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WO2025114411A1 - Nouvelle méthode de traitement de troubles cérébraux ou neurologiques - Google Patents

Nouvelle méthode de traitement de troubles cérébraux ou neurologiques Download PDF

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Publication number
WO2025114411A1
WO2025114411A1 PCT/EP2024/083856 EP2024083856W WO2025114411A1 WO 2025114411 A1 WO2025114411 A1 WO 2025114411A1 EP 2024083856 W EP2024083856 W EP 2024083856W WO 2025114411 A1 WO2025114411 A1 WO 2025114411A1
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Prior art keywords
brain
fab
trastuzumab
cancer
her2
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English (en)
Inventor
Guilhem BOUSQUET
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Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Sorbonne Paris Nord
Universite Paris Cite
Original Assignee
Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Sorbonne Paris Nord
Universite Paris Cite
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Publication of WO2025114411A1 publication Critical patent/WO2025114411A1/fr
Pending legal-status Critical Current
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    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin

Definitions

  • the present invention relates to an antigen binding fragment which binds to an antigen for use in the treatment of brain or neurologic disorders in a subject in need thereof.
  • BBB
  • Tucatinib is a tyrosine kinase inhibitor blocking the intracellular part of HER2 signaling pathway, with a better BBB diffusion compared to other anti-HER2 tyrosine kinase inhibitors [8], with promising cerebral responses [9,10], Trastuzumab-deruxtecan, an antibody-drug conjugate, has led to a 73% rate of intracranial responses, with 27% of complete cerebral responses [11,12], However, despite these promising initial results, both drugs have a time-limited efficacy [10-12], probably largely explained by efflux systems of the BBB [13],
  • the efflux of IgG was only saturable with Fc and notF(ab’)2 antibodies [17], However, the inventors hypothesized that a Fc receptor (FcRn) expressed by endothelial cells from the BBB was responsible for this efflux.
  • FcRn Fc receptor expressed by endothelial cells from the BBB was responsible for this efflux.
  • the inventors engineered a fragment antigen-binding (Fab) of trastuzumab and characterized it in preclinical models for translational therapeutic purpose. More, they implemented original rat models with catheter implanted into the cistema magna.
  • trastuzumab demonstrated the safety and equal efficacy with trastuzumab in vitro, and in vivo using a patient-derived xenograft model of HER2 overexpressing breast cancer.
  • the brain-to-blood efflux of Fab was up to 10 times lower than for trastuzumab, associated with a two-fold higher brain penetration compared to trastuzumab.
  • trastuzumab Fab as effective as the native IgG, and capable of doubling brain penetration and significantly reducing brain-to-blood efflux after intra-cerebrospinal fluid injection.
  • This Fab could thus be a new and original effective weapon in the treatment of HER2 breast cancer brain metastases, and thus used as a proof of concept of the powerful use of antigen binding fragment (antibody Fab derivatives) in brain or neurologic disorders.
  • the present invention relates to an antigen binding fragment which binds to an antigen for use in the treatment of brain or neurologic disorders in a subject in need thereof.
  • An antigen binding fragment which binds to an antigen for use in the treatment of brain or neurologic disorders in a subject in need thereof.
  • the antigen biding fragment is selected from the group consisting of a Fab, a F(ab)’2, a single domain antibody, a ScFv, a Sc(Fv)2, a diabody, a triabody, a tetrabody, an unibody, a minibody, a maxibody, a small modular immunopharmaceutical (SMIP), minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody as an isolated complementary determining region (CDR), and fragments which comprise or consist of the VL or VH chains of an antibody.
  • SMIP small modular immunopharmaceutical
  • antigen binding fragment of an antibody, as used herein, refers to one or more fragments of an intact antibody that retain the ability to specifically binds to a given antigen (e.g., [antigen]).
  • Antigen biding functions of an antibody can be performed by fragments of an intact antibody.
  • binding fragments encompassed within the term antigen binding fragment of an antibody include a Fab fragment, a monovalent fragment consisting of the VL,VH,CL and CHI domains; a Fab’ fragment, a monovalent fragment consisting of the VL,VH,CL,CH1 domains and hinge region; a F(ab’)2 fragment, a bivalent fragment comprising two Fab’ fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of VH domains of a single arm of an antibody; a single domain antibody (sdAb) fragment (Ward et al., 1989 Nature 341 :544-546), which consists of a VH domain or a VL domain; and an isolated complementary determining region (CDR).
  • Fab fragment a monovalent fragment consisting of the VL,VH,CL and CHI domains
  • a Fab’ fragment a monovalent fragment consisting of the VL,VH,CL,CH1 domains and hinge region
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by an artificial peptide linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (ScFv); see, e.g., Bird et al., 1989 Science 242:423-426; and Huston et al., 1988 proc. Natl. Acad. Sci. 85:5879-5883).
  • dsFv is a VH::VL heterodimer stabilised by a disulfide bond.
  • Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2.
  • Such single chain antibodies include one or more antigen binding portions or fragments of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • a unibody is another type of antibody fragment lacking the hinge region of IgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of traditional IgG4 antibodies and has a univalent binding region rather than the bivalent biding region of IgG4 antibodies.
  • Antigen binding fragments can be incorporated into single domain antibodies, SMTP, maxibodies, minibodies, intrabodies, diabodies, triabodies and tetrabodies (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136).
  • diabodies tribodies or tetrabodies refers to small antibody fragments with multivalent antigen-binding sites (2, 3 or four), which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL).
  • Antigen binding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) Which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al., 1995 Protein Eng. 8(10); 1057-1062 and U.S. Pat. No. 5,641,870).
  • the Fab of the present invention can be obtained by treating an antibody which specifically reacts with [antigen] with a protease, papaine. Also, the Fab can be produced by inserting DNA encoding Fab of the antibody into a vector for prokaryotic expression system, or for eukaryotic expression system, and introducing the vector into a procaryote or eucaryote (as appropriate) to express the Fab.
  • the F(ab')2 of the present invention can be obtained treating an antibody which specifically reacts with [antigen] with a protease, pepsin. Also, the F(ab')2 can be produced by binding Fab' described below via a thioether bond or a disulfide bond.
  • the Fab' of the present invention can be obtained treating F(ab')2 which specifically reacts with [antigen] with a reducing agent, dithiothreitol.
  • the Fab' can be produced by inserting DNA encoding Fab' fragment of the antibody into an expression vector for prokaryote, or an expression vector for eukaryote, and introducing the vector into a prokaryote or eukaryote (as appropriate) to perform its expression.
  • the scFv of the present invention can be produced by obtaining cDNA encoding the VH and VL domains as previously described, constructing DNA encoding scFv, inserting the DNA into an expression vector for prokaryote, or an expression vector for eukaryote, and then introducing the expression vector into a prokaryote or eukaryote (as appropriate) to express the scFv.
  • CDR grafting involves selecting the complementary determining regions (CDRs) from a donor scFv fragment, and grafting them onto a human scFv fragment framework of known three-dimensional structure (see, e. g., W098/45322; WO 87/02671; US5,859,205; US5,585,089; US4,816,567; EP0173494).
  • Domain Antibodies are the smallest functional binding units of antibodies - molecular weight approximately 13 kDa - and correspond to the variable regions of either the heavy (VH) or light (VL) chains of antibodies. Further details on domain antibodies and methods of their production are found in US 6,291,158; 6,582,915; 6,593,081; 6,172,197; and 6,696,245; US 2004/0110941; EP 1433846, 0368684 and 0616640; WO 2005/035572, 2004/101790, 2004/081026, 2004/058821, 2004/003019 and 2003/002609, each of which is herein incorporated by reference in its entirety.
  • UniBodies are another antibody fragment technology, based upon the removal of the hinge region of IgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of a traditional IgG4 antibody and has a univalent binding region rather than a bivalent binding region. Furthermore, because UniBodies are about smaller, they may show better distribution over larger solid tumors with potentially advantageous efficacy. Further details on UniBodies may be obtained by reference to WO 2007/059782, which is incorporated by reference in its entirety.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • a subject denotes a mammal, such as a rodent, a feline, a canine, and a primate.
  • a subject according to the invention is a human. More particularly, the subject is suffering from a brain or neurologic disorders. Cancer brain disorders
  • the antigen binding fragment can be used for the treatment of cancer brain disorders.
  • a cancer brain disorder means a cancer brain localization which means that a cancer is localized in the brain.
  • cancer brain disorders can derive from different primary cancers selected from the group consisting in adrenal cortical cancer, anal cancer, bile duct cancer (e.g. perihilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma), breast cancer (e.g.
  • ductal carcinoma in situ infiltrating ductal carcinoma, infiltrating lobular carcinoma, lobular carcinoma in situ, gynecomastia
  • Castleman disease e.g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia
  • cervical cancer colorectal cancer
  • endometrial cancer e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adenocarcinoma, clear cell
  • esophagus cancer gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g.
  • kidney cancer e.g. renal cell cancer
  • laryngeal and hypopharyngeal cancer e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma
  • lung cancer e.g. small cell lung cancer, non-small cell lung cancer
  • mesothelioma plasmacytoma, nasal cavity and paranasal sinus cancer
  • esthesioneuroblastoma midline granuloma
  • nasopharyngeal cancer neuroblastoma
  • oral cavity and oropharyngeal cancer ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancer (e.g.
  • uterine cancer e.g. uterine leiomyosarcoma
  • leukaemia like acute myeloid leukaemia, acute lymphoid leukaemia, chronic myelomonocytic leukemia (CMML)
  • CMML chronic myelomonocytic leukemia
  • MDS myelodysplastic syndrome
  • cancer brain disorders are neuro-oncologic disorders.
  • cancer brain disorders are cancer brain metastasis.
  • cancer brain metastasis are breast cancer brain metastasis.
  • the antigen which is recognized by the antigen binding fragment of the invention can be all antigen useful in cancer therapy like HER2, EGFR, VEGF, VEGFR2, PDGFRalpha, PD1, PDL1, CTLA4, TROP-2, CD 19, CD3, CD20, CD22, CD30, CD33, CD38, CD52, GD2, RANKL, SLAMF7, MELAN- A.
  • the antigen is HER2.
  • the antigen biding fragment is derived from the trastuzumab or the pertuzumab.
  • the antigen biding fragment is derived from the bevacizumab, the ranibizumab, the nivolumab, the pembrolizumab, the avelumab, the dostarlimab, the blinatumomab, the rituximab, the inotuzumab, the brentuximab, the gemtuzumab, the daratumumab, the lemtuzumab, the hul4.18K322A, the denosumab, the elotuzumab or the GA733.
  • the antigen biding fragment is derived from the trastuzumab and the variable heavy chain has the sequences as set for in the SEQ ID NO: 1 and the variable light chain has the sequences as set for in the SEQ ID NO: 2.
  • the VH/CH1 chain has the sequence as set for in the SEQ ID NO: 3 and the VL/CL chain has the sequences as set for in the SEQ ID NO: 4.
  • the VH/CH1 sequence of the antigen binding fragment derived for the Trastuzumab is linked to a GST sequence thanks to a linker and has a sequence as set for in the SEQ ID NO: 5.
  • the antigen binding fragment can be used for the treatment of neurologic disorders.
  • a neurological disorder can be a brain damage, frontal lobe damage, parietal lobe damage, temporal lobe damage, occipital lobe damage, brain dysfunction (for example aphasia, dysgraphia, dysarthria, apraxia, agnosia, amnesia, spinal cord disorders, peripheral neuropathy and other peripheral nervous system disorders), delirium and dementia such as Alzheimer's disease, dizziness and vertigo, stupor and coma, head injury, stroke (CVA, cerebrovascular attack), multiple sclerosis and other demyelinating diseases, infections of the brain or spinal cord.
  • brain dysfunction for example aphasia, dysgraphia, dysarthria, apraxia, agnosia, amnesia, spinal cord disorders, peripheral neuropathy and other peripheral nervous system disorders
  • delirium and dementia such as Alzheimer's disease, dizziness and vertigo, stupor and coma
  • head injury stroke (CVA, cerebrovascular attack
  • the antigen which is recognized by the antigen binding fragment of the invention can be all antigen useful in neurologic disorder therapy like Beta-amyloid, LINGO-1, alpha4-integrin, CD117.
  • the antigen can be the Beta-amyloid.
  • the antigen can be LINGO-1 or alpha4-integrin and the antigen biding fragment is derived from the natalizumab. If the neurologic disorder is the amyotrophic lateral sclerosis (ALS), the antigen can be CD117 and the antigen biding fragment is derived from the masitinib.
  • ALS amyotrophic lateral sclerosis
  • the invention in another aspect, relates to a therapeutic composition comprising an antigen binding fragment for use in the treatment of brain or neurologic disorders in a subject in need thereof.
  • the antigen binding fragment or the therapeutic composition of the invention are administrated in a therapeutically effective amount.
  • Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • the term "therapeutically effective amount” or “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • a therapeutically effective amount of the inhibitor or the composition of the present invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the inhibitor or the composition of the present invention to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the inhibitor or the composition are outweighed by the therapeutically beneficial effects.
  • the efficient dosages and dosage regimens for the inhibitor or the composition of the present invention depend on the disease or condition to be treated and may be determined by the persons skilled in the art.
  • a physician having ordinary skill in the art may readily determine and prescribe the effective amount of the inhibitor or the composition of the invention required.
  • the physician could start doses of the inhibitor or the composition of the present invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable dose of the inhibitor or the composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen.
  • Such an effective dose will generally depend upon the factors described above.
  • a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease.
  • the ability of a compound to inhibit cancer may, for example, be evaluated in an animal model system predictive of efficacy in human tumors.
  • this property of a composition may be evaluated by examining the ability of the compound to induce cytotoxicity by in vitro assays known to the skilled practitioner.
  • a therapeutically effective amount of a therapeutic compound may decrease tumor size, or otherwise ameliorate symptoms in a subject.
  • One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
  • An exemplary, non-limiting range for a therapeutically effective amount of an antigen binding fragment of the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg.
  • An exemplary, non-limiting range for a therapeutically effective amount of an antigen binding fragment of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg. Administration may e.g.
  • Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time.
  • the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans, for example using a labeled antigen binding fragment of the present invention, fragment.
  • an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more subdoses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the monoclonal antibodies of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects.
  • An effective dose of an antigen binding fragment of the present invention may also be administered using a weekly, biweekly or triweekly dosing period.
  • the dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established.
  • treatment according to the present invention may be provided as a daily dosage of an antigen binding fragment of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45,
  • Administration may be oral, topical, intravenous, intranasal, parenteral, intraocular, intramuscular, intraperitoneal, intrathecal, intra-cerebrospinal fluid, intratumoral or subcutaneous, and for instance administered proximal to the site of the target.
  • Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time.
  • the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans.
  • an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the oligomers of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects.
  • An effective dose of the antigen binding fragment of the present invention may also be administered using a weekly, biweekly or triweekly dosing period.
  • the dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established.
  • treatment according to the present invention may be provided as a daily dosage of the antigen binding fragment of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7,
  • the quantity of the antigen binding fragment administered to a subject in need thereof is between 10 4 to 10 9 fragment per kg.
  • the quantity of antigen binding fragment injected is 10 6 or 10 7 fragment per kg.
  • the antigen binding fragment of the invention can be administrated is 1, 2, 3, 4 or 5 times to the subject in need thereof.
  • “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • compositions for example, the form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the subject, etc.
  • compositions of the invention can be formulated for a oral, topical, intravenous, intranasal, parenteral, intraocular, intramuscular, intraperitoneal, intrathecal, intra- cerebrospinal fluid, intratumoral or subcutaneous administration and the like.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze- dried compositions.
  • these may be in organic solvent such as DMSO, ethanol which upon addition, depending on the case, of sterilized water or physiological saline permit the constitution of injectable solutions.
  • compositions include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.
  • the antigen binding fragment of the invention are delivered in a manner consistent with conventional methodologies associated with management of the disease or disorder for which treatment is sought.
  • an effective amount of the antigen binding fragment of the invention administered to a subject in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder.
  • Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) are generally designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be easily made.
  • Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)).
  • MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • the physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.
  • the antigen binding fragment of the invention or the pharmaceutical composition of the invention may comprise a further therapeutic active agent.
  • the present invention also relates to a kit comprising an inhibitor according to the invention and a further therapeutic active agent.
  • active agent relates to a chemical substance inducing an effect such as a therapeutic or a preventive effect. It may be a bioactive chemical compound from a drug or the drug itself. Active agent can be a single molecule or a mixture of several substances.
  • anti-cancer agents may be added to the pharmaceutical composition or used in combination with the inhibitor of the invention in the case of the treatment of a cancer.
  • Anti-cancer agents or conventional cancer therapies can be surgery, radiotherapy, chemotherapy, or combinations thereof.
  • Anti-cancer agents include other anti-cancer antibodies, cytotoxic agents, chemotherapeutic agents, anti -angiogenic agents, anti-cancer immunogens, cell cycle control/apoptosis regulating agents, hormonal regulating agents, and other agents described below.
  • the inhibitor or composition of the present invention is used in combination with a chemotherapeutic agent.
  • chemotherapeutic agent refers to chemical compounds that are effective in inhibiting tumor growth.
  • examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its ad
  • calicheamicin especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33:183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino
  • paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • antihormonal agents that act to regulate or inhibit honnone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • the inhibitor or composition of the present invention is used in combination with a targeted cancer therapy.
  • Targeted cancer therapies are drugs or other substances that block the growth and spread of cancer by interfering with specific molecules ("molecular targets") that are involved in the growth, progression, and spread of cancer.
  • Targeted cancer therapies are sometimes called “molecularly targeted drugs,” “molecularly targeted therapies,” “precision medicines,” or similar names.
  • the targeted therapy consists of administering the subj ect with a tyrosine kinase inhibitor.
  • tyrosine kinase inhibitor refers to any of a variety of therapeutic agents or drugs that act as selective or non-selective inhibitors of receptor and/or non-receptor tyrosine kinases. Tyrosine kinase inhibitors and related compounds are well known in the art and described in U.S Patent Publication 2007/0254295, which is incorporated by reference herein in its entirety.
  • a compound related to a tyrosine kinase inhibitor will recapitulate the effect of the tyrosine kinase inhibitor, e.g., the related compound will act on a different member of the tyrosine kinase signaling pathway to produce the same effect as would a tyrosine kinase inhibitor of that tyrosine kinase.
  • tyrosine kinase inhibitors and related compounds suitable for use in methods of embodiments of the present invention include, but are not limited to, dasatinib (BMS-354825), PP2, BEZ235, saracatinib, gefitinib (Iressa), sunitinib (Sutent; SU11248), erlotinib (Tarceva; OSI-1774), lapatinib (GW572016; GW2016), canertinib (CI 1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib (Gleevec; STI571), leflunomide (SU101), vandetanib (Zactima; ZD6474), MK-2206 (8-[4-aminocyclobutyl)phenyl]-9-phenyl-l,2,4-triazolo[3,4-
  • the tyrosine kinase inhibitor is a small molecule kinase inhibitor that has been orally administered and that has been the subject of at least one Phase I clinical trial, more preferably at least one Phase II clinical, even more preferably at least one Phase III clinical trial, and most preferably approved by the FDA for at least one hematological or oncological indication.
  • inhibitors include, but are not limited to, Gefitinib, Erlotinib, Lapatinib, Canertinib, BMS- 599626 (AC-480), Neratinib, KRN-633, CEP-11981, Imatinib, Nilotinib, Dasatinib, AZM- 475271, CP-724714, TAK-165, Sunitinib, Vatalanib, CP-547632, Vandetanib, Bosutinib, Lestaurtinib, Tandutinib, Midostaurin, Enzastaurin, AEE-788, Pazopanib, Axitinib, Motasenib, OSI-930, Cediranib, KRN-951, Dovitinib, Seliciclib, SNS-032, PD-0332991, MKC-I (Ro- 317453; R-440), Sorafenib, ABT
  • the antigen binding fragment or composition of the present invention is used in combination with an immunotherapeutic agent.
  • immunotherapeutic agent refers to a compound, composition or treatment that indirectly or directly enhances, stimulates or increases the body's immune response against cancer cells and/or that decreases the side effects of other anticancer therapies. Immunotherapy is thus a therapy that directly or indirectly stimulates or enhances the immune system's responses to cancer cells and/or lessens the side effects that may have been caused by other anti-cancer agents. Immunotherapy is also referred to in the art as immunologic therapy, biological therapy biological response modifier therapy and biotherapy.
  • immunotherapeutic agents examples include, but are not limited to, cytokines, cancer vaccines, monoclonal antibodies and non-cytokine adjuvants.
  • the immunotherapeutic treatment may consist of administering the subject with an amount of immune cells (T cells, NK, cells, dendritic cells, B cells).
  • Immunotherapeutic agents can be non-specific, i.e. boost the immune system generally so that the human body becomes more effective in fighting the growth and/or spread of cancer cells, or they can be specific, i.e. targeted to the cancer cells themselves immunotherapy regimens may combine the use of nonspecific and specific immunotherapeutic agents.
  • Non-specific immunotherapeutic agents are substances that stimulate or indirectly improve the immune system.
  • Non-specific immunotherapeutic agents have been used alone as a main therapy for the treatment of cancer, as well as in addition to a main therapy, in which case the non-specific immunotherapeutic agent functions as an adjuvant to enhance the effectiveness of other therapies (e.g. cancer vaccines).
  • Non-specific immunotherapeutic agents can also function in this latter context to reduce the side effects of other therapies, for example, bone marrow suppression induced by certain chemotherapeutic agents.
  • Non-specific immunotherapeutic agents can act on key immune system cells and cause secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the agents can themselves comprise cytokines.
  • Nonspecific immunotherapeutic agents are generally classified as cytokines or non-cytokine adjuvants.
  • cytokines have found application in the treatment of cancer either as general non-specific immunotherapies designed to boost the immune system, or as adjuvants provided with other therapies. Suitable cytokines include, but are not limited to, interferons, interleukins and colony-stimulating factors. Interferons (IFNs) contemplated by the present invention include the common types of IFNs, IFN-alpha (IFN-a), IFN-beta (IFN-P) and IFN- gamma (IFN-y). IFNs can act directly on cancer cells, for example, by slowing their growth, promoting their development into cells with more normal behavior and/or increasing their production of antigens thus making the cancer cells easier for the immune system to recognise and destroy.
  • IFNs Interferons
  • IFN-a IFN-alpha
  • IFN-P IFN-beta
  • IFN-y IFN-gamma
  • IFNs can act directly on cancer cells, for example, by slowing their growth, promoting
  • IFNs can also act indirectly on cancer cells, for example, by slowing down angiogenesis, boosting the immune system and/or stimulating natural killer (NK) cells, T cells and macrophages.
  • Recombinant IFN-alpha is available commercially as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation).
  • Interleukins contemplated by the present invention include IL-2, IL-4, IL-11 and IL-12. Examples of commercially available recombinant interleukins include Proleukin® (IL-2; Chiron Corporation) and Neumega® (IL- 12; Wyeth Pharmaceuticals). Zymogenetics, Inc.
  • Colony-stimulating factors contemplated by the present invention include granulocyte colony stimulating factor (G-CSF or filgrastim), granulocyte-macrophage colony stimulating factor (GM-CSF or sargramostim) and erythropoietin (epoetin alfa, darbepoietin). Treatment with one or more growth factors can help to stimulate the generation of new blood cells in subjects undergoing traditional chemotherapy.
  • CSF colony stimulating factor
  • Various-recombinant colony stimulating factors are available commercially, for example, Neupogen® (G-CSF; Amgen), Neulasta (pelfilgrastim; Amgen), Leukine (GM-CSF; Berlex), Procrit (erythropoietin; Ortho Biotech), Epogen (erythropoietin; Amgen), Arnesp (erytropoietin).
  • G-CSF Neupogen®
  • Amgen Neulasta
  • Leukine GM-CSF
  • Berlex Procrit
  • Procrit erythropoietin
  • Ortho Biotech Epogen
  • Epogen erythropoietin
  • Arnesp erytropoietin
  • Combination compositions and combination administration methods of the present invention may also involve "whole cell” and "adoptive" immunotherapy methods.
  • such methods may comprise infusion or re-infusion of immune system cells (for instance tumor-infiltrating lymphocytes (TILs), such as CC2+ and/or CD8+ T cells (for instance T cells expanded with tumor-specific antigens and/or genetic enhancements), antibody-expressing B cells or other antibody-producing or - presenting cells, dendritic cells (e.g., dendritic cells cultured with a DC-expanding agent such as GM-CSF and/or Flt3-L, and/or tumor-associated antigen-loaded dendritic cells), anti-tumor NK cells, so-called hybrid cells, or combinations thereof.
  • TILs tumor-infiltrating lymphocytes
  • CC2+ and/or CD8+ T cells for instance T cells expanded with tumor-specific antigens and/or genetic enhancements
  • antibody-expressing B cells or other antibody-producing or - presenting cells for instance dendritic cells cultured with a DC-expanding agent such as GM-CSF and
  • Cellular “vaccines” in clinical trials that may be useful in such aspects include CanvaxinTM, APC-8015 (Dendreon), HSPPC-96 (Antigenics), and Melacine® cell lysates. Antigens shed from cancer cells, and mixtures thereof (see for instance Bystryn et al., Clinical Cancer Research Vol. 7, 1882-1887, July 2001), optionally admixed with adjuvants such as alum, may also be components in such methods and combination compositions.
  • Radiotherapy may comprise radiation or associated administration of radiopharmaceuticals to a patient.
  • the source of radiation may be either external or internal to the patient being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)).
  • Radioactive elements that may be used in practicing such methods include, e.g., radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium-i l l.
  • the antigen binding fragment or composition of the present invention is used in combination with an antibody that is specific for a costimulatory molecule.
  • antibodies that are specific for a costimulatory molecule include but are not limited to anti-CTLA4 antibodies (e.g. Ipilimumab), anti-PDl antibodies, anti-PDLl antibodies, anti- TIMP3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies or anti- B7H6 antibodies.
  • the second agent is an agent that induces, via ADCC, the death of a cell expressing an antigen to which the second agent binds.
  • the agent is an antibody (e.g. of IgGl or IgG3 isotype) whose mode of action involves induction of ADCC toward a cell to which the antibody binds.
  • NK cells have an important role in inducing ADCC and increased reactivity of NK cells can be directed to target cells through use of such a second agent.
  • the second agent is an antibody specific for a cell surface antigens, e.g., membrane antigens.
  • the second antibody is specific for a tumor antigen as described above (e.g., molecules specifically expressed by tumor cells), such as CD20, CD52, ErbB2 (or HER2/Neu), CD33, CD22, CD25, MUC-1, CEA, KDR, aVp3, etc., particularly lymphoma antigens (e.g., CD20).
  • a tumor antigen as described above (e.g., molecules specifically expressed by tumor cells), such as CD20, CD52, ErbB2 (or HER2/Neu), CD33, CD22, CD25, MUC-1, CEA, KDR, aVp3, etc., particularly lymphoma antigens (e.g., CD20).
  • a tumor antigen as described above (e.g., molecules specifically expressed by tumor cells), such as CD20, CD52, ErbB2 (or HER2/Neu), CD33, CD22, CD25, MUC-1, CEA, KDR, aVp3, etc., particularly lymphoma antigens (
  • Antibodies of interest for the methods of the invention act through ADCC, and are typically selective for tumor cells, although one of skill in the art will recognize that some clinically useful antibodies do act on non-tumor cells, e.g. CD20.
  • CD20 There are a number of antigens and corresponding monoclonal antibodies for the treatment of B cell malignancies.
  • One popular target antigen is CD20, which is found on B cell malignancies.
  • Rituximab is a chimeric unconjugated monoclonal antibody directed at the CD20 antigen.
  • CD20 has an important functional role in B cell activation, proliferation, and differentiation.
  • the CD52 antigen is targeted by the monoclonal antibody alemtuzumab, which is indicated for treatment of chronic lymphocytic leukemia.
  • CD22 is targeted by a number of antibodies, and has recently demonstrated efficacy combined with toxin in chemotherapyresistant hairy cell leukemia.
  • Monoclonal antibodies targeting CD20 also include tositumomab and ibritumomab.
  • Monoclonal antibodies useful in the methods of the invention, which have been used in solid tumors include without limitation edrecolomab and trastuzumab (herceptin).
  • Edrecolomab targets the 17-1 A antigen seen in colon and rectal cancer, and has been approved for use in Europe for these indications.
  • Trastuzumab targets the HER- 2/neu antigen. This antigen is seen on 25% to 35% of breast cancers. Trastuzumab is thought to work in a variety of ways: downregulation of HER-2 receptor expression, inhibition of proliferation of human tumor cells that overexpress HER-2 protein, enhancing immune recruitment and ADCC against tumor cells that overexpress HER-2 protein, and downregulation of angiogenesis factors.
  • Alemtuzumab (Campath) is used in the treatment of chronic lymphocytic leukemia; colon cancer and lung cancer; Gemtuzumab (Mylotarg) finds use in the treatment of acute myelogenous leukemia; Ibritumomab (Zevalin) finds use in the treatment of non-Hodgkin's lymphoma; Panitumumab (Vectibix) finds use in the treatment of colon cancer. Cetuximab (Erbitux) is also of interest for use in the methods of the invention.
  • the antibody binds to the EGF receptor (EGFR) and has been used in the treatment of solid tumors including colon cancer and squamous cell carcinoma of the head and neck (SCCHN).
  • Another aspect of the present invention relates to i) an antigen binding fragment, and ii) at least one further therapeutic active agent according to the invention, as a combined preparation for simultaneous, separate or sequential use in the treatment of brain or neurologic disorders in a subject in need thereof.
  • the invention relates to i) an antigen binding fragment, and ii) at least one further therapeutic active agent according to the invention for simultaneous, separate or sequential use in the treatment of brain or neurologic disorders in a subject in need thereof.
  • the term “simultaneous use” denotes the use of an antigen binding fragment and at least one therapeutic active agent occurring at the same time.
  • the term “separate use” denotes the use of an antigen binding fragment and at least one therapeutic active agent not occurring at the same time.
  • sequential use denotes the use of an antigen binding fragment and at least one therapeutic active agent occurring by following an order.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 2 Pharmacokinetic studies after intra-CSF administration of anti-HER2 antibodies in rats.
  • Table 1 Pharmacokinetic data after intra-CSF administration of anti-HER2 antibodies in rats using a two-compartment model.
  • kio diffusion coefficient from CSF to brain
  • kn diffusion coefficient from CSF to blood
  • k2o diffusion coefficient from serum towards physiological elimination.
  • Kpu partition coefficients from CSF to brain from 0 to 4h after injection in the total brain (Kpu,ubrain.o-4h), in CE1 deeper area (Kpu,ucEi,o-4h), in CE2 posterior area (Kpu,ucE2,o-4h ), and in CE3 superficial area (Kpu,ucE3,o-4h).
  • a first step of proof of concept was necessary to validate the absence of efflux of Fab fragments, using two commercialized anti-VEGF antibodies, bevacizumab and ranibizumab.
  • Enzyme-linked Immunosorbent Assay procedures for bevacizumab and ranibizumab concentration assessment in serum, brain and cerebrospinal fluid.
  • Bevacizumab and ranibizumab were provided by the pharmacy of Avicenne Hospital.
  • ranibizumab concentration assessment we developed a protocol based on the procedure implemented by Paintaud et al to assess bevacizumab serum concentration in patients 18.
  • Microtiter 96-well plates were prepared by incubating 100 pL VEGF165 at a concentration of 0.25 mg/L in the coating buffer (1 mol/L carbonate-bicarbonate buffer, pH 9.6) overnight at +4°C. The plates were washed four times with PBS containing 0.05% Tween 20. The remaining protein-binding sites were blocked by 2 hours incubation at room temperature with 200 pL blocking buffer (PBS containing 1% BSA).
  • Reading was performed at two wavelengths (492 and 620 nm) using an ELISA plate reader (CLARIOstare, BMG Labtech).
  • the absorbance at 620nm corresponds to the background signal linked to the plate and was subtracted from the absorbance at 492 nm.
  • bevacizumab we used an ELISA procedure already validated for clinical practice 15,16,18,19. We used an anti -human IgG Fc specific as secondary antibody, coupled with peroxidase (Sigma).
  • the rat is first anesthetized with a cocktail of isofluorane 3% and a mixture of buprenorphine at 0,3mg/mL (0,05mg/kg), ketamine at lOOmg/mL (90mg/kg), xylazine at 100 mg/mL (lOmg/kg). It is then placed on a stereotactic frame with the head positioned in order to achieve a flat skull. On sterile conditions, the operating area is shaved and cleaned using dermic betadine. Skin is incised on 2-3 cm length from the dorsal midline of the skull to the occipital crest. Using blunt scissor, skin is disjoining from the skull.
  • the skull is then exposed by gently scraping the periosteum with a scalpel.
  • a 0.7mm hole is drilled into the interparietal bone, 1 millimeter ahead of the occipital crest on the sagittal midline, at an angle of 60-70° from the horizontal plan, in the caudal direction, at 5mm depth.
  • Catheter (SAI infusion Technologies) is then place into the hole and fixed at the skull with a drop of tissue glue (histoacryl, Braun). Cutaneous plan is then sutured, and the end of the catheter is placed under the skin.
  • the objective is to analyze the intrathecal pharmacokinetics after injection of ranibizumab or bevacizumab antibodies.
  • Two antibody solutions were prepared. 14 pg of bevacizumab and ranibizumab solutions were diluted in saline (0.9%) to achieve a total volume of 100 uL. Each solution was injected into the cistema magna using the catheter, in a total of 10 rats (5 rats per group). Then, 50 pL of cerebrospinal fluid and blood were taken at 0 (before injection of the solution), 30 minutes, 1 hour, 3 and 4 hours after injection. Once collected, the blood tube was centrifuged at 10,000 rpm at 4°C for 10 minutes to isolate the serum. The cerebrospinal fluid and serum were then analyzed with the developed ELISA technique (described in the result section). Engineering of anti-HER2 Fab fragments
  • Fab#l For the Fab fragment we have engineered (Fab#l), light chain and heavy chain of the Fab were synthesized separately and then assembled into Chinese hamster ovary (CHO) cells.
  • the light chain was synthesized in pcDNA3.2 vector, using the sequence corresponding to the light chain of trastuzumab and the heavy chain was synthesized in pFUSEss-CHIg-hGl vector (Invitrogen, USA).
  • a stop codon was added at the end of each sequence to avoid adding other elements to the sequence of interest.
  • E.Coli XLl-Blue bacteria Sub-cloning in expression vector was first realized in E.Coli XLl-Blue bacteria.
  • E.Coli were first transformed with genes or vectors separately by heat shock at 42°C for 30 seconds followed by 2 minutes in ice, to amplify them. Bacteria were then incubated in a rich SOC medium (Invitrogen, USA) for 1 hour at 37°C at 230 rpm and then cultured in a liquid environment containing the appropriate selection antibiotic. The tubes were incubated at 37°C overnight.
  • plasmid DNA was recovered with the Wizard Plus SV Minipreps DNA Purification System Kit (Promega, USA), which allows the production of a clarified lysat and the purification of plasmid DNA.
  • a PCR was performed with the purified plasmids.
  • the products were digested with Dpnl (New England BioLabs, France) for 2 hours in order to get rid of bacterial DNA. Products were purified again before being controlled on 1% agarose gel (Invitrogen, USA).
  • genes and vectors were treated 2 hours with T4-DNA polymerase (New England BioLabs, France) to generate blunt ends.
  • E.Coli were transformed by heat shock with the gene of the fab light chain or the heavy chain, their vector and its specific primers. Each gene and its corresponding vectors were assembled with specific primer.
  • a colony-based PCR was performed. Each colony collected was amplified and PCR products were controlled on 2% agarose gel (Invitrogen, USA). For each product the plasmid DNA was recovered, sequenced, and analyzed by alignment with the expected sequence.
  • Insertion in synthesis vector was performed to synthesize the Fab.
  • the CHO cells were seeded 24 hours before transfection with 70 to 90% confluence in 6 wells plates.
  • the two plasmids (the light chain and heavy chain) were placed together in the culture medium, with a transfection agent (Sigma- Aldrich) at a 3: 1 ratio. They were incubated for 15 minutes at 20°C before being added to each well containing CHO cells. After 24 hours, the antibiotics were added to lead to 50% mortality of the cells and then gradually increased to 100% mortality of non-transfected cells. After several days, the culture medium was removed and purified by column chromatography. A SDS-PAGE gel was then performed to ensure the weight of the Fab.
  • a Western Blot was performed with anti-trastuzumab antibody (R&D SYSTEM).
  • the amino-acid sequence of the Fab fragments was controlled using a tandem mass spectrometry (MS/MS).
  • MS/MS tandem mass spectrometry
  • the stained protein bands corresponding to the recombinant Fab were excised from the SDS-PAGE and cut into small pieces using a scalpel and proteins were in-gel digested as previously described (30).
  • Peptide samples were analyzed with a QTOF mass spectrometer (Impact HD, Bruker) equipped with the CaptiveSpray ion source (Bruker).
  • the QTOF was coupled to a nano liquid chromatography (Ultimate 3000, ThermoFisher Scientific) running with two buffers: 0.1% formic acid in water (A) and 0.1% formic acid in 80% acetonitrile (B). Chromatographic separation was carried out on a C18 reverse phase column (75 pm, 150 mm, 120 A Wide Pore, Bruker) with a gradient elution at a flow rate of 300 nL/min during 60 min. The system was operated with automatic switching between MS and MS/MS modes using a data-dependent acquisition (DDA) method for peptide fragmentation. MS/MS spectra were then processed with the Data Analysis software (Bruker).
  • DDA data-dependent acquisition
  • peptides have been identified automatically using the MASCOT software (Matrix Science, London, UK) and the SwissProt database (www. expasy.org) with the following parameters: carbamidomethylation for cysteine residues; potential oxidation for methionine residues; tolerance on mass measurements of 20 ppm in MS mode and 0.1 Da in MS/MS mode; enzymatic cleavage by trypsin with one missed cleavages allowed. The species of origin was restricted to the Mammalia.
  • peptides not automatically identified were then manually sequenced on the basis of their MS/MS spectra using the annotate tool in the Data Analysis software (Bruker).
  • Two breast cancer cell lines were used, BT474 with HER2 overexpression and MDA- MB-231 which does not overexpress HER2.
  • the cell lines were obtained from ATCC. These cells were cultured at 37 °C under normoxic conditions (20 % of 02 and 5 % of CO2) in RPMI 1640 medium supplemented with 10 % of foetal calf serum and 1 % antibiotics.
  • trastuzumab or Fab anti-HER2 fragments were assessed on the BT-474 and MDA-MB-231 cell lines.
  • the two cell lines were grown separately on culture slides (BD FalconTM).
  • Five micrograms of commercial trastuzumab (Roche) or anti-HER2 Fab fragments coupled with Alexa Fluor 488 fluorophore (using APEXTM Alexa FluorTM 488 Antibody Labeling Kit, Invitrogen) were diluted in 300 pL of PBS and incubated for 1 hour with each type of human cancer cell line. Then, the PBS was removed and the cells were rinsed to remove unbound antibodies.
  • the cells were fixed in acetone at 4°C, the nuclei were stained with DAPI (Vector Laboratories, Vectashield, H-1200)) and fluorescence staining was observed at 400x magnification. The experiment was conducted five times independently, and a minimum of 100 cells were analysed.
  • DAPI Vector Laboratories, Vectashield, H-1200
  • BT474 cells were cultured in RPMI1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 pg/mL streptomycin. Cells were maintained at 37°C in a humidified atmosphere of 5% CO2. All media and cell culture supplements were from Life Technologies. Collected cells were seeded (300,000 cells/tube). Cells were incubated overnight at 4°C with 600 pL of D- phosphate-buffered saline (PBS) supplemented with 0.1% BSA and 5% normal goat serum (NGS, Life Technologies) and containing increasing concentrations of trastuzumab or anti- HER2Fab antibodies.
  • PBS D- phosphate-buffered saline
  • NGS normal goat serum
  • BT474 cells were seeded in 96-well tissue culture plates at a density of 5x103 cells per well. After 24 hours of incubation, the cells were exposed to increasing concentrations of anti-HER2 Fab fragment or trastuzumab (0 to 8 pg/mL) for 72 additional hours. Cell viability was determined by the colorimetric conversion of yellow, water-soluble tetrazolium MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl- tetrazolium-bromide; Sigma), to purple, water-insoluble formazan.
  • BT474 cells were seeded in 96-well tissue culture plates at a density of 5x103 cells per well. Then, 8 pg/mL of anti-HER2 Fab or trastuzumab were added and cells were counted each day for 5 consecutive days. Experiments were performed in triplicate, untreated cells being used as positive controls. Results were expressed as percent of cell viability compared to untreated cells ay Day 1.
  • Nude mice purchased from Janvier (Centre-Elevage-Janvier, France), were maintained in specific pathogen-free animal housing (SMBH, Bobigny, agreement n°C9300801). After the tumor biopsy had been performed, one sample was transported in RPMI-1640 and subcutaneously grafted in 6-week-old NMRI-nude mice, under xylasin (10mg/kg)/ketamin (lOOmg/kg) anaesthesia. The Ministry of Research and Ethics Committee for experimental animal studies approved this study (APAFIS#17190-2018101814245111).
  • a daily clinical score was recorded and tumor growth measured weekly until tumor weight reached the ethically recommended limit of less than 10% of mouse weight (Directive 2010/63/EU of the European Parliament and the Council of 22 September 2010 on the protection of animals used for scientific purposes; Official Journal of the European Union L 276/33).
  • Ultrasonography was performed twice a week on treated and untreated mice with an AplioXT device (Toshiba, Japan) to assess tumor response.
  • Tumours were dissected and divided into three parts: one part was immediately snap-frozen in liquid nitrogen, one part was formalin-fixed (fixing agent AFA, CARLO ERBA Reagents) and paraffin-embedded, one part was glutaraldehyde-fixed and Epon resin-embedded.
  • Necrosis areas were evaluated on H&E colored, 2 pm-thick paraffin sections, at 40 magnifications, and expressed in percentage of necrosis area/total tumoral area.
  • Antiproliferative effect was observed by Ki67 immunostaining in 5 um-thick sections, on 5 different non necrotic fields at 200 magnifications, and quantified by percentage of positive cells/HPF.
  • Anti -angiogenic effect was assessed using CD31 immunostaining in 5 um-thick sections, on 5 different non necrotic fields at 200 magnifications, and quantified by number of positive endothelial cells/HPF.
  • Pro apoptotic effect was assessed by cleaved-caspase 3 immunostaining in 5 um-thick sections, on 5 different non necrotic fields at 200 magnifications, and quantified by percentage of positive cells/HPF. Staining was performed using an indirect immunoperoxydase method with a rabbit anti -Human Ki67 antibody (dilution 1 : 100, abeam), a rat anti-Mouse CD31 antibody (dilution 1 :20, Dianova), and a rabbit anti-Human Cleaved caspase-3 antibody (dilution 1 :50, Cell Signaling Technology) as primary antibodies.
  • Quantification of mRNA expression of adrenomedullin and BNP was performed with RT-qPCR assay using GoScriptTM-Reverse-Transcription System (Promega, France), GoTaq® qPCR Master Mix (Promega, France), and taqman primers and probes for Adrenomedullin (mM00437438_Gl, Thermofisher) and BNP (mM01255770_gl, Thermofisher). A total mix volume of 120 pL for retrotranscription and 324 pL per probe for RT-qPCR was used. Assays were read at 95° for 60 cycles on the Biorad Real-Time Detection System.
  • TBP Hs99999910_ml
  • GAPDH Hs02786624_gl
  • ThermoFisher Scientific was used as the endogenous control for normalization. Data were normalized on the reference gene, using CFX manager software and expression levels were calculated using the 2-ACq method. All Cq value >40 was not retained for analysis.
  • Immunochemistry was performed on tumor xenografts 5 pm -thick paraffin sections with an indirect immunoperoxydase method using rabbit anti -Human HER2 (dilution 1 : 100, cloneSP3, Spring Bioscience) as the primary monoclonal antibody. Tissue sections were analyzed under an Olympus AX 70 microscope at X400 magnification.
  • Reagent mixes (with Hs00223586_cn ERBB2 as the primer and TaqMan® Copy Number Reference Assay, human, RNase P, Life Technologies) were prepared using standard Taqman primer/probe chemistry with a 2 X ddPCR Mastermix (BioRad, Laboratories), a 20 X primer/probe (900/250 nM), and 5 pL of sample DNA template in a final volume of 20 pL.
  • the reagent mixture was loaded into an eight-channel droplet generator (BioRad, Laboratories). 70 pL of droplet generation oil were loaded for each channel and after generation of water-in-oil droplets the droplets were transferred to a 96-well PCR plate and placed in a Biorad thermocycler.
  • ELISA Enzyme-linked Immunosorbent Assay
  • trastuzumab were provided by the pharmacy of Avicenne Hospital. For trastuzumab concentration assessment, we used an ELISA procedure already validated for clinical practice. Blood and CSF samples
  • trastuzumab and anti-HER2 Fab were prepared in saline solution (0.9%) in a total volume of lOOpL.
  • 1400pg of trastuzumab and anti-HER2 Fab were prepared in saline solution in a total volume of lOOpL.
  • Brains were macroscopically divided in 3 longitudinal sections: The central section was formalin-fixed and paraffin-embedded (FFPE) for immunohistochemical analyses. One lateral part was frozen for immunohistochemical analyses, and the second lateral part was immediately divided into three distinct areas (cortical, central and posterior) and frozen for further ELISA assessment.
  • FFPE paraffin-embedded
  • frozen brains were cut by a cryotome into fine slivers of 5 micron, then rabbit anti -trastuzumab primary antibody (MAB95471 R&D System) diluted at 1/10, and anti-rabbit secondary antibody coupled to peroxidase were added. Each section was examined at *400 magnification.
  • ELISA antibody concentration assessment in brain we used the same ELISA assessment developed and described above. Each part (cortical, central, posterior) was previously prepared using protein extraction agent (N-PERTM Neuronal Protein Extraction Reagent, Thermo Fisher Scientific, USA), with a ratio of 1g tissue for lOmL of N-PER reagent. Samples were homogenized in N-PEER reagent for 10 minutes, and centrifugated at 10 000 RPM for 10 minutes at 4°. Supernatant were collected for further ELISA analysis. Appropriate controls were implemented, on rat brains who did not receive any antibody injection.
  • protein extraction agent N-PERTM Neuronal Protein Extraction Reagent, Thermo Fisher Scientific, USA
  • trastuzumab and of the anti-HER2 Fab#2 in rats were assessed independently with a population approach using Monolix 2020R1 (Lixoft, Antony, France).
  • a two-compartment model with two first-order elimination rates and an absorption compartment was implemented to describe the concentration data in serum and CSF. Parameters followed an exponential model for between - subject variabilities, unless standard deviations estimates or shrinkage were considered too high, in which case a typical population value was estimated with no variability.
  • Proportional error models were used to describe residual variabilities in serum and CSF. Brain diffusions of trastuzumab and anti-HER2 Fab#2 were assessed comparing typical values of parameters describing CSF-to-serum flow and elimination in CSF.
  • FcRn receptor is expressed by endothelial cells of the blood-brain barrier in human and rat brain.
  • FcRn is a transmembrane protein composed of two subunits: a large transmembrane alpha chain with 3 extracellular domains (al, a2, a3), also called the subunit P51, and the P2 microglobulin subunit [22] (data not shown).
  • FcRn is ubiquitously expressed across different cells, tissues and species, including endothelial cells of the mammalian BBB [22], Using the Uniprot database [23], we compared the amino acid sequence of human, mouse and rat FcRn (data not shown). Between human and rat, the homology is 67.1% for the entire protein composed of 484 amino acids. It is 64.6% for the P51 subunit and 69.7% for the P2microglobulin subunit. In contrast, the IgG fixation site corresponding to 10 amino acids 24 is highly conserved between species (data not shown).
  • ranibizumab CSF concentration rapidly decreased overtime, we hypothesized that it penetrated the brain parenchyma.
  • ranibizumab was identified in the cerebellum close to Purkinje cells (data not shown), a region where VEGF is physiologically expressed and acts as a factor of neurogenesis and neuronal maturation 25.
  • Engineer of anti-HER2 Fab antibodies were used.
  • trastuzumab Fab Two different anti-HER2 Fab fragments of trastuzumab have been engineered using a comparable synthesis approach. The sequence was determined using the known Fab sequence of trastuzumab. The first Fab fragment (anti-HER2 Fab#l) was produced in our research unit and extracted from the culture supernatant. Using SDS-PAGE, we identified two ⁇ 25 kDa fragments corresponding to the light and heavy chains of the Fab fragment (data not shown). Using western-Blot and an anti-trastuzumab antibody recognizing the kappa light chain, we confirmed it was a trastuzumab Fab (data not shown).
  • the second fragment (anti-HER2 Fab#2) was produced by Biotem Industry and initially led to 1.82mg of a 45 kDa purified antibody.
  • the analysis by SDS-PAGE under reducing conditions identified two fragments of ⁇ 23 kDa and ⁇ 25 kDa, corresponding respectively to the light and heavy chains of the Fab (data not shown).
  • Analysis by SDS-PAGE under non-reducing conditions identified a fragment ⁇ 40-45 kDa, corresponding to Fab in its complete form (data not shown).
  • the protein sequence was then analyzed using mass spectrometry (data not shown).
  • Mascot software which recognized 88% of the constant sequence of light chain (CL), and 22% of the constant sequence of heavy chain (CH). Remaining amino-acids were identified manually, like for the variable sequence using the Data Analysis software (Bruker), leading to a 100% sequence recognition.
  • Anti-HER2 Fab antibodies have in vitro effects comparable to those of trastuzumab.
  • Anti-HER2 Fab antibodies have in vivo effects comparable to those of trastuzumab.
  • Tumor cell apoptosis assessed using cleaved-caspase 3 immunostaining, was very sparse both in treated and untreated mice (data not shown).
  • a comparative pharmacological study was conducted on blood samples obtained before each intravenous injection (at DayO, Day7, Dayl4 and Day 21), and 30 minutes after the first injection. Concentrations at 30 minutes were 45 mg/L and 10 mg/L respectively for trastuzumab and for the Fab#2. Trastuzumab steady-state concentration was of 1 mg/L while undetectable for the Fab#2 (data not shown). Trastuzumab half-life was estimated at 1.5 days. We could not calculate it for the Fab#2, probably because its serum half-life in mice is very short and because we did not have enough sampling between 30 minutes and Day7.
  • anti-HER2 Fab antibodies had in vitro and in vivo anti -turn or effects comparable to those of trastuzumab.
  • Anti-HER2 Fab antibodies and trastuzumab have minimal cardiac toxicity.
  • the anti-HER2 Fab antibody does not efflux from brain to blood after intra-thecal injection in rats.
  • the anti-HER2 Fab antibody does penetrate into the deeper brain parenchyma.
  • Kpu,ubrain AUC U , brain /AUC u ,csf
  • the Kpu,ubrain was 12.3% for trastuzumab and 22.7% for Fab#2, in accordance with the result of the modeling approach (Figure 2F).

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Abstract

La présente invention concerne le traitement de troubles cérébraux ou neurologiques. Dans cette étude, les inventeurs ont supposé qu'un récepteur Fc (FcRn) exprimé par des cellules endothéliales provenant de la BHE était responsable de cet efflux. En tant que preuve de concept, les inventeurs ont modifié un fragment de liaison à l'antigène (Fab) du trastuzumab et l'ont caractérisé par des modèles précliniques à des fins thérapeutiques translationnelles. Ils ont démontré la sécurité et une efficacité égale avec le trastuzumab in vitro, et in vivo à l'aide d'un modèle de xénogreffe dérivé d'un patient de HER2 surexprimant le cancer du sein. Ils ont été modifiés avec succès et ont soigneusement permis le développement préclinique d'un Fab de trastuzumab comme efficace en tant qu'IgG native, et pouvant doubler la pénétration cérébrale et de réduire significativement l'efflux cerveau vers le sang après injection de fluide intra-cérébrospinal. Ce Fab pourrait ainsi être une arme efficace nouvelle et originale dans le traitement de métastases cérébrales du cancer du sein HER2, et ainsi utilisé en tant que preuve de concept de l'utilisation puissante du fragment de liaison à l'antigène (dérivés Fab d'anticorps) dans les troubles cérébraux ou neurologiques. Ainsi, la présente invention concerne un fragment de liaison à l'antigène qui se lie à un antigène destiné à être utilisé dans le traitement de troubles cérébraux ou neurologiques chez un sujet en ayant besoin.
PCT/EP2024/083856 2023-11-29 2024-11-28 Nouvelle méthode de traitement de troubles cérébraux ou neurologiques Pending WO2025114411A1 (fr)

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