WO2017013231A1 - Épitopes t et b dans la protéine de surface spermatique sp17 à titre de vaccins contre le cancer et de cibles d'anticorps - Google Patents

Épitopes t et b dans la protéine de surface spermatique sp17 à titre de vaccins contre le cancer et de cibles d'anticorps Download PDF

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WO2017013231A1
WO2017013231A1 PCT/EP2016/067468 EP2016067468W WO2017013231A1 WO 2017013231 A1 WO2017013231 A1 WO 2017013231A1 EP 2016067468 W EP2016067468 W EP 2016067468W WO 2017013231 A1 WO2017013231 A1 WO 2017013231A1
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cancer
antigen
antibody
peptide
cells
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Sue D. XIANG
Magdalena Plebanski
Arne Heyerick
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate

Definitions

  • the present invention relates to peptide antigens corresponding to autoantigenic and therapeutic epitopes present on human sperm surface protein Sp17, in addition to therapeutic and preventive methods employing such peptides and pharmaceutical compositions comprising same, including vaccine and antibody compositions.
  • Immunotherapy strategies including anti-cancer vaccines, are considered to be less toxic and more specific than many current treatments for cancer, including ovarian cancer, and are also regarded as having great potential to significantly benefit cancer patients in need thereof.
  • tumor associated antigens have been identified in ovarian cancer cells, including cerebellar degeneration-related protein cdr2, p53, HER2/neu, mesothelin, folate receptor-alpha, cancer testis antigens, such as NY-ESO-1 , sperm surface protein Sp17, cancer antigen CA-125 and MUC1 (Kandalaft L. et a!., 201 1 ).
  • Sp17 comprises 151 amino acids that are highly conserved (i.e. 94% homology between mouse and human), highly expressed in spermatozoa, and presented as a cancer antigen in various models and studies (Arnaboldi F. et al., 20 4). Aside from being expressed in the testis, Sp17 is aberrantly expressed in cancers of unrelated histological origin, including multiple myeloma, ovarian cancer, nervous system tumors, and esophageal squamous cell cancer.
  • Sp17-derived sequences have shown great potential as candidate vaccines for use in immunotherapy, including the use of adjuvants such as CpG, which was demonstrated in a syngeneic murine model of ovarian cancer where CpG-adjuvant Sp17 vaccines exhibited both therapeutic and prophylactic activity, and also increased overall levels of CTL responses, thereby suggesting that antigen specific responses were induced (Chiriva-lnternati M. et al., 2010).
  • Sp17 acts as an autoantigen in humans, this protein is highly immunogenic in vivo.
  • Sp17 protein based vaccines have shown promising protective and therapeutic efficacy in several animal cancer models.
  • unnecessary antigenic load in a protein, and production limitations commonly associated with recombinant protein have hindered vaccine development using the protein target.
  • peptide vaccines engineered using short peptide fragments displaying immunogenic epitopes in combination with an optimal carrier/adjuvant offer an attractive alternative therapeutic strategy, possibly avoiding undesirable anti-allergenic responses.
  • peptide-based vaccines may take advantage of emergent computational paradigms that involve immunoinformatic prediction, thereby facilitating the identification of T cell and B cell epitopes within protein antigens, in particular from cancer (auto) antigens which can be developed into vaccine and antibody targets for a variety of therapeutic uses.
  • Sp17-derived peptides have been previously described in connection with autoantigens, for instance, in cancer treatments, diagnosis, and/or vaccination (W01995/0 5764; WO2002/068451 ; WO2013/040071 ; WO2014/127006).
  • the present invention relates to a fragment of human Sp17 (hSp17) corresponding to amino acids 1 1 1 -142 (i.e. hSp17 1 - 42 , having a sequence of KEKEEVAAVKSQAAFRGHIAREEAKKMKTNSL; SEQ ID NO:1 ), which acts as a strongly immunogenic or immunodominant portion of hSp17.
  • This fragment provides new peptide sequences, each useful as an immunogen, especially for cancer vaccine compositions.
  • the hSp17 fragment is described herein in the context of a therapeutic mouse model, as further characterized using human cells or tissues such as those obtained from clinical samples.
  • the presently disclosed therapeutic peptides can include 1 , 2, 3, or more amino acid substitutions within hSp17 111-142 , preferably conservative amino acid substitutions, and/or may consist of an hSpl 7i i i-i 42 fragment containing from 9 to 31 amino acids.
  • Exemplary peptide sequences include hSpl 7m-i 2 4 (corresponding to KEKEEVAAVKIQAA; SEQ ID NO:2), hSp17 12 i-i 38 (corresponding to IQAAFRGHIAREEAKKMK; SEQ ID NO:3), hSp17 134-142 (corresponding to AKKMKTNSL; SEQ ID NO:4), and hSp17 1 l 5- i 3 3 (EVAAVKIQAAFRGHIAREE; SEQ ID NO:5; corresponding to IQ motif of hSp17; Wen et al., 1999).
  • These peptide sequences may be included in a longer peptide that incorporates the sequence of said antigenic fragment, and which further includes from 1 to 10 additional amino acids at the N- terminus and/or the C-terminus.
  • the hSp17 111-142 peptide and the above-mentioned derived sequences are useful as immunogens and in the preparation of pharmaceutical compositions, in particular anti-cancer vaccine compositions, and also in methods for treating or preventing cancer (in particular ovarian cancer), whereby Sp17epoc is preferably administered using a liquid or solid formulation further comprising a carrier, excipients, and/or adjuvants.
  • Sp17epoc is preferably administered using a liquid or solid formulation further comprising a carrier, excipients, and/or adjuvants.
  • antibodies directed against Sp17 ep0 c represent useful pharmaceutical compositions, in particular anti-cancer compositions, which are preferably administered using a liquid or solid formulation further comprising a carrier, excipients, and/or adjuvants.
  • These vaccine and antibody compositions are useful in methods for treating or preventing cancer, preferably ovarian cancer,
  • a further therapeutic approach relates to methods for generating hSp-17- specific immune effector cells ex vivo, comprising pulsing antigen presenting cells with Sp1 7epoc, and contacting the pulsed antigen presenting cells with immune effector cells for a time sufficient to stimulate Sp17-reactive immune effector cells under conditions permissive for proliferation of Sp17-reactive immune effector cells for generating Sp1 7- specific immune effector cells.
  • the antigen presenting cells can be dendritic cells and the immune effector cells can be cytotoxic T lymphocytes.
  • the present invention provides methods of treating cancer by administering to a patient in need thereof a composition comprising a compound based on Sp17 epoc that is a Sp17 ep0 c agent, for instance, a Sp17 ep0 c-based vaccine or an antibody binding Sp17 epoc , preferably when Sp17 epoc is detected in cancer cells (preferably on the cell surface).
  • the present invention provides methods of administering Sp17 ep0 c agent together with an agonist therapeutic agent that is directed at one or more adjuvant, one or more anti-cancer agent, or one or more immune effector cells in a subject.
  • Sp17 ep0 c expression !eve! is determined before, substantia!ly simultaneously with, and/or after administration of one or more doses of a Sp17 ep0 c agent.
  • a human sample preferably including one or more biopsies or tumor ascites (as circulating cells, peritumoral cells, and/or intratumoral cells), in particular by determining expression levels using an anti-Sp17 ep0 c antibody according to the present invention.
  • the present methods are applied or administered to subjects that have been diagnosed with cancer, in particular from a cancer that is selected from the group of hematologic malignances including acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, multiple myeloma, ASDS-related lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, Langerhans cell histiocytosis, and myeloproliferative neoplasms.
  • a cancer that is selected from the group of hematologic malignances including acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, multiple myeloma, ASDS-related lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, Langerhans cell
  • the cancer is selected from solid tumors including breast carcinoma, squamous cell carcinoma, colon cancer, head and neck cancer, lung cancer, genitourinary cancer, ovarian cancer, rectal cancer, gastric cancer, sarcoma, melanoma, and esophageal cancer.
  • a Sp17 ep0 c agent is or comprises a Sp17 epoc antibody, an aptamer, or other non-immunoglobulin compound that specifically binds to Sp17 epoc .
  • a Sp17 epoc antibody is or comprises an antibody agent that specifically binds to Sp17 epoc on surfaces of cancer cells, in some embodiments, the antibody agent is or comprises a polyclonal antibody, a monoclonal antibody, a humanized or human antibody, or includes antigen binding elements of such antibody.
  • the antibody agent is or comprises Sp17 ep oc binding elements thereof (i.e.
  • an antibody agent is a multi-specific agent, such as a bispecific antibody, in particular binding specifically to Sp17 epoc and to a cancer antigen, including CD19, CD20, CD22, CD38, CD52, CD137, CD33, CD138, CD254, CD261 , CD262, CD309, CD319, CD326, PD-1 , PD-L1 , VEGF, EGFR, and HER3.
  • a cancer antigen including CD19, CD20, CD22, CD38, CD52, CD137, CD33, CD138, CD254, CD261 , CD262, CD309, CD319, CD326, PD-1 , PD-L1 , VEGF, EGFR, and HER3.
  • the Sp17 epoc agent may be administered in combination with one or more other agents and/or therapeutic regimens commonly used in the treatment of cancer, in particular those cancer presenting Sp17 epoc -expressing cells as determined above.
  • the Sp17 epoc agent and/or therapeutic regimens involve the use and/or administration of chemotherapeutic agents, radiotherapy, immunotherapeutic agents, and/or inhibitors of kinases and of other cancer-relevant pathways.
  • the present invention further provides a variety of kits and/or articles of manufacture containing components relevant to administering Sp17 ep0 c agents for therapeutic uses and/or detection of Sp17 epoc expression, particularly the expression within and/or on the surfaces of cancer cells, and within patient samples (such as biopsies, tumor ascites, sera, plasma, and other biological materials).
  • FSG. 2 Immunogenicity of recombinant mouse Sp17 (rmSp17) protein combined with a CpG adjuvant in HLA-A2/Kb mice.
  • Mice injected with equal volume of PBS served as controls ("naive"). 14 days after the last immunization, spienocytes were restimuiated with recall antigen peptides in IFN- ⁇ , IL-4 and IL-17 ELISPOT assays; individual mouse samples were tested in triplicate.
  • SI Simulation Index
  • SFU spot Forming Unit
  • SD Standard Deviation
  • SI SFU of the antigen (rmSp17) response in vaccinated mice/SFU for the same antigen response in naive mice.
  • FIG. 3 Immunogenicity of hSp1 7 peptide-based vaccines either combined with a CpG adjuvant or adjuvanted/carried by polystyrene nanoparticles (PSNPs).
  • PSNPs polystyrene nanoparticles
  • FIG. 3A IFN- ⁇ responses to CpG-adjuvanted, hSp17-derived peptides.
  • FIG. 3A IFN- ⁇ responses to CpG-adjuvanted, hSp17-derived peptides.
  • FIG. 3B Antigen specific antibody production (IgG) by CpG-adjuvanted, hSp17-derived peptides.
  • FIG. 3C IFN- ⁇ and IL-17 T cell responses to PSNPs-adjuvanted, hSp17-derived peptides.
  • FIG. 3D Antigen specific antibody production by PSNPs-adjuvanted, hSp17-derived peptides.
  • SI SI of SFU ⁇ SD
  • FIG. 4 B cell epitope recognition using different adjuvant system.
  • hSp17m_i 42 - PSNPs vaccine formulation containing 36 pg /ml of hSp1 7 111 _ 142 peptide and 1 % PSNPs
  • FIG. 4A and FIG. 4B Two series of peptide fragments within hSp17m_i 42 sequence and hSpl 7i i i_i42 itself were used to compete for the antibody reactivity in sera produced by both vaccine formulations for epitope recognition.
  • FIG. 4C Antibody cross-species reactivity to mSp17 fragments. Five mSp1 7 peptides were used to compete for antibody reactivity in sera produced by both vaccine formulations. Data presented as average OD450 nm ⁇ SD (assayed in triplicate).
  • FIG. 5 Immunogenicity of formulations wherein hSpl 7m-i42 is delivered with CpG or PSNP as an adjuvant in different mice strains.
  • FIG. 5B Anti-hSp17 1 1 1 -14 2 specific antibody (IgG) production. * *** p ⁇ 0.0001 .
  • FIG. 6 Comparison of the immunogenicity of rmSP17 protein and hSp17ii-
  • 10 days after the final immunization splenocyte !FN-y and IL-17 responses to the immunogen itself were measured in triplicate by ELISPOT assays.
  • FIG. 6B Antibody cross-reactivity to rmSp17.
  • Serum from C57BL/6 mice immunized with hSp17 111-142 (100 pg/dosage) and rmSp17 protein (100 pg /dosage) both adjuvanted with either CpG (25 pg/dosage) or PSNP (1 - %/dosage), were assayed in ELISA coated with rmSp17 protein (5 pg/ml) to test the cross-reactivity of these sera to the rmSp17 protein.
  • FIG. 7 Sp17 affects tumor development in mouse models and can be exploited for cancer vaccination and immunotherapy.
  • FIG. 7B anti-ID8 lysate specific antibody production in mice groups as established in FIG. 7A indicating a statistically significant increase in both treatment groups CpG-adjuvanted rmSp17 ( ** p ⁇ 0.01 ) and hSp17i i i.i 42 (*** p ⁇ 0.001 ) when compared to PBS treatment (data presented as the average OD 450 nm ⁇ SD, in triplicate assay).
  • FIG. 7C CFSE staining to compare the growth rates of the Sp17 + and Sp17 " ID8 cells by flow cytometry. Data presented as mean fluorescent intensity (MFI) of CFSE at different time points.
  • MFI mean fluorescent intensity
  • FIG. 7D Flow cytometry analysis of Sp17 co-expression with PD-L1 and STAT3 on M4-ID8 cells. Fluorescence due to Sp17 expression is comparatively higher for both STAT3-positive and PDL1 -positive M4-ID8 cells when compared to STAT3-negative or to PDL1 -negative M4-ID8 cells (data also confirmed in human ovarian cancer cell lines SKOV3 and OVCA433).
  • FIG. 7E Relationship between Sp17 expression & tumorgenicity in vivo. Survival curve for tumor induction by Sp17-positive (Sp17 + ) and Sp17-negative (Sp17 ⁇ ) ID8 cells in a mouse C57BL/6-ID8 model.
  • ID8 cells were inoculated at the dosage of 4 million (4M for both Sp17 + and Sp17 " ), 8 million (8M, Sp17-negative only) and 10 million cells (10M, Sp17-negative only).
  • FIG. 7F Survival curve comparing the tumorgenicity of ID8 and M4-SD8 ovarian cancer cell lines.
  • FIG. 8 Antitumor activity of different anti-hSp17 targeting antibodies and their peptide recognition specificities. Antitumor activities by mouse sera FIG. 8A or commercial antibodies FIG. 8C.
  • Serum was obtained from mice immunized with either CpG or PSNPs adjuvanted hSp17in -142 peptides (two immunizations, 2 weeks apart). Antibody recognition of Sp17 peptide fragments by serum, FIG. 8B or commercial antibodies, FIG. 8D.
  • administration refers to the administration of a composition to a subject.
  • Administration to an animal subject can be accomplished via a plurality of routes.
  • administration may be bronchial (including by bronchial instillation), buccal, enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (for example, intrahepatic, intratumoral, and the like), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal and vitreal.
  • bronchial including by bronchial instillation
  • buccal enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (for example, intrahe
  • Administration may also involve intermittent dosing.
  • administration may be by continuous dosing (e.g., perfusion) for at least a predetermined period of time.
  • antibody therapy is commonly administered parenterally, e.g. by intravenous, subcutaneous, or intratumoral injection, for instance, particularly when high doses within a tumor are desired).
  • agent may refer to a compound or entity of any chemical class including, for example, polypeptides, nucleic acids, saccharides, small molecules, metals, or combinations thereof.
  • agents include small molecules, drugs, antibodies, antibody fragments, aptamers, nucleic acids including but not limited to small interfering RNAs, antisense oligonucleotides, ribozymes, peptides, peptide mimetics, etc.
  • An agent may be or comprise a polymer.
  • antibody refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen.
  • the skilled person is aware that intact antibodies produced in nature are approximately 150 kD tetrameric agents comprised of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about
  • Each heavy chain is comprised of at least four domains (each about 1 10 amino acids long), an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CH1 , CH2, and the carboxy-terminal CH3 (located at the base of the Y's stem).
  • VH amino-terminal variable
  • CH1 , CH2 amino-terminal variable
  • CH3 carboxy-terminal CH3
  • Each light chain is comprised of two domains - an amino-terminal variable (VL) domain, followed by a carboxy-terminal constant (CL) domain, separated from one another by another "switch".
  • Intact antibody tetramers are comprised of two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed.
  • Naturally produced antibodies are also glycosylated, typically on the CH2 domain, and each domain has a structure characterized by an "immunoglobulin fold" formed from two beta sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other in a compressed antiparallel beta barrel.
  • Each variable domain contains three hypervariable loops known as "complement determining regions” (CDR1 , CDR2, and CDR3; as understood in the art, for example determined according to Kabat numbering scheme) and four somewhat invariant "framework" regions (FR1 , FR2, FR3, and FR4).
  • the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three-dimensional space so that they create a single hypervariable antigen-binding site located at the tip of the Y structure.
  • the Fc region of naturally occurring antibodies binds to elements of the complement system, and also to receptors on effector cells, including for example effector cells that mediate cytotoxicity.
  • affinity and/or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification.
  • the antibodies produced and/or utilized in accordance with the present invention include glycosylated Fc domains; including Fc domains with modified or engineered such glycosylation.
  • any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences as found in natural antibodies can be referred to and/or used as an "antibody", whether such polypeptide is naturally produced (e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology.
  • an antibody is polyclonal or oligoclonal, that is generated as a panel of antibodies, each associated to a single antibody sequence and binding a more or less distinct epitopes within an antigen (such as different epitopes within).
  • Polyclonal or oligoclonal antibodies can be provided in a single preparation for medical uses as described in the literature (Kearns JD et al., 2015).
  • an antibody is monoclonal.
  • an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies.
  • the antibody sequence elements can be humanized, primatized, or chimeric, as known in the art.
  • antibody as used herein, can (unless otherwise stated or clear from context) refer to any of the art-known or developed constructs or formats for utilizing antibody structural and functional features in an alternative presentation, for instance as antigen-binding fragments, as described below.
  • an antibody utilized in accordance with the present invention is in a format selected from, but not limited to, intact IgG, IgE and IgM, bi- or multi- specific antibodies (e.g., Zybodies®,), single chain variable domains (scFv), polypeptide-Fc fusions, a Fab fragment, a F(ab')2 fragment, a single chain variable fragment (scFv), a scFv-Fc fragment, a single chain antibody (scAb), an aptamer, cameloid antibodies, or a nanobody.
  • bi- or multi-specific antibodies e.g., Zybodies®, single chain variable domains (scFv), polypeptide-Fc fusions, a Fab fragment, a F(ab')2 fragment, a single chain variable fragment (scFv), a scFv-Fc fragment, a single chain antibody (scAb), an aptamer, cameloid antibodies, or a nanobody.
  • An antibody or antigen binding fragment thereof may be a rabbit, mouse, chimeric, humanized or fully human antibody or antigen-binding fragment thereof.
  • a provided antibody or antigen-binding fragment thereof may be of an IgG, IgA, IgE, or IgM isotype, as most suitable for a given use.
  • a provided antibody or antigen- binding fragment thereof is an IgG isotype, more particularly an lgG1 , lgG2, lgG3, or lgG4 isotype, masked antibodies (e.g., Probodies®), or fusion proteins with polypeptides that allow expression and exposure on the cell surface (as scFv within constructs for obtaining artificial T cell receptors that are used to graft the specificity of a monoclonal antibody onto a T cell).
  • an antibody does not contain a covalent modification, for instance, a glycan attachment that it would have if produced naturally.
  • an antibody may contain a covalent modification, for instance, the attachment of a glycan, a payload such as a detectable moiety, a therapeutic moiety, a catalytic moiety and the like, or other pendant group, for instance, poly-ethylene glycol.
  • the term "antigen” refers to an agent that elicits an immune response and/or that binds to a T cell receptor, for instance, when presented by an MHC molecule.
  • An antigen eliciting a humoral response involves the production of antigen-specific antibodies as shown in the Examples.
  • a specific Sp17 ep0 c may be used for screening antibody libraries and identifying candidate antibody sequences that can be further characterized.
  • the term "antigen-binding fragment” refers to an agent that specifically binds to a Sp17 epoc peptide, preferably an Sp17in-i 42 sequence.
  • this term encompasses any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding.
  • exemplary antigen-binding fragments include, but are not limited to, Small Modular ImmunoPharmaceuticals ("SMIPsTM " ), single chain antibodies, cameloid antibodies, single domain antibodies (e.g., shark single domain antibodies), single chain or Tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies®, minibodies, BiTE®s, ankyrin repeat proteins or DARPINs®, Avimers®, a DART, a TCR-like antibody, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, a TrimerX®, MicroProteins, Centyrins®, CoVX bodies, BiCyclic peptides, Kunitz domain derived antibody constructs, or any other antibody fragments so long as they exhibit the desired antigen binding activity.
  • SIPsTM Small Modular
  • an antigen-binding fragment also encompasses alternative protein structures such as stapled peptides, antibody-like binding peptidomimetics, antibodylike binding scaffold proteins, monobodies, and other known non-antibody protein scaffolds (e.g. in Helma J et a!., 2015).
  • an antigen-binding fragment is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity- determining region (CDR).
  • CDR complementarity- determining region
  • an antigen-binding fragment is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to one found in an anti-Sp17 ep0c sequence; amino acid sequence, and in particular in anti-Sp17 epoc -HCDR3 sequence.
  • an included CDR is substantially identical to an anti-Sp17in-i 4 2 amino acid sequence, and in particular to the anti-Sp17 111-142 -HCDR3 sequence, in that it is either identical in sequence or contains 1 , 2, 3, 4, or more amino acid substitutions that do not change its binding or biological activity.
  • biological sample typically refers to a sample obtained or derived from a biological source of interest, for instance, a tissue or organism or cell culture.
  • a biological source of interest for instance, a tissue or organism or cell culture.
  • One source of interest can be an animal or human organism.
  • the biological sample may comprise one or more biological tissues or fluids.
  • cancer As used herein, the terms “cancer”, “tumor”, and “carcinoma”, are used interchangeably herein to refer to cells that exhibit relatively abnormal, uncontrolled, and/or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation.
  • cells of interest for detection or treatment in the present application include precancerous (benign), malignant, pre-metastatic, metastatic, and non-metastatic cells.
  • precancerous benign
  • malignant pre-metastatic
  • metastatic metastatic
  • non-metastatic cells The present disclosure may be applicable to any and all cancers.
  • Non-limiting examples include hematopoietic cancers such as leukemias, lymphomas (Hodgkins and non-Hodgkins), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, ovarian, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, breast cancer, gastro-intestinal cancers and nervous system cancers, and benign lesions such as papillomas.
  • hematopoietic cancers such as leukemias, lymphomas (Hodgkins and non-Hodgkins), myelo
  • the term "combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens, such as exposure to two or more therapeutic agents.
  • two or more agents may be administered simultaneously.
  • such agents may be administered sequentially; otherwise, such agents are administered in overlapping dosing regimens.
  • the term "comparable” refers to two or more agents, entities, situations, effects, sets of conditions, and the like that may not be identical to one another but that are sufficiently similar to permit comparison between them such that conclusions may reasonably be drawn based on differences or similarities observed.
  • Such comparable sets of conditions, effects, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features.
  • composition or method described herein as “comprising” one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. It is also understood that any composition or method described as “comprising” (or which "comprises") one or more named elements or steps also describes the corresponding, more limited composition or method “consisting essentially of (or which "consists essentially of) the same named elements or steps, meaning that the composition or method inc!udes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method,
  • the term "dosage form" refers to a physically discrete unit of an active agent including a therapeutic or diagnostic agent, for administration to a subject.
  • Each unit contains a predetermined quantity of active agent.
  • such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen).
  • a dosage amount or a whole fraction thereof
  • the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.
  • the term "dosing regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.
  • a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
  • a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length.
  • a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses.
  • all doses within a dosing regimen are of the same unit dose amount.
  • different doses within a dosing regimen are of different amounts.
  • a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount.
  • a dosing regimen may comprise a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount and it is correlated with a desired or beneficial outcome when administered across a relevant population, namely a therapeutic dosing regimen.
  • epitope refers to a portion of an antigen that is bound by an antibody.
  • an epitope is conformational in that it is comprised of portions of an antigen that are not covalently contiguous in the antigen but that are near to one another in three- dimensional space when the antigen is in a relevant conformation.
  • conformational epitopes are those comprised of amino acid residues that are not contiguous in Sp17 111-142 sequence
  • linear epitopes are those comprised of amino acid residues that are contiguous in Sp17m.i 4 2 sequence.
  • the term "patient” or “subject” refers to any organism to which a provided composition is or may be administered, for example, for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals including but not limited to mammals such as mice, rats, rabbits, non-human primates, and/or humans. In some preferred embodiments, a patient is a human. In some embodiments, a patient is suffering from or susceptible to one or more disorders or conditions. A patient may display one or more symptoms of a disorder or condition, or may have been diagnosed with one or more disorders or conditions (such as cancer, or presence of one or more tumors). In some embodiments, the patient is receiving or has received certain therapy to diagnose and/or to treat such disease, disorder, or condition.
  • the term "pharmaceutically acceptable" applied to the carrier, diluent, or excipient used to formulate a composition as disclosed herein means that the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.
  • the term "pharmaceutical composition” refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers.
  • a pharmaceutical compositions may be formulated for administration in solid or liquid form, including those adapted for oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, for example, those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous, intradermal, intratumoral, or epidural injection as a sterile solution or suspension, or sustained- release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to skin, lungs, or oral cavity; intravaginally, intrarectally, sublingually, ocularly, transdermally, nasally, pulmonary, and to other mucosal surfaces.
  • solid tumor refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign or malignant. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas (including cancers arising from transformed cells of mesenchymal origin in tissues such as cancellous bone, cartilage, fat, muscle, vascular, hematopoietic, or fibrous connective tissues, carcinomas (including tumors arising from epithelial cells), melanomas, lymphomas, mesothelioma, neuroblastoma, retinoblastoma, and the like.
  • sarcomas including cancers arising from transformed cells of mesenchymal origin in tissues such as cancellous bone, cartilage, fat, muscle, vascular, hematopoietic, or fibrous connective tissues
  • carcinomas including tumors arising from epithelial cells
  • melanomas lymphomas
  • mesothelioma neuroblast
  • the term "therapeutically effective amount” refers to an amount, for instance, of an agent or of a pharmaceutical composition, that is sufficient when administered to a population suffering from or susceptible to a disease and/or condition in accordance with a therapeutic dosing regimen for treating such disease and/or condition,
  • a therapeutically effective amount is one that reduces the incidence and/or severity of, stabilizes, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition.
  • a "therapeutically effective amount” does not in fact require successful treatment be achieved in a particular subject.
  • treatment refers to any administration of an Sp17 epoc agent (e.g., Sp17 epoc -based vaccine or anti- Sp17 ep0c antibody) that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms.
  • an Sp17 epoc agent e.g., Sp17 epoc -based vaccine or anti- Sp17 ep0c antibody
  • mice can be immunized with a human Sp17 ep0 c (hSp17 epoc , such as Sp17in -14 2, with or without the use of an adjuvant such CpG or nanoparticles, as described in the Examples) to generate panels of B cells suitable for cell fusion using the mouse splenocytes and a mouse myeloma cell line, after confirming the presence of hSp17 epoc immunoreactivity in the serum of immunized mice.
  • hSp17 epoc such as Sp17in -14 2
  • an adjuvant such CpG or nanoparticles
  • human sera can be screened for identifying human subjects already presenting such hSp17 ep oc immunoreactivity and human B cell samples can be isolated from such human subjects for generating hybridomas using known techniques.
  • the corresponding clonal cells and hybridomas can then be used for screening clones expressing monoclonal antibodies that bind to peptides representing hSpl 7 epoc (or fragments thereof) and/or for cancer relevant functional activities, such as the lysis of mouse and/or human cancer cell clones (as shown in the Examples with ovarian cancer cell lines).
  • binding to hSp17 epoc peptide can be determined by screening the culture supernatants derived from hybridoma cells using an antibody capture ELSSA directly against human and murine Sp17 ep0 c peptides.
  • positive clones can be also screened for binding to the entire recombinant human Sp17 protein and/or for binding to cells that are stained within human primary and metastatic tumors and tissues (for example, from ovarian cancer samples compared to normal ovarian samples) and/or in a wide panel of commonly studied human cancer cell lines (for ovarian cancer SKOV3, ID8, OVCA433) by cytochemistry or by flow cytometry, using as control Sp17-negative cell lines (as shown in the examples, in murine Sp 7-positive and Sp17-negative ID8 cell lines).
  • Anti-Sp17 ep0 c body binding activity can be further quantified using standardized analytical methods, such as methods based on Biocore assay, using peptides corresponding to Sp17 ep0 c that are covalently linked to the surface of a Sensor Chip CMS via primary amine coupling.
  • a sample containing the candidate anti-hSp17 ep0 c monoclonal antibody is contacted with the receptor surface to establish binding on-rate and off-rate properties and binding affinities.
  • Libraries of antigen-dinging sequence and or other protein libraries can be similarly screened.
  • positive outcomes from such initial binding screening can be further validated, after verifying monoclonality, in a secondary screen for binding and biological interaction with cancer cells (such as ovarian cancer primary cells or ovarian derived cell lines) and/or the absence of binding or other biological activity for other epitopes present in hSp17, in other proteins or other cells (such as normal tissues).
  • cancer cells such as ovarian cancer primary cells or ovarian derived cell lines
  • the Examples disclose several representative approaches involving the use of sera and antibodies for determining the lytic, cytotoxic activity on Sp17-positive and Sp17-negative ID8 cell lines in addition to other cell Sines.
  • the protein sequence within such anti-hSpl 7 epoc candidates can then be cloned in the most appropriate protein scaffold (for instance, immunoglobulin-derived or non-immunoglobulin derived) for evaluating the additional features that can make such agent most suitable for therapeutic use, for example, cytotoxicity, stability, biodistribution, recombinant production in cell lines for pharmaceutical uses, lack of aggregation, means for purification, and the like.
  • the most appropriate protein scaffold for instance, immunoglobulin-derived or non-immunoglobulin derived
  • the one or more candidate anti-hSp17 e 0 c monoclonal antibodies can be characterized for their complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) against human or mouse cancer cell lines (such as human SKOV3 ovarian cancer cell line).
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the candidate one or more anti-hSp17 epoc monoclonal antibodies When compared to a control antibody (such as isotype control antibody or an antibody for unrelated antigen or Sp17 epitope), present a dose-response cytotoxicity over a range of concentrations such as 0.01 , 0.1 , 1 and 10 pg/ml concentration, using fresh normal human serum (NHS; for CDC assay) or freshly isolated PBMCs from healthy donor (for ADCC assay) as the source of complement or effector cells respectively.
  • the lactase dehydrogenase (LDH) released in culture media upon cell lysis can be measured following a colorimetric reaction. The amount of color formed is proportional to the number of lysed cells.
  • the one or more candidate anti-hSp17 ep0 c monoclonal antibodies can be further evaluated for a direct therapeutic activity in immunocompetent or immunodeficient animal cancer models, for instance, as described in the Examples with respect to ovarian cancer, in which cancer cell lines are inoculated intravenously or intraperitoneally.
  • the properties of the one or more candidate anti-hSpl 7 epoc monoclonal antibodies can be compared against other therapies, such as conventional first-line chemotherapeulic agents, like carboplatin and paclitaxel, or other antibodies binding antigens present on ovarian cancer cells.
  • outcome criteria including the development of ovarian cancer, weight, immune effector and therapeutic functions (ADCC, CDC and potential to enhance immunity through the use in combination with checkpoint inhibitors targeting the same cells), tumor ascites formation, or other clinical score can be regularly monitored in each group of control and treated mice (with or without sacrificing them) over one or more months.
  • the one or more candidate anti-hSp17 epoc monoclonal antibodies can be tested alone or in combination with suitable anti-cancer drugs for evaluating additional valuable effects, for example, synergism, decreasing negative side effects or drug resistance, reducing the effective dose or the period of treatment, reducing metastasis or recurrence, increasing the percentage of responders to the therapy.
  • the formulation of the one or more candidate anti-hSpl 7 epoc monoclonal antibodies with the anti-cancer drug can be the same (e.g. within the same liquid solution) or share the same carrier (e.g. nanoparticles loaded with both drugs). Overall viability and cancer recurrence can provide (together with pharmaceutical criteria such as biodistribution and safety) a pre-clinical evaluation of the one or more candidate anti- hSpl 7 epoc monoclonal antibodies for a potential therapeutic use prior to human testing.
  • the one or more candidate anti-hSp17 epoc monoclonal antibodies can be characterized by sequencing the variable regions characterizing each antibody and identifying the relevant CDR sequences.
  • the entire antibody variable sequences (or the isolated CDRs from heavy and light chains) can be recloned within the (no-)immunoglobulin protein scaffold most appropriate in terms of recombinant production, stability during storage, pharmacokinetics, immunological properties, tissue penetration, conjugation with ( radiochemicals, size or other criteria that are relevant for the final medical use of resulting protein (either for therapeutic or diagnostic uses).
  • the one or more candidate anti-hSpl 7 ep oc monoclonal antibody can be also comprised in a single, multispecific (e.g. bispecific) construct together with other antigen-binding sequences that bind other cancer targets including checkpoint inhibitors, or antigen characterizing cancer cells of general or specific subtypes (e.g. PD-1 , PD-L1 , CTLA-4, LAG3, TIM3, CD28, CD20, EGFR, CD52, OX40, CD137, HER2 and the like; for a review, see Redman J et a!., 2015).
  • PD-1 , PD-L1 , CTLA-4, LAG3, TIM3, CD28, CD20, EGFR, CD52, OX40, CD137, HER2 and the like for a review, see Redman J et a!., 2015.
  • a provided antibody or antigen-binding fragment thereof is included in an agent that further comprises a conjugated payload such as a therapeutic or diagnostic agent.
  • the agent is considered and/or referred to as an "immunoconjugate".
  • the present invention provides anti-hSp17 ep0 c sequences that identify antibodies or antigen-binding fragments thereof.
  • such sequences identify antibodies or antigen-binding fragments thereof that bind an epitope in hSp17 ep oc, and optionally, also a corresponding epitope of Cynomologous monkey and/or murine Sp1 7e P o C! > either as isolated proteins or on the surface of cells expressing hSp17 ep0 c (such as ovarian cancer cells or other cancer cells and cell lines).
  • the present invention also provides nucleic acid molecules encoding an isolated antibody or antigen-binding fragment thereof comprising anti Sp1 7 epoc , amino acid sequences.
  • nucleic acid molecules may contain codon-optimized nucleic acid sequences, and/or may be included in expression cassettes within appropriate nucleic acid vectors for the expression in host cells such as, for example, bacterial, yeast, insect, murine, simian, or human cells.
  • host cells may comprise heterologous nucleic acid molecules (such as DNA vectors) that express the antibody or antigen-binding fragment thereof (comprising anti Sp17 ep0 c amino acid sequences).
  • the present disclosure further provides methods of preparing an isolated antibody or antigen-binding fragment thereof as described herein, for instance, comprising anti-Sp17 ep0 c amino acid sequences.
  • Such methods may comprise culturing a host cell that comprises nucleic acids (e.g., heterologous nucleic acids that may comprise and/or be delivered to the host cell via vectors).
  • the host cells can be arranged and constructed so that antibody or antigen-binding fragment thereof is secreted from host cells such that the antibody can be isolated from cell culture supernatants and/or exposed on the cell surface (for instance, if such anti-Sp17 ep0 c amino acid sequences are intended to be used in the context of, or together with, such cells, e.g. artificial T cell receptors grafting an antibody specificity onto T cells).
  • compositions comprising an Sp17 ep0 c agent for use according to the present invention may be prepared for storage and/or delivery using any of a variety of techniques and/or technologies known to those skilled in the art.
  • the Sp17 ep0 c agent is administered according to a dosing regimen approved by a regulatory authority such as the United States Food and Drug Administration (FDA) and/or the European Medicines Agency (EMEA), e.g., for a given indication.
  • FDA United States Food and Drug Administration
  • EMEA European Medicines Agency
  • use of Sp17 ep0 c agent may permit reduced dosing, for example, a lower amount of active compound in one or more doses, a smaller number of doses, and/or a reduced frequency of doses) of an approved agent used in combination with the Sp17 ep oc agent.
  • dosing and administration of the Sp17 ep0 c agent utilizes an active agent having a desired degree of purity combined with one or more physiologically acceptable carriers, excipients or stabilizers in any or variety of forms.
  • physiologically acceptable carriers include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions including injectable and infusible solutions, dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.
  • a preferred form may depend on the intended mode of administration and/or therapeutic application, typically in the form of injectable or infusible solutions, such as compositions similar to those used for treating of human subjects with vaccines or antibodies.
  • ingredient(s) of the composition according to the invention can be prepared with carriers that protect the agent(s) against rapid release and/or degradation, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as polyanhydrides, polyglycolic acid, polyorthoesters, and polylactic acid.
  • each active agent is formulated, dosed, and administered in therapeutically effective amount using pharmaceutical compositions and dosing regimens that are consistently with good medical practice and appropriate for the relevant agent(s) including antibody agents.
  • compositions containing active agents according to the invention can be administered by any appropriate method known in the art, including, without limitation, oral, mucosal, by- inhalation, topical, buccal, nasal, rectal, or parenteral, for instance intravenous, infusion, intratumoral, intranodal, subcutaneous, intraperitoneal, intramuscular, intradermal, transdermal, or other modes of administration involving a physical breach of a tissue of a subject and subsequent administration of pharmaceutical composition through such breach.
  • a dosing regimen for a Sp17 ep0 c agent may involve intermittent or continuous (e.g. by perfusion or slow release system) administration, for example to achieve a particular desired pharmacokinetic profile or other pattern of exposure in one or more tissues or fluids of interest in the subject.
  • different agents administered in combination may be administered via different routes of delivery and/or according to different schedules.
  • Factors to be considered when optimizing routes and/or dosing schedule for a given therapeutic regimen may include, for example, the particular cancer being treated (such as type, stage, location), the clinical condition of a subject (including age, overall health, weight), the site of delivery of the agent, the nature of the agent (e.g. an antibody or other protein-based compound), the mode and/or route of administration of the agent, the presence or absence of combination therapy, and other factors familiar to medical practitioners.
  • a specific route of delivery may impact the required dose amount.
  • focused delivery such as intratumoral delivery
  • one or more features of a particular pharmaceutical composition and/or of a utilized dosing regimen may be modified over time (increasing or decreasing amount of active in any individual dose, increasing or decreasing time intervals between doses, etc.), for instance, in order to optimize a desired therapeutic effect or response (e.g., a therapeutic or biological response that is related to Sp17 ep0 c expression on cell surface).
  • the type, amount, and frequency of dosing of active agents in accordance with the present invention in governed by safety and efficacy requirements that apply when relevant agent(s) is/are administered to a mammal, preferably a human.
  • an exemplary desirable therapeutic response may involve, but is not !imited to, inhibition of and/or decreased tumor growth, tumor size, metastasis, one or more of the symptoms and side effects that are associated with the tumor, as well as increased apoptosis of cancer cells, therapeutically relevant decrease or increase of one or more cell marker or circulating markers and the like.
  • Such criteria can be readily assessed by any of a variety of immunological, cytological, and other methods that are disclosed in the literature.
  • the therapeutically effective amount of Sp17 ep0 c agent alone or in combination with a further agent can be determined as being sufficient to kill cancer cells.
  • a therapeutically effective amount of an active agent or composition comprising same can be readily determined using techniques available in the art including, for example, considering one or more factors such as the disease or condition being treated, the stage of the disease, the age and health and physical condition of the mammal being treated, the severity of the disease, other previous or ongoing treatments, and the like.
  • a therapeutically effective amount is an effective dose (and/or a unit dose) of an active agent that may be at least about 0.01 pg/kg body weight, at least about 0.05 pg/kg body weight; at least about 0.1 pg/kg body weight, at least about 1 pg/kg body weight, at least about 5 pg/kg body weight, at least about 10 pg/kg body weight, or more (e.g. about 100 pg/kg body weight). It will be understood by one of skill in the art that such guidelines may be adjusted for the molecular weight of the active agent.
  • the dosage may also be varied for route of administration, the cycle of treatment, or consequently to dose escalation protocol that can be used to determine the maximum tolerated dose and dose limiting toxicity (if any) in connection to the administration of the Sp17 ep0 c agent at increasing doses.
  • compositions should typically be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration.
  • Sterile injectable solutions can be prepared by incorporating the antibody in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other required ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile filtered solution.
  • the proper fluidity of a solution can be maintained, for example, by using a coating, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • the formulation of the Sp17 ep0 c agent according to the invention is preferably sterile, as accomplished by filtration through sterile filtration membranes, and then packaged, or sold in a form suitable for bolus administration or for continuous administration.
  • injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi dose containers containing a preservative.
  • Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained- release or biodegradable formulations as discussed herein.
  • Sterile injectable formulations may be prepared using a non-toxic parenteraliy acceptable diluent or solvent, such as water or 1 ,3 butanediol.
  • a non-toxic parenteraliy acceptable diluent or solvent such as water or 1 ,3 butanediol.
  • Other parenteraliy administrable formulations that are useful include those which comprise the active ingredient in microcrysta!line form, in a liposomal preparation, or as a component of biodegradable polymer systems.
  • Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer or salt.
  • compositions for use in accordance with the present invention may include pharmaceutically acceptable dispersing agents, wetting agents, suspending agents, isotonic agents, coatings, antibacterial and antifungal agents, carriers, excipients, salts, or stabilizers are non-toxic to the subjects at the dosages and concentrations employed.
  • buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; salts containing pharmacologically acceptable anions (such as acetate, benzoate, bicarbonate, bisulfate, isothionate, lactate, lactobionate, laurate, malate, maleate, salicylate, stearate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, thiethiodode, and valerate salts); preservatives (such as octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; sodium chloride; phenol, butyl or benzyl alcohol; a Iky I parabens such as methyl or propyl paraben; catechol; resor
  • agents can be administered simultaneously or sequentially.
  • administration of one agent is specifically timed relative to administration of another agent.
  • desired relative dosing regimens for agents administered in combination may be assessed or determined empirically, for example using ex vivo, in vivo and/or in vitro models; in some embodiments, such assessment or empirical determination is made in vivo, in a particular patient or patient population (such that a correlation is made).
  • the Sp17 ep0 c agent and one or more active agents utilized in practice of the present invention are administered according to an intermittent dosing regimen comprising at least two cycles.
  • one or more doses of different agents may be interdigitated with one another.
  • One or more doses of the second agent is administered a period of time after (or before) a dose of Sp17 ep0 c agent.
  • the Sp17 ep0 c agent is provided as a container with a label.
  • suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition that is effective for treating the condition 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).
  • the formulation is packaged in clear glass vials with a rubber stopper and an aluminum seal.
  • the label on, or associated with, the container indicates that the composition is used for treating the condition of choice.
  • Such container can be included in an article of manufacture that may further comprise a separate container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution.
  • a pharmaceutically acceptable buffer such as phosphate-buffered saline, Ringer's solution and dextrose solution.
  • St may further include other materials desirable from a commercial and user standpoint.
  • the article of manufacture may allow providing each or the agent in an intravenous formulation as a sterile aqueous solution containing a total of 2 mg, 5 mg, 10 mg, 20 mg, 50 mg, or more that are formulated, with appropriate diluents and buffers, at a final concentration of 0.1 mg/ml, 1 mg/ml, 10 mg/ml, or at an higher concentration.
  • the Sp17 epoc agent (especially in the form of a vaccine or anti-Sp17 ep0 c antibody) can be provided within the kits-of-parts in the form of lyophilized is to be reconstituted with any appropriate aqueous solution that provided or not with the kits, or other types of dosage unit using any compatible pharmaceutical carrier.
  • One or more unit dosage forms of the isolated antibody or antigen-binding fragment thereof comprising the Sp17 ep0 c agent may be provided in a pack or dispenser device.
  • a pack or device may, for example, comprise metal or plastic foil, such as a blister pack.
  • kits-of-parts may further comprise buffers, diluents, filters, needles, syringes, and package inserts with instructions for use in the treatment of cancer.
  • the instructions that are associated to the article of manufacture and/or the kits of the invention may be in the form of a label, a leaflet, a publication, a recording, a diagram, or any other means that can be used to inform about the correct use and/or monitoring of the possible effects of the Sp17 ep0 c agents, formulations, and other materials in the article of manufacture and/or in the kit.
  • Mouse recombinant Sp17 protein was provided by Dr. Chiriva- Snternati (Texas Tech University, USA).
  • Six 32-mer Song human Sp17 overlapping peptides (hSp17i. 3 2, hSp17 23 -54, hSpl 7 67 -98, hSp178 9- i 2o, hSp17 45-7 6 and hSp17m. 42 ) were designed (FIG. 1 ) and commercially synthesized by e.g. GenScript (NJ, USA) or Mimotopes (Clayton, Australia).
  • Vaccine formulations [00090] rmSp17 protein and six of the overlapping peptides from the human Sp17 protein sequence, namely hSp17i. 32 , hSp17 2 3-54, hSp17C 4 5-76, hSp17 6 7-98, hSp17 8 g-i2o and Sp17iii-i4 2 ) were used as vaccine antigens.
  • Each of the individual peptides were either mixed with CpG (ODN 1826, Invivogen, USA) directly or conjugated to 40-50 nm carboxylated polystyrene nanoparticles (PSNPs, Polysciences Inc., Warrington, USA). Each vaccine antigen was mixed with the indicated amount of CpG in PBS prior to injection.
  • Peptide conjugation to PSNPs was optimized for each peptide in order to achieve the best conjugation efficiency and size (40-60 nm), following the conjugation procedures as described previously (Xiang S. et al., 2013), using two conjugation buffer systems (MES and PBS) and a range of pH conditions (pH 5.5 to pH 8) that were tested for each peptide conjugated to PSNPs.
  • MES and PBS conjugation buffer systems
  • pH conditions pH 5.5 to pH 8
  • EDC 1 -ethyl-3-(3-dimethylaminopropryl carbodiimide hydrochloride
  • Sulfo-NHS 50 mM final
  • PierceTM Thermo Fisher Scientific, Waltham, Massachusetts, USA
  • the excess activation agents EDC and Sulfo-NHS
  • EDC and Sulfo-NHS were removed from the preactivation mix using a gel filtration column (Zeba spin desalting column following manufacturer instructions; ThermoFisher Scientific); the buffer was exchanged at the same time via the column (with optimized buffer conditions for each antigen), before adding the peptide antigen for a further 2 hours.
  • the final conjugation mix was then dialyzed against PBS in 1 kDa dialysis membrane. All hSp17 peptides were successfully conjugated to the PSNPs in an optimal size range ( ⁇ 60 nm) and efficiency (% peptide effectively conjugated to the PSNPs ⁇ 60%). Final conjugation efficiency was determined by BCATM protein assay (PierceTM Micro BCA protein assay, Thermo Fisher Scientific) and sizes were measured by Zetasizer (Malvern Instruments Ltd, Worcestershire, UK).
  • Each vaccine dose (-100 ⁇ ) contained 50-100 pg peptides with 20 pg CpG or -1 % solid of PSNPs in PBS.
  • the amounts of peptide antigen injected were matched for both formulations by adjusting the injection volume for each experiment.
  • mice were typically boosted with the same formulation 7-10 days apart (see description and comments to the respective Figure for each experiment). 10-14 days following the final immunization, mice were euthanized by C0 2 asphyxiation, their spleens removed and splenocytes harvested for subsequent immunogenicity assays (ELISPOT). Serum was also collected from each mouse prior to immunization and at cull for detecting antigen specific antibodies using ELISA.
  • ELISPOT immunogenicity assays
  • Antigen specific CD4, CD8 or Th17 T cell responses were evaluated by IL-4, IFN-Y and IL-17 ELISPOT assays. Briefly, 96-well filtration plates (MSIP or MAIP plates, Millipore, Billerica, MA) were coated with 100 ⁇ /well of either anti-mouse IFN-y (AN18,
  • CM fetal calf serum
  • 2 mM glutamine 100 pg/ml streptomycin and 100 units/ml penicillin
  • 0.2 mM ⁇ -mercaptoethanol 20 mM Hepes (all from Gibco, Life Technologies, California, USA)
  • Splenocytes 50 ⁇ taken from immunized mice (2 x 10 7 cells/ml, either individual or pooled) were added to triplicate wells and incubated with 50 ⁇ of recall antigens (rmSp17 protein or Sp17 peptides) at various concentrations (2.5 - 25 pg/ml for all potential CD8 epitopes and 25 - 100 pg/ml for long peptides and protein) in a 37°C incubator filled with 5% CO 2 for a minimum of 16 hours for IFN-
  • biotinylated detection antibodies [anti-mouse IFN- ⁇ biotinylated mAb R4-6A2 (Mabtech); anti-mouse IL-17 biotin (Mabtech); rat anti- mouse IL-4 biotin (BD), all at 1 pg/ml] at room temperature for 1-2 hours.
  • steptavidin-alkaline phosphatase to detect IFN- ⁇ and IL-17, MabTech
  • Extravidin-ALP for detection of IL-4, Sigma-Aldrich
  • SI stimulation index
  • An antigen-specific response was considered to be positive only when the SI > 2 and net SFU ⁇ 20 per million cells.
  • Serum samples were collected from vaccinated animals prior to immunization and when euthanized; each sample was assayed for antigen-specific antibody production by ELISA. Briefly, 96-well plates were coated with rmSp17 protein or peptides diluted in carbonate/bicarbonate coating buffer (5 pg/ml, 50 ⁇ I/well) and incubated overnight at 4°C. After washing with PBS/0.05% Tween-20 and blocking with 5% skim milk, serial dilutions of mouse sera were added and incubated at 37°C for 2 hours or 4°C overnight.
  • HRP-conjugated sheep anti-mouse IgG (Amersham) was added and allowed to incubate at 37°C for another 1 hour.
  • the reaction was developed using TMB substrate (Invitrogen, USA) and stopped with 1 M HCI, before reading the absorbance at 450 nm (OD 450 nm).
  • Antibody endpoint titers represent the degree to which the serum could be diluted and still contain detectable amounts of antibody, and were calculated as the serum dilution at which the OD 45 onm was equal to the mean OD of the serum of naive mice + 3 standard deviations (SD).
  • Competition ELISA were performed by incubating the test serum (usually at 1 :100 - 1 :400 dilutions, depending on the antibody titers) with competing peptides (serially diluted from 10 pg/well) at 1 :1 ratio (volume:volume) in a 96-well tissue culture plate for 1 hour in a 37°C incubator. Following incubation, 50 ⁇ of competition mix from each well were transferred to corresponding ELISA plates that were pre-coated (and blocked) with the same competing peptide. Standard ELISA protocol was carried out as described above. Changes in OD 45 onm value reflect cross-reaction between antibody and competing peptides.
  • Sp17 has an immunogenic region containing both B cell and Th1 cell epitopes
  • mice were immunized twice intradermally with hSp17 1-32l hSp17 2 3-5 4 , hSp17 45-76 , hSp17 67 -98, hSp17 8 9-i2o, or hSp17 111-142 peptides with CpG adjuvant, as well as recombinant murine Sp17 protein (rmSp17).
  • rmSp17 recombinant murine Sp17 protein
  • a positive response was determined by the stimulation index (SI) of the SFU of recall peptide in vaccinated mouse/SFU for the same peptide in naive mouse.
  • SI stimulation index
  • the SI > 2 considered as a positive response.
  • the homology was adjusted between mouse and human because of the identical amino acid shift in those regions.
  • ⁇ indicated strong positive responses with Sl>10.
  • CpG (20 ⁇ g/mouse) adjuvanted hSpl7 peptides (hSpl7 5 32 , hSpl7 hSpl7 45 76 , hSpl7 67 . 98 , hSpl7 89 . 120 , and hSpl7 i n 50 ⁇ / ⁇ 5 ⁇ ) vaccine formulations intradermally, 2 weeks apart.
  • splenocytes were tested in triplicate for reactivity to each of the 13 predicted HLA-A2.1 restricted CD8 T cell epitopes in IFN-vEUSPOT assays. & no binding to hSpl7 67 98 . @ no binding to hSpl7 g9 . 12 o .
  • TABLE 2 shows summary results from 5 independent experiments. A positive response was determined by the stimulation index (SI) of the SFU of recall peptide in vaccinated mouse/SFU for the same peptide in naive mouse. A SI > 2 was considered as a positive response.
  • SI stimulation index
  • hSp17 1 2 i-i 38 for CpG-adjuvanted hSp17ni_ 142 vaccine formulation
  • hSp17 3 - i 42 or hSp 7 34 -i42 for PSNPs-adjuvanted, hSp17-derived peptides
  • TNSL 139-142 amino acids
  • the change in immunogenicity by 121-138 amino acids may have occurred by promoting exposure of a region otherwise masked by the rest of the peptide.
  • none of the murine equivalent peptides could efficiently block the binding to hSp17m_i 4 2 by antibodies induced by either hSp17m_ 4 2 formulation (FIG. 4C).
  • IFN- ⁇ response induced by the hSp17iii-i 42 peptide was significantly higher in C56BL/6 mice than in HLA-A2.1 mice for the same formulation tested, and the response to the CpG adjuvant formulation was higher than that to the PSNP adjuvant formulation in both mice strains (FIG. 5A).
  • both formulations induced similar levels of IgG in HLA-A2.1 mice, which were also comparable to the CpG adjuvanted formulation in C57BL/6 mice.
  • the PSNP adjuvant formulation induced much lower antibody responses in C57BL/6 mice (FIG. 5B).
  • the use of a PSNP based delivery platform for the hSp17 i-i 2 peptide also resulted in a different antibody IgG subtype being induced compared to the CpG adjuvant formulation.
  • the CpG adjuvant formulation predominantly induced lgG2a and lgG2b, whereas the PSNP-conjugated formulation induced lgG1 in C57BL/6 mice.
  • the pattern was slightly different in HLA-A2.1 mice, with the CpG adjuvant formulation inducing lgG2a, and the PSNPs-conjugated formulation inducing both lgG2a and lgG1 subtypes (FIG. 5C).
  • FIG. 6 shows that both formulations induced Th1 , but not Th17 antigen- specific responses to the immunizing antigen (FIG. 6A).
  • serum from hSp17i 11-142 peptide based vaccines (CpG or PSNP adjuvant formulations) showed different levels of cross-reactivity to the rmSp17 protein (coating with the rmSp17 protein in ELISA; FIG. 6B).
  • hSp17 i.e. the reference hSp17iii-i 42 peptide, or the fragments and variants that were tested in this Example 1 , and as further defined as hSp17 ep oc.
  • hSp17 fragments in combination with one or more noninflammatory PSNPs as a vaccine delivery system, promotes a qualitatively different response to CpG, particularly in the nature of the antibody response elicited. Consequently, this approach also is particularly advantageous for establishing panels of B cells to be used for generating hybridomas and selecting monoclonal antibodies that present the desired specificity for hSp17 ep0 c peptides and isotype.
  • EXAMPLE 2 Cancer-specific Activities of Compounds Targeting hSpl 7
  • Materials & Methods [0001 5] Cell lines and Cell Preparations
  • the mouse ovarian MOSEC cell line clone ID8 more commonly known as ID8 cells, were kindly provided by Dr. Katherine Roby (University of Kansas Medical Center, USA; Roby K. et al., 2000).
  • the ID8 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) or RPMI supplemented with 4% FBS and 1x ITS (Insulin-Transferrin-Selenium; Mediatech/Corning). ID8 cells were selected on the basis of Sp17 expression by limiting dilution method, and the single cell clones were tested for Sp17 expression by flow cytometry.
  • DMEM Dulbecco's modified Eagle's medium
  • RPMI RPMI supplemented with 4% FBS and 1x ITS (Insulin-Transferrin-Selenium; Mediatech/Corning).
  • ID8 cells were selected on the basis of Sp17 expression by limiting dilution method, and the single cell clones were tested
  • the Sp17 + and Sp17-negative ID8 clones were expanded in cell culture for generating cell preparations of Sp17 + and Sp17-negative ID8 clones.
  • Mice were inoculated with Sp17 + ID8 clones cell preparations, tumor mass was then isolated from each diseased mouse, and cultured further in vitro before reinoculated in another group of mice. Again, tumor cells were isolated from the tumor mass, and cultured in vitro.
  • This new ID8-originated cell line was designated "M4-ID8", and characterized as a highly tumorigenic variant of ID8 M4-ID8 cells..
  • the ID8 murine ovarian cancer model was first established in C57BL/6 mice. Briefly, 4x10 6 ID8 murine OC cells in PBS (100 ⁇ ) were inoculated intraperitoneally at the low right flank (abdomen) area. Three weeks after the initial inoculation, mice were treated with various vaccine formulations injected intradermally at the base of the tail. Each treatment group was repeated in triplicate with the same formulation 3 weeks apart. Mice were monitored daily for the first appearance of a tumor, and measured daily for weight and abdominal circumference. Survival was followed until the circumference reached 100 mm (due to the build-up of ascites fluid), or until killed earlier due to complications arising from the tumor.
  • Serum antibody were collected at the day of cull for each mouse, pooled and assayed (at 1 :100 dilution) for antigen specific reactivity to ID8 ce!l !ysate.
  • Each vaccine dose (-100 ⁇ ) contained 50-100 pg peptides with 20 pg CpG or -1 % solid of PSNPs in PBS were prepared and administered as described in Example 1.
  • CFSE Carboxyfluorescein succinimidyl ester
  • Flow cytometry analysis of Sp17 co-expression with PDL1 and STAT3 was performed using 5x10 5 M4-ID8 cells stained with PE labeled anti-PDI_1 (rat anti-mouse CD274; BD Biosciences, cat. No. 558091 ) and Alexa Fluor® 647 labeled mouse anti- STAT3 (pY705; BD Biosciences, cat. No. 557815), or indirectly stained using mouse anti-Sp17 (A-12; SantaCruz Cat. No. sc-365325) followed by FITC-conjugated secondary antibody. Controls included labeled isotypes and secondary antibody alone. After staining and washing, cells were fixed in 1 % PFA. Flow cytometry data was acquired with BDTM LSR II Cell Analyzer (BD Biosciences), and analyzed using Flowjo analysis software (TreeStar).
  • PE labeled anti-PDI_1 rat anti-mouse CD274; BD Biosciences, cat.
  • the hSp17i .i 42 -specific sera were produced using mice immunized with either CpG- or PSNPs-adjuvanted hSp17 1 11 -142 peptide (each mouse received two immunizations, administered 2 weeks apart) and tested at 1 :200 final dilution.
  • the commercial anti-human Sp17 agents that were tested at 1 Mg/ml final concentration were: goat anti-Sp17 (N-17; SantaCruz® Cat. no. sc-66643), rabbit anti-Sp17 (SPA17; ProteintechTM cat. no. 13367-1 -AP), mouse anti-Sp17 (EP6496; Abeam® cat. No. 172626), or mouse anti-hSp17 (A-12; SantaCruz Cat. No. sc-365325).
  • M4-ID8 cells or the human ovarian cancer cell line SKOV3 (ATCC cat. No. HTB-77TM) were seeded at 5000 cells/well.
  • a control either naive mouse serum or isotype-matching antibodies were used as control samples at the same concentration of anti-Sp17 agents .Each condition was tested in triplicate.
  • Cell viability at 24 hours was determined by the calcein release assay. In brief, 24h after exposure to the anti- Sp17 agent or control agent, cells were washed and Calcein AM (2 pg/ml; ThermoFisher) was added to each well (100 ⁇ /well in PBS). After a 30 minute incubation, the release of fluorescent calcein into the culture was measured on a plate reader set at 485 nm for excitation and 530 nm for emission.
  • Example 1 The biological activities and responses described in Example 1 demonstrate that hSp17iii-i 42 contains sequences that are unexpectedly immunogenic and highly protective in animal models of human cancers, both in the presence or absence of an adjuvant, such as CpG. Accordingly, these sequences can be advantageously used for preparing peptides and related pharmaceutical formulations for medical uses and methods, in particular those involving the administration of therapeutic vaccines and/or protective against cancer.
  • the CpG-adjuvanted, hSp17 i-i 42 formulation can be compared directly with CpG-adjuvanted, rmSp17 formulation in the murine ID8 model for ovarian cancer (OC).
  • Tumor cells (4x10 6 ID8 tumor cells) are injected 21 days before the first therapeutic vaccination, followed by another 3 repeated treatments, 3 weeks apart.
  • Control (naive) mice are mock treated with PBS. Both rmSp17 full protein and hSp 7-i i i.-i42 peptide treatments successfully and significantly delay tumor progression (e.g. by -20 days; FIG. 7A).
  • hSp17in-i42 fragments e.g. those defined as hSp17 epoc
  • Animals receiving either protein or peptide treatment also revealed significantly increased antibody reactivity to the ID8 lysate in serum samples, thereby demonstrating the elicitation of tumor relevant immunity (FIG. 7B).
  • the therapeutic efficacy for the human hSp17in-i42 peptide in a mouse OC model can be explained by the fact that the identified 9-mer strongly immunogenic B cell epitope in the human AKKMKTNSL (amino acids 134-142) and murine VKKMKSDKN (amino acids 132-140) Sp17 shares seven residues that are either identical (4 residues, shown underlined) or exhibit only a conservative change.
  • T cell epitopes KEKEEVAAVKIQAA amino acids 11 1 -124) and IQAAFRGIAREEAKKMK (amino acids 121 -138) also contain conservative changes and stretches of identity with their murine equivalents (REQEEAAALKIQSL and IQSLFRGHVAREEVKKMK), which are useful in human studies as well as the sequence of the IQ motif EVAAVKIQAAFRGIAREE (calmodulin-binding recognition sequence) that is present in hSp17 (amino acids 1 15- 133; Wen et a!., 1999).
  • the more tumorigenic clonal population (the M4-ID8 cells) was analyzed on the basis of Sp17 co-expression with two important markers for ovarian cancer (OC): PD-L1 and STAT3.
  • OC patients showing higher expression of PDL1 on OC cells have poorer prognosis than those with lower expression (Hamanishi J et al., 2007) and the expression of PDL1 on tumor cells has been associated with tumor escape (Iwai Y et al., 2002).
  • the transcription factor STAT3 has a dual role in cancer, but confers resistance to chemotherapy and other cancer drugs to OC cells (Duan Z et al., 2006).
  • Sp17 is co-expressed with high levels of PDL1 and STAT3 (FIG. 7D). Therefore, targeting Sp17-positive cells would mean simultaneously targeting cancer cells associated with resistance to chemotherapy and resistance to destruction by T cells or NK cells.
  • Sp17 appears therefore an essential feature for cancerous tumor growth in vivo, especially for ovarian cancer. This property can be exploited in various ways, for instance for combining Sp 7-targeting agents with other treatments to eliminate the most forms of aggressive cancer cells and/or for targeting potential residual Sp17- negative cancer ce!!s after targeting Sp17-positive cancer cells. Indeed, the presence of antibodies against hSp17 111-142 in OC-bearing animals that responded to the therapeutic vaccination with hSpl 7m-i42 (Example 1 ), revealed that such antibodies might directly promote the specific killing of tumor cells.
  • the resulting anti-hSp17 for Sp17 antibodies are useful for hSp17 immunodetection, but not as anti-cancer agents.
  • the Sp17 epitope mapping for commercial antibodies indicates a strong preference for epitopes located in Sp17- derived peptides other than hSp17 1 n-i4 2 (such as hSp17i. 32 , hSp17 23- 5 , hSp17 5- 76 , and/or hSp17 67 - 98 peptides).
  • the present data permit the identification of improved anti-Sp17 agents for treating cancers such as ovarian cancer, in particular by using monoclonal antibodies that selectively target cells exposing cancer-specific human Sp17 epitopes (anti- Sp17 ep oc)- Anti-Sp 7 ep oc can be suitably used as anti-cancer agents alone or in combination with other anti-cancer agents that target other cancer-relevant antigens (such as other cell surface antigens, immune checkpoint inhibitors, etc.) radio- or chemotherapy, or other anti-cancer drugs, such as those listed in WHO Model List of Essential Medicines (19 th edition, April 2015).
  • Variable regions or selected CDR sequences from anti-Sp17 ep0 c can be integrated in protein scaffolds or modules that are based on natural immunoglobulins (such as full IgG scFV, Nanobodies, Fab, etc.) or non-immunoglobulin scaffolds for protein binding (such as affibodies, DARPins, Anticalin, Monobody, etc.).
  • Anii-hSp1 7epoc may be used for treating cancers including ovarian cancer (OC), which is the !eading cause of death from gynecologica! malignancy, which requires new therapeutic approaches.
  • OC ovarian cancer
  • OC cells Different from known targeted therapies involving cancer cell specific monoclonal antibodies that can increase patient survival for breast, lung, and other cancer indications, only a limited proportion of OC cells presents specifically express cell surface targets such as those commonly found with other cancers, such Her-2/neu or MUC1 . Additionally, OC cell variability within an individual patient, typically leads to growth of drug resistant cells following chemotherapy and cellular evasion of immune mechanisms of tumor elimination (Chester, C, et al., 2015; Lloyd K et al., 2015).
  • Anti-hSp17 ep oc may also broadly target OC patients, and specifically target elements of OC cells crucial to their in vivo survival and for their ability to escape other known therapeutic approaches, by eliminating the cells most likely to cause untreatable recurrences.
  • anti-Sp 7 ep0 c may target cancer cells in which hSp17 ep oc is present with proteins associated with drug resistance like STAT3 (Lee H-J et al., 2014) or immune evasion, like PDL1 (Adachi K and Tamada K, 2015; Beatty G et al., 2015).
  • the present invention demonstrates how sera, raised using hSp17 ep0 c, and thus enriched in anti-hSp17 ep0 c antibodies, is also very effective in directly targeting and destroying murine and human OC cells. This particular result is not obtainable using presently available commercial monoclonal antibodies that detect hSp17 via other regions, which have been shown to be otherwise ineffective.
  • the inventive aspects disclosed herein can be advantageously exploited for developing a variety of new therapeutic strategies against tumors, including tumors associated with ovarian cancer, whereby such strategies beneficially overcome the often toxic and not fully effective therapeutic regimens used currently.

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Abstract

Cette invention concerne une protéine de surface spermatique (Sp17) comprenant des séquences qui sont à la fois immunogènes et protectrices dans des modèles animaux de cancers humains, à la fois en présence ou en l'absence d'un adjuvant tel que CpG. Ces séquences Sp17 sont en outre utiles pour préparer des peptides et des compositions pharmaceutiques associées pour divers procédés et usages thérapeutiques concernant l'administration de vaccins à base de Sp17 ou de préparations d'anticorps dirigés contre l'apparition du cancer et/ou pour prévenir le cancer, notamment le cancer de l'ovaire.
PCT/EP2016/067468 2015-07-21 2016-07-21 Épitopes t et b dans la protéine de surface spermatique sp17 à titre de vaccins contre le cancer et de cibles d'anticorps Ceased WO2017013231A1 (fr)

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US12319747B2 (en) 2023-07-03 2025-06-03 Medicovestor, Inc. Methods of using anti-SP17 immunotherapeutics
US12364777B2 (en) 2023-10-20 2025-07-22 Medicovestor, Inc. Homodimeric antibodies for use in treating cancers and methods of use
US12371494B2 (en) 2023-12-26 2025-07-29 Medicovestor, Inc. Methods of manufacturing dimeric antibodies
US12600795B2 (en) 2023-12-26 2026-04-14 Medicovestor, Inc. Oligomeric IgG for immunotherapeutics and diagnostics
US12616756B2 (en) 2024-08-16 2026-05-05 Medicovestor, Inc. Dimeric antibodies

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12319747B2 (en) 2023-07-03 2025-06-03 Medicovestor, Inc. Methods of using anti-SP17 immunotherapeutics
US12364777B2 (en) 2023-10-20 2025-07-22 Medicovestor, Inc. Homodimeric antibodies for use in treating cancers and methods of use
US12371494B2 (en) 2023-12-26 2025-07-29 Medicovestor, Inc. Methods of manufacturing dimeric antibodies
US12600795B2 (en) 2023-12-26 2026-04-14 Medicovestor, Inc. Oligomeric IgG for immunotherapeutics and diagnostics
US12616756B2 (en) 2024-08-16 2026-05-05 Medicovestor, Inc. Dimeric antibodies

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