Compartments, particularly Fc-modified biological agents for local delivery to the CNS

Abstract

The present invention relates to a polypeptide comprising a 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 a compartment, particularly the central nervous system. The Fc region contains mutations I253N and H435Q, which result in reduced affinity for the neonatal Fc receptor (FcRn), and an H at position 310, which results in increased brain-to-serum concentrations of the polypeptide.

Description

Technical Field

[0001] The present invention relates to a locally delivered biological pharmaceutical characterized by an Fc polypeptide having a low affinity for the neonatal Fc receptor (FcRn), specifically for use in neurological diseases.

Background Art

[0002] Immunotherapy is one of the most promising directions in the treatment of brain tumors. Interleukin (IL)-12 is an inflammatory cytokine and has a strong antitumor effect on brain tumors in preclinical models. Based on the results of promising preclinical trials, clinical trials were rapidly initiated in the late 1990s as a systemic treatment with intravenous (i.v.) application of IL-12. However, serious adverse events were reported in phase II clinical trials, and 12 out of 17 patients were hospitalized and 2 died. These adverse effects were subsequently attributed to the rapid induction of high systemic levels of interferon (IFN)-γ, an effector cytokine downstream of IL-12.

[0003] Considering the toxicity of systemically applied IL-12 and the need for high concentrations at the tumor site, tightly controlling the IL-12 level in tissues is an essential prerequisite for clinical application. Local administration to the brain has recently become possible by using new neurosurgical techniques such as convection-enhanced delivery (CED). However, local intracranial delivery does not exclude subsequent systemic leakage.

[0004] Mouse IL-12Fc, a single-chain fusion protein of IL-12, and the crystallizable fragment (Fc) of immunoglobulin G (IgG) show increased pharmacological stability, increased bioavailability, and decreased passive leakage from the brain compared to unmodified recombinant IL-12. However, after local delivery to the brain, it is actively effluxed 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 splenic red pulp macrophages, preventing degradation and extending the serum half-life of Fc-containing molecules and serum albumin. Thus, IL-12Fc shows increased systemic accumulation compared to unmodified IL-12.

[0005] IgG Fc residues known to be involved in FcRn binding (isoleucine 253 - Ile253, histidine 310 - His310, and histidine 435 - His435), 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).

[0006] For example, Bitonti et al. reported that mutating the residues Ile253, His310, and His435 in the Fc domain of wild-type IgG to Ala253, Ala310, and Ala310, respectively, results in abrogation of FcRn binding at pH 6 (Bitonti et al., Proceedings of the National Academy of Sciences (2004) 101(26):9763 - 9768).

[0007] However, substitution of an amino acid to alanine is a common biochemical method for screening the functional role at a given position within a target protein. Apart from this one specific mutation (AAA), this paper does not disclose any other mutations from which conclusions can be drawn regarding the resulting binding properties to FcRn. Furthermore, this paper addresses the FcRn-mediated transport of an Fc fusion protein containing erythropoietin (Epo), a glycoprotein hormone agent that stimulates erythropoiesis, in the lungs of non-human primates. This paper does not mention anything regarding the applicability of the results to fusion polypeptides containing IL-12 and the administration of Fc fusion polypeptides to the brain, respectively.

[0008] There are publications that actually address fusion polypeptides containing IL-12 and methods for increasing their serum half-lives.

[0009] For example, Jung et al. describe the production and antitumor activity of a fusion polypeptide containing IL-12 and a human IgG4-based heterodimeric Fc with an A107 mutation pair that reduces the affinity to the Fcγ receptor (Jung et al., Oncoimmunology, Vol. 7(7):e1438800).

[0010] However, it is clear that FcRn is not equivalent to FcγR because the Fc gamma receptor (FcγR) family is a functional group of proteins characterized by the constant region of the antibody, i.e., differences in structure but binding to the Fc portion, non-overlapping binding sites in the Fc portion, localization in different compartments of the cell (intracellular vs. extracellular), pH-dependent binding (acidic vs. neutral), and overall function.

[0011] In another example of the state of the art, a comparison is made between recombinant IL-12 and IL-12Fc with respect to tissue retention and leakage into the systemic circulation (Beffinger et al., Neuro-Oncology (2017), 19(Supplement 6), vi273). There, the authors state that IL-12Fc showed higher intracerebral concentrations 24 hours after intracranial application compared to recombinant IL-12.

[0012] However, this study does not disclose a fusion polypeptide having a mutation in the Fc region of IgG or the effect on the binding to FcRn.

[0013] Cooper et al. studied the role of FcRn in IgG efflux from rat brain by local delivery of two variants of recombinant human IgG1 mAb having either an increase in FcRn binding (from asparagine 434 to alanine in IgG1, N434A) or a decrease in FcRn binding (from histidine 435 to alanine in IgG1, 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 the 434 and 435 amino acid positions, respectively. This study was conducted in rats using human antibodies.

[0014] Regarding the binding characteristics of Fc mutants to mouse and human forms of FcRn, Andersen et al. disclosed five different Fc mutants having mutations at the levels of Ile253, His310, and His435, namely, 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 the mutant having both H310A and H435Q mutations (IAQ).

[0015] Even though the last two studies mentioned herein indicate that FcRn plays an important role in IgG efflux from rat brain and disclose distinct mutants with reduced affinity for FcRn respectively, none of these studies serves as a basis for evaluating how the presence of IL - 12Fc affects the binding to FcRn. Furthermore, the concept of generating a maximum brain - to - blood concentration gradient is not disclosed.

[0016] Based on the above state of the art, the object of the present invention is to expand the therapeutic window of a specific compartment, specifically a pharmaceutical delivered locally to the brain, and to prevent both efflux from and systemic accumulation in said compartment, specifically the brain, thereby increasing the compartment-to-serum ratio, specifically the brain-to-serum ratio. This object is achieved by the claims of the present specification.

[0017] In the context of the present specification, the term crystallizable fragment (Fc) region refers to the fraction of an IgG antibody comprising two identical heavy chain fragments covalently linked by disulfide bonds or to a single heavy chain fragment. The heavy chain fragments are composed of the constant domains (C H 2 and C H 3 domains).

[0018] In the context of the present specification, 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 for numbering the amino acid residues of the Fc region. The EU numbering scheme is a standard widely adopted for numbering residues in antibodies in a consistent manner. The amino acid sequence is given from the amino terminus 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). Lower case letters for positions in the amino acid sequence refer to the corresponding D- or (2R)-amino acids.

[0019] The amino acid residues I253, H310 and H435 are C H 2-C HIt is located at the 3-domain interface and is conserved across IgG subclasses within a species and between IgG molecules found in both rodents and humans, except for R435 in human IgG3 (Miyakawa et al., RNA (2008) 14:1154-1163). According to the present invention, the modified Fc region or fragments thereof can be derived from IgG1, IgG2 or IgG4 immunoglobulins and need to contain at least amino acid residues 253, 310 and 435 of the Fc domain of immunoglobulin G (IgG) according to the EU numbering system. In the context of this specification, IL-12 refers to interleukin 12.

[0020] In the context of this specification, hIL-12 relates to human IL-12.

[0021] In the context of this specification, mIL-12 relates to mouse IL-12.

[0022] In the context of this specification, rmIL-12 relates to recombinant mouse IL-12.

[0023] In the context of this specification, rhIL-12 relates to recombinant human IL-12. In the context of this specification, IL-12Fc WT relates to IL-12 linked to the wild-type unmodified Fc region, specifically by the fusion of p40 and p35 with a Gly-Ser linker or by the addition of an IgG4 tag.

[0024] In the context of this specification, mIL-12hFc WT relates to mouse IL-12 linked to the human wild-type Fc region of IgG4 containing the serine 228 to proline (S228P) mutation and the NHQ mutation.

[0025] In the context of this specification, mIL-12hFc NHQ relates to mouse IL-12 linked to the human wild-type Fc region of IgG4 containing proline from serine 228, similar to the NHQ mutation.

[0026] In the context of this specification, the mIL-12hFc:anti-PD-L1 bifunctional molecule relates to mouse IL-12 dimerized with a half-molecule (one heavy chain and one light chain) of a fully human or humanized PD-L1-binding IgG1 antibody linked to human IgG1 Fc. The Fc portion of the resulting molecule contains the NHQ mutation.

[0027] In the context of this specification, FcRn tg refers to a mouse strain that lacks functional mouse FcRn and carries a transgene for the expression of the human FcRnα chain under the control of a natural human regulatory element described by the allele symbol Tg(FCGRT)32Dcr.

[0028] In the context of the present invention, the 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, the IL-12 polypeptide has an amino acid sequence that includes the p35 and p40 sequences or homologs thereof as part of the same continuous amino acid chain. In the above continuous amino acid chain, only the N-terminal polypeptide (p40) functional homolog retains the signal peptide. In another embodiment, the IL-12 polypeptide includes two different amino acid chains, one containing the p35 sequence and the other containing the p40 sequence, both having individual signal peptides. The 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 above IL-12 polypeptide, most specifically the stimulation of T effector cells acting via perforin.

[0029] In the context of this specification, the terms "sequence identity" and "percentage of sequence identity" refer to a value determined by comparing two aligned sequences. Methods for aligning sequences for comparison are well known in the art. Alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., Vol. 2: 482 (1981), the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol., Vol. 48: 443 (1970), the similarity search method of Pearson and Lipman, Proc. Nat. Acad. Sci., Vol. 85: 2444 (1988), or by computerized implementations of these algorithms, including but not limited to CLUSTAL, GAP, BESTFIT, BLAST, FASTA, and TFASTA. Software for performing BLAST analysis is publicly available, for example, through the National Center for Biotechnology-Information (http: / / blast.ncbi.nlm.nih.gov / ).

[0030] An example of amino acid sequence comparison is the BLASTP algorithm with default settings: expectation threshold: 10; word size: 3; maximum matches in query range: 0; matrix: BLOSUM62; gap costs: existence: 11, extension: 1; composition adjustment: use conditional composition score matrix adjustment. One such example for nucleic acid sequence comparison is the BLASTN algorithm with default settings: expectation threshold: 10; word size: 28; maximum matches in query range: 0; match / mismatch score: 1.-2; gap costs: use linear.

[0031] Unless otherwise specified, the sequence identity values provided in this specification refer to values obtained using the program's BLAST suite (Altschul et al., J. Mol. Biol., Vol. 215: 403-410 (1990)), using the default parameters identified above for protein and nucleic acid comparison, respectively.

[0032] In the context of this specification, IL-10 refers to interleukin 10. In certain embodiments, IL-10 is employed in the treatment of inflammation, autoimmune inflammation, dementia or stroke. In certain embodiments, neutralizing IL-10 is employed in the treatment of pulmonary paracoccidioidomycosis.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] In the context of this specification, TNFα refers to tumor necrosis factor alpha and is 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.

[0039] In the context of this specification, CTLA-4 refers to cytotoxic T lymphocyte-associated protein 4 and is 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.

[0040] In the context of this specification, TGFβ refers to transforming growth factor beta. In certain embodiments, blocking TGFβ is employed in the treatment of cancer and infectious diseases. In certain embodiments, TGFβ is employed in the treatment of inflammation, autoimmune inflammation, dementia, and stroke. In certain embodiments, TGFβ antagonists are employed in the treatment of cystic fibrosis.

[0041] 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.

[0042] 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.

[0043] In the context of this specification, 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.

[0044] 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.

[0045] In the context of this specification, CD95 refers to Fas, which is also known as FasR, apoptosis antigen 1, APO-1, APT, or TNF receptor superfamily member 6. In certain embodiments, blocking CD95 is employed in the treatment of cancer.

[0046] In the context of this specification, IL-1RA refers to interleukin 1 receptor antagonist. In certain embodiments, IL-1RA is employed in the treatment of inflammation, autoimmune inflammation, rheumatoid arthritis, gout, pseudogout, dementia, and stroke. In certain embodiments, blocking IL-1RA is employed in the treatment of rheumatoid arthritis.

[0047] 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.

[0048] In the context of this specification, IL-13 refers to interleukin 13. In certain embodiments, IL-13 is employed in the treatment of inflammation, autoimmune inflammation, dementia, and stroke. In certain embodiments, neutralizing anti-IL-13 is employed in the treatment of severely uncontrolled asthma. In certain embodiments, blocking and / or neutralizing IL-13 is employed in the treatment of chronic rhinosinusitis with nasal polyps. In certain embodiments, an IL-13 antagonist is employed in the treatment of idiopathic pulmonary fibrosis.

[0049] In the context of this specification, TSLP refers to thymic stromal lymphopoietin, a protein belonging to the cytokine family. In certain embodiments, neutralizing TSLP is employed in the treatment of allergic asthma. In certain embodiments, blocking and / or neutralizing TSLP is employed in the treatment of chronic rhinosinusitis with nasal polyps.

[0050] In the context of this specification, SIRPα refers to signal regulatory protein alpha. In certain embodiments, SIRPα is employed in the treatment of cancer.

[0051] In the context of this specification, 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.

[0052] In the context of this specification, 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 employed in the treatment of cancer. In certain embodiments, blocking GM-CSF is employed in the treatment of multiple sclerosis.

[0053] 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), and refers to the receptor for granulocyte-macrophage colony-stimulating that stimulates the production of white blood cells. In certain embodiments, blocking GM-CSFR is used in the treatment of rheumatoid arthritis.

[0054] 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.

[0055] In the context of this specification, CD80 refers to B7-1 and is also known as B7.1. In certain embodiments, CD80 is employed in the treatment of cancer.

[0056] In the context of this specification, CD86 refers to B7-2 and is also known as B7.2. In certain embodiments, CD86 is used in the treatment of cancer.

[0057] 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.

[0058] In the context of this specification, 4-1BBL refers to the ligand of 4-1BB and is 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.

[0059] In the context of this specification, EphrinA1 refers to EFNA1. In certain embodiments, EphrinA1 is employed in the treatment of cancer.

[0060] In the context of this specification, EphrinB2 refers to EFNB2. In certain embodiments, EphrinB2 is employed in the treatment of cancer.

[0061] In the context of this specification, EphrinB5 refers to EFNB5. In certain embodiments, EphrinB5 is employed in the treatment of cancer.

[0062] In the context of this specification, PD-L1 refers to programmed cell death ligand 1 and is 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, blockade of PD-L1 is employed in the treatment of uveal melanoma. In certain embodiments, blockade of PD-1 is employed in the treatment of lung cancer.

[0063] 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 to histone is employed in the treatment of cancer.

[0064] In the context of this specification, CXCL10 refers to C-X-C motif chemokine 10 and is also known as interferon gamma-induced protein 10 (IP-10) or small inducible cytokine B10. In certain embodiments, CXCL10 is employed in the treatment of cancer.

[0065] In the context of this specification, PD-1 refers to programmed cell death protein 1 and is also known as CD279. In certain embodiments, binding to PD-1 is employed in the treatment of cancer. In certain other embodiments, binding to PD-1 is employed in the treatment of dementia. In certain embodiments, blocking of PD-1 is employed in the treatment of uveal melanoma. In certain embodiments, blocking of PD-1 is employed in the treatment of lung cancer.

[0066] In the context of this specification, TREM2 refers to a triggering receptor expressed on myeloid cells 2. In certain embodiments, blocking TREM2 is employed in the treatment of inflammation, autoimmune inflammation, dementia, and stroke.

[0067] 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.

[0068] In the context of this specification, IL-6R refers to the interleukin-6 receptor. In certain embodiments, blocking IL-6R is employed in the treatment of inflammation, autoimmune inflammation, rheumatoid arthritis, juvenile idiopathic arthritis, and adult-onset Still's disease. In certain embodiments, blocking and / or neutralizing IL-6R is employed in the treatment of diseases caused by coronavirus disease 2019 (COVID-19) and / or severe acute respiratory syndrome coronavirus (SARS-CoV). 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.

[0069] In certain embodiments, blocking CD27 is employed in the treatment of inflammation or autoimmune inflammation.

[0070] In certain embodiments, activating CD27 is employed in the treatment of cancer.

[0071] In certain embodiments, blocking CD25 is employed in the treatment of inflammation, autoimmune inflammation, and multiple sclerosis.

[0072] In certain embodiments, binding CD25 is employed in the treatment of cancer.

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

[0074] In the context of this specification, Nogo-A refers to an axon elongation 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 context of this specification, IL-12Rb1 refers to the interleukin-12 receptor beta 1 subunit. In certain embodiments, blocking IL-12Rb1 is employed in the treatment of inflammation, autoimmune inflammation, dementia, and stroke.

[0076] In the context of this specification, CD47 refers to the 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) and is 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).

[0078] In the context of this specification, EGFR refers to the epidermal growth factor receptor and is also known as ErbB-1. In certain embodiments, blocking EGFR is employed in the treatment of cancer.

[0079] In the context of this specification, EGFRvIII refers to the vIII mutant of the epidermal growth factor receptor and is also known as the vIII mutant of ErbB-1. In certain embodiments, blocking EGFRvIII is used in the treatment of cancer.

[0080] In the context of this specification, Her2 refers to the receptor tyrosine-protein kinase erbB-2 and is also known as CD340 or the proto-oncogene Neu. In certain embodiments, blocking Her2 is employed in the treatment of cancer.

[0081] In the context of this specification, PDGFR refers to platelet-derived growth factor receptor (PDGF-R). In certain embodiments, blocking PDGF-R is used for the treatment of cancer.

[0082] In the context of this specification, FGFR refers to fibroblast growth factor receptor. In certain embodiments, blocking FGFR is employed for the treatment of cancer.

[0083] In the context of this specification, IL-4RA refers to interleukin 4 receptor and is also known as IL-4R or CD124. In certain embodiments, blocking IL-4RA is employed for the treatment of cancer. In certain embodiments, blocking IL-4R is employed for the treatment of asthma.

[0084] In the context of this specification, TfR refers to transferrin receptor. In certain embodiments, binding to TfR is employed for the treatment of inflammation, autoimmune inflammation, dementia, traumatic CNS injury, cancer, and stroke.

[0085] In the context of this specification, LfR refers to lactoferrin receptor and is also known as omentin or intestinal lactoferrin receptor. In certain embodiments, binding to LfR is employed for the treatment of inflammation, autoimmune inflammation, dementia, traumatic CNS injury, cancer, and stroke.

[0086] In the context of this specification, IR refers to insulin receptor. In certain embodiments, binding to IR is employed for the treatment of inflammation, autoimmune inflammation, dementia, traumatic CNS injury, cancer, and stroke.

[0087] In the context of this specification, LDL-R refers to low-density lipoprotein receptor. In certain embodiments, binding to LDL-R is employed for the treatment of inflammation, autoimmune inflammation, dementia, traumatic CNS injury, cancer, and stroke.

[0088] In the context of this specification, LRP-1 refers to low density lipoprotein receptor-related protein 1 (LRP1), and is 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.

[0089] In the context of this specification, CD133 refers to prominin-1. In certain embodiments, binding CD133 is employed in the treatment of cancer.

[0090] In the context of this specification, CD111 refers to poliovirus receptor-related 1 (PVRL1), and is also known as nectin-1. In certain embodiments, binding CD111 is employed in the treatment of cancer.

[0091] In the context of this specification, VEGFR refers to the 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.

[0092] 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.

[0093] 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.

[0094] In the context of this specification, IL-10R refers to the interleukin 10 receptor, and is also known as the receptor for cytokine synthesis inhibitory factor. In certain embodiments, blocking IL-10R is employed in the treatment of cancer.

[0095] In the context of this specification, IL-13Rα2 refers to interleukin-13 receptor subunit alpha-2 and is also known as CD213A2. In certain embodiments, binding to IL-13Rα2 is employed in the treatment of cancer. In certain embodiments, IL-13Rα2 is employed in the treatment of cancer.

[0096] In certain embodiments, binding to α-synuclein is employed in the treatment of Parkinson's disease.

[0097] In the context of this specification, CSF1R refers to colony stimulating factor 1 receptor (CSF1R) and is also known as macrophage colony stimulating factor receptor (M-CSFR) and CD115. In certain embodiments, blocking CSF1R is employed in the treatment of cancer.

[0098] In the context of this specification, GITR refers to glucocorticoid-induced TNFR-related protein and is also known as TNF receptor superfamily member 18 (TNFRSF18) or activation-induced TNFR family receptor or AITR. In certain embodiments, binding to GITR is employed in the treatment of cancer.

[0099] 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.

[0100] In the context of this specification, TIM-3 refers to T cell immunoglobulin and mucin domain-containing 3 and is also known as hepatitis A virus cellular receptor 2 (HAVCR2). In certain embodiments, blocking TIM-3 is employed in the treatment of cancer.

[0101] 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.

[0102] In the context of this specification, TIGIT refers to a T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif domains. In certain embodiments, blocking TIGIT is employed in the treatment of cancer.

[0103] 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.

[0104] In the context of this specification, VISTA refers to the V domain Ig suppressor of T cell activation. In certain embodiments, blocking VISTA is employed in the treatment of cancer.

[0105] In the context of this specification, CD96 refers to T cell activation and increased late expression, also known as TACTILE. In certain embodiments, blocking CD96 is employed in the treatment of cancer.

[0106] In the context of this specification, 4-1BB refers to CD137, also known as TNFR superfamily member 9, or is induced by lymphocyte activation or ILA. In certain embodiments, binding of 4-1BB is employed in the treatment of cancer.

[0107] In the context of this specification, 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.

[0108] In the context of this specification, IL-1 refers to a member of the IL-1 cytokine family. In certain embodiments, the blockade of IL-1 is employed in the treatment of multiple sclerosis.

[0109] In the context of this specification, IL-1R refers to a receptor for cytokines of the IL-1 cytokine family. In certain embodiments, the blockade of IL-1R is employed in the treatment of multiple sclerosis.

[0110] 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.

[0111] 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.

[0112] In the context of this specification, EphB2 refers to ephrin type-B receptor 2 and is also known as ERK. In certain embodiments, blocking EphB2 is employed in the treatment of cancer.

[0113] 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.

[0114] 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.

[0115] In the context of this specification, OX40 refers to TNF receptor superfamily member 4 and is also known as CD134 or the OX40 receptor. In certain embodiments, binding OX40 is employed in the treatment of cancer.

[0116] In the context of this specification, LINGO-1 refers to leucine-rich repeat and immunoglobulin-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.

[0117] In the context of this specification, L1CAM refers to L1 cell adhesion molecule and is also known as L1. In certain embodiments, blocking L1 is employed in the treatment of multiple sclerosis, traumatic brain CNS injury or stroke.

[0118] In the context of this specification, NCAM refers to neural cell adhesion molecule. In certain embodiments, blocking NCAM is employed in the treatment of multiple sclerosis, traumatic brain CNS injury or stroke.

[0119] 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).

[0120] In the context of this specification, SIGMAR-1 refers to sigma-1 receptor. In certain embodiments, blocking SIGMAR-1 is employed in the treatment of amyotrophic lateral sclerosis (ALS).

[0121] In the context of this specification, SIGMAR-2 refers to sigma-2 receptor. In certain embodiments, blocking SIGMAR-2 is employed in the treatment of amyotrophic lateral sclerosis (ALS).

[0122] In the context of this specification, TDP-43 refers to TAR DNA-binding protein 43. In certain embodiments, binding to TDP-43 is employed in the treatment of amyotrophic lateral sclerosis (ALS).

[0123] In the context of this specification, amyloid β refers to amyloid beta. In certain embodiments, binding to amyloid β is employed in the treatment of Alzheimer's disease (AD).

[0124] In the context of this specification, Tau refers to the tau protein. In certain embodiments, binding to Tau is employed in the treatment of Alzheimer's disease (AD).

[0125] 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.

[0126] 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.

[0127] 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.

[0128] In the context of this specification, 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.

[0129] In the context of this specification, VGCC refers to voltage-gated calcium channels, also known as voltage-dependent calcium channels (VDCC). In certain embodiments, blocking VGCC is employed in the treatment of multiple sclerosis.

[0130] 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.

[0131] In the context of this specification, TTR refers to transthyretin. In certain embodiments, blocking TTR is employed in the treatment of transthyretin amyloidosis.

[0132] 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.

[0133] In the context of this specification, JCV refers to the JC virus or John Cunningham virus. In certain embodiments, blocking the major capsid protein VP1 (viral protein 1) of JCV is employed in the treatment of progressive multifocal leukoencephalopathy (PML).

[0134] In the context of this specification, C9orf72 refers to the protein encoded by the open reading frame 72 gene on chromosome 9. In certain embodiments, C9orf72 is employed in the treatment of dementia. In certain embodiments, blocking C9orf72 is employed in the treatment of dementia.

[0135] In the context of this specification, 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.

[0136] 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.

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

[0138] 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.

[0139] In this specification, 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.

[0140] In the context of this specification, TRAIL refers to TNF-related apoptosis-inducing ligand and is also known as CD253 or tumor necrosis factor superfamily, member 10. In certain embodiments, TRAIL is employed in the treatment of cancer.

[0141] 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.

[0142] In the context of this specification, IL-3 refers to interleukin 3. In certain embodiments, IL-3 is employed in the treatment of cancer.

[0143] 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, blocking IL-5 is employed in the treatment of asthma. In certain embodiments, blocking IL-5 is employed in the treatment of chronic obstructive pulmonary disease (COPD).

[0144] In the context of this specification, IL-8 refers to interleukin 8 and is 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, the blockade of IL-8 is employed in the treatment of pulmonary edema. In certain embodiments, an IL-8 antagonist is employed in the treatment of cystic fibrosis.

[0145] In the context of this specification, IL-17 refers to interleukin 17. In certain embodiments, the neutralization of IL-17 is employed in the treatment of uveitis.

[0146] In the context of this specification, IL-17A refers to interleukin 17A. In certain embodiments, the neutralization of IL-17A is employed in the treatment of rheumatoid arthritis and / or psoriatic arthritis and / or ankylosing spondylitis.

[0147] In the context of this specification, IL-18 refers to interleukin 18 and is also known as interferon-gamma inducing factor. In certain embodiments, IL-18 is employed in the treatment of cancer.

[0148] In the context of this specification, IL-21 refers to interleukin 21. In certain embodiments, IL-21 is employed in the treatment of cancer.

[0149] In the context of this specification, IL-21R refers to the interleukin 21 receptor. In certain embodiments, the blockade of IL-21R is employed in the treatment of allergic asthma.

[0150] 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.

[0151] 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.

[0152] In the context of this specification, CD20 refers to 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.

[0153] In the context of this specification, CCL5 refers to chemokine (C-C motif) ligand 5. In certain embodiments, CCL5 is employed in the treatment of cancer.

[0154] In the context of this specification, CCL21 refers to chemokine (C-C motif) ligand 21. In certain embodiments, CCL21 is employed in the treatment of cancer.

[0155] In the context of this specification, CCL10 refers to chemokine (C-C motif) ligand 10, and is also known as CCL9 or chemokine (C-C motif) ligand 9. In certain embodiments, CCL10 is employed in the treatment of cancer.

[0156] In the context of this specification, CCL16 refers to chemokine (C-C motif) ligand 16. In certain embodiments, CCL16 is employed in the treatment of cancer.

[0157] In the context of this specification, CX3CL1 refers to chemokine (C-X3-C motif) ligand 1, and is also known as fractalkine. In certain embodiments, CX3CL1 is employed in the treatment of cancer.

[0158] In the context of this specification, CXCL16 refers to chemokine (C-X-C motif) ligand 16. In certain embodiments, CXCL16 is employed in the treatment of cancer.

[0159] In the context of this specification, NF-kB refers to the nuclear factor kappa-light-chain enhancer of activated B cells. In certain embodiments, NF-kB antagonists are employed in the treatment of cystic fibrosis.

[0160] 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 in the treatment of inflammation, autoimmune inflammation, arthritis, and osteoarthritis. In certain embodiments, blockade of NGF may be employed in the treatment of osteoarthritis. In the context of this specification, the term antibody refers to a type G antibody (IgG), any antigen-binding fragment or single chain thereof, and related or derived constructs. An entire antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy-chain variable region (V H ) and a heavy-chain constant region (C H ). The heavy-chain constant region is composed of three domains, C H 1, C H 2, and C H 3. Each light chain is composed of a light-chain variable region (abbreviated herein as V L ) and a light-chain constant region (C L ). The light-chain constant region is composed of one domain, C L . The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an 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 the entire antibody comprising two H chains and two L chains, but also aberrant antibodies comprising only one H chain and one L chain, or even antibodies consisting of only one H chain.

[0161] In the context of this specification, the term specifically binds refers to binding with high affinity / Kd ≤ 10E -8 mol / l.

[0162] In the context of this specification, the term "antibody-like molecule" refers to a molecule containing at least a part of the Fc fragment of an IgG antibody and at least one target-binding element directly or indirectly fused to the Fc fragment, and in particular a heavy chain and light chain variable region, a single-chain variable fragment, a bispecific retargeting protein, or a bispecific T cell engager. An antibody-like molecule can specifically bind to another molecule or target with a high affinity / Kd ≤ 10E -8 mol / l. An antibody-like molecule binds to its target in the same way as the specific binding of an antibody.

[0163] One skilled in the art will recognize that the present invention does not require that an antibody or antibody-like molecule contain or be fused to an Fc region.

[0164] In the context of this specification, the term dissociation constant (K D ) refers to the equilibrium constant that measures the tendency of a complex composed of [almost two] different components to dissociate reversibly into its components. This complex can be, for example, an antibody-antigen complex AbAg composed of an antibody Ab and an antigen Ag. K D is expressed in molar concentration [mol / l] and corresponds to the concentration of [Ab] at which half of the binding sites of [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 by the following formula:

[0165] [Number]

[0166] [Ab]: Concentration of antibody; [Ag]: Concentration of antigen; [AbAg]: Concentration of antibody-antigen complex In the context of this specification, the terms off-rate (Koff; [1 / sec]) and on-rate (Kon; [1 / sec * M]) are used in the meanings known in the fields of chemistry and physics, and they refer to the rate constants that measure the dissociation (Koff) or association (Kon) of an antibody with its target antigen. K off and Kon can be determined experimentally using methods well established in the art. Methods for determining the Koff and Kon of an antibody employ surface plasmon resonance. This is the principle behind biosensor systems such as the Biacore® or ProteOn® systems. They can also calculate the dissociation constant K D using the following formula:

[0167] [Number]

[0168] 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. This can be done using a biosensor system such as the Biacore® or ProteOn® systems.

[0169] In the context of this specification, high-grade glioma (HGG) refers to gliomas of WHO grade IV or glioblastoma multiforme. In the context of this specification, the Fc region referred to as "NHQ" refers to the Fc region in which positions 253, 310, and 435 (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. Thus, the Fc region referred to as "IAQ" refers to the Fc region having I at position 253, A at position 310, and Q at position 435 (i.e., an Fc region having mutations H310A and H435Q). Table 1 shows some examples of modified Fc regions.

[0170] The present invention provides a polypeptide comprising a crystallizable fragment (Fc) region of IgG for use in the prevention or treatment of diseases affecting the central nervous system. The Fc region is modified to have a reduced affinity for the neonatal Fc receptor (FcRn). The Fc region contains the mutations I253N and H435Q, and an H at position 310. The polypeptide is administered to the brain.

[0171] In certain embodiments, the polypeptide according to the invention further comprises IL-12.

[0172] Administration to the brain can be carried out by intracranial delivery. The intracranial delivery can be continuous or intermittent or non-recurrent. The expression "administration to the brain" also means including flushing the resection cavity after surgery. The administration can be intrathecal or intracerebral.

[0173] The modification of the Fc region results in a decrease in the serum-to-intracerebral concentration ratio of the polypeptide. The decrease in the serum-to-intracerebral concentration has the advantage that a high local concentration is achieved in the brain while negative side effects due to a high systemic concentration are prevented.

[0174] In certain embodiments, the serum or plasma-to-intracerebral concentration ratio of the polypeptide is less than a predetermined threshold. The predetermined threshold is the FcRn tg measurable 24 hours after intracranial injection, specifically intracranial bolus injection or CED, into the striatum of a mouse, a. at most 2 / 3 of the serum or plasma-to-intracerebral concentration ratio of the same polypeptide containing an unmodified Fc region, specifically IL-12FcWT, b. at most 1 / 8 of the serum or plasma-to-intracerebral concentration ratio of the same polypeptide containing neither an Fc region nor a peptide linker, specifically rhIL-12 selected from.

[0175] FcRn tgMeasure 24 hours after intracranial injection of 1 μg at 1 μl / min using a 26sG Hamilton syringe with a smooth tip or CED into the striatum of the mouse (using a 27G smooth tip needle made of fused silica with a 1 mm step at the tip, inner diameter 0.1 mm, wall thickness 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; total volume 5 μl, total amount 1 μg).

[0176] The fusion polypeptide according to the first aspect of the present invention has a lower serum-to-intracerebral concentration ratio than IL-12 (IL-12Fc WT) linked to an unmodified Fc region. IL-12Fc WT has a long serum half-life due to FcRn-mediated recycling in circulating blood.

[0177] The fusion polypeptide according to the first aspect of the present invention has a lower serum-to-intracerebral concentration ratio than rhIL-12, which shows high passive leakage from the brain.

[0178] In certain embodiments, the reduced affinity of the polypeptide for FcRn is a. K that characterizes the binding of FcRn to the same polypeptide containing an unmodified Fc region D is increased by at least 2-fold compared to D , and b. Ka that is increased by at least 1.5-fold compared to K that characterizes the binding of FcRn to the same polypeptide containing a differently modified Fc region D , i.e., one mutant selected from IAQ (having mutations H310A and H45Q) and AAA (having mutations I253A, H310A, and H435A) D , and is characterized by a dissociation constant (K ) selected from D .

[0179] In certain embodiments, K D is increased by at least 3-fold compared to K D that characterizes the binding of FcRn to the same polypeptide containing an unmodified Fc region. In certain embodiments, K Dis at least 4-fold increased compared to K that characterizes the binding of FcRn to the same polypeptide containing an unmodified Fc region. In certain embodiments, K D is at least 5-fold increased compared to K that characterizes the binding of FcRn to the same polypeptide containing an unmodified Fc region. D is at least 4-fold increased compared to K that characterizes the binding of FcRn to the same polypeptide containing an unmodified Fc region. D is at least 5-fold increased compared to K that characterizes the binding of FcRn to the same polypeptide containing an unmodified Fc region.

[0180] In certain embodiments, K D is at least 2-fold increased compared to K that characterizes the binding of FcRn to the same polypeptide containing an Fc region that has been modified differently. In certain embodiments, the Fc region that has been modified differently is an Fc region having I at position 253, A at position 310, and Q (IAQ) at position 435. In certain embodiments, the Fc region that has been modified differently is an Fc region having A at position 253, A at position 310, and A (AAA) at position 435. D In certain embodiments, intracranial delivery is performed by convection enhanced delivery (CED) or a variant thereof. CED refers to a technique for directly delivering drugs into 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 normal bolus injection and diffusion-driven infusion regimens is the pressure gradient created by raising the injection until bulk flow within the tissue is reached. Here, it is the duration rather than the infusion rate that determines the extent of the tissue reached. This approach enables the delivery of macromolecular drugs that normally do not enter the brain to reach high concentrations effectively within the brain (tumor) tissue.

[0181] In certain embodiments, intracranial delivery is performed by convection enhanced delivery (CED) or a variant thereof. CED refers to a technique for directly delivering drugs into 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 normal bolus injection and diffusion-driven infusion regimens is the pressure gradient created by raising the injection until bulk flow within the tissue is reached. Here, it is the duration rather than the infusion rate that determines the extent of the tissue reached. This approach enables the delivery of macromolecular drugs that normally do not enter the brain to reach high concentrations effectively within the brain (tumor) tissue.

[0182] In certain embodiments, intracranial delivery is effected 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 under the arachnoid membrane, either into the brain (e.g., via an Ommaya reservoir) or into the subarachnoid space in the spinal cord. Non-limiting examples are intrathecal delivery for treating leptomeningeal carcinomatosis and primary Her2 / neu-positive brain tumors, as well as CD20-positive CNS lymphoma and intraocular lymphoma with trastuzumab or rituximab, respectively. Another example is intrathecal administration of anti-NogoA antibody for the treatment of acute spinal cord injury, multiple sclerosis, or stroke. This approach enables the delivery of macromolecular drugs that normally do not enter the brain and effectively reach high concentrations in the leptomeninges or brain parenchyma.

[0183] In certain embodiments, intracranial delivery is effected by the intraventricular delivery of the above polypeptide. Intraventricular administration refers to the direct administration of a drug into the cerebrospinal fluid (CSF) in the ventricular cavity using a catheter.

[0184] In certain embodiments, intracranial delivery is effected by in situ production of the above polypeptide. In situ production relates exclusively or substantially to the local production of a polypeptide within the brain or a brain tumor. As non-limiting examples, local production can result from DNA formulations, mRNA, modified mRNA, self-replicating mRNA, viral vectors, encapsulated modified producer cells, or modified T cells. Spatial control over local production can be achieved by the local delivery of a molecule or vector encoding the polypeptide or by the 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 the local or systemic administration of an agent that acts as a transcriptional repressor reliever or transcriptional activator of a conditional expression cassette. Examples include, but are not limited to, ecdysone receptor / invertebrate retinoid x receptor-based inducible gene expression systems or tetracycline-regulated transcriptional regulators.

[0185] In certain embodiments, intracranial delivery is effected by systemic delivery of cells modified to produce the polypeptide having homing ability to a tumor or the CNS. The polypeptide can be produced in a constitutive or inducible manner. Examples include, but are not limited to, modified T cells or mesenchymal stem cells.

[0186] In certain embodiments, intracranial delivery is effected by release from an implanted slow-release / extended-release / sustained-release / delivery-controlled formulation. In the context of this specification, such a formulation relates to a dosage form designed to release a drug at a predetermined rate in order to maintain a constant drug concentration for a specific period while minimizing side effects. Those skilled in the art are aware of various suitable formulations. Non-limiting examples are liposomes, drug-polymer conjugates, hydrogels, wafers or coated nanoparticles.

[0187] In certain embodiments, intracranial delivery is effected by intranasal delivery of the polypeptide.

[0188] In certain embodiments, intracranial delivery is effected by receptor-mediated transcytosis of the polypeptide. Non-limiting examples are bispecific constructs that bind to the TfR, as well as targets found in diseased brain parenchyma, specifically Aβ plaques in Alzheimer's disease (AD).

[0189] In certain embodiments, the disease affecting the central nervous system is a malignant disease.

[0190] In certain embodiments, the disease affecting the central nervous system is glioma. In certain embodiments, the disease affecting the central nervous system is high-grade glioma (HGG).

[0191] In certain embodiments, the disease affecting the central nervous system is a secondary brain tumor, also known as a brain metastasis.

[0192] In certain embodiments, the disease affecting the central nervous system is ischemic brain injury or cerebral infarction, stroke, cerebral hypoxia-ischemia, intracranial embolism or intracranial thrombosis.

[0193] In certain embodiments, the disease affecting the central nervous system is epilepsy, traumatic brain injury.

[0194] In certain embodiments, the disease affecting the central nervous system is spinal cord injury, dementia, Parkinson's disease (PD), Lewy body, Alzheimer's disease (AD), frontotemporal dementia (FTD), familial frontotemporal dementia (FTD), or amyotrophic lateral sclerosis (ALS).

[0195] In certain embodiments, the disease affecting the central nervous system is transmissible spongiform encephalopathy, specifically Creutzfeldt-Jakob disease (CJD), kuru, scrapie, bovine spongiform encephalopathy (BSE). 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). In certain embodiments, the disease affecting the central nervous system is a genetic disorder, specifically Huntington's disease. In certain embodiments, the disease affecting the central nervous system is a genetic disorder, specifically autism, autism spectrum disorder (ASD), for example, Asperger's syndrome.

[0196] In certain embodiments, the disease affecting the central nervous system is hereditary leukodystrophy, specifically metachromatic leukodystrophy, Krabbe disease, Canavan disease, X-linked adrenoleukodystrophy, Alexander disease. In certain embodiments, the disease affecting the central nervous system is a genetic metabolic disorder, specifically Tay-Sachs disease or Wilson's disease.

[0197] In certain embodiments, the disease affecting the central nervous system is a mental disorder, specifically amnesia, attention deficit hyperactivity disorder, psychosis, anxiety disorder, bipolar disorder, depression, mania, intellectual developmental disorder, general developmental delay, post-traumatic stress disorder, acute stress disorder, dissociative disorder.

[0198] 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. 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). 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), Rasmussen's encephalitis.

[0199] In certain embodiments, the disease affecting the central nervous system is infectious encephalomyelitis caused by a virus, specifically rabies virus, human herpes virus, virus causing rash, insect-borne virus, tick-borne virus, human immunodeficiency virus (HIV).

[0200] In certain embodiments, the disease affecting the central nervous system is infectious encephalomyelitis caused by bacteria or infectious encephalomyelitis caused by parasites.

[0201] In certain embodiments, the disease affecting the central nervous system is progressive multifocal leukoencephalopathy (PML) caused by JC polyomavirus (usually abbreviated as JCPyV or JCV).

[0202] In certain embodiments, the disease affecting the central nervous system is post-infectious encephalomyelitis.

[0203] In certain embodiments, the disease affecting the central nervous system is age-related macular degeneration (wet AMD) associated with angiogenesis and diabetic macular edema or retinitis pigmentosa.

[0204] In a further aspect of the present invention, the polypeptide according to the present invention is used for the prevention or treatment of diseases affecting the lung, which diseases are 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, glanders, pneumonic plague, anthrax, invasive fungal diseases in the lung, pulmonary paracoccidioidomycosis, interstitial lung disease, idiopathic pulmonary fibrosis, and chronic rhinosinusitis with nasal polyps.

[0205] In certain embodiments, the disease affecting the lung is coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0206] In certain embodiments, the disease affecting the lung is severe acute respiratory syndrome (SARS).

[0207] In certain embodiments, the disease affecting the lung is severe acute respiratory syndrome (SARS) caused by a virus, specifically a coronavirus.

[0208] In certain embodiments, the disease affecting the lung is asthma, allergic asthma, severe uncontrolled asthma, or a combination thereof.

[0209] In certain embodiments, the disease affecting the lung is chronic obstructive pulmonary disease (COPD).

[0210] In certain embodiments, the disease affecting the lung is fibrosis, cystic fibrosis, pulmonary fibrosis, or a combination thereof.

[0211] In certain embodiments, the disease affecting the lung is influenza caused by the influenza virus.

[0212] In certain embodiments, the disease affecting the lung is sarcoidosis (also known as Besnier-Boeck-Schaumann disease).

[0213] In certain embodiments, the disease affecting the lung is lung cancer.

[0214] In certain embodiments, in general terms, the disease affecting the lung is caused by a virus, bacterium, fungus, or parasite.

[0215] In certain embodiments, the disease affecting the lung is tuberculosis caused by Mycobacterium tuberculosis (commonly abbreviated as M. tuberculosis or M. tb).

[0216] In certain embodiments, the disease affecting the lung is a respiratory infection caused by the respiratory syncytial virus human orthopneumovirus (human respiratory syncytial virus, or HRSV, or simply RSV as it is also known).

[0217] In certain embodiments, the disease affecting the lung is bubonic plague caused by the bacterium Yersinia pestis.

[0218] In certain embodiments, the disease affecting the lung is pneumonic plague caused by Yersinia pestis.

[0219] In certain embodiments, the disease affecting the lung is anthrax, an infection caused by the bacterium Bacillus anthracis.

[0220] In certain embodiments, the disease affecting the lung is an invasive fungal disease (also known as fungal lung disease) caused by pulmonary fungal pathogens such as Aspergillus, Cryptococcus, Pneumocystis, and epidemic fungi.

[0221] In certain embodiments, the disease affecting the lung is paracoccidioidomycosis (typically abbreviated as PCM) caused by the fungus Paracoccidioides brasiliensis.

[0222] In certain embodiments, the disease affecting the lung is chronic rhinosinusitis with nasal polyps (typically abbreviated as CRSwNP), a subgroup of chronic rhinosinusitis (CRS).

[0223] In certain embodiments, the disease affecting the lung is pulmonary edema.

[0224] In certain embodiments, the disease affecting the lung is interstitial lung disease.

[0225] In certain embodiments, the disease affecting the lung is idiopathic pulmonary fibrosis.

[0226] 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.

[0227] In certain embodiments, the disease affecting the joint is rheumatoid arthritis (RA). In certain embodiments, the disease affecting the joint is juvenile rheumatoid arthritis.

[0228] In certain embodiments, the disease affecting the joint is gout, a form of inflammatory arthritis caused by a persistent elevation of uric acid levels in the blood. In certain embodiments, the disease affecting the joint is pseudogout.

[0229] In certain embodiments, the disease affecting the joint is osteoarthritis (OA) resulting from the destruction of articular cartilage and the underlying bone.

[0230] In certain embodiments, the disease affecting the joint is chronic hemophilic synovitis.

[0231] In certain embodiments, the disease affecting the joint is psoriatic arthritis, a long-term inflammatory arthritis that occurs in people suffering from psoriasis, an autoimmune disease.

[0232] In certain embodiments, the disease affecting the joint is ankylosing spondylitis (also known as Bekhterev's disease, Bechterew's disease, or morbus Bechterew).

[0233] In a further aspect of the invention, the polypeptide according to the invention is used for the prevention or treatment of diseases affecting the eye, said diseases being selected from uveal melanoma and uveitis.

[0234] In certain embodiments, the disease affecting the eye is uveal melanoma, an eye cancer (melanoma) involving the iris, ciliary body, or choroid (collectively referred to as the uvea).

[0235] In certain embodiments, the disease affecting the eye is uveitis, i.e., an inflammation of the uvea.

[0236] It is understood that the polypeptide of the invention can be used simultaneously and / or sequentially for the prevention or treatment of multiple diseases or combinations of diseases disclosed herein.

[0237] In certain embodiments, the Fc region is a chimeric Fc region comprising a human or humanized amino acid sequence.

[0238] In certain embodiments, the Fc region is a human or humanized Fc region.

[0239] The Fc region contains the mutations I253N and H435Q, and an H at position 310.

[0240] In certain embodiments, the Fc region is or comprises a sequence characterized by SEQ ID NO: 004 (NHQ).

[0241] A broader aspect of the invention provides a polypeptide comprising a crystallizable fragment (Fc) region of IgG for use in the prevention or treatment of a disease. The Fc region has a modification that results in a reduced affinity for the neonatal Fc receptor (FcRn), and the polypeptide is delivered by local administration to a tissue affected by the disease.

[0242] In certain embodiments, the polypeptide is delivered to the eye by intravitreal administration.

[0243] In certain embodiments, the polypeptide is delivered to the joint by intra-articular administration.

[0244] In certain embodiments, the polypeptide is delivered to the lung via inhalation.

[0245] The present invention further comprises a crystallizable fragment (Fc) region of IgG, preferably further -IL-12; or -a polypeptide that binds to any one of VEGFR, Ang2, TNFα, IL-17, PD-1, PD-L1, more preferably a polypeptide that binds to any one of VEGFR, Ang2, TNFα, IL-17 A polypeptide for use in the prevention or treatment of a disease affecting the eye, specifically a neonatal disease affecting the eye, comprising the above Fc region, wherein the Fc region has a modification that results in a reduced affinity for the neonatal Fc receptor (FcRn), the Fc comprises the mutations I253N and H435Q, and H (NHQ) at position 310, and the polypeptide is delivered to the eye by intravitreal administration.

[0246] The present invention further comprises a crystallizable fragment (Fc) region of IgG, preferably further -IL-12; or A polypeptide that binds to any one of -TNFα, IL-1RA, IL-6R, IL-6, CD27, IL-22, IL-17, CD27, more preferably a polypeptide that binds to any one of TNFα, IL-1RA, IL-6R, IL-6, CD27 A polypeptide for use in the prevention or treatment of a disease affecting a joint, comprising the above, wherein the Fc region has a modification that results in a reduced affinity for the neonatal Fc receptor (FcRn), the Fc contains the mutations I253N and H435Q, and H at position 310, and the polypeptide is delivered to the joint by intra-articular administration, provides a polypeptide.

[0247] The present invention further comprises a crystallizable fragment (Fc) region of IgG, preferably further comprising -IL-12; or -IL-10; or 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, for use in the prevention or treatment of a disease affecting the lung, comprising the above, wherein the Fc region has a modification that results in a reduced affinity for the neonatal Fc receptor (FcRn), the Fc contains the mutations I253N and H435Q, and H at position 310, and the polypeptide is delivered to the lung by inhalation, provides a polypeptide.

[0248] The present invention further provides a fusion polypeptide for use as a medicament, comprising a crystallizable fragment (Fc) region of IgG, specifically further comprising IL-12, wherein the Fc region has a modification that results in a reduced affinity for the neonatal Fc receptor (FcRn), the Fc contains the mutations I253N and H435Q, and H at position 310.

[0249] In certain embodiments, the crystallizable fragment (Fc) region of the polypeptide for use in preventing or treating a disease is or comprises SEQ ID NO: 004 (NHQ). In certain embodiments, the crystallizable fragment (Fc) region of the fusion polypeptide for use as a medicament is or comprises SEQ ID NO: 004 (NHQ).

[0250] After topical administration, the reduced affinity for FcRn ensures a decrease in transport into the circulating blood and systemic enrichment, thereby reducing the systemic toxic side effects of any of the polypeptides.

[0251] The present invention further 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 preventing or treating a disease that affects the central nervous system. The antibody or antibody-like molecule comprises modifications I253 and NH435Q that result in a reduced affinity for the neonatal Fc receptor (FcRn), and an Fc region having an H at position 310. The antibody or antibody-like molecule is administered to the central nervous system, specifically the brain.

[0252] 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 the OX40 receptor, for use in preventing or treating a disease that affects the central nervous system. The antibody or antibody-like molecule comprises an Fc region having a modification that results in a reduced affinity for the neonatal Fc receptor (FcRn). The antibody or antibody-like molecule is administered to the brain.

[0253] The present invention further provides a polypeptide comprising a crystallizable fragment (Fc) region of IgG. The Fc region has a modification that results in a decreased affinity for the neonatal Fc receptor (FcRn) as compared to the affinity of the same polypeptide containing an unmodified Fc region. The Fc contains the mutations I253N and H435Q, and an H at position 310. In certain embodiments, this polypeptide according to the invention further comprises IL-12.

[0254] In certain embodiments, the polypeptide is selected from a fusion protein comprising an effector polypeptide and the above Fc region; or an antibody or antibody-like molecule comprising or linked to the above Fc region.

[0255] In certain embodiments, the antibody or antibody-like molecule is a bispecific construct capable of binding two antigens simultaneously.

[0256] In certain embodiments, the polypeptide is an antibody or antibody-like molecule comprising or linked to the above Fc region, preferably the antibody or antibody-like molecule is a bispecific construct capable of binding two antigens simultaneously, specifically the bispecific antibody or antibody-like molecule binds to PD-L1 and the IL-12 receptor in an agonist mode.

[0257] Those skilled in the art will recognize that in the case of an antibody, the antibody itself already contains an Fc region. In the case of an antibody-like molecule, the antibody-like molecule is linked to the Fc region. In certain embodiments, the effector polypeptide a. a cytokine or hormone or growth factor, b. a cytokine receptor or hormone receptor or growth factor receptor, or c. a metabolite has a function and is known to have a therapeutic or prophylactic effect against a disease, specifically a disease affecting the central nervous system.

[0258] In certain embodiments, the effector polypeptide can specifically bind to the extracellular matrix (ECM) and is known to have a therapeutic or prophylactic effect against a disease, specifically a disease affecting the central nervous system. In certain embodiments, the effector polypeptide can specifically bind to RNA and is known to have a therapeutic or prophylactic effect against a disease, specifically a disease affecting the central nervous system.

[0259] 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, 4-1BBL, 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.

[0260] In certain embodiments, the antibody or antibody-like molecule is selected from antibodies or antibody-like molecules that specifically bind agonistically or antagonistically to 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, 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, C9orf72.

[0261] The antibody or antibody-like molecule according to the above aspect of the present invention can be an antibody-like molecule derived from the recognition site or a full antibody of the physiological ligand of PD-1 or PD-L1 or PD-L2. Such an antibody or antibody-like molecule competes with the physiological ligand for binding to PD-1 or PD-L1 or PD-L2, respectively. Specifically, a non-agonistic PD-1 antibody or antibody-like molecule, or a non-agonistic PD-L1 antibody or antibody-like molecule, or a non-agonistic PD-L2 antibody or antibody-like molecule does not result in attenuated T cell activity when bound to PD-1 on the surface of T cells.

[0262] In some embodiments, the non-agonistic PD-1 antibody or antibody-like molecule used in the present invention, when bound to PD-1, can sterically block the interaction between PD-1 and its binding partners PD-L1 and / or PD-L2.

[0263] In some embodiments, the non-agonistic PD-1 antibody or antibody-like molecule is a gamma immunoglobulin that binds to PD-1 and does not elicit a physiological response of the PD-1 interaction with PD-L1 and / or PD-L2, which are the binding partners of PD-1.

[0264] In some embodiments, the non-agonistic PD-L1(PD-L2) antibody or antibody-like molecule is a gamma immunoglobulin that binds to PD-L1(PD-L2) and does not elicit a physiological response of the PD-1 interaction with PD-L1 and / or PD-L2, which are the binding partners of PD-1.

[0265] Non-limiting examples of PD-1 antibodies are the clinically approved antibodies pembrolizumab (CAS number 1374853-91-4) and nivolumab (CAS number 946414-94-4).

[0266] Non-limiting examples of PD-L1 antibodies are the clinically approved antibodies atezolizumab (CAS number 1380723-44-3), durvalumab (CAS number 1428935-60-7) and avelumab (CAS number 1537032-82-8).

[0267] Non-limiting examples of PD-1 / PD-L1 or PD-L2 antibodies currently in clinical development are the antibodies MDX-1105 / BMS-936559 or AMP-224. Non-limiting examples of antibodies that specifically bind to IL-12 / 23 are ustekinumab (CAS number 815610-63-0).

[0268] In certain embodiments, the antibody or antibody-like molecule is an antibody that specifically binds to PD-L1.

[0269] In some embodiments, the agonistic OX40 antibody or antibody-like molecule used in the present invention can trigger a signaling cascade in OX40-expressing cells upon binding to OX40 and in the absence of OX40 ligand.

[0270] Non-limiting examples of OX40 antibodies are the antibodies PF-04518600 / PF-8600m, BMS-986178, GSK3174998, MOXR0916, INCAGN01949, bavolimab / MEDI0562, which are currently in clinical development.

[0271] In certain embodiments, the antibody or antibody-like molecule is an antibody that specifically binds to OX40.

[0272] In some embodiments, the antibody or antibody-like molecule used in the present invention can block the interaction between CD47 and SIRPα signals that impede the phagocytosis of cancer cells.

[0273] 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.

[0274] In certain embodiments, the antibody or antibody-like molecule is an antibody that specifically binds to Nogo-A.

[0275] In certain embodiments, the antibody or antibody-like molecule is a bispecific construct that can bind two antigens simultaneously.

[0276] In certain embodiments, the antibody or antibody-like molecule is an antibody against histone present in the necrotic core of a tumor, armed with IL-12. In certain cases, the armed antibody is an immunocytokine. Non-limiting examples of armed antibodies as immunocytokines are NHS-IL-12, NHS-IL2LT, huBC1-IL-12.

[0277] In certain embodiments, the Fc region is or comprises a sequence characterized by SEQ ID NO: 004 (NHQ).

[0278] 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 the binding between the polypeptide containing Fc and FcRn, and thus mimic the technical effects of the invention. The combination with the FcRn blocking antibody can enhance the described advantages of the polypeptide comprising the modified Fc region according to the invention.

[0279] In certain embodiments of any aspect of the invention, the Fc region is the Fc region of immunoglobulin G (IgG). IgG is a major effector molecule of the human humoral immune response. There are four different subgroups of human IgG designated IgG1, IgG2, IgG3, and IgG4. The four subclasses show more than 95% homology in the amino acid sequence of the constant domain of the heavy chain, but differ with respect to the structure and flexibility of the hinge region, particularly the number of inter-heavy chain disulfide bonds in this domain. The structural differences between IgG subclasses are also reflected in their susceptibility to proteolytic enzymes, specifically papain, plasmin, trypsin, and pepsin.

[0280] In certain embodiments of any aspect of the invention, the Fc region is the Fc region of IgG4. Only one isoform of human IgG4 is known. In contrast to human IgG1, IgG2, and IgG3, human IgG4 does not activate complement. Furthermore, IgG4 is less sensitive to proteolytic enzymes compared to IgG2 and IgG3. Contrary to these expectations, in fact, IgG1 full-length antibody constructs having the mutations I253N and H435Q were surprisingly found to be characterized by a lower affinity for FcRn, as exemplified by a lower plasma-to-brain ratio determined compared to the corresponding IgG4 full-length antibody constructs.

[0281] Similarly, within the scope of the present invention, there is included the use for treating or preventing a malignant neoplastic disease, specifically a solid tissue tumor, more specifically a glioma, in a patient in need thereof, comprising administering to the patient a polypeptide comprising a modified Fc region according to one of the aspects of the present invention as described above, or a nucleic acid encoding the polypeptide, or a viral vector comprising a nucleic acid encoding the polypeptide.

[0282] Similarly, there is provided a dosage form for the prevention or treatment of a malignant neoplastic disease, specifically a solid tissue tumor, more specifically a glioma, comprising a polypeptide comprising a modified Fc region according to one of the aspects of the present invention as described above, or a nucleic acid encoding the polypeptide, or a viral vector comprising a nucleic acid encoding the polypeptide.

[0283] It should be understood that whenever options for a single separable feature are presented herein as "embodiments", such options can be freely combined to form distinct embodiments of the invention disclosed herein.

[0284] The present invention is further illustrated by the following examples and drawings, from which further alternative embodiments and advantages can be drawn. These examples are intended to illustrate the invention but not to limit its scope. BRIEF DESCRIPTION OF THE DRAWINGS

[0285]

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Mode for Carrying Out the Invention

[0286] Example 1: Materials and Methods Animals C57BL / 6J mice were obtained from Charles River. mFcRn - / - hFcRn tg(32) (FcRn tgMice were obtained from Jackson Laboratory (stock number 014565). All animals were housed under specific pathogen-free (SPF) conditions, with free access to food and water, on a 12-hour light / dark cycle, in accordance with the facility's guidelines.

[0287] Tumor cell line GL-261 cells were provided by A. Fontana of Experimental Main Epidemiology at the University of Zurich in Zurich, Switzerland, and cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum and L-glutamine (all from Thermo Fisher Scientific). Mouse 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 Streptococcus 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 KO cells were purified by flow cytometry by gating PD-L1-negative cells after 48-hour IFN-γ stimulation (10 ng / ml). Single clones were further amplified, and the loss of PD-L1 expression was reconfirmed by flow cytometry before use in experiments.

[0288] Surgical procedure For glioma inoculation, mice aged 6 - 10 weeks 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 stereotactic fixation robot (Neurostar). Briefly, a blunt-ended syringe (Hamilton; 75N, 26s / 2’’ / 2.5μl) was placed 1.5 mm lateral and 1 mm anterior to bregma. The needle was lowered through the burr hole to a depth of 4 mm below the dura mater surface and retracted 1 mm to form a small reservoir. The injection was performed at a volume of 2 μl at 1 μl / min. The needle was left in place for 2 minutes and then retracted at 1 mm / min. The burr hole was closed with bone wax (Aesculap, Braun), and the scalp wound was sealed with tissue adhesive (Indermil, Henkel). The anesthesia was discontinued using a mixture of flumazenil (Labatec Pharma AG) and buprenorphine (Indivior Schweiz AG), and atipamezole was injected 20 minutes later (Janssen). Carprofen (Pfizer AG) was used for perioperative analgesia.

[0289] Seven to fourteen days later, osmotic pumps (model 2004, 0.25 μl / h; Alzet) were filled with mouse IL-12Fc (12.5 μg / kg / 24 h) or PBS alone and primed at 37°C in PBS. The mice were anesthetized as described above, the previous burr hole for glioma injection was located, the bone wax and periosteal bone were removed, and the infusion cannula was lowered through a 3-mm burr hole into the estimated center of the tumor. Using Vacutainer tubes and following the manufacturer's instructions (Becton, Dickinson and Company), serum samples were collected every 2 days by blood sampling from the tail vein starting on day -1 of pump implantation.

[0290] For comparison of the IL-12 and IL-12Fc WT serum-to-intracerebral concentration ratios after bolus injection, mice were anesthetized and intracranially injected into the right hemisphere using a stereotactic fixation 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). The dosage was calculated based on the HEK-Blue IL-12 bioactivity assay. After 24 hours, the animals were sacrificed by controlled CO2 asphyxiation. Blood samples were collected by cardiac puncture and the mice were perfused with 20 ml of ice-cold PBS. Serum was isolated as described above and the brain tissue was snap-frozen in liquid nitrogen.

[0291] For comparison of the IL-12 WT and IL-12Fc NHQ serum-to-intracerebral concentration ratios after bolus injection, mice were anesthetized and intracranially injected into the right hemisphere using a stereotactic fixation robot (Neurostar) as described above. Mice received 1 μg of human IL-12Fc WT or IL-12Fc NHQ. After 24 hours, the animals were sacrificed by controlled CO2 asphyxiation. Blood samples were collected by cardiac puncture and the mice were perfused with 20 ml of ice-cold PBS. Serum was isolated as described above and the brain tissue was snap-frozen in liquid nitrogen.

[0292] For convective enhanced delivery (CED) of proteins into the brain, mice were anesthetized and stereotaxically injected into the right hemisphere using a stereotactic fixation robot (Neurostar), and a catheter was used that was made with a 27G blunt-ended needle with 1-mm steps at the tip made of fused silica with an inner diameter of 0.1 mm and a wall thickness of 0.0325 mm. Briefly, burr holes were made at positions 1 mm anterior and posterior and 2 mm medial and lateral to bregma. The catheter was lowered into the burr hole to a depth of 3.5 mm under the dura surface. Injections were made at 0.2 μl / min for a volume of 5 μl, then at 0.5 μl / min for 2 μl, and at 0.8 μl / min for 2 μl. The needle was left in place for 2 minutes at the predetermined position and then 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, the animals were sacrificed by controlled CO2 asphyxiation. The ipsilateral cerebral hemisphere was snap-frozen in liquid nitrogen.

[0293] In vivo bioluminescence imaging Mice bearing tumors 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 dark room of a Xenogen IVIS Lumina III (PerkinElmer) imaging system, and luminescence was recorded for 1–2 minutes with medium binning (4). Subsequently, the data were analyzed using Living Image 4.7.1 software (PerkinElmer). A circular region of interest (ROI; 1.5 cm in diameter) was delineated around the tumor site, and the photon flux in this region was read out and plotted.

[0294] BLI and systematic group allocation Twenty days after transplantation of GL-261luc glioma cells, animals with tumors were distributed into experimental groups with equivalent mean BLI.

[0295] Blood collection Blood samples were collected 10 minutes before CED, or 6 hours, 24 hours, 72 hours, and 7 days after CED injection. 20 - 50 μL of blood was collected from the tail vein into microtainers containing dried K2-EDTA (Becton, Dickinson and Company). After centrifugation at 10,000 g for 5 minutes, the plasma was transferred to fresh tubes and frozen.

[0296] FcRn ELISA IL-12 Fc variant, or recombinant human IgG4 anti-GFP antibody (clone 515, AbD Serotec) functioning as a control, was coated onto microtiter plates (Greiner Bio-One). Histidine-tagged FcRn (R&D Systems) was incubated with increasing concentrations in ELISA diluent (Mabtech) at pH = 6.0. FcRn was detected by biotinylated anti-His antibody (clone 13 / 45 / 31-2, Dianova), streptavidin-conjugated horseradish peroxidase (Mabtech), and colorimetric substrate (Chromogen-TMB, Thermo Fisher Scientific). The optical density at a wavelength of 450 nm was measured using a spectrophotometer (Molecular Devices).

[0297] 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 (Tree Star).

[0298] 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). The cells were incubated for 17 hours with increasing amounts of IL-12, IL-12Fc WT, or variants designed for decreased 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).

[0299] Detection of human IL-12 in the brain, plasma, and serum after injection into the brain, and calculation of the serum or plasma to brain concentration ratio Samples were diluted in PBS containing 0.05% Tween-20 and 0.1% BSA, and IL-12 levels were evaluated by ELISA (Mabtech) for hIL-12p70. To calculate the serum or plasma to brain concentration ratio, the IL-12 concentration in serum or plasma was reported in pg / ml, while the concentration in the brain was calculated by dividing the total amount of IL-12 extracted from the brain corrected for the efficiency of protein extraction by the hemisphere weight (pg / mg brain tissue).

[0300] Detection of human IgG in the brain and plasma after intracerebral injection and calculation of the plasma to brain concentration ratio Samples were diluted in PBS containing 0.05% Tween-20 and 0.1% BSA, and IgG levels were evaluated 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 the plasma to brain ratio, the concentrations of human IgG in plasma and brain were reported in pg / ml.

[0301] Production of Human IgG1 Variant, IgG4 Variant hIL-12hFc:aPD-L1 NHQ and mIL-12hFc:aPD-L1 NHQ The IgG4 variant was expressed in transiently transfected human embryonic kidney (HEK) cell cultures. The IgG1 variant, hIL-12hFc:aPD-L1 NHQ and mIL-12hFc:aPD-L1 NHQ were produced by transiently transfected Chinese hamster ovary (CHO) or cell cultures. Briefly, the culture supernatants were harvested 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 cut-off). The proteins were stored in 20 mM histidine, 150 mM NaCl, pH = 6.0 buffer. The quality was evaluated by gel electrophoresis (SDS-PAGE) and then Coomassie staining was performed according to standard protocols. Nivolumab, atezolizumab, ipilimumab and rituximab are commercially available IFN-γ production by lymphocytes stimulated with IL-12Fc.

[0302] Human peripheral blood mononuclear cells (PBMC) were stimulated for 24 hours with increasing concentrations of IL-12, IL-12Fc or IL-12Fc variant with reduced FcRn affinity in the presence of 100 ng / ml anti-CD3 antibody. IFN-γ levels in the supernatants were measured by ELISA (Mabtech) according to the manufacturer's instructions.

[0303] Isolation of Brain Proteins After euthanizing and carefully removing the calvarium, the brain was 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 and then passed through a 20G needle and finally sonicated for 20 seconds. Samples were centrifuged at 15,000 g for 10 minutes 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.

[0304] All human and mouse IL-12Fc variants were expressed in HEK293T. Variants retaining protein G affinity were purified from the culture supernatant by affinity chromatography using protein G sepharose (Biovision) and overnight dialysis with PBS. Variants that lost protein G affinity were precipitated with 50% saturated ammonium sulfate (VI), and the precipitate was then dissolved in PBS and purified by purification on a ceramic hydroxyapatite (CHT) column (type II, 40 μm Bio-Rad). After protein G or CHT chromatography, samples were further purified by ion exchange chromatography using diethylaminoethanol-linked sepharose (HiTrap DEAE Sepharose FF column, GE Healthcare) as an anion exchanger on an AKTA purifier chromatography system (GE Healthcare). Finally, all IL-12Fc variants were purified by size exclusion chromatography (GE Healthcare) using 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 cut-off. Protein purity was verified by SDS-PAGE electrophoresis followed by staining with Coomassie Brilliant Blue (VWR Life Science). Protein concentration was measured using a Pierce BCA assay kit (Thermo Fisher Scientific) and an ELISA for IL-12p70 (Becton, Dickinson and Company).

[0305] Phosphorylation of STAT-4 by lymphocytes stimulated with IL-12Fc Human peripheral blood mononuclear cells (PBMCs) were stimulated for 1 hour with 10 ng / ml of IL-12, IL-12Fc or an IL-12Fc variant with reduced FcRn affinity in the presence of 100 ng / ml anti-CD3. Cells were then lysed using Pierce RIPA buffer (Thermo Fisher Scientific). Samples were analyzed by SDS-Page electrophoresis, then transferred using a 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 an ECL transparent substrate (Bio-Rad Laboratories, Inc.) and a BioRad MPCD imager (Bio-Rad Laboratories, Inc.).

[0306] Surface plasmon resonance SPR was performed using a ProteOn XPR36 system (Bio-Rad Laboratories, Inc.) with a human recombinant biotinylated FcRn (Immunitrack) coated onto a ProteOn NLC sensor chip to approximately 80 response units (RU). The IL-12Fc variant was run at concentrations decreasing in 3-fold steps from 729 nM to 9 nM in 10 mM sodium citrate buffer pH = 6.0. The dissociation time was 600 seconds. Analysis was performed using ProteOn Manager software (Bio-Rad Laboratories, Inc.) with data normalization to injection time, spot-to-spot background subtraction and built-in artifact removal functions. Kd was calculated using an equilibrium analysis model.

[0307] Thermal shift assay Briefly, a 0.2 mg / ml protein sample was mixed with Sypro Orange Protein stain (Sigma-Aldrich) diluted 1:1000 and electrophoresed on a CFX384 thermocycler (Biorad) with a temperature increase of 0.2 °C every 30 s from 20 °C to 95 °C, reading fluorescence for detection. 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.

[0308] Statistical analysis Statistical analysis was performed using Graphpad Prism 5 software. Outliers were removed 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.

[0309] Flow cytometry PD-L1 binding assay GL261:lucE9 or GL261:lucE9:PD-L1KO cells were cultured overnight with mouse interferon-gamma added at a final concentration of 20 ng / mL. The next day, the cells were washed with DPBS. Trypsin-EDTA (Invitrogen 25300-054) was added to the flask and immediately removed again. The cells were left for 2 - 5 minutes to detach from the flask. They were washed with medium and centrifuged at 350 g for 5 minutes at 4 °C. Then, the cells were plated at 100,000 cells / well in a round-bottom 96-well plate and washed twice with DPBS.

[0310] Staining was performed at 25 μL per well with PBS containing either Zombie Aqua Fixable Viability Kit (BioLegend) diluted 1:200 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 the washing step with PBS, the cells were incubated for 30 minutes at 4 °C in the dark in PBS with secondary antibody anti-human IgG-Fc-PE (Biolegend, catalog number 409304, lot B260868) at 0.2 mg / mL or anti-mouse PD-L1-BV421 (Biolegend, catalog number 124315; lot B228149) control antibody (data not shown). Cells were washed twice with PBS, filtered through a 40 μm mesh, and acquired using an LSRII Fortessa flow cytometer (Becton, Dickinson and Company). Data analysis was performed using FlowJo version 10.6 (Tree Star).

[0311] Survival analysis The neurological symptoms of animals with tumors were examined, and body weight was measured once a week until day 21 after tumor cell transplantation. After day 21, the monitoring frequency was increased to daily checks and once-weekly bioluminescence imaging (BLI). Animals were euthanized by asphyxiation with controlled CO2 when they reached predefined termination criteria (weight loss exceeding 20% of the maximum body weight and / or moribund state) according to the veterinary authorities of the Canton of Zurich (ZH194 / 19).

[0312] Example 2: Intracranial injection of human IL-12 has higher systemic leakage than hIL-12Fc IL-12Fc is highly promising for the local treatment of brain tumors. However, for use in clinical trials, a human version of IL-12Fc that exhibits similar properties is required. To obtain a human analog of mouse IL-12IgG3, the inventors 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) and does not activate the complement system. To test the leakage and stability of human IL-12Fc (hIL-12Fc) versus recombinant human IL-12 (rhIL-12), the inventors 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, the inventors analyzed the human IL-12 concentration in the lysates and sera of the ipsilateral hemisphere to gain a more detailed understanding of stability and retention (residual concentration) at the injection site and the rate of leakage into the bloodstream (Figure 1B). For each mouse, the inventors calculated the ratio of the concentration in serum 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 superior tissue retention compared to rhIL-12, as the inventors observed a considerably lower ratio (Figure 1C). For local GB treatment, the human IL-12Fc fusion cytokine is considered an excellent compound compared to its natural counterpart due to its high tissue retention, stability, and solubility.

[0313] Example 3: FcRn binding results in systemic accumulation of IL-12Fc The neonatal Fc receptor (FcRn)-based endosomal recycling system in endothelial cells and red splenic macrophages prevents the rapid degradation and clearance of IgG. After endocytosis promoted by the acidic pH of endosomes, FcRn binds to IgG and is recycled to the cell surface, where neutral pH induces release. When injected locally, IL-12Fc can leak from the brain in an FcRn-mediated manner due to its Fc tag. Leakage from the brain causes serum accumulation of IL-12Fc, which can ultimately reach toxic levels. To test whether FcRn-based recycling actually promotes the accumulation of hIL-12Fc in serum, the inventors utilized transgenic mice expressing human FcRn on a mouse FcRn-deficient background (FcRntg). Although human FcRn has a weak affinity for mouse IgG, only mouse IgG recycling is impaired in this mouse model because it promotes normal albumin recycling. Thus, mouse IL-12Fc has considerably less binding to FcRn in these FcRn-humanized mice. Therefore, the inventors compared the serum mIL-12 levels of wild-type (wt) and FcRntg mice with gliomas that had been treated with local mouse IL-12Fc via osmotic minipumps. Indeed, after 1 week, the inventors observed an increase in IL-12 levels in FcRntg mice that was not as prominent in wt mice (Figure 2A), followed by an increase in IFN-γ levels (Figure 2D). The inventors even observed an increase in IL-12 levels in the serum of some mice as early as 1 day after pump implantation (Figure 2B). As a result, this led to a significant increase in the serum concentration of IFN-γ in wt mice but not in the FcRntg cohort (Figure 2C). Similar to mouse IL-12Fc, human IL-12Fc can also leak and accumulate, with the potential for systemic side effects.Furthermore, IFN-γ is one of the major mediators of IL-12-related side effects (Leonard et al., page 1997, Blood 90:2541-2548), and its persistent systemic presence can be toxic (Weiss et al., 2007, Expert Opin Biol Ther 7:1705-1721). In summary, the inventors conclude that even a small amount of IL-12Fc leakage from the treatment site is sufficient to induce detectable serum IFN-γ levels.

[0314] Example 4: Generation of human IL-12Fc variants designed for improved tissue retention The observation that reduced FcRn binding potentially abrogates efflux from the brain and results in dramatically reduced recycling upon leakage from the brain can be utilized to increase the safety margin of hIL-12Fc. Accordingly, the inventors set out to reduce the binding of the Fc portion of hIL-12Fc to human FcRn. By increasing the positive charge at the FcRn-binding interface of the Fc portion, this interaction - and thus recycling - at acidic pH can be abrogated, which has been shown to decrease the serum half-life of immunoglobulins. The inventors introduced a number of mutations into hIL-12Fc at the FcRn-binding site (Table 1) with the aim of decreasing its serum half-life in the case of leakage.

[0315] The inventors generated three IL-12Fc variants with mutations similar to those of previously published antibodies with reduced FcRn affinity, designated IAQ, AHH, and AAA. Furthermore, the inventors replaced the isoleucine at position 253 not with alanine, which represents a simple shortening of the side chain, but instead changed it to asparagine (I253N). Asparagine is a polar amino acid and its side chain has a length similar to that of isoleucine. The inventors also modified the histidine at position 310 to alanine, and the histidine at position 435 to glutamine, alanine, or glutamic acid.

[0316] All variants were expressed in human embryonic kidney 293T cell (HEK293T) cultures. The expression levels of all variants were similar.

[0317] Example 5: Human IL-12Fc variants have similar protein stability First, the inventors verified whether the changes introduced into the Fc affected the overall protein stability. For this purpose, the inventors measured the denaturation temperature for each of the variants in a thermal shift assay 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 as low as about 57 °C (Figure 3B).

[0318] Example 6: Human IL-12Fc variants maintain their biological activity Even though the inventors aimed to reduce the binding of hIL-12Fc to FcRn, the inventors 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 the IL-12 signaling components and a downstream enzyme that catalyzes a colorimetric reaction. Only the NAQ variant showed an approximately 2-fold decrease in activity compared to IL-12Fc, while all others showed EC50 values within the range of IL-12Fc WT (Figure 4A). Importantly, IL-12Fc had an activity equivalent to rIL-12 in vitro.

[0319] To further verify the activity of the IL-12Fc variants, the inventors activated peripheral blood mononuclear cells (PBMCs) using three different hIL-12Fc variants, namely IAQ, AHQ, and NHQ, and then analyzed STAT-4 phosphorylation (Figure 4B). Even more importantly, this STAT-4 phosphorylation was translated into a strong production of IFN-γ after 24 hours (Figure 4C), indicating that all variants retained the activity of rhIL-12.

[0320] Example 7: Human IL-12Fc variants differ in their binding to Protein G Protein A and G affinity chromatography is one of the standard methods used for the purification of recombinant antibodies and Fc fusion proteins. Modifying the interface between Fc and FcRn is known to abrogate Protein A binding, an observation that the inventors also confirmed with IL-12Fc variants. To confirm the feasibility of production in a scale-up process, the inventors determined to verify the possibility of purifying IL-12Fc variants via a Protein G affinity column. Most of the inventors' variants retained affinity for Protein G, but unexpectedly for the inventors, all variants containing both I253N and the H310A mutation were not suitable for Protein G purification (Table 2). This effect was independent of the additional mutation at position 435. For further studies, the inventors have focused on variants with retained Protein G affinity.

[0321] Example 8: Human IL-12Fc variants have reduced FcRn affinity To verify the affinity of IL-12Fc variants for FcRn, the inventors used surface plasmon resonance (SPR), a label-free method for characterizing protein-protein interactions. The inventors 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 have reduced affinity for human FcRn, with the NHQ variant showing the strongest decrease (about 8-fold lower). The inventors used a commercially available human monoclonal anti-GFP IgG4 antibody as a control.

[0322] Furthermore, the inventors supported these data with ELISA data on the NHQ constructs, using IL-12Fc WT, anti-GFP IgG4, and the published variant IAQ as references. Both IAQ and NHQ showed reduced binding, with NHQ having the lowest affinity (Figure 5B). Thus, the inventors concluded that the substituted NHQ combinations appear 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), which suggested that the mutation by the combination of H310A and H435Q was responsible for the strongest reduction in binding to FcRn at low pH.

[0323] Example 9: Introduction of NHQ mutations reduces systemic exposure to locally delivered hIL-12Fc The inventors hypothesized that a decrease in FcRn affinity would increase the retention of hIL-12Fc in the CNS while simultaneously impeding its systemic accumulation. This was addressed in a similar manner to the comparison of hIL-12Fc WT and recombinant human IL-12 (Figure 1B). FcRn tg Mice were injected once with 1 μg of IL-12Fc WT or an NHQ variant, and IL-12 was measured in the ipsilateral cerebral hemisphere and serum by ELISA. Mice injected with the NHQ variant showed a decrease in the serum-to-brain ratio compared to mice injected with hIL-12Fc WT (Figure 6A). The inventors hypothesized that this could be the effect of both increased retention in the CNS and attenuated systemic accumulation by FcRn-mediated recycling.

[0324] Furthermore, using CED instead of bolus injection, the inventors compared the concentrations of hIL-12Fc WT, IAQ, AAA, and NHQ in plasma with the hemisphere injected 24 hours after CED and observed that the NHQ variant exhibited the most significantly reduced plasma-to-brain ratio (Figure 6B), even in an optimized delivery setting compared to bolus injection. Such an increase in CNS retention may potentially improve the safety profile of local IL-12Fc therapy in conjunction with a decrease in systemic exposure.

[0325] Example 10: IL-12Fc variant NHQ has higher brain tissue retention than other low-affinity variants The inventors measured tissue retention after intracranial delivery of the protein. For this purpose, the inventors injected 1 μg of unmodified IL-12Fc WT, i.e., two previously published variants with reduced FcRn affinity, i.e., IAQ and AAA, and NHQ, which has the lowest FcRn affinity as measured by the inventors (Figure 5A). Instead of bolus injection of the protein solution, to ensure maximum perfusion of the brain hemisphere, the inventors used a CED protocol with a step catheter and a ramp-up injection regimen. To study the effect of different modifications at the FcRn binding interface in the most physiological setting, the inventors used FcRn tg mice. As described above, FcRn is important for both efflux from the CNS and accumulation of Fc-containing molecules in serum. As an attempt to decouple the two effects and focus only on preventing transport from the CNS, the inventors measured the amount of protein remaining in the brain 6 hours after CED. The mice were euthanized, 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 has superior tissue retention compared to IL-12Fc WT. Importantly, it was also better than the other two variants, IAQ and AAA, with reduced FcRn affinity. Surprisingly, there was no significant difference between IAQ and AAA and IL-12Fc WT.

[0326] Example 11: Anti-tumor 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 the in vivo anti-tumor effect in mouse models. To test the effect of the reduced affinity for FcRn, the inventors fused single-chain mouse IL-12 to the same human IgG4 Fc as for hIL-12Fc (Figure 8A).

[0327] 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). Subsequently, IFNγ can lead to the upregulation of PD-L1 on myeloid cells and tumor cells in a process called adaptive resistance (O’Rourke et al., Sci. Transl. Med. (2017) (9), eaaa0984.). Therefore, the inventors inferred that PD-L1 serves as an inductive anchor for further increasing IL-12 tissue retention.

[0328] To evaluate the efficacy of IL-12Fc in combination with locally applied anti-PD-L1 antibody therapy, a bispecific Fc fusion molecule was created. It combines mIL-12hFc, an anti-PD-L1 half-antibody, and 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 is a clinically approved antibody and is derived from atezolizumab, which is cross-reactive with mouse and human PD-L1 (US Patent No. 8,217,149 B2) (Figure 8A).

[0329] The inventors confirmed the bioactivity of the mIL-12hFc:aPD-L1 NHQ molecule in vitro: For IL-12 functionality, an IL-12-responsive reporter cell line was used, and IL-12 induced secreted alkaline phosphatase, which then catalyzed a colorimetric reaction (Figure 8B). Binding to cells expressing PD-L1 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 had NHQ variants in their C H 2 domain and C H 3 domain, and thus, when confirmed by surface plasmon resonance, FcRn binding was abrogated and they had a relatively high K D value compared to unmodified anti-PD-L1 antibodies (Figure 8D).

[0330] After in vitro characterization, the inventors subsequently measured its properties in vivo. Using the mouse glioma model GL-261, the anti-tumor effect and systemic distribution were monitored in vivo. Briefly, mice with tumors 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 impact was monitored by clinical scoring (Figure 9B). To evaluate leakage and drainage during CED, systemic IL-12 and IFNγ levels were measured in plasma at various time points (Figure 9C). Animals administered rmIl-12 or mIL-12hFc wt showed a rapid increase in systemic IL-12 signal and a subsequent rapid increase in IFNγ at the time of CED, while animals administered mIL-12hFc NHQ or mIL-12hFc:aPD-L1 NHQ had a rapid return to baseline and a strongly reduced systemic IL-12 signal that clearly reduced the IFNγ signal (Figure 9C). The difference in tissue retention between mIL-12hFc wt and mIL-12hFc NHQ leads to a lower systemic IL-12 signal already 6 hours after CED1 (Figure 9D). Regarding the clinical course of the treated animals, all groups receiving the IL-12 construct showed a significant increase in survival 3 weeks after tumor inoculation, even at an extremely late stage of disease progression compared to the control group (Figure 9E). Notably, the treatment response in the groups receiving the NHQ construct (mIL-12hFc NHQ or mIL-12hFc:aPD-L1 NHQ) responded at least equally well to treatment compared to the groups receiving mIL-12hFc wt or rmIL-12, but showed a marked decrease in systemic IL-12 and IFNg.

[0331] Example 12: Measurement of the affinity of IL-12Fc and IgG variants for hFcRn To further evaluate the impact of low FcRn affinity, which favorably affects the plasma-to-brain ratio in local delivery to the CNS, IAQ, AAA, and NHQ variants were compared to unmodified antibodies (Figure 10). The inventors selected human IgG1 directed against PD-L1 (Figure 10A and Figure 10B, atezolizumab) and a human anti-influenza A IgG4 antibody (Figure 10C and Figure 10D, Flu HA3.1, US Patent Application Publication No. 2014 / 0370032A1). The finding that hIL-12Fc is functional, has higher tissue retention than rhIL-12, and can increase the safety margin by stopping systemic recycling in case of leakage may have broad implications for the local administration of any Fc-containing molecule. These modifications enable safe and effective local delivery of any antibody or Fc-fusion molecule for the local treatment of neurological diseases.

[0332] Administration of therapeutic agents to the CNS via the systemic route (either os or i.v.) is difficult mainly because of the BBB - compared to the rest of the body - and only a very small selection of today's therapeutic agents actually reach the brain. Unfortunately, antibodies and Fc-containing biologicals, specifically Fc fusion proteins, do not easily cross the BBB and are further actively effluxed. Enabling the transport of antibodies across the BBB into the brain parenchyma has been extensively studied, for example, by utilizing receptor-mediated transcytosis for transferrin. Cytokines have a short half-life in the circulating blood, a high risk of side effects, and narrow their therapeutic opportunity window. Cytokines can be conjugated to antibodies that home to the tumors where they accumulate, specifically NHS-IL-12. Even after subcutaneous administration, these antibodies induce an IFNγ response when moving to the tumors via the bloodstream. Initially, the systemic delivery of IL-12 was evaluated for the treatment of non-brain cancers. However, these clinical trials had to be terminated early because intravenous application at effective doses caused severe adverse events including death. One of the main reasons is thought to be the induction of IFNγ by IL-12.

[0333] The serum half-life and solubility of protein therapeutics can be improved by directly fusing the crystallizable fragment (Fc) of an antibody with the therapeutic moiety. For direct local application to anatomically distinct sites, this can result in undesirable effects. One of these can be the efflux of Fc-containing molecules via FcRn from immunoprivileged anatomical sites, specifically the brain, and their serum accumulation similar to IgG recycling.

[0334] The inventors observed that local administration of an IL-12Fc fusion cytokine into the brain caused FcRn-dependent efflux of IL-12Fc into the circulation via the BBB. IL-12Fc accumulated in the blood and caused potentially dangerous IFNγ production.

[0335] The inventors found that IL-12Fc with reduced FcRn affinity was functional and had higher tissue retention than recombinant IL-12 and unmodified IL-12Fc. Compared to the brain tissue retention experiment, the NHQ mutant showed higher brain tissue retention compared to IL-12Fc WT, as well as the IAQ and AAA variants (see Figure 7). Surprisingly, IAQ and AAA, two variants in which FcRn binding was reported to be dramatically reduced, were no different from unmodified IL-12Fc, suggesting that to obtain a biological difference, the FcRn affinity must be reduced beyond a certain threshold, which is only achieved by NHQ modification. Alternatively, it cannot be excluded that the NHQ mutation introduces other features that improve tissue retention in an FcRn-independent manner.

[0336] This leads to an improvement in the safety profile and widens the therapeutic window for IL-12Fc therapy of brain tumors. Furthermore, the findings of the present inventors can be translated to any Fc-containing therapeutic agent, mainly therapeutic antibodies, which have a strong theoretical basis for local intracranial administration. Such an application route is preferred because it is less effective when administered systemically and potentially has a poor effect of crossing the BBB, or because the desired therapeutic effect should be locally confined. Local therapy with biological agents optimized for such delivery eliminates systemic toxicity and can thus improve the safety profile of the drug.

[0337]

Table 1

[0338]

Table 2

[0339]

Table 3

[0340] The combined bispecific molecule can consist of molecules described 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 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.

Claims

1. A polypeptide comprising a crystallizable fragment (Fc) region of IgG for use in the prevention or treatment of a disease affecting the central nervous system (CNS), wherein the Fc region has a modification that results in a reduced affinity for the neonatal Fc receptor (FcRn), the Fc region has an H at position 310, the modification is the mutations I253N and H435Q according to the EU numbering system, the polypeptide is administered to the brain.

2. The serum or plasma to brain concentration ratio of the polypeptide is measurable 24 hours after intracranial injection into the striatum of ｔｇ mice and is FcRn ｔｇ measurable 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. at most 1 / 8 of the serum or plasma to brain concentration ratio of the same polypeptide comprising neither an Fc region nor a peptide linker A polypeptide for use in the prevention or treatment of a disease affecting the central nervous system according to claim 1, which is less than a predetermined threshold selected from the group consisting of

3. the reduction in the affinity of the polypeptide for FcRn is a. The dissociation constant (K Ｄ ) characterizing the binding of FcRn to the same polypeptide containing an unmodified Fc region, which is increased by at least 2-fold compared to K Ｄ , and b. K that characterizes the binding of FcRn to the same polypeptide containing one mutant selected from Fc regions having different modifications, namely IAQ (having mutations H310A and H45Q) and AAA (having mutations I253A, H310A and H435A) Ｄ K increased by at least 1.5-fold compared to Ｄ K selected from Ｄ A polypeptide for use in the prevention or treatment of a disease affecting the central nervous system according to claim 1 or 2, characterized by

4. administration to the brain is a. single, intermittent or continuous local infusion including convection enhanced delivery (CED), b. intrathecal or intraventricular administration, c. in situ generation of the polypeptide, d. release from an implantable sustained release formulation, e. molecular transport to the CNS, f. cell transport to the CNS, or g. transport to the CNS after intranasal application An intracranial delivery effected by a method selected from the group consisting of, a polypeptide for use in the treatment or prevention of a disease affecting the central nervous system according to any one of claims 1 to 3.

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.

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 SEQ ID NO: 4 (NHQ).

8. A polypeptide for use in the prevention or treatment of a disease affecting the eye, comprising a crystallizable fragment (Fc) region of IgG, wherein the Fc region has a modification that results in a reduced affinity for the neonatal Fc receptor (FcRn), the Fc region has H at position 310, the modification is the mutations I253N and H435Q according to the EU numbering system, the polypeptide is a polypeptide that is delivered to the eye by intravitreal administration. **Claim 9** A polypeptide for use in the prevention or treatment of a disease affecting the joints, comprising a crystallizable fragment (Fc) region of IgG, wherein the Fc region has a modification that results in a reduced affinity for the neonatal Fc receptor (FcRn), the Fc region has H at position 310, the modification is the mutations I253N and H435Q according to the EU numbering system, the polypeptide is a polypeptide that is delivered to the joints by intra-articular administration. **Claim 10** A polypeptide for use in the prevention or treatment of a disease affecting the lungs, comprising a crystallizable fragment (Fc) region of IgG, wherein the Fc region has a modification that results in a reduced affinity for the neonatal Fc receptor (FcRn), the Fc region has H at position 310, the modification is the mutations I253N and H435Q according to the EU numbering system, the polypeptide is a polypeptide that is delivered to the lungs by inhalation. **Claim 11** A polypeptide for use as a medicament, comprising a crystallizable fragment (Fc) region of IgG, wherein the Fc region has a modification that results in a reduced affinity for the neonatal Fc receptor (FcRn), the Fc region has H at position 310, the modification is the mutations I253N and H435Q according to the EU numbering system, a polypeptide. **Claim 12** A polypeptide for use in the prevention or treatment of a disease according to any one of claims 8 to 11, wherein the Fc region is or comprises SEQ ID NO: 4 (NHQ). **Claim 13** A polypeptide comprising an Fc region of IgG, wherein the Fc region has a modification that results in a reduced affinity for the neonatal Fc receptor (FcRn), the Fc region has H at position 310, the modification is the mutations I253N and H435Q according to the EU numbering system, a polypeptide. **Claim 14** The polypeptide according to claim 13, wherein the polypeptide is an antibody or antibody-like molecule that includes or is linked to the Fc region.

15. The polypeptide according to claim 13 or 14, wherein the Fc region is or includes the sequence of SEQ ID NO: 4 (NHQ).

16. The polypeptide according to any one of claims 13 to 15, for use in the treatment of a disease selected from brain cancer, stroke, dementia, Parkinson's disease, Alzheimer's disease, multiple sclerosis, epilepsy, and traumatic CNS injury.

17. The polypeptide according to claim 10 or any one of claims 13 to 15, for use in the treatment of a disease selected from coronavirus disease 2019, diseases caused by severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome, asthma, allergic asthma, severely uncontrolled asthma, fibrosis, cystic fibrosis, pulmonary fibrosis, chronic obstructive pulmonary disease, influenza, pulmonary edema, sarcoidosis, lung cancer, tuberculosis, human orthopneumovirus, pneumonic plague, anthrax, invasive fungal diseases in the lung, respiratory syncytial virus, pulmonary paracoccidioidomycosis, interstitial lung disease, idiopathic pulmonary fibrosis, and chronic rhinosinusitis with nasal polyps.

18. The polypeptide according to claim 9 or any one of claims 13 to 15, for use in the treatment of a disease selected from rheumatoid arthritis, juvenile rheumatoid arthritis, gout, pseudogout, osteoarthritis, chronic hemophilic synovitis, psoriatic arthritis, and ankylosing spondylitis.

19. The polypeptide according to claim 8 or any one of claims 13 to 15, for use in the treatment of a disease selected from uveal melanoma, uveitis, and wet age-related macular degeneration.

20. A nucleic acid encoding the polypeptide according to any one of claims 13 to 19 or a viral vector comprising the nucleic acid.

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