Fc-modified biologics for localized delivery to compartments, particularly the CNS

Abstract

The present invention relates to a polypeptide comprising the crystallizable fragment (Fc) region of IgG for use in the prevention or treatment of diseases, particularly diseases affecting the central nervous system. The polypeptide is administered locally to the affected compartment, particularly the central nervous system. The Fc region has a modification that reduces affinity for the neonatal Fc receptor (FcRn), resulting in increased brain to serum concentrations of the polypeptide.

Description

[Technical Field]

[0001] The present invention relates to locally delivered biological pharmaceuticals characterized by Fc polypeptides with low affinity for the neonatal Fc receptor (FcRn), particularly for use in neurological disorders. [Background technology]

[0002] Currently, the incidence of neurological diseases in Europe and the United States exceeds 200 cases per 100,000 people and is expected to increase further due to the aging of the population. Advances in preclinical research have provided many promising targets for the local treatment of neurological diseases, including cytokine therapy (e.g., IL-12 in brain tumors and IL-10 in MS), as well as neutralizing antibodies (e.g., against interleukin (IL)-12 / 23p40 in MS and against tumor necrosis factor alpha (TNFα) in Parkinson's disease and Alzheimer's disease) or immune checkpoint blockade molecules (e.g., blockade of the PD-1 / PD-L1 axis in brain tumors).

[0003] Immunotherapy is one of the most promising directions in brain tumor treatment. Interleukin (IL)-12 is a proinflammatory cytokine that has potent antitumor effects against brain tumors in preclinical models. Based on promising preclinical results, clinical trials using intravenously administered IL-12 as a systemic treatment were rapidly initiated in the late 1990s. However, serious adverse events were reported in phase II clinical trials, resulting in hospitalization of 12 of 17 patients and death in 2. These adverse effects have subsequently been attributed to the rapid induction of high systemic levels of interferon (IFN)-γ, a downstream effector cytokine of IL-12.

[0004] Considering the toxicity of systemically applied IL-12 and the need for high concentrations at tumor sites, strict control of IL-12 levels in tissues is an essential prerequisite for clinical application. Local administration to the brain has recently become possible using new neurosurgical techniques such as convection-enhanced delivery (CED). However, local intracranial delivery does not preclude subsequent systemic leakage.

[0005] Murine IL-12Fc, a single-chain fusion protein of IL-12 and a crystallizable fragment of immunoglobulin G (Fc), exhibits increased pharmacological stability, increased bioavailability, and reduced passive leakage from the brain compared to unmodified recombinant IL-12. However, after local delivery to the brain, it is actively excreted across the blood-brain barrier (BBB) ​​by the neonatal Fc receptor (FcRn), which mediates the efflux of all proteins containing the Fc region from the cerebrospinal fluid. FcRn is also active in endothelial cells and red pulp macrophages, preventing degradation and extending the serum half-life of Fc-containing molecules and serum albumin. Therefore, compared to unmodified IL-12, IL-12Fc exhibits increased systemic accumulation.

[0006] The IgG Fc residues known to be involved in FcRn binding (isoleucine 253-I253, histidines 310-H310 and histidines 435-H435) and the pH dependence of the interaction between these residues and FcRn are known from the state of the art (Pyzik et al., Frontiers in Immunology (2019) 10:1540).

[0007] For example, Bitonti et al. reported that mutating residues I253, H310, and H435 in the Fc domain of wild-type IgG to alanines 253-A253, A310, and A435 (AAA), respectively, abolished FcRn binding at pH 6 (Bitonti et al., Proceedings of the National Academy of Sciences (2004) 101(26):9763-9768).

[0008] However, substitution of amino acids with alanine is a common biochemical method for screening the functional role of a given position in a protein of interest. Apart from this one specific mutation (AAA), this paper does not disclose any other mutations from which conclusions can be drawn about the resulting binding properties to FcRn. Furthermore, this paper addresses the FcRn-mediated transport of Fc fusion proteins containing erythropoietin (Epo), a glycoprotein hormone that stimulates red blood cell production, in the lungs of non-human primates. This paper makes no mention of the applicability of the results to fusion polypeptides containing IL-12 or the administration of Fc fusion polypeptides to the brain.

[0009] There are publications that demonstrate fusion polypeptides containing IL-12 and methods to increase their serum half-life.

[0010] For example, Jung et al. describe the generation and antitumor activity of a fusion polypeptide comprising IL-12 and a human IgG4-based heterodimeric Fc with an A107 mutation pair that reduces affinity to the Fcγ receptor (Jung et al., Oncoimmunology, Vol. 7(7):e1438800).

[0011] However, it is clear that FcRn is not equivalent to FcγR, as the Fc gamma receptor (FcγR) family is a functional group of proteins characterized by structural but distinct binding to the constant region of antibodies, i.e., the Fc portion, non-overlapping binding sites in the Fc portion, localization in different cellular compartments (intracellular vs. extracellular), pH-dependent binding (acidic vs. neutral), and overall function.

[0012] In another example of the state of the art, a comparison is made between recombinant IL-12 and IL-12Fc with regard to tissue retention and leakage into the systemic circulation (Beffinger et al., Neuro-Oncology (2017), 19(Suppl. 6), vi273), where the authors state that IL-12Fc showed higher brain concentrations 24 hours after intracranial application compared to recombinant IL-12.

[0013] However, this study does not disclose fusion polypeptides with mutations in the Fc region of IgG or their effect on binding to FcRn.

[0014] Cooper et al. investigated the role of FcRn in IgG efflux from the rat brain by local delivery of two variants of a recombinant human IgG1 mAb with either increased FcRn binding (IgG1 asparagine 434 to alanine, N434A) or decreased FcRn binding (IgG1 histidine 435 to alanine, H435A) compared to the Fc of wild-type IgG (Cooper et al., Brain Research (2013) 1534:13-21). The mutants were obtained by incorporating mutations at amino acid positions 434 and 435, respectively. The study was performed in rats using human antibodies.

[0015] Regarding the binding properties of Fc mutants to mouse and human forms of FcRn, Andersen et al. disclosed five different Fc mutants with mutations at the levels of Ile253, His310, and His435: H435Q, H435R, H310A, I253A, and H310A / H435Q (Andersen et al., Journal of Biological Chemistry (2012) 287(27):22927-22937). The variant characterized by the lowest affinity for human FcRn was a mutant with both the H310A and H435Q mutations (IAQ).

[0016] Although the last two studies mentioned herein demonstrated that FcRn plays an important role in IgG export from the rat brain and each disclosed a distinct mutant with reduced affinity for FcRn, neither of these studies served as a basis for assessing how the presence of IL-12Fc affected binding to FcRn. Furthermore, the concept of generating a maximal brain-to-blood concentration gradient was not disclosed. Summary of the Invention [Problem to be solved by the invention]

[0017] Based on the above-mentioned state of the art, the object of the present invention is to provide means and methods for expanding the therapeutic window of pharmaceuticals delivered locally to a specific compartment, specifically the brain, and preventing both elimination from said compartment, specifically the brain, and systemic accumulation, thereby increasing the compartment-to-serum ratio, specifically the brain-to-serum ratio. This object is achieved by the claims herein. [Means for solving the problem]

[0018] In the present context, the term fragment crystallizable (Fc) region refers to the fraction of an IgG antibody that contains two identical heavy chain fragments linked by disulfide bonds or covalently to a single heavy chain fragment. The heavy chain fragment contains the constant domains (C in IgG antibody isotypes). H 2 and C H It consists of three domains.

[0019] In the present context, the EU numbering system (Edelman et al., Proceedings of the National Academy of Sciences of the United States of America (1969) 63(1):78-85) is used to number the amino acid residues in the Fc region. The EU numbering scheme is a widely adopted standard for numbering residues in antibodies in a consistent manner.

[0020] Amino acid sequences are given from the amino to the carboxyl terminus. Capital letters for sequence positions refer to L-amino acids in the one-letter code (Stryer, Biochemistry, 3rd Edition, p. 21). Lowercase letters for amino acid sequence positions refer to the corresponding D- or (2R)-amino acids.

[0021] Amino acid residues I253, H310 and H435 are CH 2-C H It is located at the three-domain interface and, with the exception of R435 in human IgG3, is conserved across IgG subclasses within a species and between IgG molecules found in both rodents and humans (Miyakawa et al., RNA (2008) 14:1154-1163). According to the present invention, the modified Fc region or fragment thereof can be derived from an IgG1, IgG2, or IgG4 immunoglobulin and must include at least amino acid residues 253, 310, and 435 of the Fc domain of immunoglobulin G (IgG) according to the EU numbering system.

[0022] In the present context, IL-12 refers to interleukin-12. In the present context, hIL-12 relates to human IL-12.

[0023] In the present context, mIL-12 relates to murine IL-12. In the present context, rhIL-12 relates to recombinant human IL-12.

[0024] In the present context, rmIL-12 relates to recombinant murine IL-12. In the present context, IL-12Fc WT relates to IL-12 linked to a wild-type unmodified Fc region, in particular by fusion of p40 and p35 with a Gly-Ser-linker or by addition of an IgG4 tag.

[0025] In the present context, mIL-12hFc WT relates to murine IL-12 linked to the human wild-type Fc region of IgG4 containing the S228P mutation.

[0026] In the present context, mIL-12hFc NHQ relates to murine IL-12 linked to a human wild-type Fc region of IgG4 containing serine 228 to proline-S228P, similar to the NHQ mutation.

[0027] In the context of this specification, mIL-12hFc:anti-PD-L1 bifunctional molecule refers to murine IL-12 linked to a human IgG1 Fc and dimerized with half molecules (one heavy chain and one light chain) of a fully human PD-L1-binding IgG1 antibody. The Fc portion of the resulting molecule contains an NHQ mutation.

[0028] In the context of this specification, FcRn tg relates to a mouse strain lacking functional mouse FcRn and carrying a transgene for expression of the human FcRn α-chain under the control of native human regulatory elements, described by the allele symbol Tg(FCGRT)32Dcr.

[0029] In the context of the present invention, an IL-12 polypeptide is a polypeptide having an amino acid sequence that includes the sequence of p35 (Uniprot ID 29459) or a functional homolog thereof and the sequence of p40 (Uniprot ID 29460) or a functional homolog thereof. In one embodiment, an IL-12 polypeptide has an amino acid sequence that includes p35 and p40 sequences or their homologs as part of the same continuous amino acid stretch. In the continuous amino acid stretch, only the N-terminal polypeptide (p40) functional homolog retains its signal peptide. In another embodiment, an IL-12 polypeptide comprises two different amino acid stretches, one containing the p35 sequence and the other containing the p40 sequence, both of which have their respective signal peptides. An IL-12 polypeptide has the biological activity of IL-12. The biological activity of IL-12 in the context of the present invention includes the stimulation of NK or T cells by the IL-12 polypeptide, most specifically the stimulation of T effector cells acting via perforin.

[0030] In the present context, the terms sequence identity and sequence identity percentage refer to the value determined by comparing two aligned sequences.The method of aligning sequences for comparison is well known in the art.The alignment of sequences for comparison can be carried out by Smith and Waterman, Adv.Appl.Math., 2:482 (1981) local homology algorithm, Needleman and Wunsch, J.Mol.Biol., 48:443 (1970) global alignment algorithm, Pearson and Lipman, Proc.Nat.Acad.Sci., 85:2444 (1988) similarity search method, or by computerized implementation of these algorithms, including but not limited to CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available through, for example, the National Center for Biotechnology Information (http: / / blast.ncbi.nlm.nih.gov / ).

[0031] An example of a comparison of amino acid sequences is the BLASTP algorithm, which uses default settings: expected threshold: 10; word size: 3; maximum matches in query range: 0; matrix: BLOSUM62; gap costs: presence: 11, extension: 1; composition adjustment: conditional composition score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm, which uses default settings: expected threshold: 10; word size: 28; maximum matches in query range: 0; match / mismatch score: 1.-2; gap costs: linear.

[0032] Unless otherwise specified, sequence identity values ​​provided herein refer to values ​​obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol., 215:403-410 (1990)), using the default parameters identified above for protein and nucleic acid comparisons, respectively.

[0033] In the present context, IL-10 refers to interleukin 10. In certain embodiments, IL-10 is employed to treat inflammation, autoimmune inflammation, dementia, or stroke. In certain embodiments, neutralizing IL-10 is employed to treat pulmonary paracoccidioidomycosis.

[0034] In the context of this specification, IL-2 refers to interleukin 2. In certain embodiments, IL-2 is employed in the treatment of cancer and infectious diseases.

[0035] In the context of this specification, IL-7 refers to interleukin 7. In certain embodiments, IL-7 is employed in the treatment of cancer and infectious diseases.

[0036] In the context of this specification, IFNγ refers to interferon gamma. In certain embodiments, IFNγ is employed in the treatment of cancer and infectious diseases.

[0037] In the context of this specification, IL-15 refers to interleukin 15. In certain embodiments, IL-15 is employed in the treatment of cancer and infectious diseases.

[0038] In the context of this specification, IL-23 refers to interleukin 23. In certain embodiments, IL-23 is employed in the treatment of cancer and infectious diseases.

[0039] In the context of this specification, TNFα refers to tumor necrosis factor alpha, also known as cachexin or cachectin. In certain embodiments, TNFα is employed in the treatment of cancer and infectious diseases. In certain embodiments, blocking TNFα is employed in the treatment of inflammation, autoimmune inflammation, and arthritis. In certain embodiments, blocking TNFα is employed in the treatment of uveitis. In certain embodiments, blocking TNFα is employed in the treatment of rheumatoid arthritis. In certain embodiments, blocking TNFα is employed in the treatment of sarcoidosis. In certain embodiments, blocking TNFα is employed in the treatment of cystic fibrosis.

[0040] In the context of this specification, CTLA-4 refers to cytotoxic T lymphocyte-associated protein 4, also known as CD152. In certain embodiments, blocking CTLA-4 is employed in the treatment of cancer. In certain embodiments, blocking CTLA-4 is employed in the treatment of lung cancer.

[0041] In the present context, TGFβ refers to transforming growth factor beta.In some embodiments, blocking TGFβ is used to treat cancer and infectious diseases.In some embodiments, TGFβ is used to treat inflammation, autoimmune inflammation, dementia and stroke.In some embodiments, TGFβ antagonist is used to treat cystic fibrosis.

[0042] In the context of this specification, TGFα refers to transforming growth factor alpha. In certain embodiments, TGFα antagonists are employed in the treatment of cystic fibrosis.

[0043] In the context of this specification, TGFβRII refers to transforming growth factor beta receptor II. In certain embodiments, blocking TGFβRII or using TGFβRII-Fc is employed in the treatment of cancer and infectious diseases.

[0044] In the present context, GDNF refers to glial cell line-derived neurotrophic factor. In certain embodiments, GDNF is employed in the treatment of multiple sclerosis, Parkinson's disease, dementia, stroke, and genetic disorders.

[0045] In the context of this specification, IL-35 refers to interleukin 35. In certain embodiments, IL-35 is employed in the treatment of inflammation, autoimmune inflammation, dementia, and stroke.

[0046] In the context of this specification, CD95 refers to Fas, also known as FasR, apoptosis antigen 1, APO-1, APT, or TNFR superfamily member 6. In certain embodiments, blocking CD95 is employed in the treatment of cancer.

[0047] In the present context, IL-1RA refers to interleukin 1 receptor antagonist.In some embodiments, IL-1RA is used to treat inflammation, autoimmune inflammation, rheumatoid arthritis, gout, pseudogout dementia and stroke.In some embodiments, blocking IL-1RA is used to treat rheumatoid arthritis.

[0048] In the context of this specification, IL-4 refers to interleukin 4. In certain embodiments, IL-4 is employed in the treatment of inflammation, autoimmune inflammation, dementia, and stroke.

[0049] In the present context, IL-13 refers to interleukin-13. In certain embodiments, IL-13 is used to treat inflammation, autoimmune inflammation, dementia, and stroke. In certain embodiments, neutralizing anti-IL-13 is used to treat severe, uncontrolled asthma. In certain embodiments, blocking and / or neutralizing IL-13 is used to treat chronic rhinosinusitis with nasal polyps. In certain embodiments, IL-13 antagonists are used to treat idiopathic pulmonary fibrosis.

[0050] In the present context, TSLP refers to thymic stromal lymphopoietin, a protein belonging to the cytokine family.In certain embodiments, neutralizing TSLP is used to treat allergic asthma.In certain embodiments, blocking and / or neutralizing TSLP is used to treat chronic rhinosinusitis with nasal polyps.

[0051] In the context of this specification, SIRPα refers to signal-regulatory protein alpha. In certain embodiments, SIRPα is employed in the treatment of cancer.

[0052] In the present context, G-CSF refers to granulocyte-colony stimulating factor (G-CSF or GCSF), also known as colony-stimulating factor 3 (CSF3). In certain embodiments, G-CSF is employed in the treatment of cancer.

[0053] In the present context, GM-CSF refers to granulocyte-macrophage colony-stimulating factor (GM-CSF), also known as colony-stimulating factor 2 (CSF2). In certain embodiments, GM-CSF is used to treat cancer. In certain embodiments, blocking GM-CSF is used to treat multiple sclerosis.

[0054] In the context of this specification, GM-CSFR refers to granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR), also known as CD116 (cluster of differentiation 116), which refers to the receptor for granulocyte-macrophage colony-stimulating factor, which stimulates the production of white blood cells. In certain embodiments, blocking GM-CSFR is used to treat rheumatoid arthritis.

[0055] In the context of this specification, OX40L refers to the ligand of OX40, also known as the ligand of CD134. In certain embodiments, OX40L is employed in the treatment of cancer.

[0056] In the context of this specification, CD80 refers to B7-1, also known as B7.1. In certain embodiments, CD80 is employed in the treatment of cancer.

[0057] In the context of this specification, CD86 refers to B7-2, also known as B7.2. In certain embodiments, CD86 is used in the treatment of cancer.

[0058] In the context of this specification, GITRL refers to TNFSF18, AITRAIL, TL6, TNLG2A, TNF superfamily member 18. In certain embodiments, GITRL is employed in the treatment of cancer.

[0059] In the context of this specification, 4-1BBL refers to the ligand of 4-1BB, also known as the ligand of ILA or the ligand of CD137 or the ligand of TNFR superfamily member 9. In certain embodiments, 4-1BB is employed in the treatment of cancer.

[0060] In the context of this specification, EphrinA1 refers to EFNA1. In certain embodiments, EphrinA1 is employed in the treatment of cancer.

[0061] In the context of this specification, EphrinB2 refers to EFNB2. In certain embodiments, EphrinB2 is employed in the treatment of cancer.

[0062] In the context of this specification, EphrinB5 refers to EFNB5. In certain embodiments, EphrinB5 is employed in the treatment of cancer.

[0063] In the context of this specification, PD-L1 refers to programmed cell death ligand 1, also known as CD274 or B7 homolog 1 or B7-H1. In certain embodiments, PD-L1 blockade is employed in the treatment of cancer. In certain embodiments, PD-L1 blockade is employed in the treatment of uveal melanoma. In certain embodiments, PD-1 blockade is employed in the treatment of lung cancer.

[0064] In the context of this specification, histone refers to proteins belonging to the histone families H1 / H5, H2A, H2B, H3, and H4. In certain embodiments, binding histones is employed in the treatment of cancer.

[0065] In the context of this specification, CXCL10 refers to C-X-C motif chemokine 10, also known as interferon gamma-inducible protein 10 (IP-10) or small inducible cytokine B 10. In certain embodiments, CXCL10 is employed in the treatment of cancer.

[0066] In the context of this specification, PD-1 refers to programmed cell death protein 1, also known as CD279. In certain embodiments, binding PD-1 is employed in the treatment of cancer. In certain other embodiments, binding PD-1 is employed in the treatment of dementia. In certain embodiments, blocking PD-1 is employed in the treatment of uveal melanoma. In certain embodiments, blocking PD-1 is employed in the treatment of lung cancer.

[0067] In the context of this specification, TREM2 refers to triggering receptor expressed on myeloid cells 2. In certain embodiments, blocking TREM2 is employed to treat inflammation, autoimmune inflammation, dementia, and stroke.

[0068] In the context of this specification, IL-6 refers to interleukin 6. In certain embodiments, blocking IL-6 is employed in the treatment of inflammation, autoimmune inflammation, dementia, and stroke.

[0069] In the context of this specification, IL-6R refers to the interleukin 6 receptor. In certain embodiments, blocking IL-6R is used to treat inflammation, autoimmune inflammation, rheumatoid arthritis, juvenile idiopathic arthritis, and adult-onset Still's disease. In certain embodiments, blocking and / or neutralizing IL-6R is used to treat diseases caused by coronavirus disease 2019 (COVID-19) and / or severe acute respiratory syndrome coronavirus (SARS-CoV).

[0070] In the context of this specification, Cx3cr1 refers to CX3C chemokine receptor 1, also known as fractalkine receptor or G protein-coupled receptor 13 (GPR13). In certain embodiments, binding Cx3cr1 is employed in the treatment of cancer, dementia, inflammation, autoimmune inflammation, and stroke.

[0071] In certain embodiments, blocking CD27 is employed in the treatment of inflammation or autoimmune inflammation.

[0072] In certain embodiments, activating CD27 is employed in the treatment of cancer. In certain embodiments, blocking CD25 is employed in the treatment of inflammation, autoimmune inflammation and multiple sclerosis.

[0073] In certain embodiments, binding CD25 is employed in the treatment of cancer. In certain embodiments, activating CD28 is employed in the treatment of cancer.

[0074] In the present context, Nogo-A refers to an axonal outgrowth inhibitor, also known as NOGO or NSP or NSP-CL reticulon 4. In certain embodiments, blocking Nogo-A is employed in the treatment of autoimmune inflammation, traumatic CNS injury, and stroke.

[0075] In the present context, IL-12Rb1 refers to the interleukin-12 receptor beta 1 subunit. In certain embodiments, blocking IL-12Rb1 is employed to treat inflammation, autoimmune inflammation, dementia, and stroke.

[0076] In the context of this specification, CD47 refers to an integrin-associated protein (IAP). In certain embodiments, blocking CD47 is employed in the treatment of cancer.

[0077] In the context of this specification, CD147 refers to basigin (BSG), also known as extracellular matrix metalloproteinase inducer (EMMPRIN). In certain embodiments, blocking CD147 is employed in the treatment of coronavirus disease 2019 (COVID-19). In certain embodiments, blocking CD147 is employed in the treatment of diseases caused by severe acute respiratory syndrome coronavirus (SARS-CoV). In the context of this specification, EGFR refers to epidermal growth factor receptor, also known as ErbB-1. In certain embodiments, blocking EGFR is employed in the treatment of cancer.

[0078] In the context of this specification, EGFRvIII refers to the vIII mutant of epidermal growth factor receptor, also known as the vIII mutant of ErbB-1. In certain embodiments, blocking EGFRvIII is used to treat cancer.

[0079] In the present context, Her2 refers to the receptor tyrosine-protein kinase erbB-2, also known as CD340 or the proto-oncogene Neu. In certain embodiments, blocking Her2 is employed in the treatment of cancer.

[0080] In the context of this specification, PDGFR refers to the platelet-derived growth factor receptor (PDGF-R). In certain embodiments, blocking PDGF-R is used in the treatment of cancer.

[0081] In the present context, FGFR refers to fibroblast growth factor receptor. In certain embodiments, blocking FGFR is employed in the treatment of cancer.

[0082] In the present context, IL-4RA refers to the interleukin 4 receptor, also known as IL-4R or CD124. In certain embodiments, blocking IL-4RA is employed in the treatment of cancer. In certain embodiments, blocking IL-4R is employed in the treatment of asthma.

[0083] In the context of this specification, TfR refers to the transferrin receptor. In certain embodiments, binding TfR is employed in the treatment of inflammation, autoimmune inflammation, dementia, traumatic CNS injury, cancer, and stroke.

[0084] In the present context, LfR refers to the lactoferrin receptor, also known as omentin or intestinal lactoferrin receptor. In certain embodiments, binding LfR is employed to treat inflammation, autoimmune inflammation, dementia, traumatic CNS injury, cancer, and stroke.

[0085] In the context of this specification, IR refers to the insulin receptor. In certain embodiments, binding the IR is employed in the treatment of inflammation, autoimmune inflammation, dementia, traumatic CNS injury, cancer, and stroke.

[0086] In the present context, LDL-R refers to the low-density lipoprotein receptor. In certain embodiments, binding LDL-R is employed in the treatment of inflammation, autoimmune inflammation, dementia, traumatic CNS injury, cancer, and stroke.

[0087] In the present context, LRP-1 refers to low-density lipoprotein receptor-related protein 1 (LRP1), also known as alpha-2-macroglobulin receptor (A2MR) or apolipoprotein E receptor (APOER) or CD91. In certain embodiments, binding LRP-1 is employed in the treatment of inflammation, autoimmune inflammation, dementia, traumatic CNS injury, cancer, and stroke.

[0088] In the context of this specification, CD133 refers to prominin-1. In certain embodiments, binding CD133 is employed in the treatment of cancer.

[0089] In the context of this specification, CD111 refers to poliovirus receptor-related 1 (PVRL1), also known as nectin-1. In certain embodiments, binding CD111 is employed in the treatment of cancer.

[0090] In the present context, VEGFR refers to a receptor for vascular endothelial growth factor. In certain embodiments, blocking VEGFR is employed in the treatment of cancer or wet AMD, diabetic macular edema, or retinitis pigmentosa.

[0091] In the context of this specification, VEGF-A refers to vascular endothelial growth factor A. In certain embodiments, blocking VEGF-A is employed in the treatment of cancer or wet AMD, diabetic macular edema, retinitis pigmentosa, or chronic hemophilic synovitis.

[0092] In the context of this specification, Ang-2 refers to angiopoietin 2. In certain embodiments, blocking VEGF-A is employed in the treatment of cancer or wet AMD, diabetic macular edema or retinitis pigmentosa.

[0093] In the context of this specification, IL-10R refers to the interleukin-10 receptor, also known as the receptor for cytokine synthesis inhibitor. In certain embodiments, blocking IL-10R is employed in the treatment of cancer.

[0094] In the present context, IL-13Rα2 refers to the interleukin-13 receptor subunit alpha-2, also known as CD213A2. In certain embodiments, binding IL-13Rα2 is employed in the treatment of cancer. In certain embodiments, IL-13Rα2 is employed in the treatment of cancer.

[0095] In certain embodiments, binding alpha-synuclein is employed in the treatment of Parkinson's disease.

[0096] In the present context, CSF1R refers to colony-stimulating factor 1 receptor (CSF1R), also known as macrophage colony-stimulating factor receptor (M-CSFR) and CD115. In certain embodiments, blocking CSF1R is employed in the treatment of cancer.

[0097] In the present context, GITR refers to glucocorticoid-induced TNFR-related protein, also known as TNFR superfamily member 18 (TNFRSF18) or activation-induced TNFR family receptor or AITR. In certain embodiments, binding GITR is employed in the treatment of cancer.

[0098] In the context of this specification, CD22 refers to cluster of differentiation 22. In certain embodiments, blocking CD22 is employed in the treatment of neurodegenerative diseases, autoimmune inflammation, dementia, and stroke.

[0099] In the context of this specification, TIM-3 refers to T-cell immunoglobulin and mucin domain-containing 3, also known as hepatitis A virus cellular receptor 2 (HAVCR2). In certain embodiments, blocking TIM-3 is employed in the treatment of cancer.

[0100] In the context of this specification, LAG-3 refers to lymphocyte activation gene 3. In certain embodiments, blocking LAG-3 is employed in the treatment of cancer. In certain embodiments, blocking LAG-3 is employed in the treatment of lung cancer.

[0101] In the present context, TIGIT refers to an Ig and T cell immunoreceptor having an immunoreceptor tyrosine-based inhibitory motif domain. In certain embodiments, blocking TIGIT is used to treat cancer.

[0102] In the context of this specification, BTLA refers to B and T lymphocyte attenuator, also known as CD272. In certain embodiments, blocking BTLA is employed in the treatment of cancer.

[0103] In the context of this specification, VISTA refers to a V-domain Ig suppressor of T-cell activation. In certain embodiments, blocking VISTA is employed in the treatment of cancer.

[0104] In the context of this specification, CD96 refers to T cell activation, increased late expression, also known as TACTILE. In certain embodiments, blocking CD96 is employed in the treatment of cancer.

[0105] In the context of this specification, 4-1BB refers to CD137, also known as TNFR superfamily member 9, or induced by lymphocyte activation or ILA. In certain embodiments, binding of 4-1BB is employed in the treatment of cancer.

[0106] In the present context, CCL-2 refers to chemokine (C-C motif) ligand 2 (CCL2), also known as monocyte chemoattractant protein 1 (MCP1) or small inducible cytokine A2. In certain embodiments, CCL-2 is employed in the treatment of cancer, stroke, and dementia. In certain embodiments, blocking CCL-2 is employed in the treatment of autoimmune inflammation and cancer.

[0107] In the present context, IL-1 refers to a member of the IL-1 cytokine family. In certain embodiments, blockade of IL-1 is employed in the treatment of multiple sclerosis.

[0108] In the present context, IL-1R refers to the receptor for cytokines of the IL-1 cytokine family. In certain embodiments, blockade of IL-1R is employed in the treatment of multiple sclerosis.

[0109] In the context of this specification, EphA2 refers to Ephrin type-A receptor 2. In certain embodiments, blocking EphA2 is employed in the treatment of cancer.

[0110] In the context of this specification, EphA3 refers to Ephrin type-A receptor 3. In certain embodiments, blocking EphA3 is employed in the treatment of cancer.

[0111] In the context of this specification, EphB2 refers to ephrin type B receptor 2, also known as ERK. In certain embodiments, blocking EphB2 is employed in the treatment of cancer.

[0112] In the context of this specification, EphB3 refers to ephrin type B receptor 3. In certain embodiments, blocking EphB3 is employed in the treatment of cancer.

[0113] In the context of this specification, EphB4 refers to ephrin type B receptor 4. In certain embodiments, blocking EphB4 is employed in the treatment of cancer.

[0114] In the context of this specification, OX40 refers to TNFR superfamily member 4, also known as CD134 or OX40 receptor. In certain embodiments, binding OX40 is employed in the treatment of cancer.

[0115] In the context of this specification, LINGO-1 refers to leucine-rich repeat and immunoglobin-like domain-containing protein 1. In certain embodiments, blocking LINGO-1 is employed in the treatment of multiple sclerosis, traumatic brain CNS injury, or stroke.

[0116] In the present context, L1CAM refers to L1 cell adhesion molecule, also known as L1. In certain embodiments, blocking L1 is employed in the treatment of multiple sclerosis, traumatic brain CNS injury, or stroke.

[0117] In the present context, NCAM refers to neural cell adhesion molecule. In certain embodiments, blocking NCAM is used to treat multiple sclerosis, traumatic brain CNS injury or stroke.

[0118] In the context of this specification, SOD-1 refers to superoxide dismutase 1. In certain embodiments, blocking SOD-1 is employed in the treatment of amyotrophic lateral sclerosis (ALS).

[0119] In the context of this specification, SIGMAR-1 refers to the sigma-1 receptor. In certain embodiments, blocking SIGMAR-1 is employed in the treatment of amyotrophic lateral sclerosis (ALS).

[0120] In the context of this specification, SIGMAR-2 refers to the sigma-2 receptor. In certain embodiments, blocking SIGMAR-2 is employed in the treatment of amyotrophic lateral sclerosis (ALS).

[0121] In the context of this specification, TDP-43 refers to TAR DNA-binding protein 43. In certain embodiments, binding TDP-43 is employed in the treatment of amyotrophic lateral sclerosis (ALS).

[0122] In the context of this specification, amyloid beta refers to amyloid beta. In certain embodiments, binding amyloid beta is employed in the treatment of Alzheimer's disease (AD).

[0123] In the present context, Tau refers to the Tau protein. In certain embodiments, binding Tau is employed in the treatment of Alzheimer's disease (AD).

[0124] In the context of this specification, IFNα refers to interferon-α. In certain embodiments, IFNα is employed in the treatment of cancer and infectious diseases.

[0125] In the context of this specification, IFNβ refers to interferon-beta. In certain embodiments, IFNβ is employed in the treatment of cancer and infectious diseases.

[0126] In the context of this specification, TRPM4 refers to transient receptor potential cation channel subfamily M member 4. In certain embodiments, blocking TRPM4 is employed in the treatment of multiple sclerosis.

[0127] In the present context, ASIC1 refers to acid-sensing ion channel 1, also known as amiloride-sensitive cation channel 2, neuron (ACCN2) or brain sodium channel 2 (BNaC2). In certain embodiments, blocking ASIC1 is employed in the treatment of multiple sclerosis.

[0128] In the present context, VGCC refers to voltage-gated calcium channels, also known as voltage-dependent calcium channels (VDCCs). In certain embodiments, blocking VGCCs is employed in the treatment of multiple sclerosis.

[0129] In the context of this specification, CB1 refers to cannabinoid receptor type 1, also known as cannabinoid receptor 1. In certain embodiments, blocking CB1 is employed in the treatment of multiple sclerosis.

[0130] In the context of this specification, TTR refers to transthyretin. In certain embodiments, blocking TTR is employed in the treatment of transthyretin amyloidosis.

[0131] In the context of this specification, HTT refers to the huntingtin protein. In certain embodiments, blocking HTT is employed in the treatment of Huntington's disease.

[0132] In the context of this specification, JCV refers to JC virus or John Cunningham virus. In certain embodiments, blocking the major capsid protein VP1 (viral protein 1) of JCV is employed to treat progressive multifocal leukoencephalopathy (PML).

[0133] In the present context, C9orf72 refers to the protein encoded by the chromosome 9 open reading frame 72 gene.In some embodiments, C9orf72 is used to treat dementia.In some embodiments, blocking C9orf72 is used to treat dementia.

[0134] In the present context, BDNF refers to brain-derived neurotrophic factor. In certain embodiments, BDNF is employed in the treatment of multiple sclerosis, Parkinson's disease, dementia, stroke, and genetic disorders.

[0135] In the context of this specification, NRTN refers to neurturin. In certain embodiments, NRTN is employed in the treatment of multiple sclerosis, Parkinson's disease, dementia, stroke, and genetic disorders.

[0136] In the present context, ARTN refers to Artemin. In certain embodiments, ARTN is employed in the treatment of multiple sclerosis, Parkinson's disease, dementia, stroke, and genetic disorders.

[0137] In the context of this specification, PSPN refers to Persephin. In certain embodiments, PSPN is employed in the treatment of multiple sclerosis, Parkinson's disease, dementia, stroke, and genetic disorders.

[0138] As used herein, CNTF refers to ciliary neurotrophic factor. In certain embodiments, CNTF is employed in the treatment of multiple sclerosis, Parkinson's disease, dementia, stroke, and genetic disorders.

[0139] In the context of this specification, TRAIL refers to TNF-related apoptosis-inducing ligand, also known as CD253 or tumor necrosis factor superfamily, member 10. In certain embodiments, TRAIL is employed in the treatment of cancer.

[0140] In the context of this specification, HA refers to hemagglutinin (or haemagglutinin), a homotrimeric glycoprotein found on the surface of influenza viruses. In certain embodiments, neutralizing HA is employed in the treatment of influenza.

[0141] In the context of this specification, IL-3 refers to interleukin 3. In certain embodiments, IL-3 is employed in the treatment of cancer.

[0142] In the context of this specification, IL-5 refers to interleukin 5. In certain embodiments, IL-5 is employed in the treatment of cancer. In certain embodiments, IL-5 blockade is employed in the treatment of asthma. In certain embodiments, IL-5 blockade is employed in the treatment of chronic obstructive pulmonary disease (COPD).

[0143] In the context of this specification, IL-8 refers to interleukin 8, also known as chemokine (C-X-C motif) ligand 8 or CXCL8. In certain embodiments, IL-8 is employed in the treatment of cancer. In certain embodiments, IL-8 blockade is employed in the treatment of pulmonary edema. In certain embodiments, IL-8 antagonists are employed in the treatment of cystic fibrosis.

[0144] In the present context, IL-17 refers to interleukin 17. In certain embodiments, neutralization of IL-17 is employed in the treatment of uveitis. In the present context, IL-17A refers to interleukin 17A. In certain embodiments, neutralization of IL-17A is employed in the treatment of rheumatoid arthritis and / or psoriatic arthritis and / or ankylosing spondylitis.

[0145] In the context of this specification, IL-18 refers to interleukin 18, also known as interferon-gamma inducer. In certain embodiments, IL-18 is employed in the treatment of cancer.

[0146] In the context of this specification, IL-21 refers to interleukin 21. In certain embodiments, IL-21 is employed in the treatment of cancer.

[0147] In the present context, IL-21R refers to the receptor for interleukin 21. In certain embodiments, blockade of IL-21R is employed in the treatment of allergic asthma.

[0148] In the context of this specification, IL-22 refers to interleukin 22. In certain embodiments, neutralizing IL-22 is employed in the treatment of rheumatoid arthritis.

[0149] In the context of this specification, IL-25 refers to interleukin 25 (also known as interleukin 17E or IL-17E). In certain embodiments, neutralizing IL-25 is employed in the treatment of allergic asthma.

[0150] In the context of this specification, CD20 refers to the B lymphocyte antigen CD20. In certain embodiments, CD20 binding antibodies are employed in the treatment of interstitial lung disease. In certain embodiments, CD20 binding antibodies are employed in the treatment of cancer. In the context of this specification, CCL5 refers to chemokine (CC motif) ligand 5. In certain embodiments, CCL5 is employed in the treatment of cancer.

[0151] In the context of this specification, CCL21 refers to chemokine (CC motif) ligand 21. In certain embodiments, CCL21 is employed in the treatment of cancer.

[0152] In the context of this specification, CCL10 refers to chemokine (C-C motif) ligand 10, also known as CCL9 or chemokine (C-C motif) ligand 9. In certain embodiments, CCL10 is employed in the treatment of cancer.

[0153] In the context of this specification, CCL16 refers to chemokine (CC motif) ligand 16. In certain embodiments, CCL16 is employed in the treatment of cancer.

[0154] In the context of this specification, CX3CL1 refers to chemokine (C-X3-C motif) ligand 1, also known as fractalkine. In certain embodiments, CX3CL1 is employed in the treatment of cancer.

[0155] In the context of this specification, CXCL16 refers to chemokine (CXC motif) ligand 16. In certain embodiments, CXCL16 is employed in the treatment of cancer.

[0156] In the context of this specification, NF-kB refers to nuclear factor kappa-light-chain-enhancer of activated B cells. In certain embodiments, NF-kB antagonists are employed in the treatment of cystic fibrosis.

[0157] In the context of this specification, NRA refers to non-rheumatoid arthritis. In certain embodiments, anti-nerve growth factor (NGF) antibodies or antibody-like molecules may be employed to treat inflammation, autoimmune inflammation, arthritis, and osteoarthritis. In certain embodiments, blockade of NGF may be employed to treat osteoarthritis. In the context of this specification, the term antibody refers to an antibody of type G (IgG), any antigen-binding fragment or single chain thereof, and related or derived constructs. A whole antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (V H ) and the heavy chain constant region (C H The heavy chain constant region is composed of C H 1. C H 2 and C H Each light chain is composed of three domains: a light chain variable region (referred to herein as V L ) and the light chain constant region (C L The light chain constant region consists of one domain, C L The variable regions of the heavy and light chains contain the binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system. In the context of this specification, the term antibody is meant to include not only whole antibodies containing two H chains and two L chains, but also abnormal antibodies containing only one H chain and one L chain, or even antibodies consisting of only one H chain.

[0158] In the context of this specification, the term specifically binds refers to binding with high affinity / Kd≦10E -8 Refers to bonding in mol / l.

[0159] In the context of the present specification, the term "antibody-like molecule" refers to a molecule containing at least a portion of the Fc fragment of an IgG antibody and at least one target binding element fused directly or indirectly to the Fc fragment, particularly heavy and light chain variable regions, single chain variable fragments, dual affinity retargeting proteins, or bispecific T cell engagers. Antibody-like molecules have high affinity / Kd≦10E -8 They are capable of specifically binding to another molecule or target in mol / l. Antibody-like molecules bind to their target in a manner similar to the specific binding of an antibody.

[0160] Those skilled in the art will recognize that the present invention requires that the antibody or antibody-like molecule comprise or be fused to an Fc region.

[0161] In the context of this specification, the dissociation constant (K D The term K refers to the equilibrium constant that measures the tendency of a complex composed of (mostly two) different components to reversibly dissociate into its components. This complex can be, for example, an antibody-antigen complex AbAg composed of an antibody Ab and an antigen Ag. D is expressed in molar concentration [mol / l] and corresponds to the concentration of [Ab] at which half of the binding sites for [Ag] are occupied. In other words, the concentration of unbound [Ab] is equal to the concentration of the [AbAg] complex. The dissociation constant can be calculated using the following formula:

[0162]

number

[0163] [Ab]: antibody concentration; [Ag]: antigen concentration; [AbAg]: antibody-antigen complex concentration In the context of this specification, the off-rate (Koff; [1 / sec]) and on-rate (Kon; [1 / sec]) * The terms K (M) are used in the sense known in the arts of chemistry and physics to refer to the rate constants that measure the dissociation (K) or association (K) of an antibody with its target antigen. off and K.on can be determined experimentally using methods well established in the art. A method for determining the Koff and Kon of an antibody employs surface plasmon resonance. This is the principle behind biosensor systems such as the Biacore® or ProteOn® systems. They also calculate the dissociation constant K using the following equation: D You can ask for:

[0164]

number

[0165] In the context of this specification, K D can also be determined by equilibrium analysis of experimental data determined using methods well established in the art, which can be performed using biosensor systems such as Biacore® or ProteOn® systems.

[0166] In the present context, high-grade glioma (HGG) refers to WHO grade IV glioma or glioblastoma multiforme.

[0167] In the context of this specification, an Fc region designated "NHQ" refers to an Fc region in which positions 253, 310, and 435 (as designated by the EU numbering system) contain the indicated amino acid residues, i.e., N at position 253, H at position 310, and Q at position 435. This corresponds to an Fc region with two mutations, I253N and H435Q. Accordingly, an Fc region designated "IAQ" refers to an Fc region with I at position 253, A at position 310, and Q at position 435 (i.e., an Fc region with mutations H310A and H435Q). Table 1 shows some examples of modified Fc regions.

[0168] A first aspect of the present invention provides a fusion polypeptide comprising IL-12 and a fragment crystallizable (Fc) region of IgG for use in the prevention or treatment of diseases affecting the central nervous system. The Fc region is modified to reduce affinity for the neonatal Fc receptor (FcRn). The polypeptide is administered to the brain.

[0169] Administration to the brain can be carried out by intracranial delivery. Intracranial delivery can be continuous or intermittent or non-recurrent. The term "administration to the brain" also includes rinsing the resection cavity after surgery. Administration can be intrathecal or intraparenchymal.

[0170] Modification of the Fc region results in a reduction in the serum-to-brain concentration ratio of the polypeptide, which has the advantage that high local concentrations are achieved in the brain while preventing negative side effects due to high systemic concentrations.

[0171] In certain embodiments, the serum or plasma to brain concentration ratio of the polypeptide is below a predetermined threshold. tg Intracranial injection into the striatum of mice, specifically, intracranial bolus injection or CED, can be measured 24 hours after a. serum or plasma to brain concentration ratio of the same polypeptide comprising an unmodified Fc region, specifically IL-12FcWT, that is at most 2 / 3; b. The same polypeptide without the Fc region or peptide linker, specifically, rhIL-12, has a serum or plasma-to-brain concentration ratio that is up to 1 / 8 that of rhIL-12 is selected from.

[0172] FcRn tg Measurements are performed 24 hours after intracranial injection of 1 μg at 1 μl / min into the striatum of mice using a blunt-ended 26sG Hamilton syringe or CED (using a 27G blunt needle with a 1 mm step tip made of fused silica with an internal diameter of 0.1 mm and a wall thickness of 0.0325 mm and a ramp-up injection regimen of 0.2 μl / min for 5 minutes, 0.5 μl / min for 4 minutes, and 0.8 μl / min for 2.5 minutes; a total volume of 5 μl and a total amount of 1 μg).

[0173] The fusion polypeptide according to the first aspect of the present invention has a lower serum-to-brain concentration ratio than IL-12 linked to an unmodified Fc region (IL-12Fc WT), which has a long serum half-life due to FcRn-mediated recycling in the circulation.

[0174] The fusion polypeptide according to the first aspect of the invention has a lower serum to brain concentration ratio than rhIL-12, which exhibits high passive leakage from the brain.

[0175] In certain embodiments, the decreased affinity of the polypeptide for FcRn is a. K characterizing the binding of FcRn to the same polypeptide containing the unmodified Fc region D At least two-fold increase in K compared to D , and b. K that characterizes FcRn binding to the same polypeptide containing differently modified Fc regions D At least 1.5-fold increased Ka compared to D , namely one mutant selected from IAQ (having mutations H310A and H45Q) and AAA (having mutations I253A, H310A and H435A). Dissociation constant (K D ) is characterized by

[0176] In certain embodiments, K D is the K that characterizes the binding of FcRn to the same polypeptide containing the unmodified Fc region. D In certain embodiments, K D is the K that characterizes the binding of FcRn to the same polypeptide containing the unmodified Fc region. D In certain embodiments, K D is the K that characterizes the binding of FcRn to the same polypeptide containing the unmodified Fc region. D at least a five-fold increase compared to

[0177] In certain embodiments, K D K characterizes the binding of FcRn to the same polypeptide containing the above Fc region with different modifications. D at least a two-fold increase compared to

[0178] In certain embodiments, the differentially modified Fc region is an Fc region having an I at position 253, an A at position 310, and a Q at position 435 (IAQ).

[0179] In certain embodiments, the differentially modified Fc region is an Fc region having an A at position 253, an A at position 310, and an A at position 435 (AAA).

[0180] In certain embodiments, intracranial delivery is achieved by convection-enhanced delivery (CED) or its variants. CED refers to a technique for delivering drugs directly to the brain (tumor) parenchyma. The CED technique involves minimally invasive surgical irradiation of the brain, followed by placement of a small-diameter catheter directly into the brain, thereby bypassing the blood-brain barrier. The main difference from conventional bolus injection and diffusion-driven infusion regimens is the pressure gradient created by increasing the injection until it reaches bulk flow within the tissue. Here, the duration, rather than the injection rate, determines the range of tissue reached. This approach allows the delivery of macromolecular drugs that normally do not enter the brain to effectively reach high concentrations in brain (tumor) tissue.

[0181] In certain embodiments, intracranial delivery is performed by intrathecal delivery. Intrathecal administration refers to the direct administration of a drug into the cerebrospinal fluid (CSF). Intrathecal administration is defined as the application of a substance below the subarachnoid membrane, either in the brain (e.g., via an Ommaya reservoir) or into the subarachnoid space in the spinal cord. A non-limiting example is intrathecal delivery for the treatment of leptomeningeal carcinomatosis and primary Her2 / neuron-positive brain tumors, as well as CD20-positive CNS lymphoma and intraocular lymphoma, using trastuzumab or rituximab, respectively. Another example is the intrathecal administration of anti-NogoA antibodies for the treatment of acute spinal cord injury, multiple sclerosis, or stroke. This approach allows the delivery of macromolecular drugs that normally do not enter the brain, effectively reaching high concentrations in the leptomeninges or brain parenchyma.

[0182] In certain embodiments, intracranial delivery is achieved by intraventricular delivery of the polypeptide. Intraventricular administration refers to administering a drug directly into the cerebrospinal fluid (CSF) into the intraventricular space using a catheter.

[0183] In certain embodiments, intracranial delivery is achieved by in situ production of the polypeptide. In situ production refers to the local production of the polypeptide exclusively or substantially within the brain or brain tumor. By way of non-limiting example, local production can occur from DNA formulations, mRNA, modified mRNA, self-replicating mRNA, viral vectors, encapsulated modified producer cells, or modified T cells. Spatial control of local production can be achieved by local delivery of a molecule or vector encoding the polypeptide or by local activation of polypeptide production. Local production by local delivery of a molecule or vector encoding the polypeptide, and subsequent local activation of polypeptide production, can be achieved by local or systemic administration of an agent that acts as a transcriptional derepressor or a transcriptional activator of a conditional expression cassette. Examples include, but are not limited to, an ecdysone receptor / invertebrate retinoid x receptor-based inducible gene expression system or a tetracycline-regulated transcriptional regulator.

[0184] In some embodiments, intracranial delivery is carried out by systemic delivery of cells modified to produce the above-mentioned polypeptide with tumor or CNS homing ability. The polypeptide can be produced constitutively or inducibly. Examples include, but are not limited to, modified T cells or mesenchymal stem cells.

[0185] In certain embodiments, intracranial delivery is achieved by release from an implanted slow-release / extended-release / sustained-release / controlled-release formulation. In the context of this specification, such formulations refer to dosage forms designed to release the drug at a predetermined rate to maintain a constant drug concentration for a specific period of time while minimizing side effects. Those skilled in the art are aware of a variety of suitable formulations. Non-limiting examples are liposomes, drug-polymer conjugates, hydrogels, wavers, or coated nanoparticles.

[0186] In certain embodiments, intracranial delivery is achieved by intranasal delivery of the polypeptide.

[0187] In certain embodiments, intracranial delivery is achieved by receptor-mediated transcytosis of the polypeptide. A non-limiting example is a bispecific construct that binds to TfR, a target found in diseased brain parenchyma, specifically Aβ plaques in Alzheimer's disease (AD).

[0188] In certain embodiments, the disease affecting the central nervous system is a malignant disease. In certain embodiments, the disease affecting the central nervous system is glioma.

[0189] In certain embodiments, the disease affecting the central nervous system is high-grade glioma (HGG).

[0190] In certain embodiments, the disease affecting the central nervous system is a secondary brain tumor, also known as brain metastasis.

[0191] In certain embodiments, the disease affecting the central nervous system is ischemic brain injury. In certain embodiments, the disease affecting the central nervous system is cerebral infarction, stroke, cerebral hypoxia-ischemia, intracranial embolism or intracranial thrombosis.

[0192] In certain embodiments, the disease affecting the central nervous system is epilepsy. In certain embodiments, the disease affecting the central nervous system is traumatic brain injury.

[0193] In certain embodiments, the disease affecting the central nervous system is a spinal cord injury. In certain embodiments, the disease affecting the central nervous system is dementia.

[0194] In certain embodiments, the disease affecting the central nervous system is Parkinson's disease (PD).

[0195] In certain embodiments, the disease affecting the central nervous system is dementia with Lewy bodies.

[0196] In certain embodiments, the disease affecting the central nervous system is Alzheimer's disease (AD). In certain embodiments, the disease affecting the central nervous system is familial Alzheimer's disease (AD).

[0197] In certain embodiments, the disease affecting the central nervous system is frontotemporal dementia (FTD).

[0198] In certain embodiments, the disease affecting the central nervous system is familial frontotemporal dementia (FTD).

[0199] In certain embodiments, the disease affecting the central nervous system is amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease.

[0200] In certain embodiments, the disease affecting the central nervous system is a transmissible spongiform encephalopathy, specifically Creutzfeldt-Jakob disease (CJD), kuru, scrapie, or bovine spongiform encephalopathy (BSE).

[0201] In certain embodiments, the disease affecting the central nervous system is a genetic disorder. In certain embodiments, the disease affecting the central nervous system is a genetic disorder, specifically cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL).

[0202] In certain embodiments, the disease affecting the central nervous system is a genetic disorder, specifically Huntington's disease.

[0203] In certain embodiments, the disease affecting the central nervous system is a genetic disorder, in particular autism, an autism spectrum disorder (ASD), such as Asperger's syndrome.

[0204] In certain embodiments, the disease affecting the central nervous system is a hereditary leukodystrophy, specifically metachromatic leukodystrophy, Club disease, Canavan disease, X-linked adrenoleukodystrophy, or Alexander disease.

[0205] In certain embodiments, the disease affecting the central nervous system is an inherited metabolic disorder, specifically Tay-Sachs disease or Wilson's disease.

[0206] In certain embodiments, the disease affecting the central nervous system is a psychiatric disorder, specifically memory loss, attention deficit hyperactivity disorder, psychosis, anxiety disorder, bipolar disorder, depression, mania, intellectual disability, global developmental delay, post-traumatic stress disorder, acute stress disorder, or dissociative disorder.

[0207] In certain embodiments, the disease affecting the central nervous system is epilepsy. In certain embodiments, the disease affecting the central nervous system is autoimmune encephalitis.

[0208] In certain embodiments, the disease affecting the central nervous system is multiple sclerosis. In certain embodiments, the disease affecting the central nervous system is neuromyelitis optica (NMO).

[0209] In certain embodiments, the disease affecting the central nervous system is autoimmune encephalitis, specifically anti-NMDAR encephalitis, limbic encephalitis, LGI1 / CASPR2 antibody encephalitis, Hashimoto's encephalopathy, acute disseminated encephalomyelitis (ADEM), Binswanger's disease (subcortical leukoencephalopathy), or Rasmussen's encephalitis.

[0210] In certain embodiments, the disease affecting the central nervous system is infectious encephalomyelitis caused by a virus, particularly rabies virus, human herpes virus, a virus that causes a rash, an insect-borne virus, a tick-borne virus, or human immunodeficiency virus (HIV).

[0211] In certain embodiments, the disease affecting the central nervous system is infectious encephalomyelitis caused by bacteria.

[0212] In certain embodiments, the disease affecting the central nervous system is infectious encephalomyelitis caused by a parasite.

[0213] In certain embodiments, the disease affecting the central nervous system is progressive multifocal leukoencephalopathy (PML), which is caused by the JC polyomavirus (commonly abbreviated as JCPyV or JCV).

[0214] In certain embodiments, the disease affecting the central nervous system is post-infectious encephalomyelitis. In certain embodiments, the disease affecting the central nervous system is neovascularization-related age-related macular degeneration (wet AMD) and diabetic macular edema or retinitis pigmentosa.

[0215] In a further aspect of the invention, the polypeptide according to the invention is used for the prevention or treatment of a disease affecting the lungs, said disease being selected from coronavirus disease 2019, severe acute respiratory syndrome, asthma, allergic asthma, severe uncontrolled asthma, fibrosis, cystic fibrosis, pulmonary fibrosis, chronic obstructive pulmonary disease, influenza, pulmonary edema, sarcoidosis, lung cancer, tuberculosis, human orthopneumovirus, bubonic plague, pneumonic plague, anthrax, invasive fungal diseases of the lungs, pulmonary paracoccidioidomycosis, interstitial lung disease, idiopathic pulmonary fibrosis, and chronic rhinosinusitis with nasal polyps.

[0216] In certain embodiments, the disease affecting the lungs is coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0217] In certain embodiments, the disease affecting the lungs is severe acute respiratory syndrome (SARS).

[0218] In certain embodiments, the disease affecting the lungs is severe acute respiratory syndrome (SARS), which is caused by a virus, specifically a coronavirus.

[0219] In certain embodiments, the disease affecting the lungs is asthma, allergic asthma, severe uncontrolled asthma, or a combination thereof.

[0220] In certain embodiments, the disease affecting the lungs is chronic obstructive pulmonary disease (COPD).

[0221] In certain embodiments, the disease affecting the lungs is fibrosis, cystic fibrosis, pulmonary fibrosis, or a combination thereof.

[0222] In certain embodiments, the disease affecting the lungs is influenza, which is caused by the influenza virus.

[0223] In certain embodiments, the disease affecting the lungs is sarcoidosis (also known as Besnier-Boeck-Schaumann disease).

[0224] In certain embodiments, the disease affecting the lungs is lung cancer. In certain embodiments, in general terms, the disease affecting the lungs is caused by a virus, bacteria, fungus, or parasite.

[0225] In certain embodiments, the disease affecting the lungs is tuberculosis, caused by mycobacterium tuberculosis (commonly abbreviated as M. tuberculosis or M.tb).

[0226] In certain embodiments, the disease affecting the lungs is a respiratory tract infection caused by the syncytial virus human orthopneumovirus (also known as human respiratory syncytial virus, or HRSV, or simply RSV).

[0227] In certain embodiments, the disease affecting the lungs is bubonic plague, which is caused by the bacterium Yersinia pestis.

[0228] In certain embodiments, the disease affecting the lungs is pneumonic plague, caused by Yersinia pestis.

[0229] In certain embodiments, the disease affecting the lungs is anthrax, an infection caused by the bacterium Bacillus anthracis.

[0230] In certain embodiments, the disease affecting the lungs is an invasive fungal disease (also known as fungal lung disease) caused by pulmonary fungal pathogens such as Aspergillus, Cryptococcus, Pneumocystis, and epidemic fungi.

[0231] In certain embodiments, the disease affecting the lungs is pulmonary paracoccidioidomycosis (typically abbreviated as PCM), which is caused by the fungus Paracoccidioides brasiliensis.

[0232] In certain embodiments, the disease affecting the lungs is chronic rhinosinusitis with nasal polyps (typically abbreviated as CRSwNP), a subgroup of chronic rhinosinusitis (CRS).

[0233] In certain embodiments, the disease affecting the lungs is pulmonary edema. In certain embodiments, the disease affecting the lungs is an interstitial lung disease.

[0234] In certain embodiments, the disease affecting the lungs is idiopathic pulmonary fibrosis. In a further aspect of the invention, the polypeptide according to the invention is used for the prevention or treatment of a disease affecting at least one joint, said disease being selected from rheumatoid arthritis, juvenile rheumatoid arthritis, gout, pseudogout, osteoarthritis, chronic hemophilic synovitis, psoriatic arthritis, and ankylosing spondylitis.

[0235] In certain embodiments, the disease affecting the joints is rheumatoid arthritis (RA). In certain embodiments, the disease affecting the joints is juvenile rheumatoid arthritis.

[0236] In certain embodiments, the disease affecting the joints is gout, a form of inflammatory arthritis caused by persistently elevated levels of uric acid in the blood, hi certain embodiments, the disease affecting the joints is pseudogout.

[0237] In certain embodiments, the disease affecting the joints is osteoarthritis (OA), which results from the destruction of articular cartilage and the underlying bone.

[0238] In certain embodiments, the disease affecting the joints is chronic hemophilic synovitis. In certain embodiments, the disease affecting the joints is psoriatic arthritis, a long-term inflammatory arthritis that occurs in people with the autoimmune disease psoriasis.

[0239] In certain embodiments, the disease affecting the joints is ankylosing spondylitis (also known as Bekhterev's disease, Bechterew's disease, or morbus Bechterew).

[0240] In a further aspect of the invention, the polypeptide according to the invention is used for the prevention or treatment of a disease affecting the eye, said disease being selected from uveal melanoma and uveitis.

[0241] In certain embodiments, the disease affecting the eye is uveal melanoma, which is a cancer of the eye (melanoma) that involves the iris, ciliary body, or choroid (collectively called the uvea).

[0242] In certain embodiments, the disease affecting the eye is uveitis, i.e., inflammation of the uvea.

[0243] It is understood that the polypeptides of the present invention can be used simultaneously and / or sequentially to prevent or treat more than one disease or a combination of diseases disclosed herein. In certain embodiments, the Fc region is a chimeric Fc region comprising a human or humanized amino acid sequence.

[0244] In certain embodiments, the Fc region is a human or humanized Fc region. In certain embodiments, the Fc region has a mutation at position 253 relative to SEQ ID NO: 1. In certain embodiments, the Fc region has the mutation I253A. In certain embodiments, the Fc region has the mutation I253N.

[0245] In certain embodiments, the Fc region has a mutation at position 435 relative to SEQ ID NO: 1. In certain embodiments, the Fc region has the mutation H435Q.

[0246] In certain embodiments, the Fc region does not have a mutation at position 435 relative to SEQ ID NO: 1. Thus, the Fc region has an H at position 435.

[0247] In certain embodiments, the Fc region does not have a mutation at 310 relative to SEQ ID NO: 1. Thus, the Fc region has an H at position 310.

[0248] In certain embodiments, the Fc region comprises: - mutations I253A and H435Q, as well as H at position 310 (AHQ); - mutations I253N and H435Q, as well as H at position 310 (NHQ); - mutations I253A, H310A and H435Q (AAQ); - mutations I253N, H310A and H435Q (NAQ); - mutation I253A, and H at positions 310 and 435 (AHH); - mutation I253N, and H at positions 310 and 435 (NHH); - mutations I253A and H310A, and H at position 435 (AAH); - mutations I253N and H310A, as well as H at position 435 (NAH); - mutations I253N, H310A and H435A (NAA); - mutations I253N, H310A and H435E (NAE); - mutations I253A, H310A and H435A (AAA); or -Mutations I253A, H310A and H435E (AAE) Includes.

[0249] In certain embodiments, the Fc region comprises: - mutations I253N and H435Q, as well as H at position 310 (NHQ); - mutations I253A, H310A and H435Q (AAQ); - mutations I253N, H310A and H45Q (NAQ); - mutations I253N, H310A and H435E (NAE); or -Mutations I253A, H310A and H435E (AAE) Includes.

[0250] In certain embodiments, the Fc region comprises the mutations I253N and H435Q, and an H at position 310.

[0251] In certain embodiments, the Fc region comprises the mutations I253A, H310A and H435Q (AAQ).

[0252] In certain embodiments, the Fc region comprises the mutations I253N, H310A and H435Q (NAQ).

[0253] In certain embodiments, the Fc region comprises the mutations I253N, H310A and H435E (NAE).

[0254] In certain embodiments, the Fc region comprises the mutations I253A, H310A and H435E (AAE).

[0255] In certain embodiments, the Fc region is or comprises a sequence characterized by SEQ ID NO:002 (IAQ), SEQ ID NO:003 (AHQ), SEQ ID NO:004 (NHQ), SEQ ID NO:005 (AAQ), SEQ ID NO:006 (NAQ), SEQ ID NO:007 (AHH), SEQ ID NO:008 (NHH), SEQ ID NO:009 (AAH), SEQ ID NO:010 (NAH), SEQ ID NO:011 (NAA), SEQ ID NO:012 (NAE), SEQ ID NO:013 (AAA), or SEQ ID NO:014 (AAE).

[0256] In certain embodiments, the Fc region is or comprises a sequence characterized by SEQ ID NO: 004 (NHQ), SEQ ID NO: 005 (AAQ), SEQ ID NO: 006 (NAQ), SEQ ID NO: 012 (NAE), or SEQ ID NO: 014 (AAE).

[0257] In certain embodiments, the Fc region is or comprises the sequence characterized by SEQ ID NO: 004 (NHQ).

[0258] In certain embodiments, the Fc region is or comprises the sequence characterized by SEQ ID NO: 006 (NAQ).

[0259] In certain embodiments, the Fc region is or comprises the sequence characterized by SEQ ID NO: 012 (NAE).

[0260] In certain embodiments, the Fc region is or comprises the sequence characterized by SEQ ID NO: 014 (AAE). Polypeptides containing a crystallizable fragment (Fc) region for use in treatment - Patent Application 20070122999 A broader aspect of the present invention provides a polypeptide comprising a crystallizable fragment (Fc) region of IgG for use in the prevention or treatment of disease, wherein the Fc region has a modification that results in reduced affinity for the neonatal Fc receptor (FcRn), and the polypeptide is delivered by local administration to tissue affected by the disease.

[0261] In certain embodiments, the polypeptide is delivered to the eye by intraocular administration. In certain embodiments, the polypeptide is delivered to the joint by intra-articular administration.

[0262] In certain embodiments, the polypeptide is delivered to the lung via inhalation. The present invention further includes a crystallizable fragment (Fc) region of IgG, preferably further comprising: IL-12; or - a polypeptide that binds to any one of VEGFR, Ang2, TNFα, IL-17, PD-1, and PD-L1, more preferably a polypeptide that binds to any one of VEGFR, Ang2, TNFα, and IL-17. wherein the Fc region has a modification that results in reduced affinity for the neonatal Fc receptor (FcRn), and the Fc comprises mutations I253N and H435Q, and H(NHQ) at position 310, and wherein the polypeptide is delivered to the eye by intraocular administration.

[0263] The present invention further includes a crystallizable fragment (Fc) region of IgG, preferably further comprising: IL-12; or - a polypeptide that binds to any one of TNFα, IL-1RA, IL-6R, IL-6, CD27, IL-22, IL-17, and CD27, more preferably a polypeptide that binds to any one of TNFα, IL-1RA, IL-6R, IL-6, and CD27 wherein the Fc region has a modification that results in reduced affinity for the neonatal Fc receptor (FcRn), and the Fc comprises mutations I253N and H435Q, and an H at position 310, and wherein the polypeptide is delivered to the joint by intra-articular administration.

[0264] The present invention further includes a crystallizable fragment (Fc) region of IgG, preferably further comprising: IL-12; or IL-10; or and a polypeptide that binds to any one of IL-4RA, TNFα, IL-5, IL-6R, PD-1, PD-L1, CTLA-4, IL-8, IL-21R, CD25, CD20, NF-kB; more preferably, a polypeptide that binds to any one of IL-4RA, TNFα, IL-5, IL-6R, PD-1, PD-L1, CTLA-4, wherein the Fc region has a modification that results in reduced affinity for the neonatal Fc receptor (FcRn), and the Fc comprises the mutations I253N and H435Q, and an H at position 310, and wherein the polypeptide is delivered to the lung by inhalation.

[0265] The present invention further provides a fusion polypeptide for use as a pharmaceutical comprising a crystallizable fragment (Fc) region of IgG, in particular further comprising IL-12, wherein said Fc region has a modification that results in reduced affinity for the neonatal Fc receptor (FcRn), said Fc comprising the mutations I253N and H435Q, and an H at position 310.

[0266] In certain embodiments, the fragment crystallizable (Fc) region of a polypeptide for use in the prevention or treatment of disease is or comprises SEQ ID NO: 004 (NHQ). In certain embodiments, the fragment crystallizable (Fc) region of a fusion polypeptide for use as a pharmaceutical is or comprises SEQ ID NO: 004 (NHQ).

[0267] After local administration, the reduced affinity for FcRn ensures reduced transport into the circulation and reduced systemic concentration, thereby reducing any systemic toxic side effects of the polypeptide.

[0268] In certain embodiments, the polypeptide is ai effector polypeptides and ii. The Fc region a fusion protein comprising: b. An antibody or antibody-like molecule comprising or linked to an Fc region as described above is selected from.

[0269] Further embodiments of polypeptides comprising a fragment crystallizable (Fc) region for use in treatment can be found in the "Articles" section below.

[0270] Targeting the PD-1 / PD-L1 axis for therapeutic use Another aspect of the present invention provides an antibody or antibody-like molecule that specifically binds to programmed cell death protein 1 (PD-1) or programmed cell death ligand 1 (PD-L1) for use in the prevention or treatment of diseases affecting the central nervous system. The antibody or antibody-like molecule comprises an Fc region with a modification that results in reduced affinity for the neonatal Fc receptor (FcRn). The antibody or antibody-like molecule is administered to the central nervous system, specifically the brain.

[0271] Anti-OX40 for use in treatment Another aspect of the present invention provides an antibody or antibody-like molecule that specifically binds to tumor necrosis factor receptor superfamily member 4 (TNFRSF4), also known as CD134, OX40, or OX40 receptor, for use in the prevention or treatment of diseases affecting the central nervous system. The antibody or antibody-like molecule comprises an Fc region with a modification that results in reduced affinity for the neonatal Fc receptor (FcRn). The antibody or antibody-like molecule is administered to the brain.

[0272] Anti-CD47 used in treatment Another aspect of the present invention provides an antibody or antibody-like molecule that specifically binds to CD47, also known as integrin-associated protein (IAP), for use in the prevention or treatment of diseases affecting the central nervous system. Yet another aspect of the present invention provides a ligand for CD47, specifically SIRPα or thrombospondin-1 (TSP-1) fused to an Fc region. The antibody or antibody-like molecule or Fc-fusion molecule comprises an Fc region with a modification that results in reduced affinity for the neonatal Fc receptor (FcRn). The antibody or antibody-like molecule or Fc-fusion molecule is administered to the brain.

[0273] Anti-Nogo-A for use in treatment Another aspect of the present invention provides an antibody or antibody-like molecule that specifically binds to Nogo-A for use in the prevention or treatment of diseases affecting the central nervous system. Yet another aspect of the present invention provides a ligand of Nogo-A, specifically the Nogo-66 receptor, also known as Nogo-66 receptor 1 (NgR1), fused to an Fc region. The antibody or antibody-like molecule or Fc-fusion molecule comprises an Fc region with a modification that results in reduced affinity for the neonatal Fc receptor (FcRn). The antibody or antibody-like molecule or Fc-fusion molecule is administered to the brain.

[0274] T-CELL ENGINEERING BISPECIFIC ANTIBODIES FOR USE IN TREATMENT - Patent application Another aspect of the present invention provides T cells involving a bispecific antibody or antibody-like molecule that specifically binds to a tumor-associated antigen (TAA) on cancer cells and simultaneously binds to CD3 on T cells, providing T cell receptor-independent polyclonal activation when bound to the TAA, for use in the prevention or treatment of diseases affecting the central nervous system. Yet another aspect of the present invention provides a bispecific antibody or antibody-like molecule that specifically binds to PD-L1 and simultaneously binds to 4-1BB on T cells. Yet a further aspect of the present invention provides a bispecific antibody or antibody-like molecule that specifically binds to PD-L1 and simultaneously binds to CD28 on T cells. The antibody or antibody-like molecule or Fc-fusion molecule comprises an Fc region with a modification that results in reduced affinity for the neonatal Fc receptor (FcRn). The antibody or antibody-like molecule or Fc-fusion molecule is administered to the brain.

[0275] Armed and Targeted Antibodies for Use in Treatment Another aspect of the present invention provides armed antibodies or antibody-like molecules that specifically bind to tumor-associated antigens (TAA) on cancer cells, antigens present in the tumor vasculature, or antigens present in the necrotic core of tumors. The antibodies or antibody-like molecules also carry effector molecules, specifically cytokines, radioisotopes, or cytotoxic substances, for use in the prevention or treatment of diseases affecting the central nervous system. The armed antibodies or antibody-like molecules or Fc-fusion molecules contain an Fc region with modifications that result in reduced affinity for the neonatal Fc receptor (FcRn). The antibodies or antibody-like molecules or Fc-fusion molecules are administered to the brain.

[0276] Tumor- or tissue-conditional antibodies Another aspect of the present invention provides an antibody or antibody-like molecule that specifically binds to a first tumor-associated antigen (TAA) on cancer cells, an antigen present in the tumor microenvironment, an antigen present in the necrotic core of a tumor, or an antigen present in a target tissue. The antibody or antibody-like molecule may also include a second effector molecule, specifically a cytokine, or may be a bispecific or multispecific antibody having at least one antibody or antibody-like molecule that binds to a second antigen different from the first antigen for use in the prevention or treatment of diseases affecting the central nervous system. In such a construct, the second antibody or antibody-like domain is masked and cannot bind to its target antigen. The masking domain is linked to the second antibody or antibody-like construct via a protease-sensitive linker peptide, which is cleaved in the tumor or target tissue by proteases found primarily or exclusively therein. The masking domain may be an antibody or antibody-like molecule that binds to the first antigen. Upon cleavage of the shielding domain in the target tissue or tumor microenvironment, the second antibody or antibody-like molecule binds to its target antigen, or in the case of a cytokine or chemokine, to its receptor. The tumor- or tissue-conditioning antibody or antibody-like molecule or Fc-fusion molecule comprises an Fc region with a modification that results in reduced affinity for the neonatal Fc receptor (FcRn). The antibody or antibody-like molecule or Fc-fusion molecule is administered to the brain.

[0277] Those skilled in the art will recognize that in the case of antibodies, the antibody itself already contains an Fc region. In the case of antibody-like molecules, the antibody-like molecule or Fc-fusion molecule is linked to an Fc region.

[0278] Further embodiments of antibodies or antibody-like molecules or Fc-fusion molecules comprising a fragment crystallizable (Fc) region for use in treatment can be found in the "Articles" section below.

[0279] The affinity for FcRn was reduced (K D Polypeptides containing an Fc region (enhanced A second aspect of the present invention provides a polypeptide comprising an Fc region of an IgG, wherein the Fc region comprises a modification that results in a decreased affinity for the neonatal Fc receptor (FcRn) compared to the affinity of the same polypeptide comprising an unmodified Fc region.

[0280] In certain embodiments, the decreased affinity of the polypeptide for FcRn is a. The dissociation constant (K) characterizing the binding of FcRn to the same polypeptide containing the unmodified Fc region D ) at least two-fold increase in K D , and b. K characterizing the binding of FcRn to the same polypeptide containing differently modified Fc regions, i.e., one mutant selected from IAQ (with mutations H310A and H45Q) and AAA (with mutations I253A, H310A, and H435A). D At least 1.5-fold increased K compared to D K selected from D It is characterized by:

[0281] In certain embodiments, K D is the K that characterizes the binding of FcRn to the same polypeptide containing the unmodified Fc region. D In certain embodiments, K D is the K that characterizes the binding of FcRn to the same polypeptide containing the unmodified Fc region. D In certain embodiments, K D is the K that characterizes the binding of FcRn to the same polypeptide containing the unmodified Fc region. D at least a five-fold increase compared to

[0282] In certain embodiments, K D K characterizes the binding of FcRn to the same polypeptide containing the Fc region with the different modifications described above. D at least a two-fold increase compared to

[0283] In certain embodiments, the polypeptide is ai effector polypeptides and ii. The Fc region a fusion protein comprising: b. An antibody or antibody-like molecule comprising or linked to an Fc region as described above is selected from.

[0284] Those skilled in the art will recognize that in the case of antibodies, the antibody itself already contains an Fc region, and in the case of antibody-like molecules, the antibody-like molecule is linked to an Fc region.

[0285] In certain embodiments, the effector polypeptide is a. cytokine or hormone or growth factor; b. a cytokine receptor, hormone receptor, or growth factor receptor, or C. metabolites It is known that these compounds have the functions of: (1) providing therapeutic or preventive effects against diseases, specifically diseases that affect the central nervous system;

[0286] In certain embodiments, the effector polypeptide is capable of specifically binding to the extracellular matrix (ECM) and is known to have a therapeutic or preventative effect against diseases, particularly diseases affecting the central nervous system. In certain embodiments, the effector polypeptide is capable of specifically binding to RNA and is known to have a therapeutic or preventative effect against diseases, particularly diseases affecting the central nervous system.

[0287] In certain embodiments, the effector polypeptide is selected from the group consisting of IL-12, IL-10, IL-2, IL-7, IFNα, IFNβ, IFNγ, IL-15, TNFα, CTLA-4, TGFβ, TGFβRII, GDNF, IL-35, CD95, IL-1RA, IL-4, IL-13, IL-33, IL-23, SIRPα, G-CSF, GM-CSF, OX40L, CD80, CD86, GITRL, 4-1 The effector polypeptide is selected from the group comprising BBL, EphrinA1, EphrinB2, EphrinB5, BDNF, C9orf72, NRTN, ARTN, PSPN, CNTF, TRAIL, IL-4, IL-3, IL-1, IL-5, IL-8, IL-18, IL-21, CCL5, CCL21, CCL10, CCL16, CX3CL1, CXCL16, and specifically, the effector polypeptide is IL-12.

[0288] In certain embodiments, the antibody or antibody-like molecule agonistically or antagonistically inhibits PD-L1, TNFα, Histone, IFNγ, CXCL10, CTLA4, PD-1, CD3, OX40, CD20, CD22, CD25, CD28, TREM2, IL-6, CX3CR1, Nogo-A, CD27, IL-12, IL-12Rb1, IL-23, IL-17, CD47, TGFβ, EGFR, EGFRvIII, Her2, PDGFR, TGFR, FGFR, IL-4RA, TfR, LfR, IR, LDL-R, LRP-1, CD133, CD111, VEGFR, VEGF-A, Ang-2, IL-10, The antibody or antibody-like molecule may be selected from the group consisting of an antibody or antibody-like molecule that specifically binds to IL-10R, IL-13Rα2, α-synuclein, CSF1R, G-CSF, GM-CSF, GITR, TIM-3, LAG-3, TIGIT, BTLA, VISTA, CD96, CD147, 4-1BB, CCL2, IL-1 or IL-1R, EphA2, EphA3, EphB2, EphB3, EphB4, LINGO-1, L1CAM, NCAM, SOD-1, SIGMAR-1, SIGMAR-2, TDP-43, Aβ, Tau, IFNα, IFNβ, TRPM4, ASIC1, VGCCs, CB1, TTR, HTT, JCV, and C9orf72.

[0289] The antibodies or antibody-like molecules according to the above aspects of the invention can be antibody-like molecules derived from the recognition site or whole antibody of the physiological ligands of PD-1, PD-L1, or PD-L2. Such antibodies or antibody-like molecules compete with the physiological ligands for binding to PD-1, PD-L1, or PD-L2, respectively. Specifically, non-agonistic PD-1 antibodies or antibody-like molecules, or non-agonistic PD-L1 antibodies or antibody-like molecules, or non-agonistic PD-L2 antibodies or antibody-like molecules do not result in attenuated T cell activity when bound to PD-1 on the surface of T cells.

[0290] In some embodiments, the non-agonistic PD-1 antibodies or antibody-like molecules used in the invention, when bound to PD-1, are capable of sterically blocking the interaction of PD-1 with its binding partners PD-L1 and / or PD-L2.

[0291] In some embodiments, the non-agonist PD-1 antibody or antibody-like molecule is a gamma immunoglobulin that binds to PD-1 and does not elicit the physiological response of PD-1 interaction with its binding partners PD-L1 and / or PD-L2.

[0292] In some embodiments, the non-agonist PD-L1 (PD-L2) antibody or antibody-like molecule is a gamma immunoglobulin that binds to PD-L1 (PD-L2) and does not elicit the physiological response of PD-1 interaction with its binding partners, PD-L1 and / or PD-L2.

[0293] Non-limiting examples of PD-1 antibodies are the clinically approved antibodies pembrolizumab (CAS No. 1374853-91-4) and nivolumab (CAS No. 946414-94-4).

[0294] Non-limiting examples of PD-L1 antibodies are the clinically approved antibodies atezolizumab (CAS No. 1380723-44-3), durvalumab (CAS No. 1428935-60-7) and avelumab (CAS No. 1537032-82-8).

[0295] Non-limiting examples of PD-1 / PD-L1 or PD-L2 antibodies currently in clinical development are antibodies MDX-1105 / BMS-936559 or AMP-224. A non-limiting example of an antibody that specifically binds to IL-12 / 23 is ustekinumab (CAS No. 815610-63-0).

[0296] In certain embodiments, the antibody or antibody-like molecule is an antibody that specifically binds to PD-L1.

[0297] In some embodiments, the agonistic OX40 antibodies or antibody-like molecules used in the present invention are capable of triggering a signaling cascade in OX40-expressing cells upon binding to OX40 and in the absence of an OX40 ligand.

[0298] Non-limiting examples of OX40 antibodies are antibodies currently in clinical development: PF-04518600 / PF-8600m BMS-986178, GSK3174998, MOXR0916, INCAGN01949, vavolimab / MEDI0562.

[0299] In certain embodiments, the antibody or antibody-like molecule is an antibody that specifically binds to OX40.

[0300] In some embodiments, the antibody or antibody-like molecule used in the present invention is capable of blocking the interaction between CD47 and SIRPα signaling, which prevents phagocytosis of cancer cells.

[0301] Non-limiting examples of CD47 blocking antibodies or SIRPα fusion proteins are Hu5F9-G4, CC-90002 / INBRX-103, IBI188, OSE-172, NI-1801, DSP107, TTI-622, TTI-621, ALX148 and SRF231.

[0302] In certain embodiments, the antibody or antibody-like molecule is an antibody that specifically binds to Nogo-A.

[0303] In certain embodiments, the antibody or antibody-like molecule is a bispecific construct capable of simultaneously binding two antigens.

[0304] In certain embodiments, the antibody or antibody-like molecule is a trispecific construct. In certain embodiments, the antibody or antibody-like molecule is a multispecific construct.

[0305] In certain embodiments, the antibody or antibody-like molecule is an antibody against a histone present in the necrotic core of a tumor, which is armed with IL-12 or IL-2. In certain cases, the armed antibody is an immunocytokine. Non-limiting examples of armed antibodies as immunocytokines are NHS-IL-12, NHS-IL2LT, Hu14.18-IL2, HuKS-IL2, and huBC1-IL-12.

[0306] In certain embodiments, the Fc region is a chimeric Fc region comprising a human amino acid sequence. In certain embodiments, the Fc region is a human Fc region.

[0307] In certain embodiments, the Fc region has a mutation at position 253. In certain embodiments, the Fc region has the mutation I253A. In certain embodiments, the Fc region has the mutation I253N.

[0308] In certain embodiments, the Fc region has a mutation at position 435. In certain embodiments, the Fc region has the mutation H435Q.

[0309] In certain embodiments, the Fc region does not have a mutation at position 435. Thus, the Fc region has an H at position 435.

[0310] In certain embodiments, the Fc region does not have a mutation at position 310. Thus, the Fc region has an H at position 310.

[0311] In certain embodiments, the Fc region comprises: - mutations I253A and H435Q, as well as H at position 310 (AHQ); - mutations I253N and H435Q, as well as H at position 310 (NHQ); - mutations I253A, H310A and H435Q (AAQ); - mutations I253N, H310A and H435Q (NAQ); - mutation I253A, and H at positions 310 and 435 (AHH); - mutation I253N, and H at positions 310 and 435 (NHH); - mutations I253A and H310A, and H at position 435 (AAH); - mutations I253N and H310A, as well as H at position 435 (NAH); - mutations I253N, H310A and H435A (NAA); - mutations I253N, H310A and H435E (NAE); - mutations I253A, H310A and H435A (AAA); or -Mutations I253A, H310A and H435E (AAE) Includes.

[0312] In certain embodiments, the Fc region comprises: - mutations I253N and H435Q, as well as H at position 310 (NHQ); - mutations I253A, H310A and H435Q (AAQ); - mutations I253N, H310A and H435Q (NAQ); - mutations I253N, H310A and H435E (NAE); or -Mutations I253A, H310A and H435E (AAE) Includes.

[0313] In certain embodiments, the Fc region comprises the mutations I253N and H435Q, and an H at position 310.

[0314] In certain embodiments, the Fc region comprises the mutations I253A, H310A and H435Q (AAQ).

[0315] In certain embodiments, the Fc region comprises the mutations I253N, H310A and H435Q (NAQ).

[0316] In certain embodiments, the Fc region comprises the mutations I253N, H310A and H435E (NAE).

[0317] In certain embodiments, the Fc region comprises the mutations I253A, H310A and H435E (AAE).

[0318] In certain embodiments, the Fc region is or comprises a sequence characterized by SEQ ID NO:002 (IAQ), SEQ ID NO:003 (AHQ), SEQ ID NO:004 (NHQ), SEQ ID NO:005 (AAQ), SEQ ID NO:006 (NAQ), SEQ ID NO:007 (AHH), SEQ ID NO:008 (NHH), SEQ ID NO:009 (AAH), SEQ ID NO:010 (NAH), SEQ ID NO:011 (NAA), SEQ ID NO:012 (NAE), SEQ ID NO:013 (AAA), or SEQ ID NO:014 (AAE).

[0319] In certain embodiments, the Fc region is or comprises a sequence characterized by SEQ ID NO: 004 (NHQ), SEQ ID NO: 005 (AAQ), SEQ ID NO: 006 (NAQ), SEQ ID NO: 012 (NAE), or SEQ ID NO: 014 (AAE).

[0320] In certain embodiments, the Fc region is or comprises the sequence characterized by SEQ ID NO: 004 (NHQ).

[0321] In certain embodiments, the Fc region is or comprises the sequence characterized by SEQ ID NO: 006 (NAQ).

[0322] In certain embodiments, the Fc region is or comprises the sequence (NAE) characterized by SEQ ID NO:012.

[0323] In certain embodiments, the Fc region is or comprises the sequence characterized by SEQ ID NO: 014 (AAE).

[0324] nucleic acid Another aspect of the invention provides a nucleic acid encoding a polypeptide according to the above aspect of the invention.

[0325] virus Another aspect of the present invention provides a viral vector comprising a nucleic acid according to the above aspect of the invention. The viral vector may be a replicating or non-replicating virus suitable for application to a patient during treatment.

[0326] In certain embodiments of any aspect of the invention, a polypeptide comprising a modified Fc region according to the invention is used in combination with an FcRn-blocking antibody. The FcRn-blocking antibody can inhibit binding between an Fc-containing polypeptide and FcRn, thus mimicking the technical effect of the invention. Combination with an FcRn-blocking antibody can enhance the described advantages of a polypeptide comprising a modified Fc region according to the invention.

[0327] In certain embodiments of any aspect of the invention, the Fc region is an Fc region of immunoglobulin G (IgG). IgG is the primary effector molecule of the human humoral immune response. There are four distinct subgroups of human IgG, designated IgG1, IgG2, IgG3, and IgG4. The four subclasses share over 95% homology in the amino acid sequence of the heavy chain constant domain but differ in the structure and flexibility of the hinge region, particularly the number of inter-heavy chain disulfide bonds in this domain. Structural differences between IgG subclasses are also reflected in their susceptibility to proteolytic enzymes, specifically papain, plasmin, trypsin, and pepsin.

[0328] In certain embodiments of any aspect of the present invention, the Fc region is an IgG4 Fc region. There is only one known isoform of human IgG4. In contrast to human IgG1, IgG2, and IgG3, human IgG4 does not activate complement. Furthermore, IgG4 is less susceptible to proteolytic enzymes than IgG2 and IgG3. Contrary to these expectations, it has been surprisingly found that IgG1 full-length antibody constructs with the mutations I253N and H435Q are actually characterized by lower affinity for FcRn, as exemplified by higher dissociation constants and plasma-to-brain ratios determined compared to the corresponding IgG4 full-length antibody constructs.

[0329] Similarly, within the scope of the present invention is the use for treating or preventing malignant neoplastic diseases, particularly solid tissue tumors, more particularly gliomas, in a patient in need thereof, comprising administering to the patient a polypeptide comprising a modified Fc region according to one of the above-described aspects of the invention, or a nucleic acid encoding the polypeptide, or a viral vector comprising a nucleic acid encoding the polypeptide.

[0330] Similarly, a dosage form for the prevention or treatment of malignant neoplastic diseases, particularly solid tissue tumors, more particularly gliomas, is provided, comprising a polypeptide comprising a modified Fc region according to one of the above-described aspects of the invention, or a nucleic acid encoding the polypeptide, or a viral vector comprising a nucleic acid encoding the polypeptide.

[0331] Wherever options for single separable features are presented herein as "embodiments," it should be understood that such options can be freely combined to form separate embodiments of the invention disclosed herein.

[0332] The present invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be derived, which are intended to illustrate the invention but not to limit its scope. [Brief explanation of the drawings]

[0333] [Figure 1A] Human IL-12Fc has better tissue retention than IL-12. A. Schematic structure of murine IL-12Fc. rhIL-12 - recombinant human IL-12, hIL-12Fc - human IL-12Fc. IgG4 Fc - crystallizable region of a fragment of human IgG4. B. Schematic of the experiment. IL-12 in IL-12Fc was injected into the striatum of FcRntg mice. 24 hours later, the amount of injected protein remaining was assessed in the brain and compared to the amount present in the serum. C. Ratio of serum to brain IL-12 levels assessed by ELISA. ELISA measured hIL-12 as a general measure of IL-12Fc. Unpaired Student's t-test. **p<0.005. Mean ± SD. [Figure 1B]Human IL-12Fc has better tissue retention than IL-12. A. Schematic structure of murine IL-12Fc. rhIL-12 - recombinant human IL-12, hIL-12Fc - human IL-12Fc. IgG4 Fc - crystallizable region of a fragment of human IgG4. B. Schematic of the experiment. IL-12 in IL-12Fc was injected into the striatum of FcRntg mice. 24 hours later, the amount of injected protein remaining was assessed in the brain and compared to the amount present in the serum. C. Ratio of serum to brain IL-12 levels assessed by ELISA. ELISA measured hIL-12 as a general measure of IL-12Fc. Unpaired Student's t-test. **p<0.005. Mean ± SD. [Figure 1C] Human IL-12Fc has better tissue retention than IL-12. A. Schematic structure of murine IL-12Fc. rhIL-12 - recombinant human IL-12, hIL-12Fc - human IL-12Fc. IgG4 Fc - crystallizable region of a fragment of human IgG4. B. Schematic of the experiment. IL-12 in IL-12Fc was injected into the striatum of FcRntg mice. 24 hours later, the amount of injected protein remaining was assessed in the brain and compared to the amount present in the serum. C. Ratio of serum to brain IL-12 levels assessed by ELISA. ELISA measured hIL-12 as a general measure of IL-12Fc. Unpaired Student's t-test. **p<0.005. Mean ± SD. [Figure 2A]IL-12Fc is released from the brain via FcRn. A. Wt and FcRntg mice bearing brain tumors were implanted with osmotic pumps that delivered 12.5 μg / kg / day of murine IL-12Fc directly to the tumor lesion. Murine IL-12 levels measured in serum using a bead-based array. Unpaired Student's t-test for wt mIL-12Fc vs. FcRntg mIL-12Fc groups. Mean ± SD. One-way ANOVA with Tukey's multiple comparison test. B. Mice treated as in Figure 2A. The amount of IL-12 present in the circulation 24 hours after the start of treatment, as measured in serum using a bead-based array. Mean ± SD. C. Mice treated as in Figure 2A. The level of IFNγ in the circulation 24 hours after the start of treatment, as measured in serum using a bead-based array. Mean ± SD. D. IFNγ levels at day 7, experiment A, mean ± SD. [Figure 2B] IL-12Fc is released from the brain via FcRn. A. Wt and FcRntg mice bearing brain tumors were implanted with osmotic pumps that delivered 12.5 μg / kg / day of murine IL-12Fc directly to the tumor lesion. Murine IL-12 levels measured in serum using a bead-based array. Unpaired Student's t-test for wt mIL-12Fc vs. FcRntg mIL-12Fc groups. Mean ± SD. One-way ANOVA with Tukey's multiple comparison test. B. Mice treated as in Figure 2A. The amount of IL-12 present in the circulation 24 hours after the start of treatment, as measured in serum using a bead-based array. Mean ± SD. C. Mice treated as in Figure 2A. The level of IFNγ in the circulation 24 hours after the start of treatment, as measured in serum using a bead-based array. Mean ± SD. D. IFNγ levels at day 7, experiment A, mean ± SD. [Figure 2C]IL-12Fc is released from the brain via FcRn. A. Wt and FcRntg mice bearing brain tumors were implanted with osmotic pumps that delivered 12.5 μg / kg / day of murine IL-12Fc directly to the tumor lesion. Murine IL-12 levels measured in serum using a bead-based array. Unpaired Student's t-test for wt mIL-12Fc vs. FcRntg mIL-12Fc groups. Mean ± SD. One-way ANOVA with Tukey's multiple comparison test. B. Mice treated as in Figure 2A. The amount of IL-12 present in the circulation 24 hours after the start of treatment, as measured in serum using a bead-based array. Mean ± SD. C. Mice treated as in Figure 2A. The level of IFNγ in the circulation 24 hours after the start of treatment, as measured in serum using a bead-based array. Mean ± SD. D. IFNγ levels at day 7, experiment A, mean ± SD. [Figure 2D] IL-12Fc is released from the brain via FcRn. A. Wt and FcRntg mice bearing brain tumors were implanted with osmotic pumps that delivered 12.5 μg / kg / day of murine IL-12Fc directly to the tumor lesion. Murine IL-12 levels measured in serum using a bead-based array. Unpaired Student's t-test for wt mIL-12Fc vs. FcRntg mIL-12Fc groups. Mean ± SD. One-way ANOVA with Tukey's multiple comparison test. B. Mice treated as in Figure 2A. The amount of IL-12 present in the circulation 24 hours after the start of treatment, as measured in serum using a bead-based array. Mean ± SD. C. Mice treated as in Figure 2A. The level of IFNγ in the circulation 24 hours after the start of treatment, as measured in serum using a bead-based array. Mean ± SD. D. IFNγ levels at day 7, experiment A, mean ± SD. [Figure 3A] Protein stability measured using a thermal shift assay. Proteins were incubated in PBS (A) or artificial cerebrospinal fluid (aCSF, B). Five measurements per IL-12Fc variant. Whiskers represent minimum and maximum spread. [Figure 3B]Protein stability measured using a thermal shift assay. Proteins were incubated in PBS (A) or artificial cerebrospinal fluid (aCSF, B). Five measurements per IL-12Fc variant. Whiskers represent minimum and maximum spread. [Figure 4A] Mutations in the Fc fragment of IL-12Fc do not affect the biological activity of IL-12. A. Bioactivity of IL-12 measured using the HEK-Blue™ IL-12 assay. EC50—the effective concentration that produces 50% of the maximum signal from HEK-Blue™ IL-12 reporter cells stimulated with IL-12Fc in the range of 0–50 ng / ml, with two replicates per concentration. Secreted alkaline phosphatase activity was measured using a colorimetric method. Each point represents the result of an independent experiment. Mean ± SD. B. Phosphorylation of STAT-4 in peripheral blood mononuclear cells (PBMCs) stimulated for 1 hour with 100 ng / ml anti-CD3 and 10 ng / ml recombinant IL-12, IL-12Fc WT, or three variants designed to reduce FcRn affinity. Mean ± SD. C. IFNγ production by PBMCs stimulated for 24 h with 100 ng / ml anti-CD3 and the indicated concentrations of recombinant IL-12, IL-12Fc WT, or three variants designed to reduce FcRn affinity. [Figure 4B]Mutations in the Fc fragment of IL-12Fc do not affect the biological activity of IL-12. A. Bioactivity of IL-12 measured using the HEK-Blue™ IL-12 assay. EC50—the effective concentration that produces 50% of the maximum signal from HEK-Blue™ IL-12 reporter cells stimulated with IL-12Fc in the range of 0–50 ng / ml, with two replicates per concentration. Secreted alkaline phosphatase activity was measured using a colorimetric method. Each point represents the result of an independent experiment. Mean ± SD. B. Phosphorylation of STAT-4 in peripheral blood mononuclear cells (PBMCs) stimulated for 1 hour with 100 ng / ml anti-CD3 and 10 ng / ml recombinant IL-12, IL-12Fc WT, or three variants designed to reduce FcRn affinity. Mean ± SD. C. IFNγ production by PBMCs stimulated for 24 h with 100 ng / ml anti-CD3 and the indicated concentrations of recombinant IL-12, IL-12Fc WT, or three variants designed to reduce FcRn affinity. [Figure 4C] Mutations in the Fc fragment of IL-12Fc do not affect the biological activity of IL-12. A. Bioactivity of IL-12 measured using the HEK-Blue™ IL-12 assay. EC50—the effective concentration that produces 50% of the maximum signal from HEK-Blue™ IL-12 reporter cells stimulated with IL-12Fc in the range of 0–50 ng / ml, with two replicates per concentration. Secreted alkaline phosphatase activity was measured using a colorimetric method. Each point represents the result of an independent experiment. Mean ± SD. B. Phosphorylation of STAT-4 in peripheral blood mononuclear cells (PBMCs) stimulated for 1 hour with 100 ng / ml anti-CD3 and 10 ng / ml recombinant IL-12, IL-12Fc WT, or three variants designed to reduce FcRn affinity. Mean ± SD. C. IFNγ production by PBMCs stimulated for 24 h with 100 ng / ml anti-CD3 and the indicated concentrations of recombinant IL-12, IL-12Fc WT, or three variants designed to reduce FcRn affinity. [Figure 5A]Human IL-12Fc variants have reduced affinity for FcRn. A. Surface plasmon resonance (SPR) measurements of FcRn affinity between surface-immobilized human recombinant FcRn and IL-12Fc variants in solution. Affinity measured at pH 6.0. Data normalized to IL-12Fc WT. B. IL-12Fc variants binding to human FcRn. Measured by ELISA at pH 6.0. Mean ± SD. [Figure 5B] Human IL-12Fc variants have reduced affinity for FcRn. A. Surface plasmon resonance (SPR) measurements of FcRn affinity between surface-immobilized human recombinant FcRn and IL-12Fc variants in solution. Affinity measured at pH 6.0. Data normalized to IL-12Fc WT. B. IL-12Fc variants binding to human FcRn. Measured by ELISA at pH 6.0. Mean ± SD. [Figure 6A] IL-12Fc concentration ratio in the blood and injected hemisphere. A. 1 μg of IL-12Fc WT or NHQ variant was injected into the striatum of FcRntg mice. 24 h later, the amount of IL-12 was assessed by ELISA in the injected hemisphere and serum, and their ratios were calculated and normalized to those in the IL-12Fc WT group. Four mice per group. Unpaired Student's t-test. *p<0.05. Mean ± SD. B. 1 μg of IL-12Fc WT, IAQ, AAA, or NHQ was injected into the striatum of FcRntg mice using convection-enhanced delivery (CED). 24 h later, the amount of IL-12 was assessed by ELISA in the injected hemisphere and plasma, and their ratios were calculated and normalized to those in the IL-12Fc WT group. Seven to eight mice per group. One-way ANOVA with Tukey's multiple comparison test. Mean ± SD. [Figure 6B]IL-12Fc concentration ratio in the blood and injected hemisphere. A. 1 μg of IL-12Fc WT or NHQ variant was injected into the striatum of FcRntg mice. 24 h later, the amount of IL-12 was assessed by ELISA in the injected hemisphere and serum, and their ratios were calculated and normalized to those in the IL-12Fc WT group. Four mice per group. Unpaired Student's t-test. *p<0.05. Mean ± SD. B. 1 μg of IL-12Fc WT, IAQ, AAA, or NHQ was injected into the striatum of FcRntg mice using convection-enhanced delivery (CED). 24 h later, the amount of IL-12 was assessed by ELISA in the injected hemisphere and plasma, and their ratios were calculated and normalized to those in the IL-12Fc WT group. Seven to eight mice per group. One-way ANOVA with Tukey's multiple comparison test. Mean ± SD. [Figure 7] Brain retention after local treatment with IL-12Fc variants. FcRntg mice were injected with 1 μg of IL-12Fc WT, IAQ, AAA, or NHQ into the striatum using convection-enhanced delivery (CED). The amount of IL-12Fc remaining in brain tissue was measured 6 hours post-injection by ELISA and normalized to IL-12Fc WT. One-way ANOVA with Tukey's multiple comparison test. Abnormal removal. Mean ± SD. [Figure 8A]A. Schematic structures of native and rmIL-12, mIL-12hIgG4 wt, mIL-12hIgG4 NHQ, and mIL-12hIgG1:anti-hPD-L1 NHQ. B. Biological activity of murine IL-12 constructs measured using the HEK-Blue™ IL-12 assay. HEK-Blue™ reporter cells were stimulated with IL-12 or IL-12Fc variants ranging from 0 to 50 ng / mL, using 5-8 dilution steps, with two replicates per concentration. Activity is measured using a colorimetric method using secreted alkaline phosphatase activity. X-axis values: concentration plotted in pmol / mL as the corresponding molecular weight of IL-12. Representative of two independent experiments. C. Binding to PD-L1 on cells compared to intact anti-PD-L1 (atezolizumab) antibody. GL-261:luc or PD-L1-deficient GL-261:luc (PD-L1 KO) cells were stimulated with mouse interferon-gamma (IFNγ) to stimulate PD-L1 expression and stained with anti-PD-L1 antibody or h / mIL-12hFc:aPD-L1 NHQ variant. Cell-bound antibody was detected using an anti-human IgG-PE secondary antibody. D. Affinity of NHQ variants for FcRn compared to WT as measured by surface plasmon resonance (SPR). Surface-immobilized human recombinant FcRn and PD-L1 binders in solution phase. Affinity measured at pH=6. Affinity constant KD in nM. [Figure 8B]A. Schematic structures of native and rmIL-12, mIL-12hIgG4 wt, mIL-12hIgG4 NHQ, and mIL-12hIgG1:anti-hPD-L1 NHQ. B. Biological activity of murine IL-12 constructs measured using the HEK-Blue™ IL-12 assay. HEK-Blue™ reporter cells were stimulated with IL-12 or IL-12Fc variants ranging from 0 to 50 ng / mL, using 5-8 dilution steps, with two replicates per concentration. Activity is measured using a colorimetric method using secreted alkaline phosphatase activity. X-axis values: concentration plotted in pmol / mL as the corresponding molecular weight of IL-12. Representative of two independent experiments. C. Binding to PD-L1 on cells compared to intact anti-PD-L1 (atezolizumab) antibody. GL-261:luc or PD-L1-deficient GL-261:luc (PD-L1 KO) cells were stimulated with mouse interferon-gamma (IFNγ) to stimulate PD-L1 expression and stained with anti-PD-L1 antibody or h / mIL-12hFc:aPD-L1 NHQ variant. Cell-bound antibody was detected using an anti-human IgG-PE secondary antibody. D. Affinity of NHQ variants for FcRn compared to WT as measured by surface plasmon resonance (SPR). Surface-immobilized human recombinant FcRn and PD-L1 binders in solution phase. Affinity measured at pH=6. Affinity constant KD in nM. [Figure 8C]A. Schematic structures of native and rmIL-12, mIL-12hIgG4 wt, mIL-12hIgG4 NHQ, and mIL-12hIgG1:anti-hPD-L1 NHQ. B. Biological activity of murine IL-12 constructs measured using the HEK-Blue™ IL-12 assay. HEK-Blue™ reporter cells were stimulated with IL-12 or IL-12Fc variants ranging from 0 to 50 ng / mL, using 5-8 dilution steps, with two replicates per concentration. Activity is measured using a colorimetric method using secreted alkaline phosphatase activity. X-axis values: concentration plotted in pmol / mL as the corresponding molecular weight of IL-12. Representative of two independent experiments. C. Binding to PD-L1 on cells compared to intact anti-PD-L1 (atezolizumab) antibody. GL-261:luc or PD-L1-deficient GL-261:luc (PD-L1 KO) cells were stimulated with mouse interferon-gamma (IFNγ) to stimulate PD-L1 expression and stained with anti-PD-L1 antibody or h / mIL-12hFc:aPD-L1 NHQ variant. Cell-bound antibody was detected using an anti-human IgG-PE secondary antibody. D. Affinity of NHQ variants for FcRn compared to WT as measured by surface plasmon resonance (SPR). Surface-immobilized human recombinant FcRn and PD-L1 binders in solution phase. Affinity measured at pH=6. Affinity constant KD in nM. [Figure 8D]A. Schematic structures of native and rmIL-12, mIL-12hIgG4 wt, mIL-12hIgG4 NHQ, and mIL-12hIgG1:anti-hPD-L1 NHQ. B. Biological activity of murine IL-12 constructs measured using the HEK-Blue™ IL-12 assay. HEK-Blue™ reporter cells were stimulated with IL-12 or IL-12Fc variants ranging from 0 to 50 ng / mL, using 5-8 dilution steps, with two replicates per concentration. Activity is measured using a colorimetric method using secreted alkaline phosphatase activity. X-axis values: concentration plotted in pmol / mL as the corresponding molecular weight of IL-12. Representative of two independent experiments. C. Binding to PD-L1 on cells compared to intact anti-PD-L1 (atezolizumab) antibody. GL-261:luc or PD-L1-deficient GL-261:luc (PD-L1 KO) cells were stimulated with mouse interferon-gamma (IFNγ) to stimulate PD-L1 expression and stained with anti-PD-L1 antibody or h / mIL-12hFc:aPD-L1 NHQ variant. Cell-bound antibody was detected using an anti-human IgG-PE secondary antibody. D. Affinity of NHQ variants for FcRn compared to WT as measured by surface plasmon resonance (SPR). Surface-immobilized human recombinant FcRn and PD-L1 binders in solution phase. Affinity measured at pH=6. Affinity constant KD in nM. [Figure 9A]Optimized IL-12 Fc fusions for local treatment of brain cancer result in reduced systemic exposure without affecting therapeutic efficacy. A. Experimental schedule by days after tumor injection. Animals bearing GL-261:luc brain tumors were systematically assigned to treatment groups of equivalent tumor burden via bioluminescence imaging (BLI) on day 20 and treated with buffer only (control) or 1 μg of rmIL-12, mIL-12hFc:anti-PD-L1 bifunctional molecule, mIL-12hFc WT, or mIL-12hFc NHQ via convection-enhanced delivery (CED) on days 21 and 28 after tumor implantation. Blood samples were collected for plasma at time points 0, 6, 24, and 72 hours and 7 days after CED injection, as well as 14 days after the second CED injection. B. Tumor progression during treatment monitored by bioluminescence imaging. Average radiance (p / s / cm² / sr) is plotted from regions of interest (ROIs) for individual animals grouped by treatment cohort. Treatment with CED is indicated by the vertical dotted line. C. Plasma levels of IL-12 (black line, left Y-axis) and IFNγ (gray line, right Y-axis) in response to treatment, measured at indicated time points by bead-based cytokine array. Treatment with CED is indicated by the vertical dotted line. D. FcRn affinity-dependent differences in plasma IL-12 levels 6 hours after CED on day 21. Mice injected with mIL-12hFc WT and mIL-12hFc NHQ. Data from experiments shown in A-C. Kaplan-Meyer analysis of survival of treated mice from E-A-D. 6-7 mice per group. [Figure 9B]Optimized IL-12 Fc fusions for local treatment of brain cancer result in reduced systemic exposure without affecting therapeutic efficacy. A. Experimental schedule by days after tumor injection. Animals bearing GL-261:luc brain tumors were systematically assigned to treatment groups of equivalent tumor burden via bioluminescence imaging (BLI) on day 20 and treated with buffer only (control) or 1 μg of rmIL-12, mIL-12hFc:anti-PD-L1 bifunctional molecule, mIL-12hFc WT, or mIL-12hFc NHQ via convection-enhanced delivery (CED) on days 21 and 28 after tumor implantation. Blood samples were collected for plasma at time points 0, 6, 24, and 72 hours and 7 days after CED injection, as well as 14 days after the second CED injection. B. Tumor progression during treatment monitored by bioluminescence imaging. Average radiance (p / s / cm² / sr) is plotted from regions of interest (ROIs) for individual animals grouped by treatment cohort. Treatment with CED is indicated by the vertical dotted line. C. Plasma levels of IL-12 (black line, left Y-axis) and IFNγ (gray line, right Y-axis) in response to treatment, measured at indicated time points by bead-based cytokine array. Treatment with CED is indicated by the vertical dotted line. D. FcRn affinity-dependent differences in plasma IL-12 levels 6 hours after CED on day 21. Mice injected with mIL-12hFc WT and mIL-12hFc NHQ. Data from experiments shown in A-C. Kaplan-Meyer analysis of survival of treated mice from E-A-D. 6-7 mice per group. [Figure 9C]Optimized IL-12 Fc fusions for local treatment of brain cancer result in reduced systemic exposure without affecting therapeutic efficacy. A. Experimental schedule by days after tumor injection. Animals bearing GL-261:luc brain tumors were systematically assigned to treatment groups of equivalent tumor burden via bioluminescence imaging (BLI) on day 20 and treated with buffer only (control) or 1 μg of rmIL-12, mIL-12hFc:anti-PD-L1 bifunctional molecule, mIL-12hFc WT, or mIL-12hFc NHQ via convection-enhanced delivery (CED) on days 21 and 28 after tumor implantation. Blood samples were collected for plasma at time points 0, 6, 24, and 72 hours and 7 days after CED injection, as well as 14 days after the second CED injection. B. Tumor progression during treatment monitored by bioluminescence imaging. Average radiance (p / s / cm² / sr) is plotted from regions of interest (ROIs) for individual animals grouped by treatment cohort. Treatment with CED is indicated by the vertical dotted line. C. Plasma levels of IL-12 (black line, left Y-axis) and IFNγ (gray line, right Y-axis) in response to treatment, measured at indicated time points by bead-based cytokine array. Treatment with CED is indicated by the vertical dotted line. D. FcRn affinity-dependent differences in plasma IL-12 levels 6 hours after CED on day 21. Mice injected with mIL-12hFc WT and mIL-12hFc NHQ. Data from experiments shown in A-C. Kaplan-Meyer analysis of survival of treated mice from E-A-D. 6-7 mice per group. [Figure 9D]Optimized IL-12 Fc fusions for local treatment of brain cancer result in reduced systemic exposure without affecting therapeutic efficacy. A. Experimental schedule by days after tumor injection. Animals bearing GL-261:luc brain tumors were systematically assigned to treatment groups of equivalent tumor burden via bioluminescence imaging (BLI) on day 20 and treated with buffer only (control) or 1 μg of rmIL-12, mIL-12hFc:anti-PD-L1 bifunctional molecule, mIL-12hFc WT, or mIL-12hFc NHQ via convection-enhanced delivery (CED) on days 21 and 28 after tumor implantation. Blood samples were collected for plasma at time points 0, 6, 24, and 72 hours and 7 days after CED injection, as well as 14 days after the second CED injection. B. Tumor progression during treatment monitored by bioluminescence imaging. Average radiance (p / s / cm² / sr) is plotted from regions of interest (ROIs) for individual animals grouped by treatment cohort. Treatment with CED is indicated by the vertical dotted line. C. Plasma levels of IL-12 (black line, left Y-axis) and IFNγ (gray line, right Y-axis) in response to treatment, measured at indicated time points by bead-based cytokine array. Treatment with CED is indicated by the vertical dotted line. D. FcRn affinity-dependent differences in plasma IL-12 levels 6 hours after CED on day 21. Mice injected with mIL-12hFc WT and mIL-12hFc NHQ. Data from experiments shown in A-C. Kaplan-Meyer analysis of survival of treated mice from E-A-D. 6-7 mice per group. [Figure 9E]Optimized IL-12 Fc fusions for local treatment of brain cancer result in reduced systemic exposure without affecting therapeutic efficacy. A. Experimental schedule by days after tumor injection. Animals bearing GL-261:luc brain tumors were systematically assigned to treatment groups of equivalent tumor burden via bioluminescence imaging (BLI) on day 20 and treated with buffer only (control) or 1 μg of rmIL-12, mIL-12hFc:anti-PD-L1 bifunctional molecule, mIL-12hFc WT, or mIL-12hFc NHQ via convection-enhanced delivery (CED) on days 21 and 28 after tumor implantation. Blood samples were collected for plasma at time points 0, 6, 24, and 72 hours and 7 days after CED injection, as well as 14 days after the second CED injection. B. Tumor progression during treatment monitored by bioluminescence imaging. Average radiance (p / s / cm² / sr) is plotted from regions of interest (ROIs) for individual animals grouped by treatment cohort. Treatment with CED is indicated by the vertical dotted line. C. Plasma levels of IL-12 (black line, left Y-axis) and IFNγ (gray line, right Y-axis) in response to treatment, measured at indicated time points by bead-based cytokine array. Treatment with CED is indicated by the vertical dotted line. D. FcRn affinity-dependent differences in plasma IL-12 levels 6 hours after CED on day 21. Mice injected with mIL-12hFc WT and mIL-12hFc NHQ. Data from experiments shown in A-C. Kaplan-Meyer analysis of survival of treated mice from E-A-D. 6-7 mice per group. [Figure 10A]Antibodies. A. Surface plasmon resonance (SPR) measurements of FcRn affinity of surface-immobilized human recombinant FcRn and IgG1 variants in solution. Affinity measured at pH 6.0. Data normalized to an IgG1 antibody with unmodified Fc (WT). Three additional IgG1 clinical-grade antibodies with unmodified Fc moieties (IgG1_01 ipilimumab, IgG1_02 atezolizumab, IgG1_03 rituximab) were used as additional references. Mean ± SD. Affinity measured at pH 6.0. Data normalized to an IgG4 antibody with unmodified Fc (WT). A second IgG4 antibody (nivolumab) with an unmodified Fc moiety was used as an additional reference (IgG4). Mean ± SD. B. 1 μg of IgG1 WT, IAQ, AAA, or NHQ variants was injected into the striatum of FcRntg mice using convection-enhanced delivery (CED). Twenty-four hours later, the amount of human IgG in the injected hemisphere and plasma was assessed by ELISA. The ratio was calculated and normalized to the ratio of the IL-12Fc WT group. Five mice per group. Mean ± SD. C. Surface plasmon resonance (SPR) measurement of FcRn affinity between surface-immobilized human recombinant FcRn and IgG4 variants in solution. Affinity measured at pH 6.0. Data normalized to the IgG4 antibody with unmodified Fc (WT). A second IgG4 antibody (nivolumab) with an unmodified Fc portion was used as an additional reference (IgG4). Mean ± SD. D. 1 μg of IgG4 WT, IAQ, AAA, or NHQ variants was injected into the striatum of FcRntg mice using convection-enhanced delivery (CED). Twenty-four hours later, the amount of human IgG in the injected hemisphere and plasma was assessed by ELISA. The ratio was calculated and normalized to the ratio of the IL-12Fc WT group. Five mice per group. Mean ± SD. [Figure 10B]Antibodies. A. Surface plasmon resonance (SPR) measurements of FcRn affinity of surface-immobilized human recombinant FcRn and IgG1 variants in solution. Affinity measured at pH 6.0. Data normalized to an IgG1 antibody with unmodified Fc (WT). Three additional IgG1 clinical-grade antibodies with unmodified Fc moieties (IgG1_01 ipilimumab, IgG1_02 atezolizumab, IgG1_03 rituximab) were used as additional references. Mean ± SD. Affinity measured at pH 6.0. Data normalized to an IgG4 antibody with unmodified Fc (WT). A second IgG4 antibody (nivolumab) with an unmodified Fc moiety was used as an additional reference (IgG4). Mean ± SD. B. 1 μg of IgG1 WT, IAQ, AAA, or NHQ variants was injected into the striatum of FcRntg mice using convection-enhanced delivery (CED). Twenty-four hours later, the amount of human IgG in the injected hemisphere and plasma was assessed by ELISA. The ratio was calculated and normalized to the ratio of the IL-12Fc WT group. Five mice per group. Mean ± SD. C. Surface plasmon resonance (SPR) measurement of FcRn affinity between surface-immobilized human recombinant FcRn and IgG4 variants in solution. Affinity measured at pH 6.0. Data normalized to the IgG4 antibody with unmodified Fc (WT). A second IgG4 antibody (nivolumab) with an unmodified Fc portion was used as an additional reference (IgG4). Mean ± SD. D. 1 μg of IgG4 WT, IAQ, AAA, or NHQ variants was injected into the striatum of FcRntg mice using convection-enhanced delivery (CED). Twenty-four hours later, the amount of human IgG in the injected hemisphere and plasma was assessed by ELISA. The ratio was calculated and normalized to the ratio of the IL-12Fc WT group. Five mice per group. Mean ± SD. [Figure 10C]Antibodies. A. Surface plasmon resonance (SPR) measurements of FcRn affinity of surface-immobilized human recombinant FcRn and IgG1 variants in solution. Affinity measured at pH 6.0. Data normalized to an IgG1 antibody with unmodified Fc (WT). Three additional IgG1 clinical-grade antibodies with unmodified Fc moieties (IgG1_01 ipilimumab, IgG1_02 atezolizumab, IgG1_03 rituximab) were used as additional references. Mean ± SD. Affinity measured at pH 6.0. Data normalized to an IgG4 antibody with unmodified Fc (WT). A second IgG4 antibody (nivolumab) with an unmodified Fc moiety was used as an additional reference (IgG4). Mean ± SD. B. 1 μg of IgG1 WT, IAQ, AAA, or NHQ variants was injected into the striatum of FcRntg mice using convection-enhanced delivery (CED). Twenty-four hours later, the amount of human IgG in the injected hemisphere and plasma was assessed by ELISA. The ratio was calculated and normalized to the ratio of the IL-12Fc WT group. Five mice per group. Mean ± SD. C. Surface plasmon resonance (SPR) measurement of FcRn affinity between surface-immobilized human recombinant FcRn and IgG4 variants in solution. Affinity measured at pH 6.0. Data normalized to the IgG4 antibody with unmodified Fc (WT). A second IgG4 antibody (nivolumab) with an unmodified Fc portion was used as an additional reference (IgG4). Mean ± SD. D. 1 μg of IgG4 WT, IAQ, AAA, or NHQ variants was injected into the striatum of FcRntg mice using convection-enhanced delivery (CED). Twenty-four hours later, the amount of human IgG in the injected hemisphere and plasma was assessed by ELISA. The ratio was calculated and normalized to the ratio of the IL-12Fc WT group. Five mice per group. Mean ± SD. [Figure 10D]Antibodies. A. Surface plasmon resonance (SPR) measurements of FcRn affinity of surface-immobilized human recombinant FcRn and IgG1 variants in solution. Affinity measured at pH 6.0. Data normalized to an IgG1 antibody with unmodified Fc (WT). Three additional IgG1 clinical-grade antibodies with unmodified Fc moieties (IgG1_01 ipilimumab, IgG1_02 atezolizumab, IgG1_03 rituximab) were used as additional references. Mean ± SD. Affinity measured at pH 6.0. Data normalized to an IgG4 antibody with unmodified Fc (WT). A second IgG4 antibody (nivolumab) with an unmodified Fc moiety was used as an additional reference (IgG4). Mean ± SD. B. 1 μg of IgG1 WT, IAQ, AAA, or NHQ variants was injected into the striatum of FcRntg mice using convection-enhanced delivery (CED). Twenty-four hours later, the amount of human IgG in the injected hemisphere and plasma was assessed by ELISA. The ratio was calculated and normalized to the ratio of the IL-12Fc WT group. Five mice per group. Mean ± SD. C. Surface plasmon resonance (SPR) measurement of FcRn affinity between surface-immobilized human recombinant FcRn and IgG4 variants in solution. Affinity measured at pH 6.0. Data normalized to the IgG4 antibody with unmodified Fc (WT). A second IgG4 antibody (nivolumab) with an unmodified Fc portion was used as an additional reference (IgG4). Mean ± SD. D. 1 μg of IgG4 WT, IAQ, AAA, or NHQ variants was injected into the striatum of FcRntg mice using convection-enhanced delivery (CED). Twenty-four hours later, the amount of human IgG in the injected hemisphere and plasma was assessed by ELISA. The ratio was calculated and normalized to the ratio of the IL-12Fc WT group. Five mice per group. Mean ± SD. DETAILED DESCRIPTION OF THE INVENTION

[0334] Example 1: Materials and Methods animal C57BL / 6J mice were obtained from Charles River. - / - hFcRn tg(32) (FcRn tg) mice were obtained from Jackson Laboratory (stock number 014565). All animals were housed under specific pathogen-free (SPH) conditions in accordance with institutional guidelines, with food and water available ad libitum and a 12-h light / dark cycle. All animal experiments were performed in accordance with institutional guidelines and approved by the Swiss Cantonal Veterinary Office (license number 246 / 2015).

[0335] Tumor cell lines GL-261 cells were provided by A. Fontana, Department of Experimental Main Epidemiology, University of Zurich, Zurich, Switzerland, and were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum and L-glutamine (all from Thermo Fisher Scientific). The murine GL-261 brain tumor cell line (syngeneic to C57BL / 6) was stably transfected with pGl3-ctrl and pGKPuro (Promega) and selected with puromycin (Sigma-Aldrich) to generate luciferase-stable GL-261 cells. To generate GL-261:luc PD-L1 KO tumor cells, cells were transiently transfected with a streptococcal Cas9 P2A GFP-single guide RNA (sgRNA) expression vector (pX458; Addgene) modified to express the following sgRNA: 5'-GTATGGCAGCAACGTCACGA-3'. Three days after transfection, GFP-positive, PD-L1 knockout cells were purified by flow cytometry by gating on PD-L1-negative cells after 48 hours of IFN-γ stimulation (10 ng / ml). Single clones were further expanded and reconfirmed for loss of PD-L1 expression by flow cytometry before use in experiments.

[0336] Surgical procedure For glioma inoculation, 6- to 10-week-old mice were anesthetized with a mixture of fentanyl (Helvepharm AG), midazolam (Roche Pharma AG), and medetomidine (Orion Pharma AG). GL261 cells were injected intracranially (i.c.) into the right hemisphere using a stereotaxic robot (Neurostar). Briefly, a blunt-end syringe (Hamilton; 75N, 26s / 2'' / 2.5µl) was positioned 1.5mm lateral to bregma and 1mm bronchially. The needle was lowered into the burr hole to a depth of 4mm below the dural surface and retracted 1mm to form a small reservoir. Injections were performed with a volume of 2µl at 1µl / min. The needle was left in place for 2min and then retracted at 1mm / min. The burr hole was closed with bone wax (Aesculap, Braun), and the scalp wound was sealed with tissue adhesive (Indermil, Henkel). Anesthesia was interrupted with a mixture of flumazenil (Labatec Pharma AG) and buprenorphine (Indivior Schweiz AG), followed 20 minutes later by an injection of atipamezole (Janssen). Perioperative analgesia was provided with Carprofen (Pfizer AG).

[0337] After 7–14 days, osmotic pumps (Model 2004, 0.25 μl / h; Alzet) were filled with murine IL-12Fc (12.5 μg / kg / 24 h) or PBS alone and primed in PBS at 37°C. Mice were anesthetized as described above, the previous burr hole for glioma injection was positioned, bone wax and periosteal bone were removed, and an infusion cannula was lowered through a 3 mm burr hole into the estimated center of the tumor. Serum samples were collected every 2 days by tail vein blood sampling using Vacutainer tubing according to the manufacturer's instructions (Becton, Dickinson and Company), starting on day -1 after pump implantation.

[0338] To compare IL-12 and IL-12Fc WT serum-to-brain concentration ratios after bolus injection, mice were anesthetized and intracranially injected into the right hemisphere using a stereotaxic robot (Neurostar) as described above for tumor cell injection. Mice received 100 ng of recombinant human IL-12 (Prospec) or the same amount of IL-12Fc (69 ng / mouse). Dose was calculated based on a HEK-Blue IL-12 bioactivity assay. After 24 hours, animals were sacrificed by controlled CO2 asphyxiation. Blood samples were collected by cardiac puncture, and mice were perfused with 20 ml of ice-cold PBS. Serum was isolated as described above, and brain tissue was snap-frozen in liquid nitrogen.

[0339] To compare the serum-to-brain concentration ratios of IL-12 WT and IL-12Fc NHQ after bolus injection, mice were anesthetized and intracranially injected into the right hemisphere using a stereotaxic robot (Neurostar) as described above for tumor cell injection. Mice received 1 μg of human IL-12Fc WT or IL-12Fc NHQ. After 24 hours, animals were sacrificed by controlled CO2 asphyxiation. Blood samples were collected by cardiac puncture, and mice were perfused with 20 ml of ice-cold PBS. Serum was isolated as described above, and brain tissue was snap-frozen in liquid nitrogen.

[0340] For convection-enhanced delivery (CED) of proteins into the brain, mice were anesthetized and intracranial injections were performed into the right hemisphere using a stereotaxic robot (Neurostar). A catheter was fabricated from fused silica with an internal diameter of 0.1 mm and a wall thickness of 0.0325 mm, using a 27G blunt-end needle with a 1 mm step at the tip. Briefly, a burr hole was created 1 mm anterior-posterior and 2 mm mediolateral to bregma. The catheter was lowered into the burr hole to a depth of 3.5 mm below the dura surface. Injections were performed with a volume of 5 μl at 0.2 μl / min, followed by 2 μl at 0.5 μl / min, and 2 μl at 0.8 μl / min. The needle was left in place for 2 minutes before being withdrawn at 1 mm / min. Mice received 1 μg of recombinant human IL-12Fc WT, IL-12Fc IAQ, IL-12Fc AAA, IL-12Fc NHQ, or rmIL-12, mIL-12hFc WT, mIL-12hFc HNQ, mIL-12hFc:PD-L1 NHQ, Flu HA3.1 WT, Flu HA3.1 IAQ, Flu HA3.1 AAA, Flu HA3.1 NHQ, or atezolizumab WT, atezolizumab IAQ, atezolizumab AAA, or atezolizumab NHQ. Six hours later, animals were sacrificed by controlled CO2 asphyxiation. The ipsilateral hemisphere was snap-frozen in liquid nitrogen.

[0341] In vivo bioluminescence imaging Tumor-bearing mice were injected with d-luciferin (150 mg / kg body weight; XenoLight d-luciferin potassium salt; BioVision 7903-1G; 15 mg / mL in PBS). The animals were transferred to the darkroom of a Xenogen IVIS Lumina III (PerkinElmer) imaging system, and luminescence was recorded for 1–2 min with medium binning (4). Data were then analyzed using Living Image 4.7.1 software (PerkinElmer). A circular region of interest (ROI; 1.5 cm diameter) was defined around the tumor site, and the photon flux in this region was read and plotted.

[0342] BLI and systematic group allocation Twenty days after implantation of GL-261luc glioma cells, tumor-bearing animals were distributed into experimental groups with comparable mean BLI.

[0343] Blood collection Blood samples were collected 10 min before CED or 6, 24, 72 h, and 7 days after CED injection. 20–50 μL of blood was collected from the tail vein into a microtuber containing dried K2-EDTA (Becton, Dickinson and Company). After centrifugation at 10,000 g for 5 min, plasma was transferred to a fresh tube and frozen.

[0344] FcRn ELISA IL-12 Fc variants or a recombinant human IgG4 anti-GFP antibody (clone 515, AbD Serotec) serving as a control were coated onto microwell plates (Greiner Bio-One). Histidine-conjugated FcRn (R&D Systems) was incubated at increasing concentrations in ELISA diluent (Mabtech) at pH 6.0. FcRn was detected with a biotinylated anti-His antibody (clone 13 / 45 / 31-2, Dianova), streptavidin-conjugated horseradish peroxidase (Mabtech), and a colorimetric substrate (Chromogen-TMB, Thermo Fisher Scientific). The optical density at 450 nm was measured using a spectrophotometer (Molecular Devices).

[0345] Bead-based cytokine array Serum levels of mIL-12 and mIFNγ were measured using the Legendplex Mouse Inflammation Panel (Biolegend) according to the manufacturer's instructions. Samples were acquired using an LSRII Fortessa (Becton, Dickinson and Company). Data analysis was performed using FlowJo version 10.6 (Treestar).

[0346] HEK-Blue IL-12 bioactivity assay HEK-Blue IL-12 cells (InvivoGen) were seeded onto flat-bottom 96-well plates (Corning) at a density of 50,000 cells / well in medium containing normocin (InvivoGen). Cells were incubated for 17 hours with increasing amounts of IL-12, IL-12Fc WT, or a variant designed for reduced FcRn affinity. The medium was collected and incubated for 2 hours in the presence of Quanti-Blue detection reagent (InvivoGen). Absorbance was measured at 640 nm using a tabletop spectrophotometer (Molecular Devices).

[0347] Detection of human IL-12 in brain, plasma, and serum after brain injection and calculation of serum or plasma to brain concentration ratios Samples were diluted in PBS containing 0.05% Tween-20 and 0.1% BSA, and IL-12 levels were assessed by ELISA (Mabtech) for hIL-12p70. To calculate serum or plasma to brain concentration ratios, IL-12 concentrations in serum or plasma were reported in pg / ml, while brain concentrations were calculated as the total amount of IL-12 extracted from the brain, corrected for the efficiency of protein extraction, divided by the weight of the hemisphere (pg / mg brain tissue).

[0348] Detection of human IgG in brain and plasma after intracerebral injection and calculation of plasma-to-brain concentration ratio Samples were diluted in PBS containing 0.05% Tween-20 and 0.1% BSA, and IgG levels were assessed by ELISA. Briefly, plates were coated with polyclonal donkey anti-human IgG (Jackson ImmunoResearch) blocked with PBS containing 0.05% Tween-20 and 0.1% BSA. Analytes were detected with polyclonal goat anti-human IgG (Sigma-Aldrich) and amplified with polyclonal donkey anti-goat HRP-conjugated antibody (Promega). For calculation of plasma-to-brain ratios, the concentrations of human IgG in plasma and brain were reported in pg / ml.

[0349] Production of human IgG1 variant, IgG4 variant hIL-12hFc:aPD-L1 NHQ and mIL-12hFc:aPD-L1 NHQ IgG4 variants were expressed in transiently transfected human embryonic kidney (HEK) cell cultures. IgG1 variants, hIL-12hFc:aPD-L1 NHQ and mIL-12hFc:aPD-L1 NHQ, were produced by transiently transfected Chinese hamster ovary (CHO) or cell cultures. Briefly, culture supernatants were collected, and the proteins were purified by affinity chromatography (protein G). The proteins were further purified by ion exchange (IEC) and size exclusion chromatography (SEC). The proteins were concentrated using a spin column (Sartorius, 30 kDa cutoff). The proteins were stored in 20 mM histidine, 150 mM NaCl, pH 6.0 buffer. The quality was assessed by gel electrophoresis (SDS-PAGE), followed by Coomassie staining according to standard protocols. Nivolumab, atezolizumab, ipilimumab, and rituximab are commercially available.

[0350] IFN-γ production by lymphocytes stimulated with IL-12Fc Human peripheral blood mononuclear cells (PBMCs) were stimulated with increasing concentrations of IL-12, IL-12Fc, or IL-12Fc variants with reduced FcRn affinity in the presence of 100 ng / ml anti-CD3 antibody for 24 h. IFN-γ levels in the supernatants were measured by ELISA according to the manufacturer's instructions (Mabtech).

[0351] Brain protein isolation After euthanasia and careful removal of the calvaria, brains were isolated. The cerebellum and olfactory bulbs were removed, the hemispheres were separated along the midline, and the injected (ipsilateral) hemisphere was snap-frozen in liquid nitrogen. Brain lysates were prepared by homogenization in ice-cold lysis buffer (Cell Signaling) containing Halt protease inhibitor cocktail (Thermo Fisher Scientific). 0.1 ml of lysis buffer was added per 10 mg of brain tissue. The brain tissue was minced with scissors, then passed through a 20G needle and finally sonicated for 20 seconds. The samples were centrifuged for 10 minutes at 15,000 g at 4°C, and the supernatant was transferred to a fresh tube. Protein concentration was measured using the Pierce BCA Assay Kit (Thermo Fisher Scientific), and this data was used to correct for protein extraction efficiency in each experiment.

[0352] All human and mouse IL-12 Fc variants were expressed in HEK239T cells. Variants that retained protein G affinity were purified from culture supernatants by affinity chromatography using protein G Sepharose (Biovision) and overnight dialysis against PBS. Variants that lost protein G affinity were purified by precipitation with 50% saturation of ammonium sulfate (VI), followed by dissolution of the precipitate in PBS and purification on a ceramic hydroxyapatite (CHT) column (type II, 40 μm Bio-Rad). After protein G or CHT chromatography, the samples were further purified by ion exchange chromatography using diethylaminoethanol-coupled Sepharose (HiTrap DEAE Sepharose FF column, GE Healthcare) as anionite on an AKTA Pure chromatography system (GE Healthcare). Finally, all IL-12 Fc variants were purified by size exclusion chromatography (GE Healthcare) on a pre-packed Superose 6 column (GE Healthcare) on an AKTA chromatography system (GE Healthcare). The dimer fraction was concentrated using a Vivaspin 2 ml spin column (GE Healthcare) with a 30 kDa cutoff. Protein purity was verified by SDS-PAGE electrophoresis followed by staining with Coomassie Brilliant Blue (VWR Life Sciences). Protein concentration was measured using the Pierce BCA Assay Kit (Thermo Fisher Scientific) and an ELISA for IL-12p70 (Becton, Dickinson and Company).

[0353] Phosphorylation of STAT-4 by lymphocytes stimulated with IL-12Fc Human peripheral blood mononuclear cells (PBMCs) were stimulated with 10 ng / ml of IL-12, IL-12Fc, or IL-12Fc variants with reduced FcRn affinity in the presence of 100 ng / ml anti-CD3 for 1 hour. Cells were then lysed using Pierce RIPA buffer (Thermo Fisher Scientific). Samples were analyzed by SDS-Page electrophoresis, then transferred using the Trans-Blot Turbo Blotting system (Bio-Rad Laboratories, Inc.), and stained with anti-STAT4 pY693 (clone 38 / p-Stat4, Becton, Dickinson and Company). Band visualization was performed using ECL transparency substrate (Bio-Rad Laboratories, Inc.) and a BioRad MPCD imager (Bio-Rad Laboratories, Inc.).

[0354] Surface plasmon resonance SPR was performed using a ProteOn XPR36 system (Bio-Rad Laboratories, Inc.) with human recombinant biotinylated FcRn (Immunitrack) coated onto a ProteOn NLC sensor chip to approximately 80 response units (RU). IL-12 Fc variants were run in 10 mM sodium citrate buffer, pH 6.0, at concentrations decreasing from 729 nM to 9 nM in three-fold steps. The dissociation time was 600 seconds. Analysis was performed using ProteOn Manager software (Bio-Rad Laboratories, Inc.) with data normalization to injection time, interspot background subtraction, and built-in artifact removal. Kd was calculated using an equilibrium analysis model.

[0355] Thermal shift assay Briefly, 0.2 mg / ml protein samples were mixed with 1:1000 diluted Sypro Orange Protein stain (Sigma-Aldrich) and run on a CFX384 thermocycler (Biorad) from 20°C to 95°C with a temperature increase of 0.2°C every 30 seconds, using fluorescence as the readout. The denaturation temperature was determined as the first derivative of fluorescence over temperature. Experiments were performed in PBS and artificial cerebrospinal fluid (aCSF; 125 mM NaCl, 26 mM NaHCO3, 1.25 mM NaH2PO3, and 2.5 mM KCl) as solvents.

[0356] statistical analysis Statistical analysis was performed using Graphpad Prism 5 software. Outliers were excluded from the final analysis according to the Grubb test (49). Two groups were compared using the Student's t test. One-way ANOVA and Tukey's multiple comparison test were used to compare more than two groups.

[0357] Flow cytometry PD-L1 binding assay GL261:lucE9 or GL261:lucE9:PD-L1KO cells were cultured overnight with mouse interferon-gamma at a final concentration of 20 ng / mL. The next day, cells were washed with DPBS. Trypsin-EDTA (Invitrogen 25300-054) was added to the flask and immediately removed again. The cells were allowed to detach from the flask for 2–5 minutes. They were then washed with medium and centrifuged at 350 × g for 5 minutes at 4°C. Cells were then plated at 100,000 cells / well into a round-bottom 96-well plate and washed twice with DPBS.

[0358] Staining was performed with 25 μL per well of PBS containing a 1:200 dilution of Zombie Aqua Fixable Viability Kit (BioLegend) and either human anti-PD-L1 (atezolizumab) or m / hIL-12hFc:aPD-L1 NHQ at a final concentration of 0.1 mg / mL. Cells were stained for 20 minutes at 4°C in the dark. After a PBS wash step, cells were incubated with the secondary antibody anti-human IgG-Fc-PE (Biolegend, catalog no. 409304, lot B260868) or anti-mouse PD-L1-BV421 (Biolegend, catalog no. 124315; lot B228149) control antibody at 0.2 mg / mL in PBS for 30 minutes at 4°C in the dark. Cells were washed twice with PBS, filtered through a 40 μm mesh, and acquired using an LSRII Fortessa flow cytometer (BD). Data analysis was performed using FlowJo version 10.6 (Tree Star).

[0359] Survival analysis Tumor-bearing animals were examined for neurological symptoms and weighed weekly until day 21 after tumor cell implantation. After day 21, monitoring frequency was increased to daily checks and weekly bioluminescence imaging (BLI). In accordance with the county's veterinary authorities (ZH194 / 19), animals were euthanized by controlled CO2 asphyxiation upon reaching predefined discontinuation criteria (weight loss >20% of maximum body weight and / or moribund status).

[0360] Example 2: Intracranial injection of human IL-12 has higher systemic leakage than hIL-12Fc IL-12Fc for the local treatment of brain tumors shows great promise. However, for use in clinical trials, a human version of IL-12Fc is required that should exhibit similar properties. To obtain a human analog to murine IL-12IgG3, we fused single-chain human IL-12 to the crystallizable fragment (Fc) of human immunoglobulin G4 (hIgG4) (Figure 1A). Similar to mIgG3, hIgG4 does not support antibody-dependent cell-mediated cytotoxicity (ADCC) or activate the complement system. To test the leakage and stability of human IL-12Fc (hIL-12Fc) versus recombinant human IL-12 (rhIL-12), we injected a single bolus into the striatum of transgenic mice expressing human FcRn on a mouse FcRn-deficient background (FcRntg) (Postow et al., 2015, N Engl J Med 372:2006-2017; Kamran et al., 2016, Expert Opin Biol Ther 16:1245-1264). After 24 hours, we analyzed human IL-12 concentrations in lysates and serum from the ipsilateral hemisphere to learn more about the stability and retention (residual concentrations) at the injection site and the rate of leakage into the bloodstream (Figure 1B). For each mouse, we calculated the ratio of serum concentration to the concentration at the injection site as an estimate of tissue retention. Compared to serum levels and local concentrations at the injection site, hIL-12Fc showed better tissue retention than rhIL-12, as we observed a significantly lower ratio (Figure 1C). For local GB treatment, the human IL-12Fc fusion cytokine appears to be a superior compound compared to its native counterpart due to its high tissue retention, stability, and solubility.

[0361] Example 3: FcRn binding leads to systemic accumulation of IL-12Fc The neonatal Fc receptor (FcRn)-based endosomal recycling system in endothelial cells and red pulp macrophages prevents the rapid degradation and clearance of IgG. After pinocytosis, facilitated by the acidic pH of the endosome, FcRn binds IgG and recycles it to the cell surface, where neutral pH induces its release. When injected locally, IL-12Fc can leak from the brain in an FcRn-mediated manner due to its Fc tag. Brain leakage can lead to serum accumulation of IL-12Fc, eventually reaching toxic levels. To test whether FcRn-based recycling actually promotes the accumulation of hIL-12Fc in serum, we utilized transgenic mice expressing human FcRn on a murine FcRn-deficient background (FcRntg). Although human FcRn has weak affinity for mouse IgG, it promotes normal albumin recycling, so only mouse IgG recycling is impaired in this mouse model. Thus, mouse IL-12Fc binds significantly less to FcRn in these FcRn-humanized mice. Therefore, we compared serum mIL-12 levels in glioma-bearing wild-type (wt) and FcRntg mice that had been treated with local mouse IL-12Fc via osmotic minipumps. Indeed, after one week, we observed an increase in IL-12 levels in wt mice (Figure 2A), but not in FcRntg mice (Figure 2D), followed by an increase in IFN-γ levels. We even observed an increase in IL-12 levels in the serum of some mice as early as one day after pump implantation (Figure 2B). Consequently, this resulted in a significant increase in serum IFN-γ levels in wt mice, but not in the FcRntg cohort (Figure 2C). Similar to mouse IL-12Fc, human IL-12Fc is also likely to leak and accumulate, potentially leading to systemic side effects.Furthermore, IFN-γ is one of the major mediators of IL-12-associated side effects (Leonard et al., 1997, Blood 90:2541-2548), and its persistent systemic presence can be toxic (Weiss et al., 2007, Expert Opin Biol Ther 7:1705-1721). Taken together, we conclude that even minute leakage of IL-12Fc from the treatment site is sufficient to induce detectable serum IFN-γ levels.

[0362] Example 4: Generation of human IL-12 Fc variants designed for improved tissue retention The observation that reduced FcRn binding potentially abolishes brain egress and results in dramatically reduced recycling upon brain leakage can be exploited to increase the safety margin of hIL-12Fc. Therefore, the present inventors set out to reduce the binding of the Fc portion of hIL-12Fc to human FcRn. It has been shown that by increasing the positive charge of the FcRn-binding interface of the Fc portion, this interaction—and therefore recycling—at acidic pH can be abolished, which reduces the serum half-life of the immunoglobulin. The present inventors introduced a number of mutations into hIL-12Fc at its FcRn-binding site with the aim of reducing its serum half-life in the event of leakage (Table 1).

[0363] We created three IL-12Fc variants with mutations similar to those of previously published antibodies with reduced FcRn affinity, designated IAQ, AHH, and AAA. Furthermore, we substituted the isoleucine at position 253 with an asparagine (I253N) instead of an alanine, which represents a simple shortening of the side chain. Asparagine is a polar amino acid, and its side chain has a similar length to that of isoleucine. We also modified the histidine at position 310 to alanine and the histidine at position 435 to glutamine, alanine, or glutamic acid.

[0364] All variants were expressed in human embryonic kidney 293T cell (HEK293T) cultures, and the expression levels of all variants were similar.

[0365] Example 5: Human IL-12 Fc variants have similar protein stability First, we examined whether the changes introduced into the Fc affected overall protein stability. To this end, we measured the denaturation temperature for each of the variants in thermal shift assays performed in PBS and artificial cerebrospinal fluid (aCSF). The denaturation temperatures of all variants oscillated around 60°C (Figure 3A). Measurements performed in aCSF confirmed that all variants had similar stability, although the overall denaturation temperature was lower, at approximately 57°C (Figure 3B).

[0366] Example 6: Human IL-12 Fc variants maintain their biological activity Although we aimed to reduce the binding of hIL-12Fc to FcRn, we could not rule out that these changes affected the biological activity of IL-12. This was first tested using a HEK cell line stably transfected with IL-12 signaling components and downstream enzymes catalyzing colorimetric reactions. Only the NAQ variant showed an approximately two-fold reduction in activity compared to IL-12Fc, while all others exhibited EC50s in the range of IL-12Fc WT (Figure 4A). Importantly, IL-12Fc had comparable activity to rIL-12 in vitro.

[0367] To further validate the activity of IL-12Fc variants, we activated peripheral blood mononuclear cells (PBMCs) with three different hIL-12Fc variants, i.e., IAQ, AHQ, and NHQ, and then analyzed STAT-4 phosphorylation (Figure 4B). More importantly, this STAT-4 phosphorylation translated into robust production of IFN-γ after 24 h (Figure 4C), demonstrating that all variants retained the activity of rhIL-12.

[0368] Example 7: Human IL-12 Fc variants differ in binding to Protein G Protein A and G affinity chromatography is one of the standard methods used to purify recombinant antibodies and Fc fusion proteins. Modification of the interface between Fc and FcRn is known to abolish Protein A binding, an observation we confirmed with IL-12Fc variants. To confirm the feasibility of production in a scale-up process, we decided to examine the possibility of purifying IL-12Fc variants through a Protein G affinity column. Most of our variants retained affinity for Protein G, but to our surprise, all variants containing both the I253N and H310A mutations were unsuitable for Protein G purification (Table 2). This effect was independent of the additional mutation at position 435. For further study, we focused on variants with retained Protein G affinity.

[0369] Example 8: Human IL-12 Fc variants have reduced FcRn affinity To verify the affinity of the IL-12Fc variants for FcRn, we used surface plasmon resonance (SPR), a label-free method for characterizing protein-protein interactions. We immobilized human FcRn and measured the binding of IL-12Fc variants at various concentrations in the lysosomal pH range (pH = 6.0) (43). As shown in Figure 5A, most of the modified IL-12Fc variants exhibited reduced affinity for human FcRn, with the NHQ variant showing the strongest reduction (approximately 8-fold lower). We used a commercially available human monoclonal anti-GFP IgG4 antibody as a control.

[0370] Furthermore, we corroborated these data with ELISA data for the NHQ constructs, using IL-12Fc WT, anti-GFP IgG4, and the published variant IAQ as a reference. Both IAQ and NHQ showed reduced binding, with NHQ having the lowest affinity (Figure 5B). Therefore, we concluded that the NHQ combination of substitutions appears to most dramatically reduce binding to FcRn. This is in contrast to the results of Kenanova and colleagues (Kenanova et al., 2005, Cancer Research 65:622-631), who suggested that the combined mutations H310A and H435Q were responsible for the strongest reduction in binding to FcRn at low pH.

[0371] Example 9: Introduction of the NHQ mutation reduces systemic exposure to locally delivered hIL-12Fc We hypothesized that reduced FcRn affinity would increase the retention of hIL-12Fc in the CNS while preventing its systemic accumulation. This was addressed in a similar manner comparing hIL-12Fc WT and recombinant human IL-12 (Figure 1B). tg Mice were injected with 1 μg of IL-12Fc WT or NHQ variant, and IL-12 was measured in the ipsilateral brain hemisphere and serum by ELISA. Mice injected with the NHQ variant showed a reduced serum-to-brain ratio compared with mice injected with hIL-12Fc WT (Figure 6A). We hypothesize that this may be due to both increased retention in the CNS via FcRn-mediated recycling and attenuated systemic accumulation.

[0372] Furthermore, using CED instead of bolus injection, we compared plasma concentrations of hIL-12Fc WT, IAQ, AAA, and NHQ in the injected hemisphere 24 h after CED and observed that the NHQ variant exhibited the most significantly reduced plasma-to-brain ratio (Figure 6B), even at optimized delivery settings compared to bolus injection. This increased CNS retention, combined with reduced systemic exposure, may potentially improve the safety profile of local IL-12Fc therapy.

[0373] Example 10: IL-12Fc variant NHQ has higher brain tissue retention than other low affinity variants Finally, we measured tissue retention after intracranial delivery of the protein. To this end, we injected 1 μg of unmodified IL-12Fc WT, two previously published variants with reduced FcRn affinity, i.e., IAQ and AAA, and NHQ, the variant with the lowest FcRn affinity according to our measurements (Figure 5A). Instead of a bolus injection of the protein solution, we used a CED protocol with a step catheter and a ramp-up injection regimen to ensure maximal perfusion of the cerebral hemisphere. To study the impact of different modifications at the FcRn binding interface in the most physiological setting, we used the FcRn binding protocol. tg We employed mice. As previously mentioned, FcRn is important for both CNS egress and serum accumulation of Fc-containing molecules. In an attempt to dissociate the two effects and focus solely on preventing CNS transport, we measured the amount of protein remaining in the brain 6 hours after CED. Mice were euthanized and perfused with PBS. Total protein in the ipsilateral hemisphere was isolated, and hIL-12 was measured by ELISA. As shown in Figure 7, IL-12Fc NHQ had superior tissue retention compared to IL-12Fc WT. Importantly, it also had better tissue retention than two other variants with reduced FcRn affinity, IAQ and AAA. Surprisingly, IAQ and AAA were not significantly different from IL-12Fc WT.

[0374] Example 11: Antitumor effect in vivo Human IL-12 has only low cross-reactivity with the mouse IL-12 receptor. This means that surrogate molecules must be used to study in vivo antitumor effects in mouse models. To test the effect of reduced affinity for FcRn, we fused single-chain mouse IL-12 to the same human IgG4 Fc as for hIL-12Fc (Figure 8A).

[0375] IL-12 induces the expression of IFNγ in target cells, such as T cells and NK cells (Tugues et al., Cell death and differentiation (2015) 22:237-246). IFNγ, in turn, can lead to the upregulation of PD-L1 on myeloid and tumor cells in a process called adaptive resistance (O'Rourke et al., Sci. Transl. Med. (2017) (9): eaaa0984). Therefore, we reasoned that PD-L1 serves as a guidance anchor to further increase IL-12 tissue retention.

[0376] To evaluate the efficacy of IL-12 Fc in combination with topically applied anti-PD-L1 antibody therapy, we constructed a bispecific Fc fusion molecule. It combines mIL-12 hFc, an anti-PD-L1 half antibody, and an hIgG1 Fc containing the NHQ mutation. The knob-into-hole method was used for heterodimeric heavy chain assembly (Ridgway et al., Protein Eng (1996) 9:617-621). The anti-PD-L1 half molecule was derived from atezolizumab, a clinically approved antibody that cross-reacts with mouse and human PD-L1 (U.S. Patent No. 8,217,149 B2) (Figure 8A).

[0377] We confirmed the biological activity of the mIL-12hFc:aPD-L1 NHQ molecule in vitro: for IL-12 functionality, an IL-12-sensitive reporter cell line was used, in which IL-12 induces secreted alkaline phosphatase, which in turn catalyzes a colorimetric reaction (Figure 8B). Binding to PD-L1-bound cells was confirmed by flow cytometry to detect binding of the heterodimeric bifunctional construct to PD-L1 on the cell surface (Figure 8C). The bifunctional heterodimeric constructs exhibited their C H 2 domain and C H 3 domain, and therefore abrogated FcRn binding, as confirmed by surface plasmon resonance, resulting in a relatively high K compared to unmodified anti-PD-L1 antibodies. D values ​​(Figure 8D).

[0378] Following in vitro characterization, we continued to measure its properties in vivo. Antitumor efficacy and systemic distribution were monitored in vivo using the mouse glioma model GL-261. Briefly, tumor-bearing mice were intracranially injected twice via CED with rmIL-12, mIL-12hFc:aPD-L1 NHQ, mIL-12hFc WT or NHQ, or vehicle control (injection buffer only) (Figure 9A). Changes in tumor size were monitored using bioluminescence imaging, and clinical effects were monitored by clinical scoring (Figure 9B). To assess leakage and shedding during CED, systemic IL-12 and IFNγ levels were measured in plasma at various time points (Figure 9C). Animals receiving rmIL-12 or mIL-12hFc wt showed a rapid increase in systemic IL-12 signaling and IFNγ immediately following CED, whereas animals receiving mIL-12hFc NHQ or mIL-12hFc:aPD-L1 NHQ showed a strongly reduced systemic IL-12 signaling that rapidly returned to baseline and significantly reduced IFNγ signaling (Figure 9C). The difference in tissue retention between mIL-12hFc wt and mIL-12hFc NHQ led to a lower systemic IL-12 signal already 6 hours after CED1 (Figure 9D). Regarding the clinical course of treated animals, all groups receiving IL-12 constructs showed a significant increase in survival 3 weeks after tumor inoculation compared to the control group, even at an exceptionally late stage when disease was extremely advanced (Figure 9E). Notably, the treatment response in the groups receiving the NHQ constructs (mIL-12hFc NHQ or mIL-12hFc:aPD-L1 NHQ) was at least equally good compared to the groups receiving mIL-12hFc wt or rmIL-12, but showed a marked reduction in systemic IL-12 and IFNg.

[0379] Example 12: Affinity measurements of IL-12Fc and IgG variants for hFcRn To further evaluate the impact of low FcRn affinity in favorably influencing the plasma-to-brain ratio for local delivery to the CNS, the IAQ, AAA, and NHQ variants were compared with unmodified antibodies (Figure 10). We selected a human IgG1 (Figures 10A and 10B, atezolizumab) and a human anti-influenza A IgG4 antibody (Figures 10C and 10D, Flu HA3.1, U.S. Patent Application Publication No. 2014 / 0370032A1) directed against PD-L1.

[0380] The finding that hIL-12Fc is functional, has greater tissue retention than rhIL-12, and can increase the margin of safety by halting systemic recycling in the event of leakage may have broad implications for the local administration of any Fc-containing molecule. These modifications could enable safe and effective local delivery of any antibody or Fc-fusion molecule for the local treatment of neurological disorders.

[0381] Administration of therapeutics to the CNS via systemic routes (either intravenously or intravenously) is challenging, primarily due to the BBB (compared to the rest of the body), and only a select few therapeutics today actually reach the brain. Unfortunately, antibodies and Fc-containing biologics, specifically Fc fusion proteins, do not readily cross the BBB and are actively excreted. Enabling antibody transport across the BBB into the brain parenchyma has been extensively studied, for example, by utilizing receptor-mediated transcytosis for transferrin. Cytokines have short circulatory half-lives, a high risk of side effects, and a narrow therapeutic window. Cytokines can be linked to antibodies that home to tumors where they accumulate, specifically NHS-IL-12. Even after subcutaneous administration, these antibodies induce an IFNγ response as they migrate to tumors via the bloodstream. Initially, systemic delivery of IL-12 was evaluated for the treatment of non-brain cancers. However, these clinical trials had to be prematurely terminated because intravenous administration at effective doses caused serious adverse events, including death. One of the main reasons is thought to be the induction of IFNγ by IL-12.

[0382] The serum half-life and solubility of protein therapeutics can be improved by directly fusing the therapeutic moiety with the crystallizable fragment (Fc) of an antibody. Due to direct local application to distinct anatomical sites, this can lead to undesirable effects. One of these may be the FcRn-mediated efflux of Fc-containing molecules from immune-privileged anatomical sites, specifically the brain, and their serum accumulation, similar to IgG recycling.

[0383] We observed that local administration of IL-12Fc fusion cytokines into the brain triggered FcRn-dependent secretion of IL-12Fc across the BBB into the circulation, where it accumulated and triggered potentially dangerous IFNγ production.

[0384] We found that IL-12Fc with reduced FcRn affinity was functional and had higher tissue retention than recombinant IL-12 and unmodified IL-12Fc. When compared in brain tissue retention experiments, the NHQ mutant was the only one to show improved retention compared to IL-12Fc WT. Surprisingly, the two variants reported to have dramatically reduced FcRn binding, IAQ and AAA, did not differ from unmodified IL-12Fc, suggesting that to achieve biological differentiation, FcRn affinity must be reduced beyond a certain threshold, which only the NHQ modification achieves. Alternatively, we cannot exclude that the NHQ mutation introduces other features that improve tissue retention in an FcRn-independent manner.

[0385] This leads to an improved safety profile and broadens the therapeutic window for IL-12Fc therapy of brain tumors. Furthermore, our findings can be translated to any Fc-containing therapeutic, primarily therapeutic antibodies, for which there is a strong rationale for local intracranial administration. This route of administration is preferred because systemically administered drugs have poor efficacy, potentially due to poor cross-sectional effects across the BBB, or because the desired therapeutic effect should be contained locally. Local therapy with such delivery-optimized biologics can eliminate systemic toxicity and thus improve the drug's safety profile.

[0386] [Table 1]

[0387] [Table 2]

[0388] [Table 3]

[0389] Combined bispecific molecules may consist of the molecules set forth as SEQ ID NO:15 and SEQ ID NO:21, SEQ ID NO:16 and SEQ ID NO:21, SEQ ID NO:17 and SEQ ID NO:22, SEQ ID NO:18 and SEQ ID NO:22, SEQ ID NO:17 and SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:18 and SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:19 and SEQ ID NO:22, SEQ ID NO:20 and SEQ ID NO:22, SEQ ID NO:19 and SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:20 and SEQ ID NO:23 and SEQ ID NO:24.

[0390] item 1. A polypeptide comprising the crystallizable fragment (Fc) region of IgG for use as a medicament (use in medicine), the Fc region comprises a modification that results in reduced affinity for the neonatal Fc receptor (FcRn); The polypeptide is delivered to the tissue affected by the disease by local administration.

[0391] 2. The above polypeptide -By intracranial administration, by intrathecal administration, or -by intraocular administration 2. The polypeptide for use as a pharmaceutical according to item 1, wherein the polypeptide is delivered to a patient.

[0392] 3. A polypeptide for use as a pharmaceutical according to any one of the preceding items, wherein the polypeptide is administered by intracranial administration, and the serum or plasma to brain concentration ratio of the polypeptide is tg It can be measured 24 hours after intracranial bolus injection into the striatum of mice. a. serum or plasma to brain concentration ratio that is at most 2 / 3 of the same polypeptide containing an unmodified Fc region; or b. Serum or plasma to brain concentration ratios up to 1 / 8 of the same polypeptide without an Fc region or peptide linker A polypeptide that is below a predetermined threshold selected from:

[0393] 4. The polypeptide is administered by intracerebroventricular or intrathecal administration, and the serum or plasma to CSF ​​concentration ratio of the polypeptide is increased by FcRn tg It can be measured 24 hours after intracerebroventricular or intrathecal injection in mice. c. A serum or plasma to CSF ​​concentration ratio of the same polypeptide containing an unmodified Fc region that is at most 2 / 3; d. Up to 1 / 8 the serum or plasma to CSF ​​concentration ratio of the same polypeptide without the Fc region or peptide linker 2. The polypeptide for use as a pharmaceutical according to any one of the preceding items, wherein the polypeptide has a specific activity below a predetermined threshold selected from the group consisting of:

[0394] 5. The decrease in the affinity of the polypeptide for FcRn is a. The dissociation constant (K) characterizing the binding of FcRn to the same polypeptide containing the unmodified Fc regionD ) at least two-fold, specifically at least three-fold, more specifically at least four-fold, and even more specifically at least five-fold increased K D , and b. K characterizing the binding of FcRn to the same polypeptide containing differently modified Fc regions, i.e., one mutant selected from IAQ (with mutations H310A and H435Q) and AAA (with mutations I253A, H310A, and H435A). D at least 1.5-fold, specifically at least 2-fold, increased K D K selected from D 2. A polypeptide for use as a medicament according to any one of the preceding items, characterized by:

[0395] 6. Intracranial delivery a. Single, intermittent, or continuous local infusion, including convection-enhanced delivery (CED); b. Intrathecal administration c. in situ production of said polypeptide; d. Release from implantable sustained release formulations; e.Molecular transport to the CNS, f. Cellular transport to the CNS, or g. Transport to the CNS after intranasal application The polypeptide for use as a pharmaceutical according to any one of the preceding items, wherein the polypeptide is administered by a method selected from the group consisting of:

[0396] 7. A polypeptide for use as a medicament according to any one of the preceding items for the treatment or prevention of a disease affecting the central nervous system.

[0397] 8. The polypeptide for use as a medicament according to item 7, wherein the disease affecting the central nervous system is a malignant disease, particularly glioma, more particularly high-grade glioma (HGG).

[0398] 9. A polypeptide for use as a pharmaceutical according to any one of the preceding items, wherein the Fc region is a human Fc region or a chimeric Fc region comprising a human amino acid sequence and has a mutation at position 253, specifically I253A or I253N, more specifically I253N.

[0399] 10. The polypeptide for use as a medicament according to item 9, wherein the Fc region does not have a mutation at position 310.

[0400] 11. The Fc region is -mutations H310A and H435Q (IAQ); - mutations I253A and H435Q, as well as H at position 310 (AHQ); - mutations I253N and H435Q, as well as H at position 310 (NHQ); - mutations I253A, H310A and H435Q (AAQ); - mutations I253N, H310A and H435Q (NAQ); - mutation I253A, and H at positions 310 and 435 (AHH); - mutation I253N, and H at positions 310 and 435 (NHH); - mutations I253A and H310A, and H at position 435 (AAH); - mutations I253N and H310A, as well as H at position 435 (NAH); - mutations I253N, H310A and H435A (NAA); - mutations I253N, H310A and H435E (NAE); - mutations I253A, H310A and H435A (AAA); or -Mutations I253A, H310A and H435E (AAE) 2. A polypeptide for use as a pharmaceutical according to any one of the preceding items, comprising:

[0401] 12. The Fc region is - mutations I253N and H435Q, as well as H at position 310 (NHQ); - mutations I253A, H310A and H435Q (AAQ); - mutations I253N, H310A and H435Q (NAQ); - mutations I253N, H310A and H435E (NAE); or -Mutations I253A, H310A and H435E (AAE) 10. A polypeptide for use as a medicament according to any one of the preceding items, comprising:

[0402] 13. A polypeptide for use as a medicament according to any one of the preceding items, wherein the Fc region is or comprises a sequence characterized by SEQ ID NO: 002 (IAQ), SEQ ID NO: 003 (AHQ), SEQ ID NO: 004 (NHQ), SEQ ID NO: 005 (AAQ), SEQ ID NO: 006 (NAQ), SEQ ID NO: 007 (AHH), SEQ ID NO: 008 (NHH), SEQ ID NO: 009 (AAH), SEQ ID NO: 010 (NAH), SEQ ID NO: 011 (NAA), SEQ ID NO: 012 (NAE), SEQ ID NO: 013 (AAA) or SEQ ID NO: 014 (AAE).

[0403] 14. A polypeptide for use as a medicament according to any one of the preceding items, wherein the Fc region is or comprises a sequence characterized by SEQ ID NO: 004 (NHQ), SEQ ID NO: 005 (AAQ), SEQ ID NO: 006 (NAQ), SEQ ID NO: 012 (NAE) or SEQ ID NO: 014 (AAE).

[0404] 15. The polypeptide is ai effector polypeptide, and ii. The Fc region a fusion protein comprising: b. An antibody or antibody-like molecule containing the above Fc region 2. A polypeptide for use as a pharmaceutical according to any one of the preceding items, selected from:

[0405] 16. The polypeptide is a. Bispecific, trispecific or multispecific antibodies or antibody-like molecules, in particular i. CD3 and tumor-associated antigens, ii. Agonistic histone and IL12 receptors; iii.PD-L1 and 4-1BB, iv.PD-L1 and CD28, v. PD-L1 and IL-12 receptors in an agonistic manner, or vi. Agonistic tumor-associated antigen and IL-12 receptor a bispecific antibody or antibody-like molecule that specifically binds to b. an armed antibody or antibody-like molecule comprising an effector polypeptide; or c. a tumor- or tissue-conditioning antibody or antibody-like molecule comprising a shielding domain and a cleavable, protease-sensitive linker peptide 2. A polypeptide for use as a pharmaceutical according to any one of the preceding items, selected from:

[0406] 17. The polypeptide is a bispecific, trispecific, or multispecific antibody or antibody-like molecule, in particular a bispecific antibody or antibody-like molecule that specifically binds to PD-L1, comprising: i. effector polypeptides, ii. IL-12Fc, iii. A combination of a molecule characterized by a sequence selected from SEQ ID NOs: 015-016 and a molecule characterized by the sequence of SEQ ID NO: 021; iv. A combination of a molecule characterized by a sequence selected from SEQ ID NOs: 017 to 020 and a molecule characterized by the sequence of SEQ ID NO: 022, or v. A combination of a molecule characterized by a sequence selected from SEQ ID NOs: 017 to 020, a molecule characterized by the sequence of SEQ ID NO: 023, and a molecule characterized by the sequence of SEQ ID NO: 024. 17. The polypeptide for use as a pharmaceutical according to Item 16, comprising:

[0407] 18. A polypeptide is administered by intracranial administration, and the serum or plasma to brain concentration ratio of the polypeptide is increased by FcRn tg It can be measured 24 hours after intracranial bolus injection into the striatum of mice. a. serum or plasma to brain concentration ratio that is up to 1 / 8 of the same polypeptide containing an unmodified Fc region; or b. Serum or plasma to brain concentration ratio of the same polypeptide without the Fc region or peptide linker is up to 1 / 20 17. The polypeptide for use as a pharmaceutical according to item 16, wherein the polypeptide has a molecular weight less than a predetermined threshold selected from the group consisting of:

[0408] 19. The effector polypeptide is hIL-12, hIL-10, hIL-2, hIL-7, IFNα, IFNβ, IFNγ, hIL-15, TNFα, CTLA-4, TGFβ, TGFβRII, GDNF, h IL-35, CD95, hIL-1RA, hIL-4, hIL-13, SIRPα, G-CSF, GM-CSF, OX40L, CD80, CD86, GITRL, 4-1BBL, EphrinA1, EphrinB2, E 19. The polypeptide for use as a medicament according to any one of items 15 to 18, wherein the effector polypeptide is selected from phrinB5, BDNF, C9orf72, NRTN, ARTN, PSPN, CNTF, TRAIL, IL-4, IL-3, IL-1, IL-5, IL-8, IL-18, IL-21, CCL5, CCL21, CCL10, CCL16, CX3CL1, and CXCL16, and specifically the effector polypeptide is hIL-12.

[0409] 20. The antibody or antibody-like molecule is selected from the group consisting of PD-L1, TNFα, histone, IFNγ, CXCL10, CTLA4, PD-1, and OX40. CD3, CD25, CD28, TREM2, IL-6, CX3CR1, CD25, Nogo-A, CD27, IL-12, IL-12Rb1, IL-23, CD47, TGFβ, EGFR, EGFRvIII, Her2, PDGFR, TGFR, FGFR, IL-4RA, TfR, LfR, IR, L DL-R, LRP-1, CD133, CD111, VEGFR, VEGF-A, Ang-2, IL-10, IL-10R, IL-13Rα2, α-synuclein, CSF1R, GITR, TIM-3, LAG-3, TIGIT, BTLA, VISTA, CD96, 4-1BB, CCL2, IL-1 19. The polypeptide for use as a pharmaceutical according to any one of Items 15 to 18, wherein the antibody or antibody-like molecule is selected from the group consisting of an antibody or antibody-like molecule that specifically binds to IL-1R, EphA2, EphA3, EphB2, EphB3, EphB4, LINGO-1, L1CAM, NCAM, SOD-1, SIGMAR-1, SIGMAR-2, TDP-43, Aβ, Tau, IFNα, IFNβ, TRPM4, ASIC1, VGCCs, CB1, TTR, HTT, JCV, or C9orf72, specifically the antibody or antibody-like molecule is an antibody that specifically binds to PD-L1, OX40, CD47, or Nogo-A.

[0410] 21. An antibody or antibody-like molecule that specifically binds to OX40 in an agonistic manner, comprising the crystallizable fragment (Fc) region of IgG, for use in the prevention or treatment of a disease affecting the central nervous system, the Fc region comprises a modification that results in reduced affinity for the neonatal Fc receptor (FcRn); The antibody or antibody-like molecule is an antibody or antibody-like molecule that is administered to the brain.

[0411] 22. The serum or plasma to brain concentration ratio of the antibody or antibody-like molecule is FcRn tg It can be measured 24 hours after intracranial bolus injection or CED into the striatum of mice. a. serum or plasma to brain concentration ratio of the same polypeptide containing an unmodified Fc region is at most 2 / 3; b. Serum or plasma to brain concentration ratios up to 1 / 8 of the same polypeptide without the Fc region or peptide linker 22. The antibody or antibody-like molecule for use in the prevention or treatment of a disease affecting the central nervous system according to item 21, wherein the antibody or antibody-like molecule has a titer below a predetermined threshold selected from the group consisting of:

[0412] 23. The reduction in affinity of the antibody or antibody-like molecule for FcRn is a. The dissociation constant (K) characterizing the binding of FcRn to the same antibody or antibody-like molecule containing the unmodified Fc region D ) at least two-fold, specifically at least three-fold, more specifically at least four-fold, and even more specifically at least five-fold increased K D , and b. K characterizing the binding of FcRn to the same antibody or antibody-like molecule containing a differently modified Fc region, i.e., one mutant selected from IAQ (with mutations H310A and H435Q) and AAA (with mutations I253A, H310A, and H435A). D at least 1.5-fold, specifically at least 2-fold, increased K D K selected from D 23. The antibody or antibody-like molecule for use in the prevention or treatment of diseases affecting the central nervous system according to any one of items 21 to 22, characterized in that:

[0413] 24. The intracranial delivery is a. Single, intermittent, or continuous local infusion, including convection-enhanced delivery (CED); b. in situ production of said polypeptide; c Intrathecal or intracerebroventricular administration; d. Release from implantable sustained release formulations; e.Molecular transport to the CNS, f cellular transport to the CNS, or g. Transport to the CNS after intranasal application 24. The antibody or antibody-like molecule for use in the treatment or prevention of a disease affecting the central nervous system according to any one of items 21 to 23, wherein the treatment or prevention is carried out by a method selected from the group consisting of:

[0414] 25. The antibody or antibody-like molecule for use in the treatment or prevention of a disease affecting the central nervous system according to any one of items 21 to 24, wherein the disease affecting the central nervous system is a malignant disease, particularly a glioma, more particularly a high-grade glioma (HGG).

[0415] 26. The antibody or antibody-like molecule for use in the treatment or prevention of a disease affecting the central nervous system according to any one of items 21 to 25, wherein the Fc region is a human Fc region or a chimeric Fc region comprising a human amino acid sequence and has a mutation at position 253 [Kabat numbering system], specifically I253A or I253N, more specifically I253N.

[0416] 27. The antibody or antibody-like molecule for use in the prevention or treatment of a disease affecting the central nervous system according to item 26, wherein the Fc region does not have a mutation at position 310.

[0417] 28. The Fc region is -mutations H310A and H435Q (IAQ); - mutations I253A and H435Q, as well as H at position 310 (AHQ); - mutations I253N and H435Q, as well as H at position 310 (NHQ); - mutations I253A, H310A and H435Q (AAQ); - mutations I253N, H310A and H435Q (NAQ); - mutation I253A, and H at positions 310 and 435 (AHH); - mutation I253N, and H at positions 310 and 435 (NHH); - mutations I253A and H310A, and H at position 435 (AAH); - mutations I253N and H310A, as well as H at position 435 (NAH); - mutations I253N, H310A and H435A (NAA); - mutations I253N, H310A and H435E (NAE); - mutations I253A, H310A and H435A (AAA); or -Mutations I253A, H310A and H435E (AAE) 28. The antibody or antibody-like molecule for use in the prevention or treatment of a disease affecting the central nervous system according to any one of items 21 to 27, comprising:

[0418] 29. The Fc region is - mutations I253N and H435Q, as well as H at position 310 (NHQ); - mutations I253A, H310A and H435Q (AAQ); - mutations I253N, H310A and H435Q (NAQ); - mutations I253N, H310A and H435E (NAE); or -Mutations I253A, H310A and H435E (AAE) 29. The antibody or antibody-like molecule for use in the prevention or treatment of a disease affecting the central nervous system according to any one of items 21 to 28, comprising:

[0419] 30. The antibody or antibody-like molecule for use in the prevention or treatment of a disease affecting the central nervous system according to any one of items 21 to 29, wherein the Fc region is or comprises a sequence characterized by SEQ ID NO: 002 (IAQ), SEQ ID NO: 003 (AHQ), SEQ ID NO: 004 (NHQ), SEQ ID NO: 005 (AQ), SEQ ID NO: 006 (NAQ), SEQ ID NO: 007 (AHH), SEQ ID NO: 008 (NHH), SEQ ID NO: 009 (AAH), SEQ ID NO: 010 (NAH), SEQ ID NO: 011 (NAA), SEQ ID NO: 012 (NAE), SEQ ID NO: 013 (AAA), or SEQ ID NO: 014 (AAE).

[0420] 31. The Fc region is or comprises a sequence characterized by SEQ ID NO: 004 (NHQ), SEQ ID NO: 005 (AAQ), SEQ ID NO: 006 (NAQ), SEQ ID NO: 012 (NAE) or SEQ ID NO: 014 (AAE); 31. An antibody or antibody-like molecule for use in the prevention or treatment of a disease affecting the central nervous system according to any one of items 21 to 30.

Claims

1. A fusion polypeptide comprising IL-12 and a crystallizable fragment (Fc) region of IgG for use in the prevention or treatment of a disease affecting the central nervous system (CNS), comprising: the Fc region comprises a modification that results in reduced affinity for the neonatal Fc receptor (FcRn); the polypeptide is administered to the brain; Wherein, according to the EU numbering system: - the Fc region comprises an I at position 253 and the modifications are the mutations H310A and H435Q, - the modifications are the mutations I253A, H310A, and H435A, - said Fc region comprises H at position 310 and said modifications are the mutations I253N and H435Q, - said Fc region comprises H at position 310 and said modifications are the mutations I253A and H435Q, - the modifications are the mutations I253A, H310A, and H435Q, - said Fc region comprises H at position 310 and H at position 435, and said modification is the mutation I253A, - said Fc region comprises H at position 310 and H at position 435, and said modification is the mutation I253N, - the Fc region comprises H at position 435 and the modifications are the mutations I253A and H310A, or - the modifications are the mutations I253A, H310A, and H435E, Fusion polypeptides.

2. The serum or plasma to brain concentration ratio of the polypeptide is ｔｇ It can be measured 24 hours after intracranial injection into the striatum of mice. a. at most 2 / 3 of the serum or plasma to brain concentration ratio of the same polypeptide comprising an unmodified Fc region; or b. A serum or plasma to brain concentration ratio that is up to 1 / 8 of the same polypeptide without an Fc region or a peptide linker 2. The polypeptide for use in the prevention or treatment of a disease affecting the central nervous system according to claim 1, wherein the level of the polypeptide is less than a predetermined threshold selected from the group consisting of:

3. The reduced affinity of the polypeptide for FcRn is a. The dissociation constant (K) that characterizes the binding of FcRn to the same polypeptide containing an unmodified Fc region Ｄ ) at least two-fold increased K Ｄ , and b. K that characterizes the binding of FcRn to the same polypeptide containing differently modified Fc regions, i.e., one mutant selected from IAQ (having the mutations H310A and H435Q according to the EU numbering system) and AAA (having the mutations I253A, H310A, and H435A according to the EU numbering system). Ｄ At least 1.5-fold increased K Ｄ K selected from Ｄ 3. A polypeptide for use in the prevention or treatment of diseases affecting the central nervous system according to claim 1 or 2, characterized in that:

4. The intracranial injection a. Single, intermittent or continuous local infusion, including convection-enhanced delivery (CED); b. Intrathecal or intracerebroventricular administration; c. in situ production of said polypeptide; d. Release from implantable sustained release formulations; e. molecular transport into the CNS; f. Cellular delivery to the CNS; or g. Transport to the CNS after intranasal application 3. The polypeptide for use in the treatment or prevention of a disease affecting the central nervous system according to claim 2, wherein the treatment or prevention is carried out by a method selected from the group consisting of:

5. The polypeptide for use in the treatment or prevention of a disease affecting the central nervous system according to any one of claims 1 to 4, wherein the disease affecting the central nervous system is a malignant disease.

6. The polypeptide for use in the prevention or treatment of a disease affecting the central nervous system according to any one of claims 1 to 5, wherein the Fc region is a human Fc region or a chimeric Fc region comprising a human amino acid sequence and has a mutation at position 253.

7. The polypeptide for use in the prevention or treatment of a disease affecting the central nervous system according to any one of claims 1 to 6, wherein the Fc region is or comprises a sequence characterized by SEQ ID NO:2 (IAQ), SEQ ID NO:3 (AHQ), SEQ ID NO:4 (NHQ), SEQ ID NO:5 (AAQ), SEQ ID NO:6 (NAQ), SEQ ID NO:7 (AHH), SEQ ID NO:8 (NHH), SEQ ID NO:9 (AAH), SEQ ID NO:10 (NAH), SEQ ID NO:11 (NAA), SEQ ID NO:12 (NAE), SEQ ID NO:13 (AAA) or SEQ ID NO:14 (AAE).

8. The polypeptide for use in the prevention or treatment of a disease affecting the central nervous system according to any one of claims 1 to 7, wherein the Fc region is or comprises a sequence characterized by SEQ ID NO: 4 (NHQ), SEQ ID NO: 5 (AAQ), SEQ ID NO: 6 (NAQ), SEQ ID NO: 12 (NAE) or SEQ ID NO: 14 (AAE).

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