Compositions and methods comprising iga antibody constructs

Abstract

To provide therapeutics, pharmaceutical compositions comprising IgA constant region and methods.SOLUTION: The disclosure provides an antibody construct comprising: (a) an immunoglobulin A (IgA) heavy chain domain; (b) a CD47 binding domain; and (c) an antigen binding domain, where the IgA heavy chain domain specifically binds to the FcαR on an immune effector cell, the CD47 binding domain inhibits the binding of the CD47 expressed on a target cell to a signal regulatory protein α (SIRPα) on the immune effector cell, the antigen binding domain binds to the antigen on the target cell, and the antibody construct has a higher binding affinity to the antigen as compared with CD47.SELECTED DRAWING: Figure 9

Description

[Background technology]

[0001] cross reference

[0001] This application is a joint venture with European Patent Application EP1820318 filed on October 29, 2018. This application claims the benefit of U.S. Provisional Application No. 62 / 752,641, filed Oct. 30, 2018, which is hereby incorporated by reference in its entirety.

[0002]

[0002] Monoclonal antibodies of the IgG isotype targeting tumor antigens have been developed in a variety of It has been proven to be an effective treatment for malignant tumors. Over the years, more and more monoclonal antibodies targeting different tumor antigens have been approved for use in cancer treatment. However, their clinical efficacy, especially in monotherapy, is still not sufficient. There is an interest in developing new alternative antibody therapies with improved clinical efficacy. Summary of the Invention

[0003] In one aspect, the present invention provides a method for the production of a medicament comprising the steps of: (a) an immunoglobulin A (IgA) heavy chain domain; and (c) an antigen-binding domain, wherein the IgA heavy chain domain specifically binds to FcαR on an immune effector cell, the CD47-binding domain inhibits binding between CD47 expressed on a target cell and signal regulatory protein alpha (SIRP α) on the immune effector cell, the antigen-binding domain binds to an antigen on the target cell, and the antibody construct has a higher binding affinity for the antigen compared to CD47.

[0004] In some embodiments, the CD47 binding domain comprises a first light chain variable domain and and at least one of a first heavy chain variable domain. In some embodiments, the antigen binding domain comprises at least one of a second light chain variable domain and a second heavy chain variable domain. In some embodiments, the construct comprises the first light chain variable domain, the first light chain variable domain comprising a variable light chain complementarity determining region 1 (CDR-L1), a variable light chain complementarity determining region 2 (CDR L2), and a variable light chain complementarity determining region 3 (CDR-L3), wherein the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 13 or a variant thereof having up to two amino modifications in SEQ ID NO: 13, the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 14 or a variant thereof having up to two amino modifications in SEQ ID NO: 14, and the CDR-L3 comprises the amino acid sequence of SEQ ID NO: 15 or a variant thereof having up to two amino modifications in SEQ ID NO: 15. In some embodiments, the construct comprises said first light chain variable domain, wherein said first light chain variable domain comprises an amino acid sequence having 90% sequence identity to SEQ ID NO:26.

[0005] In some embodiments, the construct comprises the first heavy chain variable domain, The first heavy chain variable domain comprises a variable heavy chain complementarity determining region 1 (CDR-H1), a variable heavy chain complementarity determining region 2 (CDR-H2) and a variable heavy chain complementarity determining region 3 (CDR-H3), wherein the CDR-H1 comprises the amino acid sequence of SEQ ID NO:4 or a variant thereof with up to two amino modifications in SEQ ID NO:4, the CDR-H2 comprises the amino acid sequence of SEQ ID NO:5 or a variant thereof with up to two amino modifications in SEQ ID NO:5, and the CDR-H3 comprises the amino acid sequence of SEQ ID NO:6 or a variant thereof with up to two amino modifications in SEQ ID NO:6.

[0006] In some embodiments, the construct comprises the first heavy chain variable domain, The first heavy chain variable domain has an amino acid sequence having 90% sequence identity to SEQ ID NO:23. Contains arrays.

[0007] In some embodiments, the construct comprises the second light chain variable domain, The second light chain variable domain comprises CDR-L1, CDR-L2, and CDR-L3, wherein the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 10 or a variant thereof with up to two amino modifications in SEQ ID NO: 10, the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 11 or a variant thereof with up to two amino modifications in SEQ ID NO: 11, and the CDR-L3 comprises the amino acid sequence of SEQ ID NO: 12 or a variant thereof with up to two amino modifications in SEQ ID NO: 12. In some embodiments, the construct comprises the second light chain variable domain, wherein the second light chain variable domain comprises the amino acid sequence of SEQ ID NO: 25.

[0008] In some embodiments, the construct comprises the second heavy chain variable domain, The second heavy chain variable domain comprises CDR-H1, CDR-H2 and CDR-H3, wherein the CDR-H1 comprises the amino acid sequence of SEQ ID NO:1 or a variant thereof having up to two amino modifications in SEQ ID NO:1, the CDR-H2 comprises the amino acid sequence of SEQ ID NO:2 or a variant thereof having up to two amino modifications in SEQ ID NO:2, and the CDR-H3 comprises the amino acid sequence of SEQ ID NO:3 or a variant thereof having up to two amino modifications in SEQ ID NO:3.

[0009] In some embodiments, the construct comprises the second heavy chain variable domain, The second heavy chain variable domain comprises an amino acid sequence having 90% sequence identity to SEQ ID NO:22.

[0010]

[0010] In some embodiments, an antibody construct comprises at least one of: a first polypeptide comprising the first heavy chain variable domain and a second heavy chain variable domain, wherein the C-terminus of the second heavy chain variable domain is linked to the N-terminus of the first heavy chain variable domain; and a second polypeptide comprising the first light chain variable domain and a second light chain variable domain, wherein the C-terminus of the second light chain variable domain is linked to the N-terminus of the first light chain variable domain.

[0011] In some embodiments, the first or second polypeptide further comprises a linker peptide linking the variable domains. In some embodiments, the linker peptide comprises the sequence of SEQ ID NO: 44. In some embodiments, the first polypeptide comprises the sequence of SEQ ID NO: 29. In some embodiments, the C-terminus of the first polypeptide is linked to an (IgA) heavy chain region. In some embodiments, the linkage is via an IgA CH1 constant domain.

[0012] In some embodiments, the second polypeptide comprises the sequence of SEQ ID NO: 31. In some embodiments, the antibody construct comprises a first polypeptide and a second polypeptide. In some embodiments, the antibody construct comprises at least one of: a first polypeptide in which the C-terminus of a first heavy chain variable domain is linked to the N-terminus of a second heavy chain variable domain; or a second polypeptide in which the C-terminus of a first light chain variable domain is linked to the N-terminus of a second light chain variable domain.

[0013] In some embodiments, the linkage is by a linker peptide. In some embodiments, the linker peptide comprises the sequence of SEQ ID NO: 44. In some embodiments, the first polypeptide comprises the sequence of SEQ ID NO: 30. In some embodiments, the C-terminus of the first polypeptide is linked to an (IgA) heavy chain region.

[0014] In some embodiments, the link is via the IgA CH1 constant domain. In some embodiments, the second polypeptide comprises the sequence of SEQ ID NO:32. In some embodiments, the antibody construct comprises a first polypeptide and a second polypeptide. In some embodiments, the antibody construct comprises a first polypeptide comprising a single chain variable fragment (scFv), the scFv comprising said second heavy chain variable domain linked to said second light chain variable domain. In some embodiments, the construct comprises a linker peptide linking said variable domains. In some embodiments, the linker peptide comprises the sequence of SEQ ID NO:45. In some embodiments, the first polypeptide comprises a first light chain variable domain; or an scFv linked to a first heavy chain variable domain. In some embodiments, the link is by a linker peptide. In some embodiments, the linker peptide comprises the sequence of SEQ ID NO:44. In some embodiments, the antibody construct comprises a first polypeptide comprising a scFv linked to a first light chain variable domain, the first polypeptide comprising a sequence having at least 90% identity to SEQ ID NO:35.

[0015] In some embodiments, the antibody construct comprises a first polypeptide comprising an scFv linked to a first heavy chain variable domain, the first polypeptide comprising a sequence having at least 90% identity to SEQ ID NO: 33. In some embodiments, the antibody construct comprises a first polypeptide comprising a single chain variable fragment (scFv), the scFv comprising a first heavy chain variable domain linked to a first light chain variable domain. In some embodiments, the antibody construct comprises a linker peptide linking said variable domains. In some embodiments, the linker peptide comprises the sequence of SEQ ID NO: 45. In some embodiments, the first polypeptide comprises an scFv linked to a second light chain variable domain; or a second heavy chain variable domain.

[0016] In some embodiments, the linkage is by a linker peptide. In some embodiments, the linker peptide comprises the sequence of SEQ ID NO: 44. In some embodiments, the antibody construct comprises a first polypeptide comprising an scFV linked to a second light chain variable domain, the first polypeptide comprising a sequence having 90% identity to SEQ ID NO: 36. In some embodiments, the antibody construct comprises a first polypeptide comprising an scFV linked to a second heavy chain variable domain, the first polypeptide comprising a sequence having 90% identity to SEQ ID NO: 34. In some embodiments, the IgA heavy chain domain comprises an IgA heavy chain constant domain.

[0017] In some embodiments, the IgA heavy chain constant domain is an IgA1 constant domain or a variant thereof. In some embodiments, the IgA1 constant domain comprises at least one of an IgA1 CH2 region and an IgA1 CH3 region or a variant thereof. In some embodiments, the IgA1 constant region comprises an IgA1 CH1 region or a variant thereof. In some embodiments, the IgA heavy chain constant domain is an IgA2 constant domain or a variant thereof. In some embodiments, the IgA2 constant domain comprises at least one of an IgA2 CH2 region and an IgA2 CH3 region or a variant thereof. In some embodiments, the IgA2 constant region further comprises an IgA2 It further comprises a CH1 region.

[0018] In some embodiments, the antigen is CD20, GD2, mesothelin, CD38, CD19, EGFR, HER2, PD-L1, or CD25. In some embodiments, the IgA heavy chain constant domain lacks at least one or two naturally occurring glycosylation sites compared to the corresponding wild-type IgA. In some embodiments, the construct lacks at least two naturally occurring glycosylation sites, the at least two naturally occurring glycosylation sites being at least one IgA CH2 region or IgA In some embodiments, at least two naturally occurring glycosyltransferases are present in the CH3 region. The sylation sites are two naturally occurring N-linked glycosylation sites. In some embodiments, the IgA heavy chain constant domain lacks at least two naturally occurring asparagine (N) amino acid residues compared to the corresponding wild-type IgA.

[0019] In some embodiments, the IgA heavy chain constant domain comprises an amino acid substitution of at least two naturally occurring asparagine (N) amino acid residues, or a substitution of both residues. In some embodiments, the IgA heavy chain constant domain lacks at least one naturally occurring cysteine ​​(C) amino acid residue compared to the corresponding wild-type IgA. In some embodiments, the IgA heavy chain constant domain comprises an amino acid substitution of at least one naturally occurring cysteine ​​(C) amino acid residue. In some embodiments, the IgA heavy chain constant domain comprises a non-conservative amino acid substitution of at least one naturally occurring cysteine ​​(C) amino acid residue. In some embodiments, the IgA heavy chain constant domain comprises an amino acid deletion of at least one naturally occurring cysteine ​​(C) amino acid residue.

[0020] In some embodiments, the IgA heavy chain constant domain lacks at least one naturally occurring tyrosine (Y) amino acid residue compared to the corresponding wild-type IgA. In some embodiments, the IgA heavy chain constant domain comprises an amino acid substitution of at least one naturally occurring cysteine ​​(Y) amino acid residue. In some embodiments, the IgA heavy chain constant domain comprises a deletion of at least one naturally occurring cysteine ​​(Y) amino acid residue. In some embodiments, the IgA heavy chain constant domain lacks at least one naturally occurring threonine (T) amino acid residue compared to the corresponding wild-type IgA.

[0021] In some embodiments, the IgA heavy chain constant domain comprises an amino acid substitution of at least one naturally occurring threonine (T) amino acid residue. In some embodiments, the IgA heavy chain constant domain comprises a deletion of at least one naturally occurring threonine (T) amino acid residue. In some embodiments, the IgA heavy chain constant domain lacks at least one naturally occurring isoleucine (I) amino acid residue compared to the corresponding wild-type IgA. In some embodiments, the IgA heavy chain constant domain comprises an amino acid substitution of at least one naturally occurring isoleucine (I) amino acid residue. In some embodiments, the IgA heavy chain constant domain comprises a deletion of at least one naturally occurring isoleucine (I) amino acid residue.

[0022] In some embodiments, the IgA heavy chain constant domain lacks at least one naturally occurring proline (P) amino acid residue compared to the corresponding wild-type IgA. In some embodiments, the IgA heavy chain constant domain comprises an amino acid substitution of at least one naturally occurring proline (P) amino acid residue. In some embodiments, the IgA heavy chain constant domain comprises a deletion of at least one naturally occurring proline (P) amino acid residue.

[0023]

[0023] In some embodiments, the construct exhibits a longer circulating half-life compared to a corresponding antibody construct comprising a corresponding wild-type IgA heavy chain constant region.

[0024] In some embodiments, the antibody construct comprises an attenuated CD47 binding domain that has a lower affinity for CD47 compared to the corresponding wild-type CD47 binding domain.

[0024]

[0025] In some embodiments, the antibody construct exhibits reduced aggregation compared to a corresponding antibody construct comprising a corresponding wild-type IgA heavy chain constant region. In some embodiments, the antibody construct exhibits reduced aggregation with serum proteins compared to a corresponding antibody construct comprising a corresponding wild-type IgA heavy chain constant region.

[0025]

[0026] In some embodiments, the IgA heavy chain constant region comprises one or more albumin binding domains. In some embodiments, the antibody construct has a longer half-life than a corresponding antibody construct that does not include one or more albumin binding domains.

[0026]

[0027] In some embodiments, the IgA heavy chain constant domain comprises an amino acid sequence having 90% sequence identity to a sequence selected from any one of SEQ ID NOs: 37-41.

[0027]

[0028] In some embodiments, the construct further comprises a hinge region, hi some embodiments, the hinge region comprises an IgA hinge amino acid sequence, or a variant or fragment thereof.

[0028]

[0029] In some embodiments, the hinge region comprises a human IgA hinge amino acid sequence or a variant or fragment thereof. In some embodiments, the hinge is an IgA1 hinge or an IgA2 hinge, or a variant or fragment thereof. In some embodiments, the antibody construct further comprises a light chain constant region. In some embodiments, the light chain constant region is a kappa constant region. In some embodiments, the light chain constant region is a lambda constant region. In some embodiments, the light chain constant region is linked to the first light chain variable domain or the first heavy chain variable domain.

[0029]

[0030] In one aspect, provided herein is an antibody construct comprising: (a) an immunoglobulin A (IgA) heavy chain constant domain; (b) a CD47 binding domain; and (c) an antigen binding domain, wherein the IgA heavy chain domain specifically binds to FcαR on an immune effector cell, the CD47 binding domain inhibits binding between CD47 expressed on a target cell and signal regulatory protein alpha (SIRP alpha) on the immune effector cell, the antigen binding domain binds to an antigen on the target cell, and the antibody construct has a higher binding affinity for the antigen compared to CD47.

[0030]

[0031] In some embodiments, the CD47 binding domain comprises a first light chain variable domain and a first heavy chain variable domain. In some embodiments, the antigen binding domain comprises a second light chain variable domain and a second heavy chain variable domain. In some embodiments, the first light chain variable domain is of the kappa type. In some embodiments, the second light chain variable domain is of the lambda type.

[0031]

[0032] In some embodiments, the first light chain variable domain is of the lambda type. In some embodiments, the second light chain variable domain is of the kappa type. In some embodiments, the first light chain variable domain is of the kappa type and comprises the variable light chain complementarity determining region 1 (CDR-L1) amino acid sequence of SEQ ID NO: 16, the variable light chain complementarity determining region 2 (CDR L2) amino acid sequence of SEQ ID NO: 17, and the variable light chain complementarity determining region 3 (CDR-L3) amino acid sequence of SEQ ID NO: 18. In some embodiments, the first light chain variable domain comprises an amino acid sequence having 90% sequence identity to SEQ ID NO: 27.

[0032]

[0033] In some embodiments, the second light chain variable domain is of the lambda type comprising the variable light chain complementarity determining region 1 (CDR-L1) amino acid sequence of SEQ ID NO: 19, the variable light chain complementarity determining region 2 (CDR L2) amino acid sequence of SEQ ID NO: 20, and the variable light chain complementarity determining region 3 (CDR-L3) amino acid sequence of SEQ ID NO: 21. In some embodiments, the second light chain variable domain comprises an amino acid sequence having 90% sequence identity to SEQ ID NO: 28. In some embodiments, the first heavy chain variable domain and the second heavy chain variable domain are the same.

[0033]

[0034] In some embodiments, the first heavy chain variable domain and the second heavy chain variable domain comprise a variable heavy chain complementarity determining region 1 (CDR-H1) amino acid sequence of SEQ ID NO:7, a variable heavy chain complementarity determining region 2 (CDR-H2) amino acid sequence of SEQ ID NO:8, and a variable heavy chain complementarity determining region 3 (CDR-H3) amino acid sequence of SEQ ID NO:9.

[0034]

[0035] In some embodiments, the first heavy chain variable domain and the second heavy chain variable domain comprise an amino acid sequence having 90% sequence identity to SEQ ID NO:24. In some embodiments, the first light chain variable domain further comprises a kappa constant region. In some embodiments, the second light chain variable domain further comprises a lambda constant region. In some embodiments, the first light chain variable domain further comprises a lambda constant region. In some embodiments, the second light chain variable domain further comprises a kappa constant region. In some embodiments, the kappa constant region comprises an amino acid sequence having 95% sequence identity to SEQ ID NO:42. In some embodiments, the lambda constant region comprises an amino acid sequence having 95% sequence identity to SEQ ID NO:43. In some embodiments, the IgA heavy chain constant domain comprises at least one of an IgA CH2 region and an IgA CH3 region or a variant thereof.

[0035]

[0036] In some embodiments, the IgA heavy chain constant domain further comprises an IgA CH1 region or a variant thereof. In some embodiments, the CD47 binding domain is linked to the IgA constant domain by an IgA CH1 domain. In some embodiments, the antigen binding domain is linked to the IgA constant domain by an IgA CH1 domain. In some embodiments, the IgA heavy chain constant domain is an IgA1 constant region or a variant thereof. In some embodiments, the IgA1 constant region comprises an IgA1 CH2 region and an IgA1 CH3 region.

[0036]

[0037] In some embodiments, the IgA1 constant region further comprises an IgA1 CH1 region. In some embodiments, the IgA heavy chain constant domain is an IgA2 constant region or a variant thereof. In some embodiments, the IgA2 constant region is an IgA2 CH2 region and an IgA2 CH3 region. In some embodiments, the IgA2 constant region comprises an Ig In some embodiments, the antigen further comprises a CD20, GD2, Mesothelin, CD38, CD19, EGFR, HER2, PD-L1, or CD25.

[0037]

[0038] In some embodiments, the IgA heavy chain constant domain lacks one, two or three naturally occurring glycosylation sites compared to the corresponding wild-type IgA. In some embodiments, the sites are in at least one of the IgA CH2 region or the IgA CH3 region. In some embodiments, the construct lacks two naturally occurring N-linked glycosylation sites. In some embodiments, the IgA heavy chain constant domain lacks a naturally occurring asparagine (N) amino acid residue compared to the corresponding wild-type IgA.

[0038]

[0039] In some embodiments, the IgA heavy chain constant domain comprises an amino acid substitution of at least one naturally occurring asparagine (N) amino acid residue, or a substitution of at least two naturally occurring asparagine (N) amino acid residues. In some embodiments, the IgA heavy chain constant domain lacks at least one naturally occurring cysteine ​​(C) amino acid residue compared to the corresponding wild-type IgA. In some embodiments, the IgA heavy chain constant domain comprises an amino acid substitution of at least one naturally occurring cysteine ​​(C) amino acid residue. In some embodiments, the IgA heavy chain constant domain comprises a non-conservative amino acid substitution of at least one naturally occurring cysteine ​​(C) amino acid residue.

[0039]

[0040]

[0041] In some embodiments, the IgA heavy chain constant domain comprises an amino acid deletion of at least one naturally occurring cysteine ​​(C) amino acid residue. In some embodiments, the IgA heavy chain constant domain lacks at least one naturally occurring tyrosine (Y) amino acid residue compared to the corresponding wild-type IgA. In some embodiments, the IgA heavy chain constant domain comprises an amino acid substitution of at least one naturally occurring cysteine ​​(Y) amino acid residue. In some embodiments, the IgA heavy chain constant domain comprises a deletion of at least one naturally occurring cysteine ​​(Y) amino acid residue.

[0040]

[0042] In some embodiments, the IgA heavy chain constant domain lacks at least one naturally occurring threonine (T) amino acid residue compared to the corresponding wild-type IgA. In some embodiments, the IgA heavy chain constant domain comprises an amino acid substitution of at least one naturally occurring threonine (T) amino acid residue. In some embodiments, the IgA heavy chain constant domain comprises a deletion of at least one naturally occurring threonine (T) amino acid residue. In some embodiments, the IgA heavy chain constant domain lacks at least one naturally occurring isoleucine (I) amino acid residue compared to the corresponding wild-type IgA.

[0041]

[0043] In some embodiments, the IgA heavy chain constant domain comprises an amino acid substitution of at least one naturally occurring isoleucine (I) amino acid residue. In some embodiments, the IgA heavy chain constant region comprises a deletion of at least one naturally occurring isoleucine (I) amino acid residue. In some embodiments, the IgA heavy chain constant domain lacks at least one naturally occurring proline (P) amino acid residue compared to the corresponding wild-type IgA. In some embodiments, the IgA heavy chain constant region comprises an amino acid substitution of at least one naturally occurring proline (P) amino acid residue.

[0042]

[0044] In some embodiments, the IgA heavy chain constant domain comprises a deletion of at least one naturally occurring proline (P) amino acid residue. In some embodiments, the antibody construct exhibits a longer circulating half-life compared to a corresponding antibody construct comprising a corresponding wild-type IgA heavy chain constant region. In some embodiments, the antibody construct exhibits a longer half-life compared to a corresponding antibody construct comprising a corresponding wild-type IgA heavy chain constant region. In some embodiments, the antibody construct exhibits reduced aggregation compared to a corresponding antibody construct comprising a corresponding wild-type IgA heavy chain constant region. In some embodiments, the antibody construct exhibits reduced aggregation with serum proteins compared to a corresponding antibody construct comprising a corresponding wild-type IgA heavy chain constant region.

[0043]

[0045] In some embodiments, the IgA heavy chain constant region comprises one or more albumin binding domains. In some embodiments, the antibody construct has a longer half-life than a corresponding antibody construct that does not comprise one or more albumin binding domains. In some embodiments, the IgA heavy chain constant region further comprises a hinge region. In some embodiments, the hinge region comprises an IgA hinge amino acid sequence or a variant or fragment thereof.

[0044]

[0046] In some embodiments, the hinge region comprises a human IgA hinge amino acid sequence or a variant or fragment thereof. In some embodiments, the hinge is an IgA1 hinge or an IgA2 hinge, or a variant or fragment thereof. In some embodiments, the antibody construct comprises an immunoadhesion molecule, The immunological agent further comprises an imaging agent, a therapeutic agent, or a cytotoxic agent. In some embodiments, the imaging agent is a radioactive label, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, or biotin. In some embodiments, the therapeutic agent or cytotoxic agent is an antimetabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an antiangiogenic agent, an antimitotic agent, an anthracycline, a toxin, or an apoptotic agent. In some embodiments, the immune effector cell is a neutrophil.

[0045]

[0047] In some embodiments, the antibody construct inhibits signal regulatory protein alpha (SIRP alpha) function on immune effector cells only if the antigen binding domain also binds to a target cell expressing CD47. In some embodiments, the antibody construct is bispecific. In some embodiments, the CD47 binding domain is a Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, diabody, linear antibody, monoclonal antibody, or monoclonal antibody. In some embodiments, the antigen binding domain is a Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, diabody, linear antibody, single domain antibody (sdAb), or camelid VHH domain. In some embodiments, the IgA heavy chain domain is a heterodimer of two IgA heavy chain constant domains. In some embodiments, the heterodimerization is by knobs-into-holes coupling, salt bridge / electrostatic complementarity. coupling, CrossMab coupling, strand-exchange manipulation domain In some embodiments, the method includes, but is not limited to, using a combination of techniques, such as, for example, a cellular or human endothelial cell line (e.g., a cellular or human endothelial cell line), ... The antigen-binding domain binds to an antigen of a cancer cell or a pathogen. In some embodiments, the pathogen is a pathogen, a microorganism, or a virus.

[0046]

[0048] In one aspect, provided herein is an antibody construct comprising: (a) an immunoglobulin A (IgA) heavy chain domain; (b) a CD47 binding domain; and (c) an antigen binding domain, wherein the IgA heavy chain domain specifically binds to FcαR on an immune effector cell, the CD47 binding domain inhibits binding of CD47 expressed on a target cell to signal regulatory protein alpha (SIRP alpha) on the immune effector cell, the antigen binding domain binds to an antigen on the target cell, the antigen being CD20, GD2, mesothelin, CD38, CD19, EGFR, HER2, PD-L1 or CD25, and wherein the antibody construct has a higher binding affinity for the antigen compared to CD47.

[0047]

[0049] In one aspect, provided herein is a multispecific immune effector cell engager molecule comprising: (a) an immunoglobulin A (IgA) heavy chain domain; (b) a CD47 binding domain; and (c) an antigen binding domain, wherein the IgA heavy chain domain is The multispecific immune effector cell engager molecule specifically binds to FcαR on immune effector cells, the CD47 binding domain inhibits binding between CD47 expressed on target cells and signal regulatory protein alpha (SIRPα) on immune effector cells, the antigen binding domain binds to an antigen on the target cell, and the antibody construct has a higher binding affinity for the antigen compared to CD47.

[0048]

[0050] In one aspect, provided herein is a pharmaceutical composition comprising any one of the above antibody constructs and a pharma- ceutically acceptable carrier, adjuvant or diluent. In some embodiments, the pharmaceutical composition further comprises at least one additional therapeutic agent. In some embodiments, the additional therapeutic agent is an imaging agent, a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor, a costimulatory molecule blocker, an adhesion molecule blocker, an anti-cytokine antibody or functional fragment thereof, methotrexate, cyclosporine, rapamycin, FK506, a detectable label or reporter, a TNF antagonist, an antirheumatic drug, a muscle relaxant, a narcotic, a nonsteroidal anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antibacterial agent, an antipsoriatic drug, a corticosteriod, an anabolic steroid, erythropoietin, immunization, an immunoglobulin, an immunosuppressant, a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, epinephrine or an analog thereof, a cytokine, or a cytokine antagonist.

[0049]

[0051] In one aspect, provided herein is an isolated nucleic acid encoding the antibody construct of any one of the above aspects.

[0052] In one aspect, provided herein is a vector comprising the isolated nucleic acid described above.

[0050]

[0053] In one aspect, there is provided herein an in vitro cell comprising an isolated nucleic acid as disclosed above.

[0054] In one aspect, provided herein is an in vitro cell expressing the antibody construct of any one of the above aspects.

[0051]

[0055] In one aspect, provided herein is a method of treating a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising an antibody construct, the antibody construct comprising: (b) an immunoglobulin A (IgA) heavy chain domain; (b) a CD47 binding domain; and (c) an antigen binding domain, wherein the IgA heavy chain domain specifically binds to FcαR on an immune effector cell, the CD47 binding domain inhibits binding of CD47 expressed on a target cell to signal regulatory protein alpha (SIRP α) on the immune effector cell, the antigen binding domain binds to an antigen on the target cell, and the antibody construct has a higher binding affinity for the antigen compared to CD47.

[0052]

[0056] In some embodiments, inhibition of CD47 binding to SIRPα inhibits CD47 expression in target cells. Increase phagocytosis and clearance. In some embodiments, the composition is administered by oral, intralesional, intravenous treatment, or by subcutaneous, intramuscular, intraarterial, intravenous, intracavitary, intracranial, or intraperitoneal injection. In some embodiments, the composition is administered daily, weekly, biweekly, monthly, bimonthly, once every three months, once every six months, or once every twelve months. In some embodiments, the subject has cancer. In some embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia, acute myeloid leukemia, biliary tract cancer, B-cell leukemia, B-cell lymphoma, biliary tract cancer, bone cancer, brain cancer, breast cancer, triple-negative breast cancer, cervical cancer, Burkitt's lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastric cancer, gastrointestinal cancer, glioma, hairy cell leukemia, head and neck cancer, Hodgkin's lymphoma, liver cancer, lung cancer, medullary thyroid cancer, melanoma, multiple myeloma, ovarian cancer, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, pulmonary tract cancer, renal cancer, sarcoma, skin cancer, testicular cancer, urothelial cancer, and bladder cancer.

[0053]

[0057] In some embodiments, the subject is a human. In some embodiments, the method comprises administering an effective amount of at least one additional therapeutic agent. In some embodiments, the additional therapeutic agent is an imaging agent, a chemotherapeutic agent, a kinase inhibitor, a costimulatory molecule blocker, an adhesion molecule blocker, a second antibody or antigen-binding fragment thereof, a drug, a toxin, an enzyme, a cytotoxic agent, an anti-angiogenic agent, a pro-apoptotic agent, an antibiotic, a hormone, an immune modulator, a cytokine, a chemokine, an antisense oligonucleotide, a small interfering RNA (siRNA), methotrexate, cyclosporine, rapamycin, FK506, a detectable label or reporter, a TNF antagonist, an antirheumatic agent, a muscle relaxant, a narcotic, a nonsteroidal anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocking agent, an antibacterial agent, an antipsoriatic agent, a corticosteroid, an anabolic steroid, erythropoietin, immunization, an immunoglobulin, an immunosuppressant, a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, or an antipsychotic agent.

[0054]

[0058] In one aspect, provided herein is a method of inducing a neutrophil-mediated immune response against a target cell comprising contacting the target cell with an effective amount of an antibody construct, the antibody construct comprising: (a) an immunoglobulin A (IgA) heavy chain domain; (b) a CD47 binding domain; and (c) an antigen binding domain; the IgA heavy chain domain specifically binds to FcαR on a neutrophil, and the CD47 binding domain specifically binds to CD47 expressed on the target cell and to FcαR on the neutrophil. wherein the antigen-binding domain inhibits binding of the antibody to signal regulatory protein alpha (SIRP alpha), the antigen-binding domain binds to an antigen on a target cell, and the antibody construct has a higher binding affinity for the antigen compared to CD47, thereby inducing a neutrophil-mediated immune response.

[0055]

[0059] In some embodiments, the neutrophil-mediated immune response includes phagocytosis of target cells or lysis of target cells. In some embodiments, the antigen-presenting cell is a cancer cell or a viral cell. In some embodiments, the cancer cell is a lymphocyte.

[0056]

[0060] In some embodiments, the CD47 binding domain comprises a first light chain variable domain comprising variable light chain complementarity determining region 1 (CDR-L1), variable light chain complementarity determining region 2 (CDR-L2) and variable light chain complementarity determining region 3 (CDR-L3), wherein CDR-L1, CDR-L2 and CDR-L3 comprise an amino acid sequence selected from Table A.

[0057]

[0061] In some embodiments, the CD47 binding domain comprises a first heavy chain variable domain comprising a variable light chain complementarity determining region 1 (CDR-H1), a variable light chain complementarity determining region 2 (CDR H2) and a variable light chain complementarity determining region 3 (CDR-H3), wherein CDR-H1, CDR-H2 and CDR-H3 comprise an amino acid sequence selected from Table A.

[0058]

[0062] In some embodiments, the antigen binding domain comprises a second light chain variable domain comprising variable light chain complementarity determining region 1 (CDR-L1), variable light chain complementarity determining region 2 (CDR-L2) and variable light chain complementarity determining region 3 (CDR-L3), wherein CDR-L1, CDR-L2 and CDR-L3 comprise an amino acid sequence selected from Table B.

[0059]

[0063] In some embodiments, the antigen binding domain comprises a second heavy chain variable domain comprising a variable light chain complementarity determining region 1 (CDR-H1), a variable light chain complementarity determining region 2 (CDR H2) and a variable light chain complementarity determining region 3 (CDR-H3), wherein CDR-H1, CDR-H2 and CDR-H3 comprise an amino acid sequence selected from Table B.

[0060]

[0064] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the disclosure are utilized, and the accompanying drawings, in which:

[0065] 1A-1E show exemplary antibody constructs described herein. Methods for preparing exemplary constructs are described in the Examples. [Brief description of the drawings]

[0061] [Figure 1A] FIG. 1A shows bivalent dimeric constructs based on exemplary IgA constant domain scaffolds (in the exemplary constructs described herein, IgA heavy chain scaffolds of IgA1, IgA2, or modified IgA2 (also called IgA2.0 or IgA3.0) with reduced glycosylation and / or better expression profile are illustrated), with specificity determined by the VH and kappa VL regions attached to the IgA scaffold. The constructs can bind to CD47 and an exemplary antigen on antigen presenting cells (in the exemplary case, CD20). [Figure 1B] Figure 1B shows a bivalent heterodimer consisting of a common heavy chain and both kappa and lambda light chains, with specificity determined solely by the VL regions of both the kappa and lambda regions, one of which specifically binds to CD47 and the other to an exemplary antigen on an antigen-presenting cell (in this exemplary case, CD20). [Figure 1C] Figure 1C shows a construct based on DVD-IgA2, a tetravalent homodimer. The specificity is determined by the combination of two VH / VL pairs. An additional VH is linked to the original VH, and an additional VL is linked to the original VL, which are linked to the existing VH and VL by peptide linkers. [Figure 1D] Figure ID shows the tetravalent homodimer DVD-IgA3.0-scFv_LC, where IgA3.0 is an IgA optimized for glycosylation and manufacturability. Specificity is determined by the combination of scFvs linked to the original VL. [Figure 1E]FIG. 1E shows DVD-IgA3.0-scFv_HC, a tetravalent homodimer. Specificity is determined by the combination of scFvs linked to the original VH. In the embodiments described herein, the construct specifically binds to CD47 and antigens on antigen-presenting cells (in exemplary cases, CD20, HER2, GD2, mesothelin, EGFR, etc.). In the antibody construct designs described herein in any of FIGS. 1A-1E, the antigen-binding region and the CD47-binding region can be derived from commercially available antibodies or other known antibodies. In certain embodiments, the antibody construct is designed to have a CD47-binding domain or region that has a lower binding affinity to CD47 than the antigen-binding domain or region in the construct. [Figure 2A]

[0066] Figures 2A-2K show the optimal heavy chain:light chain ratios required for expression of the IgA antibody constructs described herein in HEK293-Freestyle (HEK293F) cells. The X-axis shows the ratio of DNA encoding the heavy chain (HC) to DNA encoding the light chain (KLC) in combination with pAdvantage-pAdv (Promega) at a total concentration of 1ug / mL in a volume of 2mL. The amount of pAdv DNA is always equal to the HC DNA. The Y-axis shows the protein concentration measured for the individual antibody constructs. Protein concentrations were determined for each of the bispecific antibodies in Tables 1 and 2. For DVD-IgA diabody #1 (Figure 2A) and DVD-IgA diabody #2 (Figure 2B), DVD-IgA-scFv_LC diabody #5 (Figure 2C), DVD-IgA-scFv_LC diabody #6 (Figure 2D), DVD-IgA-scFv_HC diabody #3 (Figure 2E), DVD-IgA-scFv_HC diabody #4 (Figure 2F), the protein concentration is determined by kappa-IgA ELISA. For kappa-kappa IgA (Figures 2G-2H) and lambda-lambda IgA (Figures 2I-2J), the protein concentration is measured by kappa / lambda-IgA ELISA. OD415 value for kappa-lambda IgA (Figure 2K), since the concentration could not be measured due to lack of standard. The method is described in section 3 of the Examples. FIG. 2A shows the optimal heavy chain:light chain ratio required for expression of DVD-IgA bispecific antibody #1 in HEK293-Freestlye (HEK293F) cells. [Figure 2B] FIG. 2B shows the optimal heavy chain:light chain ratio required for expression of DVD-IgA bispecific antibody #2 in HEK293-Freestlye (HEK293F) cells. [Figure 2C] FIG. 2C shows the optimal heavy chain:light chain ratio required for expression of DVD-IgA-scFv_LC bispecific antibody #5 in HEK293-Freestlye (HEK293F) cells. [Figure 2D] FIG. 2D shows the optimal heavy chain:light chain ratio required for expression of DVD-IgA-scFv_LC bispecific antibody #6 in HEK293-Freestlye (HEK293F) cells. [Figure 2E] FIG. 2E shows the optimal heavy chain:light chain ratio required for expression of DVD-IgA-scFv_HC bispecific antibody #3 in HEK293-Freestlye (HEK293F) cells. [Figure 2F] FIG. 2F shows the optimal heavy chain:light chain ratio required for expression of DVD-IgA-scFv_HC bispecific antibody #4 in HEK293-Freestlye (HEK293F) cells. [Figure 2G] FIG. 2G shows the optimal heavy chain:light chain ratio required for expression of kappa-kappa IgA in HEK293-Freestyle (HEK293F) cells. [Figure 2H] FIG. 2H shows the optimal heavy chain:light chain ratio required for expression of kappa-kappa IgA in HEK293-Freestyle (HEK293F) cells. [Figure 2I] FIG. 2I shows the optimal heavy chain:light chain ratio required for expression of lambda-lambda IgA in HEK293-Freestyle (HEK293F) cells. [Figure 2J] FIG. 2J shows the optimal heavy chain:light chain ratio required for expression of lambda-lambda IgA in HEK293-Freestyle (HEK293F) cells. [Figure 2K] FIG. 2K shows the optimal heavy chain:light chain ratio required for expression of kappa-lambda IgA in HEK293-Freestyle (HEK293F) cells in terms of OD415 values. [Figure 3A]

[0067] 3A-3B show the purification of an exemplary recombinant IgA antibody construct described herein. Figure 3A illustrates the affinity purification elution profile of an exemplary bispecific DVD-IgA molecule. [Figure 3B] Figure 3B shows the purity of bispecific antibodies #1 and #5 on a non-reducing SDS-PAGE gel at the expected molecular weight. The gel shows low levels of unassociated kappa light chain (35 kDa for #1; 50 kDa for #5). Methods are described in section 3 of the Examples. [Figure 4]

[0068] Figure 4 shows the measured concentrations of six representative antibody constructs (DVD-IgA) described herein (e.g., listed in Table 1). All six bispecific IgA molecules were produced in HEK293F cells and concentrations were measured in the supernatants by ELISA. [Figure 5A]

[0069] Figures 5A-5D show the gating strategy for flow cytometry analysis and identification of cells expressing IgA antibody constructs. Figure 5A shows the selection of live cell populations based on cell size and shape. [Figure 5B] FIG. 5B shows further selection of a single cell from the population of live cells. [Figure 5C] FIG. 5C shows the gating strategy for control IgA-negative cells after staining with anti-IgA PE-labeled antibody. [Figure 5D] FIG. 5D shows the analysis and selection of IgA positive cells by staining with anti-IgA PE-labeled antibody. [Figure 6A]

[0070] Figures 6A-6B show the binding analysis of the antibody constructs described herein (e.g., Tables 1 and 2) by flow cytometry. Figure 6A shows the characterization of the SKBR3 cell line used. Both SKBR3 WT and SKBR3-CD20 cells are stained with IgA antibodies against CD20 (Obi = Obinutuzumab complementation region) or CD47 (clone 2.3D11). Unstained cells and secondary antibody only are controls. [Figure 6B]FIG. 6B shows the binding of bispecific antibodies to SKBR3 WT and SKBR3-CD20 cells. The X-axis of the left panel of FIG. 6B identifies antibodies, e.g., anti-CD47 antibody 2.3D11 alone, antibody #1, antibody #2, antibody #3, antibody #4, antibody #5, and antibody #6. The X-axis of the right panel of FIG. 6B identifies antibodies, e.g., anti-CD20 Obi antibody alone, antibody #1, antibody #2, antibody #3, antibody #4, antibody #5, and antibody #6. Supernatants containing individual bispecific antibodies #1-6 were tested on both SKBR3 WT (left panel) and SKBR3-CD20 (right panel) cells in the absence (black) or presence (gray) of CD47 blocking antibodies. Bispecific antibodies #2, #3, and #5 show the effect of CD47 blocking, demonstrating that CD20 binding is enhanced by additional CD47 binding. [Figure 7A]

[0071] Figures 7A-7B show antibody-dependent cell-mediated cytotoxicity (ADCC) analysis of bispecific antibodies. SKBR3-CD20 cells were used as target cells in PMN ADCC assays. Antibodies IgA3.0-Obi and IgA3.0-2.3D11 were used either alone or in combination, and bispecific IgA antibody #2 was tested in parallel. The combination (Obi+2.3D11) and bispecific antibodies were pre-blocked with mIgG1 CD47 PerCP-Cy5.5 antibody. Pre-blocking of CD47 reduces the killing observed for the combination and bispecific. The functional effect of bispecific blocking of CD47 enhances CD20-dependent ADCC. FIG. 7A shows Obi-IgA and 2.3D11-IgA dependent ADCC activity on PMNs using either SKBR3 (CD20- / -) WT (top panel) or SKBR3 CD20(+) (bottom panel) as target cells. SKBR3 WT and CD20 cells show no ADCC with IgG1. SKBR3 WT shows no specific lysis with Obi-IgA alone, but 2.3D11 induces ADCC. In SKBR3-CD20(+) cells, Obi-IgA induces further ADCC in combination with 2.3D11-IgA, which was partially reverted by pre-blockade of CD47 with mIgG1-anti-CD47 PerCP-Cy5.5. The x-axis shows antibody concentration in ug / mL. [Figure 7B]Figure 7B shows SKBR3-CD20(+) cells in a PMN ADCC assay to test the ADCC activity of bispecific antibody #2, DVD-IgA Obi-2.3D11. As controls, IgA3.0-Obi and IgA3.0-2.3D11 were used either alone or in combination. The combination (Obi+2.3D11) showed the best ADCC, which could be efficiently blocked by preincubating SKBR3-CD20(+) cells with mIgG1 CD47 PerCP-Cy5.5 antibody. Bispecific antibody #2 showed a similar killing efficiency compared to the combination. This effect could only be partially reduced by pre-blocking with mIgG1 anti-CD47 PerCP-Cy5.5. Thus, the functional effect of blocking CD47 in the same molecule, bispecific IgA, greatly enhances CD20-dependent ADCC. [Figure 8A]

[0072] 8A-8D show an analysis of CD47 binding by antibody constructs.

[0073] Figure 8A shows the gating strategy for red blood cells. CD235a+ cells were selected and gated on a secondary IgA antibody as a simple negative gate. CD47 is highly positive on red blood cells. [Figure 8B] Figure 8B shows the gating strategy for platelets. CD61+ cells were selected and gated with a secondary IgA antibody as the only negative gate. CD47 is highly positive on platelets. [Figure 8C] FIG. 8C shows flow cytometry analysis of the binding of the antibody constructs described herein to red blood cells. [Figure 8D] Figure 8D shows flow cytometry analysis of the binding of the antibody constructs described herein to platelets. Figure 8C shows that analysis of CD47 binding to erythrocytes shows low levels of CD47 binding to erythrocytes by bispecific antibody #4 (Obi-scFv:2.3D11_HC). No binding to erythrocytes was observed with all other bispecifics. Figure 8D shows that no platelet binding of the bispecific antibodies was observed. [Figure 9]

[0074] FIG. 9 shows an example illustrating the mechanism of action of the antibody construct described herein. The antibody construct of the present disclosure comprises an antigen binding domain, a CD47 binding domain, and an IgA heavy chain constant domain. The antibody construct binds to an antigen on a target cell via the antigen binding domain, binds to CD47 on the same target cell via the CD47 binding domain, and binds to an Fc receptor on an immune effector cell (e.g., neutrophil) via the IgA heavy chain constant domain. Binding to CD47 inhibits the interaction between SIRPα on the immune effector cell and CD47 on the target cell, thereby inhibiting the "don't eat me" signal and inducing an immune effector cell response against the target cell. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062]

[0075] The following description and examples are intended to illustrate the aspects of the present disclosure in detail. It should be understood that the present disclosure is not limited to the specific aspects described herein and can therefore be modified. Those skilled in the art will recognize that there are many variations and modifications of the present disclosure, which are encompassed within the scope of the present disclosure.

[0063]

[0076] All terms are intended to be understood as understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0064]

[0077] The section headings used herein are for organizational purposes only and are not intended to be limiting. It should not be construed as limiting the subject matter covered.

[0078] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein for clarity in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment.

[0065] definition

[0079] The following definitions are for the benefit of those skilled in the art, are subject to this application, and should not be attributed to any related or unrelated cases, such as any co-owned patent or application. Any method and material similar or equivalent to those described herein can be used in carrying out the test of the present disclosure, but preferred materials and methods are those described herein. Therefore, the terminology used herein is only intended to describe specific embodiments, and is not intended to be limiting.

[0066]

[0080] In this application, the use of the singular includes the plural unless otherwise specified. It should be noted that as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless otherwise specified. Furthermore, the use of the term "including" as well as other forms such as "include," "include (singular)," and "included" is not limiting.

[0067]

[0081] References herein to "some aspects," "an aspect," "one aspect," or "other aspects" mean that a particular feature, structure, or characteristic described in connection with an aspect is included in at least some aspects of the disclosure, but not necessarily in all aspects.

[0068]

[0082] As used in this specification and claim(s), the terms "comprising" (and any form of including, such as "comprise" and "comprise"), "having" (and any form of including, such as "having" and "having"), "including" (and any form of including, such as "including" and "comprise"), or "containing" (and any form of including, such as "containing" and "comprise") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Additionally, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0069]

[0083] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations, as practiced in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In other examples, the amount "about 10" encompasses any amount between 10 and 9-11. In still other examples, the term "about" with respect to a reference numerical value can also encompass a range of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. Alternatively, particularly with respect to biological systems or processes, the term "about" can mean within an order of magnitude of the value, preferably within 5-fold, and more preferably within 2-fold. When a particular value is described in the application and claims, the term "about" should be assumed to mean within an acceptable error range of the particular value, unless otherwise specified.

[0070]

[0084] As used herein, "antigen on antigen-presenting cells" refers to antigenic substances that are associated with antigen presentation and may induce an immune response in the host. For example, an antigen is any antigenic substance that is produced or overexpressed in a pathogenic cell, such as a cancer cell. An antigen is a protein, peptide, or polysaccharide. An antigen-presenting cell as described herein is a cell that presents an antigen, for example, in the form of a peptide on a histocompatibility molecule. A cancer cell, such as a tumor cell that presents a tumor antigen, is a representative antigen-presenting cell. A tumor antigen is a representative antigen in the constructs described herein, where the tumor antigen is produced in a tumor cell. It may, for example, induce an immune response in the host. Alternatively, for the purposes of this disclosure, a tumor antigen can be a protein that is expressed by both healthy cells and tumor cells, but is a suitable therapeutic target because it identifies a particular tumor type or is overexpressed in a particular tumor type. In an embodiment, the tumor antigen is CD20, GD2, CD38, CD19, EGFR, HER2, PD-L1, CD25, CD33, BCMA, CD44, alpha-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER3, folate binding protein, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MAGE-A1, MUC16, h5T4, PSMA, TAG-72, EGFRvIII, CD123 or VEGF-R2. In one embodiment, the tumor antigen is EGFRvIII, which is the target of therapeutic agents for treating myeloid malignancies, such as glioblastoma or glioblastoma multiforme (GBM). In another embodiment, the tumor antigen is CD20. In yet another embodiment, the tumor antigen is GD2. In yet other embodiments, the tumor antigen is mesothelin.

[0071]

[0085] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or is immunologically reactive with a particular antigen. Antibodies can include, for example, polyclonal antibodies, monoclonal antibodies, engineered antibodies, and antigen-binding fragments thereof. Antibodies can be, for example, murine antibodies, chimeric antibodies, humanized antibodies, heteroconjugate antibodies, bispecific antibodies, diabodies, triabodies, or tetrabodies. Antigen binding Examples of the fragments include Fab', F(ab')2, Fab, Fv, rlgG, scFv, hcAb (heavy chain antibody), single domain antibody, V HH , V NAR, sdAb, or nanobody. As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of B cells and binding to the same epitope. In contrast, a "polyclonal antibody" refers to a population of antibodies produced by different B cells and binding to different epitopes of the same antigen. A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. The VH and VL regions have a similar general structure, each region containing four framework regions, the sequences of which are relatively conserved. The framework regions are connected by three complementarity determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable region" of the antibody responsible for antigen binding. In the constructs described herein, the antibody comprises an IgA heavy chain domain or region that forms a scaffold for attachment of the CD47 binding domain and / or the antigen binding domain. The IgA constant region domain is based on the IgA1 or IgA2 constant region domain, which in some cases have been further modified for improved glycosylation profile, improved manufacturability, ease of heterodimer formation, or adapted / selective Fc receptor binding.

[0072]

[0086] As used herein, a "recombinant antibody" refers to an antibody that is produced from two different species or Antibodies that contain amino acid sequences from different sources, including synthetic molecules. Non-limiting examples include antibodies that contain non-human CDRs and human variable region frameworks or constant or Fc regions, antibodies with binding domains from two different monoclonal antibodies, or antibodies that contain mutations of one or more amino acid residues to increase or decrease the biological activity or binding of a portion of the antibody. In certain embodiments, recombinant antibodies are produced from recombinant DNA molecules or are synthesized. In certain embodiments, the antibodies described herein are polypeptides (one or more) encoded by one or more polynucleotides.

[0073]

[0087] As used herein, "recognize" or "bind" or "selective for" refers to the association or binding between an antigen-binding domain and an antigen. As used herein, "antigen" refers to an antigenic substance capable of eliciting an immune response in a host. The antigenic substance can be a molecule, such as a costimulatory molecule, capable of eliciting an immune response in a host.

[0074]

[0088] As used herein, "antibody construct" refers to a construct containing an antigen binding domain and an Fc domain.

[0089] As used herein, "binding domain" refers to an antibody or a non-antibody domain.

[0075]

[0090] As used herein, "antigen binding domain" refers to an antibody-derived or non-antibody-derived binding domain that can bind to an antigen. The antigen binding domain can be a tumor antigen binding domain or a binding domain that can bind to an antigen (e.g., a molecule) on an antigen-presenting cell. If more than one antigen binding domain is present in a given conjugate or antibody construct, the antigen binding domain can be numbered (e.g., a first antigen binding domain, a second antigen binding domain, a third antigen binding domain, etc.). Different antigen binding domains in the same conjugate or construct can target the same or different antigens (e.g., a first antigen binding domain that can bind to a tumor antigen, a second antigen binding domain that can bind to a molecule on an antigen-presenting cell (APC antigen), and a third antigen binding domain that can bind to an APC antigen).

[0076]

[0091] As used herein, an "antibody antigen-binding domain" refers to a binding domain derived from an antibody capable of binding to an antigen.

[0092] As used herein, "Fc domain" refers to an Fc domain derived from an antibody or a non-antibody derived Fc domain capable of binding to an Fc receptor. As used herein, "Fc domain" and "Fc-containing domain" can be used interchangeably.

[0077]

[0093] As used herein, "target binding domain" refers to a construct containing an antibody-derived or non-antibody-derived antigen-binding domain capable of binding to an antigen.

[0094] As used herein, the abbreviations of the natural monoenantiomeric amino acids are conventional and can be as follows: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine ​​(C, Cys); glutamic acid (E, Glu); glutamine (Q, Gin); glycine (G, Gly); histidine (H, His); isoleucine (I, He); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); valine (V, Val). Unless otherwise specified, X can represent any amino acid. In some aspects, X is asparagine (N), glutamine (Q), histidine (H), lysine (K), or arginine (A). It can be Nin(R).

[0078]

[0095] The phrase "pharmaceutical acceptable" is employed herein to refer to compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject, e.g., humans and animals, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0079]

[0096] As used herein, the phrase "pharmaceutical acceptable excipient" or "pharmaceutical acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, Olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffers; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0080]

[0097] Antigens can induce immune responses. Antigens can be proteins, polysaccharides, lipids, or glycolipids, which can be recognized by immune cells, such as T cells or B cells. When immune cells are exposed to one or more of these antigens, a rapid cell division and differentiation response can be induced, leading to the clonal formation of the exposed T cells and B cells. B cells can differentiate into plasma cells, which can produce antibodies that selectively bind to the antigen.

[0081]

[0098] The terms "cancer", "tumor" and "proliferative disease" refer to a physiological condition in mammals that is characterized by deregulated cell growth. Cancer is a class of diseases in which a group of cells exhibit uncontrolled or unwanted growth. Cancer cells can also spread to other sites, which can result in the formation of metastases. The spread of cancer cells in the body can occur, for example, via lymph or blood. Uncontrolled growth, invasion and metastasis formation are also referred to as malignant properties of cancer. These malignant properties distinguish cancer from benign tumors, which typically do not invade or metastasize.

[0082]

[0099] "Antigen recognition moiety" or "antibody recognition domain" refers to a molecule or a portion of a molecule that specifically binds to an antigen. In one embodiment, the antigen recognition moiety is an antibody, an antibody-like molecule or a fragment thereof, and the antigen is a tumor antigen or an infectious disease antigen.

[0083]

[0100] The terms "fragment of an antibody", "antibody fragment", "functional fragment of an antibody" and "antibody fragment" are used interchangeably. The terms "antigen-binding domain" and "antigen-binding domain" or grammatical equivalents thereof are used interchangeably herein to mean one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (see generally, Holliger et al., Nat. Biotech. .,23(9):1126-1129(2005)). Antibody fragments desirably contain, for example, one or more CDRs, a variable region (or a portion thereof), a constant region (or a portion thereof), or a combination thereof. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the stalk region; (iii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; and (iv) a single-chain Fv (scFv), which is a monovalent molecule consisting of two domains of an Fv fragment (i.e., VL and VH) joined by a synthetic linker that allows the two domains to be synthesized as a single polypeptide chain (see, e.g., Bird et al., Science,242:423-426(1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16:778 (1998); and (v) diabodies, which are dimers of polypeptide chains, in which each polypeptide chain comprises a VH connected to a VL by a peptide linker that is too short to allow pairing between the VH and VL on the same polypeptide chain, thereby promoting pairing between complementary domains on different VH-VL polypeptide chains to result in a dimeric molecule with two functional antigen-binding sites. Antibody fragments are known in the art and are described in more detail, for example, in U.S. Patent No. 8,603,950. Other antibody fragments can include variable fragments of heavy chain antibodies (VHH).

[0084]

[0101] The term "conservative amino acid substitution" or "conservative mutation" refers to a group of amino acids that share a common property. Conservative mutations refer to the substitution of one amino acid by another amino acid that has a similar effect on the overall protein structure. A functional method for defining common characteristics between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such an analysis, groups of amino acids can be defined as those in which the amino acids in the group preferentially exchange with each other and are therefore most similar to each other in their effect on the overall protein structure (Schulz, GE and Schirmer, RH, supra). Examples of conservative mutations include amino acid substitutions of amino acids within the above subgroups, such as lysine for arginine and vice versa, so that a positive charge can be maintained; glutamic acid for aspartic acid and vice versa, so that a negative charge can be maintained; serine for threonine, so that a free -OH can be maintained; and glutamine for asparagine, so that a free -NH2 can be maintained. Alternatively, or in addition, a therapeutic agent can include the amino acid sequence of a reference protein with at least one non-conservative amino acid substitution.

[0085]

[0102] The term "non-conservative mutation" or "non-conservative amino acid substitution" refers to a mutation that occurs in a different group. Non-conservative amino acid substitutions include inter-group amino acid substitutions, e.g., lysine for tryptophan, phenylalanine for serine, etc. In this case, the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the Therapeutic. The non-conservative amino acid substitution can improve the biological activity of the Therapeutic, such that the biological activity of the Therapeutic is improved compared to the wild-type Therapeutic. IgA antibody

[0103] Immunoglobulin A (IgA) is known for its antimicrobial role. It is abundant at mucosal sites in a dimer form. As a monomer, it is the second most common antibody present in serum. IgA contains two subclasses, IgA1 and IgA2, which bind with similar affinity to the myeloid IgA receptor (FcαRI, CD89). In some embodiments, IgA antibodies have a superior ability to recruit neutrophils for antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, IgA antibodies require a lower effector:target (E:T) ratio. In some embodiments, IgA antibodies reduce tumor-opsonizing antibody concentrations compared to other types of antibodies (e.g., IgG). In general, cancer therapeutic antibodies work through a combination of both direct and indirect immune-mediated effects. These effects include complement activation-induced cytotoxicity, antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent cellular cytotoxicity (ADCC). ADCC can be mediated by the activation of different Fc receptor-expressing cells, including natural killer (NK) cells, macrophages, and neutrophils. Of these Fc receptor-expressing effector cells, macrophages and neutrophils express FcαRI, which is necessary to bind IgA antibodies, and therefore can kill tumor cells by ADCP or ADCC.

[0086]

[0104] In some embodiments, the IgA antibody inhibits immune cell-mediated (e.g., neutrophil-mediated) proliferation of pathogenic cells, such as tumor cells, following IgA antibody-immune cell engagement. ) induces phagocytosis or trogocytosis. This mechanism of tumor cell killing is primarily mediated by interaction with the Fc receptor for IgA (FcαRI; CD89), which is the best-characterized IgA receptor. FcαRI is expressed on monocytes, macrophages, granulocytes, a subset of dendritic cells, and Kupffer cells and binds both monomeric and dimeric IgA isoforms with intermediate affinity. IgA binding to FcαRI mediates effector functions such as phagocytosis, oxidative burst, cytokine release, antigen presentation, and ADCC. In humans, two IgA isotypes, IgA1 and IgA2, and three allotypes, IgA2m(1), IgA2m(2), and IgA2n, have been identified. In some embodiments, IgA antibodies induce polymorphonuclear cell (PMN)-mediated ADCC more efficiently than IgG antibodies.

[0087]

[0105] IgA has two subclasses (IgA1 and IgA2) and is monomeric and It can be produced as a dimeric form as well as a secreted form. In some embodiments, the IgA antibody can be monomeric. In some embodiments, the IgA antibody can comprise one or more IgA1 amino acid sequences. In some embodiments, the IgA antibody can comprise one or more IgA2 amino acid sequences. In some embodiments, the IgA antibody can comprise one or more IgA1 amino acid sequences and one or more IgA2 amino acid sequences.

[0088]

[0106] In some embodiments, the antibody construct is an IgA1 or Ig! antibody. The IgA2 antibody comprises a constant region domain derived from an IgA antibody. In some cases, the IgA2 antibody is an allotype of IgA2 antibody, i.e., IgA2m(1), IgA2(m)2, or IgA2n. In some embodiments, the IgA2m(1) antibody is a Caucasian IgA2m(1) antibody. In some embodiments, the IgA2m(2) antibody is an African IgA2m(2) antibody or an Asian IgA2m(2) antibody. In some embodiments, the IgA2 antibody is modified for improved manufacturability, preferred glycosylation, improved solubility, or improved activity, such as selective Fc receptor binding. Exemplary IgA2-based constant regions or uses in the antibody constructs described herein, such as IgA2.0 and IgA3.0, with such improvements are provided herein. Further such modifications based on the knowledge of one skilled in the art are within the scope of the constructs described herein.

[0089]

[0107] In some embodiments, the antibody construct described herein comprises at least 6 In some embodiments, the IgA antibody comprises a heavy chain constant region that comprises at least 50, 60, 70, 75, 80, 85, 90, 95, or 99% IgA amino acids. The light chain constant region comprises amino acids.

[0090]

[0108] In some embodiments, the antibody construct described herein comprises one or more I In some embodiments, the antibody constructs described herein comprise a heavy chain constant region comprising one or more amino acids of an IgG CH3 region, an IgG CH2, or an IgG CH1 region, or any combination thereof. In some embodiments, the antibody constructs described herein comprise a light chain region domain comprising an IgA CH1 region. In some embodiments, the antibody constructs described herein comprise a heavy chain constant region comprising one or more amino acids of an IgG CH3 region, an IgG CH2, or an IgG CH1 region, or any combination thereof. In some embodiments, the antibody constructs described herein comprise a heavy chain constant region comprising an IgA CH3 region, an IgA CH2, and an IgA CH1 region, or any combination thereof. In some embodiments, the antibody constructs described herein comprise a light chain region domain comprising an IgA CH1 region. In some embodiments, the antibody constructs described herein comprise a heavy chain constant region comprising one or more amino acids of an IgG CH3 region, an IgG CH2, or an IgG CH1 region, or any combination thereof. The heavy chain constant region comprises a CH1 region, a CH2 region, or any combination thereof.

[0091]

[0109] In some embodiments, the antibody construct described herein comprises an IgG variable The antibody constructs described herein comprise a light chain variable region, for example, a light chain variable region fused or connected to an IgA constant region, optionally by the use of a linker. In some embodiments, the antibody constructs described herein comprise an IgG heavy chain variable region. In some embodiments, the antibody constructs described herein comprise an IgG light chain variable region and an IgG heavy chain variable region.

[0092]

[0110] In some embodiments, the antibody construct described herein is a humanized antibody. In some embodiments, the antibody constructs described herein may comprise one or more domains derived from a chimeric antibody, a murine antibody, a camelid antibody or a shark antibody. In some embodiments, the antibody constructs described herein may comprise domains derived exclusively from a human antibody.

[0093]

[0111] In some embodiments, the antibody constructs described herein are bispecific. The antibody may be a trispecific antibody or a multispecific antibody construct. In some embodiments, the antibody construct described herein may be a trispecific antibody. In some embodiments, the antibody construct described herein may be a multispecific antibody.

[0094]

[0112] In some embodiments, the antibody constructs described herein are bispecific. The first antigen may be an antibody. In some embodiments, the antibody construct described herein engages CD47 and one or more antigens simultaneously on the cell surface. In some examples, the binding of the antibody construct described herein to two different antigens is sequential. For example, the binding of the construct to the first antigen occurs first, thereby limiting the space explored by the arm of the second antibody. As a result, a significant increase in the local concentration of the second antigen may occur, which can facilitate the binding of the arm of the second antibody. In some cases, such a first antigen is an antigen that is selectively expressed or overexpressed on antigen-presenting cells, and the second antigen is CD47, which is also expressed on antigen-presenting cells. In some cases, the construct binds to the second antigen with a lower binding strength than the first antigen, thereby allowing the binding to the first antigen to dictate cell specificity.

[0095]

[0113] In some embodiments, the construct comprises an Fc domain of IgA or a variant thereof. In some embodiments, the antibody constructs described herein comprise a heavy chain constant region comprising a CH3, a CH2, and a CH1 domain. In some embodiments, the antibody constructs described herein comprise a light chain constant region comprising a CH1.

[0096]

[0114] In some embodiments, the construct induces complement dependent cytotoxicity (CDC). In some embodiments, the construct induces polymorphonuclear neutrophil (PMN)-mediated tumor cell lysis. In some embodiments, the IgA antibody induces programmed cell death (PCD) via a caspase-independent pathway. In some embodiments, the antibody construct induces antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the IgA antibody induces neutrophil-mediated antibody-dependent cell-mediated cytotoxicity (ADCC).

[0097]

[0115] In some embodiments, the antibody constructs described herein include the corresponding I IgA antibodies may have a superior ability to recruit neutrophils for antibody-dependent cell-mediated cytotoxicity (ADCC) compared to IgG antibodies. In some embodiments, IgA antibodies may require a lower effector:target (E:T) ratio. In some embodiments, the antibody constructs described herein may require a lower tumor-opsonizing antibody concentration compared to other types of antibodies (e.g., IgG). In some embodiments, the antibody constructs described herein can induce neutrophil-mediated phagocytosis or trogocytosis of tumor cells following IgA antibody-neutrophil engagement. This tumor cell killing mechanism is primarily mediated by interaction with the Fc receptor for IgA (FcαRI; CD89), which is the best-characterized IgA receptor. FcαRI is expressed on monocytes, macrophages, granulocytes, a subset of dendritic cells, and Kupffer cells, and binds both monomeric and dimeric IgA isoforms with intermediate affinity. IgA binding to FcαRI mediates effector functions such as phagocytosis, oxidative burst, cytokine release, antigen presentation, and ADCC. In humans, two IgA isotypes, IgA1 and IgA2, and three allotypes, IgA2m(1), IgA2m(2), and IgA2n, have been identified. In some embodiments, IgA antibodies induce polymorphonuclear cell (PMN)-mediated ADCC more efficiently than IgG antibodies.

[0098]

[0116] In some embodiments, the antibody constructs described herein are They do not bind to cells, platelets, and / or red blood cells. For example, in some embodiments, IgA antibodies can have low immunogenicity.

[0099]

[0117] In some embodiments, the antibody constructs described herein are therapeutic antibodies. In some embodiments, the antibody constructs described herein can be recombinant antibodies. In some embodiments, the antibody constructs described herein are made in a cell line. In some embodiments, the cell line is CHO. In some embodiments, the cell line is SP20. In some embodiments, the cell line is a HEK239 cell line. In some embodiments, the HEK293 cell line is HEK293F.

[0100] IgA antibody modification

[0118] One or more amino acid substitutions in one or more IgA constant region domains and / or one or more Described herein are antibody constructs as described herein that contain the above amino acid deletions.

[0101]

[0119] In some embodiments, the constructs based on the IgA antibodies described herein The amino acid numbering of is shown according to the IMGT unique numbering for C-DOMAIN and C-LIKE-DOMAIN (as disclosed in "IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains." Dev Comp Immunol. 2005;29(3):185-203, the entire contents of which are incorporated herein by reference).

[0102]

[0120] In some embodiments, the construct comprises at least four glycosyltransferases in the constant region. In some embodiments, the antibody construct comprises a deletion of at least three N-linked glycosylation sites in the constant region of the antibody. In some embodiments, the antibody construct comprises a deletion of at least three N-linked glycosylation sites in the constant region of the antibody and at least one O-linked glycosylation site in the constant region of the antibody.

[0103]

[0121] In some embodiments, the antibody construct comprises an IgA constant region comprising a deleted tailpiece. In some embodiments, the construct comprises deletion of 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4 C-terminal amino acids. In some embodiments, the construct comprises deletion of 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4 C-terminal amino acids. In some embodiments, the C-terminal amino acids are from amino acids 131 to 148 of the IgA2 antibody, numbered according to the IMGT scheme.

[0104]

[0122] In some embodiments, the antibody construct is numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a deletion of amino acids 131-148, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a deletion of amino acids 147-148, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a deletion of amino acids 146-148, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a deletion of amino acids 145-148, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a deletion of amino acids 144-148, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a deletion of amino acids 143-148, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a deletion of amino acids 142-148, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a deletion of amino acids 141-148, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a deletion of amino acids 140-148, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a deletion of amino acids 139-148, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a deletion of amino acids 138-148, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a deletion of amino acids 137-148, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a deletion of amino acids 136 to 148, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a deletion of amino acids 135 to 148, numbered according to the IMGT scheme.In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a deletion of amino acids 134-148, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a deletion of amino acids 133-148, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a deletion of amino acids 132-148, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a deletion of amino acids 131-148, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a deletion of amino acids 131-148, numbered according to the IMGT scheme. The construct contains an IgA2-based constant region containing a deletion of amino acids P131 to Y148, numbered according to the IMGT scheme.

[0105]

[0123] In some embodiments, the IgA antibody comprises a C-terminal asparagine (N) amino acid In some embodiments, the mutation is a non-conservative amino acid substitution. In some embodiments, the mutation deletes the glycosylation site of the C-terminal asparagine (N) amino acid of IgA. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a mutation at N135, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a non-conservative mutation at N135, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising an N135Q mutation, numbered according to the IMGT scheme.

[0106]

[0124] In some embodiments, the antibody construct is numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a non-conservative mutation at N45.2, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising an N45.2G mutation, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region having an extended circulating half-life compared to an antibody construct comprising an IgA2-based constant region without a mutation at the N45.2 amino acid.

[0107]

[0125] In some embodiments, the antibody construct is numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a non-conservative mutation at P124, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a P124R, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising an IgA2-based constant region comprising a P124R mutation, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region has an extended circulating half-life compared to an antibody construct comprising an IgA2-based constant region without a mutation at the P124 amino acid. In some embodiments, the antibody construct comprises an IgA2-based constant region has an increased stability compared to an antibody construct comprising an IgA2-based constant region without a mutation at the P124 amino acid.

[0108]

[0126] In some embodiments, the antibody construct is numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a non-conservative mutation at C92, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a C92S mutation, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a C92S mutation, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region having reduced aggregation compared to an IgA2 antibody not having a mutation at the C92 amino acid. In some embodiments, the antibody construct comprises an IgA2-based constant region having reduced aggregation with serum proteins compared to an IgA2 antibody not having a mutation at the C92 amino acid. In some embodiments, the antibody construct has reduced aggregation in vitro or in vivo compared to an IgA2 antibody not having a mutation at the C92 amino acid.

[0109]

[0127] In some embodiments, the antibody construct is numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a non-conservative mutation at N120, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising an N120T mutation, numbered according to the IMGT scheme. In one embodiment, the IgA2 antibody has an extended circulating half-life compared to an IgA2 antibody that does not have a mutation at the N120 amino acid.

[0110]

[0128] In some embodiments, the antibody construct is numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a non-conservative mutation at I121, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a non-conservative mutation at I121, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising an I121L, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody has an extended circulating half-life compared to an IgA2 antibody that does not have a mutation at the N338 amino acid.

[0111]

[0129] In some embodiments, the antibody construct is numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a non-conservative mutation at T122, numbered according to the IMGT scheme. ...

[0112]

[0130] In some embodiments, the antibody construct is numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a non-conservative mutation at C147, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region comprising a deletion of amino acid C147, numbered according to the IMGT scheme. In some embodiments, the IgA2 antibody has reduced aggregation compared to an IgA2 antibody that does not have a mutation at the C147 amino acid.

[0113]

[0131] In some embodiments, the antibody construct is numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region with a non-conservative mutation at Y148, numbered according to the IMGT scheme. In some embodiments, the antibody construct comprises an IgA2-based constant region with a deletion of amino acid Y148, numbered according to the IMGT scheme.

[0114]

[0132] In some embodiments, the IgA antibody comprises one or more albumin binding domains. In some embodiments, one or more albumin binding domains are fused to the light or heavy chain of the IgA constant region. In some embodiments, one or more albumin binding domains are fused to the heavy chain of the IgA constant region. In some embodiments, one or more albumin binding domains are fused to the C-terminal region of the CH3 region of the heavy chain of the IgA constant region. In some embodiments, the IgA2 antibody has an extended circulating half-life compared to an IgA2 antibody that does not include one or more albumin binding domains. In some embodiments, the antibody construct comprises an IgA2-based constant region that comprises one or more albumin binding domains and has a circulating half-life within 1%, 5%, or 10% of the circulating half-life of the corresponding IgG antibody. In some embodiments, the antibody construct comprises an IgA2-based constant region that comprises one or more albumin binding domains and has a circulating half-life longer than the half-life of the corresponding IgG antibody.

[0115]

[0133] In some embodiments, the IgA antibody is Cancer Res. 2015;76(2):403-17; Meyer S.et al. mAbs. 2016;8(1):87-98; or Leusen J.et al. Contains one or more of the mutations described in Molecular Immunology. 2015;68:35-39.

[0116]

[0134] In some embodiments, one or more mutations or deletions compared to a reference IgA sequence results in an increase or decrease in the circulating half-life of the antibody construct. In some embodiments, the one or more mutations or deletions result in an increase in the circulating half-life of the antibody construct. For example, the one or more mutations can increase the serum half-life of the antibody construct in humans by up to 21 days or more. Furthermore, the one or more mutations can increase the serum half-life of the antibody construct in mice by up to 9 days or more. In some embodiments, the one or more mutations can increase the serum half-life of the antibody construct to a level comparable to the serum half-life of an immunoglobulin G (IgG) molecule. In some embodiments, the one or more mutations or deletions result in a decrease in the circulating half-life of the antibody construct.

[0117]

[0135] In some embodiments, the one or more mutations increase the serum half-life of the antibody construct by at least In some embodiments, the one or more mutations can extend the serum half-life of the antibody construct by at least about 7 days to about 30 days or more. In some embodiments, the one or more mutations can extend the serum half-life of the antibody construct by at least about 7 days. In some embodiments, the one or more mutations can extend the serum half-life of the antibody construct by up to about 30 days. In some embodiments, the one or more mutations can extend the serum half-life of the antibody construct by at least about 7 days to about 8 days, about 7 days to about 9 days, about 7 days to about 10 days, about 7 days to about 15 days, about 7 days to about 20 days, about 7 days to about 25 days, about 7 days to about 30 days, about 8 days to about 9 days, about 8 days to about 10 days, about 8 days to about 15 days, about 8 days to about 20 days, about 8 days to about 25 days, about 8 days to about 30 days, about 9 days to about 1 0 days, about 9 to about 15 days, about 9 to about 20 days, about 9 to about 25 days, about 9 to about 30 days, about 10 to about 15 days, about 10 to about 20 days, about 10 to about 25 days, about 10 to about 30 days, about 15 to about 20 days, about 15 to about 25 days, about 15 to about 30 days, about 20 to about 25 days, about 20 to about 30 days, or about 25 to about 30 days. In some embodiments, one or more mutations can extend the serum half-life of the antibody construct by about 7 days, about 8 days, about 9 days, about 10 days, about 15 days, about 20 days, about 25 days, or about 30 days.

[0118]

[0136] In some embodiments, the antibody constructs exhibit increased stability. In embodiments, the one or more mutations and / or the one or more deletions result in increased stability of the antibody construct compared to a corresponding IgA antibody that does not contain the one or more mutations and / or the one or more deletions.

[0119]

[0137] In some embodiments, the antibody construct exhibits reduced aggregation. Antibody aggregation is characterized by: It is a more common manifestation of physical instability. Protein aggregates generally have reduced activity and, more importantly, greater immunogenic potential due to epitope diversity and / or structural changes. Immunoglobulin aggregates are known to cause severe renal failure and anaphylactoid reactions, such as headache, fever, and chills. Therefore, it is advantageous to reduce aggregation in antibody therapy. In addition, aggregate levels in commercial intravenous immunoglobulin products are limited to less than 5% based on World Health Organization (WHO) standards. In some embodiments, one or more mutations result in reduced aggregation. In some embodiments, one or more mutations and / or one or more deletions result in reduced aggregation of the antibody construct compared to the corresponding IgA antibody that does not contain one or more mutations and / or one or more deletions.

[0120]

[0138] In some embodiments, the antibody construct provided herein comprises at least about In some embodiments, the antibody constructs provided herein have aggregate levels in the range of at least about 0.1%. In some embodiments, the antibody constructs provided herein have aggregate levels in the range of up to about 5%. In some embodiments, the antibody constructs provided herein have aggregate levels in the range of about 0. having an aggregate level in the range of 1% to about 0.5%, about 0.1% to about 1%, about 0.1% to about 2%, about 0.1% to about 3%, about 0.1% to about 4%, about 0.1% to about 5%, about 0.5% to about 1%, about 0.5% to about 2%, about 0.5% to about 3%, about 0.5% to about 4%, about 0.5% to about 5%, about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 2% to about 3%, about 2% to about 4%, about 2% to about 5%, about 3% to about 4%, about 3% to about 5%, or about 4% to about 5%. In some embodiments, the antibody constructs provided herein have aggregate levels in the range of about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, or about 5%.

[0121]

[0139] The therapeutic antibodies disclosed herein may contain one or more amino acid substitutions. Instead, it may contain synthetic amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-

[0140] Dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclo These include pentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0122] CD47 and SIRPα

[0141] Signal regulatory protein α (SIRP-α) is widely expressed on the membrane of myeloid cells SIRP-α is a protein that interacts with CD47 (cluster of differentiation 47), a protein that is widely expressed on many cell types in the body. The interaction of SIRP-α with CD47 prevents the embracing of "self" cells that would normally be recognized by the immune system. It has been observed that in acute myeloid leukemia and some solid tumor cancers, high CD47 expression on tumor cells can act as a negative prognostic factor for survival. Strategies focused on disrupting the interaction of CD47 with SIRP-α, for example, administering agents that mask either CD47 or SIRP-α, may be potential anticancer therapies.

[0123]

[0142] In addition, inhibitory receptor signal-regulatory protein alpha (SIRPα) or Checkpoint inhibition of its ligand CD47 has proven highly effective in preclinical models in combination with IgG1 anticancer therapy, and some of these CD47-SIRPa interaction blockers are already being tested in clinical trials for hematological and solid cancers (www.clinicaltrials.gov identification numbers: NCT02216409; NCT02678338, NCT02641002; NCT02367196, NCT02890368; NCT02663518, NCT02953509). SIRPα is more or less selectively present on myeloid cells, limiting ADCP / ADCC by macrophages and neutrophils, respectively. Its ubiquitously expressed ligand CD47 acts as a "don't eat me" signal and is often found overexpressed on cancer cells to inhibit phagocytosis and clearance by macrophages. Indeed, in clinical settings, the expression level of CD47 on cancer cells has been found to inversely correlate with clinical response to anticancer antibody therapy. In several preclinical models, CD47-SIRPα blockers have been shown to inhibit the expression of IgG mAbs targeting various tumor antigens, such as cetuximab and CD47-SIRPα has been proven to be a promising target for enhancing cancer immunotherapy when combined with sirolimus and trastuzumab. However, the enhancing effect of this additional CD47-SIRPα checkpoint inhibition has not yet been examined for IgA antibodies against specific tumor antigens. Because CD47 is ubiquitously expressed on human cells, widespread inhibition of CD47 may present a risk of adverse effects. Therefore, effective local targeting of CD47 to cancer cells is desirable. In some embodiments, the therapeutic agent described herein has low affinity binding to CD47; thus preventing unwanted binding to cells other than cancer cells. In certain embodiments of the constructs described herein, such low affinity binding of CD47 combined with high affinity binding to antigens expressed on antigen-binding cells allows antigen binding to dictate the cell specificity of the construct.

[0124]

[0143] Provided herein is a therapeutic agent comprising an IgA constant region domain, as shown in FIG. Thus, a therapeutic agent is provided that binds to CD47 and an additional tumor associated antigen. Further provided herein is a method of treating a subject in need thereof comprising administering to the subject a therapeutic dose of a therapeutic agent disclosed herein. Further provided herein is a pharmaceutical composition comprising a therapeutic agent disclosed herein and a pharma- ceutical acceptable carrier.

[0125] Treatment

[0144] Therapeutic agents comprising the antibody constructs described herein are provided.

[0145] In the embodiments described herein, an immune effector cell engager molecule and Described herein is an antibody construct that acts as an immune effector cell engager. Such an immune effector cell engager comprises an immunoglobulin A (IgA) heavy chain domain based on an IgA construct as described herein, a CD47 binding domain as described herein; and an antigen binding domain expressed on an antigen presenting cell; wherein the IgA heavy chain domain specifically binds to FcαR on an immune effector cell, thereby engaging said immune effector cell, the antigen binding domain binds to an antigen on a target antigen presenting cell, and the CD47 binding domain inhibits the binding of CD47 expressed on the target antigen presenting cell to signal regulatory protein alpha (SIRP alpha) on the immune effector cell, thereby enabling the immune effector cell to destroy the antigen presenting cell, and the antibody construct has a higher binding affinity for the antigen compared to CD47, thereby selectively binding to the antigen presenting cell.

[0126]

[0146] In certain embodiments, the method comprises contacting a target cell with an effective amount of an antibody construct. a method of inducing a neutrophil-mediated immune response against a target antigen-presenting cell, wherein the antibody construct comprises an immunoglobulin A (IgA) heavy chain domain; a CD47-binding domain; and an antigen-binding domain; The antigen binding domain binds to an antigen on a target cell, the IgA heavy chain domain specifically binds to FcαR on a neutrophil, thereby recruiting the neutrophil to the target cell, the CD47 binding domain inhibits binding of CD47 expressed on a target cell to signal regulatory protein alpha (SIRP α) on the neutrophil, thereby promoting killing of the target cell by the neutrophil, and the antibody construct has a higher binding affinity for the antigen compared to CD47, thereby inducing a neutrophil-mediated immune response, thereby selectively binding to antigen-presenting target cells.

[0127]

[0147] In some cases, the target cell is a cancer cell that presents a tumor antigen. There are four general groups of tumor antigens: (i) viral tumor antigens, which can be identical for any viral tumor of this type; (ii) oncogenic tumor antigens, which can be specific for the patient and the tumor; (iii) transplant-type alloantigens or tumor-specific transplantation antigens, which can be different in every individual type of tumor, but can be identical in different tumors caused by the same virus; and (iv) tumor-specific tumor antigens, which can be specific for every individual type of tumor, but can be identical in different tumors caused by the same virus. ) embryonic antigens. As a result of the discovery of tumor antigens, they have become important in the development of novel cancer treatments that can specifically target cancer. This has led to the development of antibodies against these tumor antigens. In addition to the development of antibodies against tumor antigens for cancer treatment, antibodies have also been developed to target immune cells and stimulate an immune response.

[0128]

[0148] Disclosed herein, in certain embodiments, are therapeutic agents that comprise an IgA constant region. In some cases, the therapeutic agent is an IgA antibody. The IgA constant region can be an IgA1 constant region. The IgA constant region can be an IgA2 constant region. For example, the therapeutic agent can be an IgA1 antibody or an IgA2 antibody. In some embodiments, the therapeutic agent does not bind to B cells, T cells, platelets, and / or red blood cells. For example, the therapeutic agent has low immunogenicity.

[0129]

[0149] The therapeutic agent can be a bispecific or multispecific antibody. In some cases, the therapeutic agent co-engages with two antigens on the cell surface. The therapeutic agent can co-engage with two different antigens. In some cases, the binding of the therapeutic agent to two different antigens is sequential. For example, the binding of the therapeutic agent to the first antigen occurs first, thereby limiting the space explored by the arm of the second antibody. As a result, a significant increase in the local concentration of the second antigen can occur, which can facilitate the binding of the arm of the second antibody.

[0130]

[0150] In some embodiments, the therapeutic agent binds to the C antigen with a lower affinity compared to binding to the antigen. D47. In some cases, the therapeutic agent reduces CD47 binding of cancer cells. For example, the therapeutic agent can inhibit the interaction between human CD47 and signal regulatory protein alpha (SIRPα). Furthermore, inhibition of the interaction between human CD47 and SIRPα can increase the therapeutic potential of the therapeutic agent. Inhibition of the interaction between human CD47 and SIRPα can increase the phagocytosis and clearance of cancer cells at tumor sites. For example, the cancer cells can be IgA-opsonized cancer cells. The therapeutic agent can be an IgA bispecific antibody comprising a low affinity CD47 arm. In some embodiments, the therapeutic agent described herein has low affinity binding to CD47, which prevents the therapeutic agent from binding to CD47 on cells other than cancer cells. In some cases, the low affinity CD47 arm of the therapeutic agent described herein binds to tumor cells expressing CD47. In some examples, the low affinity CD47 arm of the therapeutic agent described herein does not bind to cells expressing CD47 that are not tumor cells. The Therapeutic Agent can be an IgA bispecific antibody that disrupts the CD47-SIRPα signal. In some instances, the Therapeutic Agent has a lower binding affinity (K d ) and contains an attenuated CD47-binding domain that binds to CD47 at .

[0131]

[0151] In some embodiments, the therapeutic agent is at least about 0.01 micromolar (μ Binding affinity (K M ) of approximately 999 μM or higher d In some embodiments, the therapeutic agent binds to CD47 with a binding affinity (K d In some embodiments, the Therapeutic Agent binds to CD47 with a binding affinity (K dIn some embodiments, the therapeutic agent binds to CD47 at about 0.01 μM to about 0.1 μM, about 0.01 μM to about 0.5 μM, about 0.01 μM to about 1 μM, about 0.01 μM to about 5 μM, about 0.01 μM to about 10 μM, about 0.01 μM to about 50 μM, about 0.01 μM to about 100 μM, about 0.01 μM to about 200 μM, about 0.01 μM to about 300 μM, about 0.01 μM to about 500 μM, about 0.01 μM to about 999 μM, about 0.1 μM to about 0.5 μM , about 0.1 μM to about 1 μM, about 0.1 μM to about 5 μM, about 0.1 μM to about 10 μM, about 0.1 μM to about 50 μM, about 0.1 μM to about 100 μM, about 0.1 μM to about 200 μM, about 0.1 μM to approximately 300 μM, approximately 0.1 μM to approximately 500 μM, approximately 0.1 μM to approximately 999 μM, approximately 0.5 μM to approximately 1 μM, approximately 0.5 μM to approximately 5 μM, approximately 0.5 μM to approximately 10 μM, approximately 0 .5μM to about 50μM, about 0.5μM to about 100μM, about 0.5μM to about 200μM, about 0.5μM to about 300μM, about 0.5μM to about 500μM, about 0.5μM to approximately 999μM, approximately 1μM to approximately 5μM, approximately 1μM to approximately 10μM, approximately 1μM to approximately 50μM, approximately 1μM to approximately 100μM, approximately 1μM to approximately 200μM , about 1 μM to about 300 μM, about 1 μM to about 500 μM, about 1 μM to about 999 μM, about 5 μM to about 10 μM, about 5 μM to about 50 μM, about 5 μM to about 100 μM, approximately 5 μM to approximately 200 μM, approximately 5 μM to approximately 300 μM, approximately 5 μM to approximately 500 μM, approximately 5 μM to approximately 999 μM, approximately 10 μM to approximately 50 μM, approximately 10 μM Approximately 100μM, approximately 10μM to approximately 200μM, approximately 10μM to approximately 300μM, approximately 10μM to approximately 500μM, approximately 10μM to approximately 999μM, approximately 50μM to approximately 10 0μM, about 50μM to about 200μM, about 50μM to about 300μM, about 50μM to about 500μM, about 50μM to about 999μM, about 100μM to about 200μM , about 100 μM to about 300 μM, about 100 μM to about 500 μM, about 100 μM to about 999 μM, about 200 μM to about 300 μM, about 200 μM to about 500 μM, about 200 μM to about 999 μM, about 300 μM to about 500 μM, about 300 μM to about 999 μM, or about 500 μM to about 999 μM. dIn some embodiments, the therapeutic agent binds to CD47 with a binding affinity (K) of about 0.01 μM, about 0.1 μM, about 0.5 μM, about 1 μM, about 5 μM, about 10 μM, about 50 μM, about 100 μM, about 200 μM, about 300 μM, about 500 μM, or about 999 μM. d ) and binds to CD47.

[0132]

[0152] The constructs described herein are either known or generated de novo. Any CD47 binding domain derived from a CD47 targeting antibody or molecule may be included. These CD47 binding regions may be directly incorporated into the constructs described herein, or they may be further attenuated to reduce CD47 binding strength before being incorporated into the constructs described herein. Representative CDR regions of antibodies that can be used for CD47 targeting are provided in Table A below.

[0133]

[0153]

[0134] [Table 1]

[0135]

[0154] In some embodiments, the therapeutic agent is at about 1 mM to about 1000 millimolar (mM) Binding affinity (K d In some embodiments, the therapeutic agent binds to CD47 with a binding affinity (K d In some embodiments, the Therapeutic Agent binds to CD47 with a binding affinity (K dIn some embodiments, the therapeutic agent binds to CD47 at about 1 mM to about 5 mM, about 1 mM to about 10 mM, about 1 mM to about 50 mM, about 1 mM to about 100 mM, about 1 mM to about 200 mM, about 1 mM to about 300 mM, about 1 mM to about 400 mM, about 1 mM to about 500 mM, about 1 mM to about 600 mM, about 1 mM to about 800 mM, about 1 mM to about 1000 mM, about 5 mM to about 10 mM, about 5 mM to about 50 mM, about 5 mM to about 600 mM, about 5 mM to about 800 mM, about 1 mM to about 1000 mM, about 5 mM to about 10 mM, about 5 mM to about 50 mM, about 5 mM to about 10 ... about 50 mM, about 5 mM to about 100 mM, about 5 mM to about 200 mM, about 5 mM to about 300 mM, about 5 mM to about 400 mM, about 5 mM to about 500 mM, about 5 mM to about 600 mM, about 5 mM to about 800 mM, about 5 mM to about 1000 mM, about 10 mM to about 50 mM, about 10 mM to about 100 mM, about 10 mM to about 200 mM, about 10 mM to about 300 mM, about 10 mM to about 40 0 mM, about 10 mM to about 500 mM, about 10 mM to about 600 mM, about 10 mM to about 800 mM, about 10 mM to about 1000 mM, about 50 mM to about 100 mM, about 50 mM to about 200 mM, about 50 mM to about 300 mM, about 50 mM to about 400 mM, about 50 mM to about 500 mM, about 50 mM to about 600 mM, about 50 mM to about 800 mM, about 50 mM to about 1000 mM, about 100 mM to about 200 mM, about 100 mM to about 300 mM, about 100 mM to about 400 mM, about 100 mM to about 500 mM, about 100 mM to about 600 mM, about 100 mM to about 800 mM, about 100 mM to about 1000 mM, about 200 mM to about 300 mM, about 200 mM to about 40 0 mM, about 200 mM to about 500 mM, about 200 mM to about 600 mM, about 200 mM to about 800 mM, about 200 mM to about 1000 mM, about 300 mM to about 400 mM, about 300 mM to about 500 mM, about 300 mM to about 600 mM, about 300 mM to about 800 mM, about 300 mM to about 1000 mM, about 400 mM to about 500 Binding affinity (K dIn some embodiments, the therapeutic agent binds to CD47 with a binding affinity (K) of about 1 mM, about 5 mM, about 10 mM, about 50 mM, about 100 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM, about 600 mM, about 800 mM, or about 1000 mM. d ) and binds to CD47.

[0136]

[0155] In some cases, the therapeutic agent binds to a tumor-associated antigen. The antibody may be an IgA bispecific antibody comprising an affinity tumor-associated antigen arm. The tumor-associated antigen may be CD20. The tumor-associated antigen may be GD2. The tumor-associated antigen may be mesothelin. The tumor-associated antigen may be selected from the group consisting of GD2, CD38, CD19, EGFR, HER2, PD-L1, and CD25. The tumor-associated antigen may be CD33, BCMA, CD44, alpha-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER3, folate binding protein, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MAGE-A1, MUC16, h5T4, PSMA, TAG-72, EGFRvIII, CD123, VEGF-R2, or any combination thereof.

[0137]

[0156] The constructs described herein are either known or generated de novo. Any antigen-binding domain derived from antigen targeting antibody or molecule can be included.The representative CDR region of antibody that can be used for antigen targeting is provided in the following table B.Those skilled in the art should know to utilize suitable antigen-binding domain from known sources to appropriately target antigen-presenting cells.

[0138]

[0157]

[0139] [Table 2]

[0140]

[0158] In some embodiments, the therapeutic agents described herein bind to CD20. CD20 is a 33-37 kD nonglycosylated phosphoprotein expressed on the surface of almost all normal and malignant B cells. CD20 knockout mice display a nearly normal phenotype, suggesting a high level of redundancy. Recently, it has been reported that CD20 deficiency in humans results in impaired T cell-independent antibody responses. In addition to functioning as a calcium channel, CD20 is hypothesized to reside in lipid rafts, i.e., cholesterol-rich microdomains in the plasma membrane. CD20 spans the membrane four times and has intracellular C- and N-termini. Two regions of CD20 are extracellular, forming a small and large loop.

[0141]

[0159] In some embodiments, the therapeutic agents described herein bind to HER2. HER2 / neu (human epidermal growth factor receptor 2 / receptor tyrosine-protein kinase erbB-2) is part of the human epidermal growth factor family. Overexpression of this protein may be shown to play an important role in the progression of cancers, such as breast cancer. HER2 / neu protein can function as a receptor tyrosine kinase and autophosphorylates upon dimerization with a binding partner. HER2 / neu can activate several signaling pathways, such as mitogen-activated protein kinase, phosphoinositide 3-kinase, phospholipase C, protein kinase C, and signal transducer and activator of transcription (STAT). Examples of antibodies that can target and inhibit HER2 / neu include trastuzumab and pertuzumab.

[0142]

[0160] In some embodiments, the therapeutic agents described herein bind to EGFR. EGFR (epidermal growth factor receptor) encodes a member of the human epidermal growth factor family. Mutations that can lead to overexpression or overactivity of EGFR can be associated with several cancers, including squamous cell carcinoma and glioblastoma. EGFR can function as a receptor tyrosine kinase, and ligand binding can induce dimerization and autophosphorylation with binding partners. Phosphorylated EGFR can then activate several downstream signaling pathways, including mitogen-activated protein kinase, phosphoinositide 3-kinase, phospholipase Cy, protein kinase C, and signal transducer and activator of transcription (STAT). Examples of antibodies that can target and inhibit EGFR include cetuximab, panitumumab, nimotuzumab, and Zalutumumab can be mentioned. One mutation variant of EGFR is EGFRvIII (epidermal growth factor receptor variant III). EGFRvIII may be the result of an EGFR gene rearrangement in which exons 2-7 of the extracellular domain are deleted. This mutation may result in a mutant receptor that cannot bind to any known ligand. The resulting receptor can engage in constitutive low-level signaling and participate in tumor progression. Examples of antibodies that can target EGFRvIII include AMG595 and ABT806.

[0143]

[0161] In some embodiments, the therapeutic agents described herein bind to mesothelin. Mesothelin was initially described as an antigen recognized by the K1 monoclonal antibody, which was generated after immunization of mice with the OVCAR-3 human ovarian cancer cell line. The mesothelin gene was subsequently cloned in 1996. The mesothelin cDNA contains an open reading frame of 1884 bp and encodes a 69 kDa precursor protein (628 amino acids). After glycosylation, the precursor is cleaved by furin at amino acids 288–293, resulting in a 40 kDa protein and a smaller 32 kDa fragment that is released from the cell. This 32 kDa shed fragment is called megakaryocyte potentiating factor (MPF). The 40 kDa protein is found on the cell surface and can be released by treatment with phosphatidylinositol-specific phospholipase C. This 40 kDa GPI-linked membrane-bound protein was named mesothelin because it is produced by normal mesothelial cells. Since malignant mesothelioma and ovarian adenocarcinoma are derived from normal mesothelial cells, it is not surprising that mesothelin is associated with these malignancies.

[0144]

[0162] The most common form of mesothelin is membrane-bound, but two variants have been found. It has been reported that variant-1, which has an insertion of 8 amino acids, is also membrane-bound. Variant-2 is shed and soluble due to the lack of the GPI anchor signal sequence. Soluble mesothelin protein is detectable in the serum of ovarian cancer patients and may provide a useful novel marker for diagnosing ovarian cancer and / or monitoring its response to treatment together with CA125 (cancer antigen 125). In addition, soluble mesothelin is elevated in the blood and effusions of mesothelioma patients, and the determination of mesothelin levels in these fluids has been approved by the US FDA, primarily as a tool for monitoring patient response and progression.

[0145]

[0163] Mesothelin interacts with CA125, a tumor antigen used in the diagnosis of ovarian cancer. It is suspected to play a role in cell adhesion and tumor metastasis through its action on mesothelin. CA125 is also called MUC16. CA125 / MUC16 is a type I transmembrane protein expressed on the cell surface of many epithelia, and its soluble form can be released into the extracellular space. Furthermore, binding of mesothelin to CA125 / MUC16 promotes pancreatic cancer cell motility and invasion through MMP-7 activation.

[0146]

[0164] Mesothelin is considered a differentiation antigen because its expression in normal tissues is restricted to the mesothelium. Although mesothelin has been reported to be highly expressed in a variety of tumors, including mesothelioma, ovarian, pancreatic, and lung cancers, it is highly expressed in a variety of tumors, including mesothelioma, ovarian cancer, pancreatic cancer, and lung cancer. Mesothelin can be detected by immunohistological methods on normal mesothelial cells lining pleural, pericardial, and peritoneal surfaces, but not in vital organs. It is often highly expressed in many epithelial cancers. The differential overexpression of mesothelin in tumors and its role in cell adhesion and tumor metastasis make it a suitable target for cancer therapy.

[0147]

[0165] In some embodiments, the antigen-binding portion of a therapeutic agent described herein is D2, CD38, CD19, EGFR, HER2, PD-L1, CD25, CD33, BCMA, CD44, α-folate receptor, CAIX, CD30, ROR1, CEA, EGP- 2, EGP-40, HER2, HER3, folate binding protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MUC-16, MAGE-A1, MUC16, h5T4, PSMA, TAG-72, EGFRvIII, CD123 and VEGF-R2. In some embodiments, the antigen binding domain comprises a single chain antibody fragment (scFv) comprising a variable domain light chain (VL) and a variable domain heavy chain (VH) of a target antigen-specific monoclonal antibody joined by a flexible linker, such as a glycine-serine linker or a Whitlow linker. In some embodiments, the scFv is humanized. In some embodiments, the therapeutic agent comprises a variable domain and an antibody. In some embodiments, the therapeutic agent comprises a variable domain from an IgG antibody of human, humanized, or any other non-human origin. In some embodiments, the antigen binding portion can comprise a VH and a VL directionally linked, e.g., N-to-C terminus, VH-linker-VL or VL-linker-VH. In some examples, the antigen binding domain recognizes a target epitope.

[0148]

[0166] In one embodiment, the antigen-binding portion of the therapeutic agent described herein is an antigen binding moiety that inhibits CD20 In one embodiment, the antigen binding portion of the therapeutic agent described herein is specific for CD19. In one embodiment, the antigen binding portion of the therapeutic agent described herein is specific for CD38. In one embodiment, the antigen binding portion of the therapeutic agent described herein is specific for PD-L1. In one embodiment, the antigen binding portion of the therapeutic agent described herein is specific for CD25. In one embodiment, the antigen binding portion of the therapeutic agent described herein is specific for CD33. In other embodiments, the antigen binding portion of the therapeutic agent described herein is specific for BCMA. In still other embodiments, the antigen binding portion of the therapeutic agent described herein is specific for CD44. In some embodiments, the antigen binding portion of the therapeutic agent described herein is specific for alpha-folate receptor. In some embodiments, the antigen binding portion of the therapeutic agent described herein is specific for CAIX. In one embodiment, the antigen binding portion of the therapeutic agent described herein is specific for CD30. In some embodiments, the antigen binding portion of the therapeutic agent described herein is specific for ROR1. In one embodiment, the antigen binding portion of the therapeutic agent described herein is specific for CEA. In some embodiments, the antigen-binding portion of the therapeutic described herein is specific for EGP-2. In one embodiment, the antigen-binding portion of the therapeutic described herein is specific for EGP-40. In other embodiments, the antigen-binding portion of the therapeutic described herein is specific for HER2. In yet other embodiments, the antigen-binding portion of the therapeutic described herein is specific for HER3. In yet other embodiments, the antigen-binding portion of the therapeutic described herein is specific for folate binding protein. In some embodiments, the antigen-binding portion of the therapeutic described herein is specific for GD2. In some embodiments, the antigen-binding portion of the therapeutic described herein is specific for GD3. In one embodiment, the antigen-binding portion of the therapeutic described herein is specific for IL-13R-a2. In one embodiment, the antigen-binding portion of the therapeutic described herein is specific for KDR. In one embodiment, the antigen-binding portion of the therapeutic described herein is specific for EDB-F.In other embodiments, the antigen-binding portion of the therapeutic described herein is specific for mesothelin. In yet other embodiments, the antigen-binding portion of the therapeutic described herein is specific for CD22. In one embodiment, the antigen-binding portion of the therapeutic described herein is specific for EGFR. In one embodiment, the antigen-binding portion of the therapeutic described herein is specific for MUC-1. In one embodiment, the antigen-binding portion of the therapeutic described herein is specific for MUC-16. In one embodiment, the antigen-binding portion of the therapeutic described herein is specific for MAGE-A1. In some embodiments, the antigen-binding portion of the therapeutic described herein is specific for h5T4. In some embodiments, the antigen-binding portion of the therapeutic described herein is specific for PSMA. In another embodiment, the antigen-binding portion of the therapeutic agent described herein is specific for TAG-72. In yet another embodiment, the antigen-binding portion of the therapeutic agent described herein is specific for EGFRvIII. In another embodiment, the antigen-binding portion of the therapeutic agent described herein is specific for CD123. In yet another embodiment, the antigen-binding portion of the therapeutic agent described herein is specific for VEGF-R2.

[0149]

[0167] Unmodified IgA as a therapeutic monoclonal antibody has a relatively short half-life. This may be due to a combination of lack of binding to neonatal Fc receptors (FcRn) in vivo and low levels of sialylation in recombinant production systems. The FcRn receptor has an established role in maintaining antibody half-life, and reduced binding of antibody molecules to FcRn leads to shorter half-life in serum. Furthermore, since IgA is more highly glycosylated than IgG, the enzymes that transfer sialic acid to nascent oligosaccharides (sialyltransferases) are limited. In vivo, the asialoglycoprotein receptor (ASGPR) clears IgA more quickly than IgG due to its lower level of sialylation. The development of antibody therapeutics with extended half-life in serum is desirable for a variety of reasons, including but not limited to improved therapeutic efficacy and improved administration due to sustained circulation in the bloodstream.

[0150]

[0168] Provided herein are therapeutic agents that contain one or more mutations that result in improved pharmacokinetics. (Lohse S. et al. Cancer Res. 2016; 76(2):403-17; Meyer S. et al. mAbs. 2016; 8(1):87-98). Provided herein are therapeutics comprising one or more albumin binding domains that provide improved pharmacokinetics. In some embodiments, the therapeutic comprises one or more mutations. The mutations can be conservative mutations. The mutations can be non-conservative mutations. In some embodiments, the therapeutic comprises one or more deletions. In some embodiments, the therapeutic comprises one or more albumin binding domains. The mutations or deletions can be found in the IgA constant region. Alternatively, the one or more mutations and / or the one or more deletions can be found in the IgA variable region. The one or more mutations can extend the serum half-life of the therapeutic to a level comparable to the half-life of an immunoglobulin G (IgG) molecule. For example, the one or more mutations can extend the serum half-life of the therapeutic up to 21 days or more in humans. Additionally, one or more of the mutations can extend the serum half-life of the therapeutic agent in mice up to 9 days or more.

[0151]

[0169] In some embodiments, the one or more mutations increase the serum half-life of the therapeutic agent by at least In some embodiments, the one or more mutations can extend the serum half-life of the Therapeutic Agent by at least about 7 days. In some embodiments, the one or more mutations can extend the serum half-life of the Therapeutic Agent by up to about 30 days. ..., about 7 days to about 9 days, about 7 days to about 10 days, about 7 days to about 15 days, about 7 days to about 20 days, about 7 days to about 25 days, about 7 days to about 30 days, about 8 days to about 9 days, about 8 days to about 10 days, about 8 days to about 15 days, about 8 days to about 20 days, about 8 days to about 25 days, about 8 days to about 30 days, about 9 days to about 10 days. The incubation period can be extended for about 7 days, about 8 days, about 9 days to about 10 days, about 15 days, about 20 days, about 25 days, about 9 days to about 30 days, about 10 days to about 15 days, about 10 days to about 20 days, about 10 days to about 25 days, about 10 days to about 30 days, about 15 days to about 20 days, about 15 days to about 25 days, about 15 days to about 30 days, about 20 days to about 25 days, about 20 days to about 30 days, or about 25 days to about 30 days. In some embodiments, the one or more mutations can be extended for about 7 days, about 8 days, about 9 days, about 10 days, about 15 days, about 20 days, about 25 days, or about 30 days. In some embodiments, the one or more mutations can extend the serum half-life of the therapeutic agent by about 7 days, about 8 days, about 9 days, about 10 days, about 15 days, about 20 days, about 25 days, or about 30 days.

[0152]

[0170] In some embodiments, the IgA constant region contains a mutation at position 166. The difference can be a non-conservative change. For example, the change can include replacing the asparagine at position 166 with glycine or a glycine homologue. In some cases, the amino acid substitution at position 166 extends the serum half-life of the therapeutic agent to a level equal to or greater than the substitution with glycine. The therapeutic agent can have a longer half-life than the corresponding therapeutic agent that contains an asparagine at position 166.

[0153]

[0171] In some cases, the IgA constant region contains a mutation at position 337. The difference can be a non-conservative change. For example, the change can include replacing the asparagine at position 337 with threonine or a threonine homologue. In certain embodiments, the amino acid replacement at position 337 extends the serum half-life of the therapeutic agent to be equal to or longer than replacement with asparagine. In some embodiments, the therapeutic agent has a longer half-life than the corresponding therapeutic agent that contains asparagine at position 337.

[0154]

[0172] In some embodiments, the IgA constant region contains a mutation at position 338. The difference can be a non-conservative change. For example, the change can include replacing the isoleucine at position 338 with leucine or a leucine homologue. In certain cases, the amino acid substitution at position 338 extends the serum half-life of the therapeutic agent to a level equal to or greater than that of a substitution with isoleucine. In some instances, the therapeutic agent has a longer half-life than the corresponding therapeutic agent that contains isoleucine at position 338.

[0155]

[0173] In some embodiments, the IgA constant region contains a mutation at position 339. The difference can be a non-conservative change. For example, the change can include replacing the threonine at position 339 with a serine or a serine homologue. In some cases, the therapeutic agent has a longer half-life than a corresponding therapeutic agent that includes a threonine at position 339.

[0156]

[0174] In certain embodiments, the IgA constant region lacks one or more glycosylation sites. In some cases, the IgA constant region comprises one or more albumin binding domains. In certain cases, one or more albumin binding domains are fused to the light or heavy chain of the IgA constant region. In some embodiments, the one or more albumin binding domains extend the serum half-life of the therapeutic agent. In some cases, the therapeutic agent has a longer half-life than a corresponding therapeutic agent that does not include one or more albumin binding domains.

[0157]

[0175] The development of antibody therapeutics with increased stability has the potential to maintain therapeutic activity while This is desirable because it can extend the shelf life of the therapeutic agent. In some embodiments, the therapeutic agent provided herein has the same or better stability as an IgG molecule. In some cases, the stability of the therapeutic agent is improved by introducing a mutation. In some embodiments, the IgA constant region contains a mutation at position 221. The mutation can be a non-conservative mutation. For example, the mutation can include replacing the proline at position 221 with arginine or an arginine homolog. In some examples, the amino acid substitution makes the therapeutic agent more stable than the corresponding therapeutic agent containing a proline at position 221.

[0158]

[0176] An additional inherent biophysical property of human antibodies is their tendency to aggregate. Aggregation is a more common manifestation of physical instability. Protein aggregates generally have reduced activity and, more importantly, greater immunogenic potential due to epitope diversity and / or structural changes. Immunoglobulin aggregates are known to cause severe renal failure and anaphylactoid reactions, such as headache, fever, and chills. It is therefore advantageous to reduce aggregation in antibody therapeutics. In addition, aggregate levels in commercially available intravenous immunoglobulin products are expected to be greater than World Health Organization (WHO) standards. is limited to less than 5% based on the

[0159]

[0177] Provided herein are therapeutic agents that have reduced aggregation. In some embodiments, Thus, the therapeutic agent provided herein has reduced aggregation compared to the aggregation of IgG molecules. In some embodiments, the therapeutic agent provided herein has the same or better aggregation tendency as IgG molecules. In some cases, the aggregation tendency of the therapeutic agent is improved by introducing a mutation. In some embodiments, the IgA constant region contains a mutation at position 311. The mutation can be a non-conservative mutation. For example, the mutation can include replacing the cysteine ​​at position 311 with a serine or a serine homolog. In some examples, the therapeutic agent has reduced aggregation compared to the corresponding therapeutic agent that contains a cysteine ​​at position 311.

[0160]

[0178] In some cases, the aggregation tendency of a therapeutic agent can be reduced by introducing deletions. In some cases, the IgA constant region contains a deletion at position 417. In some cases, the deletion of a cysteine ​​at position 471 can reduce the aggregation tendency of a therapeutic agent. In certain instances, a therapeutic agent has reduced aggregation compared to a corresponding therapeutic agent that does not include a deletion at position 471.

[0161]

[0179] In some embodiments, the therapeutic agents provided herein have a concentration of at least about 0. Therapeutic agents provided herein have aggregate levels ranging from 1% up to about 5%. In some embodiments, therapeutic agents provided herein have aggregate levels ranging from at least about 0.1%. In some embodiments, therapeutic agents provided herein have aggregate levels ranging from up to about 5%. In some embodiments, the Therapeutic Agents provided herein have aggregate levels in the range of about 0.1% to about 0.5%, about 0.1% to about 1%, about 0.1% to about 2%, about 0.1% to about 3%, about 0.1% to about 4%, about 0.1% to about 5%, about 0.5% to about 1%, about 0.5% to about 2%, about 0.5% to about 3%, about 0.5% to about 4%, about 0.5% to about 5%, about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 2% to about 3%, about 2% to about 4%, about 2% to about 5%, about 3% to about 4%, about 3% to about 5%, or about 4% to about 5%. In some embodiments, the therapeutic agents provided herein have aggregate levels in the range of about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, or about 5%.

[0162]

[0180] The therapeutic agents disclosed herein include a substituted amino acid sequence for one or more naturally occurring amino acids. The amino acid sequence may include synthetic amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, etc. These include β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0163]

[0181] A "multispecific antibody" is an antibody that binds to at least two targets of different structures, e.g., two An antibody that can simultaneously bind to different antigens, two different epitopes on the same antigen, or a hapten and / or antigen or epitope. A "multivalent antibody" is an antibody that can simultaneously bind to at least two targets of the same or different structure. The number indicates how many binding arms or sites the antibody has for a single antigen or epitope, i.e., monovalent, bivalent, trivalent or multivalent. The multivalency of an antibody means that the antibody can utilize multiple interactions when binding to an antigen, thus increasing the avidity of binding to the antigen. Specificity indicates the number of antigens or epitopes that the antibody can bind to, i.e., monospecific, bispecific, trispecific, multispecific. Using these definitions, a natural antibody, e.g., IgA, is bivalent because it has two binding arms, but monospecific because it binds to one epitope. A multispecific, multivalent antibody is a construct that has more than one binding region of different specificities. For example, the bispecific antibody construct disclosed herein has a CD47 binding region and an antigen binding region.

[0164]

[0182] "Bispecific antibodies" are capable of simultaneously binding to two targets of different structures. Bispecific antibodies (bsAbs) and bispecific antibody fragments (bsFabs) can have at least one arm that specifically binds, for example, to CD47 and at least one other arm that specifically binds to an antigen produced by or associated with a diseased cell, tissue, organ, or pathogen, for example, a tumor-associated antigen. A variety of bispecific antibodies can be produced using molecular engineering.

[0165]

[0183] The bispecific antibody construct or the composition described herein is administered When the amount is physiologically significant, it is said to be administered in a "therapeutically effective amount". An agent is physiologically significant if its presence causes a detectable change in the physiology of the recipient subject. In certain embodiments, the bispecific antibody construct disclosed herein is physiologically significant if its presence causes an anti-tumor response or reduces the signs and symptoms of an infectious disease state. A physiologically significant effect can also be the elicitation of a humoral and / or cellular immune response in the recipient subject that results in growth inhibition or death of target cells.

[0166]

[0184] The term "linker" refers to two or more amino acid residues joined by peptide bonds. and is used to refer to a polypeptide used to link one or more antigen-binding portions or variable domains. Such linker polypeptides are well known in the art (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad.Sci.USA 90:6444-6448;Poljak,RJ,e (see, e.g., J. Med. Sci. 1994, 1:1121-1123). In some embodiments, the linker peptide comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the linker peptide comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45.

[0167]

[0185] "Fv" or "Fv fragment" refers to a "single arm" of an immunoglobulin. "Fv" is therefore the smallest antibody fragment that contains a complete antigen recognition and binding site. A "two-chain" Fv fragment consists of a dimer of one heavy chain variable region and one light chain variable domain in tight, non-covalent association. Single-chain Fv species (scFv) contain the VH and VL domains of an immunoglobulin, which are covalently linked to each other by a linker peptide and are present in a single polypeptide chain. Typically, in scFv fragments, the light and heavy chain variable domains are associated in a dimeric structure similar to two-chain Fv species. Single-chain Fv fragments have the variable domain of the light chain located at the N-terminus of the single polypeptide chain, followed by a linker, and the variable domain of the heavy chain located at the C-terminus of the polypeptide chain, or vice versa, with the variable domain of the heavy chain located at the N-terminus. The VH and VL domains can be either VH or VL, with the light chain variable domain at the C-terminus and a linker peptide placed between them. The linker peptide can be any flexible linker known in the art, for example one made from glycine and serine residues. The domain association between the VH and VL domains can be further stabilized by introducing a disulfide bond into the conserved framework region (Reiter et al. Stabilization of the Fv fragments in recombinant immunotoxins by (See, "Disulfide bonds engineered into conserved framework regions," Biochemistry 1994, 33, 6551-5459). Such scFv fragments are also known as disulfide-stabilized scFv fragments (ds-scFv). Binding to CD47

[0186] Bispecific antibody constructs that bind to CD47 and its fragments are The bispecific antibodies act to modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with the functional activity of CD47. The functional activity of CD47 includes, by way of non-limiting example, interaction with SIRPα. An antibody is considered to fully modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with CD47-SIRPα interaction if the level of CD47-SIRPα interaction in the presence of the antibody is reduced by at least 50%, 60%, 70%, 80%, 90% or 95%, e.g., 96%, 97%, 98%, 99% or 100%, compared to the level of CD47-SIRPα interaction in the absence of binding to a bispecific antibody described herein. A bispecific antibody is considered to partially modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with CD47-SIRPα interaction if the level of CD47-SIRPα interaction in the presence of the antibody is reduced by less than 95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90%, compared to the level of CD47-SIRPα interaction in the absence of binding to a bispecific antibody described herein.

[0168]

[0187] The present disclosure relates to a method for producing a medicament comprising administering to a subject an antibody or antibody composition comprising: In some embodiments, the antibody construct is specific for a tumor antigen such as CD20. In some embodiments, the construct is a functional IgA construct capable of binding to an Fc receptor on neutrophils. The antigen-binding region of the bispecific antibody includes an Fc portion. The antigen-binding region of the bispecific antibody targets the CD47-binding region to antigen-presenting cells. The IgA Fc portion binds to neutrophils. The CD47 arm blocks, inhibits, or otherwise reduces the interaction between CD47 and SIRPα, thereby inducing a neutrophil-mediated immune response to the antigen. In some embodiments, the antigen-binding region of the bispecific antibody includes an anti-CD20 antibody sequence or an antigen-binding fragment thereof. The antigen-binding region for use in the constructs herein can target any antigen, as described elsewhere in this disclosure. Such antigen-binding regions can also be obtained from known antibodies, such as those shown in Table B.

[0169]

[0188] In some embodiments, the affinity of the antigen binding region is In some embodiments, the affinity of the CD47 binding region is decreased in the bispecific construct. For example, in a bispecific antibody, the affinity of the antigen binding region is increased and the affinity of the CD47 binding region is decreased. These differences in binding affinity of the antigen binding region and the CD47 binding region allow, for example, to improve selectivity for a target cell or group of target cells. The CD47 binding regions for use in the constructs herein can be designed de novo or can be derived from known antibodies, for example, as shown in Table A.

[0170]

[0189] In some embodiments, the affinity of the antigen-binding region is at least about 2, 4, 5 , 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 10 0-fold increase. In some embodiments, the increase is relative to a monospecific antigen-binding antibody. In some embodiments, the increase is relative to the binding affinity of the CD47 binding region to CD47 in a bispecific antibody. In some embodiments, the affinity of the CD47 binding region is reduced by at least about 2, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100-fold. In some embodiments, the reduction is relative to a monospecific CD47-binding antibody. In some embodiments, the reduction is relative to the binding affinity of the antigen-binding region to the antigen in a bispecific antibody.

[0171] How to generate antibodies General antibody technology

[0190] Techniques for preparing monoclonal antibodies against virtually any target antigen are available. , are well known in the art. See, e.g., Kohler and Milstein, Nature 256:495 (1975), and Coligan et al. (eds.), CUR See RENT PROTOCOLS IN IMMUNOLOGY, VOL.1, pp. 2.5.1-2.6.7 (John Wiley & Sons 1991). Briefly, monoclonal antibodies can be obtained by injecting a composition containing an antigen into a mouse, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies against the antigen, culturing the clones that produce antibodies against the antigen, and isolating the antibodies from the hybridoma cultures.

[0172]

[0191] MAbs are isolated from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography on Protein A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, e.g., Coligan, pp. 2.7.1-2.7.12 and 2.9.1-2.9.3. See also Baines et al., “Purification of Immunoglobulin G (IgG),” METHODS IN MOLECULAR BIOLOGY, VOL. 10. , pp. 79-104 (The Humana Press, Inc. 1992).

[0173]

[0192] First, the body is primed against an immunogen, then the antibodies are sequenced and subsequently engineered. Humanized antibodies can be prepared by techniques such as humanization and chimerization of mouse antibodies and antibody fragments. The use of humanized, chimeric or human antibody-derived antibody components obviates potential problems associated with the immunogenicity of mouse constant regions.

[0174] Chimeric antibodies

[0193] Chimeric antibodies are antibodies in which the variable regions of a human antibody are replaced with the complementarity determining regions of, for example, a murine antibody. Chimeric antibodies are recombinant proteins in which the variable regions of a mouse antibody, such as the CDRs, have been replaced by those of a mouse antibody. Chimeric antibodies exhibit reduced immunogenicity and increased stability when administered to a subject. General techniques for cloning mouse immunoglobulin variable domains are disclosed, for example, in Orlandi et al., Proc. Nat'l Acad. Sci. USA 86:3833 (1989). Techniques for constructing chimeric antibodies include , well known to those skilled in the art. See, for example, Leung et al., Hybridoma 13:469 (1994) produced the LL2 chimera by combining DNA sequences encoding the Vκ and VH domains of murine LL2, an anti-CD22 monoclonal antibody, with the respective human κ and IgG1 constant region domains.

[0175] Humanized antibodies

[0194] Techniques for producing humanized MAbs are well known in the art (see, e.g., Jones et al., J. Med. Soc. 1999, 143:1311-1325). s et al., Nature 321:522 (1986), Riechmann et al.,Nature 332:323(1988),Verhoeyen et al. al., Science 239:1534(1988), Carter et al. (See, e.g., Proc. Nat'l Acad. Sci. USA 89:4285 (1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), and Singer et al., J. Immun. 150:2844 (1993)). Chimeric or murine monoclonal antibodies can be humanized by transferring murine CDRs from the variable heavy and light chains of a murine immunoglobulin into the corresponding variable domains of a human antibody. The murine framework regions (FRs) in the chimeric monoclonal antibody are also replaced with human FR sequences. Simply transferring murine CDRs into human FRs often results in a reduction or even loss of antibody affinity, so further modifications may be required to restore the original affinity of the murine antibody. This can be achieved by replacing one or more human residues in the FR regions with their murine counterparts to obtain an antibody with good binding affinity to its epitope. See, e.g., Tempest et al., Biotechnology 9:266 (1991) and Verhoeyen et al. al., Science 239:1534 (1988). Generally, the mouse counterpart Human FR amino acid residues that are different from and that are located adjacent to or contiguous with one or more CDR amino acid residues are candidates for substitution.

[0176] Human antibodies

[0195] Combinatorial approaches or transformation with human immunoglobulin loci Methods for producing fully human antibodies using any of the transgenic animals described herein are known in the art (e.g., Mancini et al., 2004, New Microbiol. 27:315-28; Conrad and Scheller, 2004, New Microbiol. 27:315-28; 05, Comb. Chem. High Throughput Screen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Pharmacol. 3:544-50). Fully human antibodies can also be constructed by genetic or chromosomal transfection methods, as well as phage display technology, all of which are known in the art. See, e.g., McCafferty et al., Nature 348:552-553 (1990). Such fully human antibodies are expected to exhibit fewer side effects than chimeric or humanized antibodies and function in vivo as substantially endogenous human antibodies. In certain embodiments, the claimed methods and procedures can utilize human antibodies produced by such techniques.

[0177]

[0196] In one alternative, phage display technology is used to generate human antibodies. Human antibodies may be used to generate antibodies against disease-associated antigens (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res. 4:126-40). Human antibodies can be generated from normal humans or from humans exhibiting a particular disease state such as cancer (Dantas-Barbosa et al., 2005). The advantage of constructing human antibodies from diseased individuals is that the circulating antibody repertoire can be biased towards antibodies against disease-associated antigens.

[0178]

[0197] In one non-limiting example of this methodology, Dantas-Barbosa et al. (2005) constructed a phage display library of human Fab antibody fragments from osteosarcoma patients. In general, total RNA was obtained from circulating blood lymphocytes (ibid.). Recombinant Fabs were cloned from μ, γ and κ chain antibody repertoires and inserted into a phage display library (ibid.). The RNA was converted to cDNA and used to generate a Fab cDNA library using specific primers for heavy and light chain immunoglobulin sequences (Marks et al., 1991, J Mol. Biol. 222:581-97). Library construction was performed as previously described by Andris-Widhopf et al. al. (2000, PHAGE DISPLAY LABORATORY MANUAL, Barbas et al. (eds.), 1st ed., Cold Spring Harb or Laboratory Press, Cold Spring Harbor, NY pp.9.1-9.22). The final Fab fragments were digested with restriction endonucleases and inserted into a bacteriophage genome to generate a phage display library. Such libraries can be screened by standard phage display methods as known in the art (see, e.g., Pasqualini and Ruoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J. Nucl. Med. 43:159-162).

[0179]

[0198] Phage display can be performed in a variety of formats. For a review thereof, see, e.g., Johnson and Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993). Human antibodies can also be generated by in vitro activated B cells. See U.S. Patent Nos. 5,567,610 and 5,229,275, which are incorporated herein by reference in their entireties. Those skilled in the art will appreciate that these techniques are exemplary and that any known method for making and screening human antibodies or antibody fragments may be utilized.

[0180]

[0199] Another alternative is to use transgenic mice genetically engineered to produce human antibodies. These transgenic animals can be used to produce antibodies against virtually any immunogenic target using standard immunization protocols. Methods for obtaining human antibodies from transgenic mice are described in Green et al., Nature Genet. 7:1 3(1994), Lonberg et al., Nature 368:856(19 94), and Taylor et al., Int. Immun. 6:579 (1994). A non-limiting example of such a system is the XENOMOUSE® from Abgenix (Fremont, Calif.) (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23). ​​In the XENOMOUSE® and similar animals, mouse antibody genes have been inactivated and replaced with functional human antibody genes, while the remainder of the mouse immune system remains intact.

[0181]

[0200] XENOMOUSE® is a mouse model of mouse development, along with accessory genes and regulatory sequences. The XENOMOUSE® was transformed with a germline construct YAC (yeast artificial chromosome) containing a portion of the human IgH and Ig kappa loci encompassing most of the variable region sequences. The human variable region repertoire can be used to generate antibody-producing B cells, which can be processed into hybridomas by known techniques. The XENOMOUSE® immunized with the target antigen produces human antibodies by normal immune response, which can be harvested and / or produced by standard techniques as described above. Various strains of XENOMOUSE® are available, each capable of producing a different class of antibody. Transgenically produced human antibodies have been shown to have therapeutic potential while retaining the pharmacokinetic properties of normal human antibodies (Green et al., 1999). One skilled in the art will appreciate that the claimed compositions and methods are not limited to the use of the XENOMOUSE® system, but may utilize any transgenic animal genetically engineered to produce human antibodies.

[0182] Antibody cloning and production

[0201] Various techniques have been developed to clone antibodies, such as producing chimeric or humanized antibodies. Antigen-binding Vκ (variable light chain) and VH (variable heavy chain) sequences for an antibody of interest can be obtained by a variety of molecular cloning procedures, such as RT-PCR, 5'-RACE, and cDNA library screening. The V genes of the antibody from the cell expressing the mouse antibody can be cloned and sequenced by PCR amplification. To confirm its authenticity, the cloned VL and VH genes can be expressed as chimeric antibodies in cell culture as described by Orlandi et al., (Proc. Natl. Acad. Sci. USA, 86:3833 (1989)). Based on the V gene sequence, humanized antibodies can then be designed and constructed as described by Leung et al. (Mol. Immunol., 32:1413 (1995)).

[0183]

[0202] cDNA was cloned to produce mouse antibodies by standard molecular cloning techniques. It can be prepared from any known hybridoma line or transfected cell line (Sambrook et al., Molecular Cloning, A laboratory manual, 2nd ed. (1989)). Antibody Vκ sequences can be amplified using primers VK1BACK and VK1FOR (Orlandi et al., 1989) or the extension primer set described by Leung et al. (BioTechniques, 15:286 (1993)). VH sequences can be amplified using the primer pair VH1BACK / VH1FOR (Orlandi et al., 1989) or primers annealing to the constant region of mouse IgG described by Leung et al. (Hybridoma, 13:469 (1994)). Humanized V genes can be constructed by a combination of long oligonucleotide template synthesis and PCR amplification, as described by Leung et al. (Mol. Immunol., 32:1413 (1995)).

[0184]

[0203] The PCR product for Vκ was subcloned into a staging vector. The staging vectors can be cloned into a suitable expression vector, such as VKpBR, a pBR327-based staging vector, containing an Ig promoter, a signal peptide sequence and convenient restriction sites. The VH PCR products can be subcloned into a similar staging vector, such as VHpBS, which is based on pBluescript. Expression cassettes containing the Vκ and VH sequences along with the promoter and signal peptide sequences can be excised from VKpBR and VHpBS and ligated into appropriate expression vectors, such as pKh and pG1g, respectively (Leung et al., Hybridoma, 13:469 (1994)). The expression vectors can be co-transfected into suitable cells and the supernatants monitored for the production of chimeric, humanized or human antibodies. Alternatively, the Vκ and VH expression cassettes can be excised and subcloned into a single expression vector, such as pdHL2, as described by Gillie et al. Immunol. Methods 125:191 (1989) and shown in Losman et al., Cancer, 80:2660 (1997).

[0185]

[0204] In other embodiments, the expression vector is transfected in serum-free medium. The Bcl-EEE gene can be transfected into a host cell that is pre-adapted for growth and expression. Representative cell lines that can be used include Sp / EEE, Sp / ESF and Sp / ESF-X cell lines (see, for example, U.S. Patent Nos. 7,531,327; 7,537,930 and 7,608,425; the Examples section of each is incorporated herein by reference). These representative cell lines are based on the Sp2 / 0 myeloma cell line, transfected with a mutant Bcl-EEE gene, exposed to methotrexate to amplify the transfected gene sequence, and pre-adapted to a serum-free cell line for protein expression.

[0186] Antibody fragments

[0205] Antibody fragments that recognize specific epitopes can be generated by known techniques. Antibody fragments are antigen-binding portions of antibodies, such as F(ab')2, Fab', F(ab)2, Fab, Fv, scFv, etc. F(ab')2 fragments can be generated by pepsin digestion of antibody molecules, and Fab' fragments can be generated by reducing disulfide bridges of F(ab')2 fragments. Alternatively, Fab' expression libraries can be constructed (Huse et al., 1989, Science, 246:1274-1281), allowing rapid and easy identification of monoclonal Fab' fragments with the desired specificity. F(ab)2 fragments can be generated by papain digestion of antibodies.

[0187]

[0206] A single chain Fv molecule (scFv) contains a VL domain and a VH domain. The VH domain and VH domain associate to form a target binding site. These two domains are further covalently linked by a peptide linker (L). Methods for making scFv molecules and designing suitable peptide linkers are described in U.S. Patent No. 4,704,692; U.S. Patent No. 4,946,778; Raag and Whitlow, FASEB 9:73-80 (1995) and Bird and Walker, TIBTECH, 9:132-137 (1991).

[0188]

[0207] Techniques for producing single domain antibodies (DABs or VHHs) are also available, e.g. Single domain antibodies are known in the art, for example, as disclosed in Cossins et al. (2006, Prot Express Purif 51:253-259), which is incorporated herein by reference. Single domain antibodies can be obtained, for example, from camels, alpacas or llamas by standard immunization techniques. (See, for example, Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75, 2003; Maass et al., J Immunol Methods 324:13-25, 2007). VHHs can have strong antigen-binding capacity and can interact with novel epitopes that are inaccessible to conventional VH-VL pairs. (Muyldermans et al., J Immunol Methods 324:13-25, 2007). s et al., 2001). Alpaca serum IgG contains approximately 50% camelid heavy chain only IgG antibodies (HCAb) (Maass et al., 2007). Alpacas can be immunized with known antigens such as TNF-α and VHHs that bind and neutralize the target antigen can be isolated (Maass et al., 2007). PCR primers that amplify virtually all alpaca VHH coding sequences have been identified and can be used to construct an alpaca VHH phage display library, which can be used to isolate antibody fragments by standard biopanning techniques well known in the art (Maass et al., 2007). In certain embodiments, anti-pancreatic cancer VHH antibody fragments can be utilized in the claimed compositions and methods.

[0189]

[0208] Antibody fragments can be prepared by proteolytic hydrolysis of a full-length antibody, or Antibody fragments can be prepared by expression in E. coli or other hosts of DNA encoding the fragments. Antibody fragments can be obtained by pepsin or papain digestion of full-length antibodies by conventional methods. These methods are described, for example, in Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein. See also Nisonoff et al., Arch Biochem. Biophys. 89:230 (1960); Porter, Biochem. J. 73:119 (1959); Edelman et al., METHODS IN ENZYMOLOGY VOL.1, p. 422 (Academic Press 1967), and Coligan, p. 2.8.1-2.8.10 and 2.10.-2.10.4.

[0190] bispecific antibody

[0209] Bispecific antibodies have binding specificities for at least two different antigens. It is an antibody. In this case, one of the binding specificities is for a target, for example, CD47 or any fragment thereof. The second binding target is any other antigen, preferably a cell surface protein or receptor or receptor subunit.

[0191]

[0210] Methods for producing bispecific antibodies are known in the art. Recombinant production is based on the simultaneous expression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because the assortment of immunoglobulin heavy and light chains is random, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is usually performed by an affinity chromatography step. Similar procedures are described in WO 93 / 08829, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0192]

[0211] Bispecific antibodies such as kappa-lambda antibodies are disclosed in WO2012 / 023053. The contents of the above patents, which may be made using any of a variety of art recognized techniques, including those set forth herein, are incorporated by reference in their entirety.

[0193]

[0212] In another embodiment of producing bispecific antibodies, antibodies having the desired binding specificities are The variable domain (antibody-antigen binding site) can be linked to an immunoglobulin constant domain sequence. The fusion is preferably with an immunoglobulin heavy chain constant domain, including at least part of the hinge, CH2 and CH3 regions. It is preferred to have the first heavy chain constant region (CH1) containing the site necessary for light chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, optionally, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. For further details on generating bispecific antibodies, see, e.g., Suresh et al., Methods in Enzymology, 121:210 (1986).

[0194]

[0213] In some embodiments, the interface between a pair of antibody molecules in the constructs herein is , can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. A preferred interface encompasses at least a portion of the CH3 region of the IgA antibody constant domain. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory "cavity" of identical or similar size to the large side chain(s) is created on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end-products such as homodimers.

[0195]

[0214] Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. The resulting bispecific antibodies can be used as agents for the selective immobilization of enzymes.

[0196]

[0215] Making bispecific antibody fragments directly from recombinant cell culture Various isolation techniques have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form the antibody heterodimers. This method can also be used for the production of antibody homodimers. Hollinger et al., Proc. Natl. Acad. Sci. USA The "diabody" technology described by Gruber et al., J. Immunol. 90:6444-6448 (1993) provided an alternative mechanism for making bispecific antibody fragments. The fragments contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152:5368 (1994).

[0197]

[0216] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Bispecific antibodies can be used to target cytotoxic agents to cells expressing a particular antigen. These antibodies have an antigen-binding arm and an arm that binds to a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Other bispecific antibodies of interest bind to the protein antigens described herein and also to tissue factor (TF).

[0198]

[0217] Chemical cross-linking of antibody fragments, forced heterodimerization, quadroma technology, Several strategies have been used to generate such bispecific molecules, such as the fusion of antibody fragments via polypeptide linkers and the use of single domain antibodies. The availability of recombinant DNA technology has led to the generation of a large number of bispecific antibody formats (see, for example, Ridgway JB et al., (1996) Protein Eng 9:617-621). Linkers and mutations are often introduced into different regions of the antibody to force the formation of heterodimers or to connect different binding moieties into a single molecule.

[0199] chemical crosslinking

[0218] The use of chemical cross-linking reagents to covalently link two antibodies is conceptually simple. This is an approach in which antibody fragments generated from the respective parent antibodies by enzymatic digestion or by recombinant techniques are conjugated using bifunctional reagents (Glennie MJ et al., J Exp Med 1992;175:217-225). The homogeneity of the product is the main limitation of this approach, since the bispecific species must be purified from homodimers and the modification steps may alter the integrity and stability of the protein.

[0200] Quadroma

[0219] Quadromas and triomas consist of two hybridomas or one Hybridomas can be generated by fusing one of two types of B lymphocytes with (Suresh MR et al., Methods Enzymol 1986;121:210-228). In this case, the simultaneous expression of two heavy and two light chains results in the random assembly of 10 antibody combinations, with the desired bispecific antibody representing only a small fraction of the secreted antibodies. The bispecific antibody must be purified using a combination of chromatographic techniques, dramatically reducing the production yield. The main limitation is that quadromas result in bispecific antibodies of rodent origin, limiting their therapeutic potential due to immunogenicity issues.

[0201] Recombinant Bispecific Antibodies

[0220] The majority of bispecific antibody formats are linked via a polypeptide linker. They can be produced by genetic engineering techniques using antibody fragments such as scFv or Fab fragments as building blocks to be linked. Formats based on linked antibody fragments include tandem scFv (BiTE), diabodies and tandem-diabodies (Kipriyanov SM. Methods Mol Biol 2003; 207: 323-333; Korn T et al., Int J Cancer 2002; 100: 690-697). These building blocks can be further linked to immunoglobulin Fc regions to generate "IgA-like" molecules. These formats include diabody-Fc, tandem diabody-Fc, tandem diabody-CH3, (scFv)4-Fc and DVD-Ig (Lu D et al., J Immunol Methods 2003;279:219-232; Lu D et al., J Biol Chem 2005;280:19665-19672; Lu D et al., J Biol Chem 2004;279:2856-2865;Wu C et al., Nat Biotechnol 2007 25:1290-7).

[0202]

[0221] Strategies based on forcing heterodimerization of the two heavy chains have been explored. The approach, coined "knobs-into-holes," aims to force pairing of two different IgG heavy chains by introducing mutations into the CH3 domain to alter the contact interface (Ridgway JB et al., Protein Eng. 1996;9:617-621). An amino acid with a large side chain was introduced on one chain to create a "knob". Conversely, a bulky amino acid was replaced with an amino acid with a short side chain to create a "hole" in the other CH3 domain. By co-expressing these two heavy chains, more than 90% heterodimer formation ("knob-hole") was observed relative to homodimer formation ("hole-hole" or "knob-knob"). A similar concept was developed using the Strand Exchange Engineered Domain (SEED) human CH3 domain based on human IgG and human IgA sequences (Davis JH et al.,2010,PEDS 23:195-202). These engineered domains lead to the formation of heterodimeric molecules that can have two different specificities.

[0203]

[0222] Recently, an improvement of the “knob-into-hole” approach, the “CrossMa The method is described in WO2009 / 080253 A1. In addition to the "one-hole" mutations, it encompasses several exchanges of light and heavy chain domains.

[0204]

[0223] Single domain-based antibodies. Camelidae (llamas and camels) and cartilaginous fish ( The immune system of the nurse shark (Salmo gairdneri) uses a single V domain fused to Fc, showing that a single domain can confer high affinity binding to an antigen. V domains from camelids, sharks and even humans represent surrogates for antibodies but have also been used to generate bsAbs. These can be reformatted into orthodox IgG, with each arm having the potential to bind two targets via either its VH or VL domain.

[0205]

[0224] The bispecific antibodies of the present disclosure may be any of the antibodies disclosed or disclosed in this application. The nanotubes can be made by any process known in the art. Dual variable domain immunoglobulins

[0225] Bispecific antibodies contain individually encoded peptides or "segments." and which, in a single continuous chain, contain a compact tertiary structure. The component peptides are selected to be asymmetric in their assumed structure so that they do not self-associate to form homomultimers, but rather associate complementarily to adopt a stable complex resembling the parent tertiary structure. At the genetic level, these segments are encoded by interchangeable cassettes with suitable restriction sites. These standardized cassettes are fused C- or N-terminally to different recombinant proteins via linkers or hinges in an appropriate expression vector system. Polypeptide segments that do not have the ability to assemble as homodimers are derived by cleaving the parent polypeptide with a compact tertiary structure. These polypeptide segments can then be fused to one or more different functional domains at the genetic level. These different polypeptide segments, now fused to one or more functional domains, can be, for example, co-expressed, resulting in the formation of a native-like parent structure attached to the functional domain. This parent structure is formed by dimerization of the polypeptide segments derived from the original parent polypeptide. The resulting multifunctional construct appears as a compact tertiary structure attached to one or more functional domains. Once the structural subdomains have been identified, the protein is disassembled in such a way that these subdomains remain intact.As part of the present disclosure, DNA sequences, vectors, preferably bicistronic vectors, vector cassettes can be created, which contain at least one, preferably single, cloning site for inserting a DNA sequence encoding an amino acid sequence and, optionally, at least one further (poly)peptide comprised in the multifunctional polypeptide of the invention, and additionally, a DNA encoding at least one further functional domain, or alternatively, which contain suitable restriction sites for cloning a DNA sequence encoding an amino acid sequence and, optionally, a further (poly)peptide(s) comprised in the multifunctional polypeptide of the invention, and a DNA sequence encoding a functional domain, so that, in a suitable host, the DNA sequence can be expressed after insertion of a DNA sequence encoding a functional domain into said restriction site, resulting in the formation of a multifunctional polypeptide of the invention. Said vector cassettes are characterized in that they contain an inserted DNA sequence(s) encoding said functional domain(s) and a host cell transformed with at least one vector or vector cassette of the invention that can be used for the preparation of said bispecific or multifunctional polypeptide. The host cell can be a mammalian, preferably human, yeast, insect, plant or bacterial, preferably E. coli cell. A bispecific antibody may be prepared by a method comprising culturing in a suitable medium at least two host cells of the invention, wherein each of said host cells produces only one of said first and said second amino acid sequences attached to at least one further functional domain; recovering said amino acid sequences; mixing them under mild denaturing conditions; and allowing in vitro folding of a multifunctional polypeptide of the invention from said amino acid sequences. The method may be characterized in that the further amino acid sequence attached to the at least one further functional domain is produced by at least one further host cell that does not produce said first or second amino acid sequence.Furthermore, the method can be characterized in that at least one further amino acid sequence attached to at least one further functional domain is produced by the host cell of the invention which produces said first or second amino acid sequence.

[0206]

[0226] Either the second or first portion of the antibody construct described herein may be two When the first or second antibody variable domains are included, these two antibody variable domains can be VH and VL domains associated with each other. However, when the second or first antibody variable domains are included, the first and second antibody variable domains can be VH and VL domains associated with each other. It is also contemplated that the two antibody variable domains contained in either of the first or second moieties may be two VH domains or two VL regions associated with each other. When the two antibody variable domains of the first or second moiety are covalently associated with each other, the two antibody variable domains can be designed as scFv fragments, meaning that the two domains are separated from each other by a peptide linker of sufficient length to allow intermolecular association between these two domains. The design of linkers suitable for this purpose is described in the prior art, for example in the granted patents EP623679B1, US5258498, EP573551B1 and US5525491. In other words, a bispecific antibody can be a construct with a total of three antibody variable domains. One of the antibody variable domains alone, i.e., without pairing with the other antibody variable domain, (a) specifically binds to a human immune effector cell by specifically binding to an effector antigen on the human immune effector cell, or to a target cell. Meanwhile, the remaining two antibody variable domains, together, specifically bind (b) to a target antigen on a target cell or to a human immune effector cell by specifically binding to an effector antigen on a human immune effector cell, respectively. In this case, the presence of three antibody variable domains in a bispecific antibody entails unique advantages. Often, an scFv is already known and optimized that exhibits a desired binding specificity to a target antigen, and deleting one of the two antibody variable domains would eliminate or at least weaken its binding properties. Such scFvs can form part of the antibody constructs described herein. In particular, such three-domain antibodies can advantageously comprise the entire scFv, either as its effector antigen-conferring or target antigen-conferring part. Effectively, this then allows a bispecific antibody to be formed starting from a desired scFv by simply incorporating only one additional antibody variable domain into the same polypeptide chain as the scFv, in which the incorporated one additional antibody variable domain has an antigen-binding specificity different from that of the scFv.The first and second parts of the bispecific antibody may be separated from each other by a synthetic polypeptide spacer moiety that covalently (i.e., peptide-bonded) links the C-terminus of the first part to the N-terminus of the second part, or the C-terminus of the second part to the N-terminus of the first part. Thus, these bispecific antibody parts can be arranged as either N-(first part)-(second part)-C or N-(second part)-(first part)-C. In some embodiments, the binding site of the second specificity is fused to the N-terminus or C-terminus of the heavy or light chain, for example in the form of an scFv fragment or a variable single domain, resulting in a bispecific tetravalent molecule. Bispecific molecules generated by fusion of scFv fragments to mAbs exhibit high flexibility. ScFv molecules can be linked to the N-terminus as well as the C-terminus of the heavy or light chain variable domain of the mAb, generally without compromising productivity or antigen binding activity. This group of bispecific molecules also includes DVD-Igs, for which a second VH and VL domain is fused to the heavy and light chains, respectively, of a mAb, two-in-one antibodies in which the second specificity is introduced into the natural binding site of an IgG molecule, and mAb2 molecules in which the second specificity is built into the CH3 domain of the Fc region. A feature of all these molecules is the symmetry resulting from the dimeric assembly of two identical heavy chains, an inherent property of these chains.

[0207]

[0227] Heavy chain heterodimerization can be achieved by engineering the charged CH3 interface to introduce an electrostatic steering effect or by using chain-exchange engineered domain technology (SEEDbody) with a CH3 sequence composed of alternating segments from human IgA and IgG. In contrast to bispecific IgG-like molecules, these bispecific antibodies are bivalent with a size essentially identical to IgG. Fc heterodimerization has recently been applied to generate trivalent bispecific molecules fusing the VH and VL domains to the C-terminus of an engineered heavy chain (HA-TF Fc variant). Bispecific antibodies with molecular masses in the range of 50-100 kDa are characterized by the fact that they are a complex of two antibodies with variable domains. For example, two scFvs are connected in a tandem orientation (tandem scFv, taFv, tascFv) by a more or less flexible peptide linker, which can be further extended by additional scFvs to generate, for example, bispecific or trispecific triple bodies (sctb). Diabodies are composed of VHA-VLB and VH They are heterodimeric molecules composed of two antibody variable domains arranged either in the order B-VLA (VH-VL orientation) or in the order VLA-VHB and VLB-VHA (VL-VH orientation). The linker connecting the two domains in one chain is about 5 residues and leads to a head-to-tail assembly after co-expression of the two chains in one cell, thus forming a compact molecule with two functional binding sites. The diabody (Db) format was further stabilized by introducing an interchain disulfide bond (dsDb, DART molecule) or by generating single chain derivatives (scDb). ScDb can be converted into a tetravalent molecule by reducing the intermediate linker, resulting in homodimerization of the two chains. Small bispecific molecules have also been produced by fusing scFv to the heavy or light chains of Fab fragments. Additionally, tandem scFvs, diabodies and scDbs have been fused to Fc or CH3 domains to generate tetravalent derivatives. Also, scFvs can be combined with Fc or CH3 domains to generate tetravalent molecules, for example, by fusing scFvs to the N- and C-termini of an Fc fragment, or by using a knob-into-hole approach to generate bivalent scFv-Fc or scFv-CH3 molecules. A different approach to generate bispecific antibodies of the invention is the dock-and-lock method (DNL), where antibody fragments bind to human cAMP-dependent It has been fused to a homodimerization docking domain (DDD) from protein kinase A (PKA) and an anchoring domain (AD) from A kinase anchor protein (AKAP), resulting in the formation of a bispecific trivalent molecule. Many of the established bispecific antibody formats can also be combined with additional proteins and components, such as drugs, toxins, enzymes and cytokines, to allow dual targeting and delivery of the fusion partners. Furthermore, fusion to plasma proteins such as serum albumin or albumin binding moieties can be applied to extend the plasma half-life of the bispecific antibody. Structure of a bispecific antibody

[0228] In one example, a bispecific antibody comprises a binding protein comprising a first polypeptide chain. In some embodiments, the VD-H1 and VD-H2 in the binding protein can be heavy chain variable domains selected from the group consisting of mouse heavy chain variable domains, human heavy chain variable domains, CDR-grafted heavy chain variable domains, and humanized heavy chain variable domains. VD-H1 and VD-H2 can potentially bind to different antigens. C can be a heavy chain constant domain. For example, X1 is a linker peptide. For example, X1 is a linker as listed herein. In one embodiment, X2 is an IgA Fc region. In other embodiments, X2 is a variant IgA Fc region. In some embodiments, VD-H1 is capable of binding to CD47 and VD-H2 is capable of binding to an antigen. In some embodiments, VD-H1 is capable of binding to an antigen and VD-H2 is capable of binding to CD47.

[0208]

[0229] In one example, a bispecific antibody comprises a binding protein comprising a second polypeptide chain. and in this regard, the polypeptide chain can be of the structure VD-L1-(X1)n-VD-L2-C-(X2)n, where VD-L1 is a first light chain variable domain, VD-L2 is a second light chain variable domain, C is a constant domain, and X1 is a polypeptide linker. X1 represents an IgA Fc region, and n is 0 or 1. In some embodiments, VD-L1 and VD-L2 in the binding protein can be light chain variable domains selected from the group consisting of mouse light chain variable domain, human light chain variable domain, CDR-grafted light chain variable domain, and humanized light chain variable domain. VD-L1 and VD-L2 can be capable of binding different antigens. C can be a heavy chain constant domain. For example, X1 is a linker peptide. For example, X1 is a linker as listed herein. In one embodiment, X2 is an IgA Fc region. In another embodiment, X2 is a variant IgA Fc region. In some embodiments, VD-L1 can bind CD47 and VD-L2 can bind an antigen. In some embodiments, VD-L1 can bind an antigen and VD-L2 can bind CD47. In some embodiments, the bispecific antibody construct comprises both a first polypeptide chain and a second polypeptide chain. The bispecific antibodies of the present disclosure may be dual variable domain immunoglobulins (DVD-Ig) as described in Jakob 2013. TM ), which combines the target-binding domains of two monoclonal antibodies via a flexible, naturally occurring linker, resulting in a tetravalent IgG-like molecule.

[0209]

[0230] The present invention further provides a parent antibody to CD47 and a desired antigen (e.g., CD20). The present invention provides a method for producing a DVD-Ig binding protein by preselecting a first variable domain of an IgA polypeptide chain, which is a first parent antibody or an antigen-binding portion thereof, and a second parent antibody or an antigen-binding portion thereof. The method for producing a dual variable domain immunoglobulin that binds two antigens includes the steps of: a) obtaining a first parent antibody or an antigen-binding portion thereof that binds a first antigen; b) obtaining a second parent antibody or an antigen-binding portion thereof that binds a second antigen; c) constructing two copies of a first polypeptide chain, each of which is represented by the formula: VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first heavy chain variable domain obtained from said first parent antibody or an antigen-binding portion thereof; VD2 is a second heavy chain variable domain obtained from said second parent antibody or an antigen-binding portion thereof, which second parent antibody can be the same or different from the first parent antibody; C is a heavy chain constant domain; (X1)n is a linker, said (X1)n being either present or absent; and (X2)n is an IgA polypeptide chain. and (X2)n does not comprise an IgA Fc region, and wherein the (X2)n is either present or absent; and e) expressing two copies of the first and second polypeptide chains, resulting in a DVD-Ig that binds to the first antigen (e.g., CD47) and the second antigen (e.g., CD20).

[0210] Generation of antigen-binding domains and / or CD47-binding domains:

[0231] The variable domains of the DVD binding proteins are polyclonal antibodies that bind to the antigen of interest. MAbs can be derived from parent antibodies, such as monoclonal and mAbs. These antibodies can be naturally occurring, or generated by recombinant technology, or designed de novo. MAbs can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. Monoclonal antibodies can be prepared by the methods disclosed herein.

[0211]

[0232] Dual variable domain immunoglobulin (DVD-Ig) molecules are composed of two different parent models. Two different light chain variable domains (VL) from monoclonal antibodies are directly linked in tandem. , or are designed to be linked via a short linker by recombinant DNA technology, followed by a light chain constant domain and, optionally, an Fc region. Similarly, the heavy chain comprises two different heavy chain variable domains (VH) linked in tandem, followed by the constant domain CH1 and an Fc region. The variable domains can be obtained using recombinant DNA technology from a parent antibody generated by any one of the methods described herein. The variable domains can be murine heavy or light chain variable domains, CDRs, human heavy or light chain variable domains. The first and second variable domains can be directly linked to each other using recombinant DNA technology, can be linked via a linker sequence, or the two variable domains are linked. The variable domains can bind to the same antigen or can bind to different antigens. The constant domains can be linked to the two linked variable domains using recombinant DNA technology. A sequence containing linked heavy chain variable domains can be linked to a heavy chain constant domain, and a sequence containing linked light chain variable domains is linked to a light chain constant domain. The constant domains can also be human heavy chain constant domains and human light chain constant domains, respectively. The DVD heavy chain can be further linked to an IgA Fc region. The Fc region can be a native sequence Fc region, or a variant Fc region, or a human Fc region, or an Fc region from IgA1, IgA2. Two heavy chain DVD polypeptides and two light chain DVD polypeptides can be combined to form a DVD-Ig molecule.

[0212]

[0233] The design of the "dual-specific, multivalent, full length binding proteins" of the present invention primarily results in dual variable domain light chains and dual variable domain heavy chains assembling into the desired "dual-specific, multivalent, full length binding protein."

[0213] Building the DVD molecule

[0234] Dual variable domain immunoglobulin (DVD-Ig) molecules are identical but distinct The heavy chain is designed such that two different light chain variable domains (VL) from two parent monoclonal antibodies, which may be either directly linked in tandem or linked via a short linker by recombinant DNA techniques, followed by a light chain constant domain and, optionally, an IgA Fc region. Similarly, the heavy chain comprises two different heavy chain variable domains (VH) linked in tandem, followed by the constant domain CH1 and an IgA Fc region.

[0214]

[0235] The variable domain may be a parent gene produced by any one of the methods described herein. The antibody can be obtained by recombinant DNA technology. In one embodiment, the variable domain is a mouse heavy or light chain variable domain. In another embodiment, the variable domain is a CDR-grafted or humanized variable heavy or light chain domain. In one embodiment, the variable domain is a human heavy or light chain variable domain.

[0215]

[0236] In one embodiment, the first and second variable domains are produced using recombinant DNA techniques. In one embodiment, the variable domains are linked directly to each other via a linker sequence. In another embodiment, the variable domains are linked via a linker sequence. In one embodiment, two variable domains are linked. Three or more variable domains can also be linked directly or via a linker sequence. The variable domains can bind to the same antigen or can bind to different antigens. The DVD-Ig molecules of the present invention can include one immunoglobulin variable domain and one non-immunoglobulin variable domain, e.g., a ligand binding domain of a receptor or an active domain of an enzyme. The DVD-Ig molecules can also include two or more non-Ig domains.

[0216]

[0237] In one embodiment, the constant domain is constructed using recombinant DNA techniques. In one embodiment, the sequence comprising the linked heavy chain variable domains is linked to a heavy chain constant domain and the sequence comprising the linked light chain variable domains is linked to a light chain constant domain. In one embodiment, the constant domains are each linked to a human heavy chain constant domain. The DVD heavy chain is further linked to an Fc region. The Fc region can be a native sequence Fc region, or a variant Fc region. In other embodiments, the Fc region is a human Fc region. In other embodiments, the Fc region includes an Fc region from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

[0217]

[0238] In another embodiment, two heavy chain DVD polypeptides and two light chain DVD polypeptides. The peptides are combined to form the DVD-Ig molecule.

[0239] The binding proteins of the invention can be isolated by any of several techniques known in the art. For example, expression from a host cell, in which the expression vector(s) encoding the DVD heavy and DVD light chains are transfected into the host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although it is possible to express the DVD proteins of the present invention in either prokaryotic or eukaryotic host cells, the DVD proteins are expressed in eukaryotic cells, such as mammalian host cells, because such eukaryotic cells (and particularly mammalian cells) are more likely than prokaryotic cells to assemble and secrete properly folded, immunologically active DVD proteins.

[0218]

[0240] Representative mammalian host cells for expressing the recombinant antibodies of the present invention include Examples of suitable cells include Indian hamster ovary (CHO) cells (e.g., dhfr-CHO cells, as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, e.g., with a DHFR selectable marker as described in Kaufman, RJ and Sharp, PA (1982) Mol. Biol. 159:601-621), NS0 myeloma cells, COS cells, SP2 and PER.C6 cells. When a recombinant expression vector encoding a DVD protein is introduced into a mammalian host cell, the DVD protein is produced by culturing the host cell for a period of time sufficient to allow expression of the DVD protein within the host cell or secretion of the DVD protein into the medium in which the host cell is grown. The DVD protein can be recovered from the culture medium using standard protein purification methods.

[0219]

[0241] For recombinant expression of DVD proteins in the constructs described herein In a representative system, a recombinant expression vector encoding both the DVD heavy chain and the DVD light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the DVD heavy and light chain genes are each operatively linked to a CMV enhancer / AdMLP promoter regulatory element. The recombinant expression vector also carries a DHFR gene, which allows for the selection of CHO cells transfected with the vector using methotrexate selection / amplification. The selected transformed host cells are cultured to allow expression of the DVD heavy and light chains, and the intact DVD protein is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the DVD protein from the culture medium. Additionally, the present invention provides a method for synthesizing the DVD protein of the present invention by culturing the host cells of the present invention in an appropriate culture medium until the DVD protein of the present invention is synthesized. The method can further include isolating the DVD protein from the culture medium.

[0220]

[0242] A key feature of DVD-Ig is that it can be produced and purified in a similar manner to conventional antibodies. The production of DVD-Ig can be achieved by altering the sequence of the constant region or any kind of This results in a homogenous single primary product with the desired dual specific activity without chemical modification of the .Other methods for generating "bispecific", "multispecific" and "multispecific multivalent" full length binding proteins described above do not result in a single primary product, but instead result in the intracellular or secretory production of a mixture of assembled inactive proteins, monospecific proteins, multispecific proteins, multivalent proteins, full length binding proteins, and multivalent full length binding proteins with different combinations of binding sites.As an example, based on the design described by Miller and Presta (PCT Publication No. WO2001 / 077342(A1)), there are 16 possible combinations of heavy and light chains.As a result, only 6.25% of the protein may be in the desired active form, rather than as a single major or single primary product, compared to the other 15 possible combinations.Separation of the desired fully active form of the protein from the inactive and partially active forms of the protein using standard chromatographic techniques typically used in large scale manufacturing has not yet been demonstrated.

[0221]

[0243] "Dual specific, multivalent, full length binding proteins" for use in the constructs described herein The design of these "antibody antibodies" primarily results in dual variable domain light chains and dual variable domain heavy chains that assemble into the desired "bispecific, multivalent, full-length binding protein."

[0222]

[0244] At least one of the assembled and expressed dual variable domain immunoglobulin molecules At least 50%, at least 75%, and at least 90% are the desired dual specific tetravalent protein. This aspect of the invention, among other things, enhances the commercial utility of the invention. Thus, the invention encompasses methods of expressing a dual variable domain light chain and a dual variable domain heavy chain in a single cell to result in a single primary product of a "dual specific tetravalent full length binding protein."

[0223]

[0245] As used herein, the dual variable domain light chain and the dual variable domain heavy chain are isolated from a single cell. in a "primary product" of a "dual specific tetravalent full length binding protein," wherein the "primary product" is greater than 50% of all assembled protein, including the dual variable domain light chain and the dual variable domain heavy chain.

[0224]

[0246] As used herein, the dual variable domain light chain and the dual variable domain heavy chain are isolated from a single cell. in a "primary product" of a "dual specific tetravalent full length binding protein," wherein the "primary product" is greater than 75% of all assembled protein, including the dual variable domain light chain and the dual variable domain heavy chain.

[0225]

[0247] As used herein, the dual variable domain light chain and the dual variable domain heavy chain are isolated from a single cell. in a "primary product" of a "dual specific tetravalent full length binding protein," wherein the "primary product" is greater than 90% of all assembled protein, including the dual variable domain light chain and the dual variable domain heavy chain.

[0226] Kappa lambda

[0248] In some embodiments, bispecific antibodies in a kappa-lambda antibody format Provided herein are bispecific antibodies. The bispecific antibodies provided herein have a common heavy chain, two light chains - one kappa (K), one lambda (λ) -, each of the latter having a different specificity (i.e., two light chains, two specificities). The methods provided herein produce molecules with specific binding in which diversity is restricted to the VL region. These methods produce bispecific antibodies through the controlled co-expression of three chains (one VH chain, two VL chains) and purification of the bispecific antibodies.

[0227]

[0249] This type of molecule contains two copies of a unique heavy chain polypeptide, the constant kappa The bispecific antibody of the present invention is composed of a first light chain variable region fused to a constant lambda domain, and a second light chain variable region fused to a constant lambda domain. Each binding site exhibits a different antigen specificity, to which both the heavy and light chains contribute. The light chain variable region can be of the lambda or kappa family, and is preferably fused to lambda and kappa constant domains, respectively. This is preferred to avoid the generation of non-natural polypeptide junctions. However, it is also possible to obtain the bispecific antibody of the present invention by fusing a kappa light chain variable domain to a constant lambda domain for the first specificity, and a lambda light chain variable domain to a constant kappa domain for the second specificity.

[0228]

[0250] An essential step of the method is to develop a method for the preparation of antibodies that share the same heavy chain variable domain but have different antigen specificities. The identification of two antibody Fv regions (each composed of a variable light chain and a variable heavy chain domain) that bind to each other. Many methods have been described for the production of monoclonal antibodies and their fragments. (See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Press, 1999). (See, Harbor Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by reference.) A fully human antibody is an antibody molecule in which both the light and heavy chain sequences, including CDR1 and 2, are derived from human genes. The CDR3 region can be of human origin or engineered by synthetic means. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies." Human monoclonal antibodies can be produced using a variety of techniques, including trioma technology; human B-cell hybridoma technology (see Kozbor et al., 1983 Immunol Today 4:72); and EBV hybridoma technology for producing human monoclonal antibodies (Cole et al., 1983 Immunol Today 4:72). Human monoclonal antibodies are available, which can be produced by using human hybridomas (see Cote et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (see Cole et al., 1985: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pages 77-96).

[0229]

[0251] Monoclonal antibodies can be used to identify, for example, a target antigen or an immunogenic fragment thereof, The derivative or variant is produced by immunizing animals. Alternatively, animals are immunized with cells transfected with vectors containing nucleic acid molecules encoding target antigens, so that the target antigens are expressed and associated with the surface of transfected cells. Various techniques for producing xenogeneic non-human animals are well known in the art. For example, see U.S. Patent Nos. 6,075,181 and 6,150,584. These are incorporated herein by reference in their entirety.

[0230]

[0252] Alternatively, the antibody may be a polypeptide having an antibody or antigen-binding domain sequence for binding to a target antigen. These sequences are obtained by screening libraries containing sequences that have been prepared, for example, in bacteriophage as protein or peptide fusions with bacteriophage coat proteins that are expressed on the surface of assembled phage particles, with the coding DNA sequences contained within the phage particle (i.e., "phage display libraries").

[0231]

[0253] The hybridomas obtained from the myeloma / B cell fusion were then subjected to immunoreaction against the target antigen. Monoclonal antibodies are screened for reactivity to the antibody, as described, for example, by Kohler and Milstein, Nature, 256:495 (1975). The hybridoma is prepared using a hybridoma method such as that described herein. In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0232]

[0254] Kappa-lambda antibodies have the same heavy chain variable domains. Bispecific antibodies of the present invention can be generated by using an antibody library in which the light chain variable domain is the same for all library members and thus diversity is limited to the light chain variable domain. Such libraries are described, for example, in WO2010 / 135558. However, since the light chain variable domain is expressed in association with the heavy chain variable domain, both domains can contribute to antigen binding. To further facilitate this process, antibody libraries containing the same heavy chain variable domain and either diverse lambda or kappa variable light chains can be used in parallel for in vitro selection of antibodies against different antigens. This approach allows the identification of two antibodies that have a common heavy chain, but one with a lambda light chain variable domain and the other with a kappa light chain variable domain that can be used as a building block for the generation of bispecific antibodies in the complete immunoglobulin format of the present invention. The bispecific antibodies of the present invention can be of IgA isotype and their Fc parts can be modified to alter the binding properties to different Fc receptors, thus modifying the effector functions of the antibody as well as its pharmacokinetic properties. A number of methods for modification of the Fc portion have been described and can be applied to the antibodies of the invention (see, e.g., Strohl, WR Curr Opin Biotechnol 2009(6):685-91).

[0233]

[0255] Another important step of the present invention is the synthesis of a common heavy chain and two different light chains into a single cell. to allow for the assembly of the bispecific antibodies of the invention. If all polypeptides are expressed at the same level and assemble equally well to form immunoglobulin molecules, the ratio of monospecific (same light chain) and bispecific (two different light chains) should be 50%.

[0234]

[0256] Co-expression of a heavy chain and two light chains resulted in the production of three different antibodies in the cell culture supernatant. , i.e., a mixture of two monospecific bivalent antibodies and one bispecific bivalent antibody is generated. The latter must be purified from the mixture to obtain the molecule of interest. The method described herein significantly facilitates this purification procedure by the use of affinity chromatography media that specifically interact with kappa or lambda light chain constant domains, such as CaptureSelect Fab Kappa and CaptureSelect Fab Lambda affinity matrices (BAC BV, The Netherlands). This multi-step affinity chromatography purification approach is efficient and generally applicable to the antibodies of the invention. This is in clear contrast to specific purification methods that must be developed and optimized for each bispecific antibody derived from a quadroma or other cell line expressing the antibody mixture. Indeed, if the biochemical properties of the different antibodies in the mixture are similar, separation using standard chromatographic techniques, such as ion exchange chromatography, may be difficult or even impossible altogether.

[0235]

[0257] The simultaneous expression of three chains allows the production of three different antibodies, i.e. two monospecific antibodies and resulted in the assembly of one bispecific antibody. Their theoretical relative ratio should be 1:1:2, provided that the expression levels and assembly rates are similar for both light chains. The bispecific antibodies were purified using a three-step affinity chromatography procedure: (1) Protein A: captures IgA (mono- and bi-), (2) Kappa select: captures IgA containing kappa light chains (one or more), and (3) Lambda select: captures IgG containing lambda light chains. Kappa select and Lambdaselect are affinity chromatography media developed by BAC, BV and GE Healthcare.

[0236]

[0258] The purified bispecific antibodies were characterized as follows: Flow-through and elution from the purification step were analyzed by SDS-PAGE. The specificity and affinity of the κλ-bodies were determined by ELISA and surface plasmon resonance. The method of the present invention allows the identification of antibodies with affinities in the sub-nanomolar to nanomolar range without optimization. This is not evident since the diversity in the antibody libraries described herein is restricted to the light chains, which contribute little to the binding energy in standard antibodies.

[0237]

[0259] Access to two antibodies with kappa and lambda light chain variable domains To avoid the need for a constant domain, which is perceived as a limitation to the present invention, the methods described herein allow for the generation of hybrid light chains in which a lambda variable domain can be fused to a kappa constant domain and vice versa. In some embodiments, the methods for the production of bispecific and / or multispecific antibodies use a completely serum-free and chemically defined process. These methods incorporate the most widely used mammalian cell line in the pharmaceutical industry, the Chinese Hamster Ovary (CHO) cell line. The methods described therein are used to generate both semi-stable and stable cell lines. The methods can be used to produce the bispecific and / or multispecific antibodies of the invention on a small scale (e.g., in Erlenmeyer flasks) and medium scale (e.g., in 25L Wave bags). The methods are also easily applicable to larger scale production of bispecific and / or multispecific antibodies as well as antibody mixtures of the invention.

[0238] Representative antibody constructs

[0260] In some embodiments, the CD47 binding region comprises: (a) the amino acid sequence of SEQ ID NO:4 (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:5; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:6; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:6.

[0239]

[0261] In some embodiments, the CD47 binding region comprises: (a) the amino acid sequence of SEQ ID NO:7 (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 8; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 9; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 9.

[0240]

[0262] In some embodiments, the CD47 binding region comprises: (a) amino acid sequence of SEQ ID NO: 13 and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15.

[0241]

[0263] In some embodiments, the CD47 binding region comprises: (a) the amino acid sequence of SEQ ID NO: 16 and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:18.

[0242]

[0264] In some embodiments, the CD47 binding region comprises: (a) the amino acid sequence of SEQ ID NO:4 (b) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:5; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:6; and a first heavy chain variable domain comprising at least one, two, or three CDRs selected from (a) the amino acid sequence of SEQ ID NO:13. (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15; and (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16.

[0243]

[0265] In some embodiments, the CD47 binding region comprises: (a) the amino acid sequence of SEQ ID NO:7 (b) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 8; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 9; and a first light chain variable domain comprising at least one, two, or three CDRs selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18.

[0244]

[0266] In some embodiments, the CD47 binding region comprises the amino acid sequence of SEQ ID NO:23. In some embodiments, the antibody comprises a first heavy chain variable sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to

[0245]

[0267] In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 23. The first heavy chain variable sequence having 95%, 96%, 97%, 98%, or 99% identity to the reference sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody or antigen-binding fragment thereof comprising the sequence retains the ability to bind to an antigen. In some embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 23. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises a VH sequence of the amino acid sequence of SEQ ID NO: 23, including post-translational modifications of the sequence.

[0246]

[0268] In some embodiments, the CD47 binding region comprises the amino acid sequence of SEQ ID NO:26 The antibody or antigen-binding fragment thereof comprises a first light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 26. In some embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antibody or antigen-binding fragment thereof comprising the sequence retains the ability to bind to an antigen. In some embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in any one of the amino acid sequences of SEQ ID NO: 26. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises the VL sequence of SEQ ID NO: 26, including post-translational modifications of the sequence.

[0247]

[0269] In some embodiments, the CD47 binding region comprises the first heavy chain polypeptide of SEQ ID NO:23. The first light chain variable domain amino acid sequence in SEQ ID NO: 26, including the variable domain amino acid sequence and post-translational modifications of the sequence.

[0248]

[0270] In some embodiments, the CD47 binding region comprises the amino acid sequence of SEQ ID NO:24. In some embodiments, the antibody comprises a first heavy chain variable sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to

[0249]

[0271] In some embodiments, at least 90%, 91%, 92%, 93%, 94%, %, 95%, 96%, 97%, 98%, or 99% identity to the first heavy chain variable sequence, but contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence. In some embodiments, an antibody or antigen-binding fragment thereof comprising said sequence retains the ability to bind to an antigen. In some embodiments, a total of 1-10 amino acids are substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 24. In some embodiments, the substitutions, insertions or deletions occur in regions outside the CDRs (e.g., in the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises a VH sequence of the amino acid sequence of SEQ ID NO: 27, including post-translational modifications of the sequence.

[0250]

[0272] In some embodiments, the CD47 binding region comprises the amino acid sequence of SEQ ID NO:27 27. In some embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 27. In some embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antibody or antigen-binding fragment thereof comprising the sequence retains the ability to bind to the antigen. In some embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in any one of the amino acid sequences of SEQ ID NO: 27. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises the VL sequence of SEQ ID NO: 27, including post-translational modifications of the sequence.

[0251]

[0273] In some embodiments, the CD47 binding region comprises the first heavy chain polypeptide of SEQ ID NO:24. The first light chain variable domain amino acid sequence in SEQ ID NO:27, including the variable domain amino acid sequence and post-translational modifications of the sequence.

[0252]

[0274] In some embodiments, the antigen-binding region comprises: (a) the amino acid sequence of SEQ ID NO:1 (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:4; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:5.

[0253]

[0275] In some embodiments, the antigen-binding region comprises: (a) the amino acid sequence of SEQ ID NO: 10 (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:11; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:12; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:13.

[0254]

[0276] In some embodiments, the antigen-binding region comprises: (a) the amino acid sequence of SEQ ID NO:1 (b) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:2; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; and a second first light chain variable domain comprising at least one, two, or three CDRs selected from (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:10; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:11; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:12.

[0255]

[0277] In some embodiments, the antigen binding region comprises a second heavy chain variable domain.

[0278] In some embodiments, the antigen-binding region corresponds to the amino acid sequence of SEQ ID NO:22. In some embodiments, the antibody comprises a second heavy chain variable sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the first heavy chain variable sequence.

[0256]

[0279] In some embodiments, at least 90%, 91%, 92%, 93%, 94%, %, 95%, 96%, 97%, 98%, or 99% identity to the reference sequence, but contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence. In some embodiments, an antibody or antigen-binding fragment thereof comprising said sequence retains the ability to bind to an antigen. In some embodiments, a total of 1-10 amino acids are substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 22. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises a VH sequence of the amino acid sequence of SEQ ID NO: 22, including post-translational modifications of the sequence.

[0257]

[0280] In some embodiments, the antigen-binding region corresponds to the amino acid sequence of SEQ ID NO:25. The second light chain variable domain (VL) has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference sequence. In some embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antibody or antigen-binding fragment thereof comprising the sequence retains the ability to bind to the antigen. In some embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in any one of the amino acid sequences of SEQ ID NO: 25. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs). Optionally, the antibody or antigen-binding fragment thereof comprises the VL sequence of SEQ ID NO: 25, including post-translational modifications of the sequence.

[0258]

[0281] In some embodiments, the antigen binding region comprises the second heavy chain variable domain of SEQ ID NO: 22. It comprises the main amino acid sequence and the second light chain variable domain amino acid sequence in SEQ ID NO: 25, including post-translational modifications of the sequence.

[0259]

[0282] In some embodiments, the CD47 binding region comprises the CDRs from Table A and is Described herein are constructs in which the binding regions comprise the CDRs from Table B. These CDRs and binding regions can be substituted with others known in the literature to achieve the desired binding profile for recruiting immune effector cells to target antigen presenting cells.

[0260] Representative DVD Ig antibodies

[0283] In some embodiments, the antibody construct comprises a C-terminus of the second heavy chain variable domain The polypeptide includes a polypeptide linked to the N-terminus of the first heavy chain variable domain. In some embodiments, the linkage is via a linker peptide. In some embodiments, the linker peptide includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the polypeptide in which the C-terminus of the second heavy chain variable domain is linked to the N-terminus of the first heavy chain variable domain includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the C-terminus of the polypeptide is linked to an amino acid sequence encoding an IgA constant heavy chain.

[0261]

[0284] In some embodiments, the antibody construct comprises the C-terminus of the second light chain variable domain The polypeptide includes a polypeptide linked to the N-terminus of the first light chain variable domain. In some embodiments, the linkage is via a linker peptide. In some embodiments, the linker peptide includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:44. In some embodiments, the polypeptide in which the C-terminus of the second light chain variable domain is linked to the N-terminus of the first light chain variable domain includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:31. In some embodiments, the polypeptide The C-terminus of is linked to an amino acid sequence encoding the IgA constant heavy chain.

[0262]

[0285] In some embodiments, the bispecific antibody has the amino acid sequence of SEQ ID NO:29. and a second polypeptide comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:31.

[0263]

[0286] In some embodiments, the bispecific antibody comprises a first heavy chain variable domain having a C-terminus The polypeptide comprises a first polypeptide comprising a C-terminus of a first heavy chain variable domain linked to the N-terminus of a second heavy chain variable domain. In some embodiments, the linkage is via a linker peptide. In some embodiments, the linker peptide comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the polypeptide comprising a C-terminus of a first heavy chain variable domain linked to the N-terminus of a second heavy chain variable domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the C-terminus of the polypeptide is linked to an amino acid sequence encoding an IgA constant heavy chain.

[0264]

[0287] In some embodiments, the bispecific antibody comprises a first light chain variable domain C-terminal The polypeptide comprises a first polypeptide comprising a C-terminus of a first light chain variable domain linked to the N-terminus of a second light chain variable domain. In some embodiments, the linkage is via a linker peptide. In some embodiments, the linker peptide comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the polypeptide comprising a C-terminus of a first light chain variable domain linked to the N-terminus of a second light chain variable domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the C-terminus of the polypeptide is linked to an amino acid sequence encoding an IgA constant heavy chain.

[0265]

[0288] In some embodiments, the bispecific antibody has the amino acid sequence of SEQ ID NO: 30. and a second polypeptide comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:32.

[0266]

[0289] In some embodiments, the antibody construct is linked to a light or heavy chain variable domain. In some embodiments, the linker peptide comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:44 or SEQ ID NO:45.

[0267]

[0290] In some embodiments, the scFv comprises a second heavy chain variable domain and a second In some embodiments, the scFv comprises a light chain variable domain and is linked to a first light chain variable domain. In some embodiments, the scFv comprises a light chain variable domain and is linked to a first light chain variable domain (e.g., a second heavy chain variable domain). The first polypeptide comprising a first light chain variable domain and a second light chain variable domain comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:35.

[0268]

[0291] In some embodiments, the scFv comprises a second heavy chain variable domain and a second In some embodiments, the scFv comprises a light chain variable domain and is linked to a first heavy chain variable domain. In some embodiments, the scFv comprises a light chain variable domain and is linked to a first heavy chain variable domain (e.g., a second heavy chain variable domain). The first polypeptide comprising a first light chain variable domain and a second light chain variable domain comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:33.

[0269]

[0292] In some embodiments, the scFv comprises a first heavy chain variable domain and a first In some embodiments, the scFv comprises a light chain variable domain linked to a second light chain variable domain. In some embodiments, the scFv comprises a light chain variable domain linked to a second light chain variable domain (e.g., a first heavy chain variable domain). The first polypeptide comprising a first light chain variable domain and a first light chain variable domain comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:36.

[0270]

[0293] In some embodiments, the scFv comprises a first heavy chain variable domain and a first In some embodiments, the first polypeptide comprising the scFv (e.g., comprising a first heavy chain variable domain and a first light chain variable domain) linked to the second heavy chain variable domain comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:34.

[0271] Representative kappa-lambda complexes

[0294] In some embodiments, the antibody construct (e.g., kappa-lambda) comprises In some embodiments, the antibody construct (e.g., kappa-lambda body) comprises a first light chain variable domain of kappa type and a second light chain variable domain of lambda type. In some embodiments, the antibody construct (e.g., kappa-lambda body) comprises a first light chain variable domain of lambda type and a second light chain variable domain of lambda type. In some embodiments, the antibody construct (e.g., kappa-lambda body) comprises a first light chain variable domain of lambda type and a second light chain variable domain of lambda type. In some embodiments, the CD47 binding region comprises a first light chain variable domain comprising at least one, two, or three CDRs selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the CD47 binding region comprises a first light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:27.

[0272]

[0295] In some embodiments, the antigen binding domain comprises: (a) the amino acid sequence of SEQ ID NO: 19 and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, the antigen binding domain comprises a first light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 28.

[0273]

[0296] In some embodiments, the first light chain variable domain further comprises a light chain constant domain. In some embodiments, the second light chain variable domain further comprises a light chain constant domain. In some embodiments, the light chain constant domain is of the kappa type. In some embodiments, the light chain constant domain is of the lambda type. In some embodiments, the light chain constant domain is of the kappa type comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 42. In some embodiments, the light chain constant domain is of the lambda type comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 43.

[0274]

[0297] In some embodiments, the antibody construct comprises a first heavy chain variable domain and a second heavy chain variable domain. In some embodiments, the first heavy chain variable domain and the second heavy chain variable domain comprise at least one, two, or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:7; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:8; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:9. In some embodiments, the first heavy chain variable domain and the second heavy chain variable domain comprise a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:24.

[0275]

[0298] In some embodiments, the antibody constructs herein comprise an IgA heavy chain constant region. In some embodiments, the IgA heavy chain constant region comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 37. In some embodiments, the IgA heavy chain constant region comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the IgA heavy chain constant region comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 39. In some embodiments, the IgA heavy chain constant region comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the IgA heavy chain constant region comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:41.

[0276] IgA constant region

[0299] In one embodiment, the bispecific IgA antibody of the present disclosure comprises a complete IgA heavy chain constant domain. In some embodiments, the IgA heavy chain constant domain contains one or more modifications to create an asymmetric interface between the two heavy chains.

[0277]

[0300] The term "asymmetric interface" refers to an interface between two IgA heavy chain constant regions, such as the first and second IgA heavy chain constant regions. It is used to refer to the interface (as defined above) formed between antibody chains and / or between an IgA heavy chain constant region and its matching light chain, in which the contact residues in the first and second chains differ by design and include complementary contact residues. The asymmetric interface can be created by knob / hole interactions and / or salt bridge coupling (charge swap) and / or other techniques known in the art, such as the CrossMab approach to link a heavy chain to its matching light chain. A "cavity" or "hole" refers to at least one amino acid side chain that is recessed from the interface of a second polypeptide, thereby accommodating a corresponding protrusion ("knob") on the adjacent interface of a first polypeptide. A cavity (hole) is a The "import" amino acid residues ("original" amino acid residues) can be present in the original interface or can be synthetically introduced (e.g., by modifying the nucleic acid encoding the interface). Usually, the nucleic acid encoding the interface of the second polypeptide is modified to encode the cavity. To achieve this, the nucleic acid encoding at least one "original" amino acid residue in the interface of the second polypeptide is replaced with DNA encoding at least one "import" amino acid residue having a smaller side chain volume than the original amino acid residue. It will be understood that there may be more than one original residue and the corresponding import residue. The upper limit for the number of original residues to be replaced is the total number of residues in the interface of the second polypeptide. The import residues preferred for forming the cavity are usually naturally occurring amino acid residues, preferably selected from alanine (A), serine (S), threonine (T), valine (V) and glycine (G). The most preferred amino acid residues are serine, alanine or threonine, most preferably alanine. In preferred embodiments, the original residue for formation of the protrusion has a large side chain volume, such as tyrosine (Y), arginine (R), phenylalanine (F) or tryptophan (W).

[0278]

[0301] The "original" amino acid residue has a smaller or larger side chain volume than the original residue. You can import *The imported amino acid residue can be a naturally occurring or a non-naturally occurring amino acid residue, preferably the former.

[0279]

[0302] A "non-naturally occurring" amino acid residue is one that is not encoded by the genetic code. By non-naturally occurring amino acid residues is meant a residue that can be covalently linked to adjacent amino acid residues (one or more) in a polypeptide chain. Examples of non-naturally occurring amino acid residues are norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogs, e.g., as described in Eilman et al Enzym. 202:301-336 (1991). To generate such non-naturally occurring amino acid residues, the procedures of Noren et al, Science 244:182 (1989) and Eilman et al., supra, can be used. Briefly, this involves chemically activating a suppressor tRNA with the non-naturally occurring amino acid residue, followed by in vitro transcription and translation of the RNA. The method of the invention, in certain embodiments, involves replacing at least one original amino acid residue in the IgM heavy chain, although more than one original residue can be replaced. Usually, not more than all of the residues in the interface of the first or second polypeptide contain the original amino acid residue that has been replaced. The preferred original residue for replacement is "buried", meaning that the residue is substantially inaccessible to solvent. The preferred import residue is not cysteine, to prevent possible oxidation or mispairing of disulfide bonds. The protuberance is "positionable" in the cavity, meaning that the spatial location of the protuberance and cavity on the interface of the first and second polypeptides, respectively, and the size of the protuberance and cavity are such that the protuberance can be positioned in the cavity without significantly disrupting the normal association of the first and second polypeptides at the interface. Because protuberances such as Tyr, Phe, and Trp typically do not extend perpendicularly from the axis of the interface and have preferred conformations, the alignment of the protuberance with the corresponding cavity relies on modeling the protuberance / cavity pair based on a three-dimensional structure, such as obtained by X-ray crystallography or nuclear magnetic resonance (NMR). This can be achieved using techniques that are widely accepted in the art, including those of molecular modeling.

[0280]

[0303] The "original nucleic acid" is a nucleic acid that has been "modified" (i.e. modified) to encode a protrusion or cavity. By "original" or "starting" nucleic acid is meant a nucleic acid that encodes a polypeptide of interest, which may be genetically engineered or mutated. The original or starting nucleic acid may be a naturally occurring nucleic acid or may comprise a nucleic acid that has previously been modified (e.g., a humanized antibody fragment). By "modifying" is meant that the original nucleic acid is mutated by inserting, deleting or substituting at least one codon encoding an amino acid residue of interest. Usually, the codon encoding the original residue is replaced by a codon encoding the import residue. Techniques for genetically modifying DNA in this way are reviewed in Mutagenesis: a Practical Approach. M, J. McPherson, ed. (IRL Press, Oxford, UK (1991) and include, for example, site-directed mutagenesis, cassette mutagenesis and polymerase chain reaction (PGR) mutagenesis.

[0281]

[0304] The protrusions or cavities can be produced by synthetic means, e.g., recombinant techniques, in vitro. The protrusion or cavity can be "introduced" into the interface of the first or second polypeptide by peptide synthesis, the techniques for introducing non-naturally occurring amino acid residues described above, enzymatic or chemical coupling of peptides, or some combination of these techniques. The protrusion or cavity "introduced" is therefore "non-naturally occurring" or "non-natural", meaning that it is not present in nature or in the original polypeptide (e.g., a humanized monoclonal antibody).

[0282]

[0305] Preferably, the import amino acid residues for forming the protuberance are a relatively small number "Rotamer" (e.g., about 3 to 6). A "rotamer" is an energetically favorable conformation of an amino acid side chain. The number of rotamers of various amino acid residues.

[0283]

[0306] In a further embodiment, the multispecific IgA antibody of the invention comprises a complete native J chain. The J chain is a key protein in the generation of SlgA, since it promotes the polymerization of IgA and its presence in these polymers is thought to be necessary for their affinity to the SC / plgR. The multispecific IgA antibodies herein can include I chain fragments, or otherwise modified J chains, so long as the fragments or modified J chains retain the functions of the native J chain, in particular allowing efficient polymerization of IgA and binding of such polymers to the secretory component (SC) / polymeric (p)IgR. For further details on the structure-function relationship of the J chain, see, e.g., Johansen et al., 2001; j.Immunol, 167(9):5185-5192.

[0284]

[0307] To generate IgA molecules with two different heavy chains (i.e., combined nn In the case where at least one of the heavy chains is bispecific), a solution must be found for coupling two matching heavy chains with two different binding specificities to each other. Furthermore, if a light chain is required to form the binding region, a solution must be found for coupling each heavy chain with its matching light chain to provide the desired binding specificity.

[0285]

[0308] Coupling can be achieved by salt bridging versus charge switching (charge swapping or This can be achieved by charge reversal (also called charge inversion) and / or by creating knob-hole interactions between the two chains. Heavy chains can also be paired with their matching light chains by using CrossMab technology. Different approaches can also be combined to achieve optimal results.

[0286] Knob-into-hole technology

[0309] In order to improve the yield of the pentameric or hexameric bispecific binding molecules of the invention, The gA heavy chain constant region, e.g., the CH3 domain, can be modified by the "knobs-into-holes" technique, which is described in detail with some examples in WO96 / 02701 1; Ridgway, J., B., et al., Protein Eng 9 (1996) 617-621; and Merchant, A. M. et al. Nat Biotechno). 16 (1998) 677-681. In this technique, the interaction surfaces of the two IgA heavy chain constant domains are modified to increase the heterodimerization of two heavy chains with different binding specificities and / or between a heavy chain and its matching light chain. Each of the two heavy chain domains can be a "knob" and the other a "hole". The introduction of disulfide bridges stabilizes the heterodimers (Merchant, A. M. et al., Nature Biotech 16 (1998) 677-681; Atwell, S., et al., J. Moi. Biol. 270 (1997) 26-35) and increases the yield. Similarly, matching heavy and light chains can be coupled to each other by this technique (Zhu, Z.; Presta, L. G.; Zapata, G.; Carter, P. Remodeling domain interfaces to enhance heterodimerization. formation. Prof Sci. 6:781-788(1997)).

[0287]

[0310] This approach was followed by the identification of the corresponding domains of the other heavy chain in the bispecific IgA antibody. In the original interface of the Co. (e.g., Ca3) domain of one heavy chain, amino acid residues that fit into the original interface of the IgA heavy chain may be replaced with amino acid residues having a larger side chain volume, thereby creating a protrusion in the interface. This protrusion is positioned in a cavity in the interface of the corresponding domain in the other IgA heavy chain constant region. Similarly, a second IgA heavy chain can be modified by replacing amino acid residues in the interface with the corresponding domain in the constant region of the first IgA heavy chain with amino acid residues having a smaller side chain volume, thereby creating a hole (cavity) in the interface between the two heavy chain regions. Salt bridges versus charge switching (charge swapping)

[0311] Opposite charges attract each other and like charges repel each other. Charge is pH dependent and can be characterized by the pK values ​​determined for the alpha amino group (N), the alpha carboxy group (C) and the side chain of a free amino acid. When an amino acid is part of a protein or peptide, the local environment can change the pH of the side chain.

[0288]

[0312] The charge properties of an amino acid molecule can also be characterized by its isoelectric point (pi), etc. The electric point is the pH at which the overall charge of a molecule is neutral. Because amino acids differ from each other in their side chains, pi reflects differences in the pK of the side chains.

[0289]

[0313] Most amino acids (15 out of 20) have a pi close to 6, so They are considered to have a neutral overall charge. Asp and Glu are negatively charged, and His, Lys, and Arg are positively charged.

[0290]

[0314] One or more of these mutations, or sets of mutations, may be involved in the differentiation of the two different IgA heavy chains and and / or can be combined with one or more sets of knobs-hole mutations to provide the desired asymmetric interface between the IgA heavy chain and its matching light chain.

[0291]

[0315] Preferably, the asymmetric interface between two different IgA heavy chain constant regions is This can be created by up to 8, e.g., 1-8, or 1-7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2 mutations in the A heavy chain, or 2-10, or 2-9, or 2-8, or 2-7, or 2-6, or 2-5, or 2-4, or 2-3 combined mutations in the two IgA heavy chains. CrossMab technology

[0316] As mentioned above, knobs-into-holes technology or charge swapping can be used to reduce antibody weights. The correct association of light chains and their cognate heavy chains can be achieved by swapping heavy and light chain domains within the antigen-binding fragment (Fab) half of the bispecific antibody binding unit. The swapping of complete VH-CH and VL-CL domains, of only VH and VL domains, or of the bispecific binding unit half of the igA antibody. This may occur as a crossover of the CA and CL domains in the same molecule. This "crossover" preserves antigen binding affinity, but diverges the two arms so that mispairing of the light chains no longer occurs. For more details, see, for example, Sehaeffer et al., (2011) Proc Natl AcadSci USA 108(27):11187-11192 in the context of IgG antibodies.

[0292] Immunoconjugates

[0317] In certain embodiments, the antibody or fragment thereof is administered in combination with one or more therapeutic agents or It can be conjugated to a diagnostic agent. The therapeutic agent does not have to be the same, but can be different, for example, a drug and a radioisotope. For example, 131I can be incorporated into a drug that is attached to the epsilon amino group of the tyrosine and lysine residues of an antibody or fusion protein. The therapeutic agent and diagnostic agent can also be attached to, for example, reduced SH groups and / or carbohydrate side chains. Many methods are known in the art for making covalent or non-covalent conjugates of therapeutic agents or diagnostic agents with antibodies or fusion proteins, and any such known method can be utilized.

[0293]

[0318] The therapeutic or diagnostic agent binds to the hinge of the reduced antibody component via disulfide bond formation. Alternatively, such agents can be attached using heterobifunctional crosslinkers, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for such conjugation are well known in the art. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis et al., "Modification of Antibodies by Chemical Methods", MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pp. 187-230 (Wiley-Liss, Inc. 1995); Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies", MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pp. 60-84 (Cambridge University Press 1995). Alternatively, therapeutic or diagnostic agents can be conjugated via carbohydrate moieties in the Fc region of the antibody. The carbohydrate group can be used to increase the loading of the same agent that is attached to the thiol group, or the carbohydrate moiety can be used to attach a different therapeutic or diagnostic agent.

[0294]

[0319] For conjugating peptides to antibody components via antibody carbohydrate moieties The methods are well known to those skilled in the art. See, e.g., Shih et al., Int. J. Cancer 41:832 (1988); 46:1101 (1990); and Shih et al., U.S. Patent No. 5,057,313, which are incorporated herein by reference in their entireties. A typical method involves reacting an antibody component bearing an oxidized carbohydrate moiety with a carrier polymer bearing at least one free amine functional group. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.

[0295]

[0320] The antibody used as the antibody component of the immunoconjugate is an antibody fragment. In such cases, the Fc region may not be present. However, it is possible to introduce a carbohydrate moiety into the light chain variable region of a full length antibody or antibody fragment. See, for example, Leung et al. See, e.g., I., J. Immunol. 154:5919 (1995); Hansen et al., U.S. Patent No. 5,443,953 (1995); Leung et al., U.S. Patent No. 6,254,868, which are incorporated by reference in their entireties. The engineered carbohydrate moieties are used to attach therapeutic or diagnostic agents.

[0296]

[0321] In some embodiments, the chelator is an antibody, an antibody fragment, or a fusion protein. The chelating agents can be attached to conjugated proteins and used to form chelates with therapeutic or diagnostic agents such as radionuclides. Representative chelating agents include, but are not limited to, DTPA (such as Mx-DTPA), DOTA, TETA, NETA, or NOTA. Methods of conjugation and the use of chelating agents to attach metals or other ligands to proteins are well known in the art (see, e.g., U.S. Patent No. 7,563,433, the Examples section of which is incorporated herein by reference).

[0297]

[0322] In certain embodiments, the radiometal or paramagnetic ion has a long tail. They can be attached to proteins or peptides by reaction with suitable reagents, and the long tails can have multiple chelating groups attached for binding ions. Such tails can be polymers, e.g., polylysine, polysaccharides, or other derivatized or derivatizable chains having pendant groups to which chelating groups can be attached, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and similar groups known to be useful for this purpose.

[0298]

[0323] Chelates may be prepared, for example, as disclosed in U.S. Pat. No. 4,824,659. It can be directly linked to an antibody or peptide. This patent is incorporated herein by reference in its entirety. Particularly useful metal chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, which are used with diagnostic isotopes in the general energy range of 60-4000 keV, such as 125I, 131I, 123I, 124I, 62Cu, 64Cu, 18F, 111In, 67Ga, 68Ga, 99mTc, 94mTc, 11C, 13N, 15O, 76Br for radioimaging. The same chelates are useful for MM when complexed with non-radioactive metals such as manganese, iron, and gadolinium. Macrocyclic chelates such as NOTA, DOTA, and TETA are used with a variety of metals and radioactive metals, most particularly with radionuclides of gallium, yttrium, and copper, respectively. Such metal-chelate complexes can be made very stable by tailoring the ring size to the metal of interest. Other ring-type chelates, such as macrocyclic polyethers, are included that are of interest for stably binding nuclides, such as 223Ra for RAIT.

[0299]

[0324] More recently, 18F labeling methods used in PET scanning techniques, e.g., F Methods are disclosed that involve the reaction of 18F with metal or other atoms, such as aluminum.18F-Al conjugates can be directly attached to antibodies or complexed with chelating groups such as DOTA, NOTA, or NETA, which are used to label labelable constructs in pretargeting methods.Such F-18 labeling techniques are disclosed in US Patent No. 7,563,433, the Examples section of which is incorporated herein by reference.

[0300]

[0325] Other representative immunoconjugates are described by Johannson et al. 006, AIDS 20:1911-15), in which doxorubicin-conjugated P4 / D10 (anti-gp120) antibody was found to be highly effective in treating HIV-infected cells.

[0301] Additional medications

[0326] In other embodiments, the agent is a cytotoxic agent, an antiangiogenic agent, a proapoptotic agent, an antibiotic agent, Therapeutic agents such as proteins, hormones, hormone antagonists, chemokines, drugs, prodrugs, toxins, enzymes, or other agents can be used either conjugated to the subject bsAbs, ADCs and / or antibodies, or administered separately before, simultaneously with, or after the bsAbs, ADCs and / or antibodies. The drugs used can have medicinal properties selected from the group consisting of antimitotics, antikinase drugs, alkylating drugs, antimetabolites, antibiotics, alkaloid drugs, antiangiogenic drugs, proapoptotic drugs, and combinations thereof.

[0302]

[0327] Representative drugs useful include, but are not limited to, 5-fluorouracil. afatinib, aplidine, azaribine, anastrozole, anthraquinone Icrin, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan, calicheamicin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, Celebrex, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecan, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dacarbazine Satinib, dinaciclib, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicin (2P-DOX), cyano-morpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptor binding agents, etoposide (VP16), etoposide glucuronide, etoposide phosphate, exemestane, fingolimod, floxuridine (FUdR), 3',5'-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl-protein transferase inhibitors, flavopiridol, fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine, mechlorethamine, mexanthine These include: rufalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib, nitrosoureas, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine, sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (aqueous form of DTIC), transplatinum, thalidomide, thioguanine, thiopeta, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine, vincristine, vinca alkaloids, and ZD1839.

[0303]

[0328] Useful toxins include ricin, abrin, alpha toxin, saporin, and ribonucleic acid. RNases, such as onconase, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

[0304]

[0329] Useful chemokines include RANTES, MCAF, MIP1-alpha, Examples of such anti-angiogenic agents include angiostatin, baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-P1GF peptides and antibodies, anti-vascular growth factor antibodies, anti-Flk-1 antibodies, anti-Flt-1 antibodies and peptides, anti-Kras antibodies, anti-cMET antibodies, anti-MIF (macrofilament). Phage migration inhibitory factor antibody, laminin peptide, fibronectin peptide, plasminogen activator inhibitor, tissue metalloproteinase inhibitor, interferon, interleukin-12, IP-10, Gro-β, thrombospondin, 2-methoxyoestradiol, proliferin-related protein, carboxamide Triazole, CM101, marimastat, pentosan polysulfate, angiopoietin-2, interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, linomide (roquinimex), thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutane, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline may be useful.

[0305]

[0330] Useful immunomodulatory agents include cytokines, stem cell growth factors, lymphotoxins, and hematopoietic The inhibitors may be selected from factors, colony stimulating factors (CSF), interferons (IFN), erythropoietin, thrombopoietin, and combinations thereof. Particularly useful are lymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors such as interleukins (IL), colony stimulating factors such as granulocyte colony stimulating factor (G-CSF) or granulocyte macrophage colony stimulating factor (GM-CSF), interferons such as interferon-α, -β, or -λ, and stem cell growth factors such as those designated "S1 factors." Included among cytokines are growth hormones, e.g., human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, e.g., follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factors; prostaglandins, fibroblast growth factors; prolactin; placental lactogen, OB protein; tumor necrosis factor-α and -β; Mullerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factors, e.g., NGF-β; platelet growth factor; transforming growth factor transforming growth factors (TGFs), such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors: interferons, such as interferon-α, -β, and -γ; colony-stimulating factors (CSFs), such as macrophage-CSF (M-CSF); interleukins (ILs), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, Kit-ligand or FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor, and LT.

[0306]

[0331] Useful radionuclides include, but are not limited to, 111In, 177 Lu, 212Bi, 213Bi, 211At, 62Cu, 67Cu, 90Y, 125I, 131I, 32P, 33P, 47Sc, 111Ag, 67Ga, 142Pr, 153Sm, 161Tb, 166Dy, 166Ho, 186Re, 188Re, 189Re, 212Pb, 223Ra, 225Ac, 59Fe, 75Se, 77As, 89Sr, 99Mo, 105Rh, 109Pd, 143Pr, 149Pm, 169Er, 194Ir, 198Au, 199Au, 211Pb, and 227Th. Therapeutic radionuclides preferably have decay energies in the range of 20-6000 keV, preferably 60-200 keV for Auger emitters, 100-2500 keV for beta emitters, and 4000-6000 keV for alpha emitters. Maximum decay energies of useful beta particle emitting nuclides are preferably 20-5000 keV, more preferably 100-4000 keV, and most preferably 500-2500 keV. Also preferred are radionuclides that substantially decay with Auger emitting particles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In- 111, Sb-119, 1-125, Ho-161, Os-189m, and Ir-192. The decay energies of useful beta particle emitting radionuclides are preferably <1000 keV, more preferably <100 keV, and most preferably <70 keV. Radionuclides that substantially decay with the production of alpha particles are also preferred. Such radionuclides include, but are not limited to, Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213, Th-227, and Fm-255. The decay energies of useful alpha particle emitting radionuclides are preferably 2000-10000 keV, more preferably 3000-8000 keV, and most preferably 4000-7000 keV. Further possible useful radioisotopes include 11C, 13N, 15O, 75Br, 198Au, 224Ac, 126I, 133I, 77Br, 113mIn, 95Ru, 97Ru, 103Ru, 105Ru, 107Hg, 203Hg, 121mTe, 122mTe, 125mTe, 165mTe, 1657Tm, 168Tm, 197Pt, 109Pd, 105Rb, 142Pr, 143Pr, 161Tb, 166Ho, 199Au, 57Co, 58Co, 51Cr, 59Fe, 75Se, 201Tl, 225Ac, 76Br, and 169Y. Some useful diagnostic nuclides include 18F, 52Fe, 62Cu, 64Cu, 67Cu, 67Ga, 68Ga, 86Y, 89Zr, 94Tc, 94mTc, 99mTc, or 111In.

[0307]

[0332] The therapeutic agent may include a photoactive agent or dye. Compositions and other chromogens, or dyes such as porphyrins that are sensitive to visible light, have been used to detect and treat lesions by directing the appropriate light at the lesion. In therapy, this is called photoirradiation, phototherapy, or photodynamic therapy. Joni et al. (eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES (Libreria Progetto 1985; van den Bergh, Chem. Britain (1986), 22:430. Additionally, monoclonal antibodies have been coupled with photoactivated dyes to achieve phototherapy. Mew et al., J. Immunol (1983), 130:1473; ibid., Cancer Res. (1985), 45:4380; Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83:8744; ibid., Photochem. Photobiol. (1987), 46:83; Hasan et al., Prog. Clin. Biol. Res. (1989), 288:471; Tatsuta et al., Lasers Surg. Med. (1989), 9:422; Pelegrin et al., Cancer (1991), 67:2529.

[0308]

[0333] Other useful therapeutic agents include oligonucleotides, particularly preferably oncogenes and It may comprise antisense oligonucleotides directed against oncogene products, for example, bcl-2 or p53.The preferred form of therapeutic oligonucleotide is siRNA.Those skilled in the art will understand that any siRNA or interference RNA species can be attached to antibody or its fragment for delivery to target tissue.Many siRNA species against a wide variety of targets are known in the art, and any such known siRNA can be utilized in the claimed method and composition.

[0309]

[0334] Known siRNA species that may be useful include IKK-gamma (U.S. Pat. No. 7,333,636). 022828); VEGF, Flt-1 and Flk-1 / KDR (U.S. Patent No. 7,148,342); Bcl2 and EGFR (U.S. Patent No. 7,541,453); CDC20 (U.S. Patent No. 7,550,572); transducin (beta)-like 3 (U.S. Patent No. 7,576,196); KRAS (U.S. Patent No. 7,576,197); carbonic anhydrase II (U.S. Patent No. 7,579,457); complement component 3 (U.S. Patent No. 7,582,746); interleukin (IL-1) receptors (IL-2) (U.S. Patent No. 7,582,746); Examples of such proteins include those specific for IRAK4 (U.S. Pat. No. 7,592,443); survivin (U.S. Pat. No. 7,6087,070); superoxide dismutase 1 (U.S. Pat. No. 7,632,938); MET proto-oncogene (U.S. Pat. No. 7,632,939); amyloid beta precursor protein (APP) (U.S. Pat. No. 7,635,771); IGF-1R (U.S. Pat. No. 7,638,621); ICAM1 (U.S. Pat. No. 7,642,349); complement factor B (U.S. Pat. No. 7,696,344); p53 (U.S. Pat. No. 7,781,575), and apolipoprotein B (U.S. Pat. No. 7,795,421). The Examples section of each referenced patent is incorporated herein by reference.

[0310]

[0335] Additional siRNA species are available from known commercial sources, including, among many others, siRNAs are available from Sigma-Aldrich (St Louis, Mo.), Invitrogen (Carlsbad, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), Ambion (Austin, Tex.), Dharmacon (Thermo Scientific, Lafayette, Colo.), Promega (Madison, Wis.), Minis Bio (Madison, Wis.), and Qiagen (Valencia, Calif.), among others. Other publicly available sources of siRNA species include the siRNAdb database at the Stockholm Bioinformatics Centre, the MIT / ICBP These include siRNA database, the RNAi Consortium shRNA library of Broad Institute, and the Probe database of NCBI.For example, there are 30852 siRNA species in the NCBI Probe database.Those skilled in the art will understand that for any gene of interest, siRNA species has already been designed or can be easily designed using publicly available software tools.Any such siRNA species can be delivered using this DNL® complex.

[0311] Treatment

[0336] In certain aspects, disclosed herein is a method for administering to a subject a therapeutic dose of the present invention. The present invention also provides a method of treating a subject in need thereof, comprising administering a therapeutic agent or pharmaceutical composition disclosed herein. In some examples, the subject has cancer or an infectious disease. In some cases, the cancer is associated with expression of CD20, GD2, CD38, CD19, EGFR, HER2, PD-L1, CD25, CD33, BCMA, CD44, alpha-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER3, folate binding protein, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MAGE-A1, MUC16, h5T4, PSMA, TAG-72, EGFRvIII, CD123, or VEGF-R2.

[0312]

[0337] In some embodiments, the pharmaceutical composition or therapeutic agent is administered intravenously, cutaneously, or subcutaneously. In some cases, the pharmaceutical composition or therapeutic agent is administered as a single dose or in divided doses within about 48 hours of each other.In some instances, the pharmaceutical composition or therapeutic agent induces greater immune activation against cancer compared to non-cancerous tissue, as measured by the reduction in cancer cell number or volume.

[0313]

[0338] In some embodiments, disclosed herein is a method for the detection of CD20 overexpression. In some embodiments, disclosed herein is a method of administering a therapeutic agent or pharmaceutical composition described herein to a subject having a cancer associated with overexpression of GD2. In some embodiments, disclosed herein is a method of administering a therapeutic agent or pharmaceutical composition described herein to a subject having a cancer associated with overexpression of mesothelin. In some embodiments, disclosed herein is a method of administering a therapeutic agent or pharmaceutical composition described herein to a subject having a cancer associated with overexpression of CD38, CD1 The method comprises administering modified effector cells to a subject having a cancer associated with overexpression of EGFR, HER2, PD-L1, CD25, CD33, BCMA, CD44, alpha-folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER3, folate binding protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, MUC-1, MAGE-A1, MUC16, h5T4, PSMA, TAG-72, EGFRvIII, CD123 or VEGF-R2. In some cases, the cancer is a metastatic cancer. In other cases, the cancer is a recurrent or refractory cancer.

[0314]

[0339] In some cases, the cancer is a solid tumor or a hematological malignancy. In some cases, the cancer is a solid tumor. In other cases, the cancer is a hematological malignancy. In some cases, the cancer is a metastatic cancer. In some cases, the cancer is a recurrent or refractory cancer.

[0315]

[0340] In some cases, the cancer is a solid tumor. Exemplary solid tumors include: These include, but are not limited to, anal cancer; appendix cancer; bile duct cancer (i.e., cholangiocarcinoma); bladder cancer; brain cancer; breast cancer; cervical cancer; colon cancer; cancer of unknown primary site (CUP); esophageal cancer; eye cancer; fallopian tube cancer; gastrointestinal cancer; kidney cancer; liver cancer; lung cancer; medulloblastoma; melanoma; oral cancer; ovarian cancer; pancreatic cancer; parathyroid disease; penile cancer; pituitary tumor; prostate cancer; rectal cancer; skin cancer; stomach cancer; testicular cancer; pharyngeal cancer; thyroid cancer; uterine cancer; vaginal cancer; vulvar cancer; or glioblastoma.

[0316]

[0341] Glioblastoma, or glioblastoma multiforme (GBM), is an aggressive neuroepithelial brain cancer. GBM may originate from glial-type cells, astrocytes, oligodendrocyte precursor cells, or neural stem cells. Four subtypes of glioblastoma have been identified. The majority of GBMs, the classical subtype, harbor an extra copy of the epidermal growth factor receptor (EGFR) gene, and most have higher than normal expression of EGFR. In a subset of cases, EGFR amplification is accompanied by gene rearrangements, the most common of which is EGFR variant III (EGFRvIII). TP53 (p53), a gene frequently mutated in glioblastomas, is rarely mutated in the classical subtype. The proneural subtype Glioblastomas often have a high rate of alterations in TP53 (p53), PDGFRA, the gene that codes for platelet-derived growth factor receptor A, and IDH1, the gene that codes for isocitrate dehydrogenase-1. The mesenchymal subtype is characterized by a high rate of mutations or other alterations in NF1, the gene that codes for neurofibromin 1, fewer alterations in the EGFR gene, and less expression of EGFR than the other types. The neuroblastoma subtype is characterized by the expression of neuronal markers such as NEFL, GABRA1, SYT1, and SLC12A5. Other genetic alterations have been described in glioblastomas, the majority of which are clustered in two pathways, RB and PI3K / AKT. Glioblastomas have alterations in 68-78% and 88% of these pathways, respectively.

[0317]

[0342] In some instances, the cancer is a hematological malignancy. In some cases, the hematological malignancy includes lymphoma, leukemia, myeloma, or B-cell malignancy. In some cases, the hematological malignancy includes lymphoma, leukemia, or myeloma. In some instances, exemplary hematological malignancies include chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk CLL, non-CLL / SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma, non-Burkitt's high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-cell lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmocytic leukemia, and lympho-plasmocytic lymphoma. In some embodiments, the hematological malignancies include myeloid leukemia. In some embodiments, the hematological malignancies include acute myeloid leukemia (AML) or chronic myeloid leukemia (CML).

[0318]

[0343] In some examples, disclosed herein are methods for treating chronic lymphocytic leukemia ( CLL), small lymphocytic lymphoma (SLL), high-risk CLL, non-CLL / SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt lymphoma, non-Burkitt high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma A method of administering modified effector cells as described herein to a subject having a hematological malignancy selected from: primary myeloid leukemia (PMBL), immunoblastic large cell lymphoma, precursor B-cell lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis. In some cases, disclosed herein is a method of administering modified effector cells to a subject having a hematological malignancy selected from: AML or CML.

[0319]

[0344] In other cases, disclosed herein are methods for treating infection with an infectious disease. The method of administering to a subject suffering from an infectious disease can be a disease caused by bacterial, viral or fungal infection. In another example, exemplary viral pathogens include Adenoviridae, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Respiratory Syncytial Virus (RSV), JC virus, BK virus, HSV, HHV family of viruses, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae, and Togaviridae. Representative pathogenic viruses cause smallpox, influenza, mumps, measles, chickenpox, Ebola, and rubella. Representative pathogenic fungi include Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, and Stachybotrys. Representative pathogenic bacteria include Streptococcus, Pseudomonas, Shigella, Campylobacter, Staphylococcus, Helicobacter, E. coli, Rickettsia, Bacillus, Bordetella, Chlamydia, Spirochetes, and Salmonella.

[0320] Use of bispecific antibodies

[0345] Administration of therapeutic entities according to the present invention provides improved migration, delivery, tolerability, etc. It will be understood that the drug will be administered with suitable carriers, excipients, and other agents that will be incorporated into the formulation to achieve the intended effect. A large number of suitable formulations can be found in the formulary known to all medicinal chemists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, Pa (1975)), particularly Chapter 87 by Blaug, Seymour. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, ointments, gels ... Examples of suitable mixtures include Marchon Carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing Carbowax. Any of the above mixtures may be suitable for treatment and therapy according to the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation, and the formulation is physiologically compatible with the route of administration and is tolerated. For further information regarding formulations, excipients and carriers well known to pharmaceutical chemists, see also the following literature and citations therein: Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance." Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int.J.Pharm.203(1-2):1-60(2000), Charman WN “Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts.” J Pharm Sci.89(8):967-78(2000), Powell et al. “Compendium of excipients for parenteral formulations”PDA J Pharm Sci Technol.52:238-311(1998).

[0321]

[0346] The therapeutic formulations of the present invention, including the antibodies of the present invention, are useful for treating cancer, for example, as a non-limiting example. and for treating or alleviating symptoms associated with leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung and bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, urinary bladder cancer, kidney and renal pelvis cancer, oral cavity and pharynx cancer, uterine cancer, and / or melanoma. The present invention also provides methods for treating or alleviating symptoms associated with cancer. The therapeutic regimen is carried out by identifying a subject, e.g., a human patient suffering from (or at risk of developing) cancer, using standard methods.

[0322]

[0347] The effectiveness of treatment can be determined using any known method for diagnosing or treating a particular immune-related disease. Amelioration of one or more symptoms of the immune-related disease indicates that the antibody confers a clinical benefit.

[0323]

[0348] Methods for screening for antibodies with desired specificity include, but are not limited to, Non-limiting examples of immunoassay techniques include enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known in the art.

[0324]

[0349] Targets such as CD47, tumor-associated antigens, or other antigens (or fragments thereof) Antibodies against targets can be used in methods known in the art relating to the localization and / or quantification of these targets, for example, for use in measuring the levels of these targets in an appropriate physiological sample, for use in diagnostic methods, for use in protein imaging, etc. In certain embodiments, antibodies specific for any of these targets, or derivatives, fragments, analogs or homologs thereof, that contain an antibody-derived antigen-binding domain, are utilized as pharmacologically active compounds (hereinafter referred to as "therapeutic agents").

[0325]

[0350] The antibodies of the present invention can be used in immunoaffinity, chromatography or immunoprecipitation assays. Such antibodies can be used to isolate specific targets using standard techniques such as antibody screening. The antibodies (or fragments thereof) of the present invention can be used diagnostically to monitor protein levels in tissues as part of a clinical testing procedure, for example to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include: Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; examples of suitable radioactive materials include 125I, 131I, 35S, or 3H.

[0326]

[0351] The antibodies of the present invention include polyclonal, monoclonal, humanized and fully human antibodies. Antibodies can be used as therapeutic agents. Such agents are generally used to treat or prevent diseases or pathologies associated with abnormal expression or activation of a given target in a subject. An antibody preparation, preferably one with high specificity and high affinity for a target antigen, is administered to a subject and generally has an effect due to binding with its target. Administration of the antibody can neutralize, inhibit, or interfere with the signaling function of the target. Administration of the antibody can neutralize, inhibit, or interfere with the binding of the target to the endogenous ligand to which it naturally binds. For example, the antibody binds to the target and neutralizes or otherwise inhibits the interaction between CD47 and SIRPα.

[0327]

[0352] A therapeutically effective amount of an antibody of the invention generally relates to the amount needed to achieve a therapeutic objective. As mentioned above, this may be a binding interaction between the antibody and its target antigen, which in certain cases interferes with the function of the target. The amount required to be administered further depends on the binding affinity of the antibody to its specific antigen, and also on the rate at which the administered antibody is depleted from the free volume of the other subject to which it is administered. A typical range for therapeutically effective administration of the antibody or antibody fragment of the present invention can be, as a non-limiting example, about 0.1 mg / kg body weight to about 50 mg / kg body weight. Typical dosing frequency can range, for example, from twice a day to once a week.

[0328]

[0353] The antibodies or fragments thereof of the present invention may be used in the treatment of various diseases and disorders. For this purpose, the composition can be administered in the form of a pharmaceutical composition. The principles and considerations involved in the preparation of such compositions, as well as guidance for the selection of ingredients, are provided, for example, in Remington: The Science And Practice Of Pharmacy, 19th Edition (Alfonso R. Gennaro, et al., editors) Mack Pub.Co.,Easton,Pa.:1995;Drug Absorption Enhancement Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0329]

[0354] When antibody fragments are used, they are specific for the binding domain of the target protein. The smallest inhibitory fragment that binds to the target protein sequence is preferred. For example, based on the variable region sequences of an antibody, peptide molecules can be designed that retain the ability to bind to the target protein sequence. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology (see, e.g., Marasco et al., Proc. (See Natl. Acad. Sci. USA, 90:7889-7893 (1993)). Formulations may also contain more than one active compound as necessary for the particular indication being treated, preferably It is possible to contain active compounds that have complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can include an agent that enhances its function, such as a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent. Such molecules are suitably present in the combination in amounts that are effective for the intended purpose.

[0330]

[0355] The active ingredient may also be polymerized, for example, by coacervation techniques or by interfacial polymerization. The drugs can be entrapped in microcapsules prepared by, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, respectively.

[0331]

[0356] Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0357] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include those in which the antibody is Semipermeable matrices of solid hydrophobic polymers containing the protein are included, and the matrices are in the form of shaped articles, such as films or microcapsules.Examples of sustained release matrices include polyesters, hydrogels (such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, while certain hydrogels release proteins for shorter periods of time.

[0332]

[0358] The antibodies according to the invention bind to a given target (or a protein fragment thereof) in a sample. In some embodiments, the antibody contains a detectable label. The antibody is polyclonal or, more preferably, monoclonal. The whole antibody, or a fragment thereof (e.g., Fab, scFv, or F(ab)2) is used. The term "labeled" with respect to a probe or antibody is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody, and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to encompass tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present within a subject. Thus, included in the use of the term "biological sample" are blood, and fractions or components of blood, including serum, plasma, or lymph. That is, the detection methods of the present invention can be used to detect analyte mRNA, protein, or genomic DNA in biological samples in vitro and in vivo. For example, in vitro techniques for detection of analyte mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detection of analyte protein include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. In vitro techniques for detection of analyte genomic DNA include Southern hybridization. Procedures for performing immunoassays are described, for example, in “ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. Vol. 42, J.R.Crowther (ed.), Human Press, Totowa, NJ, 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Additionally, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in the subject can be detected by standard imaging techniques.

[0333] Pharmaceutical Compositions and Dosages

[0359] Disclosed herein, in certain embodiments, is a method for administering to a subject, comprising: A pharmaceutical composition comprising the therapeutic agent disclosed herein.

[0334]

[0360] In some instances, a pharmaceutical composition comprising a therapeutic agent described herein is The pharmaceutical compositions described herein are formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and adjuvants, which facilitate the processing of the active compounds into pharma- ceutical preparations. The appropriate formulation depends on the route of administration selected. A summary of pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, 19th Edition (Easton, Pa.: Mack Publishing Co., Ltd.). Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA and Lachman, L., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition (Lippincott Williams & Wilkins 1999).

[0335]

[0361] Pharmaceutical compositions may, if desired, be formulated in a conventional manner, for example, by way of example only, conventional mixing. The pharmaceutical compositions may be prepared by means of dissolving, granulating, dragee-making, elutriating, emulsifying, encapsulating, entrapping or compression processes.

[0336]

[0362] In certain embodiments, the composition also contains one or more pH adjusting or buffering agents, e.g. For example, acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris-hydroxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition in an acceptable range.

[0337]

[0363] In other embodiments, the composition also provides a composition that reduces the osmolality of the composition to an acceptable range. The composition may include one or more salts in an amount necessary to effect the synthesis of the compound of the present invention. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0338]

[0364] The pharmaceutical compositions described herein can be administered by any suitable route, including, but not limited to, In some cases, the pharmaceutical composition is administered by, but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular, intracerebral, intraventricular, intraarticular, intraperitoneal, or intracranial), intranasal, buccal, sublingual, or rectal routes of administration. In some cases, the pharmaceutical composition is formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intracerebral, intraventricular, intraarticular, intraperitoneal, or intracranial) administration.

[0339]

[0365] The pharmaceutical compositions described herein can be administered in any suitable dosage form, including, but not limited to, and / or other suitable formulations, including but not limited to aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, etc., for oral ingestion by the individual to be treated. In some embodiments, the pharmaceutical composition is formulated into a capsule. In some embodiments, the pharmaceutical composition is formulated into a solution (e.g., for IV administration). In some cases, the pharmaceutical composition is formulated as an infusion. In some cases, the pharmaceutical composition is formulated as an injection.

[0340]

[0366] The pharmaceutical solid dosage forms described herein may optionally be The composition includes a compound and one or more pharma- ceutically acceptable excipients, such as a compatible carrier, binder, filler, suspending agent, flavoring agent, sweetener, disintegration agent, dispersing agent, surfactant, lubricant, colorant, diluent, solubilizer, humectant, plasticizer, stabilizer, penetration enhancer, wetting agent, antifoaming agent, antioxidant, preservative, or one or more combinations thereof.

[0341]

[0367] In yet another respect, Remington's Pharmaceutica A film coating is provided around the composition using standard coating procedures such as those described in I Sciences, 20th Edition (2000). In some embodiments, the composition is formulated into particles (e.g., for administration by capsules), and some or all of the particles are coated. In some embodiments, the composition is formulated into particles (e.g., for administration by capsules), and some or all of the particles are microencapsulated. In some embodiments, the composition is formulated into particles (e.g., for administration by capsules), and some or all of the particles are not microencapsulated or coated.

[0342]

[0368] In certain embodiments, the compositions provided herein also inhibit microbial activity. The composition may contain one or more preservatives to prevent the formation of mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0343]

[0369] As referred to herein, a "proliferative disorder" is a disorder characterized by excessive proliferation of cells and cell matrix. This represents a unifying concept that cell turnover contributes significantly to the pathogenesis of several diseases, including cancer.

[0344]

[0370] As used herein, a "patient" or "subject" refers to a subject having a physiological condition, e.g., It refers to a mammalian subject who is diagnosed or suspected to have or develop cancer or an autoimmune condition or an infectious disease. In some embodiments, the term "patient" refers to a mammalian subject who is more likely than average to develop cancer. Representative patients can be humans, apes, dogs, pigs, cows, cats, horses, goats, sheep, rodents, and other mammals that can benefit from the therapeutic methods disclosed herein. Representative human patients can be male and / or female.

[0345]

[0371] A "patient in need thereof" or a "subject in need thereof" is used herein. In the present specification, the term "cancer" refers to a patient who is diagnosed or suspected of having a disease or injury, for example, a proliferative disorder, such as, but not limited to, cancer. In some cases, the cancer is a solid tumor or a hematological malignancy. In some cases, the cancer is a solid tumor. In other cases, the cancer is a hematological malignancy. In some cases, the cancer is a metastatic cancer. In some cases, the cancer is a recurrent or refractory cancer. In some cases, the cancer is a solid tumor. Exemplary solid tumors include, but are not limited to, anal cancer; appendix cancer; bile duct cancer (i.e., cholangiocarcinoma); bladder cancer; brain cancer; breast cancer; cervical cancer; colon cancer; cancer of unknown primary (CUP); esophageal cancer; eye cancer; fallopian tube cancer; gastrointestinal cancer; kidney cancer; liver cancer; lung cancer; medulloblastoma; melanoma; oral cancer; ovarian cancer; pancreatic cancer; parathyroid disease; penile cancer; pituitary tumor; prostate cancer; rectal cancer; skin cancer; stomach cancer; testicular cancer; pharyngeal cancer; thyroid cancer; uterine cancer; vaginal cancer; vulvar cancer; or glioblastoma. In some embodiments, the leukemia can be, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML).

[0346]

[0372] As used herein, "administering" refers to providing a composition of the present disclosure to a patient. By way of example and not limitation, administration of the composition, e.g., injection, can be by intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. One or more such routes may be used. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. Alternatively, or simultaneously, administration can be by oral route. Additionally, administration can also be by surgical deposition of a bolus or pellet of cells. The target cell population may be due to the presence of a target cell in a cell-free environment, or due to the positioning of a medical device. In one embodiment, the composition of the present disclosure may comprise an engineered cell or host cell expressing a nucleic acid sequence described herein, or a vector comprising at least one nucleic acid sequence described herein, in an amount effective to treat or prevent a proliferative disorder. A pharmaceutical composition may comprise a target cell population described herein in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise a buffer, such as neutral buffered saline, phosphate buffered saline; a carbohydrate, such as glucose, mannose, sucrose, or dextran, mannitol; a protein; a polypeptide or amino acid, such as glycine; an antioxidant; a chelating agent, such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative.

[0347]

[0373] As used herein, the terms "treatment," "treating," and the like, Legal equivalent refers to obtaining a desired pharmacological and / or physiological effect. In embodiments, the effect is therapeutic, i.e., the effect partially or completely cures the disease and / or the adverse symptoms caused by the disease. To this end, the methods described herein include admini...

Claims

1. (a) Immunoglobulin A (IgA) heavy chain domain; (b) a CD47 binding domain; and (c) an antigen-binding domain; An antibody construct comprising: the IgA heavy chain domain specifically binds to FcαR on immune effector cells; the CD47 binding domain inhibits binding of CD47 expressed on a target cell to signal regulatory protein alpha (SIRP alpha) on an immune effector cell; the antigen-binding domain binds to an antigen on a target cell; and The antibody construct has a higher binding affinity for the antigen compared to CD47. The antibody construct.

2. The antibody construct of claim 1, wherein the CD47 binding domain comprises at least one of the first light chain variable domain and the first heavy chain variable domain.

3. 3. The antibody construct of claim 1 or claim 2, wherein the antigen binding domain comprises at least one of a second light chain variable domain and a second heavy chain variable domain.

4. The construct comprises the first light chain variable domain, the first light chain variable domain comprising a variable light chain complementarity determining region 1 (CDR-L1), a variable light chain complementarity determining region 2 (CDR L2), ), and variable light chain complementarity determining region 3 (CDR-L3); said CDR-L1 comprises the amino acid sequence of SEQ ID NO: 13 or a variant thereof having up to two amino modifications in SEQ ID NO: 13, said CDR-L2 comprises the amino acid sequence of SEQ ID NO: 14 or a variant thereof having up to two amino modifications in SEQ ID NO: 14, and said CDR-L3 comprises the amino acid sequence of SEQ ID NO: 15 or a variant thereof having up to two amino modifications in SEQ ID NO:

15. An antibody construct according to claim 2 or claim 3.

5. The antibody construct according to any one of claims 2 to 4, wherein said construct comprises the first light chain variable domain, said first light chain variable domain comprising an amino acid sequence having 90% sequence identity to SEQ ID NO:

26.

6. said construct comprising said first heavy chain variable domain, said first heavy chain variable domain comprising variable heavy chain complementarity determining region 1 (CDR-H1), variable heavy chain complementarity determining region 2 (CDR-H2), and variable heavy chain complementarity determining region 3 (CDR-H3); said CDR-H1 comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof having up to two amino modifications in SEQ ID NO: 4, said CDR-H2 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof having up to two amino modifications in SEQ ID NO: 5, and said CDR-H3 comprises the amino acid sequence of SEQ ID NO: 6 or a variant thereof having up to two amino modifications in SEQ ID NO: 6, An antibody construct according to any one of claims 2 to 5.

7. The antibody construct according to any one of claims 2 to 6, wherein the construct comprises the first heavy chain variable domain, the first heavy chain variable domain comprising an amino acid sequence having 90% sequence identity to SEQ ID NO:

23.

8. said construct comprising said second light chain variable domain, said second light chain variable domain comprising CDR-L1, CDR-L2, and CDR-L3; The CDR-L1 has the amino acid sequence of SEQ ID NO: 10 or up to two amino acids in SEQ ID NO: 10 wherein said CDR-L2 comprises the amino acid sequence of SEQ ID NO: 11 or a variant thereof having up to two amino modifications in SEQ ID NO: 11, and said CDR-L3 comprises the amino acid sequence of SEQ ID NO: 12 or a variant thereof having up to two amino modifications in SEQ ID NO:

12. An antibody construct according to any one of claims 3 to 7.

9. An antibody construct according to any one of claims 3 to 8, wherein said construct comprises said second light chain variable domain, said second light chain variable domain comprising the amino acid sequence of SEQ ID NO:

25.

10. said construct comprising said second heavy chain variable domain, said second heavy chain variable domain comprising CDR-H1, CDR-H2, and CDR-H3; The antibody construct of any one of claims 3 to 9, wherein said CDR-H1 comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof with up to two amino modifications in SEQ ID NO: 1, said CDR-H2 comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof with up to two amino modifications in SEQ ID NO: 2, and said CDR-H3 comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof with up to two amino modifications in SEQ ID NO:

3.

11. The antibody construct according to any one of claims 3 to 10, wherein the construct comprises the second heavy chain variable domain, the second heavy chain variable domain comprising an amino acid sequence having 90% sequence identity to SEQ ID NO:

22.

12. a first polypeptide comprising the first heavy chain variable domain and a second heavy chain variable domain, wherein the C-terminus of the second heavy chain variable domain is linked to the N-terminus of the first heavy chain variable domain; and a second polypeptide comprising the first light chain variable domain and a second light chain variable domain, wherein the C-terminus of the second light chain variable domain is linked to the N-terminus of the first light chain variable domain; The antibody construct according to any one of claims 3 to 11, comprising at least one of the following:

13. The antibody construct of claim 12, wherein the first or second polypeptide further comprises a linker peptide connecting the variable domains.

14. The antibody construct of claim 13, wherein the linker peptide comprises the sequence of SEQ ID NO:

44.

15. The antibody construct according to any one of claims 12 to 14, wherein the first polypeptide comprises the sequence of SEQ ID NO:

29.

16. The antibody construct of any one of claims 12 to 15, wherein the C-terminus of the first polypeptide is linked to an (IgA) heavy chain region.

17. 17. The antibody construct of claim 16, wherein the linkage is via the IgA CH1 constant domain.

18. The antibody construct according to any one of claims 12 to 17, wherein the second polypeptide comprises the sequence of SEQ ID NO:

31.

19. The antibody construct according to any one of claims 12 to 18, wherein the antibody construct comprises a first polypeptide and a second polypeptide.

20. a first polypeptide in which the C-terminus of a first heavy chain variable domain is linked to the N-terminus of a second heavy chain variable domain; or a second polypeptide in which the C-terminus of the first light chain variable domain is linked to the N-terminus of the second light chain variable domain; The antibody construct of claim 3, comprising at least one of the following:

21. The antibody construct of claim 20, wherein the linkage is via a linker peptide.

22. The antibody construct of claim 21 , wherein the linker peptide comprises the sequence of SEQ ID NO:

44.

23. The antibody construct according to any one of claims 20 to 22, wherein the first polypeptide comprises the sequence of SEQ ID NO:

30.

24. The antibody construct of any one of claims 20 to 23, wherein the C-terminus of the first polypeptide is linked to an (IgA) heavy chain region.

25. 24. The antibody construct of claim 23, wherein the linkage is via the IgA CH1 constant domain.

26. The antibody construct according to any one of claims 22 to 25, wherein the second polypeptide comprises the sequence of SEQ ID NO:

32.

27. The antibody construct according to any one of claims 20 to 26, wherein the antibody construct comprises a first polypeptide and a second polypeptide.

28. The antibody construct according to any one of claims 3 to 11, wherein the antibody construct comprises a first polypeptide comprising a single chain variable fragment (scFv), said scFv comprising said second heavy chain variable domain linked to said second light chain variable domain.

29. 29. The construct of claim 28, comprising a linker peptide connecting the variable domains.

30. 30. The antibody construct of claim 29, wherein the linker peptide comprises the sequence of SEQ ID NO:

45.

31. The antibody construct of any one of claims 28 to 30, wherein the first polypeptide comprises a scFv linked to the first light chain variable domain; or the first heavy chain variable domain.

32. The antibody construct of claim 31 , wherein the linkage is via a linker peptide.

33. The antibody construct of claim 32, wherein the linker peptide comprises the sequence of SEQ ID NO:

44.

34. 34. The antibody construct of any one of claims 31 to 33, comprising a first polypeptide comprising a scFv linked to a first light chain variable domain, said first polypeptide comprising a sequence having at least 90% identity to SEQ ID NO:

35.

35. 34. The antibody construct of any one of claims 31 to 33, comprising a first polypeptide comprising a scFv linked to a first heavy chain variable domain, said first polypeptide comprising a sequence having at least 90% identity to SEQ ID NO:

33.

36. 4. The antibody construct of claim 3, wherein the antibody construct comprises a first polypeptide comprising a single chain variable fragment (scFv), the scFv comprising a first heavy chain variable domain linked to a first light chain variable domain.

37. 37. The antibody construct of claim 36, comprising a linker peptide connecting the variable domains. 。

38. The antibody construct of claim 37, wherein the linker peptide comprises the sequence of SEQ ID NO:

45.

39. The antibody construct of any one of claims 36 to 38, wherein the first polypeptide comprises a scFv linked to a second light chain variable domain; or to a second heavy chain variable domain.

40. The antibody construct of claim 39, wherein the linkage is via a linker peptide.

41. The antibody construct of claim 40, wherein the linker peptide comprises the sequence of SEQ ID NO:

44.

42. 42. The antibody construct of any one of claims 39 to 41, comprising a first polypeptide comprising a scFv linked to a second light chain variable domain, said first polypeptide comprising a sequence having 90% identity to SEQ ID NO:

36.

43. 42. The antibody construct of any one of claims 39 to 41, comprising a first polypeptide comprising a scFv linked to a second heavy chain variable domain, said first polypeptide comprising a sequence having 90% identity to SEQ ID NO:

34.

44. An antibody construct according to any one of claims 1 to 43, wherein the IgA heavy chain domain comprises an IgA heavy chain constant domain.

45. 45. The antibody construct of claim 44, wherein the IgA heavy chain constant domain is an IgA1 constant domain or a variant thereof.

46. 46. ​​The antibody construct of claim 45, wherein the IgA1 constant domain comprises at least one of the IgA1 CH2 region and the IgA1 CH3 region or a variant thereof.

47. The IgA1 constant region further comprises an IgA1 CH1 region or a variant thereof.

47. The antibody construct of claim 46.

48. 45. The antibody construct of claim 44, wherein the IgA heavy chain constant domain is an IgA2 constant domain or a variant thereof.

49. 49. The antibody construct of claim 48, wherein the IgA2 constant domain comprises at least one of the IgA2 CH2 region and the IgA2 CH3 region or a variant thereof.

50. 50. The antibody construct of claim 49, wherein the IgA2 constant region further comprises an IgA2 CH1 region. thing.

51. 51. The antibody construct of any one of claims 1 to 50, wherein the antigen is CD20, GD2, mesothelin, CD38, CD19, EGFR, HER2, PD-L1, or CD25.

52. An antibody construct according to any one of claims 44 to 51, wherein the IgA heavy chain constant domain lacks at least one or two naturally occurring glycosylation sites compared to the corresponding wild type IgA.

53. The construct lacks at least two naturally occurring glycosylation sites, and the at least two naturally occurring glycosylation sites are located in at least one IgA CH2 region or The antibody construct of claim 52, wherein said antibody construct is in the IgA CH3 region.

54. 54. The antibody construct of claim 53, wherein the at least two naturally occurring glycosylation sites are two naturally occurring N-linked glycosylation sites.

55. 55. The construct of any one of claims 44 to 54, wherein the IgA heavy chain constant domain lacks at least two naturally occurring asparagine (N) amino acid residues compared to the corresponding wild-type IgA.

56. 55. The antibody construct of any one of claims 44 to 54, wherein the IgA heavy chain constant domain comprises an amino acid substitution of at least two naturally occurring asparagine (N) amino acid residues, or a substitution of both residues.

57. 57. The antibody construct of any one of claims 44 to 56, wherein the IgA heavy chain constant domain lacks at least one naturally occurring cysteine ​​(C) amino acid residue compared to the corresponding wild type IgA.

58. 58. The antibody construct of claim 57, wherein the IgA heavy chain constant domain comprises an amino acid substitution of at least one naturally occurring cysteine ​​(C) amino acid residue.

59. 59. The antibody construct of claim 58, wherein the IgA heavy chain constant domain comprises a non-conservative amino acid substitution of at least one naturally occurring cysteine ​​(C) amino acid residue.

60. 58. The antibody construct of claim 57, wherein the IgA heavy chain constant domain comprises an amino acid deletion of at least one naturally occurring cysteine ​​(C) amino acid residue.

61. 61. The antibody construct of any one of claims 44 to 60, wherein the IgA heavy chain constant domain lacks at least one naturally occurring tyrosine (Y) amino acid residue compared to the corresponding wild type IgA.

62. 62. The antibody construct of claim 61 , wherein the IgA heavy chain constant domain comprises an amino acid substitution of at least one naturally occurring cysteine ​​(Y) amino acid residue.

63. 62. The antibody construct of claim 61 , wherein the IgA heavy chain constant domain comprises a deletion of at least one naturally occurring cysteine ​​(Y) amino acid residue.

64. 64. The antibody construct of any one of claims 44 to 63, wherein the IgA heavy chain constant domain lacks at least one naturally occurring threonine (T) amino acid residue compared to the corresponding wild type IgA.

65. 65. The antibody construct of claim 64, wherein the IgA heavy chain constant domain comprises an amino acid substitution of at least one naturally occurring threonine (T) amino acid residue.

66. 66. The antibody construct of claim 65, wherein the IgA heavy chain constant domain comprises a deletion of at least one naturally occurring threonine (T) amino acid residue.

67. 67. The antibody construct of any one of claims 44 to 66, wherein the IgA heavy chain constant domain lacks at least one naturally occurring isoleucine (I) amino acid residue compared to the corresponding wild type IgA.

68. 68. The antibody construct of claim 67, wherein the IgA heavy chain constant domain comprises an amino acid substitution of at least one naturally occurring isoleucine (I) amino acid residue.

69. 69. The antibody construct of claim 68, wherein the IgA heavy chain constant domain comprises a deletion of at least one naturally occurring isoleucine (I) amino acid residue.

70. 70. The antibody construct of any one of claims 44 to 69, wherein the IgA heavy chain constant domain lacks at least one naturally occurring proline (P) amino acid residue compared to the corresponding wild type IgA.

71. 71. The antibody construct of claim 70, wherein the IgA heavy chain constant domain comprises an amino acid substitution of at least one naturally occurring proline (P) amino acid residue.

72. 72. The antibody construct of claim 71, wherein the IgA heavy chain constant domain comprises a deletion of at least one naturally occurring proline (P) amino acid residue.

73. An antibody construct according to any one of claims 52 to 72, wherein said construct exhibits a longer circulating half-life compared to a corresponding antibody construct comprising a corresponding wild-type IgA heavy chain constant region.

74. An antibody construct according to any one of claims 1 to 73, wherein the antibody construct comprises an attenuated CD47 binding domain having a lower affinity for CD47 compared to a corresponding wild-type CD47 binding domain.

75. 75. The antibody construct of any one of claims 52 to 74, wherein the antibody construct exhibits reduced aggregation compared to a corresponding antibody construct comprising a corresponding wild type IgA heavy chain constant region.

76. 76. The antibody construct of any one of claims 52 to 75, wherein the antibody construct exhibits reduced aggregation with serum proteins compared to a corresponding antibody construct comprising a corresponding wild type IgA heavy chain constant region.

77. 77. The antibody construct of any one of claims 44 to 76, wherein the IgA heavy chain constant region comprises one or more albumin binding domains.

78. 78. The antibody construct of claim 77, wherein the antibody construct has a longer half-life than a corresponding antibody construct that does not comprise one or more albumin binding domains.

79. 79. The antibody construct of any one of claims 44 to 78, wherein the IgA heavy chain constant domain comprises an amino acid sequence having 90% sequence identity to a sequence selected from any one of SEQ ID NOs: 37 to 41.

80. An antibody construct according to any one of claims 1 to 79, wherein the construct further comprises a hinge region.

81. 81. The antibody construct of claim 80, wherein the hinge region comprises an IgA hinge amino acid sequence or a variant or fragment thereof.

82. 82. The antibody construct of claim 80 or claim 81, wherein the hinge region comprises a human IgA hinge amino acid sequence or a variant or fragment thereof.

83. The hinge is an IgA1 hinge or an IgA2 hinge, or a variant or variant thereof. An antibody construct according to any one of claims 80 to 82, which is a fragment.

84. An antibody construct according to any one of claims 1 to 83, wherein the antibody construct further comprises a light chain constant region.

85. The antibody construct of claim 84, wherein the light chain constant region is a kappa constant region.

86. The antibody construct of claim 84, wherein the light chain constant region is a lambda constant region.

87. The antibody construct according to any one of claims 84 to 86, wherein the light chain constant region is linked to the first light chain variable domain or to the first heavy chain variable domain.

88. (a) the immunoglobulin A (IgA) heavy chain constant domain; (b) a CD47 binding domain; and (c) an antigen-binding domain; An antibody construct comprising: the IgA heavy chain domain specifically binds to FcαR on immune effector cells; the CD47 binding domain inhibits binding of CD47 expressed on a target cell to signal regulatory protein alpha (SIRP alpha) on an immune effector cell; the antigen-binding domain binds to an antigen on a target cell; and The antibody construct has a higher binding affinity for the antigen compared to CD47. The antibody construct.

89. The antibody construct of claim 88, wherein the CD47 binding domain comprises a first light chain variable domain and a first heavy chain variable domain.

90. 90. The antibody construct of claim 89, wherein the antigen binding domain comprises a second light chain variable domain and a second heavy chain variable domain.

91. 91. The antibody construct of claim 90, wherein the first light chain variable domain is of the kappa type.

92. 92. The antibody construct of claim 91, wherein the second light chain variable domain is of the lambda type.

93. 91. The antibody construct of claim 90, wherein the first light chain variable domain is of the lambda type.

94. 94. The antibody construct of claim 93, wherein the second light chain variable domain is of the kappa type.

95. 93. The antibody construct according to claim 91 or claim 92, wherein the first light chain variable domain is of the kappa type and comprises the variable light chain complementarity determining region 1 (CDR-L1) amino acid sequence of SEQ ID NO: 16, the variable light chain complementarity determining region 2 (CDR L2) amino acid sequence of SEQ ID NO: 17, the variable light chain complementarity determining region 3 (CDR-L3) amino acid sequence of SEQ ID NO:

18.

96. 96. The antibody construct of claim 95, wherein the first light chain variable domain comprises an amino acid sequence having 90% sequence identity to SEQ ID NO:

27.

97. 93. The antibody construct of claim 92, wherein the second light chain variable domain is of the lambda type comprising the variable light chain complementarity determining region 1 (CDR-L1) amino acid sequence of SEQ ID NO: 19, the variable light chain complementarity determining region 2 (CDR L2) amino acid sequence of SEQ ID NO: 20, and the variable light chain complementarity determining region 3 (CDR-L3) amino acid sequence of SEQ ID NO:

21.

98. 98. The antibody construct of claim 97, wherein the second light chain variable domain comprises an amino acid sequence having 90% sequence identity to SEQ ID NO:

28.

99. The antibody construct of any one of claims 90 to 98, wherein the first heavy chain variable domain and the second heavy chain variable domain are the same.

100. 100. The antibody construct of claim 99, wherein the first heavy chain variable domain and the second heavy chain variable domain comprise the variable heavy chain complementarity determining region 1 (CDR-H1) amino acid sequence of SEQ ID NO: 7, the variable heavy chain complementarity determining region 2 (CDR-H2) amino acid sequence of SEQ ID NO: 8, and the variable heavy chain complementarity determining region 3 (CDR-H3) amino acid sequence of SEQ ID NO:

9.

101. The antibody construct of claim 100, wherein the first heavy chain variable domain and the second heavy chain variable domain comprise an amino acid sequence having 90% sequence identity to SEQ ID NO:

24.

102. The antibody construct of any one of claims 89 to 101, wherein the first light chain variable domain further comprises a kappa constant region.

103. The antibody construct of any one of claims 90 to 102, wherein the second light chain variable domain further comprises a lambda constant region.

104. The antibody construct of any one of claims 89 to 101, wherein the first light chain variable domain further comprises a lambda constant region.

105. The antibody construct of any one of claims 90 to 101 or claim 104, wherein the second light chain variable domain further comprises a kappa constant region.

106. The antibody construct according to any one of claims 102 to 105, wherein the kappa constant region comprises an amino acid sequence having 95% sequence identity to SEQ ID NO:

42.

107. The antibody construct of any one of claims 103 to 105, wherein the lambda constant region comprises an amino acid sequence having 95% sequence identity to SEQ ID NO:

43.

108. An antibody construct according to any one of claims 88 to 107, wherein the IgA heavy chain constant domain comprises at least one of the IgA CH2 region and the IgA CH3 region or a variant thereof.

109. The antibody construct of claim 108, wherein the IgA heavy chain constant domain further comprises an IgA CH1 region or a variant thereof.

110. The antibody construct of claim 109, wherein the CD47 binding domain is linked to the IgA constant domain by the IgA CH1 domain.

111. The antibody construct of claims 109 to 110, wherein the antigen binding domain is linked to the IgA constant domain by the IgA CH1 domain.

112. An antibody construct according to any one of claims 88 to 111, wherein the IgA heavy chain constant domain is an IgA1 constant region or a variant thereof.

113. The antibody construct of claim 112, wherein the IgA1 constant region comprises an IgA1 CH2 region and an IgA1 CH3 region.

114. The antibody construct of claim 113, wherein the IgA1 constant region further comprises an IgA1 CH1 region. building.

115. An antibody construct according to any one of claims 88 to 111, wherein the IgA heavy chain constant domain is an IgA2 constant region or a variant thereof.

116. The antibody construct of claim 115, wherein the IgA2 constant region comprises an IgA2 CH2 region and an IgA2 CH3 region.

117. The antibody construct of claim 116, wherein the IgA2 constant region further comprises an IgA2 CH1 region. building.

118. The antibody construct of any one of claims 88 to 117, wherein the antigen is CD20, GD2, mesothelin, CD38, CD19, EGFR, HER2, PD-L1, or CD25.

119. An antibody construct according to any one of claims 108 to 118, wherein the IgA heavy chain constant domain lacks one, two or three naturally occurring glycosylation sites compared to the corresponding wild type IgA.

120. The antibody construct of claim 119, wherein the site is in at least one of the IgA CH2 region or the IgA CH3 region.

121. The antibody construct of claim 119 or claim 120, wherein the construct lacks two naturally occurring N-linked glycosylation sites.

122. 122. The antibody construct of any one of claims 108 to 121, wherein the IgA heavy chain constant domain lacks naturally occurring asparagine (N) amino acid residues compared to the corresponding wild type IgA.

123. 123. The antibody construct of claim 122, wherein the IgA heavy chain constant domain comprises an amino acid substitution of at least one naturally occurring asparagine (N) amino acid residue, or a substitution of at least two naturally occurring asparagine (N) amino acid residues.

124. 124. The antibody construct of any one of claims 108 to 123, wherein the IgA heavy chain constant domain lacks at least one naturally occurring cysteine ​​(C) amino acid residue compared to the corresponding wild type IgA.

125. The antibody construct of claim 124, wherein the IgA heavy chain constant domain comprises an amino acid substitution of at least one naturally occurring cysteine ​​(C) amino acid residue.

126. The antibody construct of claim 125, wherein the IgA heavy chain constant domain comprises a non-conservative amino acid substitution of at least one naturally occurring cysteine ​​(C) amino acid residue.

127. The antibody construct of claim 126, wherein the IgA heavy chain constant domain comprises an amino acid deletion of at least one naturally occurring cysteine ​​(C) amino acid residue.

128. 128. The method of claim 108, wherein the IgA heavy chain constant domain lacks at least one naturally occurring tyrosine (Y) amino acid residue compared to the corresponding wild-type IgA. The antibody construct described.

129. 129. The antibody construct of claim 128, wherein the IgA heavy chain constant domain comprises an amino acid substitution of at least one naturally occurring cysteine ​​(Y) amino acid residue.

130. 130. The antibody construct of claim 129, wherein the IgA heavy chain constant domain comprises a deletion of at least one naturally occurring cysteine ​​(Y) amino acid residue.

131. 131. The antibody construct of any one of claims 108 to 130, wherein the IgA heavy chain constant domain lacks at least one naturally occurring threonine (T) amino acid residue compared to the corresponding wild type IgA.

132. 132. The antibody construct of claim 131, wherein the IgA heavy chain constant domain comprises an amino acid substitution of at least one naturally occurring threonine (T) amino acid residue.

133. The antibody construct of claim 132, wherein the IgA heavy chain constant domain comprises a deletion of at least one naturally occurring threonine (T) amino acid residue.

134. 134. The antibody construct of any one of claims 108 to 133, wherein the IgA heavy chain constant domain lacks at least one naturally occurring isoleucine (I) amino acid residue compared to the corresponding wild type IgA.

135. 135. The antibody construct of claim 134, wherein the IgA heavy chain constant domain comprises an amino acid substitution of at least one naturally occurring isoleucine (I) amino acid residue.

136. The antibody construct of claim 135, wherein the IgA heavy chain constant region comprises a deletion of at least one naturally occurring isoleucine (I) amino acid residue.

137. 137. The antibody construct of any one of claims 108 to 136, wherein the IgA heavy chain constant domain lacks at least one naturally occurring proline (P) amino acid residue compared to the corresponding wild type IgA.

138. The antibody construct of claim 137, wherein the IgA heavy chain constant region comprises an amino acid substitution of at least one naturally occurring proline (P) amino acid residue.

139. The antibody construct of claim 138, wherein the IgA heavy chain constant domain comprises a deletion of at least one naturally occurring proline (P) amino acid residue.

140. 140. The antibody construct of any one of claims 119 to 139, wherein the antibody construct exhibits a longer circulating half-life compared to a corresponding antibody construct comprising a corresponding wild type IgA heavy chain constant region.

141. An antibody construct according to any one of claims 119 to 140, wherein the antibody construct exhibits a longer half-life compared to a corresponding antibody construct comprising a corresponding wild type IgA heavy chain constant region.

142. 142. The antibody construct of any one of claims 119 to 141, wherein the antibody construct exhibits reduced aggregation compared to a corresponding antibody construct comprising a corresponding wild type IgA heavy chain constant region.

143. The antibody construct according to any one of claims 119 to 142, wherein the antibody construct exhibits reduced aggregation with serum proteins compared to a corresponding antibody construct comprising a corresponding wild-type IgA heavy chain constant region. The antibody constructs described above.

144. An antibody construct according to any one of claims 1 to 143, wherein the IgA heavy chain constant region comprises one or more albumin binding domains.

145. The antibody construct of claim 144, wherein the antibody construct has a longer half-life than a corresponding antibody construct that does not comprise one or more albumin binding domains.

146. An antibody construct according to any one of claims 88 to 145, wherein the IgA heavy chain constant region further comprises a hinge region.

147. The antibody construct of claim 146, wherein the hinge region comprises an IgA hinge amino acid sequence or a variant or fragment thereof.

148. The antibody construct of claim 147, wherein the hinge region comprises a human IgA hinge amino acid sequence or a variant or fragment thereof.

149. 149. The antibody construct of claim 147 or 148, wherein the hinge is an IgA1 hinge or an IgA2 hinge, or a variant or fragment thereof.

150. The antibody construct of any one of claims 1 to 149, wherein the antibody construct further comprises an immunoadhesion molecule, an imaging agent, a therapeutic agent, or a cytotoxic agent.

151. The antibody construct of claim 150, wherein the imaging agent is a radioactive label, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, or biotin.

152. The antibody construct of claim 150, wherein the therapeutic or cytotoxic agent is an antimetabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an antiangiogenic agent, an antimitotic agent, an anthracycline, a toxin, or an apoptotic agent.

153. 89. The antibody construct of claim 1 or claim 88, wherein the immune effector cell is a neutrophil.

154. 90. The antibody construct of claim 1 or claim 88, wherein the antibody construct inhibits signal regulatory protein alpha (SIRP a) function on an immune effector cell only if the antibody construct also binds the antigen binding domain to a target cell expressing CD47.

155. 89. The antibody construct of claim 1 or claim 88, wherein the antibody construct is bispecific.

156. 90. The antibody construct of claim 1 or claim 88, wherein the CD47 binding domain is a Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, diabody, linear antibody, single domain antibody (sdAb), or camelid VHH domain.

157. 89. The antibody construct of claim 1 or claim 88, wherein the antigen binding domain is a Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, diabody, linear antibody, single domain antibody (sdAb) or camelid VHH domain.

158. An antibody construct according to any one of claims 153 to 157, wherein the IgA heavy chain domain is a heterodimer of two IgA heavy chain constant domains.

159. The antibody construct of claim 158, wherein the heterodimerization is by knobs-into-hole coupling, salt bridge / electrostatic complementation coupling, CrossMab coupling, strand exchange engineered domain technology, or a combination thereof.

160. An antibody construct according to any one of claims 153 to 159, wherein the antigen binding domain binds to an antigen of a cancer cell or a pathogen.

161. The antibody construct of claim 160, wherein the pathogen is a pathogen, a microorganism, or a virus.

162. (a) Immunoglobulin A (IgA) heavy chain domain; (b) a CD47 binding domain; and (c) an antigen-binding domain; An antibody construct comprising: the IgA heavy chain domain specifically binds to FcαR on immune effector cells; the CD47 binding domain inhibits binding of CD47 expressed on a target cell to signal regulatory protein alpha (SIRP alpha) on an immune effector cell; the antigen-binding domain binds to an antigen on a target cell; the antigen is CD20, GD2, mesothelin, CD38, CD19, EGFR, HER2, PD-L1, or CD25, and The antibody construct has a higher binding affinity for the antigen compared to CD47. The antibody construct.

163. (a) Immunoglobulin A (IgA) heavy chain domain; (b) a CD47 binding domain; and (c) an antigen-binding domain; 1. A multispecific immune effector cell engager molecule comprising: the IgA heavy chain domain specifically binds to FcαR on immune effector cells; the CD47 binding domain inhibits binding of CD47 expressed on a target cell to signal regulatory protein alpha (SIRP alpha) on an immune effector cell; the antigen-binding domain binds to an antigen on a target cell; and The antibody construct has a higher binding affinity for the antigen compared to CD47. The multispecific immune effector cell engager molecule.

164. An antibody construct according to any one of claims 1 to 161, and a pharma- ceutically acceptable carrier, adjuvant or diluent; 23. A pharmaceutical composition comprising:

165. The pharmaceutical composition of claim 164, further comprising at least one additional therapeutic agent.

166. The pharmaceutical composition of claim 165, wherein the additional therapeutic agent is an imaging agent, a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor, a costimulatory molecule blocker, an adhesion molecule blocker, an anti-cytokine antibody or a functional fragment thereof, methotrexate, cyclosporine, rapamycin, FK506, a detectable label or reporter, a TNF antagonist, an antirheumatic drug, a muscle relaxant, a narcotic, a nonsteroidal anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocking agent, an antibacterial agent, an antipsoriatic drug, a corticosteroid, an anabolic steroid, erythropoietin, immunization, an immunoglobulin, an immunosuppressant, a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, epinephrine or an analog thereof, a cytokine, or a cytokine antagonist.

167. An isolated nucleic acid encoding an antibody construct according to any one of claims 1 to 161.

168. A vector comprising the isolated nucleic acid of claim 167.

169. 168. An in vitro cell comprising the isolated nucleic acid of claim 167.

170. An in vitro cell expressing an antibody construct according to any one of claims 1 to 161.

171. 1. A method of treating a subject in need thereof comprising administering to the subject an effective amount of a composition comprising an antibody construct, the antibody construct comprising: (a) Immunoglobulin A (IgA) heavy chain domain; (b) a CD47 binding domain; and (c) an antigen-binding domain; Including, the IgA heavy chain domain specifically binds to FcαR on immune effector cells; the CD47 binding domain inhibits binding of CD47 expressed on a target cell to signal regulatory protein alpha (SIRP alpha) on an immune effector cell; the antigen-binding domain binds to an antigen on a target cell; and The antibody construct has a higher binding affinity for the antigen compared to CD47. The method of treatment.

172. Inhibition of CD47 binding with SIRPα increases phagocytosis and clearance of target cells The method of claim 171,

173. 173. The method of any one of claims 171 to 172, wherein the composition is administered orally, intralesionally, by intravenous therapy, or by subcutaneous, intramuscular, intraarterial, intravenous, intracavitary, intracranial, or intraperitoneal injection.

174. 174. The method of any one of claims 171-173, wherein the composition is administered daily, weekly, biweekly, monthly, bimonthly, once every three months, once every six months, or once every twelve months.

175. The method of any one of claims 171 to 174, wherein the subject has cancer.

176. The method of claim 175, wherein the cancer is selected from the group consisting of acute lymphoblastic leukemia, acute myeloid leukemia, biliary tract cancer, B-cell leukemia, B-cell lymphoma, biliary tract cancer, bone cancer, brain cancer, breast cancer, triple-negative breast cancer, cervical cancer, Burkitt's lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastric cancer, gastrointestinal cancer, glioma, hairy cell leukemia, head and neck cancer, Hodgkin's lymphoma, liver cancer, lung cancer, medullary thyroid cancer, melanoma, multiple myeloma, ovarian cancer, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, pulmonary tract cancer, renal cancer, sarcoma, skin cancer, testicular cancer, urothelial cancer, and bladder cancer.

177. The method of any one of claims 171 to 176, wherein the subject is a human.

178. 178. The method of any one of claims 171 to 177, comprising administering an effective amount of at least one additional therapeutic agent.

179. The additional therapeutic agent is an imaging agent, a chemotherapeutic agent, a kinase inhibitor, a costimulatory molecule blocker, an adhesion molecule blocker, a second antibody or an antigen-binding fragment thereof, a drug, a toxin, an enzyme, or a cytotoxic agent. , antiangiogenic drugs, proapoptotic drugs, antibiotics, hormones, immune modulators, cytokines, chemokines, antisense oligonucleotides, small interfering RNA (siRNA), methotrexate, cyclosporine, rapamycin, FK506, detectable labels or reporters, TNF antagonists, antirheumatic drugs, muscle relaxants, narcotics, nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, local anesthetics, neuromuscular blocking drugs, antibacterial drugs, antipsoriatic drugs, corticosteroids, anabolic steroids, erythropoietin, immunization, immunoglobulins, immunosuppressants, growth hormones, hormone replacement drugs, radiopharmaceuticals, antidepressants, or antipsychotics.

180. 1. A method of inducing a neutrophil-mediated immune response against a target cell, comprising contacting the target cell with an effective amount of an antibody construct, The antibody construct, (a) Immunoglobulin A (IgA) heavy chain domain; (b) a CD47 binding domain; and (c) antigen-binding domain Including; the IgA heavy chain domain specifically binds to FcαR on neutrophils; the CD47 binding domain inhibits binding of CD47 expressed on a target cell to signal regulatory protein alpha (SIRP alpha) on a neutrophil; the antigen-binding domain binds to an antigen on a target cell; and The antibody construct has a higher binding affinity for the antigen compared to CD47, thereby inducing a neutrophil-mediated immune response; The method.

181. The method of claim 180, wherein the neutrophil-mediated immune response comprises phagocytosis of target cells or lysis of target cells.

182. The method of any one of claims 180 to 181, wherein the antigen-presenting cell is a cancer cell or a viral cell.

183. 183. The method of claim 182, wherein the cancer cell is a lymphocyte.

184. 163. The antibody construct of any one of claims 1 to 162, wherein the CD47 binding domain comprises a first light chain variable domain comprising variable light chain complementarity determining region 1 (CDR-L1), variable light chain complementarity determining region 2 (CDR L2) and variable light chain complementarity determining region 3 (CDR-L3), wherein CDR-L1, CDR-L2 and CDR-L3 comprise an amino acid sequence selected from Table A.

185. 185. The antibody construct of any one of claims 1 to 162 or claim 184, wherein the CD47 binding domain comprises a first heavy chain variable domain comprising variable light chain complementarity determining region 1 (CDR-H1), variable light chain complementarity determining region 2 (CDR H2) and variable light chain complementarity determining region 3 (CDR-H3), wherein CDR-H1, CDR-H2 and CDR-H3 comprise an amino acid sequence selected from Table A.

186. 186. The antibody construct according to any one of claims 1 to 162 or claims 184 to 185, wherein the antigen binding domain comprises a second light chain variable domain comprising variable light chain complementarity determining region 1 (CDR-L1), variable light chain complementarity determining region 2 (CDR L2) and variable light chain complementarity determining region 3 (CDR-L3), wherein CDR-L1, CDR-L2 and CDR-L3 comprise an amino acid sequence selected from Table B.

187. The antibody construct according to any one of claims 1 to 162 or claims 184 to 186, wherein the antigen binding domain comprises a second heavy chain variable domain comprising variable light chain complementarity determining region 1 (CDR-H1), variable light chain complementarity determining region 2 (CDR H2) and variable light chain complementarity determining region 3 (CDR-H3), wherein CDR-H1, CDR-H2 and CDR-H3 comprise an amino acid sequence selected from Table B.

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