Multispecific molecules for immunoglobulin clearance in the treatment of autoantibody-induced diseases

Abstract

The present invention relates to multispecific molecules that bind to immunoglobulins and recycling targets. Binding of the immunoglobulin to a recycling target results in degradation of the immunoglobulin and, in certain embodiments, recycling of the multispecific molecule. The multispecific molecules of the invention are believed to be useful in the treatment of autoantibody-induced diseases.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 419,549, filed October 26, 2022, which is incorporated herein by reference in its entirety.

[0002] Statement regarding electronically submitted text files This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated herein by reference in its entirety. A computer-readable copy of this Sequence Listing, created on October 25, 2023, is designated 10179-WO01-SEC_ST26 and is 72.1 kilobytes in size.

[0003] The present invention relates to the field of autoantibody-induced diseases. The present invention relates to multispecific molecules, such as bispecific scFv molecules, that bind to immunoglobulins and recycling targets. The multispecific molecules of the present invention are useful in the treatment of autoantibody-induced diseases. [Background technology]

[0004] Autoantibody-induced immune disorders affect more than 2.5% of the world's population (Lenti et al., Autoimmunity Rev. Sept. 2022;21(9):103143). Due to immune system deficiencies, antibodies generated against self-antigens, known as autoantibodies, induce pathogenic effects through various mechanisms, such as blocking function at the binding site, modifying the antigen transport mechanism, degrading the antigen, and activating complement. Autoantibodies play a central role in disease pathology, and therefore, considerable efforts are being expended to inhibit their production or deplete them from circulation.

[0005] In various autoimmune disorders, autoantibodies are primarily IgG subclasses, which bind to FcRn (neonatal Fc receptor) via their constant region (Fc) in a pH-dependent manner. This FcRn:IgG pH-dependent interaction allows internalized IgG in early endosomes to bind to FcRn, allowing the IgG to be transported back to the cell surface. IgG has a long serum half-life due to its salvage from lysosomal degradation. Therefore, blocking the FcRn:Fc interaction increases IgG shunting to the lysosomal compartment, thereby enhancing IgG degradation.

[0006] A wide variety of FcRn inhibitors have been developed and are either approved or in late-stage clinical trials for the treatment of autoantibody-mediated disorders. Blockade of the FcRn:Fc interaction has been demonstrated to reduce overall IgG levels in both clinical and preclinical animal models. Although FcRn inhibitors have demonstrated clinical efficacy by depleting circulating IgG, IgG depletion does not occur immediately after inhibitor administration. The effect of FcRn inhibitors depends on IgG endocytosis, a rate-limiting process. Clinical studies have observed that it takes approximately 3 to 4 weeks after FcRn inhibitor administration for approximately 50% depletion of total and antigen-specific IgG to be achieved. Additionally, FcRn inhibitors must be administered frequently and / or at high doses to achieve their efficacy.

[0007] To overcome this delay and induce rapid depletion of serum IgG, multispecific molecules of the present invention have been engineered. These multispecific molecules bind to immunoglobulins and recycling targets, resulting in rapid lysosomal degradation of the immunoglobulins. Multispecific molecules can be pH / Ca2+ dependent or independent. In addition to rapid immunoglobulin depletion, multispecific molecules of the present invention are expected to be advantageous in clearing immunoglobulins such as IgG by abolishing IgG complex formation, thereby resulting in reduced off-target effects. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Lenti et al.,Autoimmunity Rev.Sept.2022;21(9):103143 Summary of the Invention [Means for solving the problem]

[0009] In one aspect, the present invention relates to a multispecific molecule comprising a first binding domain and a second binding domain, wherein the first binding domain specifically binds to an immunoglobulin and the second binding domain specifically binds to a recycling target. In certain embodiments, the first binding domain is an scFv, Fv, scFab, Fab', or Fab, and the second binding domain is an scFv, Fv, scFab, Fab', or Fab. In particular embodiments, the first binding domain and / or the second binding domain is an scFv. In certain such embodiments, the first binding domain and the second binding domain are each scFv. In particular embodiments, the first binding domain and / or the second binding domain are Fv. In certain such embodiments, the first binding domain and the second binding domain are each Fv. In certain embodiments, the first binding domain and / or the second binding domain are scFab. In certain such embodiments, the first binding domain and the second binding domain are each scFab. In certain embodiments, the first binding domain and / or the second binding domain is a Fab. In certain such embodiments, the first binding domain and the second binding domain are each a Fab. In certain embodiments, the first binding domain is an scFv and the second binding domain is a Fab. In certain embodiments, the first binding domain is an scFv and the second binding domain is an scFab. In certain embodiments, the first binding domain is a Fab and the second binding domain is an scFv. In certain embodiments, the first binding domain is an scFab and the second binding domain is an scFv. In certain embodiments, the first binding domain is a Fab and the second binding domain is an scFab. In certain embodiments, the first binding domain is an scFab and the second binding domain is a Fab. In certain embodiments, the first binding domain is an scFv and the second binding domain is an Fv. In certain embodiments, the first binding domain is an Fv and the second binding domain is an scFv.In certain embodiments, the first binding domain is an scFab and the second binding domain is an Fv. In certain embodiments, the first binding domain is an Fv and the second binding domain is an scFab. In certain embodiments, the first binding domain is an Fv and the second binding domain is a Fab. In certain embodiments, the first binding domain is a Fab and the second binding domain is an Fv. In certain embodiments, the first binding domain is an scFv and the second binding domain is a Fab'. In certain embodiments, the first binding domain is a Fab' and the second binding domain is an scFv. In certain embodiments, the first binding domain is a Fab' and the second binding domain is an Fv. In certain embodiments, the first binding domain is an Fv and the second binding domain is a Fab'. In certain embodiments, the first binding domain is a Fab' and the second binding domain is an scFab. In certain embodiments, the first binding domain is an scFab and the second binding domain is a Fab'. In certain embodiments, the first binding domain and the second binding domain are each a Fab'. In certain embodiments, the first binding domain is a Fab and the second binding domain is a Fab'. In certain embodiments, the first binding domain is a Fab' and the second binding domain is a Fab.

[0010] In certain embodiments, the single-chain polypeptide comprises a first binding domain and a second binding domain.

[0011] In certain embodiments, the first binding domain and the second binding domain are linked via a linker. In certain embodiments, the linker is a polypeptide linker. In certain embodiments, the linker is an SG4S linker. In detailed embodiments, the SG4S linker comprises one SG4S linking the two binding domains. In other embodiments, the SG4S linker comprises two SG4S repeats. In certain embodiments, the SG4S linker comprises three SG4S repeats. In other embodiments, the SG4S linker comprises four SG4S repeats. In other embodiments, the SG4S linker comprises five SG4S repeats. In other embodiments, the SG4S linker comprises six SG4S repeats. In other embodiments, the SG4S linker comprises seven or more SG4S repeats. In certain embodiments, the linker is selected from the group consisting of (Gly3Ser)3 (SEQ ID NO:76), (Gly4Ser)3 (SEQ ID NO:77), (Gly3Ser)4 (SEQ ID NO:78), (Gly4Ser)4 (SEQ ID NO:79), (Gly3Ser)5 (SEQ ID NO:80), (Gly4Ser)5 (SEQ ID NO:81), (Gly3Ser)6 (SEQ ID NO:82), (Gly4Ser)6 (SEQ ID NO:83), GSADDAKKDAAKKDAAKKDDAKKDDAGS (SEQ ID NO:84), GSADDAKKDAAKKDAAKKDDAKKDDAKKDAGS (SEQ ID NO:85), The nucleic acid molecule comprises a sequence selected from the group consisting of (Gly3Gln)2 (SEQ ID NO: 86), (Gly4Gln)2 (SEQ ID NO: 87), (Gly3Gln)3 (SEQ ID NO: 88), (Gly4Gln)3 (SEQ ID NO: 89), (Gly3Gln)4 (SEQ ID NO: 90), (Gly4Gln)4 (SEQ ID NO: 91), (Gly3Gln)5 (SEQ ID NO: 92), (Gly4Gln)5 (SEQ ID NO: 93), (Gly3Gln)6 (SEQ ID NO: 94), (Gly4Gln)6 (SEQ ID NO: 95), (Gly3Ser)2 (SEQ ID NO: 96) and (Gly4Ser)2 (SEQ ID NO: 97).

[0012] In certain embodiments, the first binding domain specifically binds to an immunoglobulin. In certain embodiments, the first binding domain specifically binds to a recycling target. In certain embodiments, the second binding domain specifically binds to an immunoglobulin. In certain embodiments, the second binding domain specifically binds to a recycling target.

[0013] In certain embodiments, a multispecific molecule of the invention specifically binds to an immunoglobulin, and the bound immunoglobulin is IgG, IgA, IgE, IgD, or IgM. In certain embodiments, the bound immunoglobulin is IgG. In certain embodiments, the bound immunoglobulin is IgA. In certain embodiments, the bound immunoglobulin is IgE. In certain embodiments, the bound immunoglobulin is IgD. In certain embodiments, the bound immunoglobulin is IgM. In certain embodiments, the bound immunoglobulin is expressed on B cells. In certain embodiments, the bound immunoglobulin is expressed on plasma cells. In certain embodiments, the bound immunoglobulin is circulating in the blood.

[0014] In certain embodiments, the multispecific molecules of the invention specifically bind to a recycling target, and the recycling target is ASGR1. In certain embodiments, the recycling target is the transferrin receptor. In certain embodiments, the recycling target is the mannose-6-phosphate receptor.

[0015] In specific embodiments, the multispecific molecules of the invention deplete at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of bound immunoglobulins in vivo. In particular embodiments, the multispecific molecules deplete about 50% to about 70% of the immunoglobulins. In particular embodiments, the multispecific molecules deplete at least 50% of the immunoglobulins. In particular embodiments, the multispecific molecules deplete at least 55% of the immunoglobulins. In particular embodiments, the multispecific molecules deplete at least 60% of the immunoglobulins. In particular embodiments, the multispecific molecules deplete at least 65% of the immunoglobulins. In particular embodiments, the multispecific molecules deplete at least 70% of the immunoglobulins. In particular embodiments, the multispecific molecules deplete at least 75% of the immunoglobulins. In particular embodiments, the multispecific molecules deplete at least 80% of immunoglobulins. In particular embodiments, the multispecific molecules deplete at least 85% of immunoglobulins. In particular embodiments, the multispecific molecules deplete at least 90% of immunoglobulins. In particular embodiments, the multispecific molecules deplete at least 95% of immunoglobulins. In particular embodiments, the multispecific molecules deplete at least 98% of immunoglobulins. In particular embodiments, the multispecific molecules deplete at least 99% of immunoglobulins. In particular embodiments, the multispecific molecules deplete 100% of immunoglobulins. In particular embodiments, the immunoglobulins are depleted in mice. In particular embodiments, the immunoglobulins are depleted in non-human primates. In particular embodiments, the immunoglobulins are depleted in human patients. In particular embodiments, the immunoglobulin is IgG. In particular embodiments, the immunoglobulin is IgA. In particular embodiments, the immunoglobulin is IgE. In certain embodiments, the immunoglobulin is IgD. In certain embodiments, the immunoglobulin is IgM. In certain embodiments, the immunoglobulin is depleted in less than 96 hours after administration. In certain embodiments, the immunoglobulin is depleted in less than 72 hours after administration.In certain embodiments, the immunoglobulin is depleted within 3 to 96 hours of administration. In certain embodiments, the immunoglobulin is depleted within 3 to 72 hours of administration.

[0016] In certain embodiments, the multispecific molecules of the invention bind to the recycling target in a catabolic manner and to Ig in a non-catabolic manner.

[0017] In certain embodiments, the multispecific molecules of the invention bind to recycling targets in a catabolic manner and bind to Ig in a catabolic manner.

[0018] In certain embodiments, the multispecific molecules of the invention bind to the recycling target in a non-catabolic manner and to Ig in a catabolic manner.

[0019] In certain embodiments, the multispecific molecules of the invention remain bound to the immunoglobulin and the recycling target.

[0020] In certain embodiments, the multispecific molecules of the invention dissociate from the immunoglobulin and the recycling target in the endosomes of cells expressing the recycling target.

[0021] In certain embodiments, the multispecific molecules of the invention dissociate from the recycling target in the endosomes of cells expressing the recycling target.

[0022] In certain embodiments, the multispecific molecules of the invention dissociate from the immunoglobulin in the endosome of cells expressing the recycling target, hi certain such embodiments, the multispecific molecule remains bound to the recycling target in the endosome and is recycled to the cell surface of cells expressing the recycling target.

[0023] In another aspect, the present invention relates to an antibody that specifically binds to ASGR1, comprising a heavy chain (HC) and a light chain (LC), wherein the HC comprises a heavy chain variable region (HCVR), and the LC comprises a light chain variable region (LCVR), and the LCVR comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence given by SEQ ID NO: 1, HCDR2 comprises the amino acid sequence given by SEQ ID NO: 2, HCDR3 comprises the amino acid sequence given by SEQ ID NO: 3, LCDR1 comprises the amino acid sequence given by SEQ ID NO: 4, LCDR2 comprises the amino acid sequence given by SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence given by SEQ ID NO: 6. In certain embodiments, the HCVR comprises the amino acid sequence given by SEQ ID NO: 7. In certain embodiments, the LCVR comprises the amino acid sequence given by SEQ ID NO: 8. In certain embodiments, the HC comprises the amino acid sequence given by SEQ ID NO: 9. In certain embodiments, the LC comprises the amino acid sequence given by SEQ ID NO: 10. In particular embodiments, the antibodies of the present invention are non-catabolic.

[0024] In particular embodiments, a multispecific molecule of the invention comprises a binding domain that specifically binds to ASGR1, said binding domain comprising an HCVR and an LCVR, wherein the HCVR comprises HCDR1, HCDR2 and HCDR3, and wherein the LCVR comprises LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises the amino acid sequence given in SEQ ID NO: 1, HCDR2 comprises the amino acid sequence given in SEQ ID NO: 2, HCDR3 comprises the amino acid sequence given in SEQ ID NO: 3, LCDR1 comprises the amino acid sequence given in SEQ ID NO: 4, LCDR2 comprises the amino acid sequence given in SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence given in SEQ ID NO: 6. In particular embodiments, the HCVR comprises the amino acid sequence given in SEQ ID NO: 7. In particular embodiments, the LCVR comprises the amino acid sequence given in SEQ ID NO: 8. In particular embodiments, the multispecific molecule of the invention is non-catabolic.

[0025] The present invention provides antibodies that specifically bind to ASGR1, comprising a heavy chain (HC) and a light chain (LC), wherein the HC comprises a heavy chain variable region (HCVR), and the LC comprises a light chain variable region (LCVR), and the LCVR comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence given by SEQ ID NO: 11, HCDR2 comprises the amino acid sequence given by SEQ ID NO: 12, HCDR3 comprises the amino acid sequence given by SEQ ID NO: 13, LCDR1 comprises the amino acid sequence given by SEQ ID NO: 14, LCDR2 comprises the amino acid sequence given by SEQ ID NO: 15, and LCDR3 comprises the amino acid sequence given by SEQ ID NO: 16. In certain embodiments, the HCVR comprises the amino acid sequence given by SEQ ID NO: 17. In certain embodiments, the LCVR comprises the amino acid sequence given by SEQ ID NO: 18. In certain embodiments, the HC comprises the amino acid sequence given by SEQ ID NO: 19. In certain embodiments, the LC comprises the amino acid sequence given by SEQ ID NO: 20. In particular embodiments, the antibodies of the present invention are catabolic.

[0026] In particular embodiments, a multispecific molecule of the invention comprises a binding domain that specifically binds to ASGR1, said binding domain comprising an HCVR and an LCVR, wherein the HCVR comprises HCDR1, HCDR2 and HCDR3, and wherein the LCVR comprises LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises the amino acid sequence given by SEQ ID NO: 11, HCDR2 comprises the amino acid sequence given by SEQ ID NO: 12, HCDR3 comprises the amino acid sequence given by SEQ ID NO: 13, LCDR1 comprises the amino acid sequence given by SEQ ID NO: 14, LCDR2 comprises the amino acid sequence given by SEQ ID NO: 15, and LCDR3 comprises the amino acid sequence given by SEQ ID NO: 16. In particular embodiments, the HCVR comprises the amino acid sequence given by SEQ ID NO: 17. In particular embodiments, the LCVR comprises the amino acid sequence given by SEQ ID NO: 18. In particular embodiments, the multispecific molecule of the invention is catabolic.

[0027] The present invention provides antibodies that specifically bind to ASGR1, comprising an HCVR and an LCVR, wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, and the LCVR comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence given by SEQ ID NO: 27, HCDR2 comprises the amino acid sequence given by SEQ ID NO: 28, HCDR3 comprises the amino acid sequence given by SEQ ID NO: 29, LCDR1 comprises the amino acid sequence given by SEQ ID NO: 30, LCDR2 comprises the amino acid sequence given by SEQ ID NO: 31, and LCDR3 comprises the amino acid sequence given by SEQ ID NO: 32. In certain embodiments, the HCVR comprises the amino acid sequence given by SEQ ID NO: 35. In certain embodiments, the LCVR comprises the amino acid sequence given by SEQ ID NO: 36. In particular embodiments, the antibodies of the invention are non-catabolic.

[0028] In particular embodiments, a multispecific molecule of the invention comprises a binding domain that specifically binds to ASGR1, said binding domain comprising an HCVR and an LCVR, wherein the HCVR comprises HCDR1, HCDR2 and HCDR3, wherein the LCVR comprises LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises the amino acid sequence given by SEQ ID NO: 27, HCDR2 comprises the amino acid sequence given by SEQ ID NO: 28, HCDR3 comprises the amino acid sequence given by SEQ ID NO: 29, LCDR1 comprises the amino acid sequence given by SEQ ID NO: 30, LCDR2 comprises the amino acid sequence given by SEQ ID NO: 31, and LCDR3 comprises the amino acid sequence given by SEQ ID NO: 32. In particular embodiments, the HCVR comprises the amino acid sequence given by SEQ ID NO: 35. In particular embodiments, the LCVR comprises the amino acid sequence given by SEQ ID NO: 36. In particular embodiments, the multispecific molecule of the invention is non-catabolic.

[0029] In another aspect, the invention relates to an antibody that specifically binds to ASGR1, comprising an HCVR and an LCVR, wherein the HCVR comprises HCDR1, HCDR2, and HCDR3; and the LCVR comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence given by SEQ ID NO: 38, HCDR2 comprises the amino acid sequence given by SEQ ID NO: 39, HCDR3 comprises the amino acid sequence given by SEQ ID NO: 40, LCDR1 comprises the amino acid sequence given by SEQ ID NO: 41, LCDR2 comprises the amino acid sequence given by SEQ ID NO: 42, and LCDR3 comprises the amino acid sequence given by SEQ ID NO: 43. In certain embodiments, the HCVR comprises the amino acid sequence given by SEQ ID NO: 44. In certain embodiments, the LCVR comprises the amino acid sequence given by SEQ ID NO: 45. In particular embodiments, the antibodies of the invention are catabolic.

[0030] In particular embodiments, a multispecific molecule of the invention comprises a binding domain that specifically binds to ASGR1, said binding domain comprising an HCVR and an LCVR, wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, and wherein the LCVR comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence given in SEQ ID NO: 38, HCDR2 comprises the amino acid sequence given in SEQ ID NO: 39, HCDR3 comprises the amino acid sequence given in SEQ ID NO: 40, LCDR1 comprises the amino acid sequence given in SEQ ID NO: 41, LCDR2 comprises the amino acid sequence given in SEQ ID NO: 42, and LCDR3 comprises the amino acid sequence given in SEQ ID NO: 43. In particular embodiments, the HCVR comprises the amino acid sequence given in SEQ ID NO: 44. In particular embodiments, the LCVR comprises the amino acid sequence given in SEQ ID NO: 45. In particular embodiments, the multispecific molecule of the invention is catabolic.

[0031] The present invention provides antibodies that specifically bind to IgG, comprising an HCVR and an LCVR, wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, and the LCVR comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence given by SEQ ID NO: 21, HCDR2 comprises the amino acid sequence given by SEQ ID NO: 22, HCDR3 comprises the amino acid sequence given by SEQ ID NO: 23, LCDR1 comprises the amino acid sequence given by SEQ ID NO: 24, LCDR2 comprises the amino acid sequence given by SEQ ID NO: 25, and LCDR3 comprises the amino acid sequence given by SEQ ID NO: 26. In certain embodiments, the HCVR comprises the amino acid sequence given by SEQ ID NO: 33. In certain embodiments, the LCVR comprises the amino acid sequence given by SEQ ID NO: 34. In certain embodiments, the HC comprises the amino acid sequence given by SEQ ID NO: 51. In certain embodiments, the LC comprises the amino acid sequence given by SEQ ID NO: 52.

[0032] In certain embodiments, a multispecific molecule of the invention comprises a binding domain that specifically binds IgG, said binding domain comprising an HCVR and an LCVR, wherein the HCVR comprises HCDR1, HCDR2, and HCDR3, and wherein the LCVR comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence given by SEQ ID NO: 21, HCDR2 comprises the amino acid sequence given by SEQ ID NO: 22, HCDR3 comprises the amino acid sequence given by SEQ ID NO: 23, LCDR1 comprises the amino acid sequence given by SEQ ID NO: 24, LCDR2 comprises the amino acid sequence given by SEQ ID NO: 25, and LCDR3 comprises the amino acid sequence given by SEQ ID NO: 26. In certain embodiments, the HCVR comprises the amino acid sequence given by SEQ ID NO: 33. In certain embodiments, the LCVR comprises the amino acid sequence given by SEQ ID NO: 34.

[0033] In certain embodiments, a multispecific molecule of the invention comprises a binding domain that specifically binds to ASGR1 and a binding domain that specifically binds to an immunoglobulin. In certain embodiments, the binding domain that specifically binds to an immunoglobulin specifically binds to IgG, IgM, IgA, IgD, or IgE. In particular embodiments, the binding domain specifically binds to IgG. In other particular embodiments, the binding domain specifically binds to IgA. In certain such embodiments, the binding domain that specifically binds to ASGR1 and the binding domain that specifically binds to IgG are of the invention. In particular embodiments, the binding domain that specifically binds to ASGR1 and / or an immunoglobulin is an scFv, scFab, Fab', and / or Fab.

[0034] In a particular embodiment, the multispecific molecule of the invention comprises the amino acid sequence given by SEQ ID NO:37.

[0035] In a particular embodiment, the multispecific molecule of the invention comprises the amino acid sequence given by SEQ ID NO:46.

[0036] In a particular embodiment, the multispecific molecule of the invention comprises the amino acid sequence given by SEQ ID NO:75.

[0037] In certain embodiments, multispecific molecules of the invention comprise an HCDR1 comprising SEQ ID NO: 59, an HCDR2 comprising SEQ ID NO: 60, an HCDR3 comprising SEQ ID NO: 61, an LCDR1 comprising SEQ ID NO: 62, an LCDR2 comprising SEQ ID NO: 63, and an LCDR3 comprising SEQ ID NO: 64. In certain embodiments, multispecific molecules of the invention comprise an HCVR comprising SEQ ID NO: 71 and an LCVR comprising SEQ ID NO: 72. In certain embodiments, multispecific molecules of the invention comprise an HC comprising SEQ ID NO: 53 and an LC comprising SEQ ID NO: 54. In certain embodiments, the multispecific molecule is an antibody. In certain embodiments, the multispecific molecule is an scFab. In certain embodiments, the multispecific molecule is an scFv. In certain embodiments, the multispecific molecule further comprises a binding arm that binds to a recycling target. In certain embodiments, the recycling target is ASGR1.

[0038] In certain embodiments, multispecific molecules of the invention comprise an HCDR1 comprising SEQ ID NO: 65, an HCDR2 comprising SEQ ID NO: 66, an HCDR3 comprising SEQ ID NO: 67, an LCDR1 comprising SEQ ID NO: 62, an LCDR2 comprising SEQ ID NO: 63, and an LCDR3 comprising SEQ ID NO: 64. In certain embodiments, multispecific molecules of the invention comprise an HCVR comprising SEQ ID NO: 73 and an LCVR comprising SEQ ID NO: 72. In certain embodiments, multispecific molecules of the invention comprise an HC comprising SEQ ID NO: 55 and an LC comprising SEQ ID NO: 56. In certain embodiments, multispecific molecules are antibodies. In certain embodiments, multispecific molecules are scFabs. In certain embodiments, multispecific molecules are scFvs. In certain embodiments, multispecific molecules further comprise a binding arm that binds to a recycling target. In certain embodiments, the recycling target is ASGR1.

[0039] In certain embodiments, multispecific molecules of the invention comprise an HCDR1 comprising SEQ ID NO: 68, an HCDR2 comprising SEQ ID NO: 69, an HCDR3 comprising SEQ ID NO: 70, an LCDR1 comprising SEQ ID NO: 62, an LCDR2 comprising SEQ ID NO: 63, and an LCDR3 comprising SEQ ID NO: 64. In certain embodiments, multispecific molecules of the invention comprise an HCVR comprising SEQ ID NO: 74 and an LCVR comprising SEQ ID NO: 72. In certain embodiments, multispecific molecules of the invention comprise an HC comprising SEQ ID NO: 57 and an LC comprising SEQ ID NO: 58. In certain embodiments, the multispecific molecule is an antibody. In certain embodiments, the multispecific molecule is an scFab. In certain embodiments, the multispecific molecule is an scFv. In certain embodiments, the multispecific molecule further comprises a binding arm that binds to a recycling target. In certain embodiments, the recycling target is ASGR1.

[0040] Also provided herein are one or more nucleic acid sequences encoding an antibody of the invention or a multispecific molecule of the invention. In certain embodiments, the invention provides a DNA molecule comprising a polynucleotide encoding the HC or HCVR of an antibody or multispecific molecule of the invention. The invention also provides a DNA molecule comprising a polynucleotide encoding the LC or LCVR of an antibody or multispecific molecule of the invention. The invention also provides a DNA molecule comprising a polynucleotide encoding both the LC or LCVR of an antibody or multispecific molecule of the invention and the HC or HCVR of an antibody or multispecific molecule of the invention.

[0041] The present invention further provides a mammalian cell transformed with a DNA molecule of the present invention, wherein the transformed mammalian cell is capable of expressing an antibody or multispecific molecule of the invention.

[0042] The invention also provides a method for producing an antibody or multispecific molecule of the invention, comprising culturing mammalian cells under conditions such that the antibody or multispecific molecule is expressed, and recovering the expressed antibody or multispecific molecule. In one embodiment, the mammalian cell is transformed with a DNA molecule of the invention, such that the transformed mammalian cell is capable of expressing the antibody or multispecific molecule of the invention. The invention also provides the antibody or multispecific molecule obtained by this method.

[0043] The present invention provides multispecific molecules of the invention for use in therapy.

[0044] The present invention provides multispecific molecules of the invention for use in the treatment of autoantibody-induced diseases.

[0045] The present invention provides a multispecific molecule of the invention for the manufacture of a medicament for the treatment of an autoantibody-induced disease.

[0046] In certain embodiments, the autoantibody-induced disease is selected from the group consisting of myasthenia gravis, Guillain-Barré syndrome, epilepsy, autoimmune limbic encephalitis, spinal cord injury, pediatric autoimmune neuropsychiatric disorder associated with streptococcal infection, neuromyotonia, Morvan's syndrome, multiple sclerosis, pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, epidermolysis bullosa acquisita, pemphigoid of gestationis, mucous membrane pemphigoid, lichen sclerosus, antiphospholipid syndrome, relapsing polychondritis, autoimmune anemia, idiopathic thrombocytotic purpura, autoimmune Graves' disease, dilated cardiomyopathy, vasculitis, Goodpasture's syndrome, idiopathic membranous nephropathy, rheumatoid arthritis, and systemic lupus erythematosus.

[0047] The present invention provides methods of treating a patient having at least one autoantibody-induced disease, comprising administering to the patient an effective amount of a multispecific molecule of the invention. In certain embodiments, the patient has at least one autoantibody-induced disease. In certain embodiments, the patient has at least one of myasthenia gravis, Guillain-Barré syndrome, epilepsy, autoimmune limbic encephalitis, spinal cord injury, pediatric autoimmune neuropsychiatric disorder associated with streptococcal infection, neuromyotonia, Morvan's syndrome, multiple sclerosis, pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, epidermolysis bullosa acquisita, pemphigoid gestationis, mucous membrane pemphigoid, lichen sclerosus, antiphospholipid syndrome, relapsing polychondritis, autoimmune anemia, idiopathic thrombocytotic purpura, autoimmune Graves' disease, dilated cardiomyopathy, vasculitis, Goodpasture's syndrome, idiopathic membranous nephropathy, rheumatoid arthritis, and systemic lupus erythematosus.

[0048] The present invention also provides pharmaceutical compositions comprising a multispecific molecule of the invention and one or more pharmaceutically acceptable carriers, diluents or excipients. [Brief explanation of the drawings]

[0049] [Figure 1A] Figure 1 illustrates the difference in clearance of non-CAT and CAT anti-ASGR1 antibodies in human FcRn Tg mice. Mice were intravenously administered non-catabolic (Figure 1A) or catabolic (Figure 1B) antibodies, and total antibody concentrations were measured over time. Individual measurements are indicated by symbols for antibody doses of 0.3 mg / kg (▼), 3 mg / kg (▲), 10 mg / kg (■), and 30 mg / kg (●). Figure 1C illustrates the data from Figure 1B from time zero to 24 hours. [Figure 1B]Figure 1 illustrates the difference in clearance of non-CAT and CAT anti-ASGR1 antibodies in human FcRn Tg mice. Mice were intravenously administered non-catabolic (Figure 1A) or catabolic (Figure 1B) antibodies, and total antibody concentrations were measured over time. Individual measurements are indicated by symbols for antibody doses of 0.3 mg / kg (▼), 3 mg / kg (▲), 10 mg / kg (■), and 30 mg / kg (●). Figure 1C illustrates the data from Figure 1B from time zero to 24 hours. [Figure 1C] Figure 1 illustrates the difference in clearance of non-CAT and CAT anti-ASGR1 antibodies in human FcRn Tg mice. Mice were intravenously administered non-catabolic (Figure 1A) or catabolic (Figure 1B) antibodies, and total antibody concentrations were measured over time. Individual measurements are indicated by symbols for antibody doses of 0.3 mg / kg (▼), 3 mg / kg (▲), 10 mg / kg (■), and 30 mg / kg (●). Figure 1C illustrates the data from Figure 1B from time zero to 24 hours. [Figure 2] 1 illustrates the simultaneous binding of ASGR1 and IVIg to an immobilized multispecific molecule of the invention. [Figure 3]

[0033] Figure 1 illustrates the rapid depletion of exogenously administered human IgG in mice after administration of a catabolic bispecific scFv molecule of the invention. Mice were administered human IgG followed by administration of either a bispecific scFv molecule of the invention (bispecific scFv) or PBS. The concentration-time profile of human IgG in mouse serum was plotted by quantifying the human IgG concentration by ELISA after administration of either a bispecific scFv molecule of the invention or PBS. [Figure 4] 1 illustrates the rapid depletion of exogenously administered human IgG in cynomolgus monkeys after administration of a catabolic bispecific scFv molecule of the invention. The concentration-time profile of human IgG in cynomolgus monkey serum was plotted by quantifying human IgG concentration by ELISA after administration of either a bispecific scFv molecule of the invention or PBS. [Figure 5A]Figure 5 illustrates that bispecific scFv molecules of the invention that bind ASGR1 in a catabolic and noncatabolic manner are equally effective in depleting huIgG in vivo. Seventy-two hours after administration of huIVIg, animals were administered PBS or either a catabolic bispecific scFv molecule of the invention or a noncatabolic bispecific scFv molecule of the invention. Normalized serum (Figure 5A) and whole body (Figure 5B) radioactivity counts from mice administered I-125-labeled huIVIg are shown. [Figure 5B] Figure 5 illustrates that bispecific scFv molecules of the invention that bind ASGR1 in a catabolic and noncatabolic manner are equally effective in depleting huIgG in vivo. Seventy-two hours after administration of huIVIg, animals were administered PBS or either a catabolic bispecific scFv molecule of the invention or a noncatabolic bispecific scFv molecule of the invention. Normalized serum (Figure 5A) and whole body (Figure 5B) radioactivity counts from mice administered I-125-labeled huIVIg are shown. [Figure 6-1] Illustrated are bispecific scFv molecules of the invention that drive IgG to the liver for rapid catabolism. Mice were administered IVIg labeled with I-125 (left column) or In-111 (right column), and 72 hours later, the animals were administered either a catabolic bispecific scFv molecule of the invention or PBS. At 3, 24, or 96 hours after administration of the clearance agent, the animals were perfused and organs were harvested, and their radioactivity counts were measured and plotted. [Figure 6-2] Illustrated are bispecific scFv molecules of the invention that drive IgG to the liver for rapid catabolism. Mice were administered IVIg labeled with I-125 (left column) or In-111 (right column), and 72 hours later, the animals were administered either a catabolic bispecific scFv molecule of the invention or PBS. At 3, 24, or 96 hours after administration of the clearance agent, the animals were perfused and organs were harvested, and their radioactivity counts were measured and plotted. DETAILED DESCRIPTION OF THE INVENTION

[0050] The present invention provides multispecific molecules that bind to an immunoglobulin and a recycling target. In certain embodiments, the multispecific molecule comprises a first binding domain that specifically binds to an immunoglobulin and a second binding domain that specifically binds to a recycling target. The multispecific molecule bound to the immunoglobulin and the recycling target is internalized into the cell, followed by degradation of the immunoglobulin in the lysosome. In certain embodiments, the recycling target remains bound to the multispecific molecule and is recycled back to the cell surface. The multispecific molecule can then bind to another immunoglobulin, which it internalizes and degrades. In certain embodiments, the recycling target is recycled back to the cell surface after detaching from the multispecific molecule inside the cell.

[0051] Multispecific molecules of the present invention comprise at least two binding domains. The binding domains are antigen-binding portions of antibodies or binding domains derived from antigen-binding portions of antibodies. Any binding domain is considered suitable, provided that it specifically binds to either an immunoglobulin or a recycling target. In certain embodiments, the binding domain lacks an Fc region. Examples of binding domains include scFv, Fab, scFab, Fab', Fv, and dsFv. Multispecific molecules comprising binding domains can be in formats such as F(ab')2, (scFv-Zip)2, (scFv)2 (e.g., BiTE® molecules), diabodies, scDbs, and tandem diabodies. Multispecific molecules comprising binding domains derived from camelid antibodies are also contemplated. In certain embodiments, the multispecific molecules comprising binding domains derived from camelid antibodies comprise at least one camelid VH and at least one camelid VL, at least two camelid VHs, or at least two camelid VLs. In certain embodiments, the molecule further comprises a half-life extending (HLE) moiety. UniDab® molecules are also contemplated. The binding domains and multispecific molecules of the invention can be produced by well-known methods (see, e.g., Kipriyanov SM (2003); Recombinant Antibodies for Cancer Therapy; 207(3-26); Janssens et al, October 10, 2006; PNAS; 103(41)15130-15135).

[0052] Non-limiting examples of HLE moieties include Fc polypeptides, single-chain Fc polypeptides (scFc), albumin, albumin fragments, moieties that bind to albumin or the neonatal Fc receptor (FcRn), derivatives of fibronectin engineered to bind to albumin or a fragment thereof, peptides, single-domain protein fragments, or other polypeptides capable of increasing serum half-life. In other embodiments, the half-life extending moiety can be a non-polypeptide molecule, such as, for example, polyethylene glycol (PEG). In certain embodiments, the HLE is a single-chain Fc ("scFc").

[0053] The present invention provides bispecific scFv molecules that bind to an immunoglobulin and a recycling target, comprising an scFv that specifically binds to an immunoglobulin (the "first scFv") and an scFv that specifically binds to a recycling target (the "second scFv"), linked via a linker.

[0054] As used herein, the term "recycling target" refers to a protein that is internalized from the cell surface into the cell and then returned to the cell surface. Thus, a recycling target is one that is recycled to the cell surface. Protein internalization can occur through endocytosis, which can occur by a variety of mechanisms. In a general sense, endocytosis begins with the formation of endocytic vesicles that carry cargo that has been endocytosed into the cell, and the cargo is then delivered to early endosomes. The cargo then proceeds to late endosomes and lysosomes for degradation, to the trans-Golgi network (TGN), or to recycling endosomal carriers that return the cargo to the plasma membrane. In this context, as it relates to the present invention, cargo refers to a recycling target to which the multispecific molecule of the present invention binds, which also binds to immunoglobulins. A recycling target is a receptor that can be rapidly internalized into the cell (endosome) and recycled back to the cell surface. Examples of recycling targets include, but are not limited to, asialoglycoprotein receptor 1 (ASGR1), transferrin receptor, and mannose 6-phosphate receptor.

[0055] ASGR1 is a membrane-bound receptor expressed in hepatocytes and composed of ASGPR1 and ASGPR2 subunits. ASGR1 removes desialylated glycoproteins from the circulation by receptor-mediated endocytosis. ASGR1 has been shown to have a receptor recycling time of approximately 10-15 minutes in human cells. ASGR1 has been used for liver-specific delivery of compounds, including small molecules (see, e.g., Willoughby et al., Mol Ther. 2018 Jan 3;26(1):105-114). In certain embodiments, ASGR1 is a recycling target. ASGR1 is highly expressed on the cell surface of hepatocytes, which has extremely fast internalization and recycling rates, which can facilitate depletion of high antigen loads. Because ASGR1 is expressed in the liver and antigen catabolism occurs in the liver, the expected toxicity of using ASGR1 as a recycling target is expected to be tolerable.

[0056] The pH of early endosomes is about 6.5-6.0, the pH of late endosomes is about 5.5, and lysosomes have a pH of about 4.0. The multispecific molecules of the invention cause immunoglobulin (Ig) depletion (or clearance) in lysosomes. Immunoglobulin depletion can be measured by assays known in the art, including immunoassays, blood radioactivity, and flow cytometry assays.

[0057] In certain embodiments, the multispecific molecules are said to be catabolic ("CAT"). A catabolic molecule is a molecule that either detaches from a recycling target in an endosome (a "recycling-targeted catabolic molecule") and / or detaches from an Ig in an endosome. In certain embodiments, the recycling-targeted catabolic molecule remains bound to the Ig (e.g., a molecule that binds to ASGR1 in a catabolic manner and binds to Ig in a non-catabolic manner). In certain embodiments, the recycling-targeted catabolic molecule also dissociates from the Ig (e.g., a molecule that binds to ASGR1 in a catabolic manner and binds to Ig in a catabolic manner). In certain embodiments, the catabolic molecule dissociates from the Ig but remains bound to the recycling target (e.g., a molecule that binds to ASGR1 non-catabolic and binds to Ig catabolic). Upon detachment from the recycling target and / or Ig, the Ig is degraded in the lysosome. In certain embodiments, the recycling target is recycled back to the cell surface. In certain such embodiments, where the multispecific molecule binds to the recycling target in a non-catabolic manner and binds to the Ig in a catabolic manner, such that the multispecific molecule is recycled along with the recycling target, it is expected that lower doses of these catabolic molecules will be given to patients for the treatment of autoantibody-induced diseases.

[0058] Catabolic molecules are sensitive to the low pH in endosomes, which causes dissociation of the multispecific molecule and Ig from the recycling target (or dissociation of Ig from the multispecific molecule). Catabolic molecules may demonstrate, for example, reduced binding to ASGR1 (and / or Ig) at pH 6.0 and 2 μM calcium chloride. Molecules that exhibit high affinity for recycling targets and / or Ig at neutral pH / high calcium concentrations, but no detectable binding at acidic pH / low calcium concentrations, can accelerate the degradation (catabolism) of Ig and are therefore termed catabolic.

[0059] In certain embodiments, the multispecific molecules are said to be non-catabolic ("non-CAT"). A non-CAT molecule is a multispecific molecule that remains bound to the recycling target and Ig, and the complex (non-CAT molecule, recycling target, and Ig) is recycled back to the cell surface. In contrast to catabolic molecules, non-catabolic molecules are pH insensitive and therefore do not dissociate from the recycling target or Ig in the endosome. A non-catabolic molecule may, for example, demonstrate similar binding to ASGR1 and Ig at pH 6.0 and 2 μM calcium chloride compared to neutral pH (pH 7.4) and 2 mM calcium chloride. In certain embodiments, the recycling target and / or non-catabolic molecule and / or Ig are degraded.

[0060] According to certain embodiments of the invention, the binding domain that specifically binds to Ig (e.g., IgG) is non-catabolic, and the binding domain that specifically binds to ASGR1 is catabolic. In other embodiments, the binding domain that specifically binds to Ig (e.g., IgG) is catabolic, and the binding domain that specifically binds to ASGR1 is non-catabolic. In other embodiments, the binding domain that specifically binds to Ig (e.g., IgG) is non-catabolic, and the binding domain that specifically binds to ASGR1 is non-catabolic.

[0061] In certain embodiments, the bispecific scFv molecules of the present invention comprise an scFv that specifically binds to a recycling target and an scFv that specifically binds to an immunoglobulin. The bispecific scFv molecules of the present invention can be single-chain polypeptides comprising a first scFv-linker-second scFv. The scFv, or single-chain variable fragment, is composed of the variable domains of an antibody heavy and light chain, which can be linked together by a short peptide linker. For example, a (G4S)3 linker can be used with any number of repeats, such as 1 to 4. Each scFv can be VL-linker-VH or VH-linker-VL, from the N-terminus to the C-terminus.

[0062] According to one embodiment of the present invention, the multispecific molecules of the invention are single-chain molecules. The two domains of the binding domain, VL and VH, are encoded by separate genes, but using recombinant methods, they may be joined by an artificial linker—as described above—which allows them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule; see, e.g., Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883.

[0063] An example of a bispecific scFv molecule that can be engineered from an scFab that binds mIgG2a in a catabolic manner and that binds ASGR1 in a non-catabolic manner is SEQ ID NO: 75. To improve stability, one or more cysteines may be further introduced (see, e.g., Reiter et al., Biochemistry 1994, 33, 5451-5459), which is not expected to alter the catabolic properties of the molecule.

[0064] Conversion of the scFab molecule to a bispecific scFv molecule is not expected to alter the catabolic properties of the molecule.

[0065] Binding domains are obtained using conventional techniques known to those skilled in the art, and such binding domains are evaluated for functionality in the same manner as full-length antibodies or IgGs. Thus, for example, scFvs are fusion proteins of the variable regions of immunoglobulin heavy (VH) and light (VL) chains, usually linked by a short linker peptide. The linker is usually glycine-rich for flexibility and serine- or threonine-rich for solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. The protein retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of the linker.

[0066] Bispecific single-chain molecules are known in the art and are described in WO 99 / 54440, Mack, J. Immunol. (1997), 158, 3965-3970, Mack, PNAS, (1995), 92, 7021-7025, Kufer, Cancer Immunol. Immunother., (1997), 45, 193-197, Loffler, Blood, (2000), 95, 6, 2098-2103, Bruhl, Immunol., (2001), 166, 2420-2426, and Kipriyanov, J. Mol. Biol., (1999), 293, 41-56. Techniques described for the production of single chain antibody constructs (see, inter alia, U.S. Pat. No. 4,946,778; Kontermann and Diibel (2010), supra; and Little (2009), supra) can be applied to the production of single chain antibody constructs that specifically recognize one or more selected targets.

[0067] Bivalent (also called divalent) or bispecific single-chain variable fragments (bi-scFv or di-scFv) with format (scFv)2 can be engineered by linking two scFv molecules (e.g., with a linker as described above). Linking can be performed by creating a single polypeptide chain containing two VH and two VL regions, resulting in a tandem scFv (see, for example, Kufer P. et al., (2004) Trends in Biotechnology 22(5):238-244). Another possibility is the creation of scFv molecules containing a linker peptide such that the two variable regions are too short (e.g., about 5 amino acids) to fold together, forcing the scFvs to dimerize. In this case, the VH and VL binding domains (which bind either immunoglobulins or recycling targets) are not directly linked, but rather via a peptide linker. Thus, for example, the VH of an immunoglobulin binding domain can be fused via a peptide linker to the VL of a recycling target binding domain, which in turn is fused via a peptide linker to the VL of an immunoglobulin binding domain, a type known as a diabody (see, e.g., Hollinger, Philipp et al., (July 1993) Proceedings of the National Academy of Sciences of the United States of America 90(14):6444-8).

[0068] In certain embodiments, multispecific molecules of the invention, such as bispecific scFv molecules, comprise two binding domains (e.g., scFvs) linked to each other by a linker. The linker linking the two binding domains can be, for example, a helical linker or a flexible linker. The term "peptide linker" as used herein includes an amino acid sequence that links the amino acid sequences of one (variable and / or binding) domain and another (variable and / or binding) domain of a multispecific molecule of the invention. Among suitable peptide linkers are those described in U.S. Pat. Nos. 4,751,180 and 4,935,233 or WO 88 / 09344.

[0069] An example of a peptide linker connecting two binding domains is an SG4S linker. Multispecific molecules of the invention can have 1, 2, 3, 4, 5, or 6 repeats of the SG4S linker. For example, two SG4S repeats would result in binding domain (e.g., scFv)-SGGGGSSGGGGS-binding domain (e.g., scFv) (SGGGGSSGGGGS as given by SEQ ID NO: 98). In certain embodiments, multispecific molecules of the invention have one SG4S as a linker. In other embodiments, the linker contains 2, 3, or 4 SG4S repeats. In other embodiments, the linker contains 5 or 6 SG4S repeats. In other embodiments, the linker contains 7 or more SG4S repeats, as long as the multispecific molecule can be expressed and purified. Other examples of linkers include (Gly3Ser)3 (SEQ ID NO: 76), (Gly4Ser)3 (SEQ ID NO: 77), (Gly3Ser)4 (SEQ ID NO: 78), (Gly4Ser)4 (SEQ ID NO: 79), (Gly3Ser)5 (SEQ ID NO: 80), (Gly4Ser)5 (SEQ ID NO: 81), (Gly3Ser)6 (SEQ ID NO: 82), (Gly4Ser)6 (SEQ ID NO: 83), GSADDAKKDAAKKDAAKKDDAKKDDAGS (SEQ ID NO: 84), GSADDAKKDAAKKDAAKKDDAKKDDAKKDAGS (SEQ ID NO: 85), (Gly3G (Gly4Gln) (SEQ ID NO: 86), (Gly4Gln) (SEQ ID NO: 87), (Gly3Gln) (SEQ ID NO: 88), (Gly4Gln) (SEQ ID NO: 89), (Gly3Gln) (SEQ ID NO: 90), (Gly4Gln) (SEQ ID NO: 91), (Gly3Gln) (SEQ ID NO: 92), (Gly4Gln) (SEQ ID NO: 93), (Gly3Gln) (SEQ ID NO: 94), (Gly4Gln) (SEQ ID NO: 95), (Gly3Ser) (SEQ ID NO: 96), and (Gly4Ser) (SEQ ID NO: 97).

[0070] As used herein, an "antibody" is an immunoglobulin molecule comprising two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. The amino-terminal portion of each LC and HC contains a variable region of approximately 100-120 amino acids that is primarily responsible for antigen recognition via the CDRs contained therein. Interspersed between the CDRs are more highly conserved regions called framework regions ("FRs"). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDRs of the LC are referred to as "LCDR1, LCDR2, and LCDR3," and the three CDRs of the HC are referred to as "HCDR1, HCDR2, and HCDR3."

[0071] The CDRs contain most of the residues that form specific interactions with the antigen, and therefore the functional ability of an antibody to bind to a specific antigen is largely influenced by the amino acid residues within these six CDRs.

[0072] A binding domain characterized in the context of the present invention is a domain that specifically binds to / interacts with / recognizes a given target epitope or a given target site of a target molecule (antigen), here an immunoglobulin or recycling target. The structure and function of the first and second binding domains are based on or derived from the structure and / or function of an antibody; more particularly, these binding domains are derived from or derived from the variable heavy (VH) and variable light (VL) domains of an antibody. In a particular embodiment, the binding domain is characterized by the presence of three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VL region) and three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH region). The assignment of amino acids to CDR domains within the LCVR and HCVR regions of the antibodies of the present invention described herein is based on a known numbering convention called AHo (A. Honegger & A. Plueckthun. "Yet another numbering scheme for immunoglobulin variable domains: An automatic modeling and analysis tool", J. Mol. Biol., 309 (2001) 657-670).Other numbering conventions, such as the Kabat numbering convention (Kabat, et al., Ann. NY Acad. Sci. 190:382-93 (1971); Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242 (1991)); Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins", Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)) and / or North (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 273, 927-948 (1997)) may be used. Biology, 406, 228-256 (2011)) can be used.

[0073] As used herein, an antigen-binding domain (and / or multispecific molecule) is defined as a binding domain and / or multispecific molecule that binds to its antigen with a binding affinity of ≦10 as measured by surface plasma resonance (e.g., BIACore, GE-Healthcare Uppsala, Sweden) or equilibrium exclusion assay (KinExA, Sapidyne, Boise, ID). -6 When an antibody binds with a dissociation constant (KD) of M, it is said to "specifically bind" to that antigen.

[0074] It is envisioned that the binding domains of the present invention can be generated or obtained by phage display or library screening methods, or by grafting CDR sequences from existing monoclonal antibodies onto a scaffold. Catabolic molecules can be obtained by screening for molecules that produce catabolic activity as described herein. Engineering (e.g., in one or more CDRs) can also be used to obtain catabolic molecules. Engineering by phage display and / or histidine scanning can also be used to introduce one or more histidine residues into the CDRs. Histidine has a PK of approximately 6.5, so the acidic conditions in the endosome will disrupt binding, resulting in a catabolic molecule.

[0075] In the context of antibodies, the present invention contemplates antibodies that may have clipping of the C-terminal lysine or cysteine ​​residue of the HC.

[0076] In the context of an antibody or binding domain of the invention, N-terminal glutamine and / or N-terminal glutamic acid may be converted to pyroglutamic acid.

[0077] It is contemplated that the multispecific molecules of the invention will have a format that does not result in self-binding. For example, multispecific molecules engineered to bind IgG and recycling targets must lack an Fc region. In certain embodiments, multispecific molecules of the invention preferably demonstrate monovalent binding to immunoglobulins, as cross-linking of immunoglobulins can activate the immune system and result in side effects such as anaphylaxis.

[0078] As used herein, "multispecific" refers to a molecule comprising at least a first binding domain and a second binding domain, where the first binding domain specifically binds to one antigen (target) and the second binding domain specifically binds to another antigen (target). Thus, multispecific molecules of the present invention have specificity for at least two different antigens or targets. In certain embodiments, a multispecific molecule comprises two or more but not more than two binding domains (each binding domain specifically binding to a different target, e.g., an immunoglobulin and a recycling target), and the multispecific molecule can be referred to as a "bispecific molecule." Thus, a "bispecific scFv molecule," as used herein, refers to a multispecific molecule comprising two scFvs (binding domains) that bind to two different targets (antigens), such as an immunoglobulin and a recycling target (e.g., ASGR1), where one scFv specifically binds to the immunoglobulin and the other scFv specifically binds to the recycling target.

[0079] The multispecific molecules of the present invention bind to and deplete immunoglobulins. Immunoglobulin depletion is believed to be beneficial in treating patients with antibody-mediated autoimmune diseases (also known as autoantibody-induced diseases). As used herein, "patient" refers to a human. Autoantibody-induced diseases occur when the body's immune system is unable to distinguish between self-antigens and non-self-antigens, which causes the immune system to attack normal parts of the body, potentially resulting in damage and / or disease. For example, the immune system may begin producing antibodies that attack the body's own tissues. Examples of treatments for autoimmune diseases include anti-inflammatory drugs, corticosteroids, pain-relieving medications, immunosuppressants, physical therapy, surgery, high-dose immunosuppression, and disease-specific therapy. Other treatments used for autoimmune diseases include plasmapheresis, intravenous Ig (IVIg) administration, and immunoadsorption. However, these treatments are associated with high treatment costs and / or side effects. Clinically, ABDEG® ("antibodies that enhance IgG degradation") molecules, antibody-based FcRn inhibitors, have been shown to deplete human IgG by approximately 50-70% within 2-3 weeks of administration.

[0080] The molecules of the present invention can be readily produced in mammalian cells, non-limiting examples of which include CHO, NSO, HEK293 or COS cells. Host cells are cultured using techniques well known in the art.

[0081] In certain embodiments, the present invention provides a vector comprising a nucleic acid encoding a polypeptide of the present invention or a portion thereof. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors. A vector containing a polynucleotide sequence of interest (e.g., a polynucleotide encoding a polypeptide of a molecule and an expression control sequence) can be transferred into a host cell by well-known methods, which vary depending on the type of cellular host. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors.

[0082] The recombinant expression vector of the present invention can contain a nucleic acid of the present invention in a form suitable for expression of the nucleic acid in a host cell. The recombinant expression vector contains one or more regulatory sequences, selected based on the host cell to be used for expression, operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells (e.g., the SV40 early gene enhancer, the Rous sarcoma virus promoter, and the cytomegalovirus promoter), and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences; Voss et al., 1986, Trends Biochem. Sci. 11:287; Maniatis et al., 1987, Science 236:1237, incorporated herein by reference in its entirety), and those that direct inducible expression of nucleotide sequences in response to particular treatments or conditions (e.g., the metallothionein promoter in mammalian cells and tet- and / or streptomycin-responsive promoters in both prokaryotic and eukaryotic systems, see ibid.). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the desired level of protein expression, and the like. The expression vectors of the invention, when introduced into a host cell, can thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.

[0083] In certain embodiments, the present invention provides a host cell into which a recombinant expression vector of the present invention has been introduced. The host cell can be any prokaryotic or eukaryotic cell. Prokaryotic host cells include gram-negative or gram-positive organisms, such as E. coli or Bacillus. Higher eukaryotic cells include insect cells, yeast cells, and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells or their derivatives, such as Veggie CHO, and related cell lines that grow in serum-free medium (see Rasmussen et al., 1998, Cytotechnology 28:31) or the CHO line DXB-11, which lacks DHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20). Additional CHO cell lines include CHO-K1 (ATCC No. CCL-61), EM9 (ATCC No. CRL-1861), and UV20 (ATCC No. CRL-1862). Additional host cells include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), AM-1 / D cells (described in U.S. Pat. No. 6,210,924), HeLa cells, the BHK (ATCC CRL 10) cell line, the CV1 / EBNA cell line (ATCC CCL 70) derived from the African green monkey kidney cell line CV1 (see McMahan et al., 1991, EMBO J. 10:2821), 293, 293 EBNA, or MSR. Examples of suitable cloning and expression vectors include human embryonic kidney cells such as 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK, or Jurkat cells. Suitable cloning and expression vectors for use in bacterial, fungal, yeast, and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985).

[0084] Typically, expression vectors used in any host cell will include sequences for plasmid maintenance and sequences for cloning and expression of exogenous nucleotide sequences. In certain embodiments, these sequences, collectively referred to as "flanking sequences," will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence with donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element. The leader sequence may include the amino acid sequence of SEQ ID NO: 47 (MDMRVPAQLLGLLLLWLRGARC), which is encoded by SEQ ID NO: 48 (atggacatgagagtgcctgcacagctgctgggcctgctgctgctgtggctgagaggcgccagatgc). The leader sequence may comprise the amino acid sequence of SEQ ID NO: 49 (MAWALLLLTLLTQGTGSWA), which is encoded by SEQ ID NO: 50 (atggcctggg ctctgctgct cctcaccctc ctcactcagg gcacagggtc ctgggcc). The present invention contemplates molecular protein sequences without a leader sequence.

[0085] A variety of protein purification methods can be used to purify proteins, including but not limited to antibodies or binding domains, and such methods are known in the art.

[0086] The molecules of the invention can be biosynthesized, purified, and formulated for administration using well-known methods. For example, suitable host cells, such as HEK 293 or CHO, are either transiently or stably transfected with an expression system for secreting the antibody or binding domain using two vectors, or a predetermined HC:LC or HCVR:LC vector ratio when a single vector system encoding both the heavy and light chains is used. Suitable vectors for the expression and secretion of antibodies or binding domains from these commonly used host cells are well known. After expression and secretion of the antibody or binding domain, the medium is clarified to remove the cells, and the clarified medium is purified using any of a number of commonly used techniques. For example, the medium can be applied to a Protein A or G column equilibrated with a buffer, such as phosphate-buffered saline (pH 7.4). The column is washed to remove nonspecifically bound components. The bound antibody or binding domain is eluted, for example, with a pH gradient (e.g., 0.1 M sodium phosphate buffer pH 6.8 to 0.1 M sodium citrate buffer pH 2.5). The antibody or binding domain fractions are detected, such as by SDS-PAGE, and then pooled. Further purification is optional and depends on the intended use. The antibody or binding domain can be concentrated and / or sterile filtered using common techniques. Materials other than the antibody or binding domain, such as host cell and growth medium components, as well as soluble aggregates and multimers of the antibody or binding domain, can be effectively reduced or removed by common techniques, including size exclusion, hydrophobic interaction, cation exchange, anion exchange, affinity, or hydroxyapatite chromatography. The purity of the antibody or binding domain after these chromatography steps is typically greater than 95%. The product can be frozen at -70°C or lyophilized.

[0087] Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. For stable transfection of mammalian cells, it is known that only a small percentage of cells may integrate the foreign DNA into their genome, depending on the expression vector and transfection technique used. To identify and select such integrants, a gene encoding a selectable marker (e.g., for antibiotic resistance) is generally introduced into the host cells along with the gene of interest. Additional selectable markers include those that confer resistance to drugs, such as G418, hygromycin, and methotrexate. Cells that have been stably transfected with the introduced nucleic acid can be identified by drug selection, among other methods (e.g., cells that have incorporated the selectable marker gene will survive, while other cells will die).

[0088] Polynucleotides encoding the amino acid sequences of the molecules of the present invention can be of any length as appropriate for the desired use or function, can include one or more additional sequences, such as regulatory sequences, and / or can be part of a larger nucleic acid, such as a vector. Those skilled in the art will understand that due to the degeneracy of the genetic code, each of the polypeptide sequences disclosed herein is encoded by numerous other nucleic acid sequences. Mutations can also be introduced into nucleic acids without significantly altering the biological activity of the polypeptides they encode. For example, nucleotide substitutions can be made that lead to amino acid substitutions at non-essential amino acid residues.

[0089] The transformed cells can be cultured under conditions that promote expression of the polypeptide, and the polypeptide can be recovered by conventional protein purification procedures. Polypeptides contemplated for use herein include substantially homogeneous recombinant mammalian polypeptides that are substantially free of contaminating endogenous materials. Cells containing nucleic acids encoding the molecules of the present invention also include hybridomas.

[0090] In certain embodiments, a vector is provided comprising a nucleic acid molecule as described herein. In certain embodiments, the present invention includes a host cell comprising a nucleic acid molecule as described herein. In certain embodiments, a nucleic acid molecule encoding a molecule as described herein is provided. In certain embodiments, a pharmaceutical composition is provided comprising at least one molecule as described herein.

[0091] Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of the invention.

[0092] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino group of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, pp. 79-86), acetylation of N-terminal amines, and amidation of any C-terminal carboxyl group.

[0093] Another type of covalent modification of molecules included within the scope of the present invention involves altering the glycosylation pattern of a protein. As is known in the art, glycosylation patterns can depend both on the sequence of the protein (e.g., the presence or absence of particular glycosylated amino acid residues, discussed below) and the host cell or organism in which the protein is produced. Detailed expression systems are discussed below.

[0094] Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine (although 5-hydroxyproline or 5-hydroxylysine can also be used).

[0095] Immunoglobulins are produced by B cells and plasma cells and are important in the humoral immune response to bacteria, viruses, fungi, parasites, cellular antigens, chemicals, and synthetic substances. Immunoglobulins can be classified as IgG, IgM, IgA, IgD, or IgE based on their heavy chain constant region. Immunoglobulins are primarily expressed by B cells, which then secrete and circulate in the blood (see, e.g., Hoffman et al., Clin J Am Soc Nephrol. 2016 Jan 7;11(1):137-154). As used herein, the phrase "circulating in the blood" refers to immunoglobulins that circulate in the blood after secretion.

[0096] IgM serves as the first line of defense and provides short-term protection. IgA, also known as secretory antibody, is secreted via mucus. IgD and IgE make up a relatively small proportion of serum antibodies but still play a role in the innate immune system and against parasitic infections, respectively.

[0097] IgG is the most abundant immunoglobulin in the body, accounting for approximately 75-80% of antibodies found in plasma. IgG can activate the complement system and also has the longest lifespan of any immunoglobulin. The long half-life of IgG is due to a recycling pathway involving the neonatal fragment crystallizable receptor (FcRn). Therefore, modalities that inhibit FcRn are thought to deplete IgG through lysosomal degradation (see, e.g., Hans-Hartmut et al., J Allergy Clin Immunol. 2020 Sep;146(3):479-491). One such antibody-based FcRn inhibitor is called ABDEG (see, e.g., Challa et al., MAbs. 2013 Sep 1;5(5):655-659). Efgartigimod, an Fc fragment derived from human IgG1 modified using ABDEG technology, has been demonstrated to reduce IgG in humans (Ulrichts et al., J Clin Invest. 2018;128(10):4372-438).

[0098] The multispecific molecules of the present invention are intended to treat autoantibody-mediated diseases. Autoantibody-mediated diseases include, but are not limited to, myasthenia gravis, Guillain-Barré syndrome, epilepsy, autoimmune limbic encephalitis, spinal cord injury, pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection, neuromyotonia, Morvan's syndrome, multiple sclerosis, pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, epidermolysis bullosa acquisita, pemphigoid of gestationis, mucous membrane pemphigoid, lichen sclerosus, antiphospholipid syndrome, relapsing polychondritis, autoimmune anemia, idiopathic thrombocytopenic purpura, autoimmune Graves' disease, dilated cardiomyopathy, vasculitis, Goodpasture's syndrome, idiopathic membranous nephropathy, rheumatoid arthritis, and systemic lupus erythematosus (e.g., Wang L et al., J. Internal Medicine, 2015, 278, 369-395; Ludwig RJ et al., Front Immunol. 2017, 8, 603; and Pruss H, 2021, Nat. Rev. Immunol, 21(12), 798-813).

[0099] Multispecific molecules of the invention or pharmaceutical compositions comprising same can be administered parenterally, non-limiting examples of which are subcutaneous and intravenous administration. Intramuscular, intraarterial, intralesional, and peritoneal bolus injection are other possible routes of administration. Multispecific molecules can also be administered by infusion, e.g., intravenous or subcutaneous. Multispecific molecules of the invention can be administered to patients in single or multiple doses together with a pharmaceutically acceptable carrier, diluent, or excipient. Optionally, the composition additionally comprises one or more physiologically active agents. Pharmaceutical compositions of the invention can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), A. Loyd et al., Pharmaceutical Press) and comprise a multispecific molecule as disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0100] As used interchangeably herein, "treatment," and / or "treating," and / or "treating" are intended to refer to any process that may slow, interrupt, arrest, control, halt, or reverse the progression of the disorders described herein, but does not necessarily indicate the complete disappearance of all disorder symptoms. Treatment includes the administration of the multispecific molecules of the invention for the treatment of a disease or condition in a human that would benefit from the activity of the multispecific molecules of the invention, and includes (a) inhibiting further progression of the disease; and (b) palliating the disease, i.e., causing regression of the disease or disorder or alleviating its symptoms or complications. "Therapy" or "therapeutic agent," as used herein, refers to the treatment of a patient with at least one autoantibody-induced disease.

[0101] A therapeutically effective amount (or dose) of the multispecific molecules of the invention can be administered. As used herein, "effective amount" refers to the amount of a multispecific molecule of the invention, or a pharmaceutical composition comprising such a multispecific molecule, that will elicit the biological or medical response of, or the desired therapeutic effect on, a tissue, system, animal, mammal, or human that a researcher, physician, or other clinician is seeking. An effective amount of a multispecific molecule can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the antibody are outweighed by the beneficial effects of treatment. Such benefits include amelioration of signs or symptoms of cancer. An effective amount of a multispecific molecule of the invention can be administered in a single dose or multiple doses. In determining an effective amount for a patient, several factors will be considered by the attending physician, including, but not limited to, the patient's size (e.g., weight or mass), body surface area, age, and general health; the specific disease or disorder involved; the extent, involvement, or severity of the disease or disorder; the individual patient's response; the particular compound being administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; the use of concomitant medications; and other relevant circumstances known to the physician. [Example]

[0102] Example 1: Antibody binding as determined by surface plasmon resonance Surface plasmon resonance was used to screen for antibodies that bind to ASGR1 in a pH / Ca2+-dependent manner. Antibodies were screened at either 2 mM CaCl2 at pH 7.4 or 2 μM CaCl2 at pH 6.0 and analyzed for binding to ASGR1. By immobilizing the receptor on a chip and using antibodies as the analyte, K D value was determined.

[0103] The binding of anti-ASGR1 antibodies that bind to ASGR1 in a pH / Ca2+-dependent or -independent manner was determined by BIAcore. Antibodies that bind to ASGR1 in a non-catabolic manner comprise the HC amino acid sequence given by SEQ ID NO: 9 and the LC amino acid sequence given by SEQ ID NO: 10. Antibodies that bind to ASGR1 in a catabolic manner comprise the HC amino acid sequence given by SEQ ID NO: 19 and the LC amino acid sequence given by SEQ ID NO: 20.

[0104] The equilibrium binding affinity of the interaction between mouse ASGR1 and anti-ASGR1 antibodies was determined using a BIAcore 3000. Mouse ASGR1 protein was obtained from R&D-systems (catalog number 2755-AS / CF) and immobilized on a CM5 chip using amine coupling chemistry at a density of approximately 1500 RU. A reference flow cell was used on each CM5 chip, which was coupled with coupling buffer only. Antibodies were injected over the immobilized ASGR1 in a 2-fold serial dilution range from 1000 nM to 0.2 nM. To determine pH / Ca-sensitive binding, runs were performed using phosphate-buffered saline (PBS) containing 0.01% (v / v) Tween 20 and 0.05% azide at either pH 7.4 / 2 mM calcium chloride or pH 6.0 / 2 μM calcium chloride. Between each injection cycle, the chip was regenerated using 0.15 M NaCl, 0.1 M glycine, pH 1.5 buffer. Equilibrium dissociation constants were determined using a 1:1 interaction model using BIAevaluation.

[0105] Anti-ASGR1 antibodies that show no difference in binding at either pH 7.4 / 2 mM calcium chloride or pH 6.0 / 2 μM calcium chloride are referred to as non-catabolic antibodies (non-CAT mAbs), while antibodies that show no detectable binding at pH 6.0 / 2 μM calcium chloride are referred to as catabolic antibodies (CAT mAbs).

[0106] These data demonstrate that the CAT mAb and non-CAT mAb have affinities of 20 nM and 6 nM, respectively, for ASGR1 at pH 7.4 and 2 mM CaCl (Table 1). The non-CAT mAb exhibited a binding affinity of 5.5 nM for ASGR1 at low pH / Ca conditions, whereas the CAT mAb had no detectable binding under the conditions analyzed (Table 1).

[0107] [Table 1]

[0108] Example 2: In vivo pharmacokinetics The pharmacokinetic parameters of antibodies observed in homozygous huFcRn Tg32 mice correlate highly with those observed in nonhuman primates and humans (see, e.g., Avery et al., mAbs 2016, 8(6), 1064-1078). To determine the in vivo pharmacokinetics of anti-ASGR1 antibodies, pharmacokinetic experiments were performed in 8- to 12-week-old male or female C57BL / 6 mice harboring the human FcRn gene (homozygous huFcRn Tg32 mice; Jackson laboratory, item number 014565) and wild-type C57BL / 6 mice. Mice were intravenously administered CAT ASGR1 or non-CAT ASGR1 antibodies via the lateral tail vein in 150 μl of the respective antibody buffer. Doses ranged from 0.3 to 30 mg / kg. At the indicated time points, 50 μl of whole blood was collected using a sparse sequential blood collection scheme via submandibular vein puncture using SARSTEDT Microvette® serum separator tubes (n=3 mice / group / time point). Whole blood was allowed to clot for 20 minutes at room temperature, then centrifuged at 11,500 rpm for 15 minutes, and the resulting serum was stored at -70°C until further analysis. Noncompartmental analysis was performed on the concentration-time profile of the administered antibody. AUClast was calculated for each individual animal. Mean values ​​are presented in Table 2 with standard deviations in parentheses.

[0109] The non-CAT-WT antibody administered at 0.3 mg / kg and 3 mg / kg did not exhibit dose-proportional exposure, indicating substantial TMDD (target-mediated drug elimination) clearance (Figure 1A). The rapid decline in antibody concentrations to undetectable levels 24 hours after administration of the 0.3 mg / kg and 3 mg / kg non-CAT-WT antibody indicates rapid binding and degradation of the bound antibody by ASGR1. At the 10 mg / kg dose, the clearance profile was biphasic, with a slight decrease in clearance rate 48 hours after administration. A typical four-phase TMDD-mediated clearance was observed for the antibody administered at 30 mg / kg (Figure 1A). In contrast, the CAT antibody exhibited a modulated clearance profile compared to the non-CAT antibody. At all dose levels, the CAT antibody exhibited an enhanced rapid decline in concentrations within 24 hours after administration (Figure 1B). Twenty-four hours after administration, the CAT antibody clearance rate substantially decreased, suggesting that ASGR1-mediated clearance was minimal during this period. CAT antibodies designed to overcome TMDD were effective at low dose levels (Table 2). At doses of 0.3 mg / kg and 3 mg / kg, the CAT antibody exposure was 84.3- and 58-fold greater than that of the non-CAT antibody, respectively. At the 10 mg / kg dose, both the CAT and non-CAT antibodies had similar exposures (Table 2). However, at the 30 mg / kg dose, the CAT antibody exposure decreased to 0.4-fold that of the non-CAT antibody. The clearance characteristics of both the CAT and non-CAT antibodies suggest that ASGR1-targeting moieties may be utilized to deplete soluble antigens by crosslinking to anti-ASGR1 antibodies.

[0110] [Table 2]

[0111] These data demonstrate that at high administered doses, clearance of CAT antibodies in vivo is rapid compared to non-CAT antibodies.

[0112] Further studies demonstrate simultaneous binding to human IgG and ASGR1 by the multispecific molecules of the invention. Catabolic bispecific scFv was immobilized on a CM5 chip, and the analytes ASGR1 and IVIg were injected sequentially over the immobilized bispecific scFv. ASGR1 was injected at 50 nM and 100 nM, and IVIg was injected at 100 nM. Runs were performed in duplicate. Both runs are presented in Figure 2. Binding of IVIg to the complexed ASGR1:bispecific scFv demonstrates that the bispecific scFv of the invention can simultaneously bind ASGR1 and IVIg.

[0113] Example 3: Format and ease of manufacturing of multispecific molecules Based on the in vivo clearance of catabolic and non-catabolic anti-ASGR1 antibodies, anti-ASGR1 moieties can be used as vehicles for depleting soluble antigens. Targeting autoantibody-mediated diseases requires depletion of circulating human Ig (see, e.g., Howard et al., Neurology 2019, 92(23)). Cross-linking Ig (e.g., IgG) with ASGR1 facilitates Ig clearance).

[0114] While designing the format of the bispecific scFv molecules of the present invention that cross-link ASGR1 and human IgG, two important design principles were considered. First, because the bispecific scFv molecules of the present invention abolish the formation of IgG complexes that activate the immune system and cause anaphylaxis or other immune-mediated side effects (see, e.g., Mayadas et al., Circulation 120(20), 17 Nov. 2009:2012-2024), the moiety targeting IgG binding should be monovalent. Second, the bispecific scFv molecules of the present invention should lack human IgG fragments that can lead to self-binding. For example, if an Fc fragment is targeted for IgG depletion, the bispecific scFv molecules of the present invention should lack the Fc fragment. Similarly, if an Fc fragment is targeted for IgG depletion, the bispecific scFv molecules of the present invention should also lack the Fc fragment.

[0115] To generate bispecific scFvs, the antibody sequence was reformatted to form scFvs consisting of the antibody heavy and light chains fused by a 3x G4S linker, followed by 6x His at the C-terminus. The gene fragment for the scFv was synthesized and cloned into a mammalian stable expression pTT5-derived vector with a puromycin selection marker. Secreted scFv protein was captured directly from the conditioned medium by affinity chromatography using Ni Sepharose Excel resin (GE Healthcare Life Sciences). Further polishing was achieved using either CHT™ Ceramic Hydroxyapatite Type I 40µm resin (Bio-Rad) eluted with a linear sodium phosphate gradient or Source 15S resin (GE Healthcare Life Sciences) eluted with a linear NaCl gradient. The final protein was buffer exchanged by dialysis to the final formulation: 25mM citrate, 75mM arginine, 4% sucrose, pH 7.0. The quality of the final manufactured product was confirmed by mass spectrometry (Agilent 1260 Infinity Binary UHPLC / 6230 Time-of-Flight Mass Spectrometer), HPLC-SEC (Agilent 1100), and endotoxin testing (Charles River EndoSafe MCS). Bispecific scFvs were selected for further in vitro and in vivo experiments. The bispecific scFv molecule that binds to ASGR1 in a non-catabolic manner and to Ig in a non-catabolic manner (non-CAT bispecific scFv molecule) comprises the amino acid sequence given by SEQ ID NO: 37. The bispecific scFv molecule that binds to ASGR1 in a catabolic manner and to Ig in a non-catabolic manner (CAT bispecific scFv molecule) comprises the amino acid sequence given by SEQ ID NO: 46.

[0116] Example 4: Bispecific scFv molecules that simultaneously bind ASGR1 and human IgG To demonstrate simultaneous binding to human IgG and ASGR1 by the bispecific scFv molecules of the present invention, ASGR1 was immobilized on an SPR chip, followed by co-administration of 100 nM of the bispecific scFv molecules of the present invention (containing a binding domain that binds to ASGR1 in a catabolic manner and a binding domain that binds to IgG in a non-catabolic manner) or an anti-ASGR1 antibody (containing a binding domain that binds to ASGR1 in a catabolic manner) with 100 nM IVIg (intravenous immunoglobulin). IVIg contains a pool of immunoglobulins from a large cohort of healthy human volunteers. By immobilizing the receptor on the chip and using the antibody as the analyte, K D value was determined.

[0117] The binding of anti-ASGR1 antibodies that bind to ASGR1 in a pH / Ca2+-dependent or -independent manner was determined by BIAcore. The equilibrium binding affinities of the interactions between (i) mouse or human ASGR1 and anti-ASGR1 antibodies; and (ii) mouse or human ASGR1 and the bispecific scFv molecules of the present invention were determined using a BIAcore 3000. Mouse and human ASGR1 proteins were obtained from R&D-systems (catalog numbers 2755-AS / CF and 4394-AS / CF, respectively) and immobilized on CM5 chips using amine coupling chemistry to a density of approximately 1500 RU. A reference flow cell was used on each CM5 chip, which was coupled with coupling buffer only. Antibodies were injected over the immobilized ASGR1 in a concentration range of 1000 nM to 0.2 nM in two-fold serial dilutions. To determine pH / Ca-sensitive binding, runs were performed using either pH 7.4 / 2 mM calcium chloride or pH 6.0 / 2 μM calcium chloride in phosphate-buffered saline (PBS) containing 0.01% (v / v) Tween 20 and 0.05% azide. Between each injection cycle, the chip was regenerated using 0.15 M NaCl, 0.1 M glycine pH 1.5 buffer. Equilibrium dissociation constants were determined using a 1:1 interaction model using BIAevaluation.

[0118] These data demonstrate that the bispecific scFv molecules of the invention exhibited dose-dependent binding to immobilized ASGR1, and the binding of IVIg to the complexed ASGR1:bispecific scFv molecules of the invention indicates that the bispecific scFv molecules of the invention can simultaneously bind to ASGR1 and IVIg. The anti-hu IgG component of the bispecific scFv molecules of the invention binds to the IgG1, IgG2, and IgG4 subclasses of human IgG. Due to conversion from mAb format to scFv, the bispecific scFv molecules of the invention in scFv format exhibit reduced affinity for ASGR1 (Table 3). These data demonstrate that the bispecific scFv molecules of the invention (bispecific scFv) can simultaneously bind to ASGR1 and human IgG in the bispecific scFv format. Binding of one target to the bispecific scFv does not inhibit binding to the other target.

[0119] [Table 3]

[0120] Example 5: In vivo clearance Administration of IVIg in animals mimics the clinical scenario of circulating IgG in vivo (Schwab I and Nimmerjahn F, Nat. Rev. 2013, 13, 176-189). Moieties that block FcRn-mediated IgG recycling, such as anti-FcRn antibodies or ABDEG, require approximately 4 days to deplete approximately 70-80% of administered human IgG in mice, and ABDEG takes 2-3 weeks to remove approximately 50-70% of IgG in humans (Vaccaro C et al., Nat. Biotechnol. 23, 1283-1288 (2005); Getman KE & Balthasar JP, J. Pharm. Sci. 94, 718-729 (2005); Mezo AR et al., Proc. Natl. Acad. Sci. 2008, 105, 2337-2342; Peter U et al., J. Clin. Invest. 2018, 128(10), 4372-4386; and James FH et al. al., Neurology, 2019, 92(23)).

[0121] To determine the clearance of exogenously administered IVIg in mice and non-human primates, clearance was determined in mice and cynomolgus monkeys that were exogenously administered IVIg and a bispecific scFv molecule of the present invention that binds to ASGR1 with certain pH and calcium sensitivity.

[0122] To analyze human IgG clearance, mice were intravenously administered IVIG (Sigma, Cat. No. 56834), and 72 hours later, mice were administered either a CAT bispecific scFv molecule of the present invention (which binds ASGR1 in a catabolic manner and Ig in a non-catabolic manner) (1.67 μM) or PBS. At the indicated time points, 50 μl of whole blood was collected using a sparse sequential blood collection scheme by submandibular venipuncture using SARSTEDT Microvette® serum separator tubes (n=3 mice / group / time point). Whole blood was allowed to clot for 20 minutes at room temperature before being centrifuged at 11,500 rpm for 15 minutes, and the resulting serum was stored at -80°C until further analysis.

[0123] For cynomolgus monkey analysis, drug-naive female cynomolgus monkeys were administered an intravenous dose of the bispecific scFv molecules of the invention via the saphenous vein, and blood was collected from the femoral vein at the indicated time points into anticoagulant-free blood collection tubes (serum separator tubes). Blood was allowed to clot at ambient temperature and serum was obtained by centrifugation. Centrifugation began within 1 hour of collection. Serum was placed in polypropylene tubes and kept on dry ice before being stored at -80°C.

[0124] For quantification of huIVIG, an ELISA-based assay was used with a mouse anti-human IgG, F(ab')2 specific antibody (Jackson ImmunoResearch Labs, catalog number 209-005-097) as the capture and detection reagent.

[0125] These data demonstrate that human IgG concentrations in mice were reduced by approximately 70% within 3 hours after administration of the CAT bispecific scFv molecules of the invention (Figure 3). A similar enhanced clearance effect of the bispecific scFv molecules of the invention was observed in cynomolgus monkeys, where the bispecific scFv molecules of the invention depleted human IgG by 72% within 12 hours after administration of the bispecific scFv molecules of the invention (Figure 4).

[0126] Example 6: Generation of CAT and non-CAT bispecific scFv molecules As shown in Figure 1, anti-ASGR1 antibodies bound to the receptor in a catabolic manner showed faster in vivo clearance compared to non-catabolic anti-ASGR1 antibodies. This rapid clearance indicates that catabolic anti-ASGR1 accumulates faster in lysosomes. To analyze whether the rapid clearance of the catabolic anti-ASGR1 moiety can lead to faster clearance of human IgG when the bispecific scFv molecules of the present invention bind to ASGR1 in a catabolic manner (and to Ig in a non-catabolic manner), bispecific scFv molecules of the present invention that bind to ASGR1 in a catabolic and non-catabolic manner were tested in vivo. The bispecific scFv molecule that binds to ASGR1 in a non-catabolic manner comprises the amino acid sequence given by SEQ ID NO: 37. The bispecific scFv molecule that binds to ASGR1 in a catabolic manner comprises the amino acid sequence given by SEQ ID NO: 46.

[0127] Human IgG (IVIg) was labeled with Na125I (Perkin Elmer, catalog number NEZ033L) to determine radioactivity-based pharmacokinetics. Mice were injected with 1.67 μM human IVIg via the tail vein, and the animals' radioactivity was immediately measured using a dose calibrator (Capintec, catalog number CRC-15R) for initial (T=0) systemic activity. Mice were then injected with 1.67 μM of the bispecific scFv molecule of the present invention via the tail vein. The animals' systemic activity was read at the indicated time points, and blood samples were then taken by tail vein puncture and collected into capillary tubes. All capillary tubes were weighed before and after collection to determine the exact blood weight / volume collected. Serum samples were analyzed for radioactivity using a gamma counter.

[0128] As shown in Figure 5, the bispecific scFv molecules of the present invention, whether bound to ASGR1 in a catabolic or non-catabolic manner, exhibited similar efficacy in depleting serum IgG and its catabolism in vivo, suggesting that pH / Ca2+-dependent binding to ASGR1 did not significantly affect the bispecific scFv molecules of the present invention in terms of clearance of exogenously administered human IgG in mice.

[0129] Example 7: Bispecific scFv molecules induced IgG hepatic catabolism ASGR1 is expressed primarily in hepatocytes, both on the plasma membrane and in the cytoplasm, particularly on the limiting membrane of endosomes. Ligands that target ASGR1 are predicted to accumulate in the liver and be catabolized there.

[0130] To analyze the catabolism of exogenously administered IVIg in mice after administration of a targeting molecule, IVIg is labeled with the non-persistent and persistent radiolabels I-125 and In-111, respectively. Iodine, a non-persistent dye, is secreted from cells upon cleavage from IgG during IgG degradation and subsequently undergoes renal clearance. However, indium is a persistent dye, and therefore remains in cells even after cleavage from IgG. Therefore, similar levels of I-125 and In-111 indicate no catabolism, while different levels indicate hepatic catabolism of IgG.

[0131] Radiolabeled IVIg was administered to mice via tail vein injection, and the initial (T=0) systemic activity of the animals was measured using a dose calibrator (Capintec, catalog number CRC-15R). 72 hours later, the animals were administered either a CAT bispecific scFv molecule of the present invention (which binds to ASGR1 in a catabolic manner and to Ig in a non-catabolic manner) or PBS. The animals were read for systemic activity at the indicated time points, and then blood samples were taken by tail vein puncture and collected into capillary tubes. All capillary tubes were weighed before and after collection to determine the exact blood weight / volume collected. The radioactivity of serum samples was analyzed using a gamma counter. After the animals were perfused, the organs were subsequently removed, weighed, and radioactivity was measured.

[0132] These data, shown in Figure 6, demonstrate that by 3 hours after administration of the targeting molecule, the majority of circulating human IgG was localized to the liver, as indicated by the radioactivity of the residually labeled In-111 compared to the PBS control group. The accumulated human IgG was rapidly catabolized by hepatic lysosomes, as indicated by the radioactivity signal of the non-retained I-125 label, which was approximately 1% ID / g at all time points. Clearance of antigens, such as Ig, from the circulation via liver delivery may be less toxic than clearance of antigens by other mechanisms.

[0133] Example 8: Catabolic IgG molecules We constructed a molecule that binds to ASGR1 in a noncatabolic manner and to IgG in a catabolic manner. This binding property to ASGR1 and IgG may allow continuous recycling of the molecule by binding to ASGR1 while dissociating IgG in the endosomal compartment. The selection of mouse IgG2a as the antigen allowed us to test the constructed molecule in an in vivo disease model expressing autoantibodies of the IgG2a subclass.

[0134] Rabbits were immunized with mouse IgG2a, and B cells were collected from the spleens of immunized animals. The collected cells were screened by analyzing binding to mouse IgG2a, IgG2b, IgG2c, IgG1, and an irrelevant antigen in a FACS-based multiplex assay. Clones that specifically bound to mouse IgG2a were identified and further screened for their catabolic binding by analyzing binding at 2 μM CaCl2 at pH 6.0 and 2 mM CaCl2 at pH 7.4. For binders exhibiting the desired catabolic binding properties, antibody heavy and light chain sequences were extracted, and the binders were converted to Fab-scFc format (anti-mIgG2a). Their catabolic binding affinity for binding to mouse IgG2a was analyzed in BIAcore and Octet-based assays.

[0135] For the Octet-based assay, streptavidin biosensors were loaded with avidin-coupled mouse IgG2a, and the association and dissociation of the conjugates were performed under acidic and neutral pH conditions as described above. The biosensors were regenerated with 10 mM glycine buffer at pH 1.5.

[0136] Similarly, for the BIAcore-based assay, cross-adsorbed anti-mouse IgG (H+L) was amine-coupled to a CM5 sensor chip, and mouse IgG2a was captured as the ligand, while the resulting conjugate was injected as the analyte in either 2 mM CaCl2 at neutral pH or 2 μM CaCl2 at acidic pH. The analyte was injected in 3-fold serial dilutions ranging from a highest concentration of 900 nM. The CM5 chip was regenerated with 10 mM glycine buffer at pH 1.5. Binding analysis was performed using a 1:1 Langmuir fit in BIAevaluation.

[0137] Octet data for molecule 099 (comprising an HC comprising SEQ ID NO: 53 and an LC comprising SEQ ID NO: 54) is shown in Table 4. Molecule 099 exhibited tight binding to mouse IgG2a at neutral pH and 2 mM CaCl2, while exhibiting reduced affinity (approximately 3-fold) for mouse IgG2a at acidic pH and 2 μM CaCl2. The weaker affinity for antigen at acidic pH corresponds to more efficient release of the antigen in endosomes.

[0138] To enhance pH-dependent binding, molecule 099 was further engineered by introducing histidine residues into the CDR regions. Two resulting clones (465, comprising an HC comprising SEQ ID NO:55 and an LC comprising SEQ ID NO:56; and 463, comprising an HC comprising SEQ ID NO:57 and an LC comprising SEQ ID NO:58) demonstrated further decreased binding to mouse IgG2a at pH 6.0 and 2 μM CaCl, while retaining tight binding at neutral pH and 2 mM CaCl (Table 5). The resulting sensograms (BIAcore data) demonstrated similar catabolic binding properties to mouse IgG2a as determined by the Octet assay.

[0139] [Table 4]

[0140] [Table 5]

[0141] These data demonstrate that the tested molecules bind IgG in a catabolic manner. The molecules show reduced binding to mouse IgG2a at 2 μM CaCl at pH 6.0, while retaining tight binding at 2 mM CaCl at neutral pH.

[0142] These conjugates were converted to an scFv-scFv format by converting them to scFv and fusing them with an anti-mouse / human ASGR1 scFv (e.g., SEQ ID NO: 75). An additional cysteine(s) was introduced to improve the stability of the bispecific scFv molecules. These bispecific scFvs were analyzed for binding to mouse / human ASGR1 and mouse IgG2a in a flow-based assay, and it was determined that binding to ASGR1 and IgG2a at neutral pH was maintained upon conversion from Fab to scFv format.

[0143] array Non-catabolic anti-ASGR1 antibody HCDR1 (SEQ ID NO: 1) DYNMA Non-catabolic anti-ASGR1 antibody HCDR2 (SEQ ID NO: 2) TIIYDGGSTYYRHSVKG Non-catabolic anti-ASGR1 antibody HCDR3 (SEQ ID NO: 3) QTYFGSRDYFDY Non-catabolic anti-ASGR1 antibody LCDR1 (SEQ ID NO: 4) LTSEDIYNNLA Non-catabolic anti-ASGR1 antibody LCDR2 (SEQ ID NO: 5) YASNFQD Non-catabolic anti-ASGR1 antibody LCDR3 (SEQ ID NO: 6) LQDSEYPP Non-catabolic anti-ASGR1 antibody HCVR (SEQ ID NO: 7) [ka] Non-catabolic anti-ASGR1 antibody LCVR (SEQ ID NO: 8) DIQMTQSPTSLSASLGETVSIECLTSEDIYNNLAWYQQKPGKSPQLLISYASNFQDGVPSRFSGSGSGTQYSLKINSLESEDAATYFCLQDSEYPPTFGGGTKLELKR Non-catabolic anti-ASGR1 antibody HC (SEQ ID NO: 9) [ka] Non-catabolic anti-ASGR1 antibody LC (SEQ ID NO: 10) [ka] Catabolic anti-ASGR1 antibody HCDR1 (SEQ ID NO: 11) SYGMH Catabolic anti-ASGR1 antibody HCDR2 (SEQ ID NO: 12) VIWYDGSNKYYADSVKG Catabolic anti-ASGR1 antibody HCDR3 (SEQ ID NO: 13) DSSPYGMDV Catabolic anti-ASGR1 antibody LCDR1 (SEQ ID NO: 14) RASQGISSWLA Catabolic anti-ASGR1 antibody LCDR2 (SEQ ID NO: 15) GASSLQS Catabolic anti-ASGR1 antibody LCDR3 (SEQ ID NO: 16) QQSDSFPRT Catabolic anti-ASGR1 antibody HCVR (SEQ ID NO: 17) [ka] Catabolic anti-ASGR1 antibody LCVR (SEQ ID NO: 18) DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYCQQSDSFPRTFGQGTKVEIKR Catabolic anti-ASGR1 antibody HC (SEQ ID NO: 19) [ka] Catabolic anti-ASGR1 antibody LC (SEQ ID NO: 20) [ka] Anti-IgG HCDR1 (SEQ ID NO: 21) DTYIH Anti-IgG HCDR2 (SEQ ID NO: 22) RIDPANGNTKYDPKFQD Anti-IgG HCDR3 (SEQ ID NO: 23) NYGSNYDPMDY Anti-IgG LCDR1 (SEQ ID NO: 24) RASQNIDTNIH Anti-IgG LCDR2 (SEQ ID NO: 25) YASESIS Anti-IgG LCDR3 (SEQ ID NO: 26) QQSDTWPWT Non-catabolic anti-ASGR1 HCDR1 (SEQ ID NO: 27) DYSVH Non-catabolic anti-ASGR1 HCDR2 (SEQ ID NO: 28) IMWTGGSTAYNSALKS Non-catabolic anti-ASGR1 HCDR3 (SEQ ID NO: 29) DGDYGPDY Non-catabolic anti-ASGR1 LCDR1 (SEQ ID NO: 30) QASQDIGNWLS Non-catabolic anti-ASGR1 LCDR2 (SEQ ID NO: 31) GATSLAD Non-catabolic anti-ASGR1 LCDR3 (SEQ ID NO: 32) LQAYSAPPWT Anti-IgG HCVR (SEQ ID NO: 33) [ka] Anti-IgG LCVR (SEQ ID NO: 34) DILLTQSPAILSVSPGERVSFSCRASQNIDTNIHWYQRRTNDSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSDTWPWTFGGGTKLEIKR Non-catabolic Anti-ASGR1 HCVR (SEQ ID NO: 35) EVQLKESGPGLVQPSQTLSLTCTVSGFSLTDYSVHWVRQSPGKGLEWMGIMWTGGSTAYNSALKSRLSISRDTSKSQVFLKMNSLQTEDTAIYYCTRDGDYGPDYWGQGVMVTVSS Non-catabolic anti-ASGR1 LCVR (SEQ ID NO: 36) DIQMTQSPASLSASLEEIVTITCQASQDIGNWLSWYQQKPGKSPQLLIYGATSLADGVPSRFSGSRSGTQYSLKISRLQVEDIGIYYCLQAYSAPPWTFGGGTKLELKR Non-catabolic bispecific scFv (SEQ ID NO: 37) [ka] Catabolic Anti-ASGR1 HCDR1 (SEQ ID NO: 38) SYGMH Catabolic Anti-ASGR1 HCDR2 (SEQ ID NO: 39) VIWYDGSNKYYADSVKG Catabolic Anti-ASGR1 HCDR3 (SEQ ID NO: 40) DSSPYGMDV Catabolic Anti-ASGR1 LCDR1 (SEQ ID NO: 41) RASQGISSWLA Catabolic Anti-ASGR1 LCDR2 (SEQ ID NO: 42) GASSLQS Catabolic Anti-ASGR1 LCDR3 (SEQ ID NO: 43) QQSDSFPRT Catabolic Anti-ASGR1 HCVR (SEQ ID NO: 44) [ka] Catabolic Anti-ASGR1 LCVR (SEQ ID NO: 45) DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYCQQSDSFPRTFGQGTKVEIKR Catabolic bispecific scFv (SEQ ID NO: 46) [ka] Leader sequence (SEQ ID NO: 47) MDMRVPAQLLGLLLLWLRGARC Nucleic acid sequence encoding the leader sequence of SEQ ID NO:47 (SEQ ID NO:48) ATGGACATGAGAGTGCCTGCACAGCTGCTGGGCCTGCTGCTGCTGTGGCTGAGAGGCGCCAGATGC Leader sequence (SEQ ID NO: 49) MAWALLLLTLLTQGTGSWA Nucleic acid sequence encoding the leader sequence of SEQ ID NO:49 (SEQ ID NO:50) ATGGCCTGGGCTCTGCTGCTCCTCACCCTCCTCACTCAGGGCACAGGGTCCTGGGCC Anti-Ig HC (SEQ ID NO: 51) [ka] Anti-Ig LC (SEQ ID NO: 52) [ka] Catabolic anti-mIgG2a 099 Fab-scFc HC (SEQ ID NO: 53) [ka] Catabolic anti-mIgG2a 099 Fab-scFc LC (SEQ ID NO: 54) [ka] Catabolic anti-mIgG2a 465 Fab-scFc HC (SEQ ID NO: 55) [ka] Catabolic anti-mIgG2a 465 Fab-scFc LC (SEQ ID NO: 56) [ka] Catabolic anti-mIgG2a 463 Fab-scFc HC (SEQ ID NO: 57) [ka] Catabolic anti-mIgG2a 463 Fab-scFc LC (SEQ ID NO: 58) [ka] Catabolic anti-mIgG2a 099 HCDR1 (SEQ ID NO: 59) SDYYMC Catabolic anti-mIgG2a 099 HCDR2 (SEQ ID NO: 60) CIGAGDIHTTYYANWAKG Catabolic anti-mIgG2a 099 HCDR3 (SEQ ID NO: 61) DTYNIGGYTGDFDL Catabolic anti-mIgG2a 099, 465 and 463 LCDR1 (SEQ ID NO: 62) QASESISTWLA Catabolic anti-mIgG2a 099, 465 and 463 LCDR2 (SEQ ID NO: 63) YASTLAS Catabolic anti-mIgG2a 099, 465 and 463 LCDR3 (SEQ ID NO: 64) AGHKSYSSDDFA Catabolic anti-mIgG2a 465 HCDR1 (SEQ ID NO: 65) SDYYMC Catabolic anti-mIgG2a 465 HCDR2 (SEQ ID NO: 66) CHGAGDIHTTYYANWAKG Catabolic anti-mIgG2a 465 HCDR3 (SEQ ID NO: 67) DTYNIGGYTGDFDL Catabolic anti-mIgG2a 463 HCDR1 (SEQ ID NO: 68) SDYYHC Catabolic anti-mIgG2a 463 HCDR2 (SEQ ID NO: 69) CIGAGDIHTTYYANWAKG Catabolic anti-mIgG2a 463 HCDR3 (SEQ ID NO: 70) DTYNIGGYTGDFDL Catabolic anti-mIgG2a 099 HCVR (SEQ ID NO: 71) [ka] Catabolic anti-mIgG2a 099, 463 and 465 LCVR (SEQ ID NO: 72) ALVMTQPPASVSAAVGGTVTINCQASESISTWLAWYQQKPGQPPKLLIYYASTLASGVPSRFKGSGSGTQFTLTISGVECDDAATYYCAGHKSYSSDDFAFGGGTEVVVKG Catabolic anti-mIgG2a 465 HCVR (SEQ ID NO: 73) [ka] Catabolic anti-mIgG2a 463 HCVR (SEQ ID NO: 74) [ka] Catabolic anti-mIgG2a non-catabolic ASGR1 bispecific scFv (SEQ ID NO: 75) [ka] (Gly3Ser)3 (SEQ ID NO: 76) GGGSGGGSGGGS (Gly4Ser)3 (SEQ ID NO: 77) GGGGSGGGGSGGGGS (Gly3Ser)4 (SEQ ID NO: 78) GGGSGGGSGGGSGGGS (Gly4Ser)4 (SEQ ID NO: 79) GGGGSGGGGSGGGGSGGGGS (Gly3Ser)5 (SEQ ID NO: 80) GGGSGGGSGGGSGGGSGGGS (Gly4Ser)5 (SEQ ID NO: 81) GGGGSGGGGSGGGGSGGGGSGGGGS (Gly3Ser)6 (SEQ ID NO: 82) GGGSGGGSGGGSGGGSGGGSGGGS (Gly4Ser)6 (SEQ ID NO: 83) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GSADDAKKDAAKKDAAKKDDAKKDDAGS (SEQ ID NO: 84) GSADDAKKDAAKKDAAKKDDAKKDDAKKDAGS (SEQ ID NO: 85) (Gly3Gln)2 (SEQ ID NO: 86) GGGQGGGQ (Gly4Gln)2 (SEQ ID NO: 87) GGGGQGGGGQ (Gly3Gln)3 (SEQ ID NO: 88) GGGQGGGQGGGQ (Gly4Gln)3 (SEQ ID NO: 89) GGGGQGGGGQGGGGQ (Gly3Gln)4 (SEQ ID NO: 90) GGGQGGGQGGGQGGGQ (Gly4Gln)4 (SEQ ID NO: 91) GGGGQGGGGQGGGGQGGGGQ (Gly3Gln)5 (SEQ ID NO: 92) GGGQGGGQGGGQGGGQGGGQ (Gly4Gln)5 (SEQ ID NO: 93) GGGGQGGGGQGGGGQGGGGQGGGGQ (Gly3Gln)6 (SEQ ID NO: 94) GGGQGGGQGGGQGGGQGGGQGGGQ (Gly4Gln)6 (SEQ ID NO: 95) GGGGQGGGGQGGGGQGGGGQGGGGQGGGGQ (Gly3Ser)2 (SEQ ID NO: 96) GGGSGGGS (Gly4Ser)2 (SEQ ID NO: 97) GGGGSGGGGS SGGGGSSGGGGS (SEQ ID NO: 98)

Claims

1. 1. A multispecific molecule comprising a first binding domain and a second binding domain, wherein the first binding domain binds to an immunoglobulin and the second binding domain binds to a recycling target.

2. 2. The multispecific molecule of claim 1, wherein the first binding domain is an scFv, Fv, scFab, Fab', or Fab and the second binding domain is an scFv, Fv, scFab, Fab', or Fab.

3. 3. The multispecific molecule of claim 1, wherein the first binding domain and / or the second binding domain is an scFv.

4. The multispecific molecule of any one of claims 1 to 3, wherein the first binding domain and the second binding domain are each scFv.

5. 3. The multispecific molecule of claim 1 or 2, wherein the first binding domain and / or the second binding domain is an scFab.

6. 6. The multispecific molecule of claim 1, wherein the first binding domain and the second binding domain are each scFabs.

7. 3. The multispecific molecule of claim 1, wherein the first binding domain and / or the second binding domain is a Fab.

8. 8. The multispecific molecule of claim 1, wherein the first binding domain and the second binding domain are each a Fab.

9. 3. The multispecific molecule of claim 1, wherein the first binding domain is an scFv and the second binding domain is a Fab.

10. 3. The multispecific molecule of claim 1, wherein the first binding domain is an scFv and the second binding domain is an scFab.

11. 3. The multispecific molecule of claim 1, wherein the first binding domain is a Fab and the second binding domain is an scFv.

12. 3. The multispecific molecule of claim 1, wherein the first binding domain is an scFab and the second binding domain is an scFv.

13. 3. The multispecific molecule of claim 1, wherein the first binding domain is a Fab and the second binding domain is a scFab.

14. 3. The multispecific molecule of claim 1, wherein the first binding domain is an scFab and the second binding domain is a Fab.

15. The multispecific molecule of any one of claims 1 to 14, wherein the first binding domain and the second binding domain are linked via a linker.

16. The multispecific molecule of claim 15 , wherein the linker is a polypeptide linker.

17. The linker is SG ４ 16. The multispecific molecule of claim 15, which is an S linker.

18. The linker may be (Gly ３ Ser) ３ (SEQ ID NO: 76), (Gly ４ Ser) ３ (SEQ ID NO: 77), (Gly ３ Ser) ４ (SEQ ID NO: 78), (Gly ４ Ser) ４ (SEQ ID NO: 79), (Gly ３ Ser) ５ (SEQ ID NO: 80), (Gly ４ Ser) ５ (SEQ ID NO: 81), (Gly ３ Ser) ６ (SEQ ID NO: 82), (Gly ４ Ser) ６ (SEQ ID NO: 83), GSADDAKKDAAKKDAAKKDDAKKDDAGS (SEQ ID NO: 84), GSADDAKKDAAKKDAAKKDDAKKDDAKKDAGS (SEQ ID NO: 85), (Gly ３ Gln) ２ (SEQ ID NO: 86), (Gly ４ Gln) ２ (SEQ ID NO: 87), (Gly ３ Gln) ３ (SEQ ID NO: 88), (Gly ４ Gln) ３ (SEQ ID NO: 89), (Gly ３ Gln) ４ (SEQ ID NO: 90), (Gly ４ Gln) ４ (SEQ ID NO: 91), (Gly ３ Gln) ５ (SEQ ID NO: 92), (Gly ４ Gln) ５ (SEQ ID NO: 93), (Gly ３ Gln) ６ (SEQ ID NO: 94), (Gly ４ Gln) ６ (SEQ ID NO: 95), (Gly ３ Ser) ２ (SEQ ID NO: 96) and (Gly ４ Ser) ２ 16. The multispecific molecule of claim 15, comprising a sequence selected from the group consisting of: (SEQ ID NO: 97).

19. The multispecific molecule of any one of claims 1 to 18, wherein the immunoglobulin bound by the first domain is IgG, IgA, IgE, IgD or IgM.

20. 20. The multispecific molecule of claim 19, wherein the immunoglobulin is IgG or IgA.

21. The multispecific molecule of any one of claims 1 to 20, wherein the immunoglobulin is expressed on plasma cells or B cells.

22. The multispecific molecule of any one of claims 1 to 20, wherein the immunoglobulin is circulating in the blood.

23. The multispecific molecule of any one of claims 1 to 22, wherein the recycling target is ASGR1.

24. 24. The multispecific molecule of any one of claims 1 to 23, which depletes at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of immunoglobulins in vivo.

25. 25. The multispecific molecule of claim 24, which depletes at least 70% of immunoglobulins.

26. 26. The multispecific molecule of any one of claims 1 to 25, which depletes immunoglobulins in humans, cynomolgus monkeys or mice.

27. The multispecific molecule of any one of claims 24 to 26, wherein the immunoglobulin is depleted in less than 72 hours.

28. The multispecific molecule of any one of claims 1 to 27, which dissociates from the immunoglobulin in the endosome of a cell expressing the recycling target.

29. 29. The multispecific molecule of claim 28, which remains bound to the recycling target in the endosome and is recycled to the cell surface.

30. The multispecific molecule of any one of claims 1 to 27, which dissociates from the recycling target in the endosome of a cell expressing the recycling target.

31. A multispecific molecule according to any one of claims 1 to 30 for use in therapy.

32. A multispecific molecule according to any one of claims 1 to 30 for use in the treatment of an autoantibody-induced disease.

33. A multispecific molecule according to any one of claims 1 to 30 for the manufacture of a medicament for the treatment of an autoantibody-induced disease.

34. 34. The multispecific molecule of claim 32 or 33, wherein the autoantibody-induced disease is selected from the group consisting of myasthenia gravis, Guillain-Barré syndrome, epilepsy, autoimmune limbic encephalitis, spinal cord injury, pediatric autoimmune neuropsychiatric disorder associated with streptococcal infection, neuromyotonia, Morvan's syndrome, multiple sclerosis, pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, epidermolysis bullosa acquisita, pemphigoid of gestationis, mucous membrane pemphigoid, lichen sclerosus, antiphospholipid syndrome, relapsing polychondritis, autoimmune anemia, idiopathic thrombocytotic purpura, autoimmune Graves' disease, dilated cardiomyopathy, vasculitis, Goodpasture's syndrome, idiopathic membranous nephropathy, rheumatoid arthritis, and systemic lupus erythematosus.

35. 31. A method of treating a patient with at least one autoantibody-induced disease, comprising administering to said patient an effective amount of a multispecific molecule of any one of claims 1 to 30.

36. 36. The method of claim 35, wherein the autoantibody-induced disease is selected from the group consisting of myasthenia gravis, Guillain-Barré syndrome, epilepsy, autoimmune limbic encephalitis, spinal cord injury, pediatric autoimmune neuropsychiatric disorder associated with streptococcal infection, neuromyotonia, Morvan's syndrome, multiple sclerosis, pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, epidermolysis bullosa acquisita, pemphigoid of gestationis, mucous membrane pemphigoid, lichen sclerosus, antiphospholipid syndrome, relapsing polychondritis, autoimmune anemia, idiopathic thrombocytotic purpura, autoimmune Graves' disease, dilated cardiomyopathy, vasculitis, Goodpasture's syndrome, idiopathic membranous nephropathy, rheumatoid arthritis, and systemic lupus erythematosus.

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