Modified FCRN-binding fragments with improved half-life

Abstract

The present disclosure relates to modified FcRn-binding fragments with improved half-life, in particular fusion proteins and polypeptides comprising said modified FcRn-binding fragments, as well as methods for producing said fusion proteins and their use in therapeutic methods.

Description

[Technical field]

[0001] The present disclosure relates to modified FcRn-binding fragments with improved half-life, in particular fusion proteins and polypeptides comprising said modified FcRn-binding fragments, as well as methods for producing said fusion proteins and their use in therapeutic methods. [Background technology]

[0002] The immunoglobulin Fc region is a homodimer consisting of two sets of CH2 and CH3 domains and has been exploited to generate two-arm protein fusions with high expression yields, simplified purification processes and long serum half-lives. However, attempts to generate one-arm fusion proteins with a monomeric Fc having one set of CH2 and CH3 domains often suffer from challenges such as weak binding to FcRn or partial monomer formation.

[0003] Monovalent versions of Fc fusion proteins (Alprolix-blood coagulation factor IX fusion, Eloctate-factor VIII fusion) or monovalent antibodies (Onartuzumab-anti-cMet one-arm mAb) that have advanced to the clinic utilize Fc regions engineered to form heterodimers with conjugation or “knobs-into-holes” techniques. These, along with other heterodimeric Fc technologies, rely on robust purification processes to eliminate undesired chain pairing and obtain homogeneous fusion proteins. To explore alternative approaches aimed at simplifying product development, significant efforts have been made in engineering fusion protein platforms with monomeric Fc modalities consisting of only one set of CH2 and CH3 domains by weakening the interactions or by adding glycans to the CH3-CH3 dimer interface in Fc to generate steric hindrance. So far, most of these approaches have faced challenges on several fronts, including solubility and stability, loss of FcRn binding, or lack of homogeneity. In addition, many of the previously engineered monomeric Fc molecules were observed to have a tendency to aggregate by dynamic light scattering, highlighting the challenge of stabilizing the monomeric conformation after weakening the homodimer interface. Among the engineered monomeric Fc modalities, only two molecules have been reported to have crystal structures with apparent uniformity and stability. One of these is a monomer stabilized by the addition of a glycosylation site that blocks CH3-CH3 interactions. The other is a monomeric Fc derived from an IgG4 phage library that was rationally designed based on previous findings (Non-Patent Document 1). However, it is known that the second platform can oligomerize at high concentrations, which may impair its usefulness for pharmaceutical products that require delivery at high concentrations.

[0004] Due to the increasing priority of approved biological therapeutics and the expanding methods of their administration and use, it remains desirable to develop a robust platform of monovalent versions of Fc fusion proteins. For example, gene therapy applications and inhalable products are both rich areas of development for biological therapeutic modalities. However, both have size constraints with respect to their ability to package DNA encoding a therapeutic agent or optimal size for effective biodistribution in the lungs. Furthermore, generating smaller molecules with similar pharmacokinetic or pharmacodynamic properties to monoclonal antibodies is generally attractive to increase commercial manufacturing yields and reduce product costs.

[0005] Thus, there remains a need to provide monovalent versions of Fc fusion proteins. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Shan et al(2016)PLoS ONE 11(8):e0160345 Summary of the Invention [Means for solving the problem]

[0007] The present disclosure relates to the surprising discovery that modifications in the FcRn-CH3 dimerization interface improve the monomerization of FcRn-binding fragments, and therefore these FcRn-binding fragments have excellent exploitation potential when used in fusion proteins, polypeptides, or FcRn-binding fragment-nonprotein agent conjugates (referred to herein as "molecules") that require or desire a monovalent FcRn-binding fragment derived from the Fc region.

[0008] Thus, the present disclosure provides a fusion protein comprising an FcRn-binding fragment of an Fc region of an IgG molecule, the FcRn-binding fragment comprising phenylalanine (F) at position 351; arginine (R), lysine (K), aspartic acid (D), glutamic acid (E), phenylalanine (F), tyrosine (Y), proline (P), glycine (G), leucine (L) or methionine (M) at position 354; arginine (R) at position 366; lysine (K) at position 395; arginine (R) at position 405; and glutamic acid (E) at position 407, where the amino acid numbering is according to the EU index. It has been found that the substitution of these amino acids for the wild type residues at each of these positions improves the monomeric stability of the FcRn-binding fragment.

[0009] In another aspect, the disclosure provides a polypeptide comprising at least an FcRn-binding fragment of an Fc region of an IgG molecule, wherein the FcRn-binding fragment comprises: phenylalanine (F) at position 351; arginine (R), lysine (K), aspartic acid (D), glutamic acid (E), phenylalanine (F), tyrosine (Y), proline (P), glycine (G), leucine (L) or methionine (M) at position 354; arginine (R) at position 366; lysine (K) at position 395; arginine (R) at position 405; and glutamic acid (E) at position 407, where the amino acids are numbered according to the EU index.

[0010] In another aspect, the disclosure provides a molecule comprising a nonproteinaceous agent conjugated to an FcRn-binding fragment of an Fc region of an IgG molecule, wherein the FcRn-binding fragment comprises phenylalanine (F) at position 351; arginine (R), lysine (K), aspartic acid (D), glutamic acid (E), phenylalanine (F), tyrosine (Y), proline (P), glycine (G), leucine (L) or methionine (M) at position 354; arginine (R) at position 366; lysine (K) at position 395; arginine (R) at position 405; and glutamic acid (E) at position 407, where the numbering of the amino acids is according to the EU index.

[0011] In another aspect, the disclosure provides a nucleic acid encoding said fusion protein or polypeptide or an FcRn-binding fragment conjugated to said molecule.

[0012] In another aspect, the disclosure provides a vector comprising the nucleic acid.

[0013] In another aspect, the disclosure provides a host cell comprising the vector or nucleic acid.

[0014] In another aspect, the present disclosure provides a method for producing the fusion protein, FcRn-binding fragment polypeptide for use in the molecule by expressing and purifying the fusion protein, FcRn-binding fragment polypeptide from the host cell.

[0015] In another aspect, the disclosure provides a fusion protein, polypeptide or molecule for use in therapy.

[0016] In another aspect, the disclosure provides the use of the fusion protein, polypeptide or molecule in the manufacture of a medicament for the treatment of a disease.

[0017] In another aspect, the disclosure provides a method of treatment comprising administering a therapeutically effective amount of the fusion protein, polypeptide or molecule to a patient in need thereof. [Brief description of the drawings]

[0018] [Figure 1] [Figure 1A] Sequence alignment of the CH2 and CH3 domains of wild-type IgG4, MFc1 and MFc2 from a previous phage library campaign, a T1 variant in which the YTE mutation in the MFc1 CH2 domain was replaced with a set of CH3 mutations, and the final sequences of MFc3 and MFc4. [Figure 1B] Crystal structure of T1. [Figure 1C] Close-up of T1 detailing a small set of hydrogen bonds formed at Thr350 / Leu440 and Gln355 / Glu356. [Diagram 2] [Figure 2A] Sequence alignment of the CH2 and CH3 domains in wild-type IgG4, T1 and T1-lib for a panel of point mutation variants targeting residue S354. [Figure 2B] Representative SEC-MALS analysis showing T1 point mutants exhibiting molecular weights of approximately 26-27 kDa with good homogeneity. [Figure 2C] DSF comparison between T1 purified mutants to identify S354E and S354D with moderately high thermal stability. [Diagram 3] The crystal structure of the new monomeric Fc MFc3 in its non-glycosylated form (N297D) is shown. The superposition of MFc3 (orange) with the previously solved structure of MFc2 (or C4n) (light purple) shows that both maintain a similar monomeric Fc structure and that the S354E mutation did not cause any significant changes in the Fc region structure. As designed, due to the S354E mutation, the glutamic acid side chain protrudes into any possible dimer interactions observed in T1. [Figure 4] [Figure 4A] Structural survey of MFc3 (orange) and FcRn (blue) / β2-macroglobulin (pink) complex. [Figure 4B] Binding interface between MFc3 (orange) and FcRn (blue). Hydrogen bonds are indicated by dashed lines. [Figure 4C] MFc4 (green) and FcRn (blue) / β2-macroglobulin (pink) complex. [Figure 4D] Binding interface between MFc4 (green) and FcRn (blue). Hydrogen bonds are indicated by dashed lines. [Figure 4E] Heat map showing differential solvation energy ΔiG (kcal / mol) contribution of each MFc residue involved in the receptor binding interface. Higher positive values ​​indicate stronger solvation effect from the monomeric Fc binding surface. [Diagram 5] [Figure 5A] Schematic diagram of a monomeric Fc-based monovalent bispecific antibody. [Figure 5B] SEC-MALS analysis of Fab-MFc1-scFv and Fab-MFc4-scFv, showing a measured molecular weight of approximately 100 kDa with a polydispersity of 1.001. [Figure 5C] Simultaneous binding analysis using biolayer interferometry showing the expected binding activity from both the Fab and scFv portions to the recombinant antigen. [Figure 6][Figure 6A] In vivo mouse PK analysis of monomeric Fc-bispecific antibodies. hFcRn transgenic mouse serum clearance curves are plotted for Fab-MFc1-scFv, Fab-MFc4-scFv, Fab-MFc1 and IgG1 control based on simultaneous binding of Fab and Fc regions. [Figure 6B] PK parameters determined by non-compartmental analysis using model 201. AUCINF = area under the concentration-time curve of plasma concentration versus time graph from 0 hours to infinity; CL = clearance; Cmax = peak concentration; t1 / 2 = terminal half-life. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] definition As used herein, unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" are intended to include the plural forms of the terms they specifically refer to.

[0020] The term "about" or "approximately" refers to an acceptable error for a particular value as determined by one of ordinary skill in the art, depending in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain examples, the term "about" or "approximately" means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of the indicated value or range. Whenever the term "about" or "approximately" precedes the first number in a series of two or more numbers, it is to be understood that the term "about" or "approximately" applies to each of the numbers in the series. When "about" is used in conjunction with an amino acid position, it means within 1, 2, 3, 4, 5, or 10 amino acids of the specified position.

[0021] "Amino acid deletion" or "deleted" refers to removing an amino acid residue present in a parent sequence. Amino acids in a parent sequence may be deleted, for example, by recombinant methods known in the art. Thus, reference to a "deletion at position X" refers to the deletion of the amino acid present at position X. The deletion pattern can be described according to schema AX, where A is the one-letter code corresponding to the naturally occurring amino acid at position X, and A is the deleted amino acid residue. Thus, L234 refers to the deletion of the leucine amino acid (L) at position 234. Under such circumstances, residues 233 and 235 are next encoded in the sequence.

[0022] The term "amino acid substitution" or "substitution" refers to the replacement of an amino acid residue present in a parent sequence with another amino acid residue. An amino acid in a parent sequence may be replaced, for example, via chemical peptide synthesis or by recombinant methods known in the art. Thus, reference to a "substitution at position X" or a "substitution at position X" refers to the replacement of the amino acid present at position X with another amino acid residue. Substitution patterns can be described according to the schema AXY, where A is the one-letter code corresponding to the naturally occurring amino acid at position X and A is the replacing amino acid residue. Thus, L234F refers to the replacement of the leucine amino acid (L) at position 234 with phenylalanine (F).

[0023] "Antibody" or is used in the broadest sense to encompass a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. Immunoglobulins, such as immunoglobulin G (IgG), are an example of an antibody. The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain possesses. There are five major classes of antibodies, IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 8, E, y, and 11, respectively.

[0024] "Antigen-binding fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody-binding domains include, but are not limited to, Fv, Fab, Fab', F(ab')2, Fab'-SH, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antigen-binding fragments. For a review of specific antigen-binding domains, see Hudson et al. Nat.Med. 9:129-134(2003). For a review of scFv fragments, see, e.g., Pluckthuen, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185, and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives, see U.S. Pat. No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites, which may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0025] The EU Index was developed by Kabat et al. (Sequences of Proteins of Immunological Interest, 5 thed., 1991 NIH Pub. No. 91-3242, which is incorporated herein by reference in its entirety. The amino acid residues of the FcRn-binding fragments disclosed herein are numbered according to this numbering system.

[0026] "Fab" refers to an antibody fragment that contains a VH-CH1 and a VL-CL pairing. The term encompasses Fabs that contain non-canonical sequence variants, such as amino acid substitutions, deletions, or insertions within the Fab outside of sequence regions typically associated with high sequence variability. For example, Fab variants include Fabs that contain non-canonical amino acids or sequence changes in the VH or VL framework regions or in the CH1 or CL domains. Such changes may include the presence of non-canonical cysteines or other derivatizable amino acids, which may be used to conjugate the Fab variants to heterologous moieties. Other such changes include the presence of a non-canonical polypeptide linker, which is a polypeptide sequence that covalently bridges between two domains. For example, a Fab variant may contain a linker polypeptide that covalently links the CH1 domain to the VL domain or the CL domain to the VH domain, such that the Fab can be expressed as a single polypeptide chain.

[0027] An "Fc region" or "Fc domain" is a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. It does not have antigen binding activity but contains carbohydrate moieties and binding sites for complement receptors and Fc receptors, including the FcRn receptor (see below). The Fc region comprises the entire second constant domain CH2 (residues 231-340 of human IgG according to the EU index) and the third constant domain CH3 (residues 341-447). A reference sequence for the human IgG1 Fc region can be found under UniProtKB Accession No. P01857. A reference sequence for the human IgG4 Fc region can be found under UniProtKB Accession No. P01861.

[0028] "FcRn-binding fragment" refers to a fragment of an Fc region that binds to the FcRn receptor. An FcRn-binding fragment can include a portion of the heavy chain CH2-CH3 region or hinge-CH2-CH3 region that is involved in binding to FcRn (see Roopenian et al., Nature Rev. Immunol. 7:715-725 (2007)).

[0029] "FcRn receptor" or "FcRn" refers to an Fc receptor (where "n" indicates neonate) that is known to be involved in the transfer of maternal IgG to the fetus through the human or primate placenta or yolk sac (rabbit) and from colostrum through the small intestine to the neonate. It is also known that FcRn is involved in maintaining constant serum IgG levels by binding IgG molecules and recycling them into serum. Binding of FcRn to naturally occurring IgG1, IgG2 and IgG4 molecules is strictly pH dependent, with optimal binding at pH 6. IgG3 has a known mutation at position 435 (i.e., human IgG has R435 instead of H435 found in human IgG1, IgG2 and IgG4), which may result in reduced binding at pH 6. FcRn comprises a heterodimer of two polypeptides with molecular weights of approximately 50 kD and 15 kD, respectively. The extracellular domain of the 50 kD polypeptide was shown to be related to the major histocompatibility complex (MHC) class I α chain, and the 15 kD polypeptide was shown to be nonpolymorphic β2-microglobulin (β2-m). FcRn is also expressed in various tissues throughout species, in addition to the placenta and neonatal intestine, as well as in various types of endothelial cell lines. It is also expressed in adult vascular endothelium, muscle vasculature, and hepatic sinusoids, suggesting that endothelial cells may be most responsible for maintaining serum IgG levels in humans and mice.

[0030] "Fusion protein" refers to a chimeric polypeptide comprising a first polypeptide linked to a second polypeptide that is not naturally linked in nature. Fusion proteins can contain three or more domains.

[0031] The terms "hinge Fc region", "Fc hinge region", "hinge Fc domain" or "Fc hinge domain", as used herein, are used interchangeably and refer to the region of an IgG molecule that consists of the Fc region (residues 231 to 447 as numbered by the EU index) and the hinge region (residues 216 to 230 as numbered by the EU index) extending from the N-terminus of the Fc region.

[0032] The term "host cell" refers to a particular subject cell that has been transfected with a nucleic acid molecule or infected with a phagemid or bacteriophage, and the progeny or potential progeny of such a cell. The progeny of such cells may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in successive generations or upon integration of the nucleic acid molecule into the host cell genome.

[0033] "Linked," "fused," or "fusion" are used interchangeably. These terms refer to the joining of two or more elements or components by any means, including chemical conjugation or recombinant means.

[0034] "Polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can contain modified nucleotides, such as methylated nucleotides and their analogs.

[0035] "ScFv" refers to an antibody fragment comprising an antibody VH / VL domain pairing. An scFv comprises a polypeptide linker between the VH and VL domains. An scFv may also comprise a non-canonical amino acid sequence variant, such as an engineered cysteine. An scFv may comprise a pair of engineered cysteines for intradomain disulfide bond formation.

[0036] "Subject" refers to an animal, human or non-human, to which treatment according to the method of the present invention is provided. Veterinary and non-veterinary applications are contemplated. The term includes, but is not limited to, mammals, such as humans, other primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep and goats. Typical subjects include humans, farm animals, and household pets such as cats and dogs. A preferred subject is a human.

[0037] A "therapeutically effective amount" refers to an amount of a fusion protein, polypeptide, molecule, or pharmaceutical composition thereof effective to "treat" a disease or disorder in a subject or mammal.

[0038] "Treating" or "treatment" or "treat" refers to both (1) therapeutic measures that cure, slow, alleviate symptoms, and / or halt progression of a diagnosed pathological condition or disorder, and (2) prophylactic or preventative measures that prevent and / or slow progression of a targeted pathological condition or disorder. Thus, those in need of treatment include those who already have the disorder; those susceptible to having the disorder; and those in whom the disorder is to be prevented. In certain embodiments, a subject is successfully "treated" of a disease or condition, e.g., cancer, by the methods of the present disclosure when the patient exhibits a complete, partial, or temporary remission of the disease or condition, e.g., certain types of cancer.

[0039] "Vector" refers to a construct capable of delivering, and in some embodiments expressing, one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0040] FcRN binding fragment The Fc region is a non-antigen-binding component of the antibody that facilitates various antibody-mediated functions. One specific function is to bind to the FcRn receptor to facilitate antibody recycling and regulate antibody half-life. High affinity binding between the Fc region and FcRn at endosomal pH facilitates antibody transport out of acidic endosomes and inhibits antibody degradation in lysosomes.

[0041] The present disclosure relates to the development of FcRn-binding fragments stabilized in monomeric form that can be used to extend the half-life of various therapeutic methods. Such enhancement is achieved by enabling the FcRn-recycling property of Fc region to be achieved in monomeric form. The FcRn-binding fragments can be used to create fusion proteins to expand the repertoire of therapeutic methods that benefit from FcRn recycling.

[0042] Thus, the present disclosure provides an FcRn-binding fragment of the Fc region of an IgG molecule, the FcRn-binding fragment comprising F at position 351, R, K, D, E, F, Y, P, G, L or M at position 354, R at position 366, K at position 395, R at position 405, and E at position 407. Amino acid numbering is according to the EU index.

[0043] The examples show that these amino acid substitutions to the wild-type (wt) sequence of the Fc region of an IgG molecule improve the monomeric stability of the FcRn-binding fragment. The Fc region in a wt antibody typically exists in a dimeric form. Improved monomeric stability is advantageous when utilizing the FcRn-binding fragment in a fusion protein where it is desired that the therapeutic protein is monomeric (e.g., in gene therapy applications or for delivery by inhalation, where packing size limitations or aerosolization properties prevent administration of standard antibodies via these routes). Expression and purification of the FcRn-binding fragment for conjugation to fusion proteins, polypeptides, or non-protein agents may also improve yields in large-scale production compared to isolating the therapeutic protein with quaternary structure.

[0044] Generally, the amino acids at each specified position in the sequence are non-canonical, meaning that they are not normally found at the specified position in a wild-type Fc region, particularly a wild-type human Fc region. Amino acid modifications (e.g., substitutions, deletions, or insertions) can be engineered into the sequence using standard genetic engineering techniques well known to those of skill in the art.

[0045] Thus, the present disclosure provides an FcRn-binding fragment of the Fc region of an IgG molecule, the FcRn-binding fragment comprising the following amino acid substitutions: F at position 351, R, K, D, E, F, Y, P, G, L or M at position 354, R at position 366, K at position 395, R at position 405, and E at position 407. Amino acid numbering is according to the EU index.

[0046] Unless otherwise stated herein, reference to a particular amino acid (numbered according to the EU index) at a particular position in an Fc region means that the amino acid is a substitution at that particular position compared to the native sequence.

[0047] In some examples, the FcRn-binding fragment further comprises R, K, D, or E at position 354, and optionally R, K, D, or E at D or E (wherein the numbering is according to the EU index). The examples show that S354 supports the dimerization of the FcRn-binding fragment at high concentrations. The formation of higher order species at high concentrations may be undesirable for certain therapeutic agents administered as highly concentrated solutions by subcutaneous injection. Certain treatments may require the administration of relatively high amounts (e.g., more than 300 mg) to achieve a therapeutic effect. High amounts may require highly concentrated formulations to reduce injection volumes. Higher injection volumes are generally undesirable for patients. The formation of higher order species at high concentrations may increase viscosity, which may be problematic when the formulation is administered from a pressurized device such as a pre-filled syringe. Furthermore, higher monomer purity at high concentrations may increase production yields if higher order species are lost during the manufacturing process, for example, during filtration or fractionation. In some examples, the FcRn-binding fragment further comprises an E at position 354.

[0048] In some examples, the FcRn-binding fragment comprises from about amino acid residue 216 to about amino acid residue 447 of an IgG molecule (wherein numbering is according to the EU index). Residues 216-447 include the hinge-Fc region (residues 216-230, as numbered according to the EU index) and the Fc region (residues 231-447, as numbered according to the EU index).

[0049] In some examples, an FcRn-binding fragment comprises from about amino acid residue 231 to about amino acid residue 447 of an IgG molecule (wherein numbering is according to the EU index).

[0050] In a particular example, the FcRn-binding fragment is derived from the IgG4 subclass of IgG, but may be any other IgG subclass of a given animal. For example, in humans, the IgG classes include IgG1, IgG2, IgG3, and IgG4. In a particular example, the FcRn-binding fragment is derived from an IgG4 Fc region.

[0051] In some examples, the FcRn binding fragment contains an F at position 351, an E at position 354, an R at position 366, a K at position 395, an R at position 405, and an E at position 407 (wherein the amino acids are numbered according to the EU index).

[0052] In some examples, the FcRn-binding fragment may contain conservative amino acid substitutions for each of these amino acids at each of the following amino acid positions: F at position 351, E at position 354, R at position 366, K at position 395, R at position 405, and E at position 407 (wherein the amino acid numbering is according to the EU index).

[0053] Exemplary conservative amino acid substitutions for each of these amino acids are as follows:

[0054] [Table 1]

[0055] Conservative substitution is an amino acid substitution in a protein that changes a given amino acid to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, and size). Thus, one skilled in the art can expect that conservative amino acid substitution will produce similar benefits as the most exemplary FcRn-binding fragments described herein in terms of monomer formation and half-life modification. One skilled in the art can generate FcRn-binding fragments with conservative amino acid substitutions at one or more of positions 351, 354, 366, 395, 405, and 407 (numbered according to the EU index) and test whether these variants have similar properties (e.g., monomer stability, FcRn-binding ability, half-life properties) as the preferred FcRn-binding fragments disclosed herein by carrying out the experiments described in the Examples.

[0056] In some examples, the FcRn-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 1. In some examples, the FcRn-binding fragment has the amino acid sequence set forth in SEQ ID NO: 1. In some examples, the FcRn-binding fragment consists of the amino acid sequence set forth in SEQ ID NO: 1.

[0057] In some examples, the FcRn-binding fragment comprises a half-life extending mutation (eg, an amino acid insertion, deletion, or substitution).

[0058] In some instances, the FcRn-binding fragment has an equilibrium dissociation constant (K D). The Examples show that human IgG1 Fc region binds to FcRn with a KD of approximately 300 nM at pH 6. In contrast, the engineered FcRn-binding fragment can bind to human FcRn with a substantially improved KD at pH 6.0. The stronger binding at lower pH means that the recycling tendency from endosomes may be improved compared to FcRn-binding fragments containing the native sequence.

[0059] K D can be measured by a variety of techniques well known to those skilled in the art, including those outlined in the Examples. For example, binding of FcRn-binding fragments to purified recombinant human FcRn can be measured by biolayer interferometry. Biolayer interferometry can be performed using an Octet384 instrument (ForteBio, Menlo Park, CA). For example, 1 μg / mL biotinylated FcRn in PBS buffer (pH 7.4) or 100 mM MES buffer (pH 6.0) containing 3 mg / mL bovine serum albumin, 0.05% (vol / vol) and Tween 20 (1× Kinetics Buffer; ForteBio) is captured with a streptavidin biosensor (ForteBio). The loaded biosensor is then washed with assay buffer to remove unbound proteins, followed by measuring binding and dissociation by serial dilutions of different Fc variants or Fc fusion constructs at the desired pH. The kinetic parameters (k) were then calculated from a nonlinear fit based on a 1:1 binding kinetic model of the data using Octet software (version 7.2). on and k off ) and apparent K D is calculated using the following formula:

number

[0060] In some instances, the FcRn-binding fragment has an equilibrium dissociation constant (K D ).

[0061] In some examples, the FcRn-binding fragment has a K of about 1 nM to about 300 nM (e.g., about 1 nM to about 250 nM, about 1 nM to about 240 nM, about 1 nM to about 230 nM, about 1 nM to about 200 nM, about 1 nM to about 180 nM, about 1 nM to about 160 nM, about 1 nM to about 140 nM, about 1 nM to about 120 nM, about 1 nM to about 100 nM, about 1 nM to about 80 nM, about 1 nM to about 60 nM, about 1 nM to about 40 nM, about 1 nM to about 20 nM, or about 1 nM to about 100 nM) to human FcRn at pH 6. D In some examples, the FcRn-binding fragment binds to the FcRn-binding domain at pH 6 with a K of about 1 nM to about 10 nM. D In some examples, the FcRn-binding fragment binds to human FcRn with a K of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, or about 10 nM. D In some examples, the FcRn-binding fragment binds to human FcRn with a K of about 5 nM at pH 6.0. D In some examples, the FcRn-binding fragment binds to human FcRn with a K of 5 nM at pH 6.0. D It binds to human FcRn at

[0062] In some instances, K D The value may be determined, for example, by biolayer interferometry, as described above and in the Examples.

[0063] Antigen-binding domain In certain examples, the present disclosure provides a fusion protein comprising at least one antigen-binding domain covalently linked to an FcRn-binding fragment disclosed herein.

[0064] In some examples, the fusion protein or polypeptide comprises multiple antigen binding domains.

[0065] In some examples, the fusion protein or polypeptide comprises one, two, three, four or five antigen binding domains.

[0066] In some examples, the fusion protein or polypeptide comprises two antigen binding domains.

[0067] In some examples, the first antigen-binding domain is at the N-terminus of the FcRn-binding fragment.

[0068] In some examples, the second antigen-binding domain is at the C-terminus of the FcRn-binding fragment.

[0069] In some examples, each antigen-binding domain is at the N-terminus of the FcRn-binding fragment.

[0070] In some examples, each antigen-binding domain is at the C-terminus of the FcRn-binding fragment.

[0071] In some examples, each antigen-binding domain specifically binds to a different antigen.

[0072] In some examples, each antigen binding domain is independently selected from an Fv, Fab, Fab', F(ab')2, Fab'-SH, diabody, triabody, tetrabody, linear antibody, or scFv.

[0073] In some examples, the antigen-binding fragment is a Fab.

[0074] In some examples, the antigen-binding fragment is an scFv.

[0075] In some examples, the fusion protein or polypeptide comprises a first and a second antigen binding domain that are Fabs.

[0076] In some examples, the fusion protein or polypeptide comprises a first and a second antigen binding domain that is an scFv.

[0077] In some examples, the fusion protein or polypeptide comprises a first antigen-binding domain that is a Fab and a second antigen-binding domain that is an scFv.

[0078] The Examples demonstrate that the FcRn-binding fragments disclosed herein can be used to generate bispecific molecules.

[0079] Fc region variants In a particular example, one or more amino acid modifications can be introduced into the Fc region, thereby generating an Fc region variant. This Fc region variant can then be incorporated into the FcRn-binding fragment disclosed herein. The Fc region variant can include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0080] In some instances, the FcRn-binding fragment has some, but not all, effector functions that make it a desirable candidate for applications where the half-life of the FcRn-binding fragment in vivo is important, but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus may lack ADCC activity) but maintains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIIIA, while monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the molecule of interest may be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0081] In some examples, one or more amino acid modifications can be introduced into the Fc region to increase binding to FcRn. In some examples, the FcRn-binding fragment contains the following three mutations, numbered by the EU index: M252Y, S254T, and T256E ("YTE mutations") (see also U.S. Pat. No. 8,697,650; Dall'Acqua et al., Journal of Biological Chemistry 281(33):23514-23524 (2006)).

[0082] In certain instances, the YTE mutations increase the serum half-life of the FcRn binding fragment as compared to the native (i.e., non-YTE mutant) FcRn binding fragment. In some instances, the YTE mutations increase the serum half-life of the FcRn binding fragment by 2-fold as compared to the native (i.e., non-YTE mutant) FcRn binding fragment. In some instances, the YTE mutations increase the serum half-life of the FcRn binding fragment by 3-fold as compared to the native (i.e., non-YTE mutant) FcRn binding fragment. In some instances, the YTE mutations increase the serum half-life of the FcRn binding fragment by 4-fold as compared to the native (i.e., non-YTE mutant) FcRn binding fragment. In some instances, the YTE mutations increase the serum half-life of the FcRn binding fragment by at least 5-fold as compared to the native (i.e., non-YTE mutant) FcRn binding fragment. In some instances, the YTE mutation increases the serum half-life of the FcRn-binding fragment by at least 10-fold compared to a native (i.e., non-YTE mutant) FcRn-binding fragment. See, e.g., U.S. Patent No. 8,697,650; see also Dall'Acqua et al., Journal of Biological Chemistry 281(33):23514-23524 (2006).

[0083] In some examples, the FcRn-binding fragment is mutated to reduce effector function. In some examples, the FcRn-binding fragment with reduced effector function includes one or more substitutions of residues 238, 265, 269, 270, 297, 327 and 329 of the Fc region numbered by EU index (US Pat. No. 6,737,056). Such mutant FcRn-binding fragments include those having substitutions of two or more of the amino acids at positions 265, 269, 270, 297 and 327 of the EU index, such as the so-called "DANA" Fc region mutants having substitutions of residues 265 and 297 of the EU index (i.e., D265A and N297A according to EU numbering) (US Pat. No. 7,332,581). In a particular example, the FcRn-binding fragment includes the following two amino acid substitutions: D265A and N297A. In a particular example, the FcRn-binding fragment consists of the following two amino acid substitutions: D265A and N297A.

[0084] In a particular example, the proline at position 329 (EU index numbering) (P329) is substituted with glycine or arginine, or an amino acid residue large enough to disrupt the proline sandwich in the Fc / Fcγ receptor interface formed between P329 of Fc and tryptophan residues W87 and W110 of FcgRIII (Sondermann et al.: Nature 406, 267-273 (20 July 2000)). In a further example, the at least one additional amino acid substitution in the FcRn-binding fragment is S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S, and in yet another example, the at least one additional amino acid substitution is L234A and L235A in the Fc region of human IgG1, or S228P and L235E in the Fc region of human IgG4 (U.S. Pat. No. 8,969,526).

[0085] In some examples, the FcRn-binding fragment comprises one or more substitutions described in US Patent Application Publication No. 2005 / 0014934A1 that improve binding of the Fc region to FcRn, including those having a substitution at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, as numbered by the EC index, such as residue 434 in the Fc region (US Pat. No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0086] In some examples, the FcRn-binding fragment comprises a His435 loop mutation described in WO 2015 / 175874, which is incorporated herein by reference in its entirety, that modulates binding to FcRn. The examples show that the His435 loop mutation increases the serum half-life of the FcRn-binding fragment compared to an FcRn-binding fragment comprising native amino acid residues at positions 432-437, numbered according to the EU index.

[0087] In some examples, the Fc region comprises the following substitutions: C at position 432; H, R, P, T, K, S, A, M, or N at residue 433; Y, N, R, W, H, F, S, M, or T at residue 434; H at residue 435; L, Y, F, R, I, K, M, V, H, S, or T at residue 436; and C at residue 437. In some examples, the in vivo half-life of the modified FcRn-binding fragment is increased compared to a native (i.e., non-YTE mutant) FcRn-binding fragment. Increasing the in vivo half-life of biologically active molecules has many advantages, including reduced dosage and / or frequency of administration of these molecules, for example, in vaccines, passive immunotherapy, and other therapeutic and prophylactic methods.

[0088] In some examples, the FcRn binding fragment variant comprises the following substitutions: C at position 432; S, H, R, P, T, K, A, M or N at residue 433; Y, R, W, H or F at residue 434; H at residue 435; L, R, I, K, M, V or H at residue 436; and C at residue 437 (wherein numbering is according to the EU index). In some examples, the FcRn binding fragment comprises: C at position 432; S at position 433; W or Y at position 434; H at position 435; L at position 436, and C at position 437 (wherein numbering is according to the EU index). In some examples, the FcRn binding fragment variant comprises C at position 432; S at position 433, Y at position 434; H at position 435; L at position 436, and C at position 437 (wherein numbering is according to the EU index). WO 2015 / 175874 and the Examples herein show that this particular combination of mutations increases the pH-dependent binding of the Fc region to FcRn and increases pH-dependent FcRn-mediated recycling, thereby improving half-life.

[0089] In some examples, the FcRn-binding fragment variant comprises a deletion of the amino acid at position 438 according to the EU index numbering. The examples surprisingly show that deleting this amino acid from the sequence produces an FcRn-binding fragment with at least equivalent half-life extension properties without the developability profile. In some examples, the FcRn-binding fragment comprises a deletion of Q438 (wherein the numbering is according to the EU index).

[0090] Treatment methods The fusion proteins, polypeptides or molecules disclosed herein can be used in methods of treatment, such as methods of treating cancer. Methods of treatment are also provided, comprising administering to a subject in need of treatment a therapeutically effective amount of the fusion proteins, polypeptides or molecules disclosed herein. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, such as the decision regarding dosage, is within the responsibility of general practitioners and other physicians.

[0091] nucleic acid The present disclosure provides polynucleotides comprising nucleic acid sequences encoding the FcRn-binding fragments of the fusion proteins, polypeptides or molecules disclosed herein. These polynucleotides may be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or the non-coding strand (antisense strand). In a particular example, the DNA is cDNA used to generate the FcRn-binding fragments of the non-naturally occurring recombinant fusion proteins, polypeptides or molecules.

[0092] In certain instances, the polynucleotide is isolated. In certain instances, the polynucleotide is substantially pure. In certain instances, the polynucleotide comprises a coding sequence for a mature polypeptide (e.g., a leader sequence that functions as a secretory sequence to control transport of the polypeptide out of the cell) fused in the same reading frame to a polynucleotide (natural or heterologous) that aids in the expression and secretion of the polypeptide from a host cell, for example. A polypeptide having a leader sequence is a preprotein, and can have a leader sequence that is cleaved by the host cell to form the mature form of the polypeptide. In some aspects, the polynucleotide is modified to optimize codon usage for a particular host cell.

[0093] In certain aspects, the polynucleotide comprises a sequence encoding a fusion protein, FcRn-binding fragment of a polypeptide or molecule, fused in the same reading frame to a heterologous marker sequence that allows, for example, purification of the encoded polypeptide. For example, the marker sequence can be a hexahistidine tag provided by the pQE-9 vector, which provides for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence can be a hemagglutinin (HA) tag derived from influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used.

[0094] Polynucleotides may contain modifications in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain changes that produce silent substitutions, additions, or deletions, but do not change the properties or activities of the encoded polypeptide. In some embodiments, polynucleotide variants are created by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants may also be generated for a variety of reasons, such as, for example, to optimize codon expression for a particular host (changing codons in human mRNA to those preferred by bacterial hosts, such as E. coli).

[0095] Also provided are vectors and cells comprising the polynucleotides described herein. When constructed (by synthesis, site-directed mutagenesis, or otherwise), a polynucleotide sequence encoding a particular isolated polypeptide of interest can be inserted into an expression vector and operably linked to expression control sequences suitable for protein expression in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of biologically active polypeptides in a suitable host. As is well known in the art, to achieve high expression levels of a transfected gene in a host, the gene must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host.

[0096] In a particular example, a recombinant expression vector is used to amplify and express DNA encoding the FcRn-binding fragment of a fusion protein, polypeptide, or molecule disclosed herein. A recombinant expression vector is a replicable DNA construct that has a synthetic or cDNA-derived DNA fragment encoding the polypeptide chain of a fusion protein, polypeptide, or FcRn-binding fragment of a molecule operably linked to suitable transcriptional or translational regulatory elements derived, for example, from mammalian, microbial, viral, or insect genes. A transcription unit generally comprises a collection of (1) genetic elements that have a regulatory role in gene expression, such as a transcriptional promoter or enhancer, (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, and (3) appropriate transcriptional and translational initiation and termination sequences, as described in more detail below. Such regulatory elements may include operator sequences that control transcription. A wide variety of expression host / vector combinations are available. Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression control sequences derived from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, for example, plasmids derived from E. coli, such as pCR1, pBR322, pMB9 and their derivatives, broader host cell plasmids such as M13, and filamentous single-stranded DNA phages.

[0097] Suitable host cells for expressing the FcRn-binding fragments of the fusion proteins, polypeptides or molecules disclosed herein include prokaryotes, yeast, insect or higher eukaryotic cells under the control of a suitable promoter. Prokaryotes include gram-negative or gram-positive organisms, such as E. coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems can also be used. Cloning and expression vectors suitable for use with bacterial, fungal, yeast and mammalian cell hosts are described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985), the relevant disclosure of which is incorporated herein by reference. Further information regarding methods of protein production, including antibody production, can be found, for example, in U.S. Patent Application Publication No. 2008 / 0187954, U.S. Patent Nos. 6,413,746 and 6,660,501, and WO 04 / 009823, each of which is incorporated by reference in its entirety.

[0098] The FcRn-binding fragment of the fusion protein, polypeptide or molecule produced by the transformed host can be purified by any suitable method. Such standard methods include chromatography (e.g., ion exchange, affinity, and size column chromatography), centrifugation, differential solubility, or any other standard protein purification technique. Affinity tags such as hexahistidine, maltose binding domain, influenza coat sequence, and glutathione-S-transferase can be attached to the protein to allow easy purification by passing through a suitable affinity column. The isolated protein can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

[0099] In another aspect, the disclosure provides a method for making a fusion protein, polypeptide or FcRn-binding fragment as defined herein. The method comprises: (i) mutating a nucleic acid sequence encoding the FcRn-binding fragment by replacing the codons at amino acid positions 351, 354, 366, 395, 405 and 407 with codons encoding the amino acids at each position as described herein. The numbering of the amino acid positions is according to the EU index. The method further comprises expressing the mutagenized nucleic acid sequence; and isolating the expressed fusion protein, polypeptide or FcRn-binding fragment.

[0100] In some examples, the method comprises the further step of reacting the FcRn-binding fragment with a non-proteinaceous agent to form a molecule, as disclosed herein.

[0101] Pharmaceutical Compositions The present disclosure extends to compositions comprising the fusion proteins, polypeptides or molecules described herein, in particular pharmaceutical compositions (or diagnostic compositions) comprising a fusion protein, polypeptide or molecule of the present disclosure and a pharmaceutical excipient, diluent or carrier.

[0102] The composition will usually be supplied as part of a sterile pharmaceutical composition which will usually include a pharma- ceutically acceptable carrier.The pharmaceutical composition of the invention may further comprise a pharma- ceutically acceptable adjuvant in the context of a vaccine formulation.

[0103] The present disclosure also extends to methods of preparing said compositions, e.g., the preparation of a pharmaceutical or diagnostic composition, comprising mixing together a molecule of the present disclosure, e.g., a hydrolyzed molecule of the present disclosure, with the addition of one or more pharma- ceutically acceptable excipients, diluents or carriers.

[0104] The fusion proteins, polypeptides or molecules of the present disclosure may be the sole active ingredient in a pharmaceutical or diagnostic composition or may be associated with other active ingredients.

[0105] A pharmaceutical composition suitably contains a therapeutically effective amount of a fusion protein, polypeptide or molecule according to the present disclosure. The therapeutically effective amount can be estimated initially in cell culture assays or in animal models, usually rodents, rabbits, dogs, pigs or primates. Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes of administration in humans.

[0106] The compositions may be administered alone to a patient or may be administered in combination (eg, simultaneously, sequentially or separately) with other agents, drugs or hormones.

[0107] A pharma- ceutically acceptable carrier should not itself induce the production of antibodies harmful to the individual receiving the composition and should not be toxic.

[0108] Pharmaceutically acceptable carriers in therapeutic compositions may further contain liquids such as water. Auxiliary substances such as wetting and emulsifying agents or pH buffering substances may also be included in such compositions. Such carriers allow the pharmaceutical compositions to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries and suspensions for ingestion by the patient.

[0109] Suitable administration forms include those suitable for parenteral administration, such as by injection or infusion, for example, by bolus injection or continuous infusion. If the product is for injection or infusion, it may take the form of a suspension, solution or emulsion, and may contain compounding agents, such as suspending agents, preservatives, stabilizers and / or dispersing agents. Alternatively, the molecules of the present disclosure may be in a dry form to be reconstituted with an appropriate sterile liquid before use.

[0110] In formulations according to the present disclosure, preferably the pH of the final formulation is not similar to the isoelectric point of the fusion protein, polypeptide or molecule, for example, if the pH of the formulation is 7, a pI of 8-9 or higher may be appropriate.

[0111] The pharmaceutical compositions of the present invention can be administered by any number of routes, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous (see, e.g., WO 98 / 20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, vaginal or rectal routes. The pharmaceutical compositions of the present invention can also be administered using a hypodermic spray. Typically, the therapeutic compositions can be prepared as injectables, either as solutions or suspensions. Solid forms suitable for solution or suspension in liquid vehicles prior to injection can also be prepared.

[0112] Direct delivery of the composition can generally be accomplished by injection, subcutaneous, intraperitoneal, intravenous or intramuscular delivery, or delivered to the interstitial space of a tissue. The composition can also be administered to a lesion. Dosage treatment can be a single dose schedule or a multiple dose schedule.

[0113] A thorough discussion of pharma- ceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Publishing Company, NJ 1991). EXAMPLES

[0114] Example 1 In the field of drug discovery, there has been remarkable progress in the development of paradigm-shifting antibodies and antibody-derived therapeutics, along with groundbreaking advances in both disease biology and antibody technology. 1,2 Synergies between research advances in immuno-oncology and the development of novel bispecific and multispecific targeting platforms are generating many promising possibilities for driving current successes in cancer therapy. 3-7The importance of minimizing toxicity while achieving clinical efficacy highlights the need to fine-tune targeting valency from the default bivalent formats of immunoglobulin G (IgG) or fragment crystallizable (Fc) fusion proteins. Increasing evidence indicates that bispecific and multispecific formats with monovalent targeting arms are required to reduce nonspecific cell killing, cytokine release, and undesired receptor cross-linking and improve receptor agonism and trafficking. 8-11 T cell engagers or natural killer (NK) cell engagers, such as bispecific T cell engagers (BiTe), dual affinity retargeting proteins (DART), bispecific killer cell engagers (BiKE), and trispecific killer cell engagers (TriKE), have shown clinical promise for monovalent bispecific and multispecific targeting, but suffer from short half-lives. Heterodimeric Fc engineering has made it possible to extend the half-life of these engagers by developing technologies such as knob-in-hole, CrossmAb, and DuetmAb that can guide the correct chain pairing for bispecific and multispecific targeting. 11-13

[0115] Successful generation of conformationally stable monomeric Fc antibody fragments with "tunable" serum half-lives would open new possibilities for antibody and Fc fusion therapeutics. Many of the more than 180 therapeutic proteins approved by the US Food and Drug Administration have the potential to be adapted into active, long-lasting fusion proteins. 14-18Immune-cell engagers, antibody-drug conjugates, immunocytokine fusions, and other therapeutic proteins can be tailor-designed to provide monovalent targeting in both monospecific and multispecific formats for enhanced activity and reduced toxicity. Previous efforts to engineer monomeric Fc, defined as a set of CH2 and CH3 domains, have proven difficult due to extensive interactions that must be disrupted at the CH3-CH3 dimer interface. At high concentrations, a monomer-dimer equilibrium has been observed for many mutants. 19,20 Among engineered monomeric Fc modalities, only two molecules have been reported to have crystal structures with apparent homogeneity and stability. 13,21 One of these is a monomer that is stabilized by the addition of a glycosylation site that blocks the CH3-CH3 interaction. 21 The other, generated by our group, is a monomeric Fc derived from an IgG4 phage library that was rationally designed based on previous findings. 13

[0116] To expand the application of the monomeric Fc platform for the next wave of protein therapeutics, we decided to address three interrelated key aspects of this endeavor: (1) tunable serum half-life, (2) diverse construction of monovalent bispecific molecules, and (3) facile structural exploration of the interaction of fetal Fc receptor (FcRn) with Fc variants. The pH-dependent Fc-FcRn interaction is a key factor responsible for the extended serum half-life of antibodies and their derivatives. FcRn utilizes antibody molecules and carries them through acidic endosomal vesicles, protecting them from lysosomal degradation and releasing them outside the cell due to weak binding at neutral pH. 22,23 Monomeric Fc variants are expected and observed to have reduced apparent binding to FcRn due to loss of dimer avidity. 13,21Previously, we circumvented the loss of FcRn binding by incorporating the YTE (M252Y / S254T / T256E) mutation into the phage library template design, and the resulting monomeric Fc molecules provided a greater than 10-fold improvement in FcRn binding affinity compared to their counterparts without YTE. 13,24

[0117] Here we report a structure-guided approach to engineer monomeric Fc molecules amenable to modification of half-life extension beyond that achieved by previously engineered YTE mutations. This is the first proof-of-concept monomeric bispecific molecular design showing that only one copy of the CH2-CH3 domain can significantly improve serum half-life in vivo. Co-crystal structures of these monomeric Fc molecules with FcRn reveal details of the interface that may serve as a basis for constructing other half-life extensions.

[0118] result Disruption of the CH3-CH3 interface based on structural insight. In a previous study, we generated a monomeric Fc variant C4 (renamed MFc1) from a rationally designed IgG4 phage library containing a set of mutations in the CH3 domain to completely stabilize the disruption of the Fc dimer interface (Figure 1a). To extend the suitability of these dimer-disrupting mutations with half-life extending mutations other than YTE, we began our efforts with test variant T1 by replacing the YTE mutation in the CH2 domain of the monomeric Fc sequence with a new set of half-life modifying mutations in CH3 around residues 432-438. This set of mutations was selected because it met two criteria: First, the new set of mutations would be useful to explore ways to further extend the half-life based on findings from a previous phage library campaign showing that the YTE mutations may improve FcRn binding more than the YTE mutations. 25 Second, the extensive nature of this set of mutations will act as a "stress test" for the ability of monomeric Fc to sustain disruptions at the Fc dimerization interface.

[0119] We solved the crystal structure of T1 and analyzed it together with the crystal structure of dimeric IgG4 Fc and the previously solved structure of monomeric Fc, MFc2 (or C4n without the YTE mutation; PDB ID: 5HVW). 13 The CH3 domains of MFc2 and IgG4 Fc (PDB ID: 4C54) were superimposed with T1 with root mean square deviations (RMSD) of approximately 0.7 and 0.5 Å on the Cα atoms, respectively (when excluding the last 11 residues that form an artificial chain exchange in the MFc2 structure). Despite the high degree of similarity in the CH3 domains, T1 unexpectedly exhibited homodimer formation (Fig. 1b). However, the dimer interface was significantly different from that of wild-type IgG4. A small set of hydrogen bonds involving amino acids Thr350 / Leu440 and Gln355 / Glu356 was formed between the chains, indicating a possible breakdown in monomeric Fc formation (Fig. 1c). When examining the newly formed T1 dimer interface superimposed with MFc2, it was observed that one of the mutations in MFc2, namely Arg366, shifted position in its side chain. This shift allowed Phe351 from each chain to "reach" and form hydrophobic stacking interactions. Moreover, the Arg366 mutation established few new hydrogen bonds that stabilized the dimer (Fig. 1c). From detailed examination of the structure, we infer that the Arg366 shift was enabled in part by the presence of space on the Ser354 side chain. Although analytical methods showed a relatively well-behaved T1 protein, the crystal structure suggests that despite extensive disruption of the dimer interface, it remains possible that at high protein concentrations the engineered Fc may participate in dimer formation.

[0120] Structure-based engineering and characterization of binding and biophysical properties. The newly formed interactions observed in the T1 crystal structure created an opportunity to construct a more adaptable monomeric Fc molecule. Structural inspection of the T1 dimer interface (Fig. 1c) suggested that replacing the serine at position 354 with an amino acid with a larger side chain might prevent the Arg366 side chain from paving the way for hydrophobic interactions involving Phe351. This substitution might also hinder the resulting hydrogen bond formation. Interestingly, Ser354 was chosen as one of the interface mutation positions in the original phage library template, even though our first monomeric Fc construct, MFc1, had generated mutations at all target positions except Ser354 (Fig. 1a). 13 Therefore, we designed a small panel of improved mutations at position 354 to test whether the monomer formation could be fully stabilized by introducing a bulkier side chain or electrostatic repulsion. T1 variants (T1-lib) containing substitutions at position 354 with charged residues (R, K, D, E) and bulky polar and non-polar residues (F, Y, P, Q, L, M) were constructed (Fig. 2a) and subsequently purified by Protein A affinity chromatography. SEC-MALS analysis revealed that almost all T1-lib improved variants were monomeric (Fig. 2b).

[0121] To select the most stable monomer among the T1-lib variants, we used the hydrophobic (T h ) We used differential scanning fluorometry (DSF), which has been established as an orthogonal screening tool to assess thermal denaturation as a function of residue exposure. 32,33 Monitor the thermal denaturation of T1-lib variants and determine the transition temperature of hydrophobic exposure, T h In particular, the type of amino acid substitutions was significantly different from that of the control, as acidic residues (Glu and Asp) produced transition temperatures up to 3°C higher than basic residues (Arg and Lys). h This affected the ranking (Figure 2c).

[0122] Structure and properties of monomeric Fc structures (MFc3 and MFc4) with S354E. Based on the SEC-MALS and DSF results, the S354E mutation was selected to explore the general applicability of the MFc platform. First, MFc3, an S354E point mutant of MFc1, was generated together with its non-glycosylated variant (N297D) for crystal structure confirmation to confirm compatibility with the original monomeric Fc construct (Fig. 1a). Crystals of the non-glycosylated MFc3 protein grew readily and diffracted to 2.4 Å. The solved structure showed that the MFc3 molecule maintained a monomeric state at high protein concentrations (Fig. 3). Superposition with the previously published monomeric Fc structure of MFc2 showed that the S354E mutation did not cause any significant changes in the structure of the CH2 or CH3 domains of Fc. 13 Apart from a slight change in the R405 side chain position, all other original sets of dimer-disrupting mutations in MFc1 aligned almost perfectly with each other. Importantly, the crystal structure showed that, as designed, the glutamic acid side chain substitution at position 354 indeed protrudes and disrupts the type of interaction observed in T1.

[0123] In accordance with our goal of constructing a set of monomeric Fc variants to modulate FcRn-mediated circulatory half-life, we focused on functional and structural characterization of FcRn interactions with our monomeric Fc molecules. Using recombinant FcRn binding assays, we found that MFc3 was significantly different from MFc1, with an equilibrium dissociation constant (K D ) (Table A). This finding suggested that the S354E substitution does not alter the interaction with FcRn. To confirm this binding mode, the MFc3 / FcRn complex was prepared at low pH, subsequently purified and crystallized, and diffraction data were collected at 2.6 Å resolution (FIG. 4a). The closest Fc-FcRn complex structure available for comparison was solved by our group at 3.8 Å and consisted of human IgG1 Fc (with the YTE set of mutations) bound to human FcRn in complex with human serum albumin (PDB ID: 4N0U). 29Comparison of the two interfaces showed that the modes of interaction were nearly identical, with minor differences that most likely arose from differences in the resolution of side chains in the electron density maps. This structural and functional invariance of the Fc-FcRn interaction was expected because the S354E mutation is more than 20 Å away from the FcRn interface.

[0124] Table A. Equilibrium binding of monomeric Fc variants to human FcRn in recombinant 1:1 binding format

[0125] [Table 2]

[0126] To assess whether the S354E mutation could indeed be used to drive T1 into a monomeric state, we crystallized T1-S354E (MFc4) in complex with FcRn (Figure 4b). The solved MFc4 / FcRn complex structure showed that MFc4 is indeed monomeric. Despite the fact that the exploration of crystallization conditions for the MFc3 / FcRn and MFc4 / FcRn complexes was performed independently, the crystals grew from the same conditions and showed nearly identical cell parameters and space groups. As expected from previous studies, the sequence change from MFc3 to MFc4 resulted in a significant increase in the recombinant FcRn binding affinity from 300 nM to 5 nM (Table A). 25 For these molecules, pH-dependent FcRn binding was observed, with a ∼50-fold decrease in binding upon a change from pH 6 to neutral pH (data not shown). The overall structures of the MFc4 / FcRn and MFc3 / FcRn complexes were superimposed with an RMSD of 0.35 Å over 3,700 non-hydrogen atoms, suggesting a high degree of similarity. A comparative view of the structures of the MFc3 / FcRn and MFc4 / FcRn complexes revealed clear details of the Fc-FcRn interactions and provided a structural explanation for the observed differences in FcRn binding. The newly introduced Tyr434 and Leu436 in MFc4 increase the interface area by 500 Å. 2 From approximately 600 Å 2While increasing the cleavage potential of FcRn, we also added some hydrophobicity to the interaction (Fig. 4c, 4d). Residue Leu135 in FcRn, which was known to contribute to hydrophobicity at the Fc / FcRn interface, 29 This was implicated by the presence of Tyr434 and Leu436 in MFc4.

[0127] Using PDBePISA analysis, differential solvation energy heat maps were used to capture the energy contributions from individual residues in the binding pockets of MFc3 / FcRn and MFc4 / FcRn (Figure 4e). As a measure of the extent of the binding interface, differential solvation energy calculations reflect the portions of the surface of the structure that are inaccessible to solvent. 30,31 The strongest contributors to the binding interface from MFc4 were found to be Ile253, Tyr434, and Leu436, in contrast to residues Ile253 and Thr254 in MFc3. The total solvation energy change was 5.09 kcal / mol for MFc4, indicating significantly stronger hydrophobic interactions than MFc3, with a total ΔiG of 3.14 kcal / mol (Figure 4e). In comparison, we performed the same calculations only on the other available human YTE IgG1 Fc-FcRn complex and found a similar ΔiG of 2.67 kcal / mol, consistent with the binding affinity measurements.

[0128] The MFc4 / FcRn complex structure also provided structural insight into wild-type human Fc / FcRn interactions, which were not available previously for two reasons. First, the wild-type human Fc / FcRn interaction is relatively weak, making purification of the complex difficult, if not impossible. Second, the crystallization tendency of dimeric Fc (hence the name "crystallizable fragment") is so high that most attempts result in crystals containing only Fc (unpublished data). Before our stable monomeric Fc variants were available, we had to rely on rat Fc or FcYTE to improve the affinity between Fc and FcRn, and increase the residence time of the bound state for crystal formation, together with albumin to disrupt Fc crystal lattice formation. 29With the help of MFc3 and MFc4 structural complexes with FcRn and their two distal sets of mutations, it became possible to obtain a better understanding between the wild-type binding interface and their mutation sets. For example, in the wild-type interface near residues 252, 254 and 256, where the YTE (M252Y / S254T / T256E) mutations are present, the interface solvation energy map showed that Met252 only has a moderate contribution to the FcRn interface and Thr256 is practically not involved (Fig. 4e). The most significant FcRn interaction from the 252-254-256 loop was provided by Ser254. This analysis also explains why the YTE mutation set can improve the affinity between Fc and FcRn and why its S254T substitution showed the strongest contribution.

[0129] Construction of monomeric bispecific molecules. These monomeric Fc molecules can be easily used as building blocks to design monovalent dual-targeting Fc fusion proteins. Previously, we used the MFc1 variant to generate onartuzumab Fab-MFc1 fusion proteins. 8,13 In this study, we designed the first example of a monovalent bispecific targeting molecule using a monomeric Fc construct. With a single plasmid construct, we conjugated the same Fab domain from onartuzumab to the N-terminus of MFc1 or MFc4 together with the C-terminal single-chain variable fragment (scFv) of an antibody targeting programmed cell death ligand 1 (PD-L1) (Figure 5a). The constructs were transfected for transient expression in HEK293 suspension cultures (expression titer approx. 90 mg / L) and the protein was subsequently purified with a one-step protein A purification. SEC-MALS analysis suggested that the protein was monodisperse with an expected molecular weight of 100 kDa (Figure 5b). The dual targeting activity of the monomeric bispecific molecules was confirmed in a sandwich format on the Octet platform (Figure 5c). The bispecific molecules also maintained their corresponding FcRn binding at pH 6, 2-3 times that of the Fc domain alone.

[0130] Improved in vivo half-life with next-generation monomeric Fc. We achieved a significant improvement in in vitro FcRn binding in the MFc4 variant compared to MFc1. We also wanted to evaluate whether this improvement translates into an extended serum half-life in vivo. The newly generated Fab-MFc4-scFv and Fab-MFc1-scFv, with molecular weights (100 kDa) far exceeding the typical renal filtration clearance size (approximately 60 kDa), 34 It is an ideal molecule to evaluate the implications of improving the in vivo half-life of FcRn binding. We performed in vivo pharmacokinetic (PK) studies in hemizygous human FcRn (TG276) transgenic mice. This mouse model is a well-studied model that reflects Fc mutations and demonstrable PK effects on human FcRn binding from standard IgG1 with a serum half-life of about 18 hours. 13,25,35 Mice were administered 2.5 mg / kg of the fusion protein and serum protein concentrations were measured by enzyme-linked immunosorbent assay (ELISA). The Fab-MFc4-scFv bispecific protein had higher serum levels than Fab-MFc1-scFv (Figure 6a). PK parameters were analyzed and determined, and the clearance rate and terminal half-life of Fab-MFc4-scFv were significantly greater than those of Fab-MFc1-scFv, almost two-fold. This indicates that the stronger MFc4-mediated human FcRn binding contributes to enhanced serum protein recycling compared to MFc1. As expected, the increase in molecular size contributed to the decreased clearance from Fab-MFc1 to the bispecific molecule Fab-MFc1-scFv.

[0131] Consideration Monovalent antibodies or fusion proteins based on monomeric Fc have the potential to confer IgG-like serum properties to an expanded class of protein therapeutics. Following the successful engineering of stable monomeric Fc, we addressed the challenge to build on and expand the utility of the MFc platform to achieve several key properties.

[0132] First, we wanted to establish a more universal monomeric Fc molecule that includes alternative Fc mutations for half-life tuning, including the potential for further half-life extension. Second, we wanted to show that the MFc platform could indeed remain monomeric and stable for desired targeting strategies that would enable novel therapeutic applications, including bispecific molecular targeting, immuno-oncology, and receptor-mediated transcytosis. 4-7,9 A monovalent bispecific drug format built around a monomeric Fc may offer the advantage of complete ablation of effector functions as well as reduced toxicity and off-target sinks, design features optimal for T-cell and other immune cell engagers. Finally, we aimed to validate computational approaches that would enable the development of future monomeric Fc designs. To achieve these goals, we attempted to harness the power of structure-guided molecular design.

[0133] We previously showed that the YTE mutation in MFc1 can improve FcRn binding affinity, compensating for the reduced binding affinity. 13 Based on the output from previous Fc phage library and engineering studies, we identified mutation set T1 (Figure 1) with promising enhancement of FcRn binding at pH 6.0, providing an ideal test case to evaluate the suitability of monomer-stabilizing mutations for half-life mutations in the CH3 domain as opposed to YTE mutations from the CH2 domain.

[0134] The crystal structure of the T1 protein suggested that the engineered Fc may participate in newly packed dimer formation at high protein concentrations. Although we did not find a direct contribution by the new set of half-life extension mutations to this new form of Fc dimerization, we believe that these mutations play a role in enabling induced dimerization under tight-packing conditions. Close examination of this dimer interface led to our rational design to expand the side chain of the residue at position 354. From a series of mutations replacing the serine residue, glutamic acid was selected based on its excellent thermostability profile (Figure 2). Using solution analysis and crystallography, we showed that S354E was able to maintain a monomeric Fc structure, with the glutamic acid side chain protruding as designed (Figure 3). Confirmation of the adaptability and activity of S354E to the monomeric Fc structure was observed in the co-crystal structure where MFc3 and MFc4 maintained the monomeric state and maintained extensive engagement with FcRn (Figures 4a, 4c). This allowed us to easily generate co-crystal structures to probe the Fc-FcRn interface, demonstrating for the first time that quantifiable differences exist in their binding interactions. Using differential solvation energy calculations of the binding interface, we were able to quantify the enhanced FcRn binding due to the extended hydrophobic interactions in MFc4 (Figure 4b-e).

[0135] The availability of monomeric Fc molecules with variable FcRn binding capabilities has made it possible to validate the use of the MFc platform to construct monovalent bispecific molecular targeting. We have generated Fab-MFc-scFv molecules with both MFc1 and MFc4 with demonstrable monomeric conformational purity and dual targeting activity. With molecular sizes well above the renal filtration cutoff, these molecules are well suited to evaluate in vivo pharmacokinetic outcomes from improved FcRn binding. We found that the Fab-MFc4-scFv bispecific molecule maintained serum levels higher than Fab-MFc1-scFv and closer to those of standard IgG1 antibodies. These results also indicate that these MFc constructs can contribute to tunable serum protein recycling, providing a versatile bispecific platform for developing next wave technologies to aid in therapeutic advances.

[0136] Disruption of the Fc dimer to expose the CH3 domain is not a new phenomenon. The IgG4 scaffold was chosen for our MFc platform due to the facile engagement of Fab-arm exchange of IgG4 molecules that alternate the Fc between a dimer-monomer equilibrium. 36 Furthermore, in order to mount a strong and specific immune response, T cell epitopes need to be processed and presented by antigen-presenting cells. 37 To alleviate concerns that the newly introduced mutations may form novel T cell epitopes, we performed in silico T cell epitope predictions around the monomer-forming mutations in MFc1 and MFc4. We observed an overall lower predicted binding rate for these mutations, indicating a lower immunogenicity risk. 38

[0137] The ultimate success of curative treatments will rely on many collaborative efforts to address the balance of efficacy vs. toxicity, and serum half-life vs. tissue penetration, along with a deep understanding of immune and translational science. The MFc platform offers a timely opportunity to further expand the search for immune cell engagers, as it has the ability and flexibility to mimic IgG-like serum properties while presenting monovalent and bispecific targeting motifs to avoid any undesired Fc receptor-mediated cytotoxicity and off-target sinks.

[0138] method Ethics Statement: Protocols (MI-13-0012) requiring the use of animals in these studies were reviewed and approved by AstraZeneca's Animal Care and Use Committee and comply with the animal welfare standards of the United States Department of Agriculture, the Guide for the Care and Use of Laboratory Animals, and the Assessment and Accreditation of Laboratory Animal Care.

[0139] Antibody cloning, expression and purification. All antibody positions are listed according to the Kabat numbering convention for variable domains and the EU numbering convention for CH2-CH3 domains. 41,42 All chemicals were of analytical grade. Oligonucleotides were purchased from Eurofins MWG Operon (Louisville, KY). Plasmids encoding mAb-J were generated using the In-Fusion HD cloning kit from Takara Bio (Mountain View, CA) and variable heavy and variable light chain sequences were encoded into an in-house IgG1 mammalian expression vector. Point mutations were introduced by site-directed mutagenesis using the QuikChange Multi Lightning mutagenesis kit (Agilent Technologies, Santa Clara, CA).

[0140] Variants were transiently transfected into the human embryonic kidney cell line HEK293FT using 293Fectin transfection reagent (Life Technologies, Carlsbad, CA). Cells were grown in FreeStyle 293-F expression medium (Life Technologies). Expressed antibodies were purified from cell supernatants by affinity chromatography using HiTrap Protein A columns (GE Healthcare Life Sciences, Marlborough, MA). Antibodies were eluted with Pierce IgG elution buffer (Thermo Fisher Scientific, Waltham, MA) and neutralized with 1 M Tris, pH 8.0. Antibodies were dialyzed into phosphate-buffered saline (PBS), pH 7.2. Monomer content for all antibodies was determined to be >95% by analytical SEC.

[0141] SEC-MALS and analytical ultracentrifugation. Purified Fc clones and fusion proteins at concentrations of 1 mg / mL or higher were analyzed by SEC on an 1100 HPLC system (Agilent, Santa Clara, CA) at room temperature using a TSK-GEL G2000SWXL column (Tosoh Biosciences, Tokyo, Japan) with a bed volume of 14 mL. Samples were eluted isocratically in PBS for 20 min at a flow rate of 1 mL / min. Eluted proteins were detected by UV absorbance at a wavelength of 280 nm. Data analysis was performed using ChemStation software (version A.02.10). Column calibration was performed using a series of molecular weight standards ranging from 10 to 500 kDa (Bio-Rad, Hercules, CA). In-line SEC-MALS was performed. Sample measurements were performed on a Dawn Heleos II MALS equipped with an Optilab Rex refractometer (Wyatt Technologies, Santa Barbara, CA). The molecular weight of each protein within a defined chromatographic peak was calculated using Astra, version 6.1 (Wyatt Technologies).

[0142] For analytical ultracentrifugation analysis, samples and reference buffers were loaded into 12 mm double sector cells with Epon center pieces and then placed in an An-50 Ti rotor for ultracentrifugation at 50,000 rpm using an Optima XL-I centrifuge (Beckman-Coulter, Indianapolis, IN) set at 20 °C. Sedimentation data collected at 280 nm for scans 2–160 were analyzed with Sedfit software (version 16.1c) to generate c(s) distributions. 43,44 The partial specific volume was set to 0.73 mL / g. The solution concentration and viscosity values ​​of PBS were set to 1.00523 g / mL and 1.019 mPa s, respectively, using calculated values ​​from the Sednterp program (version 20130813). 45 Based on the Swedberg equation, monomeric Fc with a molecular weight of 27 kDa is expected to have a sedimentation coefficient of 1.7 to 2.4 S (Swedberg units), assuming a friction ratio of 1.3 to 1.8 (globular to extended shape).

[0143] Crystallization, data collection, and structure determination. Prior to crystallization, Protein A purified T1, MFc3, and MFc4 were further purified by ion exchange chromatography on a Q HP 5 mL prepacked column (GE Healthcare Life Sciences) equilibrated with 25 mM Tris-HCl buffer, pH 8, and by SEC using a Superdex 200 Increase 10 / 300 GL column (GE Healthcare Life Sciences) pre-equilibrated with 25 mM Tris-HCl, pH 8, and 100 mM NaCl. Culture medium of harvested recombinant heterodimeric FcRn was pH adjusted for affinity purification on an IgG Sepharose column (GE Healthcare Life Sciences). FcRn was purified on a Q HP column (GE Healthcare Life Sciences) and then dialyzed into 30 mM sodium acetate buffer, pH 5.2, and 1% molar deletion of FcRn was complexed with MFc3 and MFc4, and the complex was purified by SEC using the same Superdex 200 column equilibrated with 30 mM sodium acetate, pH 5.2, and 100 mM NaCl. The complex composition was confirmed by SDS PAGE.

[0144] Initial crystallization trials of all proteins and protein complexes were performed by sitting drop vapor diffusion at 20°C. Crystallization drops were dispensed by a Phoenix robot (Art Robbins Instruments) into 96-well crystallization plates (Intelli-plate 102-0001-20; Art Robbins Instruments, Sunnyvale, CA) and consisted of equal volumes of protein and reservoir buffer. To crystallize T1 and MFc3 alone, a commercial screen (Hampton Research, Aliso Viejo, CA; Molecular Dimensions, Suffolk, UK) was used. For crystallization of FcRn complex proteins, we created a new screen consisting of a combination of low pH conditions included in the commercial screen. Diffraction quality crystals were grown in a crystallization optimization step in hanging drop format from the following crystallization solutions: T1: 0.01 M zinc sulfate heptahydrate; 0.1 M morpholineethanesulfonic acid (MES) monohydrate, pH 6.5, and 25% (w / v) PEG 550 MME, protein concentration 5.5 mg / mL. MFc4 / FcRn complex: 0.2 M magnesium chloride hexahydrate, 1 M sodium iodide, 0.1 M MES, pH 6, and 20% PEG 6000, protein concentration 6.35 mg / mL. MFc3 / FcRn complex: 0.2 M magnesium chloride hexahydrate, 30% 1,5-diaminopentane dihydrochloride, 0.1 M MES, pH 6, and 20% PEG 6000, protein concentration 6 mg / mL. Crystals of MFc3 were collected directly from the original sitting drop plate in conditions consisting of 0.8% anesthetic alkaloids (2% w / v lidocaine hydrochloride monohydrate, 2% w / v procaine hydrochloride, 2% w / v proparacaine hydrochloride, 2% w / v tetracaine hydrochloride), 0.1 M MOPS (acid) and sodium HEPES pH 7.5, and a 50% v / v mix of precipitants (40% v / v ethylene glycol, 20% w / v PEG8000), protein concentration 7 mg / mL. All crystals collected for X-ray analysis were flash cooled by immersion in liquid nitrogen.Diffraction data were collected from single crystals on beamline BL9-2 at the Stanford Synchrotron Radiation Lightsource equipped with a Pilatus 6M PAD detector (Paul Scherer Institute, Villigen, Switzerland) with an oscillation range of 180°, an increment of 0.5°, and an exposure of 0.8 s per image. Diffraction data were processed using the program XDS. 46 All crystallographic calculations were performed using the CCP4 software suite (version 7.0). 47 The molecular replacement procedure was carried out using the Molrep program. 48 The structure was fine-tuned using Refmac5, and the model was adjusted using the "O" program. 49,50 Diagrams including structures were prepared using PyMOL (Schroedinger, New York, NY).

[0145] Octet binding analysis. Binding measurements of monomeric Fc and its fusion proteins to in-house purified recombinant human FcRn were performed by biolayer interferometry on an Octet384 instrument (ForteBio, Menlo Park, CA). Biotinylated FcRn at 1 μg / mL in PBS buffer (pH 7.4) or 100 mM MES buffer (pH 6.0) containing 3 mg / mL bovine serum albumin, 0.05% (vol / vol) and Tween 20 (1× Kinetics Buffer; ForteBio) was captured with a streptavidin biosensor (ForteBio). The loaded biosensor was washed with assay buffer to remove unbound proteins, and binding and dissociation were subsequently measured by serial dilutions of the different Fc variants or Fc fusion constructs. Kinetic parameters (k on and k off ) and apparent affinity (K D ) is calculated using the following formula:

number

[0146] Simultaneous binding measurements of Fab-MFc-scFv molecules to recombinant antigen proteins were also performed. Biotinylated cMet protein was captured at 5 μg / mL on a streptavidin biosensor (ForteBio) in PBS buffer (pH 7.2) using 1× Kinetics Buffer. The binding step included 300 nM Fab-MFc-scFv with buffer control followed by binding to antigen 2 with buffer control.

[0147] In vivo PK in hFcRn transgenic mice. Human FcRn transgenic mice used in this study are an F1 cross of mouse FcRn-deficient B6.129X1-Fcgrttm1Dcr / DcrJ and human FcRn cDNA transgenic line B6.Cg-Fcgrttm1Dcr Tg(CAG-FCGRT)276Dcr / DcrJ. Gender-matched (6-16 weeks old) mice were administered a bolus intravenous dose of 2.5 mg / kg monomeric Fc fusion protein on day 0. Eight mice per protein were used, and two groups of mice (groups A and B) were bled at different time points. Blood samples were obtained from the retroorbital plexus using a capillary pipette at different time points throughout the entire 2-3 week study. All animals remained healthy throughout the study. A quantitative ELISA was used to monitor serum concentrations of the test antibodies. Briefly, 96-well plates were coated with 2 μg / mL of cMet extracellular domain. Plates coated with 5 μg / mL of cMet were incubated overnight at 4° C., blocked with 3% bovine serum albumin in PBS-Tween, and then incubated with diluted serum samples at different time points. 4Dilutions of goat anti-human Fc-specific horseradish peroxidase-conjugated antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) were used for detection. Absorbance at 450 nm was measured after development with 3,3',5,5'-tetramethylbenzidine substrate (KPL, Gaithersburg, MD) according to the manufacturer's instructions. Standard curves were generated for each antibody variant. The linear portion of the standard curve was generated in Prism (version 6; GraphPad software, La Jolla, CA) and then used to quantify human anti-cMet fusion protein in serum samples. Noncompartmental PK data analysis was performed using Phoenix 64 WinNonlin 6.3 (Pharsight, Mountain View, CA). Maximum observed peak plasma concentrations were determined by inspecting the observed data using WinNonlin. Terminal elimination half-life was determined using the formula ln(2) / λz, where λz is the slope of the terminal portion of the natural log concentration-time curve, determined by linear regression of at least the last three time points. Systemic exposure was determined using the linear / log trapezoidal rule to plot the plasma concentration versus time graph from the start of dosing to the last measurable concentration (AUC last The area under the curve (AUC) of the plasma concentration versus time graph from time 0 to infinity (AUC ∞ ) was calculated as follows: AUC last +C last / λz (In the formula, C last is the last quantifiable concentration). Clearance (CL) is the ratio of dose to area under ∞ and the steady-state volume of distribution was calculated as follows: (AUMC ∞ ×CL) / AUC ∞ (In the formula, AUMC ∞ (AUC from the first instant extrapolated to infinity) PK parameters were summarized statistically and presented as mean values.

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[0149] Sequence mFc4 (SEQ ID NO: 1) [Chemical formula]

Claims

1. A molecule comprising an FcRn-binding fragment of an Fc region of an IgG molecule, wherein the molecule comprising the FcRn-binding fragment is a fusion protein comprising the FcRn-binding fragment, is a polypeptide comprising the FcRn-binding fragment, or comprises a non-proteinaceous agent conjugated to the FcRn-binding fragment, wherein the FcRn-binding fragment is a. F ​​in 351st place b. R, K, D, E, F, Y, P, G, L or M at position 354 c. R at 366th place d. K in 395th place e. R at position 405; and f. E in 407th place Includes wherein the amino acid numbering is according to the EU index. A molecule comprising an FcRn-binding fragment of the Fc region of an IgG molecule.

2. 2. A molecule comprising an FcRn-binding fragment of the Fc region of an IgG molecule according to claim 1, wherein the FcRn-binding fragment comprises an R, K, D or E at position 354, or comprises a D or E at position 354 (wherein the numbering of the amino acids is according to the EU index).

3. A molecule comprising an FcRn-binding fragment of the Fc region of an IgG molecule according to claim 2, wherein the FcRn-binding fragment comprises an E at position 354 (wherein the numbering is according to the EU index).

4. 4. A molecule comprising an FcRn-binding fragment of the Fc region of an IgG molecule according to any one of claims 1 to 3, wherein the FcRn-binding fragment comprises from about amino acid residue 216 to about amino acid residue 446 of the IgG molecule, where numbering is according to the EU index.

5. A molecule comprising an FcRn-binding fragment of the Fc region of an IgG molecule according to claim 4, wherein the FcRn-binding fragment comprises from about amino acid residue 236 to about amino acid residue 446 of the IgG molecule (wherein the numbering is according to the EU index).

6. A molecule comprising an FcRn-binding fragment of the Fc region of an IgG molecule according to any one of claims 1 to 3, wherein the IgG molecule is IgG4.

7. A molecule comprising an FcRn-binding fragment of the Fc region of an IgG molecule according to any one of claims 1 to 3, wherein the FcRn-binding fragment further comprises a half-life extending mutation.

8. 8. A molecule comprising an FcRn-binding fragment of the Fc region of an IgG molecule according to claim 7, wherein the FcRn-binding fragment comprises a Y at position 252, a T at position 254, and an E at position 256 (wherein the numbering is according to the EU index).

9. The Fc region comprises the following amino acids: a. C at positions 432 and 437; b. S, H, R, P, T, K, A, M or N at position 433; c. Y, N, R, W, H, F, S, M or T at position 434; d. H at position 435; and e. L, Y, F, R, I, K, M, V, H, S or T at position 436 Includes (where the numbering follows the EU index), A molecule comprising an FcRn-binding fragment of the Fc region of the IgG molecule of claim 7.

10. The FcRn binding fragment comprises: a. Y, R, W, H or F at position 434; and b. L, R, I, K, M, V or H at position 436 Includes (where the numbering follows the EU index), A molecule comprising an FcRn-binding fragment of the Fc region of the IgG molecule of claim 9.

11. The FcRn binding fragment has the following amino acid sequence: a. C at positions 432 and 437; b. S at position 433; c. Y at position 434; d. H at position 435; and e. L at 436th place Includes (where the numbering follows the EU index), A molecule comprising an FcRn-binding fragment of the Fc region of the IgG molecule of claim 10.

12. A molecule comprising an FcRn-binding fragment of the Fc region of an IgG molecule according to any one of claims 1 to 3, wherein said FcRn-binding fragment comprises a deletion of an amino acid at position 438 (wherein the numbering is according to the EU index).

13. A molecule comprising an FcRn-binding fragment of the Fc region of an IgG molecule according to any one of claims 1 to 3, wherein Q438 (EU index numbering) of the FcRn-binding fragment is deleted.

14. 4. A molecule comprising an FcRn-binding fragment of the Fc region of an IgG molecule according to any one of claims 1 to 3, wherein the FcRn-binding fragment comprises an E inserted immediately after residue 437 (wherein the numbering is according to the EU index).

15. The molecule comprising the FcRn-binding fragment is the fusion protein comprising the FcRn-binding fragment or the polypeptide comprising the FcRn-binding fragment, and comprises an FcRn-binding fragment of the Fc region of an IgG molecule described in any one of claims 1 to 3, comprising at least one antigen-binding domain.

16. A molecule comprising an FcRn-binding fragment of the Fc region of an IgG molecule described in claim 15, comprising one antigen-binding domain.

17. A molecule comprising an FcRn-binding fragment of the Fc region of an IgG molecule according to claim 15, comprising two antigen-binding domains.

18. A molecule comprising an FcRn-binding fragment of the Fc region of an IgG molecule described in claim 15, comprising at least one antigen-binding domain, wherein a first antigen-binding domain is at the N-terminus of the FcRn-binding fragment.

19. A molecule comprising an FcRn-binding fragment of the Fc region of an IgG molecule described in claim 15, comprising at least two antigen-binding domains, a second antigen-binding domain being at the C-terminus of the FcRn-binding fragment.

20. The molecule comprising an FcRn-binding fragment of the Fc region of an IgG molecule of claim 15, wherein each antigen-binding domain is independently selected from an Fv, Fab, Fab', F(ab')2, Fab'-SH, diabody, triabody, tetrabody, linear antibody, and scFv.

21. The molecule comprising the FcRn-binding fragment is the fusion protein comprising the FcRn-binding fragment or the polypeptide comprising the FcRn-binding fragment, and comprises an FcRn-binding fragment of the Fc region of an IgG molecule described in any one of claims 1 to 3, comprising a non-IgG protein domain.

22. The molecule comprising an FcRn-binding fragment of an Fc region of an IgG molecule according to claim 21 , wherein the non-IgG domain is an immunomodulator, a receptor, a hormone, an enzyme or a drug.

23. A molecule comprising an FcRn-binding fragment of an Fc region of an IgG molecule according to any one of claims 1 to 3, wherein the molecule comprising the FcRn-binding fragment comprises a non-proteinaceous agent conjugated to the FcRn-binding fragment, the non-proteinaceous agent being a nucleic acid, a lipid, a glycolipid, a polysaccharide, a drug, a radioisotope, a chelating metal, a nanoparticle, a reporter group, a fluorescent compound or a compound detectable by NMR or ESR spectroscopy.

24. The molecule comprising an FcRn-binding fragment of an Fc region of an IgG molecule according to claim 23, wherein the nucleic acid is DNA, RNA, siRNA, RNAi or microRNA.

25. The molecule comprising an FcRn-binding fragment of an Fc region of an IgG molecule according to claim 23 , wherein the drug is a cytotoxic agent, a chemotherapeutic agent, an anti-tumor drug, an angiogenesis inhibitor or a pro-apoptotic agent.

26. A molecule comprising an FcRn-binding fragment of the Fc region of an IgG molecule according to claim 1, wherein the FcRn-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:

1.

27. A nucleic acid comprising a nucleotide sequence encoding a molecule comprising an FcRn-binding fragment of an Fc region of an IgG molecule according to any one of claims 1 to 3, wherein the molecule comprising the FcRn-binding fragment is the fusion protein comprising the FcRn-binding fragment or the polypeptide comprising the FcRn-binding fragment.

28. A vector comprising the nucleic acid of claim 27.

29. 29. A host cell comprising the nucleic acid of claim 27 or the vector of claim 28.

30. A nucleic acid comprising a nucleotide sequence encoding an FcRn-binding fragment of an Fc region of an IgG molecule, the FcRn-binding fragment comprising: a. F ​​in 351st place b. R, K, D, E, F, Y, P, G, L or M at position 354 c. R at 366th place d. K in 395th place e. R at position 405; and f. E in 407th place Includes wherein the amino acid numbering is according to the EU index. Nucleic acid.

31. A vector comprising the nucleic acid of claim 30.

32. 32. A host cell comprising the nucleic acid of claim 30 or the vector of claim 31.

33. A method for producing a molecule comprising an FcRn-binding fragment of an Fc region of an IgG molecule according to any one of claims 1 to 3, wherein the molecule comprising the FcRn-binding fragment is a fusion protein comprising the FcRn-binding fragment or a polypeptide comprising the FcRn-binding fragment, the method comprising the steps of expressing the fusion protein or the polypeptide from a host cell according to claim 29, and purifying the fusion protein or the polypeptide.

34. A pharmaceutical composition comprising a molecule comprising an FcRn-binding fragment of the Fc region of an IgG molecule according to any one of claims 1 to 3, a pharma- ceutically acceptable carrier and a diluent.

35. 35. A pharmaceutical composition according to claim 34 for use in therapy.

36. Use of a molecule comprising an FcRn-binding fragment of the Fc region of an IgG molecule according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment of a disease.

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