Humanized anti-human CD89 antibodies and uses thereof

Abstract

This disclosure describes humanized antibodies that can bind to the extracellular portion of human CD89 (human FcaRI) on human CD89-expressing cells and prevent human IgA from binding to human CD89 when the antibodies are bound to the cells. This disclosure also describes the use of such antibodies in combating certain diseases.

Description

[Technical Field]

[0001] The present invention relates to the field of antibodies and uses of such antibodies. The antibodies are particularly useful for preventing the binding of human IgA to human CD89. The antibodies are particularly useful for targeting human effector cells. [Background technology]

[0002] Receptors for the Fc portion of immunoglobulins (FcRs) are present on various types of immune cells and provide a link between the humoral and cellular branches of the immune system. Interaction between antibodies and FcRs provides cells expressing FcRs with antigen-specific recognition properties. The interaction can initiate a variety of responses, including endocytosis, phagocytosis, transcytosis, exocytosis, superoxide generation, antibody-dependent cellular cytotoxicity, and release of cytokine inflammatory mediators. Immunoglobulin receptors and their role in the immune response have been extensively investigated.

[0003] Currently, five classes of antibody constant regions are recognized: IgG, IgA, IgD, IgE, and IgM. IgA plays a role in the innate immune system. It is involved in preventing the invasion of microorganisms and foreign proteins into mucosal surfaces. IgA can also neutralize toxins and infectious organisms. Furthermore, IgA has anti-inflammatory properties and can inhibit functions such as IgG-induced cytokine release and phagocytosis.

[0004] Different classes of antibody constant regions can interact with different Fc receptors. An Fc receptor capable of binding IgA is CD89. Human CD89 can bind to the heavy chain constant regions of human IgA1 and human IgA2. CD89 is a glycosylated transmembrane receptor and is also known as FcαRI. CD89 has two extracellular domains, EC1 and EC2, a transmembrane domain, and an intracellular domain. The interaction between IgA and CD89 is mediated by the EC1 extracellular domain. For the reference sequence, see NP_001991.1 (Immunoglobulin alpha Fc receptor isoform precursor). Reference is made solely for the purpose of identifying the human CD89 gene / protein. It is not intended to limit the human CD89 described herein to the specific sequence of the database entry. Natural variants of human CD89 are within the scope of the present invention. Recombinant human CD89 is also within the scope of the present invention if it is capable of binding IgA and the antibodies described herein. CD89 is present on the cell surface of myeloid cells, including neutrophils, eosinophils, and most monocytes and macrophages. However, the receptor is not expressed on mast cells and intestinal macrophages. CD89 expression has been found to be constitutive and independent of the presence of IgA ligands.

[0005] CD89 can interact with monomeric IgA, polymeric IgA, and IgA complexes. Monomeric IgA binds to CD89 transiently, whereas polymeric IgA and IgA complexes are thought to bind avidly to CD89. CD89 can play a role in both pro- and anti-inflammatory responses. To respond to IgA binding, the receptor must associate with another factor, most likely a dimeric form of the FcRγ chain. Ligand binding to CD89 can initiate various biological processes. Cellular functions promoted by ligand binding to CD89 also depend on the associated FcRγ chain.

[0006] Cross-linking of CD89 receptors on cells can be achieved by binding of IgA antibodies, IgA immune complexes, or anti-CD89 antibodies. Cross-linking triggers an immune response that can have positive or negative effects, depending on the context, particularly the binding. CD89-specific antibodies can be used as tools / drugs to regulate immune responses. For example, individuals suffering from chronic inflammatory diseases could benefit from methods to inhibit immune responses. CD89 receptors are expressed on various cell types, including neutrophils, eosinophils, monocytes, and macrophages. In particular, individuals suffering from diseases associated with CD89-expressing cells and / or IgA-related diseases could benefit from treatment with CD89 antibodies. Currently, there are no known treatments for diseases involving neutrophils. Summary of the Invention

[0007] In one aspect, the disclosure provides a humanized anti-human CD89 antibody capable of binding to the extracellular portion of human CD89, comprising the amino acid sequence: EVQLLESGGG LVQPGGSLRL SCAASGLTFS SYGMSWVRQA PGKGLEX1VX2TIX3GX4GDITYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDY DYDYAMDYWG QGTLVTVSS wherein: X1 is L or W X2 is A or S X3 is N, S X4 is Q, T, or N; a heavy chain variable region that contains 0, 1, 2, or 3 amino acid variations, insertions, deletions, substitutions, additions, or a combination thereof, relative to the designated amino acid sequence at positions other than X1, X2, X3, and X4; Amino acid sequence: DIQMTQSPSS LSASVGDRVT ITCRASQDII NYLNWYQQKP GKZ1Z2KLLIYY TSRLHSGVPS RFSGSGSGTDZ3TLTISSLQP EDFATYZ4CQQ GKTLPYTFGQ GTKLEIK a light chain variable region comprising: Z1 is A or T Z2 is V or P Z3 is Y or F Z4 is Y or F; and a light chain variable region, wherein the light chain variable region contains 0, 1, 2, or 3 amino acid variations, insertions, deletions, substitutions, additions, or a combination thereof, at positions other than Z1, Z2, Z3, and Z4, relative to the designated amino acid sequence.

[0008] In a preferred embodiment, X1 is L, X2 is A, X3 is S, X4 is T, Z1 is A, Z2 is V, Z3 is Y, and Z4 is Y.

[0009] In another preferred embodiment, X1 is L, X2 is A, X3 is S, X4 is Q, Z1 is A, Z2 is V, Z3 is Y, and Z4 is Y.

[0010] In one aspect, the disclosure provides an antibody capable of binding to the extracellular portion of human CD89, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 138 or 139 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain having the amino acid sequence of SEQ ID NO: 122 with 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid insertions, deletions, substitutions, or additions.

[0011] The antibodies described herein can bind to the extracellular portion of human CD89 on human CD89-expressing cells and, when bound to the cells, can prevent the binding of human IgA to human CD89.

[0012] In a further aspect, the antibodies described herein can bind to the extracellular portion of human CD89 on human CD89-expressing HEK293F cells. A Budapest Treaty deposit of human CD89-expressing HEK293F cells was deposited by Depositor Swiss Pharma International AG, Waldmannstr. 8, 8001 Zurich, Switzerland, under reference to 293F CG89 clone 2 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Braunschweig, Germany, and has the accession number: DSM ACC3341.

[0013] In one aspect, the present disclosure provides nucleic acid molecule(s) encoding the antibodies disclosed herein or antigen-binding fragments disclosed herein. Nucleic acids encoding the variable regions disclosed herein, preferably the heavy and / or light chain variable regions of the humanized anti-human CD89 antibodies described herein, are also provided.

[0014] In one aspect, the present disclosure provides a vector comprising a nucleic acid molecule described herein. In one aspect, the present disclosure provides a cell (host cell) comprising an antibody, nucleic acid molecule(s), and / or vector disclosed herein. Preferably, the host cell is a mammalian cell, an insect cell, a plant cell, a bacterial cell, or a yeast cell. More preferably, the host cell is a human cell. Preferably, the host cell is a hybridoma cell, a Chinese hamster ovary (CHO) cell, an NSO cell, or a PER-C6™ cell (European Collection of Cell Cultures (ECACC) 96022940, WO 1997 / 000326, Pau et al., 2001. Vaccine 19:2716-2721).

[0015] In one aspect, the disclosure provides a method for producing an antibody disclosed herein. The method preferably includes harvesting the antibody. Preferably, the antibody is produced using and harvested from a cell. Preferably, the cell is a hybridoma cell, a Chinese Hamster Ovary (CHO) cell, an NSO cell, or a PER-C6™ cell. In another preferred embodiment, the antibody is produced synthetically.

[0016] One aspect of the present disclosure provides a pharmaceutical composition comprising the disclosed antibody or antigen-binding fragment thereof, nucleic acid, and / or cell. Preferably, the composition or antibody or antigen-binding fragment thereof disclosed herein is for use in the manufacture of a medicament. Preferably, the medicament is for the treatment or prevention of a chronic inflammatory disease.

[0017] In one aspect, the present disclosure provides a method for treating a chronic inflammatory disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof, nucleic acid molecule or vector, or pharmaceutical composition disclosed herein.

[0018] In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof for use in treating a chronic inflammatory disease.

[0019] In one aspect, the present disclosure provides an antibody that can bind to or binds to the extracellular portion of human CD89 on human CD89-expressing cells, and that, when bound to the cells, can prevent or prevent human IgA from binding to human CD89, and does not reduce cell viability of the cells by more than 60% after overnight incubation at 37° C. Preferably, the change in viability or expression after overnight incubation at 37° C. referred to herein refers to the change relative to a control value when incubated under the same conditions but without the antibody. The viability experiment is preferably performed in a suitable medium, such as a serum-free medium.

[0020] In a further aspect, the present disclosure provides an antibody that can bind to the extracellular portion of human CD89 on human CD89-expressing cells, and that when bound to the cells, can prevent human IgA from binding to human CD89, and that does not increase phosphatidylserine expression in the cells by more than 20% after overnight incubation at 37°C compared to the same cells incubated under the same conditions but without the antibody.

[0021] In one aspect, the present disclosure provides an antibody, which can bind to the extracellular portion of human CD89 on human CD89-expressing cells and can prevent human IgA from binding to human CD89 when bound to the cells at 37°C in the absence of NaN3, and cannot displace monomeric human IgA or heat-aggregated IgA when bound to the cells at 4°C in the presence of NaN3.

[0022] In one aspect, the present disclosure provides an antibody that can bind to the extracellular portion of human CD89 on a human CD89-expressing cell and, when bound to the cell, can prevent human IgA from binding to human CD89, and binds 20% or less to a recombinant human CD89 molecule in which amino acids 22-46 of human CD89 have been replaced with amino acids 22-46 of cynomolgus CD89.

[0023] In one aspect, the present disclosure provides an antibody that can bind to the extracellular portion of human CD89 on a human CD89-expressing cell and, when bound to the cell, can prevent human IgA from binding to human CD89, and binds 20% or less to a chimeric human CD89 molecule in which amino acids 47-71 of human CD89 have been replaced with amino acids 47-71 of cynomolgus CD89.

[0024] In one aspect, the present disclosure provides an antibody that can bind to the extracellular portion of human CD89 on a human CD89-expressing cell and, when bound to the cell, can prevent human IgA from binding to human CD89, and binds 20% or less to a chimeric human CD89 molecule in which amino acids 72-96 of human CD89 have been replaced with amino acids 72-96 of cynomolgus CD89.

[0025] In one aspect, the present disclosure provides an antibody that can bind to the extracellular portion of human CD89 on a human CD89-expressing cell and, when bound to the cell, can prevent binding of human IgA to human CD89, and has 20% or less reduction in binding to a chimeric human CD89 molecule in which amino acids 97-121 of human CD89 are replaced with amino acids 97-121 of cynomolgus CD89.

[0026] In one aspect, the disclosure provides an antibody that can bind to the extracellular portion of human CD89 on a human CD89-expressing cell and, when the antibody is bound to the cell, can prevent human IgA from binding to human CD89, and binds 20% or less to a chimeric human CD89 molecule in which amino acids 58, 59, 73, 74, 76, 106, and 107 of human CD89 have been replaced with amino acids 58, 59, 73, 74, 76, 106, and 107, respectively, of cynomolgus monkey CD89.

[0027] In one aspect, the present disclosure provides an antibody capable of binding to the extracellular portion of human CD89, comprising a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 29-31 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 32-34 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions.

[0028] In a further aspect, the disclosure provides an antibody capable of binding to the extracellular portion of human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 27 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 28 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions.

[0029] In one aspect, the present disclosure provides an antibody capable of binding to the extracellular portion of human CD89, comprising a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 45 to 47, with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 48 to 50, with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions.

[0030] In a further aspect, the disclosure provides an antibody capable of binding to the extracellular portion of human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 43 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 44 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions.

[0031] In one aspect, the present disclosure provides an antibody capable of binding to the extracellular portion of human CD89, comprising a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 69-71 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 72-74 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions.

[0032] In a further aspect, the disclosure provides an antibody capable of binding to the extracellular portion of human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 67 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 68 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions.

[0033] In one aspect, the present disclosure provides an antibody capable of binding to the extracellular portion of human CD89, comprising a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 77 to 79 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 80 to 82 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions.

[0034] In a further aspect, the disclosure provides an antibody capable of binding to the extracellular portion of human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 75 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 76 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions.

[0035] In one aspect, the present disclosure provides an antibody capable of binding to the extracellular portion of human CD89, comprising a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 53 to 55 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 56 to 58 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions.

[0036] In a further aspect, the disclosure provides an antibody capable of binding to the extracellular portion of human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 51 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 52 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions.

[0037] In one aspect, the present disclosure provides an antibody capable of binding to the extracellular portion of human CD89, preferably the antibody is capable of binding to the EC1 extracellular domain of CD89. The present disclosure may be configured as follows. [Section 1] 1. A humanized anti-human CD89 antibody capable of binding to the extracellular portion of human CD89, comprising the amino acid sequence: EVQLLESGGG LVQPGGSLRL SCAASGLTFS SYGMSWVRQA PGKGLEX 1 VX 2 T IX 3 GX 4 GDITYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDY DYDYAMDYWG QGTLVTVSS wherein: X 1 is L or W X 2 is A or S X 3 But N, S X 4 is Q, T, or N, the heavy chain variable region is X 1 、X 2 、X 3 , and X 4 a heavy chain variable region comprising 0, 1, 2, or 3 amino acid variations, insertions, deletions, substitutions, additions, or combinations thereof, with respect to the indicated amino acid sequence at positions other than Amino acid sequence: DIQMTQSPSS LSASVGDRVT ITCRASQDII NYLNWYQQKP GKZ 1 Z 2 KLLIYY TSRLHSGVPS RFSGSGSGTD Z 3 TLTISSLQP EDFATYZ 4 CQQ GKTLPYTFGQ GTKLEIK a light chain variable region comprising: Z 1 is A or T Z 2 is V or P Z 3 is Y or F Z4 is Y or F, the light chain variable region is Z 1 、Z 2 、Z 3 and Z 4 and a light chain variable region that contains 0, 1, 2, or 3 amino acid variations, insertions, deletions, substitutions, additions, or combinations thereof, with respect to the indicated amino acid sequence at positions other than the indicated amino acid sequence. [Section 2] Item 1. The humanized anti-human CD89 antibody according to Item 1, wherein the heavy chain variable region has been deamidated and repaired. [Section 3] the heavy chain variable region has the amino acid sequence: EVQLLESGGG LVQPGGSLRL SCAASGLTFS SYGMSWVRQA PGKGLELVAT ISGX 4 GDITYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDY DYDYAMDYWG QGTLVTVSS, where X 4 is Q, T, or N, 3. The humanized anti-human CD89 antibody of paragraph 1 or 2, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 122, which contains 0, 1, 2, or 3 amino acid variations, insertions, deletions, substitutions, additions, or a combination thereof, relative to the indicated amino acid sequence. [Section 4] The antibody according to any one of Items 1 to 3, wherein the antibody has an IgG1 or IgG4 isotype. [Section 5] The humanized anti-human CD89 antibody of any one of Aspects 1 to 4, wherein the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 142 or 143, which has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain having the amino acid sequence of SEQ ID NO: 136, which has 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid insertions, deletions, substitutions, or additions. [Section 6] The antibody according to any one of Items 1 to 5, wherein the antibody has a higher affinity for the extracellular portion of human CD89 compared to a chimeric antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 94 and a light chain having the amino acid sequence of SEQ ID NO: 99. [Section 7] The antibody according to any one of Items 1 to 6, wherein the antibody, when bound to the cells, is capable of binding to the extracellular portion of human CD89 on human CD89-expressing cells and preventing the binding of human IgA to human CD89. [Section 8] The antibody according to item 7, wherein the cells are human CD89-expressing HEK293F cells (deposited under the number DSM ACC3341). [Section 9] Item 9. One or more nucleic acid molecules encoding the antibody or antigen (human CD89)-binding fragment thereof according to any one of Items 1 to 8. [Section 10] A nucleic acid molecule encoding the variable region of the antibody according to any one of items 1 to 8. [Section 11] A vector comprising the nucleic acid molecule of Item 9 or 10. [Section 12] A cell comprising the antibody according to any one of Items 1 to 8, one or more nucleic acid molecules according to any one of Items 9 and 10, and / or the vector according to Item 11, wherein the cell is a mammalian cell, an insect cell, a plant cell, a bacterial cell, or a yeast cell, and more preferably a human cell. [Section 13] A method for producing the antibody according to any one of Items 1 to 8, the method comprising harvesting (recovering) the antibody, wherein the antibody is preferably produced using and harvested (recovered) from cells, and the cells are preferably hybridoma cells, Chinese hamster ovary (CHO) cells, NSO cells, or PER-C6 (trademark) cells. [Section 14] A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of Items 1 to 8, one or more nucleic acid molecules according to any one of Items 9 and 10, and / or the vector according to Item 11. [Section 15] The antibody or antigen-binding fragment thereof according to any one of Items 1 to 8, one or more nucleic acid molecules according to any one of Items 9 and 10, and / or the vector according to Item 11, for use in the treatment or prevention of a chronic inflammatory disease. [Section 16] A method for treating a chronic inflammatory disease in a subject, the method comprising administering to the subject in need of treatment for the chronic inflammatory disease a therapeutically effective amount of the antibody or antigen-binding fragment thereof described in any one of Items 1 to 8, one or more nucleic acid molecules described in any one of Items 9 and 10, and / or the vector described in Item 11. [Brief explanation of the drawings]

[0038] [Figure 1] Binding of mouse anti-human CD89 antibody (supernatant) to membrane-bound full-length human CD89 on HEK293F cells using flow cytometry. The dashed line represents background (i.e., no binding of mouse anti-human CD89 antibody). Mean ± SD (n=2) is shown. [Figure 2] Effect of mouse anti-human CD89 antibody (supernatant) on the binding of serum human IgA to rhuCD89 on HEK293F cells (A, ELISA) or membrane-bound human CD89 (B, FACS). Mean ± SD (n=2) is shown. [Figure 3] Binding characteristics of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies to rhuCD89 on HEK293F cells (A, ELISA) or membrane-bound human CD89 (B, FACS). Mean ± SD (n=2) is shown. [Figure 4A] Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies on the binding of serum human IgA to rhuCD89 on HEK293F cells (A, ELISA) or membrane-bound human CD89 (B, FACS). Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies on the binding of secreted human IgA to membrane-bound human CD89 on HEK293F cells (C). Mean ± SD (n=2) is shown. [Figure 4B] Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies on the binding of serum human IgA to rhuCD89 on HEK293F cells (A, ELISA) or membrane-bound human CD89 (B, FACS). Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies on the binding of secreted human IgA to membrane-bound human CD89 on HEK293F cells (C). Mean ± SD (n=2) is shown. [Figure 4C]Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies on the binding of serum human IgA to rhuCD89 on HEK293F cells (A, ELISA) or membrane-bound human CD89 (B, FACS). Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies on the binding of secreted human IgA to membrane-bound human CD89 on HEK293F cells (C). Mean ± SD (n=2) is shown. [Figure 5A] The effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibody (at 10 μg / mL) on (A, C) preventing the binding of non-aggregated or heat-aggregated serum human IgA to membrane-bound human CD89-expressing HEK293F cells, (B, D) replacing non-aggregated or heat-aggregated serum human IgA previously saturated with the cells, and (E) inducing cell death (cell viability and phosphatidylserine expression) in the cells. The effect of CD89 / IgA-blocking mouse anti-human CD89 antibody was investigated (A, B) under metabolically inactive conditions (i.e., in the presence of NaN3 at cold ambient temperature (4°C)) and (C, D, E) under metabolically active conditions (i.e., in the absence of NaN3 at physiological ambient temperature (37°C)). Mean ± SD (n = 2) is shown. [Figure 5B] The effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibody (at 10 μg / mL) on (A, C) preventing the binding of non-aggregated or heat-aggregated serum human IgA to membrane-bound human CD89-expressing HEK293F cells, (B, D) replacing non-aggregated or heat-aggregated serum human IgA previously saturated with the cells, and (E) inducing cell death (cell viability and phosphatidylserine expression) in the cells. The effect of CD89 / IgA-blocking mouse anti-human CD89 antibody was investigated (A, B) under metabolically inactive conditions (i.e., in the presence of NaN3 at cold ambient temperature (4°C)) and (C, D, E) under metabolically active conditions (i.e., in the absence of NaN3 at physiological ambient temperature (37°C)). Mean ± SD (n = 2) is shown. [Figure 5C]The effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibody (at 10 μg / mL) on (A, C) preventing the binding of non-aggregated or heat-aggregated serum human IgA to membrane-bound human CD89-expressing HEK293F cells, (B, D) replacing non-aggregated or heat-aggregated serum human IgA previously saturated with the cells, and (E) inducing cell death (cell viability and phosphatidylserine expression) in the cells. The effect of CD89 / IgA-blocking mouse anti-human CD89 antibody was investigated (A, B) under metabolically inactive conditions (i.e., in the presence of NaN3 at cold ambient temperature (4°C)) and (C, D, E) under metabolically active conditions (i.e., in the absence of NaN3 at physiological ambient temperature (37°C)). Mean ± SD (n = 2) is shown. [Figure 5D] The effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibody (at 10 μg / mL) on (A, C) preventing the binding of non-aggregated or heat-aggregated serum human IgA to membrane-bound human CD89-expressing HEK293F cells, (B, D) replacing non-aggregated or heat-aggregated serum human IgA previously saturated with the cells, and (E) inducing cell death (cell viability and phosphatidylserine expression) in the cells. The effect of CD89 / IgA-blocking mouse anti-human CD89 antibody was investigated (A, B) under metabolically inactive conditions (i.e., in the presence of NaN3 at cold ambient temperature (4°C)) and (C, D, E) under metabolically active conditions (i.e., in the absence of NaN3 at physiological ambient temperature (37°C)). Mean ± SD (n = 2) is shown. [Figure 5E] The effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibody (at 10 μg / mL) on (A, C) preventing the binding of non-aggregated or heat-aggregated serum human IgA to membrane-bound human CD89-expressing HEK293F cells, (B, D) replacing non-aggregated or heat-aggregated serum human IgA previously saturated with the cells, and (E) inducing cell death (cell viability and phosphatidylserine expression) in the cells. The effect of CD89 / IgA-blocking mouse anti-human CD89 antibody was investigated (A, B) under metabolically inactive conditions (i.e., in the presence of NaN3 at cold ambient temperature (4°C)) and (C, D, E) under metabolically active conditions (i.e., in the absence of NaN3 at physiological ambient temperature (37°C)). Mean ± SD (n = 2) is shown. [Figure 6]Effect of prototypic commercial mouse anti-human CD89 antibodies clone MIP8a, clone A59, and clone A3 on preventing / inhibiting binding of non-aggregated (A) or heat-aggregated (B) serum human IgA to membrane-bound human CD89 on HEK293F cells. Mean ± SD (n=2) shown. [Figure 7] Binding of purified CD89 / IgA-blocking mouse anti-human CD89 antibody (at 10 μg / mL) to membrane-bound human CD89 on ex vivo human neutrophilic granulocytes (A, mean ± SD from five different healthy donors), HEK293F cells (B, mean ± SD (n=2)), and monocytic U937 cells (C, mean ± SD (n=2)). [Figure 8-1] Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies on the phagocytosis of serum human IgA-coated latex beads by human CD89-expressing ex vivo primary human neutrophil granulocytes. The dashed line represents the phagocytosis of IgA-coated beads alone (i.e., no antibody added). Mean ± SD (n = 2) is shown from three different healthy donors (1, 2, and 3). [Figure 8-2] Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies on the phagocytosis of serum human IgA-coated latex beads by human CD89-expressing ex vivo primary human neutrophil granulocytes. The dashed line represents the phagocytosis of IgA-coated beads alone (i.e., no antibody added). Mean ± SD (n = 2) is shown from three different healthy donors (1, 2, and 3). [Figure 8-3] Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies on the phagocytosis of serum human IgA-coated latex beads by human CD89-expressing ex vivo primary human neutrophil granulocytes. The dashed line represents the phagocytosis of IgA-coated beads alone (i.e., no antibody added). Mean ± SD (n = 2) is shown from three different healthy donors (1, 2, and 3). [Figure 9A-1]Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibody (at 20 μg / mL) on serum human IgA-coated Sepharose bead-induced two-dimensional migration of human CD89-expressing ex vivo primary human neutrophilic granulocytes (A, mean ± SD (n = 3)), chemotactic activity from the corresponding supernatants of the cells, and chemoattractant LTB4 levels from the corresponding supernatants of the cells (C, n = 1). Dashed lines represent (A) two-dimensional granulocyte migration, (B) granulocyte chemotaxis, and (C) granulocyte LTB4 production induced by IgA-coated Sepharose beads alone (i.e., without the addition of antibodies). Data for (A), (B), and (C) are shown from three healthy donors (1, 2, and 3). ND = not determined. [Figure 9A-2] Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibody (at 20 μg / mL) on serum human IgA-coated Sepharose bead-induced two-dimensional migration of human CD89-expressing ex vivo primary human neutrophilic granulocytes (A, mean ± SD (n = 3)), chemotactic activity from the corresponding supernatants of the cells, and chemoattractant LTB4 levels from the corresponding supernatants of the cells (C, n = 1). Dashed lines represent (A) two-dimensional granulocyte migration, (B) granulocyte chemotaxis, and (C) granulocyte LTB4 production induced by IgA-coated Sepharose beads alone (i.e., without the addition of antibodies). Data for (A), (B), and (C) are shown from three healthy donors (1, 2, and 3). ND = not determined. [Figure 9A-3]Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibody (at 20 μg / mL) on serum human IgA-coated Sepharose bead-induced two-dimensional migration of human CD89-expressing ex vivo primary human neutrophilic granulocytes (A, mean ± SD (n = 3)), chemotactic activity from the corresponding supernatants of the cells, and chemoattractant LTB4 levels from the corresponding supernatants of the cells (C, n = 1). Dashed lines represent (A) two-dimensional granulocyte migration, (B) granulocyte chemotaxis, and (C) granulocyte LTB4 production induced by IgA-coated Sepharose beads alone (i.e., without the addition of antibodies). Data for (A), (B), and (C) are shown from three healthy donors (1, 2, and 3). ND = not determined. [Figure 9B-1] Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibody (at 20 μg / mL) on serum human IgA-coated Sepharose bead-induced two-dimensional migration of human CD89-expressing ex vivo primary human neutrophilic granulocytes (A, mean ± SD (n = 3)), chemotactic activity from the corresponding supernatants of the cells, and chemoattractant LTB4 levels from the corresponding supernatants of the cells (C, n = 1). Dashed lines represent (A) two-dimensional granulocyte migration, (B) granulocyte chemotaxis, and (C) granulocyte LTB4 production induced by IgA-coated Sepharose beads alone (i.e., without the addition of antibodies). Data for (A), (B), and (C) are shown from three healthy donors (1, 2, and 3). ND = not determined. [Figure 9B-2]Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibody (at 20 μg / mL) on serum human IgA-coated Sepharose bead-induced two-dimensional migration of human CD89-expressing ex vivo primary human neutrophilic granulocytes (A, mean ± SD (n = 3)), chemotactic activity from the corresponding supernatants of the cells, and chemoattractant LTB4 levels from the corresponding supernatants of the cells (C, n = 1). Dashed lines represent (A) two-dimensional granulocyte migration, (B) granulocyte chemotaxis, and (C) granulocyte LTB4 production induced by IgA-coated Sepharose beads alone (i.e., without the addition of antibodies). Data for (A), (B), and (C) are shown from three healthy donors (1, 2, and 3). ND = not determined. [Figure 9B-3] Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibody (at 20 μg / mL) on serum human IgA-coated Sepharose bead-induced two-dimensional migration of human CD89-expressing ex vivo primary human neutrophilic granulocytes (A, mean ± SD (n = 3)), chemotactic activity from the corresponding supernatants of the cells, and chemoattractant LTB4 levels from the corresponding supernatants of the cells (C, n = 1). Dashed lines represent (A) two-dimensional granulocyte migration, (B) granulocyte chemotaxis, and (C) granulocyte LTB4 production induced by IgA-coated Sepharose beads alone (i.e., without the addition of antibodies). Data for (A), (B), and (C) are shown from three healthy donors (1, 2, and 3). ND = not determined. [Figure 9C-1]Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibody (at 20 μg / mL) on serum human IgA-coated Sepharose bead-induced two-dimensional migration of human CD89-expressing ex vivo primary human neutrophilic granulocytes (A, mean ± SD (n = 3)), chemotactic activity from the corresponding supernatants of the cells, and chemoattractant LTB4 levels from the corresponding supernatants of the cells (C, n = 1). Dashed lines represent (A) two-dimensional granulocyte migration, (B) granulocyte chemotaxis, and (C) granulocyte LTB4 production induced by IgA-coated Sepharose beads alone (i.e., without the addition of antibodies). Data for (A), (B), and (C) are shown from three healthy donors (1, 2, and 3). ND = not determined. [Figure 9C-2] Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibody (at 20 μg / mL) on serum human IgA-coated Sepharose bead-induced two-dimensional migration of human CD89-expressing ex vivo primary human neutrophilic granulocytes (A, mean ± SD (n = 3)), chemotactic activity from the corresponding supernatants of the cells, and chemoattractant LTB4 levels from the corresponding supernatants of the cells (C, n = 1). Dashed lines represent (A) two-dimensional granulocyte migration, (B) granulocyte chemotaxis, and (C) granulocyte LTB4 production induced by IgA-coated Sepharose beads alone (i.e., without the addition of antibodies). Data for (A), (B), and (C) are shown from three healthy donors (1, 2, and 3). ND = not determined. [Figure 9C-3]Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibody (at 20 μg / mL) on serum human IgA-coated Sepharose bead-induced two-dimensional migration of human CD89-expressing ex vivo primary human neutrophilic granulocytes (A, mean ± SD (n = 3)), chemotactic activity from the corresponding supernatants of the cells, and chemoattractant LTB4 levels from the corresponding supernatants of the cells (C, n = 1). Dashed lines represent (A) two-dimensional granulocyte migration, (B) granulocyte chemotaxis, and (C) granulocyte LTB4 production induced by IgA-coated Sepharose beads alone (i.e., without the addition of antibodies). Data for (A), (B), and (C) are shown from three healthy donors (1, 2, and 3). ND = not determined. [Figure 10A-1] (A) Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies on serum human IgA binding to human CD89-expressing ex vivo primary human neutrophil granulocytes and (B) their corresponding serum human IgA-mediated lactoferrin production (a degranulation marker) from the neutrophil granulocytes. The dashed lines represent (A) granulocyte binding and (B) their corresponding lactoferrin production induced by IgA-coated plates alone (i.e., without the addition of antibody). Mean ± SD (n=2) from three healthy donors (1, 2, and 3) in (A) and (B) are shown. [Figure 10A-2] (A) Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies on serum human IgA binding to human CD89-expressing ex vivo primary human neutrophil granulocytes and (B) their corresponding serum human IgA-mediated lactoferrin production (a degranulation marker) from the neutrophil granulocytes. The dashed lines represent (A) granulocyte binding and (B) their corresponding lactoferrin production induced by IgA-coated plates alone (i.e., without the addition of antibody). Mean ± SD (n=2) from three healthy donors (1, 2, and 3) in (A) and (B) are shown. [Figure 10A-3](A) Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies on serum human IgA binding to human CD89-expressing ex vivo primary human neutrophil granulocytes and (B) their corresponding serum human IgA-mediated lactoferrin production (a degranulation marker) from the neutrophil granulocytes. The dashed lines represent (A) granulocyte binding and (B) their corresponding lactoferrin production induced by IgA-coated plates alone (i.e., without the addition of antibody). Mean ± SD (n=2) from three healthy donors (1, 2, and 3) in (A) and (B) are shown. [Figure 10B-1] (A) Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies on serum human IgA binding to human CD89-expressing ex vivo primary human neutrophil granulocytes and (B) their corresponding serum human IgA-mediated lactoferrin production (a degranulation marker) from the neutrophil granulocytes. The dashed lines represent (A) granulocyte binding and (B) their corresponding lactoferrin production induced by IgA-coated plates alone (i.e., without the addition of antibody). Mean ± SD (n=2) from three healthy donors (1, 2, and 3) in (A) and (B) are shown. [Figure 10B-2] (A) Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies on serum human IgA binding to human CD89-expressing ex vivo primary human neutrophil granulocytes and (B) their corresponding serum human IgA-mediated lactoferrin production (a degranulation marker) from the neutrophil granulocytes. The dashed lines represent (A) granulocyte binding and (B) their corresponding lactoferrin production induced by IgA-coated plates alone (i.e., without the addition of antibody). Mean ± SD (n=2) from three healthy donors (1, 2, and 3) in (A) and (B) are shown. [Figure 10B-3](A) Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies on serum human IgA binding to human CD89-expressing ex vivo primary human neutrophil granulocytes and (B) their corresponding serum human IgA-mediated lactoferrin production (a degranulation marker) from the neutrophil granulocytes. The dashed lines represent (A) granulocyte binding and (B) their corresponding lactoferrin production induced by IgA-coated plates alone (i.e., without the addition of antibody). Mean ± SD (n=2) from three healthy donors (1, 2, and 3) in (A) and (B) are shown. [Figure 11] Cross-competition of unlabeled purified CD89 / IgA-blocking mouse anti-human CD89 antibodies (at 10 μg / mL) with PE-conjugated commercial mouse anti-CD89 antibody clone MIP8a (A, CD89 / IgA blocker), clone A59 (B, CD89 / IgA non-blocker), and clone A3 (C, CD89 / IgA non-blocker) for membrane-bound human CD89 on HEK293F cells. Shown are the mean ± SD (n = 2 at least). [Figure 12A] Detailed analysis of unlabeled purified CD89 / IgA-blocking mouse anti-human CD89 antibodies cross-competing with PE-conjugated commercial mouse anti-CD89 antibody clone MIP8a (A, CD89 / IgA blocker), clone A59 (B, CD89 / IgA non-blocker), and clone A3 (C, CD89 / IgA non-blocker) for membrane-bound human CD89 on HEK293F cells. Mean ± SD (n = 2 at least) is shown. [Figure 12B] Detailed analysis of unlabeled purified CD89 / IgA-blocking mouse anti-human CD89 antibodies cross-competing with PE-conjugated commercial mouse anti-CD89 antibody clone MIP8a (A, CD89 / IgA blocker), clone A59 (B, CD89 / IgA non-blocker), and clone A3 (C, CD89 / IgA non-blocker) for membrane-bound human CD89 on HEK293F cells. Mean ± SD (n = 2 at least) is shown. [Figure 12C]Detailed analysis of unlabeled purified CD89 / IgA-blocking mouse anti-human CD89 antibodies cross-competing with PE-conjugated commercial mouse anti-CD89 antibody clone MIP8a (A, CD89 / IgA blocker), clone A59 (B, CD89 / IgA non-blocker), and clone A3 (C, CD89 / IgA non-blocker) for membrane-bound human CD89 on HEK293F cells. Mean ± SD (n = 2 at least) is shown. [Figure 13A] (A) Schematic diagram of wild-type human CD89, wild-type bovine Fcγ2R, and the chimeric human / bovine FcR constructs derived therefrom. [Figure 13B] (B) Membrane-bound human full-length CD89 ("human EC1-EC2-CD89", i.e., [ka] ), membrane-bound chimeric human EC1-CD89 / bovine EC2-Fcγ2R ("human EC1-CD89", i.e., [ka] ), membrane-bound chimeric bovine EC1-Fcγ2R / human EC2-CD89 ("human EC2-CD89", i.e., [ka] ), and membrane-bound bovine full-length Fcγ2R ("bovine Fcγ2R", i.e., [ka] Binding of rabbit anti-human CD89 polyclonal antibody and non-agglutinated or heat-agglutinated serum human IgA to the antibody (n=1). [Figure 13C](C) Binding of purified CD89 / IgA-blocking mouse anti-human CD89 antibody to membrane-bound human full-length CD89 ("human EC1-EC2-CD89"), membrane-bound chimeric human EC1-CD89 / bovine EC2-Fcγ2R ("human EC1-CD89"), membrane-bound chimeric bovine EC1-Fcγ2R / human EC2-CD89 ("human EC2-CD89"), and membrane-bound bovine full-length Fcγ2R ("bovine Fcγ2R") on HEK293F cells (n=1). The gray dotted line represents background (i.e., no binding of mouse anti-human CD89 antibody). [Figure 14] (A) Binding of rabbit anti-human CD89 polyclonal antibody and non-aggregated or heat-aggregated serum human IgA to membrane-bound cynomolgus monkey full-length CD89. (B) Binding of purified CD89 / IgA-blocking mouse anti-human CD89 antibody to membrane-bound cynomolgus monkey full-length CD89. The gray dotted line represents background (i.e., no binding of mouse anti-human CD89 antibody). [Figure 15A] (A) Schematic diagram of wild-type human CD89, wild-type cynomolgus CD89, and the chimeric human / cynomolgus CD89 constructs derived therefrom. [Figure 15B] (B) Membrane-bound human full-length CD89 ("human EC1-CD89" i.e., [ka] ), membrane-bound chimeric human / cynomolgus CD89 hotspot ("ΔThr58, Gln59, ΔArg73, Arg74, Lys76, ΔHis106, Tyr107 human EC1-CD89", i.e., [ka] ), membrane-bound chimeric human / cynomolgus CD89-I ("ΔGln22-Lys46 human EC1-CD89", i.e., [ka] ), membrane-bound chimeric human / cynomolgus CD89-II ("ΔIle47-Ile71 human EC1-CD89", i.e., [ka] ), membrane-bound chimeric human / cynomolgus CD89-III ("ΔGly72-Gly96 human EC1-CD89", i.e., [ka] ), membrane-bound chimeric human / cynomolgus CD89-IV ("ΔArg97-Gly121 human EC1-CD89", i.e., [ka] ), and membrane-bound chimeric cynomolgus full-length CD89 ("cynomolgus EC1-CD89", i.e., [ka] Binding of rabbit anti-human CD89 polyclonal antibody and non-agglutinated or heat-agglutinated serum human IgA to the antibody (n=1). [Figure 15C] (C) Membrane-bound human full-length CD89 ("human EC1-CD89"), membrane-bound chimeric human / cynomolgus CD89 hotspot ("ΔThr58, Gln59, ΔArg73, Arg74, Lys76, ΔHis106, Tyr107 human EC1-CD89"), membrane-bound chimeric human / cynomolgus CD89-I ("ΔGln22-Lys46 human EC1-CD89"), membrane-bound chimeric human / cynomolgus CD89-II ("Δ Figure 1 shows binding of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies to membrane-bound chimeric human / cynomolgus CD89-III ("ΔGly72-Gly96 human EC1-CD89"), membrane-bound chimeric human / cynomolgus CD89-IV ("ΔArg97-Gly121 human EC1-CD89"), and membrane-bound chimeric cynomolgus full-length CD89 ("cynomolgus EC1-CD89") (n=1). The dashed gray line represents background (i.e., no binding of mouse anti-human CD89 antibody). [Figure 16]Binding characteristics of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibodies to rhuCD89 on HEK293F cells (A, ELISA) or membrane-bound human CD89 (B, FACS). Mean ± SD (n=2) is shown. [Figure 17A] Effect of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibodies on the binding of non-aggregated (A) or heat-aggregated (B) serum human IgA to membrane-bound human CD89 on HEK293F cells. Effect of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibodies on the binding of secreted human IgA to membrane-bound human CD89 on HEK293F cells (C). Mean ± SD (n=2) is shown. [Figure 17B] Effect of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibodies on the binding of non-aggregated (A) or heat-aggregated (B) serum human IgA to membrane-bound human CD89 on HEK293F cells. Effect of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibodies on the binding of secreted human IgA to membrane-bound human CD89 on HEK293F cells (C). Mean ± SD (n=2) is shown. [Figure 17C] Effect of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibodies on the binding of non-aggregated (A) or heat-aggregated (B) serum human IgA to membrane-bound human CD89 on HEK293F cells. Effect of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibodies on the binding of secreted human IgA to membrane-bound human CD89 on HEK293F cells (C). Mean ± SD (n=2) is shown. [Figure 18] Binding of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibodies (at 10 μg / mL) to membrane-bound human CD89 on ex vivo human neutrophil granulocytes (mean ± SD from three different healthy donors). [Figure 19-1]Effect of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibodies on the phagocytosis of serum human IgA-coated latex beads by human CD89-expressing ex vivo primary human neutrophil granulocytes. The dashed line represents the phagocytosis of IgA-coated beads alone (i.e., no antibody added). Mean ± SD (n=2) is shown from three different healthy donors (1, 2, and 3). [Figure 19-2] Effect of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibodies on the phagocytosis of serum human IgA-coated latex beads by human CD89-expressing ex vivo primary human neutrophil granulocytes. The dashed line represents the phagocytosis of IgA-coated beads alone (i.e., no antibody added). Mean ± SD (n=2) is shown from three different healthy donors (1, 2, and 3). [Figure 19-3] Effect of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibodies on the phagocytosis of serum human IgA-coated latex beads by human CD89-expressing ex vivo primary human neutrophil granulocytes. The dashed line represents the phagocytosis of IgA-coated beads alone (i.e., no antibody added). Mean ± SD (n=2) is shown from three different healthy donors (1, 2, and 3). [Figure 20A-1] Effect of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibody (at 20 μg / mL) on serum human IgA-coated Sepharose bead-induced two-dimensional migration of human CD89-expressing ex vivo primary human neutrophil granulocytes (A, mean ± SD (n = 3)), chemotactic activity from the corresponding supernatants of the cells (B, mean ± SD (n = 3)), and chemoattractant LTB4 levels from the corresponding supernatants of the cells (C, mean ± SD (n = 2)). Dashed lines represent (A) two-dimensional granulocyte migration, (B) granulocyte chemotaxis, and (C) granulocyte LTB4 production induced by IgA-coated Sepharose beads alone (i.e., without the addition of antibodies). Data are shown from two (B) to three ((A) and (C)) healthy donors (1, 2, and 3). [Figure 20A-2]Effect of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibody (at 20 μg / mL) on serum human IgA-coated Sepharose bead-induced two-dimensional migration of human CD89-expressing ex vivo primary human neutrophil granulocytes (A, mean ± SD (n = 3)), chemotactic activity from the corresponding supernatants of the cells (B, mean ± SD (n = 3)), and chemoattractant LTB4 levels from the corresponding supernatants of the cells (C, mean ± SD (n = 2)). Dashed lines represent (A) two-dimensional granulocyte migration, (B) granulocyte chemotaxis, and (C) granulocyte LTB4 production induced by IgA-coated Sepharose beads alone (i.e., without the addition of antibodies). Data are shown from two (B) to three ((A) and (C)) healthy donors (1, 2, and 3). [Figure 20A-3] Effect of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibody (at 20 μg / mL) on serum human IgA-coated Sepharose bead-induced two-dimensional migration of human CD89-expressing ex vivo primary human neutrophil granulocytes (A, mean ± SD (n = 3)), chemotactic activity from the corresponding supernatants of the cells (B, mean ± SD (n = 3)), and chemoattractant LTB4 levels from the corresponding supernatants of the cells (C, mean ± SD (n = 2)). Dashed lines represent (A) two-dimensional granulocyte migration, (B) granulocyte chemotaxis, and (C) granulocyte LTB4 production induced by IgA-coated Sepharose beads alone (i.e., without the addition of antibodies). Data are shown from two (B) to three ((A) and (C)) healthy donors (1, 2, and 3). [Figure 20B-1]Effect of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibody (at 20 μg / mL) on serum human IgA-coated Sepharose bead-induced two-dimensional migration of human CD89-expressing ex vivo primary human neutrophil granulocytes (A, mean ± SD (n = 3)), chemotactic activity from the corresponding supernatants of the cells (B, mean ± SD (n = 3)), and chemoattractant LTB4 levels from the corresponding supernatants of the cells (C, mean ± SD (n = 2)). Dashed lines represent (A) two-dimensional granulocyte migration, (B) granulocyte chemotaxis, and (C) granulocyte LTB4 production induced by IgA-coated Sepharose beads alone (i.e., without the addition of antibodies). Data are shown from two (B) to three ((A) and (C)) healthy donors (1, 2, and 3). [Figure 20B-2] Effect of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibody (at 20 μg / mL) on serum human IgA-coated Sepharose bead-induced two-dimensional migration of human CD89-expressing ex vivo primary human neutrophil granulocytes (A, mean ± SD (n = 3)), chemotactic activity from the corresponding supernatants of the cells (B, mean ± SD (n = 3)), and chemoattractant LTB4 levels from the corresponding supernatants of the cells (C, mean ± SD (n = 2)). Dashed lines represent (A) two-dimensional granulocyte migration, (B) granulocyte chemotaxis, and (C) granulocyte LTB4 production induced by IgA-coated Sepharose beads alone (i.e., without the addition of antibodies). Data are shown from two (B) to three ((A) and (C)) healthy donors (1, 2, and 3). [Figure 20C-1]Effect of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibody (at 20 μg / mL) on serum human IgA-coated Sepharose bead-induced two-dimensional migration of human CD89-expressing ex vivo primary human neutrophil granulocytes (A, mean ± SD (n = 3)), chemotactic activity from the corresponding supernatants of the cells (B, mean ± SD (n = 3)), and chemoattractant LTB4 levels from the corresponding supernatants of the cells (C, mean ± SD (n = 2)). Dashed lines represent (A) two-dimensional granulocyte migration, (B) granulocyte chemotaxis, and (C) granulocyte LTB4 production induced by IgA-coated Sepharose beads alone (i.e., without the addition of antibodies). Data are shown from two (B) to three ((A) and (C)) healthy donors (1, 2, and 3). [Figure 20C-2] Effect of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibody (at 20 μg / mL) on serum human IgA-coated Sepharose bead-induced two-dimensional migration of human CD89-expressing ex vivo primary human neutrophil granulocytes (A, mean ± SD (n = 3)), chemotactic activity from the corresponding supernatants of the cells (B, mean ± SD (n = 3)), and chemoattractant LTB4 levels from the corresponding supernatants of the cells (C, mean ± SD (n = 2)). Dashed lines represent (A) two-dimensional granulocyte migration, (B) granulocyte chemotaxis, and (C) granulocyte LTB4 production induced by IgA-coated Sepharose beads alone (i.e., without the addition of antibodies). Data are shown from two (B) to three ((A) and (C)) healthy donors (1, 2, and 3). [Figure 20C-3]Effect of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibody (at 20 μg / mL) on serum human IgA-coated Sepharose bead-induced two-dimensional migration of human CD89-expressing ex vivo primary human neutrophil granulocytes (A, mean ± SD (n = 3)), chemotactic activity from the corresponding supernatants of the cells (B, mean ± SD (n = 3)), and chemoattractant LTB4 levels from the corresponding supernatants of the cells (C, mean ± SD (n = 2)). Dashed lines represent (A) two-dimensional granulocyte migration, (B) granulocyte chemotaxis, and (C) granulocyte LTB4 production induced by IgA-coated Sepharose beads alone (i.e., without the addition of antibodies). Data are shown from two (B) to three ((A) and (C)) healthy donors (1, 2, and 3). [Figure 21-1] Effect of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibodies on serum human IgA-mediated lactoferrin production (a degranulation marker) from human CD89-expressing ex vivo primary human neutrophil granulocytes. The dashed line represents lactoferrin production induced by IgA-coated plates alone (i.e., no antibody added). Mean ± SD (n=2) is shown from two healthy donors (1 and 2). [Figure 21-2] Effect of purified CD89 / IgA-blocking chimeric mouse / human anti-human CD89 antibodies on serum human IgA-mediated lactoferrin production (a degranulation marker) from human CD89-expressing ex vivo primary human neutrophil granulocytes. The dashed line represents lactoferrin production induced by IgA-coated plates alone (i.e., no antibody added). Mean ± SD (n=2) is shown from two healthy donors (1 and 2). [Figure 22A] The Budapest Treaty deposit of human CD89-expressing HEK293F cells has accession number: DSM ACC3341 and identification reference: 293F CD89 clone 2. Deposit form: (A) Receipt of original deposit (B) Viability status. [Figure 22B]The Budapest Treaty deposit of human CD89-expressing HEK293F cells has accession number: DSM ACC3341 and identification reference: 293F CD89 clone 2. Deposit form: (A) Receipt of original deposit (B) Viability status. [Figure 23] Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibody on serum human IgA-coated latex bead-induced NET release by human CD89-expressing ex vivo primary human neutrophil granulocytes. The dashed line represents NET release induced by IgA-coated beads alone (i.e., no antibody added). Mean ± SD (n = 3) from eight different healthy donors (1–8) is shown. [Figure 24] Effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies on induction of cell death (percentage of propidium iodide (PI)) in unprimed and LPS-primed ex vivo primary human neutrophil granulocytes expressing membrane-bound human CD89. Mean ± SD (n = 1 or 2) is shown from three healthy donors (1, 2, and 3). [Figure 25] Binding characteristics of (supernatants) of the CD89 / IgA-blocking humanized anti-human CD89 antibody 10E7 (i.e., the humanized VH1,2,3 and VL1,2,3,4 versions derived from the VH and VL regions of the CD89 / IgA-blocking murine anti-human antibody 10E7 combined with the human constant IgG4 / κ region) to rhuCD89 on HEK293F cells (A, ELISA) and membrane-bound human CD89 (B, FACS). The CD89 / IgA-blocking chimeric anti-human CD89 antibody 10E7 (i.e., chimeric IgG4 / κ) was used as a reference. Mean ± SD (n=2) is shown. [Figure 26]Characterization of the binding of purified CD89 / IgA-blocking "CDR2 deamidation repaired" (i.e., VH3SQVL3 10E7 and VH3STVL3 10E7) humanized anti-human CD89 antibody 10E7 versions VH3SQVL3 and VH3STVL3 (i.e., humanized VH3 and VL3 versions derived from the VH and VL regions of the CD89 / IgA-blocking murine anti-human antibody 10E7 combined with the human constant IgG4 kappa region) to rhuCD89 on HEK293F cells (A, ELISA) and membrane-bound human CD89 (B, FACS). The CD89 / IgA-blocking chimeric anti-human CD89 antibody 10E7 (i.e., chimeric IgG4 / kappa 10E7) was used as a reference. Mean ± SD (n=2) is shown. [Figure 27] Effect of purified CD89 / IgA-blocking "CDR2 deamidation repair" (i.e., VH3SQVL3 10E7 and VH3STVL3 10E7) humanized anti-human CD89 antibody 10E7 versions VH3SQVL3 and VH3STVL3 (i.e., humanized VH3 and VL3 versions derived from the VH and VL regions of the CD89 / IgA-blocking murine anti-human antibody 10E7 combined with the human constant IgG4 kappa region) on the binding of serum human IgA (A) and secreted human IgA (B) to membrane-bound human CD89 on HEK293F cells. CD89 / IgA-blocking chimeric anti-human CD89 antibody 10E7 (i.e., chimeric IgG4 / kappa 10E7) was used as a reference. Mean ± SD (n=2) is shown. [Figure 28] Binding of purified CD89 / IgA-blocking "CDR2 deamidation repaired" humanized IgG4 / kappa anti-human CD89 monoclonal antibodies 10E7 versions VH3SQVL3 and VH3STVL3 (at 10 μg / mL) to membrane-bound human CD89 on ex vivo human neutrophil granulocytes (mean ± SD from three different healthy donors). [Figure 29-1]Effect of purified CD89 / IgA-blocking "CDR2 deamidation repair" humanized IgG4 / kappa anti-human CD89 monoclonal antibody 10E7 versions VH3SQVL3 and VH3STVL3 on phagocytosis of serum-infused human IgA-coated latex beads by ex vivo primary human neutrophil granulocytes expressing membrane-bound human CD89. The dashed line represents phagocytosis of IgA-coated beads alone (i.e., no antibody added). Mean ± SD (n=2) is shown from three different healthy donors (1, 2, and 3). [Figure 29-2] Effect of purified CD89 / IgA-blocking "CDR2 deamidation repair" humanized IgG4 / kappa anti-human CD89 monoclonal antibody 10E7 versions VH3SQVL3 and VH3STVL3 on phagocytosis of serum-infused human IgA-coated latex beads by ex vivo primary human neutrophil granulocytes expressing membrane-bound human CD89. The dashed line represents phagocytosis of IgA-coated beads alone (i.e., no antibody added). Mean ± SD (n=2) is shown from three different healthy donors (1, 2, and 3). [Figure 29-3] Effect of purified CD89 / IgA-blocking "CDR2 deamidation repair" humanized IgG4 / kappa anti-human CD89 monoclonal antibody 10E7 versions VH3SQVL3 and VH3STVL3 on phagocytosis of serum-infused human IgA-coated latex beads by ex vivo primary human neutrophil granulocytes expressing membrane-bound human CD89. The dashed line represents phagocytosis of IgA-coated beads alone (i.e., no antibody added). Mean ± SD (n=2) is shown from three different healthy donors (1, 2, and 3). [Figure 30-1]Effect of purified CD89 / IgA-blocking "CDR2 deamidation repair" humanized IgG4 / kappa anti-human CD89 monoclonal antibody 10E7 versions VH3SQVL3 and VH3STVL3 on serum human IgA-mediated lactoferrin production (a degranulation marker) from ex vivo primary human neutrophil granulocytes expressing membrane-bound human CD89. The dashed line represents lactoferrin production induced by IgA-coated plates alone (i.e., no antibody added). Mean ± SD (n=2) from five healthy donors (1-5) is shown. [Figure 30-2] Effect of purified CD89 / IgA-blocking "CDR2 deamidation repair" humanized IgG4 / kappa anti-human CD89 monoclonal antibody 10E7 versions VH3SQVL3 and VH3STVL3 on serum human IgA-mediated lactoferrin production (a degranulation marker) from ex vivo primary human neutrophil granulocytes expressing membrane-bound human CD89. The dashed line represents lactoferrin production induced by IgA-coated plates alone (i.e., no antibody added). Mean ± SD (n=2) from five healthy donors (1-5) is shown. [Figure 30-3] Effect of purified CD89 / IgA-blocking "CDR2 deamidation repair" humanized IgG4 / kappa anti-human CD89 monoclonal antibody 10E7 versions VH3SQVL3 and VH3STVL3 on serum human IgA-mediated lactoferrin production (a degranulation marker) from ex vivo primary human neutrophil granulocytes expressing membrane-bound human CD89. The dashed line represents lactoferrin production induced by IgA-coated plates alone (i.e., no antibody added). Mean ± SD (n=2) from five healthy donors (1-5) is shown. [Figure 30-4]Effect of purified CD89 / IgA-blocking "CDR2 deamidation repair" humanized IgG4 / kappa anti-human CD89 monoclonal antibody 10E7 versions VH3SQVL3 and VH3STVL3 on serum human IgA-mediated lactoferrin production (a degranulation marker) from ex vivo primary human neutrophil granulocytes expressing membrane-bound human CD89. The dashed line represents lactoferrin production induced by IgA-coated plates alone (i.e., no antibody added). Mean ± SD (n=2) from five healthy donors (1-5) is shown. [Figure 30-5] Effect of purified CD89 / IgA-blocking "CDR2 deamidation repair" humanized IgG4 / kappa anti-human CD89 monoclonal antibody 10E7 versions VH3SQVL3 and VH3STVL3 on serum human IgA-mediated lactoferrin production (a degranulation marker) from ex vivo primary human neutrophil granulocytes expressing membrane-bound human CD89. The dashed line represents lactoferrin production induced by IgA-coated plates alone (i.e., no antibody added). Mean ± SD (n=2) from five healthy donors (1-5) is shown. [Figure 31A] (A) Experimental setup of the in vivo LABD mouse model and treatment regimen with the CD89 / IgA-blocking mouse anti-human CD89 antibody 10E7. [Figure 31B] (B) In vivo effect of purified CD89 / IgA-blocking mouse anti-human CD89 antibody 10E7 on the influx of human CD89-expressing mouse neutrophil granulocytes induced by anti-mouse collagen XVII-human IgA antibody at the injection site. Mean ± SD is shown. Black circles and black squares represent PBS injection in the left ear and anti-mouse collagen XVII-human IgA injection in the right ear, respectively, of each individual mouse. *P<0.05 (two-tailed unpaired Student's t-test), ns=not significant. DETAILED DESCRIPTION OF THE INVENTION

[0039] The present disclosure describes antibodies capable of binding to the extracellular portion of human CD89 (human FcαRI) on human CD89-expressing cells. The antibodies described herein are useful for preventing human IgA binding to human CD89 when the antibodies are bound to the cells. Several antibodies capable of binding to the CD89 receptor have been generated. Monoclonal antibodies that bind to the EC1 domain of CD89 can block IgA binding, whereas antibodies that bind to the EC2 domain do not appear to prevent IgA from binding to the receptor. Antibodies that specifically interfere with the IgA-binding site on CD89 are known in the art. For example, MIP8a, 2D11, or MY43, described in Morton et al., J. Exp. Med. 1999 Jun 7;189(11):1715-22 and Shen LA, J Leukoc Biol. 1992 Apr;51(4):373-8, are used. MIP8a is a murine monoclonal antibody that binds to human CD89 and has a murine IgG1 constant region. Antibodies that can bind to CD89, such as MIP8a, can induce neutrophil death (Wehrli et al., J Immunol. 2014 Dec 1;193(11):5649-59).

[0040] In one aspect, the present invention provides a new humanized antibody that can bind to the extracellular portion of human CD89 (human FcαRI) on human CD89-expressing cells and prevent the binding of human IgA to human CD89 when the antibody is bound to the cells.

[0041] In one aspect, the present invention provides a new antibody that can bind to the extracellular portion of human CD89 (human FcαRI) on human CD89-expressing cells, and when the antibody is bound to the cells, can prevent human IgA from binding to human CD89, and induces less cell death in human CD89-expressing cells compared to the antibody MIP8a.

[0042] The term "antibody" typically refers to an immunoglobulin molecule consisting of two identical pairs of polypeptide chains, each pair having one "heavy" (H) chain and one "light" (L) chain. Human light chains are classified as kappa (κ) and lambda (λ). Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region of IgD, IgG, and IgA consists of three domains, CH1, CH2, and CH3, while the heavy chain constant region of IgM and IgE consists of four domains, CH1, CH2, CH3, and CH4. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The light and heavy chain variable regions together form the antibody binding site and define the specificity for the epitope. Various methods for assigning amino acids to regions or domains in an antibody are known in the art. Well-known methods include the Kabat method and the Chothia method (Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)); Chothia et al. Conformations of immunoglobulin hypervariable regions in Nature 1989;342(6252):877-83).The assignment of amino acids to each region or domain of the present disclosure follows the Kabat definition.

[0043] The term "antibody" encompasses murine, humanized, deimmunized, human, and chimeric antibodies, as well as antibodies that are multimeric forms of antibodies, such as dimers, trimers, or higher multimers of monomeric antibodies. Antibodies also encompass monospecific, bispecific, or multispecific antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site of the required specificity. It also encompasses antibodies linked or attached to non-antibody moieties. Furthermore, the term "antibody" is not limited by any particular method of producing the antibody. For example, it includes monoclonal, recombinant, and polyclonal antibodies. The present invention provides the antibodies described herein. Additionally, the present invention provides portions, derivatives, and / or analogs of the antibodies disclosed herein. Portions, derivatives, and / or analogs retain the antigen-binding properties of the antibody in kind, but not necessarily quantity. Non-limiting examples of portions and / or derivatives include portions of antibodies that are antigen-binding portions, typically containing one or more variable domains of the antibody. Non-limiting examples are various Fab fragments. Portions may also be so-called single-domain antibody fragments. Single-domain antibody fragments (sdAbs, called nanobodies by their developer, Ablynx) are antibody fragments containing a single monomeric variable antibody domain. Like whole antibodies, they can selectively bind to specific antigens. With molecular weights of only 12–15 kDa, single-domain antibody fragments are much smaller than typical antibodies (150–160 kDa), which consist of two heavy and two light chains. They are even smaller than Fab fragments (approximately 50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (approximately 25 kDa, two variable regions, one light chain and one heavy chain). Single-domain antibody fragments themselves are not much smaller than conventional antibodies (typically 90–100 kDa). Single-domain antibody fragments are engineered from heavy-chain antibodies, primarily found in camelids; these are called VHH fragments (Nanobodies®). Some fish also possess heavy-chain-only antibodies (IgNAR, "immunoglobulin new antigen receptor"), from which single-domain antibody fragments called VNAR fragments can be derived. An alternative approach is to split the dimeric variable domain from common immunoglobulin G (IgG) of human or mouse origin into monomers.While most research on single-domain antibodies is currently based on heavy chain variable domains, nanobodies derived from light chains have also been shown to specifically bind target epitopes. Non-limiting examples of antibody moieties contain the heavy and / or light chain variable domains of an antibody or its equivalent. Non-limiting examples of such moieties are VHHs, human domain antibodies (dAbs), and unibodies. Preferred antibody moieties or derivatives have at least the heavy and light chain variable domains of the antibodies described herein. Non-limiting examples of derivatives or moieties are F(ab) fragments and single-chain Fv fragments. Functional moieties of bispecific antibodies include the antigen-binding portion of the bispecific antibody, or derivatives and / or analogs of the binding portion.

[0044] A "single-chain antibody" (scFv) has a single polypeptide chain comprising a VL domain connected to a VH domain, where the VL and VH domains pair to form a monovalent molecule. Single-chain antibodies can be prepared according to methods known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). A "diabody" has two chains, each comprising a heavy-chain variable region connected to a light-chain variable region on the same polypeptide chain connected by a short peptide linker, where the two regions on the same chain do not pair with each other but pair with complementary domains on the other chain to form a bispecific molecule. Methods for preparing diabodies are known in the art (see, for example, Holliger P. et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448, and Poljak RJ et al., (1994) Structure 2:1121-1123). Domain antibodies (dAbs) are small functional binding units of antibodies that correspond to the variable region of either the heavy or light chain of an antibody. Domain antibodies are well expressed in bacteria, yeast, and mammalian cell systems. Further details of domain antibodies and methods for their production are known in the art (see, for example, U.S. Patent Nos. 6,291,158, 6,582,915, 6,593,081, WO04 / 003019, and WO03 / 002609). Nanobodies are derived from the heavy chain of an antibody. Nanobodies typically contain a single variable domain and two constant domains (CH2 and CH3) and retain the antigen-binding ability of the original antibody. Nanobodies can be prepared by methods known in the art (see, for example, U.S. Pat. No. 6,765,087, U.S. Pat. No. 6,838,254, and WO06 / 079372). Unibodies have one light chain and one heavy chain of an IgG4 antibody. Unibodies can be generated by removing the hinge region of an IgG4 antibody. Further details of unibodies and methods for their preparation can be found in WO2007 / 059782.

[0045] The list of analogs to antibodies grows every year, and the current extensive knowledge of the variable domain sequences and 3D structures of many different antibodies allows one skilled in the art to convert an antibody of the invention into one or other antibody analogs, portions or derivatives.

[0046] In addition to the binding molecule, the molecules of the invention may further comprise moieties to increase the in vivo half-life of the molecule, such as polyethylene glycol (PEG), human serum albumin, glycosylation groups, fatty acids, and dextran. Such additional moieties may be conjugated to or otherwise combined with the binding moiety using methods well known in the art.

[0047] Also provided are chimeric antigen receptors (CARs) comprising variable domains of the antibodies described herein. CARs are engineered receptors that combine a new specificity (typically the antigen-binding portion of an antibody or a derivative thereof) with immune cells directed against target cells. The receptor is called chimeric because it is a fusion of portions from different sources (T lymphocytes that have been genetically modified to express one or more chimeric antigen receptors (CARs; see, e.g., Eshhar, U.S. Pat. No. 7,741,465; Eshhar, U.S. Patent Application Publication No. 2012 / 0093842). In some embodiments, the antibodies disclosed herein can be coupled to an active compound, e.g., a toxin. Additionally, the disclosed antibodies or antigen-binding fragments can be coupled to a label, e.g., a fluorescent protein, a chemical label, an organic dye, a colored particle, or an enzyme. The antibodies disclosed herein can be coupled to a drug to form an antibody-drug conjugate (ADC). The present invention provides antibody analogs, antibody portions, and antibody derivatives, even when the molecule is coupled to or incorporated into another molecule.

[0048] In some embodiments, the antibodies disclosed herein are chimeric antibodies. The term "chimeric antibody" refers to an antibody that contains amino acid sequences derived from two different species, such as human and mouse, typically a combination of mouse variable regions (from heavy and light chains) and human constant regions (heavy and light chains). A non-limiting example of generating such a chimeric antibody is described in the Examples (Example 6). In this chimeric antibody, the mouse IgG1 / kappa constant region is replaced with a human IgG / kappa constant domain.

[0049] In a preferred embodiment, the antibodies disclosed herein are humanized antibodies. The term "humanized antibody" refers to an antibody that contains some or all of the CDRs from a non-human animal antibody, while the framework and constant regions of the antibody contain amino acid residues derived from human antibody sequences. Humanized antibodies are typically produced by grafting CDRs from a mouse antibody onto human framework sequences, followed by back-substitution of certain human framework residues for the corresponding mouse residues from the source antibody. Humanized antibodies can also be generated in silico using the amino acid sequence of an antibody of non-human origin and Protein Design Lab (PDL) technology (U.S. Patent No. 569376, U.S. Patent No. 5693671, U.S. Patent No. 5585089). The advantage of humanized antibodies is increased in vivo tolerance compared to their mouse counterparts.

[0050] The term "deimmunized antibody" also refers to an antibody of non-human origin, typically in one or more variable regions, from which one or more epitopes that have a high tendency to constitute human T-cell and / or B-cell epitopes have been removed, in order to reduce immunogenicity. The amino acid sequence of the epitope may be completely or partially removed. Typically, however, the amino acid sequence is modified by substituting one or more of the amino acids that constitute the epitope with one or more other amino acids, thereby changing the amino acid sequence to one that does not constitute a human T-cell and / or B-cell epitope. The amino acids are optionally substituted with amino acids present at the corresponding positions in the corresponding human variable heavy or light chain.

[0051] In a preferred embodiment, the anti-CD89 antibodies disclosed herein are humanized.

[0052] As used herein, a "deamidated" amino acid sequence refers to an amino acid sequence in which an amino acid prone to deamidation is replaced with an amino acid that is not prone to deamidation or is not prone to deamidation. Deamidation is a chemical reaction in which the amide functional group in the side chain of the amino acid asparagine or glutamine is removed or converted to another functional group. Typically, asparagine (Asn) is converted to aspartic acid or isoaspartic acid. Glutamine (Gln) may be converted to glutamic acid or pyroglutamic acid. The Asn-Gly site is most prone to deamidation, and asparagine is more easily deamidated than glutamine. Deamidation of asparagine and / or glutamine can alter the structure of an antibody, as well as its stability and / or function. In antibodies, this reaction is undesirable because it can alter the antibody structure, stability, or function (i.e., antibody-antigen binding) and can lead to antibody degradation. It is possible to predict amino acids in antibody variable regions that are prone to deamidation (Sydow et al. PLoS ONE 2014; 9(6):e100736). Thus, residues at risk of deamidation can be identified, and these sites can then be repaired by replacing the deamidation-prone amino acids with amino acids that are less prone to deamidation.

[0053] In a preferred embodiment, the humanized anti-CD89 antibodies disclosed herein are deamidated. In one preferred embodiment, the deamidation-prone asparagine is replaced with a serine (S), threonine (T), or glutamine (Q) amino acid.

[0054] In some embodiments, the antibodies disclosed herein are human antibodies. The term "human antibody" refers to an antibody consisting of amino acid sequences exclusively of human immunoglobulin sequences. A human antibody may contain mouse carbohydrate chains if produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell. Human antibodies can be prepared in a variety of ways known in the art. Chimeric, humanized, deimmunized, and human antibodies are within the scope of the present invention.

[0055] Antibodies capable of binding to human CD89 bind to the receptor under conditions typically used for antibody binding. When the antibody and human CD89 receptor are contacted with each other under conditions suitable for antibody binding, the antibody binds to the human CD89 receptor. The antibody binds to membrane-bound human CD89 expressed on HEK293F cells deposited under the number DSM ACC3341, while the antibody does not significantly bind to HEK293F cells that do not express human CD89 on the cell membrane. Binding of the antibody to human CD89-expressing cells can be detected by methods known to those skilled in the art. For example, by using a secondary antibody bearing a fluorescent label, labeled cells are measured using flow cytometry (FACS).

[0056] CD89 is an Fc receptor capable of binding IgA. This receptor is also known as FcαRI. Human CD89 can bind to the heavy chain constant regions of human IgA1 and human IgA2. CD89 is a glycosylated transmembrane receptor with two extracellular domains, EC1 and EC2, a transmembrane domain, and an intracellular domain. The interaction between IgA and CD89 is mediated by the EC1 extracellular domain. For the reference sequence, see NP_001991.1 (Immunoglobulin alpha Fc receptor isoform precursor). The reference is made solely for the purpose of identifying the human CD89 gene / protein. It is not intended to limit the human CD89 described herein to the specific sequence of the database entry. Natural variants of human CD89 capable of binding IgA and the antibodies described herein are within the scope of the present invention. Recombinant human CD89, if capable of binding IgA and the antibodies described herein, is also within the scope of the present invention.

[0057] CD89 is present on the cell surface of myeloid cells, including neutrophils, eosinophils, and most monocytes and macrophages. The receptor is not present on mast cells and intestinal macrophages. CD89 expression has been found to be constitutive and independent of the presence of IgA ligands. The term "human CD89-expressing cells" refers to cells that express human CD89. Exemplary cells are neutrophils, eosinophils, monocytes, and / or macrophages.

[0058] Cross-linking of CD89 receptors on cells can be achieved by binding of IgA antibodies, IgA immune complexes, or anti-CD89 antibodies. IgA binding can trigger an immune response that can have positive and negative effects.

[0059] The term "extracellular" literally means outside the cell. The term "extracellular portion" refers to the portion of a molecule that is outside the cell membrane. This portion of the molecule may be available for interaction with other molecules outside the cell. The human CD89 receptor has two extracellular domains, namely, EC1 and EC2. These domains may interact with molecules outside the cell, such as IgA antibodies. IgA is known to bind to the EC1 domain of the human CD89 receptor.

[0060] Human CD89-expressing cells are cells that express the human CD89 molecule. Preferably, the molecule is present on the cell membrane of the cell. Examples of cells that express human CD89 include, but are not limited to, neutrophils, eosinophils, monocytes, and / or macrophages. Furthermore, a modified HEK293F cell line that expresses human CD89 on its cell membrane has been deposited under the number DSM ACC3341.

[0061] The term "preventing binding" refers to the ability of the antibodies or antigen-binding fragments thereof disclosed herein to prevent IgA from interacting with the human CD89 receptor. When an anti-CD89 antibody or antibody fragment thereof binds to the human CD89 receptor, IgA can no longer bind to the human CD89 receptor. Preferably, binding of the anti-CD89 antibodies disclosed herein blocks or reduces the ability of IgA to bind to the human CD89 receptor by at least 50%, preferably at least 60%, at least 70%, at least 80%, or at least 90%. Prevention, blocking, or reduction of IgA binding to CD89 is preferably measured by the methods described in the Examples. Preferably, CD89-expressing cells are used. Preferably, the cells stably express human CD89. The antibody of interest is titrated against the CD89-expressing cells. The cells are then incubated with IgA. After washing, cell-bound IgA is detected using a labeled antibody against IgA, preferably a fluorescently labeled antibody. IgA binding to the membranes of human CD89-expressing cells can be measured using a flow cytometer (FACS). The amount of bound IgA indicates the blocking capacity of the titrated antibody. Low IgA binding at a particular antibody concentration indicates a stronger blocking capacity of the antibody. A preferred method is described in the Examples, where the results are shown in Figures 4a and 4b. Preferably, the test antibody is titrated on human CD89-coated wells or on human CD89-expressing HEK293F cells. The blocking capacity is then easily determined by comparing the titration curve with that obtained with a positive control, such as MIP8a. The blocking percentage is usually given as a percentage compared to the blockade of MIP8a under otherwise identical conditions. Comparison of the percentage binding of the test antibody and the control antibody MIP8a is preferably performed at an antibody concentration at which the test antibody reaches at least 90% of its blocking capacity. In Figure 4b, this is an antibody concentration of approximately 1 μg / ml. In this example, the blocking percentage of antibody 8F3 is calculated to be about ((800-200) / 800) x 100 = about 75%. The blocking percentage of antibody 16D6 is calculated to be about ((800-450) / 800) x 100 = about 45%.A control IgG1 that does not bind to CD89 typically does not prevent IgA from binding to CD89.

[0062] Immunoglobulin A is an antibody isotype found primarily in blood and serous mucosal secretions. Human IgA has two subclasses, IgA1 and IgA2, and can be produced in monomeric and dimeric forms. The dimeric form is the most common form. The secretory form of dimeric IgA is also called secretory IgA. The secretory form of IgA is partially sterically hindered in binding to FcαRI because some of the FcαRI-binding site of sIgA is obscured by a segment of the cleaved polymeric Ig receptor (called the secretory component after cleavage), which aids in the secretion of sIgA into the intestinal lumen. Before binding to the polymeric receptor, dimeric IgA (dIgA) binds to FcαRI with nearly the same affinity as monomeric IgA. The term "heat-aggregated IgA" refers to complexes of IgA formed by heating IgA. These aggregates can mimic immune complexes, such as circulating immune complexes. The heat-aggregated IgA complexes can be produced by any method known to those skilled in the art.

[0063] The term "cell death" refers to an event in which a living cell ceases to perform its function. Cell death can result from a variety of causes, such as apoptosis, programmed cell death, mitotic catastrophe, ischemic cell death, and / or immunogenic cell death. The term "cell viability" relates to the ability of a cell to perform a specific function, such as metabolism, growth, replication, some form of responsiveness, and adaptability. Cell death and cell viability can be assessed by a number of suitable assays known to those skilled in the art, such as the MultiTox-Glo or MultiTox-Fluor multiplex cytotoxicity assay (Promega, Madison, Wisconsin) or the Live / Dead Cell Assay (Abcam, Cambridge, Massachusetts). Dye exclusion is frequently used as a means to determine dead cells. Dyes such as trypan blue do not readily cross the membranes of viable cells but do enter dead cells that are unable to maintain the integrity of their cell membranes. Suitable methods for determining cell viability are described in the Examples section.

[0064] Antibodies that specifically interfere with the IgA-binding site on CD89 are known in the art. For example, MIP8a, 2D11, or MY43, described in Morton et al., J. Exp. Med. 1999 Jun 7; 189(11):1715-22 and Shen LA, J Leukoc Biol. 1992 Apr; 51(4):373-8, are used. MIP8a is a mouse monoclonal antibody that binds to human CD89 and has a mouse IgG1 constant region. MIP8a has been shown to induce neutrophil death, as described in Wehrli et al., J Immunol. 2014 Dec 1; 193(11):5649-59.

[0065] In one aspect, the disclosure provides a humanized anti-human CD89 antibody capable of binding to the extracellular portion of human CD89, comprising the amino acid sequence: EVQLLESGGG LVQPGGSLRL SCAASGLTFS SYGMSWVRQA PGKGLEX1VX2TIX3GX4GDITYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDY DYDYAMDYWG QGTLVTVSS wherein: X1 is L or W X2 is A or S X3 is N, S X4 is Q, T, or N; a heavy chain variable region that comprises, with respect to the designated amino acid sequence, preferably 0 to 3, preferably 0 to 2, preferably 0 to 1, and preferably 0 amino acid variations, insertions, deletions, substitutions, additions, or a combination thereof, at positions other than X1, X2, X3, and X4; Amino acid sequence: DIQMTQSPSS LSASVGDRVT ITCRASQDII NYLNWYQQKP GKZ1Z2KLLIYY TSRLHSGVPS RFSGSGSGTD Z3TLTISSLQP EDFATYZ4CQQ GKTLPYTFGQ GTKLEIK a light chain variable region comprising: Z1 is A or T Z2 is V or P Z3 is Y or F Z4 is Y or F; and a light chain variable region that contains, with respect to the indicated amino acid sequence, preferably 0 to 3, preferably 0 to 2, preferably 0 to 1, and preferably 0 amino acid variations, insertions, deletions, substitutions, additions, or a combination thereof, at positions other than Z1, Z2, Z3, and Z4.

[0066] In some embodiments, X1, X2, X3, and X4 are L, A, S, and T, or L, A, S, and Q, or L, A, N, and N, or W, A, N, and N, or W, S, N, and N; and Z1, Z2, Z3, and Z4 are A, V, Y, and Y.

[0067] In other embodiments, X1, X2, X3, and X4 are L, A, N, and N, or W, A, N, and N, or W, S, N, and N, and Z1, Z2, Z3, and Z4 are A, P, F, and Y.

[0068] In further embodiments, X1, X2, X3, and X4 are L, A, N, and N, or W, A, N, and N, or W, S, N, and N; and Z1, Z2, Z3, and Z4 are A, P, Y, and Y.

[0069] In further embodiments, X1, X2, X3, and X4 are L, A, N, and N, or W, A, N, and N, or W, S, N, and N; and Z1, Z2, Z3, and Z4 are T, V, Y, and F.

[0070] In a preferred embodiment, X1, X2, X3, and X4 are L, A, S, and T, and Z1, Z2, Z3, and Z4 are A, V, Y, and Y. In another preferred embodiment, X1, X2, X3, and X4 are L, A, S, and Q, and Z1, Z2, Z3, and Z4 are A, V, Y, and Y.

[0071] In a preferred embodiment, the anti-human CD89 antibody disclosed herein has the amino acid sequence EVQLLESGGG LVQPGGSLRL SCAASGLTFS SYGMSWVRQA PGKGLELVAT IX3GX4GDITYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDY DYDYAMDYWG QGTLVTVSS, the heavy chain variable region comprises an amino acid sequence comprising, with respect to the designated amino acid sequence, preferably 0 to 3, preferably 0 to 2, preferably 0 to 1, and preferably 0 amino acid variations, insertions, deletions, substitutions, additions, or a combination thereof, at positions other than X3 and X4; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 122, which contains 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions.

[0072] In a preferred embodiment, X3 is S and X4 is T.

[0073] In another preferred embodiment, X3 is S and X4 is Q.

[0074] The heavy chains disclosed herein contain 10, preferably 9, preferably 8, preferably 7, preferably 6, preferably 5, preferably 4, preferably 3, preferably 2, preferably 1, or preferably 0 amino acid insertions, deletions, substitutions, or additions. Of the 0 to 10 amino acid insertions, deletions, substitutions, or additions, no more than 3 are present in the heavy chain variable region. The heavy chain variable regions disclosed herein contain 3, preferably 2, preferably 1, or preferably 0 amino acid insertions, deletions, substitutions, or additions. In a preferred embodiment, the 0 to 3 amino acid insertions, deletions, or substitutions are present at positions other than X1, X2, X3, and X4, and the amino acid insertions, deletions, substitutions, or additions in the heavy chain variable region, if present, are in the framework regions. If present, the amino acid insertion, deletion, substitution, or addition in the heavy chain variable region is preferably not an amino acid insertion, deletion, substitution, or addition selected from leucine (L) at position 5, arginine (R) at position 19, alanine (A) at position 40, glycine (G) at position 42, glycine (G) at position 44, serine (S) at position 75, asparagine (N) at position 84, arginine (R) at position 87, alanine (A) at position 88, valine (V) at position 93, and leucine (L) at position 114 of the heavy chain variable region.

[0075] The light chains disclosed herein contain 8, preferably 7, preferably 6, preferably 5, preferably 4, preferably 3, preferably 2, preferably 1, and preferably 0 amino acid insertions, deletions, substitutions, or additions. Of the 0-8 amino acid insertions, deletions, substitutions, or additions, no more than 5 are in the light chain constant region and no more than 3 are in the light chain variable region. The light chain variable regions disclosed herein contain 3, preferably 2, preferably 1, and preferably 0 amino acid insertions, deletions, substitutions, or additions. In a preferred embodiment, the 0-3 amino acid insertions, deletions, substitutions, or additions are at positions other than Z1, Z2, Z3, and Z4, and the amino acid insertions, deletions, substitutions, or additions in the light chain variable region, if present, are in the framework regions. If present, the amino acid insertion, deletion, substitution, or addition in the light chain variable region is preferably not an amino acid insertion, deletion, substitution, or addition selected from proline (P) at position 8, valine (V) at position 15, arginine (R) at position 18, threonine (T) at position 22, glycine (G) at position 41, lysine (K) at position 42, aspartic acid (D) at position 70, threonine (T) at position 72, isoleucine (I) at position 75, serine (S) at position 77, glutamine (Q) at position 79, glutamine (Q) at position 80, threonine (T) at position 83, and glutamine (Q) at position 100 of the light chain variable region.

[0076] In one aspect, the disclosure provides a humanized antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 138 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 122 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions.

[0077] In a further aspect, the disclosure provides a humanized antibody that binds to CD89, the antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 142 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, wherein there are no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the heavy chain, and a light chain having the amino acid sequence of SEQ ID NO: 136 with 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid insertions, deletions, substitutions, or additions, wherein there are preferably no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the light chain. Preferably, the humanized antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 142 and a light chain variable region having the amino acid sequence of SEQ ID NO: 136. An exemplary antibody having these characteristics is antibody 10E7 VH3STVL3.

[0078] In one aspect, the disclosure provides a humanized antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 139 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 122 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions.

[0079] In a further aspect, the present disclosure provides a humanized antibody that binds to CD89, the antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 143 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, wherein there are no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the heavy chain, and a light chain having the amino acid sequence of SEQ ID NO: 136 with 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid insertions, deletions, substitutions, or additions, wherein there are preferably no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the light chain. Preferably, the humanized antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 143 and a light chain variable region having the amino acid sequence of SEQ ID NO: 136. An exemplary antibody having these characteristics is antibody 10E7 VH3SQVL3.

[0080] In one aspect, the disclosure provides a humanized antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 119 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 122 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions.

[0081] In a further aspect, the present disclosure provides a humanized antibody that binds to CD89, the antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 133 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, wherein there are no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the heavy chain, and a light chain having the amino acid sequence of SEQ ID NO: 136 with 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid insertions, deletions, substitutions, or additions, wherein there are preferably no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the light chain. Preferably, the humanized antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 133 and a light chain variable region having the amino acid sequence of SEQ ID NO: 136. An exemplary antibody with these characteristics is antibody 10E7 VH3VL3.

[0082] In one aspect, the disclosure provides a humanized antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 119 with zero, one, two, or three amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 123 with zero, one, two, or three amino acid insertions, deletions, substitutions, or additions.

[0083] In a further aspect, the present disclosure provides a humanized antibody that binds to CD89, the antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 133 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, wherein there are no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the heavy chain, and a light chain having the amino acid sequence of SEQ ID NO: 137 with 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid insertions, deletions, substitutions, or additions, wherein there are preferably no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the light chain. Preferably, the humanized antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 133 and a light chain variable region having the amino acid sequence of SEQ ID NO: 137. An exemplary antibody with these characteristics is antibody 10E7 VH3VL4.

[0084] In one aspect, the disclosure provides a humanized antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 117 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 120 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions.

[0085] In a further aspect, the present disclosure provides a humanized antibody that binds to CD89, the antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 131 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, wherein there are no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the heavy chain, and a light chain having the amino acid sequence of SEQ ID NO: 134 with 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid insertions, deletions, substitutions, or additions, wherein there are preferably no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the light chain. Preferably, the humanized antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 131 and a light chain variable region having the amino acid sequence of SEQ ID NO: 134. An exemplary antibody with these characteristics is antibody 10E7 VH1VL1.

[0086] In one aspect, the disclosure provides a humanized antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 117 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 121 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions.

[0087] In a further aspect, the present disclosure provides a humanized antibody that binds to CD89, wherein the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 131 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, wherein there are no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the heavy chain, and a light chain having the amino acid sequence of SEQ ID NO: 135 with 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid insertions, deletions, substitutions, or additions, wherein there are preferably no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the light chain. Preferably, the humanized antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 131 and a light chain variable region having the amino acid sequence of SEQ ID NO: 135. An exemplary antibody with these characteristics is antibody 10E7 VH1VL2.

[0088] In one aspect, the disclosure provides a humanized antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 117 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 122 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions.

[0089] In a further aspect, the present disclosure provides a humanized antibody that binds to CD89, the antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 131 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, wherein there are no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the heavy chain, and a light chain having the amino acid sequence of SEQ ID NO: 136 with 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid insertions, deletions, substitutions, or additions, wherein there are preferably no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the light chain. Preferably, the humanized antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 131 and a light chain variable region having the amino acid sequence of SEQ ID NO: 136. An exemplary antibody with these characteristics is antibody 10E7 VH1VL3.

[0090] In one aspect, the disclosure provides a humanized antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 117 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 123 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions.

[0091] In a further aspect, the present disclosure provides a humanized antibody that binds to CD89, the antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 131 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, wherein there are no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the heavy chain, and a light chain having the amino acid sequence of SEQ ID NO: 137 with 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid insertions, deletions, substitutions, or additions, wherein there are preferably no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the light chain. Preferably, the humanized antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 131 and a light chain variable region having the amino acid sequence of SEQ ID NO: 137. An exemplary antibody with these characteristics is antibody 10E7 VH1VL4.

[0092] In one aspect, the disclosure provides a humanized antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 118 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 120 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions.

[0093] In a further aspect, the present disclosure provides a humanized antibody that binds to CD89, the antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 132 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, wherein there are no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the heavy chain, and a light chain having the amino acid sequence of SEQ ID NO: 134 with 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid insertions, deletions, substitutions, or additions, wherein there are preferably no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the light chain. Preferably, the humanized antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 132 and a light chain variable region having the amino acid sequence of SEQ ID NO: 134. An exemplary antibody with these characteristics is antibody 10E7 VH2VL1.

[0094] In one aspect, the disclosure provides a humanized antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 118 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 121 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions.

[0095] In a further aspect, the present disclosure provides a humanized antibody that binds to CD89, the antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 132 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, wherein there are no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the heavy chain, and a light chain having the amino acid sequence of SEQ ID NO: 135 with 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid insertions, deletions, substitutions, or additions, wherein there are preferably no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the light chain. Preferably, the humanized antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 132 and a light chain variable region having the amino acid sequence of SEQ ID NO: 135. An exemplary antibody with these characteristics is antibody 10E7 VH2VL2.

[0096] In one aspect, the disclosure provides a humanized antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 118 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 122 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions.

[0097] In a further aspect, the present disclosure provides a humanized antibody that binds to CD89, the antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 132 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, wherein there are no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the heavy chain, and a light chain having the amino acid sequence of SEQ ID NO: 136 with 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid insertions, deletions, substitutions, or additions, wherein there are preferably no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the light chain. Preferably, the humanized antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 132 and a light chain variable region having the amino acid sequence of SEQ ID NO: 136. An exemplary antibody with these characteristics is antibody 10E7 VH2VL3.

[0098] In one aspect, the disclosure provides a humanized antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 118 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 123 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions.

[0099] In a further aspect, the present disclosure provides a humanized antibody that binds to CD89, the antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 132 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, wherein there are no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the heavy chain, and a light chain having the amino acid sequence of SEQ ID NO: 137 with 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid insertions, deletions, substitutions, or additions, wherein there are preferably no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the light chain. Preferably, the humanized antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 132 and a light chain variable region having the amino acid sequence of SEQ ID NO: 137. An exemplary antibody with these characteristics is antibody 10E7 VH2VL4.

[0100] In one aspect, the disclosure provides a humanized antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 119 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 120 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions.

[0101] In a further aspect, the present disclosure provides a humanized antibody that binds to CD89, the antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 133 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, wherein there are no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the heavy chain, and a light chain having the amino acid sequence of SEQ ID NO: 134 with 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid insertions, deletions, substitutions, or additions, wherein there are preferably no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the light chain. Preferably, the humanized antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 133 and a light chain variable region having the amino acid sequence of SEQ ID NO: 134. An exemplary antibody with these characteristics is antibody 10E7 VH3VL1.

[0102] In one aspect, the disclosure provides a humanized antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 119 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 121 with 0, 1, 2, or 3 amino acid insertions, deletions, substitutions, or additions.

[0103] In a further aspect, the present disclosure provides a humanized antibody that binds to CD89, the antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 133 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, wherein there are no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the heavy chain, and a light chain having the amino acid sequence of SEQ ID NO: 135 with 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid insertions, deletions, substitutions, or additions, wherein there are preferably no more than 3, preferably no more than 2, preferably no more than 1, and preferably no amino acid insertions, deletions, substitutions, or additions in the variable region of the light chain. Preferably, the humanized antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain having the amino acid sequence of SEQ ID NO: 133 and a light chain variable region having the amino acid sequence of SEQ ID NO: 135. An exemplary antibody with these characteristics is antibody 10E7 VH3VL2.

[0104] In one aspect, the humanized antibodies disclosed herein have a higher affinity for the extracellular portion of human CD89 compared to a chimeric antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 94 and a light chain having the amino acid sequence of SEQ ID NO: 99.

[0105] In one aspect, the present invention provides an antibody capable of binding to the extracellular portion of human CD89 (human FcαRI) on human CD89-expressing cells, preventing human IgA from binding to human CD89 when bound to the cells, and inducing less cell death in human CD89-expressing cells compared to antibody MIP8a. Preferably, the antibody induces 10% less cell death compared to antibody MIP8a. More preferably, the antibody induces 20% less cell death compared to antibody MIP8a. More preferably, the antibody induces 40% less cell death compared to antibody MIP8a. The cell death-inducing property of the antibody is preferably determined using human CD89-expressing HEK293F cells deposited under number DSM ACC3341.

[0106] Targeted effector cells can be lysed after binding of anti-human CD89 antibodies. The antibodies disclosed herein are useful for targeting cells expressing human CD89 without causing widespread cell death or lysis of the target cells. Such a characteristic is useful for maintaining target cells alive. Surviving cells can respond to CD89 antibody binding, possibly by altered signaling. Surviving CD89-expressing cells can respond to the lack of IgA binding to CD89 due to the blocking properties of the antibodies disclosed herein. Cells bearing the antibodies disclosed herein may be available to respond to other stimuli, pathogens, and / or immune effects that are IgA-independent.

[0107] In some embodiments, the present disclosure provides antibodies that can bind to the extracellular portion of human CD89 on human CD89-expressing cells and, when bound to the cells, can prevent human IgA from binding to human CD89 and do not reduce cell viability of the cells by more than 60% after overnight incubation at 37° C. Preferably, binding of the antibody to the cells does not reduce cell viability of the cells by more than 50%, or more than 40%, or more than 30%, or more than 20%, or more than 10%, or less, after overnight incubation at 37° C. The cell viability-protecting properties of the antibody are preferably determined using human CD89-expressing HEK293F cells deposited under number DSM ACC3341.

[0108] Cells are typically cultured at 37°C. The effect of a stimulus on the cells may be seen directly or after an incubation period. Some stimuli require cell signaling before an effect is visible. The term "overnight incubation at 37°C" indicates that the cells are incubated with the stimulus overnight. Overnight can mean, for example, 12-16 hours or 8-24 hours, after which a characteristic of the cells, such as cell viability or phosphatidylserine expression, is measured.

[0109] In some embodiments, the present disclosure provides antibodies that can bind to the extracellular portion of human CD89 on human CD89-expressing cells, that can prevent human IgA from binding to human CD89 when bound to the cells, and that do not increase phosphatidylserine expression in the cells by more than 20% after overnight incubation at 37° C. Preferably, binding of the antibody to the cells does not increase phosphatidylserine expression in the cells by more than 20%, more preferably by more than 10%, and more preferably by more than 5% after overnight incubation at 37° C. The effect of the antibody on phosphatidylserine expression is preferably determined using human CD89-expressing HEK293F cells deposited under number DSM ACC3341.

[0110] Phosphatidylserine is a phospholipid and a component of the cell membrane. It plays a role in cell signaling and is correlated with cell death and apoptosis. Phosphatidylserine expression in the cell membrane can be used as a marker of cell death. Phosphatidylserine expression in the cell membrane can be measured by methods known to those skilled in the art. Phosphatidylserine is actively retained facing the cytoplasmic (inner) side of the cell membrane. However, when cells undergo apoptosis, phosphatidylserine is no longer restricted to the cytoplasmic side. Instead, it is rapidly exchanged between the two sides. Phosphatidylserine expression is typically determined by detecting phosphatidylserine on the outer surface of the cell membrane. Phosphatidylserine levels can be compared with control cells, e.g., untreated cells. Exemplary methods are described in detail in the Examples section.

[0111] Phosphatidylserine expression can be determined by incubating cells with anti-phosphatidylserine antibody. In parallel, cells can be incubated with a negative control, such as anti-human CD19 antibody. After washing and fixing the cells, membrane phosphatidylserine expression can be measured using a flow cytometer (FACS).

[0112] In some embodiments, an antibody is provided that is capable of binding to the extracellular portion of human CD89 on human CD89-expressing cells, preventing human IgA from binding to human CD89 when bound to the cells in the absence of NaN3 at 37° C., and not displacing monomeric human IgA or heat-aggregated IgA when greater than 90% bound to the cells in the presence of NaN3 at 4° C. The effect of the antibody on IgA displacement is preferably determined using human CD89-expressing HEK293F cells deposited under number DSM ACC3341.

[0113] In a preferred embodiment, the antibody is a humanized antibody.

[0114] Sodium azide is used in combination with a low temperature of 4°C to inhibit the metabolic activity of cells. Sodium azide is a reversible inhibitor of mitochondrial respiration. Inhibition of the metabolic activity of cells refers to a decrease in the mitochondrial respiration of cells. In some embodiments, the antibodies disclosed herein cannot replace more than 90% of human IgA on metabolically inhibited cells.

[0115] In one embodiment, the present disclosure provides an antibody that binds to recombinant human CD89 molecules at 20% or less, where amino acids 22-46 of human CD89 have been replaced with amino acids 22-46 of cynomolgus monkey CD89, but the antibody can bind to the extracellular portion of human CD89 on human CD89-expressing cells and prevent human IgA from binding to human CD89 when bound to the cells. Preferably, the antibody binds to the recombinant human CD89 molecule at 10% or less. An exemplary antibody having these characteristics is antibody 20B4. The substituted amino acids Gln22 to Lys46 in this CD89 molecule are part of the EC1 domain of CD89 (SEQ ID NO: 23). In some embodiments, binding of the antibody to the cells induces less cell death in human CD89-expressing cells compared to MIP8a. In some embodiments, binding of the antibody to the cells does not reduce cell viability by more than 60% after overnight incubation at 37°C. In some embodiments, binding of the antibody to the cells does not increase phosphatidylserine expression in the cells by more than 20% after overnight incubation at 37°C.

[0116] The IgA system differs among various species, including humans, mice, and rabbits. For example, there is no identified mouse homolog of the human CD89 gene. CD89 homologs have been identified in rats and cows. The present disclosure includes examples of antibodies that bind to CD89 on human cells expressing human CD89 and on human cells expressing chimeric CD89 molecules. A human / cynomolgus CD89 molecule has portions of the human CD89 molecule and portions of cynomolgus CD89. The portions are combined such that the general protein structure remains intact.

[0117] The cynomolgus monkey (Macaca fascicularis) CD89 gene has a similar intron / exon structure to human CD89 and exhibits 86% homology with the human gene (Rogers et al. 2004, Immunology). Substitution of amino acids in human CD89 with the corresponding amino acids in cynomolgus monkey CD89 can be used to test the specificity and cross-reactivity of antibodies. Substitutions in cynomolgus monkey CD89 can contribute to the identification of epitopes for anti-human CD89 antibodies.

[0118] In one embodiment, the present disclosure provides an antibody that binds to a chimeric CD89 molecule at 20% or less, in which amino acids 47-71 of human CD89 have been replaced with amino acids 47-71 of cynomolgus monkey CD89, and that can bind to the extracellular portion of human CD89 on human CD89-expressing cells and prevent human IgA from binding to the cells when the antibody is bound to the cells. Preferably, the antibody binds to the recombinant human CD89 molecule at 10% or less. Preferably, the antibody binds to the recombinant human CD89 molecule at 5% or less. Exemplary antibodies with these characteristics are antibodies 20B4, 8F3, 30C7, and 16D6. Amino acids Ile47 to Ile71 are part of the EC1 domain of CD89 (SEQ ID NO: 24). IgA can bind to this portion of the CD89 receptor. Therefore, antibodies that bind to this portion of the CD89 receptor potentially interfere with the interaction of IgA with CD89.

[0119] In one embodiment, the present disclosure provides an antibody that binds to a chimeric CD89 molecule at 20% or less, in which amino acids 72-96 of human CD89 have been replaced with amino acids 72-96 of cynomolgus monkey CD89, and that can bind to the extracellular portion of human CD89 on human CD89-expressing cells and prevent human IgA from binding to the cells when the antibody is bound to the cells. Preferably, the antibody binds to the recombinant human CD89 molecule at 10% or less. Preferably, the antibody binds to the recombinant human CD89 molecule at 5% or less. Exemplary antibodies with these characteristics are antibodies 8F3, 10E7, and 16D6. Amino acids Gly72 to Gly96 are part of the EC1 domain of CD89 (SEQ ID NO: 25). This portion of the EC1 domain is thought to include the FG loop, which is predicted to be located at the bottom of EC1, close to the cell membrane.

[0120] In one embodiment, the present disclosure provides an antibody whose binding to a chimeric CD89 molecule is not reduced by more than 20%, wherein amino acids 97-121 of human CD89 are exchanged with amino acids 97-121 of cynomolgus monkey CD89, and the antibody can bind to the extracellular portion of human CD89 on human CD89-expressing cells and prevent human IgA from binding to human CD89 when bound to the cells. Preferably, the binding of the antibody to the recombinant human CD89 molecule is not reduced by more than 10%. Exemplary antibodies having these characteristics are antibodies 20B4, 8F3, 30C7, 10E7, and 16D6. Amino acids Arg97 to Gly121 are part of the EC1 domain of CD89 (SEQ ID NO: 26). For example, an MIP8a antibody can bind to this portion of the EC1 domain.

[0121] In one embodiment, the present disclosure provides an antibody that binds to a chimeric CD89 molecule at 20% or less, in which amino acids 58, 59, 73, 74, 76, 106, and 107 of human CD89 have been replaced with amino acids 58, 59, 73, 74, 76, 106, and 107 of cynomolgus monkey CD89, respectively, and that can bind to the extracellular portion of human CD89 on human CD89-expressing cells and prevent human IgA from binding to the cells when the antibody is bound to the cells. Preferably, the antibody binds to the recombinant human CD89 molecule at 10% or less. Preferably, the antibody binds to the recombinant human CD89 molecule at 5% or less. Exemplary antibodies with these characteristics are antibodies 8F3, 10E7, and 16D6. Amino acids Thr58 and Gln59 are believed to be involved in human IgA-CD89 binding.

[0122] In some embodiments, binding of the antibody to the cells induces less cell death in human CD89-expressing cells compared to MIP8a. In some embodiments, binding of the antibody to the cells does not reduce cell viability of the cells by more than 60% after overnight incubation at 37° C. In some embodiments, binding of the antibody to the cells does not increase phosphatidylserine expression in the cells by more than 20% after overnight incubation at 37° C.

[0123] The antibodies described herein can bind to the extracellular portion of human CD89 on human CD89-expressing HEK293F cells. Exemplary HEK293F cells expressing membrane-bound human CD89 have been deposited under the Budapest Treaty under the number DSM ACC3341. These HEK293F cells stably express human CD89 on their cell surface. Preferably, these cells can be used to compare the effects of different antibodies targeting human CD89. Preferably, the percentage of cells that undergo cell death after binding of a CD89 antibody is studied. Using a cell line that stably expresses human CD89 typically reduces expression differences between cells and allows for better comparison of experimental conditions compared to transiently transfected cells.

[0124] One aspect of the present disclosure provides an antibody that binds to human CD89, comprising a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 29 to 31, with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 32 to 34, with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions. Preferably, the antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 29 to 31, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 32 to 34.

[0125] In a further aspect, the disclosure provides an antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 27 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 28 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions. In preferred embodiments, the 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions are located in framework regions of the light and / or heavy chain variable regions. Preferably, the antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 27 and a light chain variable region having the amino acid sequence of SEQ ID NO: 28. An exemplary antibody having these characteristics is antibody 8F3. Antibodies having these CDRs may bind to epitopes in the EC1 domain of CD89, in particular to portions of the sequences of SEQ ID NO:24 and SEQ ID NO:25.

[0126] In one aspect, the present disclosure provides an antibody that binds to human CD89, comprising a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 45 to 47, with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 48 to 50, with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions. Preferably, the antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 45 to 47, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 48 to 50.

[0127] In a further aspect, the disclosure provides an antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 43 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 44 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions. In preferred embodiments, the 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions are located in framework regions of the light and / or heavy chain variable regions. Preferably, the antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 43 and a light chain variable region having the amino acid sequence of SEQ ID NO: 44. An exemplary antibody having these characteristics is antibody 10E7. Antibodies having these CDRs may bind to an epitope in the EC1 domain of CD89, in particular a portion of the sequence of SEQ ID NO:25.

[0128] In one aspect, the present disclosure provides an antibody that binds to human CD89, comprising a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 69-71 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 72-74 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions. Preferably, the antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 69-71 and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 72-74.

[0129] In a further aspect, the disclosure provides an antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 67 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 68 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions. In preferred embodiments, the 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions are located in framework regions of the light and / or heavy chain variable regions. Preferably, the antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 67 and a light chain variable region having the amino acid sequence of SEQ ID NO: 68. An exemplary antibody having these characteristics is antibody 20B4. Antibodies having these CDRs may bind to epitopes in the EC1 domain of CD89, in particular to portions of the sequences of SEQ ID NO:23 and SEQ ID NO:24.

[0130] In one aspect, the present disclosure provides an antibody that binds to human CD89, comprising a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 77 to 79, with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 80 to 82, with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions. Preferably, the antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 77 to 79, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 80 to 82.

[0131] In a further aspect, the disclosure provides an antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 75 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 76 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions. In preferred embodiments, the 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions are located in framework regions of the light and / or heavy chain variable regions. Preferably, the antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 75 and a light chain variable region having the amino acid sequence of SEQ ID NO: 76. An exemplary antibody having these characteristics is antibody 30C7. Antibodies having these CDRs may bind to an epitope in the EC1 domain of CD89, in particular a portion of the sequence of SEQ ID NO:24.

[0132] In one aspect, the present disclosure provides an antibody that binds to human CD89, comprising a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 53 to 55, with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 56 to 58, with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions. Preferably, the antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 53 to 55, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 56 to 58.

[0133] In a further aspect, the disclosure provides an antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 51 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 52 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions. In preferred embodiments, the 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions are located in framework regions of the light and / or heavy chain variable regions. Preferably, the antibody capable of binding to the extracellular portion of human CD89 comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 51 and a light chain variable region having the amino acid sequence of SEQ ID NO: 52. An exemplary antibody having these characteristics is antibody 16D6. Antibodies having these CDRs may bind to epitopes in the EC1 domain of CD89, in particular to portions of the sequences of SEQ ID NO:24 and SEQ ID NO:25.

[0134] Binding of an antibody referred to by sequence herein to a cell can induce less cell death in human CD89-expressing cells when compared to MIP8a. In some embodiments, binding of the antibody to the cell does not reduce cell viability of the cell by more than 60% after overnight incubation at 37°C. In some embodiments, binding of the antibody to the cell does not increase phosphatidylserine expression of the cell by more than 20% after overnight incubation at 37°C.

[0135] Provided is an antibody that binds to human CD89, comprising a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 37 to 39 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 40 to 42 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions.

[0136] A preferred embodiment provides an antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 35 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 36 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions. In preferred embodiments, the amino acid insertions, deletions, substitutions, or additions are located in framework regions of the light and / or heavy chain variable regions. An exemplary antibody having these characteristics is antibody 9H7.

[0137] One embodiment provides an antibody that binds to human CD89, comprising a heavy chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 61 to 63 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 64 to 66 with 0, 1, or 2 amino acid insertions, deletions, substitutions, or additions.

[0138] A preferred embodiment provides an antibody that binds to human CD89, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 59 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions, and a light chain variable region having the amino acid sequence of SEQ ID NO: 60 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or additions. In preferred embodiments, the amino acid insertions, deletions, substitutions, or additions are located in framework regions of the light and / or heavy chain variable regions. An exemplary antibody having these characteristics is antibody 26D6.

[0139] The anti-CD89 antibodies or antigen-binding fragments thereof of the present disclosure preferably comprise the heavy chain variable region and light chain variable region described herein. Such antibodies have favorable characteristics. Of course, it is possible to generate variants of such original antibodies by modifying one or more amino acids therein. Many such variants will behave more or less similarly when compared to the original. Such variants are also within the scope of the present disclosure.

[0140] Variants may have amino acid substitutions, insertions, deletions, or additions relative to the sequence of the original antibody. An amino acid substitution is the replacement of an amino acid with another amino acid. Preferably, an amino acid is previously placed with an amino acid having similar chemical properties, often referred to as a conservative substitution. An amino acid deletion results in the deletion of one or more amino acids from the sequence. An amino acid insertion results in one or more additional amino acids in the sequence. An amino acid addition results in one or more amino acids at the beginning or end of the amino acid sequence.

[0141] A non-limiting example of such a modification is an antibody containing pyroglutamate instead of glutamate. Other non-limiting examples of such modifications are the insertion, deletion, inversion, and / or substitution of one or more amino acids compared to the original antibody. Preferably, the amino acid substitution, insertion, deletion, or addition is outside the CDR of the variable domain. Preferably, the amino acid substitution, insertion, deletion, or addition is within the framework regions of the variable region and / or in the constant region of the antibody. CD89 binding of the variants can be tested as described herein.

[0142] In some embodiments, the constant region of an antibody of the invention is the constant region of an IgG, IgA, IgD, IgE, or IgM antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody. The constant region may contain modifications such as amino acid substitutions to confer specific properties to the constant region. For example, mutations in the IgG4 hinge region to make the antibody more stable against half-molecule exchange. Other modifications affect the half-life of the antibody, add or remove glycosylation sites, improve production, improve the homogeneity of antibody products produced in large-scale fermenters, etc.

[0143] The antibodies of the present invention are preferably murine IgG1, human IgG1 mutated in the constant region to reduce or prevent complement activation or Fc receptor interaction, or human IgG4, or human IgG4 mutated to prevent half-molecule exchange with other IgG4 molecules. In a preferred embodiment, the antibodies disclosed herein are humanized IgG4, or humanized IgG4 mutated to prevent half-molecule exchange with other IgG4 molecules, or humanized IgG1 mutated in the constant region to reduce or prevent complement activation or Fc receptor interaction.

[0144] Some variation in the constant regions of the antibodies disclosed herein is tolerated. Typically, about 0 to 10 amino acid substitutions are tolerated in the constant region. Often, more than 10 amino acid changes are tolerated. Antibodies of the present invention may have a heavy chain constant region (CH1-CH2-CH3) with 0 to 15, preferably 0 to 10, more preferably 0 to 5, more preferably 5, 4, 3, 2, 1, or 0 amino acid substitutions relative to a naturally occurring heavy chain constant region (CH1-CH2-CH3). Such antibodies may have a light chain constant region with 0 to 5, preferably 5, 4, 3, 2, 1, or 0 amino acid substitutions relative to a naturally occurring light chain constant region.

[0145] Some variation in IgG4 occurs naturally and / or is tolerated without altering the immunological properties of the resulting antibody. Antibodies with IgG4 constant regions or mutated IgG1 constant regions will have at least most of the pharmacological properties of antibodies, but will not fix complement and therefore will not induce in vivo depletion of cells to which they bind. Preferably, the constant region is a human antibody constant region (chimera).

[0146] Preferably, the constant region is a region that is deficient in complement activation, and is preferably a human IgG4 constant region or a mutated human IgG1 constant region. In preferred embodiments, the humanized antibodies disclosed herein have an IgG4 or IgG1 isotype.

[0147] CD89 binding by the antibodies and antigen-binding fragments thereof disclosed herein can be confirmed by any of a number of suitable assays known to those of skill in the art. Such assays include, for example, affinity assays such as Western blots, radioimmunoassays, FACS, and ELISA (enzyme-linked immunosorbent assays). The Examples (e.g., Example 2(a)) detail some of the many assays that can be used to measure CD89 binding and methods for determining the relative binding affinity of antibodies to human CD89.

[0148] The term "binding molecule" encompasses (1) antibodies, (2) antigen-binding fragments of antibodies, and (3) derivatives of antibodies, each as defined herein. The terms "binds to CD89" or "capable of binding to CD89" or "binding to CD89," as defined herein, refer to binding of a binding molecule to the human CD89 receptor in an in vitro assay, e.g., BIAcore™ (surface plasmon resonance) or Octet® (biolayer interferometry). A binding molecule is present in an amount of approximately 1 x 10 -6 M or less, approximately 1×10 -7 M or less, approximately 1×10 -8 M or less, approximately 1×10 -9 M or less, approximately 1×10 -10 M or less, approximately 1×10 -11 M or less, or about 1 x 10 -12 Binding affinity (K D ) Preferably, the binding is specific, meaning that CD89 or an epitope thereof is specifically bound by the binding molecule. Affinity is a measure of the strength of binding to a particular antigen or epitope. Specific binding or "specific recognition," as used herein, means a binding affinity of at most 1 x 10E -6 M, preferably at most 1 × 10E -7 M, 1×10E- 8 M, or most preferably at most 1 x 10E -9 Affinity of M (K D ) is defined as a bond.

[0149] As described herein below, the K D is preferably less than 1.5 nM, such as less than 1.4 nM, less than 1.3 nM, less than 1.2 nM, less than 1.1 nM, or less than 1 nM. D is the K of the parent murine antibody, which is approximately 1.7 nM D is surprisingly small in comparison.

[0150] "K DThe term "K" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction and is used to describe the binding affinity between a ligand (such as an antibody) and a protein (such as CD89). The smaller the equilibrium dissociation constant, the tighter the ligand binds or the higher the affinity between the ligand and the protein. D can be measured by assays based on surface plasmon resonance and biolayer interferometry. The term "anti-CD89 antibody", as defined herein, refers to an antibody capable of binding to CD89, preferably human CD89.

[0151] "K on " and "K off The terms "K" and "K" refer to the rate constants of a ligand (such as an antibody) for association (on-rate) and dissociation (off-rate), respectively, from its target protein (such as CD89). on ” characterizes the rate at which a ligand (such as an antibody) binds to its target protein (such as CD89), and is expressed as “K off " characterizes the rate at which a ligand (such as an antibody) dissociates from its target protein (such as CD89).

[0152] The ability of the antibodies and antigen-binding fragments thereof disclosed herein to block the interaction of IgA with the CD89 receptor can be confirmed by a number of suitable assays known to those of skill in the art. Such assays include, for example, the affinity assays ELISA and FACS. The examples provided (e.g., Example 2(b)) detail two of the many assays, FACS and ELISA, that can be used to test the ability of anti-CD89 antibodies to block the binding of IgA to the CD89 receptor.

[0153] To test the IgA-blocking characteristics of an antibody using an ELISA assay, recombinant CD89 is coated onto a plate. The coated plate is then blocked using a blocking buffer to prevent nonspecific binding. The plate with the recombinant CD89 is then incubated with the antibody of interest and / or hybridoma supernatant. IgA is then added to the wells containing CD89. After washing, the amount of bound IgA is measured using ELISA technology. The amount of bound IgA indicates the blocking ability of the tested antibody, whereby low IgA binding indicates strong blocking ability of the antibody. CD89-expressing cells can also be used to test the IgA-blocking characteristics of an antibody by FACS assay. Preferably, the cells stably express human CD89. The CD89-expressing cells are incubated with the antibody of interest or the hybridoma supernatant. The cells are then incubated with IgA. After washing, the cell-bound IgA is labeled using a secondary antibody against IgA, preferably a fluorescent secondary antibody. The binding of IgA to the membrane of human CD89-expressing cells can be measured using a flow cytometer (FACS). The amount of bound IgA indicates the blocking ability of the tested antibody, whereby low binding of IgA indicates a strong blocking ability of the antibody.

[0154] To analyze whether the purified anti-human CD89 antibodies disclosed herein can displace IgA previously saturated with human CD89, those skilled in the art can use several known suitable assays. One suitable test method is disclosed in the Examples section. In this assay, IgA is bound to CD89-expressing cells. The anti-CD89 antibody is then added to the cells. The amount of IgA still bound to the cells can be measured by FACS analysis. The assay is described in detail in Example 2. This and other assays can be used to measure human IgA displacement by anti-human CD89 antibodies. Displacement can be measured using metabolically active cells (e.g., incubated overnight at 37°C) or metabolically inactive cells (e.g., incubated at 4°C in the presence of sodium azide).

[0155] In a further aspect, the present disclosure provides nucleic acid molecule(s) encoding the antibodies or antigen-binding fragments disclosed herein. Nucleic acid molecules encoding the variable regions disclosed herein are also provided. The nucleic acids used in the present disclosure are typically, but not exclusively, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Based on the genetic code, those skilled in the art can determine the nucleic acid sequence encoding the antibody variants disclosed herein. Based on the degeneracy of the genetic code, 64 codons can be used to encode 20 amino acids and a translation termination signal. As known to those skilled in the art, codon usage bias in different organisms can affect gene expression levels. Various computational tools are available to those skilled in the art to optimize codon usage depending on the organism in which the desired nucleic acid will be expressed.

[0156] In a further aspect, the present disclosure provides vectors comprising the nucleic acid sequence molecules described herein. As used herein, the term "vector" refers to a nucleic acid molecule, such as a plasmid, bacteriophage, or animal virus, capable of introducing a heterologous nucleic acid sequence into a host cell. A vector according to the present invention allows for the expression or production of an antibody of the present invention encoded by a heterologous nucleic acid sequence in a host cell. Vectors used according to the present invention may be derived from, for example, an animal virus, including, but not limited to, vaccinia virus (including attenuated derivatives such as modified vaccinia virus Ankara, MVA), Newcastle disease virus (NDV), adenovirus, or retrovirus. A vector according to the present invention preferably comprises an expression cassette comprising a promoter suitable for initiating transcription of an antibody of the present invention in a selected host cell. Examples of promoters suitable for expression of a polypeptide according to the present invention in a eukaryotic host cell include, but are not limited to, the β-actin promoter, immunoglobin promoter, 5S RNA promoter, or virus-derived promoters such as cytomegalovirus (CMV), Rous sarcoma virus (RSV), and simian virus 40 (SV40) promoters for mammalian hosts.

[0157] When the nucleic acid molecule(s) disclosed herein are expressed in a cell, the cell can produce an antibody according to the present disclosure. Thus, in one embodiment, a cell is provided comprising an antibody, nucleic acid molecule(s), and / or vector according to the present disclosure. The host cell can be a mammalian cell, an insect cell, a plant cell, a bacterial cell, or a yeast cell. The cell is preferably an animal cell, preferably a mammalian cell, and most preferably a human cell. Examples of mammalian cell lines suitable as host cells include hybridoma cells, Chinese hamster ovary (CHO) cells, NSO cells, or PER-C6™ cells. A suitable cell for the purposes of the present disclosure is any cell capable of containing, and preferably producing, the antibody and / or the nucleic acid. The present disclosure further encompasses a cell culture comprising the cell.

[0158] The term "host cell" refers to a cell into which an expression vector expressing an anti-human CD89 antibody described herein has been introduced. The term encompasses not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in successive generations, either due to environmental influences or mutations, such progeny may not be identical to the parent cell but are still included within the scope of the term "host cell."

[0159] The antibodies disclosed herein can be produced by any method known to those of skill in the art. In a preferred embodiment, the antibodies are produced using cells, preferably hybridoma cells, CHO cells, NS0 cells, or PER-C6™ cells. In a particularly preferred embodiment, the cells are CHO cells, and preferably the cells are cultured in serum-free medium. This includes harvesting the antibody from the culture. The antibody is preferably purified from the medium, preferably the antibody is affinity purified. Alternatively, the antibody can be synthetically produced.

[0160] Various institutions and companies have developed cell lines for the large-scale production of antibodies, for example, for clinical use. These cells are also used for other purposes, such as the production of proteins. Cell lines developed for the industrial-scale production of proteins and antibodies are further referred to herein as industrial cell lines. Accordingly, preferred embodiments of the present disclosure provide for the use of cell lines developed for the large-scale production of such antibodies.

[0161] The antibodies according to the present invention exhibit several activities that can be advantageously used in therapeutic and non-therapeutic uses. In particular, the antibodies according to the present invention are useful for treating individuals. Preferably, the antibodies of the present invention are useful for treating or preventing immune-related diseases. In some embodiments, the antibodies according to the present invention are preferably used in therapy, preferably human therapy. In some embodiments, the antibodies disclosed herein can be used for research purposes, for example, in in vitro experiments, cell culture, organ culture, and in vivo models.

[0162] Methods for the treatment or prevention of chronic inflammatory diseases (CIDs) have also been described. Examples of CIDs include inflammatory bowel diseases (IBDs) such as ulcerative colitis or Crohn's disease, chronic obstructive pulmonary disease (COPD), asthma, allergic and non-allergic rhinitis, food allergies such as celiac disease, and skin diseases such as linear IgA bullous disease or dermatitis herpetiformis. One of the common features contributing to the tissue destruction observed in CIDs is the local accumulation of polymorphonuclear cells, more specifically neutrophils and / or eosinophils. Polymorphonuclear cells are leukocytes characterized by the presence of granules in their cytoplasm.

[0163] Binding of IgA to its receptor CD89 can trigger a cascade of events, including immune cell activation, ultimately leading to the migration, accumulation, and infiltration of polymorphonuclear cells. Because cross-linking of CD89 by IgA-immune complexes potently recruits and activates neutrophils, the presence of abnormal IgA can lead to an exacerbated pro-inflammatory response, resulting in tissue damage. This may play a role in various CIDs characterized by elevated serum (autologous) IgA levels, such as IgA nephropathy, Henoch-Schonlein purpura, ankylosing spondylitis, Sjögren's syndrome, alcoholic cirrhosis, celiac disease, asthma, IBD, rheumatoid arthritis, linear IgA bullous disease, and dermatitis herpetiformis (Aleyd et al. Immunol Rev 2015;268:123-138). Interfering with the binding between IgA and its receptor CD89 can inhibit the signaling cascade and polymorphonuclear cell accumulation. Therefore, CID can be treated or prevented by administering an effective amount of the antibody of the present invention to a patient in need of such treatment. Blocking the interaction between IgA and receptors on polymorphonuclear cells such as neutrophils can stop the inflammatory response. Therefore, patients with CID can benefit from treatment using the antibodies disclosed herein.

[0164] The present invention provides a method for treating a subject suffering from an inflammatory disease, comprising administering to the subject a therapeutically effective amount of an antibody disclosed herein. Methods for preparing a medicament for treating a subject suffering from an inflammatory disease are also provided. The present disclosure describes a method for preventing immune cell activation by blocking the binding between IgA and CD89.

[0165] The present disclosure further includes pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof disclosed herein, or nucleic acids encoding them, or cells comprising the antibodies or antigen-binding fragments thereof disclosed herein, or nucleic acids encoding them. Pharmaceutical compositions are provided comprising a polypeptide according to the present invention or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent, and / or excipient. Such compositions are particularly suitable for use as pharmaceuticals. The compositions may be in any suitable form, including liquid, semi-solid, and solid dosage forms. The dose and schedule of the selected formulation may be determined by standard procedures well known to those skilled in the art. Such procedures include extrapolating and extrapolating dosing schedules to form animal models and then determining optimal dosages in human clinical dose-ranging studies. The dosage in a pharmaceutical composition will vary depending on several factors, such as the desired release and pharmacological characteristics.

[0166] As used herein, a "subject" is a human or an animal. Subjects include, but are not limited to, mammals such as humans, pigs, ferrets, seals, rabbits, cats, dogs, cows, and horses, and birds such as chickens, ducks, geese, and turkeys. In preferred embodiments of the present invention, the subject is a mammal. In particularly preferred embodiments, the subject is a human.

[0167] The term "antigen-binding fragment" of an antibody refers to one or more portions of a full-length antibody that retain the ability of the antibody to bind to the same antigen (i.e., human CD89). The term "antigen-binding fragment" also encompasses portions of antibodies that are part of a larger molecule formed by non-covalent or covalent association, or that have one or more additional molecular entities. Examples of additional molecular entities include amino acids, peptides, or proteins, such as the streptavidin core region, which can be used to generate tetrameric scFv molecules (Kipriyanov et al. Hum Antibodies Hybridomas 1995;6(3):93-101). Exemplary antigen-binding fragments are the VH and / or VL of an antibody. Antigen-binding fragments include Fab, F(ab'), F(ab')2, complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, and other antigen-recognizing immunoglobulin fragments. In some instances, the term "antibody" as used herein can be understood to include antigen-binding fragments thereof.

[0168] The term "human antibody" refers to an antibody that consists of amino acid sequences exclusively of human immunoglobulin sequences. A human antibody may contain murine carbohydrate chains if produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell. Human antibodies can be prepared in a variety of ways known in the art.

[0169] The term "epitope" refers to a portion of an antigen that can specifically bind to an antibody or T-cell receptor, or otherwise interact with a molecule. "Epitope" is also referred to in the art as "antigenic determinant." Epitopes generally consist of chemically active surface groupings of molecules, such as amino acids, carbohydrates, or sugar side chains. Epitopes can be "linear" or "non-linear / conformational." Once a desired epitope is determined (e.g., by epitope mapping), antibodies can be generated against that epitope. Antibody generation and characterization can also provide information about the desired epitope. From this information, it is then possible to screen antibodies for those that bind to the same epitope, for example, by performing cross-competition studies to find antibodies that competitively bind with each other, i.e., antibodies that compete for binding to the antigen.

[0170] As used herein, "comprise" and its conjugations are used in their open-ended sense, meaning that the items following the word are included, but not items not specifically mentioned are excluded. Additionally, the verb "consisting of" may be replaced with "consisting essentially of." This means that a compound or ancillary compound defined herein may contain additional components other than those specifically identified, where such additional components do not alter the inherent characteristics of the invention.

[0171] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0172] The word "approximately" or "about," when used in connection with a numerical value (approximately 10, about 10), preferably means that the value may be 1% more or less than 10 of the given value.

[0173] As used herein, the terms "treatment," "treat," and "treating" refer to reversing, alleviating, delaying the onset of, or inhibiting the progression of a disease or disorder described herein, or one or more symptoms thereof. In some embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, e.g., to prevent or delay their recurrence.

[0174] For purposes of clarity and conciseness of description, features may be described herein as part of the same or separate embodiments, however, it will be understood that the scope of the present invention may include embodiments having all or any combination of the described features.

[0175] All patents and literature references cited herein are incorporated by reference in their entirety.

[0176] The present invention is further described in the following examples, which do not limit the scope of the invention but merely serve to clarify it. [Example]

[0177] Example 1. Generation of CD89 / IgA-blocking mouse anti-human CD89 monoclonal antibodies (a) Generation of HEK293F cells transiently expressing surface human CD89 for immunization. A cDNA encoding the human full-length CD89 protein (Swiss-Prot number P24071.1, see SEQ ID NO: 1) was optimized for mammalian expression and synthesized by GENEART, Regensburg, Germany (see SEQ ID NO: 2). This cDNA was subcloned into a pcDNA3.1-derived expression plasmid. This full-length human CD89 plasmid was transiently transfected into FreeStyle™ 293F cells (Life Technologies) using the FreeStyle™ 293 Expression System (Life Technologies). After two days, these HEK293F cells were harvested, washed with sterile phosphate-buffered saline (PBS), and incubated for 2 days at 4°C for 1 hour.

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[0178] (b) Generation of HEK293F cells stably expressing surface human CD89 for screening. A cDNA encoding human full-length CD89 protein (Swiss-Prot No. P24071.1, see SEQ ID NO: 1) was optimized for mammalian expression and synthesized by GENEART, Regensburg, Germany (see SEQ ID NO: 2). This cDNA was subcloned into a pcDNA3.1-derived expression plasmid. This full-length human CD89 plasmid was transfected into FreeStyle™ 293F cells (Life Technologies) using the FreeStyle™ 293 Expression System (Life Technologies). HEK293F cell clone number 2, stably transfected with human full-length CD89, was selected using 125 μg / mL G418 / Geneticin (Gibco). Human CD89 surface expression on transfected HEK293F cells was confirmed by flow cytometry using a 1:20 diluted PE-conjugated mouse anti-human CD89 antibody (clone MIP8a, BioRad).

[0179] (c) Immunization and generation of mouse anti-human CD89 monoclonal antibodies Four BALB / c mice (female, 6-8 weeks old, Charles River Laboratories) were

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[0180] (d) Screening for the presence of mouse anti-human CD89 monoclonal antibodies Starting from days 12–14 after each fusion, supernatants from growing hybridomas were screened for the presence of IgG class mouse anti-human CD89 antibodies (i.e., "high affinity" IgG as opposed to "low affinity" IgM) using an ELISA with recombinant C-terminal polyhistidine-tagged human (extracellular) CD89 (rhuCD89, Sino Biological) as the target protein. To this end, rhuCD89 was coated at 0.5 μg / mL (25 ng / 50 μL / well) in PBS onto half-section 96-well EIA plates (Corning) for 16–24 h at 4–8 °C. After extensive washing with PBS / 0.05% Tween 20, the plates were blocked with PBS / 0.05% Tween 20 / 1% bovine serum albumin (BSA, Roche) for 1 h at room temperature. Subsequently, the plates were incubated with 50 μL of undiluted hybridoma supernatant / well for 1 h at room temperature. In parallel, 50 μL of culture medium (opti-MEM® I with GlutaMax / 10% FCS) and 50 μL of 10 μg / mL (diluted in medium) mouse anti-human CD89 antibody clone MIP8a (BioRad) were run as negative and positive controls, respectively. After extensive washing with PBS / 0.05% Tween 20, antibody binding to rhuCD89 was determined using a 1:5,000 diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG Fcγ-specific antibody (Jackson ImmunoResearch) for 1 h at room temperature, followed by a ready-to-use solution of TMB substrate (Invitrogen) for colorimetric detection. After adding 1 M H2SO4, antibody binding (optical density) to rhuCD89 was measured using a microplate reader (iMark, BioRad) at a wavelength of 450 nm (reference wavelength of 655 nm).

[0181] From days 12-14 post-fusion, supernatants from growing hybridomas were also screened and confirmed for production of IgG class mouse anti-human CD89 antibodies (i.e., "high affinity" IgG as opposed to "low affinity" IgM) using FACS with membrane-bound human CD89 as the target protein. To this end, HEK293F cells (clone no. 2, see Example 1(b) above) stably transfected with human full-length CD89 were diluted to 10x10 in ice-cold PBS containing 0.1% BSA (Sigma) / 0.05% NaN (PBS / BSA / NaN) supplemented with 50µg / mL human IgG (to block potential Fcγ receptors, Sigma). 6 The cells were then incubated at 4°C for 10 minutes at 10 µL / tube (i.e., 0.1 x 10 cells / mL). 6 These cells (100 μL each) were incubated with 100 μL of undiluted hybridoma supernatant per tube at 4°C for 30 minutes. In parallel, 100 μL of culture medium (opti-MEM® I with GlutaMax / 10% FCS), 100 μL of 10 μg / mL mouse IgG1 isotype control (BD Biosciences), and 100 μL of 10 μg / mL mouse IgG2a isotype control (BD Biosciences) were run as negative controls, and 100 μL of 10 μg / mL mouse anti-human CD89 antibody clone MIP8a (BioRad) was run as a positive control. Untransfected wild-type (WT) HEK293F cells (i.e., negative for membrane-bound human CD89 expression) were also run as negative control cells to determine antibody specificity. After extensive washing with PBS / BSA / NaN3, cells were then incubated with a 1:200 diluted PE-conjugated goat anti-mouse IgG Fcγ-specific antibody (Jackson ImmunoResearch) for 30 minutes at 4°C. After extensive washing with PBS / BSA / NaN3, cells were fixed in 2% formaldehyde in PBS / BSA / NaN3 for 30 minutes at 4°C. Binding of the antibody to membrane human CD89 (geometric mean fluorescence intensity) was measured using a flow cytometer (FACSCalibur, BD Biosciences).

[0182] Dual-CD89-positive (i.e., rhuCD89+ by ELISA (data not shown) and membrane CD89+ HEK293F cells by FACS (Figure 1)) hybridomas were expanded and cryopreserved. Supernatants from these dual-CD89-positive hybridomas showed no reactivity with untransfected (i.e., negative for membrane-bound human CD89 expression) WT HEK293F cells. As shown in Figure 1, this approach yielded 21 mouse anti-human CD89-specific antibody-producing hybridomas. Supernatants from these mouse anti-human CD89-specific antibody-producing hybridomas were subsequently tested for their ability to block the binding of serum human IgA on its receptor, CD89 (see Example 1(e) below).

[0183] (e) Screening for the presence of CD89 / IgA-blocking mouse anti-human CD89 monoclonal antibodies To analyze the effect of mouse anti-human CD89 antibodies on the binding of serum human IgA to human CD89, ELISA and FACS analysis were used to determine the ability of mouse anti-human CD89 antibodies to sterically hinder the interaction between serum human IgA and human CD89.

[0184] ELISA: rhuCD89 was coated at 0.5 μg / mL (25 ng / 50 μL / well) in PBS using half-area 96-well EIA plates (Corning) for 16–24 h at 4–8°C. After extensive washing with PBS / 0.05% Tween 20, the plates were blocked with PBS / 0.05% Tween 20 / 1% BSA (Roche) for 1 h at room temperature. Subsequently, the plates were incubated with 25 μL of undiluted hybridoma supernatant / well for 30 h at room temperature. In parallel, 25 μL of culture medium (opti-MEM® I with GlutaMax / 10% FCS) and 25 μL of mouse anti-human CD89 antibody clone MIP8a (BioRad) at 20 μg / mL (diluted in medium) were run as negative and positive controls, respectively. After this (i.e., without washing), 25 μL of purified human (serum-derived) IgA (Bethyl Laboratories) at 2 μg / mL (diluted in culture medium) was added to the wells and incubated for an additional 30 minutes at room temperature. After extensive washing with PBS / 0.05% Tween 20, binding of serum human IgA to rhuCD89 was measured with 1 μg / mL biotin-conjugated F(ab')2 fragment anti-human goat serum IgA α-chain-specific antibody (Jackson ImmunoResearch) for 1 hour at room temperature. After extensive washing with PBS / 0.05% Tween 20, HRP-conjugated streptavidin (Jackson ImmunoResearch) diluted 1:10,000 was added and incubated for 1 h at room temperature, followed by the addition of a ready-to-use solution of TMB substrate (Invitrogen) for colorimetric detection. After the addition of 1 M H2SO4, the binding (optical density) of serum human IgA to rhuCD89 was measured at a wavelength of 450 nm (reference wavelength of 655 nm) using a microplate reader (iMark, BioRad).

[0185] FACS: HEK293F cells stably transfected with human full-length CD89 (clone no. 2, see Example 1(b) above) were cultured at 10 x 10 in ice-cold PBS containing 0.1% BSA (Sigma) / 0.05% NaN (PBS / BSA / NaN).6 Then, 10 μL / tube (i.e., 0.1 × 10 cells / mL) was added. 6 These cells (100 μL each) were incubated with 50 μL of undiluted hybridoma supernatant per tube at 4°C for 30 minutes. In parallel, 50 μL of culture medium (opti-MEM® I with GlutaMax / 10% FCS), 50 μL of 20 μg / mL mouse IgG1 isotype control (BD Biosciences), and 50 μL of 20 μg / mL mouse IgG2a isotype control (BD Biosciences) were run as negative controls, and 50 μL of 20 μg / mL mouse anti-human CD89 antibody clone MIP8a (BioRad) was run as a positive control. After this (i.e., without washing), 50 μL of 20 μg / mL purified human (serum-derived) IgA (Bethyl Laboratories) (diluted in culture medium) was added to the cells and incubated for an additional 30 minutes at 4°C. After extensive washing with PBS / BSA / NaN3, binding of serum human IgA to membrane human CD89 was determined for 30 minutes at 4°C using 5 μg / mL biotin-conjugated F(ab')2 fragment goat anti-human serum IgA α-chain-specific antibody (Jackson ImmunoResearch). After extensive washing with PBS / BSA / NaN3, PE-conjugated streptavidin (Jackson ImmunoResearch) diluted 1:200 was added and incubated for 30 minutes at 4°C. After extensive washing with PBS / BSA / NaN3, cells were fixed in 2% formaldehyde in PBS / BSA / NaN3 for 30 minutes at 4°C. Binding of serum human IgA to membrane human CD89 (geometric mean fluorescence intensity) was measured using a flow cytometer (FACSCalibur, BD Biosciences).

[0186] As shown in Figure 2A, six of the 21 examined supernatants from mouse anti-human CD89-specific antibody-producing hybridomas (i.e., 8F3, 9H7, 10E7, 26D6, 20B4, and 30C7) showed strong / complete blocking of serum human IgA binding to rhuCD89, whereas one of the 21 examined supernatants from a mouse anti-human CD89-specific antibody-producing hybridoma (i.e., 16D6) showed intermediate / partial blocking of serum human IgA binding to rhuCD89. For reference purposes, purified mouse anti-human CD89 antibody clone MIP8a, a known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), was run in parallel and showed strong / complete blocking of serum human IgA binding to rhuCD89.

[0187] As shown in Figure 2B, six of the 21 examined supernatants from mouse anti-human CD89-specific antibody-producing hybridomas (i.e., 8F3, 9H7, 10E7, 26D6, 20B4, and 30C7) showed strong / complete blocking of serum human IgA binding to membrane-bound human CD89, whereas one of the 21 examined supernatants from a mouse anti-human CD89-specific antibody-producing hybridoma (i.e., 16D6) showed intermediate / partial blocking of serum human IgA binding to membrane-bound human CD89. For reference purposes, purified mouse anti-human CD89 antibody clone MIP8a (BioRad), a known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), was run in parallel and showed strong / complete blocking of serum human IgA binding to membrane-bound human CD89.

[0188] Mouse antibodies were purified from the supernatants of the CD89 / IgA-blocked mouse anti-human CD89-specific antibody-producing hybridomas using a Protein G column (GE Healthcare). The heavy and light chains were isotyped using the IsoStrip™ Mouse Monoclonal Antibody Isotyping Kit (Roche). All purified CD89 / IgA-blocked mouse anti-human CD89-specific antibodies (i.e., 8F3, 9H7, 10E7, 16D6, 26D6, 20B4, and 30C7) were found to be IgG1 / κ. Additionally, LPS levels were determined using an LAL chromogenic endpoint assay (Hycult Biotech). All purified CD89 / IgA-blocked mouse anti-human CD89-specific antibodies (i.e., 8F3, 9H7, 10E7, 16D6, 26D6, 20B4, and 30C7) contained <0.005 EU LPS / μg of mouse IgG. These purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies were then extensively tested for their relative binding affinity to human CD89, their blocking effect on serum human IgA binding to human CD89, and their blocking effect on serum human IgA-mediated phagocytosis and migration by human CD89-expressing primary human neutrophil granulocytes, as described in Examples 2 and 3. In addition, the excellent specificity of these purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies was investigated by cross-competition with known commercial CD89 / IgA-blocking and non-blocking mouse anti-human CD89-specific antibodies, by human CD89 / bovine Fcγ2R domain mapping, by cross-species binding to cynomolgus CD89, and by human / cynomolgus CD89 epitope mapping, as described in Example 4.

[0189] Example 2. Binding Characterization of CD89 / IgA Blocking Mouse Anti-Human CD89 Monoclonal Antibodies (a) Relative binding affinity of CD89 / IgA-blocking mouse anti-human CD89 antibodies to human CD89 ELISA and FACS analysis were used to determine the relative binding affinity of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies to human CD89.

[0190] ELISA: rhuCD89 was coated at 0.5 μg / mL (25 ng / 50 μL / well) in PBS using half-area 96-well EIA plates (Corning) for 16–24 h at 4–8 °C. After extensive washing with PBS / 0.05% Tween 20, the plates were blocked with PBS / 0.05% Tween 20 / 1% BSA (Roche) for 1 h at room temperature. Subsequently, the plates were incubated with 50 μL of titrated purified mouse anti-human CD89 antibody / well (in blocking buffer) for 1 h at room temperature. In parallel, 50 μL of titrated purified mouse anti-human CD89 antibody clone MIP8a (BioRad) (in blocking buffer) was run as a positive control. After extensive washing with PBS / 0.05% Tween 20, antibody binding to rhuCD89 was determined using a 1:5,000 diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG Fcγ-specific antibody (Jackson ImmunoResearch) for 1 h at room temperature, followed by a ready-to-use solution of TMB substrate (Invitrogen) for colorimetric detection. After adding 1 M H2SO4, antibody binding (optical density) to rhuCD89 was measured at a wavelength of 450 nm (reference wavelength of 655 nm) using a microplate reader (iMark, BioRad).

[0191] FACS: HEK293F cells stably transfected with human full-length CD89 (clone no. 2, see Example 1(b) above) were cultured at 10x10 in ice-cold PBS containing 0.1% BSA (Sigma) / 0.05% NaN3 (PBS / BSA / NaN3) supplemented with 50 μg / mL human IgG (to block potential Fcγ receptors, Sigma). 6 The cells were then incubated at 4°C for 10 minutes at 10 µL / tube (i.e., 0.1 x 10 cells / mL). 6These cells (100 μL each) were incubated with 100 μL of titrated purified mouse anti-human CD89 antibody (in PBS / BSA / NaN3) per tube for 30 minutes at 4°C. In parallel, 100 μL of titrated purified mouse IgG1 isotype control (BD Biosciences) and 100 μL of titrated purified mouse anti-human CD89 antibody clone MIP8a (BioRad) (in PBS / BSA / NaN3) were run as negative and positive controls, respectively. After extensive washing with PBS / BSA / NaN3, the cells were then incubated with a 1:200 diluted PE-conjugated goat anti-mouse IgG Fcγ-specific antibody (Jackson ImmunoResearch) for 30 minutes at 4°C. After extensive washing with PBS / BSA / NaN3, the cells were fixed in 2% formaldehyde in PBS / BSA / NaN3 for 30 minutes at 4°C. Binding of antibodies to membrane human CD89 (geometric mean fluorescence intensity) was measured using a flow cytometer (FACSCalibur, BD Biosciences).

[0192] As shown in Figure 3A, all purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies bound to rhuCD89 in a dose-dependent manner. Based on their binding profiles, the following relative affinity ranking was found (from high affinity to low affinity): 9H7 = 26D6 = 20B4 > 8F3 = 10E7 = 30C7 (= MIP8a) > 16D6. For reference purposes, purified mouse anti-human CD89 antibody clone MIP8a, a known CD89 / IgA-blocking agent (Zhang et al. Clin Exp Immunol 2000;121:106-111), was run in parallel and also showed dose-dependent binding to rhuCD89.

[0193] As shown in Figure 3B, all purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies bound to membrane human CD89 in a dose-dependent manner. Based on their binding profiles, the following relative affinity ranking was found (from high affinity to low affinity): 9H7 = 26D6 = 20B4 (=MIP8a) > 8F3 = 10E7 = 30C7 > 16D6, which was consistent with the relative affinity ranking found in ELISA. For reference purposes, purified mouse anti-human CD89 antibody clone MIP8a, a known CD89 / IgA-blocking agent (Zhang et al. Clin Exp Immunol 2000;121:106-111), was run in parallel and also showed dose-dependent binding to membrane human CD89.

[0194] (b) CD89 / IgA blocking ability of mouse anti-human CD89 antibodies To analyze the extent of CD89 / IgA blocking of purified mouse anti-human CD89 antibodies, the ability of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies to sterically interfere with the interaction of human IgA with human CD89 was determined using ELISA and FACS analysis.

[0195] ELISA: rhuCD89 was coated at 0.5 μg / mL (25 ng / 50 μL / well) in PBS using half-area 96-well EIA plates (Corning) for 16–24 h at 4–8 °C. After extensive washing with PBS / 0.05% Tween 20, the plates were blocked with PBS / 0.05% Tween 20 / 1% BSA (Roche) for 1 h at room temperature. Subsequently, the plates were incubated with 25 μL of titrated purified mouse anti-human CD89 antibody / well (in blocking buffer) for 30 h at room temperature. In parallel, 25 μL of titrated purified mouse anti-human CD89 antibody clone MIP8a (BioRad) (in blocking buffer) was run as a positive control. After this (i.e., without washing), 25 μL of purified human (serum-derived) IgA (Bethyl Laboratories) at 2 μg / mL (diluted in blocking buffer) was added to the wells and incubated for an additional 30 minutes at room temperature. After extensive washing with PBS / 0.05% Tween 20, binding of serum human IgA to rhuCD89 was measured with 1 μg / mL biotin-conjugated F(ab')2 fragment anti-human goat serum IgA α-chain-specific antibody (Jackson ImmunoResearch) for 1 hour at room temperature. After extensive washing with PBS / 0.05% Tween 20, HRP-conjugated streptavidin (Jackson ImmunoResearch) diluted 1:10,000 was added and incubated for 1 h at room temperature, followed by the addition of a ready-to-use solution of TMB substrate (Invitrogen) for colorimetric detection. After the addition of 1 M H2SO4, the binding (optical density) of serum human IgA to rhuCD89 was measured at a wavelength of 450 nm (reference wavelength of 655 nm) using a microplate reader (iMark, BioRad).

[0196] FACS: HEK293F cells stably transfected with human full-length CD89 (clone no. 2, see Example 1(b) above) were cultured at 10x10 in ice-cold PBS containing 0.1% BSA (Sigma) / 0.05% NaN3 (PBS / BSA / NaN3) supplemented with 50 μg / mL human IgG (to block potential Fcγ receptors, Sigma). 6 The cells were then incubated at 4°C for 10 minutes at 10 µL / tube (i.e., 0.1 x 10 cells / mL). 6 These cells (100 μL each) were incubated with 50 μL of titrated purified mouse anti-human CD89 antibody (in PBS / BSA / NaN3) per tube for 30 minutes at 4°C. In parallel, 50 μL of titrated purified mouse IgG1 isotype control (BD Biosciences) (in PBS / BSA / NaN3) and 50 μL of titrated purified mouse anti-human CD89 antibody clone MIP8a (BioRad) (in PBS / BSA / NaN3) were run as negative and positive controls, respectively. After this (i.e., without washing), 50 μL of 20 μg / mL (diluted in PBS / BSA / NaN3) purified human (serum-derived) IgA (Bethyl Laboratories) was added to the cells and incubated for an additional 30 minutes at 4°C. After extensive washing with PBS / BSA / NaN3, binding of serum human IgA to membrane human CD89 was determined for 30 minutes at 4°C using 5 μg / mL biotin-conjugated F(ab')2 fragment goat anti-human serum IgA α-chain-specific antibody (Jackson ImmunoResearch). After extensive washing with PBS / BSA / NaN3, PE-conjugated streptavidin (Jackson ImmunoResearch) diluted 1:200 was added and incubated for 30 minutes at 4°C. After extensive washing with PBS / BSA / NaN3, cells were fixed in 2% formaldehyde in PBS / BSA / NaN3 for 30 minutes at 4°C. Binding of serum human IgA to membrane human CD89 (geometric mean fluorescence intensity) was measured using a flow cytometer (FACSCalibur, BD Biosciences).

[0197] As shown in Figure 4A, all purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies dose-dependently prevented serum human IgA binding to rhuCD89. Based on their CD89 / IgA-blocking profiles, the following ranking was found (from strongest to weakest CD89 / IgA blocking): 9H7 = 26D6 = 20B4 > 8F3 = 10E7 = 30C7 (=MIP8a) > 16D6. Interestingly, there appeared to be a strong positive relationship between the extent to which these investigated purified mouse anti-human CD89 antibodies sterically blocked serum human IgA binding to rhuCD89 (this example) and their respective relative binding affinities for rhuCD89 (see Example 2(a) above). For reference purposes, purified mouse anti-human CD89 antibody clone MIP8a, a known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), was run in parallel and also showed dose-dependent blockade of serum human IgA binding to rhuCD89.

[0198] As shown in Figure 4B, all purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies dose-dependently prevented serum human IgA binding to membrane human CD89. Based on their CD89 / IgA-blocking profiles, the following ranking was found (from strongest to weakest): 9H7 = 26D6 = 20B4 (=MIP8a) > 8F3 = 10E7 = 30C7 > 16D6, which was consistent with the degree of CD89 / IgA blocking found in ELISA. Interestingly, there appeared to be a strong positive relationship between the extent to which these investigated purified mouse anti-human CD89 antibodies sterically blocked serum human IgA binding to membrane human CD89 (this example) and their respective relative binding affinities for membrane human CD89 (see Example 2(a) above). For reference purposes, purified mouse anti-human CD89 antibody clone MIP8a, a known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), was run in parallel and also showed dose-dependent blockade of serum human IgA binding to membrane human CD89.

[0199] To analyze the extent of CD89 / IgA blocking of purified mouse anti-human CD89 antibodies, the ability of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies to sterically hinder the interaction of secreted human IgA with human CD89 was determined using FACS analysis.

[0200] HEK293F cells stably transfected with human full-length CD89 (clone no. 2, see Example 1(b) above) were cultured at 10 × 10 in ice-cold PBS containing 0.1% BSA (Sigma) / 0.05% NaN (PBS / BSA / NaN). 6 Then, 10 μL / tube (i.e., 0.1 × 10 cells / mL) was added. 6These cells (100 μL each) were incubated with or without 50 μL of titrated purified mouse anti-human CD89 antibody (in PBS / BSA / NaN3) per tube for 30 minutes at 4°C. In parallel, 50 μL of titrated purified mouse anti-human CD89 antibody clone MIP8a (BioRad) (in PBS / BSA / NaN3) was run as a positive control. After this (i.e., without washing), 50 μL of 0.16 μM (diluted in PBS / BSA / NaN3) purified human (colostrum-derived) IgA (BioRad) was added to the cells and incubated for an additional 30 minutes at 4°C. After extensive washing with PBS / BSA / NaN3, binding of secreted human IgA to membrane human CD89 was determined for 30 minutes at 4°C using 5 μg / mL biotin-conjugated F(ab')2 fragment goat anti-human serum IgA α-chain-specific antibody (Jackson ImmunoResearch). After extensive washing with PBS / BSA / NaN3, PE-conjugated streptavidin (Jackson ImmunoResearch) diluted 1:200 was added and incubated for 30 minutes at 4°C. After extensive washing with PBS / BSA / NaN3, cells were fixed in 4% formaldehyde in PBS / BSA / NaN3 for 30 minutes at 4°C. Binding of secreted human IgA to membrane human CD89 (geometric mean fluorescence intensity) was measured using a flow cytometer (FACSCalibur, BD Biosciences).

[0201] As shown in Figure 4C, all purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies dose-dependently prevented the binding of secreted human IgA to membrane human CD89. Based on their CD89 / IgA-blocking profiles, the following ranking was found (from strongest to weakest): 9H7 = 26D6 = 20B4 (=MIP8a) > 8F3 = 10E7 = 30C7 > 16D6, which was consistent with the degree of CD89 / IgA blocking using serum human IgA found by FACS (Figure 4B). Interestingly, there appeared to be a strong positive relationship between the extent to which these investigated purified mouse anti-human CD89 antibodies sterically blocked secreted human IgA binding to membrane human CD89 (this example) and their respective relative binding affinities for membrane human CD89 (see Example 2(a) above). For reference purposes, purified mouse anti-human CD89 antibody clone MIP8a, a known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), was run in parallel and also showed dose-dependent blockade of secreted human IgA binding to membrane human CD89.

[0202] Human CD89 has low / moderate affinity for monomeric human IgA

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[0203] HEK293F cells stably transfected with human full-length CD89 (clone no. 2, see Example 1(b) above) were cultured at 10 × 10 in ice-cold PBS containing 0.1% BSA (Sigma) / 0.05% NaN (PBS / BSA / NaN) supplemented with 50 μg / mL human IgG (to block potential Fcγ receptors, Sigma). 6 The cells were then added at 10 μL / tube (i.e., 0.1 × 10 cells / mL) and placed at 4°C for 10 minutes. 6These cells (20 μg / mL in PBS / BSA / NaN3) were incubated with or without 50 μL of purified mouse anti-human CD89 antibody at 20 μg / mL for 30 minutes at 4°C. In parallel, 50 μL of purified mouse IgG1 isotype control (BD Biosciences) at 20 μg / mL in PBS / BSA / NaN3 and 50 μL of purified mouse anti-human CD89 antibody clone MIP8a (a well-known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), BioRad) at 20 μg / mL in PBS / BSA / NaN3 were run as negative and positive controls, respectively. Additionally, 50 μL of purified mouse anti-human CD89 antibody clone A59 (a well-known CD89 / IgA non-blocking agent (Monteiro et al. J Immunol 1992;148:1764-1770), BD Biosciences) at 20 μg / mL (in PBS / BSA / NaN3) and 50 μL of purified mouse anti-human CD89 antibody clone A3 (a well-known CD89 / IgA non-blocking agent (Monteiro et al. J Immunol 1992;148:1764-1770), Santa Cruz Biotechnology) at 20 μg / mL (in PBS / BSA / NaN3) were run as additional negative controls. After this (i.e., without washing), 50 μL of purified non-aggregated or heat-aggregated human (serum-derived) IgA (Bethyl Laboratories) at 20 μg / mL (diluted in PBS / BSA / NaN3) was added to the cells and incubated for an additional 30 minutes at 4°C. After extensive washing with PBS / BSA / NaN3, binding of non-aggregated or heat-aggregated serum human IgA to membrane human CD89 was determined for 30 minutes at 4°C using 5 μg / mL biotin-conjugated F(ab')2 fragment goat anti-human serum IgA α-chain-specific antibody (Jackson ImmunoResearch). After extensive washing with PBS / BSA / NaN3, PE-conjugated streptavidin (Jackson ImmunoResearch) diluted 1:200 was added and incubated for 30 minutes at 4°C.After extensive washing with PBS / BSA / NaN3, cells were fixed in 2% formaldehyde in PBS / BSA / NaN3 for 30 min at 4°C. Binding (geometric mean fluorescence intensity) of non-aggregated or heat-aggregated serum human IgA to membrane human CD89 was measured using a flow cytometer (FACSCalibur, BD Biosciences).

[0204] As shown in Figure 5A, all of our purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies prevented the binding of heat-aggregated serum human IgA to membrane human CD89 to a similar extent as that seen with non-aggregated serum human IgA. For reference purposes, purified mouse anti-human CD89 antibody clone MIP8a, a known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), was run in parallel and also showed blocking of the binding of heat-aggregated serum human IgA to membrane human CD89 to a similar extent as that seen with non-aggregated serum human IgA. Surprisingly, purified mouse anti-human CD89 antibodies clone A59 and clone A3, both of which are known CD89 / IgA non-blocking agents (Monteiro et al. J Immunol 1992;148:1764-1770), partially blocked the binding of heat-aggregated serum human IgA to membrane human CD89 to an extent similar to that found with non-aggregated serum human IgA.

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[0205] Collectively, these results demonstrated that the CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 8F3, 9H7, 10E7, 16D6, 26D6, 20B4, and 30C7 prevented the binding of monomeric, dimeric, trimeric, tetrameric, or higher multimeric serum human IgA (i.e., nonaggregated and heat-aggregated IgA) and dimeric secretory human IgA to membrane human CD89. For a summary, see Table 1A (i.e., serum human IgA blocking).

[0206] (c) Replacement of serum human IgA by CD89 / IgA-blocking mouse anti-human CD89 antibody To analyze whether purified CD89 / IgA-blocking mouse anti-human CD89 antibodies were able to displace previously saturated serum human IgA onto human CD89, the effect of purified mouse anti-human CD89 antibodies on the displacement of serum human IgA onto membrane-bound human CD89 on HEK293F cells was determined using FACS analysis.

[0207] HEK293F cells stably transfected with human full-length CD89 (clone no. 2, see Example 1(b) above) were cultured at 10 × 10 in ice-cold PBS containing 0.1% BSA (Sigma) / 0.05% NaN (PBS / BSA / NaN) supplemented with 50 μg / mL human IgG (to block potential Fcγ receptors, Sigma). 6 The cells were then added at 10 μL / tube (i.e., 0.1 × 10 cells / mL) and placed at 4°C for 10 minutes. 6These cells (100 μL each) were incubated with 50 μL of purified non-aggregated or heat-aggregated human (serum-derived) IgA (Bethyl Laboratories) at 20 μg / mL (in PBS / BSA / NaN3) for 30 minutes at 4° C. After this (i.e., without washing), 50 μL of purified mouse anti-human CD89 antibody at 20 μg / mL (in PBS / BSA / NaN3) was added to the cells and incubated for an additional 30 minutes at 4° C. In parallel, 50 μL of purified mouse IgG1 isotype control (BD Biosciences) at 20 μg / mL (in PBS / BSA / NaN3) and 50 μL of purified mouse anti-human CD89 antibody clone MIP8a (a well-known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), BioRad) at 20 μg / mL (in PBS / BSA / NaN3) were run as negative and positive controls, respectively. Additionally, 50 μL of purified mouse anti-human CD89 antibody clone A59 (a well-known CD89 / IgA non-blocking agent (Monteiro et al. J Immunol 1992;148:1764-1770), BD Biosciences) at 20 μg / mL (in PBS / BSA / NaN3) and 50 μL of purified mouse anti-human CD89 antibody clone A3 (a well-known CD89 / IgA non-blocking agent (Monteiro et al. J Immunol 1992;148:1764-1770), Santa Cruz Biotechnology) at 20 μg / mL (in PBS / BSA / NaN3) were run as additional negative controls. After extensive washing with PBS / BSA / NaN3, binding of non-aggregated or heat-aggregated serum human IgA to membrane human CD89 was determined using 5 μg / mL biotin-conjugated F(ab')2 fragment goat anti-human serum IgA α chain-specific antibody (Jackson ImmunoResearch) for 30 min at 4°C. After extensive washing with PBS / BSA / NaN3, PE-conjugated streptavidin (Jackson ImmunoResearch) diluted 1:200 was added and incubated for 30 min at 4°C.After extensive washing with PBS / BSA / NaN3, cells were fixed in 2% formaldehyde in PBS / BSA / NaN3 for 30 min at 4°C. Binding (geometric mean fluorescence intensity) of non-aggregated or heat-aggregated serum human IgA to membrane human CD89 was measured using a flow cytometer (FACSCalibur, BD Biosciences).

[0208] As shown in Figure 5B, all of our purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies displaced non-agglutinated serum human IgA on previously saturated membrane human CD89 to various degrees. Based on their degree of serum human IgA displacement, the following ranking was found (from strong to weak human IgA displacement): 9H7 = 26D6 (=MIP8a) > 20B4 > 30C7 > 8F3 = 10E7 > 16D6. For reference purposes, purified mouse anti-human CD89 antibody clone MIP8a, a known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), was run in parallel and also displaced non-agglutinated serum human IgA on previously saturated membrane human CD89. In contrast, purified mouse anti-human CD89 antibodies clone A59 and clone A3, both of which are well-known CD89 / IgA non-blocking agents (Monteiro et al. J Immunol 1992;148:1764-1770), did not displace non-agglutinated serum human IgA from previously saturated membrane human CD89.

[0209] As shown in Figure 5B, all of our purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies (except 16D6) also displaced heat-aggregated serum human IgA on previously saturated membrane human CD89 to varying degrees, and to a degree slightly less than that found with nonaggregated serum human IgA. Based on their degree of serum human IgA displacement, the following ranking was found (from strong to weak human IgA displacement): 9H7 = 26D6 (= MIP8a) > 20B4 > 30C7 > 8F3 = 10E7. For reference purposes, purified mouse anti-human CD89 antibody clone MIP8a, a known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), was run in parallel and also displaced non-aggregated serum human IgA from pre-saturated membrane human CD89, albeit to a slightly lesser extent than seen with non-aggregated serum human IgA. In contrast, purified mouse anti-human CD89 antibodies clone A59 and clone A3, both known CD89 / IgA non-blockers (Monteiro et al. J Immunol 1992;148:1764-1770), did not displace heat-aggregated serum human IgA from pre-saturated membrane human CD89.

[0210] These results demonstrated that the CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 8F3, 9H7, 10E7, (16D6), 26D6, 20B4, and 30C7 exhibited displacement of monomeric, dimeric, and trimeric serum human IgA (i.e., non-aggregated IgA) and, to a lesser extent, tetrameric or higher multimeric serum human IgA (i.e., heat-aggregated IgA) on previously saturated membrane human CD89. See Table 1A for a summary. [Table 1]

[0211] Because all of the above experiments (see Examples 2(b) and 2(c) above) were performed under (flow cytometry) metabolically inactive conditions (i.e., at cold ambient temperature (4°C) and in the presence of NaN, a reversible inhibitor of mitochondrial respiration that prevents capping, shedding, and internalization of the antibody-antigen complex after antibody receptor binding), the ability of our purified CD89 / IgA-blocking mouse anti-human CD89 antibody to sterically hinder the interaction of unaggregated and heat-aggregated human serum IgA with membrane-bound human CD89 under metabolic (active) conditions was investigated as follows.

[0212] Human IgA blocking setup: HEK293F cells stably transfected with human full-length CD89 (clone no. 2, see Example 1(b) above) were cultured at 1.70 x 10 in FreeStyle™ 293 culture medium (Life Technologies) supplemented with 125 μg / mL G418 / Geneticin (Gibco). 6 cells / mL and placed at 4°C. Then, 400 μL / tube (i.e., 0.7 × 10 6These cells (200 μg / mL) were incubated with or without 50 μL of purified mouse anti-human CD89 antibody at 100 μg / mL (in FreeStyle™ 293 culture medium) for 30 minutes at 4° C. In parallel, 50 μL of purified mouse IgG1 isotype control (BD Biosciences) at 100 μg / mL (in FreeStyle™ 293 culture medium) and 50 μL of purified mouse anti-human CD89 antibody clone MIP8a (a well-known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), BioRad) at 100 μg / mL (in FreeStyle™ 293 culture medium) were run as negative and positive controls, respectively. Additionally, 50 μL of purified mouse anti-human CD89 antibody clone A59 (a known CD89 / IgA non-blocking agent (Monteiro et al. J Immunol 1992;148:1764-1770), BD Biosciences) at 100 μg / mL (in FreeStyle™ 293 culture medium) and 50 μL of purified mouse anti-human CD89 antibody clone A3 (a known CD89 / IgA non-blocking agent (Monteiro et al. J Immunol 1992;148:1764-1770), Santa Cruz Biotechnology) at 100 μg / mL (in PBS / BSA / NaN3) were run as additional negative controls. After this (i.e., without washing), 50 μL of purified non-aggregated or heat-aggregated human (serum-derived) IgA (Bethyl Laboratories) at 100 μg / mL (in FreeStyle™ 293 culture medium) was added to the cells and incubated for an additional 24 hours at 37°C in a 5% CO incubator. After extensive washing with PBS / BSA / NaN3, binding of non-aggregated or heat-aggregated serum human IgA to membrane human CD89 was determined for 30 minutes at 4°C using 5 μg / mL biotin-conjugated F(ab')2 fragment goat anti-human serum IgA α-chain-specific antibody (Jackson ImmunoResearch).After extensive washing with PBS / BSA / NaN3, PE-conjugated streptavidin (Jackson ImmunoResearch) diluted 1:200 was added and incubated for 30 minutes at 4°C. After extensive washing with PBS / BSA / NaN3, cells were fixed in 2% formaldehyde in PBS / BSA / NaN3 for 30 minutes at 4°C. Binding (geometric mean fluorescence intensity) of non-aggregated or heat-aggregated serum human IgA to membrane human CD89 was measured using a flow cytometer (FACSCalibur, BD Biosciences).

[0213] Human IgA replacement set-up: HEK293F cells stably transfected with human full-length CD89 (clone no. 2, see Example 1(b) above) were cultured at 1.70 x 10 in FreeStyle™ 293 culture medium (Life Technologies) supplemented with 125 μg / mL G418 / Geneticin (Gibco). 6 cells / mL and placed at 4°C. Then, 400 μL / tube (i.e., 0.7 × 10 6These cells (100 μg / mL, 100 μg / mL, 100 μg / mL, 100 μg / mL, 100 μg / mL, 100 μg / mL, 100 μg / mL, 100 μg / mL, 100 μg / mL, 100 μL of purified non-aggregated or heat-aggregated human (serum-derived) IgA (Bethyl Laboratories) were incubated for 30 minutes at 4° C. After this (i.e., without washing), 50 μL of purified mouse anti-human CD89 antibody (100 μg / mL, 100 μg / mL, 100 μg / mL, 100 μL of purified mouse anti-human CD89 antibody (100 μL, 100 μg / mL ... In parallel, 50 μL of purified mouse IgG1 isotype control (BD Biosciences) at 100 μg / mL (in FreeStyle™ 293 culture medium) and 50 μL of purified mouse anti-human CD89 antibody clone MIP8a (a well-known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), BioRad) at 100 μg / mL (in FreeStyle™ 293 culture medium) were run as negative and positive controls, respectively. Additionally, 50 μL of purified mouse anti-human CD89 antibody clone A59 (a known CD89 / IgA non-blocking agent (Monteiro et al. J Immunol 1992;148:1764-1770), BD Biosciences) at 100 μg / mL (in FreeStyle™ 293 culture medium) and 50 μL of purified mouse anti-human CD89 antibody clone A3 (a known CD89 / IgA non-blocking agent (Monteiro et al. J Immunol 1992;148:1764-1770), Santa Cruz Biotechnology) at 100 μg / mL (in PBS / BSA / NaN3) were run as additional negative controls. After extensive washing with PBS / BSA / NaN3, binding of non-aggregated or heat-aggregated serum human IgA to membrane human CD89 was determined for 30 min at 4°C using 5 μg / mL biotin-conjugated F(ab')2 fragment goat anti-human serum IgA α chain-specific antibody (Jackson ImmunoResearch).After extensive washing with PBS / BSA / NaN3, PE-conjugated streptavidin (Jackson ImmunoResearch) diluted 1:200 was added and incubated for 30 minutes at 4°C. After extensive washing with PBS / BSA / NaN3, cells were fixed in 2% formaldehyde in PBS / BSA / NaN3 for 30 minutes at 4°C. Binding (geometric mean fluorescence intensity) of non-aggregated or heat-aggregated serum human IgA to membrane human CD89 was measured using a flow cytometer (FACSCalibur, BD Biosciences).

[0214] As shown in Figure 5C, the purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 8F3, 9H7, 10E7, 26B6, 20B4, and 30C7 potently inhibited nonaggregated and heat-aggregated serum human IgA binding to membrane human CD89 under metabolically active conditions. The purified CD89 / IgA-blocking mouse anti-human CD89-specific antibody 16D6 showed partial blocking of nonaggregated serum human IgA binding to membrane human CD89, but the mouse anti-human CD89 antibody 16D6 failed to block the binding of heat-aggregated serum human IgA binding to membrane human CD89 under metabolically active conditions. For reference purposes, purified mouse anti-human CD89 antibody clone MIP8a, a known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), was run in parallel and also showed potent blocking of non-aggregated and heat-aggregated serum human IgA binding to membrane human CD89 under metabolically active conditions. Surprisingly, purified mouse anti-human CD89 antibodies clone A59 and clone A3, both of which are known CD89 / IgA non-blockers (Monteiro et al. J Immunol 1992;148:1764-1770), showed partial blocking of non-aggregated serum human IgA binding to membrane human CD89, but mouse anti-human CD89 antibodies clone A59 and clone A3 were unable to block heat-aggregated serum human IgA binding to membrane human CD89 under metabolically active conditions.

[0215] As shown in Figure 5D, the purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 8F3, 9H7, 10E7, 26B6, 20B4, and 30C7 potently displaced nonaggregated and heat-aggregated serum human IgA from pre-saturated membrane human CD89 under metabolically active conditions. The purified CD89 / IgA-blocking mouse anti-human CD89-specific antibody 16D6 was able to partially displace nonaggregated serum human IgA from pre-saturated membrane human CD89, but the mouse anti-human CD89 antibody 16D6 was unable to displace heat-aggregated serum human IgA from pre-saturated membrane human CD89 under metabolically active conditions. For reference purposes, purified mouse anti-human CD89 antibody clone MIP8a, a known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), was run in parallel and also potently displaced non-aggregated and heat-aggregated serum human IgA bound to previously saturated membrane human CD89 under metabolically active conditions. Surprisingly, purified mouse anti-human CD89 antibodies clone A59 and clone A3, both known CD89 / IgA non-blockers (Monteiro et al. J Immunol 1992;148:1764-1770), were able to partially displace non-aggregated serum human IgA from previously saturated membrane human CD89, but were unable to displace heat-aggregated serum human IgA from previously saturated membrane human CD89 under metabolically active conditions.

[0216] These results demonstrated that the CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 8F3, 9H7, 10E7, 26D6, 20B4, and 30C7 inhibited the binding of monomeric, dimeric, and trimeric serum human IgA (i.e., non-aggregated IgA) and tetrameric or higher polymeric serum human IgA (i.e., heat-aggregated IgA) to membrane human CD89 under metabolically active conditions. In addition, the CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 8F3, 9H7, 10E7, 26D6, 20B4, and 30C7 also displaced monomeric, dimeric, and trimeric serum human IgA (i.e., non-aggregated IgA) and tetrameric or higher polymeric serum human IgA (i.e., heat-aggregated IgA) from previously saturated membrane human CD89 under metabolically active conditions. For a summary, see Table 1B. [Table 2]

[0217] The CD89 / IgA-blocking mouse anti-human CD89 antibody clone MIP8a has been described to induce human cell death in CD89-expressing human neutrophil granulocytes (Wehrli et al. J Immunol 2014, 193:5649-5659). Depending on the inflammatory microenvironment, caspase-dependent (typically observed during apoptosis) or caspase-independent (non-apoptotic) cell death was induced in these human neutrophil granulocytes upon exposure to the bivalent mouse anti-human CD89 antibody clone MIP8a. Therefore, the effects of the generated CD89 / IgA-blocking mouse anti-human CD89 antibodies 8F3, 9H7, 10E7, 16D6, 26D6, 20B4, and 30C7 on human CD89-mediated cell death were investigated using HEK293F cells stably transfected with human full-length CD89 as target cells.

[0218] HEK293F cells stably transfected with human full-length CD89 (clone no. 2, see Example 1(b) above) were cultured at 1.25 × 10 in FreeStyle™ 293 culture medium (Life Technologies) supplemented with 125 μg / mL G418 / Geneticin (Gibco). 6 cells / mL and placed at 4°C. Then, 400 μL / tube (i.e., 0.5 × 10 6These cells (200 μg / mL) were incubated with or without 50 μL of purified mouse anti-human CD89 antibody at 100 μg / mL (in FreeStyle™ 293 culture medium) for 30 minutes at 4° C. In parallel, 50 μL of purified mouse IgG1 isotype control (BD Biosciences) at 100 μg / mL (in FreeStyle™ 293 culture medium) and 50 μL of purified mouse anti-human CD89 antibody clone MIP8a (a well-known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), BioRad) at 100 μg / mL (in FreeStyle™ 293 culture medium) were run as negative and positive cell death induction controls, respectively. Additionally, 50 μL of purified mouse anti-human CD89 antibody clone A59 (a known CD89 / IgA non-blocking agent (Monteiro et al. J Immunol 1992;148:1764-1770), BD Biosciences) at 100 μg / mL (in FreeStyle™ 293 culture medium) and 50 μL of purified mouse anti-human CD89 antibody clone A3 (a known CD89 / IgA non-blocking agent (Monteiro et al. J Immunol 1992;148:1764-1770), Santa Cruz Biotechnology) at 100 μg / mL (in PBS / BSA / NaN) were run as additional controls. Subsequently (i.e., without washing), 50 μL of FreeStyle™ 293 culture medium was added to the cells and they were further incubated at 37°C in a 5% CO incubator for 24 hours. After this 24-hour incubation, cells were stained with 0.02% trypan blue (Sigma-Aldrich) to distinguish between live and dead cells. The percentage of live cells was then counted using a Burker hemocytometer. Additionally, after extensive washing with PBS / BSA / NaN3, the expression of the phospholipid phosphatidylserine in the cell membrane, a known marker of apoptosis or cell death, was determined with 10 μg / mL Alexa Fluor® 488-conjugated mouse anti-phosphatidylserine antibody (Merck Millipore) in PBS / BSA / NaN3 for 30 minutes at 4°C.In parallel, Alexa Fluor® 488-conjugated mouse anti-human CD19 antibody (BD Biosciences) was run as a negative control. After extensive washing with PBS / BSA / NaN3, cells were fixed in 2% formaldehyde in PBS / BSA / NaN3 for 30 minutes at 4°C. Membrane phosphatidylserine expression (geometric mean fluorescence intensity) was measured using a flow cytometer (FACSCalibur, BD Biosciences).

[0219] As shown in Figure 5E, the purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 9H7 and 26B6 caused cell death in human CD89-expressing HEK293F cells, as evidenced by a decrease in cell viability (measured by trypan blue exclusion assay) and an increase in phosphatidylserine expression levels after treatment with both mouse anti-human CD89-specific antibodies. As expected, the commercial CD89 / IgA-blocking mouse anti-human CD89 antibody clone MIP8a also induced cell death in these human CD89-expressing HEK293F cells, and this cell death induction was comparable to that observed with the mouse anti-human CD89-specific antibodies 9H7 and 26B6. Surprisingly, the purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 8F3, 10E7, 16D6, 20B4, and 30C7 did not induce significant cell death in these human CD89-expressing HEK293F cells, nor did the commercial CD89 / IgA-nonblocking mouse anti-human CD89 antibodies clone A59 and clone A3.

[0220] These results demonstrated that the CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 9H7 and 26B6 induced cell death in membrane human CD89-expressing cells, whereas the CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 8F3, 10E7, 16D6, 20B4, and 30C7 did not induce cell death in membrane human CD89-expressing cells.

[0221] (d) The extent of CD89 / IgA blocking capacity of commercial prototype mouse anti-human CD89 antibodies Human CD89 consists of a short cytoplasmic tail, a transmembrane region, and two extracellular (EC) Ig-like domains. A short interdomain hinge region allows these two Ig-like EC domains to fold at approximately 90° angles relative to each other (Ding et al. J Biol Chem 2003;278:27966-27970). The binding site for human IgA on human CD89 is located in the membrane-distal Ig-like EC1 domain, but not in the membrane-proximal Ig-like EC2 domain (Wines et al. J Immunol 1999;162:2146-2153, Morton J Exp Med 1999;189:1715-1722, Lu et al. Protein Sci 2014;23:378-386). Thus, it is generally accepted that mouse anti-human CD89 antibodies that bind to the EC1 domain of human CD89 can block human IgA binding on human CD89, but that mouse anti-human CD89 antibodies that bind to the EC2 domain of human CD89 cannot block human IgA binding on human CD89 (Morton et al. Arch Immunol Ther Exp 2001;49:217-229, Bakema et al. Immunol Rev 2011;4:612-624). More specifically, the prototypical CD89 / IgA-blocking mouse anti-human CD89 antibody clone MIP8a recognizes an epitope within the EC1 domain of human CD89 (Lu et al. Protein Sci 2014;23:378-386), whereas the prototypical CD89 / IgA-non-blocking mouse anti-human CD89 antibody clones A59 and A3 recognize epitopes within the EC2 domain of human CD89 (Morton J Exp Med 1999;189:1715-1722) and within the EC1-EC2 domain boundary of human CD89, respectively (Morton J Exp Med 1999;189:1715-1722).

[0222] Surprisingly, purified, well-known CD89 / IgA non-blocking (Monteiro et al. J Immunol 1992;148:1764-1770) mouse anti-human CD89 antibodies clone A59 and clone A3 (when tested at 10 μg / mL) showed partial but significant binding of both unaggregated and heat-aggregated serum human IgA to membrane human CD89.

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[0223] HEK293F cells stably transfected with human full-length CD89 (clone no. 2, see Example 1(b) above) were cultured at 10 × 10 in ice-cold PBS containing 0.1% BSA (Sigma) / 0.05% NaN (PBS / BSA / NaN) supplemented with 50 μg / mL human IgG (to block potential Fcγ receptors, Sigma). 6 The cells were then incubated at 4°C for 10 minutes at 10 µL / tube (i.e., 0.1 x 10 cells / mL). 6These cells (100 μL each) were incubated with 50 μL of titrated purified mouse anti-human CD89 antibody clone A59 (BD Biosciences) and clone A3 (Santa Cruz Biotechnology) in PBS / BSA / NaN3 for 30 minutes at 4°C. In parallel, 50 μL of titrated purified mouse IgG1 isotype control (BD Biosciences) in PBS / BSA / NaN3 and 50 μL of purified mouse anti-human CD89 antibody clone MIP8a (BioRad) in PBS / BSA / NaN3 were run as negative and positive controls, respectively. After this (i.e., without washing), 50 μL of 20 μg / mL (diluted in PBS / BSA / NaN3) purified non-aggregated or heat-aggregated human (serum-derived) IgA (Bethyl Laboratories) was added to the cells and incubated for an additional 30 minutes at 4°C. After extensive washing with PBS / BSA / NaN3, binding of non-aggregated or heat-aggregated serum human IgA to membrane human CD89 was determined using 5 μg / mL biotin-conjugated F(ab')2 fragment goat anti-human serum IgA α-chain-specific antibody (Jackson ImmunoResearch) for 30 minutes at 4°C. After extensive washing with PBS / BSA / NaN3, PE-conjugated streptavidin (Jackson ImmunoResearch) diluted 1:200 was added and incubated for 30 minutes at 4°C. After extensive washing with PBS / BSA / NaN3, cells were fixed in 2% formaldehyde in PBS / BSA / NaN3 for 30 minutes at 4°C. Binding (geometric mean fluorescence intensity) of non-aggregated or heat-aggregated serum human IgA to membrane human CD89 was measured using a flow cytometer (FACSCalibur, BD Biosciences).

[0224] As shown in Figures 6A and 6B, both mouse anti-human CD89 specific antibodies clone 59 and A3 partially dose-dependently inhibited the binding of non-aggregated and heat-aggregated serum human IgA to membrane human CD89 (i.e., up to

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[0225] These results demonstrated that the mouse anti-human CD89-specific antibodies clones 59 and A3, a prototype antibody recognizing an epitope within the EC2 domain of human CD89 (Morton J Exp Med 1999;189:1715-1722) and an epitope within the boundary of the EC1-EC2 domain of human CD89 (Morton J Exp Med 1999;189:1715-1722), respectively, can specifically and partially inhibit the binding of monomeric, dimeric, trimeric, tetrameric, or higher multimeric serum human IgA (i.e., unaggregated and heat-aggregated IgA) to membrane human CD89. Conceivably, the mouse anti-human CD89-specific antibodies clones 59 and A3, after binding to the EC2 domain or the EC1-EC2 interface, may alter the folding of membrane human CD89 in such a way that binding of serum human IgA to human CD89 is "favored" or suboptimal (as opposed to steric hindrance by anti-human CD89 antibodies that recognize the IgA-binding site on human CD89 (i.e., the EC1 domain)).

[0226] Example 3. Biological characterization of CD89 / IgA-blocking mouse anti-human CD89 monoclonal antibodies using ex vivo human CD89-expressing primary human neutrophil granulocytes (a) Binding of a CD89 / IgA-blocking mouse anti-human CD89 antibody to primary human neutrophil granulocytes expressing human CD89. FACS analysis was used to determine the binding of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies to human CD89-expressing primary human neutrophil granulocytes.

[0227] Primary human neutrophilic granulocytes were isolated from peripheral blood of healthy donors (after informed consent) using Lymphoprep™ (Axis-Shield) gradient centrifugation followed by lysis of red blood cells in NH4Cl lysis buffer. For comparison, HEK293F cells stably transfected with human full-length CD89 (clone no. 2, see Example 1(b) above) and the human CD89-expressing monocytic U937 cell line (a generous gift from Dr. R.T. Urbanus, Department of Haematology, University Medical Centre Utrecht, NL) were investigated in parallel. After washing with PBS, granulocytes were cultured at 10 x 10 in ice-cold PBS containing 0.1% BSA (Sigma-Aldrich, PBS / BSA) supplemented with 50 μg / mL human IgG (to block potential Fcγ receptors, Sigma-Aldrich). 6 The cells were then added at 10 μL / tube (i.e., 0.1 × 10 cells / mL) and placed at 4°C for 10 minutes. 6These cells (100 μL) were incubated with 100 μL of purified mouse anti-human CD89 antibody at 10 μg / mL (in PBS / BSA) for 30 minutes at 4°C. In parallel, 100 μL of purified mouse IgG1 isotype control (Biolegend) at 10 μg / mL (in PBS / BSA) was run as a negative control, and 100 μL of purified mouse anti-human CD89 antibody clone MIP8a (BioRad) at 10 μg / mL (in PBS / BSA), clone A59 (BD Biosciences) at 10 μg / mL (in PBS / BSA), and clone A3 (Santa Cruz Biotechnology) at 10 μg / mL (in PBS / BSA) were run as positive controls. After extensive washing with PBS / BSA, cells were then incubated with a 1:200 diluted PE-conjugated goat anti-mouse IgG Fcγ-specific antibody (Jackson ImmunoResearch) for 30 minutes at 4° C. After extensive washing with PBS / BSA, cells were fixed in 2% formaldehyde in PBS / BSA for 30 minutes at 4° C. Binding of the antibody (geometric mean fluorescence intensity) to membrane human CD89 from ex vivo human neutrophil granulocytes was measured using a flow cytometer (cyan, Beckman Coulter).

[0228] As shown in Figure 7A, all of our purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies (at 10 μg / mL) bound to membrane human CD89 from ex vivo human neutrophil granulocytes isolated from multiple donors (n=5). Based on their binding profiles to neutrophil granulocytes, the following ranking was found (from high to low binding strength): 9H7 = 26D6 (= MIP8a) > 10E7 = 30C7 = 20B4 (= A59 = A3) > 8F3 = 16D6, which was unexpectedly and significantly different from the binding ranking found with these purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies (at 10 μg / mL) against membrane-length human CD89 from HEK293F cells (see Figure 7B), i.e., 8F3 = 9H7 = 10E7 = 26D6 = 20B4 = 30C7 (= MIP8a = A59 = A3) > 16D6. In addition, the binding ranking found with purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies (at 10 μg / mL) to membrane human CD89 from monocytic U937 cells (see Figure 7C), i.e., 9H7 = 26D6 (= MIP8a) > 10E7 = 30C7 (= A59 = A3) > 8F3 = 16D6 = 20B4, appears similar to the previously described binding ranking found with purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies (at 10 μg / mL) to membrane human CD89 from ex vivo neutrophil granulocytes (except for 20B4).

[0229] These results demonstrated that the CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 8F3, 9H7, 10E7, 16D6, 26D6, 20B4, and 30C7 recognized epitopes on membrane human CD89 from ex vivo human neutrophil granulocytes, membrane human full-length CD89 from HEK293F cells, and membrane human CD89 from monocytic U937 cells. However, the binding rankings found with these CD89 / IgA-blocking mouse anti-human CD89-specific antibodies were significantly different when ex vivo human CD89-expressing human neutrophil granulocytes (from high to low binding intensity: 9H7 = 26D6 (= MIP8a) > 10E7 = 30C7 = 20B4 (= A59 = A3) > 8F3 = 16D6) versus human full-length CD89-expressing HEK293F cells (from high to low binding intensity: 8F3 = 9H7 = 10E7 = 26D6 = 20B4 = 30C7 (= MIP8a = A59 = A3) > 16D6) were used as target cells in this comparative study, suggesting the potential for (lack of) recognition of multiple membrane human CD89 isoforms on these cells. In fact, primary human neutrophil granulocytes and monocytes are known to express two alternative splicing variant transcripts of human CD89, in addition to the full-length CD89 (also called FcαRIa.1) (Patry at al. J Immunol 1996;156:4442-4448, Pleass et al. Biochem J 1996;318:771-777, Togo et al. FEBS Letters 2003;535:20-209), which lack either (1) part of the EC2 domain of human CD89 (Gly195 to Thr216, Swiss-Prot number P24071.2) (termed FcαRIa.2 or Δ66EC2), or (2) the entire EC2 domain of human CD89 (Gly121 to Thr216, Swiss-Prot number P24071.3) (termed FcαRIa.3 or ΔEC2).The mouse anti-human CD89 specific antibodies 8F3, 9H7, 10E7, 16D6, 26D6, 20B4, and 30C7 at 10 μg / mL bound to HEK293F cells (expressing only full-length human CD89 versions) to a similar extent (except for the low affinity antibody 16D6, see also Example 2(a) above), whereas binding of 10 μg / mL mouse anti-human CD89 specific antibodies 8F3, 10E7, 16D6, 20B4, and 30C7 to primary human neutrophil granulocytes (expressing full-length human CD89, ΔEC2, and to a lesser extent Δ66EC2 versions) (within the EC2 domain of human CD89 (Morton J Exp Med 1999;189:1715-1722) and human IgA non-blocking clones A59 and A3, which recognize an epitope within the boundary of the EC1-EC2 domain of human CD89, respectively (Morton J Exp Med 1999;189:1715-1722), were used at 10 μg / mL with mouse anti-human CD89-specific antibodies 9H7 and 26D6, as was the human IgA blocking antibody MIP8a, which recognizes an epitope within the EC1 domain of human CD89 (Lu et al. Protein Sci 2014;23:378-386)), suggesting that (1) the mouse anti-human CD89-specific antibodies 8F3, 10E7, 16D6, 20B4, and 30C7 recognize epitopes either within the EC2 domain or the EC1-EC2 domain boundary of human CD89 (like the human IgA non-blocking clones A59 and A3, which cannot bind to the alternative splicing variant FcαRIa.3 due to the lack of an EC2 domain), or (2) the "normal" full-length human CD89 / FcαRIa.1 epitopes. These findings suggest that (1) the "abnormal" protein folding of these alternative splice variants (as opposed to the normal protein folding) recognizes epitopes within the EC1 domain that are inaccessible to the alternative splice variants of human CD89 (i.e., FcαRIa.2 and / or FcαRIa.3), and (2) the mouse anti-human CD89-specific antibodies 9H7 and 26D6 recognize epitopes within the EC1 domain of human CD89 (like the human IgA blocking agent MIP8a).Interestingly, the alternative splicing variant FcαRIa.3 (or ΔEC2) lacks binding to serum-derived human IgA, but the entire EC1 domain (i.e., the IgA-binding site on human CD89) is present in this splicing variant, indicating an "abnormal" protein fold of this alternative splicing variant FcαRIa.3 (in contrast to the "normal" protein fold of full-length human CD89 / FcαRIa.1). Furthermore, full-length human CD89 likely forms two distinct conformations for inside-out signaling: an inactive state versus an active state of human CD89 (Brandsma et al. Immunol Rev 2015, 268:74-87). As a result, inactive human CD89 exhibits low-affinity binding to human IgA, whereas active human CD89 exhibits high-affinity binding to human IgA (Bracke et al. Blood 2001;97:3478-3483). Therefore, the conformational change / state of full-length human CD89 (during inside-out signaling) may result in differential binding of our generated CD89 / IgA-blocking mouse anti-human CD89-specific antibody to full-length human CD89 on ex vivo human neutrophil granulocytes.

[0230] Consistently, to monocytic U937 cells, which are also known to express full-length human CD89, the ΔEC2 and Δ66EC2 versions, binding of 10 μg / mL of mouse anti-human CD89-specific antibodies 8F3, 10E7, 16D6, 20B4, and 30C7 (like the non-blocking clones A59 and A3, which recognize epitopes within the EC2 domain of human CD89 (Morton J Exp Med 1999;189:1715-1722) and within the EC1-EC2 domain boundary of human CD89, respectively) significantly reduced the binding of 10 μg / mL of mouse anti-human CD89-specific antibodies 9H7 and 26D6 (like the blocking agent MIP8a, which recognizes an epitope within the EC1 domain of human CD89 (Lu et al. Protein Sci 2014;23:378-386)) (Patry at al. J Immunol 1996;156:4442-4448, Togo et al. FEBS Letters 2003;535:20-209).

[0231] These results also demonstrated that the CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 8F3, 9H7, 10E7, 16D6, 26D6, 20B4, and 30C7 recognized non-polymorphic epitopes on membrane human CD89 from ex vivo human neutrophil granulocytes, since these antibodies bound specifically to human neutrophil granulocytes isolated from all five studied donors.

[0232] (b) Blockade of serum human IgA-mediated phagocytosis by human CD89-expressing primary human neutrophil granulocytes using a CD89 / IgA-blocking mouse anti-human CD89 monoclonal antibody. To analyze the biological activity of the purified CD89 / IgA-blocking mouse anti-human CD89 antibodies, the ability of the generated CD89 / IgA-blocking mouse anti-human CD89 antibodies to inhibit phagocytosis of serum human IgA-coated latex beads by human CD89-expressing primary human neutrophil granulocytes was determined.

[0233] Primary human neutrophil granulocytes were isolated from peripheral blood of healthy donors (after informed consent) using Lymphoprep™ (Axis-Shield) gradient centrifugation followed by lysis of red blood cells in NH4Cl lysis buffer. After washing in PBS, 2.0 × 10 neutrophils were cultured in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FCS (Sigma-Aldrich). 6 Granulocytes were resuspended at 0.2 x 10 cells / mL. 100 μL / well (i.e., 0.2 x 10 cells / mL) was then added to a 96-well flat-bottom plate. 6These cells (Greiner) were incubated with titrated purified mouse anti-human CD89 antibodies (in RPMI / 10% FCS) for 20 minutes at 4°C. In parallel, a titrated purified mouse IgG1 isotype control (Biolegend) (in RPMI / 10% FCS) was run as a negative control, and titrated purified mouse anti-human CD89 antibodies clone MIP8a (BioRad), clone A59 (BD Biosciences), and clone A3 (Santa Cruz Biotechnology) (in RPMI / 1% FCS) were run as controls. After this (i.e., without washing), 1.2 μL of purified human (serum-derived) IgA (MP Biomedicals) coated fluorescent latex beads (1 μm size and carboxylate-modified polystyrene, Sigma-Aldrich) were added to the cells at a cell-to-bead ratio of 1:60 (preparation IgA beads, see Aleyd et al. J Immunol 2014;192:2374-2383) and incubated for a further 30 minutes at 37° C. In parallel, BSA (Sigma-Aldrich) coated fluorescent latex beads at a cell-to-bead ratio of 1:60 were run as a negative control (preparation BSA beads, see Aleyd et al. J Immunol 2014;192:2374-2383). After washing in RPMI / 10% FCS and resuspension in PBS / 0.1% BSA (Sigma-Aldrich), serum human IgA-mediated phagocytosis (geometric mean fluorescence intensity used to calculate the phagocytic index according to Aleyd et al. J Immunol 2014;192:2374-2383) of fluorescent latex beads by membrane human CD89 on ex vivo human neutrophilic granulocytes was measured using a flow cytometer (cyan, Beckman Coulter).

[0234] As shown in Figure 8, although some donor-to-donor variation was observed, all purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies dose-dependently inhibited serum human IgA-mediated phagocytosis in membrane-human CD89-expressing ex vivo primary human neutrophil granulocytes isolated from three healthy individuals. Based on their degree of serum human IgA-mediated phagocytosis inhibition, the following ranking was found (from strongest to weakest): 8F3 = 9H7 = 10E7 = 26D6 = 30C7 (= MIP8a) > 20B4 > 16D16. For reference purposes, purified mouse anti-human CD89 antibody clone MIP8a, a known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), was run in parallel and also showed dose-dependent inhibition of serum human IgA-mediated phagocytosis in human CD89-expressing ex vivo primary human neutrophil granulocytes isolated from three healthy individuals, although some donor-to-donor variation was observed. In contrast, purified mouse anti-human CD89 antibodies clone A59 and clone A3, both well-known CD89 / IgA non-blocking agents (Monteiro et al. J Immunol 1992;148:1764-1770), showed no, little, or weak inhibition of serum human IgA-mediated phagocytosis in membrane human CD89-expressing ex vivo primary human neutrophil granulocytes isolated from three healthy individuals.

[0235] These results demonstrated that the CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 8F3, 9H7, 10E7, 16D6, 26D6, 20B4, and 30C7 inhibited human IgA-mediated phagocytosis of ex vivo primary human neutrophil granulocytes expressing membrane human CD89. For a summary, see Table 2. [Table 3]

[0236] (c) Blockade of serum human IgA-mediated chemotaxis and serum human IgA-mediated leukotriene B4 production from human CD89-expressing primary human neutrophil granulocytes using a CD89 / IgA-blocking mouse anti-human CD89 monoclonal antibody. To analyze the biological activity of purified CD89 / IgA-blocking mouse anti-human CD89 antibodies, we determined the ability of the generated CD89 / IgA-blocking mouse anti-human CD89 antibodies to inhibit (1) serum human IgA-mediated migration, (2) serum human IgA-mediated chemotaxis, and (3) serum human IgA-mediated neutrophil-chemoattractant leukotriene B4 (LTB4) production from human CD89-expressing primary human neutrophil granulocytes.

[0237] Two-dimensional (2-D) migration assay: Primary human neutrophil granulocytes were isolated from peripheral blood of healthy donors (after informed consent) using Lymphoprep™ (Axis-Shield) gradient centrifugation followed by lysis of red blood cells in NH4Cl lysis buffer. These primary human neutrophil granulocytes were then labeled with 1 μM fluorescent calcein AM (Molecular Probes) for 30 minutes at 37°C. After washing, these calcein AM-labeled granulocytes were cultured at 2.5 × 10 in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FCS (Sigma-Aldrich). 6 Resuspend at 0.25 x 10 cells / mL, followed by 100 µL of calcein AM-labeled granulocytes (i.e., 0.25 x 10 cells / mL) in a 96-well flat-bottom plate. 6Cells / well (Greiner) were incubated with purified mouse anti-human CD89 antibody at 20 μg / mL (in RPMI / 10% FCS) for 20 minutes at 4°C. In parallel, purified mouse IgG1 isotype control (Biolegend) at 20 μg / mL (in RPMI / 10% FCS) was run as a negative control, and purified mouse anti-human CD89 antibodies clone MIP8a (BioRad), clone A59 (BD Biosciences), and clone A3 (Santa Cruz Biotechnology) at 20 μg / mL (in RPMI / 10% FCS) were run as controls. Then, 150 μL of RPMI / 10% FCS was added per well, and the cells were allowed to restabilize for 10 minutes. After this (i.e., without washing), 10 μL of purified human (serum-derived) IgA (MP Biomedicals)-coated Sepharose 4B beads (90 μm size and cyanogen bromide activated, GE Healthcare) were gently added to the monolayer of these cells (preparation 3 μg / mL IgA beads, see Van der Steen et al. Gastroenterol 2009;137:2018-2029) and incubated for a further 40 minutes at 37° C. In parallel, BSA (Sigma-Aldrich)-coated Sepharose 4B beads were run as a negative control (preparation 3 μg / mL BSA beads, see Van der Steen et al. Gastroenterol 2009;137:2018-2029). The supernatant was then collected and used for chemotaxis assays and LBT4 ELISA (see below). Sepharose beads were washed to remove unbound / unmigrated calcein AM-labeled granulocytes. Subsequently, granulocytes were lysed in 0.2% (w / v) hexadecyltrimethylammonium bromide (Sigma-Aldrich) buffer at room temperature for 30 min, and the released calcein AM (reflecting the number of IgA-bound / migrated granulocytes) was measured in a 96-well flat-bottom plate (Greiner) using a fluorometer (FLUOstar / POLARstar, BMG Labtech). The number of IgA-bound / migrated ex vivo human neutrophil granulocytes was determined by dividing the number of lysed calcein AM-labeled granulocytes by the known number (i.e., 0–0.3 × 10).6 Quantification was performed using a standard curve in 1000 cells / well.

[0238] Chemotaxis assay: Primary human neutrophilic granulocytes were isolated from peripheral blood of healthy donors (after informed consent) using Lymphoprep™ (Axis-Shield) gradient centrifugation followed by lysis of red blood cells in NH4Cl lysis buffer. These primary human neutrophilic granulocytes were then labeled with 1 μM fluorescent calcein AM (Molecular Probes) for 30 minutes at 37°C. After washing, these calcein AM-labeled granulocytes were diluted to 1.0 × 10 in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FCS (Sigma-Aldrich). 6 Cells were resuspended at 0.05 × 10 cells / mL. To measure chemotaxis, wells in the lower compartment of a Boyden chamber (Neuro Probe) were filled with 29 μL of supernatant from IgA-coated Sepharose bead-stimulated primary human neutrophil granulocytes (i.e., from another healthy donor, see above). In parallel, RPMI / 10% FCS medium alone and 1 or 10 nM purified LTB4 (in RPMI, Sigma-Aldrich) were run as negative and positive controls, respectively. The lower compartment was then covered with a 3 μm pore size polyvinylpyrrolidone-coated polycarbonate filter (Neuro Probe), and the upper compartment was subsequently assembled into the lower compartment of the Boyden chamber. After this, 50 μL of calcein AM-labeled granulocytes (i.e., 0.05 × 10 cells / mL) were added. 6Cells / well) were added to the wells of the upper compartment. After 40 min of incubation at 37 °C, the chemotaxis of ex vivo human neutrophil granulocytes from the upper compartment toward the wells of the lower compartment was determined. In this case, granulocytes in the lower compartment were lysed in 0.1% (w / v) hexadecyltrimethylammonium bromide (Sigma-Aldrich) buffer at room temperature for 30 min, and the released calcein AM (reflecting the number of chemotactic granulocytes) was measured in a 96-well flat-bottom plate (Greiner) using a fluorometer (FLUOstar / POLARstar, BMG Labtech). The number of IgA-coated Sepharose bead-induced chemotactic ex vivo human neutrophil granulocytes was determined by counting a known number of lysed calcein AM-labeled granulocytes (i.e., 0–0.05 × 10). 6 Quantification was performed using a standard curve in 1000 cells / well.

[0239] LTB4 ELISA: LTB4 levels were measured in supernatants from ex vivo human neutrophil granulocytes stimulated with IgA-coated Sepharose beads (see above). For this purpose, a commercial LTB4 competitive ELISA kit (R&D Systems) was used according to the manufacturer's instructions.

[0240] As shown in Figure 9A, although some donor-to-donor variation was observed, all of our purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies at 20 μg / mL inhibited serum human IgA-coated Sepharose bead-mediated migration of membrane human CD89-expressing ex vivo primary human neutrophil granulocytes isolated from three healthy individuals. For reference purposes, purified mouse anti-human CD89 antibody clone MIP8a, a known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), was run in parallel and also demonstrated inhibition of serum human IgA-coated Sepharose bead-mediated migration of membrane human CD89-expressing ex vivo primary human neutrophil granulocytes isolated from three healthy individuals, although some donor-to-donor variation was observed. In contrast, purified mouse anti-human CD89 antibodies clone A59 and clone A3, both well-known CD89 / IgA non-blocking agents (Monteiro et al. J Immunol 1992;148:1764-1770), showed highly variable (i.e., none, weak, or intermediate) inhibition of serum human IgA-coated Sepharose bead-mediated migration of membrane human CD89-expressing ex vivo primary human neutrophil granulocytes.

[0241] As shown in Figure 9B, although some donor-to-donor variation was observed, all of our purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies at 20 μg / mL inhibited serum human IgA-coated Sepharose bead-mediated chemotaxis of membrane human CD89-expressing ex vivo primary human neutrophil granulocytes isolated from three healthy individuals. For reference purposes, purified mouse anti-human CD89 antibody clone MIP8a, a known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), was run in parallel and also demonstrated inhibition of serum human IgA-coated Sepharose bead-mediated chemotaxis of membrane human CD89-expressing ex vivo primary human neutrophil granulocytes isolated from three healthy individuals, although some donor-to-donor variation was observed. In contrast, purified mouse anti-human CD89 antibodies clone A59 and clone A3, both well-known CD89 / IgA non-blocking agents (Monteiro et al. J Immunol 1992;148:1764-1770), showed highly variable (i.e., none, intermediate, or strong) inhibition of serum human IgA-coated Sepharose bead-mediated chemotaxis of membrane human CD89-expressing ex vivo primary human neutrophil granulocytes.

[0242] As shown in Figure 9C, although some donor-to-donor variation was observed, all of our purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies at 20 μg / mL inhibited serum human IgA-coated Sepharose bead-mediated chemoattractant LTB4 production by membrane human CD89-expressing ex vivo primary human neutrophil granulocytes isolated from three healthy individuals. For reference purposes, purified mouse anti-human CD89 antibody clone MIP8a, a known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), was run in parallel and also demonstrated inhibition of serum human IgA-coated Sepharose bead-mediated chemoattractant LTB4 production by membrane human CD89-expressing ex vivo primary human neutrophil granulocytes isolated from three healthy individuals, although some donor-to-donor variation was observed. Surprisingly, the purified mouse anti-human CD89 antibodies clone A59 and clone A3, both of which are well-known CD89 / IgA non-blocking agents (Monteiro et al. J Immunol 1992;148:1764-1770), also showed inhibition of serum human IgA-coated Sepharose bead-mediated chemoattractant LTB4 production by ex vivo primary human neutrophil granulocytes expressing membrane human CD89.

[0243] These results demonstrated that the CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 8F3, 9H7, 10E7, 16D6, 26D6, 20B4, and 30C7 inhibited serum human IgA-mediated migration, chemotaxis, and chemoattractant LTB4 release of ex vivo primary human neutrophil granulocytes expressing membrane human CD89. See Tables 3 and 4 for a summary. [Table 4] [Table 5]

[0244] (d) Blockade of serum human IgA binding to human CD89-expressing primary human neutrophil granulocytes and serum human IgA-mediated lactoferrin production using a CD89 / IgA-blocking mouse anti-human CD89 monoclonal antibody. To analyze the biological activity of the purified CD89 / IgA-blocking mouse anti-human CD89 antibodies, we determined the ability of the generated CD89 / IgA-blocking mouse anti-human CD89 antibodies to (1) sterically hinder the interaction of human CD89-expressing primary human neutrophilic granulocytes with serum human IgA and (2) inhibit serum human IgA-mediated lactoferrin production from human CD89-expressing primary human neutrophilic granulocytes.

[0245] Primary human neutrophilic granulocytes were isolated from peripheral blood of healthy donors (after informed consent) using Lymphoprep™ (Axis-Shield) gradient centrifugation followed by lysis of red blood cells in NH4Cl lysis buffer. These primary human neutrophilic granulocytes were then labeled with 1 μM fluorescent calcein AM (Molecular Probes) for 30 minutes at 37°C. After washing, these calcein AM-labeled granulocytes were diluted to 2.0 × 10 in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FCS (Sigma-Aldrich). 6 Resuspend at 0.2 × 10 cells / mL, followed by 100 µL of calcein AM-labeled granulocytes (i.e., 0.2 × 10 6 Cells / well) were incubated with titrated (in RPMI / 10% FCS) purified mouse anti-human CD89 antibodies for 20 minutes at 4°C. In parallel, a titrated (in RPMI / 10% FCS) purified mouse IgG1 isotype control (Biolegend) was run as a negative control, and titrated (in RPMI / 10% FCS) purified mouse anti-human CD89 antibodies clone MIP8a (BioRad), clone A59 (BD Biosciences), and clone A3 (Santa Cruz Biotechnology) were run as controls. After this (i.e., without washing), 100 μL of these cells (i.e., 0.2 × 10 6Cells (180 μL / well) were added to 96-well flat-bottom ELISA plates (Nunc-Immuno MaxiSorp), which were pre-coated with either 10 μg / mL purified human (serum-derived) IgA (MP Biomedicals) at 100 μL / well or 10 μg / mL BSA (Sigma-Aldrich, used as a negative control) at 100 μL / well. After 30 minutes of incubation at 37°C, supernatants (180 μL / well) were harvested to remove unbound granulocytes, and these supernatants (after several centrifugal clearance steps) were used to measure lactoferrin production levels (used as a degranulation marker, see below). After washing the plates, granulocytes were lysed in 0.2% (w / v) hexadecyltrimethylammonium bromide (Sigma-Aldrich) buffer at room temperature for 30 min, and the released calcein AM (reflecting the number of IgA-binding granulocytes) was measured in a 96-well flat-bottom plate (Greiner) using a fluorometer (FLUOstar / POLARstar, BMG Labtech). The number of IgA-binding ex vivo human neutrophil granulocytes was determined by counting the number of lysed calcein AM-labeled granulocytes (i.e., 0–0.3 × 10). 6 Quantification was performed using a standard curve in 1000 cells / well.

[0246] Lactoferrin production (representing the degree of degranulation) was measured in the supernatants of primary human neutrophil granulocytes stimulated with plate-bound serum human IgA (see above). To this end, 96-well flat-bottom ELISA plates (Nunc-Immuno MaxiSorp) were coated with 100 μL / well of rabbit anti-human lactoferrin antibody (1:5000, Sigma-Aldrich) for 16–24 h at 4–8°C. After extensive washing with PBS / 0.05% Tween 20, the plates were blocked with 200 μL / well of PBS / 0.05% Tween 20 / 0.5% BSA (Sigma-Aldrich) at room temperature for 1 h. The plates were then incubated with 100 μL / well of supernatant at a 1:2 dilution (in blocking buffer) for 1 h at 37°C. After extensive washing with PBS / 0.05% Tween 20, the plates were incubated with alkaline phosphatase-conjugated rabbit anti-human lactoferrin detection antibody (1:2500, MP Biomedicals) for 1 hour at 37°C. After the addition of p-nitrophenyl phosphate (Sigma-Aldrich), the optical density was measured at a wavelength of 405 nm using a microplate reader (iMArk, Bio-Rad). Purified human lactoferrin (Sigma-Aldrich) was used as a standard to calculate the amount of lactoferrin released by serum human IgA-stimulated ex vivo human neutrophil granulocytes.

[0247] As shown in Figure 10A, although some donor-to-donor variation was observed, all of our purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies (except 16D6) dose-dependently inhibited the binding of human CD89-expressing ex vivo primary human neutrophil granulocytes isolated from three healthy individuals to serum human IgA. Based on their degree of inhibition, the following ranking was found (from strongest to weakest): 9H7 = 10E7 = 26D6 = 30C7 (=MIP8a) > 8F3 > 20B4. For reference purposes, purified mouse anti-human CD89 antibody clone MIP8a, a known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), was run in parallel and demonstrated dose-dependent inhibition of serum human IgA binding of human CD89-expressing ex vivo primary human neutrophil granulocytes isolated from three healthy individuals, although some donor-to-donor variation was observed. In contrast, purified mouse anti-human CD89 antibody clones A59 and A3, both known non-CD89 / IgA blockers (Monteiro et al. J Immunol 1992;148:1764-1770), showed no inhibition of serum human IgA binding of human CD89-expressing ex vivo primary human neutrophil granulocytes isolated from three healthy individuals.

[0248] As shown in Figure 10B, although some donor-to-donor variation was observed, all of our purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies (except 16D6) dose-dependently inhibited IgA-mediated lactoferrin production by human CD89-expressing ex vivo primary human neutrophil granulocytes isolated from three healthy individuals. Based on their degree of inhibition, the following ranking was found (from strongest to weakest): 9H7 = 10E7 = 26D6 = 30C7 (=MIP8a) > 8F3 > 20B4. Interestingly, the degree to which these investigated purified mouse anti-human CD89 antibodies inhibited serum human IgA-mediated lactoferrin production by human neutrophil granulocytes mirrored the degree of their respective inhibition of the binding of human CD89-expressing human neutrophil granulocytes to serum human IgA (compare Figure 10B with Figure 10A). For reference purposes, purified mouse anti-human CD89 antibody clone MIP8a, a known CD89 / IgA blocker (Zhang et al. Clin Exp Immunol 2000;121:106-111), was run in parallel and, although some donor-to-donor variation was observed, demonstrated dose-dependent inhibition of IgA-mediated lactoferrin production by human CD89-expressing ex vivo primary human neutrophil granulocytes isolated from three healthy individuals. In contrast, purified mouse anti-human CD89 antibodies clone A59 and clone A3, both known CD89 / IgA non-blockers (Monteiro et al. J Immunol 1992;148:1764-1770), did not demonstrate inhibition of IgA-mediated lactoferrin production by human CD89-expressing ex vivo primary human neutrophil granulocytes isolated from three healthy individuals.

[0249] These results demonstrated that the CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 8F3, 9H7, 10E7, 26D6, 20B4, and 30C7 inhibited the binding of human CD89-expressing ex vivo primary human neutrophil granulocytes to serum human IgA and their corresponding IgA-mediated lactoferrin production (a marker of degranulation). For a summary, see Table 5. [Table 6]

[0250] (e) Blockade of serum human IgA-mediated neutrophil extracellular traps (NETs) from human CD89-expressing primary human neutrophil granulocytes using a CD89 / IgA-blocking mouse anti-human CD89 monoclonal antibody. To analyze the biological activity of the purified CD89 / IgA-blocking mouse anti-human CD89 antibodies, we determined the ability of the generated CD89 / IgA-blocking mouse anti-human CD89 antibodies to inhibit serum human IgA-mediated release of NETs from human CD89-expressing primary human neutrophil granulocytes.

[0251] Primary human neutrophil granulocytes were isolated from peripheral blood of multiple healthy donors (after informed consent) using Lymphoprep™ (Axis-Shield) gradient centrifugation followed by lysis of red blood cells in NH4Cl lysis buffer. After washing in PBS, 0.5 × 10 neutrophils were cultured in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FCS (Sigma-Aldrich). 6 Granulocytes were resuspended at 1.0 x 10 cells / mL. 5Cells / 200 μL / well (in a 96-well U-bottom plate (Greiner)) were incubated with 20 μg / mL of purified mouse anti-human CD89 antibody for 20 minutes at 4°C. In parallel, a purified mouse IgG1 isotype control (Biolegend) was run as a negative control. After this (i.e., without washing), 3.0 μL of purified human (serum-derived) IgA (MP Biomedicals)-coated non-fluorescent latex beads (0.9 μm size and carboxylate-modified polystyrene, Sigma-Aldrich) were added to the cells at a cell-to-bead ratio of 1:300 (preparation IgA beads, see Aleyd et al. J Immunol 2014;192:2374-2383) and incubated for an additional 30 minutes at 37°C. In parallel, a negative control was run using BSA (Sigma-Aldrich) coated non-fluorescent latex beads at a cell-to-bead ratio of 1:300 (preparation BSA beads, see Aleyd et al. J Immunol 2014;192:2374-2383). After this, cells were washed twice in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FCS (Sigma-Aldrich), and then the cells were diluted to 1.0 × 10 5 Cells were transferred at 200 μL / well to a 96-well flat-bottom black plate (FLUOTRAC™ 200, Greiner) and incubated for an additional 3 hours at 37°C. Where indicated, 100 μg / mL of DNAse I was added. The release of extracellular DNA was then investigated by adding 2.5 μg / mL of SYTOX® Green (Invitrogen). Fluorescence intensity was measured using a fluorometer (FLUOstar / POLARstar, BMG Labtech).

[0252] As shown in Figure 23, although some donor-to-donor variation was observed, all of our purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies inhibited serum human IgA-mediated NET release from human CD89-expressing ex vivo primary human neutrophil granulocytes isolated from eight healthy individuals.

[0253] These results demonstrated that the CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 8F3, 9H7, 10E7, 16D6, 26D6, 20B4, and 30C7 inhibited human IgA-mediated NET release from human CD89-expressing ex vivo primary human neutrophil granulocytes.

[0254] (f) Induction of cell death in human CD89-expressing primary human neutrophil granulocytes using a CD89 / IgA-blocking mouse anti-human CD89 monoclonal antibody. The CD89 / IgA-blocking mouse anti-human CD89 antibody clone MIP8a has been described to induce human cell death in CD89-expressing human neutrophilic granulocytes (Wehrli et al. J Immunol 2014, 193:5649-5659). Depending on the inflammatory microenvironment, caspase-dependent (typically observed during apoptosis) or caspase-independent (non-apoptotic) cell death was induced in these human neutrophilic granulocytes upon exposure to the bivalent mouse anti-human CD89 antibody clone MIP8a. Therefore, the effects of the generated CD89 / IgA-blocking mouse anti-human CD89 antibodies 8F3, 9H7, 10E7, 16D6, 26D6, 20B4, and 30C7 on human CD89-mediated cell death were investigated using non-primed (mimicking a non-inflammatory state) and LPS-primed (mimicking an inflammatory state) human CD89-expressing primary human neutrophilic granulocytes as target cells.

[0255] Primary human neutrophil granulocytes were isolated from peripheral blood of multiple healthy donors (after informed consent) using Lymphoprep™ (Axis-Shield) gradient centrifugation followed by lysis of red blood cells in NH4Cl lysis buffer. After washing in PBS, 2.0 × 10 neutrophils were cultured in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FCS (Sigma-Aldrich). 6Granulocytes were resuspended at 2.0 × 10 cells / mL. The cells were then either unprimed or LPS-primed (100 ng / mL Ultrapure LPS from E. coli 0111:B4, Invivogen) for 5 minutes at 37°C. After washing, the granulocytes were resuspended at 2.0 × 10 cells / mL in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FCS (Sigma-Aldrich). 6 Resuspend at 0.5 x 10 cells / mL, followed by 250 µL of granulocytes (i.e., 0.5 x 10 cells / mL) in a 96-well flat-bottom plate. 6 Cells / well (Falcon) were incubated for 5 hours at 37°C with a combination of 10 μg / mL purified mouse anti-human CD89 antibody and 10 μg / mL cross-linking goat anti-mouse IgG specific antibody (Southern Biotech). In parallel, purified mouse IgG1 isotype control (Biolegend) and purified mouse anti-human CD89 antibody clone MIP8a (BioRad) were run as negative and positive controls, respectively. After extensive washing with ice-cold PBS containing 0.1% BSA (Sigma-Aldrich, PBS / BSA), granulocytes were stained with 3 μM red fluorescent DNA counterstain propidium iodide for 30–60 minutes at 4°C. After extensive washing with PBS / BSA, cells were fixed in 2% formaldehyde in PBS / BSA for 30 minutes at 4°C. The percentage of cell death (based on propidium iodide staining) in ex vivo human neutrophil granulocytes was determined using a flow cytometer (FACSCalibur or Fortessa, BD Biosciences).

[0256] As shown in Figure 24, none of our purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies nor the commercial CD89 / IgA-blocking mouse anti-human CD89 antibody clone MIP8a induced cell death in unprimed, human CD89-expressing, ex vivo primary human neutrophil granulocytes isolated from three healthy individuals. In contrast, the commercial CD89 / IgA-blocking mouse anti-human CD89 antibody clone MIP8a induced significant cell death in LPS-primed, human CD89-expressing, ex vivo primary human neutrophil granulocytes, although some donor-to-donor variation was observed. However, none of our purified mouse anti-human CD89 / IgA-blocking mouse CD89-specific antibodies induced cell death in LPS-primed, human CD89-expressing, ex vivo primary human neutrophil granulocytes.

[0257] These results demonstrated that the CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 8F3, 9H7, 10E7, 16D6, 26D6, 20B4, and 30C7 did not induce cell death in human CD89-expressing ex vivo primary human neutrophil granulocytes under non-inflammatory (i.e., non-primed) and inflammatory (i.e., LPS-primed) conditions.

[0258] (g) Blockade of the experimentally induced human IgA-mediated autoimmune skin disorder, linear IgA bullous disease, using the CD89 / IgA-blocking murine anti-human CD89 monoclonal antibody 10E7. To analyze the biological activity of the purified CD89 / IgA-blocking murine anti-human CD89 antibody 10E7, we determined the ability of the generated CD89 / IgA-blocking murine anti-human CD89 antibody 10E7 to inhibit the experimentally induced human IgA-mediated autoimmune skin disorder linear IgA bullous disease (LABD).

[0259] LABD is a chronic skin disease associated with IgA autoantibodies, characterized by subepidermal blisters with dense inflammatory infiltrates dominated by CD89-expressing neutrophil granulocytes (Van der Steen et al. J Immunol 2012;189:1594-1601). In patients with LABD, linear IgA deposits are typically found at the dermal-epidermal junction, and these IgA autoantibodies are primarily directed against the transmembrane hemidesmosomal antigen BP180 / collagen XVII, which induces sustained neutrophil granulocyte recruitment via cross-linking of human CD89 (Otten et al. Curr Mol Med 2014;14:69-95). As a result, the constant activation (i.e., by reactive oxygen species and pro-inflammatory cytokine production) and infiltration (i.e., by local chemoattractant LTB4 release) of CD89-expressing neutrophil granulocytes causes severe tissue damage and exacerbation of symptoms in LABD patients.

[0260] In vivo LABD mouse model: In double transgenic human CD89 / human IgA (Tg huCD89 / huIgA) mice lacking the mouse CD89 homolog, human CD89 expression, regulation, interaction with human IgA, and function mimic the human situation (Van Egmond et al, Blood 1999;93:4387-4394). These Tg huCD89 / huIgA (evenly distributed female and male) mice express membrane human CD89 on circulating mouse neutrophil granulocytes (Van Egmond et al., Blood 1999;93;4387-4394). The mice were injected subcutaneously with 10 μL of 7 mg / mL anti-mouse collagen XVII human IgA (autologous) antibody (Prof. Dr. M. van Egmond, Dept. Molecular Cell Biology and Immunology, VUmc, Amsterdam, NL) in the right ear or 10 μL of PBS in the left ear on days 0, 2, 4, 6, 8, 10, and 12. The influx of human CD89-expressing mouse neutrophil granulocytes at the injection site was monitored with or without treatment with the purified CD89 / IgA-blocking mouse anti-human CD89 antibody 10E7. To this end, 100 μL of 1.5 mg / mL purified CD89 / IgA-blocking mouse anti-human CD89 antibody 10E7 was injected intraperitoneally on days 7 and 11 (see Figure 31A for the treatment regimen). In parallel, 100 μL of 1.5 mg / mL mouse IgG1 isotype control (Biolegend) was run as a negative control. On day 14, mice were sacrificed, and ears were excised and subsequently snap-frozen in liquid nitrogen. These ear tissue specimens were cryosectioned (6 μm) and fixed in acetone for 10 minutes at room temperature. These air-dried cryosections were then incubated with Alexa Fluor® 488-conjugated rat anti-mouse Ly-6G (GR-1 staining, neutrophil granulocyte marker, eBioscience) diluted 1:400 for 1 hour at room temperature. After washing with PBS, nuclei were counterstained with 1 μg / mL DAPI (Invitrogen) for 5 min at room temperature. Tile scans to acquire images of the whole ear were performed using a Vectra Polaris microscope with the following settings:DAPI MSI 0.43ms, FITC 81.70ms, and 20x magnification. GR-1 staining of frozen sections was analyzed using ImageJ / Fiji software. Total area of ​​mouse ear (μm 2 ) and the area of ​​specific GR-1 staining (μm 2 ) was measured. Quantification was performed using the GR-1 area (μm 2 ) to the total area (μm 2 The GR-1 / total area ratio was determined using the following formula:

number

[0261] As shown in Figure 31B, the purified CD89 / IgA-blocking mouse anti-human CD89-specific antibody 10E7 significantly inhibited the influx of human CD89-expressing mouse neutrophil granulocytes induced by anti-mouse collagen XVII human IgA antibody at the injection site (P<0.05).

[0262] These results demonstrated that the CD89 / IgA-blocking mouse anti-human CD89-specific antibody 10E7 inhibited experimentally induced in vivo human IgA-mediated influx of human CD89-expressing mouse neutrophil granulocytes in Tg huCD89 / huIgA mice.

[0263] Example 4. Characterization of human CD89 epitopes and CD89 domains recognized by CD89 / IgA-blocking mouse anti-human CD89 monoclonal antibodies (a) Cross-competition of unlabeled CD89 / IgA-blocking mouse anti-human CD89 antibodies with PE-conjugated commercial mouse anti-CD89 antibodies clone MIP8a (CD89 / IgA blocker), clone A59 (CD89 / IgA non-blocker), and clone A3 (CD89 / IgA non-blocker) for human CD89. To analyze the specificity of the purified CD89 / IgA-blocking mouse anti-human CD89 antibodies, we determined the positions recognized by the generated CD89 / IgA-blocking mouse anti-human CD89 antibodies by cross-competition with a known CD89 / IgA-blocking agent, purified mouse anti-human CD89 antibody clone MIP8a (Zhang et al. Clin Exp Immunol 2000;121:106-111), a known CD89 / IgA-non-blocking agent, purified mouse anti-human CD89 antibody clone A59 (Monteiro et al. J Immunol 1992;148:1764-1770), and a known CD89 / IgA-non-blocking agent, purified mouse anti-human CD89 antibody clone A3 (Monteiro et al. J Immunol 1992;148:1764-1770).

[0264] Human CD89 consists of a short cytoplasmic tail, a transmembrane region, and two extracellular (EC) Ig-like domains. A short interdomain hinge region allows these two Ig-like EC domains to fold at approximately 90° angles relative to each other (Ding et al. J Biol Chem 2003;278:27966-27970). The binding site for human IgA on human CD89 is located in the membrane-distal Ig-like EC1 domain, but not in the membrane-proximal Ig-like EC2 domain (Wines et al. J Immunol 1999;162:2146-2153, Morton J Exp Med 1999;189:1715-1722, Lu et al. Protein Sci 2014;23:378-386). Thus, it is generally accepted that mouse anti-human CD89 antibodies that bind to the EC1 domain of human CD89 can block human IgA binding on human CD89, but that mouse anti-human CD89 antibodies that bind to the EC2 domain of human CD89 cannot block human IgA binding on human CD89 (Morton et al. Arch Immunol Ther Exp 2001;49:217-229, Bakema et al. Immunol Rev 2011;4:612-624). More specifically, the prototypical CD89 / IgA-blocking mouse anti-human CD89 antibody clone MIP8a recognizes an epitope within the EC1 domain of human CD89 (Lu et al. Protein Sci 2014;23:378-386), whereas the prototypical CD89 / IgA-non-blocking mouse anti-human CD89 antibody clones A59 and A3 recognize epitopes within the EC2 domain of human CD89 (Morton J Exp Med 1999;189:1715-1722) and within the EC1-EC2 domain boundary of human CD89, respectively (Morton J Exp Med 1999;189:1715-1722). The ability of the generated purified CD89 / IgA-blocking mouse anti-human CD89 antibodies to compete with known CD89 / IgA-blocking and non-blocking mouse anti-human CD89 antibodies (i.e., clone MIP8a, clone A59, and clone A3) for membrane human CD89 was determined by FACS analysis.

[0265] HEK293F cells stably transfected with human full-length CD89 (clone no. 2, see Example 1(b) above) were cultured at 10 × 10 in ice-cold PBS containing 0.1% BSA (Sigma) / 0.05% NaN (PBS / BSA / NaN) supplemented with 50 μg / mL human IgG (to block potential Fcγ receptors, Sigma). 6 The cells were then added at 10 μL / tube (i.e., 0.1 × 10 cells / mL) and placed at 4°C for 10 minutes. 6 These cells (100 μL each) were incubated with or without 100 μL of purified mouse anti-human CD89 antibody at 10 μg / mL (in PBS / BSA / NaN3) for 30 minutes at 4°C. In parallel, 100 μL of purified mouse IgG1 isotype control (BD Biosciences) at 10 μg / mL (in PBS / BSA / NaN3) was run as a negative control, and 100 μL of purified mouse anti-human CD89 antibody clone MIP8a (BioRad) at 10 μg / mL (in PBS / BSA / NaN3), clone A59 (BD Biosciences) at 10 μg / mL (in PBS / BSA / NaN3), or clone A3 (Santa Cruz Biotechnology) at 10 μg / mL (in PBS / BSA / NaN3) were run as positive controls. After this time (i.e., without washing), 5 μL of undiluted PE-conjugated mouse anti-human CD89 antibodies clone MIP8a (BioRad), clone A59 (BD Biosciences), and clone A3 (Santa Cruz Biotechnology) was added to the cells and incubated for an additional 30 minutes at 4°C. After extensive washing with PBS / BSA / NaN3, the cells were fixed in 2% formaldehyde in PBS / BSA / NaN3 for 30 minutes at 4°C. Binding (geometric mean fluorescence intensity) of PE-conjugated mouse anti-human CD89 antibodies clone MIP8a, clone A59, and clone A3 to membrane human CD89 was measured using a flow cytometer (FACSCalibur, BD Biosciences).

[0266] As shown in Figure 11A, preincubation with 10 μg / mL of unlabeled, purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 9H7, 26D6, and 30C7 completely blocked (>90%) the subsequent binding of the commercial PE-conjugated CD89 / IgA-blocking mouse anti-human CD89 antibody clone MIP8a to membrane human CD89, whereas preincubation with 10 μg / mL of unlabeled, purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 8F3, 10E7, 16D6, and 20B4 did not block (<25%) the subsequent binding of the commercial PE-conjugated CD89 / IgA-blocking mouse anti-human CD89 antibody clone MIP8a to membrane human CD89. For reference purposes, preincubation with 10 μg / mL of unlabeled purified mouse anti-human CD89 / IgA-blocking mouse anti-human CD89 antibody clone MIP8a also completely (>90%) blocked the subsequent binding of its PE-conjugated antibody counterpart to membrane human CD89, whereas preincubation with 10 μg / mL of unlabeled purified well-known mouse non-blocking CD89 / IgA anti-human CD89 antibody clones A59 and A3 did not block (<25%) the subsequent binding of the commercial PE-conjugated CD89 / IgA-blocking mouse anti-human CD89 antibody clone MIP8a to membrane human CD89.

[0267] As shown in Figure 11B, preincubation with 10 μg / mL of unlabeled purified CD89 / IgA-blocking mouse anti-human CD89 specific antibodies 9H7, 26D6, and 20B4 partially blocked the subsequent binding of the commercial PE-conjugated CD89 / IgA-blocking mouse anti-human CD89 antibody clone A59 to membrane human CD89.

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[0268] As shown in Figure 11C, preincubation with 10 μg / mL of unlabeled purified CD89 / IgA-blocking mouse anti-human CD89 specific antibodies 9H7, 26D6, and 20B4 partially blocked the subsequent binding of the commercial PE-conjugated CD89 / IgA-blocking mouse anti-human CD89 antibody clone A3 to membrane human CD89.

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[0269] To analyze the extent of this cross-competition, we also performed preincubations with titrated cross-competing unlabeled purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies (as opposed to using only 10 μg / mL), followed by incubations with PE-conjugated mouse anti-human CD89 antibody clone MIP8a, clone A59, and clone A3 using HEK293F cells stably transfected with human full-length CD89 (clone no. 2, see Example 1(b) above).

[0270] As shown in Figure 12A, all "MIP8a" cross-competing unlabeled purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies (i.e., 9H7, 26D6, and 30C7) dose-dependently blocked the binding of the commercial PE-conjugated CD89 / IgA-blocking mouse anti-human CD89 antibody clone MIP8a to membrane human CD89. Based on their "MIP8a" cross-competition profiles, the following ranking was found (from strong "MIP8a-PE" blockade to weak "MIP8a-PE" blockade): 9H7 = 26D6 (= MIP8a) > 30C7. For a summary, see Table 6.

[0271] As shown in Figure 12B, all "A59" cross-competing unlabeled purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies (i.e., 9H7, 26D6, and 20B4) dose-dependently blocked the binding of the commercial PE-conjugated CD89 / IgA-nonblocking mouse anti-human CD89 antibody clone A59 to membrane human CD89. Based on their "A59" cross-competition profiles, the following ranking was found (from strong "A59-PE" blocking to weak "A59-PE" blocking): (A59 = A3) > 9H7 = 26D6 = 20B4 (= MIP8a). For a summary, see Table 6.

[0272] As shown in Figure 12C, all "A3" cross-competing unlabeled purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies (i.e., 9H7, 26D6, and 20B4) dose-dependently blocked the binding of the commercial PE-conjugated CD89 / IgA-nonblocking mouse anti-human CD89 antibody clone A3 to membrane human CD89. Based on their "A3" cross-competition profiles, the following ranking was found (from strong "A3-PE" blockade to weak "A3-PE" blockade): (A59 = A3) > 9H7 = 26D6 = 20B4 (= MIP8a). For a summary, see Table 6. [Table 7]

[0273] These results demonstrated that the CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 8F3, 10E7, 16D6, 20B4, and 30C7 bound to a different human CD89 epitope than that recognized by the commercial mouse anti-human CD89 antibodies clone MIP8a, clone A59, or clone A3, since their cross-competition profiles were significantly different from those obtained with these commercial mouse anti-human CD89 antibodies. Furthermore, these results demonstrated that the CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 8F3, 10E7, and 16D6 (three antibodies with identical cross-competition profiles) versus the CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 9H7 and 26B6 as a group (both antibodies with identical cross-competition profiles) versus the CD89 / IgA-blocking mouse anti-human CD89-specific antibody 20B4 versus the CD89 / IgA-blocking mouse anti-human CD89-specific antibody 30C7 bound to non-identical CD89 epitopes because their cross-competition profiles were significantly different. In addition, these results demonstrated that the CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 9H7 and 26B6 as a group (both antibodies with identical cross-competition profiles) versus the commercial mouse anti-human CD89 antibody clone MIP8a appeared to bind to similar CD89 epitopes because their cross-competition profiles were identical. These results also demonstrated that the CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 8F3, 10E7, and 16D6 appeared to bind to similar CD89 epitopes because their cross-competition profiles were identical. Finally, these results demonstrated that the CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 9H7 and 26B6 appeared to bind to similar CD89 epitopes because their cross-competition profiles were identical.

[0274] (b) Binding of CD89 / IgA-blocking mouse anti-human CD89 antibodies to membrane-length human CD89 and various membrane-chimeric human CD89 / bovine Fcγ2R constructs (EC1 vs. EC2 domain mapping of human CD89). To analyze the specificity of the purified CD89 / IgA-blocking mouse anti-human CD89 antibodies, the location of the epitope recognized by the generated CD89 / IgA-blocking mouse anti-human CD89 antibodies was determined by domain mapping. The ability of the CD89 / IgA-blocking mouse anti-human CD89 antibodies to bind to the EC1 or EC2 domain of human CD89 expressed on the surface of HEK293F cells was determined by FACS analysis.

[0275] Structurally, human CD89 and bovine Fcγ2R are highly homologous and closely related to each other (Ravetch et al. Annu Rev Immunol 1991;9:457-492, Zhang et al. J Immunol 1995;155:1534-1541). Therefore, a chimeric human / bovine receptor was designed by exchanging the Ig-like EC1 and EC2 domains between these two receptor proteins (see also Figure 13A). Functionally, human CD89 and bovine Fcγ2R are completely different in that human CD89 binds human IgA but not bovine IgG2, whereas bovine Fcγ2R binds bovine IgG2 but not human IgA. The following human CD89 constructs were generated and transiently expressed: (1) a membrane-length human CD89 construct that contained both the Ig-like EC1 and Ig-like EC2 domains of human CD89 (see SEQ ID NO: 1), thus designated "human EC1-EC2-CD89"; (2) a membrane-chimeric Ig-like EC1 domain of human CD89 combined with the Ig-like EC2 domain of bovine Fcγ2R construct (see SEQ ID NO: 3 and SEQ ID NO: 4, i.e., combined with the bovine transmembrane intracellular region or the human transmembrane intracellular region, respectively), thus designated "human EC1-CD89"; and (3) a membrane-chimeric Ig-like EC1 domain of bovine Fcγ2R combined with the Ig-like EC2 domain of human CD89 (see SEQ ID NO: 7), thus designated "human EC2-CD89". Additionally, a full-length bovine Fcγ2R construct was also generated, which contains both the Ig-like EC1 and Ig-like EC2 domains of bovine Fcγ2R (see SEQ ID NO: 9) and is therefore designated "bovine Fcγ2R." cDNAs encoding the above-described "human EC1-EC2-D89," "human EC1-CD89," "human EC2-CD89," and "bovine Fcγ2R" constructs were optimized for mammalian expression and synthesized by GENEART, Regensburg, Germany (see SEQ ID NOs: 2, 5, 6, 8, and 10, respectively). These cDNAs were subcloned into pcDNA3.1-derived expression plasmids.

[0276] FreeStyle™ 293F cells (Invitrogen) were transiently transfected with "human CD89," "human EC1-CD89," "human EC2-CD89," and "bovine Fcγ2R" constructs using the FreeStyle™ 293 Expression System (Invitrogen). After 48 and / or 72 hours, binding of a CD89 / IgA-blocking mouse anti-human CD89 antibody to the aforementioned chimeric human / bovine receptors on the transfected cells was analyzed by FACS analysis. To this end, transiently transfected HEK293F cells were cultured at 10 × 10 in ice-cold phosphate-buffered saline containing 0.1% BSA (Sigma) / 0.05% NaN (PBS / BSA / NaN) supplemented with 50 μg / mL human IgG (to block Fcγ receptors, Sigma). 6 The cells were then added at 10 μL / tube (i.e., 0.1 × 10 cells / mL) and incubated at 4°C for 10 minutes. 6 These cells (100 μL each) were incubated with or without 100 μL of purified mouse anti-human CD89 antibody at 10 μg / mL (in PBS / BSA / NaN3) for 30 minutes at 4°C. In parallel, 100 μL of purified mouse IgG1 isotype control (BD Biosciences) at 10 μg / mL (in PBS / BSA / NaN3) was run as a negative control, and 100 μL of purified mouse anti-human CD89 antibody clone MIP8a (BioRad), clone A59 (BD Biosciences), or clone A3 (Santa Cruz Biotechnology) at 10 μg / mL (in PBS / BSA / NaN3) was run as a positive control. After extensive washing with PBS / BSA / NaN3, the cells were then incubated with a 1:200 diluted PE-conjugated goat anti-mouse IgG Fcγ-specific antibody (Jackson ImmunoResearch) for 30 minutes at 4°C. After extensive washing with PBS / BSA / NaN3, cells were fixed in 2% formaldehyde in PBS / BSA / NaN3 for 30 min at 4° C. Antibody binding was measured using a flow cytometer (model FACSCalibur, BD Biosciences).

[0277] In addition to the binding of the CD89 / IgA-blocking mouse anti-human CD89 antibodies described above to the chimeric human / bovine receptors, (1) the membrane surface expression level and (2) the proper folding of these chimeric human / bovine receptors were also investigated on these transiently transfected cells. In this case, transiently transfected HEK293F cells were cultured at 10 × 10 in ice-cold phosphate-buffered saline containing 0.1% BSA (Sigma) / 0.05% NaN (PBS / BSA / NaN) supplemented with 50 μg / mL human IgG (to block Fcγ receptors, Sigma). 6 The cells were then added at 10 μL / tube (i.e., 0.1 × 10 cells / mL) and incubated at 4°C for 10 minutes. 6These cells (100 μL each) were incubated for 30 minutes at 4°C with or without (1) 100 μL of purified rabbit anti-human CD89 polyclonal antibody (Sino Biological) at 2.5 μg / mL (in PBS / BSA / NaN3) and (2) 100 μL of purified non-aggregated or heat-aggregated human (serum-derived, see Example 2(b) above) IgA (Bethyl Laboratories) at 10 μg / mL (diluted in PBS / BSA / NaN3). After extensive washing with PBS / BSA / NaN3, binding of (1) rabbit anti-human CD89 polyclonal antibody and (2) non-aggregated or heat-aggregated serum human IgA to membrane human CD89 was measured with 5 μg / mL of 1:200 diluted FITC-conjugated F(ab')2 fragment goat anti-rabbit IgG heavy / light chain-specific antibody (Jackson ImmunoResearch) and biotin-conjugated F(ab')2 fragment goat anti-human serum IgA α chain-specific antibody (Jackson ImmunoResearch), respectively, for 30 min at 4°C. After extensive washing with PBS / BSA / NaN3, 1:200 diluted PE-conjugated streptavidin (Jackson ImmunoResearch) was added and incubated for 30 min at 4°C. After extensive washing with PBS / BSA / NaN3, the cells were fixed in 2% formaldehyde in PBS / BSA / NaN3 for 30 min at 4°C. Binding of (1) rabbit anti-human CD89 polyclonal antibody and (2) non-aggregated or heat-aggregated serum human IgA to the membrane chimeric human / bovine receptor was measured using a flow cytometer (model FACSCalibur, BD Biosciences).

[0278] As shown in Figure 13B, all chimeric human CD89 / bovine Fcγ2R receptors (i.e., two versions, "human EC1-CD89" and "human EC2-CD89") and full-length human CD89 were expressed on the membrane surface of transiently transfected cells, as evidenced by the binding of rabbit anti-human CD89 polyclonal antibody to these cells. Furthermore, these membrane-expressed human chimeric CD89 / bovine Fcγ2R receptors (i.e., two versions, "human EC1-CD89") and full-length human CD89 appeared to exhibit proper protein folding, as evidenced by the binding of both unaggregated and heat-aggregated serum human IgA. As expected, the chimeric human CD89 / bovine Fcγ2R receptor "human EC2-CD89," which lacks the EC1 domain (i.e., the IgA-binding site on human CD89), did not bind to unaggregated and heat-aggregated serum human IgA. As expected, rabbit anti-human CD89 polyclonal antibody, non-aggregated and heat-aggregated serum human IgA did not bind to mock-transfected or full-length bovine Fcγ2R-transfected cells.

[0279] As shown in Figure 13C, all of our purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies (i.e., 8F3, 9H7, 10E7, 16D6, 26D6, 20B4, and 30C7) showed binding to both versions of "human EC1-CD89" but not to "human EC2-CD89" in transfected 293F cells. Furthermore, all of our purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies (i.e., 8F3, 9H7, 10E7, 16D6, 26D6, 20B4, and 30C7) showed binding to cells transfected with full-length human CD89 (human EC1-EC2-CD89), but not to mock-transfected or full-length bovine Fcγ2R-transfected cells. As expected, the purified well-known CD89 / IgA-blocking mouse anti-human CD89 antibody clone MIP8a (Lu et al., Protein Sci.), which recognizes an epitope within the EC1 domain of human CD89, also showed binding to cells transfected with mock-transfected or full-length bovine Fcγ2R. 2014;23:378-386) showed binding to both versions of "human EC1-CD89" but not to "human EC2-CD89," whereas a purified, well-known CD89 / IgA non-blocking mouse anti-human CD89 antibody clone A59 (Morton J Exp Med 1999;189:1715-1722), which recognizes an epitope within the EC2 domain of human CD89, showed binding to "human EC2-CD89" but not to "human EC1-CD89." Surprisingly, a purified, well-known CD89 / IgA non-blocking mouse anti-human CD89 antibody clone A3 (Morton J Exp Med 1999;189:1715-1722), which recognizes an epitope dependent on both the EC1 and EC2 domains of human CD89, bound to "human EC2-CD89" but not to "human EC1-CD89." Finally, all of the commercial mouse anti-human CD89 specific antibodies investigated showed binding to cells transfected with full-length human CD89 (human EC1-EC2-CD89), but not to mock-transfected or full-length bovine Fcγ2R-transfected cells.

[0280] These results demonstrated that all of our CD89 / IgA-blocking mouse anti-human CD89 antibodies 8F3, 9H7, 10E7, 16D6, 26D6, 20B4, and 30C7 appeared to recognize linear and / or nonlinear / conformational epitopes within the EC1 domain of human full-length CD89 (i.e., Gln22 to Gly121, Swiss-Prot number P24071.1).

[0281] (c) Binding of CD89 / IgA-blocking mouse anti-human CD89 antibody to membrane cynomolgus monkey full-length CD89 To analyze the cross-reactivity of our purified CD89 / IgA-blocking mouse anti-human CD89 antibodies, the ability of the generated CD89 / IgA-blocking mouse anti-human CD89 antibodies to bind full-length cynomolgus monkey CD89 expressed on the surface of HEK293F cells was determined by FACS analysis.

[0282] The cDNA encoding the cynomolgus monkey CD89 protein (SEQ ID NO: 11, see NCBI reference sequence XP_005590398.1) was optimized for mammalian expression and synthesized by GENEART, Regensburg, Germany (SEQ ID NO: 12). This cDNA was subcloned into a pcDNA3.1-derived expression plasmid.

[0283] FreeStyle™ 293F cells (Invitrogen) were transiently transfected with full-length cynomolgus monkey CD89 using the FreeStyle™ 293 Expression System (Invitrogen). After 48 and / or 72 hours, cross-reactivity of CD89 / IgA-blocking mouse anti-human CD89 antibodies to surface cynomolgus monkey CD89 on transfected cells was analyzed by FACS analysis. To this end, transiently transfected HEK293F cells were cultured at 10 x 10 in ice-cold phosphate-buffered saline containing 0.1% BSA (Sigma) / 0.05% NaN (PBS / BSA / NaN) supplemented with 50 μg / mL human IgG (to block Fcγ receptors, Sigma).6 The cells were then added at 10 μL / tube (i.e., 0.1 × 10 cells / mL) and placed at 4°C for 10 minutes. 6 These cells (100 μL each) were incubated with or without 100 μL of purified mouse anti-human CD89 antibody at 10 μg / mL (in PBS / BSA / NaN3) for 30 minutes at 4°C. In parallel, 100 μL of purified mouse IgG1 isotype control (BD Biosciences) at 10 μg / mL (in PBS / BSA / NaN3) was run as a negative control, and 100 μL of purified mouse anti-human CD89 antibody clone MIP8a (BioRad), clone A59 (BD Biosciences), or clone A3 (Santa Cruz Biotechnology) at 10 μg / mL (in PBS / BSA / NaN3) was run as a positive control. After extensive washing with PBS / BSA / NaN3, the cells were then incubated with a 1:200 diluted PE-conjugated goat anti-mouse IgG Fcγ-specific antibody (Jackson ImmunoResearch) for 30 minutes at 4°C. After extensive washing with PBS / BSA / NaN3, cells were fixed in 2% formaldehyde in PBS / BSA / NaN3 for 30 min at 4° C. Antibody binding was measured using a flow cytometer (model FACSCalibur, BD Biosciences).

[0284] In addition to the binding of the CD89 / IgA-blocking mouse anti-human CD89 antibodies described above to full-length cynomolgus monkey CD89, (1) the membrane surface expression level and (2) the proper folding of full-length cynomolgus monkey CD89 were also investigated on these transiently transfected cells. To this end, transiently transfected HEK293F cells were cultured at 10 × 10 in ice-cold phosphate-buffered saline containing 0.1% BSA (Sigma) / 0.05% NaN (PBS / BSA / NaN) supplemented with 50 μg / mL human IgG (to block Fcγ receptors, Sigma). 6 The cells were then added at 10 μL / tube (i.e., 0.1 × 10 cells / mL) and incubated at 4°C for 10 minutes. 6These cells (100 μL each) were incubated for 30 minutes at 4°C with or without (1) 100 μL of purified rabbit anti-human CD89 polyclonal antibody (Sino Biological) at 2.5 μg / mL (in PBS / BSA / NaN3) and (2) 100 μL of purified non-aggregated or heat-aggregated human (serum-derived, see Example 2(b) above) IgA (Bethyl Laboratories) at 10 μg / mL (diluted in PBS / BSA / NaN3). After extensive washing with PBS / BSA / NaN3, binding of (1) rabbit anti-human CD89 polyclonal antibody and (2) non-aggregated or heat-aggregated serum human IgA to membrane cynomolgus monkey CD89 was measured with 5 μg / mL of 1:200 diluted FITC-conjugated F(ab')2 fragment goat anti-rabbit IgG heavy / light chain-specific antibody (Jackson ImmunoResearch) and biotin-conjugated F(ab')2 fragment goat anti-human serum IgA α chain-specific antibody (Jackson ImmunoResearch), respectively, for 30 min at 4°C. After extensive washing with PBS / BSA / NaN3, 1:200 diluted PE-conjugated streptavidin (Jackson ImmunoResearch) was added and incubated for 30 min at 4°C. After extensive washing with PBS / BSA / NaN3, the cells were fixed in 2% formaldehyde in PBS / BSA / NaN3 for 30 minutes at 4°C. Binding of (1) rabbit anti-human CD89 polyclonal antibody and (2) non-aggregated or heat-aggregated serum human IgA to membrane cynomolgus monkey CD89 was measured using a flow cytometer (model FACSCalibur, BD Biosciences).

[0285] As shown in Figure 14A, full-length cynomolgus CD89 was expressed on the membrane surface of transiently transfected cells, as evidenced by the binding of a cross-reactive rabbit anti-human CD89 polyclonal antibody to these cells. Furthermore, this membrane-expressed full-length cynomolgus CD89 appeared to exhibit proper protein folding, as evidenced by the binding of both cross-reactive non-agglutinated and heat-aggregated serum human IgA.

[0286] As shown in Figure 14B, the purified CD89 / IgA-blocking mouse anti-human CD89-specific antibodies 16D6 and 30C7 showed intermediate / weak cross-reactivity to full-length cynomolgus monkey CD89 on transfected 293F cells. The purified CD89 / IgA-blocking mouse anti-human CD89 antibodies 8F3, 9H7, 10E7, 26D6, and 20B4 did not recognize full-length cynomolgus monkey CD89 on transfected 293F cells. The commercially purified mouse anti-human CD89-specific antibodies clone A59 and clone A3 showed strong cross-reactivity to full-length cynomolgus monkey CD89 on transfected 293F cells, whereas the commercially purified mouse anti-human CD89-specific antibody clone MIP8a did not show any binding to full-length cynomolgus monkey CD89.

[0287] These results demonstrated that the mouse anti-human CD89 antibodies 16D6 and 30C7 likely recognized linear and / or non-linear / conformational epitopes, possibly in the EC1 domain of full-length cynomolgus CD89.

[0288] The predicted amino acid sequence of the full-length cynomolgus monkey CD89 protein (Met1 to Lys287, NCBI reference sequence: XP_005590398.1) shows 86% homology with the amino acid sequence of the full-length human CD89 protein (Met1 to Lys287, Swiss-Prot number P24071.1), and the predicted amino acid sequence of the extracellular region of cynomolgus monkey CD89 (i.e., Gln22 to Asn227, NCBI reference sequence: XP_005590398.1) shows 83% homology with the amino acid sequence of the extracellular region of human CD89 protein (i.e., Gln22 to Asn227, Swiss-Prot number P24071.1). More specifically, the predicted amino acid sequences of the EC1 domain (i.e., Gln22 to Gly121, NCBI reference sequence: XP_005590398.1), short hinge region (i.e., Leu122 to Lys125), EC2 domain (i.e., Pro126 to Asn220), and membrane-proximal "linker" region (i.e., Arg221 to Asn227) of the cynomolgus monkey CD89 protein show 72%, 100%, 93%, and 85% homology, respectively, with the corresponding amino acid sequences of the human CD89 protein.

[0289] Considering that our generated CD89 / IgA-blocking murine anti-human CD89 antibodies 8F3, 9H7, 10E7, 16D6, 26D6, 20B4, and 30C7 appear to recognize linear and / or non-linear / conformational epitopes within the EC1 domain of human full-length CD89 (i.e., Gln22 to Gly121, Swiss-Prot number P24071.1) (see Example 4(b) above). However, most of our CD89 / IgA-blocking mouse anti-human CD89 antibodies (i.e., 8F3, 9H7, 10E7, 26D6, and 20B4) do not cross-react with the low-homology (i.e., 72% amino acid sequence) EC1 domain (i.e., Gln22 to Gly121, NCBI reference sequence: XP_005590398.1) of full-length cynomolgus monkey CD89 on transfected 293F cells.

[0290] (d) Binding of CD89 / IgA-blocking mouse anti-human CD89 antibodies to various membrane chimeric human CD89 / cynomolgus monkey CD89 constructs (epitope mapping within the EC1 domain of human CD89). To analyze the specificity of the purified CD89 / IgA-blocking mouse anti-human CD89 antibodies, the epitopes recognized by the generated CD89 / IgA-blocking mouse anti-human CD89 antibodies were determined by epitope mapping. The ability of the CD89 / IgA-blocking mouse anti-human CD89 antibodies to bind to epitopes within the EC1 domain of human CD89 expressed on the surface of HEK293F cells was determined by FACS analysis.

[0291] Structurally, full-length human CD89 and full-length cynomolgus CD89 are highly homologous (i.e., amino acid sequence, see also Example 4(c) above) and closely related to each other (Rogers et al. Immunol 2004, 113:178-186). However, while all recognized epitopes with the EC1 domain of human CD89 (see Example 4(b) above), our generated CD89 / IgA-blocking mouse CD89 antibodies showed either no cross-species reactivity (i.e., 8F3, 9H7, 10E7, 26D6, and 20B4), weak cross-species reactivity (i.e., 30C7), or intermediate cross-species reactivity (i.e., 16D6) with the EC1 domain of cynomolgus CD89 (see Example 4(c) above). Therefore, a chimeric human CD89 / cynomolgus CD89 receptor was designed by exchanging a portion derived from the EC1 domain of human CD89 (i.e., a 25-amino acid long peptide) with its counterpart from the EC1 domain of cynomolgus CD89 (see also Figure 15A), and the critical region within the EC1 domain of human CD89 that is recognized by our generated CD89 / IgA-blocking mouse anti-human CD89 antibody was determined. Functionally, human CD89 and cynomolgus CD89 are similar in that both human CD89 and cynomolgus CD89 bind to human serum IgA (see Example 4(c) above). The following human CD89 constructs were generated and transiently expressed:(1) a membrane-based full-length human CD89 construct containing the full-length EC1 domain of human CD89 (see SEQ ID NO: 1), thus designated "human EC1-CD89"; (2) a membrane-based chimeric human CD89 / cynomolgus CD89 construct (I) (see SEQ ID NO: 13), thus designated "ΔGln22-Lys46 human EC1-CD89"; and (3) a membrane-based chimeric human CD89 / cynomolgus CD89 construct (II) (see SEQ ID NO: 15), thus designated "ΔGln22-Lys46 human EC1-CD89"; (4) a membrane-chimeric human CD89 / cynomolgus CD89 construct (III) (see SEQ ID NO: 17) consisting of Gly72 to Gly96 from the EC1 domain of human CD89 swapped with the reciprocal Asp72 to Gly96 from the EC1 domain of cynomolgus CD89, thus designated "ΔGly72-Gly96 human EC1-CD89," and (5) a membrane-chimeric human CD89 / cynomolgus CD89 construct (IV) (see SEQ ID NO: 19) consisting of Arg97 to Gly121 from the EC1 domain of human CD89 swapped with the reciprocal Arg97 to Gly121 from the EC1 domain of cynomolgus CD89, thus designated "ΔArg97-Gly121 human EC1-CD89." Additionally, the membrane-length cynomolgus CD89 construct contains the full-length EC1 domain of human cynomolgus CD89 (see SEQ ID NO: 11), and is therefore designated "cynomolgus EC1-CD89."

[0292] In addition to exchangi...

Claims

1. 1. A humanized anti-human CD89 antibody capable of binding to the extracellular portion of human CD89, comprising the amino acid sequence: EVQLLESGGG LVQPGGSLRL SCAASGLTFS SYGMSWVRQA PGKGLEX １ VX ２ TIX ３ GX ４ GDITYYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDY DYDYAMDYWG QGTLVTVSS (SEQ ID NO: 144) wherein: X １ is L or W X ２ is A or S X ３ is N or S X ４ is Q, T, or N; the heavy chain variable region is X １ , X ２ , X ３ , and X ４ a heavy chain variable region comprising 0, 1, 2, or 3 amino acid insertions, deletions, or substitutions, or a combination thereof, with respect to SEQ ID NO: 144, at positions other than those listed above but not within the CDR regions, wherein the amino acid sequence of the CDR1 region is SYGMS (SEQ ID NO: 45), the amino acid sequence of the CDR2 region is TINGNGDITYYPDSVKG (SEQ ID NO: 46), TISGQGDITYYPDSVKG (SEQ ID NO: 147), or TISGTGDITYYPDSVKG (SEQ ID NO: 148), and the amino acid sequence of the CDR3 region is DYDYDYAMDY (SEQ ID NO: 47); Amino acid sequence: DIQMTQSPSS LSASVGDRVT ITCRASQDII NYLNWYQQKP GKZ １ Z ２ KLLIYY TSRLHSGVPS RFSGSGSGTD Z ３ TLTISSLQP EDFATYZ ４ CQQ GKTLPYTFGQ GTKLEIK (SEQ ID NO: 145) a light chain variable region comprising: Z １ is A or T Z ２ is V or P Z ３ is Y or F Z ４ is Y or F, the light chain variable region is Z １ , Z ２ , Z ３ and Z ４ a light chain variable region comprising 0, 1, 2, or 3 amino acid insertions, deletions, or substitutions, or a combination thereof, with respect to SEQ ID NO: 145, at positions other than SEQ ID NO: 146 but not within the CDR regions, wherein the amino acid sequence of the CDR1 region is RASQDIINYLN (SEQ ID NO: 48), the amino acid sequence of the CDR2 region is YTSRLHS (SEQ ID NO: 49), and the amino acid sequence of the CDR3 region is QQGKTLPYT (SEQ ID NO: 50).

2. the heavy chain variable region having the amino acid sequence: EVQLLESGGG LVQPGGSLRL SCAASGLTFS SYGMSWVRQA PGKGLELVAT ISGX ４ GDITYYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDY DYDYAMDYWG QGTLVTVSS (SEQ ID NO: 149), wherein: X ４ is Q, T, or N; 2. The humanized anti-human CD89 antibody of claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 122, which contains 0, 1, 2, or 3 amino acid insertions, deletions, or substitutions, or a combination thereof, with respect to SEQ ID NO: 122, and wherein the 0, 1, 2, or 3 amino acid insertions, deletions, or substitutions, or a combination thereof, is not within a CDR region.

3. The antibody of claim 1 or 2, wherein the antibody has an IgG1 or IgG4 isotype.

4. 4. The humanized anti-human CD89 antibody of any one of claims 1 to 3, wherein the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 142 or 143 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, or substitutions, wherein the 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, or substitutions are not within the CDR regions; and a light chain having the amino acid sequence of SEQ ID NO: 136 with 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid insertions, deletions, or substitutions, wherein the 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acid insertions, deletions, or substitutions are not within the CDR regions.

5. 5. The antibody of claim 1, wherein the antibody has a higher affinity for the extracellular portion of human CD89 compared to a chimeric antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 94 and a light chain having the amino acid sequence of SEQ ID NO:

99.

6. The antibody is capable of binding to the extracellular portion of human CD89 on cells expressing human CD89; and When the antibody binds to the cells, the antibody is capable of preventing the binding of human IgA to human CD89. The antibody according to any one of claims 1 to 5.

7. The antibody of claim 6, wherein the cells are human CD89-expressing HEK293F cells (deposited under number DSM ACC3341).

8. One or more nucleic acid molecules encoding the antibody or antigen (human CD89) binding fragment thereof according to any one of claims 1 to 7.

9. A nucleic acid molecule encoding the variable region of the antibody according to any one of claims 1 to 7.

10. A vector comprising the nucleic acid molecule of claim 8 or 9.

11. A cell comprising an antibody according to any one of claims 1 to 7, one or more nucleic acid molecules according to claim 8 or 9, and / or a vector according to claim 10, wherein the cell is a mammalian cell, an insect cell, a plant cell, a bacterial cell, or a yeast cell.

12. The cell described in claim 11, which is a human cell.

13. A method for producing an antibody according to any one of claims 1 to 7, said method comprising: (a) providing a cell comprising an antibody according to any one of claims 1 to 7, one or more nucleic acid molecules according to claim 8 or 9, and / or a vector according to claim 10; and (b) recovering the antibody from the cells; The method comprising:

14. The method of claim 13, wherein the cell is a hybridoma cell, a Chinese hamster ovary (CHO) cell, an NS0 cell, or a PER-C6™ cell.

15. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, one or more nucleic acid molecules according to claim 8 or 9, and / or a vector according to claim 10.

16. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, one or more nucleic acid molecules according to claim 8 or 9, and / or a vector according to claim 10, for use in the treatment or prevention of a chronic inflammatory disease.

17. 11. An agent for use in treating a chronic inflammatory disease, comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, one or more nucleic acid molecules according to claim 8 or 9, and / or a vector according to claim 10.

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