Heterodimeric IgA Fc constructs and methods of use thereof

Abstract

A heterodimeric IgA Fc (IgA HetFc) construct comprising one or more amino acid mutations in the CH3 domain that enable the formation of a heterodimeric Fc with high purity and thermal stability. The IgA HetFc construct may comprise one or more target binding domains. Higher-order IgA HetFc multimers may be generated comprising multiple IgA HetFc constructs, two of which are linked by a J chain.

Description

[Technical Field]

[0001] This disclosure relates to the field of IgA-based immunotherapies, and more particularly to heterodimeric IgA Fc (IgA HetFc) constructs containing one or more binding domains, and the use of these constructs as therapeutic agents. [Background technology]

[0002] background Generally, antibody-based therapeutics include an IgG-derived framework. Ig subtypes are stable, bind to targets with high affinity, exhibit favorable pharmacokinetic behavior, and their functional effects on target and effector cells are well understood as a result of decades of focused research. However, there are limitations to the functionality of IgG-based therapies in terms of the effector cells that can be activated and the possible binding titers.

[0003] Neutrophils are an integral part of the immune system and are the most common type of white blood cell found in human blood (see Table 1). IgA is the only Ig isotype that interacts with FcαRI on neutrophils via residues at the Cα2 / Cα3 (IgA CH2 / CH3) junction of Fc. The interaction of IgA with FcαRI on neutrophils triggers a variety of pro-inflammatory responses, including the release of neutrophil extracellular traps (NETs), degranulation, and chemokine release (Heineke, 2017, Eur J Clin Invest., 47(2):184-192 (Non-patent Literature 1)). IgA may also be involved in cytotoxicity ex vivo. Neutrophils activated by IgA are associated with Her2 +++It has also been shown that BT474 cells can be killed (Borrok et al., 2015, MAbs 7:743-751 (Non-Patent Literature 2)). IgA-mediated tumor cell death via Her2 and other targets has been demonstrated ex vivo by neutrophils (Brandsma et al., 2019, Front Immunol, 10:704 (Non-Patent Literature 3)). Furthermore, IgA may be involved in inhibiting tumor growth in vivo. In particular, IgA has been shown to inhibit tumor growth in vivo in an FcαRI transgenic (Tg) mouse model (Boross et al., 2013, EMBO Mol Med, 5:1213-1226 (Non-Patent Literature 4)).

[0004] [Table 1] *Data collected from 291 adults (see Orfanakis, et al., 1970, Am J Clin Pathol, 53:647-651 (Non-Patent Literature 5))

[0005] IgA-mediated neutrophil recruitment and activation offer a novel biological function for antibody-based immunotherapy. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Heineke, 2017,Eur J Clin Invest.,47(2):184-192 [Non-Patent Document 2] Borrok et al., 2015, MAbs 7:743-751 [Non-Patent Document 3] Brandsma et al.,2019,Front Immunol,10:704 [Non-Patent Document 4] Boross et al.,2013,EMBO Mol Med,5:1213-1226 [Non-Patent Document 5] Orfanakis, et al., 1970, Am J Clin Pathol, 53:647-651 [Overview of the project]

[0007] overview HetFc (IgA HetFc) constructs comprising a first Fc polypeptide comprising a first CH3 domain sequence and a second Fc polypeptide comprising a second CH3 domain sequence, wherein the first and second CH3 domain sequences form a modified CH3 domain, the first and second CH3 domain sequences comprising amino acid mutations that promote the formation of heterodimer Fc more than homodimer Fc, the amino acid mutations in the first CH3 domain sequence being amino acid substitutions at position A6085Y selected from A6085YF, A6085YY, A6085YM, A6085YW and A6085YH, and at position T6086 T6086Y, T6086 The present invention relates to an IgA HetFc construct comprising amino acid substitutions selected from F, T6086M, T6086W, and T6086H, wherein the amino acid mutation in the second CH3 domain sequence comprises an amino acid substitution at position W6081 selected from W6081T, W6081L, W6081A, W6081V, and W6081I, the heterodimer Fc is formed with a purity of 70% or higher, and the amino acid position numbering follows IMGT numbering.

[0008] Another aspect of the present disclosure is an IgA heterodimer Fc (IgA HetFc) construct comprising a first Fc polypeptide comprising a first CH3 domain sequence and a second Fc polypeptide comprising a second CH3 domain sequence, wherein the first and second CH3 domain sequences form a modified CH3 domain, and the first and second CH3 domain sequences contain amino acid mutations that promote the formation of heterodimer Fc more than homodimer Fc. (a) The amino acid mutations in the first CH3 domain sequence include amino acid substitutions: A6085YY and T6086L, and the amino acid mutations in the second CH3 domain sequence include amino acid substitutions: L6079T, W6081L and I6088L, or (b) The amino acid mutations in the first CH3 domain sequence include amino acid substitutions: A6085YY and T6086Y, and the amino acid mutations in the second CH3 domain sequence include amino acid substitutions: L6079T, W6081L and I6088L, or (c) The amino acid mutations in the first CH3 domain sequence include amino acid substitutions: A6085YF and T6086Y, and the amino acid mutations in the second CH3 domain sequence include amino acid substitutions: L6079V, W6081L and I6088L, or (d) The amino acid mutations in the first CH3 domain sequence include amino acid substitutions: A6085YF and T6086Y, and the amino acid mutations in the second CH3 domain sequence include amino acid substitutions: L6079V, W6081T and I6088L, or (e) The amino acid mutations in the first CH3 domain sequence include amino acid substitutions: T6022V, A6085YF and T6086Y, and the amino acid mutations in the second CH3 domain sequence include amino acid substitutions: L6079V, W6081T and I6088L, or (f) The amino acid mutations in the first CH3 domain sequence include amino acid substitutions: T6022L, A6085YF and T6086Y, and the amino acid mutations in the second CH3 domain sequence include amino acid substitutions: L6079V, W6081T and I6088L, or (g) The amino acid mutations in the first CH3 domain sequence include amino acid substitutions: T6022I, A6085YF and T6086Y, and the amino acid mutations in the second CH3 domain sequence include amino acid substitutions: L6079V, W6081T and I6088L, or (h) The amino acid mutations in the first CH3 domain sequence include the amino acid substitutions: A6085YF and T6086Y, and the amino acid mutations in the second CH3 domain sequence include the amino acid substitutions: L6007F, L6079V, W6081T and I6088L, or (i) The amino acid mutations in the first CH3 domain sequence include the amino acid substitutions: H6005Y, A6085YF and T6086Y, and the amino acid mutations in the second CH3 domain sequence include the amino acid substitutions: H6005Y, L6079V, W6081T and I6088L, or (j) The amino acid mutations in the first CH3 domain sequence include the amino acid substitutions: H6005C, A6085YF and T6086Y, and the amino acid mutations in the second CH3 domain sequence include the amino acid substitutions: P6010C, L6079V, W6081T and I6088L, or (k) The amino acid mutations in the first CH3 domain sequence include the amino acid substitutions: P6010C, A6085YF and T6086Y, and the amino acid mutations in the second CH3 domain sequence include the amino acid substitutions: H6005C, L6079V, W6081T and I6088L, or (l) The amino acid mutations in the first CH3 domain sequence include the amino acid substitutions: H6005C, P6010C, A6085YF and T6086Y, and the amino acid mutations in the second CH3 domain sequence include the amino acid substitutions: H6005C, P6010C, L6079V, W6081T and I6088L, The heterodimeric Fc is formed with a purity of 70% or more, and the amino acid position numbering follows the IMGT numbering, regarding the IgA HetFc construct.

[0009] Another aspect of the present disclosure relates to a composite comprising the IgA HetFc construct described herein and one or more therapeutic agents, diagnostic agents or labeling agents.

[0010] Another aspect of the disclosure relates to an IgA HetFc multimer comprising two or more IgA HetFc constructs described herein and a J chain, wherein two of the IgA HetFc constructs are linked by the J chain.

[0011] Another aspect of the disclosure relates to a pharmaceutical composition comprising an IgA HetFc construct described herein and a pharmaceutically acceptable carrier or diluent.

[0012] Another aspect of the disclosure relates to a composite comprising an IgA HetFc construct described herein and one or more therapeutic agents, diagnostic agents or labeling agents, and a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent.

[0013] Another aspect of the disclosure relates to a pharmaceutical composition comprising an IgA HetFc multimer comprising two or more IgA HetFc constructs described herein and a J chain, and a pharmaceutically acceptable carrier or diluent.

[0014] Another aspect of the disclosure relates to an isolated polynucleotide or set of polynucleotides encoding an IgA HetFc construct described herein.

[0015] Another aspect of the disclosure relates to a set of vectors or set of vectors comprising one or more polynucleotides encoding an IgA HetFc described herein.

[0016] Another aspect of the disclosure relates to a host cell comprising one or more polynucleotides encoding an IgA HetFc described herein.

[0017] Another aspect of the disclosure relates to a method of making an IgA HetFc construct described herein, the method comprising transfecting a host cell with one or more polynucleotides encoding the IgA HetFc construct, and culturing the host cell under conditions suitable for expression of the IgA HetFc construct.

[0018] Another aspect of the present disclosure is a method for producing an IgA HetFc multimer as described herein, comprising the steps of transfecting host cells with one or more polynucleotides encoding an IgA HetFc construct containing an α-tailpiece and a polynucleotide encoding a J chain, and culturing the host cells under conditions suitable for the expression of the IgA HetFc construct and the J chain. [Brief explanation of the drawing]

[0019] [Figure 1] The diagram shows negative and positive design concepts for mutations that lead to heterodimerization of IgA Fc. [Figure 2A] This shows the unreduced CE-SDS profile of IgA Fc single-arm antibody (OAA) constructs after CaptureSelect® IgA affinity purification. IgA Fc OAA constructs containing WT IgA CH3 (variant number 32595) or stereodesigns 1, 2, 3, 4, or 6 (variant numbers 32516, 32517, 32518, 32519, and 32521, respectively). [Figure 2B] This shows the unreduced CE-SDS profiles of IgA Fc single-arm antibody (OAA) constructs after CaptureSelect® IgA affinity purification. IgA Fc OAA constructs including stereodesigns 7, 8, 9, 10, or 11 (variant numbers 33330, 33331, 33332, 33333, and 33334, respectively). [Figure 3A] The UPLC-SEC chromatograms of IgA Fc OAA constructs after CaptureSelect® IgA affinity purification are shown. The UPLC-SEC chromatogram of an IgA Fc OAA construct containing WT IgA CH3 (variant number 32595) is also shown. [Figure 3B] This shows the UPLC-SEC chromatograms of IgA Fc OAA constructs after CaptureSelect® IgA affinity purification. The UPLC-SEC chromatograms include those of IgA OAA constructs including stereodesign 1 (variant number 32516). [Figure 3C] This shows the UPLC-SEC chromatograms of IgA Fc OAA constructs after CaptureSelect® IgA affinity purification. It also includes the UPLC-SEC chromatogram of IgA OAA constructs including stereochemical design 2 (variant number 32517). [Figure 3D] This shows the UPLC-SEC chromatograms of IgA Fc OAA constructs after CaptureSelect® IgA affinity purification. The UPLC-SEC chromatograms include those of IgA OAA constructs including stereochemical design 3 (variant number 32518). [Figure 3E] This shows UPLC-SEC chromatograms of IgA Fc OAA constructs after CaptureSelect® IgA affinity purification. It also includes UPLC-SEC chromatograms of IgA OAA constructs including stereochemical design 4 (variant number 32519). [Figure 3F] This shows UPLC-SEC chromatograms of IgA Fc OAA constructs after CaptureSelect® IgA affinity purification. It also includes UPLC-SEC chromatograms of IgA OAA constructs including stereodesign 6 (variant number 32521). [Figure 3G] This shows the UPLC-SEC chromatograms of IgA Fc OAA constructs after CaptureSelect® IgA affinity purification. The UPLC-SEC chromatograms include those of IgA OAA constructs including stereodesign 7 (variant number 33330). [Figure 3H] This shows UPLC-SEC chromatograms of IgA Fc OAA constructs after CaptureSelect® IgA affinity purification. It also includes UPLC-SEC chromatograms of IgA OAA constructs including stereochemical design 8 (variant number 33331). [Figure 3I] This shows the UPLC-SEC chromatograms of IgA Fc OAA constructs after CaptureSelect® IgA affinity purification. The UPLC-SEC chromatograms include those of IgA OAA constructs including stereodesign 9 (variant number 33332). [Figure 3J] This shows the UPLC-SEC chromatograms of IgA Fc OAA constructs after CaptureSelect® IgA affinity purification. The UPLC-SEC chromatograms include those of IgA OAA constructs including stereodesign 10 (variant number 33333). [Figure 3K] This shows the UPLC-SEC chromatograms of IgA Fc OAA constructs after CaptureSelect® IgA affinity purification. The UPLC-SEC chromatograms include those of IgA OAA constructs including stereochemical design 11 (variant number 33334). [Figure 4A] The UPLC-SEC chromatogram of the IgA Fc OAA construct after purification by preparative SEC is shown. The IgA OAA construct contains WT IgA CH3 (variant number 32595). [Figure 4B] The UPLC-SEC chromatogram of the IgA Fc OAA construct after purification by preparative SEC is shown. The IgA OAA construct includes stereodesign 1 (variant number 32516). [Figure 4C] The UPLC-SEC chromatograms of the IgA Fc OAA constructs after purification by preparative SEC are shown. The IgA OAA constructs include stereodesign 2 (variant number 32517). [Figure 4D] The UPLC-SEC chromatogram of the IgA Fc OAA construct after purification by preparative SEC is shown. The IgA OAA construct includes stereodesign 3 (variant number 32518). [Figure 4E] The UPLC-SEC chromatogram of the IgA Fc OAA construct after purification by preparative SEC is shown. The IgA OAA construct includes stereodesign 6 (variant number 32521). [Figure 4F] The UPLC-SEC chromatogram of the IgA Fc OAA construct after purification by preparative SEC is shown. The IgA OAA construct includes stereodesign 7 (variant number 33330). [Figure 4G]The UPLC-SEC chromatogram of the IgA Fc OAA construct after purification by preparative SEC is shown. The IgA OAA construct includes stereodesign 8 (variant number 33331). [Figure 4H] The UPLC-SEC chromatogram of the IgA Fc OAA construct after purification by preparative SEC is shown. The IgA OAA construct includes stereodesign 9 (variant number 33332). [Figure 4I] The UPLC-SEC chromatogram of the IgA Fc OAA construct after purification by preparative SEC is shown. The IgA OAA construct includes stereodesign 10 (variant number 33333). [Figure 4J] The UPLC-SEC chromatogram of the IgA Fc OAA construct after purification by preparative SEC is shown. The IgA OAA construct includes stereodesign 11 (variant number 33334). [Figure 5A] The unreduced and reduced CE-SDS profiles of IgA Fc OAA constructs after purification by preparative SEC are shown. IgA OAA constructs containing WT IgA CH3 (variant 32595) or stereodesigns 1, 2, 3, or 6 (variant numbers 32516, 32517, 32518, and 32521, respectively). [Figure 5B] The non-reducible and reduced CE-SDS profiles of the IgA Fc OAA constructs after purification by preparative SEC are shown. These include stereodesigns 7, 8, 9, 10, or 11 (variant numbers 33330, 33331, 33332, 33333, and 33334, respectively). [Figure 6A] The image shows an overlay of a DSC thermogram on the IgA Fc OAA construct after purification by preparative SEC. The IgA Fc construct contains WT IgA CH3 (variant number 32595) or stereodesign 1, 2, 3, or 6 (variant numbers 32516, 32517, 32518, and 32521, respectively). [Figure 6B]This shows an overlay of a DSC thermogram on the IgA Fc OAA construct after purification by preparative SEC. The IgA Fc constructs include stereochemical designs 7-11 (variant numbers 33330-33334). [Figure 7A] Examples of the components and structure of an IgA HetFc binding unit are shown. The IgA HetFc skeleton is formed by the fusion of binding domains to create the IgA HetFc binding unit. The binding domain is shown as Fab for illustrative purposes, but may be various other binding domains (e.g., scFv) and combinations of binding domains. The form is shown for illustrative purposes but is not limited to this disclosure. [Figure 7B] Examples of the components and structure of an IgA HetFc binding unit are shown. An example IgA HetFc binding unit showing an IgA HetFc skeleton with two exemplary binding domains bound together. The binding domains are shown as Fab for illustrative purposes, but may be various other binding domains (e.g., scFv) and combinations of binding domains. The forms are shown for illustrative purposes but are not limited to this disclosure. [Figure 7C] Examples of the components and configurations of IgA HetFc binding units are shown. These are example IgA HetFc binding units having one to four binding domains fused to an IgA HetFc skeleton in various configurations. The binding domains are shown as Fab for illustrative purposes, but may be various other binding domains (e.g., scFv) and combinations of binding domains. The forms are shown for illustrative purposes but are not limited to this disclosure. [Figure 7D] Examples of the components and configurations of IgA HetFc binding units are shown. These are example IgA HetFc binding units having one to four binding domains fused to an IgA HetFc skeleton in various configurations. The binding domains are shown as Fab for illustrative purposes, but may be various other binding domains (e.g., scFv) and combinations of binding domains. The forms are shown for illustrative purposes but are not limited to this disclosure. [Figure 7E]Examples of the components and configurations of IgA HetFc binding units are shown. These are example IgA HetFc binding units having one to four binding domains fused to an IgA HetFc skeleton in various configurations. The binding domains are shown as Fab for illustrative purposes, but may be various other binding domains (e.g., scFv) and combinations of binding domains. The forms are shown for illustrative purposes but are not limited to this disclosure. [Figure 7F] Examples of the components and configurations of IgA HetFc binding units are shown. These are example IgA HetFc binding units having one to four binding domains fused to an IgA HetFc skeleton in various configurations. The binding domains are shown as Fab for illustrative purposes, but may be various other binding domains (e.g., scFv) and combinations of binding domains. The forms are shown for illustrative purposes but are not limited to this disclosure. [Figure 7G] Examples of the components and configurations of IgA HetFc binding units are shown. These are example IgA HetFc binding units having one to four binding domains fused to an IgA HetFc skeleton in various configurations. The binding domains are shown as Fab for illustrative purposes, but may be various other binding domains (e.g., scFv) and combinations of binding domains. The forms are shown for illustrative purposes but are not limited to this disclosure. [Figure 7H] Examples of the components and configurations of IgA HetFc binding units are shown. These are example IgA HetFc binding units having one to four binding domains fused to an IgA HetFc skeleton in various configurations. The binding domains are shown as Fab for illustrative purposes, but may be various other binding domains (e.g., scFv) and combinations of binding domains. The forms are shown for illustrative purposes but are not limited to this disclosure. [Figure 8A]An example of a higher-order IgA HetFc multimer containing two IgA HetFc binding units linked by a J chain (halftone dots) is shown. The two IgA HetFc chains are shown in gray and striped. A tailpiece assembly is shown at the center of each structure. A single orientation is shown for each assembly, but many orientations are possible. Since the J chain and Fc:Fc interactions are not selective for chain A or chain B, the orientation of the binding domain of each binding unit can also be reversed. A dimeric IgA HetFc multimer containing two bispecific IgA HetFc binding units linked by a J chain. [Figure 8B] An example of a higher-order IgA HetFc multimer containing four IgA HetFc binding units linked by a J chain (halftone dots) is shown. Two IgA HetFc chains are shown in gray and striped. A tailpiece assembly is shown at the center of each structure. A single orientation is shown for each assembly, but many orientations are possible. Since the J chain and Fc:Fc interactions are not selective for chain A or chain B, the orientation of the binding domain of each binding unit can also be reversed. A tetrameric IgA HetFc multimer containing four bispecific IgA HetFc binding units linked by a J chain. [Figure 8C] An example of a higher-order IgA HetFc multimer containing five IgA HetFc binding units linked by a J chain (halftone dots) is shown. Two IgA HetFc chains are shown in gray and striped. A tailpiece aggregate is shown at the center of each structure. A single orientation is shown for each aggregate, but many orientations are possible. Since the J chain and Fc:Fc interactions are not selective for chain A or chain B, the orientation of the binding domain of each binding unit can also be reversed. A pentameric IgA HetFc multimer containing five bispecific IgA HetFc binding units linked by a J chain. [Figure 9A] This shows the structural representation of the IgA HetFc design (stereotype 6) with chains A and B. The protein backbone is shown in cartoon form, and the side chains are shown as lines. Nonpolar hydrogens are not shown. It shows the complete IgA heterodimer Fc. [Figure 9B]This shows the structural representation of the IgA HetFc design (stereotype 6) with chains A and B. The protein backbone is shown in cartoon form, and the side chains are shown as lines. Nonpolar hydrogens are not shown. Magnified views of mutant residues centered around core positions A6085, T6086 (both in chain A) and W6081 (chain B) are shown. [Figure 10] The amino acid sequence alignments for the IgA1, IgA2m1, and IgA2m2 Fc regions are shown. [Figure 11] This variant exhibits an IgA OAA variant based on IgA HetFc, which has a mutation that eliminates FcαR binding in one or both strands of Fc. [Figure 12] This describes a modified IgA mAb based on IgA HetFc that can bind to both FcαR and FcRn. [Modes for carrying out the invention]

[0020] Detailed explanation This disclosure relates to the manipulation of the IgA Fc region by introducing amino acid mutations into the CH3 domain that promote the formation of heterodimeric IgA Fc (IgA HetFc). IgA HetFc enables the construction of IgA-based bispecific or multispecific binding proteins, as well as IgA-based multimer binding proteins. According to this disclosure, one or more amino acid mutations contained in an IgA HetFc construct enable the formation of heterodimeric Fc with a purity of at least about 70%. The IgA HetFc constructs of this disclosure are also thermally stable. For example, in certain embodiments, the CH3 domain of IgA HetFc has a melting temperature (Tm) of about 60°C or higher. In some embodiments, the CH3 domain of IgA HetFc has a Tm within 10°C (±10°C) of the Tm of the wild-type IgA CH3 domain.

[0021] The IgA HetFc constructs of this disclosure include an IgA HetFc skeleton comprising an IgA Fc region together with a hinge region; an IgA HetFc binding unit comprising an IgA skeleton and one or more binding domains; and an IgA HetFc polymer comprising a plurality (e.g., two or more) IgA HetFc binding units.

[0022] The IgA HetFc constructs of this disclosure introduce the potential for multispecificity to IgA isotypes with functions untapped by IgG. For example, in certain embodiments, IgA HetFc facilitates the production of multispecific and multimeric biologics that can recruit neutrophils via FcαRI. Since neutrophils are an integral part of the immune system and the most common type of white blood cell found in human blood, IgA-mediated neutrophil recruitment and activation offer novel biological functions to antibody-based immunotherapy. Certain embodiments of this disclosure relate to methods for using IgA HetFc binding units and IgA HetFc multimers as therapeutic agents. Certain embodiments of this disclosure relate to methods for using IgA HetFc binding units and IgA HetFc multimers as diagnostic agents.

[0023] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art.

[0024] Where used herein, the term “approximately” refers to a variation of approximately + / - 10% from a given value, unless otherwise indicated. It should be understood that such variation is always included in any given value provided herein, whether specifically mentioned or not.

[0025] The use of the terms “one (a)” or “one (an)” as used herein in conjunction with the term “including” may mean “one,” but in certain embodiments it may also mean “one or more,” “at least one,” or “one or more.”

[0026] Where used herein, the terms “comprising,” “having,” “including,” and “containing,” and their grammatical variations, are inclusive or open-ended and do not exclude additional unlisted elements and / or process steps. Where used herein in relation to a composition, use, or method, the term “essentially consisting of” indicates that additional elements and / or process steps may exist, but these additions do not substantially affect the manner in which the listed composition, method, or use functions. Where used herein in relation to a composition, use, or method, the term “consisting of” excludes the existence of additional elements and / or process steps. A composition, use, or method described herein as containing certain elements and / or steps may also, in certain embodiments, essentially consist of those elements and / or steps, and in other embodiments, whether these embodiments are specifically referenced or not.

[0027] "Fused" means that the components of the polymer described herein (e.g., an antibody or its antigen-binding fragment and an Fc domain polypeptide) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0028] As used herein, the term "single-chain" refers to a molecule containing amino acid monomers linearly linked by peptide bonds. For example, an antigen-binding fragment of an antibody may also contain a single-chain variable region (scFv).

[0029] As used herein, “IgA HetFc construct” means any IgA HetFc construct described herein, comprising an IgA HetFc skeleton (heterodimer IgA Fc), an IgA HetFc binding unit (heterodimer IgA binding unit), and an IgA HetFc polymer.

[0030] The term "functional" in relation to modified J chains means that the J chain retains the primary functions of an unmodified J chain, such as an unmodified human J chain, particularly the efficient polymerization (dimerization, tetramerization) of IgA and its ability to enable the binding of such polymers (dimers, tetramers) to secretory component (SC) / polymer (p)Ig.

[0031] When used herein in relation to a substance, the term "isolated" means that the substance has been removed from its original environment (e.g., its natural environment if it exists in nature). For example, a naturally occurring polynucleotide or polypeptide present in an animal organism is not isolated, but the same polynucleotide or polypeptide separated from some or all of the substances coexisting in the natural system is isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, but such a vector or composition is isolated in that it is not part of its natural environment.

[0032] When used herein in relation to amino acid sequences such as peptides, polypeptides, or protein sequences, the term "conservatively modified variant" means that the amino acid sequence has been modified by the substitution, addition, or removal of a single amino acid or a small proportion of amino acids in a manner that does not significantly affect the function of the sequence. For example, a conservatively modified variant may be an amino acid sequence modified by one or more conservative amino acid substitutions. Conservative substitution tables showing functionally similar amino acids are known to those skilled in the art. For example, the following eight groups each contain amino acids that are conserved substitutions with each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, for example, Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd edition (December 1993)). In certain embodiments, the IgA sequence used as the base sequence for the IgA HetFc construct may be a conservedly modified variant.

[0033] As used herein with respect to amino acid sequences, the term “substantially identical” indicates that, when optimally aligned using, for example, the methods shown below, a sequence shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a defined second amino acid sequence (or “reference sequence”). In certain embodiments, substantially identical amino acid sequences have at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the reference sequence. “Substantially identical” may also be used to refer to various types and lengths of sequences, such as full-length sequences or functional domains. The percentage of identity between two amino acid sequences can be determined by various methods well known in the art, such as commonly available computer software, e.g., Smith Waterman Alignment (Smith, TF and MS Waterman (1981) J Mol Biol 147:195-7); GeneMatcher Plus (trademark); "BestFit" incorporated into Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, MO, Ed pp353-358 (Smith and Waterman, Advances in Applied Mathematics, 482-489 10 (1981)); BLAST program (Basic Local Alignment Search Tool (Altschul, SF, W. Gish, et al. (1990) J Mol Biol)). This can be determined using 215:403-10) and its variations, including BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, and Megalign (DNASTAR) software.In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including algorithms necessary to achieve the maximum alignment over the length of the sequences being compared. Generally, with respect to amino acid sequences, the length of the comparison sequence is at least 10 amino acids. Those skilled in the art will understand that the actual length will depend on the total length of the sequence being compared and may be at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, or at least 200 amino acids, or it may be a full-length amino acid sequence. In certain embodiments, the IgA HetFc construct comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a reference amino acid sequence or fragment thereof listed in the table(s) herein.

[0034] When used in reference to recombinant amino acid sequences, the terms "derived from" and "based on" mean that the recombinant amino acid sequence is substantially identical to the sequence of the corresponding wild-type amino acid sequence. For example, an IgA Fc amino acid sequence derived from (or based on) a wild-type IgA Fc sequence is substantially identical to the wild-type IgA Fc sequence (e.g., sharing at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity).

[0035] Where used herein, the term “subject” refers to an animal, in some embodiments a mammal, that is the subject of treatment, observation, or experimentation. The animal may be a human, a non-human primate, a companion animal (e.g., a dog, a cat, etc.), a domestic animal (e.g., a cow, a sheep, a pig, a horse, etc.), or a laboratory animal (e.g., a rat, a mouse, a guinea pig, etc.).

[0036] The term “mammal” as used herein includes, but is not limited to, humans, non-human primates, dogs, cats, mice, cattle, horses, and pigs.

[0037] The term "knock-out" as used herein refers to a mutation or set of mutations at various locations in a variant that eliminates or reduces binding to a binding target.

[0038] In this document, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer within the listed range, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.

[0039] Any embodiment discussed herein is intended to be carried out with respect to any method, use, or composition disclosed herein.

[0040] Certain features, structures, and / or properties described in conjunction with embodiments disclosed herein may be combined in any preferred manner with features, structures, and / or properties described in conjunction with other embodiments disclosed herein to provide one or more further embodiments.

[0041] It should be understood that the explicit enumeration of features in one embodiment may serve as a basis for excluding features in alternative embodiments. For example, if a list of options is presented for a given embodiment or claim, it should be understood that one or more options may be removed from the list, and the shortened list may form alternative embodiments, whether or not such alternative embodiments are specifically referred to.

[0042] Terms understood by those skilled in the art of antibody technology are, unless otherwise explicitly defined herein, assigned the meanings derived in that art. Antibodies are known to have a variable region, a hinge region, and a constant domain. The structure and function of immunoglobulins are outlined, for example, in Harlow et al (Eds.), Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).

[0043] Unless otherwise specified herein, the numbering of amino acid residues in the IgA Fc region and IgA tailpiece follows the IMGT numbering system (see Lefranc, et al., 2003, Dev Comp Immunol, 27:55-77; Lefranc, et al., 2005, Dev Comp Immunol, 29:185-203). Table 2 shows the IMGT numbering and amino acid sequences of the IgA2m1 Fc CH2 and CH3 domains, along with their corresponding EU numbering (by alignment). The numbering of other IgA Fc sequences can be readily determined by those skilled in the art by simple sequence alignment with the sequences shown in Table 2 using known techniques. Table 3 shows the IMGT numbering and amino acid sequences of the IgA tailpiece.

[0044] [Table 2] TIFF2023552220000003.tif224135TIFF2023552220000004.tif226136

[0045] [Table 3]

[0046] The section headings used herein are for systematization purposes only and should not be interpreted as limiting the subject matter described.

[0047] Heterodimeric IgA Fc (IgA HetFc) constructs This disclosure relates to heterodimeric IgA Fc (IgA HetFc) constructs. An IgA HetFc construct comprises a heterodimeric Fc region derived from an IgA Fc region. The heterodimeric Fc region comprises a modified CH3 domain containing one or more asymmetric amino acid mutations that promote heterodimerization. In certain embodiments, the heterodimeric Fc region contained in an IgA HetFc construct can function as a skeleton (IgA HetFc skeleton) in which one or more binding domains can fuse to yield an IgA HetFc binding unit. In certain embodiments, multiple (e.g., two or more) IgA binding units can fuse together, for example, via a J chain, to yield an IgA HetFc multimer. In certain embodiments, other agents (e.g., therapeutic or diagnostic agents) may optionally be conjugated with the IgA HetFc construct.

[0048] IgA exists as two subtypes, IgA1 and IgA2, as well as various allotype variants (IgA2m1, IgA2m2, IgA2(n)). Of the two subtypes, IgA2 is more stable than IgA1 because a short hinge region makes it resistant to certain bacterial proteases. This short hinge also results in a rigid, non-planar structure that facilitates better multiple binding of IgA2 to antigens on the cell surface. For the purposes of this disclosure, the heterodimer Fc region of an IgA HetFc construct may be derived from an IgA1 or IgA2Fc region, including its allotype variant. In certain embodiments, the heterodimer Fc region of an IgA HetFc construct may be derived from an IgA1 Fc region. In certain embodiments, the heterodimer Fc region of an IgA HetFc construct may be derived from an IgA2 Fc region or its allotype variant. In some embodiments, the heterodimer Fc region of the IgA HetFc construct may be derived from the human IgA Fc region. In some embodiments, the heterodimer Fc region of the IgA HetFc construct may be derived from the human IgA2 or IgA2m1 Fc region.

[0049] In some embodiments, the heterodimer Fc region of the IgA HetFc construct may be derived from the human IgA2m1 Fc region. Table 4 shows the amino acid sequences of the wild-type human IgA2m1 Fc sequence and modified IgA2m1 Fc sequences that have been cleaved to remove the tailpiece and mutated to remove free cysteine ​​and glycosylation sites. The Fc sequences correspond to IMGT numbering 5001-6129 of the human IgA2m1 heavy chain. The CH3 sequence of IgA2m1 (underlined) contains amino acids 6097-6129 (IMGT numbering) of the full-length human IgA1 heavy chain (see, e.g., Chintalacharuvu, et al., 1994, J Immunol, 152:5299-5304). The sequence of the IgA tailpiece is also shown. The amino acid sequences of the IgA1 and IgA2m2 Fc regions are shown in Sequence Listing B as Sequence IDs 44 and 45. The alignment of the Fc sequences is shown in Figure 10.

[0050] [Table 4]

[0051] The terms “Fc region,” “Fc domain,” and “Fc” are used herein without distinction to define the C-terminal region of an immunoglobulin heavy chain. The Fc region typically includes a CH2 domain and a CH3 domain. The Fc region may also be considered to include a hinge region in certain embodiments. The “Fc polypeptide” of dimer Fc, as used herein, refers to one of the two polypeptides that form the dimer Fc domain, i.e., the polypeptide containing the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-assembly. For example, the Fc polypeptide of dimer IgA Fc includes an IgA CH3 domain and may also include an IgA CH2 domain.

[0052] Therefore, the Fc region of an IgA HetFc construct consists of two Fc polypeptides: a first Fc polypeptide and a second Fc polypeptide, which may be referred to herein as chain A and chain B. When each Fc region contains one first Fc polypeptide and one second Fc polypeptide (or one chain A polypeptide and one chain B polypeptide), the terms first Fc polypeptide and second Fc polypeptide (or chain A and chain B) can be used interchangeably. The first and second Fc polypeptides are in contact at a “junctional surface.” The “junctional surface” includes “contact” amino acid residues of the first Fc polypeptide that interact with one or more “contact” amino acid residues of the second Fc polypeptide.

[0053] The CH3 domain in the Fc region contains two CH3 domain sequences, one from each of the first and second Fc polypeptides of the dimer Fc. The CH2 domain contains two CH2 domain sequences, one from each of the first and second Fc polypeptides of the dimer Fc.

[0054] The IgA HetFc constructs disclosed herein include an asymmetrically modified IgA CH3 domain to form a heterodimeric Fc region. Specifically, one or more amino acid mutations are introduced into the IgA CH3 domain in an asymmetric manner to result in a heterodimeric Fc. As used herein, an asymmetric amino acid mutation is a mutation that results in an amino acid at a specific position in one Fc polypeptide that is different from an amino acid in a second Fc polypeptide at the same position. This may result in a mutation in only one of two amino acids in the first and second Fc polypeptides, or in two different amino acids of both amino acids. The IgA HetFc constructs disclosed herein include one or more asymmetric amino acid mutations in the CH3 domain.

[0055] The design of the IgA HetFc region from wild-type homodimers is demonstrated by the concepts of positive and negative design, which involve protein manipulation to maintain a balance between stability and specificity. In this case, mutations are introduced to induce heterodimer formation rather than homodimer formation when the polypeptide is expressed under cell culture conditions. These general design concepts of positive and negative design are schematically shown in Figure 1.

[0056] Negative design strategies maximize interactions unfavorable to homodimer formation by introducing a large side chain to one chain and a small side chain to the opposite chain, for example, the knobs-into-holes strategy (Ridgway, et al., 1996, Protein Eng., 9(7):617-21; Atwell, et al., 1997, J Mol Biol., 270(1):26-35), or by electrostatic manipulation that generates repulsive forces for homodimer formation, for example, the electrostatic steering strategy developed by Gunasekaran, et al. 21010, J Biol Chem., 285(25):19637-19646.

[0057] In positive design strategies, amino acid mutations are introduced into a polypeptide to maximize favorable intraprotein or interprotein interactions. Such strategies, when introducing multiple mutations that specifically stabilize a desired heterodimer while ignoring the effects on homodimers, are thought to have a net effect of better specificity for the desired heterodimer interaction than for homodimers, and therefore higher heterodimer specificity. In the context of protein manipulation, it is understood that while positive design strategies optimize the stability of desired protein interactions, specificity exceeding 90% is rarely achieved (Havranek & Harbury, 2003, Nat Struct Biol., 10(1):45-52; Bolon, et al., 2005, Proc Natl Acad Sci USA, 102(36):12724-9; Huang, et al., 2007, Protein Sci., 16(12):2770-4).

[0058] A method for designing IgA Fc heterodimers that result in stable and highly specific heterodimerization is disclosed herein. This design method combines both negative and positive design strategies with structural and computational modeling-induced protein manipulation techniques (see Example 1 herein). Computational tools and structure-function analyses used in the method for generating the IgA HetFc constructs herein include, for example, molecular dynamics analysis (MD), side-chain / back-chain repacking, knowledge base potential (KBP), cavity (hydrophobic) packing analysis (LJ, AMBER, SASA, dSASA (carbon / whole atom)), electrostatic GB calculations, and binding analysis. The calculation method for forming the mutant Fc region is also described in International Patent Publications WO2012 / 058768, WO2015 / 021540, WO2014 / 201566, WO2014 / 138994, WO2014 / 026296, WO2013 / 188984, WO2013 / 138923, WO2012 / 040833, WO2012 / 037659, and WO2011 / 063518.

[0059] In certain embodiments, the IgA HetFc construct obtained from this method has a purity of 70% or higher and stability above 60°C (measured by the melting temperature (Tm) of the CH3 domain). In certain embodiments, the IgA HetFc construct obtained from this method has a purity of 70% or higher and stability below 10°C of the CH3 domain Tm of the corresponding wild-type IgA Fc.

[0060] According to this disclosure, amino acid mutations introduced into the CH3 domain of IgA Fc promote heterodimerization compared to homodimerization. This heterodimerization compared to homodimerization is referred to herein without distinction as “purity,” “specificity,” “heterodimer purity,” or “heterodimer specificity.” It is understood that this heterodimer purity refers to the percentage of desired heterodimers formed compared to homodimer species formed in solution under standard cell culture conditions. Heterodimer purity is evaluated before selective purification of heterodimer species. In certain embodiments, purity may be evaluated after an IgA affinity purification step that is not selective to homodimer / heterodimer purification (e.g., after CaptureSelect® IgA affinity purification). For example, a 70% heterodimer purity indicates that 70% of the Fc dimers isolated from the cell culture after the IgA affinity purification step are the desired Fc heterodimers.

[0061] In certain embodiments, IgA HetFc has a purity of over 70%, for example, over 71%, over 72%, over 73%, over 74%, over 75%, over 76%, over 77%, over 78%, or over 79%. In some embodiments, IgA HetFc has a purity of over 80%, for example, over 81%, over 82%, over 83%, over 84%, over 85%, over 86%, over 87%, over 88%, or over 89%. In some embodiments, the IgA HetFc has a purity of more than about 90%, for example, more than about 91%, more than about 92%, more than about 93%, more than about 94%, more than about 95%, more than about 96%, more than about 97%, more than about 98%, or more than about 99%.

[0062] In certain embodiments, IgA HetFc has a purity between approximately 70% and 100%. In some embodiments, IgA HetFc has a purity between approximately 70% and 98%, or between approximately 70% and 97%, or between approximately 70% and 96%. In some embodiments, IgA HetFc has a purity between approximately 72% and 98%, or between approximately 74% and 98%, or between approximately 75% and 98%.

[0063] The relative amounts of heterodimers and homodimers in a sample of IgA HetFc, and therefore the purity of IgA HetFc, can be determined using a variety of techniques known in the art, including, but not limited to, size exclusion chromatography (SEC), non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), non-reducing capillary electrophoresis of sodium dodecyl sulfate (CE-SDS), and liquid chromatography-mass spectrometry (LC-MS).

[0064] In some embodiments, IgA HetFc has a purity of over 70% when determined by non-reducing CE-SDS. In some embodiments, IgA HetFc has a purity of over 70% when determined by non-reducing CE-SDS performed by a high-throughput protein expression assay using CE-SDS LabChip® GXII (Perkin Elmer, Waltham, MA). In some embodiments, IgA HetFc has a purity of over 70% when determined by non-reducing CE-SDS performed as described in Example 4 of this specification.

[0065] In some embodiments, IgA HetFc has a purity of over 70% when determined by UPLC-SEC. In some embodiments, IgA HetFc has a purity of over 70% when determined by UPLC-SEC performed on an Agilent Technologies 1260 Infinity LC system using an Agilent Technologies AdvanceBio SEC 300A column at 25°C. In some embodiments, IgA HetFc has a purity of over 70% when determined by UPLC-SEC performed as described in Example 4 of this specification.

[0066] The IgA HetFc constructs disclosed herein are thermally stable. In the context of the IgA HetFc constructs disclosed herein, “thermally stable” means that the IgA HetFc construct has a CH3 domain melting temperature (Tm) of about 60°C or higher, or has a CH3 domain Tm within 10°C (±10°C) of the Tm of the corresponding wild-type IgA CH3 domain.

[0067] In certain embodiments, IgA HetFc has a CH3 domain Tm at about 60°C or higher. In some embodiments, IgA HetFc has a CH3 domain Tm at about 62°C or higher, for example, about 63°C or higher, or about 64°C or higher, or about 65°C or higher, or about 66°C or higher, or about 67°C or higher, or about 68°C or higher, or about 69°C or higher. In some embodiments, IgA HetFc has a CH3 domain Tm at about 70°C or higher, for example, about 71°C or about 72°C or higher, or about 73°C or higher.

[0068] In certain embodiments, IgA HetFc has a CH3 domain Tm between approximately 60°C and approximately 74°C. In some embodiments, IgA HetFc has a CH3 domain Tm between approximately 62°C and approximately 74°C, or between approximately 63°C and approximately 74°C, or between approximately 64°C and approximately 74°C, or between approximately 65°C and approximately 74°C.

[0069] In certain embodiments, the IgA HetFc construct has a CH3 domain Tm within 10°C (±10°C) of the corresponding wild-type IgA CH3 domain. In some embodiments, the IgA HetFc construct has a CH3 domain Tm within 9°C (±9°C) of the corresponding wild-type IgA CH3 domain, for example, within 8°C (±8°C), or 7°C (±7°C), or 6°C (±6°C), or 5°C (±5°C) of the corresponding wild-type IgA CH3 domain.

[0070] In certain embodiments, the IgA HetFc construct has a CH3 domain Tm of about 60°C or higher, or a CH3 domain Tm within 10°C (±10°C) of the Tm of the corresponding wild-type IgA CH3 domain in the absence of any additional disulfide bonds in the CH3 domain. In certain embodiments, the IgA HetFc construct has one or more additional disulfide bonds in the CH3 domain compared to the wild-type IgA CH3 domain, but has a CH3 domain Tm of about 60°C or higher, or a CH3 domain Tm within 10°C (±10°C) of the Tm of the corresponding wild-type IgA CH3 domain in the absence of one or more disulfide bonds.

[0071] Stability, measured as Tm, can be determined using techniques known in the art, such as differential scanning calorimetry (DSC), differential scanning fluorescence quantification (DSF), circular dichroism spectroscopy (CD), and hydrogen exchange (HX). In certain embodiments, Tm is determined by DSC.

[0072] In certain embodiments, the IgA HetFc construct has a CH3 domain Tm of about 60°C or higher, or a CH3 domain Tm within 10°C (±10°C) of the Tm of the corresponding wild-type IgA CH3 domain, where Tm is determined by DSC. In some embodiments, the IgA HetFc construct has a CH3 domain Tm of about 60°C or higher, or a CH3 domain Tm within 10°C (±10°C) of the Tm of the corresponding wild-type IgA CH3 domain, where Tm is determined by DSC using NanoDSC (TA Instruments, New Castle, DE, USA). In some embodiments, the IgA HetFc construct has a CH3 domain Tm of about 60°C or higher, or a CH3 domain Tm within 10°C (±10°C) of the Tm of the corresponding wild-type IgA CH3 domain, where Tm is determined by DSC according to the protocol described in Example 6 of this specification.

[0073] In certain embodiments, IgA HetFc is (i) Having a purity of more than approximately 70%, for example, more than approximately 71%, or more than approximately 72%, or more than approximately 73%, or more than approximately 74%, or more than approximately 75%, or more than approximately 76%, or more than approximately 77%, or more than approximately 78%, or more than approximately 79%, or more than approximately 80%, or more than approximately 81%, or more than approximately 82%, or more than approximately 83%, or more than approximately 84%, or more than approximately 85%, or more than approximately 86%, or more than approximately 87%, or more than approximately 88%, or more than approximately 89%, or more than approximately 90%, or more than approximately 91%, or more than approximately 92%, or more than approximately 93%, or more than approximately 94%, or more than approximately 95%, or more than approximately 96%, or more than approximately 97%, or more than approximately 98%, or more than approximately 99%, (ii) Having CH3 domains Tm between approximately 60°C and approximately 74°C, for example, between approximately 62°C and approximately 74°C, or between approximately 63°C and approximately 74°C, or between approximately 64°C and approximately 74°C, or between approximately 65°C and approximately 74°C.

[0074] In certain embodiments, IgA HetFc is (i) Having a purity of more than approximately 70%, for example, more than approximately 71%, or more than approximately 72%, or more than approximately 73%, or more than approximately 74%, or more than approximately 75%, or more than approximately 76%, or more than approximately 77%, or more than approximately 78%, or more than approximately 79%, or more than approximately 80%, or more than approximately 81%, or more than approximately 82%, or more than approximately 83%, or more than approximately 84%, or more than approximately 85%, or more than approximately 86%, or more than approximately 87%, or more than approximately 88%, or more than approximately 89%, or more than approximately 90%, or more than approximately 91%, or more than approximately 92%, or more than approximately 93%, or more than approximately 94%, or more than approximately 95%, or more than approximately 96%, or more than approximately 97%, or more than approximately 98%, or more than approximately 99%, (ii) Having a CH3 domain Tm within 10°C (±10°C) of the corresponding wild-type IgA CH3 domain, for example, within 9°C (±9°C), or within 8°C (±8°C), or within 7°C (±7°C), or within 6°C (±6°C), or within 5°C (±5°C) of the corresponding wild-type IgA CH3 domain.

[0075] In certain embodiments, the IgA HetFc construct comprises one or more mutations that eliminate binding to a binding target, or one or more mutations that introduce binding to an embryonic Fc receptor (FcRn), or both.

[0076] Modified CH3 domain The IgA HetFc constructs described herein include a modified CH3 domain containing asymmetric amino acid mutations. Specifically, the IgA HetFc construct comprises two Fc polypeptides: a first Fc polypeptide containing a first CH3 domain sequence containing one or more amino acid mutations, and a second Fc polypeptide containing a second CH3 domain sequence containing one or more amino acid mutations, wherein at least one of the amino acid mutations in the first CH3 domain sequence is different from the amino acid mutations in the second CH3 domain sequence. The first and second CH3 domain sequences together form a modified CH3 domain. The asymmetrically introduced amino acid mutations in the first and second CH3 domain sequences result in the formation of a heterodimer Fc rather than a homodimer Fc when the two CH3 domain sequences dimerize.

[0077] As described above, in this context, "asymmetric amino acid mutation" refers to a mutation in which an amino acid at a specific position in the first CH3 domain sequence differs from an amino acid at the same position in the second CH3 domain sequence. Asymmetric mutations can result from mutations in only one of the two amino acids at the same amino acid position in each CH3 domain sequence, or from different mutations in both amino acids at the same positions in each of the first and second CH3 domain sequences. The CH3 domain sequence of IgA HetFc may contain one or more asymmetric amino acid mutations.

[0078] By using the computational strategies disclosed herein, we identified a core set of asymmetric mutations in the IgA CH3 domain to yield desired properties that promote heterodimer Fc formation. This core set of mutations is shown in Table 5.

[0079] [Table 5]

[0080] In certain embodiments, the IgA HetFc construct comprises a modified CH3 domain, wherein the amino acid mutations in the first CH3 domain sequence include an amino acid substitution at position A6085Y selected from A6085YF, A6085YY, A6085YM, A6085YW, and A6085YH, and an amino acid substitution at position T6086 selected from T6086Y, T6086F, T6086M, T6086W, and T6086H, and the amino acid mutations in the second CH3 domain sequence include an amino acid substitution at position W6081 selected from W6081T, W6081L, W6081A, W6081V, and W6081I.

[0081] In certain embodiments, the IgA HetFc construct includes a modified CH3 domain containing an amino acid mutation described for any one of the designs shown in Table 7.

[0082] In some embodiments, the amino acid substitution at position A6085Y of the first CH3 domain sequence is A6085YF, A6085YY, or A6085YW. In some embodiments, the amino acid substitution at position A6085Y of the first CH3 domain sequence is A6085YF or A6085YY.

[0083] In some embodiments, the amino acid substitution at position T6086 of the first CH3 domain sequence is T6086Y, T6086F, or T6086W. In some embodiments, the amino acid substitution at position T6086 of the first CH3 domain sequence is T6086Y.

[0084] In some embodiments, the amino acid substitution at position W6081 of the second CH3 domain sequence is W6081T or W6081L.

[0085] In certain embodiments, the IgA HetFc construct comprises a modified CH3 domain, wherein the amino acid mutations in the first CH3 domain sequence include amino acid substitutions A6085YF and T6086W, and the amino acid mutations in the second CH3 domain sequence include amino acid substitutions W6081T or W6081L.

[0086] In some embodiments, the IgA HetFc construct comprises a modified CH3 domain, wherein the amino acid mutations in the first CH3 domain sequence include amino acid substitutions A6085YF and T6086W, and the amino acid mutations in the second CH3 domain sequence include amino acid substitution W6081T.

[0087] In certain embodiments, the first CH3 domain sequence of the IgA HetFc construct may optionally further include one or more of the following: (i) an amino acid substitution at position T6022, selected from T6022V, T6022I, T6022L, and T6022A; and / or (ii) An amino acid substitution at position H6005, selected from H6005Y, H6005F, H6005M, and H6005W.

[0088] In certain embodiments, the second CH3 domain sequence of the IgA HetFc construct may optionally further include one or more of the following: (i) Amino acid substitutions selected from H6005Y, H6005F, H6005M and H6005W; and / or (ii) an amino acid substitution at position L6079, selected from L6079V, L6079T, L6079A, and L6079I; and / or (iii) an amino acid substitution at position I6088, selected from I6088L, I6088A, I6088V, and I6088T; and / or (iv) Amino acid substitution at position L6007, selected from L6007F, L6007Y, L6007M, L6007W, L6007H, and L6007I.

[0089] In certain embodiments, the IgA HetFc construct comprises a modified CH3 domain, wherein the amino acid mutations in the first CH3 domain sequence include an amino acid substitution at position A6085Y selected from A6085YF, A6085YY, A6085YM, A6085YW and A6085YH, and an amino acid substitution at position T6086 selected from T6086Y, T6086F, T6086M, T6086W and T6086H, and the amino acid mutations in the second CH3 domain sequence include an amino acid substitution at position W6081 selected from W6081T, W6081L, W6081A, W6081V and W6081I. (i) an amino acid mutation in the first CH3 domain sequence further includes an amino acid substitution at position T6022 selected from T6022V, T6022I, T6022L and T6022A; and / or (ii) The amino acid mutation in the first CH3 domain sequence further comprises an amino acid substitution at position H6005 selected from H6005Y, H6005F, H6005M and H6005W; and / or (iii) The amino acid mutation in the second CH3 domain sequence further includes an amino acid substitution at position H6005 selected from H6005Y, H6005F, H6005M and H6005W; and / or (iv) an amino acid mutation in the first CH3 domain sequence further comprising an amino acid substitution at position H6005 selected from H6005Y, H6005F, H6005M and H6005W, and an amino acid mutation in the second CH3 domain sequence further comprising an amino acid substitution at position H6005 selected from H6005Y, H6005F, H6005M and H6005W; and / or (v) Amino acid mutations in the second CH3 domain sequence further include amino acid substitutions at position L6079 selected from L6079V, L6079T, L6079A and L6079I; and / or (vi) Amino acid mutations in the second CH3 domain sequence further include amino acid substitutions at position I6088, selected from I6088L, I6088A, I6088V and I6088T; and / or (vii) The amino acid mutation in the second CH3 domain sequence further includes an amino acid substitution at position L6007, selected from L6007F, L6007Y, L6007M, L6007W, L6007H and L6007I.

[0090] In some embodiments, the amino acid mutation at position T6022 of the first CH3 domain sequence is selected from T6022V, T6022I, and T6022L.

[0091] In some embodiments, the amino acid mutation at position H6005 of the first CH3 domain sequence is H6005Y.

[0092] In some embodiments, the amino acid mutation at position H6005 of the second CH3 domain sequence is H6005Y.

[0093] In some embodiments, the amino acid mutation at position L6079 of the second CH3 domain sequence is L6079V or L6079T.

[0094] In some embodiments, the amino acid mutation at position I6088 of the second CH3 domain sequence is I6088L.

[0095] In some embodiments, the amino acid mutation at position L6007 of the second CH3 domain sequence is L6007F.

[0096] In certain embodiments, the modified CH3 domain of the IgA HetFc construct further comprises amino acid substitutions that introduce a cysteine ​​residue capable of forming a disulfide bond. In some embodiments, the modified CH3 domain of the IgA HetFc construct further comprises two cysteine ​​substitutions that introduce one disulfide bond to the CH3 domain. In some embodiments, the modified CH3 domain of the IgA HetFc construct further comprises four cysteine ​​substitutions that introduce two disulfide bonds to the CH3 domain. In some embodiments, the cysteine ​​substitutions include mutation H6005C in one CH3 domain sequence and mutation P6010C in the other CH3 domain sequence. In some embodiments, the cysteine ​​substitutions include mutations H6005C and P6010C in one CH3 domain sequence and mutations P6010C and H6005C in the other CH3 domain sequence.

[0097] Therefore, in certain embodiments, the IgA HetFc construct comprises a modified CH3 domain containing one or two introduced (i.e., unnatural) disulfide bonds. (i) One CH3 domain sequence contains the mutation H6005C and the other CH3 domain sequence contains the mutation P6010C, or (ii) One CH3 domain sequence contains mutants H6005C and P6010C, and the other CH3 domain sequence contains mutants P6010C and H6005C.

[0098] In certain embodiments, the IgA HetFc construct comprises a modified CH3 domain, wherein the amino acid mutations in the first CH3 domain sequence include an amino acid substitution at position A6085Y selected from A6085YF, A6085YY, A6085YM, A6085YW and A6085YH, and an amino acid substitution at position T6086 selected from T6086Y, T6086F, T6086M, T6086W and T6086H, and the amino acid mutations in the second CH3 domain sequence include an amino acid substitution at position W6081 selected from W6081T, W6081L, W6081A, W6081V and W6081I. (i) The first CH3 domain of the IgA HetFc construct may further optionally contain an amino acid substitution at position T6022 selected from T6022V, T6022I, T6022L and T6022A, (ii) The second CH3 domain of the IgA HetFc construct may further optionally include an amino acid substitution at position L6079 selected from L6079V, L6079T, L6079A and L6079I; and / or an amino acid substitution at position I6088 selected from I6088L, I6088A, I6088V and I6088T; and / or one or more amino acid substitutions at position L6007 selected from L6007F, L6007Y, L6007M, L6007W, L6007H and L6007I (iii) The modified CH3 domain contains one or two introduced (i.e., unnatural) disulfide bonds as described above.

[0099] In certain embodiments, the IgA HetFc construct comprises a modified CH3 domain, wherein the amino acid mutations in the first CH3 domain sequence include amino acid substitutions at positions A6085Y and T6086, and the amino acid mutations in the second CH3 domain sequence include an amino acid substitution at position W6081 and optionally one or both amino acid mutations at positions L6079 and I6088. The amino acid substitution at position A6085 is selected from A6085YF, A6085YY, A6085YM, A6085YW, and A6085YH; The amino acid substitution at position T6086 is selected from T6086Y, T6086F, T6086M, T6086W, and T6086H; The amino acid substitutions at position W6081 are selected as W6081T, W6081L, W6081A, W6081V, and W6081I; Any amino acid substitution at position L6079 is selected from L6079V, L6079T, L6079A, and L6079I; Any amino acid substitution at position I6088 is selected from I6088L, I6088A, I6088V, and I6088T.

[0100] In certain embodiments, the IgA HetFc construct includes a modified CH3 domain containing an amino acid mutation described for any one of the designs shown in Table 8. In certain embodiments, the IgA HetFc construct includes a modified CH3 domain containing an amino acid mutation described for any one of the designs shown in Table 9. In certain embodiments, the IgA HetFc construct includes a modified CH3 domain containing an amino acid mutation described for any one of the designs shown in Table 10.

[0101] In some embodiments, the amino acid substitution at position A6085Y is A6085YF, A6085YY, or A6085YW. In some embodiments, the amino acid substitution at position A6085Y is A6085YF, or A6085YY. In some embodiments, the amino acid substitution at position T6086 is T6086Y, T6086F, or T6086W. In some embodiments, the amino acid substitution at position T6086 is T6086Y. In some embodiments, the amino acid substitution at position W6081 is W6081T, or W6081L. In some embodiments, any amino acid substitution at position L6079 is L6079V, or L6079T. In some embodiments, any amino acid substitution at position I6088 is I6088L.

[0102] In some embodiments, the IgA HetFc construct comprises a modified CH3 domain, and as described in any one of the embodiments above, the amino acid mutation in the first CH3 domain sequence comprises amino acid substitutions at positions A6085Y and T6086, the amino acid mutation in the second CH3 domain sequence comprises amino acid substitutions at position W6081 and optionally one or both of positions L6079 and I6088, and either the first CH3 domain sequence or the second CH3 domain sequence, or both the first and second CH3 domain sequences, further comprises an amino acid substitution at position H6005, selected from H6005Y, H6005F, H6005M and H6005W. In some embodiments, either the first CH3 domain sequence or the second CH3 domain sequence, or both the first and second CH3 domain sequences, further comprises the amino acid substitution H6005Y.

[0103] In certain embodiments, the IgA HetFc construct comprises a modified CH3 domain, wherein the amino acid mutations in the first CH3 domain sequence include amino acid substitutions at positions A6085Y and T6086, and the amino acid mutations in the second CH3 domain sequence include an amino acid substitution at position W6081 and optionally one or more amino acid mutations at positions L6007, L6079 and I6088. The amino acid substitution at position A6085 is selected from A6085YF, A6085YY, A6085YM, A6085YW, and A6085YH; The amino acid substitution at position T6086 is selected from T6086Y, T6086F, T6086M, T6086W, and T6086H; The amino acid substitutions at position W6081 are selected as W6081T, W6081L, W6081A, W6081V, and W6081I; Any amino acid substitution at position L6007 is selected from L6007F, L6007Y, L6007M, L6007W, L6007H, and L6007I; Any amino acid substitution at position L6079 is selected from L6079V, L6079T, L6079A, and L6079I; Any amino acid substitution at position I6088 is selected from I6088L, I6088A, I6088V, and I6088T.

[0104] In some embodiments, the amino acid substitution at position A6085Y is A6085YF, A6085YY, or A6085YW. In some embodiments, the amino acid substitution at position A6085Y is A6085YF, or A6085YY. In some embodiments, the amino acid substitution at position T6086 is T6086Y, T6086F, or T6086W. In some embodiments, the amino acid substitution at position T6086 is T6086Y. In some embodiments, the amino acid substitution at position W6081 is W6081T, or W6081L. In some embodiments, the amino acid substitution at position L6007 is L6007F. In some embodiments, the amino acid mutation at position L6079 is L6079V, or L6079T. In some embodiments, the amino acid mutation at position I6088 is I6088L.

[0105] In some embodiments, the IgA HetFc construct comprises a modified CH3 domain, and as described in any one of the embodiments above, the amino acid mutation in the first CH3 domain sequence comprises amino acid substitutions at positions A6085Y and T6086, and the amino acid mutation in the second CH3 domain sequence comprises amino acid substitutions at position W6081 and optionally one or more amino acid substitutions at positions L6007, L6079 and I6088, and either the first CH3 domain sequence or the second CH3 domain sequence, or both the first and second CH3 domain sequences, further comprises an amino acid substitution at position H6005, selected from H6005Y, H6005F, H6005M and H6005W. In some embodiments, either the first CH3 domain sequence or the second CH3 domain sequence, or both the first and second CH3 domain sequences, further comprises the amino acid substitution H6005Y.

[0106] In certain embodiments, the IgA HetFc construct comprises a modified CH3 domain, and the amino acid mutations are those listed in Table 6 for any one of the variants v32516, v32517, v32518, v32521, v33330, v33331, v33332, v33333, v33334, v34688, v34689, or v34690. In some embodiments, the IgA HetFc construct comprises a modified CH3 domain, and the amino acid mutations are those listed in Table 6 for any one of the variants v32521, v33333, or v33334.

[0107] [Table 6]

[0108] In certain embodiments, the IgA HetFc constructs of this disclosure include a modified CH3 domain having the amino acid sequence shown in the CH3 domain sequences contained in SEQ ID NOs: 15 and 20; SEQ ID NOs: 16 and 20; SEQ ID NOs: 17 and 21; SEQ ID NOs: 17 and 23; SEQ ID NOs: 24 and 23; SEQ ID NOs: 25 and 23; SEQ ID NOs: 26 and 23; SEQ ID NOs: 17 and 27; SEQ ID NOs: 28 and 29; SEQ ID NOs: 30 and 31; SEQ ID NOs: 32 and 33; or SEQ ID NOs: 34 and 35. The IgA CH2 and CH3 domains can be readily identified within the SEQ ID NOs described by comparison with the IgA sequences shown in Tables 2 and 4 of this specification.

[0109] Modified CH2 domain In certain embodiments, the IgA HetFc construct further comprises a modified CH2 domain containing one or more amino acid mutations, for example, mutations that alter one or more functions of the CH2 domain. Exemplary mutations include, but are not limited to, mutations at position C5092 (which binds to the secretory compartment of WT IgA) and mutations at the glycosylation site at position N5120.

[0110] In certain embodiments, the modified CH2 contains a mutation at position C5092. In some embodiments, the mutation at position C5092 is an amino acid substitution selected from C5092S, C5092A, C5092T, C5092N, and C5092Q. In some embodiments, the mutation at position C5092 is C5092S. In certain embodiments, the modified CH2 domain contains a mutation at the glycosylation site at position N5120, which prevents glycosylation. In some embodiments, the mutation at position N5120 is the amino acid substitution N5120T.

[0111] In certain embodiments, the HetFc IgA construct includes a modified CH2 domain comprising a mutation at one or more positions C5092, N5120, I5121, and T5122. In some embodiments, the HetFc IgA construct includes a modified CH2 domain comprising one or more amino acid substitutions selected from C5092S, N5120T, I5121L, and T5122S. In some embodiments, the HetFc IgA construct includes a modified CH2 domain comprising amino acid substitutions C5092S, N5120T, I5121L, and T5122S.

[0112] In some embodiments, the modified CH2 domain includes asymmetric amino acid substitutions in the first and / or second Fc polypeptide chain. In some embodiments, the modified CH2 domain includes asymmetric amino acid substitutions that allow one chain of the CH2 domain to selectively bind to the Fc receptor. In certain embodiments, the modified CH2 domain includes asymmetric amino acid mutations that facilitate selective binding to the Fcα receptor.

[0113] The IgA HetFc constructs of this disclosure exhibit modified ligand (e.g., FcαRI) binding properties (examples of binding properties include binding specificity and equilibrium dissociation constant (K)). D ), dissociation and binding rates (k each) off and k on) may have, but is not limited to, binding affinity and / or avidity, and one of ordinary skill in the art will understand that certain modifications may be desirable to some extent depending on the end use of the IgA HetFc construct. The equilibrium dissociation constant (K D ) being defined as k off / k on is well known in the art. For certain applications, it is generally understood that an IgA HetFc construct having a low K D may be preferred over an IgA HetFc construct having a high K D . However, in some cases, the value of k on or k off may be more relevant than the value of K D . One of ordinary skill in the art can determine which kinetic parameters are most important for the use of a given IgA HetFc construct.

[0114] ]>In certain embodiments, IgA HetFc includes substitutions that reduce or eliminate binding to Fcα receptors (see, for example, Carayannopoulos, 1996, JEM, 183:1579 - 1586; Bakema, 2006, J Immunol, 176:3603 - 3610, https: / / www.pnas.org / content / 115 / 38 / E8E82). An IgA HetFc construct with reduced or eliminated binding to Fcα receptors may be useful in situations where neutrophil activation is undesirable, such as in the context of cytokine release syndrome. In this case, the IgA HetFc construct can bind to and eliminate cytokines in a subject that requires binding and elimination of cytokines while avoiding neutrophil activation. An IgA HetFc having only one FcαRI binding site may be useful for investigating the dependence of IgA - dependent neutrophil activation on the valence of FcαRI binding.

[0115] IgA HetFc may be useful for creating single Fc molecules that can bind to FcαRI and the embryonic Fc receptor (FcRn). Since the binding sites for FcαRI and FcRn are located in structurally corresponding regions of IgA and IgG, respectively (Kelton, W. et al., 2014, Chem Biol 21:1603-1609, https: / / www.sciencedirect.com / science / article / pii / S1074552114004098?via%3Dihub), the introduction of these Fc molecules into the chain is mutually exclusive, requiring heterodimeric Fc. IgA HetFc with a grafted FcRn binding site on a single chain is useful because it can not only activate neutrophils via FcαRI but also has an increased half-life due to the introduction of interaction with FcRn, thus addressing the known half-life constraints when using IgA for therapeutic benefits.

[0116] IgA HetFc may be even more useful in creating molecules that can bind monovalently to receptors, purified resins, or detection molecules. Similarly, it may be useful in creating IgA HetFc-based molecules using combinations of receptor binding sites, purification, or detection sites that would otherwise be in mutually exclusive regions of Fc. One such example would be to equip the two strands of Fc with different Fcγ receptor binding sites in a previously shown IgG / A hybrid molecule (Kelton, W. et al., 2014, Chem Biol 21:1603-1609, Borrok, MJ et al., 2015, mAbs, 7:4, 743-751, DOI:10.1080 / 19420862.2015.1047570) to create an Fcγ receptor binding profile with unique biological activity. Receptor binding sites include FcαR, FcRn, Fcγ receptor, C1q, secreted components, SSL7, streptococcal IgA-binding protein, N. meningitidis type 2 IgA1 protease, and Haemophilus influenzae type 2 IgA1 protease. Purification and detection sites include protein A, polyhistidine tags, FLAG tags, and Myc tags. For example, the introduction of a protein A binding site can be used to purify IgA HetFc-based molecules using established and widely used techniques for IgG-based therapeutics that are unsuitable for WT IgA Fc due to the lack of protein A binding.

[0117] Target-binding domain The IgA HetFc described herein may also function as a heterodimeric skeleton into which various different binding domains or other parts can be fused. In certain embodiments, this disclosure relates to IgA HetFc constructs, which are IgA HetFc binding units comprising one or more target-binding domains fused to an IgA HetFc. Target-binding domains for use in IgA HetFc binding units include various protein parts that specifically bind to a target of interest. In this context, “specifically bind” means that the binding is selective to the desired target and can be distinguished from undesirable or nonspecific interactions. The ability of the binding domain to specifically bind to the target can be measured by various techniques well known to those skilled in the art, such as enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) techniques (e.g., analyzed using BIAcore® instruments) (Liljeblad, et al., 2000, Glyco J., 17:323-329), or conventional binding assays (Heeley, 2002, Endocr Res., 28:217-229).

[0118] Examples of target-binding domains include, but are not limited to, receptors, receptor fragments (e.g., extracellular components), ligands, cytokines, and antigen-binding fragments of antibodies. In certain embodiments, the IgA HetFc binding unit includes one or more binding domains, such as antigen-binding domains, which are receptor or antibody fragments.

[0119] In certain embodiments, the IgA HetFc binding unit comprises one or more target-binding domains that are antigen-binding antibody fragments. Such antigen-binding antibody fragments may be derived from IgA or other antibody isotypes, such as IgG, IgM, IgD, or IgE. In some embodiments, the antigen-binding antibody fragments may be synthetic, chimeric, or humanized. The antigen-binding antibody fragment may include variable or highly variable regions (V) of the light and / or heavy chains of the antibody. L , V HExamples include, but are not limited to, variable fragments (Fv), Fab' fragments, F(ab')2 fragments, Fab fragments, single-chain antibodies (scAb), single-chain variable regions (scFv), VHH, complementarity-determining regions (CDR), domain antibodies (dAb), single-domain heavy-chain immunoglobulins, and single-domain light-chain immunoglobulins. Antigen-binding sites of antibodies generally include six CDRs that contribute to varying degrees to the affinity of the binding site to the antigen. There are three heavy-chain variable domain CDRs (CDRH1, CDRH2, and CDRH3) and three light-chain variable domain CDRs (CDRL1, CDRL2, and CDRL3). The extent of the CDRs and framework regions (FRs) is determined by comparing them to an aggregate database of amino acid sequences defined according to the variability between sequences and / or structural information from the antibody / antigen complex. Functional antigen-binding sites consisting of a small number of CDRs (i.e., binding specificity determined by three, four, or five CDRs) are also included within the scope of this disclosure. Fewer than the complete set of six CDRs may be sufficient to bind to some binding targets. Therefore, in some cases, a CDR consisting solely of the VH or VL domain may be sufficient for specific binding. Furthermore, certain antibodies may have binding sites to the antigen that are not associated with the CDR. Such binding sites are particularly intended herein. Antigen-binding antibody fragments may be derived from a single species, or they may be chimeric or humanized.

[0120] In certain embodiments, the binding domain includes an antigen-binding receptor fragment, such as an MHC-peptide complex-binding fragment of a T cell receptor (TCR). The TCR fragment for use in the IgA HetFc constructs herein may include an antigen-binding fragment of an αβTCR or γδTCR heterodimer. In some embodiments, the IgA HetFc constructs herein may include an antigen-binding fragment of an αβTCR heterodimer, including at least a TCR α-chain variable domain and a TCR β-chain variable domain, so that the αβTCR fragment can bind to the same type of MHC / peptide. In some embodiments, the antigen-binding TCR fragment is a single-chain TCR (scTCR) or a soluble TCR domain (see, for example, International Patent Publications WO1999 / 018129 and WO2009 / 117117). Other TCR antigen-binding fragments are known in the art and are shown, for example, in Wilson & Garcia, 1997, Curr. Opin. Struct. Biol. 7:839-848; van Boxel, et al., 2009, J. Immunol. Methods, 350:14-21; Stone, et al., 2012, Methods Enzymol., 503:189-222 and Li, et al., 2005, Nat. Biotechnol., 23:349-354.

[0121] Other target-binding domains include immunomodulatory Ig domains, non-Ig virus receptor decoys, antibody-binding and structurally mimicking non-immunoglobulin proteins such as anticarin, affilin, affibody molecules, affimers, affitins, alphabodies, avimers, DARPin, finomers, Knitz domain peptides, monobodies, and binding domains based on other manipulated skeletons such as SpA, GroEL, fibronectin, lipocalin, and the CTLA4 skeleton. Further examples of target-binding domains include ligands for desired receptors, ligand-binding moieties of receptors, lectins, and peptides that specifically bind to one or more target antigens.

[0122] In certain embodiments, the IgA HetFc binding unit includes a binding domain containing an antigen-binding fragment of a therapeutic or diagnostic antibody. In some embodiments, the target-binding domain contained in the IgA HetFc binding unit specifically binds to cell surface molecules such as proteins, lipids, or polysaccharides. In some embodiments, the binding domain contained in the IgA HetFc binding unit specifically binds to target antigens expressed on tumor cells, virus-infected cells, bacterial-infected cells, damaged red blood cells, arterial plaque cells, inflammatory tissue cells, or fibrotic tissue cells.

[0123] In certain embodiments, the target-binding domain contained in the IgA HetFc binding unit is an immune response modulator. In certain embodiments, the target-binding domain contained in the IgA HetFc binding unit specifically binds to cytokine receptors. In certain embodiments, the target-binding domain contained in the IgA HetFc binding unit specifically binds to tumor antigens. In certain embodiments, the target-binding domain contained in the IgA HetFc binding unit is an immune checkpoint protein, or specifically binds to one.

[0124] As a result of the heterodimer properties of IgA HetFc, various binding domains can be fused to one or both chains of the Fc heterodimer to form a wide range of functionally multispecific IgA HetFc binding units. An unrestricted example of such multispecific IgA HetFc binding units is shown in Figure 7. Furthermore, multiple IgA HetFc binding units can be linked together, for example, by linkage via a J chain, to form even higher-order IgA HetFc multimers. The multimeric IgA structure generally contains an IgA dimer in a tail-to-tail configuration linked by a J chain and tailpiece-to-tailpiece interactions, with additional IgA monomers linked to the dimer only via tailpiece-to-tailpiece interactions involving disulfide bonds and not in direct contact with the J chain of the complex (see, e.g., Kumar, et al., 2020, Science, 10.1126 / science.aaz5807). An example of such an IgA HetFc polymer is shown in Figure 8.

[0125] The IgA HetFc binding units according to this disclosure may be single-specific, bi-specific, triply specific, quadruply specific, or have more multiple specificities. Multiple-specific IgA HetFc binding units may specifically bind to different epitopes of a desired target molecule, or to different target molecules, or to target molecules and heterogeneous epitopes, such as heterogeneous polypeptides or solid support materials.

[0126] In some embodiments, the IgA HetFc binding unit comprises two or more target-binding domains, each having a different binding specificity. In this regard, the binding domains may bind to the same target but to different epitopes on the same target, or they may each bind to different targets.

[0127] In certain embodiments, the IgA Fc binding unit includes a target-binding domain fused to one Fc polypeptide (e.g., chain A) and either does not include a target-binding domain in the other Fc polypeptide (e.g., chain B), or includes a different target-binding domain fused to it. Therefore, chains A and B of IgA HetFc may have different Fc regions (which, as described above, have mutations in the CH3 domain that lead to heterodimerization) and may also have different binding specificities.

[0128] The term "IgA HetFc binding unit" is used herein to refer to an IgA HetFc construct having a heterodimer Fc (e.g., a pair of IgA Fc polypeptides, each containing at least an IgA CH3 domain), in which case at least one IgA Fc polypeptide is fused to a target-binding domain. In certain embodiments, both Fc polypeptides of the IgA HetFc construct are independently fused to target-binding domains. As shown in Figure 7, an IgA HetFc binding unit may include one to four target-binding domains fused to the HetFc in various different configurations. In certain embodiments, additional target-binding domains may be included in an IgA HetFc binding unit by fusion of one or more additional target-binding domains to target-binding domains fused to the IgA HetFc.

[0129] The IgA HetFc binding units provided in this disclosure may be derived from a single species, or they may be chimeric or humanized. For example, the IgA Fc polypeptide may be human, and the target-binding domain may be derived from another species, such as another mammal (e.g., mouse, rat, rabbit, non-human primate, etc.).

[0130] Figure 7 shows an exemplary configuration of an IgA HetFc construct containing a target-binding domain (IgA HetFc binding unit). In certain embodiments, the IgA HetFc binding unit contains one, two, three, or four target-binding domains fused to the IgA HetFc. In some embodiments, the IgA HetFc binding unit has a one-arm configuration in which one Fc polypeptide is fused to the target-binding domain, while the other Fc polypeptide is not fused to the target-binding domain.

[0131] In some embodiments, the IgA HetFc binding unit includes one target-binding domain fused to the N-terminus of one Fc polypeptide (e.g., chain A) and one target-binding domain fused to the N-terminus of the other Fc polypeptide (e.g., chain B) (see, for example, Figures 7B and 7C). In some embodiments, the IgA HetFc binding unit includes one target-binding domain fused to the N-terminus of one Fc polypeptide (e.g., chain A) and one target-binding domain fused to the C-terminus of the other Fc polypeptide (e.g., chain B) (see, for example, Figure 7F). In some embodiments, the IgA HetFc binding unit includes one target-binding domain fused to the C-terminus of one Fc polypeptide (e.g., chain A) and one target-binding domain fused to the C-terminus of the other Fc polypeptide (e.g., chain B) (see, for example, Figure 7D). In some embodiments, the IgA HetFc binding unit includes target-binding domains fused to both ends of one Fc polypeptide (e.g., the N-terminus and C-terminus of chain A) (see, for example, Figure 7E). In some embodiments, the IgA HetFc binding unit includes target-binding domains fused to both ends of one Fc polypeptide (e.g., the N-terminus and C-terminus of chain A) and a target-binding domain fused to one end (either the N-terminus or C-terminus) of the other Fc polypeptide (e.g., chain B) (see, for example, Figure 7G). In some embodiments, the IgA HetFc binding unit includes target-binding domains fused to both ends of one Fc polypeptide (e.g., the N-terminus and C-terminus of chain A) and a target-binding domain fused to both ends of the other Fc polypeptide (e.g., the N-terminus and C-terminus of chain B) (see, for example, Figure 7H). Other configurations including additional target-binding units fused in tandem are also possible.

[0132] In some embodiments, the IgA HetFc binding unit is bispecific, i.e., it contains two target-binding domains, each having a different specificity. In some embodiments, the IgA HetFc binding unit is triplicate, i.e., it contains three target-binding domains, each having a different specificity. In some embodiments, the IgA HetFc binding unit is quadruplespecific, i.e., it contains four target-binding domains, each having a different specificity. Even more specificity may be achieved by including several target-binding domains in tandem. In some embodiments, at least some of the target-binding domains of the bispecific, triplicate, or quadruplespecific IgA HetFc binding unit bind to the same target, but to different epitopes on the target. In some embodiments, at least some of the target-binding domains of the bispecific, triplicate, or quadruplespecific IgA HetFc binding unit bind to different target molecules.

[0133] It should be noted that the specificity of an IgA HetFc binding unit does not necessarily correlate with the number of target-binding domains it contains. For example, if both target-binding domains bind to the same target, an IgA HetFc binding unit may contain two target-binding domains but still be single-specific.

[0134] In certain embodiments, the disclosure provides higher-order IgA HetFc polymers comprising two or more IgA HetFc binding units. In certain embodiments, the higher-order IgA HetFc polymers of the disclosure comprise two, four, or five IgA HetFc binding units. In certain embodiments, at least two IgA HetFc binding units in the IgA HetFc polymer are connected by a J chain via a tailpiece. In the IgA HetFc polymers disclosed herein, the J chain may be a full-length, unmodified J chain, but may also include amino acid modifications such as substitutions, insertions, deletions, and cleavages, in particular, J chain fragments, as long as the J chain remains functional. In certain embodiments, the J chain in the IgA HetFc polymer is a modified J chain described in International Publication No. WO2015 / 153912. In certain embodiments, the J chain has the amino acid sequence shown in Sequence ID No. 48.

[0135] As described above, the IgA HetFc binding units described herein enable the assembly of multispecific IgA HetFc polymers. IgA HetFc polymers have the potential to fine-tune avidity effects, which can increase the apparent affinity of low-affinity target-binding domains and enhance associated functionality, including clustering and specificity, as well as increased titer. Figure 8 shows an exemplary configuration of an IgA HetFc polymer.

[0136] In some embodiments, an IgA HetFc polymer may be a “dimer” in that it contains two IgA HetFc binding units linked by a J chain. The IgA HetFc binding units may be single-specific, bispecific (see, for example, Figure 8A), or a combination thereof. In some embodiments, the dimeric IgA HetFc polymer of the Disclosure contains two bispecific IgA HetFc binding units, each having the same binding specificity (AB, AB). In some embodiments, the dimeric IgA HetFc polymer of the Disclosure contains two bispecific IgA HetFc binding units, each having at least one of the two binding units having a different binding specificity (e.g., AB, BC or AC, BC or AB, CD). Thus, in certain embodiments, each of the two binding units has two specificities, which may be the same (AB, AB) or different (e.g., AB, CD or AB, AC).

[0137] In some embodiments, an IgA HetFc polymer may be a "tetramer" in that it contains four IgA HetFc binding units, at least two of which are linked by a J chain. The IgA HetFc binding units may be single-specific, double-specific (see, for example, Figure 8B), or a combination thereof. In some embodiments, the tetrameric IgA HetFc polymer of this disclosure contains four double-specific binding units, each having the same binding specificity (AB, AB, AB, AB). Tetrameric IgA HetFc polymers containing IgA HetFc binding units that are either single-specific or double-specific and have different binding specificities are also intended in some embodiments.

[0138] In some embodiments, an IgA HetFc polymer may be a “pentamer” in that it contains five IgA HetFc binding units, at least two of which are linked by a J chain. The IgA HetFc binding units may be single-specific, double-specific (see, for example, Figure 8C), or a combination thereof. In some embodiments, the pentameric IgA HetFc polymer of this disclosure contains five double-specific binding units, each having the same binding specificity (AB, AB, AB, AB, AB). Pentameric IgA HetFc polymers containing IgA HetFc binding units that are either single-specific or double-specific and have different binding specificities are also intended in some embodiments.

[0139] The term "valence," as used herein, indicates the presence of a specific number of binding sites in an IgA HetFc construct. For example, the terms "divalent," "tetravalent," "hexavalent," "octavalent," and "decavalent" indicate the presence of 2, 4, 6, 8, and 10 binding sites, respectively. Thus, referring to Figure 8 herein, the dimeric IgA HetFc polymer shown in Figure 8A, containing two bispecific binding units, is tetravalent; the tetrameric IgA HetFc polymer shown in Figure 8B is octavalent (i.e., containing 4 bispecific binding units); and the pentameric IgA HetFc polymer shown in Figure 8C is decavalent (i.e., containing 5 bispecific binding units). Similarly, referring to Figure 7, the IgA HetFc binding units shown in Figures 7B, C, D, E, and F are divalent, the IgA HetFc binding unit shown in Figure 7G is trivalent, and the IgA HetFc binding unit shown in Figure 7H is tetravalent.

[0140] In IgA HetFc binding units and polymers, different components or domains may be directly fused to one another (i.e., without linkers), or one or more components or domains may be indirectly fused to adjacent components or domains via peptide linkers. Peptide linkers suitable for linking the components of multicomponent proteins are well known in the art and are selected to allow for an arrangement of components such that each can still perform its intended function.

[0141] Peptide linkers are between approximately 2 and 150 amino acid lengths. A useful linker is the glycine-serine (GlySer) linker, which is well known in the art and contains glycine and serine units combined in various orders. For example, n is at least one integer, generally between 1 and about 10, e.g., between 1 and about 8, between 1 and about 6, or between 1 and about 5 (GS) n (GSGGS) n (GGGS) n and (GGGGS) n ;n is an integer from 1 to 5 (Gly3Ser) n (Gly4Ser)1, (Gly3Ser)1(Gly4Ser) n (Gly3Ser) n (Gly4Ser) n , or (Gly4Ser) n Examples include, but are not limited to, those described above. Other useful linkers include sequences derived from immunoglobulin hinge sequences. Linkers may include all or part of hinge sequences derived from any one of the four IgG classes or TCRs, and may optionally include additional sequences. For example, a linker may include part of an immunoglobulin hinge sequence and a glycine-serine sequence. A non-limiting example is a linker containing a GlySer linker sequence that is about 10 amino acids long, such as the one described above, following the first approximately 15 residues of the IgG1 hinge.

[0142] compound Certain embodiments of this disclosure relate to complexes comprising one or more activators, such as therapeutic agents, diagnostic agents, or labeling agents, of the IgA HetFc constructs described herein (e.g., IgA HetFc skeletons, IgA HetFc binding units, or IgA HetFc polymers).

[0143] Examples of therapeutic agents include, but are not limited to, antimetabolites, alkylating agents, anthracyclines, antibiotics, mitotic inhibitors, toxins, apoptotic agents, thrombolytic agents, anti-angiogenic agents, bio-response modifiers, growth factors, radioactive materials, and macrocyclic chelates useful for conjugating radioactive metal ions. Examples of diagnostic agents include, but are not limited to, various contrast agents such as fluorescent materials, luminescent materials, and radioactive materials. Examples of labeling agents include, but are not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials.

[0144] The conjugation of a selected activator with an IgA HetFc construct can be achieved in various ways, either directly or via a linker. A linker for activator conjugation is a bifunctional or polyfunctional moiety capable of linking one or more activators to an IgA HetFc construct. A bifunctional (or monovalent) linker links a single activator to a single site on the construct, while a polyfunctional (or polyvalent) linker links two or more activators to a single site on the construct. A linker capable of linking one activator to two or more sites on an IgA HetFc construct may also be considered polyfunctional.

[0145] Conjugation can be achieved, for example, by surface lysine on the IgA HetFc construct, by reductive coupling with oxidized carbohydrates on the IgA HetFc construct, or by cysteine ​​residues on the IgA HetFc construct released by reduction of interchain disulfide bonds. Alternatively, the conjugation may involve modifications of the IgA HetFc construct to include additional cysteine ​​residues (see, e.g., U.S. Patents 7,521,541; 8,455,622 and 9,000,130) or non-natural amino acids that provide a reactive handle, such as selenomethionine, p-acetylphenylalanine, formylglycine, or p-azidomethyl-L-phenylalanine (e.g., Hofer et al., 2009, Biochemistry, 48:12047-12057; Axup et al., 2012, PNAS, 109:16101-16106; Wu et al., 2009, PNAS, 106:3000-3005; Zimmerman et al., 2014, Bioconj. This can be achieved by (see Chem., 25:351-361), enabling site-specific conjugation.

[0146] Methods for conjugating various drugs with proteins, including immunoglobulins, are known in this field (see, for example, Bioconjugate Techniques (GT Hermanson, 2013, Academic Press)).

[0147] Method for preparing polynucleotides and IgA HetFc constructs The IgA HetFc constructs described herein can be prepared using standard recombinant methods. Recombinant production of IgA HetFc constructs generally involves synthesizing one or more polynucleotides encoding the IgA HetFc construct, cloning one or more polynucleotides into a suitable vector(s), and introducing the vector(s) into a host cell suitable for expression of the IgA HetFc construct. Recombinant production of proteins is known in the art and can be achieved using standard techniques described, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, (1987 & updates), John Wiley & Sons, New York, NY; and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990).

[0148] Accordingly, certain embodiments of this disclosure relate to isolated polynucleotides, or sets of polynucleotides, that encode the IgA HetFc constructs described herein. In this context, the polynucleotides may encode all or part of the IgA HetFc constructs.

[0149] The terms “nucleic acid,” “nucleic acid molecule,” and “polynucleotide” are used herein without distinction and refer to any macromolecular form of a nucleotide of any length, either a deoxyribonucleotide or a ribonucleotide, or an analog thereof. Polynucleotides may be of genomic, cDNA, RNA, semi-synthetic or synthetic origin, or any combination thereof.

[0150] A polynucleotide that "encodes" an IgA HetFc construct is a polynucleotide that, under the control of appropriate regulatory sequences, is transcribed in vivo (in the case of DNA) and translated into a polypeptide (in the case of mRNA). The coding sequence boundary is determined by a start codon at the 5' (amino) terminus and a translation termination codon at the 3' (carboxy) terminus. The transcription termination sequence may be located 3' to the coding sequence.

[0151] One or more polynucleotides encoding an IgA HetFc construct can be inserted into a suitable expression vector(s) either directly or after one or more subcloning steps using standard ligation techniques. Examples of suitable vectors include, but are not limited to, plasmids, phagemids, cosmids, bacteriophages, baculoviruses, retroviruses, or DNA viruses. The vector is typically selected to function in the specific host cell in which it is employed; that is, the vector is compatible with the host cell's mechanisms and capable of amplifying and / or expressing the polynucleotide(s). In this regard, the selection of a suitable vector-host cell combination is within the ordinary art of those skilled in the art.

[0152] Accordingly, certain embodiments of this disclosure relate to vectors (such as expression vectors) comprising one or more polynucleotides encoding an IgA HetFc construct. The polynucleotide(s) may be contained in a single vector or in multiple vectors. In some embodiments, the polynucleotide(s) are contained in a multicistronic vector.

[0153] Typically, an expression vector contains one or more regulatory elements for plasmid maintenance and the cloning and expression of an exogenous polynucleotide sequence. Examples of such regulatory elements include promoters, enhancer sequences, origins of replication, termination sequences, donor and acceptor splice sites, leader sequences for polypeptide secretion, ribosome binding sites, polyadenylation sequences, polylinker regions for inserting polynucleotides encoding the polypeptide to be expressed, and selection markers.

[0154] Regulatory elements can be homogeneous (i.e., from the same species and / or lineage as the host cell), heterogeneous (i.e., from a species other than the host cell's species or lineage), hybrid (i.e., a combination of regulatory elements from multiple sources), or synthetic. Thus, the sources of regulatory elements may be either prokaryotes or eukaryotes, as long as their adjacent sequences function within and can be activated by the host cell mechanism employing them.

[0155] Optionally, the vector may also include a “tag” coding sequence. The tag coding sequence is a nucleic acid sequence located at the 5' or 3' end of the coding sequence that codes for a heterologous peptide sequence such as polyHis (e.g., 6×His), FLAG®, HA (hemagglutinin influenza virus), myc, metalloaffinity, avidin / streptavidin, glutathione-S-transferase (GST), or biotin tag. The tag typically remains fused to the expressed polypeptide and can function as a means of polypeptide affinity purification or detection. Optionally, the tag can then be removed from the purified polypeptide by various means, such as using a specific cleavage peptidase.

[0156] Various expression vectors are readily available from private suppliers. Alternatively, if a commercially available vector containing all the desired regulatory elements is not available, an expression vector may be constructed using a commercially available vector as a starting vector. If one or more of the desired regulatory elements are not already present in the vector, they can be obtained individually and ligated into the vector. Methods and sources for obtaining various regulatory elements are well known to those skilled in the art.

[0157] After constructing an expression vector(s) containing polynucleotides(s) encoding an IgA HetFc construct, the vector(s) can be inserted into host cells suitable for amplification and / or protein expression. Transformation of selected host cells with the expression vector can be achieved by well-known methods, including transfection, infection, calcium phosphate coprecipitation, electroporation, microinjection, lipofection, DEAE-dextran transfection, and other known techniques. The chosen method is determined in part by the type of host cell used. These methods and other suitable methods are well known to those skilled in the art (see, e.g., Sambrook, et al., ibid.).

[0158] When host cells are cultured under appropriate conditions, they express the polypeptide encoded by the vector, which can then be recovered from the culture medium (if the host cell secretes the polypeptide) or directly from the polypeptide-producing host cell (if the polypeptide is not secreted). The host cell can be a prokaryote (e.g., a bacterial cell) or a eukaryote (e.g., a yeast, fungus, plant, or mammalian cell). The selection of a suitable host cell can be easily carried out by those skilled in the art, taking into account various factors such as the desired expression level, polypeptide modifications desirable or required for activity (e.g., glycosylation or phosphorylation), and ease of folding into a bioactive molecule.

[0159] Accordingly, specific embodiments of this disclosure relate to a host cell or one or more vectors comprising a polynucleotide(s) encoding an IgA HetFc construct. In specific embodiments, the host cell is a eukaryotic cell.

[0160] For example, eukaryotic microorganisms such as filamentous fungi or yeasts may be used as host cells, including fungal and yeast strains in which the glycosylation pathway has been "humanized" (see, e.g., Gerngross, 2004, Nat. Biotech., 22:1409-1414, and Li et al., 2006, Nat. Biotech., 24:210-215). Plant cells can also be used as host cells (see, e.g., U.S. Patents No. 5,959,177; No. 6,040,498; No. 6,420,548; No. 7,125,978, and No. 6,417,429 (described in relation to PLANTIBODIES® technology)).

[0161] In some embodiments, the eukaryotic host cell is a mammalian cell. Various mammalian cell lines can also be used as host cells. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney cell line CV1 (COS-7), human fetal kidney cell line 293 (e.g., HEK293 cells described in Graham, et al., 1977, J. Gen Virol., 36:59), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, 1980, Biol. Reprod., 23:243-251), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HeLa), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor cells (MMT 060562), and TRI cells (e.g., Mather, et al., 1982, Annals). Examples of suitable cells include, but are not limited to, those described in NYAcad.Sci.,383:44-68, MRC5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (including DHFR-CHO cells described in Urlaub, et al.,1980, Proc.Natl.Acad.Sci.USA,77:4216), and myeloma cells (Y0, NS0, and Sp2 / 0, etc.). See also Yazaki and Wu,2003,Methods in Molecular Biology,Vol.248,pp.255-268(BKCLo,ed.,Humana Press,Totowa,NJ).

[0162] Specific embodiments of this disclosure relate to a method for producing an IgA HetFc construct as described herein, comprising the steps of transfecting host cells with one or more polynucleotides encoding an IgA HetFc construct, and culturing the host cells under conditions suitable for the expression of the encoded IgA HetFc construct.

[0163] Typically, IgA HetFc constructs are isolated from host cells after expression and optionally purified. Methods for isolating and purifying expressed proteins are known in the art. Standard purification methods include, for example, chromatographic techniques such as ion exchange, hydrophobic interaction, affinity, size, gel filtration, or reversed phase, which can be performed at atmospheric pressure or medium-high pressure using systems such as FPLC, MPLC, and HPLC. Other purification methods include electrophoresis, immunological methods, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques combined with protein concentration may also be useful.

[0164] Various native proteins that bind to the Fc region of antibodies are known in the art, and therefore these proteins can be used for the purification of Fc-containing proteins. For example, bacterial proteins A and G bind to the Fc region. Purification can often be made possible by specific fusion partners or affinity tags as described above. For example, antibodies can be bound to glutathione resin when GST fusion is used, or to Ni when His tags are used. +2 Purification may be performed using affinity chromatography or, if flag tags are used, immobilized anti-flag antibodies. Examples of useful purification techniques are found in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990), and Protein Purification: Principles and Practice, 3 rd This is described in Ed., Scopes, Springer-Verlag, NY (1994). The required degree of purification will vary depending on the use of the IgA HetFc construct. In some cases, purification may not be necessary.

[0165] In certain embodiments, the IgA HetFc constructs herein are purified using one or more purification methods known in the art, including but not limited to affinity chromatography, affinity chromatography with non-reducing CE-SDS, affinity purification (protein A purification column, CaptureSelect® IgA affinity purification), and size exclusion chromatography, such as ULC-SEC (see also Examples 1-6).

[0166] Post-translation modification In certain embodiments, the IgA HetFc constructs described herein may be post-translation modified.

[0167] The term "post-translational modification" and its grammatical variations, such as "post-translational modification," refers to the modification of a natural or non-natural amino acid that occurs after the amino acid has been introduced into a polypeptide chain. This term encompasses, but is not limited to, simultaneous translation in vivo modification, simultaneous translation in vitro modification (such as in cell-free translation systems), post-translational in vivo modification, and post-translational in vitro modification.

[0168] Specific examples of post-translational modifications include, but are not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or combinations thereof. Other examples include, but are not limited to, chemical modifications by known techniques including, specific chemical cleavage with cyanide bromide, trypsin, chymotrypsin, papain, V8 protease, or NaBH4; acetylation; formylation; oxidation; reduction; or metabolic synthesis in the presence of tunicamycin.

[0169] Additional post-translational modifications include the attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and the addition or removal of N-terminal methionine residues as a result of prokaryotic host cell expression.

[0170] In certain embodiments, the IgA HetFc constructs described herein may optionally be modified with detectable labels, such as enzymes, fluorescence, isotopes, or affinity labels, to enable the detection and isolation of the protein. Examples of suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dancylcloride, and phycoerythrin; examples of luminescent substances include luminol; examples of bioluminescent substances include luciferase, luciferin, and aequorin; and examples of suitable radioactive substances include radioisotopes of iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon, and fluorine.

[0171] In some embodiments, the IgA HetFc constructs described herein may be bound to a macrocyclic chelate that optionally associates with a radioactive metal ion.

[0172] In embodiments in which the IgA HetFc construct is modified by either a natural process such as post-translational processing or a chemical modification technique, the same type of modification may be present at several sites of a given polypeptide to similar or different degrees.

[0173] In certain embodiments, the IgA HetFc construct may be bound to a solid support, which may be particularly useful for immunoassays or purification of polypeptides bound to, or associated with, the proteins described herein. Examples of such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, and polypropylene.

[0174] Characterization of IgA HetFc constructs The IgA HetFc constructs described herein can be characterized in a variety of ways. For example, the purity of an IgA HetFc construct can be evaluated using techniques known in the art, including but not limited to SDS-PAGE gel, Western blotting, densitometry, mass spectrometry, size exclusion chromatography (SEC), or non-reducing capillary electrophoresis of sodium dodecyl sulfate (CE-SDS). In certain embodiments, the purity of an IgA HetFc construct is evaluated by SEC or CE-SDS.

[0175] Protein stability can also be characterized using a number of known techniques in the art, including, but not limited to, size exclusion chromatography (SEC); UV, visible, or CD spectroscopy; mass spectrometry; differential light scattering (DLS); benchtop stability assays; freeze-thaw coupled with other characterization techniques; differential scanning calorimetry (DSC); differential scanning fluorescence quantification (DSF); hydrophobic interaction chromatography (HIC); isoelectric focusing; receptor binding assays or relative protein expression levels. In certain embodiments, the stability of an IgA HetFc construct is assessed by measuring the CH3 domain melting temperature (Tm) compared to the wild-type CH3 domain Tm using techniques known in the art, such as DSC or DSF.

[0176] Where appropriate, the IgA HetFc constructs of this disclosure may also be assayed for their ability to specifically bind to a ligand, receptor, or target antigen (e.g., FcαRI, or the target antigen of the binding domain contained in the IgA HetFc construct). Various immunoassays known in the art may be used to analyze specific binding and cross-reactivity, including but not limited to competitive and non-competitive assay systems using techniques such as Western blotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradioassay, fluorescence immunoassay, and protein A immunoassay. Such assays are well-known and standardized in the art (see, for example, Ausubel, et al., eds, 1994, Current Protocols in Molecular Biology, John Wiley and Sons, Inc., New York).

[0177] IgA HetFc constructs that have been confirmed to specifically bind to a target ligand, receptor, or antigen may also be optionally assayed for affinity to the ligand, receptor, or antigen. Parameters such as binding affinity and the binding and dissociation rates of the interaction can be determined, for example, by competitive binding assays. The reaction rate parameters of IgA HetFc constructs may also be determined using surface plasmon resonance (SPR) based assays known in the art, such as BIAcore® kinetic analysis. Various SPR-based assays are known in the art (see, for example, Mullet, et al., 2000, Methods, 22:77-91; Dong, et al., 2002, Rev. Mol. Biotech., 82:303-23; Fivash, et al., 1998, Curr Opinion in Biotechnology, 9:97-101; Rich, et al., 2000, Curr Opinion in Biotechnology, 11:54-61, and U.S. Patents 6,373,577; 6,289,286; 5,322,798; 5,341,215 and 6,268,125). Fluorescence-activated cell sorting (FACS) using techniques known to those skilled in the art can also be used to characterize the binding of IgA HetFc constructs to molecules expressed on the cell surface (e.g., Fc receptors or cell surface antigens). Flow cytometers for sorting and examining living cells are well known in the art (see, for example, U.S. Patents 4,347,935; 5,464,581; 5,483,469; 5,602,039; 5,643,796 and 6,211,477). Other known flow cytometers include the FACS Vantage® system from Becton Dickinson and Company (Franklin Lakes, NJ) and the COPAS® system from Union Biometrica (Holliston, MA).A detailed explanation of binding affinity and reaction kinetics can be found in Paul, WE, ed., 1999, Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia, which focuses on antibody-immunogen interactions.

[0178] The binding properties of IgA HetFc constructs can also be characterized by in vitro functional assays to determine the downstream function of one or more FcαRIs (see, e.g., Bakema, 2006, J Immunol, 176:3603-3610).

[0179] How to use Specific embodiments of this disclosure relate to the use of the IgA HetFc constructs described herein in therapeutic or diagnostic methods. For example, the IgA constructs can be used in methods of binding to neutrophils via FcαRI and methods of activating neutrophils via FcαRI.

[0180] IgA HetFc constructs containing one or more binding domains and IgA HetFc constructs conjugated with therapeutic agents can be used in methods of treatment, such as methods for treating subjects with cancer, autoimmune diseases, immune or inflammatory disorders, or infectious diseases. Similarly, IgA constructs containing one or more binding domains and IgA HetFc constructs conjugated with targeted agents or diagnostic agents can be used in methods of diagnosis, such as methods for diagnosing subjects with cancer, autoimmune diseases, immune or inflammatory disorders, or infectious diseases.

[0181] When used in a treatment method, the IgA HetFc construct is administered to the subject in a therapeutically effective dose. The term "therapeutically effective dose," as used herein, refers to the amount of the IgA HetFc construct described herein or a composition containing the IgA HetFc construct described herein administered that would achieve the objective of the enumerated method, for example, to some extent alleviate one or more symptoms of the disease or disorder being treated. The amount of the composition described herein that would be effective in treating the disease or disorder in question can be determined by standard clinical techniques. In addition, in vitro assays may be used to help determine the optimal dose range, if applicable. The exact dose used in the formulation will also depend on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the practitioner and the circumstances of each patient.

[0182] In some embodiments in which the IgA HetFc construct is used in a treatment method, the IgA HetFc construct may be administered in combination with one or more additional therapeutic agents known to those skilled in the art for the treatment of the disease or disorder in question in a therapeutically effective amount.

[0183] The desired effects of the treatment include, but are not limited to, relief of symptoms, attenuation of any direct or indirect pathological consequences of the disease or disorder, prevention of metastasis, slowing of the rate of disease progression, remission or mitigation of the disease state, improved survival, remission, improved prognosis, or delayed relapse of the disease.

[0184] Pharmaceutical composition For therapeutic or diagnostic use, the IgA HetFc construct may be provided in the form of a composition comprising the IgA HetFc construct and a pharmaceutically acceptable carrier or diluent. The composition may be prepared by known procedures using well-known and readily available components and may be formulated for administration to a subject by, for example, oral (e.g., buccal or sublingual), topical, parenteral, rectal or vaginal routes, or by inhalation or spray. As used herein, the term “parenteral” includes injection or infusion by subcutaneous, intradermal, intra-articular, intravenous, intramuscular, intravascular, intrasternal or intrathecal routes.

[0185] The composition is typically formulated in a form suitable for administration to a target via a selected route, such as a syrup, elixir, tablet, lozenge, hard or soft capsule, pill, suppository, oily or aqueous suspension, dispersible powder or granule, emulsion, injection, or liquid. The composition may also be provided as a unit dosage form.

[0186] Pharmacochemically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used. Examples of such carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkylparabens (such as methyl or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight (less than approximately 10 amino acids) polypeptides; and serum albumin. Examples of nonionic surfactants include, but are not limited to, proteins such as gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes such as Zn-protein complexes; and nonionic surfactants such as polyethylene glycol (PEG).

[0187] In certain embodiments, the composition may be in the form of an aqueous or oily solution or suspension for sterile injection. Such solutions or suspensions may be formulated using suitable dispersants or wetting agents and / or suspending agents known in the art. The solutions or suspensions for sterile injection may contain the IgA HetFc construct in an orally acceptable, non-toxic diluent or solvent. Acceptable diluents and solvents that may be employed include, for example, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution. In addition, sterile non-volatile oils may be employed as solvents or suspension media. For this purpose, various brands of non-volatile oils, including synthetic monoglycerides or diglycerides, may be employed. In addition, fatty acids such as oleic acid are also used in the preparation of injections. Auxiliaries such as local anesthetics, preservatives, and / or buffers known in the art may also be included in the solutions or suspensions for injection.

[0188] Other pharmaceutical compositions and methods for preparing them are known in the art and are described, for example, in “Remington: The Science and Practice of Pharmacy” (formerly “Remingtons Pharmaceutical Sciences”); Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).

[0189] Kits and Products Certain embodiments of this disclosure relate to kits comprising one or more IgA HetFc constructs described herein. Packaged in separate containers, the individual components of the kit associated with such containers may be marked in a form prescribed by government agencies regulating the manufacture, use, or sale of pharmaceutical or biological products, and such marking indicates that the product is approved by the agency of manufacture, use, or sale. The kit may optionally include instructions or directives outlining the method of use or dosing regimen of the IgA HetFc construct.

[0190] If one or more components of the kit are provided as a solution, for example, an aqueous solution or a sterile aqueous solution, the container means may itself be an inhaler, syringe, pipette, eye drop container, or other similar device, and the solution may be administered to a subject or applied to and mixed with other components of the kit.

[0191] The components of the kit may also be supplied in dry or lyophilized form, and the kit may further contain a solvent suitable for reconstituting the lyophilized components. Regardless of the number or type of containers, the kits described herein may also include instruments for assisting in the administration of the composition to a patient. Such instruments may be inhalers, nasal spray devices, syringes, pipettes, forceps, measuring spoons, eye drop containers, or similar medically approved delivery vehicles.

[0192] Certain embodiments relate to a product containing materials useful for patient treatment as described herein. The product includes a container and a label or accompanying information on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, and intravenous solution bags. Containers may be formed from a variety of materials, such as glass or plastic. The container may hold a composition containing an IgA HetFc construct, either alone or in combination with another composition effective for patient treatment, and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper puncturable by a subcutaneous needle). The label or accompanying information indicates that the composition is used in a treatment suitable for the use of the composition. The product may further include a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for infusion (BWFI), phosphate-buffered water, Ringer's solution, or dextrose solution. The product may further include other materials desirable from a commercial and user perspective, including, optionally, other buffers, diluents, filters, needles, and syringes.

[0193] The following embodiments are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0194] [Table 7] TIFF2023552220000010.tif197165TIFF2023552220000011.tif91165

[0195] [Table 8] TIFF2023552220000013.tif 199165 TIFF2023552220000014.tif 201165 TIFF2023552220000015.tif 200165 TIFF2023552220000016.tif 200165 TIFF2023552220000017.tif 200165 TIFF2023552220000018.tif 199165 TIFF2023552220000019.tif 82165

[0196]

Table 9

[0197]

Table 10

[0198] Example 1: In silico selection of lead IgA heterodimer design This example describes the in silico analysis and selection of IgA Fc Cα3(CH3) mutations that may lead to heterodimerization rather than homodimerization of IgA Fc dimers.

[0199] method In a comprehensive structural analysis of the CH3:CH3 junction of IgA Fc (PDB ID: 2QEJ, Ramsland et al., 2007, Proc Natl Acad Sci USA 104:15051-15056), residues at the junction were characterized by their energetic contribution to dimerization. For this purpose, proprietary tools for analyzing structural bonding and energy properties, based on knowledge-based and physics-based possibilities, were used for static structures and 50 ns explicit molecular dynamics orbitals. Based on the results of this initial analysis, residues were selected for the introduction of mutations expected to disrupt dimerization in the first "negative design." These mutations were selected based on two main design concepts shown in Figure 1. The negative electrostatic design involved the introduction of identical charge sets and associated repulsive forces across the junction, while the negative stereodesign was based on the introduction of cavities or steric collisions at the junction. These negative designs were modeled and energetically evaluated using proprietary in-silico tools. In the second "positive design" step, additional mutations were introduced to rescue heterodimerization. Stabilization of the heterodimer complex was based on either the introduction of salt bridges by opposite charges across the junction, or the accommodation of residues with large side chains by cavities on the opposite side of the junction. Designs with the largest energy difference between homodimer and heterodimer were selected to express and evaluate.

[0200] result Table 11 shows variations in read designs based on analytical indices. Table 12 shows selected sets of in silico indices for homodimer and heterodimer read design models. Energy is relative to the wild type. Negative energy indicates a favorable interaction, and positive energy indicates an unfavorable interaction.

[0201] In particular, the stereodesign with the greatest energy difference between homodimer and heterodimer focused on mutations to large hydrophobic side chains at positions A6085Y and T6086 in chain A, and the exchange of a small residue at W6081 in the opposite chain B. Figure 9 shows an example of a read design (stereodesign 6) in which a cavity is created by introducing large hydrophobic residues at positions 6085Y and 6086 in chain A, while simultaneously exchanging threonine at W6081 in chain B. Stereodesign 6 includes two additional mutations in chain B, which are substitutions of tryptophan at position 6081 with a residue having a smaller side chain involved in creating a cavity that can accommodate the large hydrophobic residues introduced at positions 6085Y and 6086 in chain A. These three mutations together are thought to result in the main stereodesign that promotes heterodimer formation. Therefore, these three mutations (A:6085Y and 6086, B:6081) are thought to constitute the minimum core set of mutations that promote IgA Fc heterodimer formation. Specifically, the core set of mutations consists of substitutions of A6085Y and T6086 in chain A with residues containing larger and / or more hydrophobic side chains, combined with the substitution of W6081 in chain B with a residue having a smaller side chain. Larger and / or more hydrophobic residues that are expected by in silico analysis to be suitable for introducing positions 6085Y and 6086 include F, Y, M, W, and H, while smaller residues that are expected by in silico analysis to be suitable for introducing position 6081 include T, L, A, V, and I.

[0202] [Table 11]

[0203] [Table 12] TIFF2023552220000088.tif198165TIFF2023552220000089.tif163165

[0204] Example 2: Formation of a single-arm antibody (OAA) construct using heterodimer IgA Fc The mutations expected to lead to heterodimerization, as described in Example 1, were introduced into a single-arm antibody construct containing IgA Fc, and its functionality was evaluated.

[0205] method To evaluate the efficacy of mutations designed to induce heterodimer pairing of IgA Fc, we designed a single-arm IgA antibody form with a significant weight difference between its two halves. One half of the antibody consisted of an IgG1-based anti-Her2 Fab (heavy chain: SEQ ID NO: 38, light chain: SEQ ID NO: 39, Carter, et al., 1992, Proc Natl Acad Sci USA, 89:4285-4289) fused to IgA Fc in the heavy chain. A chimeric hinge containing an upper IgG1 hinge (SEQ ID NO: 40) bound to the N-terminus of an IgA2 hinge (SEQ ID NO: 41) was used to connect the IgG1 Fab to the IgA2 Fc. The sequence of IgA Fc was similar to that of the CH2 and CH3 domains of the IgA2m1 allotype (Chintalacharuvu, et al., 1994, J Immunol, 152:5299-5304). As described in Lohse et al., 2016, Cancer Res, 76:403-417, the binding site C5092 (IMGT numbering shown in Table 2) and the N5120 glycosylation site to the secretory compartment of WT IgA were mutated, the α-tailpiece was removed, and the construct terminated at G6129 (see Sequence ID 43 in Table 4).

[0206] The other half of the single-arm antibody construct consisted solely of an IgA2 hinge (SEQ ID NO: 41) fused with IgA2m1 CH2 and CH3, and did not contain Fab. It also contained the same Fc mutations as those found in the heavy chain described above. Expected to lead to pairing of the heterodimer in Example 1, the mutations listed in Table 11 were introduced into the CH3 domain of Fc in the single-arm antibody construct to obtain the variants described in Table 13. The mutations in chain A were introduced into the heavy chain containing VH and CH1(H1), while the mutations in chain B were introduced into the heavy chain containing only Fc.

[0207] [Table 13]

[0208] Example 3: Preparation of a heterodimer IgA single-arm antibody The heavy and light chain sequences of the modified IgA OAA variants designed in Examples 1 and 2 were cloned into expression vectors as shown below, expressed, and purified.

[0209] method A signal peptide (EFATMRPTWAWWLFLVLLLALWAPARG [SEQ ID NO: 49]) (Barash et al., 2002, Biochem and Biophys Res. Comm., 294:835-842) and a vector insert containing the heavy chain and light chain sequences described in Example 2 were ligated into a pTT5 vector to prepare heavy chain and light chain expression vectors. The vectors were sequenced to confirm the correct reading frame and sequence of the coding DNA.

[0210] The heavy and light chains of a modified IgA OAA variant, as well as the Fc-only chain, were co-expressed in 25 mL of Expi293F® cell culture (Thermo Fisher, Waltham, MA). Expi293® cells were cultured at 37°C on an orbital shaker rotating at 125 rpm in Expi293® expression medium (Thermo Fisher, Waltham, MA) under a humidified atmosphere of 8% CO2. 7.5 × 10⁻⁶ 7A total of 25 μg of DNA with a transfection ratio of 30:40:30 for H1:L1:H2 was transfected into a 25 mL volume containing the total number of cells. Prior to transfection, the DNA was diluted in 1.5 mL of Opti-MEM® I Reduced Serum Medium (Thermo Fisher, Waltham, MA). 80 μL of ExpiFectamine® 293 reagent (Thermo Fisher, Waltham, MA) was diluted in 1.42 mL of Opti-MEM® I Reduced Serum Medium and mixed with the DNA transfection mix after incubation for 5 minutes to a total volume of 3 mL. After 10 to 20 minutes, the DNA-ExpiFectamine® 293 reagent mixture was added to the cell culture. After incubation at 37°C for 18–22 hours, 150 μL of ExpiFectamine® 293Enhancer 1 and 1.5 mL of ExpiFectamine® 293Enhancer 2 (Thermo Fisher, Waltham, MA) were added to each culture. The cells were incubated for 5–7 days, and the supernatant was collected for protein purification.

[0211] The clear supernatant sample was diluted 1:1 with PBS and applied to 2 mL of CaptureSelect® IgA Affinity Matrix (ThermoFisher, Waltham, MA) packed in-house on an AKTA® Pure FPLC System (GE Life Sciences) Millipore Vantage L×250 column. The column was equilibrated with PBS. After loading, the column was washed with PBS and the protein was eluted with 0.1 M glycine, pH 2.5. The eluted sample was pH adjusted by adding 10% (v / v) 1 M Tris, pH 9 to obtain a final pH of 6-7. After affinity chromatography, the variants were evaluated for heterodimer purity using non-reducing CE-SDS and ULC-SEC as described in Example 4.

[0212] After concentration, to separate the heterodimeric species from the homodimeric Fc species and other impurities, a material of the variant containing a large amount of heterodimeric species was injected into an AKTA™ Pure FPLC System (GE Life Sciencies) and flowed through a Superdex 200 Increase 10 / 300 GL (GE Life Sciences) column pre-equilibrated with PBS at pH 7.4. The protein was eluted from the column at a rate of 0.75 mL / min and collected as 0.5 mL fractions. Peak fractions having a concentration of >0.5 mg / mL and a CE-SDS purity of >95% of the target protein were pooled and concentrated using a Vivaspin™ 20, 30 kDa MWCO polyethersulfone concentrator (MilliporeSigma, Burlington, MA). After sterile filtration through a 0.2 μm PALL Acrodisc™ syringe filter equipped with a Supor™ membrane, the protein was quantified based on A280nm (Nanodrop), frozen, and stored at −80 °C until further use.

[0213] Results No variants were obtained in which expression was detectable even with electrostatic design mutations, indicating the disruptive nature of these mutations. In contrast, all steric designs showed expression under the test conditions, and 10 designs were purified and further investigated (Steric 1-4, Steric 6-11). Some samples of these variants showed highly pure heterodimeric species after affinity chromatography, but generally, preparative SEC was required to obtain high purity samples due to the presence of homodimeric Fc species as well as other impurities such as half antibodies and aggregates (see Example 4). After performing preparative SEC on Steric 1-3 and Steric 6-11 designs and WT IgA Fc OAA, the yields ranged from 30 to 200 mg / L of the expression culture. Evaluation of sample purity and stability is described in Example 4, Example 5, and Example 6.

[0214] Example 4: Evaluation of Heterodimeric Purity of Lead Designs after Affinity Chromatography The OAA variant was evaluated for heterodimer purity and sample homogeneity by non-reducing CE-SDS and UPLC-SEC after CaptureSelect IgA affinity purification and before SEC purification.

[0215] Method After CaptureSelect IgA affinity purification, the purity of the samples was evaluated by non-reducing and reducing high-throughput protein expression assays using a CE-SDS LabChip® GXII (Perkin Elmer, Waltham, MA). The procedure was carried out following the HT Protein Express LabChip® User Guide version 2 with the following modifications. The antibody samples were added at either 2 μl or 5 μl (concentration range 5 - 2000 ng / μl) together with 7 μl of HT Protein Express Sample Buffer (Perkin Elmer #760328) into separate wells of a 96-well plate (BioRad, Hercules, CA). Next, the samples were denatured at 90 °C for 5 minutes and 35 μl of water was added to each sample well. The LabChip® device was operated using an HT Protein Express Chip (Perkin Elmer #760499) and an HT Protein Express 200 assay setting (14 kDa - 200 kDa).

[0216] UPLC-SEC was performed on an Agilent Technologies 1260 Infinity LC system at 25 °C using an Agilent Technologies AdvanceBio SEC 300A column. Before injection, the samples were centrifuged at 10000 g for 5 minutes and 5 μL was injected onto the column. The samples were run at a flow rate of 1 mL / min in PBS, pH 7.4 for 7 minutes and elution was monitored by UV absorbance at 190 - 400 nm. Chromatograms were extracted at 280 nm. Peak integration was performed using OpenLAB CDS ChemStation software.

[0217] Results Analysis of unreduced CE-SDS of WT IgA OAA (v32595) showed a mixture of homodimer full-size antibodies (FSAs) along with Fc and heterodimer OAA species (Figure 2). Heterodimer species were the most prominent, with each homodimer species present being less numerous. This is the expected species distribution with equimolar expression of both Fc chains in the absence of any mutations promoting heterodimerization (Ridgway, et al., 1996, Protein Eng, 9:617-621), and was also observed in ULC-SEC (Figure 3A).

[0218] Variants containing mutations that promote heterodimer formation showed different species distributions in both non-reducing CE-SDS (Figure 2) and ULC-SEC (Figure 3), particularly compared to WT IgA OAA. While FSA homodimers were absent in any of the stereodesigns shown in Figures 2 and 3, various levels of Fc homodimers and semiantibody species were found in addition to OAA heterodimers. Most notably, the stereo3 (v32518; Figure 3D) and stereo6 (v32521; Figure 3F) designs showed a significant increase in the purity of OAA heterodimer species, with stereo6 achieving >95% heterodimer purity in both CE-SDS and ULC-SEC. Conversely, stereo4 (v32519; Figure 3E) contained neither OAA heterodimers nor FSA homodimer species, only Fc homodimers and corresponding semiantibodies, suggesting a problem with the expression of the other heavy chain, likely due to the introduced mutation. The presence of small peaks at a retention time of <3 minutes indicated the presence of small amounts of high molecular weight species, such as oligomers and aggregates, in all samples.

[0219] Example 5: Evaluation of heterodimer purity of lead designs after size exclusion chromatography After SEC purification using the selected design, the samples were evaluated for sample homogeneity using non-reducing and reduced CE-SDS and UPLC-SEC as shown below.

[0220] method Non-reducing CE-SDS and ULC-SEC were performed as described in Example 4. For electrophoretic analysis under reducing conditions, the CE-SDS protocol was modified by adding 3.5 μL of DTT (1M) to 100 μL of HT Protein Express Sample Buffer.

[0221] result Figures 4 and 5 show the UPLC-SEC traces and CE-SDS electrophoresis profiles (reduced and unreduced) of heterodimer OAA samples purified by SEC as described in Example 3. UPLC-SEC analysis showed an extremely homogeneous sample containing 90%–100% heterodimer OAA species. The presence of small peaks with short retention times and shoulders with long retention times compared to the major species indicates the presence of small amounts of homodimers with WT IgA (Figure 4A), stereo1 (Figure 4B), and stereo2 (Figure 4C) designs. After SEC purification, unreduced CE-SDS showed a single major species of all variants investigated. Only three complete chains and corresponding single bands of all variants were observed by reduced CE-SDS. In particular, the light chain and the heavy chain of Fc only had similar molecular weights (23.4 kDa and 23.7 kDa) and appeared as a single band in the reduced CE-SDS profile.

[0222] Example 6: Thermal stability of lead IgA heterodimer design The purified heterodimer OAA variant samples after preparative SEC were evaluated for thermal stability by differential scanning calorimetry (DSC) as shown below.

[0223] method After preparative SEC as described in Example 3, the heterodimer OAA design samples were diluted in PBS to 0.5–1 mg / ml. For DSC analysis using NanoDSC (TA Instruments, New Castle, DE, USA), 950 μl of sample and matching buffer (PBS) were added to sample and reference 96-well plates, respectively. At the start of the DSC run, a buffer (PBS) blank injection was performed to stabilize the baseline. Next, each sample was injected and scanned from 25 to 95°C at 1°C / min under a nitrogen pressure of 60 psi. Thermograms were analyzed using NanoAnalyze software. The baseline was fitted using a sigmoid curve by subtracting the thermogram of the matching buffer from the thermogram of the sample. The data were then fitted to a two-state scale DSC model.

[0224] result DSC thermograms of WT IgA OAA (v32595) with an unmodified IgA CH3-CH3 junction showed two transitions at 74°C and 81°C (Figure 6A). The more pronounced transition at 81°C was present in all investigated designs and was attributed to unfolding of the CH2 domain, which was not mutated in either design, and unfolding of partially overlapping Fab. Conversely, the transition was observed to vary between designs and was attributed to unfolding of the CH3 domain (Figures 6A-B). Modified CH3 in stereotype 2 (v32517) was significantly more unstable compared to WT (Tm at 55°C compared to 74°C), but the design with the highest heterodimer purity showed CH3 stability close to that of WT. Transitions in stereotype 3 (v32518) and stereotype 6 (v32521) were observed at 65.9°C and 71.9°C, respectively. The two designs that showed the best thermal stability were stereo10 (v33333) and stereo11 (v33334), with CH3 unfolding transitions observed at 72.0°C and 73.6°C, respectively. Despite this high thermal stability, the heterodimer purity of these two designs, as evaluated by CE-SDS and ULC-SEC in Example 4, was lower than that of stereo3 and stereo6.

[0225] In summary, we identified combinations of IgA CH3 domain mutations that significantly led to IgA Fc heterodimer formation. The thermal stability of the CH3 domain in heterodimer variants possessing these mutations was within approximately 2°C of WT IgA CH3 for stereo6 (v32521), stereo10 (v33333), and stereo11 (v33334) designs. A summary of the characteristics of the tested stereodesigns is shown in Table 14.

[0226] [Table 14]

[0227] Example 7: Further stabilization of the IgA HetFc design To improve the thermal stability and heterodimer purity of the lead IgA HetFc design through covalent disulfide crosslinking across the junction, a cysteine ​​mutation was introduced at the CH3 junction of IgA Fc.

[0228] method The set of residues at the junction of IgA Fc was selected based on Cα and Cβ distances determined to be sufficient to accommodate the shape of the disulfide bond. Next, the selected residues were substituted with cysteine ​​residues to model the resulting covalent disulfide bond. The obtained structure was energetically evaluated using a proprietary in-silico tool.

[0229] result Cysteine ​​substitution was introduced into stereo6 designs and evaluated using a proprietary in-silico tool. Exemplary indices for the selected designs are shown in Table 15. Subsequently, cysteine ​​substitution was introduced into single and double disulfide designs for stereo6 OAA forms and into a single disulfide design for WT OAA (Table 16).

[0230] The variants shown in Table 16 are expressed and evaluated for heterodimer purity and thermal stability. It is expected that the high heterodimer purity of the Stereo 6-based designs (34688 - 34690) evaluated by UPLC-SEC and CE-SDS will be retained when compared to that of Stereo 6 (evaluated by UPLC-SEC and CE-SDS after CaptureSelect IgA purification > 90%, see Example 6). On the other hand, when compared to that of Stereo 6 (> 71 °C, see Example 6), due to the addition of one or two covalent disulfide bonds to the interface, the thermal stability of these designs measured by DSC is expected to be significantly increased. When introduced as an asymmetric single disulfide design into the WT IgA Fc (34691) with other points unchanged, the heterodimer purity evaluated by UPLC-SEC and CE-SDS is expected to be significantly improved compared to WT IgA (evaluated by UPLC-SEC and CE-SDS after CaptureSelect IgA purification > 50%, see Example 6), and the thermal stability is expected to be the same as or exceed that of WT (> 74 °C, see Example 6).

[0231] The identified disulfide designs may also be combined with other lead HetFc designs identified in Examples 1 - 6, expressed in OAA format, purified, and evaluated for heterodimer purity and thermal stability as described in Examples 2 - 6.

[0232]

Table 15

[0233]

Table 16

[0234] Example 8: Multimeric Multispecific Form Based on IgA HetFc Mutations that lead to heterodimer pairing of IgA Fc, as described in Examples 1-7, can be used to construct multimeric, multispecific variants, which can then be tested for target binding and functionality.

[0235] method Two strands of IgA1, IgA2m1, or IgA2m2 Fc containing a C-terminal tailpiece (SEQ ID NO: 46 or 47) are provided with mutations that lead to heterodimerization of the CH3 domain, as described in Examples 1-6 and Table 11, to form a core IgA HetFc skeleton. A binding domain specific to one target (e.g., Fab, scFv, VHH, immunomodulatory Ig domain, non-Ig virus receptor decoy, and as described elsewhere herein) is ligated to the N-terminal side of one IgA HetFc strand via an IgA1, IgA2, or IgG1 / IgA2 chimeric hinge, while the same hinge is used to ligate a second binding domain specific to another target to the N-terminal side of the other strand of IgA HetFc. Next, the two resulting chains are transiently expressed in a mammalian expression system along with the linking chain (J chain) and any additional polypeptide chains required to complete the IgA HetFc construct (e.g., other chains to complete the Fab used as a targeting domain). Depending on the IgA allotype used for Fc and the ratio of the J chain to the IgA Fc chain, this results in the formation of dimeric, tetrameric, or pentameric molecules (Lombana et al., 2019, MAbs, 11:1122-1138, Kumar, et al., 2020, Science, 367:1008-1014), and each IgA HetFc binding unit in the dimeric, tetrameric, or pentameric IgA HetFc multimer has two binding domains (see Figure 8). After purification by CaptureSelect® IgA affinity chromatography, the sample is evaluated for purity and particle size uniformity by one or more of the following methods: non-reducing and reduced SDS-PAGE or CE-SDS, ULC-SEC, multi-angle light scattering (MALS), or dynamic light scattering (DLS). If necessary, the sample is further purified by SEC as described in Example 3, and its sample quality is evaluated as previously described. Next, the sample is tested for target binding by one or more of the following methods: surface plasmon resonance (SPR), flow cytometry, or target-specific functional assays.

[0236] result IgA HetFc multimer variants based on IgA1 and IgA2m1 HetFc are primarily dimers, while those based on IgA2m2 HetFc exhibit dimers, tetramers, and pentamers separable by SEC. In binding studies with individual targets, an increase in apparent affinity is expected compared to monovalent binding due to the avidity provided by the multimer skeleton. This avidity effect on apparent affinity is expected to be further enhanced when both targets are present in the binding assay. Compared to IgG-based monomeric bispecific antibodies, IgA HetFc multimers with increasing binding titers (monomer < dimer < tetramer < pentamer) should show progressively improved affinity. In summary, this avidity effect is expected to lead to high specificity and high potency towards the binding target, as reflected in previously recognized functional studies (Slaga et al., 2018, Sci Transl Med, 10(463):eaat5775; International Patent Publications WO2016 / 141303 and WO2016 / 118641). When used to target viruses or bacterial pathogens, high-titer IgA HetFc polymers are expected to lead to aggregation and removal of the target(s), while multispecificity limits mutation avoidance and ensures consistently high levels of neutralization.

[0237] Example 9: Heterodimer IgA Fc containing a mutation that eliminates binding with FcαRI To evaluate the effect of the binding titer of IgA Fc-mediated FcαRI binding on functionality, IgA Fc with a single FcαRI binding site was constructed using heterodimer IgA Fc based on the mutations described in Examples 1-7.

[0238] method Mutations identified as interfering with IgA Fc:FcαRI interaction (F6116A, Posgai, MT et al., 2018, Proc Natl Acad Sci USA 115:E8882-E8891) were introduced into one or both heavy chains of stereo6-designed OAA variants (Table 17). These variants, as well as the wild-type stereo6-OAA (32521), were then expressed and purified as described in Examples 3-6. Other constructs may include combinations of mutations that achieve various FcaRI affinities for the two chains of heterodimer IgA Fc. Possible combinations are shown in Table 18. These variants can be evaluated for FcαRI binding and neutrophil activation. Schematic diagrams of variants containing two or one FcαRI binding sites, or without an FcαRI binding site, are shown in Figure 11.

[0239] [Table 17]

[0240] [Table 18]

[0241] result Variants with modified FcαRI binding sites, designed to increase, decrease, or eliminate binding, are expected to exhibit varying affinities to FcαRI and varying activity in neutrophil activation assays compared to WT IgA Fc. Knockout mutations in both strands are expected to eliminate binding and neutrophil activation, while mutations aimed at increasing FcαRI binding in both strands are expected to increase binding and neutrophil activation, resulting in the highest possible activity. All other combinations shown in Table 18 are expected to exhibit binding and neutrophil activation at levels between these limits.

[0242] Example 10: Heterodimer IgA Fc containing FcaRI and FcRn binding sites Using the mutations described in Examples 1-7 that lead to the aggregation of heterodimeric IgA Fc, we construct IgA-based variants capable of activating neutrophils via FcαRI and having an increased half-life due to the presence of an FcRn binding site.

[0243] method We grafted residues crucial for the binding of IgG Fc to the embryonic Fc receptor (FcRn) (Oganesyan, V. et al., 2014, J Biol Chem 289:7812-7824) onto a heterodimeric IgA variant to create a construct capable of binding to both FcRn and FcαRI. Since the FcαRI and FcRn binding sites are located at structurally corresponding locations on the CH2 / CH3 junctions of IgA and IgG, respectively, heterodimeric Fc is necessary (Kelton, W. et al., 2014, Chem Biol 21:1603-1609). Grafting the FcRn binding site is achieved by superimposing peptide backbone atoms of IgA and IgG Fc and identifying the IgA residues structurally corresponding to the IgG:FcRn binding region. These are then replaced with their corresponding IgG residues. Alternatively, the designs may include mutations known to alter FcRn affinity in IgG (Robbie, GJ et al., 2013, Antimicrob Agents Chemother 57:6147-6153, Yeung, YA et al., 2009, J Immunol 182:7663-7671, Hinton, PR et al., 2006, J Immunol 176:346-356, Hinton, PR et al., 2004, J Biol Chem 279:6213-6216, Dall`Acqua, WF, Kiener, PA & Wu, H., 2006, J Biol Chem 281:23514-23524). Multiple designs are energetically evaluated using a proprietary in-silico tool. These variants are expressed, purified, and then evaluated for their binding to FcαRI and FcRn, as well as for their in vitro neutrophil activation and in vivo half-life. A schematic diagram of such variants is shown in Figure 12.

[0244] result Variants that achieve binding to both FcαRI and FcRn are expected to exhibit activity in neutrophil ADCC assays and a significantly increased half-life at FcRn in an in vivo model compared to IgA Fc lacking an FcRn binding site.

[0245] Sequence List Table A provides a brief description of the sequence numbers for the clones described herein. Table B shows the amino acid sequences for each sequence number.

[0246] [Table A] TIFF2023552220000097.tif73165

[0247] [Table B] TIFF2023552220000099.tif243165TIFF2023552220000100.tif235165TIFF20235522200 00101.tif229165TIFF2023552220000102.tif235165TIFF2023552220000103.tif238165 TIFF2023552220000104.tif204165TIFF2023552220000105.tif230165TIFF20235522200 00106.tif228165TIFF2023552220000107.tif236165TIFF2023552220000108.tif228165

[0248] The methods and compositions described herein are not limited to the specific methodologies, protocols, cell lines, constructs, and reagents described herein, and should therefore be understood to be subject to modification. It should also be understood that the terminology used herein is used to describe only specific embodiments and is not intended to limit the scope of the methods and compositions described herein, which are limited only by the appended claims.

[0249] All disclosures of patents, patent applications, publications and database entries referenced herein are incorporated herein by reference in whole to the same extent as each of such individual patents, patent applications, publications and database entries is specifically and individually indicated as being incorporated by reference.

Claims

1. 1. An IgA heterodimeric Fc (IgA HetFc) construct comprising a first Fc polypeptide comprising a first CH3 domain sequence and a second Fc polypeptide comprising a second CH3 domain sequence, said first and second CH3 domain sequences forming a modified CH3 domain, the first and second CH3 domain sequences comprise amino acid mutations that promote the formation of a heterodimeric Fc over a homodimeric Fc; wherein the amino acid mutations in the first CH3 domain sequence comprise an amino acid substitution at position A6085Y selected from A6085YF, A6085YY, A6085YM, A6085YW and A6085YH, and an amino acid substitution at position T6086 selected from T6086Y, T6086F, T6086M, T6086W and T6086H; the amino acid mutation in the second CH3 domain sequence comprises an amino acid substitution at position W6081 selected from W6081T, W6081L, W6081A, W6081V, and W6081I; the heterodimeric Fc is formed with a purity of 70% or greater; The numbering of amino acid positions is according to the IMGT numbering system. The IgA heterodimeric Fc (IgA HetFc) construct.

2. the modified CH3 domain comprises: (a) has a melting temperature (Tm) of 60° C. or greater; or (b) having a melting temperature (Tm) of the corresponding wild-type IgA CH3 domain ± 10°C; 2. The IgA HetFc construct of claim 1. (a) the amino acid substitution at position A6085Y is A6085YF, A6085YY or A6085YW; and / or (b) said amino acid substitution at position T6086 is T6086Y, T6086F or T6086W; and / or (c) the amino acid substitution at position W6081 is W6081T or W6081L; 3. The IgA HetFc construct of claim 1 or 2.

4. 4. The IgA HetFc construct of any one of claims 1 to 3, wherein said amino acid substitution at position A6085Y is A6085YF or A6085YY.

5. 5. The IgA HetFc construct of any one of claims 1 to 4, wherein said amino acid substitution at position T6086 is T6086Y.

6. the amino acid mutations in the first CH3 domain sequence include the amino acid substitutions A6085YF and T6086Y; the amino acid mutation in the second CH3 domain sequence comprises the amino acid substitution W6081T or W6081L; An IgA HetFc construct according to any one of claims 1 to 3.

7. 7. The IgA HetFc construct of claim 6, wherein the amino acid mutation in the second CH3 domain sequence comprises the amino acid substitution W6081T.

8. the amino acid mutation in the second CH3 domain sequence is (a) further comprising an amino acid substitution at position L6079 selected from L6079V, L6079T, L6079A and L6079I; and / or (b) further comprising an amino acid substitution at position I6088 selected from I6088L, I6088A, I6088V and I6088T; and / or (c) further comprising an amino acid substitution at position L6007 selected from L6007F, L6007Y, L6007M, L6007W, L6007H and L6007I; and / or (d) further comprising an amino acid substitution at position H6005 selected from H6005Y, H6005F, H6005M and H6005W; An IgA HetFc construct according to any one of claims 1 to 7.

9. 8. The IgA HetFc construct of any one of claims 1 to 7, wherein said amino acid mutations in said second CH3 domain sequence further comprise the amino acid substitution L6079V.

10. 10. The IgA HetFc construct of any one of claims 1 to 9, wherein said amino acid mutations in said second CH3 domain sequence further comprise the amino acid substitution I6088L.

11. the amino acid mutation in the first CH3 domain sequence is (a) further comprising an amino acid substitution at position T6022 selected from T6022V, T6022I, T6022L and T6022A; and / or (b) further comprising an amino acid substitution at position H6005 selected from H6005Y, H6005F, H6005M and H6005W; An IgA HetFc construct according to any one of claims 1 to 10.

12. 11. The IgA HetFc construct of any one of claims 1 to 10, wherein said amino acid mutations in the first CH3 domain sequence further comprise an amino acid substitution at position T6022 selected from: T6022V, T6022I and T6022L.

13. 13. The IgA HetFc construct of any one of claims 1 to 12, wherein said amino acid mutations in said second CH3 domain sequence further comprise the amino acid substitution L6007F. (a) the amino acid mutations in the first CH3 domain sequence further comprise the amino acid substitution H6005Y; and / or (b) the amino acid mutations in the second CH3 domain sequence further comprise the amino acid substitution H6005Y. An IgA HetFc construct according to any one of claims 1 to 13.

15. 8. The IgA HetFc construct of any one of claims 1 to 7, wherein the modified CH3 domain comprises two amino acid substitutions that introduce one disulfide bond-forming cysteine ​​residue into the modified CH3 domain or four amino acid substitutions that introduce two disulfide bond-forming cysteine ​​residues into the modified CH3 domain.

16. The amino acid substitution introducing a cysteine ​​residue (a) comprising the mutation H6005C in one CH3 domain sequence and the mutation P6010C in the other CH3 domain sequence; and / or (b) comprising the mutations H6005C and P6010C in one CH3 domain sequence and the mutations P6010C and H6005C in the other CH3 domain sequence; 16. The IgA Het Fc construct of claim 15.

17. 1. An IgA heterodimeric Fc (IgA HetFc) construct comprising a first Fc polypeptide comprising a first CH3 domain sequence and a second Fc polypeptide comprising a second CH3 domain sequence, said first and second CH3 domain sequences forming a modified CH3 domain, the first and second CH3 domain sequences comprise amino acid mutations that promote the formation of a heterodimeric Fc over a homodimeric Fc; (a) the amino acid mutations in the first CH3 domain sequence comprise the amino acid substitutions: A6085YY and T6086L, and the amino acid mutations in the second CH3 domain sequence comprise the amino acid substitutions: L6079T, W6081L, and I6088L; or (b) the amino acid mutations in the first CH3 domain sequence comprise the amino acid substitutions: A6085YY and T6086Y, and the amino acid mutations in the second CH3 domain sequence comprise the amino acid substitutions: L6079T, W6081L, and I6088L; or (c) the amino acid mutations in the first CH3 domain sequence comprise the amino acid substitutions: A6085YF and T6086Y, and the amino acid mutations in the second CH3 domain sequence comprise the amino acid substitutions: L6079V, W6081L, and I6088L; or (d) the amino acid mutations in the first CH3 domain sequence comprise the amino acid substitutions: A6085YF and T6086Y, and the amino acid mutations in the second CH3 domain sequence comprise the amino acid substitutions: L6079V, W6081T, and I6088L; or (e) the amino acid mutations in the first CH3 domain sequence comprise the amino acid substitutions: T6022V, A6085YF, and T6086Y, and the amino acid mutations in the second CH3 domain sequence comprise the amino acid substitutions: L6079V, W6081T, and I6088L; or (f) the amino acid mutations in the first CH3 domain sequence comprise the amino acid substitutions: T6022L, A6085YF, and T6086Y, and the amino acid mutations in the second CH3 domain sequence comprise the amino acid substitutions: L6079V, W6081T, and I6088L; or (g) the amino acid mutations in the first CH3 domain sequence comprise the amino acid substitutions: T6022I, A6085YF, and T6086Y, and the amino acid mutations in the second CH3 domain sequence comprise the amino acid substitutions: L6079V, W6081T, and I6088L; or (h) the amino acid mutations in the first CH3 domain sequence comprise the amino acid substitutions: A6085YF and T6086Y, and the amino acid mutations in the second CH3 domain sequence comprise the amino acid substitutions: L6007F, L6079V, W6081T, and I6088L; or (i) the amino acid mutations in the first CH3 domain sequence comprise the amino acid substitutions: H6005Y, A6085YF, and T6086Y, and the amino acid mutations in the second CH3 domain sequence comprise the amino acid substitutions: H6005Y, L6079V, W6081T, and I6088L; or (j) the amino acid mutations in the first CH3 domain sequence comprise the amino acid substitutions: H6005C, A6085YF, and T6086Y, and the amino acid mutations in the second CH3 domain sequence comprise the amino acid substitutions: P6010C, L6079V, W6081T, and I6088L; or (k) the amino acid mutations in the first CH3 domain sequence comprise the amino acid substitutions: P6010C, A6085YF, and T6086Y, and the amino acid mutations in the second CH3 domain sequence comprise the amino acid substitutions: H6005C, L6079V, W6081T, and I6088L; or (l) the amino acid mutations in the first CH3 domain sequence comprise the amino acid substitutions: H6005C, P6010C, A6085YF, and T6086Y, and the amino acid mutations in the second CH3 domain sequence comprise the amino acid substitutions: H6005C, P6010C, L6079V, W6081T, and I6088L; The numbering of amino acid positions is according to the IMGT numbering system. The IgA heterodimeric Fc (IgA HetFc) construct.

18. 1. An IgA heterodimeric Fc (IgA HetFc) construct comprising a first Fc polypeptide comprising a first CH3 domain sequence and a second Fc polypeptide comprising a second CH3 domain sequence, said first and second CH3 domain sequences forming a modified CH3 domain, the first and second CH3 domain sequences comprise amino acid mutations that promote the formation of a heterodimeric Fc over a homodimeric Fc; (a) the amino acid mutations in the first CH3 domain sequence comprise the amino acid substitutions: A6085YF and T6086Y, and the amino acid mutations in the second CH3 domain sequence comprise the amino acid substitutions: L6079V, W6081T, and I6088L; or (b) the amino acid mutations in the first CH3 domain sequence comprise the amino acid substitutions: A6085YF and T6086Y, and the amino acid mutations in the second CH3 domain sequence comprise the amino acid substitutions: L6007F, L6079V, W6081T, and I6088L; or (c) the amino acid mutations in the first CH3 domain sequence comprise the amino acid substitutions: H6005Y, A6085YF, and T6086Y, and the amino acid mutations in the second CH3 domain sequence comprise the amino acid substitutions: H6005Y, L6079V, W6081T, and I6088L; the heterodimeric Fc is formed with a purity of 70% or greater; the modified CH3 domain has a melting temperature (Tm) of 60° C. or greater; The numbering of amino acid positions is according to the IMGT numbering system. The IgA heterodimeric Fc (IgA HetFc) construct.

19. 19. The IgA HetFc construct of claim 18, wherein the modified CH3 domain has a melting temperature (Tm) of the corresponding wild-type IgA CH3 domain ± 5°C.

20. 20. The IgA HetFc construct of any one of claims 1 to 19, further comprising one or more target binding domains.

21. 21. The IgA HetFc construct of claim 20, wherein the one or more target binding domains are antigen-binding antibody fragments.

22. 22. The IgA HetFc construct of claim 21, wherein each of the one or more antigen-binding antibody fragments is independently selected from a Fab and a scFv.

23. 23. The IgA HetFc construct of any one of claims 1 to 22, wherein the modified IgA CH3 domain comprises an α-tailpiece.

24. A composite comprising an IgA HetFc construct according to any one of claims 1 to 23 and one or more therapeutic, diagnostic or labelling agents.

25. An IgA HetFc multimer comprising two or more IgA HetFc constructs according to any one of claims 1 to 23 and a J chain, two of the IgA HetFc constructs are linked by the J chain; The IgA HetFc multimer.

26. A pharmaceutical composition comprising an IgA HetFc construct according to any one of claims 1 to 23, or a conjugate according to claim 24, or an IgA HetFc multimer according to claim 25, and a pharma- ceutically acceptable carrier or diluent.

27. 24. An isolated polynucleotide or set of polynucleotides encoding the IgA HetFc construct of any one of claims 1 to 23.

28. A vector set or set of vectors comprising one or more polynucleotides encoding the IgA HetFc of any one of claims 1 to 23.

29. A host cell comprising one or more polynucleotides encoding the IgA HetFc of any one of claims 1 to 23.

30. A method for producing an IgA HetFc construct according to any one of claims 1 to 23, comprising the steps of: transfecting a host cell with one or more polynucleotides encoding said IgA HetFc construct; Culturing the host cells under conditions suitable for expression of the IgA HetFc construct. The method comprising:

31. 26. A method for producing an IgA HetFc multimer according to claim 25, comprising: transfecting a host cell with one or more polynucleotides encoding the IgA HetFc construct of claim 23 and a polynucleotide encoding a J chain; Culturing the host cell under conditions suitable for expression of the IgA HetFc construct and the J chain. The method comprising:

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