CD32a-specific binding protein

Abstract

The present invention relates to binding proteins that contain ankyrin repeats, in particular so that they specifically bind to CD32a or specifically target CD32a positive cells. Nucleic acids encoding such binding proteins, nucleic acids, vectors comprising said nucleic acids, particles comprising said binding proteins, host cells comprising said nucleic acids, pharmaceutical compositions, and any of these for use in medicine and / or some gene therapy are disclosed.

Description

[Technical field]

[0001] The present invention relates to binding proteins having binding specificity for CD32a, as well as to nucleic acids encoding such binding proteins, vectors comprising said nucleic acids, said binding proteins for use in medicine and / or gene therapy; and pharmaceutical compositions comprising said binding proteins, nucleic acids or vectors. In particular, the present invention relates to said binding proteins that specifically bind to CD32a but not to CD32b. [Background technology]

[0002] CD32 (also known as FcyRll or FCGR2) is a surface receptor glycoprotein present on various immune cells, such as platelets, neutrophils, macrophages, and dendritic cells. In humans, there are three major CD32 subtypes: CD32a, CD32b, and CD32c.

[0003] CD32a is known to mediate bacterial-induced platelet responses and plays an important role in platelet activation, adhesion, and aggregation to injured blood vessels. Furthermore, CD32a on platelets is involved in heparin-induced thrombocytopenia, a life-threatening side effect of heparin treatment.

[0004] Furthermore, it is known that activation of CD32a is necessary and sufficient for the generation of T cell antitumor cell-mediated immunity. CD32a is also associated with autoimmunity and allergy. For example, it is known that CD32a induces anaphylaxis and allergic reactions. In addition, it is known that the SNP H131R of the CD32a receptor is associated with various functions. CD32a is associated with several human autoimmune diseases, such as rheumatoid arthritis and increased risk of infection.

[0005] Therefore, detection and targeting of CD32a has become of great importance. Nevertheless, the extremely high homology between the major CD32 subtypes makes it very difficult to distinguish between the different types. In the field of HIV research, CD32a has recently been identified as a possible HIV reservoir marker, but this has not been without controversy, as it is technically challenging to identify the small subset of CD32a-positive cells in the presence of CD32b. In the absence of a reliable CD32a-specific cell detection reagent, magnetic bead cell sorting methods had to be used sequentially and in multiple cycles to distinguish the different CD32-positive immune cell subsets and to isolate the minor population of CD4+CD32+ T lymphocytes from peripheral blood.

[0006] At the genetic level, commercially available qPCR primer pairs are available that distinguish between CD32a and CD32b. However, at the cellular level, and more specifically, to detect CD32a on the cell surface, a binding protein capable of distinguishing the highly homologous extracellular domains of CD32a and CD32b is required. Several CD32a antibodies are commercially available, but most also bind CD32b. Those that can be distinguished are either expressed against the intracellular portion of the protein or have not been validated for specificity to detect CD32a on the cell surface. Taken together, there are no known antibodies or antibody analogs that can selectively bind to CD32a on the cell surface. Summary of the Invention [Problem to be solved by the invention]

[0007] We designed a binding protein that can selectively recognize CD32a-positive cells in a mixture of CD32a- and CD32b-expressing cells without prior removal of the CD32b-positive cell population. The discovered binding protein specifically binds to the extracellular domain of the CD32a receptor. [Means for solving the problem]

[0008] In a first aspect, the present invention provides a binding protein comprising at least one ankyrin repeat domain, said at least one ankyrin repeat domain specifically binding to CD32a, said at least one ankyrin repeat domain being i) SEQ ID NOs: 28, 29 and 30, and ii) SEQ ID NOs: 28, 29 and 30, which contain up to 10 amino acid substitutions, independently of each other; and preferably the substitutions are independent of each other at positions 1, 3, 4, 6, 14, 15 and / or 27 of SEQ ID NO: 28, 29 and 30 and / or at up to three other positions of SEQ ID NO: 28, 29 and 30.

[0009] In a second aspect, the present invention relates to a binding protein comprising at least one ankyrin repeat domain, wherein said at least one ankyrin repeat domain specifically binds to CD32a, and said at least one ankyrin repeat domain comprises an amino acid sequence having at least 70% amino acid sequence identity with one ankyrin repeat domain selected from the group consisting of SEQ ID NOs: 1 to 27.

[0010] In a third aspect, the present invention relates to a nucleic acid comprising a nucleic acid sequence encoding a binding protein, an N-terminal capping repeat and / or a C-terminal capping repeat as described herein.

[0011] In a further aspect, the invention relates to a host cell comprising nucleic acid of the third aspect, which optionally expresses a binding protein of the invention.

[0012] In a further aspect, the present invention relates to a vector comprising the nucleic acid molecule of the third aspect.

[0013] In a further aspect, the invention relates to a particle comprising a molecule, which optionally is a binding protein as described herein.

[0014] In a further aspect, the invention relates to a composition comprising a binding protein as described herein, a nucleic acid as described herein, a host cell as described herein, a vector as described herein, a particle as described herein, or a plurality thereof.

[0015] In a further aspect, the present invention relates to a pharmaceutical composition comprising at least one of the binding proteins, nucleic acids, host cells, vectors, and / or particles described herein.

[0016] In a further aspect, the invention relates to a binding protein, a nucleic acid, a host cell, a vector, a particle, a composition or a pharmaceutical composition as described for use as a medicament.

[0017] In yet another aspect, the invention relates to a kit comprising the binding protein, nucleic acid, vector, host cell, particle, composition or pharmaceutical composition described.

[0018] In one aspect of the invention, the binding proteins, nucleic acids, vectors, particles, host cells, compositions, pharmaceutical compositions, or kits disclosed herein are for use in the treatment, prevention, or diagnosis of a condition.

[0019] In the following the content of the figures contained in this specification is described. In this context reference is also made to the above and / or following detailed description of the invention. [Brief description of the drawings]

[0020] [Figure 1]Identification of DARPin clones that bind to CD32a exposed on the cell surface. DARPin E. coli crude extracts were analyzed by flow cytometry for binding to stably expressing CD32 Sup T1 cells (SupT1-CD32a) or CD32b Sup T1 cells (SupT1-CD32b) for N3C DARPin and to stably expressing CD32 CHO cells (CHO-CD32a or CHO-CD32b) for N2C DARPin. Two DARPins that recognize CD32b and CD32a on the cell surface are 53.F1 and 53.2.D8. A: CD32a expressing cell line, B: CD32b expressing cell line. [Diagram 2] Sequence analysis of identified N3C DARPins. Sequence logo of the DARPin library covering N-cap, repeat elements and C-cap. To confirm the framework (grey) and diverse positions (black) of the generated N3C DARPin library, the output repertoire was cloned into plasmid pQE-HisHA and transformed into E. coli XL-1 Blue. Of the 33 clones derived from the VV-N3C library, 26 sequences encoded unique DARPins and sequence logos were generated using WebLogo 3 (http: / / weblogo.threeplusone.com / create.cgi). DARPin52.H6 from the VV-N2C library was not included in this analysis. [Diagram 3] Specific labeling of CD32a-positive cell populations by purified CD32a-DARPin. Purified DARPin was analyzed by flow cytometry for binding to stably expressing CD32a CHO cells (CHO-CD32a), CD32b CHO cells (CHO-CD32b) or wild-type CHO. [Figure 4]Protein sequence alignment of the extracellular domains of CD32a, CD32b, and CD32c isotypes. Alignment of the extracellular domains of human CD32a (amino acids 34 to 217 of SEQ ID NO:87), with the histidine at position 167 mutated to arginine and also referred to as "CD32a H167R" with reference to full length CD32a, or "mCD32a H134R" with reference to mature CD32a), human CD32b (amino acids 43 to 217 of SEQ ID NO:88) and human CD32c (amino acids 43 to 223 of SEQ ID NO:89). Amino acid sequence differences are highlighted in grey. The underlined histidine at position 134 (corresponding to amino acid 167 in the full-length sequence CD32a) is not thought to be involved in specific binding, since recombinant CD32a protein carrying the H167R mutation was used in the ribosome display procedure and cells overexpressing wild-type H167 CD32a protein were used in the cell-binding assay. [Diagram 5] Binding of DARPins to the chimeric SupT1-CD32b cell line. Purified DARPins were analyzed by flow cytometry for binding to the chimeric CD32b protein and to wild-type CD32b or CD32a proteins stably expressed on CHO cells compared to secondary antibody control staining. An overview of the mutations and their location in the extracellular domain of CD32b can be found in Table 2. [Figure 6] CD32a-specific gene transfer by LVs presenting CD32a-DARPins. Binding of recombinant CD32 protein to selected CD32a-DARPins fused to NiV-G protein transiently expressed on the cell surface of 293T cells. [Figure 7] Schematic configuration of Nipah virus pseudotyped lentiviral vector (modified, adapted from Frank et al., 2018). [Figure 8] Transduction with CD32a-specific DARPins. Identification of a CD32a-specific DARPin via transduction with LV vector particles produced in a non-enriched small-scale approach. [Figure 9]Transduction with Nipah pseudotyped CD32a. 53.2.F11-lentiviral vector produced in bulk comparing FCS and Panexin as cell culture supplements. [Figure 10] Affinity determination of purified DARPin 53.2.F11[TEV] for human CD32a and CD32b. Sensorgrams of varying concentrations of DARPin bound to immobilized recombinant human CD32a target protein [A] or recombinant human off-target CD32b protein [B]. Peaks caused by buffer inequity during injection were manually removed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Before the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, which may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0022] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Klbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland) and as set forth in "Pharmaceutical Substances: Syntheses, Patents, Applications" by Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999; "Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals", edited by Susan Budavari et al., CRC Press, 1996, and the United States Pharmacopeia-25 / National Formulary-20, published by the United States Pharmcopeial Convention, Inc., Rockville Md., 2001.

[0023] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology, which are described in the art (e.g., Molecular Cloning: A Laboratory Manual, 2 nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0024] Throughout this specification and the claims that follow, unless the context dictates otherwise, the word "comprise" and variations such as "comprises", "comprising" and the like are understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of other integers or steps or group of integers or steps. In the following text, the different aspects of the invention are defined in more detail. Each aspect thus defined can be combined with other aspects, unless expressly indicated to the contrary. In particular, any feature indicated as being optional, preferred or advantageous may be combined with any other feature indicated as being optional, preferred or advantageous.

[0025] In the following, elements of the present invention are described. These elements are listed with specific embodiments; however, it is understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed to limit the present invention to only the embodiments explicitly described. This specification should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed elements and / or preferred elements. Furthermore, any permutation and combination of all described elements in this application should be considered to be disclosed by the description of this application, unless the context indicates otherwise.

[0026] definition Below are provided definitions of terms frequently used herein that, in each instance of their usage, have their respective defined and preferred meanings in the remainder of the specification.

[0027] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method for individually referring to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if set forth individually herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any examples provided herein, or the use of exemplary language (e.g., "etc.") are intended merely to better illustrate the disclosure and do not limit the scope of the claims. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0028] The term "about" means approximately or approximately, and in one embodiment, in the context of numerical values ​​or ranges defined herein, means ±20%, ±10%, ±5%, or ±3% of the stated or claimed numerical value or range.

[0029] Unless expressly stated otherwise, the term "comprising" is used in the context of this specification to indicate that in addition to the members of the list introduced by "comprising", further members may optionally be present, in some cases. However, as a specific embodiment of the present disclosure, it is expected that the term "comprising" encompasses the possibility that no further members are present, i.e., for the purposes of this embodiment, "comprising" should be understood to have the meaning of "consisting of".

[0030] The term "protein" in the context of the present invention refers to a polypeptide, at least a portion of which has or can acquire a defined three-dimensional configuration by forming secondary, tertiary or quaternary structures within and / or between its polypeptide chains. When a protein is composed of two or more polypeptides, the individual polypeptide chains are linked by non-covalent or covalent bonds, such as disulfide bonds between two polypeptides. Moieties or proteins that have or can acquire a defined three-dimensional configuration by individually forming secondary or tertiary structures are called "protein domains". Such protein domains are well known to those skilled in the art.

[0031] Amino acids are the building blocks of peptides, polypeptides, and proteins. Below are the abbreviations and one-letter codes used for amino acids:

[0032] [Table 1]

[0033] According to the present disclosure, the term "peptide" includes oligopeptides and polypeptides and refers to a substance that contains about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids linked together via peptide bonds. The term "protein" or "polypeptide" refers to larger peptides, particularly peptides having at least about 151 amino acids, although the terms "peptide", "protein" and "polypeptide" are generally used synonymously herein.

[0034] In the context of the present invention, the term "polypeptide" relates to a molecule consisting of one or more chains of a plurality, i.e. two or more, amino acids linked via peptide bonds. Preferably, a polypeptide consists of more than eight amino acids linked via peptide bonds.

[0035] For the purposes of the present invention, a "variant" of an amino acid sequence (peptide, protein or polypeptide) includes an amino acid insertion variant, an amino acid addition variant, an amino acid deletion variant and / or an amino acid substitution variant. The term "variant" includes all splice variants, post-translational modification variants, conformations, isoforms and species homologs, particularly those naturally expressed by cells. The term "variant" particularly includes fragments of an amino acid sequence.

[0036] Amino acid insertion variants include insertion of a single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at a particular site in the amino acid sequence, although random insertions are also possible with appropriate screening of the resulting products. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may be at any position in the protein. Amino acid deletion variants, including deletions at the N-terminus and / or C-terminus of the protein, are also referred to as N-terminus and / or C-terminus truncation variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Modifications are preferentially located at positions of amino acid sequences that are not conserved between homologous proteins or peptides, and / or replace amino acids with other amino acids with similar properties.Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., replacement of similarly charged or uncharged amino acids.Conservative amino acid changes include the replacement of one of a family of amino acids whose side chains are related.

[0037] Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes jointly classified as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0038] The term "sequence similarity" as used in the context of the present invention indicates the percentage of amino acids that are identical or that represent conservative amino acid exchanges. The term "sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between two given sequences. The term "percent identity" is intended to indicate the percentage of identical amino acid residues between the two sequences compared, obtained after best alignment, and this percentage is purely statistical, and the differences between the two sequences are randomly distributed over their entire length. Sequence comparison between two amino acid sequences is conventionally performed by comparing these sequences after optimal alignment, said comparison being performed segment by segment or "window of comparison" to identify and compare local regions of sequence similarity. Alignment to determine sequence similarity, preferably sequence identity, is performed using tools known in the art, preferably using best sequence alignment, for example using CLC main Workbench (CLC bio) or Align, using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5. The percentage of identity is determined with reference to the full length sequence used for comparison, not just the most similar sequence or sequence stretch. Thus, an amino acid that has 100% sequence identity with 50 consecutive amino acids of a 100 amino acid long sequence used for comparison has only 50% sequence identity (assuming that there are no amino acids with identity outside the 50 consecutive amino acids).

[0039] Homologous amino acid sequences, according to the present disclosure, exhibit an identity of at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98% or at least 99% of the amino acid residues.

[0040] Preferably, the degree of similarity, preferably the degree of identity, between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence, in the context of the binding proteins of the invention, will be at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given for an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably consecutive amino acids. In a preferred embodiment, the degree of similarity or identity is given for the full length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using the best sequence alignment, for example, using Align, with standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0041] The amino acid sequence variants described herein can be easily prepared by those skilled in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or proteins with substitutions, additions, insertions or deletions is described in detail, for example, in Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be easily prepared with the aid of known peptide synthesis techniques, for example, solid phase synthesis and similar methods.

[0042] In one embodiment, the fragment or variant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence as used in the context of the present invention relates to any fragment or variant that exhibits one or more functional properties identical or similar to the amino acid sequence from which it is derived, i.e. is functionally equivalent. The term "functional fragment" or "functional variant", as used herein, refers in particular to a variant molecule or sequence that comprises an amino acid sequence in which one or more amino acids have been modified compared to the amino acid sequence of the parent molecule or sequence, and that is still capable of performing one or more functions of the parent molecule or sequence, such as binding to a target molecule. In one embodiment, the modification of the amino acid sequence of the parent molecule or sequence does not significantly affect or change the binding properties of the molecule or sequence. In different embodiments, the binding of the functional fragment or functional variant is significantly reduced, for example, the binding of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. However, in other embodiments, binding of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0043] An amino acid sequence (peptide, protein or polypeptide) "derived from" a specified amino acid sequence (peptide, protein or polypeptide) refers to the origin of the initial amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence identical, essentially identical or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof. For example, one skilled in the art will understand that antigens suitable for use herein can be modified to differ in sequence from the naturally occurring or native sequence from which they are derived while retaining the desired activity of the native sequence.

[0044] The term "recombinant" as used in recombinant protein, recombinant protein domain, recombinant binding protein, etc., means that said polypeptide is produced by the use of recombinant DNA technology well known to those skilled in the relevant art. For example, a recombinant DNA molecule (e.g., produced by gene synthesis) encoding a polypeptide can be cloned into a bacterial expression plasmid (e.g., pQE30, Qiagen), a yeast expression plasmid or a mammalian expression plasmid. For example, when such a constructed recombinant bacterial expression plasmid is inserted into a suitable bacterium (e.g., E. coli), the bacterium can produce the polypeptide encoded by the recombinant DNA. A polypeptide thus produced is called a recombinant polypeptide. Thus, the term "recombinant" in the context of the present invention means "genetically engineered". Preferably, a "recombinant" such as a recombinant cell in the context of the present invention does not exist in nature.

[0045] As used herein, the term "naturally occurring" refers to the fact that a molecule exists in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by man in the laboratory is naturally occurring.

[0046] The term "binding protein" in the context of the present invention refers to a protein comprising one or more binding domains. Furthermore, such binding proteins may comprise additional protein domains that are not binding domains, multimerization sites, polypeptide tags, polypeptide linkers and / or non-proteinaceous polymer molecules. Examples of multimerization sites are immunoglobulin heavy chain constant regions that pair to provide a functional immunoglobulin Fc domain, and leucine zippers, or polypeptides that contain free thiols that form intermolecular disulfide bonds between two such polypeptides. Examples of non-proteinaceous polymer molecules are hydroxyethyl starch (HES), polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes. A single Cys residue is used to attach other moieties to the polypeptide, for example by using maleimide chemistry well known to those skilled in the art. Preferably, said binding protein is a recombinant binding protein.

[0047] The term "repeat protein" refers to a protein / (poly)peptide comprising one or more repeat domains. Preferably, each of said repeat proteins comprises up to 4 repeat domains. More preferably, each of said repeat proteins comprises up to 2 repeat domains. Most preferably, each of said repeat proteins comprises only one repeat domain. Furthermore, said repeat proteins may comprise additional non-repeat protein domains, (poly)peptide tags, (poly)peptide linkers, enzymes that may allow detection of the repeat protein (e.g. alkaline phosphatase), or moieties that can be used for targeting (e.g. immunoglobulins or fragments thereof) and / or effector molecules.

[0048] The terms "(poly)peptide tag" or "protein tag" refer interchangeably in the context of the present invention to an amino acid sequence attached to a (poly)peptide / protein, where said amino acid sequence is useful for the purification, detection or targeting of said (poly)peptide / protein, or said amino acid sequence improves the physicochemical behavior of the polypeptide / protein, or said amino acid sequence has an effector function. The individual (poly)peptide tags, parts and / or domains of the binding protein are linked to each other directly or via a polypeptide linker. All these polypeptide tags are well known in the art and are fully available to the skilled artisan. Examples of polypeptide tags are small polypeptide sequences, such as His, HA, myc, FLAG or Strep-tags, GFP or moieties that allow the detection of said (poly)peptide / protein, such as enzymes (e.g. enzymes such as alkaline phosphatase), or moieties that can be used for targeting and / or as effector molecules (e.g. immunoglobulins or fragments thereof). The enzyme may also be a protease, such as TEV protease, the enzyme may be, for example, factor X. The (poly)peptide or protein may present tags, such as fluorescent or radiolabeled tags, primarily known in the art.

[0049] The term "polypeptide linker" in the context of the present invention refers to an amino acid sequence capable of linking, for example, two protein domains, a polypeptide tag and a protein domain, a protein domain and a non-polypeptide moiety such as polyethylene glycol, or two sequence tags. Such additional domains, tags, non-polypeptide moieties and linkers are known to those skilled in the relevant art. Particular examples of such linkers are glycine-serine-linkers and proline-threonine-linkers of variable length; preferably, said linkers have a length of 2 to 24 amino acids; more preferably, said linkers have a length of 2 to 16 amino acids.

[0050] The term "repeat domain" in the context of the present invention refers to a protein domain comprising two or more consecutive repeat units (modules) as structural units, said structural units having the same fold and stacking tightly to form, for example, a coiled-coil structure with a common hydrophobic core. Preferably, the repeat domain further comprises an N-terminal and / or a C-terminal capping unit (or module). More preferably, said N-terminal and / or C-terminal capping unit (or module) is a capping repeat.

[0051] The repeat protein may further comprise an N-terminal and / or C-terminal capping module having an amino acid sequence different from any one of said repeat modules. The term "capping module" refers to a polypeptide fused to the N-terminal or C-terminal repeat module of a repeat domain, said capping module forming tight tertiary interactions with said repeat module, thereby providing a cap that shields the hydrophobic core of said repeat module from the solvent on the side not in contact with the consecutive repeat module. The N-terminal and / or C-terminal capping module may be or be derived from a capping unit or other domain found in a naturally occurring repeat protein adjacent to the repeat unit.

[0052] The term "capping unit" in the context of the present invention refers to a naturally occurring folded (poly)peptide, which defines a specific structural unit fused at its N-terminus or C-terminus to a repeat unit, with which it forms tight tertiary interactions, thereby providing a cap that shields one hydrophobic core of the repeat unit from the solvent. Preferably, the capping unit is a capping repeat. The term "capping repeat" refers to a capping unit that has a similar or the same fold and / or sequence similarity with the adjacent repeat unit.

[0053] The term "structural unit" in the context of the present invention refers to a locally ordered portion of a polypeptide formed by three-dimensional interactions between two or more segments of secondary structure that are adjacent to each other along the polypeptide chain. Such a structural unit represents a structural motif. The term "structural motif" refers to a three-dimensional arrangement of secondary structure elements present in at least one structural unit. Structural motifs are well known to those skilled in the art. A structural unit cannot acquire a defined three-dimensional arrangement by itself; however, when arranged sequentially, for example as repeat modules in a repeat domain, adjacent units mutually stabilize each other, resulting in a coiled-coil structure.

[0054] The terms "designed repeat protein" and "designed repeat domain" refer interchangeably to repeat proteins or repeat domains, respectively, in the context of the present invention. Designed repeat proteins and designed repeat domains are synthetic and not naturally occurring. They are artificial proteins or domains, respectively, obtained by expression of corresponding designed nucleic acids. Preferably, expression is carried out in prokaryotic cells, such as eukaryotic or bacterial cells, or using cell-free in vitro expression systems. Thus, designed ankyrin repeat proteins (i.e. DARPins) correspond to the binding proteins of the present invention that contain at least one ankyrin repeat domain.

[0055] The term "repeat unit" in the context of the present invention refers to the repeat amino acid sequences of designed repeat domains, which are originally derived from repeat units (modules) of naturally occurring repeat proteins. Each repeat domain is derived from one or more repeat units of a family or subfamily or a naturally occurring repeat protein, such as the family or armadillo repeat proteins or ankyrin repeat proteins.

[0056] The term "repeat module" in the context of the present invention refers to an amino acid sequence comprising one or more naturally occurring repeat protein repeat sequence motifs, said "repeat module" (unit) being found in multiple copies and exhibiting a defined folding topology common to all said motifs that determines the folding of the protein. Such repeat units comprise framework and interacting residues. Examples of such repeat units are armadillo repeat units, leucine-rich repeat units, ankyrin repeat units, tetratricopeptide repeat units, HEAT repeat units, leucine-rich variant repeat units. Naturally occurring proteins comprising two or more such repeat units are called "naturally occurring repeat proteins". The amino acid sequences of the individual repeat units of a repeat protein may have a significant number of mutations, substitutions, additions, and / or deletions when compared to each other while substantially retaining the general pattern, i.e. motif, of the repeat units.

[0057] The term "set of repeat modules" in the context of the present invention refers to the total number of repeat modules present in a repeat domain. Such a "set of repeat modules" present in a repeat domain may comprise two or more consecutive repeat modules, may consist of only one type of repeat module present in two or more copies, or may consist of two or more different types of modules, each present in one or more copies. Such a set of repeat modules, for example comprising three repeat modules, may comprise, consecutively from N-terminus to C-terminus, repeat module 1, repeat module 2 and repeat module 3, for example as shown in FIG. 5. Repeat module 1 as shown in FIG. 5 preferably comprises amino acids 29 to 61. Repeat module 2 as shown in FIG. 5 preferably comprises amino acids 62 to 94. Repeat module 3 as shown in FIG. 5 preferably comprises amino acids 95 to 127.

[0058] Different repeat domains may have the same number of repeat modules per repeat domain or may have different numbers of repeat modules per repeat domain.

[0059] Preferably, the repeat modules comprised in the set are homologous repeat modules. In the context of the present invention, the term "homologous repeat module" refers to a repeat module in which more than 70% of the framework residues of said repeat module are homologous. Preferably, more than 80% of the framework residues of said repeat module are homologous. Most preferably, more than 90% of the framework residues of said repeat module are homologous. Computer programs for determining the percentage of homology between polypeptides, such as Fasta, Blast or Gap, are known to those skilled in the relevant art.

[0060] Repeat units may contain positions that have amino acid residues that are present in all copies of the corresponding repeat unit ("fixed positions") and positions that have different or "randomized" amino acid residues ("randomized positions"), i.e. framework and interacting residues.

[0061] An example of such a repeat unit is an ankyrin repeat unit. A naturally occurring protein that contains two or more such repeat units is called a "naturally occurring repeat protein". The amino acid sequences of the individual repeat units of a repeat protein may have a significant number of mutations, substitutions, additions, and / or deletions when compared to each other while substantially retaining the general pattern, or motif, of the repeat units.

[0062] The term "ankyrin repeat unit" in the context of the present invention refers to a repeat unit which is an ankyrin repeat. Ankyrin repeats are well known to those skilled in the art.

[0063] The term "DARPin" in the context of the present invention refers to engineered ankyrin repeat proteins, which are genetically engineered antibody mimetic proteins that exhibit highly specific and high affinity target protein binding. DARPins are well known in the art and were first described in Binz HK, et al.(2003)JMB.332 (2):489-503 and reviewed in Pluckthun A (2015).Annu.Rev.Pharmacol.Toxicol.55 (1):489-511. DARPins are based on naturally occurring ankyrin repeat proteins, but contain one or more amino acid mutations that can affect, for example, binding affinity for a given target molecule, cell surface expression, etc. Typically, DARPins contain 4-5 repeats, of which the first repeat (N-capping repeat) and the last repeat (C-capping repeat) serve to shield the hydrophobic protein core from the aqueous environment. DARPins correspond to the average size of a naturally occurring ankyrin repeat protein domain. Proteins with fewer than three repeats (i.e. capping repeats and one internal repeat) do not form sufficiently stable tertiary structures. The molecular weight of a DARPin depends on the total number of repeats, as shown in the chart below: DARPins of the invention preferably contain 2-6, more preferably 3-5, ankyrin repeat units, flanked by N- and C-capped repeats. Each ankyrin repeat unit contains approximately 33 amino acid residues.

[0064] The term "repeat sequence motif" or "repeat consensus sequence" in the context of the present invention refers to an amino acid sequence deduced from one or more repeat units. Such a repeat sequence motif comprises a framework residue position and a target interaction residue position. The framework residue position corresponds to the position of a framework residue of the repeat unit. The target interaction residue position corresponds to the position of a target interaction residue of the repeat unit. Such a repeat sequence motif comprises fixed positions and randomized positions. The term "fixed position" refers to an amino acid position in a repeat sequence motif, which position is set to a specific amino acid. Often, such fixed positions correspond to the position of a framework residue.

[0065] The term "randomized position" in the context of the present invention refers to an amino acid position in a repeat sequence motif, where two or more amino acids are allowed at said amino acid position. Often, such randomized positions correspond to the positions of target interaction residues. However, some positions of framework residues are also randomized.

[0066] The term "framework residues" in the context of the present invention refers to amino acid residues of a repeat unit, or the corresponding amino acid residues of a repeat module, which contribute to the folding topology, i.e. contribute to the folding of said repeat unit (or module) or contribute to interactions with neighboring units (or modules). Such contributions may be interactions with other residues within the repeat unit (or module), or influences on the backbone conformation of the polypeptide, such as those found in α-helices or β-sheets, or amino acid stretches forming linear polypeptides or loops.

[0067] The term "target interaction residues" in the context of the present invention refers to amino acid residues of a repeat unit, or corresponding amino acid residues of a repeat module, that contribute to the interaction with a target substance. Such contribution may be by direct interaction with the target substance or by influence on other direct interaction residues, for example by stabilizing the conformation of the polypeptide of the repeat unit (or module) to allow or enhance the interaction of the direct interaction residues with said target. Such framework and target interaction residues can be identified by analysis of structural data obtained by physicochemical methods such as X-ray crystallography, NMR and / or CD spectroscopy, or by comparison with known related structural information familiar to practitioners of structural biology and / or bioinformatics.

[0068] The term "folding topology" in the context of the present invention refers to the tertiary structure of said repeat unit or repeat module. The folding topology is determined by stretches of amino acids forming at least part of an α-helix or a β-sheet, or by stretches of amino acids forming linear polypeptides or loops, or by any combination of α-helices, β-sheets and / or linear polypeptides / loops.

[0069] The term "consecutive" in the context of the present invention refers to an arrangement in which repeat units or repeat modules are arranged in tandem. In a designed repeat protein, there are at least two, usually about 2-6, in particular at least about 6, and frequently 20 or more repeat units. In most cases, the repeat units of a repeat domain show a high degree of sequence identity (same amino acid residues at corresponding positions) or sequence similarity (different amino acid residues but with similar physicochemical properties), and some of the amino acid residues may be critical residues that are strongly conserved. However, a high degree of sequence variability by insertion and / or deletion and / or substitution of amino acids between different repeat units of a repeat domain will be possible, as long as the common folding topology of the repeat units is maintained.

[0070] In most cases, the repeat units show a high degree of sequence identity (same amino acid residues at corresponding positions) or sequence similarity (different amino acid residues but with similar physicochemical properties), and some of the amino acid residues may be key residues that are highly conserved in different repeat units found in naturally occurring proteins.

[0071] However, a high degree of sequence variability would be possible through insertions and / or deletions and / or substitutions of amino acids between the different repeat units found in naturally occurring proteins, so long as the common folding topology is maintained.

[0072] The terms "specifically binds to a target", "target specificity", "having binding specificity for a target", "specific binding" or "specifically binds" are interchangeable and in the context of the present invention refer to the binding of a binding protein or binding domain to the extracellular domain of human CD32a (amino acids 34 to 217 of SEQ ID NO: 87 (optionally the extracellular domain of the naturally occurring CD32a variants Q63R, M140V, H167R and I218V, positions are given with reference to the full length sequence according to SEQ ID NO: 87)) without any unrelated Preferably, the binding protein or binding domain of the invention binds to the extracellular domain of human CD32a (amino acids 34 to 217 of SEQ ID NO: 87 (optionally the extracellular domain of natural CD32a variants Q63R, M140V, H167R and I218V)) with a lower dissociation constant than the extracellular domain of human CD32b (amino acids 43 to 217 of SEQ ID NO: 88) or the extracellular domain of human CD32c (amino acids 43 to 223 of SEQ ID NO: 89). A lower dissociation constant is at least 10-fold lower, preferably at least 10-fold lower. 2 times lower, preferably 10 3 times lower, preferably 10 4 times lower, and preferably 10 5 In a most preferred embodiment, the dissociation constant for the extracellular domain of human CD32a (amino acids 34 to 217 of SEQ ID NO: 87 (optionally the natural variants Q63R, M140V, H167R and I218V)) is at least 10 6 , more preferably 10 7 , more preferably 10 8 , or most preferably 10 9 1-fold lower than the corresponding dissociation constant for an unrelated protein, such as for the extracellular domain of human CD32b (amino acids 43 to 217 of SEQ ID NO:88), and lower than the extracellular domain of human CD32c (amino acids 43 to 223 of SEQ ID NO:89).

[0073] The term "target" refers to an individual molecule, such as a nucleic acid molecule, a polypeptide or protein, a carbohydrate, or other naturally occurring molecule, including any portion of such an individual molecule, or a complex of two or more of such molecules. A target may be a whole cell or tissue sample, or a non-natural molecule or portion. Preferably, a target is a naturally occurring or non-naturally occurring polypeptide, or a polypeptide modified by chemical modification, such as natural or non-natural phosphorylation, acetylation, or methylation. In particular, in the present invention, the target is CD32a.

[0074] The term "CD32a" refers to a protein also known as FCGR2A. CD32a is a low affinity receptor II-a that binds to the Fc region of immunoglobulin gamma. The extracellular domain of human CD32a is highly homologous to the extracellular domains of the related proteins CD32b and CD32c. An alignment of the extracellular domains of human CD32a (amino acids 34 to 217 of SEQ ID NO:87), with the histidine at position 167 mutated to arginine, also referred to as "CD32a H167R" in reference to full-length CD32a and "mCD32a H134R" in reference to mature CD32a), human CD32b (amino acids 43 to 217 of SEQ ID NO:88), and human CD32c (amino acids 43 to 223 of SEQ ID NO:89) is shown in FIG. 4.

[0075] The term "consensus sequence" as used in the context of the present invention refers to the calculated order of the most frequent residues, either nucleotides or amino acids, found at each position in a sequence alignment between two or more sequences. It represents the result of a multiple sequence alignment in which related sequences are compared with each other and similar sequence motifs are calculated. Conserved sequence motifs are depicted as consensus sequences and show identical amino acids, i.e. amino acids that are identical between the compared sequences, conserved amino acids, i.e. amino acids that differ between the compared amino acid sequences but all amino acids belong to a particular functional or structural amino acid group, e.g. polar or neutral, and variable amino acids, i.e. amino acids that show no obvious relationship between the compared sequences. Table 1 shows the alignment of the sequences of the repeat units, including the N- and C-capping units. The alignments were made manually or an algorithm, Clustal Omega, using default settings, was used to make the alignments mentioned. The seven amino acid positions that can be substituted in some embodiments are highlighted.

[0076] The term "terminal plasma half-life" of a drug, such as a binding protein or binding domain of the invention, refers to the time required for the plasma concentration of the drug applied to a mammal to reach half after pseudo-equilibrium has been reached. This half-life is not defined as the time required to eliminate half of the dose of the drug administered to the mammal.

[0077] The term "bioactive compound" refers to a compound that, when applied to a mammal having a disease, modifies said disease. Bioactive compounds have antagonistic or agonistic properties and may be proteinaceous or non-proteinaceous bioactive compounds.

[0078] The term "compete for binding" means that two different binding domains of the present invention cannot bind to the same target simultaneously, while both can bind to the same target individually. Thus, such two binding domains compete for binding to the target. Preferably, the two competing binding domains bind to overlapping or the same binding epitope on the target. Methods such as competitive enzyme-linked immunosorbent assay (ELISA) or competitive SPR measurement (e.g., by using the Proteon device from BioRad) for determining whether two binding domains compete for binding to a target are well known to those skilled in the art.

[0079] The term "polynucleotide" or "nucleic acid" as used herein includes DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. Nucleic acids may be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the present invention, polynucleotides are preferably isolated. Nucleic acids may be contained in a vector.

[0080] The terms "vector" or "expression vector" are used interchangeably in the context of the present invention and refer to a polynucleotide or a mixture of polynucleotides and proteins that can or will introduce the collection of nucleic acids of the present invention or a nucleic acid that is part of the collection of nucleic acids of the present invention into a cell, preferably a mammalian cell. Examples of vectors include, but are not limited to, plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviruses, adenovirus-associated virus vectors (AAV), adenovirus or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC) or P1 artificial chromosomes (PAC). Said vectors include not only cloning vectors but also expression vectors. In particular, vectors are used to transport the promoter and the collection of nucleic acids of the present invention or a nucleic acid that is part of the collection of nucleic acids into a suitable host cell. An expression vector can include a "replicon" polynucleotide sequence that facilitates autonomous replication of the expression vector in a host cell. Once inside the host cell, the expression vector can replicate independently of or simultaneously with the host chromosomal DNA, generating multiple copies of the vector and its inserted DNA. If a non-replicating expression vector is used (as is often the case for safety reasons), the vector may not replicate but simply directly express the nucleic acid. Depending on the type of expression vector, the expression vector may be lost from the cell, i.e., only transiently express the neo-antigen encoded by the nucleic acid, or it may be stable within the cell. Expression vectors usually contain an expression cassette, i.e., the necessary elements that allow transcription of the nucleic acid into an mRNA molecule.

[0081] The term "viral vector" is used in the context of the present invention to refer to a single- or double-stranded nucleic acid sequence capable of assembling into an infectious viral particle. This nucleic acid sequence may be a complete or partial viral genome. In the latter case, the viral genome preferably contains one or more heterologous genes. For some viral particles, only very short sequences of the viral genome are necessary to allow the assembly of infectious viral particles. For example, only short (approximately 200 bp long) repeat sequences (usually 4.5 to 5.3 kB for adeno-associated viruses) located at the 5' and 3' of a given length of heterologous nucleic acid are required to allow the assembly of infectious adeno-associated viral particles. The minimum nucleic acid sequence for the assembly of a given virus is well known. The larger the viral genome and the smaller the minimum viral sequence for the assembly of infectious viral particles, the larger the heterologous gene that can be inserted into the viral vector.

[0082] As used herein, "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells. They are one of the most efficient methods of gene delivery vectors, as they can deliver significant amounts of genetic information to the DIMA of host cells. HIV, SIV, and FIV are all examples of lentiviruses. Lentivirus-derived vectors provide a means to achieve significant levels of gene transfer in vivo.

[0083] As used herein, "adenovirus-associated viral vector" refers to the genus Dependoparvovirus, which belongs to the Parvoviridae family. This viral vector can be used in gene therapy, infecting both dividing and quiescent cells and can persist in an extrachromosomal state without integrating into the host cell genome. Adeno-associated virus (AAV) is a non-enveloped virus that can be engineered to deliver DNA to target cells. AAV recombinant particles can be made that lack viral genes and contain DNA sequences of interest for various therapeutic applications, making it one of the safest strategies for gene therapy.

[0084] The term "encode" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, mRNA, etc., to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of a gene or cDNA, can be referred to as encoding the protein or product of that gene or cDNA.

[0085] According to the present disclosure, the term "RNA-encoded" means that when the RNA is present in the appropriate environment, such as within a cell of a target tissue, it can direct the assembly of amino acids to produce the peptide or protein that it encodes, during the process of translation. In one embodiment, the RNA can interact with the translation machinery of the cell, allowing translation of the peptide or protein. The cell can produce the encoded peptide or protein intracellularly (e.g., in the cytoplasm and / or in the nucleus), secrete the encoded peptide or protein, or produce it on its surface.

[0086] As used herein, "endogenous" refers to any substance that is derived from or produced within an organism, cell, tissue, or system.

[0087] As used herein, the term "exogenous" refers to any substance introduced from outside or produced outside an organism, cell, tissue, or system.

[0088] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence. Expression may be transient or stable. According to the present invention, the term expression also includes "abnormal expression" or "abnormal expression".

[0089] According to the present invention, a gene expression level is considered to be "altered" if the gene expression is increased or decreased by 10%, 25%, 50% or more compared to a control level. Alternatively, an expression level is considered to be "increased" or "decreased" if the gene expression is increased or decreased by at least 0.1-fold, at least 0.2-fold, at least 1-fold, at least 2-fold, at least 5-fold, or at least 10-fold or more compared to a control level.

[0090] In the context of the present invention, the phrase "control level" refers to the protein expression level detected in a control sample, and includes both "normal control level" and "disease control level". A control level may be a single expression pattern derived from a single reference population, or may be derived from multiple expression patterns. For example, a control level may be a database of expression patterns from previously tested cells. A "normal control level" refers to the gene expression level detected in a population of normal, healthy individuals, or individuals known not to be suffering from a condition associated with altered expression of CD32a (such as HIV-related disease). A normal individual is one who does not have clinical symptoms of a condition associated with altered expression of CD32a. On the other hand, a "disease control level" refers to the expression profile of CD32a-related genes found in a population suffering from a condition associated with altered expression of CD32a.

[0091] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably and refer to the joining of two or more elements or components or domains.

[0092] In the context of the present disclosure, the term "particle" refers to a structured entity formed by a molecule or a molecular complex. In one embodiment, the term "particle" refers to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure dispersed in a medium. In one embodiment, the particle is a nucleic acid-containing particle, such as a particle containing DNA, RNA or a mixture thereof.

[0093] Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged nucleic acids are involved in particle formation. This results in complexation and spontaneous formation of nucleic acid particles. In one embodiment, the nucleic acid particles are nanoparticles.

[0094] As used in this disclosure, "nanoparticles" refers to particles having an average diameter suitable for parenteral administration.

[0095] "Nucleic acid particles" can be used to deliver nucleic acids to a target site of interest (e.g., cells, tissues, organs, etc.). Nucleic acid particles can be formed from at least one cationic or cationic ionizable lipid or lipid-like substance, such as DOTAP, at least one cationic polymer, such as protamine, or a mixture thereof, and nucleic acid. Nucleic acid particles include lipid nanoparticle (LNP)-based formulations and lipoplex (LPX)-based formulations.

[0096] Without wishing to be bound by any theory, it is believed that cationic or cationically ionizable lipids or lipid-like materials and cationic polymers combine with nucleic acids to form aggregates which result in colloidally stable particles.

[0097] The particles described herein may further comprise at least one lipid or lipid-like material other than a cationic or cationically ionizable lipid or lipid-like material, at least one polymer other than a cationic polymer, or a mixture thereof.

[0098] A nucleic acid particle may contain more than one type of nucleic acid molecule, and the molecular parameters of the nucleic acid molecules may be similar or different from each other, such as with respect to molar mass, or basic structural elements such as molecular structure, capping, coding regions or other features.

[0099] The nucleic acid particles described herein, in one embodiment, may have an average diameter in the range of about 30 nm to about 1000 nm, about 50 nm to about 800 nm, about 70 nm to about 600 nm, about 90 nm to about 400 nm, or about 100 nm to about 300 nm.

[0100] The nucleic acid particles described herein, e.g., nucleic acid particles made by the processes described herein, exhibit a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, or less than or equal to about 0.2. By way of example, the nucleic acid particles can exhibit a polydispersity index in the range of about 0.1 to about 0.3 or about 0.2 to about 0.3.

[0101] The nucleic acid particles described herein can be prepared using a wide variety of methods, which may include obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like material and / or at least one cationic polymer, and mixing the colloid with nucleic acid to obtain a nucleic acid particle.

[0102] The term "colloid" as used herein refers to a type of homogeneous mixture in which the dispersed particles do not settle. The insoluble particles in the mixture are fine, ranging from 1 to 1000 nanometers in diameter. The mixture may be called a colloid or a colloidal suspension. The term "colloid" may also refer to only the particles in the mixture, rather than the entire suspension.

[0103] The term "mean diameter" refers to the average hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) by data analysis using the so-called cumulant algorithm, which results in the so-called Z-axis, which has a length dimension. 平均 , and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, IS0 13321). Here, the "average diameter", "diameter" or "size" of a particle is defined as this Z 平均 Used synonymously with the value of

[0104] The "polydispersity index", as mentioned in the definition of "average diameter", is preferably calculated based on dynamic light scattering measurements by the so-called cumulant analysis. Under certain prerequisites, it can be taken as a measure of the size distribution of the nanoparticle ensemble.

[0105] Various types of nucleic acid-containing particles have been previously described as suitable for particulate nucleic acid delivery (e.g., Kaczmarek, JC et al., 2017, Genome Medicine 9, 60). For non-viral nucleic acid delivery vehicles, nanoparticle encapsulation of nucleic acids can physically protect the nucleic acid from degradation and, depending on the specific chemical properties, aid in cellular uptake and escape from endosomes.

[0106] The present disclosure describes a particle that comprises nucleic acid, at least one cationic or cationically ionizable lipid or lipid-like substance, and / or at least one cationic polymer, and that associates with nucleic acid to form a nucleic acid particle, as well as a composition that comprises such a particle.Nucleic acid particle may comprise nucleic acid that is complexed to the particle in different forms by non-covalent interactions.The particles described herein are not virus particles, particularly infectious virus particles, i.e., they cannot infect cells virally.

[0107] Suitable cationic or cationically ionizable lipids or lipid-like substances and cationic polymers form nucleic acid particles and are included in the term "particle-forming components" or "particle-forming agents". The term "particle-forming components" or "particle-forming agents" refers to any component that associates with nucleic acid to form a nucleic acid particle. Such components include any component that can become part of a nucleic acid particle.

[0108] Cationic polymers are popular materials for nanoparticle-based delivery due to their high chemical flexibility. Typically, cationic polymers are used to electrostatically condense negatively charged nucleic acids into nanoparticles. These positively charged groups, often consisting of amines, are thought to change protonation state at pH 5.5 to 7.5, causing ionic imbalance that leads to endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine, polyethyleneimine, and naturally occurring polymers such as chitosan have all been applied in nucleic acid delivery and are suitable as cationic polymers herein. In addition, some researchers have synthesized polymers specifically for nucleic acid delivery. In particular, poly(β-amino esters) are widely used for nucleic acid delivery due to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers herein.

[0109] The term "genetic modification" includes the transfection of cells with a nucleic acid. The term "transfection" relates to the introduction of a nucleic acid, particularly RNA, into a cell. For the purposes of the present invention, the term "transfection" also includes the introduction of a nucleic acid into a cell, or the uptake of a nucleic acid by such a cell, where the cell may be present in a subject, e.g., a patient. Thus, according to the present invention, cells for transfection of a nucleic acid as described herein may be present in vitro or in vivo, e.g., the cell may form part of an organ, tissue and / or organism of a patient. According to the present invention, transfection may be transient or stable. Depending on the application of transfection, it is sufficient that the transfected genetic material is expressed transiently. RNA may be transfected into a cell to transiently express its encoded protein. Since the nucleic acid introduced in the transfection process is not usually integrated into the nuclear genome, the foreign nucleic acid is diluted or degraded by mitosis. Cells that allow episomal amplification of the nucleic acid greatly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, stable transfection must occur. Such stable transfection can occur when the nucleic acid introduced in the transfection process is integrated into the nuclear genome, and can be achieved, for example, by using viral-based or transposon-based systems for transfection. Generally, cells genetically modified to express an antigen receptor are stably transfected with a nucleic acid encoding the antigen receptor, whereas generally, the nucleic acid encoding the antigen is transiently transfected into the cells.

[0110] The term "pharmaceutical composition" relates to a formulation comprising a therapeutically active agent, preferably together with a pharma- ceutically acceptable carrier, diluent and / or excipient. Said pharmaceutical composition is useful for treating, preventing or reducing the severity of a disease or disorder by administration of said pharmaceutical composition to a subject. Pharmaceutical compositions are also known in the art as pharmaceutical formulations.

[0111] The term "pharmaceutical acceptable" as used herein preferably means non-toxicity of a material that does not interact with the action of the active agent of the pharmaceutical composition. In particular, "pharmaceutical acceptable" means approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopoeia, the European Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, and more particularly in humans.

[0112] The term "carrier" refers to a natural or synthetic organic or inorganic ingredient, in which the active ingredient is formulated to facilitate, enhance or enable application. According to the present invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to a subject. Possible carrier substances (e.g., diluents) are, for example, sterile water, Ringer's solution, lactated Ringer's solution, physiological saline, bacteriostatic saline (e.g., saline containing 0.9% benzyl alcohol), phosphate buffered saline (PBS), Hank's solution, fixed oils, polyalkylene glycols, hydrogenated naphthalenes, and biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the carrier is PBS. The resulting solution or suspension is preferably isotonic with the blood of the recipient. Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences, 17th ed., 1985, Mack Publishing Co.

[0113] The term "cell" is used in the context of the present invention to refer to a eukaryotic or prokaryotic cell, such as a bacterial cell, a yeast cell, or a mammalian cell, preferably a human, mouse, rat, rabbit, dog, monkey, or cat cell.

[0114] Aspects and Preferred Embodiments of the Invention In the following, the different aspects of the invention are defined in more detail. Each aspect thus defined can be combined with any other aspect, unless expressly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous can be combined with any other feature or features indicated as being preferred or advantageous.

[0115] In the work leading to the present invention, it was surprisingly shown that a specific binding protein is able to distinguish between the highly homologous CD32a and CD32b.

[0116] In a first aspect, the present invention provides a binding protein comprising at least one ankyrin repeat domain, wherein said at least one ankyrin repeat domain specifically binds to CD32a, said at least one ankyrin repeat domain being i) SEQ ID NOs: 28, 29 and 30, and ii) SEQ ID NOs: 28, 29 and 30, which contain up to 10 amino acid substitutions, independently of each other; The ankyrin repeat module comprises at least one ankyrin repeat module having an amino acid sequence selected from the group consisting of:

[0117] Preferably, up to 9 amino acids are substituted, more preferably up to 8 amino acids, more preferably up to 7 amino acids, more preferably up to 6 amino acids, more preferably up to 5 amino acids, more preferably up to 4 amino acids, more preferably up to 3 amino acids, more preferably up to 2 amino acids, more preferably up to 1 amino acid, and most preferably, no amino acids of SEQ ID NOs: 28, 29 and 30 are substituted.

[0118] Wherein, if 10 amino acids are replaced, then up to 10 replaced amino acids are preferably located at positions 1, 3, 4, 6, 14, 15, 27 and / or 3 further positions of SEQ ID NO:28, 29 and 30; if 9 amino acids are replaced, then up to 9 replaced amino acids are preferably located at positions 1, 3, 4, 6, 14, 15, 27 and / or 2 further positions of SEQ ID NO:28, 29 and 30; if 8 amino acids are replaced, then up to 8 amino acids are preferably located at positions 1, 3, 4, 6, 14, 15, 27 and / or 1 further position of SEQ ID NO:28, 29 and 30; if 7 amino acids are replaced, then up to 7 amino acids are preferably located at positions 1, 3, 4, 6, 14, 15, 27 of SEQ ID NO:28, 29 and 30.

[0119] Preferably, when the amino acids at positions 1, 3, 4, 6, 14 and / or 15 in SEQ ID NO: 28, 29 and / or 30 are substituted, these amino acids are any amino acid, preferably any amino acid except glycine, proline or cysteine. Preferably, when the amino acid at position 27 is substituted in SEQ ID NO: 28, 29 and / or 30, this amino acid is any amino acid, preferably histidine, asparagine or tyrosine.

[0120] In one embodiment of the first aspect, the binding protein comprises at least two repeat modules, which may be identical or different. In one embodiment of the first aspect, the binding protein comprises 2-20 repeat modules, which may be identical or different. In one embodiment of the first aspect, the binding protein comprises 3 repeat modules, which may be identical or different.

[0121] In a preferred embodiment the binding protein comprises three repeat modules, where repeat module 1 has an amino acid sequence according to SEQ ID NO: 28, repeat module 2 has an amino acid sequence according to SEQ ID NO: 29 and repeat module 3 has a 30 amino acid sequence according to SEQ ID NO: and preferably 10 or less amino acids in each module are substituted with any amino acid, preferably 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid are substituted, most preferably no amino acids are substituted.

[0122] In a preferred embodiment the binding protein comprises three repeat modules, repeat module 1 having an amino acid sequence according to SEQ ID NO: 28, repeat module 2 having an amino acid sequence according to SEQ ID NO: 29 and repeat module 3 having an amino acid sequence according to SEQ ID NO: 30, preferably in which 5 or less amino acids in each module are substituted with any amino acid, preferably 4, 3, 2 or 1 are substituted, most preferably no amino acids are substituted.

[0123] A preferred binding protein comprises at least one ankyrin repeat domain, the ankyrin repeat module being greater than or equal to 2×10 -5 Preferably, the ankyrin repeat module binds to CD32a with a dissociation constant (Kd) of 2×10 M or less as measured by surface plasmon resonance (SPR). -6 M or less, preferably 2×10 -7 M or less, 2×10 -8 M or less, or most preferably 2×10 -9 Binds to CD32a with a Kd of less than 2×10 -5 Binding proteins containing ankyrin repeat domains that bind to CD32a with a Kd of less than or equal to M are shown in the Examples.

[0124] It will be appreciated by those skilled in the art that the inclusion of two or more repeat domains that bind CD32a in the binding protein of the invention can increase the affinity of the binding protein of the invention due to an avidity effect resulting from the binding protein of the invention binding two or more CD32a antigens simultaneously, e.g., on the surface of a cell. Thus, a preferred binding protein of the invention comprising one of said repeat domains that binds CD32a has a binding affinity of 2×10 -5 Preferably, the binding proteins of the invention bind to CD32a with a Kd of 2×10 M or less as measured by SPR. -6 M or less, preferably 2×10 -7 M or less, 2×10 -8 M or less, or most preferably 2×10 -9 Binds to CD32a with a Kd of less than 2×10 -5 Binding proteins of the invention that bind to CD32a with a Kd of less than or equal to M are shown in the Examples.

[0125] Another preferred binding protein of the invention comprising two, three, four or more of said ankyrin repeat domains that bind CD32a is 2×10 -5 Preferably, the binding proteins of the invention bind to CD32a with a Kd of 2×10 M or less as measured by SPR. -6 M or less, preferably 2×10 -7 M or less, 2×10 -8 M or less, or most preferably 2×10 -9 Binds to CD32a with a Kd of less than 2×10 -5 Binding proteins of the invention that bind to CD32a with a Kd of less than or equal to M are shown in the Examples.

[0126] The ankyrin repeats of the binding proteins of the invention specifically bind to CD32a. Preferably, the binding proteins comprising at least one ankyrin repeat domain specifically bind to human CD32a.

[0127] Preferably, the binding domain of the invention is an ankyrin repeat domain or a designed ankyrin repeat domain. Examples of designed ankyrin repeat domains are given in the Examples.

[0128] In a second aspect, the present invention relates to a binding protein comprising at least one ankyrin repeat domain, wherein said at least one ankyrin repeat domain specifically binds to CD32a, and wherein said at least one ankyrin repeat domain comprises an amino acid sequence having at least 70% amino acid sequence identity with one ankyrin repeat domain selected from the group consisting of SEQ ID NOs: 1 to 27.

[0129] As defined above, the ankyrin repeat domain is 2×10 -5 Preferably, the repeat domain binds to CD32a with a Kd of 2×10 -6 M or less, preferably 2×10 -7 M or less, preferably 2×10 -8 M or less, or most preferably 2×10 -9 Binds to CD32a with a Kd of less than 2×10 -5 Binding proteins containing ankyrin repeat domains that bind to CD32a with a Kd of less than or equal to M are shown in the Examples.

[0130] A preferred binding protein of the present invention is 2×10 -5 Preferably, the binding protein binds to CD32a with a Kd of 2×10 M or less as measured by SPR. -6 M or less, preferably 2×10 -7 M or less, 2×10 -8 M or less, or most preferably 2×10 -9 Binds to CD32a with a Kd of less than 2×10 -5 Binding proteins of the invention that bind to CD32a with a Kd of less than or equal to M are shown in the Examples.

[0131] Preferably, the ankyrin repeat domain in the binding protein of the present invention comprises an amino acid sequence having at least 70% amino acid sequence identity with a randomized repeat unit or randomized position in an ankyrin repeat domain selected from the group consisting of SEQ ID NOs: 1 to 27. Preferably, the ankyrin repeat domain in the binding protein of the present invention comprises an amino acid sequence having at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% amino acid sequence identity. In a most preferred embodiment, the ankyrin repeat domain in the binding protein of the present invention comprises any of the amino acid sequences of SEQ ID NOs: 1 to 27.

[0132] In one embodiment, the binding protein of the invention comprises at least one ankyrin repeat domain and has the following sequence motif: X1DX2X3GX4TPLHLAAX5X6GHLEIVEVLLKX7GADVNA (SEQ ID NO: 31) at least one ankyrin repeat module consisting of an amino acid sequence according to X1 to X6 may be any amino acid except glycine, proline or cysteine; X7 may be histidine, asparagine or tyrosine; In one embodiment of the invention in repeat module 1, optionally up to 5, preferably 4, 3, 2 or 1 amino acids other than the position indicated by X in SEQ ID NO: 31 are substituted with any amino acid. In a preferred embodiment, up to 2 amino acids other than the position indicated by X in SEQ ID NO: 31 are substituted with any amino acid. X1 is T, E, N, S, Y, or W, preferably T; X2 is E, N, T, W, H, K, N, or I, preferably E; X3 is E, F, K, H, M, A, or T, preferably E; X4 is L, T, A, V, F, Q, or A, preferably L; X5 is L, Q, V, E, M, V, or Y, preferably L; X6 is I, F, E, Q, H, V, W, or D, preferably I; X7 is Y, N or H, preferably Y.

[0133] Thus, in a preferred embodiment of repeat module 1, X1 is T, X2 is E, X3 is E, X4 is L, X5 is L, X6 is I and X7 is Y.

[0134] In one embodiment of the present invention in repeat module 2, X1 is A, H, M, T, D, Q, S, or V, preferably A; X2 is M, Y, N, E, W, D, H, or T, preferably M; X3 is D, W, Q, S, V, F, E, or H, preferably D; X4 is T, R, Q, N, M, D, S, Y, or P, preferably T; X5 is Q, E, V, H, T, R, M, or Y, preferably Q; X6 is E, Y, W, F or A, preferably E; X7 is Y or N, preferably Y; Optionally, this sequence has an early stop codon and optionally is missing amino acids beginning at and including position 13, preferably position 14, and most preferably position 15.

[0135] Thus, in a preferred embodiment of repeat module 2, X1 is A, X2 is M, X3 is D, X4 is T, X5 is Q, X6 is E and X7 is Y.

[0136] In one embodiment of the present invention in repeat module 3, X1 is D, A, S, Q, H or Y, preferably D; X2 is F, I, H, V, Y, T, V, C, preferably F; X3 is W, A, F, V, E or G, preferably W; X4 is H, Y, L, M or F, preferably H; X5 is W, F, I, H, or R, preferably W; X6 is F, I, Q, D or L, preferably F; X7 is H, Y or N, preferably H.

[0137] Thus, in a preferred embodiment of repeat module 3, X1 is D, X2 is F, X3 is W, X4 is H, X5 is W, X6 is F and X7 is H.

[0138] Thus, in a preferred embodiment repeat module 1, X1 is T, X2 is E, X3 is E, X4 is L, X5 is L, X6 is I and X7 is Y; in repeat module 2, X1 is A, X2 is M, X3 is D, X4 is T, X5 is Q, X6 is E and X7 is Y; in repeat module 3, X1 is D, X2 is F, X3 is W, X4 is H, X5 is W, X6 is F and X7 is H.

[0139] In one embodiment, the binding protein of the present invention comprises at least two or at least three repeat modules comprising the repeat consensus sequence, which may be identical or different. In a preferred embodiment, the binding protein of the present invention comprises three repeat modules, which may be identical or different. Preferably, the repeat domain of the binding protein of the present invention comprises at least three repeat modules comprising the repeat consensus sequences of repeat module 1, repeat module 2 and repeat module 3.

[0140] In one embodiment of all aspects described herein, the binding protein of the invention further comprises an N-terminal and / or a C-terminal capping repeat domain, preferably the N-terminal and / or the C-terminal capping repeat domain has an amino acid sequence different from any one of the ankyrin repeat modules.

[0141] Preferably, the amino acid sequence of the N-terminal capping repeat domain comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 32, and the amino acid sequence of the C-terminal capping repeat domain comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 33.

[0142] Preferably, the amino acid sequence of the N-terminal capping repeat domain comprises an amino acid sequence having at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:32.

[0143] Preferably, the amino acid sequence of the C-terminal capping repeat domain comprises an amino acid sequence having at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:33.

[0144] Alternatively, the N-terminal capping repeat domain consists of an amino acid sequence according to SEQ ID NO: 32, optionally with up to 5, preferably up to 4, 3, 2 or 1 amino acids being replaced by any amino acid. The C-terminal capping repeat domain consists of an amino acid sequence according to SEQ ID NO: 33, optionally with up to 8, preferably up to 7, 6, 5, 4, 3, 2 or 1 amino acids being replaced by any amino acid.

[0145] Further preferred are binding proteins that contain any N- or C-terminal capping known in the art. Examples of binding proteins of the invention that contain N- and / or C-terminal capping repeat domains are the amino acid sequences corresponding to SEQ ID NOs: 34-60 and 61-86, respectively.

[0146] In one embodiment the binding protein further comprises another peptide or protein moiety, optionally fused to the repeat protein, hi one embodiment the binding protein further comprises a lipid or lipid-like moiety, or another non-peptide moiety.

[0147] In a preferred embodiment, the binding protein of the invention comprising at least one ankyrin repeat domain specifically binds to the extracellular domain of the CD32a receptor, preferably to the Ig-like C2 type 2 domain of the CD32a extracellular domain, more preferably the specific binding comprises binding to the Ig-like C2 type 2 domain of CD32a at leucine 168. In one embodiment, said at least one ankyrin repeat domain may compete with IgG, preferably the Fc part of IgG, for binding to CD32a.

[0148] In a preferred embodiment, the CD32a is primate CD32a, more preferably human CD32a.

[0149] In a preferred embodiment, the binding proteins of the invention distinguish between CD32a, CD32b and CD32c, and preferably do not bind to CD32b or CD32c, and most preferably do not bind to CD32b.

[0150] Also preferably, the binding protein does not bind to CD32b and has a Kd for binding to CD32b of 2×10 -5 More than M, more preferably 2×10 -4 More than M, or most preferably 2 × 10 -3 Indicated as M+.

[0151] Preferably, the binding protein of the invention comprises an antigen binding protein, preferably a recombinant antigen binding protein. Preferably, said binding protein is not an antibody or a fragment thereof, such as a Fab or scFv fragment. Most preferably, the binding protein of the invention comprises or is a DARPin.

[0152] In a further embodiment, the present invention relates to a nucleic acid comprising a nucleic acid encoding a binding protein, an N-terminal capping repeat and / or a C-terminal capping repeat as described herein.

[0153] In one embodiment, the nucleic acid sequence is operably linked to at least one transcriptional control unit. In one embodiment of the nucleic acid according to the invention, the nucleic acid additionally comprises at least an open reading frame. In another embodiment, the nucleic acid additionally comprises at least a regulatory region of a gene. Preferably, the regulatory region is a transcriptional regulatory region, more particularly, the regulatory region is selected from the group consisting of promoters, enhancers, silencers, locus control regions, and border elements. The nucleic acid according to the invention usually comprises ribonucleic acid, including mRNA, DNA, cDNA, chromosomal DNA, extrachromosomal DNA, plasmid DNA, viral DNA or RNA, also including recombinant viral vectors. In one embodiment of the nucleic acid according to the invention, the nucleic acid is DNA or RNA, and in another preferred embodiment, the nucleic acid is part of a plasmid or a recombinant viral vector. The nucleic acid according to the invention is preferably selected from any nucleic acid sequence encoding the amino acid sequence of the binding protein of the invention. Thus, all nucleic acid variants encoding the above-mentioned binding proteins of the invention with amino acid substitutions include nucleic acid variants in which the nucleotide sequence is changed due to the degeneration of the genetic code. In particular, the nucleotide sequence of the nucleic acid variant leading to improved expression of the encoded fusion protein in the selected host organism is preferred. Tables for the appropriate adjustment of nucleic acid sequences to the specific transcription / translation machinery of a host cell are known to those skilled in the art. In general, it is preferred to adapt the G / C content of a nucleotide sequence to the conditions of a particular host cell. For expression in human cells, an increase in G / C content of at least 10%, more preferably at least 20%, 30%, 50%, 70%, even more preferably 90% of the maximum G / C content (respectively encoding the binding protein of the invention) is preferred. The preparation and purification of such nucleic acids and / or derivatives are usually carried out by standard procedures.

[0154] These sequence variants preferably lead to the binding proteins of the invention having binding specificity for CD32a. Thus, the nucleic acid sequences of the invention encode all the binding protein variants of the invention. Furthermore, promoters or other expression control regions are operably linked to the nucleic acids encoding the binding proteins of the invention to regulate the expression of the binding proteins in a quantitative or tissue-specific manner.

[0155] In a further aspect, the present invention relates to a host cell comprising the nucleic acid described herein and optionally expressing the binding protein. In one embodiment, the cell is of animal, preferably vertebrate, origin, which is preferably selected from the group consisting of fish, birds, or mammals, preferably mammals.

[0156] Further provided are vectors comprising, consisting essentially of, or consisting of the nucleic acids or binding proteins described herein.

[0157] As already described, the nucleic acid encoding the binding protein of the invention may be RNA or DNA. Similarly, the nucleic acid of the invention encoding the binding protein of the invention may be linear or circularized fragment, isolated fragment, or may be inserted into a vector, preferably as a plasmid or recombinant viral DNA.

[0158] In one embodiment of all aspects, the binding protein is a molecule, optionally contained in a particle.

[0159] In one embodiment, the particles further comprise a nucleic acid, preferably the nucleic acid is RNA or DNA. The particles may deliver nucleic acid to cells in vivo as well as in vitro / ex vivo. Preferably, the particles deliver nucleic acid to immune cells, more preferably CD4 +The nucleic acid is delivered to T cells, macrophages, monocytes, platelets, neutrophils, eosinophils, basophils, and mast cells. In one embodiment, the particle is a non-viral particle. In one embodiment, the particle is a lipid-based and / or polymer-based particle. In one embodiment, the particle is a nanoparticle.

[0160] In one embodiment, the particle is functionalized with the binding protein or DARPin of the present invention on its surface.In one embodiment, the particle is functionalized with the binding protein of the present invention by linking repeat protein to at least one particle forming component.The particle carrying the cargo for genetic modification of CD32a positive cells is functionalized with the binding agent described herein, resulting in the specific delivery and modification of the cargo to CD32a positive cells.

[0161] In one embodiment of all aspects described herein, genetic modification is transient or stable.In one embodiment of all aspects described herein, genetic modification is carried out by virus-based method, transposon-based method or gene editing-based method.In one embodiment, gene editing-based method comprises CRISPR-based gene editing.

[0162] Genetic modification of cells with the binding proteins described herein or particles functionalized with DARPins to specifically target CD32-positive cells can be used ex vivo / in vitro or in vivo to deliver nucleic acids encoding antigen receptors to cells, such as CD32-positive cells, to produce cells genetically modified to express the antigen receptor. Such genetic modifications include non-viral-based DNA transfection, non-viral-based RNA transfection, such as mRNA transfection, transposon-based systems, and virus-based systems. Non-viral-based DNA transfection has a lower risk of insertional mutagenesis. Transposon-based systems can integrate transgenes more efficiently than plasmids that do not contain integration elements. Virus-based systems include the use of AAV and adenoviral vectors, as well as gamma-retroviral and lentiviral vectors. Retroviral and lentiviral vectors are enveloped particles that permanently transduce cells, while AAV and adenoviral vectors are unprotected particles that mediate transient gene delivery, at least to mitotically active cells. The display of DARPins on the surface of enveloped and unprotected viral vector particles is well established and has been previously described.

[0163] The present invention also relates to a vector which displays at least one binding protein of the invention.

[0164] For example, non-viral vectors such as lipid and / or polymer-based nanoparticles can be conjugated to CD32a-specific DARPins to bind to CD32a-positive cells. Upon binding to CD32a-positive cells, these particles are endocytosed. Their contents, e.g., nucleic acids encoding therapeutic genes, can be targeted to the nuclei of CD32a-positive cells, e.g., by including peptides containing microtubule-associated sequences (MTAS) or nuclear localization signals (NLS).

[0165] The therapeutic gene packaged in the viral or non-viral vector can be, for example, CD32a + Cytotoxic genes, or CD32a, to eliminate HIV reservoir cells + It may be an immunotherapeutic gene that confers a novel therapeutic activity to the cells. These are just some of the many different options that can all be combined with CD32a targeted particles.

[0166] By including a transposon flanking the nucleic acid and another nucleic acid, such as a plasmid, encoding a hyperactive transposase, the therapeutic nucleic acid can be efficiently integrated into the chromosome.

[0167] Another possibility is to use CRISPR / Cas9 technology to purposefully place a therapeutic gene at a specific locus: for example, if the therapeutic gene was a chimeric antigen receptor (CAR), the existing T cell receptor (TCR) would be knocked out, while the CAR would be knocked in and placed under the dynamic control of the endogenous promoter that would otherwise repress expression of the TCR.

[0168] Thus, in addition to nucleic acids encoding antigen receptors, the particles described herein can also deliver gene editing tools such as CRISPR / Cas9 (or related), or transposon systems such as sleeping beauty or piggy bag as cargo. Such tools for genome integration / editing (e.g., gene editing tools such as transposases, CRISPR / Cas9) are delivered as proteins or encoding nucleic acids (DNA or RNA). Nevertheless, delivery of mRNA is also an option to induce transient expression of therapeutic genes.

[0169] CD32a +A specific example of this is the delivery of CRISPR / Cas tools to excise and / or inactivate the integrated proviral HIV genome in chronically infected HIV positive cells. These editing mechanisms can be precisely delivered to chronically infected HIV reservoir cells by any of the vector systems described above, provided that the HIV reservoir cells display CD32a-specific DARPins on their surface.

[0170] In one embodiment of all aspects of the invention, the cells genetically modified to express a therapeutic gene are stably or transiently transfected with a nucleic acid encoding the therapeutic gene, which may or may not be integrated into the genome of the cell.

[0171] In one embodiment of all aspects of the invention, the cells described herein may be autologous, allogeneic, or syngeneic to the subject being treated. In one embodiment, the present disclosure contemplates the removal of cells from the patient and the subsequent reintroduction of cells to the patient. In one embodiment, the present disclosure does not contemplate the removal of cells from the patient. In the latter case, the entire process of genetic modification of cells is performed in vivo.

[0172] In a further aspect, the invention relates to a composition comprising a binding protein as described herein, a nucleic acid as described herein, a host cell as described herein, a particle as described herein, or a plurality thereof.

[0173] In a further aspect, the present invention relates to a pharmaceutical composition comprising at least one of the binding proteins, nucleic acids, host cells, vectors, and / or particles described herein.

[0174] The pharmaceutical composition may further comprise one or more carriers and / or excipients, all of which are preferably pharma-ceutically acceptable. According to the present invention, the pharmaceutical composition comprises an effective amount of an active agent, such as a polypeptide, a nucleic acid, a vector, or a cell described herein, to produce a desired reaction or a desired effect. The pharmaceutical composition according to the present invention is preferably sterile. The pharmaceutical composition may be provided in a homogeneous dosage form and may be prepared in a manner known per se. The pharmaceutical composition according to the present invention may be, for example, in the form of a solution or a suspension.

[0175] Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of vegetable saturated fatty acids, water, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, salts or electrolytes such as zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and wool fat. The pharmaceutical compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally or via an implanted reservoir. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the pharmaceutical composition is administered orally, intraperitoneally or intravenously. The sterile injectable form of the pharmaceutical composition of the present invention may be an aqueous or oleaginous suspension. These suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as solvents or suspending media.

[0176] The pharmaceutical composition of the present invention is preferably suitable for use in the treatment, prevention or diagnosis of a condition.Preferably, the condition is associated with the change in CD32a expression level in a subject.The change in CD32a expression level refers to the increase or decrease in the expression of CD32a compared with the control level.

[0177] In one aspect of the invention, the binding protein, nucleic acid, vector, particle, host cell, composition or pharmaceutical composition disclosed herein is used as a medicament.Preferably, the medicament is an immunomodulator, preferably in the context of an allergy, an autoimmune disease, or an immunodeficiency disorder such as caused by the human immunodeficiency virus (HIV).

[0178] The binding proteins, nucleic acids, vectors, particles, host cells, compositions or pharmaceutical compositions disclosed herein can be used for the treatment, prevention or diagnosis of a condition, preferably associated with the expression level of CD32a, preferably associated with a change in the expression level of CD32a, and / or in which targeting of CD32a is desired.

[0179] In a further aspect, the present invention relates to a kit comprising the binding protein, nucleic acid, host cell, vector, particle, composition or pharmaceutical composition disclosed herein. Preferably, the kit is for use in the treatment, prevention or diagnosis of a condition. Preferably, the kit further comprises instructions for using the kit in a method for treating, preventing or diagnosing a condition. Preferably, said condition is a condition associated with CD32a expression levels, preferably a condition associated with changes in CD32a expression levels, and / or a condition in which targeting of CD32a is desired.

[0180] In one embodiment of all aspects described herein, the condition associated with altered CD32a expression levels is an allergy, an autoimmune disease / immunodeficiency, or heparin-induced thrombocytopenia. Immunodeficiencies include HIV, rheumatoid arthritis, Huntington's disease, alpha-antitrypsin deficiency, and colon cancer, melanoma, kidney cancer, lymphoma, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), gastrointestinal tumors, lung cancer, glioma, thyroid cancer, breast cancer, prostate tumor, liver cancer, various virus-induced tumors such as papillomavirus-induced tumors (e.g., cervical cancer), adenocarcinoma, herpes virus-induced tumors (e.g., Burkitt's lymphoma, EBV-induced B-cell lymphoma), hepatitis B-induced tumors (hepatocellular carcinoma), HTLV-1 and and HTLV-2-induced lymphoma, acoustic fibromatosis, lung cancer, pharyngeal cancer, anal cancer, glioblastoma, lymphoma, rectal cancer, astrocytoma, brain tumor, gastric cancer, retinoblastoma, basal cell tumor, brain metastasis, medulloblastoma, vaginal cancer, pancreatic cancer, testicular cancer, melanoma, bladder cancer, Hodgkin's syndrome, meningioma, Schneeberger's disease, bronchial cancer, pituitary cancer, mycosis fungoides, esophageal cancer, breast cancer, schwannoma, acanthoma, Burkitt lymphoma, laryngeal cancer, thymoma, body cancer, bone cancer, non-Hodgkin's lymphoma, urethral cancer, CUP syndrome, oligodendroglioma, vulvar cancer, intestinal cancer, esophageal cancer, small intestine tumor, craniopharyngioma, ovarian cancer, liver cancer, and leukemia, or a cancer selected from skin cancer or eye cancer. Preferably, the condition is HIV or rheumatoid arthritis, more preferably HIV.

[0181] Those skilled in the art will have access to a variety of alternative techniques and procedures that will similarly enable one to successfully practice the intended invention. Working Example EXAMPLES

[0182] Identification of CD32a-DARPin Ribosome display Selection by ribosome display was performed as described by Hartmann et al. (Hartmann et al. 2018). In total, up to five selection rounds were performed. As bait protein, the extracellular domain of CD32a tagged with a C-terminal His tag (CD32a-His, Sino-Biologicals) was used, with or without biotinylation. For CD32b, counterselection was performed using recombinant CD32b protein (CD32b-His, Sino-Biologicals). The first three selection rounds were performed using immobilized biotinylated CD32a-His (Sino Biologicals, 10374-H27H1-B) and included a preselection step against neutravidin or streptavidin to exclude the selection of sticky DARPins. The fourth and fifth selection rounds were performed in solution. The fourth selection round involved off-rate selection with non-biotinylated CD32a-His protein (Sino Biologicals, 10374-H27H) to select binders with high affinity and specificity for CD32a, and the fifth selection round involved counter-selection with non-biotinylated CD32b-His (Sino Biologicals, 10259-H27H) protein.

[0183] Briefly, in the first three selection rounds, the translated VV-N2C and VV-N3C DARPin libraries were subjected to a pre-panning step with immobilized neutravidin or streptavidin (both at 66 nM). For on-target selection, the libraries were incubated with immobilized biotinylated CD32a-His (425.5 nM). The resulting DARPin libraries were amplified and used as templates for the next selection round. After the third round with immobilized target protein, the fourth and fifth rounds of selection were performed with the target in solution. In the fourth round, the libraries were first exposed to the biotinylated CD32a-His target (17 pmol) and then to a 10-fold excess of biotinylated CD32a (170 pmol) as an off-rate selection. Finally, the fifth round was divided into two approaches: (i) in one approach, the library was preselected with non-biotinylated CD32b-His protein (17 pmol) before being exposed to biotinylated CD32a-His protein (17 pmol), and (ii) in the other approach, the library was first exposed to biotinylated CD32a-His protein and then counterselected with non-biotinylated CD32b-His protein (170 pmol). After the fifth selection round, DNA fragments encoding DARPins were cloned into E. coli and analyzed for binding to CD32a by single clone analysis.

[0184] Expression of DARPin and preparation of crude lysates To test the specific binding of the DARPins selected after the fifth selection round to CD32a, the DNA fragments were cloned into the bacterial expression vector pQE-HisHA and transformed into E. coli XL 1-blue as previously described (Hartmann et al. 2018). Single clones were picked and grown overnight at 37°C in 600 μl of 2YT medium (2YT, 1% glucose, 100 μg / ml ampicillin), after which the culture was diluted with 1 ml of 2YT medium to measure the OD 600The pH was adjusted to 0.1 and expression of DARPins was induced after 1 h of incubation by the addition of 100 μL of 5.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) in 2YT medium. After 5 h of incubation at 37 °C, cells were harvested by centrifugation and the pellets were stored at -80 °C overnight. The following day, the pellets were thawed on ice and lysed by adding B-PERII solution (Thermo Scientific), followed by incubation at room temperature for 2 h. The lysed pellets were vortexed and diluted 1:10 in TBS, after which cell debris was removed by centrifugation. The supernatants containing crude DARPins were aliquoted and stored at -80 °C until use in cell binding assays. Preparation of crude lysates was always performed on ice and subjected to a maximum of three freeze-thaw cycles to avoid protein loss. A total of 285 DARPin clones from the fifth selection round were expressed in E. coli and tested for binding to cell surface expressed CD32a. Among the tested DARPin clones, promising candidates were identified and sequenced using standard sequencing techniques to obtain DNA and protein sequences.

[0185] Cell-based binding assays To analyze the CD32a-binding ability of selected DARPin clones, cell-based binding assays were performed using stable CD32a or CD32b receptor-expressing cell lines generated by lentiviral transduction and with appropriate cell surface expression confirmed by CD32 antibody staining (data not shown). 3 ~1×10 5 Cells were incubated with 5–25 μl of crude DARPin extract for 60 min at 4 °C. Following incubation, cells were washed twice, stained with fluorescently labeled antibodies detecting DARPins, and analyzed by flow cytometry. Of the 285 clones tested, 45 individual DARPin clones bound specifically to human CD32a-positive cells, whereas only two DARPins (53.F11, 53.2.D8) showed off-target binding to CD32b-positive cells (Figure 1). Of note, all identified CD32a-binding DARPins, except for one DARPin (52.H6), were derived from the VV-N3C library.

[0186] [Table 2-1] [Table 2-2]

[0187] DARPin sequence analysis Sequencing revealed that 34 of the 45 identified CD32a-specific candidates resembled the general DARPin structure, of which 27 had unique amino acid sequences (Table 1). For the remaining DARPin candidates, the typical DARPin structure was not complete (e.g., initial sequence truncation), so these candidates were not considered further. Each DARPin clone contains at least one diverse repeat domain arranged in a flange-like manner by N- and C-terminal capping repeat domains. Each repeat unit contains the same structural motif, and more than 69.8% of the framework residues of the 33 N3C DARPins are homologous to each other. The diverse amino acids at positions 1, 3, 4, 6, 14 and 15 in each diverse repeat domain can contain any amino acid except glycine, proline or cysteine. The diverse amino acid at position 27 in each diverse repeat domain can contain histidine, asparagine or tyrosine. As a result of evaluating the individual DARPin clones and the various positions of the repeat motif, the selected CD32a-DARPin contains threonine (T) at position 1, glutamic acid (E) at positions 3 and 4, leucine (L) at positions 6 and 14, isoleucine (I) at position 15, and tyrosine (Y) at position 27 in the first various repeat, alanine (A) at position 1, methionine (M) at position 3, and tyrosine (Y) at position 27 in the second various repeat. ), aspartic acid (D) at position 4, threonine (T) at position 6, glutamine (Q) at position 14, glutamic acid (E) at position 15, and tyrosine (Y) at position 27, while the third diverse repeat selectively possessed aspartic acid (D) at position 1, phenylalanine (F) at positions 3 and 15, tryptophan (W) at positions 4 and 14, and histidine (H) at positions 6 and 27 (Figure 2 and Table 1). EXAMPLES

[0188] Specific labeling of CD32a positive cell populations with purified CD32a-DARPin To identify CD32a-specific DARPins capable of discriminating between CD32a and CD32b expressing cell populations, 6 of the 34 DARPin candidates were investigated in more detail using purified CD32a-DARPin as described by Hartmann et al. (Hartmann et al. 2018) with minor modifications. Briefly, for each DARPin, a single colony was inoculated into lysogeny broth (LB) complete medium overnight at 30 °C. The next day, 100 ml of fresh LB complete medium was inoculated with 5 ml of the overnight culture. At an optical density OD of 0.7, protein expression was induced by adding 300 μM IPTG. After 4 h of incubation at 37 °C, bacteria were harvested by centrifugation and pellets were stored at -80 °C. Pellets were thawed on ice, resuspended in lysis buffer, and disrupted by sonication for mechanical extraction or mixed with B-PERII for chemical extraction. Cell debris was removed by centrifugation, the supernatant was re-pooled and glycerol buffer (2.5x) was added. Purification of DARPins on Ni-nitrilotriacetic acid (NTA) agarose columns was performed according to the manufacturer's instructions. Samples for SDS PAGE were taken at different times during this procedure. Elution fractions were dialyzed overnight at 4°C against PBS using dialysis tubing (MWCO depends on the extraction method used). Dialyzed DARPin protein was measured by Bradford protein assay and, if necessary, supplemented with glycerol and protease inhibitors for storage.

[0189] To analyze the CD32a-binding ability of selected purified DARPin proteins, cell-based binding assays were performed using transiently or stable CD32a- or CD32b-expressing cell lines. 4 ~1×10 5Cells were incubated with different amounts of purified DARPin. After incubation, cells were washed, stained with a fluorescently labeled antibody detecting the His-tag of the DARPin, and analyzed by flow cytometry. The selected purified DARPins were able to distinguish between CD32+ cells and off-target cells, although the staining intensity of the cells varied depending on the DARPin candidate (Figure 3). Binding of the available CD32a cell surface receptor appears to be achieved at DARPin concentrations below 1 μg. EXAMPLES

[0190] Mapping the binding site of CD32a-DARPin The sequences of the extracellular domains of CD32a and CD32b differ by only 12 amino acids, but the first two amino acids are close to the signal peptide and therefore are not considered in this study (Figure 4). To identify which of these amino acids contribute to CD32a DARPin binding, chimeric CD32b proteins carrying one to five CD32a-specific amino acids in the extracellular domain (Table 2) were synthesized, expressed on SupT1 cells, and used as target populations in cell-based binding assays, as described above. All chimeric CD32b proteins were well expressed on the cell surface and bound IgG-Fc to various degrees (data not shown). Interestingly, only the chimeric CD32b variant carrying a leucine instead of a serine at position 135, counting from the beginning of the extracellular domain, was able to mediate CD32a-DARPin binding (Figure 5). That this single amino acid exchange in the ECD of CD32b is sufficient to enable CD32a-DARPin binding and leads to discrimination between CD32a and CD32b by the selected DARPin was further confirmed by binding analysis of the chimeric CD32b.mut10 receptor carrying only the single point mutation S135L. In contrast, the single point mutation N138T of the chimeric CD32b.mut11, also present in the chimeric CD32b.mut4, CD32b.mut6, CD32b.mut8, and CD32b.mut9 versions, did not lead to DARPin binding. Of note, this leucine is located in the Ig-like C2 type 2 domain of CD32a, which is the binding region for the Fc portion of IgG (Maxwell et al., 1999; Ramsland et al., 2011), the native ligand of the CD32a receptor.

[0191] To analyze whether CD32a-DARPin competes with IgG antibodies for CD32a binding, we performed a competition assay. When IgG antibodies were added to the cells in advance, we showed that the binding of DARPin to CD32a+ cells was reduced to the point of being absent (data not shown). Therefore, we speculate that there is a positive correlation between the IgG-binding area and the DARPin-binding area on the surface of the CD32a receptor.

[0192] [Table 3] EXAMPLES

[0193] CD32a-specific gene transfer by LVs presenting CD32a-DARPin DARPins have previously been shown to be suitable tools to retarget lentiviral vectors to a receptor of choice (Hartmann et al., 2018; Frank et al., 2018). For this purpose, a DARPin needs to be genetically fused to the glycoprotein of Nipah virus (NiV-G). Seven identified DARPin candidates (53.2.G2, 53.2A8, 53C8, 52H6, 53.2.F11, 53D8, 53H3) were cloned into the C-terminus of N-terminally truncated and native receptor-blinded NiV-G as described by Frank et al., 2020. To analyze whether the binding capacity and specificity of the selected CD32a-DARPins were not lost upon fusion to NiV-G protein and presentation on 293T cells, we performed cell-based binding assays using 293T cells transiently expressing the various DARPin-NiV-G proteins.

[0194] Briefly, DARPin-presenting cells were generated by transient transfection of HEK293T cells using PEI. The transfection mix was prepared by mixing 400 ng of total DNA with 30 μl of DMEM without additives and adding it to 30 μl of DMEM supplemented with 2 μl of PEI solution per 24-well plate. After 15 min of incubation at room temperature, the transfection mix was added to the cells and the medium was replaced with fresh cell culture medium 6 h later. Two days after transfection, 1 × 10 5of cells were incubated with 250 ng of biotinylated recombinant CD32a or CD32b proteins. After incubation, cells were washed, stained with fluorescently labeled streptavidin and analyzed by flow cytometry. Figure 7 shows that fusion of CD32a-DARPins to NiV-G and expression in mammalian cells retains CD32a receptor binding capacity and receptor specificity. These findings are in agreement with previous results showing that selected DARPins have high specificity for CD32a (Figures 1, 3 and 6).

[0195] In the next step, receptor-targeted lentiviral vectors (CD32a-LV) displaying those DARPin candidates on the envelope and carrying GFP as a reporter were screened for their ability to mediate CD32a-specific transgene delivery, as previously described ( Frank et al., 2020 ). The schematic organization of CD32a-LV is shown in Figure 7 .

[0196] Briefly, LV vector particles were produced by transient transfection of HEK293T cells with PEI in either a small-scale format (12 wells, 0.8 μg DNA total) or a large-scale format (T175 flasks, 35 μg DNA total). For the transfection mix for small-scale production, 800 ng total DNA was mixed with 53 μl DMEM (without additives) and added to 50 μl DMEM supplemented with 3 μl PEI solution per 12-well plate. After 15 min of incubation at room temperature, the transfection mix was added to the cells. After 6 h, the medium was replaced with fresh cell culture medium. Two days after transfection, the cell supernatant containing the vector particles was harvested and either centrifuged to remove cell debris for small-scale production or filtered. The supernatant was used for transduction experiments. For LV production at large scale, refer to Frank et al., 2020.

[0197] The generated CD32a-LV particles were screened for their ability to transduce CD32a-positive (HT1080-CD32a) and -negative (HT1080-wt, HT1080-CD32b) cell lines. All seven candidates tested mediated CD32a-specific gene transfer when presented on LV particles (Figure 8). From these, the DARPins 53.2.F11 and 53.H3 were produced in large-scale production (20x T175 culture flasks) and further evaluated for transduction efficiency in SupT1 cells with or without CD32a or CD32b receptor expression (Figure 9; data not shown for CD32a.53H3-lentiviral vector). Interestingly, the use of serum-free medium (Panexin) resulted in a higher transduction rate into target cells. This finding may be explained by the presence of IgG in fetal calf serum (FCS), which may act as a competitor for the CD32a-DARPin. EXAMPLES

[0198] Kinetic measurements of selected CD32a-DARPins To determine the binding affinity of the selected DARPins to human CD32a, surface plasma resonance (SPR) measurements were performed on a Biacore T200 instrument (Cytiva). To further demonstrate the specificity of the DARPins to human CD32b, the potential binding of the DARPins to human CD32b was also analyzed.

[0199] Briefly, Neutravidin NA chips (Cytiva) were prepared according to the manufacturer's protocol and immobilized with 110 RU of human biotinylated CD32a (or 985 RU) and 114 RU of human biotinylated CD32b protein, respectively. 20 mM HEPES, 150 mM NaCl, 0.05% Tween 20 were used as running buffer. DARPin samples were injected at varying concentrations for 3 min, followed by a 15 min dissociation phase and then regeneration with 10 mM NaOH. Binding kinetics of protein-protein interactions were analyzed and fitted using Biacore T200 evaluation software. The signal of an uncoated reference flow cell was subtracted from each measurement.

[0200] In SPR experiments, the selected DARPin was specific for recombinant CD32a protein and did not cross-react with CD32b protein or neutravidin. We measured the binding kinetics of DARPin 53.2F11[TEV], the same DARPin as 53.2.F11 but carrying a TEV cleavage site between the His-tag and HA-tag, to immobilized human CD32a protein and found an affinity (KD) in the low nanomolar range (<2 × 10 -9 M), but affinity for human CD32b was not assessable (below baseline) (Figure 10). All other DARPin candidates tested (53.H3, 53.D8, 53.2.F11, 52.H6, 53.A8, 53.C8) showed similar affinity for CD32a (<2 × 10 -9 M) was observed, but binding to CD32b was always below baseline.

Claims

1. 1. A binding protein comprising at least one ankyrin repeat domain, said at least one ankyrin repeat domain specifically binding to CD32a, said at least one ankyrin repeat domain comprising: i) SEQ ID NOs: 28, 29 and 30, and ii) SEQ ID NOs: 28, 29 and 30, which contain up to 10 amino acid substitutions independently of each other. and preferably the substitutions are independent from each other at positions 1, 3, 4, 6, 14, 15 and / or 27 of the amino acid sequence according to SEQ ID NOs: 28, 29 and 30 and / or at up to three other positions of the amino acid sequence according to SEQ ID NOs: 28, 29 and 30.

2. A binding protein comprising at least one ankyrin repeat domain, said at least one ankyrin repeat domain specifically binding to CD32a, Binding protein, wherein the at least one ankyrin repeat domain comprises an amino acid sequence having at least 70% amino acid sequence identity with one ankyrin repeat domain selected from the group consisting of the amino acid sequences according to SEQ ID NOs: 1 to 27.

3. The at least one ankyrin repeat domain is represented by SEQ ID NO: 31 X1DX2X3GX4TPLHLAAX5X6GHLEIVEVLLKX7GADVNA (SEQ ID NO: 31) at least one ankyrin repeat unit consisting of an amino acid sequence according to Where: X1-X6 may be any amino acid except glycine, proline or cysteine; X7 may be histidine, asparagine or tyrosine; 3. The binding protein of claim 1 or 2, wherein up to five amino acids other than those at the positions indicated by X in SEQ ID NO: 31 are substituted with any amino acid.

4. X1 is T, E, N, S, Y, or W, preferably T; X2 is E, N, T, W, H, K, N, or I, preferably E; X3 is E, F, K, H, M, A, or T, preferably E; X4 is L, T, A, V, F, Q, or A, preferably L; X5 is L, Q, V, E, M, V, or Y, preferably L; X6 is I, F, E, Q, H, V, W, or D, preferably I; X7 is Y, N or H, preferably Y; The binding protein of claim 3.

5. X1 is A, H, M, T, D, Q, S, or V, preferably A; X2 is M, Y, N, E, W, D, H, or T, preferably M; X3 is D, W, Q, S, V, F, E, or H, preferably D; X4 is T, R, Q, N, M, D, S, Y or P, preferably T; X5 is Q, E, V, H, T, R, M, or Y, preferably Q; X6 is E, Y, W, F or A, preferably E; X7 is Y or N, preferably Y; Optionally, amino acids beginning at and including position 15 are deleted; The binding protein of claim 3.

6. X1 is D, A, S, Q, H or Y, preferably D; X2 is F, I, H, V, Y, T, V, C, preferably F; X3 is W, A, F, V, E or G, preferably W; X4 is H, Y, L, M or F, preferably H; X5 is W, F, I, H, or R, preferably W; X6 is F, I, Q, D or L, preferably F; X7 is H, Y or N, preferably H; The binding protein of claim 3.

7. 7. The binding protein according to any one of claims 3 to 6, further comprising an N-terminal and / or a C-terminal capping repeat domain, preferably the N-terminal and / or the C-terminal capping repeat domain has an amino acid sequence different from any one of said ankyrin repeat units.

8. The binding protein of any one of claims 1 to 7, wherein the binding protein comprises an antigen-binding protein, preferably which comprises or is a DARPin.

9. A nucleic acid comprising a nucleic acid encoding the binding protein according to any one of claims 1 to 8.

10. A vector comprising the nucleic acid according to claim 9 or the binding protein according to any one of claims 1 to 8.

11. A vector displaying at least one binding protein according to any one of claims 1 to 8.

12. A pharmaceutical composition comprising at least one binding protein according to any one of claims 1 to 8, or a nucleic acid according to claim 9, or a vector according to claim 10 or 11, and a pharma- ceutically acceptable carrier.

13. A binding protein according to any one of claims 1 to 8, a nucleic acid according to claim 9, a vector according to claim 10 or 11, or a pharmaceutical composition according to claim 12, for use as a medicament.

14. A binding protein according to any one of claims 1 to 8, a nucleic acid according to claim 9, a vector according to claim 10 or 11, or a pharmaceutical composition according to claim 12, for use in the treatment, prevention or diagnosis of a condition associated with an alteration of CD32a, preferably an HIV-associated condition, in a subject.

15. A kit comprising a binding protein according to any one of claims 1 to 8, a nucleic acid according to claim 9, a vector according to claim 10 or 11, or a pharmaceutical composition according to claim 12, for use in the treatment, prevention or diagnosis of a condition associated with an alteration of CD32a in a subject, preferably an HIV-associated disease.

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