immature state in the blood. Once activated, they migrate to the lymph nodes where they interact with T cells and B cells to initiate and shape the adaptive immune response. At certain development stages they grow branched projections, the dendrites that give the cell its name. Immature dendritic cells are also called veiled cells, as they possess large cytoplasmic 'veils' rather than dendrites.
  • Dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells initially transform into immature dendritic cells. These cells are characterized by high endocytic activity and low T-cell activation potential. Immature dendritic cells constantly sample the surrounding environment for pathogens such as viruses and bacteria. This is done through pattern recognition receptors (PRRs) such as the toll-like receptors (TLRs). TLRs recognize specific chemical signatures found on subsets of pathogens. Immature dendritic cells may also phagocytose small quantities of membrane from live own cells, in a process called nibbling.
  • PRRs pattern recognition receptors
  • TLRs toll-like receptors
  • dendritic cells Once they have come into contact with a presentable antigen, they become activated into mature dendritic cells and begin to migrate to the lymph node. Immature dendritic cells phagocytose pathogens and degrade their proteins into small pieces and upon maturation present those fragments at their cell surface using MHC molecules. Simultaneously, they upregulate cell-surface receptors that act as co-receptors in T-cell activation such as CD80 (B7.1), CD86 (B7.2), and CD40 greatly enhancing their ability to activate T- cells. They also upregulate CCR7, a chemotactic receptor that induces the dendritic cell to travel through the blood stream to the spleen or through the lymphatic system to a lymph node.
  • CD80 B7.1
  • CD86 B7.2
  • CD40 greatly enhancing their ability to activate T- cells.
  • CCR7 a chemotactic receptor that induces the dendritic cell to travel through the blood stream to the s
  • CLRs C- type lectin receptors
  • the invention relates to the following items:
  • the target-binding molecule according to item 1 wherein the maturation of monocytes or monocyte-derived cells results in the formation of dendritic cells and/or classically activated macrophages.
  • the target-binding molecule according to item 1 or 2 wherein the target-binding molecule is an antibody or a target-binding fragment thereof.
  • the target-binding molecule according to item 3 wherein the antibody is a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody or a multispecific antibody.
  • the target-binding molecule according to any one of items 3 to 5, wherein the antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NON, CDR2 as defined in SEQ ID NO:5 and CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO.7, CDR2 as defined in SEQ ID NO:8 and CDR3 as defined in SEQ ID NO:9; or (b) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NO: 12, CDR2 as defined in SEQ ID NO: 13 and CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO: 15, CDR2 as defined in SEQ ID NO: 16 and CDR3 as defined in SEQ ID NO: 17.
  • VH variable heavy
  • VL variable light
  • the target-binding molecule according to any one of items 3 to 6, wherein the antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 18 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:2 or SEQ ID NO: 18; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO: 19 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:3 or SEQ ID NO: 19; or (b) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO: 10 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 10; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 11.
  • VH variable heavy
  • VL variable
  • the target-binding molecule according to any one of items 3 to 7, wherein the target-binding molecule further comprises an antigenic peptide fused to a C-terminal end of the antibody, or the target-binding fragment thereof.
  • the target-binding molecule according to item 8 wherein the antigenic peptide is fused to the C-terminal end of the antibody, or the target-binding fragment thereof, via a peptide linker.
  • the target-binding molecule according to item 4 to 10 wherein the target-binding molecule is a fusion antibody, wherein the fusion antibody comprises an Fc region and two or more scFv fragments.
  • the target-binding molecule according to item 11 wherein each scFv fragment is connected to the Fc region with a peptide linker.
  • the target-binding molecule according to item 4 to 12 wherein the target-binding molecule is a multispecific antibody.
  • An antibody-antigenic peptide complex comprising the target-binding molecule according to item 14 or 15 and a molecule comprising an antigenic peptide.
  • the antibody-antigenic peptide complex according to item 16 wherein the molecule comprising the antigenic peptide comprises an epitope that is specifically bound by a Fab or scFv portion of the target-binding molecule.
  • the antibody-antigenic peptide complex according to item 17, wherein the molecule comprising the antigenic peptide is a fusion protein comprising the antigenic peptide fused to a polypeptide comprising the epitope that is specifically bound by the Fab or scFv portion of the target- binding molecule.
  • the antibody-antigenic peptide complex according to item 18 or 19, wherein the polypeptide comprising the epitope that is specifically bound by the Fab or scFv portion of the targetbinding molecule is Myc or a Myc fragment.
  • An antibody-antigenic peptide construct comprising an antibody, or a target-binding fragment thereof, specifically binding to GOLPH2 and an antigenic peptide, wherein the antigenic peptide is: a) fused to a C-terminal end of the antibody, or the target-binding fragment thereof; and/or b) comprised in a molecule that is specifically bound by a Fab or scFv portion comprised in the antibody, or the target-binding fragment thereof.
  • the antibody-antigenic peptide construct according to item 25 wherein the multispecific antibody comprises a first Fab or scFv portion specifically binding to GOLPH2 and a second Fab or scFv portion specifically binding to the molecule comprising the antigenic peptide.
  • the antibody-antigenic peptide construct according to any one of item 21 to 31, wherein the antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO.9; or (b) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO.17.
  • the antibody-antigenic peptide construct according to any one of items 21 to 32, wherein the antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NO:4, CDR2 as defined in SEQ ID NO:5 and CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NOT, CDR2 as defined in SEQ ID NO: 8 and CDR3 as defined in SEQ ID NOV; or (b) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NO: 12, CDR2 as defined in SEQ ID NO: 13 and CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO: 15, CDR2 as defined in SEQ ID NO: 16 and CDR3 as defined in SEQ ID NO: 17.
  • VH variable heavy
  • VL variable light
  • the antibody-antigenic peptide construct according to any one of items 21 to 33, wherein the antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 18 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:2 or SEQ ID NO: 18; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO: 19 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:3 or SEQ ID NO: 19; or (b) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NOTO or a sequence having 90%, preferably 95% sequence identity to SEQ ID NOTO; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NOT E
  • An immunoconjugate comprising the target
  • a cell comprising the polynucleotide according to item 36.
  • a pharmaceutical composition comprising the target-binding molecule according to any one of items 1 to 15, the antibody-antigenic peptide complex according to any one of items 16 to 20, or the antibody-antigenic peptide construct according to any one of items 21 to 34 and/or the immunoconjugate according to item 35 and further comprising a pharmaceutically acceptable earner.
  • the pharmaceutical composition according to item 39 further comprising at least one therapeutic agent.
  • the pharmaceutical composition according to item 40 wherein the therapeutic agent is at least one of a vaccine, an antigen, an adjuvant, a chemotherapeutic agent and an immune checkpoint modulator.
  • a method for inducing the maturation of monocytes and/or monocyte -derived cells in vitro comprising the steps of: a) culturing monocytes and/or monocyte-derived cells in a cell culture medium; b) adding the target-binding molecule according to any one of items 1 to 15, the antibody- antigenic peptide complex according to any one of items 16 to 20, or the antibody-antigenic peptide construct according to any one of items 21 to 34 to the cell culture of step (a); and c) obtaining matured monocytes and/or monocyte-derived cells.
  • a method for generating a cell displaying an antigenic peptide comprising the steps of: a) generating a matured monocyte or monocyte-derived cell according to the method of item 56; b) pulsing the antigenic peptide on the matured monocyte or monocyte-derived cell generated in step (a); and/or introducing a nucleic acid encoding a polypeptide comprising the antigenic peptide into the matured monocyte or monocyte-derived cell generated in step (a); and/or introducing the antigenic peptide into the matured monocyte or monocyte-derived cell as part of the target-binding molecule, the antibody-antigenic peptide complex, or the antibody-antigenic peptide construct; and c) obtaining a cell displaying an antigenic peptide.
  • a method for generating an activated T cell comprising the steps of: a) generating a cell displaying an antigenic peptide with the method according to item 57; b) contacting the cell of step (a) with a T cell, wherein the T cell comprises a T cell receptor that recognizes the antigenic peptide displayed by the cell of step (a); and c) obtaining an activated T cell.
  • a cell displaying an antigenic peptide and/or an activated T cell for use in adoptive cell transfer wherein the cell displaying the antigenic peptide has been obtained with the method according to item 57 and wherein the activated T cell has been obtained with the method according to item 58 or 59.
  • a method for inducing and/or enhancing an immune response against a specific antigen in a subject comprising the steps of: a) administering to said subject the target-binding molecule according to any one of items 1 to 15, the antibody-antigenic peptide complex according to any one of items 16 to 20, the antibody-antigenic peptide construct according to any one of items 21 to 35, the immunoconjugate according to item 36 or the pharmaceutical composition according to any one of items 40 to 42, wherein the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition comprises one or more antigenic peptide(s) derived from the specific antigen; b) inducing and/or enhancing an immune response in said subject.
  • the invention relates to a target-binding molecule specifically binding to the extracellular part of GOLPH2 on the cell surface of a monocyte or a monocyte-derived cell, wherein the binding of the target-binding molecule to the extracellular part of GOLPH2 induces the maturation of the monocyte or the monocyte-derived cell.
  • the invention is based, at least in part, on the surprising finding that target-binding molecules that bind to the extracellular part of the protein GOLPH2 on the surface of monocytes and/or monocyte-derived cells can induce the maturation of said monocytes or monocyte-derived cells.
  • FIG. l shows that GOLPH2 is not only expressed in different tumor cell lines, but also in human monocytes.
  • secretion of the soluble GOLPH2 (sGOLPH2) fragment by human monocytes is significantly lower compared to tumor cell lines. Without being bound to theory, this may be explained by lower expression of GOLPH2 in monocytes and/or by lower secretion of GOLPH2, presumably due to reduced proteolytic cleavage at the furin cleavage site in the extracellular part of GOLPH2.
  • RNA-Seq experiments revealed that monocytes that have been treated with the same anti-GOLPH2 antibody show very similar gene expression patterns as Ml macrophages (Table 4). Based on these results, it appears plausible that antibodies that bind to the extracellular part of GOLPH2 have the potential to induce the maturation of monocytes into Ml macrophages.
  • the present invention encompasses any antibody that specifically binds to the extracellular part of GOLPH2 such that it induces the maturation of monocytes, in particular the maturation of monocytes into Ml macrophages and/or dendritic cells.
  • the invention relates to the antibodies G2-2opti or G2-4 from WO 2018/091724 that have been surprisingly shown herein to induce the maturation of human and/or murine monocytes in the attached examples.
  • Antibody G2-2 from WO 2018/091724 binds in the proximity of the furin cleavage site of GOLPH2 and can target both cleaved and uncleaved GOLPH2.
  • Antibody G2-4 from WO 2018/091724 binds to a different epitope in the soluble part of GOLPH2 (sGOLPH2) and can thus only target cells comprising the uncleaved form of GOLPH2.
  • FIG.5B shows that both G2-2opti and G2-4, even though they bind to different epitopes in the extracellular part of GOLPH2, can induce the secretion of cytokines that are indicative of Ml macrophages.
  • cytokine release in the process of monocyte polarization the commercial anti-GOLPH2 antibody EPR3606 elicited similar cytokine releases in a monocyte/T cell co-culture as G2-2.
  • G2-2 showed a more pronounced cytokine release than EPR3606.
  • FOG-14C irrelevant isotype control antibody
  • GOLPH2 refers to any native GOLPH2.
  • the term includes GOLPH2 from any vertebrate source, including mammals such as primates (e.g. humans and rhesus macaques) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the term also includes naturally occurring variants of GOLPH2, e.g., splice variants or allelic variants.
  • the amino acid sequence of an exemplary human GOLPH2 protein is shown in SEQ ID NO: 1.
  • the amino acid sequence of nonlimiting exemplary mouse GOLPH protein is shown in SEQ ID NO: 22.
  • a “target-binding molecule,” as used herein, is any molecule that can specifically or selectively bind to a target molecule.
  • the target molecule may be any type of molecule, such as, without limitation, a peptide, a protein, a nucleic acid, a carbohydrate or combinations thereof.
  • the targetbinding molecule specifically binds to an epitope that is located within the target molecule.
  • epitope refers to a region of a target molecule which is recognized by a particular target-binding molecule.
  • a target-binding molecule may include or be an antibody or a fragment thereof.
  • An anti-GOLPH2 binding molecule is a molecule that binds to the GOLPH2 antigen, such as an anti-GOLPH2 antibody or fragment thereof, at a specific recognition site or part of the antigen as detailed herein. That is, target-binding molecules of the invention bind to the extracellular part of GOLPH2.
  • Other anti-GOLPH2 binding molecules may also include multivalent molecules, multispecific molecules (e.g., diabodies), fusion molecules, aptamers, avimers, or other naturally occurring or recombinantly created molecules.
  • Illustrative target-binding molecules useful in the present invention include antibody-like molecules.
  • An antibody-like molecule is a molecule that can exhibit functions by binding to a target molecule (See, e.g., Current Opinion in Biotechnology 2006, 17:653- 658; Current Opinion in Biotechnology 2007, 18: 1-10; Current Opinion in Structural Biology 1997, 7:463-469; Protein Science 2006, 15: 14-27), and includes, for example, DARPins (WO 2002/020565), Affibody (WO 1995/001937), Avimer (WO 2004/044011; WO 2005/040229), Adnectin (WO 2002/032925) and fynomers (WO 2013/135588).
  • GOLPH2 may also be referred to as an antigen and that the target-binding molecule of the invention may also be referred to as an antigen-binding molecule.
  • antigen is preferably used herein to refer to a molecule that provokes an immune response, e.g. a molecule that can be taken up and processed within an antigen-presenting cell such that an antigenic peptide derived from this antigen is presented by the antigen-presenting cell.
  • binding to defines a binding (interaction) of at least two “antigen-interaction-sites” with each other.
  • antiigen-interaction- site defines, in accordance with the present invention, a motif of a polypeptide, i.e., a part of the antibody or target-binding fragment of the present invention, which shows the capacity of specific interaction with a specific antigen or a specific group of antigens of GOLPH2. Said binding/interaction is also understood to define a “specific recognition”.
  • the term “specifically recognizing” means in accordance with this invention that the target-binding molecule is capable of specifically interacting with and/or binding to at least two amino acids of GOLPH2 as defined herein, in particular interacting with/binding to at least two amino acids within the extracellular part of GOLPH2 on the cell surface of a monocyte or a monocyte -derived cell such that the maturation of the monocyte or the monocyte-derived cell is induced.
  • target-binding molecule of the invention does not or does not essentially crossreact with (poly)peptides that are not part of or derived from the extracellular part of GOLPH2.
  • Crossreactivity of target-binding molecules in particular a panel of antibodies or target-binding fragments thereof under investigation may be tested, for example, by assessing binding of said panel of antibodies or target-binding fragments thereof under conventional conditions (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988) and Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1999)) to the (poly)peptide of interest as well as to a number of more or less (structurally and/or functionally) closely related (poly)peptides. Only those constructs (i.e.
  • GOLPH2 antibodies, target-binding fragments thereof and the like
  • bind to the certain structure of GOLPH2 as defined herein e.g., a specific part, epitope or (poly) peptide/protein of GOLPH2 as defined herein but do not or do not essentially bind to any of the other parts, epitopes or (poly) peptides of the same GOLPH2, are considered specific for the part, epitope or (poly) peptide/protein of interest and selected for further studies in accordance with the method provided herein.
  • These methods may comprise, inter alia, binding studies, blocking and competition studies with structurally and/or functionally closely related molecules.
  • binding studies also comprise FACS analysis, surface plasmon resonance (SPR, e.g. with BIAcore), analytical ultracentrifugation, isothermal titration calorimetry, fluorescence anisotropy, fluorescence spectroscopy or by radiolabeled ligand binding assays.
  • SPR
  • the target-binding molecule binds to the extracellular part of GOLPH2.
  • the “extracellular part of GOLPH2”, as used herein, refers to the extracellular C-terminal part of GOLPH2 which, when GOLPH2 is associated with the cell membrane, protrudes into the extracellular space.
  • the “extracellular part of GOLPH2” is the part of GOLPH2 that, when GOLPH2 is associated with the cell membrane, is accessible for the target-binding molecules of the invention from the extracellular space.
  • the extracellular part consists of the residues 36 to 401 of GOLPH2 as set forth in SEQ ID NO: 1.
  • the extracellular part consists of the residues 36 to 393 of GOLPH2 as set forth in SEQ ID NO:22.
  • the target-binding molecule of the invention may bind to any fragment or epitope of the extracellular part of GOLPH2.
  • GOLPH2 frequently undergoes proteolytic cleavage wherein a soluble fragment of the extracellular part of GOLPH 2 (termed soluble GOLPH2 or sGOLPH2) is cleaved off and released in the extracellular space.
  • the target-binding molecule of the invention may bind (i) to any epitope in sGOLPH2, preferably before it has been cleaved off by furin and is thus still attached to a cell, or (ii) to any epitope in the remnant extracellular part of GOLPH2 that is still bound to the membrane after sGOLPH2 has been cleaved off by furin.
  • the epitope of the remnant extracellular part of GOLPH2 may be an epitope that is only accessible after the proteolytic cleavage.
  • the target-binding molecule of the invention may bind to the same epitope of GOLPH2 as the antibody G2-2opti. That is, the targetbinding molecule of the invention may bind to a linear epitope located within the amino acid sequences of SEQ ID NOs: 23, 24 and/or 25, i.e. SSRSVDLQTRIMELEGRVRR (human, SEQ ID NO: 23), SSRSVELQTRIVELEGRVRR (murine, SEQ ID NO: 24) and/or SSRSVDLQTRIVELEGRVRR (canine, SEQ ID NO: 25), of GOLPH2.
  • SSRSVDLQTRIMELEGRVRR human, SEQ ID NO: 23
  • SSRSVELQTRIVELEGRVRR murine, SEQ ID NO: 24
  • SSRSVDLQTRIVELEGRVRR canine, SEQ ID NO: 25
  • the target-binding molecule of the invention may bind to a linear epitope located within the amino acid sequence of SEQ ID NO: 23 (SSRSVDLQTRIMELEGRVRR) of human GOLPH2 as set forth in SEQ ID NOT.
  • the epitope bound by the antibody of the invention may be within the amino acid sequence RIMELEGRVRR of SEQ ID NO: 23, or within the amino acid sequence EGRVRR of SEQ ID NO:23, 24 or 25.
  • the target-binding molecule of the invention may bind to the same epitope of GOLPH2 as the antibody G2-4 from WO 2018/091724. That is, the target-binding molecule of the invention may bind to an epitope comprised in the amino acid sequence GEDDYNMDENEAESETDKQA (SEQ ID NO:26) corresponding to amino acids 347 to 366 of human GOLPH2 as set forth in SEQ ID NO: 1.
  • PA5-18100 from Invitrogen can induce the maturation of monocytes.
  • PA5-18100 binds to the synthetic peptide sequence NLLDQREKRNHTL corresponding to amino acids 389 to 401 of human GOLPH2 as set forth in SEQ ID NO: 1 That is, the target-binding molecule of the invention may bind to an epitope comprised in the amino acid sequence NLLDQREKRNHTL (SEQ ID NO:27) corresponding to amino acids 389 to 401 of human GOLPH2 as set forth in SEQ ID NO: 1.
  • EPR3606 binds to the extracellular soluble part of GOLPH2, as has been demonstrated in FIG. 15. Accordingly, the target-binding molecule of the invention may bind to the same epitope as the commercial antibody EPR3606.
  • the target-binding molecule of the invention binds to the extracellular part of GOLPH2 on the cell surface, for example the cell surface of a monocyte or a monocyte-derived cell.
  • a protein, in particular GOLPH2 is said to be located on the surface of a cell, in particular a monocyte or monocyte-derived cell, if the protein is associated with the outer membrane of a cell (cell or plasma membrane) such that at least parts of the protein contribute to the surface of the cell and are available from the extracellular space.
  • the target-binding molecule of the invention binds to the extracellular part of GOLPH2 on the cell surface of a monocyte or a monocyte-derived cell. That is, in certain embodiments, the target-binding molecule of the invention binds to an epitope within the residues 36 to 401 of human GOLPH2 as set forth in SEQ ID NO: 1, preferably wherein GOLPH2 is located on the cell surface of a human monocyte or monocyte-derived cell.
  • the target-binding molecule of the invention binds to an epitope located within the amino acid sequences of SEQ ID NOs: 23, 24 and/or 25 of GOLPH2, preferably wherein GOLPH2 is located on the cell surface of a monocyte or monocyte-derived cell.
  • the target-binding molecule of the invention binds to an epitope located within the amino acid sequences of SEQ ID NO: 23 of GOLPH2, preferably wherein GOLPH2 is located on the cell surface of a human monocyte or monocyte-derived cell.
  • the target-binding molecule of the invention binds to an epitope located within the amino acid sequences of SEQ ID NO: 26 of human GOLPH2, preferably wherein GOLPH2 is located on the cell surface of a human monocyte or monocyte-derived cell. In other embodiments, the targetbinding molecule of the invention binds to an epitope located within the amino acid sequences of SEQ ID NO: 27 of human GOLPH2, preferably wherein GOLPH2 is located on the cell surface of a human monocyte or monocyte-derived cell.
  • Binding of the target-binding molecule of the invention to the extracellular part of GOLPH2 on the cell surface of a monocyte or a monocyte-derived cell induces the maturation of the monocyte or monocyte-derived cell.
  • the term "maturation" as used herein means a process that is required for a cell (e.g. a monocyte) to become more specialized and/or attain a fully functional state, for example its functional state in vivo. That is, in certain embodiments, binding of the target-binding molecule of the invention to the extracellular part of GOLPH2 on the cell surface of a monocyte may induce the maturation of said monocyte into a dendritic cell, in particular a matured dendritic cell. In other embodiments, binding of the target-binding molecule of the invention to the extracellular part of GOLPH2 on the cell surface of a monocyte may induce the maturation of said monocyte into a macrophage, in particular an Ml macrophage.
  • binding of the target-binding molecule of the invention to the extracellular part of GOLPH2 on the cell surface of a monocyte-derived cell may induce the maturation of said monocyte- derived cell. That is, in certain embodiments, binding of the target-binding molecule of the invention to the extracellular part of GOLPH2 on the cell surface of an immature monocyte-derived dendritic cell may induce the maturation of said immature monocyte -derived dendritic cell into a matured dendritic cell.
  • binding of the target-binding molecule of the invention to the extracellular part of GOLPH2 on the cell surface of a non-polarized MO macrophage may induce the maturation (or polarization) of said non-polarized MO macrophage into an Ml macrophage.
  • a “monocyte-derived cell” is an immature dendritic cell or an MO macrophage.
  • the invention relates to the target-binding molecule according to the invention, wherein the maturation of monocytes or monocyte -derived cells results in the formation of dendritic cells and/or classically activated macrophages.
  • the invention relates to the target-binding molecule according to the invention, wherein the maturation of monocytes or monocyte -derived cells results in the formation of matured dendritic cells and/or classically activated macrophages.
  • target-binding molecules binding to the extracellular part of GOLPH2 have the potential to induce the maturation of monocytes, in particular, to induce the maturation of monocytes into Ml macrophages and/or monocyte-derived dendritic cells.
  • the invention relates to a target-binding molecule specifically binding to the extracellular part of GOLPH2 on the cell surface of a monocyte or a monocyte-derived cell, wherein the binding of the target-binding molecule to the extracellular part of GOLPH2 induces the maturation of the monocyte or the monocyte-derived cell into an Ml macrophage.
  • the invention relates to a target-binding molecule specifically binding to the extracellular part of GOLPH2 on the cell surface of a monocyte or a monocyte-derived cell, wherein the binding of the target-binding molecule to the extracellular part of GOLPH2 induces the maturation of the monocyte or the monocyte-derived cell into a dendritic cell.
  • target-binding molecules of the invention preferably induce the maturation of monocytes and/or monocyte-derived cells into dendritic cells, in particular matured dendritic cells, while other target-binding molecules of the invention preferably induce the maturation of monocytes and/or monocyte derived cells into Ml macrophages.
  • certain target-binding molecules of the invention have the potential to induce the maturation of monocytes and/or monocyte-derived cells into dendritic cells, in particular matured dendritic cells, and Ml macrophages.
  • the same target-binding molecule may have the potential to induce the maturation of a monocyte or a monocyte-derived cell into a dendritic cell, in particular a matured dendritic cell, or an Ml macrophage.
  • the decision whether a target-binding molecule induces maturation of a monocyte or a monocyte-derived cell into a dendritic cell, in particular a matured dendritic cell, or an Ml macrophage may depend on additional factors, e.g. the incubation time of the cell with the target-binding molecule.
  • shorter incubation periods preferably result in the maturation of monocytes and/or monocyte-derived cells into Ml macrophages, while longer incubation periods preferably result in the maturation of monocytes and/or monocyte-derived cells into dendritic cells, in particular matured dendritic cells.
  • the structure of the target-binding molecule has an influence on the fate of the monocytes and/or monocyte-derived cells. That is, it has been observed by the inventors that Fab fragments derived from an antibody binding specifically to the extracellular part of GOLPH2 on the surface of a monocyte or a monocyte-derived cell preferably induce the maturation of monocytes and/or monocyte-derived cells into Ml macrophages.
  • full antibodies binding specifically to the extracellular part of GOLPH2 on the surface of a monocyte or a monocyte-derived cell preferably induce the maturation of monocytes and/or monocyte-derived cells into dendritic cells, in particular matured dendritic cells.
  • the target-binding molecule of the invention may induce the maturation of monocytes or monocyte- derived cells into immature or mature dendritic cells.
  • the targetbinding molecule of the invention may also be used for inducing the maturation of monocytes or monocyte-derived cells into cells that show characteristics of immature and mature dendritic cells.
  • a target-binding molecule is said to induce the maturation of a first cell type into a second cell type, if the binding of the target-binding molecule to the first cell type, for example to the extracellular part of a protein on the cell surface of the first cell type, initiates developmental processes, for example by activating certain signaling pathways in said first cell type, that eventually result in the maturation of the first cell type into the second cell type.
  • monocyte refers to a type of white blood cells that have two main functions in the immune system: (1) replenish resident macrophages and dendritic cells under normal states, and (2) in response to inflammation signals, monocytes can move quickly (approx. 8-12 hours) to sites of infection in the tissues and divide/differentiate into macrophages and dendritic cells to elicit an immune response. Monocytes are usually identified in stained smears by their large bilobate nucleus.
  • monocytes In addition to the expression of CD 14, monocytes also show expression of one or more of the following surface markers 125I-WVH-1, 63D3, Adipophilin, CB12, CDl la, CDl lb, CD15, CD54, Cdl63, cytidine deaminase, Flt-1.
  • monocyte includes, without limitation, the classical monocytes, the intermediate monocytes and the non-classical pro-inflammatory monocyte, which are present in human blood.
  • CD14++ CD 16+ monocyte CD14++ CD 16+ monocyte
  • non-classical pro-inflammatory monocyte refers to a cell with a lower expression of CD 14 and with high level expression of the CD16 receptor (CD14+C D16++ monocyte). The skilled person is aware of methods to determine the expression of cell surface receptors, for example by flow cytometry as described herein.
  • Monocytes are characterized by producing high amounts of pro-inflammatory cytokines such as tumor necrosis factor and interleukin- 12 in response to stimulation by microbial products.
  • monocyte -derived cell refers to any type of cell that has been derived from a monocyte, in particular from a classical monocyte, an intermediate monocyte or a non-classical pro- inflammatory monocyte. Most commonly, monocyte-derived cells may be macrophages and myeloid dendritic cells.
  • the target-binding molecule of the invention may induce the maturation of a monocyte into an MO macrophage and/or the maturation of an MO macrophage into an Ml macrophage. In other embodiments, the target-binding molecule of the invention may induce the maturation of a monocyte into an immature dendritic cell and/or the maturation of an immature dendritic cell into a mature dendritic cell.
  • macrophage refers to CD 14+ positive cells derived from the differentiation of monocytes. Macrophages are characterized in that they are phagocytes, acting both in non-specific defense (innate immunity) as well as to help initiating specific defense mechanisms (adaptive immunity) of vertebrate animals.
  • innate immunity non-specific defense
  • adaptive immunity adaptive immunity
  • macrophages One of the main roles of macrophages is to phagocytose (engulf and then digest) cellular debris and pathogens either as stationary or as mobile cells, and to stimulate lymphocytes and other immune cells to respond to the pathogen.
  • macrophages may also show expression of one or more of the following surface markers: CDl lb, F4/80(mice)/EMRl (human), Lysozyme M, MAC-l/MAC-3, 27E10, Carboxypeptidase M, Cathepsin K, CD163 and CD68. These markers may be determined by flow cytometry or immunohistochemical staining.
  • the skilled person is aware of methods to differentiate between different types of macrophages, i.e. MO, Ml and M2 macrophages.
  • the type of macrophage may be determined based on the presence of specific cell surface proteins, as described in Becker et al., PLoSONE, 2012, 7(3), e33297.
  • different types of macrophages may be discriminated according to their cytokine profde as described in Vogel et al., Journal of Neuroinflammation, 2014, 11(23) or as described in Example 8.
  • different types of macrophages may be differentiated according to their gene expression profiles as described by Orecchioni et al., Frontiers in Immunology, 2019, 10: 1084 or as described in Example 10.
  • M0 macrophage refers to a subtype of macrophages that are resting or unactivated (unpolarized).
  • the target-binding molecule of the invention induces the maturation of monocytes or monocyte-derived cells into classically activated macrophages, also called Ml macrophages or Ml inflammatory type macrophages.
  • classically activated macrophages also called Ml macrophages or Ml inflammatory type macrophages.
  • the term “classically activated macrophages” as used herein refers to a subtype of macrophages that are classically activated or exhibit an inflammatory macrophage phenotype.
  • Classically activated macrophages are activated by LPS and IFN-y, and secrete high levels of IL-l-beta, TNF-alpha, and IL-12.
  • Ml macrophages also include macrophages that exhibit a hybrid phenotype that is predominantly the Ml phenotype.
  • M2 broadly refers to macrophages that function in constructive processes like wound healing and tissue repair and include M2A and M2C macrophages. Major differences between M2A and M2C macrophages exist in wound healing.
  • myeloid dendritic cell refers to a population of dendritic cells which derive from monocytes and which include, without limitation, mDC-I and mDC-2 cells.
  • myeloid dendritic cells may also show expression of one or more of the following surface markers: ADAM19, BDCA-2, CDla, CDl lc CD21, CD86, CD208, Clusterin, Estrogen Receptor-alpha. These markers may be determined by flow cytometry or immunohistochemical staining.
  • immature and matured dendritic cell based on the presence of specific cell surface proteins or according to their cytokine secretion profile as described by Dudek et al., Frontiers in Immunology, 2013, 4:438.
  • immature and matured dendritic cells may be identified and/or discriminated according to their gene expression profiles as described by Lyons et al., npj Precision One, 2017, 1, 26 or as described in Example 10.
  • binding of the target-binding molecule of the invention to the extracellular part of GOLPH2 on the surface of a monocyte or monocyte-derived cell results in the maturation of said monocyte or monocyte-derived cell into an Ml macrophage or into a dendritic cell, in particular a matured dendritic cell.
  • the maturation state of a monocyte is determined based on the expression of the cell surface marker CD 14.
  • Monocytes are known to express higher amounts of CD 14 compared to monocyte-derived macrophages or dendritic cells (Fig.4a). That is, a target-binding molecule may be determined to induce the maturation of a monocyte or a monocyte-derived cell into a macrophage and/or a dendritic cell, if contacting of a monocyte with the target-binding molecule results in a lower expression of CD 14 as compared to the contacting of a monocyte with a comparable molecule that does not bind to the extracellular part of GOLPH2.
  • the skilled person is aware of methods to determine the expression of CD 14 on a cell.
  • the expression of CD 14 on a cell is determined by flow cytometry.
  • the maturation state of a monocyte is determined based on the expression of the cell surface marker CD40.
  • Monocytes are known to express lower amounts of CD40 compared to monocyte-derived macrophages. That is, a target-binding molecule may be determined to induce the maturation of a monocyte or a monocyte-derived cell into a macrophage, if contacting of a monocyte with the target-binding molecule results in a higher expression of CD40 as compared to the contacting of a monocyte with a comparable molecule that does not bind to the extracellular part of GOLPH2.
  • the skilled person is aware of methods to determine the expression of CD40 on a cell.
  • the expression of CD40 on a cell is determined by flow cytometry. Mohammadi et al. (Biotechnic & Histochemistry 90:6 (2015) 445-452
  • the differentiation of a monocyte into a macrophage may be determined based on the cytokine secretion profiles of these cells.
  • Macrophages are known to secrete higher amounts of the cytokines IL-lBeta, IL-6 and TNFa compared to monocytes (Fig.5).
  • IL-lBeta cytokines IL-lBeta
  • IL-6 IL-6
  • TNFa monocytes
  • the differentiation of a monocyte into a dendritic cell may be determined based on differences in the expression of cell surface markers.
  • dendritic cells are characterized by high expression of CD82 and low expression of CD37, while monocytes express lower amounts of CD82 and higher amounts of CD37 (FIG.6).
  • FOG.6 the skilled person is aware of methods to differentiate between dendritic cells and monocytes based on the expression of CD82 and CD37.
  • the expression of CD82 and CD37 may, for example, be determined by flow cytometry or by RNAseq.
  • dendritic cells For example, the determination of dendritic cells is described in Jones E.; Dendritic Cell Migration and Antigen Presentation are coordinated by the opposing functions of the tetraspanins CD82 and CD36. J of Immunology 2015 doi: 10.4049/jimmunol.1500357.
  • the differentiation state of a monocyte-derived cell is determined based on the gene expression profile.
  • Gene expression data may be obtained by any method known in the art, preferably by RNA sequencing.
  • the genes PDLIM4, CD1B, MRC1, RAP1GAP, FCER2, DUSP5, PPIC, CD1C, CCND2, STAC, CD1A, CD36, SHB, TRIB2 and PRKACB have been reported to be upregulated in immature dendritic cells and the genes CD 14, FCN1 and CCR5 have been reported to be downregulated in immature dendritic cells in comparison to untreated human monocytes.
  • a monocyte or monocyte-derived cell is determined to be differentiated into an immature dendritic cell, if at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or more than 15 of these genes are either up- or downregulated in comparison to an untreated human monocyte as described above.
  • a monocyte or monocyte-derived cell is determined to be differentiated into an immature dendritic cell, if at least 6, at least 7, at least 8, at least 9, at least 10 or all of the genes PDLIM4, CD1B, MRC1, RAP1GAP, FCER2, DUSP5, PPIC, CD1C, CCND2, STAC and CD1A are upregulated by a log2-fold change higher than 1 in comparison to an untreated human monocyte.
  • a monocyte or monocyte-derived cell is determined to be differentiated into an immature dendritic cell, if at least 3, at least 4, at least 5, at least 6, at least 7 or all of the genes PDLIM4, CD1B, MRC1, RAP1GAP, FCER2, DUSP5, PPIC and CD1C are upregulated by a log2-fold change higher than 2 in comparison to an untreated human monocyte.
  • a monocyte or monocyte-derived cell is determined to be differentiated into an immature dendritic cell, if at least 1, at least 2, at least 3 or all of the genes PDLIM4, CD1B, MRC1 and RAP1GAP are upregulated by a log2-fold change higher than 3 in comparison to an untreated human monocyte.
  • a monocyte or monocyte-derived cell is determined to be differentiated into a mature dendritic cells, if at least 15, at least 20, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35 or more than 35 of these genes are upregulated in comparison to an untreated human monocyte as described above.
  • a monocyte or monocyte-derived is determined to be differentiated into a mature dendritic cell, if at least 10, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23 or all of the genes IL6, CYP27B1, EBI3, IFIT1, HES4, DUSP5, USP18, IFI44L, IFIT3, ATF3, MX1, ISG15, GADD45A, OASL, CCL8, TRAF1, LAMP3, RGS1, CXCL8, CD44, IL7R, CD86, IFI27 and MT2A are upregulated by a log2- fold change higher than 1 in comparison to an untreated human monocyte.
  • a monocyte or monocyte-derived is determined to be differentiated into a mature dendritic cell, if at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 or all of the genes IL6, CYP27B1, EBI3, IFIT1, HES4, DUSP5, USP18, IFI44L, IFIT3, ATF3, MX1, ISG15 and GADD45A are upregulated by a log2-fold change higher than 2 in comparison to an untreated human monocyte.
  • a monocyte or monocyte-derived is determined to be differentiated into a mature dendritic cell, if at least 1, at least 2, at least 3, at least 4 or all of the genes IL6, CYP27B1, EBI3, IFIT1 and HES4 are upregulated by a log2-fold change higher than 3 in comparison to an untreated human monocyte.
  • a monocyte or monocyte -derived is determined to be differentiated into an Ml macrophage, if at least 8, at least 9, at least 10, at least 11 or more than 11 of these genes are upregulated in comparison to an untreated human monocyte as described above.
  • a monocyte or monocyte-derived is determined to be differentiated into an Ml macrophage, if at least 5, at least 6, at least 7, at least 8, at least 9 or all of the genes CD40, PTGS2, IFIT2, VCAM1, RSAD2, EHD1, TRAF1, CCND2, SOCS3 and SERPINE1 are upregulated by a log2-fold change higher than 1 in comparison to an untreated human monocyte.
  • a monocyte or monocyte -derived is determined to be differentiated into an Ml macrophage, if at least 2, at least 3, at least 4, at least 5, at least 6 or all of the genes PTGS2, IFIT2, VCAM1, RSAD2, TRAF1, CCND2, SOCS3 and SERPINE1 are upregulated by a log2-fold change higher than 1.5 in comparison to an untreated human monocyte. Even more preferably, a monocyte or monocyte-derived is determined to be differentiated into an Ml macrophage, if at least 1 or all of the genes RSAD2 and SERPINE1 are upregulated by a log2-fold change higher than 2, preferably higher than 3, in comparison to an untreated human monocyte.
  • the cell surface markers, cytokines and genes disclosed herein may be used to identify cells of a specific cell type, such as monocytes, macrophages or dendritic cells and their sub-types disclosed herein.
  • the expression of cell surface markers, the secretion of cytokines and/or gene expression in general may vary between cells of the same cell type and depends on various factors. For example, differences in the expression of cell surface markers, the secretion of cytokines and/or gene expression between two cells of the same cell type may be observed between cells that have been obtained from different donors/origins and/or have been cultured under different culturing conditions (in vitro).
  • a cell may be of a certain cell type, even if one or more cell surface markers, cytokines and/or genes are expressed or secreted differently as disclosed herein.
  • the cell surface marker CD 14 is commonly reported to be upregulated in macrophages and downregulated in dendritic cells.
  • variations in the expression of CD 14 have been reported in the art (for example in Ong SM et al; A Novel, Five-Marker Alternative to CD16-CD14 Gating to Identify the Three Human Monocyte Subsets; Front. Immunol., 26 July 2019; or Collin M and Venetia Bigley; Human dendritic cell subsets: an update. Immunology; 154(1); 2018; p.3-20).
  • the invention relates to the target-binding molecule according to the invention, wherein the target-binding molecule is an antibody or a target-binding fragment thereof.
  • the target-binding molecule of the invention may be any type of target-binding molecule that has the potential to bind to the extracellular part of GOLPH2 such that it induces the maturation of a monocyte or a monocyte-derived cell. That is, in certain embodiments, the target-binding molecule of the invention is an antibody, or a target-binding portion thereof, a bispecific antibody, or a targetbinding portion thereof, a designed ankyrin repeat protein (D ARPIN), an aptamer or another antibody mimetic, such as affibody molecules, affilins, affimers, affitins, alphabodies, anticalins, avimers, fynomers, kunitzdomain peptides, monobodies.
  • D ARPIN ankyrin repeat protein
  • the target-binding molecule of the invention is an antibody, or a targetbinding fragment thereof.
  • antibody is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fully-human antibodies and antibody fragments so long as they exhibit the desired target-binding activity.
  • Antibodies within the present invention may also be chimeric antibodies, recombinant antibodies, target-binding fragments of recombinant antibodies or humanized antibodies.
  • the term “monoclonal antibody”, as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Monoclonal antibodies are advantageous in that they may be synthesized by a hybridoma culture, essentially uncontaminated by other immunoglobulins. The term “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. As mentioned above, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method described by Kohler, Nature 256 (1975), 495.
  • polyclonal antibody refers to an antibody which was produced among or in the presence of one or more other, non-identical antibodies.
  • polyclonal antibodies are produced from a B-lymphocyte in the presence of several other B-lymphocytes which produced nonidentical antibodies.
  • polyclonal antibodies are obtained directly from an immunized animal.
  • Fully-human antibody refers to an antibody which comprises human immunoglobulin protein sequences only.
  • a fully human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell or in a hybridoma derived from a mouse cell.
  • murine antibody or “murine antibody” refers to an antibody which comprises mouse/murine immunoglobulin protein sequences only.
  • a “fully-human antibody” may contain rat carbohydrate chains if produced in a rat, in a rat cell, in a hybridoma derived from a rat cell.
  • the term “rat antibody” refers to an antibody that comprises rat immunoglobulin sequences only.
  • Fully-human antibodies may also be produced, for example, by phage display which is a widely used screening technology which enables production and screening of fully human antibodies. Also, phage antibodies can be used in context of this invention. Phage display methods are described, for example, in US 5,403,484, US 5,969, 108 and US 5,885,793. Another technology which enables development of fully-human antibodies involves a modification of mouse hybridoma technology. Mice are made transgenic to contain the human immunoglobulin locus in exchange for their own mouse genes (see, for example, US 5,877,397).
  • chimeric antibodies refers to an antibody which comprises a variable region of the present invention fused or chimerized with an antibody region (e.g., constant region) from another, human or non-human species (e.g., mouse, horse, rabbit, dog, cow, chicken).
  • an antibody region e.g., constant region
  • human or non-human species e.g., mouse, horse, rabbit, dog, cow, chicken.
  • the term antibody also relates to recombinant human antibodies, heterologous antibodies and heterohybrid antibodies.
  • recombinant (human) antibody includes all human sequence antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes; antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies have variable and constant regions (if present) derived from human germline immunoglobulin sequences.
  • Such antibodies can, however, be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • a “heterologous antibody” is defined in relation to the transgenic non-human organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic non-human animal, and generally from a species other than that of the transgenic non-human animal.
  • heterohybrid antibody refers to an antibody having light and heavy chains of different organismal origins.
  • an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody.
  • heterohybrid antibodies include chimeric and humanized antibodies.
  • humanized antibodies also relate to humanized antibodies.
  • "Humanized" forms of non-human (e.g. murine or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other target-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • humanized antibody may comprise residues, which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • a humanized antibody has one or more amino acids introduced into it from a source which is non-human still retain the original binding activity of the antibody.
  • Methods for humanization of antibodies/antibody molecules are further detailed in Jones et al. (Nature 321 (1986), 522-525); Reichmann et al. (Nature 332 (1988), 323-327) and Verhoeyen et al. (Science 239 (1988), 1534-1536).
  • Specific examples of humanized antibodies, e.g. antibodies directed against EpCAM are known in the art, see e.g. LoBuglio (Proceedings of the American Society of Clinical Oncology Abstract (1997), 1562) and Khor (Proceedings of the American Society of Clinical Oncology Abstract (1997), 847).
  • a popular method for humanization of antibodies involves CDR grafting, where a functional targetbinding site from a non-human ‘donor’ antibody is grafted onto a human ‘acceptor’ antibody.
  • CDR grafting methods are known in the art and described, for example, in US 5,225,539, US 5,693,761 and US 6,407,213.
  • Another related method is the production of humanized antibodies from transgenic animals that are genetically engineered to contain one or more humanized immunoglobulin loci which are capable of undergoing gene rearrangement and gene conversion (see, for example, US 7,129,084).
  • antibody molecules or target-binding fragments thereof are provided, which are humanized and can successfully be employed in pharmaceutical compositions.
  • the term “antibody” relates to full immunoglobulin molecules as well as to parts of such immunoglobulin molecules (i.e., “target-binding fragment thereof’). Furthermore, the term relates, as discussed above, to modified and/or altered antibody molecules. The term also relates to recombinantly or synthetically generated/synthesized antibodies. The term also relates to intact antibodies as well as to antibody fragments thereof, like, separated light and heavy chains, Fab, Fv, Fab’, Fab’-SH, F(ab’)2. The term antibody also comprises but is not limited to fully-human antibodies, chimeric antibodies, humanized antibodies, CDR-grafted antibodies and antibody constructs, like single chain Fvs (scFv) or antibody-fusion proteins.
  • scFv single chain Fvs
  • a "target-binding fragment" of an antibody refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the target to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g. scFv), and multispecific antibodies formed from antibody fragments.
  • Single-chain Fv or “scFv” antibody fragments have, in the context of the invention, the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • the target-binding molecule of the invention may be an scFv fragment or a chimeric antibody comprising an scFv fragment.
  • the scFv fragment may be derived from the antibodies G2-2 or G2-4.
  • the generation of scFv fragments is further disclosed in Example 20.
  • the variable heavy and the variable light chain of an antibody may be fused with a peptide linker.
  • the peptide linker may be a flexible linker as disclosed herein.
  • the linker may have the sequence ASGGGGSGGGGSGGGGS (SEQ ID NO:51)
  • the scFv fragment may be derived from G2-2 or G2-2opti and may have the sequence set forth in SEQ ID NO:69 or SEQ ID NO:70. In certain embodiments, the scFv fragment may be derived from G2-4 and may have the sequence set forth in SEQ ID NO:71.
  • a “Fab fragment” as used herein is comprised of one light chain and the CHI and variable regions of one heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • An "Fc” region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
  • a "Fab 1 fragment” contains one light chain and a portion of one heavy chain that contains the VH domain and the CHI domain and also the region between the CHI and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab') 2 molecule.
  • a “F(ab')2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains.
  • a F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.
  • the "Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions.
  • Antibodies, antibody constructs, antibody fragments, antibody derivatives (all being Ig-derived) to be employed in accordance with the invention or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook (1989), loc. cit.
  • the term “Ig- derived domain” particularly relates to (poly)peptide constructs comprising at least one CDR.
  • Fragments or derivatives of the recited Ig-derived domains define (poly) peptides which are parts of the above antibody molecules and/or which are modified by chemical/biochemical or molecular biological methods.
  • Corresponding methods are known in the art and described inter alia in laboratory manuals (see Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 2nd edition (1989) and 3rd edition (2001); Gerhardt et al., Methods for General and Molecular Bacteriology ASM Press (1994); Lefkovits, Immunology Methods Manual: The Comprehensive Sourcebook of Techniques; Academic Press (1997); Golemis, Protein-Protein Interactions: A Molecular Cloning Manual Cold Spring Harbor Laboratory Press (2002)).
  • CDR as employed herein relates to “complementary determining region”, which is well known in the art.
  • the CDRs are parts of immunoglobulins that determine the specificity of said molecules and make contact with a specific ligand.
  • the CDRs are the most variable part of the molecule and contribute to the diversity of these molecules.
  • CDR-H depicts a CDR region of a variable heavy chain and CDR-L relates to a CDR region of a variable light chain.
  • VH means the variable heavy chain and VL means the variable light chain.
  • the CDR regions of an Ig-derived region may be determined as described in Kabat “Sequences of Proteins of Immunological Interest”, 5th edit. NIH Publication no. 91-3242 U.S. Department of Health and Human Services (1991); Chothia J. Mol. Biol. 196 (1987), 901-917 or Chothia Nature 342 (1989), 877-883.
  • the "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, a, y, and p, respectively.
  • the antibody molecule described herein above is selected from the group consisting of a full antibody (immunoglobulin, like an IgGl, an IgG2, an IgG2a, an IgG2b, an IgAl, an IgGA2, an IgG3, an IgG4, an IgA, an IgM, an IgD or an IgE), F(ab)-, Fab’-SH-, Fv-, Fab’-, F(ab’)2-fragment, a chimeric antibody, a CDR-grafted antibody, a fully human antibody, a bivalent antibody-construct, an antibody-fusion protein, a synthetic antibody, bivalent single chain antibody, a trivalent single chain antibody and a multivalent single chain antibody.
  • a full antibody immunoglobulin, like an IgGl, an IgG2, an IgG2a, an IgG2b, an IgAl, an IgGA2, an IgG3, an IgG4, an Ig
  • the target-binding molecule may be a monoclonal antibody. In any of the embodiments described herein, the target-binding molecule may be a human, humanized, or chimeric antibody. In any of the embodiments described herein, the target-binding molecule may be an antibody fragment that binds GOLPH2. In any of the embodiments described herein, the target-binding molecule may be an IgGl, IgG2a or IgG2b, IgG3, IgG4, IgM, IgAl, IgA2, IgD or IgE antibody.
  • isotype refers to the antibody class (e.g., IgM or IgGl) that is encoded by heavy chain constant region genes.
  • the antibodies can be full length or can include only a target-binding fragment such as the antibody constant and/or variable domain of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD or IgE or could consist of a Fab fragment, a F(ab')2 fragment and a Fv fragment.
  • the antibody of the invention comprises an Fc region. In certain embodiments, the antibody of the invention comprises an Fc region that can elicit an immune effector function. That is, in certain embodiments, the antibody of the invention comprises an Fc region that can bind to an Fey receptor.
  • An "antibody that binds to an epitope" within a defined region of a protein, e.g. the extracellular part of GOLPH2 is an antibody that requires the presence of one or more of the amino acids within that region for binding to the protein.
  • the antibody of the invention may bind to the extracellular part of human GOLPH2. In some embodiments, the antibody may bind to the extracellular part of murine GOLPH2 and/or to canine GOLPH2.
  • anti-GOLPH2 antibody and "an antibody specifically binding to GOLPH2" or simply “antibody” as used herein refer to an antibody that is capable of binding GOLPH2, in particular the extracellular part of GOLPH2, with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting GOLPH2.
  • the extent of binding of an anti- GOLPH2 antibody to an unrelated, non-GOLPH2 protein is less than about 10% of the binding of the antibody to GOLPH2 as measured, e.g., by a radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • an antibody that specifically binds to GOLPH2 has a dissociation constant (Kd) of ⁇ 1 pM, ⁇ 100 nM, ⁇ 10 nM, ⁇ 5 nm, ⁇ 4 nM, ⁇ 3 nM, ⁇ 2 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g., 10’ 8 M or less, e.g. from 10" 8 M to 10" 13 M, e.g., from 10" 9 M to 10" 13 M).
  • Kd dissociation constant
  • an "antibody that binds to an epitope" within a defined region of a protein is identified by mutation analysis, in which amino acids of the protein are mutated, and binding of the antibody to the resulting altered protein (e.g., an altered protein comprising the epitope) is determined to be at least 20% of the binding to unaltered protein.
  • an "antibody that binds to an epitope" within a defined region of a protein is identified by mutation analysis, in which amino acids of the protein are mutated, and binding of the antibody to the resulting altered protein (e.g., an altered protein comprising the epitope) is determined to be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the binding to unaltered protein.
  • binding of the antibody is determined by FACS, WB or by a suitable binding assay such as ELISA.
  • Ka is measured using surface plasmon resonance assays using a BIACGRE®-2000 or a BIACORE ®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized antigen CM5 chips at -10 response units (RU).
  • CM5 carboxymethylated dextran biosensor chips
  • EDC N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • NHS N- hydroxysuccinimide
  • Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 pg/ml (-0.2 pM) before injection at a flow rate of 5 pl/m incite to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of antibody (0.58 nM to 200 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20TM) surfactant (PEST) at 25°C at a flow rate of approximately 25 pl/min.
  • TWEEN-20TM polysorbate 20
  • association rates (kon) and dissociation rates (kq /') are calculated using a simple one-to-one Langmuir binding model (BIACORE ® T100 Evaluation Software) by simultaneously fitting the association and dissociation sensorgrams.
  • the equilibrium dissociation constant (Kd) is calculated as the ratio kofflkon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999).
  • the antibody or target-binding fragment thereof as provided in the context of the present invention is not particularly limited as long as it is an “anti-GOLPH2 antibody or a target-binding fragment thereof’ as defined above.
  • the antibody may be any antibody which specifically binds to/specifically recognizes/interacts with the extracellular part of GOLPH2 on the cell surface of a monocyte or a monocyte-derived cell such that the maturation of the monocyte or the monocyte- derived cell is induced.
  • the invention also provides antibodies binding to the same region of GOLPH2, i.e. the extracellular part of GOLPH2, and carrying out the same function as any of the specific antibodies provided herein.
  • an anti-GOLPH2 antibody may bind to an epitope of GOLPH2 that is conserved among GOLPH2 from different species.
  • the antibody of the invention may bind to a defined epitope within the GOLPH2 extracellular part.
  • the antibody of the invention may bind to an epitope within the amino acid sequence of SEQ ID NO: 23, 24, 25, 26 and/or 27.
  • the specificity of the antibody or target-binding fragment of the present invention may not only be expressed by the nature of the amino acid sequence of the antibody or the target-binding fragment as defined above but also by the epitope or part of the antigen to which the antibody is capable of binding to.
  • the present invention relates, in one embodiment, to an anti-GOLPH2 antibody or a targetbinding fragment thereof which recognizes the same epitope or part of GOLPH2 as an antibody of the invention, for example the monoclonal antibodies G2-2, G2-4 and EPR3606 or the polyclonal antibody PA5-18100.
  • Vero cells infected with 3 moi are incubated after 20 h with varying concentrations of the antibody in question as the competitor for 1 hour.
  • the antibody of the present invention is applied in a constant concentration of 100 nM and its binding is flow-cytometrically detected using a fluorescence-labelled antibody directed against the constant domains of the antibody of the invention. Binding that conducts anti-proportional to the concentration of the antibody in question is indicative for that both antibodies recognize the same epitope.
  • many other assays are known in the art which may be used.
  • the invention relates to the target-binding molecule according to the invention, wherein the antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO.9; or (b) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO.17.
  • VH variable heavy
  • VL variable light chain sequence comprising CDR3 as defined in SEQ ID NO.9
  • the invention relates to the target-binding molecule according to the invention, wherein the antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NON, CDR2 as defined in SEQ ID NO:5 and CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO.7, CDR2 as defined in SEQ ID NO: 8 and CDR3 as defined in SEQ ID NO:9; or (b) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NO: 12, CDR2 as defined in SEQ ID NO: 13 and CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO: 15, CDR2 as defined in SEQ ID NO: 16 and CDR3 as defined in SEQ ID NO: 17.
  • VH variable heavy
  • VL variable light
  • the invention relates to the target-binding molecule according to the invention, wherein the antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 18 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:2 or SEQ ID NO: 18; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO: 19 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:3 or SEQ ID NO: 19; or (b) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO: 10 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 10; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 11.
  • VH variable heavy
  • VL
  • an antibody provided herein is based, in part, on the development of antibody G2-2 and/or G2-2opti from WO 2018/091724, which binds to an epitope within amino acids 36 to 55 of human GOLPH2 protein.
  • an antibody provided herein binds to an epitope within amino acids 36 to 55 of human GOLPH2.
  • an antibody provided herein comprises one or more CDR sequences of antibody G2-2.
  • the invention provides an anti-GOLPH2 antibody comprising at least one, two, three, four, five, or six CDRs selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9.
  • the invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6.
  • the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6.
  • the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9.
  • the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, and CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5.
  • the antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6.
  • an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) CDR-H3 comprising an amino acid sequence selected from SEQ ID NO: 6; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9.
  • the invention provides an antibody comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9.
  • an anti-GOLPH2 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 18.
  • VH heavy chain variable domain
  • a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 18 contains substitutions (e.g, conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-GOLPH2 antibody comprising that sequence retains the ability to bind to GOLPH2.
  • a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 2 or SEQ ID NO: 18. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 2 or SEQ ID NO: 18. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In a preferred embodiment, a total of 3 amino acids in SEQ ID NO: 2 have been substituted to optimize the expression in mammalian cells (SEQ ID NO: 18).
  • the anti-GOLPH2 antibody comprises the VH sequence of SEQ ID NO: 2 or SEQ ID NO: 18, including post-translational modifications of that sequence.
  • the VH comprises one, two or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (b) CDRH2 comprising the amino acid sequence of SEQ ID NO: 5, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6.
  • an anti-GOLPH2 antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 19.
  • VL light chain variable domain
  • a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 19 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti- GOLPH2 antibody comprising that sequence retains the ability to bind to GOLPH2.
  • a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 3 or SEQ ID NO: 19.
  • a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 3 or SEQ ID NO: 19.
  • the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).
  • a total of 8 amino acids in SEQ ID NO: 3 have been substituted to optimize the expression in mammalian cells (SEQ ID NO: 19).
  • the anti-GOLPH2 antibody comprises the VL sequence of SEQ ID NO: 3 or SEQ ID NO: 19, including post-translational modifications of that sequence.
  • the VL comprises one, two or three CDRs selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9.
  • an anti-GOLPH2 antibody comprising a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.
  • the antibody comprises the VH and VL sequences in SEQ ID NO: 18 and SEQ ID NO: 19, respectively, including post-translational modifications of those sequences.
  • an anti-GOLPH2 antibody comprises a humanized form of an antibody comprising the VH and VL sequences in SEQ ID NO: 18 and SEQ ID NO: 19, respectively.
  • the antibody comprises the VH and VL sequences in SEQ ID NO: 2 and SEQ ID NO: 3, respectively, including post-translational modifications of those sequences.
  • an anti-GOLPH2 antibody comprises a humanized form of an antibody comprising the VH and VL sequences in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
  • an antibody provided herein binds to an epitope within amino acids 347 to 366 of human GOLPH2.
  • an antibody provided herein comprises one or more CDR sequences of antibody G2-4.
  • the invention provides an anti-GOLPH2 antibody comprising at least one, two, three, four, five, or six CDRs selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.
  • the invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14.
  • the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14.
  • the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.
  • the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13.
  • the antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) CDR- H2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14.
  • an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (iii) CDR-H3 comprising an amino acid sequence selected from SEQ ID NO: 14; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.
  • the invention provides an antibody comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.
  • an anti-GOLPH2 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10.
  • VH heavy chain variable domain
  • a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 10 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-GOLPH2 antibody comprising that sequence retains the ability to bind to GOLPH2.
  • a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 10. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 10. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In a preferred embodiment, a total of 3 amino acids in SEQ ID NO: 10 have been substituted to optimize the expression in mammalian cells.
  • the anti-GOLPH2 antibody comprises the VH sequence of SEQ ID NO: 10, including post-translational modifications of that sequence.
  • the VH comprises one, two or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (b) CDRH2 comprising the amino acid sequence of SEQ ID NO: 13, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14.
  • an anti-GOLPH2 antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11.
  • VL light chain variable domain
  • a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 11 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-GOLPH2 antibody comprising that sequence retains the ability to bind to GOLPH2.
  • the anti-GOLPH2 antibody comprises the VL sequence of SEQ ID NO: 11, including post-translational modifications of that sequence.
  • the VL comprises one, two or three CDRs selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.
  • an anti-GOLPH2 antibody comprising a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.
  • the antibody comprises the VH and VL sequences in SEQ ID NO: 10 and SEQ ID NO: 11, respectively, including post-translational modifications of those sequences.
  • an anti-GOLPH2 antibody comprises a humanized form of an antibody comprising the VH and VL sequences in SEQ ID NO: 10 and SEQ ID NO: 11, respectively.
  • the antibody of the invention comprises a human light chain and heavy chain constant region. In other embodiments, the antibody of the invention comprises a murine light chain and heavy chain constant regions. Examples for human and murine constant regions of GOLPH2 antibodies are provided in WO 2018/091724.
  • the anti-GOLPH2 antibody according to any of the above embodiments, in particular the antibody G2-2opti, comprises a heavy chain constant region sequence comprising the amino acid sequence of SEQ ID NO: 20 (human IgGl heavy chain) or SEQ ID NO: 21 (human hinge region fused to murine CH2 and CH3 domains).
  • an anti-GOLPH2 antibody comprises a heavy chain constant region sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 20 or SEQ
  • an anti-GOLPH2 antibody according to any of the above embodiments is a monoclonal antibody, including a human antibody.
  • an anti- GOLPH2 antibody is an antibody fragment, e.g, a Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment.
  • the antibody is a substantially full length antibody, e.g., an IgGl antibody, IgG2a antibody or other antibody class or isotype as defined herein.
  • an anti-GOLPH2 antibody may incorporate any of the features, singly or in combination, as described below.
  • an antibody that binds to the same part or epitope as an anti- GOLPH2 antibody provided herein.
  • an antibody is provided that binds to the same epitope as an anti-GOLPH2 antibody comprising a VH sequence of SEQ ID NO: 18 and a VL sequence of SEQ ID NO: 19 respectively.
  • an antibody is provided that binds to the same epitope as an anti-GOLPH2 antibody comprising a VH sequence of SEQ ID NO: 2 and a VL sequence of SEQ ID NO: 3, respectively.
  • an antibody is provided that binds to the same epitope as an anti-GOLPH2 antibody comprising a VH sequence of SEQ ID NO: 10 and a VL sequence of SEQ ID NO: 11 respectively.
  • the antibody may be the commercial polyclonal anti-GOLPH2 antibody PA5- 18100 from Invitrogen. PA5-18100 has been raised against the synthetic peptide sequence NLLDQREKRNHTL (SEQ ID NO:27), which corresponds to amino acids 389 to 401 of human GOLPH2 as set forth in SEQ ID NO: 1.
  • the invention encompasses targetbinding molecules that bind to an epitope within the amino acid sequence NLLDQREKRNHTL, which corresponds to amino acids 389 to 401 of human GOLPH2 as set forth in SEQ ID NO: 1.
  • This targetbinding molecule may not exclusively be the polyclonal antibody PA5-18100, but may be any targetbinding molecule and, in particular, any antibody or target-binding fragment thereof, that specifically binds to an epitope within the amino acid sequence NLLDQREKRNHTL, which corresponds to amino acids 389 to 401 of human GOLPH2 as set forth in SEQ ID NO: 1.
  • the antibody may be the commercial rabbit monoclonal anti-GOLPH2 antibody EPR3606 from Abeam. That is, the target-binding molecule according to the invention may bind to the same epitope in the extracellular part of GOLPH2 as the antibody EPR3606.
  • Perfect (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • target-binding molecules specifically binding to the extracellular part of GOLPH2 on the cell surface of monocytes and/or monocyte-derived cells induce the maturation of the monocytes and/or the monocyte-derived cells into antigen-presenting cells, in particular Ml macrophages and dendritic cells, in particular matured dendritic cells.
  • the antibodies of the invention can further be used to specifically deliver antigens or antigenic peptides to Ml macrophages and/or dendritic cells, in particular matured dendritic cells.
  • An antigen or antigenic peptide may be attached to the target-binding molecule of the invention in any way, provided that the attached antigenic peptide does not inhibit binding of the target-binding molecule to the extracellular part of GOLPH2.
  • antigenic peptides are more efficiently displayed by Ml macrophages and/or matured dendritic cells, if the antigenic peptides are attached to a targetbinding molecule that is internalized into the cell upon binding to the extracellular part of GOLPH2.
  • the antigenic peptide is attached to a target-binding molecule that binds specifically to the extracellular part of GOLPH2 and is internalized into the cell upon binding to the extracellular part of GOLPH2.
  • the antigenic peptide is preferably attached to a target-binding molecule that specifically binds to an epitope located within the amino acid sequences of SEQ ID NOs: 23, 24 and/or 25 of GOLPH2.
  • the target-binding molecule is an antibody.
  • the antibody is G2-2opti from WO 2018/091724 or any antibody comprising at least one, at least two, at least three, at least four, at least five or all six CDR sequences of antibody G2-2
  • target-binding molecules that specifically bind to an epitope located within the amino acid sequences of SEQ ID NOs: 23 (human), 24 (murine) and/or 25 (canine) of GOLPH2 have the advantage that they can reach GOLPH2 -positive cells more efficiently than target-binding molecules that specifically bind to the soluble part of GOLPH2.
  • the targetbinding molecule according to the invention preferably comprises the antibody is G2-2opti from WO 2018/091724 or any antibody comprising at least one, at least two, at least three, at least four, at least five or all six CDR sequences of antibody G2-2 from WO 2018/091724.
  • the invention relates to the target-binding molecule according to the invention, wherein the target-binding molecule further comprises an antigenic peptide fused to the C- terminal end of the antibody, or the target-binding fragment thereof.
  • the antigenic peptide may be attached to an antibody or a target-binding fragment thereof. It is commonly known in the art that the N-terminal end of the light and heavy chain of an antibody are involved in antigen binding. Thus, it is preferred that antigenic peptides are attached to the C-terminal end of an antibody or a target-binding fragment thereof. Accordingly, in certain embodiments, the antigenic peptide may be attached to the C-terminal end of the antibody of the invention, or the targetbinding fragment thereof.
  • the antigenic peptide may be fused to the C-terminal end of the antibody, or the target-binding fragment thereof, directly or via a linker.
  • the invention relates to the target-binding molecule according to the invention, wherein the antigenic peptide is fused to the C- terminal end of the antibody, or the target-binding fragment thereof, via a peptide linker.
  • peptide linker refers to a peptide comprised of amino acids.
  • a range of suitable peptide linkers will be known to the person of skill in the art.
  • the peptide linker is 50 amino acids in length or less, for example 20 amino acids or less, such as about 15 amino acids or less, such as about 10 amino acids or less.
  • the linker is rich in glycine and serine amino acids. That is, in certain embodiments, the linker comprises at least one, at least two, at least three, at least four or at lest five glycine or serine amino acids. In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or all amino acids in the linker are glycine or serine amino acids.
  • the linker may comprise a peptidase cleavage site to facilitate processing of the antigenic peptide inside a cell.
  • the peptidase cleavage site may be a cathepsin cleavage site.
  • the peptidase cleavage site may be a Cathepsin B cleavage site.
  • the peptide linker may comprise the sequence GFLGSGFLGS (SEQ ID NO:75)
  • the antigenic peptide is fused to an antibody.
  • the antigenic peptide is fused to the C-terminal end of the heavy and/or light chain of the antibody.
  • the invention relates to the target-binding molecule according to the invention, wherein the antigenic peptide is fused to the C-terminal end of a heavy chain and/or light chain of an antibody.
  • a peptide such as an antigenic peptide
  • a protein such as a proteinaceous target-binding molecule, in particular an antibody or a target-binding fragment thereof.
  • fused refers to two nucleic acids fused together so that the resulting protein is expressed as a single protein.
  • the 3' nucleic acid residue of the coding sequence encoding the antibody, or the target-binding fragment thereof is bonded to the 5' nucleic acid residue of the coding sequence encoding the antigenic peptide, either directly or via a linker.
  • an antigenic peptide is said to be fused to a protein, such as an antibody or an antibody fragment, if the N- terminal amino acid of the antigenic peptide forms a peptide bond with the C-terminal amino acid of at least one polypeptide comprised in an antibody, or a target-binding fragment thereof, according to the invention.
  • a protein such as an antibody or an antibody fragment
  • Methods of molecular biology to fuse an antigenic peptide to a protein at the DNA level are known in the art.
  • the construction of an antibody fused to an antigenic peptide is further demonstrated in Example 27.
  • antibodies fused to antigenic peptides may be constructed by molecular cloning. That is, a restriction enzyme cleavage site may be introduced at the C-terminal end in the coding sequence(s) encoding the heavy and/or light chain of the antibody.
  • codons encoding amino acids at or near the C-terminal end of the heavy and/or light chain of an antibody may be modified such that a restriction enzyme cleavage site is introduced without changing the amino acid sequence of the heavy and/or light chain of the antibody.
  • the restriction enzyme cleavage site is recognized by the enzyme Smal.
  • nucleotide sequence CCCGGG (SEQ ID NO:74) may be introduced at the 3’ end of a nucleic acid molecule encoding an antibody light and/or heavy chain.
  • Many IgG type antibodies comprise the motif PGK at the C-terminal end of the heavy chain.
  • the codons encoding PG may be modified to obtain the Smal restriction site CCCGGG (SEQ ID NO:74).
  • the resulting nucleic acid molecule(s) encoding the heavy and/or light chain of the antibody may then be combined with a nucleic acid molecule encoding a polypeptide comprising one or more antigenic peptides by molecular cloning via a restriction site, such as Smal.
  • the nucleic acid molecule encoding the polypeptide comprising the one or more antigenic peptides preferably comprises a compatible restriction site near the 5’ end of the nucleic acid molecule.
  • a nucleic acid molecule encoding a heavy and/or light chain of an antibody, wherein the heavy and/or light chain is fused, optionally by a suitable linker, to a polypeptide comprising one or more antigenic peptides may be directly obtained by chemical synthesis, as known in the art.
  • the target-binding molecule of the invention is the antibody G2-2-L (see FIG. 13).
  • the antibody G2-2-L is based on the antibody G2-2opti as disclosed herein and further comprises antigenic peptides fused to the C-terminal ends of its light and heavy chains.
  • the light/heavy chain of the antibody is separated from the antigenic peptide by a peptidase cleavage site, more preferably, wherein the peptidase cleavage site is a Cathepsin cleavage site, even more preferably a Cathepsin B cleavage site.
  • the target-binding molecule G2-2-L may comprise four polypeptides comprising antigenic peptides, wherein one polypeptide is fused to each light and heavy chain C-terminal end of the antibody, respectively.
  • the four polypeptides comprising the antigenic peptides may be identical or may be different in sequence. It is further to be understood that each C- terminal end of the antibody may be fused to a polypeptide comprising multiple antigenic peptides. It is further to be understood, that antigenic peptides may be fused only to the C-terminal ends of the heavy chains or only to the C-terminal ends of the light chains of the antibody.
  • the antigenic peptides comprised in G2-2-L may be any antigenic peptide disclosed herein or may be derived from any antigen disclosed herein.
  • the antigenic peptide may be derived from Human papillomavirus type 16 (HPV-16).
  • HPV-16 Human papillomavirus type 16
  • the antigenic peptide may be derived from the E7 protein of HPV-16.
  • the antigenic peptide derived from the E7 protein of HPV-16 may have the sequence PTLHEYMLDLQPE (SEQ ID NO:54) or RAHYNIVTF (SEQ ID NO:55).
  • the antigenic peptide may be derived from Influenza virus.
  • the antigenic peptide may be derived from the Ml protein of Influenza virus.
  • the antigenic peptide derived from the Ml protein of Influenza virus may have the sequence GILGFVFTLT (SEQ ID NO: 56)
  • the antigenic peptide may be derived from SARS coronavirus 2.
  • the antigenic peptide may be derived from the nucleocapsid phosphoprotein of SARS coronavirus 2.
  • the antigenic peptide derived from the nucleocapsid phosphoprotein of SARS coronavirus 2 may have the sequence LLLLDRLNQLESKMS (SEQ ID NO: 57).
  • the antigenic peptide may be derived from the ORF7a protein of SARS coronavirus 2.
  • the antigenic peptide derived from ORF7a of SARS coronavirus 2 may have the sequence VYQLRARSV (SEQ ID NO: 58) or ITLCFTLKR (SEQ ID NO: 59)
  • the antigenic peptide may be derived from Epstein-Barr virus (human gammaherpesvirus 4, EBV).
  • the antigenic peptide may be derived from the latent membrane protein2 (LMP2A) of EBV.
  • LMP2A latent membrane protein2
  • the antigenic peptide derived from LMP2A of EBV may have the sequence CLGGLLTMV (SEQ ID NO: 60).
  • the antigenic peptide may be derived from the protein BSLF2/BMFL1 of EBV.
  • the antigenic peptide derived from BSLF2/BMFL1 of EBV may have the sequence GLCTLVAML (SEQ ID NO: 61).
  • the antigenic peptide may be derived from the Ovalbumin protein.
  • the antigenic peptide derived from Ovalbumin may have the sequence SIINFEKL (SEQ ID NO: 62) or AAHAEINEA (SEQ ID NO:63).
  • the antigenic peptide may be a neoepitope derived from the murine colon cancer cell line MC38.
  • the neoepitope derived from MC38 may be comprised in the sequence LFRAAQLANDVVLQIMEHLELASMTNMELMSSIVVISASIIVFNLLELEG (SEQ ID NO:64) or HLELASMTNMELMSSIVGSKILTFDRL (SEQ ID NO:65),
  • the antigenic peptide may be derived from the antigen tyrosinase-related protein 2 (TRP2).
  • TRP2 antigen tyrosinase-related protein 2
  • the antigenic peptide derived from tyrosinase-related protein 2 may have the sequence SVYDFFVWL (SEQ ID NO: 66).
  • the antigenic peptide may be derived from the antigen glycoprotein 100 (gplOO). In certain embodiments, the antigenic peptide derived from gplOO may have the sequence KVPRNQDWL (SEQ ID NO: 67).
  • two or more antigenic peptides may be comprised in a single polypeptide that is fused to the C-terminal end of a heavy and/or light chain of an antibody.
  • the two or more peptides may be from the same or different origin.
  • the polypeptide may comprise two antigenic peptides derived from HPV-16.
  • the polypeptide may comprise the sequence GFLGSGFLGSTPTLHEYMLDLQPEGSRAHYNIVTF (SEQ ID NO: 47).
  • the polypeptide may comprise antigenic peptides derived from HPV-16 and Influenza virus.
  • the polypeptide may comprise the sequence GFLGSGFLGSGSTPTLHEYMLDLQPEGSGILGFVFTLT (SEQ ID NO:48).
  • the polypeptide may comprise antigenic peptides derived from SARS coronavirus 2 and EBV. In certain embodiments, the polypeptide may comprise the sequence GFLGSGFLGSGLLLLDRLNQLESKMSGSGLCTLVAMLCLGGLLTMV (SEQ ID NO:49).
  • the polypeptide may comprise two antigenic peptides derived from SARS coronavirus 2.
  • the polypeptide may comprise the sequence GFLGSGFLGSVYQLRARSVGSITLCFTLKR (SEQ ID NO:50).
  • the polypeptide may comprise two antigenic peptides derived from Ovalbumin.
  • the polypeptide may comprise the sequence GFLGSGFLGSSIINFEKLGSAAHAEINEA (SEQ ID NO:43).
  • the polypeptide may comprise an MC38 neoepitope. In certain embodiments, the polypeptide may comprise the sequence
  • GFLGSGFLGSLFRAAQLANDVVLQIMEHLELASMTNMELMSSIVVISASIIVFNLLELEG SEQ ID NO:44
  • GFLGSGFLGSHLELASMTNMELMSSIVGSKILTFDRL SEQ ID NO:45
  • the polypeptide may comprise antigenic peptides derived from TRP2 and gplOO. In certain embodiments, the polypeptide may comprise the sequence GFLGSGFLGSSVYDFFVWLGSKVPRNQDWL (SEQ ID NO: 46).
  • the invention relates to the target-binding molecule according to the invention, wherein the target-binding molecule is a fusion antibody, wherein the fusion antibody comprises an Fc region and two or more scFv fragments.
  • the target-binding molecule according to the invention may be a chimeric antibody or a fusion antibody that comprises fragments of two or more antibodies or antibody fragments.
  • the chimeric or fusion antibody may have the format (scFv)2-Fc. That is, the target-binding molecule according to the invention may comprise an Fc region of a first antibody and two or more scFv fragments fused to the Fc region.
  • the two or more scFv fragments may be identical or may be different in amino acid sequence. Further, at least one of the scFv fragments may be derived from the same antibody from which the Fc fragment has been obtained. In other embodiments, the Fc region and the scFv fragments may be derived from different antibodies.
  • the chimeric or fusion antibody is a monospecific antibody of the format (SCFV) 2 -FC. That is, the chimeric or fusion antibody may comprise two scFv fragments fused to the N- terminal end of the Fc region, wherein the two scFv fragments bind to the same antigen and, preferably, to the same epitope.
  • the monospecific antibody of the format (scFv) 2 -Fc comprises two scFv fragments that bind to the extracellular part of GOLPH2 on the surface of monocytes or monocyte -derived cells such that it induces the maturation of said monocytes or monocyte-derived cells.
  • the invention relates to the antibody G2-2HX (human Fc region, SEQ ID NO:72) or G2-2MX (murine Fc region, SEQ ID NO:73) (see FIG.13).
  • G2-2HX and G2-2MX comprise two scFv fragments derived from the antibody G2-2opti disclosed herein.
  • the two scFv fragments (SEQ ID NO:70) are fused to the N-terminal end of a human (SEQ ID NO:38) or murine Fc region (SEQ ID NO:37).
  • the invention relates to the antibody G2-2HX-L (human Fc region) or G2- 2MX-L (murine Fc region) (see FIG. 13).
  • G2-2HX-L and G2-2MX-L are constructed as described above and further comprise polypeptides comprising one or more antigenic peptides fused to the C- terminal ends of the Fc regions as disclosed herein.
  • the chimeric or fusion antibody is a bispecific antibody of the format (scFv) 2 - Fc. That is, the chimeric or fusion antibody comprises two scFv fragments fused to the N-terminal end of the Fc region, wherein the two scFv fragments bind to different antigens.
  • a bispecific antibody of the format (scFv) 2 -Fc may comprise a first scFv fragments that binds specifically to the extracellular part of GOLPH2 on the surface of monocytes or monocyte -derived cells such that it induces the maturation of said monocytes or monocyte-derived cells and a second scFv fragment that binds to another antigen.
  • the other antigen may be an antigenic peptide, an immune checkpoint molecule or a ligand of an immune checkpoint molecule as disclosed herein.
  • the chimeric or fusion antibody is a bispecific antibody of the format (scFv) 2 - Fc-(scFv) 2 . That is, the chimeric or fusion antibody may comprise two scFv fragments fused to the N- terminal end of the Fc region and two scFv fragments fused to the C-terminal end of the Fc region. Preferably, the two scFv fragments fused to the N-terminal end of the Fc region bind specifically to a first epitope and the two scFv fragments fused to the C-terminal end of the Fc region bind specifically to a second epitope.
  • the first epitope may be an epitope in the extracellular part of GOLPH2.
  • the second epitope may be an epitope in an antigenic peptide, an immune checkpoint molecule or a ligand of an immune checkpoint molecule as disclosed herein.
  • the invention relates to the antibody G2-2HXQ (see FIG. 13).
  • G2-2HXQ comprises two scFv fragment derived from the antibody G2-2opti disclosed herein, wherein the two scFv fragments are fused to the N-terminal end of the Fc region set forth in SEQ ID NO:38.
  • G2-2HXQ comprises two scFv fragments that bind specifically to the antigen Myc, wherein the two scFv fragments are fused to the C-terminal end of the Fc region set forth in SEQ ID NO:38.
  • the invention relates to the target-binding molecule according to the invention, wherein each scFv fragment is connected to the Fc region with a peptide linker.
  • the scFv fragments may be connected to the N-terminal or C-terminal end of the Fc region via a peptide linker.
  • peptide linker generally refers to an amino acid based linker that connects two polypeptides, e.g., an Fc region and an scFv.
  • the linker may have any length or sequence. However, it is preferred that the linker has a length between 2 and 50 amino acid residues.
  • the linker that fuses the N-terminal end of an Fc region to an scFv is preferably a longer flexible linker. It has been surprisingly shown by the inventors that so-called “long-neck” antibodies can bind to the extracellular part of GOLPH2 with increased affinity compared to classical antibodies (see FIG. 12 A&B).
  • the linker connecting an scFv fragment to the N-terminal end of an Fc region preferably has a length between 5 and 50 amino acid residues, preferably between 10 and 30 amino acid residues, more preferably between 15 and 25 amino acid residues. Further, it is preferred that the linker connecting an scFv fragment to the N-terminal end of an Fc region preferably is a flexible linker. Flexible linkers known in the art are rich in serine and glycine residues. Thus, the flexible linker connecting an scFv fragment to the N-terminal end of an Fc region preferably comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% serine and/or glycine residues.
  • the linker connecting an scFv fragment to the N-terminal end of an Fc region has the sequence GSSSSSGSSSSGSSGRS (SEQ ID NO:52) or GSSSSSGSSSSGSSGGS (SEQ ID NO:53).
  • the linker connecting an scFv fragment to the C-terminal end of an Fc region may have a length between 2 and 50 amino acid residues, between 2 and 30 amino acid residues, between 2 and 20 amino acid residues, between 2 and 15 amino acid residues or between 2 and 10 amino acid residues.
  • the linker may be rich in glycine or serine residues. That is, the linker connecting an scFv fragment to the C-terminal end of an Fc region may comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% serine and/or glycine residues.
  • the linker connecting an scFv fragment to the C-terminal end of an Fc region may have the sequence GGGS (SEQ ID NO:68).
  • the Fc region comprised in target-binding molecules having the format (scFv)2-Fc or (scFv)2-Fc- (scFyh may comprise an Fc region with a sequence as set forth in SEQ ID NO:37 (murine) or SEQ ID NO:38 (human).
  • obtaining the chimeric antibodies described above comprises a step of molecular cloning (see Examples 20 and 25).
  • the invention relates to the target-binding molecule according to the invention, wherein the antibody is a bispecific or a multispecific antibody.
  • multispecific antibody or “bispecific antibody”, as used herein, refer to an antibody that has binding domains specific for two or more different, preferably non-overlapping, antigens or epitopes within a single antibody molecule. Such antigens or epitopes may be on the same or different targets. If the antigens or epitopes are on different targets, such targets may be on the same cell or on different cells or cell types. It will be appreciated that other molecules in addition to the canonical antibody structure can be constructed with two binding specificities. It will further be appreciated that antigen binding by bispecific antibodies can be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Bispecific antibodies can also be constructed by recombinant means.
  • bispecific antibodies may be produced with the method described by Paul S, Connor J, Nesspor T, et al. An efficient process of generating bispecific antibodies via controlled Fab-arm exchange using culture supernatants. Protein Expr Purif. 2016;121: 133-140. doi: 10.1016/j .pep.2016.01.014.
  • the invention relates to the target-binding molecule according to the invention, wherein the bispecific antibody comprises a Fab or an scFv portion specifically binding to an immune checkpoint molecule.
  • the bispecific or multispecific antibody of the invention comprises two Fab or scFv portions, respectively.
  • a “Fab portion” as used herein is comprised of one light chain and the CHI and variable regions of one heavy chain.
  • An “scFv portion” as used herein is comprised of the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • one or more Fab or scFv portions may be conjugated to an Fc fragment of an antibody.
  • the bi- or multispecific antibody of the invention comprises a first Fab or scFv portion that specifically binds to the extracellular part of GOLPH2 and a second Fab or scFv portion that specifically binds to another target molecule, for example to an immune checkpoint molecule.
  • the invention relates to the target-binding molecule according to the invention, wherein the multispecific antibody comprises a first Fab or scFv portion specifically binding to the extracellular part of GOLPH2 and at least one further Fab or scFv portion specifically binding to an immune checkpoint molecule, a ligand of an immune checkpoint molecule and/or a molecule comprising an antigenic peptide.
  • an immune response in a subject can be elicited or further be increased by administering a bi- or multispecific antibody that specifically binds to the extracellular part of GOLPH2 with one Fab or scFv portion and, specifically binds to an immune checkpoint molecule or a ligand of an immune checkpoint molecule, with another Fab or scFv portion.
  • immune checkpoint molecule is intended to include a group of proteins on the cell surface of immune cells, such as CD4+ and/or CD8+ T cells, dendritic cells, NK cells and macrophages but also on certain tumor cells, that modulate immune responses.
  • an immune check point protein may be either inhibitory, e.g. CTLA4 and PD-1 , or stimulatory, e.g. 0X40 and CD137.
  • immune checkpoint molecules include, without limitation, PD-1, CTLA4, 0X40 (CD134), CD137, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, BTLA and A2aR.
  • the bi- or multispecific antibody of the invention comprises a Fab or scFv portion that blocks an inhibitory check point molecule on the surface of a T cell.
  • inhibitory check point molecules on the surface of a T cell are CTLA4, PD-1, PD-L1, LAG3 and TIM3.
  • the bi- or multispecific antibody comprises a first Fab or scFv portion that specifically binds to the extracellular part of GOLPH2 and a second Fab or scFv portion that specifically binds to CTLA4.
  • the second Fab or scFv portion binds to CTLA4 such that it blocks CTLA4 and prevents it from binding to its cognate receptor.
  • the second Fab or scFv fragment may be any Fab or scFv that blocks CTLA4.
  • the Fab or scFv fragment is obtained or derived from an existing antibody against CTLA4. In certain embodiments, this antibody is Ipilimumab or Tremelimumab.
  • the bi- or multispecific antibody comprises a first Fab or scFv portion that specifically binds to the extracellular part of GOLPH2 and a second Fab or scFv portion that specifically binds to PD-1.
  • the second Fab or scFv portion binds to PD-1 such that it blocks PD-1 and prevents it from binding to its cognate receptor.
  • the second Fab or scFv fragment may be any Fab or scFv that blocks PD-1.
  • the Fab or scFv fragment is obtained or derived from an existing antibody against PD-1. In certain embodiments, this antibody is Nivolumab. Pembrolizumab, Cemiplimab or Spartalizumab.
  • the bi- or multispecific antibody of the invention comprises a Fab or scFv portion that blocks a ligand of an inhibitory check point molecule on the surface of an antigen presenting cell.
  • ligands of inhibitory check point molecules on the surface of an antigen presenting cell are CD80 (ligand of CTLA4 and/or PD-L1), CD86 (ligand of CTLA4), PD-L1 (ligand of PD-1), PD-L2 (ligand of PD-1) and GAL9 (ligand of TIM3).
  • the multispecific antibody of the invention may bind to the extracellular part of GOLPH2 and a ligand of an inhibitory check point molecule on the same antigen presenting cell or on different antigen presenting cells.
  • the bi- or multispecific antibody comprises a first Fab or scFv portion that specifically binds to the extracellular part of GOLPH2 and a second Fab or scFv portion that specifically binds to PD-L1.
  • the second Fab or scFv portion binds to PD-L1 such that it blocks the binding of PD-L1 to its receptor PD-1.
  • the second Fab or scFv fragment may be any Fab or scFv that blocks the binding of PD-L1 to the receptor PD-1.
  • the Fab or scFv fragment is obtained or derived from an existing antibody against PD-L1. In certain embodiments, this antibody is Atezolizumab, Durvalumab or Avelumab.
  • the bi- or multispecific antibody of the invention comprises a Fab or scFv portion that activates a stimulatory check point molecule on the surface of a T cell.
  • stimulatory check point molecules on the surface of a T cell are CD28, ICOS, SLAM, CD2, CD27, 0X40, 4- IBB, CD30, GITR, CD40L, DR3, CD 122 and LIGHT.
  • the bi- or multispecific antibody comprises a first Fab or scFv portion that specifically binds to the extracellular part of GOLPH2 and a second Fab or scFv portion that specifically binds to 4-1BB.
  • the second Fab or scFv portion binds to 4-1BB such that it activates 4- IBB.
  • the second Fab or scFv fragment may be any Fab or scFv that activates 4- IBB.
  • the Fab or scFv fragment is obtained or derived from an existing antibody against 4-1BB. In certain embodiments, this antibody is Urelumab or Utomilumab.
  • the invention relates to the target-binding molecule according to the invention, wherein the immune checkpoint molecule is selected from a group consisting of: CTLA4, PD-1, PD-L1, LAG3, TIM3, CD28, ICOS, SLAM, CD2, CD27, 0X40, 4-1BB, CD30, GITR, CD40L, DR3, CD 122 and LIGHT; and/or wherein the ligand of the immune checkpoint molecule is selected from a group consisting of: CD80, CD86, PD-L1, PD-L2 and GAL9.
  • the invention relates to a bispecific antibody, wherein the antibody comprises a first Fab portion that specifically binds to the extracellular part of GOLPH2 and a second Fab portion that specifically binds to a check point molecule, in particular wherein the check point molecule is PD-1 or CTLA4.
  • the invention relates to a bispecific antibody, wherein the antibody comprises a first Fab portion that specifically bind to the extracellular part of GOLPH2 and a second Fab portion that specifically binds to a ligand of a check point molecule, in particular wherein the ligand of the check point molecule is PD-L1.
  • the invention relates to a bispecific chimeric antibody in which two different scFv portions are fused to an Fc region of an antibody.
  • the invention relates to a bispecific chimeric antibody, wherein the antibody comprises a first scFv portion that specifically bind to the extracellular part of GOLPH2 and a second scFv portion that specifically binds to a check point molecule, in particular wherein the check point molecule is PD-1 or CTLA4.
  • the invention relates to a bispecific chimeric antibody, wherein the antibody comprises a first scFv portion that specifically bind to the extracellular part of GOLPH2 and a second scFv portion that specifically binds to a ligand of a check point molecule, in particular wherein the ligand of the check point molecule is PD-L1.
  • the invention relates to an antibody-antigenic peptide complex comprising the target-binding molecule according to the invention and a molecule comprising an antigenic peptide.
  • the target-binding molecule of the invention may be complexed with an antigenic peptide. That is, the target-binding molecule of the invention may be a bi- or multispecific antibody comprising a first Fab or scFv portion specifically binding to the extracellular part of GOLPH2 and a second Fab or scFv portion specifically binding to a molecule comprising an antigenic peptide.
  • the molecule comprising the antigenic peptide may be any molecule that can be specifically bound by a Fab or scFv portion of a bi- or multispecific antibody of the invention.
  • the molecule comprising the antigenic peptide is a polypeptide, wherein the polypeptide comprises an epitope that is specifically bound by a Fab or scFv portion comprised in the bi- or multispecific antibody of the invention.
  • the invention relates to the antibody-antigenic peptide complex according to the invention, wherein the molecule comprising the antigenic peptide comprises an epitope that is specifically bound by a Fab or scFv portion of the target-binding molecule.
  • the molecule comprising the antigenic peptide consists of the antigenic peptide. That is, the antigenic peptide may be specifically bound by the Fab or scFv portion of the bi- or multispecific antibody of the invention. In other embodiments, the antigenic peptide may be fused to another polypeptide that is specifically bound by the Fab or scFv portion of the bi- or multispecific antibody of the invention.
  • the invention relates to the antibody- antigenic peptide complex according to the invention, wherein the molecule comprising the antigenic peptide is a fusion protein comprising the antigenic peptide fused to a polypeptide comprising an epitope that is specifically bound by the Fab or scFv portion of the target-binding molecule.
  • the antigenic peptide may be fused to any polypeptide that is specifically bound by the Fab or scFv portion of the bi- or multispecific antibody of the invention.
  • the polypeptide that is fused to the antigenic peptide is an intracellular protein or a fragment of an intracellular protein.
  • fusing the antigenic peptide to an intracellular protein, or a fragment thereof reduces the risk of cross reactivity of the second Fab or scFv portion of the bi- or multispecific antibody of the invention with proteins present on the surface of other cells.
  • the invention relates to the antibody-antigenic peptide complex according to the invention, wherein the antigenic peptide is fused to a polypeptide comprising an epitope that is specifically bound by the Fab or scFv portion of the target-binding molecule via a peptide linker.
  • the antigenic peptide may be bound to the polypeptide comprising the epitope that is specifically bound by the Fab or scFv portion of the bi- or multispecific antibody of the invention either directly or via a peptide linker.
  • peptide linker refers to a peptide comprised of amino acids. A range of suitable peptide linkers will be known to the person of skill in the art. In one embodiment, the peptide linker is 50 amino acids in length or less, for example 20 amino acids or less, such as about 15 amino acids or less, such as about 10 amino acids or less. In certain embodiments, the linker is rich in glycine and serine amino acids.
  • the linker comprises at least one, at least two, at least three, at least four or at lest five glycine or serine amino acids. In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or all amino acids in the linker are glycine or serine amino acids. In certain embodiments, the linker is glycyl-serine (GS).
  • GS glycyl-serine
  • the molecule comprising the antigenic peptide may comprise more than one antigenic peptide. That is, the molecule comprising the antigenic peptide may comprise 1, 2, 3, 4, 5, 6, 7, 8 ,9, 10 or more than 10 antigenic peptides. The more than one antigenic peptide may be separated by peptide linkers of may be directly linked to each other.
  • the polypeptide comprising the epitope that is specifically bound by the Fab or the scFv portion of the bi- or multispecific antibody of the invention may be any polypeptide, in particular any intracellular polypeptide or fragment thereof.
  • the polypeptide comprising the epitope that is specifically bound by the Fab or the scFv portion of the bi- or multispecific antibody of the invention may be a synthetic polypeptide.
  • the epitope that is specifically bound by the Fab or the scFv portion of the bi- or multispecific antibody of the invention may be a peptide tag.
  • Numerous peptide tags have been disclosed in the art that can be specifically bound by a Fab and/or scFv portion.
  • Non-limiting examples of peptide tags that may be comprised in a polypeptide that is specifically bound by a Fab or scFv portion include Myc-tag, HA-tag, FLAG-tag and V5-tag.
  • a “peptide tag” as used herein may be any peptide, in particular any peptide that can be specifically bound by a Fab or scFv portion.
  • the fragment EQKLISEEDL (SEQ ID NO:28) of the human Myc protein can be fused to an antigenic peptide and that the resulting fusion protein can form a complex with a multispecific antibody comprising a first Fab or scFv portion specifically binding to the extracellular part of GOLPH2 and a second Fab or scFv portion specifically binding to an epitope comprised within the Myc fragment.
  • the invention relates to the antibody-antigenic peptide complex according to the invention, wherein the polypeptide comprising an epitope that is specifically bound by the Fab or scFv portion of the target-binding molecule is Myc or a Myc fragment.
  • the bi- or multispecific antibody of the invention comprises a Fab or scFv portion that specifically binds to an epitope within the amino acid sequence EQKLISEEDL (SEQ ID NO:28) of the human Myc protein.
  • the Fab or scFv portion specifically binding to Myc comprises a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO:32 or 40; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO:36.
  • the Fab or scFv portion specifically binding to Myc comprises a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NO:30, CDR2 as defined in SEQ ID NO:31 and CDR3 as defined in SEQ ID NO:32 or 42; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO:34, CDR2 as defined in SEQ ID NO:35 and CDR3 as defined in SEQ ID NO: 36.
  • VH variable heavy
  • VL variable light
  • the scFv portion specifically binding to Myc comprises the amino acid sequence of SEQ ID NO:39 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:39.
  • the scFv portion specifically binding to Myc is a humanized form of the scFv comprising the amino acid sequence of SEQ ID NO: 39. That is, the scFv portion specifically binding to Myc may be derived from a human framework region comprising a heavy chain CDR1 as defined in SEQ ID NO:30, a heavy chain CDR2 as defined in SEQ ID NO:31 and a heavy chain CDR3 as defined in SEQ ID NO:32 or 40; and a light chain CDR1 as defined in SEQ ID NO:34, a light chain CDR2 as defined in SEQ ID NO:35 and a light chain CDR3 as defined in SEQ ID NO:36
  • the Fab fragments specifically binding to Myc comprises a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO:29, or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 29, and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO:33, or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:33.
  • VH variable heavy
  • VL variable light
  • the Fab fragment specifically binding to Myc is a humanized Fab fragment.
  • the humanized Fab fragment specifically binding to Myc may comprise a human framework region comprising a heavy chain CDR1 as defined in SEQ ID NO:30, a heavy chain CDR2 as defined in SEQ ID NO:31 and a heavy chain CDR3 as defined in SEQ ID NO:32 or 40; and a light chain CDR1 as defined in SEQ ID NO:34, a light chain CDR2 as defined in SEQ ID NO:35 and a light chain CDR3 as defined in SEQ ID NO: 36.
  • the invention relates to an antibody-antigenic peptide construct comprising an antibody, or a target-binding fragment thereof, specifically binding to GOLPH2 and an antigenic peptide, wherein the antigenic peptide is: a) fused to a C-terminal end of the antibody, or the target-binding fragment thereof; and/or b) comprised in a molecule that is specifically bound by a Fab or scFv portion comprised in the antibody, or the target-binding fragment thereof.
  • the invention relates to an antibody that specifically binds to GOLPH2, in particular the extracellular part of GOLPH2, and further comprises an antigenic peptide.
  • the antibody may be any antibody that binds to GOLPH2, in particular to the extracellular part of GOLPH2.
  • the antibody may be any type of antibody described elsewhere herein, in particular a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody, a bispecific antibody or a multispecific antibody.
  • the invention relates to an antibody-antigenic peptide construct according to the invention, wherein the antibody is a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody or a multispecific antibody.
  • the antibody may be any antibody binding to an epitope within the amino acid sequences SEQ ID NO: 23 to 27.
  • the antibody may be or may be derived from the antibodies G2-2, G2-4, PA5-18100 or EPR3606 as described herein.
  • the antigenic peptide may be attached to the antibody in different ways.
  • the antigenic peptide may be fused to the C-terminal end of an antibody binding specifically to GOLPH2.
  • the antigenic peptide may be fused to an antibody binding specifically to GOLPH2 as described elsewhere herein. That is, in a particular embodiment, the invention relates to an antibody-antigenic peptide construct according to the invention, wherein the antigenic peptide is fused to a C-terminal end of the antibody or the target-binding fragment thereof via a peptide linker.
  • the invention relates to an antibody-antigenic peptide construct according to the invention, wherein the antigenic peptide is fused to the C-terminal end of a heavy chain and/or light chain of an antibody.
  • the antibody of the invention may be complexed with a molecule comprising an antigenic peptide. That is, the antibody may be a bi- or multispecific antibody comprising a Fab or scFv portion that specifically binds to the molecule comprising the antigenic peptide.
  • the invention relates to the antibody-antigenic peptide construct according to the invention, wherein the antibody is a multispecific antibody.
  • the invention relates to the antibody-antigenic peptide construct according to the invention, wherein the multispecific antibody comprises a first Fab or scFv portion specifically binding to GOLPH2 and a second Fab or scFv portion specifically binding to a molecule comprising an antigenic peptide.
  • the antibody of the invention may be a bi- or multispecific antibody, wherein the bi- or multispecific antibody specifically binds to at least two different targets, in particular the extracellular part of GOLPH2 and a molecule comprising an antigenic peptide.
  • the molecule comprising the antigenic peptide is the entire antigen the antigenic peptide has been derived from. That is, the antigen is specifically bound by a Fab or scFv portion comprised in the bi- or multispecific antibody. In other embodiments, the molecule comprising the antigenic peptide is the antigenic peptide. That is, the antigenic peptide is specifically bound by a Fab or scFv portion comprised in the bi- or multispecific antibody.
  • the molecule comprising the antigenic peptide is a fusion protein comprising an antigen or an antigenic peptide fused to a second polypeptide, wherein the second polypeptide comprises an epitope that is specifically bound by a Fab or scFv portion comprised in the bi- or multispecific antibody.
  • the invention relates to the antibody-antigenic peptide construct according to the invention, wherein the molecule comprising the antigenic peptide is a fusion protein comprising the antigenic peptide fused to a polypeptide comprising an epitope that is specifically bound by the second Fab or scFv portion.
  • the invention relates to the antibody-antigenic peptide construct according to the invention, wherein the antigenic peptide is fused to the polypeptide comprising the epitope that is specifically bound by the second Fab or scFv portion via a peptide linker. That is, the antigenic peptide and the polypeptide comprising the epitope that is specifically bound by the Fab or scFv portion of the bi- or multispecific antibody of the invention may be fused directly or may be fused by a peptide linker, as described elsewhere herein.
  • the invention relates to the antibody-antigenic peptide construct according to the invention, wherein the polypeptide comprising the epitope that is specifically bound by the second Fab or scFv portion is Myc or a Myc fragment and wherein the second Fab or scFv portion specifically binds to an epitope within Myc or said Myc fragment.
  • the polypeptide comprising the epitope that is specifically bound by the Fab or scFv portion of the antibody may have any origin. That is, the polypeptide may be a synthetic polypeptide or may be a naturally occurring polypeptide.
  • the polypeptide comprising the epitope that is specifically bound by the Fab or scFv portion of the antibody is not limiting and may be any polypeptide that is specifically bound by the Fab or scFv porion of the bi- or multispecific antibody.
  • the polypeptide comprising the epitope that is specifically bound by the second Fab or scFv portion is a fragment of an intracellular protein.
  • the polypeptide comprising the epitope that is specifically bound by the second Fab or scFv portion comprises a peptide tag, such as a Myc-tag, an HA-tag, a FLAG-tag or a V5-tag.
  • the polypeptide comprising the epitope that is specifically bound by the second Fab or scFv portion is a fragment of Myc protein, in particular the fragment EQKLISEEDL (SEQ ID NO:28).
  • the invention relates to the antibody-antigenic peptide construct according to the invention, wherein the multispecific antibody comprises a further Fab or scFv portion specifically binding to an immune checkpoint molecule or a ligand of an immune checkpoint molecule.
  • the invention relates to the antibody-antigenic peptide construct according to the invention, wherein the immune checkpoint molecule is selected from a group consisting of: CTLA4, PD-1, PD-L1, LAG3, TIM3, CD28, ICOS, SLAM, CD2, CD27, 0X40, 4-1BB, CD30, GITR, CD40L, DR3, CD 122, LIGHT, TIGIT, VISTA, B7-H3 and BTLA; and/or wherein the ligand of the immune checkpoint molecule is selected from a group consisting of: CD80, CD86, PD-L1, PD-L2 and GAL9.
  • the immune checkpoint molecule is selected from a group consisting of: CTLA4, PD-1, PD-L1, LAG3, TIM3, CD28, ICOS, SLAM, CD2, CD27, 0X40, 4-1BB, CD30, GITR, CD40L, DR3, CD 122, LIGHT, TIGIT, VISTA
  • the antibody-antigenic peptide construct according to the invention may be a bispecific antibody comprising a first Fab or scFv portion binding specifically to the extracellular part of GOLPH2.
  • the antibody-antigenic peptide construct may comprise a second Fab or scFv portion binding specifically to a molecule comprising an antigenic peptide.
  • the antibody-antigenic peptide construct may comprise at least one further antigenic peptide fused to a C-terminal end of the antibody.
  • the antibody-antigenic peptide construct may comprise two or more antigenic peptides, wherein at least one antigenic peptide is comprised in a molecule that is specifically bound by a Fab or scFv portion of the antibody and at least one antigenic peptide is fused to a C-terminal end of the antibody.
  • the at least one antigenic peptide may be fused to the C-terminal end of the first light chain of the antibody and/or the C-terminal end of the second light chain of the antibody and/or the C-terminal end of the first heavy chain of the antibody and/or the C-terminal end of the second heavy chain of the antibody.
  • the antibody- antigenic peptide construct may be a bispecific antibody comprising antigenic peptides fused to up to four C-terminal ends of the antibody and, additionally, at least one antigenic peptide comprised in a molecule that is specifically bound by a Fab or scFv portion of the bispecific antibody.
  • the two or more antigenic peptides comprised in the antibody-antigenic peptide construct may be identical or may differ in their amino acid sequence.
  • the antibody-antigenic peptide construct according to the invention may be a bispecific antibody comprising a first Fab or scFv portion binding specifically to the extracellular part of GOLPH2.
  • the antibody-antigenic peptide construct may comprise a second Fab or scFv portion binding specifically to an immune checkpoint molecule, in particular any one of the immune checkpoint molecules disclosed herein.
  • the antibody-antigenic peptide construct may comprise at least one antigenic peptide fused to a C-terminal end of the antibody.
  • the at least one antigenic peptide may be fused to the C-terminal end of the first light chain of the antibody and/or the C-terminal end of the second light chain of the antibody and/or the C-terminal end of the first heavy chain of the antibody and/or the C-terminal end of the second heavy chain of the antibody. That is, the bispecific antibody may be a bispecific antibody comprising up to four antigenic peptides fused to the C-terminal ends of the antibody. In cases where the antibody-antigenic peptide construct comprises more than one antigenic peptide, the two or more antigenic peptides may be identical or may differ in their amino acid sequence.
  • the bispecific antibody described above may have any format.
  • the antibody may comprise an Fc region and two Fab portions, wherein the first Fab portion binds specifically to the extracellular part of GOLPH2 and the second Fab portion binds specifically to an immune checkpoint molecule or to a molecule comprising an antigenic peptide.
  • the antibody may comprise an Fc region and two scFv portions, wherein the first scFv portion binds specifically to the extracellular part of GOLPH2 and the second scFv portion binds specifically to an immune checkpoint molecule or to a molecule comprising an antigenic peptide.
  • the antibody-antigenic peptide construct of the invention may further comprise a tri- or tetraspecific antibody.
  • the tri- or tetraspecific antibody may be based on any of the bispecific antibodies disclosed above and additionally comprise one or two scFv portions fused to the Fc region of the bispecific antibody, in particular to the C-terminal end(s) of one or both heavy chains.
  • the one or two scFv portions fused to the C-terminal end(s) of the Fc-region specifically bind to a molecule comprising an antigenic peptide.
  • the two scFv portions may be identical, e.g. bind to the same epitope, or may differ from each other, e.g. bind to different epitopes.
  • the tri- or tetraspecific antibody of the invention comprises an Fc region, two Fab portions and one or two scFv portions, wherein the two scFv portions are preferably fused to the C-terminal end of the Fc region.
  • the tri- or tetraspecific antibody of the invention comprises an Fc region and three or four scFv portions, wherein two scFv portions are fused to the N-terminal end of the Fc region and one or two scFv portions are fused to the C-terminal end of the Fc region.
  • the antibody-antigenic peptide construct disclosed above comprises at least one Fab or scFv portion binding specifically to the extracellular part of GOLPH 2.
  • at least one Fab or scFv portion specifically binds to an epitope within the peptide sequences SEQ ID NO:23 to 27 comprised in the extracellular part of GOLPH2.
  • at least on Fab or scFv portion comprised in the antibody-antigenic peptide construct is derived from antibodies G2-2, G2- 2opti and/or G2-4.
  • the invention relates to the antibody-antigenic peptide construct according to the invention, wherein antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO.9; or (b) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO.17.
  • VH variable heavy
  • VL variable light chain sequence comprising CDR3 as defined in SEQ ID NO.9
  • the invention relates to the antibody-antigenic peptide construct according to the invention, wherein the antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NON, CDR2 as defined in SEQ ID NO:5 and CDR3 as defined in SEQ ID N0:6; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO.7, CDR2 as defined in SEQ ID NO: 8 and CDR3 as defined in SEQ ID NO:9; or (b) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NO: 12, CDR2 as defined in SEQ ID NO: 13 and CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO: 15, CDR2 as defined in SEQ ID NO: 16 and CDR3 as defined in SEQ ID NO: 17.
  • VH variable heavy
  • VL
  • invention further encompasses humanized antibodies comprising the CDR sequences disclosed herein.
  • the invention relates to the antibody-antigenic peptide construct according to the invention, wherein the antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 18 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:2 or SEQ ID NO: 18; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 19 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:3 or SEQ ID NO: 19; or (b) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO: 10 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 10; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 11.
  • VH variable heavy
  • the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct according to the invention comprises one or more antigenic peptides.
  • antigenic peptide refers to a peptide, which is prone to induce/elicit, increase, prolong or maintain an immune response in a subject to whom it is administered.
  • an antigenic peptide may be a peptide that is specifically recognized by a B- or T-cell of the subject the antigenic peptide is administered to.
  • Antigenic peptides are preferably peptides that can be presented by an MHC molecule on the cell surface of an antigen-presenting cell.
  • antigenic peptides are peptides that can be displayed by an MHC class I or MHC class II molecules on the cell surface of an antigen-presenting cell.
  • Peptides that are specifically bound by MHC class I molecules preferably have a size of 8 to 10 amino acids in length.
  • Peptides that are specifically bound by MHC class II molecules preferably have a size of 15 to 24 amino acids in length.
  • the antigenic peptide of the invention may be of any origin. However, it is preferred that the antigenic peptide is of mammalian, in particular human, fungal, bacterial or viral origin. In certain embodiments, the antigenic peptide is a tumor antigen or is derived from a tumor antigen.
  • tumor antigen comprises tumor-specific antigens and tumor- associated antigens.
  • tumor antigen or “tumor protein” designates herein an antigenic substance produced in tumor cells, and sometimes also in normal cells, and which can trigger an immune response upon administration in a subject.
  • overexpressed selfantigens such as HER2/neu and its variant dHER2, p53, Wilm's Tumor 1 , Ephrin receptor, Proteinase-3, Mucin-1 , Mesothelin, EGFR, CD20
  • cancer-testis (CT) antigens such as MAGE-1, BAGE, GAGE, NY-ESO-1
  • mutational antigens also known as neo-antigens (such as mutants from MUM-1 , bcr-abl, ras, b-raf, p53, CDK-4, CDC27, beta-catenin, alpha-actenin-4); tissue-specific differentiation antigens (such as the melanoma antigens Melan A/MART-1, tyrosinase, TRPl/pg75, TRP2, gplOO and gangliosides GM3, GM2,
  • IL-13Ra2 Interleukin- 13 receptor subunit alpha-2
  • IL13RA2 is a membrane bound protein that in humans is encoded by the IL13RA2 gene.
  • IL13RA2 has been reported as a potential immunotherapy target (see Beard et al.; Clin Cancer Res; 72(11); 2012).
  • the high expression of IL13RA2 has further been associated with invasion, liver metastasis and poor prognosis in colorectal cancer (Barderas et al.; Cancer Res; 72(11); 2012).
  • the tumor antigen is selected from the group consisting of 5-a-reductase, a- fetoprotein (“AFP”), AM-1, APC, April, B melanoma antigen gene (“BAGE”), -catenin, Bel 12, bcr- abl, Brachyury, CA-125, caspase-8 (“CASP-8”, also known as “FLICE”), Cathepsins, CD 19, CD20, CD21 /complement receptor 2 (“CR2”), CD22/BL-CAM, CD23/FcaRII, CD33, CD35/complement receptor 1 (“CR1”), CD44/PGP-1, CD45/leucocyte common antigen (“LCA”), CD46/membrane cofactor protein (“MCP”), CD52/CAMPATH-1, CD55/decay accelerating factor (“DAF”), CD59/protectin, CDC27, CDK4, carcinoembryonic antigen (“CEA”), c-myc, cyclooxygenas
  • AFP
  • interleukin- 13 receptor (“IL-13R”), inducible nitric oxide synthase (“iNOS”), Ki67, KIAA0205, K- ras, H-ras, N-ras, KSA, LKLR-FUT, melanoma antigen-encoding gene 1 (“MAGE-1”), melanoma antigen-encoding gene 2 (“MAGE-2”), melanoma antigen-encoding gene 3 (“MAGE-3”), melanoma antigen-encoding gene 4 (“MAGE-4”), mammaglobin, MAP 17, Melan-A/melanoma antigen recognized by T-cells- 1 (“MART-1”), mesothelin, MIC A/B, MT-MMPs, mucin, testes-specific antigen NY-ESO-1, osteonectin, pl5, P170/MDR1, p53, p97/melanotransferrin, PAI-1, platelet- derived growth factor (“
  • the antigenic peptide is a viral antigen or is derived from a viral antigen.
  • viral antigen refers to any viral component having antigenic properties, i.e. being able to provoke an immune response in an individual.
  • the viral antigen may be derived from a viral ribonucleoprotein or an envelope protein.
  • the viral antigen is derived from a virus selected from the group consisting of adenovirus, Arbovirus, Astrovirus, Coronavirus, Coxsackievirus, Crimean-Congo hemorrhagic fever virus, cytomegalovirus (“CMV”), dengue virus, Ebola virus, Epstein-Barr virus (“EBV”), Foot-and-mouth disease virus, Guanarito virus, Hendra virus, herpes simplex virus-type 1 (“HSV-1”), herpes simplex virus-type 2 (“HSV-2”), human herpesvirus-type 6 (“HHV-6”), human herpesvirus-type 8 (“HHV-8”), hepatitis A virus (“HAV”), hepatitis B virus (“HBV”), hepatitis C virus (“HCV”), hepatitis D virus (“HDV”), hepatitis E virus (“HEV”), human immunodeficiency virus (“HIV”), influenza virus, Japanese encephalitis virus, Junin virus, Las
  • the antigenic peptide is a microbial antigen or is derived from a microbial antigen.
  • microbial antigen refers to any microbial component having antigenic properties, i.e. being able to provoke an immune response in an individual.
  • the antigenic peptide is a bacterial antigen or is derived from a bacterial antigen.
  • bacterial antigen refers to any bacterial component having antigenic properties, i.e. being able to provoke an immune response in an individual.
  • the bacterial antigen may be derived from the cell wall or cytoplasm membrane of a bacterium.
  • the bacterial antigen is derived from a bacterium selected from the group consisting of Bacillus anthracis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Burkholderia mallei, Burkholderia pseudomallei, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diptheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli, Escherichia coli) 157:H
  • the antigenic peptide is a fungal antigen or is derived from a fungal antigen.
  • the fungal antigen is derived from a fungus selected from the group consisting of Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Blastomyces dermatitidis, Candida albicans, Candida dubliniensis, Candida glabrata, Candida parapsilosis, Candida rugosa, Candida tropicalis, Cryptococcus albidus, Cryptococcus gattii, Cryptococcus laurentii, Cryptococcus neoformans, Histoplasma capsulatum, Microsporum canis, Pneumocystis carinii, Pneumocystis jirovecii, Sporothrix schenckii, Stachbotrys chartarum, Tinea barbae, Tinea captitis, Tinea corporis,
  • the antigenic peptide is a zooparasite antigen or is derived from a zooparasite antigen.
  • zooparasite antigen refers to any component of a parasite of an animal having antigenic properties, i.e. being able to provoke an immune response in an individual. Said parasite may be a flea, louse, or worm.
  • the zooparasite antigen is derived from a parasite selected from the group consisting of Anisakis spp.
  • Babesia spp. Baylisascaris procyonis, Cryptosporidium spp., Cyclospora cayetanensis, Diphyllobothrium spp., Dracunculus medinensis, Entamoeba histolytica, Giardia duodenalis, Giardia intestinalis, Giardia lamblia, Leishmania sp., Plasmodium falciparum, Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Taenia spp., Toxoplasma gondii, Trichinella spiralis, and Trypanosoma cruzi.
  • an antigenic peptide is a fragment or a derivative of an antigen.
  • an antigen is a naturally occurring molecule, preferably a protein. Antigens may be taken up by antigen-presenting cells, such as Ml macrophages or matured dendritic cells, processed and presented on the surface of the antigen-presenting cell by an MHC molecule.
  • the antigenic peptide of the invention may be any fragment or derivative of an antigen that can be displayed by an MHC class I or MHC class II molecule.
  • the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct of the invention is said to comprise an antigenic peptide
  • the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct may comprise an entire antigen, a fragment of said antigen or only an antigenic peptide derived from that antigen, e.g. a peptide that can be presented by an MHC molecule.
  • the antigenic peptide may be derived from Human papillomavirus type 16 (HPV-16).
  • HPV-16 Human papillomavirus type 16
  • the antigenic peptide may be derived from the E7 protein of HPV-16.
  • the antigenic peptide derived from the E7 protein of HPV-17 may have the sequence PTLHEYMLDLQPE (SEQ ID NO:54) or RAHYNIVTF (SEQ ID NO:55).
  • the antigenic peptide may be derived from Influenza virus.
  • the antigenic peptide may be derived from the Ml protein of Influenza virus.
  • the antigenic peptide derived from the Ml protein of Influenza virus may have the sequence GILGFVFTLT (SEQ ID NO: 56)
  • the antigenic peptide may be derived from SARS coronavirus 2.
  • the antigenic peptide may be derived from the nucleocapsid phosphoprotein of SARS coronavirus 2.
  • the antigenic peptide derived from the nucleocapsid phosphoprotein of SARS coronavirus 2 may have the sequence LLLLDRLNQLESKMS (SEQ ID NO: 57).
  • the antigenic peptide may be derived from the ORF7a protein of SARS coronavirus 2.
  • the antigenic peptide derived from ORF7a of SARS coronavirus 2 may have the sequence VYQLRARSV (SEQ ID NO: 58) or ITLCFTLKR (SEQ ID NO: 59)
  • the antigenic peptide may be derived from Epstein-Barr virus (human gammahervesvirus 4, EBV).
  • the antigenic peptide may be derived from the latent membrane protein2 (LMP2A) of EBV.
  • LMP2A latent membrane protein2
  • the antigenic peptide derived from LMP2A of EBV may have the sequence CLGGLLTMV (SEQ ID NO: 60).
  • the antigenic peptide may be derived from the protein BSLF2/BMFL1 of EBV.
  • the antigenic peptide derived from BSLF2/BMFL1 of EBV may have the sequence GLCTLVAML (SEQ ID NO: 61).
  • the antigenic peptide may be derived from the Ovalbumin protein.
  • the antigenic peptide derived from Ovalbumin may have the sequence SIINFEKL (SEQ ID NO: 62) or AAHAEINEA (SEQ ID NO:63).
  • the antigenic peptide may be a neoepitope derived from the nurine colon cancer cell line MC38.
  • the neoepitope derived from MC38 may be comprised in the sequence LFRAAQLANDVVLQIMEHLELASMTNMELMSSIVVISASIIVFNLLELEG (SEQ ID NO:64) or HLELASMTNMELMSSIVGSKILTFDRL (SEQ ID NO:65),
  • the antigenic peptide may be derived from the antigen tyrosinase-related protein 2 (TRP2).
  • TRP2 antigen tyrosinase-related protein 2
  • the antigenic peptide derived from tyrosinase-related protein 2 may have the sequence SVYDFFVWL (SEQ ID NO: 66).
  • the antigenic peptide may be derived from the antigen glycoprotein 100 (gplOO). In certain embodiments, the antigenic peptide derived from gplOO may have the sequence KVPRNQDWL (SEQ ID NO: 67).
  • the target-binding molecule, antibody-antigenic peptide complex or antibody-antigenic peptide construct of the invention may comprise one or more entire antigens.
  • an antigen may be fused to the C-terminal end of a target-binding molecule or antibody and/or may be fused to a polypeptide comprising an epitope that specifically bound by a Fab or scFv portion of an antibody of the invention.
  • the target-binding molecule, antibody-antigenic peptide complex or antibody-antigenic peptide construct of the invention comprises one or more antigenic peptide(s).
  • the target-binding molecule, antibody-antigenic peptide complex or antibody-antigenic peptide construct of the invention comprises fragments or derivatives of antigens, in particular fragments or derivatives of antigens that can be displayed by an MHC class I or MHC class II molecule.
  • two or more antigenic peptides may be combined in a polypeptide.
  • the two or more antigenic peptides that are combined in a polypeptide may derive from the same or different protein and/or species or may derive from different proteins and/or species.
  • Two antigenic peptides comprised in a single polypeptide may be separated by a linker, for example the linker glycyl-serine (GS).
  • GS linker glycyl-serine
  • a polypeptide comprising one or more antigenic peptides may comprise sequences that allow coupling of the polypeptide to the target-binding molecule of the invention. That is, the polypeptide may comprise an epitope that can be specifically bound by the target-binding molecule of the invention.
  • the polypeptide may comprise an epitope that can be specifically bound by a Fab or scFv fragment comprised in the target-binding molecule of the invention.
  • the epitope may be derived from the Myc protein and have the sequence EQKLISEEDL (SEQ ID NO: 28).
  • the polypeptide comprising one or more antigenic peptides may comprise a peptide linker for conjugating the polypeptide to a C-terminal end of the light and/or heavy chain of an antibody.
  • the linker may comprise the sequence GFLGSGFLGS (SEQ ID NO:75).
  • amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody.
  • Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g, target -binding.
  • antibody variants having one or more amino acid substitutions are provided.
  • Sites of interest for substitutional mutagenesis include the CDRs and FRs.
  • Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions.” More substantial changes are provided in Table 1 under the heading of "exemplary substitutions,” and as further described below in reference to amino acid side chain classes.
  • Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
  • Amino acids may be grouped according to common side-chain properties:
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody).
  • a parent antibody e.g. a humanized or human antibody
  • the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody.
  • An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).
  • Alterations may be made in CDRs, e.g., to improve antibody affinity. Such alterations may be made in CDR "hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity.
  • Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al.
  • affinity maturation diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis).
  • a secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity.
  • Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized.
  • CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling.
  • CDR H3 and CDR-L3 in particular are often targeted.
  • substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen.
  • conservative alterations e.g., conservative substitutions as provided herein
  • Such alterations may be outside of CDR "hotspots" or SDRs.
  • each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • a useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244: 1081-1085.
  • a residue or group of target residues e.g., charged residues such as arg, asp, his, lys, and glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions.
  • a crystal structure of an antigenantibody complex is used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution.
  • Variants may be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N- terminal methionyl residue.
  • Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
  • an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated.
  • Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
  • the carbohydrate attached thereto may be altered.
  • Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997).
  • the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.
  • antibody variants having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fe region residues); however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).
  • Examples of cell lines capable of producing defucosylated antibodies include Lecl3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al., especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells ( see, e.g., Yamane-Ohnuki et al. Bioteeh. Bioeng. 87: 614 (2004); Kanda, Y. et al., Bioteehnol. Bioeng., 94(4):680-688 (2006); and W02003/085 107).
  • Antibody variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided.
  • Such antibody variants may have improved CDC function.
  • Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
  • one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant.
  • the Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.
  • the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious.
  • In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • NK cells express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII and Fc(RIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).
  • Nonlimiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 ( see, e.g. Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc.
  • Antibody effector functions refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
  • non-radioactive assays methods may be employed (see, for example, ACTITM non radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI).
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. sci. USA 95:652-656 (1998).
  • Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.
  • a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al., Blood 101: 1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)).
  • FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).
  • Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056).
  • Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).
  • Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).
  • an antibody variant comprises a Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).
  • alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
  • CDC Complement Dependent Cytotoxicity
  • Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus are described in US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn.
  • Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826).
  • cysteine engineered antibodies e.g., "thioMAbs”
  • one or more residues of an antibody are substituted with cysteine residues.
  • the substituted residues occur at accessible sites of the antibody.
  • reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein.
  • any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; Al 18 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.
  • Cysteine engineered antibodies may be generated as described, e.g., in U.S. Patent No. 7,521,541.
  • an antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available.
  • the moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers.
  • water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol),
  • PEG
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
  • conjugates of an antibody and non-proteinaceous moiety that may be selectively heated by exposure to radiation are provided.
  • the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)).
  • the radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the non-proteinaceous moiety to a temperature at which cells proximal to the antibody- non-proteinaceous moiety are killed.
  • Anti-GOLPH2 antibodies provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.
  • an antibody of the invention is tested for its antigen binding activity, e.g., by known methods such as ELISA, BIACore®, FACS, immunofluorescence or immunohistochemistry.
  • competition assays may be used to identify an antibody that competes with any of the antibodies described herein for binding to the extracellular part of GOLPH2.
  • a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) that is bound by an antibody described herein.
  • epitope e.g., a linear or a conformational epitope
  • Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).
  • immobilized GOLPH2 is incubated in a solution comprising a first labeled antibody that binds to GOLPH2 (e.g., any of the antibodies described herein) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to GOLPH2.
  • a first labeled antibody that binds to GOLPH2 e.g., any of the antibodies described herein
  • second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to GOLPH2.
  • immobilized GOLPH2 is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to GOLPH2, excess unbound antibody is removed, and the amount of label associated with immobilized GOLPH2 is measured.
  • an antibody may be conjugated to a "receptor” (such as streptavidin) for utilization in tumor pre-targeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a drug or radionucleotide).
  • a receptor such as streptavidin
  • any of the anti-GOLPH2 antibodies provided herein is useful for detecting the presence of GOLPH2 in a biological sample.
  • the term “detecting” as used herein encompasses quantitative or qualitative detection.
  • a “biological sample” comprises, e.g., a cell or tissue (e.g., biopsy material, including cancerous or potentially cancerous lymphoid tissue, such as lymphocytes, lymphoblasts, monocytes, myelomonocytes, and mixtures thereof).
  • labeled anti-GOLPH2 antibodies include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron- dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction.
  • Exemplary labels include, but are not limited to, the radioisotopes 32 P, 14 C, 125 1, 3 H, and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Patent No.
  • luciferin 2,3 -dihydrophthalazinediones
  • horseradish peroxidase HRP
  • alkaline phosphatase B- galactosidase
  • glucoamylase lysozyme
  • saccharide oxidases e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase
  • heterocyclic oxidases such as uricase and xanthine oxidase
  • an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.
  • a label is a positron emitter.
  • Positron emitters include but are not limited to 68 Ga, 18 F, 64 Cu, 86 Y, 76 Br, 89 Zr, and 124 1.
  • a positron emitter is 89 Zr.
  • the invention relates to a method for producing the target-binding molecule according to the invention, in particular the antibody according to the invention. That is, the present invention also relates to the production of specific antibodies against native polypeptides and recombinant polypeptides of GOLPH2. This production is based, for example, on the immunization of animals, like mice. However, also other animals for the production of antibody/antisera are envisaged within the present invention. For example, monoclonal and polyclonal antibodies can be produced by rabbit, mice, goats, donkeys and the like.
  • the polynucleotide encoding a correspondingly chosen polypeptide of GOLPH2 can be subcloned into an appropriated vector, wherein the recombinant polypeptide is to be expressed in an organism being able for an expression, for example in bacteria.
  • the expressed recombinant protein can be intra-peritoneally injected into a mouse and the resulting specific antibody can be, for example, obtained from the mice serum being provided by intracardiac blood puncture.
  • the present invention also envisages the production of specific antibodies against native polypeptides and recombinant polypeptides by using a DNA vaccine strategy as exemplified in the appended examples.
  • DNA vaccine strategies are well-known in the art and encompass liposome-mediated delivery, by gene gun or jet injection and intramuscular or intradermal injection.
  • antibodies directed against a polypeptide or a protein or an epitope of GOLPH2, in particular the extracellular part of GOLPH2 or epitopes thereof can be obtained by directly immunizing the animal by directly injecting intramuscularly the vector expressing the desired polypeptide or a protein or an epitope of GOLPH2, in particular the epitope or extracellular part of GOLPH2 that is recognized by the antibodies of the invention.
  • the amount of obtained specific antibody can be quantified using an ELISA, which is also described herein below.
  • Further methods for the production of antibodies are well known in the art, see, e.g. Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988.
  • the specified antibodies and the corresponding epitope or part of GOLPH2 bind to one another and do not bind in a significant amount to other components present in a sample.
  • Specific binding to a target analyte under such conditions may require a binding moiety that is selected for its specificity for a particular target analyte.
  • a variety of immunoassay formats may be used to select antibodies specifically reactive with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with an analyte.
  • anti-GOLPH2 antibody also includes bi- or multispecific antibody that bind specifically to GOLPH2 and to one or more additional target, e.g. an immune checkpoint molecule or a molecule comprising an antigenic peptide.
  • additional target e.g. an immune checkpoint molecule or a molecule comprising an antigenic peptide.
  • anti-GOLPH2 antibody also includes the mono-, bi- or multispecific antibodies of the invention that comprise one or more antigenic peptides.
  • the target-binding molecule according to the invention may be an anti-GOLPH2 antibody.
  • the antibody-antigenic peptide complex or the antibody-antigenic peptide construct according to the invention may comprise an anti-GOLPH2 antibody.
  • the invention relates to an immunoconjugate comprising the targetbinding molecule according to the invention, the antibody-antigenic peptide complex according to the invention or the antibody-antigenic peptide construct according to the invention and a cytotoxic agent or a prodrug of a cytotoxic agent.
  • the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct of the invention may be non-conjugated or may be conjugated to a cytotoxic agent or a prodrug of a cytotoxic agent.
  • FIGs. 1 and 2 show that both an antibody binding to the extracellular part of GOLPH2, as well as different immunoconjugates of the same antibody have the potential to induce the maturation of monocytes. Accordingly, it is plausible that the fusion of a cytotoxic agent or a prodrug of a cytotoxic agent does not interfere or, at least, not significantly interferes with the function of the target-binding molecule, the antibody-antigenic peptide complex or the antibody- antigenic peptide construct of the invention.
  • the decision if the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct of invention is conjugated to a cytotoxic agent or a prodrug of a cytotoxic agent may depend on the intended use of the target-binding molecule, the antibody- antigenic peptide complex or the antibody-antigenic peptide construct.
  • the targetbinding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct is used for inducing the maturation of monocytes and/or monocyte-derived cells in a subject suffering from a GOLPH2 -positive cancer
  • the immunoconjuagte can kill GOLPH2-positive cancer cells and, at the same time, induce the maturation of monocytes and/or monocyte derived cells.
  • the target-binding molecule, the antibody-antigenic peptide complex or the antibody- antigenic peptide construct of the invention is used as an adjuvant in a vaccination therapy of a subject, i.e. to improve the antigen presentation in said subject, it may be desirable to administer the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct of the invention in a non-conjugated form.
  • the immunoconjugate is an antibody-drug conjugate.
  • antibodydrug conjugate refers to an antibody or Fc-containing polypeptide having specificity for at least one type of malignant cell, a drug, and a linker coupling the drug to e.g. the antibody.
  • the linker is cleavable or non-cleavable in the presence of the malignant cell; wherein the antibody-drug conjugate kills the malignant cell.
  • the antibody-drug conjugate may be generated chemically or enzymatically, for example with a transglutaminase or sortase enzyme.
  • the invention relates to a polynucleotide encoding the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct according to the invention.
  • the polynucleotide may be any nucleic acid sequence capable of encoding the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct according to the invention, such as single-stranded or double-stranded DNA, the sense or antisense strand of a DNA molecule, or RNA molecules, and the like.
  • the person skilled in the art knows how to derive a polynucleotide sequence coding for a protein and how to isolate or produce such a nucleic acid sequence using standard techniques of molecular biology.
  • the polynucleotide encoding the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct according to the invention is an isolated nucleic acid.
  • An "isolated nucleic acid” refers to a nucleic acid molecule that has been separated from a component of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extra chromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • the polynucleotide encoding the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct according to the invention is an isolated nucleic acid encoding an anti-GOLPH2 antibody.
  • isolated nucleic acid encoding an anti-GOLPH2 antibody refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
  • the polynucleotide of the invention may be included in an expression construct such as a vector, plasmid, virus/phagemid, artificial chromosome, cosmid, and further constructs known to the skilled person in order to provide for expression of the sequence of the protein of the invention.
  • the term "vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
  • an expression vector comprises the polynucleotide to be expressed, which is operably linked to one or more control sequences (e.g., promoter, transcriptional stop signal, translational stop signal, etc.) capable of directing the expression of the polypeptide in the desired host cell.
  • control sequences e.g., promoter, transcriptional stop signal, translational stop signal, etc.
  • the promoter may be an inducible or constitutive, general or cell specific promoter. The selection of promoters, vectors and other elements is a matter of routine design within the level of ordinary skill in the art and many different such control sequences are described in the literature and available through commercial suppliers.
  • the choice of the vector will typically depend on the choice of the host cell into which the vector will be introduced.
  • the polynucleotide or expression vector may be introduced into cells by various ways, e.g., using a virus as a carrier or by transfection including e.g. by chemical transfectants (such as Metafectene, Lipofectamine, Fugene, etc.), electroporation, calcium phosphate co-precipitation and direct diffusion of DNA. Suitable transfection techniques are known to the skilled person and the method of choice will vary depending on the host cell to be transfected. Transfection of a cell may yield stable cells or cell lines, if the transfected polynucleotide or expression vector is integrated into the genome, or by using episomal replicating plasmids, i.e. that the inheritance of the extrachromosomal plasmid is controlled by control elements that are integrated into the cell genome. In addition, unstable (transient) cells or cell lines, wherein the transfected DNA exists in an extrachromosomal form can be produced.
  • chemical transfectants such as Metafectene, Lipofectamine, Fugen
  • the expression vector may further comprise a selectable marker, which provides for positive selection of transfected cells, i.e. transfected cells exhibit resistance to the selection and are able to grow, whereas non-transfected cells generally die.
  • selectable markers include puromycin, zeocin, neomycin (neo) and hygromycin B, which confer resistance to puromycin, zeocin, aminoglycoside G- 418 and hygromycin, respectively.
  • other selection methods known to the skilled person may also be suitable.
  • the invention relates to a cell comprising the polynucleotide according to the invention.
  • the invention furthermore relates to a host cell comprising the polynucleotide of the invention.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transfected or transduced cell are included herein.
  • the invention relates to a method of producing a target-binding molecule, an antibody- antigenic peptide complex or an antibody-antigenic peptide construct, in particular an antibody, comprising culturing the host cell of the invention, wherein the host cell comprises the polynucleotide of the invention. That is, in a particular embodiment, the invention relates to a method of producing the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct according to the invention, the method comprising a step of culturing the host cell of the invention under conditions suitable to allow efficient production of the target-binding molecule of the invention.
  • Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567.
  • isolated nucleic acid encoding an anti-GOLPH2 antibody described herein is provided.
  • Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody).
  • one or more vectors e.g., expression vectors
  • a host cell comprising such nucleic acid is provided.
  • a host cell comprises (e.g., has been transfected or transduced with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody.
  • the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., YO, NSO, Sp20).
  • a method of making an anti-GOLPH2 antibody comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
  • nucleic acid encoding an antibody is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
  • nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.
  • antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed.
  • U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. See also Charlton, Methods in Molecular Biology, Vai. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli.
  • the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gemgross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
  • Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures can also be utilized as hosts. See, e.g., US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIESTM technology for producing antibodies in transgenic plants).
  • Vertebrate cells may also be used as hosts.
  • mammalian cell lines that are adapted to grow in suspension may be useful.
  • Other examples of useful mammalian host cell lines are macaque kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Viral. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod.
  • CV 1 macaque kidney cells
  • VERO-76 African green macaque kidney cells
  • HELA human cervical carcinoma cells
  • MDCK canine kidney cells
  • BBL 3 A canine kidney cells
  • W138 human liver cells
  • Hep G2 human liver cells
  • MMT 060562 mouse mammary tumor
  • TRI cells as described, e.g., in Mather et al., Annals N. Y Aead. Sei. 383:44-68 (1982); MRC 5 cells; and FS4 cells.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR CHO cells (Urlaub et al., Proc. Natl.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct according to the invention and/or the immunoconjugate according to the invention and further comprising a pharmaceutically acceptable carrier.
  • the present invention furthermore relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct or the immunoconjugate of the invention and a pharmaceutically acceptable carrier.
  • pharmaceutical formulation or “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • compositions comprising the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate as described herein are prepared by mixing such target-binding molecule, antibody-antigenic peptide complex, antibody- antigenic peptide construct or immunoconjugate having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • pharmaceutically acceptable carriers Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3- pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arg
  • sHASEGP soluble neutral-active hyaluronidase glycoproteins
  • rHuPH20 HYLENEX®, Baxter International, Inc.
  • Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968.
  • a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
  • Exemplary lyophilized antibody or immunoconjugate formulations are described in US Patent No. 6,267,958.
  • Aqueous antibody or immunoconjugate formulations include those described in US Patent No. 6,171,586 and W02006/044908, the latter formulations including a histidine-acetate buffer.
  • the invention relates to a pharmaceutical composition according to the invention further comprising at least one therapeutic agent.
  • Target-binding molecules, antibody-antigenic peptide complexes, antibody-antigenic peptide constructs or immunoconjugates of the invention may be used either alone or in combination with other agents in a therapy.
  • a target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention may be co-administered with at least one additional therapeutic agent.
  • combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant.
  • Target-binding molecules, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugates of the invention can also be used in combination with radiation or laser therapy.
  • a target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional, intrauterine or intravesical administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
  • Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or immunoconjugate, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
  • the formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
  • any of the target-binding molecules, antibody-antigenic peptide complexes, antibody-antigenic peptide constructs or immunoconjugates provided herein may be used in methods, e.g., therapeutic methods.
  • Target-binding molecules, antibody-antigenic peptide complexes, antibody-antigenic peptide constructs or immunoconjugates of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the target-binding molecule or immunoconjugate need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question.
  • the effective amount of such other agents depends on the amount of target-binding molecule, antibody-antigenic peptide complex, antibody- antigenic peptide construct or immunoconjugate present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
  • the appropriate dosage of a target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate, the severity and course of the disease, whether the target-binding molecule, antibody-antigenic peptide complex, antibody- antigenic peptide construct or immunoconjugate is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the target-binding molecule, antibody- antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate, and the discretion of the attending physician.
  • the target-binding molecule, antibody-antigenic peptide complex, antibody- antigenic peptide construct or immunoconjugate is suitably administered to the patient at one time or over a series of treatments.
  • about 1 pg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of target-binding molecule, antibody-antigenic peptide complex, antibody- antigenic peptide construct or immunoconjugate can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • One typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • One exemplary dosage of the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate would be in the range from about 0.05 mg/kg to about 10 mg/kg.
  • one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient.
  • Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the target-binding molecule).
  • An initial higher loading dose, followed by one or more lower doses may be administered.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.
  • any of the above formulations or therapeutic methods may be carried out using a target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct immunoconjugate of the invention.
  • an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the disorder and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is a target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention.
  • the label or package insert indicates that the composition is used for treating the condition of choice.
  • the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises a target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.
  • the article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.
  • the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, fdters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution or dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution or dextrose solution.
  • BWFI bacteriostatic water for injection
  • the invention relates to a pharmaceutical composition according to the invention, wherein the therapeutic agent is at least one of a vaccine, an antigen, an adjuvant, a chemotherapeutic agent and an immune checkpoint modulator.
  • the pharmaceutical composition of the invention may comprise the target-binding molecule, antibody- antigenic peptide complex, antibody-antigenic peptide construct or the immunoconjugate of the invention and an additional therapeutic agent.
  • the pharmaceutical composition may comprise the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate according to the invention and a vaccine.
  • vaccine refers to an antigenic composition usually comprising an infectious factor or a portion of an infectious factor, such as an antigen, preferably in combination with an immune adjuvant, administered into the body to elicit an immune response.
  • the antigenic portion may be a microorganism such as a virus or bacterium; a natural product purified from a microorganism; or a synthetic or genetically engineered protein, peptide, polysaccharide, or similar product.
  • the antigenic portion of the vaccine is comprised of a T cell epitope.
  • the vaccine comprised in the pharmaceutical composition of the invention is a cancer vaccine.
  • cancer vaccine refers to a vaccine that induces an immune response against a particular cancer.
  • Cancer vaccines can be categorized as: antigen vaccines, whole cell vaccines, dendritic cell vaccines, DNA vaccines and anti-idiotype vaccines.
  • FDA licensed cancer prevention vaccines include (1) vaccine to protect against infection with the human papilloma virus (HPV) to prevent cervical cancer, (2) hepatitis B vaccine to protect against infection with the human Hepatitis B virus to prevent hepatocellular carcinoma, and (3) melanoma vaccine for canines.
  • cancer vaccines examples include whole tumor cells, tumor cell lysates, tumor cell derived RNAs, tumor cell derived proteins, tumor cell derived peptides, tumor cell derived carbohydrates, tumor cell derived lipids, and tumor cell derived DNA sequences. These tumor cells could be derived from a patient's own tumor or tumor from an unrelated donor.
  • One potential advantage of cell-based vaccines is that they contain a wide range of antigens.
  • a cancer vaccine may prevent further growth of existing cancer, protect against recurrence of treated cancer, or eliminate cancer cells not already removed by other treatments.
  • “Whole cell tumor vaccines”, also referred to as “whole tumor vaccines” comprise tumor cells which may be autologous or allogeneic for the patient. These cells comprise cancer antigens which can stimulate the body's immune system. As compared to the administration of individual cancer antigens, a whole cell exposes a large number of cancer specific (unique or up-regulated) antigens to the patient's immune system. This stimulation of the immune system means that the patient is better able to prevent the subsequent growth or establishment of a tumor.
  • Whole cell tumor vaccines typically comprise tumor cells which have been modified in vitro, e.g., irradiated and dead tumor cells are preferred in many applications, although live tumor cells may be used in the vaccine.
  • the whole cell vaccine may comprise intact cells but a cell lysate may alternatively be used, and “whole” cell should be understood with this in mind.
  • the use of such a lysate (or intact cell preparation) means that the vaccine will comprise in excess of 10 antigens, typically in excess of 30 antigens.
  • the pharmaceutical composition may comprise the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention and an antigen.
  • antigen or "ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.
  • antigens can be derived from recombinant or genomic DNA.
  • any DNA which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein.
  • an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response.
  • an antigen need not be encoded by a "gene" at all.
  • an antigen can be generated synthesized or can be derived from a biological sample.
  • a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.
  • the antigen may be a tumor antigen.
  • tumor antigen indicates a molecule (e.g. a protein or peptide) that is expressed by a tumor cell and either (a) differs qualitatively from its counterpart expressed in normal cells, or (b) is expressed at a higher level in tumor cells than in normal cells.
  • a tumor antigen can differ from (e.g.
  • tumor antigens are not expressed by normal cells, or are expressed at a level at least about two-fold higher (e.g. about two-fold, three-fold, five-fold, ten- fold, 20- fold, 40-fold, 100- fold, 500-fold, 1,000-fold, 5,000-fold, or 15,000-fold higher) in a tumor cell than in the tumor cell's normal counterpart.
  • Tumor antigens include, without limitation, naturally occurring tumor antigens and modified forms thereof that induce an immune response in a subject, and further include antigens associated with tumor cells and antigens that are specific to tumor cells and modified forms of the foregoing that induce an immune response in a subject.
  • the term tumor antigen further encompasses antigens that correspond to proteins that are correlated with the induction of tumors such as oncogenic virus antigens (e.g., human papilloma virus antigens).
  • Exemplary tumor antigens include, without limitation, HER2/neu and BRCA1 antigens for breast cancer, MART-l/MelanA (melanoma antigen), Fra-1 (breast cancer), NY-BR62, NY-BR85, hTERT, gplOO, tyrosinase, TRP-1, TRP-2, CDK-4, -catenin, MUM-I, Caspase-8, KIAA0205, SART-
  • MAGE family melanoma antigens
  • BAGE family melanoma antigens
  • DAGE/PRAME family such as DAGE- 1
  • GAGE family melanoma antigens
  • RAGE family such as RAGE-I
  • SMAGE family NAG, TAG-72, CAI 25
  • mutated proto-oncogenes such as p21ras
  • mutated tumor suppressor genes such as p53, tumor associated viral antigens (e.g., HPV E6 and E7)
  • the SSX family HOM-MEL-55, NY-COL-2, HOM-HD-397, HOM- RCC-1.14, HOM-HD- 21, HOM-NSCLC-11, HOM-MEL-2.4, HOM-TES-11, RCC-3.1.3, NY-ESO-I, and the SCP family.
  • MAGE family include, but are not limited to, MAGE-1, MAGE-
  • MAGE-3, MAGE-4, MAGE-6, MAGE-11 and MAGE-12 are members of the GAGE family.
  • GAGE-1 include, but are not limited to, GAGE-1, GAGE-6.
  • the tumor antigen can also be, but is not limited to human epithelial cell mucin (Muc-1 ; a 20 amino acid core repeat for the Muc-1 glycoprotein, present on breast cancer cells and pancreatic cancer cells), MUC-2, MUC-3, MUC-18, carcino-embryonic antigen (CEA), the raf oncogene product, CA- 125, GD2, GD3, GM2, TF, sTn, gp75, EBV-LMP 1 & 2, prostate- specific antigen (PSA), prostatespecific membrane antigen (PSMA), GnT-V intron V sequence (N-acetylglucosaminyltransferase V intron V sequence), Prostate Ca psm, MUM-I- B (melanoma ubiquitious mutated gene product), alpha-fetoprotein (AFP), COI 7-1 A, GA733, gp72, -HCG, gp43, HSP-70, pi 7 mel, H
  • modified forms of the tumor antigens described above which induce an immune response in a subject.
  • Modified forms of naturally occurring tumor antigens can advantageously have reduced pathogenicity and/or enhanced immunogenicity as compared with the naturally occurring antigen.
  • the antigen that is co-administered with the target-binding molecule, antibody-antigenic peptide complex or antibody-antigenic peptide construct may be comprised in the target-binding molecule, antibody-antigenic peptide complex or antibody-antigenic peptide construct and/or may be administered separately.
  • the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate according to the invention may be co-administered with an antigen or a vaccine comprising an antigen even if the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate already comprises the an antigen or an antigenic peptide derived from an antigen.
  • the antigen or antigenic peptide that is comprised in the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate according to the invention may be identical to the antigen or may be derived from the antigen that is co-administered with the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate separately.
  • the antigen or antigenic peptide that is comprised in the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate according to the invention may be different from the antigen that is co-administered with the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate.
  • Combination of the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention with a vaccine or an antigen may result in the development of more effective vaccines for the treatment of cancer and infectious diseases.
  • the pharmaceutical composition may comprise the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention and one or more adjuvants.
  • adjuvant refers to an agent that nonspecifically increases an immune response to a particular antigen, thereby reducing the quantity of antigen necessary in any given vaccine and/or the frequency of injection necessary in order to generate an adequate immune response to the antigen of interest.
  • Suitable adjuvants for use herein include, but are not limited to, poly IC; synthetic oligodeoxynucleotides (ODNs) with a CpG motif; modified polyinosinic:polycytidylic acid (Poly-IC) including, but not limited to, Poly-IC/LC (Hiltonol) and Poly-IC12U (Ampligen); Poly-K; carboxymethyl cellulose (CMC); Adjuvant 65 (containing peanut oil, mannide monooleate, an aluminum monostearate); Freund's complete or incomplete adjuvant; mineral gels such as aluminum hydroxide, aluminum phosphate, and alum; surfactants such as hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecylammonium bromide, N,N- dioctadecyl-N',N"-bis(2- hydroxymethyl)propanediamine, methoxyhe
  • the adjuvants of the present invention may include nucleic acids based on inosine and cytosine such as poly I:poly C; poly IC; poly dC; poly dl; poly dIC; Poly-IC/LC; Poly-K; and Poly-IC 12U as well as oligodeoxynucleotides (ODNs) with a CpG motif, CMC and any other combinations of complementary double stranded IC sequences or chemically modified nucleic acids such as thiolated poly IC as described in U.S. Patent Nos. 6,008,334; 3,679,654 and 3,725,545.
  • ODNs oligodeoxynucleotides
  • Combination of the target-binding molecule, antibody-antigenic peptide complex or antibody- antigenic peptide construct of the invention with one or more adjuvant may result in the development of a more effective adjuvant that can be used in vaccination therapy.
  • the pharmaceutical composition may comprise the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention and a chemotherapeutic agent.
  • chemotherapeutic agent refers to a cytotoxic agent which is of use in chemotherapy of cancer.
  • a chemotherapeutic agent may relate to an alkylating agent, such as cyclophosphamide, mechlorethamine, chlorambucil, melphalan, dacarbazine, nitrosoureas, and temozolomide, or to an anthracy cline, such as daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, or to a cytoskeletal disruptor, such as paclitaxel, docetaxel, abraxane, and taxotere, or to an epothilone, or to a histone deacetylase inhibitor, such as vorinostat and romidepsin, or to an inhibitor of topoisomerase 1, such as irinotecan and topotecan, or to an inhibitor of topoisomerase H, such as etoposide, teniposide, and tafluposide, or
  • Combination of the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or the immunoconjugate of the invention with a chemotherapeutic agent may result in more efficient cancer therapy.
  • tumor antigens that are released by cells that have been killed by the immunoconjugate and/or the chemotherapeutic agent may be displayed more efficiently by professional antigen-presenting cells, such as dendritic cells and macrophages, and thus illicit and/or enhance an immune response against the tumor.
  • the pharmaceutical composition may comprise the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention and an immune checkpoint modulator.
  • immune checkpoint modulator refers to an agent used in cancer immunotherapy.
  • a checkpoint inhibitor preferably blocks an inhibitory immune checkpoint and thus restores immune system function, for example, an inhibitor of the immune checkpoint molecule CTLA-4, such as ipilimumab, or an inhibitor of PD-1, such as nivolumab or pembrolizumab, or an inhibitor of PD-L1, such as atezolizumab, avelumab, and durvalumab.
  • a checkpoint inhibitor relates to an antibody which targets a molecule involved in an immune checkpoint.
  • immune checkpoint modulator further encompasses agents that activate a stimulatory immune checkpoint. Examples of inhibitory and stimulating checkpoint molecules are provided herein.
  • Combination of the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or the immunoconjugate of the invention with an immune checkpoint modulator may result in an increased immune response against a subject’s tumor. While the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or the immunoconjugate of the invention results in increased display of tumor antigens by professional antigen-presenting cells, a check point inhibitor may prevent inhibition of T cells that may become activated by the professional antigen-presenting cells.
  • the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition according to the invention for use in inducing the maturation of monocytes and/or monocyte-derived cells in a subject.
  • the invention relates to the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein the maturation of monocytes or monocyte -derived cells results in the formation of dendritic cells and/or classically activated macrophages in said subject.
  • the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may be used for inducing the maturation of monocytes and/or monocyte derived cells in a subject. It has been demonstrated herein that the target-binding molecule the antibody-antigenic peptide complex, the antibody-antigenic peptide construct and the immunoconjugate of the invention have the potential to induce the maturation of monocytes or monocyte-derived cells into classically activated (Ml) macrophages and dendritic cells, in particular matured dendritic cells. Accordingly, the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may be used for inducing and/or enhancing an immune response in a subject.
  • Ml classically activated
  • the invention relates to an antibody, or an antigen-binding fragment thereof, specifically binding to the extracellular part of GOLPH2 for use in inducing and/or enhancing an immune response in a subject.
  • the invention relates to an antibody, or an antigen-binding fragment thereof, specifically binding to the extracellular part of GOLPH2 for use in treating a disease or disorder associated with an impaired immune system in a subject.
  • a ’’disease or disorder associated with an impaired immune system relates to an abnormal condition of the human or animal body that is characterized by a depressed ability of a subject’s immune system to mount an immune response to an antigen.
  • the ’’disease or disorder associated with an impaired immune system may be an acquired or a congenital disease or disorder. The skilled person is aware of methods to determine whether a subject has an impaired immune system.
  • An "individual” or “subject” is a mammal.
  • Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non human primates such as macaques), rabbits, and rodents (e.g., mice and rats).
  • domesticated animals e.g., cows, sheep, cats, dogs, and horses
  • primates e.g., humans and non human primates such as macaques
  • rabbits e.g., mice and rats
  • rodents e.g., mice and rats.
  • the individual or subject is a human.
  • the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein the subject is at risk of developing cancer, suffering from cancer or recovering from cancer.
  • the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may be used for inducing the maturation of monocytes and/or monocyte -derived cells in any subject.
  • the subject is a subject that is at risk of developing cancer, suffering from cancer or recovering from cancer.
  • a subject may be determined to be at risk of developing cancer based on the subject’s age and/or medical history.
  • a subject’s risk of developing cancer may be determined based on tests, such as genetic tests or biomarker tests. The skilled person is aware of methods to determine whether or not a subject is at risk of developing a certain type of cancer.
  • a subject is suffering from cancer, if the subject shows unregulated growth of tumor or cancerous cells.
  • Several ways of diagnosing cancer in a subject including for example imaging techniques, have been described in the art.
  • a subject is recovering from cancer, if the subject was previously diagnosed with cancer, but wherein the cancer has been treated by any means known in the art.
  • a subject recovering from cancer may be treated with the target-binding molecule, the antibody-antigenic peptide complex, the antibody- antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention to kill remaining tumor or cancerous cells or to prevent the reoccurrence of the cancer.
  • the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition may have two distinct functions, which is (i) inducing the maturation of monocytes and/or monocyte- derived cells and (ii) directly targeting GOLPH2 positive cancer cells. That is, in certain embodiments, the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may be used to induce the maturation of monocytes and/or monocyte-derived cells in a subject that is at risk of developing cancer, suffering from cancer or recovering from cancer.
  • the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may be used to increase the number of antigen-presenting cells in that subject to improve antigen presentation and (a) support an immune response against the subject’s tumor and cancerous cells in subjects suffering from cancer or (b) prevent the emergence of tumor or cancerous cells in subjects at risk of developing or recovering from cancer.
  • a target-binding molecule, an antibody- antigenic peptide complex, an antibody-antigenic peptide construct, or a pharmaceutical composition comprising a target-binding molecule is administered to the subject at risk of developing cancer, suffering from cancer or recovering from cancer.
  • the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition according to the invention may comprise an antigen or an antigenic peptide that is known to be presented by the subject’s tumor or cancerous cells to increase the subject’s immune response against the tumor or cancerous cells.
  • the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may be used to induce the maturation of monocytes and/or monocyte-derived cells in a subject suffering or recovering from a GOLPH2 positive cancer.
  • the targetbinding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may not only improve an immune response against the subject’s tumor or cancerous cells, but may also directly attack the tumor cells.
  • an immunoconjugate comprising a cytotoxic agent or a pharmaceutical composition comprising an immunoconjugate comprising a cytotoxic agent is administered to the subject suffering from or recovering from a GOLPH2 -positive cancer.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation.
  • examples of cancer include, but are not limited to, carcinoma, liver cancer, hepatocellular cancer, gastric cancer, lung cancer, esophageal cancer, breast cancer, prostate cancer, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia.
  • the invention relates to an antibody, or an antigen-binding fragment thereof, specifically binding to the extracellular part of GOLPH2 for use in treating cancer.
  • the invention relates to an antibody, or an antigen-binding fragment thereof, specifically binding to the extracellular part of GOLPH2 for use in treating cancer, wherein the binding of the antibody, or the antigen-binding fragment thereof, to the extracellular part of GOLPH2 induces the maturation of monocytes into macrophages and/or dendritic cells.
  • GOLPH2 -positive cell refers to a cell that expresses full length or partial GOLPH2 on its surface.
  • the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein the subject is immunocompromised.
  • immunocompromised refers to a subject with an innate, acquired, or induced inability to develop a normal immune response.
  • An immunocompromised subject therefore, has a weakened or impaired immune system relative to one of a normal subject.
  • a subject with a weakened or impaired immune system has an "immunodeficiency" or “immunocompromised condition,” which is associated with a primary or secondary deficiency, induced or non-induced, in one or more of the elements of the normal immune defense system.
  • an immunodeficiency is associated with a deficiency of a subject to develop professional -antigen presenting cells, such as dendritic cells and/or macrophages.
  • An immunocompromised condition is commonly due to a medical treatment, e.g., radiation therapy, chemotherapy or other immunosuppressing treatment, such as induced by treatment with steroids, cyclophosphamide, azathioprine, methotrexate, cyclosporine or rapamycin, in particular in relation to cancer treatment or the treatment or prevention of transplant rejection.
  • a medical treatment e.g., radiation therapy, chemotherapy or other immunosuppressing treatment, such as induced by treatment with steroids, cyclophosphamide, azathioprine, methotrexate, cyclosporine or rapamycin, in particular in relation to cancer treatment or the treatment or prevention of transplant rejection.
  • the presence of an immunocompromised condition in a subject can be diagnosed by any suitable technique known to persons of skill the art. Strong indicators that an immunocompromised condition may be present is when rare diseases occur or the subject gets ill from organisms that do not normally cause diseases, especially if the subject gets repeatedly infected.
  • lymphocyte proliferation test may be conducted to determine if the lymphocytes can respond to stimuli. The failure to respond to stimulants correlates with an immunocompromised condition. Antibody levels and complement levels can also be determined for diagnosing the presence of an immunocompromised condition.
  • the target-binding molecule of the invention can induce the maturation of monocytes and monocyte-derived cells into professional antigen-presenting cells, such as Ml macrophages and mature dendritic cells.
  • professional antigen-presenting cells such as Ml macrophages and mature dendritic cells.
  • the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate and/or the pharmaceutical composition of the invention can be used for increasing the number of professional antigen-presenting cells in an immunocompromised subject and thus improve the function of the immune system in these subjects.
  • the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate and/or the pharmaceutical composition of the invention may be used for inducing the maturation of monocytes and/or monocyte -derived cells in an immunocompromised subject, wherein the immunocompromised subject was diagnosed a decreased number of professional antigen- presenting cells, in particular Ml macrophages and/or mature dendritic cells.
  • the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein the subject is immunocompromised as a result of a chemotherapy, a radiotherapy, or an infection, in particular wherein the infection is aninfection with a human immunodeficiency virus.
  • the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition according to the invention for use in inducing and/or enhancing an immune response to an antigen.
  • the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate and/or the pharmaceutical composition of the invention may be used for inducing and/or enhancing an immune response to an antigen. That is, it has been demonstrated herein that the target-binding molecule, the antibody-antigenic peptide complex, the antibody- antigenic peptide construct, the immunoconjugate and/or the pharmaceutical composition of the invention can induce the maturation of monocytes and monocyte-derived cells.
  • the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate and/or the pharmaceutical composition of the invention can increase the number of professional antigen-presenting cells in a subject and thus induce and/or enhance an immune response to an antigen in said subject.
  • immune response refers to both humoral and cell-mediated immune responses.
  • the humoral branch of the immune system involves interaction of B cells with antigen and their subsequent proliferation and differentiation into antibody-secreting plasma cells.
  • the antibody functions as the effector molecule of the humoral response by binding to antigen and neutralizing it or facilitating its elimination.
  • Antibodies can cross-link the antigen, forming clusters that are more readily digested by phagocytic cells, such as macrophages. Binding of antibody to antigen can also activate the complement system resulting in lysis of the cell to which the antibody binds including foreign organisms.
  • Antibody can also neutralize toxins or viral particles by coating them and preventing binding to host cells.
  • the cell-mediated branch of the immune response occurs when effector T cells are generated in response to antigen.
  • T helper cells TH
  • CTLs cytotoxic T lymphocytes
  • Lymphokines secreted by TH cells can activate various phagocytic cells to phagocytose and kill microorganisms.
  • Activated cytotoxic T lymphocytes participate in cell-mediated immune reactions by killing altered self-cells, virally infected cells and tumor cells.
  • Macrophages mainly present antigens to T helper cells, while mature dendritic cells present antigens to T helper cells, cytotoxic T lymphocytes and B cells.
  • macrophages are also involved in the clearance of antigens that are bound by B cell-produced antibodies, it is plausible that both macrophages and dendritic cells are involved in humoral and cell-mediated immune responses.
  • the invention relates to the target-binding molecule, the immunoconjugate or the pharmaceutical composition according to the invention for use in inducing and/or enhancing a humoral and/or a cell-mediated immune response to an antigen.
  • the invention relates to the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition according to the invention for use in inducing and/or enhancing a humoral immune response to an antigen.
  • the invention relates to the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition according to the invention for use in inducing and/or enhancing a cell-mediated immune response to an antigen.
  • the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention are said to induce an immune response in a subject, if they initiate an immune response that was not present before the induction stage.
  • the target-binding molecule, the antibody-antigenic peptide complex, the antibody- antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention are said to enhance an immune response in a subject, if they maintain a desired response at the same level before the enhancement stage or increasing the desired response over a period of time.
  • the term “induce” also includes the term “enhance”.
  • the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein the immune response against the antigen is induced and/or enhanced with the help of an antigenic peptide that is comprised in the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct.
  • the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct of the invention may be used to deliver an antigen or an antigenic peptide to an antigen-presenting cell and to facilitate the presentation of the antigenic peptide by the antigen- presenting cell.
  • the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition according to the invention may be used to induce and/or enhance an immune response against a known antigen.
  • the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein the antigen has been released by a physical therapeutic intervention.
  • the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may induce and/or enhance an immune response to any type of antigen. That is, the antigen may be administered to a subject to induce and/or enhance an immune response in said subject, for example during a vaccination therapy. In other embodiments, the antigen, or a fragment or derivative thereof, may be part of the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct. In certain embodiments, the antigen is an endogenous antigen that is produced by the subject. This endogenous antigen may be released by natural processes or in response to a physical therapeutic intervention.
  • an antigen may be released by a physical therapeutic intervention and may in addition be administered, either the entire antigen or an antigen peptide derived from said antigen, to a subject separately or comprised in the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention.
  • the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein the physical therapeutic intervention is cryotherapy, surgery, radiotherapy and/or laser therapy.
  • cryotherapy refers to the use of extreme cold produced by liquid nitrogen (or argon gas) to destroy abnormal tissue.
  • Cryotherapy may be used to treat external tumors, such as those on the skin.
  • liquid nitrogen is applied directly to the cancer cells with a cotton swab or spraying device.
  • Cryotherapy may also be used to treat tumors inside the body (internal tumors and tumors in the bone).
  • liquid nitrogen or argon gas is circulated through a hollow instrument called a cryoprobe, which is placed in contact with the tumor.
  • cryosurgery or “cyroablation”.
  • the term “surgery”, as used herein, means any therapeutic procedure that involves methodical action of the hand or of the hand with an instrument, on the body of a human or other mammal, to produce a curative or remedial.
  • the term “surgery” preferably means the resection of a tumor.
  • radiotherapy means exposure to radiation from a radioactive substance used in the treatment of disease (especially cancer).
  • laser therapy or “laser surgery” refers to a type of surgery that uses special light beams instead of tools such as scalpels.
  • Laser light may be delivered either continuously or intermittently and may be used with fiber optics to treat areas of the body that are often hard to reach.
  • Common lasers used in cancer treatment include carbon dioxide (CO2) lasers, neodymium :yttrium -aluminum - garnet (Nd:YAG) lasers, laser-induced interstitial thermotherapy (LITT) and Argon lasers.
  • Physical therapeutic interventions result in the destruction of cells or tissue and thus in the release of molecule from the destroyed cells or tissues.
  • Any molecule that is release from a cell or tissue in response to a physical therapeutic intervention can potentially serve as an antigen.
  • the physical therapeutic intervention is directed to tumor cells or tissues and the antigens that are released from the tumor cells or tissues comprise tumor antigens.
  • Some of the antigens that are released from tumor cells or tissues may be presented by matured monocytes that have been treated with the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention to induce and/or enhance an immune response against these antigens.
  • the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may be administered to the subject receiving the physical therapeutic intervention before, during or after the physical therapeutic intervention. That is, the target-binding molecule, the immunoconjugate or the pharmaceutical composition of the invention may be administered to the subject several days, for example 1, 2, 3, 4 or 5 days, before or after the physical therapeutic intervention. In certain embodiments, the target-binding molecule, the immunoconjugate or the pharmaceutical composition of the invention is administered multiple times before, during and/or after the physical therapeutic intervention.
  • the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein the antigen is a tumor antigen or a pathogen-derived antigen.
  • the antigen is a tumor antigen. That is, the antigen may be a tumor antigen that is administered to a subject, for example as a cancer vaccine, or may be an endogenous tumor antigen that has been released by a tumor or cancerous cell, for example in response to a physical therapeutic intervention. Further, an antigenic peptide derived from a tumor antigen may be comprised in the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate of the invention. Exemplary tumor antigens that may induce and/or enhance an immune response in a subject are discussed above.
  • the antigen may be a pathogen-derived antigen.
  • a pathogen-derived antigen may be used to induce and/or enhance an immune response against the pathogen that it has been derived from.
  • the pathogen-derived antigen may be a single molecule that has been derived from a pathogen, such as peptides, proteins, including glycoproteins, carbohydrates, phospholipids, phosphoproteins, phospholipoproteins, and fragments of the foregoing.
  • the pathogen- derived antigen may be derived directly from the pathogen or may be chemically synthesized.
  • the pathogen-derived antigen may also be a portion or a lysate of the pathogen.
  • the pathogen-derived antigen may be an inactivated or attenuated form of the pathogen.
  • pathogen as used herein comprises, without limitation, bacteria, viruses, fungi and protozoa.
  • the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition according to the invention for use as an adjuvant in a vaccination therapy.
  • An adjuvant is a pharmacological or immunological agent that modifies the effect of other agents.
  • Adjuvants may be added to a vaccine to boost the immune response to produce more antibodies and longer-lasting immunity, thus minimizing the dose of antigen needed.
  • Adjuvants may also be used to enhance the efficacy of a vaccine by helping to modify the immune response to particular types of immune system cells: for example, by activating T cells instead of antibody-secreting B cells depending on the purpose of the vaccine.
  • agents like aluminum salts or squalene-in-water emulsions that have the ability to boost the immune answer towards defined antigens have been used and are approved as adjuvants in vaccination therapies.
  • Other adjuvants have been tested or are in clinical development, for example Poly-IC (also Poly-ICLC), a synthetic derivate of dsRNA, CpG, synthetic phosphorothioate- linked DNA oligonucleotides with optimized CpG motifs or IFA, a mineral or paraffin oil plus surfactant, to name a few (Coffman RL, Sher A, Seder RA. Vaccine adjuvants: Putting innate immunity to work. Immunity 2010;33:492-503).
  • mice CpG has been used successfully to enhance the innate and adaptive response of the rodents immune system against a given antigen. This has been exploited for vaccination and generation of humoral responses, e.g. monoclonal antibodies.
  • humoral responses e.g. monoclonal antibodies.
  • human adjuvants In human, no comparable adjuvants are in clinical use. A major obstacle with human adjuvants is the complexity that most of the adjuvants formulations carry. Consequently, the majority of hitherto tested adjuvants have not found their way into the clinic for human use (Coffman RL, Sher A, Seder RA. Vaccine adjuvants: Putting innate immunity to work. Immunity 2010;33:492-503.).
  • the target-binding molecule, the immunoconjugate or the pharmaceutical composition of the invention have the potential to improve the presentation of antigens by professional antigen-presenting cells.
  • the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention can be used as an adjuvant in a vaccination therapy.
  • the invention relates to the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein the adjuvant initiates or enhances the function of antigen-presenting cells.
  • the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein the vaccination therapy comprises administration of a viral antigen, a microbial antigen or a tumor antigen.
  • the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may be co-administered and/or formulated with any antigen against which an immune response is desired.
  • Vaccines comprising an antigen and the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may comprise one or more additional adjuvants or may comprise the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention as the sole adjuvant.
  • the skilled person is aware of methods to formulate compositions, i.e. vaccines, comprising the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention.
  • the invention relates to the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention for use as an adjuvant in a vaccination therapy, wherein the vaccine comprises one or more tumor antigens and, optionally, one or more additional adjuvants.
  • the vaccine comprises one or more tumor antigens and, optionally, one or more additional adjuvants.
  • the one or more tumor antigen may be any combination of known tumor antigens, for example any combination of the tumor antigens discussed herein.
  • the invention relates to the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention for use as an adjuvant in a vaccination therapy, wherein the vaccine comprises one or more pathogen-derived antigens and, optionally, one or more additional adjuvants.
  • the invention relates to the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention for use as an adjuvant in a vaccination therapy, wherein the vaccine comprises one or more viral antigens and, optionally, one or more additional adjuvants.
  • viral antigen refers to any molecule (e.g., protein, peptide, lipid, carbohydrate, etc.) solely or predominantly expressed by a virus, such that the antigen is associated with the virus.
  • the viral antigen may be an antigen expressed by any virus.
  • the viral antigen may also be an inactivated or attenuated form of the virus.
  • the viral antigen may be a viral antigen of only one type of virus, such that the viral antigen is associated with or characteristic of only one type of virus.
  • the viral antigen may be a viral antigen (e.g. may be characteristic) of more than one type of virus.
  • the viral antigen may be expressed by a virus selected from the group consisting of herpes viruses, pox viruses, hepadnaviruses, papilloma viruses, adenoviruses, coronoviruses, orthomyxoviruses, paramyxoviruses, flaviviruses, and caliciviruses.
  • a virus selected from the group consisting of herpes viruses, pox viruses, hepadnaviruses, papilloma viruses, adenoviruses, coronoviruses, orthomyxoviruses, paramyxoviruses, flaviviruses, and caliciviruses.
  • the invention relates to the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention for use as an adjuvant in a vaccination therapy, wherein the vaccine comprises one or more microbial antigens and, optionally, one or more additional adjuvants.
  • microbial antigen refers to any microbial component having antigenic properties, i.e. being able to provoke an immune response in an individual.
  • microbial antigen encompasses “bacterial antigens” and “fungal antigens”.
  • bacterial antigen refers to any bacterial component having antigenic properties, i.e. being able to provoke an immune response in an individual.
  • the bacterial antigen may be derived from the cell wall or cytoplasm membrane of a bacterium.
  • fungal antigen refers to any fungal component having antigenic properties, i.e. being able to provoke an immune response in an individual.
  • the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein at least one antigenic peptide derived from the viral antigen, the microbial antigen or the tumor antigen is comprised in the targetbinding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct.
  • the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may be used as an adjuvant in a vaccination therapy, wherein the separate co-administration of the antigen is not required. That is, an immune response against a specific antigen may be induced and/or enhanced by administering a target-binding molecule, an antibody-antigenic peptide complex or an antibody-antigenic peptide construct of the invention comprising an antigenic peptide, a fragment or derivative of an antigen or an entire antigen.
  • the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, or the immunoconjugate comprising at least one antigenic peptide, antigen fragment or derivative or antigen can function both as an adjuvant and as an antigen.
  • the invention relates to the use of an antibody specifically binding to the extracellular part of GOLPH2, or an antigen-binding fragment thereof, as an adjuvant in a vaccination therapy.
  • the invention relates to the use according to the invention, wherein the antibody is a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody or a multispecific antibody.
  • the invention relates to the use according to the invention, wherein the antibody is the antibody-antigenic peptide construct, the antibody-antigenic peptide complex or the fusion antibody according to the invention.
  • the invention relates to the use according to the invention, wherein the adjuvant initiates or enhances the function of antigen-presenting cells.
  • the invention relates to the use according to the invention, wherein the vaccination therapy comprises administration of a viral antigen, a microbial antigen or a tumor antigen.
  • the invention relates to the use according to the invention, wherein at least one antigenic peptide derived from the viral antigen, the microbial antigen or the tumor antigen is comprised in the antibody-antigenic peptide construct according to the invention or the antibody- antigenic peptide complex according to the invention.
  • the target -binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention are administered to a subject in need thereof in an effective amount.
  • An "effective amount" of an agent e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. It is to be understood that the effective amounts may differ from one use to another. However, the skilled person is aware of reasonable ranges of amounts and able to identify the effective amount by routine experimentation.
  • the invention relates to a method for inducing the maturation of monocytes and/or monocyte-derived cells in vitro, the method comprising the steps of: a) culturing monocytes and/or monocyte-derived cells in a cell culture medium; b) adding the target-binding molecule, the antibody-antigenic peptide complex, the antibody- antigenic peptide construct or the immunoconjugate according to the invention to the cell culture of step (a); and c) obtaining matured monocytes and/or monocyte-derived cells.
  • the invention further refers to an in vitro method for inducing the maturation of monocytes and/or monocyte-derived cells.
  • monocytes and/or monocyte -derived may be cultured in a container comprising a suitable cell culture medium.
  • suitable containers such as flasks or plates, and media for the culturing of monocytes and/or monocyte derived cells. Further examples are described in Example 2.
  • the monocytes and/or monocyte-derived cells may be cultured at any cell density.
  • the monocytes and/or monocyte-derived cells may be cultured at a cell density between IO 2 and IO 7 cells/mL, preferably at a cell density between IO 3 and IO 6 cells/mL.
  • the skilled person is aware of methods for obtaining monocytes and/or monocyte-derived cells from a sample, for example a blood or bone marrow sample, as described in Example 1.
  • the monocytes and/or monocyte-derived cells are contacted with the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate of the invention.
  • the skilled person is aware of methods to determine the concentration of the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate that is required for inducing the maturation of monocytes and/or monocyte -derived cells.
  • concentrations between 0.01 and 10 pM, preferably between 0.05 and 10 pM, more preferably between 0.1 and 10 pM and most preferably between 0.5 and 10 pM of the target-binding molecule may be added to the cells in the cell culture.
  • the monocytes and/or monocyte-derived cells may be contacted with the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate of the invention for any amount of time that is sufficient for the monocytes and/or monocyte-derived cells to mature.
  • the monocytes and/or monocyte-derived cells are contacted with the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate for 2 to 7 days to obtain matured monocytes and/or monocyte-derived cells.
  • the monocytes and/or monocyte-derived cells are contacted with the target-binding molecule, the antibody-antigenic peptide complex, the antibody- antigenic peptide construct or the immunoconjugate for 3 to 6 days to obtain matured monocytes and/or monocyte-derived cells.
  • the monocytes and/or monocyte-derived cells are contacted with the target-binding molecule, the antibody-antigenic peptide complex, the antibody- antigenic peptide construct or the immunoconjugate for 4 days to obtain matured monocytes and/or monocyte-derived cells.
  • the invention relates to a method for generating a cell displaying an antigenic peptide, the method comprising the steps of: a) generating a matured monocyte or monocyte-derived cell according to the method of the invention; b) pulsing the antigenic peptide on the matured monocyte or monocyte-derived cell generated in step (a); and/or introducing a nucleic acid encoding a polypeptide comprising the antigenic peptide into the matured monocyte or monocyte-derived cell generated in step (a); and/or introducing the antigenic peptide into the matured monocyte or monocyte-derived cell as part of the target-binding molecule, the complex, or the construct; and c) obtaining a cell displaying an antigenic peptide.
  • an antigenic peptide may be pulsed on the matured monocyte or monocyte -derived cell that have been generated with the method of the invention or a nucleic acid encoding an antigenic peptide may be introduced into the matured monocyte or monocyte-derived cell.
  • the cell displaying an antigenic peptide is preferably a matured monocyte or monocyte-derived cell. That is, the cell displaying the antigenic peptide may be a professional antigen-presenting cell that has been obtained with the method of the invention, i.e. by contacting a monocyte or monocyte-derived cell with the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate of the invention.
  • the cell displaying the antigenic peptide is an Ml macrophage that has been obtained with the method of the invention.
  • the cell displaying the antigenic peptide is a mature dendritic cell that has been obtained with the method of the invention.
  • Example 14 further describes the pulsing of an antigenic peptide on matured monocytes.
  • the antigenic peptides that are pulsed on the matured monocytes are preferably peptides that can be displayed by an MHC molecule on the surface of the matured monocyte. That is, the peptides preferably have a length of 8 to 25 amino acid residues.
  • MHC class I molecules preferably display peptides with a length of 8 to 10 amino acid residues. Accordingly, in certain embodiments, the antigenic peptide is an MHC class I peptide with a length of 8 to 10 amino acid residues.
  • MHC class II molecules preferably display peptides with a length of 13 to 25 amino acid residues. Accordingly, in certain embodiments, the antigenic peptide is an MHC class II peptide with a length of 13 to 25 amino acid residues. Accordingly, in certain embodiments, the peptide that is pulsed on the matured monocyte or monocyte-derived cell is an MHC class I or and MHC class II peptide.
  • a polynucleotide encoding an antigenic peptide may be delivered to the matured monocyte or monocyte-derived cell.
  • the skilled person is aware of methods to introduce foreign nucleic acids into a cell.
  • transfection or transduction methods may be used to deliver a foreign nucleic acid encoding an antigenic peptide into a matured monocyte.
  • the foreign nucleic acid encoding the antigenic peptide may comprise further elements. That is, the nucleic acid may encode a larger polypeptide comprising the antigenic peptide.
  • the larger polypeptide encoded by the polynucleotide comprises one or more MHC class I and/or MHC class II peptides.
  • the nucleic acid may be transcribed and translated into a larger polypeptide, which is then degraded into smaller peptides inside the matured monocyte or monocyte -derived cell. These smaller peptides, for example the antigenic peptide, may then be displayed by MHC molecules on the surface of the matured monocyte or monocyte-derived cell.
  • the nucleic acid may comprise regulatory elements that allow for expression of the nucleic acid in the matured monocyte.
  • antigenic peptide refers to a peptide comprising a structure that is recognized by the immune system of a subject.
  • Non-limiting examples of antigenic peptides are a peptide that is recognized by a B or T-cell, e.g. via binding to a T-cell receptor, or a peptide that binds to an antibody or antibody fragment, or a peptide that stimulates an immune response in a subject.
  • the antigenic peptide may be a tumor antigen or a pathogen-derived antigen as discussed herein.
  • the antigenic peptide is an MHC class I or MHC class II peptide.
  • the antigenic peptide may be delivered to the antigen-presenting cell, such as an Ml macrophage or a matured dendritic cell, as part of the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate of the invention. It has been described herein that antigenic peptides can be delivered directly to an Ml macrophage or a matured dendritic cell with the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate of the invention.
  • the antigen-presenting cell such as an Ml macrophage or a matured dendritic cell
  • the invention relates to a method for generating an activated T cell, the method comprising the steps of: a) generating a cell displaying an antigenic peptide with the method according to the invention; b) contacting the cell of step (a) with a T cell, wherein the T cell comprises a T cell receptor that recognizes the antigenic peptide displayed by the cell of step (a); and c) obtaining an activated T cell.
  • the cell displaying an antigenic peptide may be used for generating activated T cells.
  • a cell displaying an antigen peptide wherein the cell has been obtained with the methods of the invention, may be contacted with a T cell comprising a T cell receptor that recognizes the antigenic peptide displayed on the cell.
  • the T cell may be any type of T cell. However, it is preferred that the T cell is a T helper (CD4+) T cell or a cytotoxic T lymphocyte (CD8+ T cell).
  • the cell displaying the antigenic peptide may be a mature dendritic cell that has been obtained by contacting a monocyte or an immature dendritic cell with the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate of the invention.
  • the dendritic cell may display an antigenic peptide that has been pulsed on the surface of the dendritic cell.
  • the dendritic cell may display an antigenic peptide that has been introduced into the dendritic cell in the form of a polynucleotide.
  • the dendritic cell may display an antigenic peptide that has been delivered to the dendritic cell with the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate of the invention.
  • the antigenic peptide may be an MHC class I peptide that is displayed on the surface of the dendritic cell by an MHC class I molecule.
  • the dendritic cell displaying this antigenic peptide may be contacted with and activate a cytotoxic T lymphocyte.
  • the antigenic peptide may be an MHC class II peptide that is displayed on the surface of the dendritic cell by an MHC class II molecule.
  • the dendritic cell displaying this antigenic peptide may be contacted with and activate a T helper cell.
  • the cell displaying an antigenic peptide may be an Ml macrophage that has been obtained by contacting a monocyte or monocyte-derived cell with the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate of the invention.
  • the Ml macrophage may display an antigenic peptide that has been pulsed on the surface of the Ml macrophage.
  • the Ml macrophage may display an antigenic peptide that has been introduced into the Ml macrophage in the form of a polynucleotide.
  • the Ml macrophage may display an antigenic peptide that has been delivered to the Ml macrophage with the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate of the invention.
  • the antigenic peptide may be an MHC class II peptide that is displayed on the surface of the Ml macrophage by an MHC class II molecule.
  • the Ml macrophage displaying this antigenic peptide may be contacted with and activate a T helper cell.
  • T cell as used herein will be understood by those skilled in the art.
  • the term includes, but is not limited to CD8 + or CD4 + T cells capable of lysing target cells or providing effector or helper functions, such as cytokine secretion, which can result in the death of target cells or the generation or enhancement of anti-target effector activity.
  • the term “activated” as used herein relates to specific mechanisms of activation of T cells.
  • the general “two-signal model” of activation of T cells is characterized by a first signal provided by binding of the TCR to a short peptide presented by the major histocompatibility complex (MHC) on another cell ensuring that only a T cell with a TCR specific to that peptide is activated.
  • the partner cell is usually a professional antigen presenting cell (APC), for example a dendritic cell or an Ml macrophage.
  • APC professional antigen presenting cell
  • the peptides presented to CD8+ T cells by MHC class I molecules are 8-10 amino acids in length; the peptides presented to CD4+ T cells by MHC class II molecules are longer, as the ends of the binding cleft of the MHC class II molecule are open.
  • the second and co-stimulatory signal required for T cell activation involves interaction of CD28 on the T cell with CD80 or CD86 (B7 family genes) on the antigen-presenting cell. The second signal licenses the T-cell to respond to an antigen.
  • a T cell may be determined to be activated if it expresses antigens indicative of T cell activation (that is, T cell activation markers).
  • T cell activation markers include, but are not limited to, CD25, CD26, CD30, CD38, CD69, CD70, CD71, ICOS, OX-40 and 4-1BB.
  • the expression of activation markers can be measured by techniques known to those of skill in the art, including, for example, western blot analysis, northern blot analysis, RT-PCR, immunofluorescence assays, and fluorescence activated cell sorter (FACS) analysis.
  • a T cell can be determined to be activated based on the cytokine secretion pattern. The skilled person is aware that certain cytokines are secreted in higher amounts by activated T cells.
  • T cell receptor refers to a heterodimeric antigen binding receptor derived from a T lymphocyte, comprising an alpha/beta polypeptide dimer or a gamma/delta polypeptide dimer, each dimer comprising a variable region, a constant region, and an antigen binding site.
  • a TCR is said to recognize an antigenic peptide, if the interaction between the TCR and the antigenic peptide- MHC complex is sufficient to activate the cell comprising the TCR.
  • the invention relates to the method according to the invention, wherein the cell displaying an antigenic peptide and the T cell have been obtained from the same subject.
  • the T cell and the antigen presenting cell used in the method of the invention may be of any origin. That is, in certain embodiments, the T cell and the antigen-presenting cell may have been obtained from different subjects. In other embodiments, the T cell and the antigen-presenting cell may have been obtained from the same subject. In such a case, T cells and monocytes and/or monocyte-derived cells may be isolated from a blood sample that has been obtained from said subject.
  • the invention relates to a cell displaying an antigenic peptide and/or an activated T cell for use in adoptive cell transfer, wherein the cell displaying the antigenic peptide and the activated T cell have been obtained with the methods according to the invention.
  • adoptive cell transfer refers to any transfer of immune cells into a subject.
  • adoptive cell transfer is used to trigger cytotoxic immune responses that will destroy tumor cells.
  • the term comprises the direct transfer of immune cells such as T cells, monocytes, macrophages or dendritic cells into the subject.
  • the immune cells are intended to recognize and kill the tumor cells in addition to the subject's own anti-tumor response.
  • Adoptive transfer of activated dendritic cells comprises the stimulation of dendritic cells obtained with the method of the invention to activate a cytotoxic response towards the tumor cells.
  • Dendritic cells can be stimulated for example by pulsing them with an antigen or transfecting them with a viral vector.
  • the stimulated dendritic cells may be infused into the subject to initiate a cytotoxic immune response against the tumor cells.
  • Adoptive transfer of activated macrophages comprises the stimulation of Ml macrophages obtained with the method of the invention to activate a cytotoxic response towards the tumor cells.
  • Ml macrophages can be stimulated for example by pulsing them with an antigen or transfecting them with a viral vector.
  • the stimulated dendritic cells may be infused into the subject to initiate a cytotoxic immune response against the tumor cells.
  • T cells that have been activated with the method of the invention are transferred into a subject. It is preferred that the T cells have been activated with an antigen that is known to be present in the subject receiving the adoptive cell transfer.
  • the T cells may have been activated with a tumor antigen that is known to be expressed by tumor or cancerous cells in the subject receiving the adoptive cell transfer.
  • mature dendritic cells or Ml macrophages that have been obtained with the method of the invention may be pulsed with an antigenic peptide that has been previously determined to be present on tumor cells of a subject.
  • the resulting antigendisplaying cells may then be contacted with a population of T cells that have been obtained from said subject to activate and proliferate the portion of the T cells that recognize the tumor antigen.
  • the activated T cells may then be transferred back into the subject to initiate an immune response against the tumor cells.
  • adoptive cell transfer is used in the treatment of cancer.
  • the antigenic peptide that is displayed on the mature dendritic cell or the Ml macrophage is a tumor antigen, for example one of the tumor antigens listed herein.
  • the invention relates to a method for inducing and/or enhancing an immune response against a specific antigen in a subject, the method comprising the steps of: a) administering to said subject the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition according to the invention, wherein the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition comprises an antigenic peptide derived from the specific antigen; b) inducing and/or enhancing an immune response in said subject.
  • the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may be used to induce and/or enhance an immune response against a specific antigen in a subject.
  • the specific antigen may be determined by fusing or complexing this antigen, or a fragment or derivative thereof, to the target-binding molecule of the invention. It is to be understood that not all fragments or derivatives of an antigen can be displayed by an antigen-presenting cell. However, the skilled person is aware of fragments or derivatives of antigens that can be displayed by antigen-presenting cells such that an immune response against the antigen can be induced or enhanced. A non-exhaustive list of antigens is provided herein.
  • G2-2, G2- 2M, G2-2-ADC and BsMabG2-2/Myc refer to G2-2opti, G2-2optiM, G2-2opti-ADC and BsMabG2-2opti/Myc, respectively.
  • FIG.l Quantification of secreted GOLPH2 (sGOLPH2 ng/ml) of tumor cell lines and human monocytes.
  • 1A Tumor cell lines of the human hepatocellular carcinoma (HCC) HuH7, the lung adeno carcinoma H1975 and the colorectal cancer (CRC) HCT116.
  • IB human monocytes upon various treatments.
  • FIG.2 Metabolic activity test of tumor cells and human white blood cells upon G2-2-ADC treatment.
  • 2A Metabolic activity of human monocytes, the HCC cell line HuH7 and CRC cell line HCT116 upon treatment with G2-2 linked to the anthracycline analogue PNU.
  • 2B Metabolic activity of human monocytes and T-cells upon treatment with G2-2 linked to the pyrrolobenzodiazepine PBD.
  • FIG.3 Comparison of G2-2 effects on the metabolic activity of human and murine monocytes.
  • 3A Dose dependent effect on the metabolic activity of human monocytes (EC50: 0.6 pM).
  • 3B Dose dependent effect on the metabolic activity of murine bone marrow derived monocytes (EC50: 0.06 pM); Measurements performed after 96 hrs of G2-2 treatment.
  • FIG.4 Surface Expression of Differentiation Proteins upon antibody treatment of Monocytes.
  • 4A Flow cytometry showing CD 141, CD 14, CD86 and CD40 expression levels on human monocytes after treatment with G2-2 or control antibody.
  • 4B Flow cytometry showing CD 14, CD86 and CD40 expression on the human monocytic leukemia cell line THP-1 after treatment with G2-2 or control antibody with or without IFN-y.
  • FIG.5 Cytokine release upon treatment of human monocytes.
  • the monocytes were either exposed to antibodies (ISO, G2-2) or polarized to a M0, Ml or M2 phenotype by cytokines treatment.
  • 5A Cytokine expression of monocytes from three donors upon different treatment schemes.
  • 5B Cytokines expression of monocytes from one donor upon treatment with membrane-near binding G2-2, G2-2M and more C-terminal (G2-4) GOLPH2 binding antibodies.
  • FIG.6 RNA-Seq analysis upon treatment of human monocytes. Alternative-box blot analysis of CD82 and CD37. Abbreviations: B before treatment; U untreated sample; G treatment with 0.5 pg/ml G2-2 and I for the isotype control antibody.
  • FIG.7 Monocytes and autologous T cells co-culture assay. 7A Human monocytes treated with G2-2 or a control antibody for 4 days were then co-cultured with autologous T-cells for two days until culture pictures were taken. 7B Absolute cell numbers of CD8 T cells and CD4 T cells are depicted 7C-E After day two of co-culture cells were harvested for flow cytometry analysis.
  • 7C,D The bars represent the MFI of CD25 and ICOS on the surface of CD8 and CD4 T-cells.
  • 7E CD 141 expression levels on monocytes 7F Cell culture supernatant of day two of co-culture experiments were collected for cytokine measurement. The fold changes are expressed as ratio of G2-2 treatment effect compared to either an isotype antibody (G2-2/ISO) or an untreated control (G2-2/control).
  • FIG.8 Effect of G2-2M treatment on antigen presenting (APC) properties of murine bone marrow derived monocytes.
  • Flow cytometry analysis shows MFI values as bar diagram of CD 11b, CD 11c, CD8a and percent values of H2Kb-SIINFEKL positive CD8 + cells, a marker for peptide presentation after peptide loading to monocytes.
  • FIG.9 Effect of BsMabG2-2/Myc loaded with Myc-SIINFEKL peptide (SEQ ID NO:41) in the murine MC38-OVA model.
  • 9A Tumor growth in C57BL/6 mice upon treatment with PBS or Myc- SIINFEKL peptide (SEQ ID NO:41) loaded antibodies BsMabG2-2/Myc or anti-Myc.
  • 9B Survival curve of this MC38-OVA model.
  • FIG.10 Effect of BsMabG2-2/Myc loaded with Myc-SIINFEKL (SEQ ID NO:41) on antigen presentation of an immortalized murine macrophage cell line.
  • 10A Bar diagram of expression of SIINFEKL peptide in MHC I context upon treatment with the antibodies anti-Myc or BsMabG2- 2/Myc or each antibody loaded with Myc-SIINFEKL peptide (SEQ ID NO:41). Shown are absolute MFI measurements in flow cytometry. 10B depicts the same experiment as in A as percent values.
  • FIG 11 Antigen presentation of human Monocytes upon treatment with BsMabG2-2/Myc loaded with Myc-Ml peptide (SEQ ID NO:42) and consecutive T-cell activation.
  • 11B shows bar diagrams of T cell activation in percent of all CD8 + cells upon co-culture with monocytes for 72 hours.
  • 11C and HD depict CD82 cell surface expression on monocytes upon treatment with either BsMabG2-2/Myc loaded with Myc-Ml peptide or with respective controls after co-culture with T-cells.
  • FIG.12 Epitope binding and effects on monocytes of G2-2MX.
  • 12A,B depict ELISA binding curves of G2-2M versus G2-2MX to either human (12A, SEQ ID NO:23) or murine (12B, SEQ ID NO:24) GOLPH2 (aa36 to 55).
  • 12C displays bar diagrams of metabolic activity of human monocytes. There is a four to eight fold difference in antibody concentration needed for similar metabolic activity of G2-2 and G2-2MX.
  • 12D,E depict surface expression of different membranous expression markers upon G2- 2MX treatment and respective control treatments.
  • FIG.13 Cartoons of the various antibodies and antibody-antigenic peptide constructs described in the patent application
  • FIG.14 Surface Expression of differentiation marker proteins upon different antibody treatment of co-cultured Monocytes and T cells.
  • 14A Flow cytometry showing CD 14 and CD40 expression levels on human monocytes after treatment with G2-2, EPR3606 (abeam Cat. No. ab239985) or an irrelevant isotype matched control antibody for five days. The positivity of the respective monocytes is expressed as geometric mean of fluorescence intensity (gMEAN MFI) of flow cytometry analysis as described in Example 28.
  • 14B Autologous T cells added to the monocytic culture on day three were analyzed after 48 hours for the activation marker HLA-DR by flow cytometry.
  • 14C Cell culture supernatant of the co-culture experiments were collected for cytokine measurement. The fold changes are expressed as ratio of G2-2 treatment effect compared to an isotype antibody (G2-2/ISO) or EPR3606 treatment effect compared to an isotype antibody (EPR3606/ISO) as described in Example 29.
  • FIG.15 Binding of G2-2 and EPR3606 (abeam) to GOLPH2 protein.
  • 15A An ELISA showing differential binding to extracellular GOLPH2 (aa 36-401), soluble GOLPH2 (aa 56-401) and the membrane-near peptide of GOLPH2 (aa36-55).
  • EPR3606 apparently binds to GOLPH2 downstream of the Furin cleavage site. Furin cleavage occurs between amino acids 55 and 56.
  • PBMCs Peripheral blood monocytes
  • Histoplaque 30 ml Histoplaque
  • Monocytes were separated by negative selection using magnetic bead depletion (Stemcell, EasySep human monocyte isolation kit, Cat. No 19359).
  • T cells were separated by negative selection using magnetic bead depletion (Miltenyi Biotec, Pan T cell isolation kit, Cat. No. 130-096-535). Cells in the flow through were harvested, washed in phosphate buffered saline with ImM EDTA (PBS Sigma- Aldrich TMS-012) and cultured in medium as described in Example 2.
  • Mouse bone marrow monocytes were isolated from the femur and tibia of C57BL/6 mice. The bones were rinsed in 70% ethanol (Carl Roth Cat. No. 7301.1) for 90 seconds and washed with PBS before flushing the bones with cell culture medium to mobilize the bone marrow. The bone marrow was fdtered through a sterile 70 pm cell strainer (Coring Cat. No. CLS431751-50EA), the cells are washed twice in medium and cultured in 48-well plates.
  • mice OT-1 T cells Male C57BL/6-OTl-Ly5.1 mice containing transgenic inserts for mouse Tcra-V2 and Tcrb-V5 genes and the congenic marker Ly5.1 were bred in-house at the DBM animal facility, University Basel. Their transgenic T cell receptor recognizes ovalbumin peptide residues 257- 264 (OVA257-264) in the context of H2Kb (CD8 co-receptor interaction with MHC class I). This results in MHC class I-restricted, ovalbumin-specific, CD8+ T cells (OT-I cells). Thus, the CD8+ T cells of these mice primarily recognize OVA257-264 when presented by the MHC I molecule. CD 8+ T cells of these mice were harvested from the spleen using a negative isolation kit (Stemcell, Cat. No. 19853) and cultured in complete IMDM as described in Example 2.
  • a negative isolation kit Stem, Cat. No. 19853
  • Example 2 Cell culture of human and murine tumor cell lines, monocytes and T cells
  • Human monocytes were cultured in ImmunoCultTM-SF Macrophage Medium (Stemcell Technologies Cat. No.10961). This medium contains Iscove's MDM, bovine serum albumin, insulin and transferrin. For some assays, culture medium was supplemented with human recombinant M-CSF (Stemcell Technologies Cat. No. 78057), Interferon gamma (Stemcell Technologies Cat. No. 78020), human recombinant IL-4 (Stemcell Technologies Cat. No 78045) or LPS (Merck Cat. No. L4524).
  • M-CSF Stem Technologies Cat. No. 78057
  • Interferon gamma Stemcell Technologies Cat. No. 78020
  • human recombinant IL-4 Stemcell Technologies Cat. No 7804
  • LPS Merck Cat. No. L4524
  • Human T cells were cultured in RPMI 1640 containing (Gibco Cat. No. 11875093) with 10% allogeneic serum, 5 ml 100 x Sodium Pyruvate (Gibco Cat. No 11360-039), 5 ml 100 x MEM non- essential amino acids (Gibco Cat. No. 11140-035), 50 pM (0.5ml) 1000 x beta-mercaptoethanol 50 mM, 100 U/ml Penicillin and 0.1 mg/ml Streptomycin (Sigma Aldrich Cat. No. P433).
  • the human monocytic leukaemia cell line THP-1 (Sigma-Aldrich Cat. No. 88081201) was maintained in DMEM (Gibco DMEM high glucose, Cat. No. 11960085) supplemented with 10% FCS (Seraglob Sera Pro FBS Cat. No. S40500E), 2 mM L-Glutamine (Gibco Cat. No. A2916801), 100 U/ml Penicillin and 0.1 mg/ml Streptomycin (Sigma Aldrich Cat. No. P433).
  • THP-1 cells were seeded in ImmunoCult-SF Macrophage Medium (Stemcell Technologies Cat. No. 10961) supplemented with 10% FCS (Seraglob Sera Pro FBS Cat. No. S40500E), 100 U/ml Penicillin and 0.1 mg/ml Streptomycin (Sigma Aldrich Cat. No. P433).
  • Mouse T cells isolated from the spleen of C57BL/6-OTl-Ly5.1 mice were cultured in complete IMDM containing 500 ml IMDM-GlutaMAX (Gibco Cat. No. 31980-022), 50 ml heat inactivated fetal bovine serum, 5 ml 100 x Sodium Pyruvate (Gibco Cat. No 11360-039), 5 ml 100 x MEM non- essential amino acids (Gibco Cat. No. 11140-035), 50 pM (0.5ml) 1000 x beta-mercaptoethanol 50 mM, 100 U/ml Penicillin and 0.1 mg/ml Streptomycin (Sigma Aldrich Cat. No. P433).
  • Immortalized murine bone marrow derived macrophages established as described in De Nardo, Kalvakolanu DV, Latz E in Immortalization of Murine Bone Marrow-Derived Macrophages in Methods in molecular biology (Clifton, N.J.) May 2018 DOI: 10.1007/978-l-4939-7837-3_4 were cultured in RPMI 1640 (Gibco Cat. No. 11875093) supplemented with 10% FCS (Seraglob Sera Pro FBS Cat. No. S40500E), 2 mM L-Glutamine (Gibco Cat. No. A2916801), 100 U/ml Penicillin and 0.1 mg/ml Streptomycin (Sigma Aldrich Cat. No. P433).
  • HCT116 a human colon cancer cell line
  • RPMI Gibco Cat. No. 11875093
  • FCS Erglob Sera Pro FBS Cat. No. S40500E
  • 2 mM glutamine Gibco Cat. No. A2916801
  • Hl 975 a human lung adeno carcinoma cell line and HuH7, a human hepatocellular carcinoma cell line were grown in DMEM (Gibco DMEM high glucose, Cat. No. 11960085) supplemented with 10% FCS (Seraglob Sera Pro FBS Cat. No. S40500E).
  • FCS Sembo Sera Pro FBS Cat. No. S40500E
  • the murine colon cancer cell line MC38-OVA, expressing ovalbumin was maintained in DMEM (Gibco DMEM high glucose, Cat. No. 11960085) supplemented with 10% FCS (Seraglob Sera Pro FBS Cat. No. S40500E), 2 mM L-Glutamine (Gibco Cat. No. A2916801), 100 U/ml Penicillin and 0.1 mg/ml Streptomycin (Sigma Aldrich Cat. No. P433).
  • Example 3 Measurement of sGOLPH2 protein levels
  • Tumor cells (HCT116, H1975, HuH7) were seeded in 6 well plates at a concentration of 5 x 10 5 per well.
  • Human monocytes and T cells were isolated as described in Example 1, seeded in 48 well plates at a concentration of 0.25 x 10 6 per well and grown at 37 °C in a humidified incubator in 5% CO2.
  • the culture medium ImmunoCultTM-SF Macrophage Medium (Stemcell Technologies Cat. No. 109) was supplemented with 50 ng/ml human recombinant GM-CSF (Stemcell Technologies Cat. No. 78190) or human recombinant M-CSF (Stemcell Technologies Cat. No. 78057.1) or 0.5 mg/ml of G2-2opti or isotype control antibody (ISO) and cells were cultured for 10 days.
  • the isotype control antibody used in these Examples comprises a heavy chain variable region with a sequence set forth in SEQ ID NO:29 and a light chain variable region with a sequence set forth in SEQ ID NO:33.
  • the isotype control antibody used in these Examples comprises a heavy chain CDR1 as set forth in SEQ ID NO:30, a heavy chain CDR2 as set forth in SEQ ID NO:31, a heavy chain CDR3 as set forth in SEQ ID NO:32, a light chain CDR1 as set forth in SEQ ID NO:34, a light chain CDR2 as set forth in SEQ ID NO: 35 and a light chain CDR3 as set forth in SEQ ID NO: 36.
  • FIG. 1 shows that high amounts of soluble GOLPH2 (sGOLPH2) were released from the tumor cell lines HuH7, H1975 and HCT116. In contrast, significantly lower amounts of sGOLPH2 were released by human monocytes.
  • Example 4 Influence of G2-2 drug-conjugates on the viability of different cell types
  • the antibody drug conjugate (ADC) G2-2-PNU was produced as G2-2opti IgGl as described in PCT/EP2017/079870 (Seq ID NOs: 18, 19 and 20).
  • ADC antibody drug conjugate
  • G2-2-vc-PBD valine at position 205 of the kappa light chain constant region was substituted by cysteine (V205C).
  • cysteine V205C
  • TCEP Tris(2-carboxyethyl)phosphine
  • TCEP Tris(2-carboxyethyl)phosphine
  • TCEP Tris(2-carboxyethyl)phosphine
  • TCEP Tris(2-carboxyethyl)phosphine
  • TCEP Tris(2-carboxyethyl)phosphine
  • TCEP Tris(2-carboxyethyl)phosphine
  • TCEP Tris(2-carboxyethyl)phosphine
  • Monocytes and T cells from three different healthy donors were isolated by negative selection as described in Example 1.
  • Cells were seeded in 96 well plates at a concentration of I MO 3 per well and grown at 37 °C in a humidified incubator in a 5% CO2 atmosphere overnight in culture medium described in Example 2.
  • HCT116 and HuH7 cells were grown as described in Example 2 and seeded in 6 well plates at a concentration of 5 x 10 5 per well.
  • FIG.2A shows that the addition of a G2-2 antibody-drug conjugate to the tumor cell lines HCT116 and HuH7 resulted in decreased viability of the tumor cell lines.
  • addition of the same antibody-drug conjugate to human monocytes resulted in increased metabolic activity of the cells.
  • no effect was seen when the antibody-drug conjugate was added to human T cells (FIG.2B).
  • G2-2M G2-2 variable regions and heavy chain constant region
  • isotype control antibody the variable regions of the heavy and light chain of G2-2opti were substituted by variable regions of an anti Myc-Tag antibody, known as 9E10 (SEQ ID NO:29 and SEQ ID NO:33); Evan GI, Lewis GK et al. 1985 Mol Cell Biol (5), 3610-3616 and Schiwek W, Buxbaum B et al. FEBS letters 414 (1997) 33-38) and annotated here as ISOM.
  • Monocytes from a healthy donor were isolated by negative selection as described in Example 1, plated on 96-well plates in 90 pl ImmunoCultTM-SF Macrophage Medium (Stemcell Technologies Cat. No. 10961) at a density of O.l x lO 6 cells per well and grown at 37 °C in a humidified incubator in a 5% CO 2 .
  • Mouse bone marrow monocytes were isolated from C57BL/6, mice and cultured in Macrophage Base Medium DXF (PromoCell Cat. No. C-28057).
  • FIG.3 shows that also the unconjugated antibodies G2-2opti and G2-2M have the ability to increase the viability of human and mouse-derived monocytes.
  • Monocytes from three different healthy donors were isolated by negative selection as described in Example 1 and cultured as described in Example 2.
  • Culture medium was supplemented with either 0.5 mg/ml G2-2, isotype control antibody, 50 ng/ml human M-CSF for M0 monocytes, 50 ng/ml M-CSF and 50 ng/ml IFN-y and lOng/ml LPS for Ml monocytes, 50 ng/ml M-CSF and lOng/ml IL4 for M2 monocytes for 5 days.
  • Cell culture supernatant was collected, stored at -20°C and analyzed as described in Example 8.
  • Cells were harvested in 2 mM EDTA PBS and stained with Live/Dead dye (Biolegend, Cat. No.
  • FIG.4A shows that certain differentiation proteins are either upregulated or downregulated upon addition of the antibody G2-2opti to human monocytes.
  • Example 7 Surface expression of differentiation proteins upon antibody treatment ofTHP-1 cells
  • Human monocytic leukaemia cells THP-1 cultured as described in Example 2, were seeded in a 24- well-plate 0.2 x 10 6 cells per well containing ImmunoCult-SF Macrophage Medium. Next day the culture medium was changed and supplemented with either 0.5 mg/ml G2-2opti, human isotype IgGl, 50 ng/ml human INF-y (Peprotech Cat. No. F2617) plus 0.5 mg/ml G2-2opti or 50 ng/ml human INF- y plus 0.5 mg/ml human isotype IgGl. All cells were cultured for 72 hours at 37°C, 5% CO2 in a tissue culture incubator.
  • CD14-BV605 Biolegend, clone: M5E2
  • CD86-BV711 Biolegend, clone: IT2.2
  • CD40-APC-Cy7 Biolegend, clone: 5C3
  • FIG.4B shows that in the presence of IFN-y the expression of the proteins CD 14, CD40 and CD86 is upregulated.
  • Example 8 Cytokine secretion of human monocytes or monocyte-derived cells
  • the 41-plex consisted of EGF, FGF-2, Eotaxin, TGF-a, G-CSF, Flt-3L, GM-CSF, Fractalkine, IFNa2, IFN-y, GRO, IL-10, MCP-3, IL-12P40, MDC, IL-12P70, PDGF-AA, IL-13, PDGF-BB, IL-15, sCD40L, IL-17A, IL-IRA, IL-la, IL-9, IL-1B, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP-la (CCL3), MIP-1B (CCL4), RANTES (CCL5), TNFa, TNFB, and VEGF.
  • IL-18 test was added by Eve Technologies. The assay sensitivities of these markers range from 0.3 - 26.3 pg/mL. Measurements were done in duplicate and the mean of Individual ana
  • Cytokine cocktail M0 contains 50 ng/ml human M-CSF and induces the formation of resting M0 macrophages.
  • Cytokine cocktail Ml contains 50 ng/ml M-CSF, 50 ng/ml IFN-y and lOng/ml LPS and induces the polarization into classically activated Ml macrophages.
  • Cytokine cocktail M2 contains 50 ng/ml M-CSF and lOng/ml IL-4 and induces the polarization into alternatively activated M2 macrophages.
  • Monocytes from a healthy donor were isolated by negative selection as described in Example 1 and cultured as described in Example 2.
  • Culture medium was supplemented with either 0.5 mg/ml G2- 2opti, G2-2M, G2-4, isotype control antibody or left untouched for 5 days.
  • Cell culture supernatant was collected, stored at -20°C and analyzed at Eve Technology as described in Example 8 using the LuminexTM 100 system (Luminex, Austin, TX, USA).
  • LuminexTM 100 system Luminex, Austin, TX, USA
  • MILLIPLEX Human Cytokine/Chemokine 13-plex kit (Millipore, St. Charles, MO, USA) was used according to the manufacturer's protocol.
  • the 13-plex consisted of GM-CSF, IFN-y, IL-lbeta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12(p70), IL-13, MCP-1 and TNF-alpha.
  • the assay sensitivities of these markers range from 0.1 - 9.5 pg/mL for the 13-plex. Measurements were done in duplicates, the mean is shown in a bar diagram in Figure 5 B.
  • FIG.5A shows that human monocytes that have been treated with the antibody G2-2opti release comparable amounts of cytokines as human monocytes that have been treated with a cytokine cocktail that induces polarization into Ml macrophages. This data indicates that human monocytes that have been treated with the antibody G2-2opti mature into Ml macrophages.
  • FIG.5B shows that the treatment of human monocytes with the antibodies G2-2opti and G2-4 results in an increased secretion of the cytokines IL-lbeta, IL-6 and TNF-alpha.
  • RNA of human monocytes was isolated using TRIzol reagent (Life technologies, Cat. 15596026) according to manufacturers protocol. In brief, growth medium of cultured monocytes was removed and floating cells pelleted by centrifugation. The supernatant was discarded and cell pellet added to the adherent cells on the plate. 0.3 m of TRIzol Reagent per 1 x 10 6 cells were added directly to the culture dish, pipetted up and down and incubated for 5 minutes at room temperature. 0.2 m of chloroform per 1 mb of TRIzol Reagent was added, and incubated for 3 minutes. Samples were centrifuged and the colorless upper aqueous phase, containing the RNA transferred to a new tube.
  • RNAse-free water 0.5 mb of isopropanol was added, incubated for 10 minutes and centrifuged. The precipitated RNA pellet was washed with 75% Ethanol, air dried for 5-10 minutes and resuspended in RNAse-free water.
  • A 0.5 pg/ml G2-2opti
  • I isotype control antibody
  • U left untouched
  • CD82 and CD37 according to Jones E.; Dendritic Cell Migration and Antigen Presentation are coordinated by the opposing functions of the tetraspanins CD82 and CD36. J of Immunology 2015 doi: 10.4049/jimmunol. 1500357.
  • Activated murine Monocytes as shown in Table 4 according to Orecchioni, M. et al.; Macrophage Polarization: Different Gene Signatures in M1(LPS+) vs. Classically and M2(LPS-) vs. Alternatively Activated Macrophages. Frontiers in Immunology 2019 Vol 10, Article ID 1084.
  • Table 2 shows that monocytes that have been treated with the antibody G2-2opti have a gene expression profile that to a large extent overlaps with a published gene expression profile of monocyte-derived dendritic cells.
  • Table 3 further shows that monocytes that have been treated with the antibody G2-2opti have a gene expression profile that to a large extent overlaps with a published gene expression profile of matured dendritic cells.
  • FIG.6 shows that the treatment of human monocytes with the antibody G2-2opti results in the upregulation of CD82 and the downregulation of CD37, which is again indicative for matured dendritic cells.
  • treating the monocytes with an isotype control antibody had no significant impact on the expression of CD82 and CD37 in comparison to an untreated control.
  • Table 3 shows that treatment of monocytes with the antibody G2-2opti can also result in a gene expression profile that, to a large extent, overlaps with a published gene expression profile of classically activated Ml macrophages. This indicates, that the treatment of human monocytes with the antibody G2-2opti can induce the maturation of monocytes either into matured dendritic cells or in classically activated Ml macrophages.
  • Example 11 Co-culture of monocytes with T cells
  • Monocytes from healthy human donors were isolated by negative selection and cultured as described in Examples 1 and 2. Either G2-2opti or ISO in a concentration of 0.5 mg/ml were added to the culture medium for 4 days. Autologous T cells, acquired by negative isolation and cultured as described in Examples 1 and 2 were added in the presence of soluble anti-CD3 antibody (0.3 pg/ml) (clone HIT3a) on the top of monocytes for another 48 hours in co-culture.
  • soluble anti-CD3 antibody 0.3 pg/ml
  • Cell culture supernatants were collected for cytokine measurement, and cells were harvested in 2 mM EDTA in PBS for flow cytometry analysis.
  • Cells were first stained with Live/Dead dye (Biolegend, Cat. 77474) and incubated with human FcR inhibitor (Invitrogen, Cat. 4350496) for 20 min on ice. After washing with FACS buffer (PBS supplemented with 2% FCS, 0.1%sodium azide, 2 mM EDTA) twice, cells were stained with monoclonal antibodies for 30 min on ice, in brief antibodies used were CD4+ (Invitrogen, clone OKT4) and CD8+ (Becton Dickinson, clone SKI)
  • FIG.7A shows that T cells only aggregate if co-cultured with human monocytes that have been treated with the antibody G2-2opti.
  • the absolute number of T cells in the co-culture was higher if the monocytes in the co-culture had been previously treated with the antibody G2-2opti compared to monocytes that had been previously treated with the isotype control antibody.
  • Example 12 Expression of activation markers in T cells upon co-culture with monocytes
  • Anti-human monoclonal antibodies used were CD3-PE-CF584 (BD Biosciences, clone: UCHT1), CD4-PerCP-Cy5.5 (Biolegend, clone: SK3), CD8-APC-eFluor 780 (Biolegend, clone: SKI), CD25- BV605 (Biolgend, clone: BC96), ICOS- FITC (Biolegend, clone: ISA-3). CD25 and ICOS were examined based on live CD8 T cell (CD3+CD4-CD8+) and CD4 T cell (CD3+CD4+CD8-)
  • FIGs.7C-D show that the expression of the activation markers CD25 and ICOS was upregulated both in CD4+ and CD8+ T cells following co-culturing with human monocytes that had been treated with the antibody G2-2opti. In contrast, no upregulation of these activation was observed following coculturing with human monocytes that had been treated with the control antibody ISO.
  • FIG.7E shows that treatment of monocytes (CD3-CD4-CD8-) with the antibody G2-2opti results in the upregulation of CD 141, which is a marker for dendritic cells. This finding indicates that the treatment of monocytes with G2-2opti induces the maturation of monocytes into dendritic cells.
  • Example 13 Secretion of cytokines by co-cultured cells
  • Example 8 Cell culture supernatants collected as described in Example 6 were send to Eve Technologies for cytokine measurement as described in Example 8.
  • a MILLIPLEX Human Cytokine/Chemokine 13- plex kit (Millipore, St. Charles, MO, USA) was used according to the manufacturer's protocol. The 13-plex consisted of GM-CSF, I IFN-y, IL-lbeta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12(p70), IL- 13, MCP-1 and TNF-alpha. The assay sensitivities of these markers range from 0.1 - 9.5 pg/mL for the 13-plex. Measurements were done in duplicates, the mean is shown in a bar diagram in Figure 7F.
  • FIG.7F shows that multiple cytokines were secreted in significantly higher amounts when monocytes had been treated with the antibody G2-2opti in comparison to monocytes that had been treated with the control antibody ISO.
  • Negative isolated mouse bone marrow monocytes acquired as described in Example 1 and cultured as described in Example 2 were treated with G2-2M or ISOM (0.5 mg/ml) for 48 hours, then OVA257- 264 peptide (SIINFEKL) (10 pM) (Eurogentec Cat. No. AS-60193-1) was loaded for 3 hours or 20 hours. After washing out the unbonded SIINFEKL peptide cells were harvested in 2 mM EDTA PBS and stained with Live/Dead dye (Biolegend Cat. No. 77474) and murine FcR inhibitor CD16/CD32 (BD Biosciences Mouse BD Fc Block, clone 2.4G2) for 20 min on ice.
  • SIINFEKL OVA257- 264 peptide
  • Anti-mouse monoclonal antibodies used were CD8a-FITC (Biolegend, clone: KT15), CDl lb-BV605 (Biolegend, clone: MI/70), CDl lc-PE-Cy7 (Biolegend, clone: N418), H-2Kb-SIINFEKL-PE (Biolegend, clone: 25- D1.6). All the surface markers were examined based on CD1 lb+ live cells.
  • the controlled Fab arm exchange technique was applied by mutating Phenylalanine at position 405 in CH3 of the human G2-2optiIgGl to Leucine (F405L) and Lysine at position 409 in CH3 of the human anti-Myc IgGl (ISO see Example 5) to Asparagine (K409R) as described in Paul S, Connor J, Nesspor T, et al. An efficient process of generating bispecific antibodies via controlled Fab-arm exchange using culture supernatants. Protein Expr Purif. 2016;121: 133-140. doi: 10.1016/j .pep.2016.01.014.
  • Both antibodies were produced separately in CHO or in 293HEK cells by transfection of vectors coding for VL and VH of G2-2-F405L or 9E10-K409R respectively.
  • IgG was purified from cell culture media by protein A affinity chromatography.
  • Both purified antibodies were mixed at equal molar concentration and reduced for five hours with 2- Mercaptoethylamine-HCl (Thermo Scientific, article 20408) at 37°C.
  • 2-MEA 2-mercaptoethylamine-HCl
  • gel filtration was performed. Re-oxidation and formation of bispecific antibody was done by incubating over night at room temperature.
  • the bispecific G2-2/anti-Myc monoclonal antibodies (BsMabG2-2/Myc) were sterile filtrated, measured and loaded with Myc-SIINFEKL peptide (SEQ ID NO:41) or Myc-Influenza Ml peptide (SEQ ID NO:42) (synthesized by Genscript, NY, USA) at 10- fold molar excess overnight at 4°C. Unbound peptide was removed by size exclusion chromatography and peptide loaded bispecific antibody was again sterile filtrated, quantified and kept in PBS at 4°C.
  • Example 16 Tumor growth inhibition by antibody-peptide treated mice in a MC38-OVA model.
  • mice were vaccinated i.p. at day 24 and day 12 prior to s.c. implantation of 1 x 10 6 murine colon cancer cells MC38-OVA.
  • Vaccination comprised in group 1 BsMabG2-2/Myc 12.5 mg/kg loaded with the peptide MYC-SIINFEKL, in group 2 anti-Myc hlgGl (ISO) 12.5 mg/kg loaded with the peptide MYC-SIINFEKL and in group 3 PBS.
  • the efficacy was quantified by tumor volume measurements every other day. At day 34 after tumor implant, all mice from control groups 2 and 3 had been sacrificed due to high tumor volumes.
  • the humane endpoint was set above 1000 3 mm with a maximum at 1500 mm 3 .
  • Example 17 Antigen-display by antibody-peptide treated murine monocytes
  • Immortalized murine bone marrow derived macrophages were seeded in 48 well plates with 0.2 x 10 6 cells per well. Two hours later, cells were incubated with either_BsMabG2-2/Myc loaded with MYC- SIINFEKL peptide or anti-Myc IgGl(ISO) loaded with MYC-SIINFEKL peptide or G2-2optiM IgG2a (G2-2M) without peptide or anti-Myc IgG2a (ISOM) without peptide or left untouched for 24 hours. Cells were harvested in 2 mM EDTA PBS and stained with Live/Dead dye (Biolegend Cat. No.
  • FIG. 10 shows results from two independent experiments. There is an over 3.5 -fold increased presentation upon treatment with_BsMabG2-2/Myc peptide loaded compared to ISO peptide loaded.
  • Example 18 Antigen presentation by antibody-peptide treated human monocytes
  • Monocytes from three human healthy donors were isolated by negative selection as described in Example 1. Monocytes were seeded 0.25 x 10 6 cells per well in a 48 well plate in duplicate. Two hours later, either BsMabG2-2/Myc loaded with Myc-Ml peptide or anti-Myc hlgGl loaded with Myc-Ml peptide or an equimolar amount of Myc-Ml peptide was added to each well. 48 hours after treatment start cells were harvested and prepared for FACS staining as described in Example 11. Supernatant was stored for cytokine analysis. Duplicate wells were left untouched and further treated as described in Example 19.
  • Antibodies used for Flow cytometry were anti human Influenza M1/HLA-A2 complex antibody (Dendritics, Cat. No. DDX0270A647). After washing with FACS buffer twice, cells were analysis by BD LSRFortessa. All the surface markers were examined based on live single cells gating.
  • Fig 11A shows presentation of Ml peptide in the HLA-A2 context
  • Example 19 T-cell activation upon co-culture with Ml presenting autologous monocytes
  • Human Monocytes were treated for 48 hours with bispecific peptide loaded antibody or controls as described in Example 18. Thereafter, autologous T-cells, isolated as described in Example 1, were added to the monocytes in a ratio monocytes to T-cells of 1 to 3. To foster T-cells, the Macrophage Base was supplemented with 10% of autologous serum. At day three of co-culture, supernatants were collected for cytokine measurement and cells were harvested in 2 mM EDTA PBS and stained with Live/Dead dye (Biolegend, Cat. No. 77474) and human FcR inhibitor (Invitrogen Cat. No. 4350496) for 20 min on ice.
  • HLA-A2/M1-APC Endritics, clone 405H1.01
  • CDl lc-FITC Biolegend, clone 3.9
  • CD8a-BV605 Biolegend, clone RPA-T8
  • CD82-PerCP-Cy5.5 Biolegend, clone ASL-24
  • CD25-BV421 Biolegend, clone BC96
  • CD69-PE- Cy5 Biolegend, clone FN50.
  • FIG 11C shows significantly increased expression of CD82 on monocytes treated with BsMabG2-2/Myc loaded with the Myc-Ml peptide.
  • the heavy chain variable region of antibody G2-2opti (SEQ ID NO: 18) was linked by a flexible linker (SEQ ID NO:51) to the light chain variable region of G2-2opti (SEQ ID NO: 19).
  • the resulting single chain Fv (scFV) was named scG2-2 (SEQ ID NO:70).
  • the scG2-2 was attached via a Serine rich linker (SEQ ID NO:52 and/or 53) to the hinge region of (i) a murine IgG2a Fc region (SEQ ID NO:37) for the construct G2-2MX (SEQ ID NO:73), or (ii) to a human IgGl Fc region (SEQ ID NO:38) for the construct G2-2HX (SEQ ID NO:72).
  • ELISA was carried out in 96 well plates coated overnight with Streptavidin (Sigma-Aldrich Cat. No. SA 101) at a concentration of 2 pg/ml and loaded with 4 pg/ml biotinylated peptide the next morning.
  • Peptides used were human GOLPH2 aa 36 to 55 (SEQ ID NO:23) or murine GOLPH2 aa 36 to 55 (SEQ ID NO: 24) or a control peptide. After 30 minutes incubation unbound peptide was washed away and wells were blocked with 1% Gelatin (Sigma- Aldrich Cat. No G9391) for 1 hour.
  • Example 22 Proliferation of human PBMCs upon different antibody concentrations
  • Monocytes from three different healthy donors were isolated by negative selection as described in Example 1.
  • Cells were seeded in 96 well plates at a concentration of U I O’’ per well and grown at 37 °C in a humidified incubator in a 5% CO2 atmosphere overnight in culture medium described in Example 2.
  • the next day 10 pl of twofold serial dilutions of antibodies G2-2opti, G2-2M, G2-2MX or ISO in PBS were added, resulting in final antibody concentrations ranging from 0.98 pg/ml to 7.812 pg/ml. Each dilution was done in quatruplicate. After 72 hours, plates were removed from the incubator, supernatant of two wells per test were harvested for cytokine analysis.
  • PrestoBlueTM cell viability reagent a resazurin-based cell permeable solution (Life Technologies Cat. No. A 13262) was added to each well and transferred back to the incubator for 30 minutes. In viable cells resazurin is converted to fluorescent resorufm. Fluorescence was measured on a SpectraMax ® GeminiTM EM microplate reader (Molecular Devices, USA) with an excitation wavelength of 560 nm and emission wavelength of 590 nm. Data of ISO served as control and were set as 100% for each concentration. FIG.12C shows that there is a four to eight fold difference in antibody concentration needed to achieve similar metabolic activity of human monocytes.
  • Example 23 TNF-alpha secretion of human PBMCs upon treatment with different antibodies in different concentrations
  • Example 22 The supernatants of human monocytes treated as described in Example 22 were send to Eve Technologies for TNF-alpha measurement by MILLIPLEX technology as described in Example 8. Measurements were done in duplicates, the mean as a function of the antibody concentration used is shown in a bar diagram in Figure 12F.
  • Example 24 Surface expression of CD82, CD 141, CD86 and CD40 upon G2-2MX Human monocytes were isolated as described in Example 1, seeded in 48 well plates as described in Example 2 and treated either with G2-2opti, G2-2MX, control antibody, or left untouched. The antibodies G2-2opti and ISO were used at a concentration of 0.5 mg/ml, the antibody G2-2MX was used at 0.05 mg/ml. After 72 hours cells were harvested and prepared for flow cytometry as described in Example 19.
  • FIG. 12 shows bar diagrams of CD141, CD86, CD40 and CD82 surface expression on human monocytes anlaysed by flow cytometry.
  • FIG.12D depicts MFI data and FIG.12E percent values. Both, G2-2 and G2-2MX led to enhanced surface expression. Notably, G2-2MX was used at a 10 fold less concentration.
  • the single chain 3DX SEQ ID NO: 39 as described in Fujiwara K, Poikonen K et al. A single-chain antibody/epitope system for functional analysis of protein-protein interactions. Biochemistry. 2002;41(42): 12729-12738. doi: 10. 102 l/bi0263309) was synthesized by Genescript (New Jersey). 3DX is linked to the C-terminal Glycine of the heavy chain Fc CH3 of G2-2MX (SEQ ID NO:37) via a short Glycine-Serine linker (GGGS, SEQ ID NO:68). The resulting construct is named G2-2MXQ respectively and is depicted as scheme in Figure 13. The construct is expressed in CHO or 293HEK cells.
  • the resulting protein a bispecific bivalent single chain Fv-Fc-single chain antibody is purified from cell culture media by protein A affinity chromatography and sterile filtered.
  • Cloning of G2- 2HXQ was done accordingly by linking 3DX to the C-terminal Glycine of the heavy chain Fc CH3 of G2-2HX (SEQ ID NO:38).
  • Example 27 Cloning of construct G2-2-L At the C-Terminus of the kappa light chain of G2-2opti IgGl a Smal restriction site is added to allow for versatile cloning of different peptides to the light chain.
  • This construct is named G2-2-L-L and its scheme depicted in Figure 13.
  • a Sma I restriction site has been fit into the nucleotide sequence coding for the last Proline and Glycine residues of the human IgGl heavy chain (SEQ ID:N0 20).
  • This construct is named G2-2-L-H. as depicted in scheme of G2-2-L.
  • Peptides for experiments in murine cells and models include ovalbumin aa257-264 and ovalbumin aa329-338 (SEQ ID NO:43), MC38 Neo-Epitopes REPSI, ADPGK, DPAGT1 (SEQ ID NO:44), MC38 Neo-Epitopes ADPGK, RPL18 (SEQ ID NO:55), melanoma TRP2, pMELlOO (SEQ ID NO:46) and HPV-16 E7 aa5-18, aa 49-57 (SEQ ID NO:47).
  • Peptides for experiments in human cells and models included HPV-16 E7(aa5-18) and Influenza Ml aa 58-66 (SEQ ID NO:48), SARS Cov2 Nucleoprotein aa 222-235 and EBV (SEQ ID NO:49), SARS Cov2 (SEQ ID NO:50). All peptides were synthesized by Genscript.
  • the different constructs are expressed in CHO or 293HEK cells.
  • the resulting antibody peptide conjugates are purified from cell culture media by protein A affinity chromatography and sterile filtered.
  • Monocytes from a healthy human donor were isolated by negative selection and cultured as described in Examples 1 and 2 in 48 well chamber plates with 0.25 x 10 6 cells per well. Either G2-2 or EPR3606 (abeam Cat No. ab 109628) or ISO (an irrelevant isotype matched control antibody) in a concentration of 0.5 mg/ml were added to the culture medium for 3 days. Autologous T cells, acquired by negative selection using magnetic bead depletion (Stemcell EasySep human CD8 T cell isolation kit, Cat. No. 17953) and cultured as described in Examples 1 and 2 were added in a ratio of 1: 1 on the top of monocytes for another 48 hours in co-culture supplemented with 2% autologous serum.
  • T cells and cell culture supernatants were separated from the monocytic cells layer. After centrifugation, the supernatant was collected for cytokine measurement and T cells were saved for downstream application.
  • T cells and monocytes were harvested separately in 2 mM EDTA in PBS for flow cytometry analysis. Monocytes were incubated with human FcR inhibitors (anti CD64 BioLegend Cat. 305002; anti CD32 Stem Cell Technologies Cat. 60012; anti CD16 BioLegend Cat. 302002) for 30 min on ice. T cells and monocytes were washed with FACS buffer (PBS supplemented with 2% FCS, 0.1% sodium azide, 2 mM EDTA) twice, and stained with monoclonal antibodies for 30 min on ice.
  • FACS buffer PBS supplemented with 2% FCS, 0.1% sodium azide, 2 mM EDTA
  • Antibodies used were CD14-APC (BioLegend, clone M5E2), CD40-AF488 (BioLegend, clone 5C3) and HLA-DR-PB (BioLegend, clone L243). Flow cytometry analysis was performed on a Cytoflex (Beckman Coulter).
  • Example 8 Cell culture supernatants collected as described in Example 28 transferred on dry ice to Eve Technologies for cytokine measurement as described in Example 8.
  • a MILLIPLEX Human Cytokine/Chemokine 15-plex kit (HDF15, Millipore, St. Charles, MO, USA) was used according to the manufacturer's protocol.
  • the 13-plex comprised GM-CSF, I IFN-y, IL-lbeta, ILIRa, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL12(p40), IL-12(p70), IL-13, MCP-1 and TNF-alpha.
  • the assay sensitivities of these markers range from 0.1 - 9.5 pg/mL for the 15-plex. Measurements were done in duplicates, the mean is shown in a bar diagram in Figure 14C.
  • Example 30 Differential binding of antibody EPR3606 (abeam) and G2-2 on GOLPH2 protein and peptide analysed by ELISA
  • Antibody EPR3606 from patent application CN 108 324 959 A was bought from abeam (Cat. No. ab239985) to test for differential binding on GOLPH2 as compared to G2-2. Specifically, experiments were conducted to test whether EPR3606 binds to soluble GOLPH2.
  • 293HEK cells were transfected with two different cDNAs containing a GP73 AN variant including a C-terminal lOxHIS-Tag and a secretion signal at the N-terminus.
  • One cDNA construct comprised GP73 AN AA 36 to 401 with a R52A mutation, resulting in a non-cleavable variant of the whole extracellular GP73 (eGOLPH2-His R52A).
  • the second cDNA comprised GP73 AN AA 56 to 401, soluble GOLPH2 (sGOLPH2-His).
  • ELISA was carried out in a 96 well plate (Greiner, Cat. No. 655061) blocked with 1% gelatine in PBS (Sigma #G9391) for one hour and coated overnight with 2pg/ml recombinant Streptavidin (Sigma #S0677). The next morning wells were loaded with 4pg/ml biotinylated human GOLPH2 Peptide AA 36-55 (GenScript), incubated for one hour, washed and developed with 80, 40, 20, 10 or 5ng/ml anti GOLPH2 EPR3606 antibody (Abeam Cat. No. ab239985) followed by secondary anti rabbit-HRP antibody (Jackson Immuno Cat. No. 111-036-047). Chromogenic substrate (ECL Cytiva Amersham Cat. No. 10155854) was applied to detect peroxidase. The reaction was stopped and absorbance was read at 450 nm (FIG. 15A).
  • an ELISA was carried out on a Nickel coated 96 well plate (Thermo Scientific Cat. No. 15442) loaded overnight with lOpg/ml recombinant sGOLPH2-His R52A or sGOLPH2-His protein.
  • EPR3606 Abeam #ab239985
  • anti rabbit-HRP antibody Jackson Immuno, Cat. No. 111-036-047
  • G2-2 in a concentration of 80, 40, 20, 10 or 5ng/ml followed by anti human-HRP antibody (Jackson Immuno #109-036-097).
  • Chromogenic substrate was applied to detect peroxidase, the reaction was stopped and absorbance was read at 450 nm (FIG. 15A).

Landscapes

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

Abstract

The present invention relates to target-binding molecules, preferably antibodies or target-binding fragments thereof, that specifically bind to the extracellular part GOLPH2, or to an antigenic portion thereof, wherein the binding of the target-binding molecule to the extracellular part of GOLPH2, when expressed on a monocyte or monocyte-derived cell, induces the maturation of said monocyte or monocyte-derived cell. The invention further relates to immunoconjugates comprising the target-binding molecules, in particular the anti-GOLPH2 antibodies, or target-binding fragments thereof. The target-binding molecules and immunoconjugates of the invention may be administered alone, as a therapeutic conjugate or in combination with other therapeutic agents, such as chemotherapeutic agents, antigens, vaccines or checkpoint inhibitors. The present invention also relates to nucleotide sequences encoding the target-binding molecule of the invention, as well as vectors and host cells containing the nucleotide sequences. The target-binding molecules, antibodies and compositions of the invention are useful in initiating and/or enhancing an immune response in a subject. Further, the target-binding molecule may be used in methods for generating antigen-presenting cells and activated T cell.

Description

ANTI-GOLPH2 ANTIBODIES FOR MACROPHAGE AND DENDRITIC CELL DIFFERENTIATION
The present invention is in the field of immunotherapy. In particular, the invention relates to targetbinding molecules, preferably antibodies or target-binding fragments thereof, that specifically bind to the extracellular part of the protein GOLPH2, or to an antigenic portion thereof. It was surprisingly found by the inventors that binding of the target-binding molecule to the extracellular part of GOLPH2, when expressed on the cell surface of a monocyte or a monocyte-derived cell, induces the maturation of said monocyte or monocyte-derived cell. Furthermore, the inventors unexpectedly found that antibodies directed against GOLPH2, if conjugated or complexed with an antigen, elicit specific and reproducible presentation of this antigen on the surface of myeloid immune cells, which in turn results in effective and lasting T-cell stimulation. The invention further relates to immunoconjugates comprising the target-binding molecules, complexes or constructs, in particular the anti- GOLPH2 antibodies, or target-binding fragments thereof. The target-binding molecules and immunoconjugates of the invention may be administered alone, as a therapeutic conjugate or in combination with other therapeutic agents, such as chemotherapeutic agents, antigens, vaccines or checkpoint inhibitors. The present invention also relates to nucleotide sequences encoding the targetbinding molecule of the invention, as well as vectors and host cells containing the nucleotide sequences. The target-binding molecules, antibodies and compositions of the invention are useful in initiating and/or enhancing an immune response in a subject. Further, the target-binding molecule may be used in methods for generating antigen-presenting cells and activated T cell. This versatile approach can be exploited in a variety of vaccine therapies, such that an antigen of choice, be it against an infectious disease or a tumor may be utilized for raising an immune response in the vaccinated individual.
BACKGROUND OF THE INVENTION
Golgi protein-73 (GP73), alternatively named Golgi Membrane Protein 1 (G0LM1), or Golgi- associated-Phosphoprotein 2 (GOLPH2) is a single pass transmembrane type II protein. Its genuine function is unknown. The genomic sequence of GOLPH2 predicts 11 exons and two splicing variants. The transcript variant 1 (NM_016548.3) is 3100nt in length and contains exons 2 to 11, while transcript variant 2 (NM_177937.2) is 3092nt in length and contains exons 1, and 3 to 11. Both variants encode the same open reading frame. The biological significance of these variants is currently not clear; see Kim et al., Golgi phosphoprotein 2 in physiology and in diseases. Cell & Bioscience 2012 2:31. Under steady-state conditions, GOLPH2 is an integral membrane protein of the cis- and medial-Golgi apparatus. However, GOLPH2 can cycle out of the cis Golgi to endosomes and the cell surface; see Puri S et al. Cycling of early Golgi proteins via the cell surface and endosomes upon lumenal pH disruption Traffic 2002, 3:641-653.
There is evidence that the endosomal trafficking of GOLPH2 allows for proprotein convertase furin mediated cleavage, resulting in its release into the extracellular space, and provides a molecular explanation for its presence as a serum biomarker for hepatocellular carcinoma (HCC); see Bachert C et al., Endosomal trafficking and proprotein convertase cleavage of cis Golgi protein GOLPH2 produces marker for hepatocellular carcinoma Traffic 2007, 8: 1415-1423; Marrero JA et al., GOLPH2, a resident Golgi glycoprotein, is a novel serum marker for hepatocellular carcinoma J Hepatol 2005, 43: 1007-1012; Mao Y et al., Golgi protein 73 (GOLPH2) is a valuable serum marker for hepatocellular carcinoma Gut 2010, 59: 1687-1693); Li X et al., Serum golgi phosphoprotein 2 level: a better marker than alpha-fetoprotein for diagnosing early hepatocellular carcinoma Hepatology 2009, 50: 1682 or Zhu et al., Biomarkers for hepatocellular carcinoma: progression in early diagnosis, prognosis, and personalized therapy Biomark Res 2013, 1: 10. GOLPH2 has been shown to be highly expressed in several malignancies including hepatocellular, cholangiocellular, esophageal, renal, prostate and various other carcinomas but not in adjacent non-tumor tissue. Patients with GOLPH2- positive HCC have a higher tumor grade than patients with GOLPH2 -negative HCCs. In bile duct carcinomas (BDC) GOLPH2 expression correlates with better overall survival whereas in HCC GOLPH2 overexpression has been found to be associated with increased risk of metastasis, a higher probability of recurrence and a worse survival; see Riener et al. Golgi phosphoprotein 2 (GOLPH2) expression in liver tumors and its value as a serum marker in hepatocellular carcinomas. Hepatology 2009, 49: 1602-1609 and Ye et al. 2016, Cancer Cell 30, 444-458 September 12, 2016.
Antibodies for targeting GOLPH2 have been described in WO 2014/144355 A2, CN105699653 A and CN105734059 A. The use of antibodies to inhibit GOLPH2 to enhance cell-mediated immunity in cancer patients has been described in WO2012/112798 Al. GOLPH2 has been proposed as a biomarker for diagnosis of lung cancer WO 2011/093675 A2 or as a test in systemic inflammatory conditions e.g. sepsis WO2013/083781 A2.
Recently, antibodies have been described in WO 2018/091724 that bind to an extracellular part of GOLPH2 and carry out two independent functions. First, the binding of the antibodies to an epitope spanning the furin cleavage site of GOLPH2 prevents the release of soluble GOLPH2 (sGOLPH2) into the extracellular space. Second, in instances where sGOLPH2 has been cleaved off already, the antibodies can bind to a remnant part of GOLPH2 on the cell surface. Binding of these antibodies to the remnant part of GOLPH2 has been shown to result in internalization of the antibodies into the cell, which can represent a novel way of delivering therapeutic agents to GOLPH2 -positive cells.
Besides cancer, GOLPH2 is expressed on normal epithelial cells and other tissues to varying degrees. On monocytes, GOLPH2 has been described in the context of melanoma (Donizy P, et al., Golgi- related proteins GOLPH2 (GP73/GOLM1) and GOLPH3 (GOPP1/MIDAS) in cutaneous melanoma: Patterns of expression and prognostic significance. Int J Mol Sci. 2016; 17). There, GOLPH2 positive macrophages have been identified as tumor-associated macrophages (TAMs). The general importance of TAMs has been recognized and novel drugs to alter their phenotype from a so-called M2 macrophage towards less pro-tumorigenic macrophage are in development (Hu, Guorong et al. “Nanoparticles Targeting Macrophages as Potential Clinical Therapeutic Agents Against Cancer and Inflammation.” Frontiers in immunology Vol. 10 1998. 21 Aug. 2019).
Monocytes are a type of leukocyte, or white blood cell. Monocytes represent the largest type of leukocytes and can differentiate into macrophages and myeloid lineage dendritic cells. As a part of the vertebrate innate immune system, monocytes also influence the process of adaptive immunity. There are at least three subclasses of monocytes in human blood based on their phenotypic receptors.
Monocytes are produced by the bone marrow from precursors called monoblasts, bipotent cells that differentiated from hematopoietic stem cells. Monocytes circulate in the bloodstream for about one to three days and then typically move into tissues throughout the body where they differentiate into macrophages and dendritic cells. They constitute between three and eight percent of the leukocytes in the blood. About half of the body's monocytes are stored as a reserve in the spleen in clusters in the red pulp's Cords of Billroth. Moreover, monocytes are the largest corpuscle in blood.
Monocytes which migrate from the bloodstream to other tissues will then differentiate into tissue resident macrophages or dendritic cells. Macrophages are responsible for protecting tissues from foreign substances, but are also suspected to be important in the formation of important organs like the heart and brain. They are cells that possess a large smooth nucleus, a large area of cytoplasm, and many internal vesicles for processing foreign material.
Monocytes and their macrophage and dendritic cell progeny serve three main functions in the immune system. These are phagocytosis, antigen presentation, and cytokine production. Phagocytosis is the process of uptake of microbes and particles followed by digestion and destruction of this material. Monocytes can perform phagocytosis using intermediary (opsonising) proteins such as antibodies or complement that coat the pathogen, as well as by binding to the microbe directly via patternrecognition receptors that recognize pathogens. Monocytes are also capable of killing infected host cells via antibody-dependent cell-mediated cytotoxicity. Vacuolization may be present in a cell that has recently phagocytized foreign matter.
Microbial fragments that remain after such digestion can serve as antigens. The fragments can be incorporated into MHC molecules and then trafficked to the cell surface of monocytes (and macrophages and dendritic cells). This process is called antigen presentation and it can lead to activation of T lymphocytes, which then mount a specific immune response against the antigen.
Macrophages are a type of white blood cell of the immune system, that engulfs and digests cellular debris, foreign substances, microbes, cancer cells, and anything else that does not have the type of proteins specific to healthy body cells on its surface in a process called phagocytosis.
These large phagocytes are found in essentially all tissues, where they patrol for potential pathogens by amoeboid movement. They take various forms (with various names) throughout the body (e.g., histiocytes, Kupffer cells, alveolar macrophages, microglia, and others), but all are part of the mononuclear phagocyte system. Besides phagocytosis, they play a critical role in nonspecific defense (innate immunity) and also help initiate specific defense mechanisms (adaptive immunity) by recruiting other immune cells such as lymphocytes. For example, they are important as antigen presenters to T cells. In humans, dysfunctional macrophages cause severe diseases such as chronic granulomatous disease that result in frequent infections.
Beyond increasing inflammation and stimulating the immune system, macrophages also play an important anti-inflammatory role and can decrease immune reactions through the release of cytokines. Macrophages that encourage inflammation are called Ml macrophages, whereas those that decrease inflammation and encourage tissue repair are called M2 macrophages.
There are several activated forms of macrophages. In spite of a spectrum of ways to activate macrophages, there are two main groups designated Ml and M2. Ml "killer" macrophages are activated by LPS and IFN-y, and secrete high levels of IL-12 and low levels of IL-10. Ml macrophages have pro-inflammatory, bactericidal, and phagocytic functions. In contrast, the M2 "repair" designation (also referred to as alternatively activated macrophages) broadly refers to macrophages that function in constructive processes like wound healing and tissue repair, and those that turn off damaging immune system activation by producing anti-inflammatory cytokines like IL- 10. M2 is the phenotype of resident tissue macrophages, and can be further elevated by IL-4. M2 macrophages produce high levels of IL-10, TGF-beta and low levels of IL-12. Tumor-associated macrophages are mainly of the M2 phenotype, and seem to promote tumor growth.
Dendritic cells (DCs) are antigen-presenting cells of the mammalian immune system. Their main function is to process antigen material and present it on the cell surface to the T cells of the immune system. They act as messengers between the innate and the adaptive immune systems.
Dendritic cells are present in those tissues that are in contact with the external environment, such as the skin (where there is a specialized dendritic cell type called the Langerhans cell) and the inner lining of the nose, lungs, stomach and intestines. They can also be found in an immature state in the blood. Once activated, they migrate to the lymph nodes where they interact with T cells and B cells to initiate and shape the adaptive immune response. At certain development stages they grow branched projections, the dendrites that give the cell its name. Immature dendritic cells are also called veiled cells, as they possess large cytoplasmic 'veils' rather than dendrites.
Dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells initially transform into immature dendritic cells. These cells are characterized by high endocytic activity and low T-cell activation potential. Immature dendritic cells constantly sample the surrounding environment for pathogens such as viruses and bacteria. This is done through pattern recognition receptors (PRRs) such as the toll-like receptors (TLRs). TLRs recognize specific chemical signatures found on subsets of pathogens. Immature dendritic cells may also phagocytose small quantities of membrane from live own cells, in a process called nibbling. Once they have come into contact with a presentable antigen, they become activated into mature dendritic cells and begin to migrate to the lymph node. Immature dendritic cells phagocytose pathogens and degrade their proteins into small pieces and upon maturation present those fragments at their cell surface using MHC molecules. Simultaneously, they upregulate cell-surface receptors that act as co-receptors in T-cell activation such as CD80 (B7.1), CD86 (B7.2), and CD40 greatly enhancing their ability to activate T- cells. They also upregulate CCR7, a chemotactic receptor that induces the dendritic cell to travel through the blood stream to the spleen or through the lymphatic system to a lymph node. Here they act as antigen-presenting cells: they activate helper T-cells and killer T-cells as well as B-cells by presenting them with antigens derived from the pathogen, alongside non-antigen specific costimulatory signals. Dendritic cells can also induce T-cell tolerance (unresponsiveness). Certain C- type lectin receptors (CLRs) on the surface of dendritic cells, some functioning as PRRs, help instruct dendritic cells as to when it is appropriate to induce immune tolerance rather than lymphocyte activation. In view of the outstanding importance of macrophages and dendritic cells in the immune system, there is a need in the art for therapeutic agents that induce the maturation of monocytes into macrophages and dendritic cells. Such therapeutic agents may be valuable tools in the treatment of conditions that are characterized by a low number of antigen-presenting cells in the body and may be used to trigger immune responses against various antigens.
In particular, there is a need in the art for therapeutic agents that induce the formation of dendritic cells.
In addition, there is a need in the art for therapeutic agents that induce the maturation of monocytes into pro-inflammatory Ml macrophages and/or the re-polarization of tumor-associated M2 macrophages into pro-inflammatory Ml macrophages.
Finally, there is a need in the art for novel vaccination strategies in human therapeutics that are safe and efficacious.
The above technical problems are solved by the embodiments as defined in the claims.
SUMMARY OF THE INVENTION
That is, the invention relates to the following items:
1. A target-binding molecule specifically binding to the extracellular part of GOLPH2 on the cell surface of a monocyte or a monocyte-derived cell, wherein the binding of the target-binding molecule to the extracellular part of GOLPH2 induces the maturation of the monocyte or the monocyte-derived cell.
2. The target-binding molecule according to item 1, wherein the maturation of monocytes or monocyte-derived cells results in the formation of dendritic cells and/or classically activated macrophages.
3. The target-binding molecule according to item 1 or 2, wherein the target-binding molecule is an antibody or a target-binding fragment thereof.
4. The target-binding molecule according to item 3, wherein the antibody is a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody or a multispecific antibody. The target-binding molecule, according to item 3 or 4, wherein the antibody, or the targetbinding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO.9; or (b) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO.17. The target-binding molecule according to any one of items 3 to 5, wherein the antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NON, CDR2 as defined in SEQ ID NO:5 and CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO.7, CDR2 as defined in SEQ ID NO:8 and CDR3 as defined in SEQ ID NO:9; or (b) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NO: 12, CDR2 as defined in SEQ ID NO: 13 and CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO: 15, CDR2 as defined in SEQ ID NO: 16 and CDR3 as defined in SEQ ID NO: 17. The target-binding molecule according to any one of items 3 to 6, wherein the antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 18 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:2 or SEQ ID NO: 18; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO: 19 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:3 or SEQ ID NO: 19; or (b) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO: 10 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 10; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 11. The target-binding molecule according to any one of items 3 to 7, wherein the target-binding molecule further comprises an antigenic peptide fused to a C-terminal end of the antibody, or the target-binding fragment thereof. The target-binding molecule according to item 8, wherein the antigenic peptide is fused to the C-terminal end of the antibody, or the target-binding fragment thereof, via a peptide linker. The target-binding molecule according to item 8 or 9, wherein the antigenic peptide is fused to the C-terminal end of a heavy chain and/or light chain of an antibody. The target-binding molecule according to item 4 to 10, wherein the target-binding molecule is a fusion antibody, wherein the fusion antibody comprises an Fc region and two or more scFv fragments. The target-binding molecule according to item 11, wherein each scFv fragment is connected to the Fc region with a peptide linker. The target-binding molecule according to item 4 to 12, wherein the target-binding molecule is a multispecific antibody. The target-binding molecule according to item 13, wherein the multispecific antibody comprises a first Fab or scFv portion specifically binding to the extracellular part of GOLPH2 and at least one further Fab or scFv portion specifically binding to an immune checkpoint molecule, a ligand of an immune checkpoint molecule and/or a molecule comprising an antigenic peptide. The target-binding molecule according to item 14, wherein the immune checkpoint molecule is selected from a group consisting of: CTLA4, PD-1, PD-L1, LAG3, TIM3, CD28, ICOS, SLAM, CD2, CD27, 0X40, 4-1BB, CD30, GITR, CD40L, DR3, CD122, LIGHT, TIGIT, VISTA, B7- H3 and BTLA; and/or wherein the ligand of the immune checkpoint molecule is selected from a group consisting of: CD80, CD86, PD-L1, PD-L2 and GAL9. An antibody-antigenic peptide complex comprising the target-binding molecule according to item 14 or 15 and a molecule comprising an antigenic peptide. The antibody-antigenic peptide complex according to item 16, wherein the molecule comprising the antigenic peptide comprises an epitope that is specifically bound by a Fab or scFv portion of the target-binding molecule. The antibody-antigenic peptide complex according to item 17, wherein the molecule comprising the antigenic peptide is a fusion protein comprising the antigenic peptide fused to a polypeptide comprising the epitope that is specifically bound by the Fab or scFv portion of the target- binding molecule. The antibody-antigenic peptide complex according to item 17 or 18, wherein the antigenic peptide is fused to the polypeptide comprising the epitope that is specifically bound by the Fab or scFv portion of the target-binding molecule via a peptide linker. The antibody-antigenic peptide complex according to item 18 or 19, wherein the polypeptide comprising the epitope that is specifically bound by the Fab or scFv portion of the targetbinding molecule is Myc or a Myc fragment. An antibody-antigenic peptide construct comprising an antibody, or a target-binding fragment thereof, specifically binding to GOLPH2 and an antigenic peptide, wherein the antigenic peptide is: a) fused to a C-terminal end of the antibody, or the target-binding fragment thereof; and/or b) comprised in a molecule that is specifically bound by a Fab or scFv portion comprised in the antibody, or the target-binding fragment thereof. The antibody-antigenic peptide construct according to item 21, wherein the antigenic peptide is fused to a C-terminal end of the antibody, or the target-binding fragment thereof, via a peptide linker. The antibody-antigenic peptide construct according to item 21 or 22, wherein the antigenic peptide is fused to the C-terminal end of a heavy chain and/or light chain of an antibody. The antibody-antigenic peptide construct according to any one of items 21 to 23, wherein the antibody is a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody or a multispecific antibody. The antibody-antigenic peptide construct according to item 24, wherein the antibody is a multispecific antibody. The antibody-antigenic peptide construct according to item 25, wherein the multispecific antibody comprises a first Fab or scFv portion specifically binding to GOLPH2 and a second Fab or scFv portion specifically binding to the molecule comprising the antigenic peptide. The antibody-antigenic peptide construct according to item 26, wherein the molecule comprising the antigenic peptide is a fusion protein comprising the antigenic peptide fused to a polypeptide comprising an epitope that is specifically bound by the second Fab or scFv portion. The antibody-antigenic peptide construct according to item 27, wherein the antigenic peptide is fused to the polypeptide comprising the epitope that is specifically bound by the second Fab or scFv portion via a peptide linker. The antibody-antigenic peptide construct according to item 27 or 28, wherein the polypeptide comprising the epitope that is specifically bound by the second Fab or scFv portion is Myc or a Myc fragment and wherein the second Fab or scFv portion specifically binds to an epitope within Myc or said Myc fragment. The antibody-antigenic peptide construct according to any one of items 21 to 29, wherein the multispecific antibody comprises a further Fab or scFv portion specifically binding to an immune checkpoint molecule or a ligand of an immune checkpoint molecule. The antibody-antigenic peptide construct according to item 30, wherein the immune checkpoint molecule is selected from a group consisting of: CTLA4, PD-1, PD-L1, LAG3, TIM3, CD28, ICOS, SLAM, CD2, CD27, 0X40, 4-1BB, CD30, GITR, CD40L, DR3, CD122, LIGHT, TIGIT, VISTA, B7-H3 and BTLA; and/or wherein the ligand of the immune checkpoint molecule is selected from a group consisting of: CD80, CD86, PD-L1, PD-L2 and GAL9. The antibody-antigenic peptide construct according to any one of item 21 to 31, wherein the antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO.9; or (b) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO.17. The antibody-antigenic peptide construct according to any one of items 21 to 32, wherein the antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NO:4, CDR2 as defined in SEQ ID NO:5 and CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NOT, CDR2 as defined in SEQ ID NO: 8 and CDR3 as defined in SEQ ID NOV; or (b) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NO: 12, CDR2 as defined in SEQ ID NO: 13 and CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO: 15, CDR2 as defined in SEQ ID NO: 16 and CDR3 as defined in SEQ ID NO: 17. The antibody-antigenic peptide construct according to any one of items 21 to 33, wherein the antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 18 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:2 or SEQ ID NO: 18; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO: 19 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:3 or SEQ ID NO: 19; or (b) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NOTO or a sequence having 90%, preferably 95% sequence identity to SEQ ID NOTO; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NOT E An immunoconjugate comprising the target-binding molecule according to any one of items 1 to 15, the antibody-antigenic peptide complex according to any one of items 16 to 20, or the antibody-antigenic peptide construct according to any one of items 21 to 34 and a cytotoxic agent or a prodrug of a cytotoxic agent. A polynucleotide encoding the target-binding molecule according to any one of items 1 to 15, the antibody-antigenic peptide complex according to any one of items 16 to 20, or the antibody- antigenic peptide construct according to any one of items 21 to 34. A cell comprising the polynucleotide according to item 36. A method for producing the target-binding molecule according to any one of items 1 to 15, the antibody-antigenic peptide complex according to any one of items 16 to 20, or the antibody- antigenic peptide construct according to any one of items 21 to 34, the method comprising a step of culturing the cell according to item 37. A pharmaceutical composition comprising the target-binding molecule according to any one of items 1 to 15, the antibody-antigenic peptide complex according to any one of items 16 to 20, or the antibody-antigenic peptide construct according to any one of items 21 to 34 and/or the immunoconjugate according to item 35 and further comprising a pharmaceutically acceptable earner. The pharmaceutical composition according to item 39 further comprising at least one therapeutic agent. The pharmaceutical composition according to item 40, wherein the therapeutic agent is at least one of a vaccine, an antigen, an adjuvant, a chemotherapeutic agent and an immune checkpoint modulator. The target-binding molecule according to any one of items 1 to 15, the antibody-antigenic peptide complex according to any one of items 16 to 20, or the antibody-antigenic peptide construct according to any one of items 21 to 34, the immunoconjugate according to item 35 or the pharmaceutical composition according to any one of items 39 to 41 for use in inducing the maturation of monocytes and/or monocyte-derived cells in a subject. The target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to item 42, wherein the maturation of monocytes or monocyte-derived cells results in the formation of dendritic cells and/or classically activated macrophages in said subject. The target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to item 42 or 43, wherein the subject is at risk of developing cancer, suffering from cancer or recovering from cancer. The target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to item 42 or 43, wherein the subject is immunocompromised. The target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to item 45, wherein the subject is immunocompromised as a result of a chemotherapy, a radiotherapy, or an infection, in particular wherein the infection is an infection with a human immunodeficiency virus. The target-binding molecule according to any one of items 1 to 15, the antibody-antigenic peptide complex according to any one of items 16 to 20 or the antibody-antigenic peptide construct according to any one of items 21 to 34, the immunoconjugate according to item 33 or the pharmaceutical composition according to any one of items 39 to 41 for use in inducing and/or enhancing an immune response to an antigen. The target-binding molecule, the complex, the construct, the immunoconjugate or the pharmaceutical composition for use according to item 47, wherein the immune response against the antigen is induced and/or enhanced with an antigenic peptide that is comprised in the targetbinding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct. The target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to item 47 or 48, wherein the antigen has been released by a physical therapeutic intervention. The target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to item 49, wherein the physical therapeutic intervention is cryotherapy, surgery, radiotherapy and/or laser therapy. The target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to any one of items 47 to 50, wherein the antigen is a tumor antigen or a pathogen-derived antigen. The target-binding molecule according to any one of items 1 to 15, the antibody-antigenic peptide complex according to any one of items 16 to 20, or the antibody-antigenic peptide construct according to any one of items 21 to 34, the immunoconjugate according to item 35 or the pharmaceutical composition according to any one of items 39 to 41 for use as an adjuvant in a vaccination therapy. The target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to item 52, wherein the adjuvant initiates or enhances the function of antigen-presenting cells. The target-binding molecule, the immunoconjugate or the pharmaceutical composition for use according to item 52 or 53, wherein the vaccination therapy comprises administration of a viral antigen, a microbial antigen or a tumor antigen. The target-binding molecule, the immunoconjugate or the pharmaceutical composition for use according to item 54, wherein at least one antigenic peptide derived from the viral antigen, the microbial antigen or the tumor antigen is comprised in the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct. A method for inducing the maturation of monocytes and/or monocyte -derived cells in vitro, the method comprising the steps of: a) culturing monocytes and/or monocyte-derived cells in a cell culture medium; b) adding the target-binding molecule according to any one of items 1 to 15, the antibody- antigenic peptide complex according to any one of items 16 to 20, or the antibody-antigenic peptide construct according to any one of items 21 to 34 to the cell culture of step (a); and c) obtaining matured monocytes and/or monocyte-derived cells. A method for generating a cell displaying an antigenic peptide, the method comprising the steps of: a) generating a matured monocyte or monocyte-derived cell according to the method of item 56; b) pulsing the antigenic peptide on the matured monocyte or monocyte-derived cell generated in step (a); and/or introducing a nucleic acid encoding a polypeptide comprising the antigenic peptide into the matured monocyte or monocyte-derived cell generated in step (a); and/or introducing the antigenic peptide into the matured monocyte or monocyte-derived cell as part of the target-binding molecule, the antibody-antigenic peptide complex, or the antibody-antigenic peptide construct; and c) obtaining a cell displaying an antigenic peptide. A method for generating an activated T cell, the method comprising the steps of: a) generating a cell displaying an antigenic peptide with the method according to item 57; b) contacting the cell of step (a) with a T cell, wherein the T cell comprises a T cell receptor that recognizes the antigenic peptide displayed by the cell of step (a); and c) obtaining an activated T cell. The method according to item 58, wherein the cell displaying an antigenic peptide and the T cell have been obtained from the same subject. A cell displaying an antigenic peptide and/or an activated T cell for use in adoptive cell transfer, wherein the cell displaying the antigenic peptide has been obtained with the method according to item 57 and wherein the activated T cell has been obtained with the method according to item 58 or 59.
61. A method for inducing and/or enhancing an immune response against a specific antigen in a subject, the method comprising the steps of: a) administering to said subject the target-binding molecule according to any one of items 1 to 15, the antibody-antigenic peptide complex according to any one of items 16 to 20, the antibody-antigenic peptide construct according to any one of items 21 to 35, the immunoconjugate according to item 36 or the pharmaceutical composition according to any one of items 40 to 42, wherein the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition comprises one or more antigenic peptide(s) derived from the specific antigen; b) inducing and/or enhancing an immune response in said subject.
Accordingly, in one embodiment, the invention relates to a target-binding molecule specifically binding to the extracellular part of GOLPH2 on the cell surface of a monocyte or a monocyte-derived cell, wherein the binding of the target-binding molecule to the extracellular part of GOLPH2 induces the maturation of the monocyte or the monocyte-derived cell.
That is, the invention is based, at least in part, on the surprising finding that target-binding molecules that bind to the extracellular part of the protein GOLPH2 on the surface of monocytes and/or monocyte-derived cells can induce the maturation of said monocytes or monocyte-derived cells.
FIG. l shows that GOLPH2 is not only expressed in different tumor cell lines, but also in human monocytes. However, secretion of the soluble GOLPH2 (sGOLPH2) fragment by human monocytes is significantly lower compared to tumor cell lines. Without being bound to theory, this may be explained by lower expression of GOLPH2 in monocytes and/or by lower secretion of GOLPH2, presumably due to reduced proteolytic cleavage at the furin cleavage site in the extracellular part of GOLPH2.
To the surprise of the inventors, treatment of monocytes with an anti-GOLPH2 antibody or antibodydrug conjugate resulted in increased metabolic activity of monocytes, while treatment of tumor cells with the same immunoconjugates resulted in decreased viability of these tumor cells (FIGs.2 and 3). Further experiments revealed that human monocytes that have been treated with an anti-GOLPH2 antibody that specifically binds to the extracellular part of GOLPH2 show very similar cytokine secretion patterns as monocytes that have been treated with a cytokine cocktail that induces the maturation of monocytes into Ml macrophages (FIG.5). In addition, RNA-Seq experiments revealed that monocytes that have been treated with the same anti-GOLPH2 antibody show very similar gene expression patterns as Ml macrophages (Table 4). Based on these results, it appears plausible that antibodies that bind to the extracellular part of GOLPH2 have the potential to induce the maturation of monocytes into Ml macrophages.
It has been shown by the inventors that the treatment of human monocytes with an anti-GOLPH2 antibody results in increased expression of CD82 and reduced expression of CD37 in these cells, an expression pattern that is indicative of dendritic cells (FIG.6). Further RNA-Seq experiments revealed that monocytes that have been treated with an anti-GOLPH2 antibody show similar gene expression patterns as monocyte-derived dendritic cells (Table 2) and, in particular, matured monocyte-derived dendritic cells (Table 3). Thus, it further appears plausible that anti-GOLPH2 antibodies have the potential to induce the maturation of monocytes into dendritic cells and, in particular, into mature dendritic cells.
Further experiments showed that the co-culture of monocytes with antigen-specific CD8+ T cells resulted in an expansion of the CD8+ T cells when the monocytes had been treated with the anti- GOLPH2 antibody of the invention in the presence of an antigenic peptide (FIG.8). These results demonstrate that monocytes that have been treated with the anti-GOLPH2 antibody of the invention present antigenic peptides more efficiently than monocytes that have not been treated with the anti- GOLPH2 antibody of the invention and thus have a higher potential to activate T cells.
The present invention encompasses any antibody that specifically binds to the extracellular part of GOLPH2 such that it induces the maturation of monocytes, in particular the maturation of monocytes into Ml macrophages and/or dendritic cells. In certain embodiments, the invention relates to the antibodies G2-2opti or G2-4 from WO 2018/091724 that have been surprisingly shown herein to induce the maturation of human and/or murine monocytes in the attached examples.
Antibody G2-2 from WO 2018/091724 binds in the proximity of the furin cleavage site of GOLPH2 and can target both cleaved and uncleaved GOLPH2. Antibody G2-4 from WO 2018/091724 binds to a different epitope in the soluble part of GOLPH2 (sGOLPH2) and can thus only target cells comprising the uncleaved form of GOLPH2. FIG.5B shows that both G2-2opti and G2-4, even though they bind to different epitopes in the extracellular part of GOLPH2, can induce the secretion of cytokines that are indicative of Ml macrophages. Accordingly, it is plausible that antibodies binding to the extracellular part of GOLPH2 have the potential to induce the maturation of monocytes, irrespective of the exact epitope in the extracellular part of GOLPH2 these antibodies bind to. Further, it has been shown that the addition of the commercial anti-GOLPH2 antibody EPR3606 to monocytes results in the induction of CD40 expression, which is indicative for maturation into Ml macrophages and dendtritic cells (FIG.14A). In addition, it has been shown that monocytes that have been treated with EPR3606 activate CD8+ T cells more efficiently than monocytes that have been treated with a control antibody (FIG. 14B).
With regard to cytokine release in the process of monocyte polarization, the commercial anti-GOLPH2 antibody EPR3606 elicited similar cytokine releases in a monocyte/T cell co-culture as G2-2. In some aspects, namely GM-CSF and Interferon gamma, G2-2 showed a more pronounced cytokine release than EPR3606. For other cytokines similar effects were observed for both anti-GOLPH2 antibodies compared to the irrelevant isotype control antibody (FIG-14C).
The term "GOLPH2," as used herein, refers to any native GOLPH2. The term includes GOLPH2 from any vertebrate source, including mammals such as primates (e.g. humans and rhesus macaques) and rodents (e.g., mice and rats), unless otherwise indicated. The term also includes naturally occurring variants of GOLPH2, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human GOLPH2 protein is shown in SEQ ID NO: 1. The amino acid sequence of nonlimiting exemplary mouse GOLPH protein is shown in SEQ ID NO: 22.
A “target-binding molecule,” as used herein, is any molecule that can specifically or selectively bind to a target molecule. The target molecule may be any type of molecule, such as, without limitation, a peptide, a protein, a nucleic acid, a carbohydrate or combinations thereof. Preferably, the targetbinding molecule specifically binds to an epitope that is located within the target molecule. The term “epitope” as used herein refers to a region of a target molecule which is recognized by a particular target-binding molecule. A target-binding molecule may include or be an antibody or a fragment thereof. An anti-GOLPH2 binding molecule is a molecule that binds to the GOLPH2 antigen, such as an anti-GOLPH2 antibody or fragment thereof, at a specific recognition site or part of the antigen as detailed herein. That is, target-binding molecules of the invention bind to the extracellular part of GOLPH2. Other anti-GOLPH2 binding molecules may also include multivalent molecules, multispecific molecules (e.g., diabodies), fusion molecules, aptamers, avimers, or other naturally occurring or recombinantly created molecules. Illustrative target-binding molecules useful in the present invention include antibody-like molecules. An antibody-like molecule is a molecule that can exhibit functions by binding to a target molecule (See, e.g., Current Opinion in Biotechnology 2006, 17:653- 658; Current Opinion in Biotechnology 2007, 18: 1-10; Current Opinion in Structural Biology 1997, 7:463-469; Protein Science 2006, 15: 14-27), and includes, for example, DARPins (WO 2002/020565), Affibody (WO 1995/001937), Avimer (WO 2004/044011; WO 2005/040229), Adnectin (WO 2002/032925) and fynomers (WO 2013/135588).
It has to be noted that, within the present invention, GOLPH2 may also be referred to as an antigen and that the target-binding molecule of the invention may also be referred to as an antigen-binding molecule. However, the term “antigen” is preferably used herein to refer to a molecule that provokes an immune response, e.g. a molecule that can be taken up and processed within an antigen-presenting cell such that an antigenic peptide derived from this antigen is presented by the antigen-presenting cell.
The term “specifically binding to” as used in the context of the present invention defines a binding (interaction) of at least two “antigen-interaction-sites” with each other. The term “antigen-interaction- site” defines, in accordance with the present invention, a motif of a polypeptide, i.e., a part of the antibody or target-binding fragment of the present invention, which shows the capacity of specific interaction with a specific antigen or a specific group of antigens of GOLPH2. Said binding/interaction is also understood to define a “specific recognition”. The term “specifically recognizing” means in accordance with this invention that the target-binding molecule is capable of specifically interacting with and/or binding to at least two amino acids of GOLPH2 as defined herein, in particular interacting with/binding to at least two amino acids within the extracellular part of GOLPH2 on the cell surface of a monocyte or a monocyte -derived cell such that the maturation of the monocyte or the monocyte-derived cell is induced.
The term “specific binding” or “specific interaction” as used in accordance with the present invention further means that the target-binding molecule of the invention does not or does not essentially crossreact with (poly)peptides that are not part of or derived from the extracellular part of GOLPH2. Crossreactivity of target-binding molecules, in particular a panel of antibodies or target-binding fragments thereof under investigation may be tested, for example, by assessing binding of said panel of antibodies or target-binding fragments thereof under conventional conditions (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988) and Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1999)) to the (poly)peptide of interest as well as to a number of more or less (structurally and/or functionally) closely related (poly)peptides. Only those constructs (i.e. antibodies, target-binding fragments thereof and the like) that bind to the certain structure of GOLPH2 as defined herein, e.g., a specific part, epitope or (poly) peptide/protein of GOLPH2 as defined herein but do not or do not essentially bind to any of the other parts, epitopes or (poly) peptides of the same GOLPH2, are considered specific for the part, epitope or (poly) peptide/protein of interest and selected for further studies in accordance with the method provided herein. These methods may comprise, inter alia, binding studies, blocking and competition studies with structurally and/or functionally closely related molecules. These binding studies also comprise FACS analysis, surface plasmon resonance (SPR, e.g. with BIAcore), analytical ultracentrifugation, isothermal titration calorimetry, fluorescence anisotropy, fluorescence spectroscopy or by radiolabeled ligand binding assays.
Accordingly, specificity can be determined experimentally by methods known in the art and methods as described herein. Such methods comprise, but are not limited to Western Blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans.
Within the present invention, the target-binding molecule binds to the extracellular part of GOLPH2. The “extracellular part of GOLPH2”, as used herein, refers to the extracellular C-terminal part of GOLPH2 which, when GOLPH2 is associated with the cell membrane, protrudes into the extracellular space. In certain embodiments, the “extracellular part of GOLPH2” is the part of GOLPH2 that, when GOLPH2 is associated with the cell membrane, is accessible for the target-binding molecules of the invention from the extracellular space. In case of human GOLPH2, the extracellular part consists of the residues 36 to 401 of GOLPH2 as set forth in SEQ ID NO: 1. In case of murine GOLPH2, the extracellular part consists of the residues 36 to 393 of GOLPH2 as set forth in SEQ ID NO:22.
The target-binding molecule of the invention may bind to any fragment or epitope of the extracellular part of GOLPH2. As mentioned above, GOLPH2 frequently undergoes proteolytic cleavage wherein a soluble fragment of the extracellular part of GOLPH 2 (termed soluble GOLPH2 or sGOLPH2) is cleaved off and released in the extracellular space. Accordingly, the target-binding molecule of the invention may bind (i) to any epitope in sGOLPH2, preferably before it has been cleaved off by furin and is thus still attached to a cell, or (ii) to any epitope in the remnant extracellular part of GOLPH2 that is still bound to the membrane after sGOLPH2 has been cleaved off by furin. In addition, the epitope of the remnant extracellular part of GOLPH2 may be an epitope that is only accessible after the proteolytic cleavage.
The antibody G2-2opti from WO 2018/091724 has been shown in the appended Examples to induce the maturation of monocytes. Thus, in certain embodiments, the target-binding molecule of the invention may bind to the same epitope of GOLPH2 as the antibody G2-2opti. That is, the targetbinding molecule of the invention may bind to a linear epitope located within the amino acid sequences of SEQ ID NOs: 23, 24 and/or 25, i.e. SSRSVDLQTRIMELEGRVRR (human, SEQ ID NO: 23), SSRSVELQTRIVELEGRVRR (murine, SEQ ID NO: 24) and/or SSRSVDLQTRIVELEGRVRR (canine, SEQ ID NO: 25), of GOLPH2. Preferably, the target-binding molecule of the invention may bind to a linear epitope located within the amino acid sequence of SEQ ID NO: 23 (SSRSVDLQTRIMELEGRVRR) of human GOLPH2 as set forth in SEQ ID NOT. In certain embodiments, the epitope bound by the antibody of the invention may be within the amino acid sequence RIMELEGRVRR of SEQ ID NO: 23, or within the amino acid sequence EGRVRR of SEQ ID NO:23, 24 or 25.
Further, the target-binding molecule of the invention may bind to the same epitope of GOLPH2 as the antibody G2-4 from WO 2018/091724. That is, the target-binding molecule of the invention may bind to an epitope comprised in the amino acid sequence GEDDYNMDENEAESETDKQA (SEQ ID NO:26) corresponding to amino acids 347 to 366 of human GOLPH2 as set forth in SEQ ID NO: 1.
The inventors further observed that the commercial polyclonal anti-GOLPH2 antibody PA5-18100 from Invitrogen can induce the maturation of monocytes. PA5-18100 binds to the synthetic peptide sequence NLLDQREKRNHTL corresponding to amino acids 389 to 401 of human GOLPH2 as set forth in SEQ ID NO: 1 That is, the target-binding molecule of the invention may bind to an epitope comprised in the amino acid sequence NLLDQREKRNHTL (SEQ ID NO:27) corresponding to amino acids 389 to 401 of human GOLPH2 as set forth in SEQ ID NO: 1.
Further, the inventors have identified the commercial polyclonal anti-GOLPH2 antibody EPR3606 from Abeam to induce the maturation of monocytes. EPR3606 binds to the extracellular soluble part of GOLPH2, as has been demonstrated in FIG. 15. Accordingly, the target-binding molecule of the invention may bind to the same epitope as the commercial antibody EPR3606.
The target-binding molecule of the invention binds to the extracellular part of GOLPH2 on the cell surface, for example the cell surface of a monocyte or a monocyte-derived cell. A protein, in particular GOLPH2, is said to be located on the surface of a cell, in particular a monocyte or monocyte-derived cell, if the protein is associated with the outer membrane of a cell (cell or plasma membrane) such that at least parts of the protein contribute to the surface of the cell and are available from the extracellular space.
The target-binding molecule of the invention binds to the extracellular part of GOLPH2 on the cell surface of a monocyte or a monocyte-derived cell. That is, in certain embodiments, the target-binding molecule of the invention binds to an epitope within the residues 36 to 401 of human GOLPH2 as set forth in SEQ ID NO: 1, preferably wherein GOLPH2 is located on the cell surface of a human monocyte or monocyte-derived cell. In a particular embodiment, the target-binding molecule of the invention binds to an epitope located within the amino acid sequences of SEQ ID NOs: 23, 24 and/or 25 of GOLPH2, preferably wherein GOLPH2 is located on the cell surface of a monocyte or monocyte-derived cell. In a preferred embodiment, the target-binding molecule of the invention binds to an epitope located within the amino acid sequences of SEQ ID NO: 23 of GOLPH2, preferably wherein GOLPH2 is located on the cell surface of a human monocyte or monocyte-derived cell. In other embodiments, the target-binding molecule of the invention binds to an epitope located within the amino acid sequences of SEQ ID NO: 26 of human GOLPH2, preferably wherein GOLPH2 is located on the cell surface of a human monocyte or monocyte-derived cell. In other embodiments, the targetbinding molecule of the invention binds to an epitope located within the amino acid sequences of SEQ ID NO: 27 of human GOLPH2, preferably wherein GOLPH2 is located on the cell surface of a human monocyte or monocyte-derived cell.
Binding of the target-binding molecule of the invention to the extracellular part of GOLPH2 on the cell surface of a monocyte or a monocyte-derived cell induces the maturation of the monocyte or monocyte-derived cell. The term "maturation" as used herein means a process that is required for a cell (e.g. a monocyte) to become more specialized and/or attain a fully functional state, for example its functional state in vivo. That is, in certain embodiments, binding of the target-binding molecule of the invention to the extracellular part of GOLPH2 on the cell surface of a monocyte may induce the maturation of said monocyte into a dendritic cell, in particular a matured dendritic cell. In other embodiments, binding of the target-binding molecule of the invention to the extracellular part of GOLPH2 on the cell surface of a monocyte may induce the maturation of said monocyte into a macrophage, in particular an Ml macrophage.
Alternatively, binding of the target-binding molecule of the invention to the extracellular part of GOLPH2 on the cell surface of a monocyte-derived cell may induce the maturation of said monocyte- derived cell. That is, in certain embodiments, binding of the target-binding molecule of the invention to the extracellular part of GOLPH2 on the cell surface of an immature monocyte-derived dendritic cell may induce the maturation of said immature monocyte -derived dendritic cell into a matured dendritic cell. In other embodiments, binding of the target-binding molecule of the invention to the extracellular part of GOLPH2 on the cell surface of a non-polarized MO macrophage may induce the maturation (or polarization) of said non-polarized MO macrophage into an Ml macrophage. Accordingly, in certain embodiments, a “monocyte-derived cell” is an immature dendritic cell or an MO macrophage. In a particular embodiment, the invention relates to the target-binding molecule according to the invention, wherein the maturation of monocytes or monocyte -derived cells results in the formation of dendritic cells and/or classically activated macrophages.
In a particular embodiment, the invention relates to the target-binding molecule according to the invention, wherein the maturation of monocytes or monocyte -derived cells results in the formation of matured dendritic cells and/or classically activated macrophages.
In view of the above, it is plausible that target-binding molecules binding to the extracellular part of GOLPH2 have the potential to induce the maturation of monocytes, in particular, to induce the maturation of monocytes into Ml macrophages and/or monocyte-derived dendritic cells.
That is, in certain embodiments, the invention relates to a target-binding molecule specifically binding to the extracellular part of GOLPH2 on the cell surface of a monocyte or a monocyte-derived cell, wherein the binding of the target-binding molecule to the extracellular part of GOLPH2 induces the maturation of the monocyte or the monocyte-derived cell into an Ml macrophage. In other embodiments, the invention relates to a target-binding molecule specifically binding to the extracellular part of GOLPH2 on the cell surface of a monocyte or a monocyte-derived cell, wherein the binding of the target-binding molecule to the extracellular part of GOLPH2 induces the maturation of the monocyte or the monocyte-derived cell into a dendritic cell.
It is to be understood that some target-binding molecules of the invention preferably induce the maturation of monocytes and/or monocyte-derived cells into dendritic cells, in particular matured dendritic cells, while other target-binding molecules of the invention preferably induce the maturation of monocytes and/or monocyte derived cells into Ml macrophages. However, it is further to be understood that certain target-binding molecules of the invention have the potential to induce the maturation of monocytes and/or monocyte-derived cells into dendritic cells, in particular matured dendritic cells, and Ml macrophages.
It is further to be understood that the same target-binding molecule may have the potential to induce the maturation of a monocyte or a monocyte-derived cell into a dendritic cell, in particular a matured dendritic cell, or an Ml macrophage. The decision whether a target-binding molecule induces maturation of a monocyte or a monocyte-derived cell into a dendritic cell, in particular a matured dendritic cell, or an Ml macrophage may depend on additional factors, e.g. the incubation time of the cell with the target-binding molecule. For example, it has been observed by the inventors that shorter incubation periods preferably result in the maturation of monocytes and/or monocyte-derived cells into Ml macrophages, while longer incubation periods preferably result in the maturation of monocytes and/or monocyte-derived cells into dendritic cells, in particular matured dendritic cells.
Further, it has been observed by the inventors that the structure of the target-binding molecule has an influence on the fate of the monocytes and/or monocyte-derived cells. That is, it has been observed by the inventors that Fab fragments derived from an antibody binding specifically to the extracellular part of GOLPH2 on the surface of a monocyte or a monocyte-derived cell preferably induce the maturation of monocytes and/or monocyte-derived cells into Ml macrophages. On the other hand, it has been observed by the inventors that full antibodies binding specifically to the extracellular part of GOLPH2 on the surface of a monocyte or a monocyte-derived cell preferably induce the maturation of monocytes and/or monocyte-derived cells into dendritic cells, in particular matured dendritic cells.
The target-binding molecule of the invention may induce the maturation of monocytes or monocyte- derived cells into immature or mature dendritic cells. However, it is to be understood that the targetbinding molecule of the invention may also be used for inducing the maturation of monocytes or monocyte-derived cells into cells that show characteristics of immature and mature dendritic cells.
Within the present invention, a target-binding molecule is said to induce the maturation of a first cell type into a second cell type, if the binding of the target-binding molecule to the first cell type, for example to the extracellular part of a protein on the cell surface of the first cell type, initiates developmental processes, for example by activating certain signaling pathways in said first cell type, that eventually result in the maturation of the first cell type into the second cell type.
The term "monocyte", as used herein, refers to a type of white blood cells that have two main functions in the immune system: (1) replenish resident macrophages and dendritic cells under normal states, and (2) in response to inflammation signals, monocytes can move quickly (approx. 8-12 hours) to sites of infection in the tissues and divide/differentiate into macrophages and dendritic cells to elicit an immune response. Monocytes are usually identified in stained smears by their large bilobate nucleus. In addition to the expression of CD 14, monocytes also show expression of one or more of the following surface markers 125I-WVH-1, 63D3, Adipophilin, CB12, CDl la, CDl lb, CD15, CD54, Cdl63, cytidine deaminase, Flt-1.
The term monocyte includes, without limitation, the classical monocytes, the intermediate monocytes and the non-classical pro-inflammatory monocyte, which are present in human blood. A "classical monocyte", as used herein, refers to a type of monocyte cell characterized by high level expression of the CD 14 cell surface receptor (CD14++ monocyte). An “intermediate monocytes”, as used herein, refers to a type of monocyte cell characterized by a high level expression of CD 14 and additional expression of CD 16 (CD14++ CD 16+ monocyte). A "non-classical pro-inflammatory monocyte", as used herein, refers to a cell with a lower expression of CD 14 and with high level expression of the CD16 receptor (CD14+C D16++ monocyte). The skilled person is aware of methods to determine the expression of cell surface receptors, for example by flow cytometry as described herein.
Monocytes are characterized by producing high amounts of pro-inflammatory cytokines such as tumor necrosis factor and interleukin- 12 in response to stimulation by microbial products.
The term “monocyte -derived cell”, as used herein, refers to any type of cell that has been derived from a monocyte, in particular from a classical monocyte, an intermediate monocyte or a non-classical pro- inflammatory monocyte. Most commonly, monocyte-derived cells may be macrophages and myeloid dendritic cells. In certain embodiments, the target-binding molecule of the invention may induce the maturation of a monocyte into an MO macrophage and/or the maturation of an MO macrophage into an Ml macrophage. In other embodiments, the target-binding molecule of the invention may induce the maturation of a monocyte into an immature dendritic cell and/or the maturation of an immature dendritic cell into a mature dendritic cell.
The term "macrophage", as used herein, refers to CD 14+ positive cells derived from the differentiation of monocytes. Macrophages are characterized in that they are phagocytes, acting both in non-specific defense (innate immunity) as well as to help initiating specific defense mechanisms (adaptive immunity) of vertebrate animals. One of the main roles of macrophages is to phagocytose (engulf and then digest) cellular debris and pathogens either as stationary or as mobile cells, and to stimulate lymphocytes and other immune cells to respond to the pathogen.
In addition to the expression of CD 14, macrophages may also show expression of one or more of the following surface markers: CDl lb, F4/80(mice)/EMRl (human), Lysozyme M, MAC-l/MAC-3, 27E10, Carboxypeptidase M, Cathepsin K, CD163 and CD68. These markers may be determined by flow cytometry or immunohistochemical staining.
The skilled person is aware of methods to differentiate between different types of macrophages, i.e. MO, Ml and M2 macrophages. For example, the type of macrophage may be determined based on the presence of specific cell surface proteins, as described in Becker et al., PLoSONE, 2012, 7(3), e33297. Alternatively, different types of macrophages may be discriminated according to their cytokine profde as described in Vogel et al., Journal of Neuroinflammation, 2014, 11(23) or as described in Example 8. Further, different types of macrophages may be differentiated according to their gene expression profiles as described by Orecchioni et al., Frontiers in Immunology, 2019, 10: 1084 or as described in Example 10.
The term “M0 macrophage” as used herein refers to a subtype of macrophages that are resting or unactivated (unpolarized).
In certain embodiments, the target-binding molecule of the invention induces the maturation of monocytes or monocyte-derived cells into classically activated macrophages, also called Ml macrophages or Ml inflammatory type macrophages. The term “classically activated macrophages” as used herein refers to a subtype of macrophages that are classically activated or exhibit an inflammatory macrophage phenotype. Classically activated macrophages are activated by LPS and IFN-y, and secrete high levels of IL-l-beta, TNF-alpha, and IL-12. Ml macrophages also include macrophages that exhibit a hybrid phenotype that is predominantly the Ml phenotype.
The term “M2” broadly refers to macrophages that function in constructive processes like wound healing and tissue repair and include M2A and M2C macrophages. Major differences between M2A and M2C macrophages exist in wound healing.
The term "myeloid dendritic cell", as used herein, refers to a population of dendritic cells which derive from monocytes and which include, without limitation, mDC-I and mDC-2 cells. In addition to a low/ negative expression of CD 14, myeloid dendritic cells may also show expression of one or more of the following surface markers: ADAM19, BDCA-2, CDla, CDl lc CD21, CD86, CD208, Clusterin, Estrogen Receptor-alpha. These markers may be determined by flow cytometry or immunohistochemical staining.
The skilled person is aware of methods to differentiate between immature and matured dendritic cell based on the presence of specific cell surface proteins or according to their cytokine secretion profile as described by Dudek et al., Frontiers in Immunology, 2013, 4:438. Alternatively, immature and matured dendritic cells may be identified and/or discriminated according to their gene expression profiles as described by Lyons et al., npj Precision One, 2017, 1, 26 or as described in Example 10.
Within the present invention, it is preferred that binding of the target-binding molecule of the invention to the extracellular part of GOLPH2 on the surface of a monocyte or monocyte-derived cell results in the maturation of said monocyte or monocyte-derived cell into an Ml macrophage or into a dendritic cell, in particular a matured dendritic cell.
In certain embodiments, the maturation state of a monocyte is determined based on the expression of the cell surface marker CD 14. Monocytes are known to express higher amounts of CD 14 compared to monocyte-derived macrophages or dendritic cells (Fig.4a). That is, a target-binding molecule may be determined to induce the maturation of a monocyte or a monocyte-derived cell into a macrophage and/or a dendritic cell, if contacting of a monocyte with the target-binding molecule results in a lower expression of CD 14 as compared to the contacting of a monocyte with a comparable molecule that does not bind to the extracellular part of GOLPH2. The skilled person is aware of methods to determine the expression of CD 14 on a cell. Preferably, the expression of CD 14 on a cell is determined by flow cytometry.
In certain embodiments, the maturation state of a monocyte is determined based on the expression of the cell surface marker CD40. Monocytes are known to express lower amounts of CD40 compared to monocyte-derived macrophages. That is, a target-binding molecule may be determined to induce the maturation of a monocyte or a monocyte-derived cell into a macrophage, if contacting of a monocyte with the target-binding molecule results in a higher expression of CD40 as compared to the contacting of a monocyte with a comparable molecule that does not bind to the extracellular part of GOLPH2. The skilled person is aware of methods to determine the expression of CD40 on a cell. Preferably, the expression of CD40 on a cell is determined by flow cytometry. Mohammadi et al. (Biotechnic & Histochemistry 90:6 (2015) 445-452
In certain embodiments, the differentiation of a monocyte into a macrophage may be determined based on the cytokine secretion profiles of these cells. Macrophages are known to secrete higher amounts of the cytokines IL-lBeta, IL-6 and TNFa compared to monocytes (Fig.5). Thus, the skilled person is aware of methods to discriminate between monocytes and macrophages based on the secretion of these cytokines. Further, the skilled person is aware of methods for quantifying the cytokines secreted by these cells.
In certain embodiments, the differentiation of a monocyte into a dendritic cell may be determined based on differences in the expression of cell surface markers. For example, dendritic cells are characterized by high expression of CD82 and low expression of CD37, while monocytes express lower amounts of CD82 and higher amounts of CD37 (FIG.6). Accordingly, the skilled person is aware of methods to differentiate between dendritic cells and monocytes based on the expression of CD82 and CD37. The expression of CD82 and CD37 may, for example, be determined by flow cytometry or by RNAseq. For example, the determination of dendritic cells is described in Jones E.; Dendritic Cell Migration and Antigen Presentation are coordinated by the opposing functions of the tetraspanins CD82 and CD36. J of Immunology 2015 doi: 10.4049/jimmunol.1500357.
Preferably, the differentiation state of a monocyte-derived cell is determined based on the gene expression profile. Gene expression data may be obtained by any method known in the art, preferably by RNA sequencing.
Gene expression data for immature and mature dendritic cells has been reported by Lyons, Y.A. et al.; Immune cell profiling in cancer: molecular approaches to cell-specific identification, npj Precision One 1, 26 (2017) doi: 10.1038/s41698-017-0031-0. In this study, the genes PDLIM4, CD1B, MRC1, RAP1GAP, FCER2, DUSP5, PPIC, CD1C, CCND2, STAC, CD1A, CD36, SHB, TRIB2 and PRKACB have been reported to be upregulated in immature dendritic cells and the genes CD 14, FCN1 and CCR5 have been reported to be downregulated in immature dendritic cells in comparison to untreated human monocytes. Accordingly, in the present invention, a monocyte or monocyte-derived cell is determined to be differentiated into an immature dendritic cell, if at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or more than 15 of these genes are either up- or downregulated in comparison to an untreated human monocyte as described above. Preferably, a monocyte or monocyte-derived cell is determined to be differentiated into an immature dendritic cell, if at least 6, at least 7, at least 8, at least 9, at least 10 or all of the genes PDLIM4, CD1B, MRC1, RAP1GAP, FCER2, DUSP5, PPIC, CD1C, CCND2, STAC and CD1A are upregulated by a log2-fold change higher than 1 in comparison to an untreated human monocyte. More preferably, a monocyte or monocyte-derived cell is determined to be differentiated into an immature dendritic cell, if at least 3, at least 4, at least 5, at least 6, at least 7 or all of the genes PDLIM4, CD1B, MRC1, RAP1GAP, FCER2, DUSP5, PPIC and CD1C are upregulated by a log2-fold change higher than 2 in comparison to an untreated human monocyte. Even more preferably, a monocyte or monocyte-derived cell is determined to be differentiated into an immature dendritic cell, if at least 1, at least 2, at least 3 or all of the genes PDLIM4, CD1B, MRC1 and RAP1GAP are upregulated by a log2-fold change higher than 3 in comparison to an untreated human monocyte.
Further, the genes IL6, CYP27B1, EBI3, IFIT1, HES4, DUSP5, USP18, IFI44L, IFIT3, ATF3, MX1, ISG15, GADD45A, OASL, CCL8, TRAF1, LAMP3, RGS1, CXCL8, CD44, IL7R, CD86, IFI27, MT2A, GBP1, IFIH1, IRF7, CD200, BIRC3, CD83, TNFRSF9, CCR7, TNFAIP6, CD80, TUBB2A, IDO1 and NFKBIA have been reported to be upregulated in mature dendritic cells in comparison to untreated human monocytes. Accordingly, in the present invention, a monocyte or monocyte-derived cell is determined to be differentiated into a mature dendritic cells, if at least 15, at least 20, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35 or more than 35 of these genes are upregulated in comparison to an untreated human monocyte as described above. Preferably, a monocyte or monocyte-derived is determined to be differentiated into a mature dendritic cell, if at least 10, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23 or all of the genes IL6, CYP27B1, EBI3, IFIT1, HES4, DUSP5, USP18, IFI44L, IFIT3, ATF3, MX1, ISG15, GADD45A, OASL, CCL8, TRAF1, LAMP3, RGS1, CXCL8, CD44, IL7R, CD86, IFI27 and MT2A are upregulated by a log2- fold change higher than 1 in comparison to an untreated human monocyte. More preferably, a monocyte or monocyte-derived is determined to be differentiated into a mature dendritic cell, if at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 or all of the genes IL6, CYP27B1, EBI3, IFIT1, HES4, DUSP5, USP18, IFI44L, IFIT3, ATF3, MX1, ISG15 and GADD45A are upregulated by a log2-fold change higher than 2 in comparison to an untreated human monocyte. Even more preferably, a monocyte or monocyte-derived is determined to be differentiated into a mature dendritic cell, if at least 1, at least 2, at least 3, at least 4 or all of the genes IL6, CYP27B1, EBI3, IFIT1 and HES4 are upregulated by a log2-fold change higher than 3 in comparison to an untreated human monocyte.
Gene expression data for Ml macrophages has been reported by Orecchioni, M. et al.; Macrophage Polarization: Different Gene Signatures in M1(LPS+) vs. Classically and M2(LPS-) vs. Alternatively Activated Macrophages. Frontiers in Immunology 2019 Vol 10, Article ID 1084. In this study, the genes CD40, PTGS2, ICAM1, IFIT2, VCAM1, RSAD2, EHD1, TRAF1, CCND2, SOCS3, ADORA2A, SERPINE1, GPR84, NFKBIZ and IL12A have been reported to be upregulated in Ml macrophages in comparison to untreated monocytes. Accordingly, in the present invention, a monocyte or monocyte -derived is determined to be differentiated into an Ml macrophage, if at least 8, at least 9, at least 10, at least 11 or more than 11 of these genes are upregulated in comparison to an untreated human monocyte as described above. Preferably, a monocyte or monocyte-derived is determined to be differentiated into an Ml macrophage, if at least 5, at least 6, at least 7, at least 8, at least 9 or all of the genes CD40, PTGS2, IFIT2, VCAM1, RSAD2, EHD1, TRAF1, CCND2, SOCS3 and SERPINE1 are upregulated by a log2-fold change higher than 1 in comparison to an untreated human monocyte. More preferably, a monocyte or monocyte -derived is determined to be differentiated into an Ml macrophage, if at least 2, at least 3, at least 4, at least 5, at least 6 or all of the genes PTGS2, IFIT2, VCAM1, RSAD2, TRAF1, CCND2, SOCS3 and SERPINE1 are upregulated by a log2-fold change higher than 1.5 in comparison to an untreated human monocyte. Even more preferably, a monocyte or monocyte-derived is determined to be differentiated into an Ml macrophage, if at least 1 or all of the genes RSAD2 and SERPINE1 are upregulated by a log2-fold change higher than 2, preferably higher than 3, in comparison to an untreated human monocyte.
The cell surface markers, cytokines and genes disclosed herein may be used to identify cells of a specific cell type, such as monocytes, macrophages or dendritic cells and their sub-types disclosed herein. However, it is to be understood that the expression of cell surface markers, the secretion of cytokines and/or gene expression in general may vary between cells of the same cell type and depends on various factors. For example, differences in the expression of cell surface markers, the secretion of cytokines and/or gene expression between two cells of the same cell type may be observed between cells that have been obtained from different donors/origins and/or have been cultured under different culturing conditions (in vitro). Thus, a cell may be of a certain cell type, even if one or more cell surface markers, cytokines and/or genes are expressed or secreted differently as disclosed herein.
For example, the cell surface marker CD 14 is commonly reported to be upregulated in macrophages and downregulated in dendritic cells. However, variations in the expression of CD 14 have been reported in the art (for example in Ong SM et al; A Novel, Five-Marker Alternative to CD16-CD14 Gating to Identify the Three Human Monocyte Subsets; Front. Immunol., 26 July 2019; or Collin M and Venetia Bigley; Human dendritic cell subsets: an update. Immunology; 154(1); 2018; p.3-20).
Further methods have been described in the art to differentiate between monocytes, macrophages and dendritic cells (for example as summarized by Coillard A and Segura E; In vivo Differentiation of Human Monocytes. Front. Immunol., 13 August 2019). In addition, commercial kits exist that allow identification and/or isolation of specific cell types. Thus, it is to be understood that the skilled person is aware of various methods to identify monocytes and to determine, whether a monocyte matured into a dendritic cell, a macrophage or a specific sub-type thereof.
In a particular embodiment, the invention relates to the target-binding molecule according to the invention, wherein the target-binding molecule is an antibody or a target-binding fragment thereof.
The target-binding molecule of the invention may be any type of target-binding molecule that has the potential to bind to the extracellular part of GOLPH2 such that it induces the maturation of a monocyte or a monocyte-derived cell. That is, in certain embodiments, the target-binding molecule of the invention is an antibody, or a target-binding portion thereof, a bispecific antibody, or a targetbinding portion thereof, a designed ankyrin repeat protein (D ARPIN), an aptamer or another antibody mimetic, such as affibody molecules, affilins, affimers, affitins, alphabodies, anticalins, avimers, fynomers, kunitzdomain peptides, monobodies. In a preferred embodiment, the target-binding molecule of the invention is an antibody, or a targetbinding fragment thereof. In general, the term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fully-human antibodies and antibody fragments so long as they exhibit the desired target-binding activity. Antibodies within the present invention may also be chimeric antibodies, recombinant antibodies, target-binding fragments of recombinant antibodies or humanized antibodies.
The term “monoclonal antibody”, as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Monoclonal antibodies are advantageous in that they may be synthesized by a hybridoma culture, essentially uncontaminated by other immunoglobulins. The term "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. As mentioned above, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method described by Kohler, Nature 256 (1975), 495.
The term “polyclonal antibody”, as used herein, refers to an antibody which was produced among or in the presence of one or more other, non-identical antibodies. In general, polyclonal antibodies are produced from a B-lymphocyte in the presence of several other B-lymphocytes which produced nonidentical antibodies. Usually, polyclonal antibodies are obtained directly from an immunized animal.
The term “fully-human antibody” as used herein refers to an antibody which comprises human immunoglobulin protein sequences only. A fully human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell or in a hybridoma derived from a mouse cell. Similarly, “mouse antibody” or “murine antibody” refers to an antibody which comprises mouse/murine immunoglobulin protein sequences only. Alternatively, a “fully-human antibody” may contain rat carbohydrate chains if produced in a rat, in a rat cell, in a hybridoma derived from a rat cell. Similarly, the term “rat antibody” refers to an antibody that comprises rat immunoglobulin sequences only. Fully-human antibodies may also be produced, for example, by phage display which is a widely used screening technology which enables production and screening of fully human antibodies. Also, phage antibodies can be used in context of this invention. Phage display methods are described, for example, in US 5,403,484, US 5,969, 108 and US 5,885,793. Another technology which enables development of fully-human antibodies involves a modification of mouse hybridoma technology. Mice are made transgenic to contain the human immunoglobulin locus in exchange for their own mouse genes (see, for example, US 5,877,397).
The term “chimeric antibodies”, refers to an antibody which comprises a variable region of the present invention fused or chimerized with an antibody region (e.g., constant region) from another, human or non-human species (e.g., mouse, horse, rabbit, dog, cow, chicken).
The term antibody also relates to recombinant human antibodies, heterologous antibodies and heterohybrid antibodies. The term "recombinant (human) antibody" includes all human sequence antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes; antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions (if present) derived from human germline immunoglobulin sequences. Such antibodies can, however, be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
A "heterologous antibody" is defined in relation to the transgenic non-human organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic non-human animal, and generally from a species other than that of the transgenic non-human animal.
The term "heterohybrid antibody" refers to an antibody having light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody. Examples of heterohybrid antibodies include chimeric and humanized antibodies.
The term antibody also relates to humanized antibodies. "Humanized" forms of non-human (e.g. murine or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other target-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Often, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non- human residues. Furthermore, humanized antibody may comprise residues, which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acids introduced into it from a source which is non-human still retain the original binding activity of the antibody. Methods for humanization of antibodies/antibody molecules are further detailed in Jones et al. (Nature 321 (1986), 522-525); Reichmann et al. (Nature 332 (1988), 323-327) and Verhoeyen et al. (Science 239 (1988), 1534-1536). Specific examples of humanized antibodies, e.g. antibodies directed against EpCAM, are known in the art, see e.g. LoBuglio (Proceedings of the American Society of Clinical Oncology Abstract (1997), 1562) and Khor (Proceedings of the American Society of Clinical Oncology Abstract (1997), 847).
A popular method for humanization of antibodies involves CDR grafting, where a functional targetbinding site from a non-human ‘donor’ antibody is grafted onto a human ‘acceptor’ antibody. CDR grafting methods are known in the art and described, for example, in US 5,225,539, US 5,693,761 and US 6,407,213. Another related method is the production of humanized antibodies from transgenic animals that are genetically engineered to contain one or more humanized immunoglobulin loci which are capable of undergoing gene rearrangement and gene conversion (see, for example, US 7,129,084).
Accordingly, in the context of this invention, antibody molecules or target-binding fragments thereof are provided, which are humanized and can successfully be employed in pharmaceutical compositions.
Accordingly, in context of the present invention, the term “antibody” relates to full immunoglobulin molecules as well as to parts of such immunoglobulin molecules (i.e., “target-binding fragment thereof’). Furthermore, the term relates, as discussed above, to modified and/or altered antibody molecules. The term also relates to recombinantly or synthetically generated/synthesized antibodies. The term also relates to intact antibodies as well as to antibody fragments thereof, like, separated light and heavy chains, Fab, Fv, Fab’, Fab’-SH, F(ab’)2. The term antibody also comprises but is not limited to fully-human antibodies, chimeric antibodies, humanized antibodies, CDR-grafted antibodies and antibody constructs, like single chain Fvs (scFv) or antibody-fusion proteins.
A " target-binding fragment" of an antibody refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the target to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g. scFv), and multispecific antibodies formed from antibody fragments.
“Single-chain Fv” or “scFv” antibody fragments have, in the context of the invention, the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. Techniques described for the production of single chain antibodies are described, e.g., in Pltickthun in The Pharmacology of Monoclonal Antibodies, Rosenburg and Moore eds. Springer-Verlag, N.Y. (1994), 269-315.
In certain embodiments, the target-binding molecule of the invention may be an scFv fragment or a chimeric antibody comprising an scFv fragment. In certain embodiments, the scFv fragment may be derived from the antibodies G2-2 or G2-4. The generation of scFv fragments is further disclosed in Example 20. The variable heavy and the variable light chain of an antibody may be fused with a peptide linker. In certain embodiments, the peptide linker may be a flexible linker as disclosed herein. In certain embodiments, the linker may have the sequence ASGGGGSGGGGSGGGGS (SEQ ID NO:51)
In certain embodiments, the scFv fragment may be derived from G2-2 or G2-2opti and may have the sequence set forth in SEQ ID NO:69 or SEQ ID NO:70. In certain embodiments, the scFv fragment may be derived from G2-4 and may have the sequence set forth in SEQ ID NO:71.
A “Fab fragment” as used herein is comprised of one light chain and the CHI and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
An "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. A "Fab1 fragment" contains one light chain and a portion of one heavy chain that contains the VH domain and the CHI domain and also the region between the CHI and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab') 2 molecule.
A "F(ab')2 fragment" contains two light chains and two heavy chains containing a portion of the constant region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.
The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.
Antibodies, antibody constructs, antibody fragments, antibody derivatives (all being Ig-derived) to be employed in accordance with the invention or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook (1989), loc. cit. The term “Ig- derived domain” particularly relates to (poly)peptide constructs comprising at least one CDR. Fragments or derivatives of the recited Ig-derived domains define (poly) peptides which are parts of the above antibody molecules and/or which are modified by chemical/biochemical or molecular biological methods. Corresponding methods are known in the art and described inter alia in laboratory manuals (see Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 2nd edition (1989) and 3rd edition (2001); Gerhardt et al., Methods for General and Molecular Bacteriology ASM Press (1994); Lefkovits, Immunology Methods Manual: The Comprehensive Sourcebook of Techniques; Academic Press (1997); Golemis, Protein-Protein Interactions: A Molecular Cloning Manual Cold Spring Harbor Laboratory Press (2002)).
The term “CDR” as employed herein relates to “complementary determining region”, which is well known in the art. The CDRs are parts of immunoglobulins that determine the specificity of said molecules and make contact with a specific ligand. The CDRs are the most variable part of the molecule and contribute to the diversity of these molecules. There are three CDR regions CDR1, CDR2 and CDR3 in each V domain. CDR-H depicts a CDR region of a variable heavy chain and CDR-L relates to a CDR region of a variable light chain. VH means the variable heavy chain and VL means the variable light chain. The CDR regions of an Ig-derived region may be determined as described in Kabat “Sequences of Proteins of Immunological Interest”, 5th edit. NIH Publication no. 91-3242 U.S. Department of Health and Human Services (1991); Chothia J. Mol. Biol. 196 (1987), 901-917 or Chothia Nature 342 (1989), 877-883.
The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, a, y, and p, respectively.
Accordingly, in the context of the present invention, the antibody molecule described herein above is selected from the group consisting of a full antibody (immunoglobulin, like an IgGl, an IgG2, an IgG2a, an IgG2b, an IgAl, an IgGA2, an IgG3, an IgG4, an IgA, an IgM, an IgD or an IgE), F(ab)-, Fab’-SH-, Fv-, Fab’-, F(ab’)2-fragment, a chimeric antibody, a CDR-grafted antibody, a fully human antibody, a bivalent antibody-construct, an antibody-fusion protein, a synthetic antibody, bivalent single chain antibody, a trivalent single chain antibody and a multivalent single chain antibody.
In any of the embodiments described herein, the target-binding molecule may be a monoclonal antibody. In any of the embodiments described herein, the target-binding molecule may be a human, humanized, or chimeric antibody. In any of the embodiments described herein, the target-binding molecule may be an antibody fragment that binds GOLPH2. In any of the embodiments described herein, the target-binding molecule may be an IgGl, IgG2a or IgG2b, IgG3, IgG4, IgM, IgAl, IgA2, IgD or IgE antibody. As used herein, "isotype" refers to the antibody class (e.g., IgM or IgGl) that is encoded by heavy chain constant region genes. The antibodies can be full length or can include only a target-binding fragment such as the antibody constant and/or variable domain of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD or IgE or could consist of a Fab fragment, a F(ab')2 fragment and a Fv fragment.
In certain embodiments, the antibody of the invention comprises an Fc region. In certain embodiments, the antibody of the invention comprises an Fc region that can elicit an immune effector function. That is, in certain embodiments, the antibody of the invention comprises an Fc region that can bind to an Fey receptor. An "antibody that binds to an epitope" within a defined region of a protein, e.g. the extracellular part of GOLPH2, is an antibody that requires the presence of one or more of the amino acids within that region for binding to the protein.
In accordance with the above, the antibody of the invention may bind to the extracellular part of human GOLPH2. In some embodiments, the antibody may bind to the extracellular part of murine GOLPH2 and/or to canine GOLPH2.
The terms "anti-GOLPH2 antibody" and "an antibody specifically binding to GOLPH2" or simply “antibody” as used herein refer to an antibody that is capable of binding GOLPH2, in particular the extracellular part of GOLPH2, with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting GOLPH2. In one embodiment, the extent of binding of an anti- GOLPH2 antibody to an unrelated, non-GOLPH2 protein is less than about 10% of the binding of the antibody to GOLPH2 as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to GOLPH2 has a dissociation constant (Kd) of < 1 pM, < 100 nM, <10 nM, < 5 nm, < 4 nM, <3 nM, <2 nM, <1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10’8 M or less, e.g. from 10"8 M to 10"13 M, e.g., from 10"9 M to 10"13 M).
In certain embodiments, an "antibody that binds to an epitope" within a defined region of a protein is identified by mutation analysis, in which amino acids of the protein are mutated, and binding of the antibody to the resulting altered protein (e.g., an altered protein comprising the epitope) is determined to be at least 20% of the binding to unaltered protein. In some embodiments, an "antibody that binds to an epitope" within a defined region of a protein is identified by mutation analysis, in which amino acids of the protein are mutated, and binding of the antibody to the resulting altered protein (e.g., an altered protein comprising the epitope) is determined to be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the binding to unaltered protein. In certain embodiments, binding of the antibody is determined by FACS, WB or by a suitable binding assay such as ELISA.
In one embodiment, Ka is measured using surface plasmon resonance assays using a BIACGRE®-2000 or a BIACORE ®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized antigen CM5 chips at -10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS) according to the supplier' s instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 pg/ml (-0.2 pM) before injection at a flow rate of 5 pl/m incite to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of antibody (0.58 nM to 200 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PEST) at 25°C at a flow rate of approximately 25 pl/min. Association rates (kon) and dissociation rates (kq /') are calculated using a simple one-to-one Langmuir binding model (BIACORE ® T100 Evaluation Software) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio kofflkon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106 M 1 s 1 by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25 °C of a 20 nM anti -antigen antibody in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO TM spectrophotometer (Thermo Spectronic) with a stirred cuvette.
The antibody or target-binding fragment thereof as provided in the context of the present invention is not particularly limited as long as it is an “anti-GOLPH2 antibody or a target-binding fragment thereof’ as defined above. In particular, the antibody may be any antibody which specifically binds to/specifically recognizes/interacts with the extracellular part of GOLPH2 on the cell surface of a monocyte or a monocyte-derived cell such that the maturation of the monocyte or the monocyte- derived cell is induced. Accordingly, the invention also provides antibodies binding to the same region of GOLPH2, i.e. the extracellular part of GOLPH2, and carrying out the same function as any of the specific antibodies provided herein.
In certain embodiments, an anti-GOLPH2 antibody may bind to an epitope of GOLPH2 that is conserved among GOLPH2 from different species. As detailed herein, the antibody of the invention may bind to a defined epitope within the GOLPH2 extracellular part. In particular, the antibody of the invention may bind to an epitope within the amino acid sequence of SEQ ID NO: 23, 24, 25, 26 and/or 27.
The specificity of the antibody or target-binding fragment of the present invention may not only be expressed by the nature of the amino acid sequence of the antibody or the target-binding fragment as defined above but also by the epitope or part of the antigen to which the antibody is capable of binding to. Thus, the present invention relates, in one embodiment, to an anti-GOLPH2 antibody or a targetbinding fragment thereof which recognizes the same epitope or part of GOLPH2 as an antibody of the invention, for example the monoclonal antibodies G2-2, G2-4 and EPR3606 or the polyclonal antibody PA5-18100. In order to test whether a target-binding molecule in question and the target-binding molecule of the present invention recognize the same epitope, the following competition study may be carried out: Vero cells infected with 3 moi (multiplicity of infection) are incubated after 20 h with varying concentrations of the antibody in question as the competitor for 1 hour. In a second incubation step, the antibody of the present invention is applied in a constant concentration of 100 nM and its binding is flow-cytometrically detected using a fluorescence-labelled antibody directed against the constant domains of the antibody of the invention. Binding that conducts anti-proportional to the concentration of the antibody in question is indicative for that both antibodies recognize the same epitope. However, many other assays are known in the art which may be used.
In certain embodiments, the invention relates to the target-binding molecule according to the invention, wherein the antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO.9; or (b) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO.17.
In certain embodiments, the invention relates to the target-binding molecule according to the invention, wherein the antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NON, CDR2 as defined in SEQ ID NO:5 and CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO.7, CDR2 as defined in SEQ ID NO: 8 and CDR3 as defined in SEQ ID NO:9; or (b) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NO: 12, CDR2 as defined in SEQ ID NO: 13 and CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO: 15, CDR2 as defined in SEQ ID NO: 16 and CDR3 as defined in SEQ ID NO: 17.
In certain embodiments, the invention relates to the target-binding molecule according to the invention, wherein the antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 18 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:2 or SEQ ID NO: 18; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO: 19 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:3 or SEQ ID NO: 19; or (b) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO: 10 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 10; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 11.
Certain embodiments provided herein are based, in part, on the development of antibody G2-2 and/or G2-2opti from WO 2018/091724, which binds to an epitope within amino acids 36 to 55 of human GOLPH2 protein. In some embodiments, an antibody provided herein binds to an epitope within amino acids 36 to 55 of human GOLPH2. In some embodiments, an antibody provided herein comprises one or more CDR sequences of antibody G2-2.
Accordingly, in some embodiments, the invention provides an anti-GOLPH2 antibody comprising at least one, two, three, four, five, or six CDRs selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9.
In one embodiment, the invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6. In one embodiment, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6. In another embodiment, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9. In a further embodiment, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, and CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5. In a further embodiment, the antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6.
In another embodiment, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and (iii) CDR-H3 comprising an amino acid sequence selected from SEQ ID NO: 6; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9. In another embodiment, the invention provides an antibody comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9.
In another embodiment, an anti-GOLPH2 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 18. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 18 contains substitutions (e.g, conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-GOLPH2 antibody comprising that sequence retains the ability to bind to GOLPH2. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 2 or SEQ ID NO: 18. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 2 or SEQ ID NO: 18. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In a preferred embodiment, a total of 3 amino acids in SEQ ID NO: 2 have been substituted to optimize the expression in mammalian cells (SEQ ID NO: 18). Optionally, the anti-GOLPH2 antibody comprises the VH sequence of SEQ ID NO: 2 or SEQ ID NO: 18, including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (b) CDRH2 comprising the amino acid sequence of SEQ ID NO: 5, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6.
In another embodiment, an anti-GOLPH2 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 19. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 19 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti- GOLPH2 antibody comprising that sequence retains the ability to bind to GOLPH2. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 3 or SEQ ID NO: 19. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 3 or SEQ ID NO: 19. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In a preferred embodiment, a total of 8 amino acids in SEQ ID NO: 3 have been substituted to optimize the expression in mammalian cells (SEQ ID NO: 19). Optionally, the anti-GOLPH2 antibody comprises the VL sequence of SEQ ID NO: 3 or SEQ ID NO: 19, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three CDRs selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9.
In another embodiment, an anti-GOLPH2 antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In a preferred embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 18 and SEQ ID NO: 19, respectively, including post-translational modifications of those sequences. In another preferred embodiment, an anti-GOLPH2 antibody comprises a humanized form of an antibody comprising the VH and VL sequences in SEQ ID NO: 18 and SEQ ID NO: 19, respectively. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 2 and SEQ ID NO: 3, respectively, including post-translational modifications of those sequences. In another embodiment, an anti-GOLPH2 antibody comprises a humanized form of an antibody comprising the VH and VL sequences in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
Certain embodiments provided herein are based, in part, on the development of antibody G2-4 from WO 2018/091724, which binds to an epitope in human soluble GOLPH2. In some embodiments, an antibody provided herein binds to an epitope within amino acids 347 to 366 of human GOLPH2. In some embodiments, an antibody provided herein comprises one or more CDR sequences of antibody G2-4.
Accordingly, in some embodiments, the invention provides an anti-GOLPH2 antibody comprising at least one, two, three, four, five, or six CDRs selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.
In one embodiment, the invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14. In another embodiment, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17. In a further embodiment, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13. In a further embodiment, the antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) CDR- H2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14.
In another embodiment, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (iii) CDR-H3 comprising an amino acid sequence selected from SEQ ID NO: 14; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.
In another embodiment, the invention provides an antibody comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.
In another embodiment, an anti-GOLPH2 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 10 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-GOLPH2 antibody comprising that sequence retains the ability to bind to GOLPH2. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 10. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 10. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In a preferred embodiment, a total of 3 amino acids in SEQ ID NO: 10 have been substituted to optimize the expression in mammalian cells. Optionally, the anti-GOLPH2 antibody comprises the VH sequence of SEQ ID NO: 10, including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12, (b) CDRH2 comprising the amino acid sequence of SEQ ID NO: 13, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14.
In another embodiment, an anti-GOLPH2 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 11 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-GOLPH2 antibody comprising that sequence retains the ability to bind to GOLPH2. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 11. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 11. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In a preferred embodiment, a total of 8 amino acids in SEQ ID NO: 11 have been substituted to optimize the expression in mammalian cells. Optionally, the anti-GOLPH2 antibody comprises the VL sequence of SEQ ID NO: 11, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three CDRs selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.
In another embodiment, an anti-GOLPH2 antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In a preferred embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 10 and SEQ ID NO: 11, respectively, including post-translational modifications of those sequences. In another preferred embodiment, an anti-GOLPH2 antibody comprises a humanized form of an antibody comprising the VH and VL sequences in SEQ ID NO: 10 and SEQ ID NO: 11, respectively.
In certain embodiments, the antibody of the invention comprises a human light chain and heavy chain constant region. In other embodiments, the antibody of the invention comprises a murine light chain and heavy chain constant regions. Examples for human and murine constant regions of GOLPH2 antibodies are provided in WO 2018/091724.
In a further embodiment, the anti-GOLPH2 antibody according to any of the above embodiments, in particular the antibody G2-2opti, comprises a heavy chain constant region sequence comprising the amino acid sequence of SEQ ID NO: 20 (human IgGl heavy chain) or SEQ ID NO: 21 (human hinge region fused to murine CH2 and CH3 domains). In another embodiment, an anti-GOLPH2 antibody comprises a heavy chain constant region sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 20 or SEQ
ID NO: 21.
In a further embodiment of the invention, an anti-GOLPH2 antibody according to any of the above embodiments is a monoclonal antibody, including a human antibody. In one embodiment, an anti- GOLPH2 antibody is an antibody fragment, e.g, a Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a substantially full length antibody, e.g., an IgGl antibody, IgG2a antibody or other antibody class or isotype as defined herein.
In a further embodiment, an anti-GOLPH2 antibody according to any of the above embodiments may incorporate any of the features, singly or in combination, as described below.
In a further embodiment, provided herein are antibodies that bind to the same part or epitope as an anti- GOLPH2 antibody provided herein. In a preferred embodiment, an antibody is provided that binds to the same epitope as an anti-GOLPH2 antibody comprising a VH sequence of SEQ ID NO: 18 and a VL sequence of SEQ ID NO: 19 respectively. In certain embodiments, an antibody is provided that binds to the same epitope as an anti-GOLPH2 antibody comprising a VH sequence of SEQ ID NO: 2 and a VL sequence of SEQ ID NO: 3, respectively. In another preferred embodiment, an antibody is provided that binds to the same epitope as an anti-GOLPH2 antibody comprising a VH sequence of SEQ ID NO: 10 and a VL sequence of SEQ ID NO: 11 respectively.
In certain embodiments, the antibody may be the commercial polyclonal anti-GOLPH2 antibody PA5- 18100 from Invitrogen. PA5-18100 has been raised against the synthetic peptide sequence NLLDQREKRNHTL (SEQ ID NO:27), which corresponds to amino acids 389 to 401 of human GOLPH2 as set forth in SEQ ID NO: 1. Thus, in certain embodiments, the invention encompasses targetbinding molecules that bind to an epitope within the amino acid sequence NLLDQREKRNHTL, which corresponds to amino acids 389 to 401 of human GOLPH2 as set forth in SEQ ID NO: 1. This targetbinding molecule may not exclusively be the polyclonal antibody PA5-18100, but may be any targetbinding molecule and, in particular, any antibody or target-binding fragment thereof, that specifically binds to an epitope within the amino acid sequence NLLDQREKRNHTL, which corresponds to amino acids 389 to 401 of human GOLPH2 as set forth in SEQ ID NO: 1.
In certain embodiments, the antibody may be the commercial rabbit monoclonal anti-GOLPH2 antibody EPR3606 from Abeam. That is, the target-binding molecule according to the invention may bind to the same epitope in the extracellular part of GOLPH2 as the antibody EPR3606. "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
It has been shown by the inventors that target-binding molecules specifically binding to the extracellular part of GOLPH2 on the cell surface of monocytes and/or monocyte-derived cells induce the maturation of the monocytes and/or the monocyte-derived cells into antigen-presenting cells, in particular Ml macrophages and dendritic cells, in particular matured dendritic cells. To the surprise of the inventors, it has been shown that the antibodies of the invention can further be used to specifically deliver antigens or antigenic peptides to Ml macrophages and/or dendritic cells, in particular matured dendritic cells.
An antigen or antigenic peptide may be attached to the target-binding molecule of the invention in any way, provided that the attached antigenic peptide does not inhibit binding of the target-binding molecule to the extracellular part of GOLPH2.
Without being bound to theory, it is believed that antigenic peptides are more efficiently displayed by Ml macrophages and/or matured dendritic cells, if the antigenic peptides are attached to a targetbinding molecule that is internalized into the cell upon binding to the extracellular part of GOLPH2. Thus, in certain embodiments, it is preferred that the antigenic peptide is attached to a target-binding molecule that binds specifically to the extracellular part of GOLPH2 and is internalized into the cell upon binding to the extracellular part of GOLPH2. It is known in the art that antibodies that specifically bind to an epitope located within the amino acid sequences of SEQ ID NOs: 23 (human), 24 (murine) and/or 25 (canine) of GOLPH2 are internalized into the cell upon binding to their target. Thus, in certain embodiments, the antigenic peptide is preferably attached to a target-binding molecule that specifically binds to an epitope located within the amino acid sequences of SEQ ID NOs: 23, 24 and/or 25 of GOLPH2. In certain embodiments, the target-binding molecule is an antibody. In certain embodiments, the antibody is G2-2opti from WO 2018/091724 or any antibody comprising at least one, at least two, at least three, at least four, at least five or all six CDR sequences of antibody G2-2 Furthermore, target-binding molecules that specifically bind to an epitope located within the amino acid sequences of SEQ ID NOs: 23 (human), 24 (murine) and/or 25 (canine) of GOLPH2 have the advantage that they can reach GOLPH2 -positive cells more efficiently than target-binding molecules that specifically bind to the soluble part of GOLPH2. Thus, antibodies binding specifically to an epitope located within the amino acid sequences of SEQ ID NOs: 23 (human), 24 (murine) and/or 25 (canine) of GOLPH2 may be more efficacious in vivo, as these antibodies are not captured by soluble GOLPH2 in the bloodstream before reaching a GOLPH2-positive cell. For that reason, the targetbinding molecule according to the invention preferably comprises the antibody is G2-2opti from WO 2018/091724 or any antibody comprising at least one, at least two, at least three, at least four, at least five or all six CDR sequences of antibody G2-2 from WO 2018/091724.
In a particular embodiment, the invention relates to the target-binding molecule according to the invention, wherein the target-binding molecule further comprises an antigenic peptide fused to the C- terminal end of the antibody, or the target-binding fragment thereof.
The antigenic peptide may be attached to an antibody or a target-binding fragment thereof. It is commonly known in the art that the N-terminal end of the light and heavy chain of an antibody are involved in antigen binding. Thus, it is preferred that antigenic peptides are attached to the C-terminal end of an antibody or a target-binding fragment thereof. Accordingly, in certain embodiments, the antigenic peptide may be attached to the C-terminal end of the antibody of the invention, or the targetbinding fragment thereof.
The antigenic peptide may be fused to the C-terminal end of the antibody, or the target-binding fragment thereof, directly or via a linker. Thus, in a particular embodiment, the invention relates to the target-binding molecule according to the invention, wherein the antigenic peptide is fused to the C- terminal end of the antibody, or the target-binding fragment thereof, via a peptide linker.
The term “peptide linker”, as used herein, refers to a peptide comprised of amino acids. A range of suitable peptide linkers will be known to the person of skill in the art. In one embodiment, the peptide linker is 50 amino acids in length or less, for example 20 amino acids or less, such as about 15 amino acids or less, such as about 10 amino acids or less. In certain embodiments, the linker is rich in glycine and serine amino acids. That is, in certain embodiments, the linker comprises at least one, at least two, at least three, at least four or at lest five glycine or serine amino acids. In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or all amino acids in the linker are glycine or serine amino acids.
Alternatively, the linker may comprise a peptidase cleavage site to facilitate processing of the antigenic peptide inside a cell. In certain embodiments, the peptidase cleavage site may be a cathepsin cleavage site. In certain embodiments, the peptidase cleavage site may be a Cathepsin B cleavage site. In certain embodiments, the peptide linker may comprise the sequence GFLGSGFLGS (SEQ ID NO:75)
In certain embodiments, the antigenic peptide is fused to an antibody. Preferably, the antigenic peptide is fused to the C-terminal end of the heavy and/or light chain of the antibody. Accordingly, in a particular embodiment, the invention relates to the target-binding molecule according to the invention, wherein the antigenic peptide is fused to the C-terminal end of a heavy chain and/or light chain of an antibody.
The skilled person is aware of methods to fuse a peptide, such as an antigenic peptide, to a protein, such as a proteinaceous target-binding molecule, in particular an antibody or a target-binding fragment thereof. The term “fused” as used herein refers to two nucleic acids fused together so that the resulting protein is expressed as a single protein. In particular, the 3' nucleic acid residue of the coding sequence encoding the antibody, or the target-binding fragment thereof, is bonded to the 5' nucleic acid residue of the coding sequence encoding the antigenic peptide, either directly or via a linker. Thus, an antigenic peptide is said to be fused to a protein, such as an antibody or an antibody fragment, if the N- terminal amino acid of the antigenic peptide forms a peptide bond with the C-terminal amino acid of at least one polypeptide comprised in an antibody, or a target-binding fragment thereof, according to the invention. Methods of molecular biology to fuse an antigenic peptide to a protein at the DNA level are known in the art. The construction of an antibody fused to an antigenic peptide is further demonstrated in Example 27.
In particular, antibodies fused to antigenic peptides may be constructed by molecular cloning. That is, a restriction enzyme cleavage site may be introduced at the C-terminal end in the coding sequence(s) encoding the heavy and/or light chain of the antibody. In certain embodiments, codons encoding amino acids at or near the C-terminal end of the heavy and/or light chain of an antibody may be modified such that a restriction enzyme cleavage site is introduced without changing the amino acid sequence of the heavy and/or light chain of the antibody. In certain embodiments, the restriction enzyme cleavage site is recognized by the enzyme Smal. That is, the nucleotide sequence CCCGGG (SEQ ID NO:74) may be introduced at the 3’ end of a nucleic acid molecule encoding an antibody light and/or heavy chain. Many IgG type antibodies comprise the motif PGK at the C-terminal end of the heavy chain. In such cases, the codons encoding PG may be modified to obtain the Smal restriction site CCCGGG (SEQ ID NO:74). The resulting nucleic acid molecule(s) encoding the heavy and/or light chain of the antibody may then be combined with a nucleic acid molecule encoding a polypeptide comprising one or more antigenic peptides by molecular cloning via a restriction site, such as Smal. For that, the nucleic acid molecule encoding the polypeptide comprising the one or more antigenic peptides preferably comprises a compatible restriction site near the 5’ end of the nucleic acid molecule. Alternatively, a nucleic acid molecule encoding a heavy and/or light chain of an antibody, wherein the heavy and/or light chain is fused, optionally by a suitable linker, to a polypeptide comprising one or more antigenic peptides, may be directly obtained by chemical synthesis, as known in the art.
In certain embodiments, the target-binding molecule of the invention is the antibody G2-2-L (see FIG. 13). The antibody G2-2-L is based on the antibody G2-2opti as disclosed herein and further comprises antigenic peptides fused to the C-terminal ends of its light and heavy chains. Preferably, the light/heavy chain of the antibody is separated from the antigenic peptide by a peptidase cleavage site, more preferably, wherein the peptidase cleavage site is a Cathepsin cleavage site, even more preferably a Cathepsin B cleavage site. That is, the target-binding molecule G2-2-L may comprise four polypeptides comprising antigenic peptides, wherein one polypeptide is fused to each light and heavy chain C-terminal end of the antibody, respectively. The four polypeptides comprising the antigenic peptides may be identical or may be different in sequence. It is further to be understood that each C- terminal end of the antibody may be fused to a polypeptide comprising multiple antigenic peptides. It is further to be understood, that antigenic peptides may be fused only to the C-terminal ends of the heavy chains or only to the C-terminal ends of the light chains of the antibody. The antigenic peptides comprised in G2-2-L may be any antigenic peptide disclosed herein or may be derived from any antigen disclosed herein.
In certain embodiments, the antigenic peptide may be derived from Human papillomavirus type 16 (HPV-16). In particular, the antigenic peptide may be derived from the E7 protein of HPV-16. In particular, the antigenic peptide derived from the E7 protein of HPV-16 may have the sequence PTLHEYMLDLQPE (SEQ ID NO:54) or RAHYNIVTF (SEQ ID NO:55).
In certain embodiments, the antigenic peptide may be derived from Influenza virus. In particular, the antigenic peptide may be derived from the Ml protein of Influenza virus. In certain embodiments, the antigenic peptide derived from the Ml protein of Influenza virus may have the sequence GILGFVFTLT (SEQ ID NO: 56) In certain embodiments, the antigenic peptide may be derived from SARS coronavirus 2. In particular, the antigenic peptide may be derived from the nucleocapsid phosphoprotein of SARS coronavirus 2. In certain embodiments, the antigenic peptide derived from the nucleocapsid phosphoprotein of SARS coronavirus 2 may have the sequence LLLLDRLNQLESKMS (SEQ ID NO: 57). Alternatively, the antigenic peptide may be derived from the ORF7a protein of SARS coronavirus 2. In certain embodiments, the antigenic peptide derived from ORF7a of SARS coronavirus 2 may have the sequence VYQLRARSV (SEQ ID NO: 58) or ITLCFTLKR (SEQ ID NO: 59)
In certain embodiments, the antigenic peptide may be derived from Epstein-Barr virus (human gammaherpesvirus 4, EBV). In particular, the antigenic peptide may be derived from the latent membrane protein2 (LMP2A) of EBV. In certain embodiments, the antigenic peptide derived from LMP2A of EBV may have the sequence CLGGLLTMV (SEQ ID NO: 60). Alternatively, the antigenic peptide may be derived from the protein BSLF2/BMFL1 of EBV. In certain embodiments, the antigenic peptide derived from BSLF2/BMFL1 of EBV may have the sequence GLCTLVAML (SEQ ID NO: 61).
In certain embodiments, the antigenic peptide may be derived from the Ovalbumin protein. In certain embodiments, the antigenic peptide derived from Ovalbumin may have the sequence SIINFEKL (SEQ ID NO: 62) or AAHAEINEA (SEQ ID NO:63).
In certain embodiments, the antigenic peptide may be a neoepitope derived from the murine colon cancer cell line MC38. In certain embodiments, the neoepitope derived from MC38 may be comprised in the sequence LFRAAQLANDVVLQIMEHLELASMTNMELMSSIVVISASIIVFNLLELEG (SEQ ID NO:64) or HLELASMTNMELMSSIVGSKILTFDRL (SEQ ID NO:65),
In certain embodiments, the antigenic peptide may be derived from the antigen tyrosinase-related protein 2 (TRP2). In certain embodiments, the antigenic peptide derived from tyrosinase-related protein 2 may have the sequence SVYDFFVWL (SEQ ID NO: 66).
In certain embodiments, the antigenic peptide may be derived from the antigen glycoprotein 100 (gplOO). In certain embodiments, the antigenic peptide derived from gplOO may have the sequence KVPRNQDWL (SEQ ID NO: 67).
It is to be understood that two or more antigenic peptides may be comprised in a single polypeptide that is fused to the C-terminal end of a heavy and/or light chain of an antibody. The two or more peptides may be from the same or different origin. In certain embodiments, the polypeptide may comprise two antigenic peptides derived from HPV-16. In certain embodiments, the polypeptide may comprise the sequence GFLGSGFLGSTPTLHEYMLDLQPEGSRAHYNIVTF (SEQ ID NO: 47).
In certain embodiments, the polypeptide may comprise antigenic peptides derived from HPV-16 and Influenza virus. In certain embodiments, the polypeptide may comprise the sequence GFLGSGFLGSGSTPTLHEYMLDLQPEGSGILGFVFTLT (SEQ ID NO:48).
In certain embodiments, the polypeptide may comprise antigenic peptides derived from SARS coronavirus 2 and EBV. In certain embodiments, the polypeptide may comprise the sequence GFLGSGFLGSGLLLLDRLNQLESKMSGSGLCTLVAMLCLGGLLTMV (SEQ ID NO:49).
In certain embodiments, the polypeptide may comprise two antigenic peptides derived from SARS coronavirus 2. In certain embodiments, the polypeptide may comprise the sequence GFLGSGFLGSVYQLRARSVGSITLCFTLKR (SEQ ID NO:50).
In certain embodiments, the polypeptide may comprise two antigenic peptides derived from Ovalbumin. In certain embodiments, the polypeptide may comprise the sequence GFLGSGFLGSSIINFEKLGSAAHAEINEA (SEQ ID NO:43).
In certain embodiments, the polypeptide may comprise an MC38 neoepitope. In certain embodiments, the polypeptide may comprise the sequence
GFLGSGFLGSLFRAAQLANDVVLQIMEHLELASMTNMELMSSIVVISASIIVFNLLELEG (SEQ ID NO:44) or GFLGSGFLGSHLELASMTNMELMSSIVGSKILTFDRL (SEQ ID NO:45).
In certain embodiments, the polypeptide may comprise antigenic peptides derived from TRP2 and gplOO. In certain embodiments, the polypeptide may comprise the sequence GFLGSGFLGSSVYDFFVWLGSKVPRNQDWL (SEQ ID NO: 46).
In a particular embodiment, the invention relates to the target-binding molecule according to the invention, wherein the target-binding molecule is a fusion antibody, wherein the fusion antibody comprises an Fc region and two or more scFv fragments.
The target-binding molecule according to the invention may be a chimeric antibody or a fusion antibody that comprises fragments of two or more antibodies or antibody fragments. In particular, the chimeric or fusion antibody may have the format (scFv)2-Fc. That is, the target-binding molecule according to the invention may comprise an Fc region of a first antibody and two or more scFv fragments fused to the Fc region. The two or more scFv fragments may be identical or may be different in amino acid sequence. Further, at least one of the scFv fragments may be derived from the same antibody from which the Fc fragment has been obtained. In other embodiments, the Fc region and the scFv fragments may be derived from different antibodies.
In certain embodiments, the chimeric or fusion antibody is a monospecific antibody of the format (SCFV)2-FC. That is, the chimeric or fusion antibody may comprise two scFv fragments fused to the N- terminal end of the Fc region, wherein the two scFv fragments bind to the same antigen and, preferably, to the same epitope. Preferably, the monospecific antibody of the format (scFv)2-Fc comprises two scFv fragments that bind to the extracellular part of GOLPH2 on the surface of monocytes or monocyte -derived cells such that it induces the maturation of said monocytes or monocyte-derived cells.
In certain embodiments, the invention relates to the antibody G2-2HX (human Fc region, SEQ ID NO:72) or G2-2MX (murine Fc region, SEQ ID NO:73) (see FIG.13). G2-2HX and G2-2MX comprise two scFv fragments derived from the antibody G2-2opti disclosed herein. The two scFv fragments (SEQ ID NO:70) are fused to the N-terminal end of a human (SEQ ID NO:38) or murine Fc region (SEQ ID NO:37).
In certain embodiments, the invention relates to the antibody G2-2HX-L (human Fc region) or G2- 2MX-L (murine Fc region) (see FIG. 13). G2-2HX-L and G2-2MX-L are constructed as described above and further comprise polypeptides comprising one or more antigenic peptides fused to the C- terminal ends of the Fc regions as disclosed herein.
In other embodiments, the chimeric or fusion antibody is a bispecific antibody of the format (scFv)2- Fc. That is, the chimeric or fusion antibody comprises two scFv fragments fused to the N-terminal end of the Fc region, wherein the two scFv fragments bind to different antigens. For example, a bispecific antibody of the format (scFv)2-Fc may comprise a first scFv fragments that binds specifically to the extracellular part of GOLPH2 on the surface of monocytes or monocyte -derived cells such that it induces the maturation of said monocytes or monocyte-derived cells and a second scFv fragment that binds to another antigen. The other antigen may be an antigenic peptide, an immune checkpoint molecule or a ligand of an immune checkpoint molecule as disclosed herein.
In other embodiments, the chimeric or fusion antibody is a bispecific antibody of the format (scFv)2- Fc-(scFv)2. That is, the chimeric or fusion antibody may comprise two scFv fragments fused to the N- terminal end of the Fc region and two scFv fragments fused to the C-terminal end of the Fc region. Preferably, the two scFv fragments fused to the N-terminal end of the Fc region bind specifically to a first epitope and the two scFv fragments fused to the C-terminal end of the Fc region bind specifically to a second epitope. The first epitope may be an epitope in the extracellular part of GOLPH2. The second epitope may be an epitope in an antigenic peptide, an immune checkpoint molecule or a ligand of an immune checkpoint molecule as disclosed herein.
In certain embodiments, the invention relates to the antibody G2-2HXQ (see FIG. 13). G2-2HXQ comprises two scFv fragment derived from the antibody G2-2opti disclosed herein, wherein the two scFv fragments are fused to the N-terminal end of the Fc region set forth in SEQ ID NO:38. Further G2-2HXQ comprises two scFv fragments that bind specifically to the antigen Myc, wherein the two scFv fragments are fused to the C-terminal end of the Fc region set forth in SEQ ID NO:38.
In a particular embodiment, the invention relates to the target-binding molecule according to the invention, wherein each scFv fragment is connected to the Fc region with a peptide linker.
That is, the scFv fragments may be connected to the N-terminal or C-terminal end of the Fc region via a peptide linker.
The term “peptide linker” as used herein generally refers to an amino acid based linker that connects two polypeptides, e.g., an Fc region and an scFv. The linker may have any length or sequence. However, it is preferred that the linker has a length between 2 and 50 amino acid residues.
The linker that fuses the N-terminal end of an Fc region to an scFv is preferably a longer flexible linker. It has been surprisingly shown by the inventors that so-called “long-neck” antibodies can bind to the extracellular part of GOLPH2 with increased affinity compared to classical antibodies (see FIG. 12 A&B).
Thus, the linker connecting an scFv fragment to the N-terminal end of an Fc region preferably has a length between 5 and 50 amino acid residues, preferably between 10 and 30 amino acid residues, more preferably between 15 and 25 amino acid residues. Further, it is preferred that the linker connecting an scFv fragment to the N-terminal end of an Fc region preferably is a flexible linker. Flexible linkers known in the art are rich in serine and glycine residues. Thus, the flexible linker connecting an scFv fragment to the N-terminal end of an Fc region preferably comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% serine and/or glycine residues. In certain embodiments, the linker connecting an scFv fragment to the N-terminal end of an Fc region has the sequence GSSSSSGSSSSGSSGRS (SEQ ID NO:52) or GSSSSSGSSSSGSSGGS (SEQ ID NO:53).
The linker connecting an scFv fragment to the C-terminal end of an Fc region may have a length between 2 and 50 amino acid residues, between 2 and 30 amino acid residues, between 2 and 20 amino acid residues, between 2 and 15 amino acid residues or between 2 and 10 amino acid residues. The linker may be rich in glycine or serine residues. That is, the linker connecting an scFv fragment to the C-terminal end of an Fc region may comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% serine and/or glycine residues. In certain embodiments the linker connecting an scFv fragment to the C-terminal end of an Fc region may have the sequence GGGS (SEQ ID NO:68).
The Fc region comprised in target-binding molecules having the format (scFv)2-Fc or (scFv)2-Fc- (scFyh may comprise an Fc region with a sequence as set forth in SEQ ID NO:37 (murine) or SEQ ID NO:38 (human).
The skilled person is aware of methods for generating the chimeric antibodies described above. Preferably, obtaining the chimeric antibodies described above comprises a step of molecular cloning (see Examples 20 and 25).
In a particular embodiment, the invention relates to the target-binding molecule according to the invention, wherein the antibody is a bispecific or a multispecific antibody.
The terms "multispecific antibody" or "bispecific antibody", as used herein, refer to an antibody that has binding domains specific for two or more different, preferably non-overlapping, antigens or epitopes within a single antibody molecule. Such antigens or epitopes may be on the same or different targets. If the antigens or epitopes are on different targets, such targets may be on the same cell or on different cells or cell types. It will be appreciated that other molecules in addition to the canonical antibody structure can be constructed with two binding specificities. It will further be appreciated that antigen binding by bispecific antibodies can be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Bispecific antibodies can also be constructed by recombinant means. (Strohlein and Heiss, Future Oncol. : 1387-94 (2010); Mabry and Snavely, IDrugs. 73:543-9 (2010)). In particular, bispecific antibodies may be produced with the method described by Paul S, Connor J, Nesspor T, et al. An efficient process of generating bispecific antibodies via controlled Fab-arm exchange using culture supernatants. Protein Expr Purif. 2016;121: 133-140. doi: 10.1016/j .pep.2016.01.014. In certain embodiments, the invention relates to the target-binding molecule according to the invention, wherein the bispecific antibody comprises a Fab or an scFv portion specifically binding to an immune checkpoint molecule.
In certain embodiments, the bispecific or multispecific antibody of the invention comprises two Fab or scFv portions, respectively. A “Fab portion” as used herein is comprised of one light chain and the CHI and variable regions of one heavy chain. An “scFv portion” as used herein is comprised of the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. In case of a bi- or multispecific antibody, one or more Fab or scFv portions may be conjugated to an Fc fragment of an antibody. In certain embodiments, the bi- or multispecific antibody of the invention comprises a first Fab or scFv portion that specifically binds to the extracellular part of GOLPH2 and a second Fab or scFv portion that specifically binds to another target molecule, for example to an immune checkpoint molecule.
In a particular embodiment, the invention relates to the target-binding molecule according to the invention, wherein the multispecific antibody comprises a first Fab or scFv portion specifically binding to the extracellular part of GOLPH2 and at least one further Fab or scFv portion specifically binding to an immune checkpoint molecule, a ligand of an immune checkpoint molecule and/or a molecule comprising an antigenic peptide.
Specific binding of the antibody of the invention to the extracellular part of GOLPH2 results in the maturation and thus in improved antigen presentation of antigen-presenting cells, such as Ml macrophages and/or dendritic cells, in particular matured dendritic cells. Thus, it is plausible that an immune response in a subject can be elicited or further be increased by administering a bi- or multispecific antibody that specifically binds to the extracellular part of GOLPH2 with one Fab or scFv portion and, specifically binds to an immune checkpoint molecule or a ligand of an immune checkpoint molecule, with another Fab or scFv portion.
The term "immune checkpoint molecule" is intended to include a group of proteins on the cell surface of immune cells, such as CD4+ and/or CD8+ T cells, dendritic cells, NK cells and macrophages but also on certain tumor cells, that modulate immune responses. It will be appreciated by persons skilled in the art that an immune check point protein may be either inhibitory, e.g. CTLA4 and PD-1 , or stimulatory, e.g. 0X40 and CD137. Exemplary immune checkpoint molecules include, without limitation, PD-1, CTLA4, 0X40 (CD134), CD137, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, BTLA and A2aR.
In certain embodiments, the bi- or multispecific antibody of the invention comprises a Fab or scFv portion that blocks an inhibitory check point molecule on the surface of a T cell. Examples for inhibitory check point molecules on the surface of a T cell are CTLA4, PD-1, PD-L1, LAG3 and TIM3.
In certain embodiments, the bi- or multispecific antibody comprises a first Fab or scFv portion that specifically binds to the extracellular part of GOLPH2 and a second Fab or scFv portion that specifically binds to CTLA4. Preferably, the second Fab or scFv portion binds to CTLA4 such that it blocks CTLA4 and prevents it from binding to its cognate receptor. The second Fab or scFv fragment may be any Fab or scFv that blocks CTLA4. In certain embodiments, the Fab or scFv fragment is obtained or derived from an existing antibody against CTLA4. In certain embodiments, this antibody is Ipilimumab or Tremelimumab.
In certain embodiments, the bi- or multispecific antibody comprises a first Fab or scFv portion that specifically binds to the extracellular part of GOLPH2 and a second Fab or scFv portion that specifically binds to PD-1. Preferably, the second Fab or scFv portion binds to PD-1 such that it blocks PD-1 and prevents it from binding to its cognate receptor. The second Fab or scFv fragment may be any Fab or scFv that blocks PD-1. In certain embodiments, the Fab or scFv fragment is obtained or derived from an existing antibody against PD-1. In certain embodiments, this antibody is Nivolumab. Pembrolizumab, Cemiplimab or Spartalizumab.
In other embodiments, the bi- or multispecific antibody of the invention comprises a Fab or scFv portion that blocks a ligand of an inhibitory check point molecule on the surface of an antigen presenting cell. Examples for ligands of inhibitory check point molecules on the surface of an antigen presenting cell are CD80 (ligand of CTLA4 and/or PD-L1), CD86 (ligand of CTLA4), PD-L1 (ligand of PD-1), PD-L2 (ligand of PD-1) and GAL9 (ligand of TIM3). The multispecific antibody of the invention may bind to the extracellular part of GOLPH2 and a ligand of an inhibitory check point molecule on the same antigen presenting cell or on different antigen presenting cells.
In certain embodiments, the bi- or multispecific antibody comprises a first Fab or scFv portion that specifically binds to the extracellular part of GOLPH2 and a second Fab or scFv portion that specifically binds to PD-L1. Preferably, the second Fab or scFv portion binds to PD-L1 such that it blocks the binding of PD-L1 to its receptor PD-1. The second Fab or scFv fragment may be any Fab or scFv that blocks the binding of PD-L1 to the receptor PD-1. In certain embodiments, the Fab or scFv fragment is obtained or derived from an existing antibody against PD-L1. In certain embodiments, this antibody is Atezolizumab, Durvalumab or Avelumab.
In further embodiments, the bi- or multispecific antibody of the invention comprises a Fab or scFv portion that activates a stimulatory check point molecule on the surface of a T cell. Examples for stimulatory check point molecules on the surface of a T cell are CD28, ICOS, SLAM, CD2, CD27, 0X40, 4- IBB, CD30, GITR, CD40L, DR3, CD 122 and LIGHT.
In certain embodiments, the bi- or multispecific antibody comprises a first Fab or scFv portion that specifically binds to the extracellular part of GOLPH2 and a second Fab or scFv portion that specifically binds to 4-1BB. Preferably, the second Fab or scFv portion binds to 4-1BB such that it activates 4- IBB. The second Fab or scFv fragment may be any Fab or scFv that activates 4- IBB. In certain embodiments, the Fab or scFv fragment is obtained or derived from an existing antibody against 4-1BB. In certain embodiments, this antibody is Urelumab or Utomilumab.
Thus, in a particular embodiment, the invention relates to the target-binding molecule according to the invention, wherein the immune checkpoint molecule is selected from a group consisting of: CTLA4, PD-1, PD-L1, LAG3, TIM3, CD28, ICOS, SLAM, CD2, CD27, 0X40, 4-1BB, CD30, GITR, CD40L, DR3, CD 122 and LIGHT; and/or wherein the ligand of the immune checkpoint molecule is selected from a group consisting of: CD80, CD86, PD-L1, PD-L2 and GAL9.
In certain embodiments, the invention relates to a bispecific antibody, wherein the antibody comprises a first Fab portion that specifically binds to the extracellular part of GOLPH2 and a second Fab portion that specifically binds to a check point molecule, in particular wherein the check point molecule is PD-1 or CTLA4. In certain embodiments, the invention relates to a bispecific antibody, wherein the antibody comprises a first Fab portion that specifically bind to the extracellular part of GOLPH2 and a second Fab portion that specifically binds to a ligand of a check point molecule, in particular wherein the ligand of the check point molecule is PD-L1.
In certain embodiments, the invention relates to a bispecific chimeric antibody in which two different scFv portions are fused to an Fc region of an antibody. In certain embodiments, the invention relates to a bispecific chimeric antibody, wherein the antibody comprises a first scFv portion that specifically bind to the extracellular part of GOLPH2 and a second scFv portion that specifically binds to a check point molecule, in particular wherein the check point molecule is PD-1 or CTLA4. In certain embodiments, the invention relates to a bispecific chimeric antibody, wherein the antibody comprises a first scFv portion that specifically bind to the extracellular part of GOLPH2 and a second scFv portion that specifically binds to a ligand of a check point molecule, in particular wherein the ligand of the check point molecule is PD-L1.
In a particular embodiment, the invention relates to an antibody-antigenic peptide complex comprising the target-binding molecule according to the invention and a molecule comprising an antigenic peptide.
In certain embodiments, the target-binding molecule of the invention may be complexed with an antigenic peptide. That is, the target-binding molecule of the invention may be a bi- or multispecific antibody comprising a first Fab or scFv portion specifically binding to the extracellular part of GOLPH2 and a second Fab or scFv portion specifically binding to a molecule comprising an antigenic peptide.
The molecule comprising the antigenic peptide may be any molecule that can be specifically bound by a Fab or scFv portion of a bi- or multispecific antibody of the invention. Preferably, the molecule comprising the antigenic peptide is a polypeptide, wherein the polypeptide comprises an epitope that is specifically bound by a Fab or scFv portion comprised in the bi- or multispecific antibody of the invention. Thus, in a particular embodiment, the invention relates to the antibody-antigenic peptide complex according to the invention, wherein the molecule comprising the antigenic peptide comprises an epitope that is specifically bound by a Fab or scFv portion of the target-binding molecule.
In certain embodiments, the molecule comprising the antigenic peptide consists of the antigenic peptide. That is, the antigenic peptide may be specifically bound by the Fab or scFv portion of the bi- or multispecific antibody of the invention. In other embodiments, the antigenic peptide may be fused to another polypeptide that is specifically bound by the Fab or scFv portion of the bi- or multispecific antibody of the invention. Thus, in a particular embodiment, the invention relates to the antibody- antigenic peptide complex according to the invention, wherein the molecule comprising the antigenic peptide is a fusion protein comprising the antigenic peptide fused to a polypeptide comprising an epitope that is specifically bound by the Fab or scFv portion of the target-binding molecule.
The antigenic peptide may be fused to any polypeptide that is specifically bound by the Fab or scFv portion of the bi- or multispecific antibody of the invention. Preferably, the polypeptide that is fused to the antigenic peptide is an intracellular protein or a fragment of an intracellular protein. Without being bound to theory, it is believed that fusing the antigenic peptide to an intracellular protein, or a fragment thereof, reduces the risk of cross reactivity of the second Fab or scFv portion of the bi- or multispecific antibody of the invention with proteins present on the surface of other cells.
In a particular embodiment, the invention relates to the antibody-antigenic peptide complex according to the invention, wherein the antigenic peptide is fused to a polypeptide comprising an epitope that is specifically bound by the Fab or scFv portion of the target-binding molecule via a peptide linker.
That is, the antigenic peptide may be bound to the polypeptide comprising the epitope that is specifically bound by the Fab or scFv portion of the bi- or multispecific antibody of the invention either directly or via a peptide linker. The term “peptide linker”, as used herein, refers to a peptide comprised of amino acids. A range of suitable peptide linkers will be known to the person of skill in the art. In one embodiment, the peptide linker is 50 amino acids in length or less, for example 20 amino acids or less, such as about 15 amino acids or less, such as about 10 amino acids or less. In certain embodiments, the linker is rich in glycine and serine amino acids. That is, in certain embodiments, the linker comprises at least one, at least two, at least three, at least four or at lest five glycine or serine amino acids. In certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or all amino acids in the linker are glycine or serine amino acids. In certain embodiments, the linker is glycyl-serine (GS).
It is to be noted that the molecule comprising the antigenic peptide may comprise more than one antigenic peptide. That is, the molecule comprising the antigenic peptide may comprise 1, 2, 3, 4, 5, 6, 7, 8 ,9, 10 or more than 10 antigenic peptides. The more than one antigenic peptide may be separated by peptide linkers of may be directly linked to each other.
As described above, the polypeptide comprising the epitope that is specifically bound by the Fab or the scFv portion of the bi- or multispecific antibody of the invention may be any polypeptide, in particular any intracellular polypeptide or fragment thereof. In other embodiments, the polypeptide comprising the epitope that is specifically bound by the Fab or the scFv portion of the bi- or multispecific antibody of the invention may be a synthetic polypeptide.
In certain embodiments, the epitope that is specifically bound by the Fab or the scFv portion of the bi- or multispecific antibody of the invention may be a peptide tag. Numerous peptide tags have been disclosed in the art that can be specifically bound by a Fab and/or scFv portion. Non-limiting examples of peptide tags that may be comprised in a polypeptide that is specifically bound by a Fab or scFv portion include Myc-tag, HA-tag, FLAG-tag and V5-tag. A “peptide tag” as used herein may be any peptide, in particular any peptide that can be specifically bound by a Fab or scFv portion.
It has been shown by the inventors that the fragment EQKLISEEDL (SEQ ID NO:28) of the human Myc protein can be fused to an antigenic peptide and that the resulting fusion protein can form a complex with a multispecific antibody comprising a first Fab or scFv portion specifically binding to the extracellular part of GOLPH2 and a second Fab or scFv portion specifically binding to an epitope comprised within the Myc fragment. Thus, in a particular embodiment, the invention relates to the antibody-antigenic peptide complex according to the invention, wherein the polypeptide comprising an epitope that is specifically bound by the Fab or scFv portion of the target-binding molecule is Myc or a Myc fragment.
Thus, in certain embodiments, the bi- or multispecific antibody of the invention comprises a Fab or scFv portion that specifically binds to an epitope within the amino acid sequence EQKLISEEDL (SEQ ID NO:28) of the human Myc protein. Preferably, the Fab or scFv portion specifically binding to Myc comprises a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO:32 or 40; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO:36.
In certain embodiments, the Fab or scFv portion specifically binding to Myc comprises a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NO:30, CDR2 as defined in SEQ ID NO:31 and CDR3 as defined in SEQ ID NO:32 or 42; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO:34, CDR2 as defined in SEQ ID NO:35 and CDR3 as defined in SEQ ID NO: 36.
In certain embodiments, the scFv portion specifically binding to Myc comprises the amino acid sequence of SEQ ID NO:39 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:39.
In another embodiment, the scFv portion specifically binding to Myc is a humanized form of the scFv comprising the amino acid sequence of SEQ ID NO: 39. That is, the scFv portion specifically binding to Myc may be derived from a human framework region comprising a heavy chain CDR1 as defined in SEQ ID NO:30, a heavy chain CDR2 as defined in SEQ ID NO:31 and a heavy chain CDR3 as defined in SEQ ID NO:32 or 40; and a light chain CDR1 as defined in SEQ ID NO:34, a light chain CDR2 as defined in SEQ ID NO:35 and a light chain CDR3 as defined in SEQ ID NO:36
In certain embodiments, the Fab fragments specifically binding to Myc comprises a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO:29, or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 29, and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO:33, or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:33.
In another embodiment, the Fab fragment specifically binding to Myc is a humanized Fab fragment. That is, the humanized Fab fragment specifically binding to Myc may comprise a human framework region comprising a heavy chain CDR1 as defined in SEQ ID NO:30, a heavy chain CDR2 as defined in SEQ ID NO:31 and a heavy chain CDR3 as defined in SEQ ID NO:32 or 40; and a light chain CDR1 as defined in SEQ ID NO:34, a light chain CDR2 as defined in SEQ ID NO:35 and a light chain CDR3 as defined in SEQ ID NO: 36.
In a particular embodiment, the invention relates to an antibody-antigenic peptide construct comprising an antibody, or a target-binding fragment thereof, specifically binding to GOLPH2 and an antigenic peptide, wherein the antigenic peptide is: a) fused to a C-terminal end of the antibody, or the target-binding fragment thereof; and/or b) comprised in a molecule that is specifically bound by a Fab or scFv portion comprised in the antibody, or the target-binding fragment thereof.
That is, the invention relates to an antibody that specifically binds to GOLPH2, in particular the extracellular part of GOLPH2, and further comprises an antigenic peptide.
The antibody may be any antibody that binds to GOLPH2, in particular to the extracellular part of GOLPH2. The antibody may be any type of antibody described elsewhere herein, in particular a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody, a bispecific antibody or a multispecific antibody. Thus, in a particular embodiment, the invention relates to an antibody-antigenic peptide construct according to the invention, wherein the antibody is a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody or a multispecific antibody.
In certain embodiments, the antibody may be any antibody binding to an epitope within the amino acid sequences SEQ ID NO: 23 to 27. In certain embodiments, the antibody may be or may be derived from the antibodies G2-2, G2-4, PA5-18100 or EPR3606 as described herein.
The antigenic peptide may be attached to the antibody in different ways. For example, in certain embodiments, the antigenic peptide may be fused to the C-terminal end of an antibody binding specifically to GOLPH2. The antigenic peptide may be fused to an antibody binding specifically to GOLPH2 as described elsewhere herein. That is, in a particular embodiment, the invention relates to an antibody-antigenic peptide construct according to the invention, wherein the antigenic peptide is fused to a C-terminal end of the antibody or the target-binding fragment thereof via a peptide linker.
In a particular embodiment, the invention relates to an antibody-antigenic peptide construct according to the invention, wherein the antigenic peptide is fused to the C-terminal end of a heavy chain and/or light chain of an antibody.
Alternatively, or in addition, the antibody of the invention may be complexed with a molecule comprising an antigenic peptide. That is, the antibody may be a bi- or multispecific antibody comprising a Fab or scFv portion that specifically binds to the molecule comprising the antigenic peptide. Thus, in a particular embodiment, the invention relates to the antibody-antigenic peptide construct according to the invention, wherein the antibody is a multispecific antibody.
In a particular embodiment, the invention relates to the antibody-antigenic peptide construct according to the invention, wherein the multispecific antibody comprises a first Fab or scFv portion specifically binding to GOLPH2 and a second Fab or scFv portion specifically binding to a molecule comprising an antigenic peptide.
That is, the antibody of the invention may be a bi- or multispecific antibody, wherein the bi- or multispecific antibody specifically binds to at least two different targets, in particular the extracellular part of GOLPH2 and a molecule comprising an antigenic peptide.
In certain embodiments, the molecule comprising the antigenic peptide is the entire antigen the antigenic peptide has been derived from. That is, the antigen is specifically bound by a Fab or scFv portion comprised in the bi- or multispecific antibody. In other embodiments, the molecule comprising the antigenic peptide is the antigenic peptide. That is, the antigenic peptide is specifically bound by a Fab or scFv portion comprised in the bi- or multispecific antibody. In other embodiments, the molecule comprising the antigenic peptide is a fusion protein comprising an antigen or an antigenic peptide fused to a second polypeptide, wherein the second polypeptide comprises an epitope that is specifically bound by a Fab or scFv portion comprised in the bi- or multispecific antibody. Thus, in a particular embodiment, the invention relates to the antibody-antigenic peptide construct according to the invention, wherein the molecule comprising the antigenic peptide is a fusion protein comprising the antigenic peptide fused to a polypeptide comprising an epitope that is specifically bound by the second Fab or scFv portion.
In a particular embodiment, the invention relates to the antibody-antigenic peptide construct according to the invention, wherein the antigenic peptide is fused to the polypeptide comprising the epitope that is specifically bound by the second Fab or scFv portion via a peptide linker. That is, the antigenic peptide and the polypeptide comprising the epitope that is specifically bound by the Fab or scFv portion of the bi- or multispecific antibody of the invention may be fused directly or may be fused by a peptide linker, as described elsewhere herein.
In a particular embodiments the invention relates to the antibody-antigenic peptide construct according to the invention, wherein the polypeptide comprising the epitope that is specifically bound by the second Fab or scFv portion is Myc or a Myc fragment and wherein the second Fab or scFv portion specifically binds to an epitope within Myc or said Myc fragment.
As described elsewhere herein, the polypeptide comprising the epitope that is specifically bound by the Fab or scFv portion of the antibody may have any origin. That is, the polypeptide may be a synthetic polypeptide or may be a naturally occurring polypeptide. The polypeptide comprising the epitope that is specifically bound by the Fab or scFv portion of the antibody is not limiting and may be any polypeptide that is specifically bound by the Fab or scFv porion of the bi- or multispecific antibody. In certain embodiments, the polypeptide comprising the epitope that is specifically bound by the second Fab or scFv portion is a fragment of an intracellular protein. In certain embodiments, the polypeptide comprising the epitope that is specifically bound by the second Fab or scFv portion comprises a peptide tag, such as a Myc-tag, an HA-tag, a FLAG-tag or a V5-tag. In other embodiments, the polypeptide comprising the epitope that is specifically bound by the second Fab or scFv portion is a fragment of Myc protein, in particular the fragment EQKLISEEDL (SEQ ID NO:28). Variable regions of Fab portions or scFv portions that specifically bind to Myc are disclosed herein.
In a particular embodiment, the invention relates to the antibody-antigenic peptide construct according to the invention, wherein the multispecific antibody comprises a further Fab or scFv portion specifically binding to an immune checkpoint molecule or a ligand of an immune checkpoint molecule.
In a particular embodiment, the invention relates to the antibody-antigenic peptide construct according to the invention, wherein the immune checkpoint molecule is selected from a group consisting of: CTLA4, PD-1, PD-L1, LAG3, TIM3, CD28, ICOS, SLAM, CD2, CD27, 0X40, 4-1BB, CD30, GITR, CD40L, DR3, CD 122, LIGHT, TIGIT, VISTA, B7-H3 and BTLA; and/or wherein the ligand of the immune checkpoint molecule is selected from a group consisting of: CD80, CD86, PD-L1, PD-L2 and GAL9. That is, in certain embodiments, the antibody-antigenic peptide construct according to the invention may be a bispecific antibody comprising a first Fab or scFv portion binding specifically to the extracellular part of GOLPH2. In addition, the antibody-antigenic peptide construct may comprise a second Fab or scFv portion binding specifically to a molecule comprising an antigenic peptide. Optionally or in addition, the antibody-antigenic peptide construct may comprise at least one further antigenic peptide fused to a C-terminal end of the antibody. That is, the antibody-antigenic peptide construct may comprise two or more antigenic peptides, wherein at least one antigenic peptide is comprised in a molecule that is specifically bound by a Fab or scFv portion of the antibody and at least one antigenic peptide is fused to a C-terminal end of the antibody. The at least one antigenic peptide may be fused to the C-terminal end of the first light chain of the antibody and/or the C-terminal end of the second light chain of the antibody and/or the C-terminal end of the first heavy chain of the antibody and/or the C-terminal end of the second heavy chain of the antibody. That is, the antibody- antigenic peptide construct may be a bispecific antibody comprising antigenic peptides fused to up to four C-terminal ends of the antibody and, additionally, at least one antigenic peptide comprised in a molecule that is specifically bound by a Fab or scFv portion of the bispecific antibody. The two or more antigenic peptides comprised in the antibody-antigenic peptide construct may be identical or may differ in their amino acid sequence.
In other embodiments, the antibody-antigenic peptide construct according to the invention may be a bispecific antibody comprising a first Fab or scFv portion binding specifically to the extracellular part of GOLPH2. In addition, the antibody-antigenic peptide construct may comprise a second Fab or scFv portion binding specifically to an immune checkpoint molecule, in particular any one of the immune checkpoint molecules disclosed herein. In this case, the antibody-antigenic peptide construct may comprise at least one antigenic peptide fused to a C-terminal end of the antibody. The at least one antigenic peptide may be fused to the C-terminal end of the first light chain of the antibody and/or the C-terminal end of the second light chain of the antibody and/or the C-terminal end of the first heavy chain of the antibody and/or the C-terminal end of the second heavy chain of the antibody. That is, the bispecific antibody may be a bispecific antibody comprising up to four antigenic peptides fused to the C-terminal ends of the antibody. In cases where the antibody-antigenic peptide construct comprises more than one antigenic peptide, the two or more antigenic peptides may be identical or may differ in their amino acid sequence.
The bispecific antibody described above may have any format. In particular, the antibody may comprise an Fc region and two Fab portions, wherein the first Fab portion binds specifically to the extracellular part of GOLPH2 and the second Fab portion binds specifically to an immune checkpoint molecule or to a molecule comprising an antigenic peptide. Alternatively, the antibody may comprise an Fc region and two scFv portions, wherein the first scFv portion binds specifically to the extracellular part of GOLPH2 and the second scFv portion binds specifically to an immune checkpoint molecule or to a molecule comprising an antigenic peptide.
The antibody-antigenic peptide construct of the invention may further comprise a tri- or tetraspecific antibody. In particular, the tri- or tetraspecific antibody may be based on any of the bispecific antibodies disclosed above and additionally comprise one or two scFv portions fused to the Fc region of the bispecific antibody, in particular to the C-terminal end(s) of one or both heavy chains. Preferably, the one or two scFv portions fused to the C-terminal end(s) of the Fc-region specifically bind to a molecule comprising an antigenic peptide. In embodiments where the two scFv portions are fused to the C-terminal end of each heavy chain of the antibody, the two scFv portions may be identical, e.g. bind to the same epitope, or may differ from each other, e.g. bind to different epitopes. Thus, in certain embodiments, the tri- or tetraspecific antibody of the invention comprises an Fc region, two Fab portions and one or two scFv portions, wherein the two scFv portions are preferably fused to the C-terminal end of the Fc region. In other embodiments, the tri- or tetraspecific antibody of the invention comprises an Fc region and three or four scFv portions, wherein two scFv portions are fused to the N-terminal end of the Fc region and one or two scFv portions are fused to the C-terminal end of the Fc region.
The antibody-antigenic peptide construct disclosed above comprises at least one Fab or scFv portion binding specifically to the extracellular part of GOLPH 2. In certain embodiments, at least one Fab or scFv portion specifically binds to an epitope within the peptide sequences SEQ ID NO:23 to 27 comprised in the extracellular part of GOLPH2. In certain embodiments, at least on Fab or scFv portion comprised in the antibody-antigenic peptide construct is derived from antibodies G2-2, G2- 2opti and/or G2-4.
That is, in a particular embodiment, the invention relates to the antibody-antigenic peptide construct according to the invention, wherein antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO.9; or (b) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO.17.
In a further embodiment, the invention relates to the antibody-antigenic peptide construct according to the invention, wherein the antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NON, CDR2 as defined in SEQ ID NO:5 and CDR3 as defined in SEQ ID N0:6; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO.7, CDR2 as defined in SEQ ID NO: 8 and CDR3 as defined in SEQ ID NO:9; or (b) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NO: 12, CDR2 as defined in SEQ ID NO: 13 and CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO: 15, CDR2 as defined in SEQ ID NO: 16 and CDR3 as defined in SEQ ID NO: 17.
It has to be noted that the invention further encompasses humanized antibodies comprising the CDR sequences disclosed herein.
In a further embodiment, the invention relates to the antibody-antigenic peptide construct according to the invention, wherein the antibody, or the target-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 18 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:2 or SEQ ID NO: 18; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 19 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:3 or SEQ ID NO: 19; or (b) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO: 10 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 10; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 11.
In certain embodiments disclosed herein, the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct according to the invention comprises one or more antigenic peptides. The term "antigenic peptide" as used herein refers to a peptide, which is prone to induce/elicit, increase, prolong or maintain an immune response in a subject to whom it is administered. In particular, an antigenic peptide may be a peptide that is specifically recognized by a B- or T-cell of the subject the antigenic peptide is administered to. Antigenic peptides are preferably peptides that can be presented by an MHC molecule on the cell surface of an antigen-presenting cell. In particular, antigenic peptides are peptides that can be displayed by an MHC class I or MHC class II molecules on the cell surface of an antigen-presenting cell. Peptides that are specifically bound by MHC class I molecules preferably have a size of 8 to 10 amino acids in length. Peptides that are specifically bound by MHC class II molecules preferably have a size of 15 to 24 amino acids in length.
The antigenic peptide of the invention may be of any origin. However, it is preferred that the antigenic peptide is of mammalian, in particular human, fungal, bacterial or viral origin. In certain embodiments, the antigenic peptide is a tumor antigen or is derived from a tumor antigen. As used herein, the term "tumor antigen" comprises tumor-specific antigens and tumor- associated antigens. In general, the term "tumor antigen" or "tumor protein" designates herein an antigenic substance produced in tumor cells, and sometimes also in normal cells, and which can trigger an immune response upon administration in a subject. In humans, those have been classified according to their expression pattern, function or genetic origin, and include without limitation, overexpressed selfantigens (such as HER2/neu and its variant dHER2, p53, Wilm's Tumor 1 , Ephrin receptor, Proteinase-3, Mucin-1 , Mesothelin, EGFR, CD20); cancer-testis (CT) antigens (such as MAGE-1, BAGE, GAGE, NY-ESO-1); mutational antigens, also known as neo-antigens (such as mutants from MUM-1 , bcr-abl, ras, b-raf, p53, CDK-4, CDC27, beta-catenin, alpha-actenin-4); tissue-specific differentiation antigens (such as the melanoma antigens Melan A/MART-1, tyrosinase, TRPl/pg75, TRP2, gplOO and gangliosides GM3, GM2, GD2 and GD3; the prostate cancer antigens PSMA, PSA and PAP); viral antigens which are expressed by oncoviruses (such as HPV, EBV); oncofetal antigens (such as alpha-fetoprotein AFP and carcinoembryonic antigen CEA); and universal antigens (telomerase, hTERT, survivin, mdm-2, CYP-1 Bl) (Srinivasan and Wolchok, Tumor antigens for cancer immunotherapy: therapeutic potential of xenogeneic DNA vaccines. J Transl Med. 2004 Apr 1 6;2(1): 12). Accordingly, human tumor antigens are well-known in the art. For instance, the Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2 or IL13RA2) is a membrane bound protein that in humans is encoded by the IL13RA2 gene. In a non-exhaustive manner, IL13RA2 has been reported as a potential immunotherapy target (see Beard et al.; Clin Cancer Res; 72(11); 2012). The high expression of IL13RA2 has further been associated with invasion, liver metastasis and poor prognosis in colorectal cancer (Barderas et al.; Cancer Res; 72(11); 2012).
In certain embodiments, the tumor antigen is selected from the group consisting of 5-a-reductase, a- fetoprotein (“AFP”), AM-1, APC, April, B melanoma antigen gene (“BAGE”), -catenin, Bel 12, bcr- abl, Brachyury, CA-125, caspase-8 (“CASP-8”, also known as “FLICE”), Cathepsins, CD 19, CD20, CD21 /complement receptor 2 (“CR2”), CD22/BL-CAM, CD23/FcaRII, CD33, CD35/complement receptor 1 (“CR1”), CD44/PGP-1, CD45/leucocyte common antigen (“LCA”), CD46/membrane cofactor protein (“MCP”), CD52/CAMPATH-1, CD55/decay accelerating factor (“DAF”), CD59/protectin, CDC27, CDK4, carcinoembryonic antigen (“CEA”), c-myc, cyclooxygenase-2 (“cox- 2”), deleted in colorectal cancer gene (“DCC”), DcR3, E6/E7, CGFR, EMBP, Dna78, famesyl transferase, fibroblast growth factor-8a (“FGF8a”), fibroblast growth factor-8b (“FGF8b”), FLK- 1/KDR, folic acid receptor, G250, G melanoma antigen gene family (“GAGE-family”), gastrin 17, gastrin-releasing hormone, ganglioside 2 (“GD2”)/ganglioside 3 (“GD3”)/ganglioside-monosialic acid-2 (“GM2”), gonadotropin releasing hormone (“GnRH”), UDP-GlcNAc:RlMan(al-6)R2 [GlcNAc to Man(al-6)] pi,6-N-acetylglucosaminyltransferase V (“GnT V”), GP1, gpl00/Pmel l7, gp-100-in4, gpl5, gp75/tyrosine-related protein-1 (“gp75/TRP-l”), human chorionic gonadotropin (“hCG”), heparanase, Her2/neu, human mammary tumor virus (“HMTV”), 70 kiloDalton heat-shock protein (“HSP70”), human telomerase reverse transcriptase (“hTERT”), insulin-like growth factor receptor- 1 (“IGFR-1”
), interleukin- 13 receptor (“IL-13R”), inducible nitric oxide synthase (“iNOS”), Ki67, KIAA0205, K- ras, H-ras, N-ras, KSA, LKLR-FUT, melanoma antigen-encoding gene 1 (“MAGE-1”), melanoma antigen-encoding gene 2 (“MAGE-2”), melanoma antigen-encoding gene 3 (“MAGE-3”), melanoma antigen-encoding gene 4 (“MAGE-4”), mammaglobin, MAP 17, Melan-A/melanoma antigen recognized by T-cells- 1 (“MART-1”), mesothelin, MIC A/B, MT-MMPs, mucin, testes-specific antigen NY-ESO-1, osteonectin, pl5, P170/MDR1, p53, p97/melanotransferrin, PAI-1, platelet- derived growth factor (“PDGF”), pPA, PRAME, probasin, progenipoietin, prostate-specific antigen (“PSA”), prostate-specific membrane antigen (“PSMA”), prostatic acid phosphatase (“PAP”), RAGE- 1, Rb, RCAS1, SART-1, SSX-family, STAT3, STn, TAG-72, transforming growth factor-alpha (“TGF-a”), transforming growth factor-beta (“TGF-P”), Thymosin-beta- 15, tumor necrosis factoralpha (“TNF-a”), TP1, TRP-2, tyrosinase, vascular endothelial growth factor (“VEGF”), ZAG, pl6INK4, and glutathione-S-transferase (“GST”).
In certain embodiments, the antigenic peptide is a viral antigen or is derived from a viral antigen. The term "viral antigen", as used herein, refers to any viral component having antigenic properties, i.e. being able to provoke an immune response in an individual. The viral antigen may be derived from a viral ribonucleoprotein or an envelope protein. In certain embodiments, the viral antigen is derived from a virus selected from the group consisting of adenovirus, Arbovirus, Astrovirus, Coronavirus, Coxsackievirus, Crimean-Congo hemorrhagic fever virus, cytomegalovirus (“CMV”), dengue virus, Ebola virus, Epstein-Barr virus (“EBV”), Foot-and-mouth disease virus, Guanarito virus, Hendra virus, herpes simplex virus-type 1 (“HSV-1”), herpes simplex virus-type 2 (“HSV-2”), human herpesvirus-type 6 (“HHV-6”), human herpesvirus-type 8 (“HHV-8”), hepatitis A virus (“HAV”), hepatitis B virus (“HBV”), hepatitis C virus (“HCV”), hepatitis D virus (“HDV”), hepatitis E virus (“HEV”), human immunodeficiency virus (“HIV”), influenza virus, Japanese encephalitis virus, Junin virus, Lassa virus, Machupo virus, Marburg virus, measles virus, human metapneumovirus, Molluscum contagiosum virus, mumps virus, Newcastle disease virus, Nipha virus, Norovirus, Norwalk virus, human papillomavirus (“HPV”), parainfluenza virus, parvovirus, poliovirus, rabies virus, respiratory syncytial virus (“RSV”), rhinovirus, rotavirus, rubella virus, Sabia virus, severe acute respiratory syndrome virus (“SARS”), varicella zoster virus, variola virus, West Nile virus, and yellow fever virus. In certain embodiments, the antigenic peptide is a microbial antigen or is derived from a microbial antigen. The term "microbial antigen", as used herein, refers to any microbial component having antigenic properties, i.e. being able to provoke an immune response in an individual.
In certain embodiments, the antigenic peptide is a bacterial antigen or is derived from a bacterial antigen. The term "bacterial antigen", as used herein, refers to any bacterial component having antigenic properties, i.e. being able to provoke an immune response in an individual. The bacterial antigen may be derived from the cell wall or cytoplasm membrane of a bacterium. In certain embodiments, the bacterial antigen is derived from a bacterium selected from the group consisting of Bacillus anthracis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Burkholderia mallei, Burkholderia pseudomallei, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diptheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli, Escherichia coli) 157:H7, Francisella tularensis, Haemophilus influenza, Helicobacter pylon, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitides, Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae, and Yersinia pestis.
In certain embodiments, the antigenic peptide is a fungal antigen or is derived from a fungal antigen. The term "fungal antigen", as used herein, refers to any fungal component having antigenic properties, i.e. being able to provoke an immune response in an individual. In certain embodiments, the fungal antigen is derived from a fungus selected from the group consisting of Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Blastomyces dermatitidis, Candida albicans, Candida dubliniensis, Candida glabrata, Candida parapsilosis, Candida rugosa, Candida tropicalis, Cryptococcus albidus, Cryptococcus gattii, Cryptococcus laurentii, Cryptococcus neoformans, Histoplasma capsulatum, Microsporum canis, Pneumocystis carinii, Pneumocystis jirovecii, Sporothrix schenckii, Stachbotrys chartarum, Tinea barbae, Tinea captitis, Tinea corporis, Tinea cruris, Tinea faciei, Tinea incognito, Tinea nigra, Tinea versicolor, Trichophyton rubrum and Trichophyton tonsurans.
In certain embodiments, the antigenic peptide is a zooparasite antigen or is derived from a zooparasite antigen. The term "zooparasite antigen", as used herein, refers to any component of a parasite of an animal having antigenic properties, i.e. being able to provoke an immune response in an individual. Said parasite may be a flea, louse, or worm. In certain embodiments, the zooparasite antigen is derived from a parasite selected from the group consisting of Anisakis spp. Babesia spp., Baylisascaris procyonis, Cryptosporidium spp., Cyclospora cayetanensis, Diphyllobothrium spp., Dracunculus medinensis, Entamoeba histolytica, Giardia duodenalis, Giardia intestinalis, Giardia lamblia, Leishmania sp., Plasmodium falciparum, Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Taenia spp., Toxoplasma gondii, Trichinella spiralis, and Trypanosoma cruzi.
It is to be understood that an antigenic peptide is a fragment or a derivative of an antigen. Within the present invention, an antigen is a naturally occurring molecule, preferably a protein. Antigens may be taken up by antigen-presenting cells, such as Ml macrophages or matured dendritic cells, processed and presented on the surface of the antigen-presenting cell by an MHC molecule. Thus, in a particular embodiment, the antigenic peptide of the invention may be any fragment or derivative of an antigen that can be displayed by an MHC class I or MHC class II molecule.
When the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct of the invention is said to comprise an antigenic peptide, it is to be understood that the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct may comprise an entire antigen, a fragment of said antigen or only an antigenic peptide derived from that antigen, e.g. a peptide that can be presented by an MHC molecule.
In certain embodiments, the antigenic peptide may be derived from Human papillomavirus type 16 (HPV-16). In particular, the antigenic peptide may be derived from the E7 protein of HPV-16. In particular, the antigenic peptide derived from the E7 protein of HPV-17 may have the sequence PTLHEYMLDLQPE (SEQ ID NO:54) or RAHYNIVTF (SEQ ID NO:55).
In certain embodiments, the antigenic peptide may be derived from Influenza virus. In particular, the antigenic peptide may be derived from the Ml protein of Influenza virus. In certain embodiments, the antigenic peptide derived from the Ml protein of Influenza virus may have the sequence GILGFVFTLT (SEQ ID NO: 56)
In certain embodiments, the antigenic peptide may be derived from SARS coronavirus 2. In particular, the antigenic peptide may be derived from the nucleocapsid phosphoprotein of SARS coronavirus 2. In certain embodiments, the antigenic peptide derived from the nucleocapsid phosphoprotein of SARS coronavirus 2 may have the sequence LLLLDRLNQLESKMS (SEQ ID NO: 57). Alternatively, the antigenic peptide may be derived from the ORF7a protein of SARS coronavirus 2. In certain embodiments, the antigenic peptide derived from ORF7a of SARS coronavirus 2 may have the sequence VYQLRARSV (SEQ ID NO: 58) or ITLCFTLKR (SEQ ID NO: 59)
In certain embodiments, the antigenic peptide may be derived from Epstein-Barr virus (human gammahervesvirus 4, EBV). In particular, the antigenic peptide may be derived from the latent membrane protein2 (LMP2A) of EBV. In certain embodiments, the antigenic peptide derived from LMP2A of EBV may have the sequence CLGGLLTMV (SEQ ID NO: 60). Alternatively, the antigenic peptide may be derived from the protein BSLF2/BMFL1 of EBV. In certain embodiments, the antigenic peptide derived from BSLF2/BMFL1 of EBV may have the sequence GLCTLVAML (SEQ ID NO: 61).
In certain embodiments, the antigenic peptide may be derived from the Ovalbumin protein. In certain embodiments, the antigenic peptide derived from Ovalbumin may have the sequence SIINFEKL (SEQ ID NO: 62) or AAHAEINEA (SEQ ID NO:63).
In certain embodiments, the antigenic peptide may be a neoepitope derived from the nurine colon cancer cell line MC38. In certain embodiments, the neoepitope derived from MC38 may be comprised in the sequence LFRAAQLANDVVLQIMEHLELASMTNMELMSSIVVISASIIVFNLLELEG (SEQ ID NO:64) or HLELASMTNMELMSSIVGSKILTFDRL (SEQ ID NO:65),
In certain embodiments, the antigenic peptide may be derived from the antigen tyrosinase-related protein 2 (TRP2). In certain embodiments, the antigenic peptide derived from tyrosinase-related protein 2 may have the sequence SVYDFFVWL (SEQ ID NO: 66).
In certain embodiments, the antigenic peptide may be derived from the antigen glycoprotein 100 (gplOO). In certain embodiments, the antigenic peptide derived from gplOO may have the sequence KVPRNQDWL (SEQ ID NO: 67).
The target-binding molecule, antibody-antigenic peptide complex or antibody-antigenic peptide construct of the invention may comprise one or more entire antigens. For example, an antigen may be fused to the C-terminal end of a target-binding molecule or antibody and/or may be fused to a polypeptide comprising an epitope that specifically bound by a Fab or scFv portion of an antibody of the invention. More preferably, the target-binding molecule, antibody-antigenic peptide complex or antibody-antigenic peptide construct of the invention comprises one or more antigenic peptide(s). That is, the target-binding molecule, antibody-antigenic peptide complex or antibody-antigenic peptide construct of the invention comprises fragments or derivatives of antigens, in particular fragments or derivatives of antigens that can be displayed by an MHC class I or MHC class II molecule.
It is to be understood that two or more antigenic peptides may be combined in a polypeptide. The two or more antigenic peptides that are combined in a polypeptide may derive from the same or different protein and/or species or may derive from different proteins and/or species. Two antigenic peptides comprised in a single polypeptide may be separated by a linker, for example the linker glycyl-serine (GS).
A polypeptide comprising one or more antigenic peptides may comprise sequences that allow coupling of the polypeptide to the target-binding molecule of the invention. That is, the polypeptide may comprise an epitope that can be specifically bound by the target-binding molecule of the invention. For example, the polypeptide may comprise an epitope that can be specifically bound by a Fab or scFv fragment comprised in the target-binding molecule of the invention. In certain embodiments, the epitope may be derived from the Myc protein and have the sequence EQKLISEEDL (SEQ ID NO: 28).
In other embodiments, the polypeptide comprising one or more antigenic peptides may comprise a peptide linker for conjugating the polypeptide to a C-terminal end of the light and/or heavy chain of an antibody. In certain embodiments, the linker may comprise the sequence GFLGSGFLGS (SEQ ID NO:75).
In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g, target -binding.
In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions." More substantial changes are provided in Table 1 under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
TABLE 1
Amino acids may be grouped according to common side-chain properties:
(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;
(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro;
(6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).
Alterations (e.g., substitutions) may be made in CDRs, e.g., to improve antibody affinity. Such alterations may be made in CDR "hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR H3 and CDR-L3 in particular are often targeted.
In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in CDRs. Such alterations may be outside of CDR "hotspots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.
A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigenantibody complex is used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N- terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
In certain embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.
In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fe region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; W02002/031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lecl3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al., especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells ( see, e.g., Yamane-Ohnuki et al. Bioteeh. Bioeng. 87: 614 (2004); Kanda, Y. et al., Bioteehnol. Bioeng., 94(4):680-688 (2006); and W02003/085 107).
Antibody variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.
In certain embodiments, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII and Fc(RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Nonlimiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 ( see, e.g. Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82: 1499- 1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). However, it is preferred within the present invention that the antibody of the inventions possesses its natural effector funtions.
"Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
Alternatively, non-radioactive assays methods may be employed ( see, for example, ACTI™ non radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. sci. USA 95:652-656 (1998).
Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed ( see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al., Blood 101: 1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).
Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581). Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).
In certain embodiments, an antibody variant comprises a Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).
In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826).
See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351 concerning other examples of Fc region variants.
In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g., "thioMAbs", in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; Al 18 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine engineered antibodies may be generated as described, e.g., in U.S. Patent No. 7,521,541.
In certain embodiments, an antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Nonlimiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
In another embodiment, conjugates of an antibody and non-proteinaceous moiety that may be selectively heated by exposure to radiation are provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the non-proteinaceous moiety to a temperature at which cells proximal to the antibody- non-proteinaceous moiety are killed.
Anti-GOLPH2 antibodies provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.
In one embodiment, an antibody of the invention is tested for its antigen binding activity, e.g., by known methods such as ELISA, BIACore®, FACS, immunofluorescence or immunohistochemistry.
In another embodiment, competition assays may be used to identify an antibody that competes with any of the antibodies described herein for binding to the extracellular part of GOLPH2. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) that is bound by an antibody described herein. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).
In an exemplary competition assay, immobilized GOLPH2 is incubated in a solution comprising a first labeled antibody that binds to GOLPH2 (e.g., any of the antibodies described herein) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to GOLPH2. As a control, immobilized GOLPH2 is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to GOLPH2, excess unbound antibody is removed, and the amount of label associated with immobilized GOLPH2 is measured. If the amount of label associated with immobilized GOLPH2 is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to GOLPH2. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
In yet another embodiment, an antibody may be conjugated to a "receptor" (such as streptavidin) for utilization in tumor pre-targeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a drug or radionucleotide).
In certain embodiments, any of the anti-GOLPH2 antibodies provided herein is useful for detecting the presence of GOLPH2 in a biological sample. The term "detecting" as used herein encompasses quantitative or qualitative detection. A "biological sample" comprises, e.g., a cell or tissue (e.g., biopsy material, including cancerous or potentially cancerous lymphoid tissue, such as lymphocytes, lymphoblasts, monocytes, myelomonocytes, and mixtures thereof).
In certain embodiments, labeled anti-GOLPH2 antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron- dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction.
Exemplary labels include, but are not limited to, the radioisotopes 32P, 14C, 1251, 3H, and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3 -dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, B- galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like. In another embodiment, a label is a positron emitter. Positron emitters include but are not limited to 68Ga, 18F, 64Cu, 86Y, 76Br, 89Zr, and 1241. In a particular embodiment, a positron emitter is 89Zr.
In certain embodiments, the invention relates to a method for producing the target-binding molecule according to the invention, in particular the antibody according to the invention. That is, the present invention also relates to the production of specific antibodies against native polypeptides and recombinant polypeptides of GOLPH2. This production is based, for example, on the immunization of animals, like mice. However, also other animals for the production of antibody/antisera are envisaged within the present invention. For example, monoclonal and polyclonal antibodies can be produced by rabbit, mice, goats, donkeys and the like. The polynucleotide encoding a correspondingly chosen polypeptide of GOLPH2 can be subcloned into an appropriated vector, wherein the recombinant polypeptide is to be expressed in an organism being able for an expression, for example in bacteria. Thus, the expressed recombinant protein can be intra-peritoneally injected into a mouse and the resulting specific antibody can be, for example, obtained from the mice serum being provided by intracardiac blood puncture. The present invention also envisages the production of specific antibodies against native polypeptides and recombinant polypeptides by using a DNA vaccine strategy as exemplified in the appended examples. DNA vaccine strategies are well-known in the art and encompass liposome-mediated delivery, by gene gun or jet injection and intramuscular or intradermal injection. Thus, antibodies directed against a polypeptide or a protein or an epitope of GOLPH2, in particular the extracellular part of GOLPH2 or epitopes thereof, can be obtained by directly immunizing the animal by directly injecting intramuscularly the vector expressing the desired polypeptide or a protein or an epitope of GOLPH2, in particular the epitope or extracellular part of GOLPH2 that is recognized by the antibodies of the invention. The amount of obtained specific antibody can be quantified using an ELISA, which is also described herein below. Further methods for the production of antibodies are well known in the art, see, e.g. Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988.
Thus, under designated assay conditions, the specified antibodies and the corresponding epitope or part of GOLPH2 bind to one another and do not bind in a significant amount to other components present in a sample. Specific binding to a target analyte under such conditions may require a binding moiety that is selected for its specificity for a particular target analyte. A variety of immunoassay formats may be used to select antibodies specifically reactive with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with an analyte. See Shepherd and Dean (2000), Monoclonal Antibodies: A Practical Approach, Oxford University Press and/ or Howard and Bethell (2000), Basic Methods in Antibody Production and Characterization, Crc. Pr. Inc. for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Typically, a specific or selective reaction will be at least twice background signal to noise and more typically more than 10 to 100 times greater than background. The person skilled in the art is in a position to provide for and generate specific binding molecules directed against the novel polypeptides. For specific binding-assays it can be readily employed to avoid undesired cross-reactivity, for example polyclonal antibodies can easily be purified and selected by known methods (see Shepherd and Dean, loc. cit.).
It has to be noted that the term “anti-GOLPH2 antibody” as used herein also includes bi- or multispecific antibody that bind specifically to GOLPH2 and to one or more additional target, e.g. an immune checkpoint molecule or a molecule comprising an antigenic peptide. The term “anti-GOLPH2 antibody” also includes the mono-, bi- or multispecific antibodies of the invention that comprise one or more antigenic peptides. Thus, the target-binding molecule according to the invention may be an anti-GOLPH2 antibody. Further, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct according to the invention may comprise an anti-GOLPH2 antibody.
In a particular embodiment, the invention relates to an immunoconjugate comprising the targetbinding molecule according to the invention, the antibody-antigenic peptide complex according to the invention or the antibody-antigenic peptide construct according to the invention and a cytotoxic agent or a prodrug of a cytotoxic agent.
The target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct of the invention may be non-conjugated or may be conjugated to a cytotoxic agent or a prodrug of a cytotoxic agent. FIGs. 1 and 2 show that both an antibody binding to the extracellular part of GOLPH2, as well as different immunoconjugates of the same antibody have the potential to induce the maturation of monocytes. Accordingly, it is plausible that the fusion of a cytotoxic agent or a prodrug of a cytotoxic agent does not interfere or, at least, not significantly interferes with the function of the target-binding molecule, the antibody-antigenic peptide complex or the antibody- antigenic peptide construct of the invention.
Accordingly, the decision if the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct of invention is conjugated to a cytotoxic agent or a prodrug of a cytotoxic agent may depend on the intended use of the target-binding molecule, the antibody- antigenic peptide complex or the antibody-antigenic peptide construct. For example, if the targetbinding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct is used for inducing the maturation of monocytes and/or monocyte-derived cells in a subject suffering from a GOLPH2 -positive cancer, it may be desirable to administer the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct of the invention in the form of an immunoconjugate. In this case, the immunoconjuagte can kill GOLPH2-positive cancer cells and, at the same time, induce the maturation of monocytes and/or monocyte derived cells. If, on the other hand, the target-binding molecule, the antibody-antigenic peptide complex or the antibody- antigenic peptide construct of the invention is used as an adjuvant in a vaccination therapy of a subject, i.e. to improve the antigen presentation in said subject, it may be desirable to administer the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct of the invention in a non-conjugated form.
In a certain embodiment, the immunoconjugate is an antibody-drug conjugate. The term "antibodydrug conjugate", as used herein, refers to an antibody or Fc-containing polypeptide having specificity for at least one type of malignant cell, a drug, and a linker coupling the drug to e.g. the antibody. The linker is cleavable or non-cleavable in the presence of the malignant cell; wherein the antibody-drug conjugate kills the malignant cell. The antibody-drug conjugate may be generated chemically or enzymatically, for example with a transglutaminase or sortase enzyme.
The skilled person is aware of methods to generate immunoconjugates comprising the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct of the invention. Immunoconjugates of GOLPH2 antibodies, including potential linkers and drug moieties, have been described in detail in WO 2018/091724, which is fully incorporated by reference.
In a particular embodiment, the invention relates to a polynucleotide encoding the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct according to the invention.
The polynucleotide may be any nucleic acid sequence capable of encoding the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct according to the invention, such as single-stranded or double-stranded DNA, the sense or antisense strand of a DNA molecule, or RNA molecules, and the like. The person skilled in the art knows how to derive a polynucleotide sequence coding for a protein and how to isolate or produce such a nucleic acid sequence using standard techniques of molecular biology.
In certain embodiments, the polynucleotide encoding the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct according to the invention is an isolated nucleic acid. An "isolated nucleic acid" refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extra chromosomally or at a chromosomal location that is different from its natural chromosomal location.
In certain embodiments, the polynucleotide encoding the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct according to the invention is an isolated nucleic acid encoding an anti-GOLPH2 antibody. "Isolated nucleic acid encoding an anti-GOLPH2 antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
The polynucleotide of the invention may be included in an expression construct such as a vector, plasmid, virus/phagemid, artificial chromosome, cosmid, and further constructs known to the skilled person in order to provide for expression of the sequence of the protein of the invention. The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
Techniques for modifying nucleic acid sequences for insertion into a vector e.g. by utilizing recombinant DNA methods are also well-known in the art. Generally, an expression vector comprises the polynucleotide to be expressed, which is operably linked to one or more control sequences (e.g., promoter, transcriptional stop signal, translational stop signal, etc.) capable of directing the expression of the polypeptide in the desired host cell. The promoter may be an inducible or constitutive, general or cell specific promoter. The selection of promoters, vectors and other elements is a matter of routine design within the level of ordinary skill in the art and many different such control sequences are described in the literature and available through commercial suppliers. Generally, the choice of the vector will typically depend on the choice of the host cell into which the vector will be introduced.
The polynucleotide or expression vector may be introduced into cells by various ways, e.g., using a virus as a carrier or by transfection including e.g. by chemical transfectants (such as Metafectene, Lipofectamine, Fugene, etc.), electroporation, calcium phosphate co-precipitation and direct diffusion of DNA. Suitable transfection techniques are known to the skilled person and the method of choice will vary depending on the host cell to be transfected. Transfection of a cell may yield stable cells or cell lines, if the transfected polynucleotide or expression vector is integrated into the genome, or by using episomal replicating plasmids, i.e. that the inheritance of the extrachromosomal plasmid is controlled by control elements that are integrated into the cell genome. In addition, unstable (transient) cells or cell lines, wherein the transfected DNA exists in an extrachromosomal form can be produced.
The expression vector may further comprise a selectable marker, which provides for positive selection of transfected cells, i.e. transfected cells exhibit resistance to the selection and are able to grow, whereas non-transfected cells generally die. Examples of selective markers include puromycin, zeocin, neomycin (neo) and hygromycin B, which confer resistance to puromycin, zeocin, aminoglycoside G- 418 and hygromycin, respectively. However, other selection methods known to the skilled person may also be suitable.
In a particular embodiment, the invention relates to a cell comprising the polynucleotide according to the invention.
The invention furthermore relates to a host cell comprising the polynucleotide of the invention. The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transfected or transduced cell are included herein.
Furthermore, the invention relates to a method of producing a target-binding molecule, an antibody- antigenic peptide complex or an antibody-antigenic peptide construct, in particular an antibody, comprising culturing the host cell of the invention, wherein the host cell comprises the polynucleotide of the invention. That is, in a particular embodiment, the invention relates to a method of producing the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct according to the invention, the method comprising a step of culturing the host cell of the invention under conditions suitable to allow efficient production of the target-binding molecule of the invention.
Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567. In one embodiment, isolated nucleic acid encoding an anti-GOLPH2 antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transfected or transduced with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., YO, NSO, Sp20). In one embodiment, a method of making an anti-GOLPH2 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
For recombinant production of an anti-GOLPH2 antibody, nucleic acid encoding an antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vai. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gemgross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.
Plant cell cultures can also be utilized as hosts. See, e.g., US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).
Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are macaque kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Viral. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); macaque kidney cells (CV 1); African green macaque kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N. Y Aead. Sei. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR CHO cells (Urlaub et al., Proc. Natl. Acad, cii. USA 77:4216 (1980)); and myeloma cell lines such as YO, NSO and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vai. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).
In a particular embodiment, the invention relates to a pharmaceutical composition comprising the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct according to the invention and/or the immunoconjugate according to the invention and further comprising a pharmaceutically acceptable carrier.
The present invention furthermore relates to a pharmaceutical composition comprising the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct or the immunoconjugate of the invention and a pharmaceutically acceptable carrier.
The term "pharmaceutical formulation" or “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
Pharmaceutical formulations comprising the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate as described herein are prepared by mixing such target-binding molecule, antibody-antigenic peptide complex, antibody- antigenic peptide construct or immunoconjugate having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3- pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
Exemplary lyophilized antibody or immunoconjugate formulations are described in US Patent No. 6,267,958. Aqueous antibody or immunoconjugate formulations include those described in US Patent No. 6,171,586 and W02006/044908, the latter formulations including a histidine-acetate buffer.
In a particular embodiment, the invention relates to a pharmaceutical composition according to the invention further comprising at least one therapeutic agent.
Target-binding molecules, antibody-antigenic peptide complexes, antibody-antigenic peptide constructs or immunoconjugates of the invention may be used either alone or in combination with other agents in a therapy. For instance, a target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention may be co-administered with at least one additional therapeutic agent. Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant. Target-binding molecules, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugates of the invention can also be used in combination with radiation or laser therapy.
A target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional, intrauterine or intravesical administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or immunoconjugate, which matrices are in the form of shaped articles, e.g. films, or microcapsules. The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
Any of the target-binding molecules, antibody-antigenic peptide complexes, antibody-antigenic peptide constructs or immunoconjugates provided herein may be used in methods, e.g., therapeutic methods.
Target-binding molecules, antibody-antigenic peptide complexes, antibody-antigenic peptide constructs or immunoconjugates of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The target-binding molecule or immunoconjugate need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of target-binding molecule, antibody-antigenic peptide complex, antibody- antigenic peptide construct or immunoconjugate present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
For the prevention or treatment of disease, the appropriate dosage of a target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate, the severity and course of the disease, whether the target-binding molecule, antibody-antigenic peptide complex, antibody- antigenic peptide construct or immunoconjugate is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the target-binding molecule, antibody- antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate, and the discretion of the attending physician. The target-binding molecule, antibody-antigenic peptide complex, antibody- antigenic peptide construct or immunoconjugate is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of target-binding molecule, antibody-antigenic peptide complex, antibody- antigenic peptide construct or immunoconjugate can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate would be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the target-binding molecule). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.
It is understood that any of the above formulations or therapeutic methods may be carried out using a target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct immunoconjugate of the invention.
In another embodiment of the invention, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the disorder and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention. The label or package insert indicates that the composition is used for treating the condition of choice.
Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises a target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, fdters, needles, and syringes.
In a particular embodiment, the invention relates to a pharmaceutical composition according to the invention, wherein the therapeutic agent is at least one of a vaccine, an antigen, an adjuvant, a chemotherapeutic agent and an immune checkpoint modulator.
The pharmaceutical composition of the invention may comprise the target-binding molecule, antibody- antigenic peptide complex, antibody-antigenic peptide construct or the immunoconjugate of the invention and an additional therapeutic agent.
In certain embodiments, the pharmaceutical composition may comprise the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate according to the invention and a vaccine. The term "vaccine" as used herein refers to an antigenic composition usually comprising an infectious factor or a portion of an infectious factor, such as an antigen, preferably in combination with an immune adjuvant, administered into the body to elicit an immune response. The antigenic portion may be a microorganism such as a virus or bacterium; a natural product purified from a microorganism; or a synthetic or genetically engineered protein, peptide, polysaccharide, or similar product. In a certain embodiment, the antigenic portion of the vaccine is comprised of a T cell epitope.
In certain embodiments, the vaccine comprised in the pharmaceutical composition of the invention is a cancer vaccine. The term “cancer vaccine” refers to a vaccine that induces an immune response against a particular cancer. Cancer vaccines can be categorized as: antigen vaccines, whole cell vaccines, dendritic cell vaccines, DNA vaccines and anti-idiotype vaccines. To date, there are a few FDA licensed cancer prevention vaccines. These include (1) vaccine to protect against infection with the human papilloma virus (HPV) to prevent cervical cancer, (2) hepatitis B vaccine to protect against infection with the human Hepatitis B virus to prevent hepatocellular carcinoma, and (3) melanoma vaccine for canines.
Examples of cancer vaccines as used herein include whole tumor cells, tumor cell lysates, tumor cell derived RNAs, tumor cell derived proteins, tumor cell derived peptides, tumor cell derived carbohydrates, tumor cell derived lipids, and tumor cell derived DNA sequences. These tumor cells could be derived from a patient's own tumor or tumor from an unrelated donor. One potential advantage of cell-based vaccines is that they contain a wide range of antigens. A cancer vaccine may prevent further growth of existing cancer, protect against recurrence of treated cancer, or eliminate cancer cells not already removed by other treatments.
“Whole cell tumor vaccines”, also referred to as “whole tumor vaccines” comprise tumor cells which may be autologous or allogeneic for the patient. These cells comprise cancer antigens which can stimulate the body's immune system. As compared to the administration of individual cancer antigens, a whole cell exposes a large number of cancer specific (unique or up-regulated) antigens to the patient's immune system. This stimulation of the immune system means that the patient is better able to prevent the subsequent growth or establishment of a tumor.
Whole cell tumor vaccines typically comprise tumor cells which have been modified in vitro, e.g., irradiated and dead tumor cells are preferred in many applications, although live tumor cells may be used in the vaccine. The whole cell vaccine may comprise intact cells but a cell lysate may alternatively be used, and “whole” cell should be understood with this in mind. The use of such a lysate (or intact cell preparation) means that the vaccine will comprise in excess of 10 antigens, typically in excess of 30 antigens.
In certain embodiments, the pharmaceutical composition may comprise the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention and an antigen. The term "antigen" or "ag" as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid. In a particular embodiment, the antigen may be a tumor antigen. The term "tumor antigen" as used herein indicates a molecule (e.g. a protein or peptide) that is expressed by a tumor cell and either (a) differs qualitatively from its counterpart expressed in normal cells, or (b) is expressed at a higher level in tumor cells than in normal cells. Thus, a tumor antigen can differ from (e.g. by one or more amino acid residues where the molecule is a protein), or it can be identical to, its counterpart expressed in normal cells. Some tumor antigens are not expressed by normal cells, or are expressed at a level at least about two-fold higher (e.g. about two-fold, three-fold, five-fold, ten- fold, 20- fold, 40-fold, 100- fold, 500-fold, 1,000-fold, 5,000-fold, or 15,000-fold higher) in a tumor cell than in the tumor cell's normal counterpart.
Any suitable tumor antigen may be used in the practice of the present invention. Tumor antigens include, without limitation, naturally occurring tumor antigens and modified forms thereof that induce an immune response in a subject, and further include antigens associated with tumor cells and antigens that are specific to tumor cells and modified forms of the foregoing that induce an immune response in a subject. The term tumor antigen further encompasses antigens that correspond to proteins that are correlated with the induction of tumors such as oncogenic virus antigens (e.g., human papilloma virus antigens). Exemplary tumor antigens include, without limitation, HER2/neu and BRCA1 antigens for breast cancer, MART-l/MelanA (melanoma antigen), Fra-1 (breast cancer), NY-BR62, NY-BR85, hTERT, gplOO, tyrosinase, TRP-1, TRP-2, CDK-4, -catenin, MUM-I, Caspase-8, KIAA0205, SART-
1, PRAME, and pi 5 antigens, members of the MAGE family (melanoma antigens), the BAGE family (melanoma antigens), the DAGE/PRAME family (such as DAGE- 1), the GAGE family (melanoma antigens), the RAGE family (such as RAGE-I), the SMAGE family, NAG, TAG-72, CAI 25, mutated proto-oncogenes such as p21ras, mutated tumor suppressor genes such as p53, tumor associated viral antigens (e.g., HPV E6 and E7), the SSX family, HOM-MEL-55, NY-COL-2, HOM-HD-397, HOM- RCC-1.14, HOM-HD- 21, HOM-NSCLC-11, HOM-MEL-2.4, HOM-TES-11, RCC-3.1.3, NY-ESO-I, and the SCP family. Members of the MAGE family include, but are not limited to, MAGE-1, MAGE-
2, MAGE-3, MAGE-4, MAGE-6, MAGE-11 and MAGE-12. Members of the GAGE family include, but are not limited to, GAGE-1, GAGE-6.
The tumor antigen can also be, but is not limited to human epithelial cell mucin (Muc-1 ; a 20 amino acid core repeat for the Muc-1 glycoprotein, present on breast cancer cells and pancreatic cancer cells), MUC-2, MUC-3, MUC-18, carcino-embryonic antigen (CEA), the raf oncogene product, CA- 125, GD2, GD3, GM2, TF, sTn, gp75, EBV-LMP 1 & 2, prostate- specific antigen (PSA), prostatespecific membrane antigen (PSMA), GnT-V intron V sequence (N-acetylglucosaminyltransferase V intron V sequence), Prostate Ca psm, MUM-I- B (melanoma ubiquitious mutated gene product), alpha-fetoprotein (AFP), COI 7-1 A, GA733, gp72, -HCG, gp43, HSP-70, pi 7 mel, HSP-70, gp43, HMW, HOJ-I, melanoma gangliosides, TAG-72, mutated proto-oncogenes such as p21ras, mutated tumor suppressor genes such as p53, estrogen receptor, milk fat globulin, telomerases, nuclear matrix proteins, prostatic acid phosphatase, protein MZ2-E, polymorphic epithelial mucin (PEM), folate- binding-protein LK26, truncated epidermal growth factor receptor (EGFR), Thomsen- Friedenreich (T) antigen, GM-2 and GD-2 gangliosides, polymorphic epithelial mucin, folate- binding protein LK26, human chorionic gonadotropin (HCG), pancreatic oncofetal antigen, cancer antigens 15-3, 19- 9, 549, 195, squamous cell carcinoma antigen (SCCA), ovarian cancer antigen (OCA), pancreas cancer associated antigen (PaA), EBNA (Epstein-Barr Virus nuclear antigen) 1-6, gp75, chimeric protein P210BCR-ABL, lung resistance protein (LRP) Bel -2, and Ki-67. The tumor antigens that can be used in accordance with the present invention are in no way limited to the tumor antigens listed herein.
Also encompassed by the present invention are modified forms of the tumor antigens described above which induce an immune response in a subject. Modified forms of naturally occurring tumor antigens can advantageously have reduced pathogenicity and/or enhanced immunogenicity as compared with the naturally occurring antigen.
It is to be understood that the antigen that is co-administered with the target-binding molecule, antibody-antigenic peptide complex or antibody-antigenic peptide construct may be comprised in the target-binding molecule, antibody-antigenic peptide complex or antibody-antigenic peptide construct and/or may be administered separately. That is, the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate according to the invention may be co-administered with an antigen or a vaccine comprising an antigen even if the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate already comprises the an antigen or an antigenic peptide derived from an antigen. The antigen or antigenic peptide that is comprised in the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate according to the invention may be identical to the antigen or may be derived from the antigen that is co-administered with the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate separately. Alternatively, the antigen or antigenic peptide that is comprised in the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate according to the invention may be different from the antigen that is co-administered with the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate. Combination of the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention with a vaccine or an antigen may result in the development of more effective vaccines for the treatment of cancer and infectious diseases.
In certain embodiments, the pharmaceutical composition may comprise the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention and one or more adjuvants. The term "adjuvant" as used herein refers to an agent that nonspecifically increases an immune response to a particular antigen, thereby reducing the quantity of antigen necessary in any given vaccine and/or the frequency of injection necessary in order to generate an adequate immune response to the antigen of interest. Suitable adjuvants for use herein include, but are not limited to, poly IC; synthetic oligodeoxynucleotides (ODNs) with a CpG motif; modified polyinosinic:polycytidylic acid (Poly-IC) including, but not limited to, Poly-IC/LC (Hiltonol) and Poly-IC12U (Ampligen); Poly-K; carboxymethyl cellulose (CMC); Adjuvant 65 (containing peanut oil, mannide monooleate, an aluminum monostearate); Freund's complete or incomplete adjuvant; mineral gels such as aluminum hydroxide, aluminum phosphate, and alum; surfactants such as hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecylammonium bromide, N,N- dioctadecyl-N',N"-bis(2- hydroxymethyl)propanediamine, methoxyhexadecylglyerol and pluronic polyols; polyanions such as pyran, dextran sulfate, polyacrylic acid, and carbopol; peptides such as muramyl dipeptide, dimethylglycine and tuftsin; and oil emulsions. The adjuvants of the present invention may include nucleic acids based on inosine and cytosine such as poly I:poly C; poly IC; poly dC; poly dl; poly dIC; Poly-IC/LC; Poly-K; and Poly-IC 12U as well as oligodeoxynucleotides (ODNs) with a CpG motif, CMC and any other combinations of complementary double stranded IC sequences or chemically modified nucleic acids such as thiolated poly IC as described in U.S. Patent Nos. 6,008,334; 3,679,654 and 3,725,545.
Combination of the target-binding molecule, antibody-antigenic peptide complex or antibody- antigenic peptide construct of the invention with one or more adjuvant may result in the development of a more effective adjuvant that can be used in vaccination therapy.
In certain embodiments, the pharmaceutical composition may comprise the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention and a chemotherapeutic agent. The term “chemotherapeutic agent”, as used herein, refers to a cytotoxic agent which is of use in chemotherapy of cancer. For example, a chemotherapeutic agent may relate to an alkylating agent, such as cyclophosphamide, mechlorethamine, chlorambucil, melphalan, dacarbazine, nitrosoureas, and temozolomide, or to an anthracy cline, such as daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, or to a cytoskeletal disruptor, such as paclitaxel, docetaxel, abraxane, and taxotere, or to an epothilone, or to a histone deacetylase inhibitor, such as vorinostat and romidepsin, or to an inhibitor of topoisomerase 1, such as irinotecan and topotecan, or to an inhibitor of topoisomerase H, such as etoposide, teniposide, and tafluposide, or to a kinase inhibitor, such as bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, and vismodegib, or to a nucleotide analogue, such as azacitidine, azathioprine, capecitabine, cytarabine, doxifluridine, fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and tioguanine, or to a peptide antibiotics, such as bleomycin and actinomycin, or to a platinum-based agent, such as carboplatin, cisplatin, and oxaliplatin, or to a retinoid, such as tretinoin, alitretinoin, and bexarotene, or to a vinca alkaloid derivative, such as vinblastine, vincristine, vindesine, and vinorelbine.
Combination of the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or the immunoconjugate of the invention with a chemotherapeutic agent may result in more efficient cancer therapy. In particular, tumor antigens that are released by cells that have been killed by the immunoconjugate and/or the chemotherapeutic agent may be displayed more efficiently by professional antigen-presenting cells, such as dendritic cells and macrophages, and thus illicit and/or enhance an immune response against the tumor.
In certain embodiments, the pharmaceutical composition may comprise the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or immunoconjugate of the invention and an immune checkpoint modulator. The term “immune checkpoint modulator”, as used herein, refers to an agent used in cancer immunotherapy. A checkpoint inhibitor preferably blocks an inhibitory immune checkpoint and thus restores immune system function, for example, an inhibitor of the immune checkpoint molecule CTLA-4, such as ipilimumab, or an inhibitor of PD-1, such as nivolumab or pembrolizumab, or an inhibitor of PD-L1, such as atezolizumab, avelumab, and durvalumab. In many of the embodiments, a checkpoint inhibitor relates to an antibody which targets a molecule involved in an immune checkpoint. However, the term “immune checkpoint modulator” further encompasses agents that activate a stimulatory immune checkpoint. Examples of inhibitory and stimulating checkpoint molecules are provided herein.
Combination of the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or the immunoconjugate of the invention with an immune checkpoint modulator may result in an increased immune response against a subject’s tumor. While the target-binding molecule, antibody-antigenic peptide complex, antibody-antigenic peptide construct or the immunoconjugate of the invention results in increased display of tumor antigens by professional antigen-presenting cells, a check point inhibitor may prevent inhibition of T cells that may become activated by the professional antigen-presenting cells. In a particular embodiment, the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition according to the invention for use in inducing the maturation of monocytes and/or monocyte-derived cells in a subject. In a particular embodiment, the invention relates to the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein the maturation of monocytes or monocyte -derived cells results in the formation of dendritic cells and/or classically activated macrophages in said subject.
That is, the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may be used for inducing the maturation of monocytes and/or monocyte derived cells in a subject. It has been demonstrated herein that the target-binding molecule the antibody-antigenic peptide complex, the antibody-antigenic peptide construct and the immunoconjugate of the invention have the potential to induce the maturation of monocytes or monocyte-derived cells into classically activated (Ml) macrophages and dendritic cells, in particular matured dendritic cells. Accordingly, the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may be used for inducing and/or enhancing an immune response in a subject.
Accordingly, in a particular embodiment, the invention relates to an antibody, or an antigen-binding fragment thereof, specifically binding to the extracellular part of GOLPH2 for use in inducing and/or enhancing an immune response in a subject.
In a particular embodiment, the invention relates to an antibody, or an antigen-binding fragment thereof, specifically binding to the extracellular part of GOLPH2 for use in treating a disease or disorder associated with an impaired immune system in a subject.
A ’’disease or disorder associated with an impaired immune system”, as used herein, relates to an abnormal condition of the human or animal body that is characterized by a depressed ability of a subject’s immune system to mount an immune response to an antigen. The ’’disease or disorder associated with an impaired immune system” may be an acquired or a congenital disease or disorder. The skilled person is aware of methods to determine whether a subject has an impaired immune system. An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals ( e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non human primates such as macaques), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.
In a particular embodiment, the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein the subject is at risk of developing cancer, suffering from cancer or recovering from cancer.
The target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may be used for inducing the maturation of monocytes and/or monocyte -derived cells in any subject. In certain embodiments, the subject is a subject that is at risk of developing cancer, suffering from cancer or recovering from cancer.
A subject may be determined to be at risk of developing cancer based on the subject’s age and/or medical history. In addition, a subject’s risk of developing cancer may be determined based on tests, such as genetic tests or biomarker tests. The skilled person is aware of methods to determine whether or not a subject is at risk of developing a certain type of cancer.
A subject is suffering from cancer, if the subject shows unregulated growth of tumor or cancerous cells. Several ways of diagnosing cancer in a subject, including for example imaging techniques, have been described in the art.
A subject is recovering from cancer, if the subject was previously diagnosed with cancer, but wherein the cancer has been treated by any means known in the art. A subject recovering from cancer may be treated with the target-binding molecule, the antibody-antigenic peptide complex, the antibody- antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention to kill remaining tumor or cancerous cells or to prevent the reoccurrence of the cancer.
Within the present invention, the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition may have two distinct functions, which is (i) inducing the maturation of monocytes and/or monocyte- derived cells and (ii) directly targeting GOLPH2 positive cancer cells. That is, in certain embodiments, the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may be used to induce the maturation of monocytes and/or monocyte-derived cells in a subject that is at risk of developing cancer, suffering from cancer or recovering from cancer. That is, the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may be used to increase the number of antigen-presenting cells in that subject to improve antigen presentation and (a) support an immune response against the subject’s tumor and cancerous cells in subjects suffering from cancer or (b) prevent the emergence of tumor or cancerous cells in subjects at risk of developing or recovering from cancer. In this case, it is preferred that a target-binding molecule, an antibody- antigenic peptide complex, an antibody-antigenic peptide construct, or a pharmaceutical composition comprising a target-binding molecule is administered to the subject at risk of developing cancer, suffering from cancer or recovering from cancer. In particular, the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition according to the invention may comprise an antigen or an antigenic peptide that is known to be presented by the subject’s tumor or cancerous cells to increase the subject’s immune response against the tumor or cancerous cells.
In another embodiment, the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may be used to induce the maturation of monocytes and/or monocyte-derived cells in a subject suffering or recovering from a GOLPH2 positive cancer. In these embodiments, the targetbinding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may not only improve an immune response against the subject’s tumor or cancerous cells, but may also directly attack the tumor cells. In this case, it is preferred that an immunoconjugate comprising a cytotoxic agent or a pharmaceutical composition comprising an immunoconjugate comprising a cytotoxic agent is administered to the subject suffering from or recovering from a GOLPH2 -positive cancer.
The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include, but are not limited to, carcinoma, liver cancer, hepatocellular cancer, gastric cancer, lung cancer, esophageal cancer, breast cancer, prostate cancer, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. In certain embodiments, the invention relates to an antibody, or an antigen-binding fragment thereof, specifically binding to the extracellular part of GOLPH2 for use in treating cancer.
In certain embodiments, the invention relates to an antibody, or an antigen-binding fragment thereof, specifically binding to the extracellular part of GOLPH2 for use in treating cancer, wherein the binding of the antibody, or the antigen-binding fragment thereof, to the extracellular part of GOLPH2 induces the maturation of monocytes into macrophages and/or dendritic cells.
The term "GOLPH2 -positive cell" refers to a cell that expresses full length or partial GOLPH2 on its surface.
In a particular embodiment, the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein the subject is immunocompromised.
The term "immunocompromised" as used herein refers to a subject with an innate, acquired, or induced inability to develop a normal immune response. An immunocompromised subject, therefore, has a weakened or impaired immune system relative to one of a normal subject. A subject with a weakened or impaired immune system has an "immunodeficiency" or "immunocompromised condition," which is associated with a primary or secondary deficiency, induced or non-induced, in one or more of the elements of the normal immune defense system. In a particular embodiment, an immunodeficiency is associated with a deficiency of a subject to develop professional -antigen presenting cells, such as dendritic cells and/or macrophages. An immunocompromised condition is commonly due to a medical treatment, e.g., radiation therapy, chemotherapy or other immunosuppressing treatment, such as induced by treatment with steroids, cyclophosphamide, azathioprine, methotrexate, cyclosporine or rapamycin, in particular in relation to cancer treatment or the treatment or prevention of transplant rejection. The presence of an immunocompromised condition in a subject can be diagnosed by any suitable technique known to persons of skill the art. Strong indicators that an immunocompromised condition may be present is when rare diseases occur or the subject gets ill from organisms that do not normally cause diseases, especially if the subject gets repeatedly infected. Other possibilities are typically considered, such as recently acquired infections, for example, HIV, hepatitis, tuberculosis, etc. Generally, however, definitive diagnoses are based on laboratory tests that determine the exact nature of the immunocompromised condition. Most tests are performed on blood samples. Blood contains antibodies, lymphocytes, phagocytes, and complement components - all of the major immune components that might cause immunodeficiency. A blood cell count will determine if the number of phagocytic cells or lymphocytes is below normal. Lower than normal counts of either of these two cell types correlates with an immunocompromised condition. The blood cells are also checked for their appearance. Occasionally, a subject may have normal cell counts, but the cells are structurally defective. If the lymphocyte cell count is low, further testing is usually conducted to determine whether any particular type of lymphocyte is lower than normal. A lymphocyte proliferation test may be conducted to determine if the lymphocytes can respond to stimuli. The failure to respond to stimulants correlates with an immunocompromised condition. Antibody levels and complement levels can also be determined for diagnosing the presence of an immunocompromised condition.
It has been shown in the appended Examples that the target-binding molecule of the invention can induce the maturation of monocytes and monocyte-derived cells into professional antigen-presenting cells, such as Ml macrophages and mature dendritic cells. Thus, it is plausible that the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate and/or the pharmaceutical composition of the invention can be used for increasing the number of professional antigen-presenting cells in an immunocompromised subject and thus improve the function of the immune system in these subjects. In particular, the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate and/or the pharmaceutical composition of the invention may be used for inducing the maturation of monocytes and/or monocyte -derived cells in an immunocompromised subject, wherein the immunocompromised subject was diagnosed a decreased number of professional antigen- presenting cells, in particular Ml macrophages and/or mature dendritic cells.
Thus, in a particular embodiment, the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein the subject is immunocompromised as a result of a chemotherapy, a radiotherapy, or an infection, in particular wherein the infection is aninfection with a human immunodeficiency virus.
In a particular embodiment, the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition according to the invention for use in inducing and/or enhancing an immune response to an antigen.
The target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate and/or the pharmaceutical composition of the invention may be used for inducing and/or enhancing an immune response to an antigen. That is, it has been demonstrated herein that the target-binding molecule, the antibody-antigenic peptide complex, the antibody- antigenic peptide construct, the immunoconjugate and/or the pharmaceutical composition of the invention can induce the maturation of monocytes and monocyte-derived cells. Thus, it is plausible that the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate and/or the pharmaceutical composition of the invention can increase the number of professional antigen-presenting cells in a subject and thus induce and/or enhance an immune response to an antigen in said subject.
The term “immune response” as used herein refers to both humoral and cell-mediated immune responses. The humoral branch of the immune system involves interaction of B cells with antigen and their subsequent proliferation and differentiation into antibody-secreting plasma cells. The antibody functions as the effector molecule of the humoral response by binding to antigen and neutralizing it or facilitating its elimination. Antibodies can cross-link the antigen, forming clusters that are more readily digested by phagocytic cells, such as macrophages. Binding of antibody to antigen can also activate the complement system resulting in lysis of the cell to which the antibody binds including foreign organisms. Antibody can also neutralize toxins or viral particles by coating them and preventing binding to host cells.
The cell-mediated branch of the immune response occurs when effector T cells are generated in response to antigen. Both T helper cells (TH) and cytotoxic T lymphocytes (CTLs) serve as effector cells in cell-mediated immune reactions. Lymphokines secreted by TH cells can activate various phagocytic cells to phagocytose and kill microorganisms. Activated cytotoxic T lymphocytes participate in cell-mediated immune reactions by killing altered self-cells, virally infected cells and tumor cells.
Macrophages mainly present antigens to T helper cells, while mature dendritic cells present antigens to T helper cells, cytotoxic T lymphocytes and B cells. However, since macrophages are also involved in the clearance of antigens that are bound by B cell-produced antibodies, it is plausible that both macrophages and dendritic cells are involved in humoral and cell-mediated immune responses. Thus, in certain embodiments, the invention relates to the target-binding molecule, the immunoconjugate or the pharmaceutical composition according to the invention for use in inducing and/or enhancing a humoral and/or a cell-mediated immune response to an antigen. In certain embodiments, the invention relates to the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition according to the invention for use in inducing and/or enhancing a humoral immune response to an antigen. In certain embodiments, the invention relates to the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition according to the invention for use in inducing and/or enhancing a cell-mediated immune response to an antigen.
The target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention are said to induce an immune response in a subject, if they initiate an immune response that was not present before the induction stage. The target-binding molecule, the antibody-antigenic peptide complex, the antibody- antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention are said to enhance an immune response in a subject, if they maintain a desired response at the same level before the enhancement stage or increasing the desired response over a period of time. The term “induce” also includes the term “enhance”.
In a particular embodiment, the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein the immune response against the antigen is induced and/or enhanced with the help of an antigenic peptide that is comprised in the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct.
That is, the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct of the invention may be used to deliver an antigen or an antigenic peptide to an antigen-presenting cell and to facilitate the presentation of the antigenic peptide by the antigen- presenting cell. Thus, the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition according to the invention may be used to induce and/or enhance an immune response against a known antigen.
In a particular embodiment, the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein the antigen has been released by a physical therapeutic intervention.
The target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may induce and/or enhance an immune response to any type of antigen. That is, the antigen may be administered to a subject to induce and/or enhance an immune response in said subject, for example during a vaccination therapy. In other embodiments, the antigen, or a fragment or derivative thereof, may be part of the target-binding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct. In certain embodiments, the antigen is an endogenous antigen that is produced by the subject. This endogenous antigen may be released by natural processes or in response to a physical therapeutic intervention. Different types of physical therapeutic interventions are known that induce and/or facilitate the release of antigens. Without limitation, physical therapeutic intervention that induce and/or facilitate the release of antigens comprise cryotherapy, surgery, radiotherapy and laser therapy. It is to be noted that an antigen may be released by a physical therapeutic intervention and may in addition be administered, either the entire antigen or an antigen peptide derived from said antigen, to a subject separately or comprised in the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention.
In a particular embodiment, the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein the physical therapeutic intervention is cryotherapy, surgery, radiotherapy and/or laser therapy.
The term “cryotherapy”, as used herein, refers to the use of extreme cold produced by liquid nitrogen (or argon gas) to destroy abnormal tissue. Cryotherapy may be used to treat external tumors, such as those on the skin. For external tumors, liquid nitrogen is applied directly to the cancer cells with a cotton swab or spraying device. Cryotherapy may also be used to treat tumors inside the body (internal tumors and tumors in the bone). For internal tumors, liquid nitrogen or argon gas is circulated through a hollow instrument called a cryoprobe, which is placed in contact with the tumor. Cryotherapy is also referred to as “cryosurgery” or “cyroablation”.
The term "surgery", as used herein, means any therapeutic procedure that involves methodical action of the hand or of the hand with an instrument, on the body of a human or other mammal, to produce a curative or remedial. Within the present invention, the term “surgery” preferably means the resection of a tumor.
The term “radiotherapy”, as used herein, means exposure to radiation from a radioactive substance used in the treatment of disease (especially cancer). The term “laser therapy” or “laser surgery” refers to a type of surgery that uses special light beams instead of tools such as scalpels. Laser light may be delivered either continuously or intermittently and may be used with fiber optics to treat areas of the body that are often hard to reach. Common lasers used in cancer treatment include carbon dioxide (CO2) lasers, neodymium :yttrium -aluminum - garnet (Nd:YAG) lasers, laser-induced interstitial thermotherapy (LITT) and Argon lasers.
Physical therapeutic interventions, as the ones described above, result in the destruction of cells or tissue and thus in the release of molecule from the destroyed cells or tissues. Any molecule that is release from a cell or tissue in response to a physical therapeutic intervention can potentially serve as an antigen. In certain embodiments, the physical therapeutic intervention is directed to tumor cells or tissues and the antigens that are released from the tumor cells or tissues comprise tumor antigens. Some of the antigens that are released from tumor cells or tissues may be presented by matured monocytes that have been treated with the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention to induce and/or enhance an immune response against these antigens.
The target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may be administered to the subject receiving the physical therapeutic intervention before, during or after the physical therapeutic intervention. That is, the target-binding molecule, the immunoconjugate or the pharmaceutical composition of the invention may be administered to the subject several days, for example 1, 2, 3, 4 or 5 days, before or after the physical therapeutic intervention. In certain embodiments, the target-binding molecule, the immunoconjugate or the pharmaceutical composition of the invention is administered multiple times before, during and/or after the physical therapeutic intervention.
In a particular embodiment, the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein the antigen is a tumor antigen or a pathogen-derived antigen.
In certain embodiments, the antigen is a tumor antigen. That is, the antigen may be a tumor antigen that is administered to a subject, for example as a cancer vaccine, or may be an endogenous tumor antigen that has been released by a tumor or cancerous cell, for example in response to a physical therapeutic intervention. Further, an antigenic peptide derived from a tumor antigen may be comprised in the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate of the invention. Exemplary tumor antigens that may induce and/or enhance an immune response in a subject are discussed above.
In other embodiments, the antigen may be a pathogen-derived antigen. A pathogen-derived antigen may be used to induce and/or enhance an immune response against the pathogen that it has been derived from. It is to be noted that the pathogen-derived antigen may be a single molecule that has been derived from a pathogen, such as peptides, proteins, including glycoproteins, carbohydrates, phospholipids, phosphoproteins, phospholipoproteins, and fragments of the foregoing. The pathogen- derived antigen may be derived directly from the pathogen or may be chemically synthesized. In certain embodiments, the pathogen-derived antigen may also be a portion or a lysate of the pathogen. In certain embodiments, the pathogen-derived antigen may be an inactivated or attenuated form of the pathogen. The term pathogen as used herein comprises, without limitation, bacteria, viruses, fungi and protozoa.
In a particular embodiment, the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition according to the invention for use as an adjuvant in a vaccination therapy.
An adjuvant is a pharmacological or immunological agent that modifies the effect of other agents. Adjuvants may be added to a vaccine to boost the immune response to produce more antibodies and longer-lasting immunity, thus minimizing the dose of antigen needed. Adjuvants may also be used to enhance the efficacy of a vaccine by helping to modify the immune response to particular types of immune system cells: for example, by activating T cells instead of antibody-secreting B cells depending on the purpose of the vaccine.
Until now, agents like aluminum salts or squalene-in-water emulsions (MF59 or AS03) that have the ability to boost the immune answer towards defined antigens have been used and are approved as adjuvants in vaccination therapies. Other adjuvants have been tested or are in clinical development, for example Poly-IC (also Poly-ICLC), a synthetic derivate of dsRNA, CpG, synthetic phosphorothioate- linked DNA oligonucleotides with optimized CpG motifs or IFA, a mineral or paraffin oil plus surfactant, to name a few (Coffman RL, Sher A, Seder RA. Vaccine adjuvants: Putting innate immunity to work. Immunity 2010;33:492-503).
In mice, CpG has been used successfully to enhance the innate and adaptive response of the rodents immune system against a given antigen. This has been exploited for vaccination and generation of humoral responses, e.g. monoclonal antibodies. In human, no comparable adjuvants are in clinical use. A major obstacle with human adjuvants is the complexity that most of the adjuvants formulations carry. Consequently, the majority of hitherto tested adjuvants have not found their way into the clinic for human use (Coffman RL, Sher A, Seder RA. Vaccine adjuvants: Putting innate immunity to work. Immunity 2010;33:492-503.).
It has been demonstrated herein that the target-binding molecule, the immunoconjugate or the pharmaceutical composition of the invention have the potential to improve the presentation of antigens by professional antigen-presenting cells. Thus, it is plausible that the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention can be used as an adjuvant in a vaccination therapy.
Accordingly, in a particular embodiment, the invention relates to the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein the adjuvant initiates or enhances the function of antigen-presenting cells.
In a particular embodiment, the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein the vaccination therapy comprises administration of a viral antigen, a microbial antigen or a tumor antigen.
That is, the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may be co-administered and/or formulated with any antigen against which an immune response is desired. Vaccines comprising an antigen and the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may comprise one or more additional adjuvants or may comprise the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention as the sole adjuvant. The skilled person is aware of methods to formulate compositions, i.e. vaccines, comprising the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention.
In certain embodiments, the invention relates to the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention for use as an adjuvant in a vaccination therapy, wherein the vaccine comprises one or more tumor antigens and, optionally, one or more additional adjuvants. The one or more tumor antigen may be any combination of known tumor antigens, for example any combination of the tumor antigens discussed herein.
In certain embodiments, the invention relates to the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention for use as an adjuvant in a vaccination therapy, wherein the vaccine comprises one or more pathogen-derived antigens and, optionally, one or more additional adjuvants.
In certain embodiments, the invention relates to the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention for use as an adjuvant in a vaccination therapy, wherein the vaccine comprises one or more viral antigens and, optionally, one or more additional adjuvants.
The term “viral antigen”, as used herein, refers to any molecule (e.g., protein, peptide, lipid, carbohydrate, etc.) solely or predominantly expressed by a virus, such that the antigen is associated with the virus. The viral antigen may be an antigen expressed by any virus. In certain embodiments, the viral antigen may also be an inactivated or attenuated form of the virus. The viral antigen may be a viral antigen of only one type of virus, such that the viral antigen is associated with or characteristic of only one type of virus. Alternatively, the viral antigen may be a viral antigen (e.g. may be characteristic) of more than one type of virus. For example, the viral antigen may be expressed by a virus selected from the group consisting of herpes viruses, pox viruses, hepadnaviruses, papilloma viruses, adenoviruses, coronoviruses, orthomyxoviruses, paramyxoviruses, flaviviruses, and caliciviruses.
In certain embodiments, the invention relates to the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention for use as an adjuvant in a vaccination therapy, wherein the vaccine comprises one or more microbial antigens and, optionally, one or more additional adjuvants.
The term "microbial antigen", as used herein, refers to any microbial component having antigenic properties, i.e. being able to provoke an immune response in an individual. The term “microbial antigen” encompasses “bacterial antigens” and “fungal antigens”. The term "bacterial antigen", as used herein, refers to any bacterial component having antigenic properties, i.e. being able to provoke an immune response in an individual. The bacterial antigen may be derived from the cell wall or cytoplasm membrane of a bacterium. The term "fungal antigen", as used herein, refers to any fungal component having antigenic properties, i.e. being able to provoke an immune response in an individual.
In a particular embodiment, the invention relates to the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition for use according to the invention, wherein at least one antigenic peptide derived from the viral antigen, the microbial antigen or the tumor antigen is comprised in the targetbinding molecule, the antibody-antigenic peptide complex or the antibody-antigenic peptide construct.
In other embodiments, the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may be used as an adjuvant in a vaccination therapy, wherein the separate co-administration of the antigen is not required. That is, an immune response against a specific antigen may be induced and/or enhanced by administering a target-binding molecule, an antibody-antigenic peptide complex or an antibody-antigenic peptide construct of the invention comprising an antigenic peptide, a fragment or derivative of an antigen or an entire antigen. In such cases, the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, or the immunoconjugate comprising at least one antigenic peptide, antigen fragment or derivative or antigen can function both as an adjuvant and as an antigen.
In a particular embodiment, the invention relates to the use of an antibody specifically binding to the extracellular part of GOLPH2, or an antigen-binding fragment thereof, as an adjuvant in a vaccination therapy.
In a particular embodiment, the invention relates to the use according to the invention, wherein the antibody is a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody or a multispecific antibody.
In a particular embodiment, the invention relates to the use according to the invention, wherein the antibody is the antibody-antigenic peptide construct, the antibody-antigenic peptide complex or the fusion antibody according to the invention.
In a particular embodiment, the invention relates to the use according to the invention, wherein the adjuvant initiates or enhances the function of antigen-presenting cells.
In a particular embodiment, the invention relates to the use according to the invention, wherein the vaccination therapy comprises administration of a viral antigen, a microbial antigen or a tumor antigen.
In a particular embodiment, the invention relates to the use according to the invention, wherein at least one antigenic peptide derived from the viral antigen, the microbial antigen or the tumor antigen is comprised in the antibody-antigenic peptide construct according to the invention or the antibody- antigenic peptide complex according to the invention.
For all uses listed above, it is to be understood that the target -binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention are administered to a subject in need thereof in an effective amount. An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. It is to be understood that the effective amounts may differ from one use to another. However, the skilled person is aware of reasonable ranges of amounts and able to identify the effective amount by routine experimentation.
In a particular embodiment, the invention relates to a method for inducing the maturation of monocytes and/or monocyte-derived cells in vitro, the method comprising the steps of: a) culturing monocytes and/or monocyte-derived cells in a cell culture medium; b) adding the target-binding molecule, the antibody-antigenic peptide complex, the antibody- antigenic peptide construct or the immunoconjugate according to the invention to the cell culture of step (a); and c) obtaining matured monocytes and/or monocyte-derived cells.
The invention further refers to an in vitro method for inducing the maturation of monocytes and/or monocyte-derived cells. For that, monocytes and/or monocyte -derived may be cultured in a container comprising a suitable cell culture medium. The skilled person is aware of suitable containers, such as flasks or plates, and media for the culturing of monocytes and/or monocyte derived cells. Further examples are described in Example 2. The monocytes and/or monocyte-derived cells may be cultured at any cell density. For example, the monocytes and/or monocyte-derived cells may be cultured at a cell density between IO2 and IO7 cells/mL, preferably at a cell density between IO3 and IO6 cells/mL. Further, the skilled person is aware of methods for obtaining monocytes and/or monocyte-derived cells from a sample, for example a blood or bone marrow sample, as described in Example 1.
To induce the maturation of monocytes or monocyte-derived cells, the monocytes and/or monocyte- derived cells are contacted with the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate of the invention. The skilled person is aware of methods to determine the concentration of the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate that is required for inducing the maturation of monocytes and/or monocyte -derived cells. For example, when the target-binding molecule is an antibody, concentrations between 0.01 and 10 pM, preferably between 0.05 and 10 pM, more preferably between 0.1 and 10 pM and most preferably between 0.5 and 10 pM of the target-binding molecule may be added to the cells in the cell culture.
The monocytes and/or monocyte-derived cells may be contacted with the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate of the invention for any amount of time that is sufficient for the monocytes and/or monocyte-derived cells to mature. In certain embodiments, the monocytes and/or monocyte-derived cells are contacted with the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate for 2 to 7 days to obtain matured monocytes and/or monocyte-derived cells. In certain embodiments, the monocytes and/or monocyte-derived cells are contacted with the target-binding molecule, the antibody-antigenic peptide complex, the antibody- antigenic peptide construct or the immunoconjugate for 3 to 6 days to obtain matured monocytes and/or monocyte-derived cells. In certain embodiments, the monocytes and/or monocyte-derived cells are contacted with the target-binding molecule, the antibody-antigenic peptide complex, the antibody- antigenic peptide construct or the immunoconjugate for 4 days to obtain matured monocytes and/or monocyte-derived cells.
In a particular embodiment, the invention relates to a method for generating a cell displaying an antigenic peptide, the method comprising the steps of: a) generating a matured monocyte or monocyte-derived cell according to the method of the invention; b) pulsing the antigenic peptide on the matured monocyte or monocyte-derived cell generated in step (a); and/or introducing a nucleic acid encoding a polypeptide comprising the antigenic peptide into the matured monocyte or monocyte-derived cell generated in step (a); and/or introducing the antigenic peptide into the matured monocyte or monocyte-derived cell as part of the target-binding molecule, the complex, or the construct; and c) obtaining a cell displaying an antigenic peptide.
Further, the invention relates to a method for generating a cell displaying an antigenic peptide. For that, an antigenic peptide may be pulsed on the matured monocyte or monocyte -derived cell that have been generated with the method of the invention or a nucleic acid encoding an antigenic peptide may be introduced into the matured monocyte or monocyte-derived cell.
The cell displaying an antigenic peptide is preferably a matured monocyte or monocyte-derived cell. That is, the cell displaying the antigenic peptide may be a professional antigen-presenting cell that has been obtained with the method of the invention, i.e. by contacting a monocyte or monocyte-derived cell with the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate of the invention. In certain embodiments, the cell displaying the antigenic peptide is an Ml macrophage that has been obtained with the method of the invention. In other embodiments, the cell displaying the antigenic peptide is a mature dendritic cell that has been obtained with the method of the invention.
The skilled person is aware of methods for pulsing an antigenic peptide onto a professional antigen- presenting cell, in particular an Ml macrophage or a mature dendritic cell. Example 14 further describes the pulsing of an antigenic peptide on matured monocytes.
The antigenic peptides that are pulsed on the matured monocytes are preferably peptides that can be displayed by an MHC molecule on the surface of the matured monocyte. That is, the peptides preferably have a length of 8 to 25 amino acid residues. MHC class I molecules preferably display peptides with a length of 8 to 10 amino acid residues. Accordingly, in certain embodiments, the antigenic peptide is an MHC class I peptide with a length of 8 to 10 amino acid residues. MHC class II molecules preferably display peptides with a length of 13 to 25 amino acid residues. Accordingly, in certain embodiments, the antigenic peptide is an MHC class II peptide with a length of 13 to 25 amino acid residues. Accordingly, in certain embodiments, the peptide that is pulsed on the matured monocyte or monocyte-derived cell is an MHC class I or and MHC class II peptide.
Alternatively, a polynucleotide encoding an antigenic peptide may be delivered to the matured monocyte or monocyte-derived cell. The skilled person is aware of methods to introduce foreign nucleic acids into a cell. For example, transfection or transduction methods may be used to deliver a foreign nucleic acid encoding an antigenic peptide into a matured monocyte.
It is to be understood that the foreign nucleic acid encoding the antigenic peptide may comprise further elements. That is, the nucleic acid may encode a larger polypeptide comprising the antigenic peptide. In certain embodiments, the larger polypeptide encoded by the polynucleotide comprises one or more MHC class I and/or MHC class II peptides. In such cases, the nucleic acid may be transcribed and translated into a larger polypeptide, which is then degraded into smaller peptides inside the matured monocyte or monocyte -derived cell. These smaller peptides, for example the antigenic peptide, may then be displayed by MHC molecules on the surface of the matured monocyte or monocyte-derived cell. Further, the nucleic acid may comprise regulatory elements that allow for expression of the nucleic acid in the matured monocyte.
The term “antigenic peptide,” as used herein, refers to a peptide comprising a structure that is recognized by the immune system of a subject. Non-limiting examples of antigenic peptides are a peptide that is recognized by a B or T-cell, e.g. via binding to a T-cell receptor, or a peptide that binds to an antibody or antibody fragment, or a peptide that stimulates an immune response in a subject. The antigenic peptide may be a tumor antigen or a pathogen-derived antigen as discussed herein. In certain embodiments, the antigenic peptide is an MHC class I or MHC class II peptide.
Alternatively, the antigenic peptide may be delivered to the antigen-presenting cell, such as an Ml macrophage or a matured dendritic cell, as part of the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate of the invention. It has been described herein that antigenic peptides can be delivered directly to an Ml macrophage or a matured dendritic cell with the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate of the invention.
In a particular embodiment, the invention relates to a method for generating an activated T cell, the method comprising the steps of: a) generating a cell displaying an antigenic peptide with the method according to the invention; b) contacting the cell of step (a) with a T cell, wherein the T cell comprises a T cell receptor that recognizes the antigenic peptide displayed by the cell of step (a); and c) obtaining an activated T cell.
Further, the cell displaying an antigenic peptide may be used for generating activated T cells. Forthat, a cell displaying an antigen peptide, wherein the cell has been obtained with the methods of the invention, may be contacted with a T cell comprising a T cell receptor that recognizes the antigenic peptide displayed on the cell.
The skilled person is aware of methods for contacting the matured monocytes and/or monocyte- derived cells of the invention with T cells (see also Examples 11 and 14). Within the present invention, the T cell may be any type of T cell. However, it is preferred that the T cell is a T helper (CD4+) T cell or a cytotoxic T lymphocyte (CD8+ T cell).
In certain embodiments, the cell displaying the antigenic peptide may be a mature dendritic cell that has been obtained by contacting a monocyte or an immature dendritic cell with the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate of the invention. In certain embodiments, the dendritic cell may display an antigenic peptide that has been pulsed on the surface of the dendritic cell. In certain embodiments, the dendritic cell may display an antigenic peptide that has been introduced into the dendritic cell in the form of a polynucleotide. In certain embodiments, the dendritic cell may display an antigenic peptide that has been delivered to the dendritic cell with the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate of the invention. In certain embodiments, the antigenic peptide may be an MHC class I peptide that is displayed on the surface of the dendritic cell by an MHC class I molecule. In embodiments where the antigenic peptide is an MHC class I peptide, the dendritic cell displaying this antigenic peptide may be contacted with and activate a cytotoxic T lymphocyte. In other embodiments, the antigenic peptide may be an MHC class II peptide that is displayed on the surface of the dendritic cell by an MHC class II molecule. In embodiments where the antigenic peptide is an MHC class II peptide, the dendritic cell displaying this antigenic peptide may be contacted with and activate a T helper cell.
In certain embodiments, the cell displaying an antigenic peptide may be an Ml macrophage that has been obtained by contacting a monocyte or monocyte-derived cell with the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate of the invention. In certain embodiments, the Ml macrophage may display an antigenic peptide that has been pulsed on the surface of the Ml macrophage. In certain embodiments, the Ml macrophage may display an antigenic peptide that has been introduced into the Ml macrophage in the form of a polynucleotide. In certain embodiments, the Ml macrophage may display an antigenic peptide that has been delivered to the Ml macrophage with the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct or the immunoconjugate of the invention. In certain embodiments, the antigenic peptide may be an MHC class II peptide that is displayed on the surface of the Ml macrophage by an MHC class II molecule. In embodiments where the antigenic peptide is an MHC class II peptide, the Ml macrophage displaying this antigenic peptide may be contacted with and activate a T helper cell.
The term "T cell" as used herein will be understood by those skilled in the art. The term includes, but is not limited to CD8+ or CD4+ T cells capable of lysing target cells or providing effector or helper functions, such as cytokine secretion, which can result in the death of target cells or the generation or enhancement of anti-target effector activity.
The term “activated” as used herein relates to specific mechanisms of activation of T cells. The general “two-signal model” of activation of T cells is characterized by a first signal provided by binding of the TCR to a short peptide presented by the major histocompatibility complex (MHC) on another cell ensuring that only a T cell with a TCR specific to that peptide is activated. The partner cell is usually a professional antigen presenting cell (APC), for example a dendritic cell or an Ml macrophage. The peptides presented to CD8+ T cells by MHC class I molecules are 8-10 amino acids in length; the peptides presented to CD4+ T cells by MHC class II molecules are longer, as the ends of the binding cleft of the MHC class II molecule are open. The second and co-stimulatory signal required for T cell activation involves interaction of CD28 on the T cell with CD80 or CD86 (B7 family genes) on the antigen-presenting cell. The second signal licenses the T-cell to respond to an antigen.
The skilled person is aware of methods to determine if a T cell is activated. For example, a T cell may be determined to be activated if it expresses antigens indicative of T cell activation (that is, T cell activation markers). Examples of T cell activation markers include, but are not limited to, CD25, CD26, CD30, CD38, CD69, CD70, CD71, ICOS, OX-40 and 4-1BB. The expression of activation markers can be measured by techniques known to those of skill in the art, including, for example, western blot analysis, northern blot analysis, RT-PCR, immunofluorescence assays, and fluorescence activated cell sorter (FACS) analysis. Further, a T cell can be determined to be activated based on the cytokine secretion pattern. The skilled person is aware that certain cytokines are secreted in higher amounts by activated T cells.
The term “T cell receptor,” as used herein, refers to a heterodimeric antigen binding receptor derived from a T lymphocyte, comprising an alpha/beta polypeptide dimer or a gamma/delta polypeptide dimer, each dimer comprising a variable region, a constant region, and an antigen binding site. A TCR is said to recognize an antigenic peptide, if the interaction between the TCR and the antigenic peptide- MHC complex is sufficient to activate the cell comprising the TCR.
In a particular embodiment, the invention relates to the method according to the invention, wherein the cell displaying an antigenic peptide and the T cell have been obtained from the same subject.
The T cell and the antigen presenting cell used in the method of the invention may be of any origin. That is, in certain embodiments, the T cell and the antigen-presenting cell may have been obtained from different subjects. In other embodiments, the T cell and the antigen-presenting cell may have been obtained from the same subject. In such a case, T cells and monocytes and/or monocyte-derived cells may be isolated from a blood sample that has been obtained from said subject.
In a particular embodiment, the invention relates to a cell displaying an antigenic peptide and/or an activated T cell for use in adoptive cell transfer, wherein the cell displaying the antigenic peptide and the activated T cell have been obtained with the methods according to the invention.
The term "adoptive cell transfer" as used herein refers to any transfer of immune cells into a subject. In cancer immunotherapy, adoptive cell transfer is used to trigger cytotoxic immune responses that will destroy tumor cells. The term comprises the direct transfer of immune cells such as T cells, monocytes, macrophages or dendritic cells into the subject. The immune cells are intended to recognize and kill the tumor cells in addition to the subject's own anti-tumor response.
Adoptive transfer of activated dendritic cells comprises the stimulation of dendritic cells obtained with the method of the invention to activate a cytotoxic response towards the tumor cells. Dendritic cells can be stimulated for example by pulsing them with an antigen or transfecting them with a viral vector. The stimulated dendritic cells may be infused into the subject to initiate a cytotoxic immune response against the tumor cells.
Adoptive transfer of activated macrophages comprises the stimulation of Ml macrophages obtained with the method of the invention to activate a cytotoxic response towards the tumor cells. Ml macrophages can be stimulated for example by pulsing them with an antigen or transfecting them with a viral vector. The stimulated dendritic cells may be infused into the subject to initiate a cytotoxic immune response against the tumor cells.
In certain embodiments, T cells that have been activated with the method of the invention are transferred into a subject. It is preferred that the T cells have been activated with an antigen that is known to be present in the subject receiving the adoptive cell transfer. For example, the T cells may have been activated with a tumor antigen that is known to be expressed by tumor or cancerous cells in the subject receiving the adoptive cell transfer. For that, mature dendritic cells or Ml macrophages that have been obtained with the method of the invention may be pulsed with an antigenic peptide that has been previously determined to be present on tumor cells of a subject. The resulting antigendisplaying cells may then be contacted with a population of T cells that have been obtained from said subject to activate and proliferate the portion of the T cells that recognize the tumor antigen. The activated T cells may then be transferred back into the subject to initiate an immune response against the tumor cells.
Preferably, adoptive cell transfer is used in the treatment of cancer. Thus, it is preferred that the antigenic peptide that is displayed on the mature dendritic cell or the Ml macrophage is a tumor antigen, for example one of the tumor antigens listed herein.
In a particular embodiment, the invention relates to a method for inducing and/or enhancing an immune response against a specific antigen in a subject, the method comprising the steps of: a) administering to said subject the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition according to the invention, wherein the target-binding molecule, the antibody- antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition comprises an antigenic peptide derived from the specific antigen; b) inducing and/or enhancing an immune response in said subject.
That is, the target-binding molecule, the antibody-antigenic peptide complex, the antibody-antigenic peptide construct, the immunoconjugate or the pharmaceutical composition of the invention may be used to induce and/or enhance an immune response against a specific antigen in a subject. The specific antigen may be determined by fusing or complexing this antigen, or a fragment or derivative thereof, to the target-binding molecule of the invention. It is to be understood that not all fragments or derivatives of an antigen can be displayed by an antigen-presenting cell. However, the skilled person is aware of fragments or derivatives of antigens that can be displayed by antigen-presenting cells such that an immune response against the antigen can be induced or enhanced. A non-exhaustive list of antigens is provided herein.
All publications, patent applications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference(s) should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document is authoritative.
DESCRIPTION OF FIGURES It is to be understood that throughout the figures and the description of figures the terms G2-2, G2- 2M, G2-2-ADC and BsMabG2-2/Myc refer to G2-2opti, G2-2optiM, G2-2opti-ADC and BsMabG2-2opti/Myc, respectively.
FIG.l: Quantification of secreted GOLPH2 (sGOLPH2 ng/ml) of tumor cell lines and human monocytes. 1A Tumor cell lines of the human hepatocellular carcinoma (HCC) HuH7, the lung adeno carcinoma H1975 and the colorectal cancer (CRC) HCT116. IB human monocytes upon various treatments.
FIG.2: Metabolic activity test of tumor cells and human white blood cells upon G2-2-ADC treatment. 2A Metabolic activity of human monocytes, the HCC cell line HuH7 and CRC cell line HCT116 upon treatment with G2-2 linked to the anthracycline analogue PNU. 2B Metabolic activity of human monocytes and T-cells upon treatment with G2-2 linked to the pyrrolobenzodiazepine PBD.
FIG.3: Comparison of G2-2 effects on the metabolic activity of human and murine monocytes. 3A Dose dependent effect on the metabolic activity of human monocytes (EC50: 0.6 pM). 3B Dose dependent effect on the metabolic activity of murine bone marrow derived monocytes (EC50: 0.06 pM); Measurements performed after 96 hrs of G2-2 treatment.
FIG.4: Surface Expression of Differentiation Proteins upon antibody treatment of Monocytes. 4A Flow cytometry showing CD 141, CD 14, CD86 and CD40 expression levels on human monocytes after treatment with G2-2 or control antibody. 4B Flow cytometry showing CD 14, CD86 and CD40 expression on the human monocytic leukemia cell line THP-1 after treatment with G2-2 or control antibody with or without IFN-y.
FIG.5: Cytokine release upon treatment of human monocytes. The monocytes were either exposed to antibodies (ISO, G2-2) or polarized to a M0, Ml or M2 phenotype by cytokines treatment. 5A Cytokine expression of monocytes from three donors upon different treatment schemes. 5B Cytokines expression of monocytes from one donor upon treatment with membrane-near binding G2-2, G2-2M and more C-terminal (G2-4) GOLPH2 binding antibodies.
FIG.6: RNA-Seq analysis upon treatment of human monocytes. Alternative-box blot analysis of CD82 and CD37. Abbreviations: B before treatment; U untreated sample; G treatment with 0.5 pg/ml G2-2 and I for the isotype control antibody. FIG.7: Monocytes and autologous T cells co-culture assay. 7A Human monocytes treated with G2-2 or a control antibody for 4 days were then co-cultured with autologous T-cells for two days until culture pictures were taken. 7B Absolute cell numbers of CD8 T cells and CD4 T cells are depicted 7C-E After day two of co-culture cells were harvested for flow cytometry analysis. 7C,D The bars represent the MFI of CD25 and ICOS on the surface of CD8 and CD4 T-cells. 7E CD 141 expression levels on monocytes 7F Cell culture supernatant of day two of co-culture experiments were collected for cytokine measurement. The fold changes are expressed as ratio of G2-2 treatment effect compared to either an isotype antibody (G2-2/ISO) or an untreated control (G2-2/control).
FIG.8: Effect of G2-2M treatment on antigen presenting (APC) properties of murine bone marrow derived monocytes. Flow cytometry analysis shows MFI values as bar diagram of CD 11b, CD 11c, CD8a and percent values of H2Kb-SIINFEKL positive CD8+ cells, a marker for peptide presentation after peptide loading to monocytes.
FIG.9: Effect of BsMabG2-2/Myc loaded with Myc-SIINFEKL peptide (SEQ ID NO:41) in the murine MC38-OVA model. 9A Tumor growth in C57BL/6 mice upon treatment with PBS or Myc- SIINFEKL peptide (SEQ ID NO:41) loaded antibodies BsMabG2-2/Myc or anti-Myc. 9B Survival curve of this MC38-OVA model.
FIG.10: Effect of BsMabG2-2/Myc loaded with Myc-SIINFEKL (SEQ ID NO:41) on antigen presentation of an immortalized murine macrophage cell line. 10A Bar diagram of expression of SIINFEKL peptide in MHC I context upon treatment with the antibodies anti-Myc or BsMabG2- 2/Myc or each antibody loaded with Myc-SIINFEKL peptide (SEQ ID NO:41). Shown are absolute MFI measurements in flow cytometry. 10B depicts the same experiment as in A as percent values.
FIG 11: Antigen presentation of human Monocytes upon treatment with BsMabG2-2/Myc loaded with Myc-Ml peptide (SEQ ID NO:42) and consecutive T-cell activation. HA Bar diagram of Influenza Ml peptide presentation in MHC I context upon treatment with Myc-Ml peptide alone or peptide loaded on either anti-Myc antibody or BsMabG2-2/Myc. Shown are absolute MFI measurements in flow cytometry. 11B shows bar diagrams of T cell activation in percent of all CD8+ cells upon co-culture with monocytes for 72 hours. 11C and HD depict CD82 cell surface expression on monocytes upon treatment with either BsMabG2-2/Myc loaded with Myc-Ml peptide or with respective controls after co-culture with T-cells.
FIG.12: Epitope binding and effects on monocytes of G2-2MX. 12A,B depict ELISA binding curves of G2-2M versus G2-2MX to either human (12A, SEQ ID NO:23) or murine (12B, SEQ ID NO:24) GOLPH2 (aa36 to 55). 12C displays bar diagrams of metabolic activity of human monocytes. There is a four to eight fold difference in antibody concentration needed for similar metabolic activity of G2-2 and G2-2MX. 12D,E depict surface expression of different membranous expression markers upon G2- 2MX treatment and respective control treatments. Shown are bar diagrams of CD 141, CD86, CD40 and CD82 surface expression on human monocytes treated with G2-2, G2-2MX or controls (UT untreated; ISO isotype antibody) for 72 hours analyzed by flow cytometry. MFI is shown in 12D, percentage in 12E. Of note: In 12D,E G2-2MX was used at a 10 fold lower concentration than G2-2 or ISO. 12F shows the TNF-alpha secretion of human monocytes that were treated 72 hours with the indicated antibodies.
FIG.13: Cartoons of the various antibodies and antibody-antigenic peptide constructs described in the patent application
FIG.14: Surface Expression of differentiation marker proteins upon different antibody treatment of co-cultured Monocytes and T cells. 14A Flow cytometry showing CD 14 and CD40 expression levels on human monocytes after treatment with G2-2, EPR3606 (abeam Cat. No. ab239985) or an irrelevant isotype matched control antibody for five days. The positivity of the respective monocytes is expressed as geometric mean of fluorescence intensity (gMEAN MFI) of flow cytometry analysis as described in Example 28. 14B Autologous T cells added to the monocytic culture on day three were analyzed after 48 hours for the activation marker HLA-DR by flow cytometry. 14C Cell culture supernatant of the co-culture experiments were collected for cytokine measurement. The fold changes are expressed as ratio of G2-2 treatment effect compared to an isotype antibody (G2-2/ISO) or EPR3606 treatment effect compared to an isotype antibody (EPR3606/ISO) as described in Example 29.
FIG.15: Binding of G2-2 and EPR3606 (abeam) to GOLPH2 protein. 15A An ELISA showing differential binding to extracellular GOLPH2 (aa 36-401), soluble GOLPH2 (aa 56-401) and the membrane-near peptide of GOLPH2 (aa36-55). EPR3606 apparently binds to GOLPH2 downstream of the Furin cleavage site. Furin cleavage occurs between amino acids 55 and 56. Error bars are displayed when they are longer than the height of the symbol 15B Two c-terminal His tagged GOLPH2 fragments, eGOLPH2-HIS (aa 36-401 R52A) and sGOLPH2-HIS (aa 56-401) are detected in a Western Blot analysis. EPR3606 and an anti-His antibody bind to both proteins further demonstrating that their binding site is downstream of amino acid 56.
EXAMPLES Example 1: Isolation of human and murine monocytes and human T cells
For human monocyte and T cell isolation, 30 ml blood was drawn from a healthy donor and collected in an EDTA coated tube (Sarstedt Cat. No. 01.1605.100). Peripheral blood monocytes (PBMCs) were isolated by density-gradient centrifugation with 30 ml Histoplaque (Sigma, Cat. No. 10771-500ML). Monocytes were separated by negative selection using magnetic bead depletion (Stemcell, EasySep human monocyte isolation kit, Cat. No 19359). T cells were separated by negative selection using magnetic bead depletion (Miltenyi Biotec, Pan T cell isolation kit, Cat. No. 130-096-535). Cells in the flow through were harvested, washed in phosphate buffered saline with ImM EDTA (PBS Sigma- Aldrich TMS-012) and cultured in medium as described in Example 2.
Mouse bone marrow monocytes were isolated from the femur and tibia of C57BL/6 mice. The bones were rinsed in 70% ethanol (Carl Roth Cat. No. 7301.1) for 90 seconds and washed with PBS before flushing the bones with cell culture medium to mobilize the bone marrow. The bone marrow was fdtered through a sterile 70 pm cell strainer (Coring Cat. No. CLS431751-50EA), the cells are washed twice in medium and cultured in 48-well plates.
For mouse OT-1 T cells, male C57BL/6-OTl-Ly5.1 mice containing transgenic inserts for mouse Tcra-V2 and Tcrb-V5 genes and the congenic marker Ly5.1 were bred in-house at the DBM animal facility, University Basel. Their transgenic T cell receptor recognizes ovalbumin peptide residues 257- 264 (OVA257-264) in the context of H2Kb (CD8 co-receptor interaction with MHC class I). This results in MHC class I-restricted, ovalbumin-specific, CD8+ T cells (OT-I cells). Thus, the CD8+ T cells of these mice primarily recognize OVA257-264 when presented by the MHC I molecule. CD 8+ T cells of these mice were harvested from the spleen using a negative isolation kit (Stemcell, Cat. No. 19853) and cultured in complete IMDM as described in Example 2.
Example 2: Cell culture of human and murine tumor cell lines, monocytes and T cells
Human monocytes were cultured in ImmunoCult™-SF Macrophage Medium (Stemcell Technologies Cat. No.10961). This medium contains Iscove's MDM, bovine serum albumin, insulin and transferrin. For some assays, culture medium was supplemented with human recombinant M-CSF (Stemcell Technologies Cat. No. 78057), Interferon gamma (Stemcell Technologies Cat. No. 78020), human recombinant IL-4 (Stemcell Technologies Cat. No 78045) or LPS (Merck Cat. No. L4524).
Human T cells were cultured in RPMI 1640 containing (Gibco Cat. No. 11875093) with 10% allogeneic serum, 5 ml 100 x Sodium Pyruvate (Gibco Cat. No 11360-039), 5 ml 100 x MEM non- essential amino acids (Gibco Cat. No. 11140-035), 50 pM (0.5ml) 1000 x beta-mercaptoethanol 50 mM, 100 U/ml Penicillin and 0.1 mg/ml Streptomycin (Sigma Aldrich Cat. No. P433).
The human monocytic leukaemia cell line THP-1 (Sigma-Aldrich Cat. No. 88081201) was maintained in DMEM (Gibco DMEM high glucose, Cat. No. 11960085) supplemented with 10% FCS (Seraglob Sera Pro FBS Cat. No. S40500E), 2 mM L-Glutamine (Gibco Cat. No. A2916801), 100 U/ml Penicillin and 0.1 mg/ml Streptomycin (Sigma Aldrich Cat. No. P433).
For assay performance THP-1 cells were seeded in ImmunoCult-SF Macrophage Medium (Stemcell Technologies Cat. No. 10961) supplemented with 10% FCS (Seraglob Sera Pro FBS Cat. No. S40500E), 100 U/ml Penicillin and 0.1 mg/ml Streptomycin (Sigma Aldrich Cat. No. P433).
Mouse bone marrow monocytes from C57BL/6 mice were cultured in Macrophage Base Medium DXF (PromoCell Cat. No. C-28057).
Mouse T cells isolated from the spleen of C57BL/6-OTl-Ly5.1 mice were cultured in complete IMDM containing 500 ml IMDM-GlutaMAX (Gibco Cat. No. 31980-022), 50 ml heat inactivated fetal bovine serum, 5 ml 100 x Sodium Pyruvate (Gibco Cat. No 11360-039), 5 ml 100 x MEM non- essential amino acids (Gibco Cat. No. 11140-035), 50 pM (0.5ml) 1000 x beta-mercaptoethanol 50 mM, 100 U/ml Penicillin and 0.1 mg/ml Streptomycin (Sigma Aldrich Cat. No. P433).
Immortalized murine bone marrow derived macrophages established as described in De Nardo, Kalvakolanu DV, Latz E in Immortalization of Murine Bone Marrow-Derived Macrophages in Methods in molecular biology (Clifton, N.J.) May 2018 DOI: 10.1007/978-l-4939-7837-3_4 were cultured in RPMI 1640 (Gibco Cat. No. 11875093) supplemented with 10% FCS (Seraglob Sera Pro FBS Cat. No. S40500E), 2 mM L-Glutamine (Gibco Cat. No. A2916801), 100 U/ml Penicillin and 0.1 mg/ml Streptomycin (Sigma Aldrich Cat. No. P433).
HCT116, a human colon cancer cell line, was cultured in RPMI (Gibco Cat. No. 11875093) supplemented with 10% FCS (Seraglob Sera Pro FBS Cat. No. S40500E) and 2 mM glutamine (Gibco Cat. No. A2916801).
Hl 975, a human lung adeno carcinoma cell line and HuH7, a human hepatocellular carcinoma cell line were grown in DMEM (Gibco DMEM high glucose, Cat. No. 11960085) supplemented with 10% FCS (Seraglob Sera Pro FBS Cat. No. S40500E). The murine colon cancer cell line MC38-OVA, expressing ovalbumin, was maintained in DMEM (Gibco DMEM high glucose, Cat. No. 11960085) supplemented with 10% FCS (Seraglob Sera Pro FBS Cat. No. S40500E), 2 mM L-Glutamine (Gibco Cat. No. A2916801), 100 U/ml Penicillin and 0.1 mg/ml Streptomycin (Sigma Aldrich Cat. No. P433).
Example 3 : Measurement of sGOLPH2 protein levels
Tumor cells (HCT116, H1975, HuH7) were seeded in 6 well plates at a concentration of 5 x 105 per well. Human monocytes and T cells were isolated as described in Example 1, seeded in 48 well plates at a concentration of 0.25 x 106 per well and grown at 37 °C in a humidified incubator in 5% CO2. The culture medium ImmunoCult™-SF Macrophage Medium (Stemcell Technologies Cat. No. 109) was supplemented with 50 ng/ml human recombinant GM-CSF (Stemcell Technologies Cat. No. 78190) or human recombinant M-CSF (Stemcell Technologies Cat. No. 78057.1) or 0.5 mg/ml of G2-2opti or isotype control antibody (ISO) and cells were cultured for 10 days.
The isotype control antibody used in these Examples comprises a heavy chain variable region with a sequence set forth in SEQ ID NO:29 and a light chain variable region with a sequence set forth in SEQ ID NO:33. In particular, the isotype control antibody used in these Examples comprises a heavy chain CDR1 as set forth in SEQ ID NO:30, a heavy chain CDR2 as set forth in SEQ ID NO:31, a heavy chain CDR3 as set forth in SEQ ID NO:32, a light chain CDR1 as set forth in SEQ ID NO:34, a light chain CDR2 as set forth in SEQ ID NO: 35 and a light chain CDR3 as set forth in SEQ ID NO: 36.
Supernatants of tumor cell lines were collected after 72 h, supernatants of monocytes were collected after 10 days and the sGOLPH2 protein levels were analyzed by Golgi Protein 73 (GP73) Test Kit on an UPT-3A reader (Model UPT-3A-1800) according to the manufacturer’s instructions (Hotgen Biotech, Beijing, China). Briefly, 0.1 ml supernatant is diluted in 0.1 ml buffer and 0.1 ml of this mixture is applied onto a test strip. After 15 min incubation at room temperature the test strip is inserted into the UPT-3A reader for direct measurement of sGOLPH2 concentration.
FIG. 1 shows that high amounts of soluble GOLPH2 (sGOLPH2) were released from the tumor cell lines HuH7, H1975 and HCT116. In contrast, significantly lower amounts of sGOLPH2 were released by human monocytes. The treatment of human monocytes with the antibody G2-2opti, an isotype control antibody or the cytokines GM-CSF or M-CSF, respectively, had no influence on the secretion of sGOLPH2. Without being bound to theory, it appears that the expression and/or the cleavage of GOLPH2 is less frequent in human monocytes compared to cancer cell lines. Example 4: Influence of G2-2 drug-conjugates on the viability of different cell types
The antibody drug conjugate (ADC) G2-2-PNU was produced as G2-2opti IgGl as described in PCT/EP2017/079870 (Seq ID NOs: 18, 19 and 20). For G2-2-vc-PBD valine at position 205 of the kappa light chain constant region was substituted by cysteine (V205C). To allow binding to the engineered cysteine the antibody was reduced with Tris(2-carboxyethyl)phosphine (TCEP; Sigma- Aldrich, Cat. No. C4706) and the interchain disulfide bonds were reconstituted by incubating G2-2 V205C in dhAA (dehydroascorbic acid; Sigma-Aldrich Cat. No. 261556). The maleimide-PEG4-VA- Pyrrolobenzodiazepine (PBD) (Levena Biopharma Cat. No. SET0212) was incubated at 4-fold molar excess with the activated G2-2 V205C for 1 hour.
Monocytes and T cells from three different healthy donors were isolated by negative selection as described in Example 1. Cells were seeded in 96 well plates at a concentration of I MO3 per well and grown at 37 °C in a humidified incubator in a 5% CO2 atmosphere overnight in culture medium described in Example 2. HCT116 and HuH7 cells were grown as described in Example 2 and seeded in 6 well plates at a concentration of 5 x 105 per well.
The next day, 10 pl of twofold serial dilutions of G2-2 ADC in PBS were added, resulting in final ADC concentrations ranging from 312.5 nM to 0.02nM for G2-2 V205C-PBD. Each dilution was done in duplicate. After 96 h, plates were removed from the incubator and 10 pl of PrestoBlue™ cell viability reagent a resazurin-based cell permeable solution (Life Technologies Cat. No. A 13262) was added to each well and transferred back to the incubator for 30 minutes. In viable cells resazurin is converted to fluorescent resorufm. Fluorescence was measured on a SpectraMax ® Gemini™ EM microplate reader (Molecular Devices, USA) with an excitation wavelength of 560 nm and emission wavelength of 590 nm.
FIG.2A shows that the addition of a G2-2 antibody-drug conjugate to the tumor cell lines HCT116 and HuH7 resulted in decreased viability of the tumor cell lines. Surprisingly, addition of the same antibody-drug conjugate to human monocytes resulted in increased metabolic activity of the cells. At the same time, no effect was seen when the antibody-drug conjugate was added to human T cells (FIG.2B).
Example 5 : Influence of G2-2opti on the viability of monocyes
For treatment of mouse cells the optimized G2-2 variable regions and heavy chain constant region (IgG2a) was used as described in PCT/EP2017/079870 (Seq ID NOs: 18, 19 and 21). This antibody is annotated here as G2-2M. As isotype control antibody the variable regions of the heavy and light chain of G2-2opti were substituted by variable regions of an anti Myc-Tag antibody, known as 9E10 (SEQ ID NO:29 and SEQ ID NO:33); Evan GI, Lewis GK et al. 1985 Mol Cell Biol (5), 3610-3616 and Schiwek W, Buxbaum B et al. FEBS letters 414 (1997) 33-38) and annotated here as ISOM.
Monocytes from a healthy donor were isolated by negative selection as described in Example 1, plated on 96-well plates in 90 pl ImmunoCult™-SF Macrophage Medium (Stemcell Technologies Cat. No. 10961) at a density of O.l x lO6 cells per well and grown at 37 °C in a humidified incubator in a 5% CO2.
Mouse bone marrow monocytes were isolated from C57BL/6, mice and cultured in Macrophage Base Medium DXF (PromoCell Cat. No. C-28057).
The same day, 10 pl of twofold serial dilutions of G2-2opti for human monocytes or G2-2M for mouse bone marrow monocytes in PBS were added, resulting in final antibody concentrations ranging from 3.125 pMol to 0.0488pMol for G2-2opti and 3.125 pMol to 0.0061 pMol for G2-2M. Each dilution was done in duplicate. After 96 h, plates were removed from the incubator and 10 pl of PrestoBlue™ cell viability reagent a resazurin-based cell permeable solution (Life Technologies) was added to each well and transferred back to the incubator for 30 min. In viable cells resazurin is converted to fluorescent resorufm. Fluorescence was measured on a SpectraMax ® Gemini™ EM microplate reader (Molecular Devices, Silicon Valley, CA, USA) with an excitation wavelength of 560 nm and emission wavelength of 590 nm.
FIG.3 shows that also the unconjugated antibodies G2-2opti and G2-2M have the ability to increase the viability of human and mouse-derived monocytes.
Example 6: Surface expression of differentiation proteins upon antibody treatment of human monocytes
Monocytes from three different healthy donors were isolated by negative selection as described in Example 1 and cultured as described in Example 2. Culture medium was supplemented with either 0.5 mg/ml G2-2, isotype control antibody, 50 ng/ml human M-CSF for M0 monocytes, 50 ng/ml M-CSF and 50 ng/ml IFN-y and lOng/ml LPS for Ml monocytes, 50 ng/ml M-CSF and lOng/ml IL4 for M2 monocytes for 5 days. Cell culture supernatant was collected, stored at -20°C and analyzed as described in Example 8. Cells were harvested in 2 mM EDTA PBS and stained with Live/Dead dye (Biolegend, Cat. No. 77474) and human FcR inhibitor (Invitrogen, Cat. No. 4350496) for 20 min on ice. After washing with FACS buffer (PBS supplemented with 2% FCS, 0.1%sodium azide, 2 mM EDTA) twice, cells were stained with monoclonal antibodies for 30 min on ice, in brief antibodies used are: CD141-BV421 (Biolegend, clone: M80), CD14-BV605 (Biolegend, clone: M5E2), CD86- BV711 (Biolegend, clone: IT2.2), CD40-APC-Cy7 (Biolegend, clone: 5C3). After washing with FACS buffer twice, cells were analysis by BD LSRFortessa. All the surface markers were examined based on live single cells gating.
FIG.4A shows that certain differentiation proteins are either upregulated or downregulated upon addition of the antibody G2-2opti to human monocytes.
Example 7: Surface expression of differentiation proteins upon antibody treatment ofTHP-1 cells
Human monocytic leukaemia cells THP-1, cultured as described in Example 2, were seeded in a 24- well-plate 0.2 x 106 cells per well containing ImmunoCult-SF Macrophage Medium. Next day the culture medium was changed and supplemented with either 0.5 mg/ml G2-2opti, human isotype IgGl, 50 ng/ml human INF-y (Peprotech Cat. No. F2617) plus 0.5 mg/ml G2-2opti or 50 ng/ml human INF- y plus 0.5 mg/ml human isotype IgGl. All cells were cultured for 72 hours at 37°C, 5% CO2 in a tissue culture incubator.
Cells were detached from the culture plate using 5 mM EDTA in PBS and pooled with floating cells from the supernatant, washed in PBS and treated with TruStain FcX™ (Biolegend Cat. No. 422301) for blocking of human Fc receptors for 30 minutes on ice in FACS-Buffer (PBS + 2mM EDTA + 2% FCS + 0. 1% sodium azide).
For Flow Cytometry analysis the following monoclonal antibodies were used: CD14-BV605 (Biolegend, clone: M5E2), CD86-BV711 (Biolegend, clone: IT2.2), CD40-APC-Cy7 (Biolegend, clone: 5C3).
FIG.4B shows that in the presence of IFN-y the expression of the proteins CD 14, CD40 and CD86 is upregulated.
Example 8: Cytokine secretion of human monocytes or monocyte-derived cells
Human Cytokine 42-Plex Discovery panel with IL-18. Cell culture supernatants collected as described in Example 6 were send to Eve Technologies, Alberta, Canada for cytokine measurement applying fluorescent color-coded beads pre-coated with capture antibodies targeting specific cytokines.
This multiplexing analysis was performed using the Luminex™ 100 system (Luminex, Austin, TX, USA) by Eve Technologies. Sixty-four markers were simultaneously measured in the samples using a MILLIPLEX Human Cytokine/Chemokine 41-plex kit plus IL- 18, according to the manufacturer's protocol. The 41-plex consisted of EGF, FGF-2, Eotaxin, TGF-a, G-CSF, Flt-3L, GM-CSF, Fractalkine, IFNa2, IFN-y, GRO, IL-10, MCP-3, IL-12P40, MDC, IL-12P70, PDGF-AA, IL-13, PDGF-BB, IL-15, sCD40L, IL-17A, IL-IRA, IL-la, IL-9, IL-1B, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP-la (CCL3), MIP-1B (CCL4), RANTES (CCL5), TNFa, TNFB, and VEGF. IL-18 test was added by Eve Technologies. The assay sensitivities of these markers range from 0.3 - 26.3 pg/mL. Measurements were done in duplicate and the mean of Individual analyte values is shown in Figure 5.
Human monocytes have been treated with 0.5 mg/ml of the antibody G2-2opti, an isotype control antibody, or different cytokine cocktails. Cytokine cocktail M0 contains 50 ng/ml human M-CSF and induces the formation of resting M0 macrophages. Cytokine cocktail Ml contains 50 ng/ml M-CSF, 50 ng/ml IFN-y and lOng/ml LPS and induces the polarization into classically activated Ml macrophages. Cytokine cocktail M2 contains 50 ng/ml M-CSF and lOng/ml IL-4 and induces the polarization into alternatively activated M2 macrophages.
Monocytes from a healthy donor were isolated by negative selection as described in Example 1 and cultured as described in Example 2. Culture medium was supplemented with either 0.5 mg/ml G2- 2opti, G2-2M, G2-4, isotype control antibody or left untouched for 5 days. Cell culture supernatant was collected, stored at -20°C and analyzed at Eve Technology as described in Example 8 using the Luminex™ 100 system (Luminex, Austin, TX, USA). For simultaneous measurement a MILLIPLEX Human Cytokine/Chemokine 13-plex kit (Millipore, St. Charles, MO, USA) was used according to the manufacturer's protocol. The 13-plex consisted of GM-CSF, IFN-y, IL-lbeta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12(p70), IL-13, MCP-1 and TNF-alpha. The assay sensitivities of these markers range from 0.1 - 9.5 pg/mL for the 13-plex. Measurements were done in duplicates, the mean is shown in a bar diagram in Figure 5 B.
FIG.5A shows that human monocytes that have been treated with the antibody G2-2opti release comparable amounts of cytokines as human monocytes that have been treated with a cytokine cocktail that induces polarization into Ml macrophages. This data indicates that human monocytes that have been treated with the antibody G2-2opti mature into Ml macrophages. FIG.5B shows that the treatment of human monocytes with the antibodies G2-2opti and G2-4 results in an increased secretion of the cytokines IL-lbeta, IL-6 and TNF-alpha.
Example 9: RNA isolation
RNA of human monocytes was isolated using TRIzol reagent (Life technologies, Cat. 15596026) according to manufacturers protocol. In brief, growth medium of cultured monocytes was removed and floating cells pelleted by centrifugation. The supernatant was discarded and cell pellet added to the adherent cells on the plate. 0.3 m of TRIzol Reagent per 1 x 106 cells were added directly to the culture dish, pipetted up and down and incubated for 5 minutes at room temperature. 0.2 m of chloroform per 1 mb of TRIzol Reagent was added, and incubated for 3 minutes. Samples were centrifuged and the colorless upper aqueous phase, containing the RNA transferred to a new tube. 0.5 mb of isopropanol was added, incubated for 10 minutes and centrifuged. The precipitated RNA pellet was washed with 75% Ethanol, air dried for 5-10 minutes and resuspended in RNAse-free water.
Example 10: RNAseq
Monocytes from three male and three female healthy donors were isolated by negative selection as described in Example 1. As control “before treatment” (B) RNA was isolated from cells as described in Example 9 and stored at -20°C, for all other groups cells were cultured in ImmunoCult™-SF Macrophage Medium (Stemcell Technologies Cat. No. 109) supplemented with 0.5 pg/ml G2-2opti (A) or isotype control antibody (I) or left untouched (U) at 37 °C in a humidified incubator in a 5% CO2 atmosphere for 48 hours before RNA extraction.
RNA was quantified by Fluorometry using the QuantiFluor RNA System (Cat# E3310, Promega, Madison, WI, USA). Library preparation was performed, starting from 200 ng total RNA, using the TruSeq Stranded mRNA Library Kit (Cat# 20020595, Illumina, San Diego, CA, USA) and the TruSeq RNA UD Indexes (Cat# 20022371, Illumina, San Diego, CA, USA). 15 cycles of PCR were performed.
Libraries were quality-checked on the Fragment Analyzer (Advanced Analytical, Ames, IA, USA) using the Standard Sensitivity NGS Fragment Analysis Kit (Cat. No. DNF-473, Advanced Analytical) revealing excellent quality of libraries (average concentration was 124±18 nmol/L and average library size was 346±7 base pairs). Samples were pooled to equal molarity. The pool was quantified by Fluorometry using the QuantiFluor ONE dsDNA System (Cat. No. E4871, Promega, Madison, WI, USA).
Libraries were sequenced Paired-End 51 bases (in addition: 8 bases for index 1 and 8 bases for index 2) using the NovaSeq 6000 instrument (Illumina) and the SP Flow-Cell loaded at 400pM and including l% PhiX.
Primary data analysis was performed with the Illumina RTA version 3.4.4. On average per sample: 49.9±6.2 millions pass-filter reads were collected on 1 SP Flow-Cell. Reads were aligned to the human genome (UCSC version hg38AnalysisSet) with STAR (version 2.7). Read and alignment quality was evaluated using the qQCReport function of the bioconductor package QuasR (R version 3.6.0, Bioconductor version 3.10). The featureCounts function from the rsubread package was used to count the number of reads (5 'ends) overlapping with the exons of each gene assuming an exon union model (gene annotation: ensembl version 97). Differential gene expression analysis was performed with the R package edgeR. Specifically a paired design model taking treatment (B,I,U,G) and sample ID into account was fit with the glmFit function and each contrast of interest was tested using the glmQLFTest function of the edgeR package. Results were reported as significant if the false discovery rate was below 0.05.
Selected genes were analyzed according to the following published gene sets:
“Monocyte derived Dendritic Cells” as shown in Table 2 and “Matured Dendritic Cells” as shown in Table 3 according to Lyons, Y.A. et al.; Immune cell profiling in cancer: molecular approaches to cell-specific identification, npj Precision One 1, 26 (2017) doi: 10.1038/s41698-017-0031-0.
Table 2: Geneset indicating monocyte-derived dendritic cells
Table 3: Geneset indicating matured dendritic cells
“CD82 and CD37” according to Jones E.; Dendritic Cell Migration and Antigen Presentation are coordinated by the opposing functions of the tetraspanins CD82 and CD36. J of Immunology 2015 doi: 10.4049/jimmunol. 1500357.
“Activated murine Monocytes” as shown in Table 4 according to Orecchioni, M. et al.; Macrophage Polarization: Different Gene Signatures in M1(LPS+) vs. Classically and M2(LPS-) vs. Alternatively Activated Macrophages. Frontiers in Immunology 2019 Vol 10, Article ID 1084.
Table 4: Geneset indicating activated murine monocytes
Table 2 shows that monocytes that have been treated with the antibody G2-2opti have a gene expression profile that to a large extent overlaps with a published gene expression profile of monocyte-derived dendritic cells. Table 3 further shows that monocytes that have been treated with the antibody G2-2opti have a gene expression profile that to a large extent overlaps with a published gene expression profile of matured dendritic cells. In addition, FIG.6 shows that the treatment of human monocytes with the antibody G2-2opti results in the upregulation of CD82 and the downregulation of CD37, which is again indicative for matured dendritic cells. In contrast, treating the monocytes with an isotype control antibody had no significant impact on the expression of CD82 and CD37 in comparison to an untreated control. These data indicate that the treatment of human monocytes with the antibody G2-2opti induces the maturation of monocytes into dendritic cells and, in particular, into matured dendritic cells.
Table 3 shows that treatment of monocytes with the antibody G2-2opti can also result in a gene expression profile that, to a large extent, overlaps with a published gene expression profile of classically activated Ml macrophages. This indicates, that the treatment of human monocytes with the antibody G2-2opti can induce the maturation of monocytes either into matured dendritic cells or in classically activated Ml macrophages.
Example 11 : Co-culture of monocytes with T cells
Monocytes from healthy human donors were isolated by negative selection and cultured as described in Examples 1 and 2. Either G2-2opti or ISO in a concentration of 0.5 mg/ml were added to the culture medium for 4 days. Autologous T cells, acquired by negative isolation and cultured as described in Examples 1 and 2 were added in the presence of soluble anti-CD3 antibody (0.3 pg/ml) (clone HIT3a) on the top of monocytes for another 48 hours in co-culture.
Cell culture pictures were taken at an inverted bright field microscope in 4 x magnification as shown in Figure 7A.
Cell culture supernatants were collected for cytokine measurement, and cells were harvested in 2 mM EDTA in PBS for flow cytometry analysis. Cells were first stained with Live/Dead dye (Biolegend, Cat. 77474) and incubated with human FcR inhibitor (Invitrogen, Cat. 4350496) for 20 min on ice. After washing with FACS buffer (PBS supplemented with 2% FCS, 0.1%sodium azide, 2 mM EDTA) twice, cells were stained with monoclonal antibodies for 30 min on ice, in brief antibodies used were CD4+ (Invitrogen, clone OKT4) and CD8+ (Becton Dickinson, clone SKI)
By flow cytometry analysis absolute cell numbers of CD4+ and CD8+ T cells were counted as described in Example 6 and 7.
FIG.7A shows that T cells only aggregate if co-cultured with human monocytes that have been treated with the antibody G2-2opti. At the same time, the absolute number of T cells in the co-culture was higher if the monocytes in the co-culture had been previously treated with the antibody G2-2opti compared to monocytes that had been previously treated with the isotype control antibody. Example 12: Expression of activation markers in T cells upon co-culture with monocytes
Flow cytometry analysis was performed on T cells treated and harvested as described in Example 11. Anti-human monoclonal antibodies used were CD3-PE-CF584 (BD Biosciences, clone: UCHT1), CD4-PerCP-Cy5.5 (Biolegend, clone: SK3), CD8-APC-eFluor 780 (Biolegend, clone: SKI), CD25- BV605 (Biolgend, clone: BC96), ICOS- FITC (Biolegend, clone: ISA-3). CD25 and ICOS were examined based on live CD8 T cell (CD3+CD4-CD8+) and CD4 T cell (CD3+CD4+CD8-)
FIGs.7C-D show that the expression of the activation markers CD25 and ICOS was upregulated both in CD4+ and CD8+ T cells following co-culturing with human monocytes that had been treated with the antibody G2-2opti. In contrast, no upregulation of these activation was observed following coculturing with human monocytes that had been treated with the control antibody ISO.
FIG.7E shows that treatment of monocytes (CD3-CD4-CD8-) with the antibody G2-2opti results in the upregulation of CD 141, which is a marker for dendritic cells. This finding indicates that the treatment of monocytes with G2-2opti induces the maturation of monocytes into dendritic cells.
Example 13: Secretion of cytokines by co-cultured cells
Cell culture supernatants collected as described in Example 6 were send to Eve Technologies for cytokine measurement as described in Example 8. A MILLIPLEX Human Cytokine/Chemokine 13- plex kit (Millipore, St. Charles, MO, USA) was used according to the manufacturer's protocol. The 13-plex consisted of GM-CSF, I IFN-y, IL-lbeta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12(p70), IL- 13, MCP-1 and TNF-alpha. The assay sensitivities of these markers range from 0.1 - 9.5 pg/mL for the 13-plex. Measurements were done in duplicates, the mean is shown in a bar diagram in Figure 7F.
FIG.7F shows that multiple cytokines were secreted in significantly higher amounts when monocytes had been treated with the antibody G2-2opti in comparison to monocytes that had been treated with the control antibody ISO.
Example 14: Antigen-display by antibody-treated monocytes
Negative isolated mouse bone marrow monocytes acquired as described in Example 1 and cultured as described in Example 2 were treated with G2-2M or ISOM (0.5 mg/ml) for 48 hours, then OVA257- 264 peptide (SIINFEKL) (10 pM) (Eurogentec Cat. No. AS-60193-1) was loaded for 3 hours or 20 hours. After washing out the unbonded SIINFEKL peptide cells were harvested in 2 mM EDTA PBS and stained with Live/Dead dye (Biolegend Cat. No. 77474) and murine FcR inhibitor CD16/CD32 (BD Biosciences Mouse BD Fc Block, clone 2.4G2) for 20 min on ice. After washing with FACS buffer (PBS supplemented with 2% FCS, 0.1%sodium azide, 2 mM EDTA) twice, cells were stained with monoclonal antibodies for 30 min on ice, in brief antibodies used are: Anti-mouse monoclonal antibodies used were CD8a-FITC (Biolegend, clone: KT15), CDl lb-BV605 (Biolegend, clone: MI/70), CDl lc-PE-Cy7 (Biolegend, clone: N418), H-2Kb-SIINFEKL-PE (Biolegend, clone: 25- D1.6). All the surface markers were examined based on CD1 lb+ live cells.
Example 15: Cloning and peptide loading of BsMAbbG2-2/Myc
For bispecific antibody production the controlled Fab arm exchange technique was applied by mutating Phenylalanine at position 405 in CH3 of the human G2-2optiIgGl to Leucine (F405L) and Lysine at position 409 in CH3 of the human anti-Myc IgGl (ISO see Example 5) to Asparagine (K409R) as described in Paul S, Connor J, Nesspor T, et al. An efficient process of generating bispecific antibodies via controlled Fab-arm exchange using culture supernatants. Protein Expr Purif. 2016;121: 133-140. doi: 10.1016/j .pep.2016.01.014.
Both antibodies were produced separately in CHO or in 293HEK cells by transfection of vectors coding for VL and VH of G2-2-F405L or 9E10-K409R respectively. IgG was purified from cell culture media by protein A affinity chromatography.
Both purified antibodies were mixed at equal molar concentration and reduced for five hours with 2- Mercaptoethylamine-HCl (Thermo Scientific, article 20408) at 37°C. For removal of 2-MEA, gel filtration was performed. Re-oxidation and formation of bispecific antibody was done by incubating over night at room temperature. Next morning, the bispecific G2-2/anti-Myc monoclonal antibodies (BsMabG2-2/Myc) were sterile filtrated, measured and loaded with Myc-SIINFEKL peptide (SEQ ID NO:41) or Myc-Influenza Ml peptide (SEQ ID NO:42) (synthesized by Genscript, NY, USA) at 10- fold molar excess overnight at 4°C. Unbound peptide was removed by size exclusion chromatography and peptide loaded bispecific antibody was again sterile filtrated, quantified and kept in PBS at 4°C.
Example 16: Tumor growth inhibition by antibody-peptide treated mice in a MC38-OVA model.
C57BL/6 mice were vaccinated i.p. at day 24 and day 12 prior to s.c. implantation of 1 x 106 murine colon cancer cells MC38-OVA. Vaccination comprised in group 1 BsMabG2-2/Myc 12.5 mg/kg loaded with the peptide MYC-SIINFEKL, in group 2 anti-Myc hlgGl (ISO) 12.5 mg/kg loaded with the peptide MYC-SIINFEKL and in group 3 PBS. The efficacy was quantified by tumor volume measurements every other day. At day 34 after tumor implant, all mice from control groups 2 and 3 had been sacrificed due to high tumor volumes. The humane endpoint was set above 10003 mm with a maximum at 1500 mm3.
The mean tumor volume of group 1 was 362 mm3, the difference analyzed by paired t test with two- tailed P value was statistically significant p=0.0065 ) and shown in FIG. 9A. FIG. 9B depicts survival depending on the vaccination reagent. In the control groups animals survived until day 24, thereafter all control mice died within the next ten days. All mice from group 1 survived. The comparison in survival groups analyzed by Log -rank test was statistically significant (p=0.009). Statistical analysis was performed using GraphPad Prism5 software.
Example 17: Antigen-display by antibody-peptide treated murine monocytes
Immortalized murine bone marrow derived macrophages were seeded in 48 well plates with 0.2 x 106 cells per well. Two hours later, cells were incubated with either_BsMabG2-2/Myc loaded with MYC- SIINFEKL peptide or anti-Myc IgGl(ISO) loaded with MYC-SIINFEKL peptide or G2-2optiM IgG2a (G2-2M) without peptide or anti-Myc IgG2a (ISOM) without peptide or left untouched for 24 hours. Cells were harvested in 2 mM EDTA PBS and stained with Live/Dead dye (Biolegend Cat. No. 77474) and murine FcR inhibitor CD16/CD32 (BD Biosciences Mouse BD Fc Block, clone 2.4G2) for 20 min at room temperature. After washing with FACS buffer twice, cells were stained with monoclonal antibodies for 30 min on ice, in brief antibodies used are: Anti-mouse monoclonal antibody H-2Kb-SIINFEKL-PE (Biolegend, clone: 25 -DI.6) was used to quantify presentation of SIINFEKL peptide in the H-2Kb (MHC-I) context on the surface of monocytes.
FIG. 10 shows results from two independent experiments. There is an over 3.5 -fold increased presentation upon treatment with_BsMabG2-2/Myc peptide loaded compared to ISO peptide loaded.
Example 18: Antigen presentation by antibody-peptide treated human monocytes
Monocytes from three human healthy donors were isolated by negative selection as described in Example 1. Monocytes were seeded 0.25 x 106 cells per well in a 48 well plate in duplicate. Two hours later, either BsMabG2-2/Myc loaded with Myc-Ml peptide or anti-Myc hlgGl loaded with Myc-Ml peptide or an equimolar amount of Myc-Ml peptide was added to each well. 48 hours after treatment start cells were harvested and prepared for FACS staining as described in Example 11. Supernatant was stored for cytokine analysis. Duplicate wells were left untouched and further treated as described in Example 19. Antibodies used for Flow cytometry were anti human Influenza M1/HLA-A2 complex antibody (Dendritics, Cat. No. DDX0270A647). After washing with FACS buffer twice, cells were analysis by BD LSRFortessa. All the surface markers were examined based on live single cells gating. Fig 11A shows presentation of Ml peptide in the HLA-A2 context
Example 19: T-cell activation upon co-culture with Ml presenting autologous monocytes
Human Monocytes were treated for 48 hours with bispecific peptide loaded antibody or controls as described in Example 18. Thereafter, autologous T-cells, isolated as described in Example 1, were added to the monocytes in a ratio monocytes to T-cells of 1 to 3. To foster T-cells, the Macrophage Base was supplemented with 10% of autologous serum. At day three of co-culture, supernatants were collected for cytokine measurement and cells were harvested in 2 mM EDTA PBS and stained with Live/Dead dye (Biolegend, Cat. No. 77474) and human FcR inhibitor (Invitrogen Cat. No. 4350496) for 20 min on ice. After washing with FACS buffer twice, cells were stained with monoclonal antibodies for 30 min on ice, in brief anti-human antibodies used are: HLA-A2/M1-APC (Dendritics, clone 405H1.01), CDl lc-FITC (Biolegend, clone 3.9), CD8a-BV605 (Biolegen clone RPA-T8), CD82-PerCP-Cy5.5 (Biolegend, clone ASL-24), CD25-BV421 (Biolegend, clone BC96), CD69-PE- Cy5 (Biolegend, clone FN50). FIG. 1 IB shows significantly increased expression of T cell activation markers on CD8 T cells treated with BsMabG2-2/Myc loaded with the Myc-Ml peptide (SEQ ID NO:42). FIG 11C shows significantly increased expression of CD82 on monocytes treated with BsMabG2-2/Myc loaded with the Myc-Ml peptide.
Example 20: Cloning of Long neck construct G2-2MX and G2-2HX
The heavy chain variable region of antibody G2-2opti (SEQ ID NO: 18) was linked by a flexible linker (SEQ ID NO:51) to the light chain variable region of G2-2opti (SEQ ID NO: 19). The resulting single chain Fv (scFV) was named scG2-2 (SEQ ID NO:70). The scG2-2 was attached via a Serine rich linker (SEQ ID NO:52 and/or 53) to the hinge region of (i) a murine IgG2a Fc region (SEQ ID NO:37) for the construct G2-2MX (SEQ ID NO:73), or (ii) to a human IgGl Fc region (SEQ ID NO:38) for the construct G2-2HX (SEQ ID NO:72).
Example 21 : G2-2MX peptide ELISA
ELISA was carried out in 96 well plates coated overnight with Streptavidin (Sigma-Aldrich Cat. No. SA 101) at a concentration of 2 pg/ml and loaded with 4 pg/ml biotinylated peptide the next morning. Peptides used were human GOLPH2 aa 36 to 55 (SEQ ID NO:23) or murine GOLPH2 aa 36 to 55 (SEQ ID NO: 24) or a control peptide. After 30 minutes incubation unbound peptide was washed away and wells were blocked with 1% Gelatin (Sigma- Aldrich Cat. No G9391) for 1 hour. After extensive washing antibodies G2-2M and G2-2MX were applied at 2-fold logarithmic dilutions at concentrations ranging from 12.5 nM to 0.4 pM and incubated for 90 minutes. After extensive washing bound antibodies were detected by adding goat anti-Mouse IgG (Fc specific)-Peroxidase antibody (Sigma- Aldrich #A0168) at a concentration of 5 pg/ml. After extensive washing a chromogenic substrate (TMB Amersham) was added to detect peroxidase. The reaction was stopped and absorbance was read at 450 nm. Figure 12 A shows the binding curves to human GOLPH2 peptide, B to murine GOLPH2 peptide.
Example 22: Proliferation of human PBMCs upon different antibody concentrations
Monocytes from three different healthy donors were isolated by negative selection as described in Example 1. Cells were seeded in 96 well plates at a concentration of U I O’’ per well and grown at 37 °C in a humidified incubator in a 5% CO2 atmosphere overnight in culture medium described in Example 2. The next day, 10 pl of twofold serial dilutions of antibodies G2-2opti, G2-2M, G2-2MX or ISO in PBS were added, resulting in final antibody concentrations ranging from 0.98 pg/ml to 7.812 pg/ml. Each dilution was done in quatruplicate. After 72 hours, plates were removed from the incubator, supernatant of two wells per test were harvested for cytokine analysis. To the remaining two wells per test 10 pl of PrestoBlue™ cell viability reagent, a resazurin-based cell permeable solution (Life Technologies Cat. No. A 13262) was added to each well and transferred back to the incubator for 30 minutes. In viable cells resazurin is converted to fluorescent resorufm. Fluorescence was measured on a SpectraMax ® Gemini™ EM microplate reader (Molecular Devices, USA) with an excitation wavelength of 560 nm and emission wavelength of 590 nm. Data of ISO served as control and were set as 100% for each concentration. FIG.12C shows that there is a four to eight fold difference in antibody concentration needed to achieve similar metabolic activity of human monocytes.
Example 23: TNF-alpha secretion of human PBMCs upon treatment with different antibodies in different concentrations
The supernatants of human monocytes treated as described in Example 22 were send to Eve Technologies for TNF-alpha measurement by MILLIPLEX technology as described in Example 8. Measurements were done in duplicates, the mean as a function of the antibody concentration used is shown in a bar diagram in Figure 12F.
Example 24: Surface expression of CD82, CD 141, CD86 and CD40 upon G2-2MX Human monocytes were isolated as described in Example 1, seeded in 48 well plates as described in Example 2 and treated either with G2-2opti, G2-2MX, control antibody, or left untouched. The antibodies G2-2opti and ISO were used at a concentration of 0.5 mg/ml, the antibody G2-2MX was used at 0.05 mg/ml. After 72 hours cells were harvested and prepared for flow cytometry as described in Example 19. Anti-human antibodies used are: CD82-PerCP-Cy5.5 (Biolegend, clone ASL-24), CD141-PE (Biolegend, clone M80), CD86-BV711 (Biolegend, clone: IT2.2), CD40-APC-Cy7 (Biolegend, clone: 5C3). FIG. 12 shows bar diagrams of CD141, CD86, CD40 and CD82 surface expression on human monocytes anlaysed by flow cytometry. FIG.12D depicts MFI data and FIG.12E percent values. Both, G2-2 and G2-2MX led to enhanced surface expression. Notably, G2-2MX was used at a 10 fold less concentration.
Example 25 : Cloning of construct G2-2MXQ and G2-2HXQ
The single chain 3DX SEQ ID NO: 39 as described in Fujiwara K, Poikonen K et al. A single-chain antibody/epitope system for functional analysis of protein-protein interactions. Biochemistry. 2002;41(42): 12729-12738. doi: 10. 102 l/bi0263309) was synthesized by Genescript (New Jersey). 3DX is linked to the C-terminal Glycine of the heavy chain Fc CH3 of G2-2MX (SEQ ID NO:37) via a short Glycine-Serine linker (GGGS, SEQ ID NO:68). The resulting construct is named G2-2MXQ respectively and is depicted as scheme in Figure 13. The construct is expressed in CHO or 293HEK cells. The resulting protein, a bispecific bivalent single chain Fv-Fc-single chain antibody is purified from cell culture media by protein A affinity chromatography and sterile filtered. Cloning of G2- 2HXQ was done accordingly by linking 3DX to the C-terminal Glycine of the heavy chain Fc CH3 of G2-2HX (SEQ ID NO:38).
Example 26: Peptide loading of G2-2MXQ and G2-2HXQ
Purified antibodies G2-2MXQ and G2-2HXQ were measured and loaded with Myc-SIINFEKL peptide (EQKLISEEDLGSSIINFEKL) (SEQ ID NO:41) or Myc-Influenza Ml peptide (EQKLISEEDLGSGILGFVFTLT) (SEQ ID NO:42) (synthesized by Genscript, NY, USA) at 10-fold molar excess overnight at 4°C. Unbound peptide was removed by size exclusion chromatography and peptide loaded antibody was again sterile filtrated, quantified and kept in PBS at 4°C.
Example 27 : Cloning of construct G2-2-L At the C-Terminus of the kappa light chain of G2-2opti IgGl a Smal restriction site is added to allow for versatile cloning of different peptides to the light chain. This construct is named G2-2-L-L and its scheme depicted in Figure 13. To allow cloning of peptides to the C-Terminus of the Fc heavy chain, a Sma I restriction site has been fit into the nucleotide sequence coding for the last Proline and Glycine residues of the human IgGl heavy chain (SEQ ID:N0 20). This construct is named G2-2-L-H. as depicted in scheme of G2-2-L. Two Cathepsin B cleavage sites are inserted at the N-Terminus of all peptides. Peptides for experiments in murine cells and models include ovalbumin aa257-264 and ovalbumin aa329-338 (SEQ ID NO:43), MC38 Neo-Epitopes REPSI, ADPGK, DPAGT1 (SEQ ID NO:44), MC38 Neo-Epitopes ADPGK, RPL18 (SEQ ID NO:55), melanoma TRP2, pMELlOO (SEQ ID NO:46) and HPV-16 E7 aa5-18, aa 49-57 (SEQ ID NO:47).
Peptides for experiments in human cells and models included HPV-16 E7(aa5-18) and Influenza Ml aa 58-66 (SEQ ID NO:48), SARS Cov2 Nucleoprotein aa 222-235 and EBV (SEQ ID NO:49), SARS Cov2 (SEQ ID NO:50). All peptides were synthesized by Genscript.
The different constructs are expressed in CHO or 293HEK cells. The resulting antibody peptide conjugates are purified from cell culture media by protein A affinity chromatography and sterile filtered.
Example 28: Surface expression of Dendritic Cell maturation markers
Monocytes from a healthy human donor were isolated by negative selection and cultured as described in Examples 1 and 2 in 48 well chamber plates with 0.25 x 106 cells per well. Either G2-2 or EPR3606 (abeam Cat No. ab 109628) or ISO (an irrelevant isotype matched control antibody) in a concentration of 0.5 mg/ml were added to the culture medium for 3 days. Autologous T cells, acquired by negative selection using magnetic bead depletion (Stemcell EasySep human CD8 T cell isolation kit, Cat. No. 17953) and cultured as described in Examples 1 and 2 were added in a ratio of 1: 1 on the top of monocytes for another 48 hours in co-culture supplemented with 2% autologous serum.
T cells and cell culture supernatants were separated from the monocytic cells layer. After centrifugation, the supernatant was collected for cytokine measurement and T cells were saved for downstream application. T cells and monocytes were harvested separately in 2 mM EDTA in PBS for flow cytometry analysis. Monocytes were incubated with human FcR inhibitors (anti CD64 BioLegend Cat. 305002; anti CD32 Stem Cell Technologies Cat. 60012; anti CD16 BioLegend Cat. 302002) for 30 min on ice. T cells and monocytes were washed with FACS buffer (PBS supplemented with 2% FCS, 0.1% sodium azide, 2 mM EDTA) twice, and stained with monoclonal antibodies for 30 min on ice. Antibodies used were CD14-APC (BioLegend, clone M5E2), CD40-AF488 (BioLegend, clone 5C3) and HLA-DR-PB (BioLegend, clone L243). Flow cytometry analysis was performed on a Cytoflex (Beckman Coulter).
Example 29: Cytokine secretion upon treatment of monocytes with different antibodies
Cell culture supernatants collected as described in Example 28 transferred on dry ice to Eve Technologies for cytokine measurement as described in Example 8. A MILLIPLEX Human Cytokine/Chemokine 15-plex kit (HDF15, Millipore, St. Charles, MO, USA) was used according to the manufacturer's protocol. The 13-plex comprised GM-CSF, I IFN-y, IL-lbeta, ILIRa, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL12(p40), IL-12(p70), IL-13, MCP-1 and TNF-alpha. The assay sensitivities of these markers range from 0.1 - 9.5 pg/mL for the 15-plex. Measurements were done in duplicates, the mean is shown in a bar diagram in Figure 14C.
Example 30: Differential binding of antibody EPR3606 (abeam) and G2-2 on GOLPH2 protein and peptide analysed by ELISA
Antibody EPR3606 from patent application CN 108 324 959 A was bought from abeam (Cat. No. ab239985) to test for differential binding on GOLPH2 as compared to G2-2. Specifically, experiments were conducted to test whether EPR3606 binds to soluble GOLPH2.
293HEK cells were transfected with two different cDNAs containing a GP73 AN variant including a C-terminal lOxHIS-Tag and a secretion signal at the N-terminus. One cDNA construct comprised GP73 AN AA 36 to 401 with a R52A mutation, resulting in a non-cleavable variant of the whole extracellular GP73 (eGOLPH2-His R52A). The second cDNA comprised GP73 AN AA 56 to 401, soluble GOLPH2 (sGOLPH2-His). The supernatant of 293HEK cells transfected with either eGOLPH2-His R52A (SEQ ID NO: 76) or sGOLPH2-His (SEQ ID NO: 77) Both proteins were isolated using by IMAC (immobilized metal affinity chromatography) technology. The biotinylated peptide GOLPH2 AA 36-55 was ordered at Genscript.
ELISA was carried out in a 96 well plate (Greiner, Cat. No. 655061) blocked with 1% gelatine in PBS (Sigma #G9391) for one hour and coated overnight with 2pg/ml recombinant Streptavidin (Sigma #S0677). The next morning wells were loaded with 4pg/ml biotinylated human GOLPH2 Peptide AA 36-55 (GenScript), incubated for one hour, washed and developed with 80, 40, 20, 10 or 5ng/ml anti GOLPH2 EPR3606 antibody (Abeam Cat. No. ab239985) followed by secondary anti rabbit-HRP antibody (Jackson Immuno Cat. No. 111-036-047). Chromogenic substrate (ECL Cytiva Amersham Cat. No. 10155854) was applied to detect peroxidase. The reaction was stopped and absorbance was read at 450 nm (FIG. 15A).
In parallel, an ELISA was carried out on a Nickel coated 96 well plate (Thermo Scientific Cat. No. 15442) loaded overnight with lOpg/ml recombinant sGOLPH2-His R52A or sGOLPH2-His protein. The next morning, either anti-GOLPH2 rabbit antibody EPR3606 (Abeam #ab239985) in a concentration of 80, 40, 20, 10 or 5 ng/ml was applied followed by anti rabbit-HRP antibody (Jackson Immuno, Cat. No. 111-036-047) or G2-2 in a concentration of 80, 40, 20, 10 or 5ng/ml followed by anti human-HRP antibody (Jackson Immuno #109-036-097). Chromogenic substrate was applied to detect peroxidase, the reaction was stopped and absorbance was read at 450 nm (FIG. 15A).
Example 31: Differential binding of antibody EPR3606 and G2-2 on GOLPH2 protein analysed by Western Blotting
Purified eGOLPH2-His R52A or sGOLPH2-His were boiled in Laemmli buffer (Bio-Rad, Cat. No. 1610747), 2 pg of each protein was run in triplicate on a gradient polyacrylamid gel (Bio-Rad Cat. No. 4569034) and proteins were transferred onto a nitrocellulose membrane (Fisher Scientific Cat. No. 15259794) by semi-dry transfer using the Trans-Blot® SD Cell from Bio-Rad. The membrane was blocked in 5 % bovine serum albumin (Carl Roth Cat. No. 3737.3) and 0.1 % Tween 20 (Carl Roth Cat. No. 9127.2) in PBS (Carl Roth Cat. No. 9143.2) and probed it either with 2 pg/ml EPR3606 (abeam Cat. No. ab239985) followed by anti rabbit HRP conjugated antibody (Invitrogen Cat. No. A16110) or 2 pg/ml G2-2 followed by anti human HRP conjugated antibody (Sigma Cat. No. A0170) or with 0.5 pg/ml anti His HRP conjugated antibody (BioLegend #652503). HRP conjugated antibodies were detected by Clarity Max ECL substrate (Bio-Rad, Cat. No. 1705062) and luminescence was recorded in a Bio-Rad Chemi Doc.

Claims

CLAIMS An antibody, or an antigen-binding fragment thereof, specifically binding to the extracellular part of GOLPH2 for use in treating a disease or disorder associated with an impaired immune system in a subject. The antibody, or the antigen-binding fragment thereof, for use according to claim 1, wherein binding of the antibody, or the antigen-binding fragment thereof, to the extracellular part of GOLPH2 induces and/or enhances an immune response in said subject. The antibody, or the antigen-binding fragment thereof, for use according to claim 1 or 2, wherein the subject is immunocompromised as a result of a chemotherapy, a radiotherapy, or an infection. The antibody, or the antigen-binding fragment thereof, for use according to claim 3, wherein the infection is an infection with a human immunodeficiency virus. The antibody, or the antigen-binding fragment thereof, for use according to claim 1, wherein the disease or disorder associated with an impaired immune system is cancer, in particular wherein the subject is at risk of developing cancer, suffering from cancer or recovering from cancer. The antibody, or the antigen-binding fragment thereof, for use according to any one of claims 1 to 5, wherein the binding of the antibody, or the antigen-binding fragment thereof, to the extracellular part of GOLPH2 induces the maturation of monocytes into dendritic cells and/or macrophages. The antibody, or the antigen-binding fragment thereof, for use according to any one of claims 1 to 5, wherein the binding of the antibody, or the antigen-binding fragment thereof, to the extracellular part of GOLPH2 induces the maturation of MO macrophages into Ml macrophages. The antibody, or the antigen-binding fragment thereof, for use according to any one of claims 1 to 5, wherein the binding of the antibody, or the antigen-binding fragment thereof, to the extracellular part of GOLPH2 induces the re-polarization of M2 macrophages into Ml macrophages. The antibody, or the antigen-binding fragment thereof, for use according to any one of claims 1 to 5, wherein the binding of the antibody, or the antigen-binding fragment thereof, to the extracellular part of GOLPH2 induces the maturation of monocytes into immature dendritic cells. The antibody, or the antigen-binding fragment thereof, for use according to any one of claims 1 to 9, wherein the binding of the antibody, or the antigen-binding fragment thereof, to the extracellular part of GOLPH2 on the surface of an antigen-presenting cell improves presentation of an antigen by said antigen-presenting cell. The antibody, or the antigen-binding fragment thereof, for use according to claim 10, wherein the antigen-presenting cell is a monocyte, a macrophage, preferably an Ml macrophage, or a dendritic cell, preferably a mature dendritic cell. The antibody, or the antigen-binding fragment thereof, for use according to claim 10 or 11, wherein the antigen is an antigenic peptide. The antibody, or the antigen-binding fragment thereof, for use according to claim 12, wherein the antigenic peptide is comprised in the antibody, or the antigen-binding fragment thereof. The antibody, or the antigen-binding fragment thereof, for use according to claim 12 or 13, wherein the antigenic peptide is fused to a C-terminal end of the antibody, or the antigenbinding fragment thereof. The antibody, or the antigen-binding fragment thereof, for use according to claim 14, wherein the antigenic peptide is fused to the C-terminal end of the antibody, or the antigen-binding fragment thereof, via a peptide linker. The antibody, or the antigen-binding fragment thereof, for use according to claim 14 or 15, wherein the antigenic peptide is fused to the C-terminal end of a heavy chain and/or light chain of an antibody. The antibody, or the antigen-binding fragment thereof, for use according to claim 12 or 13, wherein the antibody is a multispecific antibody comprising a first Fab or scFv portion specifically binding to the extracellular part of GOLPH2 and a second Fab or scFv portion specifically binding to a molecule comprising the antigenic peptide. The antibody, or the antigen-binding fragment thereof, for use according to claim 17, wherein the multispecific antibody forms a complex with said molecule comprising the antigenic peptide. The antibody, or the antigen-binding fragment thereof, for use according to claim 17 or 18, wherein the molecule comprising the antigenic peptide is a fusion protein comprising an antigenic peptide and a polypeptide comprising an epitope that is specifically bound by the Fab or scFv portion of the multispecific antibody. The antibody, or the antigen-binding fragment thereof, for use according to claim 19, wherein the antigenic peptide is fused to the polypeptide comprising the epitope that is specifically bound by the Fab or scFv portion of the target-binding molecule via a peptide linker. The antibody, or the antigen-binding fragment thereof, for use according to claim 19 or 20, wherein the polypeptide comprising the epitope that is specifically bound by the Fab or scFv portion of the target-binding molecule comprises a peptide tag, in particular wherein the peptide tag is a Myc-tag, an HA-tag, a FLAG-tag or a V5 tag. The antibody, or the antigen-binding fragment thereof, for use according to claim 12 or 13, wherein the antigen has been released by a physical therapeutic intervention. The antibody, or the antigen-binding fragment thereof, for use according to claim 22 wherein the physical therapeutic intervention is cryotherapy, surgery, radiotherapy and/or laser therapy. The antibody, or the antigen-binding fragment thereof, for use according to any one of claims 12 to 23 wherein the antigen is a tumor antigen or a pathogen-derived antigen. The antibody, or the antigen-binding fragment thereof, for use according to any one of claims 1 to 24, wherein the antibody is a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody and/or a multispecific antibody.
145 The antibody, or the antigen-binding fragment thereof, for use according to claim 25, wherein the multispecific antibody comprises a first Fab or scFv portion specifically binding to the extracellular part of GOLPH2 and at least one further Fab or scFv portion specifically binding to an immune checkpoint molecule or a ligand of an immune checkpoint molecule. The antibody, or the antigen-binding fragment thereof, for use according to claim 26, wherein the immune checkpoint molecule is selected from a group consisting of: CTLA4, PD-1, PD-L1, LAG3, TIM3, CD28, ICOS, SLAM, CD2, CD27, 0X40, 4-1BB, CD30, GITR, CD40L, DR3, CD 122, LIGHT, TIGIT, VISTA, B7-H3 and BTLA; and/or wherein the ligand of the immune checkpoint molecule is selected from a group consisting of: CD80, CD86, PD-L1, PD-L2 and GAL9. The antibody, or the antigen-binding fragment thereof, for use according to any one of claims 1 to 27, wherein the antibody is a fusion antibody and wherein the fusion antibody comprises an Fc region and two or more scFv fragments. The antibody, or the antigen-binding fragment thereof, for use according to claim 28, wherein each scFv fragment is connected to the Fc region with a peptide linker. The antibody, or the antigen-binding fragment thereof, for use according to any one of claims 1 to 29, wherein the antibody, or the antigen-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NOV; or (b) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO. 17. The antibody, or the antigen-binding fragment thereof, for use according to any one of claims 1 to 30, wherein the antibody, or the antigen-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NO:4, CDR2 as defined in SEQ ID NO:5 and CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO.7, CDR2 as defined in SEQ ID NO:8 and CDR3 as defined in SEQ ID NOV; or (b) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NO: 12, CDR2 as defined in SEQ ID NO: 13 and CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO: 15, CDR2 as defined in SEQ ID NO: 16 and CDR3 as defined in SEQ ID NO: 17. The antibody, or the antigen-binding fragment thereof, for use according to any one of claims 1 to 31, wherein the antibody, or the antigen-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 18 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:2 or SEQ ID NO: 18; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO: 19 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:3 or SEQ ID NO: 19; or (b) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NOTO or a sequence having 90%, preferably 95% sequence identity to SEQ ID NOTO; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 11. An antibody-antigenic peptide construct comprising an antibody, or an antigen-binding fragment thereof, specifically binding to the extracellular part of GOLPH2 and an antigenic peptide, wherein the antigenic peptide is fused to a C-terminal end of the antibody, or the targetbinding fragment thereof. The antibody-antigenic peptide construct according to claim 33, wherein the antigenic peptide is fused to a C-terminal end of the antibody, or the target-binding fragment thereof, via a peptide linker. The antibody-antigenic peptide construct according to claim 33 or 34, wherein the antigenic peptide is fused to the C-terminal end of a heavy chain and/or light chain of an antibody. The antibody-antigenic peptide construct according to any one of items 33 to 35, wherein the antibody is a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody or a multispecific antibody. The antibody-antigenic peptide construct according to any one of items 33 to 35, wherein the antibody is a fusion antibody comprising an Fc region and two or more scFv fragments. The antibody-antigenic peptide construct according to claim 36, wherein the multispecific antibody comprises a further Fab or scFv portion specifically binding to an immune checkpoint molecule or a ligand of an immune checkpoint molecule. The antibody-antigenic peptide construct according to claim 38, wherein the immune checkpoint molecule is selected from a group consisting of: CTLA4, PD-1, PD-L1, LAG3, TIM3, CD28, ICOS, SLAM, CD2, CD27, 0X40, 4-1BB, CD30, GITR, CD40L, DR3, CD122, LIGHT, TIGIT, VISTA, B7-H3 and BTLA; and/or wherein the ligand of the immune checkpoint molecule is selected from a group consisting of: CD80, CD86, PD-L1, PD-L2 and GAL9. An antibody-antigenic peptide complex comprising a multispecific antibody, wherein the multispecific antibody comprises a first Fab or scFv portion specifically binding to the extracellular part of GOLPH2 and a second Fab or scFv portion specifically binding to a molecule comprising an antigenic peptide. The antibody-antigenic peptide complex according to claim 40, wherein the second Fab or scFv portion forms a complex with said molecule comprising the antigenic peptide. The antibody-antigenic peptide complex according to claim 40 or 41, wherein the molecule comprising the antigenic peptide is a fusion protein comprising the antigenic peptide fused to a polypeptide comprising an epitope that is specifically bound by the second Fab or scFv portion. The antibody-antigenic peptide complex according to any one of claims 40 to 42, wherein the antigenic peptide is fused to the polypeptide comprising the epitope that is specifically bound by the second Fab or scFv portion via a peptide linker. The antibody-antigenic peptide complex according to claim 42 or 43, wherein the polypeptide comprising the epitope that is specifically bound by the second Fab or scFv portion comprises a peptide tag and wherein the second Fab or scFv portion specifically binds to said peptide tag, in particular wherein the peptide tag is a Myc-tag, an HA-tag, a FLAG-tag or a V5-tag. The antibody-antigenic peptide complex according to any one of claims 40 to 44, wherein the multispecific antibody comprises a further Fab or scFv portion specifically binding to an immune checkpoint molecule or a ligand of an immune checkpoint molecule. The antibody-antigenic peptide construct or complex according to claim 45, wherein the immune checkpoint molecule is selected from a group consisting of: CTLA4, PD-1, PD-L1, LAG3, TIM3, CD28, ICOS, SLAM, CD2, CD27, 0X40, 4-1BB, CD30, GITR, CD40L, DR3, CD 122, LIGHT, TIGIT, VISTA, B7-H3 and BTLA; and/or wherein the ligand of the immune checkpoint molecule is selected from a group consisting of: CD80, CD86, PD-L1, PD-L2 and
148 GAL9. A fusion antibody specifically binding to GOLPH2, wherein the fusion antibody comprises an Fc region and two or more scFv fragments. The fusion antibody according to claim 47, wherein each scFv fragment is connected to the Fc region with a peptide linker. The fusion antibody according to claim 48, wherein the peptide linker has a length between 2 and 50 amino acid residues. The fusion antibody according to claims 48 or 49, wherein the peptide linker comprises or consists of a sequence as set forth in SEQ ID NO.52 or SEQ ID NO:53. The antibody-antigenic peptide construct according to any one of claims 33 to 39, the antibody- antigenic peptide complex according to any one of claims 40 to 46 or the fusion antibody according to any one of claims 47 to 50 comprising (a) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO.9; or (b) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO.17. The antibody-antigenic peptide construct according to any one of claims 33 to 39, the antibody- antigenic peptide complex according to any one of claims 40 to 46 or the fusion antibody according to any one of claims 47 to 50 comprising (a) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NO:4, CDR2 as defined in SEQ ID NO:5 and CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO.7, CDR2 as defined in SEQ ID NO:8 and CDR3 as defined in SEQ ID NO:9; or (b) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NO: 12, CDR2 as defined in SEQ ID NO: 13 and CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO: 15, CDR2 as defined in SEQ ID NO: 16 and CDR3 as defined in SEQ ID NO: 17. The antibody-antigenic peptide construct according to any one of claims 33 to 39, the antibody- antigenic peptide complex according to any one of claims 40 to 46 or the fusion antibody according to any one of claims 47 to 50 comprising (a) a variable heavy (VH) chain sequence
149 comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 18 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:2 or SEQ ID NO: 18; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO: 19 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:3 or SEQ ID NO: 19; or (b) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO: 10 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 10; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 11. An immunoconjugate comprising the antibody-antigenic peptide construct, the antibody- antigenic peptide complex or the fusion antibody according to any one of claims 33 to 53 and a cytotoxic agent or a prodrug of a cytotoxic agent. A polynucleotide encoding the antibody-antigenic peptide construct, the antibody-antigenic peptide complex or the fusion antibody according to any one of claims 33 to 53. A cell comprising the polynucleotide according to item 55. A method for producing the antibody-antigenic peptide construct, the antibody-antigenic peptide complex or the fusion antibody according to any one of claims 33 to 53, the method comprising a step of culturing the cell according to claim 56. A pharmaceutical composition comprising the antibody-antigenic peptide construct, the antibody-antigenic peptide complex or the fusion antibody according to any one of claims 33 to 53 and/or the immunoconjugate according to claim 54 and further comprising a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 58 further comprising at least one therapeutic agent. The pharmaceutical composition according to claim 59, wherein the therapeutic agent is at least one of a vaccine, an antigen, an adjuvant, a chemotherapeutic agent and an immune checkpoint modulator. Use of an antibody specifically binding to the extracellular part of GOLPH2, or an antigenbinding fragment thereof, as an adjuvant in a vaccination therapy.
150 The use according to claim 61, wherein the antibody is a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody or a multispecific antibody. The use according to claim 61 or 62, wherein the antibody is the antibody-antigenic peptide construct, the antibody-antigenic peptide complex or the fusion antibody according to any one of claims 33 to 53. The use according to any one of claims 61 to 63, wherein the adjuvant initiates or enhances the function of antigen-presenting cells. The use according to any one of claims 61 to 64, wherein the vaccination therapy comprises administration of a viral antigen, a microbial antigen or a tumor antigen. The use according to claim 65, wherein at least one antigenic peptide derived from the viral antigen, the microbial antigen or the tumor antigen is comprised in the antibody-antigenic peptide construct according to any one of claims 33 to 39 or the antibody-antigenic peptide complex according to any one of claims 40 to 46. The use according to any of claims 61 to 66, wherein the antibody, or the antigen-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO.9; or (b) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO. 17. The use according to any of claims 61 to 67, wherein the antibody, or the antigen-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NON, CDR2 as defined in SEQ ID NO:5 and CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NOT, CDR2 as defined in SEQ ID NO:8 and CDR3 as defined in SEQ ID NO:9; or (b) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NO: 12, CDR2 as defined in SEQ ID NO: 13 and CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO: 15, CDR2 as defined in SEQ ID NO: 16 and CDR3 as defined in SEQ ID NO: 17.
151 The use according to any of claims 61 to 68, wherein the antibody, or the antigen-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 18 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:2 or SEQ ID NO: 18; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO: 19 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:3 or SEQ ID NO: 19; or (b) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NOTO or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 10; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 11. An in vitro method for inducing the maturation of monocytes and/or monocyte -derived cells, the method comprising the steps of: a) culturing monocytes and/or monocyte-derived cells in a cell culture medium; b) adding an antibody specifically binding to the extracellular part of GOLPH2, or an antigenbinding fragment thereof, to the cell culture of step (a); and c) obtaining matured monocytes and/or monocyte-derived cells. The method according to claim 70, wherein the monocyte -derived cell is an M0 macrophage or an immature dendritic cell. The method according to claim 70 or 71, wherein the antibody is a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody or a multispecific antibody. The method according to claim any one of claims 70 to 72, wherein the antibody is the antibody-antigenic peptide construct, the antibody-antigenic peptide complex or the fusion antibody according to any one of claims 33 to 53. The method according to any of claims 70 to 73, wherein the antibody, or the antigen-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO.9; or (b) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO. 17. The method according to any of claims 70 to 74, wherein the antibody, or the antigen-binding
152 fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NO:4, CDR2 as defined in SEQ ID NO:5 and CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO.7, CDR2 as defined in SEQ ID NO:8 and CDR3 as defined in SEQ ID NO:9; or (b) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NO: 12, CDR2 as defined in SEQ ID NO: 13 and CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO: 15, CDR2 as defined in SEQ ID NO: 16 and CDR3 as defined in SEQ ID NO: 17. The method according to any of claims 70 to 75, wherein the antibody, or the antigen-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 18 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:2 or SEQ ID NO: 18; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO: 19 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:3 or SEQ ID NO: 19; or (b) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NOTO or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 10; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 11. A method for generating a cell displaying an antigenic peptide, the method comprising the steps of: a) generating a matured monocyte or monocyte-derived cell according to the method of any one of claims 70 to 76; b) pulsing an antigenic peptide on the matured monocyte or monocyte-derived cell generated in step (a); and/or introducing a nucleic acid encoding a polypeptide comprising an antigenic peptide into the matured monocyte or monocyte-derived cell generated in step (a); and/or introducing an antigenic peptide into the matured monocyte or monocyte- derived cell as part of an antibody-antigenic peptide complex or an antibody- antigenic peptide construct; and c) obtaining a cell displaying an antigenic peptide. A method for generating an activated T cell, the method comprising the steps of: a) generating a cell displaying an antigenic peptide with the method according to
153 claim 77; b) contacting the cell of step (a) with a T cell, wherein the T cell comprises a T cell receptor that recognizes the antigenic peptide displayed by the cell of step (a); and c) obtaining an activated T cell. The method according to claim 78, wherein the cell displaying the antigenic peptide and the T cell have been obtained from the same subject. A cell displaying an antigenic peptide and/or an activated T cell for use in adoptive cell transfer, wherein the cell displaying the antigenic peptide has been obtained with the method according to any one of claims 70 to 76 and/or wherein the activated T cell has been obtained with the method according to claim 78 or 79. A method for inducing and/or enhancing an immune response against an antigen in a subject, the method comprising the steps of: a) administering to said subject an antibody specifically binding to the extracellular part of GOLPH2, or an antigen-binding fragment thereof; and b) inducing and/or enhancing an immune response in said subject. The method according to claim 81, wherein the antibody is a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody or a multispecific antibody. The method according to claim any one of claims 81 or 82, wherein the antibody is the antibody-antigenic peptide construct, the antibody-antigenic peptide complex or the fusion antibody according to any one of claims 33 to 53. The method according to any of claims 81 to 83, wherein the antibody, or the antigen-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO:6; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO.9; or (b) a variable heavy (VH) chain sequence comprising CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR3 as defined in SEQ ID NO. 17. The method according to any of claims 81 to 84, wherein the antibody, or the antigen-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising CDR1 as
154 defined in SEQ ID NO:4, CDR2 as defined in SEQ ID N0:5 and CDR3 as defined in SEQ ID N0:6; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO.7, CDR2 as defined in SEQ ID NO:8 and CDR3 as defined in SEQ ID NOV; or (b) a variable heavy (VH) chain sequence comprising CDR1 as defined in SEQ ID NO: 12, CDR2 as defined in SEQ ID NO: 13 and CDR3 as defined in SEQ ID NO: 14; and a variable light (VL) chain sequence comprising CDR1 as defined in SEQ ID NO: 15, CDR2 as defined in SEQ ID NO: 16 and CDR3 as defined in SEQ ID NO: 17. The method according to any of claims 81 to 85, wherein the antibody, or the antigen-binding fragment thereof, comprises (a) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 18 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:2 or SEQ ID NO: 18; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO: 19 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO:3 or SEQ ID NO: 19; or (b) a variable heavy (VH) chain sequence comprising the amino acid sequence of SEQ ID NOTO or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 10; and a variable light (VL) chain sequence comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having 90%, preferably 95% sequence identity to SEQ ID NO: 11.
155
EP21769136.9A 2020-08-27 2021-08-27 Anti-golph2 antibodies for macrophage and dendritic cell differentiation Withdrawn EP4204448A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP20193168 2020-08-27
PCT/EP2021/073781 WO2022043517A2 (en) 2020-08-27 2021-08-27 Anti-golph2 antibodies for macrophage and dendritic cell differentiation

Publications (1)

Publication Number Publication Date
EP4204448A2 true EP4204448A2 (en) 2023-07-05

Family

ID=72290795

Family Applications (1)

Application Number Title Priority Date Filing Date
EP21769136.9A Withdrawn EP4204448A2 (en) 2020-08-27 2021-08-27 Anti-golph2 antibodies for macrophage and dendritic cell differentiation

Country Status (2)

Country Link
EP (1) EP4204448A2 (en)
WO (1) WO2022043517A2 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115925810A (en) * 2022-08-20 2023-04-07 北京华诺泰生物医药科技有限公司 Polypeptide of target antigen presenting cell, nano vaccine and application

Family Cites Families (73)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR6801650D0 (en) 1968-08-20 1973-03-07 R Maes PROCESS FOR PREPARING INTERFERON GENERATOR COMPLEXES
US3725545A (en) 1971-02-03 1973-04-03 R Maes Enhancement of antibody production by nucleic acid-polycation complexes
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4737456A (en) 1985-05-09 1988-04-12 Syntex (U.S.A.) Inc. Reducing interference in ligand-receptor binding assays
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
IL85035A0 (en) 1987-01-08 1988-06-30 Int Genetic Eng Polynucleotide molecule,a chimeric antibody with specificity for human b cell surface antigen,a process for the preparation and methods utilizing the same
WO1988007089A1 (en) 1987-03-18 1988-09-22 Medical Research Council Altered antibodies
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5959177A (en) 1989-10-27 1999-09-28 The Scripps Research Institute Transgenic plants expressing assembled secretory antibodies
GB9015198D0 (en) 1990-07-10 1990-08-29 Brien Caroline J O Binding substance
US5877397A (en) 1990-08-29 1999-03-02 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
DK0590058T3 (en) 1991-06-14 2004-03-29 Genentech Inc Humanized heregulin antibody
WO1993006217A1 (en) 1991-09-19 1993-04-01 Genentech, Inc. EXPRESSION IN E. COLI OF ANTIBODY FRAGMENTS HAVING AT LEAST A CYSTEINE PRESENT AS A FREE THIOL, USE FOR THE PRODUCTION OF BIFUNCTIONAL F(ab')2 ANTIBODIES
WO1993011236A1 (en) 1991-12-02 1993-06-10 Medical Research Council Production of anti-self antibodies from antibody segment repertoires and displayed on phage
EP0714409A1 (en) 1993-06-16 1996-06-05 Celltech Therapeutics Limited Antibodies
FR2707189B1 (en) 1993-07-09 1995-10-13 Gradient Ass Method for treating combustion residues and installation for implementing said method.
US5789199A (en) 1994-11-03 1998-08-04 Genentech, Inc. Process for bacterial production of polypeptides
US5840523A (en) 1995-03-01 1998-11-24 Genetech, Inc. Methods and compositions for secretion of heterologous polypeptides
US6267958B1 (en) 1995-07-27 2001-07-31 Genentech, Inc. Protein formulation
GB9603256D0 (en) 1996-02-16 1996-04-17 Wellcome Found Antibodies
WO1998003532A1 (en) 1996-07-24 1998-01-29 Hanna Michelle M Base-protected nucleotide analogs with protected thiol groups
US6171586B1 (en) 1997-06-13 2001-01-09 Genentech, Inc. Antibody formulation
EP0994903B1 (en) 1997-06-24 2005-05-25 Genentech, Inc. Methods and compositions for galactosylated glycoproteins
US6040498A (en) 1998-08-11 2000-03-21 North Caroline State University Genetically engineered duckweed
WO1999022764A1 (en) 1997-10-31 1999-05-14 Genentech, Inc. Methods and compositions comprising glycoprotein glycoforms
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
IL138608A0 (en) 1998-04-02 2001-10-31 Genentech Inc Antibody variants and fragments thereof
WO1999054342A1 (en) 1998-04-20 1999-10-28 Pablo Umana Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
PL209786B1 (en) 1999-01-15 2011-10-31 Genentech Inc Variant of mother polypeptide containing Fc region, polypeptide containing variant of Fc region with altered affinity of Fc gamma receptor binding (Fc R), polypeptide containing variant of Fc region with altered affinity of Fc gamma neonatal receptor binding (Fc Rn), composition, isolated nucleic acid, vector, host cell, method for obtaining polypeptide variant, the use thereof and method for obtaining region Fc variant
ES2420835T3 (en) 1999-04-09 2013-08-27 Kyowa Hakko Kirin Co., Ltd. Procedure to control the activity of immunofunctional molecules
US7125978B1 (en) 1999-10-04 2006-10-24 Medicago Inc. Promoter for regulating expression of foreign genes
ATE303445T1 (en) 1999-10-04 2005-09-15 Medicago Inc METHOD FOR REGULATION OF TRANSCRIPTION OF FOREIGN GENES IN THE PRESENCE OF NITROGEN
JP4668498B2 (en) 1999-10-19 2011-04-13 協和発酵キリン株式会社 Method for producing polypeptide
AU8470301A (en) 2000-08-03 2002-02-18 Wim-Van Schooten Production of humanized antibodies in transgenic animals
AU2002218166A1 (en) 2000-09-08 2002-03-22 Universitat Zurich Collections of repeat proteins comprising repeat modules
US7064191B2 (en) 2000-10-06 2006-06-20 Kyowa Hakko Kogyo Co., Ltd. Process for purifying antibody
CN103333860B (en) 2000-10-06 2015-07-08 协和发酵麒麟株式会社 Cells producing antibody compositions
US6946292B2 (en) 2000-10-06 2005-09-20 Kyowa Hakko Kogyo Co., Ltd. Cells producing antibody compositions with increased antibody dependent cytotoxic activity
AU1325102A (en) 2000-10-16 2002-04-29 Phylos Inc Protein scaffolds for antibody mimics and other binding proteins
US20030157561A1 (en) 2001-11-19 2003-08-21 Kolkman Joost A. Combinatorial libraries of monomer domains
AU2002339845B2 (en) 2001-08-03 2009-03-26 Roche Glycart Ag Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity
MXPA04003798A (en) 2001-10-25 2004-07-30 Genentech Inc Glycoprotein compositions.
US20040093621A1 (en) 2001-12-25 2004-05-13 Kyowa Hakko Kogyo Co., Ltd Antibody composition which specifically binds to CD20
US7691568B2 (en) 2002-04-09 2010-04-06 Kyowa Hakko Kirin Co., Ltd Antibody composition-containing medicament
ATE503829T1 (en) 2002-04-09 2011-04-15 Kyowa Hakko Kirin Co Ltd CELL WITH REDUCED OR DELETED ACTIVITY OF A PROTEIN INVOLVED IN GDP-FUCOSE TRANSPORT
CA2481658A1 (en) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Method of enhancing of binding activity of antibody composition to fcy receptor iiia
AU2003236019A1 (en) 2002-04-09 2003-10-20 Kyowa Hakko Kirin Co., Ltd. Drug containing antibody composition appropriate for patient suffering from Fc Gamma RIIIa polymorphism
WO2003085118A1 (en) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Process for producing antibody composition
EA200401325A1 (en) 2002-04-09 2005-04-28 Киова Хакко Когио Ко., Лтд. CELLS WITH MODIFIED GENOM
US7361740B2 (en) 2002-10-15 2008-04-22 Pdl Biopharma, Inc. Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis
SI1572744T1 (en) 2002-12-16 2010-09-30 Genentech Inc Immunoglobulin variants and uses thereof
US7871607B2 (en) 2003-03-05 2011-01-18 Halozyme, Inc. Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases
AU2004279742A1 (en) 2003-10-08 2005-04-21 Kyowa Hakko Kirin Co., Ltd. Fused protein composition
US20070134759A1 (en) 2003-10-09 2007-06-14 Harue Nishiya Process for producing antibody composition by using rna inhibiting the function of alpha1,6-fucosyltransferase
US20050164301A1 (en) 2003-10-24 2005-07-28 Avidia Research Institute LDL receptor class A and EGF domain monomers and multimers
TR201809892T4 (en) 2003-11-05 2018-07-23 Roche Glycart Ag Antigen binding molecules with binding affinity to the Fc receptor and enhanced effector function.
JPWO2005053742A1 (en) 2003-12-04 2007-06-28 協和醗酵工業株式会社 Medicament containing antibody composition
DK1737891T3 (en) 2004-04-13 2013-03-25 Hoffmann La Roche ANTI-P-selectin ANTIBODIES
TWI380996B (en) 2004-09-17 2013-01-01 Hoffmann La Roche Anti-ox40l antibodies
KR101270829B1 (en) 2004-09-23 2013-06-07 제넨테크, 인크. Cystein engineered antibodies and conjugates
JO3000B1 (en) 2004-10-20 2016-09-05 Genentech Inc Antibody Formulations.
US20080226635A1 (en) 2006-12-22 2008-09-18 Hans Koll Antibodies against insulin-like growth factor I receptor and uses thereof
KR101378919B1 (en) 2010-01-28 2014-04-14 포항공과대학교 산학협력단 System biological method of biomarker selection for diagnosis of lung cancer, subtype of lung cancer, and biomarker selected by the same
CN103517717B (en) 2011-02-16 2017-10-17 康奈尔大学 The cell-mediated immune method of enhancing
US20150045245A1 (en) 2011-12-08 2015-02-12 Biocartis Nv Biomarkers and test panels useful in systemic inflammatory conditions
CA2865597A1 (en) 2012-03-16 2013-09-19 Covagen Ag Novel binding molecules with antitumoral activity
US9469686B2 (en) 2013-03-15 2016-10-18 Abbott Laboratories Anti-GP73 monoclonal antibodies and methods of obtaining the same
CN105699653B (en) 2014-11-25 2017-08-25 北京市肝病研究所 A kind of hypersensitization super-paramagnetism nano immune microsphere and its method for detecting GP73 antigens
CN105734059A (en) 2015-12-07 2016-07-06 中国医学科学院基础医学研究所 GP73 inhibitor and application thereof
CN110023334B (en) * 2016-11-21 2023-11-14 科雅博有限责任公司 anti-GP 73 antibodies and immunoconjugates
CN108324959A (en) * 2018-02-05 2018-07-27 山西医科大学 Anti-GOLPH2-lipoplexes liposome vectors and its preparation and application

Also Published As

Publication number Publication date
WO2022043517A2 (en) 2022-03-03
WO2022043517A3 (en) 2022-06-16

Similar Documents

Publication Publication Date Title
US11692038B2 (en) Antibodies that bind chemokine (C-C motif) receptor 8 (CCR8)
JP7068736B2 (en) Antibodies that bind to CD39 and their use
JP6875295B2 (en) TIGIT binding substance and its usage
JP2021040641A (en) Agents for treatment of cancer diseases expressing claudin
AU2020301161A1 (en) FLT3L-Fc fusion proteins and methods of use
CN112672753A (en) Fusion constructs and methods of use thereof
WO2016194992A1 (en) Combined use of immune activators
KR20180081606A (en) FGFR2 inhibitor alone or in combination with an immunostimulant in cancer therapy
JP7360441B2 (en) Mesothelin and CD137 binding molecules
JP2022552748A (en) Treatment of hematological cancers with anti-CD47 and anti-CD20
JP2022529154A (en) Anti-MERTK antibody and how to use it
US20220389394A1 (en) METHODS OF USING FLT3L-Fc FUSION PROTEINS
WO2022043517A2 (en) Anti-golph2 antibodies for macrophage and dendritic cell differentiation
KR20230156727A (en) Anti-VSIG4 antibody or antigen-binding fragment thereof and uses

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20230323

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20231017