Multi-specific molecules for use in the removal of immunoglobulin

Abstract

The present invention relates to multispecific molecules that bind to immunoglobulins and recirculating targets. Binding the immunoglobulin and the recirculation target causes the immunoglobulin to degrade, and in certain embodiments, the multispecific molecule to recirculate. The multispecific molecules of the present invention are believed to be useful in the treatment of autoantibody-induced diseases.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 419,549, filed on October 26, 2022, which is hereby incorporated by reference in its entirety.

[0003] Description of Electronically Submitted Text Files

[0004] This application contains a Sequence Listing that has been electronically submitted in XML format and is hereby incorporated by reference in its entirety. A machine - readable copy of the Sequence Listing created on October 25, 2023, is named 10179 - WO01 - SEC_ST26 and is 72.1 kilobytes in size. Technical Field

[0005] The present invention relates to the field of autoantibody - induced diseases. The present invention relates to multispecific molecules that bind immunoglobulins and recycling targets, such as bispecific scFv molecules. The multispecific molecules of the present invention are useful in the treatment of autoantibody - induced diseases. Background Art

[0006] More than 2.5% of the world's population is affected by autoantibody - driven immune diseases (Lenti et al., Autoimmunity Rev. September 2022; 21(9):103143). Due to immune system dysfunction, antibodies generated against self - antigens (referred to as autoantibodies) induce pathogenic effects through various mechanisms such as blocking function, altering antigen transport mechanisms, degrading antigens, and activating complement at the binding site. Autoantibodies play a central role in disease pathology, and thus significant efforts have been made to inhibit antibody production or deplete autoantibodies from the circulation.

[0007] In various autoimmune disorders, autoantibodies mainly belong to the IgG subclass, which binds to FcRn (neonatal Fc receptor) in a pH - dependent manner through its constant region (Fc). This pH - dependent interaction of FcRn:IgG enables internalized IgG to bind to FcRn in early endosomes and be transported back to the cell surface. Rescue from lysosomal degradation gives IgG a long serum half - life. Thus, blocking the FcRn:Fc interaction increases the shunting of IgG to the lysosomal compartment, thereby enhancing IgG degradation.

[0008] A variety of FcRn inhibitors are being developed and these inhibitors have been approved or are in late-stage clinical trials for the treatment of autoantibody-mediated disorders. It has been demonstrated that blocking the FcRn:Fc interaction reduces total IgG levels both clinically and in preclinical animals. FcRn inhibitors have demonstrated clinical efficacy by depleting circulating IgG, but the depletion of IgG does not occur instantaneously after the administration of the inhibitor. The action of FcRn inhibitors depends on the endocytosis of IgG, which is a rate-limiting process. Clinically, it has been observed that after the administration of an FcRn inhibitor, it takes approximately three to four weeks to achieve approximately 50% depletion of total IgG and antigen-specific IgG. Additionally, the FcRn inhibitor must be administered at a high frequency and / or high dose to achieve this efficacy.

[0009] To overcome this delay and induce rapid depletion of serum IgG, the multispecific molecules of the present invention were designed. These multispecific molecules bind immunoglobulins and recycling targets and achieve rapid lysosomal degradation of immunoglobulins. These multispecific molecules can be pH / Ca2+-dependent or -independent. In addition to rapidly depleting immunoglobulins, it is expected that the multispecific molecules of the present invention will have an advantage in clearing immunoglobulins such as IgG by blocking the formation of IgG-complexes, thereby reducing off-target effects. Summary of the Invention

[0010] 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 an immunoglobulin and the second binding domain specifically binds a recycling target. In certain embodiments, the first binding domain is a scFv, Fv, scFab, Fab', or Fab, and the second binding domain is a scFv, Fv, scFab, Fab', or Fab. In particular embodiments, the first binding domain and / or the second binding domain is a scFv. In certain such embodiments, the first binding domain and the second binding domain are each a scFv. In particular embodiments, the first binding domain and / or the second binding domain is an Fv. In certain such embodiments, the first binding domain and the second binding domain are each an Fv. In certain embodiments, the first binding domain and / or the second binding domain is a scFab. In certain such embodiments, the first binding domain and the second binding domain are each a 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 a scFv and the second binding domain is a Fab. In certain embodiments, the first binding domain is a scFv and the second binding domain is a scFab. In certain embodiments, the first binding domain is a Fab and the second binding domain is a scFv. In certain embodiments, the first binding domain is a scFab and the second binding domain is a scFv. In certain embodiments, the first binding domain is a Fab and the second binding domain is a scFab. In certain embodiments, the first binding domain is a scFab and the second binding domain is a Fab. In certain embodiments, the first binding domain is a 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 a scFv. In certain embodiments, the first binding domain is a 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 a 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 a 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 a 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 a scFab. In certain embodiments, the first binding domain is a 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.

[0011] In certain embodiments, the single-chain polypeptide comprises the first binding domain and the second binding domain.

[0012] In certain embodiments, the first binding domain is linked to the second binding domain via a linker. In certain embodiments, the linker is a polypeptide linker. In certain embodiments, the linker is an SG4S linker. In a particular embodiment, 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 comprises a sequence 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), (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).

[0013] In certain embodiments, the first binding domain specifically binds an immunoglobulin. In certain embodiments, the first binding domain specifically binds a recycling target. In certain embodiments, the second binding domain specifically binds an immunoglobulin. In certain embodiments, the second binding domain specifically binds a recycling target.

[0014] In certain embodiments, the multispecific molecule of the invention specifically binds an immunoglobulin, wherein 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 circulates in the blood.

[0015] In certain embodiments, the multispecific molecule of the invention specifically binds a recycling target, wherein 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.

[0016] In certain embodiments, the multispecific molecules of the invention deplete in vivo at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the bound immunoglobulins. In certain embodiments, the multispecific molecule depletes from about 50% to about 70% of the immunoglobulins. In certain embodiments, the multispecific molecule depletes at least 50% of the immunoglobulins. In certain embodiments, the multispecific molecule depletes at least 55% of the immunoglobulins. In certain embodiments, the multispecific molecule depletes at least 60% of the immunoglobulins. In certain embodiments, the multispecific molecule depletes at least 65% of the immunoglobulins. In certain embodiments, the multispecific molecule depletes at least 70% of the immunoglobulins. In certain embodiments, the multispecific molecule depletes at least 75% of the immunoglobulins. In certain embodiments, the multispecific molecule depletes at least 80% of the immunoglobulins. In certain embodiments, the multispecific molecule depletes at least 85% of the immunoglobulins. In certain embodiments, the multispecific molecule depletes at least 90% of the immunoglobulins. In certain embodiments, the multispecific molecule depletes at least 95% of the immunoglobulins. In certain embodiments, the multispecific molecule depletes at least 98% of the immunoglobulins. In certain embodiments, the multispecific molecule depletes at least 99% of the immunoglobulins. In certain embodiments, the multispecific molecule depletes 100% of the immunoglobulins. In certain embodiments, the immunoglobulins are depleted in mice. In certain embodiments, the immunoglobulins are depleted in non-human primates. In certain embodiments, the immunoglobulins are depleted in human patients. In certain embodiments, the immunoglobulin is IgG. In certain embodiments, the immunoglobulin is IgA. In certain embodiments, the immunoglobulin is IgE. In certain embodiments, the immunoglobulin is IgD. In certain embodiments, the immunoglobulin is IgM. In certain embodiments, the immunoglobulins are depleted within less than 96 hours of administration. In certain embodiments, the immunoglobulins are depleted within less than 72 hours of administration. In certain embodiments, the immunoglobulins are depleted within 3 hours to 96 hours of administration. In certain embodiments, the immunoglobulins are depleted within 3 hours to 72 hours of administration.

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

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

[0019] In certain embodiments, the multispecific molecules of the invention bind to recycling targets in a non-catabolic manner and bind to Ig in a catabolic manner.

[0020] In certain embodiments, the multispecific molecules of the invention remain bound to immunoglobulins and recycling targets.

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

[0022] In certain embodiments, the multispecific molecules of the invention dissociate from the recycling targets in the endosomes of cells expressing the recycling targets.

[0023] In certain embodiments, the multispecific molecules of the invention dissociate from immunoglobulins in the endosomes of cells expressing the recycling targets. In certain such embodiments, the multispecific molecule remains bound to the recycling target in the endosome and is recycled to the cell surface of the cell expressing the recycling target.

[0024] In another aspect, the invention relates to an antibody that specifically binds to ASGR1, the antibody 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), wherein 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 a particular embodiment, the antibody of the invention is non-catabolic.

[0025] In certain embodiments, the multispecific molecule of the invention comprises a binding domain that specifically binds to ASGR1, and wherein said binding domain comprises 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: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 specific embodiments, the multispecific molecule of the invention is non-catabolic.

[0026] The present invention provides an antibody that specifically binds to ASGR1, the antibody 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), 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 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 certain specific embodiments, the antibody of the invention is catabolic.

[0027] In certain embodiments, the multispecific molecule of the invention comprises a binding domain that specifically binds to ASGR1, and wherein said binding domain comprises 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 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 multispecific molecule of the invention is catabolic.

[0028] The invention provides an antibody that specifically binds to ASGR1, the antibody 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: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 certain embodiments, the antibody of the invention is non - catabolic.

[0029] In certain embodiments, the multispecific molecule of the invention comprises a binding domain that specifically binds to ASGR1, and wherein said binding domain comprises 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: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 certain embodiments, the multispecific molecule of the invention is non-catabolic.

[0030] In another aspect, the invention relates to an antibody that specifically binds to ASGR1, the antibody 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: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 certain embodiments, the antibody of the invention is catabolic.

[0031] In certain embodiments, the multispecific molecule of the invention comprises a binding domain that specifically binds to ASGR1, and wherein said binding domain comprises 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: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 certain embodiments, the multispecific molecule of the invention is catabolic.

[0032] The invention provides an antibody that specifically binds to IgG, the antibody 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. 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.

[0033] In certain embodiments, the multispecific molecule of the invention comprises a binding domain that specifically binds IgG, and wherein said binding domain comprises 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.

[0034] In certain embodiments, the multispecific molecule of the invention comprises a binding domain that specifically binds ASGR1 and a binding domain that specifically binds an immunoglobulin. In certain embodiments, the binding domain that specifically binds an immunoglobulin specifically binds IgG, IgM, IgA, IgD, or IgE. In certain embodiments, the binding domain specifically binds IgG. In other certain embodiments, the binding domain specifically binds IgA. In certain such embodiments, the binding domain that specifically binds ASGR1 and the binding domain that specifically binds IgG belong to the invention. In certain embodiments, the binding domain that specifically binds ASGR1 and / or an immunoglobulin is an scFv, scFab, Fab', and / or Fab.

[0035] In certain embodiments, the multispecific molecule of the invention comprises the amino acid sequence given by SEQ ID NO:37.

[0036] In certain embodiments, the multispecific molecule of the invention comprises the amino acid sequence given by SEQ ID NO:46.

[0037] In certain embodiments, the multispecific molecule of the invention comprises the amino acid sequence given by SEQ ID NO:75.

[0038] In certain embodiments, the multispecific molecule of the present invention comprises an HCDR1 containing SEQ ID NO:59, an HCDR2 containing SEQ ID NO:60, an HCDR3 containing SEQ ID NO:61, an LCDR1 containing SEQ ID NO:62, an LCDR2 containing SEQ ID NO:63, and an LCDR3 containing SEQ ID NO:64. In certain embodiments, the multispecific molecule of the present invention comprises an HCVR containing SEQ ID NO:71 and an LCVR containing SEQ ID NO:72. In certain embodiments, the multispecific molecule of the present invention comprises an HC containing SEQ ID NO:53 and an LC containing 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.

[0039] In certain embodiments, the multispecific molecule of the present invention comprises an HCDR1 containing SEQ ID NO:65, an HCDR2 containing SEQ ID NO:66, an HCDR3 containing SEQ ID NO:67, an LCDR1 containing SEQ ID NO:62, an LCDR2 containing SEQ ID NO:63, and an LCDR3 containing SEQ ID NO:64. In certain embodiments, the multispecific molecule of the present invention comprises an HCVR containing SEQ ID NO:73 and an LCVR containing SEQ ID NO:72. In certain embodiments, the multispecific molecule of the present invention comprises an HC containing SEQ ID NO:55 and an LC containing SEQ ID NO:56. 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] In certain embodiments, the multispecific molecules of the present invention comprise an HCDR1 containing SEQ ID NO:68, an HCDR2 containing SEQ ID NO:69, an HCDR3 containing SEQ ID NO:70, an LCDR1 containing SEQ ID NO:62, an LCDR2 containing SEQ ID NO:63, and an LCDR3 containing SEQ ID NO:64. In certain embodiments, the multispecific molecules of the present invention comprise an HCVR containing SEQ ID NO:74 and an LCVR containing SEQ ID NO:72. In certain embodiments, the multispecific molecules of the present invention comprise an HC containing SEQ ID NO:57 and an LC containing 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 a recycling target. In certain embodiments, the recycling target is ASGR1.

[0041] The present invention also provides one or more nucleic acid sequences encoding the antibodies or the multispecific molecules of the present invention. In certain embodiments, the present invention provides a DNA molecule comprising a polynucleotide that encodes an HC or an HCVR of the antibody or multispecific molecule of the present invention. The present invention also provides a DNA molecule comprising a polynucleotide that encodes an LC or an LCVR of the antibody or multispecific molecule of the present invention. The present invention also provides a DNA molecule comprising a polynucleotide that encodes both an LC or an LCVR and an HC or an HCVR of the antibody or multispecific molecule of the present invention.

[0042] The present invention further provides mammalian cells transformed with the DNA molecules of the present invention, wherein the transformed mammalian cells are capable of expressing the antibodies or multispecific molecules of the present invention.

[0043] The present invention also provides a process for producing the antibodies or multispecific molecules of the present invention, wherein the process comprises culturing mammalian cells under conditions such that the antibodies or multispecific molecules are expressed and recovering the expressed antibodies or multispecific molecules. In an embodiment, mammalian cells are transformed with the DNA molecules of the present invention, wherein the transformed mammalian cells are capable of expressing the antibodies or multispecific molecules of the present invention. The present invention also provides the antibodies or multispecific molecules obtained by this process.

[0044] The present invention provides the multispecific molecules of the present invention for use in therapy.

[0045] The present invention provides the multispecific molecules of the present invention for use in treating autoantibody-induced diseases.

[0046] The present invention provides the multi - specific molecules of the present invention for manufacturing drugs for treating autoantibody - induced diseases.

[0047] In certain embodiments, the autoantibody - induced diseases are selected from the group consisting of: myasthenia gravis, Guillain - Barré syndrome, epilepsy, autoimmune limbic encephalitis, spinal cord injury, pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection, neuromyotonia, Morvan syndrome, multiple sclerosis, pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, acquired epidermolysis bullosa, pemphigoid gestationis, mucous membrane pemphigoid, lichen sclerosus, antiphospholipid syndrome, relapsing polychondritis, autoimmune anemia, idiopathic thrombocytopenic purpura, autoimmune Graves' disease, dilated cardiomyopathy, vasculitis, Goodpasture syndrome, idiopathic membranous nephropathy, rheumatoid arthritis, and systemic lupus erythematosus.

[0048] The present invention provides a method for treating a patient suffering from at least one autoantibody - induced disease, the method comprising administering to the patient an effective amount of the multi - specific molecules of the present invention. In certain embodiments, the patient suffers from at least one autoantibody - induced disease. In certain embodiments, the patient suffers from at least one of the following diseases: myasthenia gravis, Guillain - Barré syndrome, epilepsy, autoimmune limbic encephalitis, spinal cord injury, pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection, neuromyotonia, Morvan syndrome, multiple sclerosis, pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, acquired epidermolysis bullosa, pemphigoid gestationis, mucous membrane pemphigoid, lichen sclerosus, antiphospholipid syndrome, relapsing polychondritis, autoimmune anemia, idiopathic thrombocytopenic purpura, autoimmune Graves' disease, dilated cardiomyopathy, vasculitis, Goodpasture syndrome, idiopathic membranous nephropathy, rheumatoid arthritis, and systemic lupus erythematosus.

[0049] The present invention also provides a pharmaceutical composition comprising the multi - specific molecules of the present invention and one or more pharmaceutically acceptable carriers, diluents, or excipients. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1A 、 Figure 1B and Figure 1C depict the differential clearance of non - CAT and CAT anti - ASGR1 antibodies in human FcRn Tg mice. Non - catabolic antibodies ( Figure 1A ) or catabolic antibodies ( Figure 1B), and the total antibody concentration is measured over time. Individual measurements are labeled as follows: antibodies at a dose of 0.3 mg / kg are labeled by (▼), antibodies at a dose of 3 mg / kg are labeled by (▲), antibodies at a dose of 10 mg / kg are labeled by (■), and antibodies at a dose of 30 mg / kg are labeled by (●). Figure 1C depicts data from Figure 1B from time zero to 24 hours.

[0051] Figure 2 depicts the simultaneous binding of ASGR1 and IVIg to the immobilized multispecific molecule of the present invention.

[0052] Figure 3 depicts the rapid depletion of exogenously administered human IgG in mice after administration of the catabolic bispecific scFv molecule of the present invention. Human IgG was administered to mice, followed by administration of the bispecific scFv molecule (bispecific scFv) or PBS of the present invention. After administration of the bispecific scFv molecule or PBS of the present invention, the concentration-time curve of human IgG in mouse serum was plotted by quantifying the human IgG concentration using ELISA.

[0053] Figure 4 depicts the rapid depletion of exogenously administered human IgG in cynomolgus monkeys after administration of the catabolic bispecific scFv molecule of the present invention. After administration of the bispecific scFv molecule or PBS of the present invention, the concentration-time curve of human IgG in cynomolgus monkey serum was plotted by quantifying the human IgG concentration using ELISA.

[0054] Figure 5A and Figure 5B depicts that the bispecific scFv molecules of the present invention that bind to ASGR1 in catabolic and non-catabolic manners are equally effective in depleting huIgG in vivo. Seventy-two hours after administration of huIVIg, PBS or the catabolic bispecific scFv molecule or the non-catabolic bispecific scFv molecule of the present invention was administered to the animals. Shown are the normalized serum ( Figure 5A ) and whole body ( Figure 5B ) radioactivity counts of mice administered huIVIg labeled with I-125.

[0055] Figure 6Depicts the bispecific scFv molecule of the invention that drives IgG to the liver for rapid catabolism. IVIg labeled with I-125 (left column) or In-111 (right column) was administered to mice, and after 72 hours, the catabolic bispecific scFv molecule of the invention or PBS was administered to the animals. The animals were perfused and the organs harvested at 3 hours, 24 hours, or 96 hours after administration of the scavenger, and the radioactivity counts of the animals were measured and plotted. Detailed Description

[0056] The present invention provides multispecific molecules that bind immunoglobulins and recycling targets. In certain embodiments, the multispecific molecule comprises a first binding domain that specifically binds an immunoglobulin and a second binding domain that specifically binds a recycling target. The multispecific molecule that binds the immunoglobulin and the recycling target is internalized into the cell, after which the immunoglobulin is degraded 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 another immunoglobulin and internalize it for degradation. In certain embodiments, the recycling target dissociates from the multispecific molecule inside the cell before recycling back to the cell surface.

[0057] The multispecific molecule of the present invention comprises 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 as long as it specifically binds 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. The multispecific molecule comprising the binding domain can be in the form of, for example, F(ab’)2, (scFv-Zip)2, (scFv)2 (e.g. molecules), diabodies, scDb, and tandem diabodies. Multispecific molecules comprising binding domains derived from camelid antibodies are also contemplated. In certain embodiments, the multispecific molecule comprising a binding domain derived from a camelid antibody comprises 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 extension (HLE) moiety. Also contemplated are Molecules. The binding domains and multispecific molecules of the present invention can be produced according to well-known methods (see, e.g., Kipriyanov S.M. (2003); Recombinant Antibodies for Cancer Therapy; 207(3 - 26); Janssens et al., October 10, 2006; PNAS [Proceedings of the National Academy of Sciences of the United States of America]; 103(41) 15130 - 15135).

[0058] Non-limiting examples of the HLE moiety include Fc polypeptides, single-chain Fc polypeptides (scFc), albumin, albumin fragments, moieties that bind to albumin or to the neonatal Fc receptor (FcRn), derivatives of fibronectin engineered to bind albumin or its fragments, peptides, single-domain protein fragments, or other polypeptides that can increase serum half-life. In other embodiments, the half-life extension moiety can be a non-polypeptide molecule, such as, for example, polyethylene glycol (PEG). In certain embodiments, the HLE is single-chain Fc (“scFc”).

[0059] The present invention provides bispecific scFv molecules that bind an immunoglobulin and a recycling target. The bispecific scFv molecule comprises an scFv that specifically binds an immunoglobulin (“first scFv”) that is linked via a linker to an scFv that specifically binds a recycling target (“second scFv”).

[0060] As used herein, “recycling target” refers to a protein that is internalized from the cell surface into the cell and then brought back to the cell surface. Thus, a recycling target is a target that is recycled to the cell surface. Internalization of the protein can occur via endocytosis, which can occur by various mechanisms. Broadly speaking, endocytosis begins with the formation of an endocytic vesicle that carries the endocytic cargo into the cell, which is then delivered to the early endosome. The cargo can then enter the late endosome and lysosome for degradation, enter the trans-Golgi network (TGN), or enter the recycling endosome carrier that brings the cargo back to the plasma membrane. In this context with respect to the present invention, the cargo refers to the recycling target bound by the multispecific molecule of the present invention, which also binds an immunoglobulin. 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.

[0061] ASGR1 is a membrane-bound receptor expressed in hepatocytes and composed of ASGPR1 and ASGPR2 subunits. ASGR1 removes desialylated glycoproteins from the circulation via 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. [Molecular Therapy] January 3, 2018; 26(1):105 - 114). In certain embodiments, ASGR1 is a recycling target. ASGR1 is highly expressed on the cell surface of hepatocytes, which have very fast internalization and recycling rates, and it can help to deplete a large antigen load. Since ASGR1 is expressed in the liver and catabolism of antigens occurs in the liver, the expected toxicity resulting from using ASGR1 as a recycling target is expected to be acceptable.

[0062] Early endosomes have a pH of about 6.5 to 6.0, late endosomes have a pH of about 5.5, and lysosomes have a pH of about 4.0. The multispecific molecules of the present invention effect depletion (or clearance) of immunoglobulins (Igs) in lysosomes. Depletion of immunoglobulins can be measured by assays known in the art, including immunoassays, radioactivity in blood, and flow cytometry assays.

[0063] In certain embodiments, the multispecific molecule is said to be catabolic ("CAT"). A catabolic molecule is a molecule that disengages from a recycling target in an endosome ("recycling target catabolic molecule") and / or a molecule that disengages from an Ig in an endosome. In certain embodiments, the recycling target catabolic molecule remains bound to the Ig (e.g., a molecule that binds to ASGR1 in a catabolic manner and binds to the Ig in a non-catabolic manner). In certain embodiments, the recycling target catabolic molecule also dissociates from the Ig (e.g., a molecule that binds to ASGR1 in a catabolic manner and binds to the 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 in a non-catabolic manner and binds to the Ig in a catabolic manner). After disengaging from the recycling target and / or Ig, the Ig is degraded in the lysosome. In certain embodiments, the recycling target recycles 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 and the multispecific molecule recycles with the recycling target, it is expected that these catabolic molecules will be administered at a lower dose in a patient to treat an autoantibody-induced disease.

[0064] Catabolic molecules are sensitive to the low pH in endosomes, which causes the dissociation of the multispecific molecule and the Ig from the recycling target (or causes the dissociation of the Ig from the multispecific molecule). The catabolic molecule may exhibit, for example, a reduced binding to ASGR1 (and / or Ig) at pH 6.0 and 2 μM calcium chloride. Molecules that exhibit high affinity for the recycling target and / or Ig at neutral pH / high calcium concentration but no detectable binding at acidic pH / low calcium concentration can degrade (catabolize) Ig more rapidly and are thus referred to as catabolic.

[0065] In certain embodiments, the multispecific molecule is referred to as non - catabolic ("non - CAT"). A non - CAT molecule is a multispecific molecule that remains bound to the recycling target and the Ig, and the complex (non - CAT molecule, recycling target, and Ig) recycles back to the cell surface. Compared to catabolic molecules, non - catabolic molecules are pH - insensitive and thus do not dissociate from the recycling target or Ig in endosomes. The non - catabolic molecule may exhibit, for example, a 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 the non - catabolic molecule and / or the Ig are degraded.

[0066] In certain embodiments of the invention, the binding domain that specifically binds Ig (e.g., IgG) is non - catabolic, while the binding domain that specifically binds ASGR1 is catabolic. In other embodiments, the binding domain that specifically binds Ig (e.g., IgG) is catabolic, while the binding domain that specifically binds ASGR1 is non - catabolic. In other embodiments, the binding domain that specifically binds Ig (e.g., IgG) is non - catabolic, and the binding domain that specifically binds ASGR1 is non - catabolic.

[0067] In certain embodiments, the bispecific scFv molecule of the invention comprises an scFv that specifically binds a recycling target and an scFv that specifically binds an immunoglobulin. The bispecific scFv molecule of the invention can be a single - chain polypeptide comprising a first scFv - linker - second scFv. The scFv or single - chain variable fragment is composed of the variable domains of the antibody heavy and light chains that can be joined together by a short peptide linker. For example, a (G4S)3 linker can be used in any repeat number (e.g., one to four). The orientation of each scFv from the N - terminus to the C - terminus can be VL - linker - VH or VH - linker - VL.

[0068] According to one embodiment of the invention, the multispecific molecule of the invention is a single-chain molecule. Although these two domains of the binding domain, VL and VH, are encoded by separate genes, the two domains can be joined by an artificial linker using recombinant methods - as described above - which enables the two domains to form a single protein chain in which the VL and VH regions pair to form a monovalent molecule; see, for example, Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883.

[0069] An example of a bispecific scFv molecule engineered from an scFab that binds mIgG2a in a catabolic manner and a molecule that binds ASGR1 in a non-catabolic manner is SEQ ID NO:75. One or more cysteines can be further introduced to improve stability (see, for example, Reiter et al., Biochemistry, 1994, 33, 5451-5459) and it is expected that the catabolic properties of the molecule will not be altered.

[0070] It is expected that the conversion of the scFab molecule to the bispecific scFv molecule will not change the catabolic properties of the molecule.

[0071] The binding domains are obtained using conventional techniques known to those skilled in the art, and the functions of the binding domains are evaluated in the same manner as for full-length antibodies or IgG. Thus, an scFv is, for example, a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin, typically linked by a short linker peptide. For flexibility, the linker is usually rich in glycine, and for solubility usually rich in serine or also threonine, and can link the N-terminus of VH and the C-terminus of VL, or vice versa. Although the constant regions are removed and a linker is introduced, the protein retains the specificity of the original immunoglobulin.

[0072] Bispecific single-chain molecules are known in the art and are described in the following: 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; Kipriyanov, J. Mol. Biol. (1999), 293, 41 - 56. The techniques described for generating single-chain antibody constructs (see in particular US Patent 4,946,778; Kontermann and Diibel (2010), supra, and Little (2009), supra) can be applied to generate single-chain antibody constructs that specifically recognize one or more selected targets.

[0073] A bivalent (also known as divalent) or bispecific single-chain variable fragment (tandem-scFv or di-scFv in the form of (scFv)2) can be engineered by linking two scFv molecules (e.g., using a linker as described above). The linking can be carried out by generating a single polypeptide chain having two VH regions and two VL regions, thereby generating a tandem scFv (see, e.g., Kufer P. et al., (2004) Trends in Biotechnology 22(5):238-244). Another possibility is to generate scFv molecules having linker peptides that are too short for the two variable regions to fold together (e.g., about five amino acids), thereby forcing scFv dimerization. In this case, the VH and VL of the binding domain (which binds to an immunoglobulin or a recycling target) are directly linked without a peptide linker. Thus, the VH of the immunoglobulin binding domain can be fused, for example via a peptide linker, to the VL of the recycling target binding domain, and the VH of the recycling target binding domain is fused via such a peptide linker to the VL of the immunoglobulin binding domain. This type is called 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).

[0074] In certain embodiments, the multispecific molecules of the invention, such as bispecific scFv molecules, comprise two binding domains (e.g., scFv) 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. According to the invention, the term "peptide linker" encompasses an amino acid sequence by which the amino acid sequences of one (variable and / or binding) domain and another (variable and / or binding) domain of the multispecific molecules of the invention are linked to each other. Suitable peptide linkers are those described in U.S. Patent 4,751,180 and 4,935,233 or WO 88 / 09344.

[0075] An example of a peptide linker connecting the two binding domains is the SG4S linker. The multispecific molecules of the present invention may have one, two, three, four, five, or six repeats of the SG4S linker. For example, two SG4S repeats would be binding domain (e.g., scFv)-SGGGGSSGGGGS-binding domain (e.g., scFv) (SGGGGSSGGGGS given by SEQ ID NO:98). In certain embodiments, the multispecific molecules of the present invention have one SG4S as the linker. In other embodiments, the linker comprises two, three, or four SG4S repeats. In other embodiments, the linker comprises five or six SG4S repeats. In other embodiments, the linker comprises seven or more SG4S repeats, provided that the multispecific molecule can be expressed and purified. Other examples of linkers include linkers comprising sequences selected from the group consisting of: (Gly3Ser)3 (SEQID NO:76), (Gly4Ser)3 (SEQ ID NO:77), (Gly3Ser)4 (SEQ ID NO:78), (Gly4Ser)4 (SEQ IDNO: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), (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 (SEQID NO:90), (Gly4Gln)4 (SEQ ID NO:91), (Gly3Gln)5 (SEQ ID NO:92), (Gly4Gln)5 (SEQID 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).

[0076] As used herein, an "antibody" is an immunoglobulin molecule comprising two heavy chains (HCs) and two light chains (LCs) interconnected by disulfide bonds. The amino-terminal portion of each LC and HC includes a variable region of about 100 - 120 amino acids, which is primarily responsible for antigen recognition via the CDRs contained therein. The CDRs are interspersed with more conserved regions called framework regions ("FRs"). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, 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."

[0077] The CDRs contain most of the residues that form specific interactions with the antigen. Thus, the functional ability of an antibody to bind a specific antigen is largely influenced by the amino acid residues within the six CDRs.

[0078] The binding domain characterized in connection with the present invention is a domain that specifically binds to a given target epitope or a given target side on a target molecule (antigen) / interacts with the target epitope or target side / recognizes the target epitope or target side, where the target epitope or target side is: an immunoglobulin or a recycling target. The structures and functions of the first binding domain and the second binding domain are based on or derived from the structure and / or function of an antibody, and more particularly, they are derived from or originate from the variable heavy chain (VH) and variable light chain (VL) domains of an antibody. In certain embodiments, 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 the 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 and A. Plückthun. "Yet another numbering scheme for immunoglobulin variable domains: An automatic modeling and analysis tool." J. Mol. Biol, 309 (2001) 657-670).It should be understood that other numbering conventions may also be used, 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, 5th ed., U.S. Department 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, 406, 228-256 (2011)).

[0079] As used herein, when a binding domain and / or a multispecific molecule binds its antigen with a dissociation constant (KD) of ≤10 -6 M, the antigen-binding domain (and / or multispecific molecule) is said to "specifically bind" its antigen, as measured, for example, by surface plasmon resonance techniques (e.g., BIACore, GE Healthcare, Uppsala, Sweden) or kinetic exclusion assay (KinExA, Sapidyne, Boise, Idaho).

[0080] It is contemplated that the binding domains of the present invention are generated or obtainable by phage display or library screening methods, or are generated or obtainable by grafting CDR sequences from pre-existing monoclonal antibodies into scaffolds. Catabolic molecules can be obtained by screening for molecules that exhibit 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, and thus binding will be disrupted under the acidic conditions in endosomes, resulting in catabolic molecules.

[0081] In the context of antibodies, the present invention contemplates antibodies that may have a cleaved C-terminal lysine or cysteine residue in the HC.

[0082] In the context of the antibodies or binding domains of the present invention, the N-terminal glutamine and / or N-terminal glutamate can be converted to pyroglutamate.

[0083] It is contemplated that the multispecific molecules according to the present invention have a form that does not cause self-binding. For example, a multispecific molecule engineered to bind IgG and a recycling target should lack an Fc region. In certain embodiments, the multispecific molecules according to the present invention preferably exhibit monovalent binding to immunoglobulins, since immunoglobulin cross-linking can activate the immune system, thereby causing side effects such as allergic reactions.

[0084] As used herein, "multispecific" refers to a molecule that comprises at least a first binding domain and a second binding domain, wherein the first binding domain specifically binds one antigen (target), and the second binding domain specifically binds another antigen (target). Thus, the multispecific molecules according to the present invention comprise specificities for at least two different antigens or targets. In certain embodiments, the multispecific molecule comprises no less than two and no more than two binding domains (each binding domain specifically binding a different target, e.g., an immunoglobulin and a recycling target), and the multispecific molecule can be referred to as a "bispecific molecule." Thus, as used herein, a "bispecific scFv molecule" refers to a multispecific molecule that binds two different targets (antigens) (such as an immunoglobulin and a recycling target (e.g., ASGR1)) and comprises two scFvs (binding domains), wherein one scFv specifically binds the immunoglobulin and the other scFv specifically binds the recycling target.

[0085] The multispecific molecules of the present invention bind and deplete immunoglobulins. Depletion of immunoglobulins is considered beneficial in treating patients suffering from antibody-mediated autoimmune diseases (also known as autoantibody-induced diseases). As used herein, "patient" refers to a human. Autoantibody-induced diseases are due to the body's immune system's inability to distinguish between self-antigens and non-self-antigens, causing the immune system to attack normal parts of the body, potentially leading to damage and / or disease. For example, the immune system may start producing antibodies that attack the body's own tissues. Examples of treatments for autoimmune diseases include anti-inflammatory drugs, corticosteroids, painkillers, immunosuppressive drugs, physical therapy, surgery, high-dose immunosuppression, and disease-specific treatments. Other therapies used in autoimmune diseases are plasmapheresis, intravenous Ig (IVIg) administration, and immunoadsorption; however, these are associated with high treatment costs and / or side effects. Clinically, the ("antibody enhancing IgG degradation") molecule (antibody-based FcRn inhibitor) has been shown to deplete human IgG by approximately 50%-70% within 2-3 weeks after administration.

[0086] The molecules of the present invention can be easily 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.

[0087] 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, episomal mammalian vectors, and expression vectors (e.g., recombinant expression vectors). The vector containing the polynucleotide sequence of interest (e.g., the polynucleotide encoding the polypeptide of the molecule and expressing a control sequence) can be transferred into the host cell by well-known methods, which vary depending on the type of cell host. Examples of vectors include, but are not limited to, plasmids, viral vectors, episomal mammalian vectors, and expression vectors (e.g., recombinant expression vectors).

[0088] The recombinant expression vector of the present invention may comprise the nucleic acid of the present invention in a form suitable for expression in a host cell. The recombinant expression vector includes 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), those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences, see Voss et al., 1986, Trends Biochem. Sci. 11:287; Maniatis et al., 1987, Science 236:1237, which are incorporated herein by reference in their entirety), and those that direct inducible expression of a nucleotide sequence in response to a particular treatment or condition (e.g., the metallothionein promoter in mammalian cells and tetracycline-responsive and / or streptomycin-responsive promoters in both prokaryotic and eukaryotic systems (see ibid.)). Those skilled in the art will understand that the design of the expression vector can depend on factors such as the choice of host cell to be transformed, the desired level of protein expression, etc. The expression vector of the present invention can be introduced into a host cell, thereby producing a protein or peptide encoded by the nucleic acid as described herein, including a fusion protein or peptide.

[0089] In certain embodiments, the present invention provides host cells that have been introduced with the recombinant expression vectors of the present invention. The host cells can be any prokaryotic or eukaryotic cells. Prokaryotic host cells include Gram-negative or Gram-positive organisms such as Escherichia coli or bacilli. 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 derivatives thereof (such as Veggie CHO) and related cell lines that grow in serum-free media (see Rasmussen et al., 1998, Cytotechnology 28:31) or DHFR-deficient CHO strain DXB-11 (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20). Additional CHO cell lines include CHO-K1 (ATCC#CCL-61), EM9 (ATCC#CRL-1861), and UV20 (ATCC#CRL-1862). Additional host cell lines 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. Patent No. 6,210,924), HeLa cells, BHK (ATCC CRL 10) cell line, CV1 / EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) (see McMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cells (such as 293, 293EBNA or MSR 293), human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from primary tissues, primary explants in vitro culture, HL-60, U937, HaK or Jurkat cells. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985).

[0090] Typically, an expression vector for use in any host cell will contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. Such sequences are collectively referred to as "flanking sequences" and, in certain embodiments, 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 containing 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 nucleic acid encoding a polypeptide to be expressed, and a selectable marker element. The leader sequence may contain the amino acid sequence of SEQ ID NO: 47 (MDMRVPAQLLGLLLLWLRGARC), which is encoded by SEQ ID NO: 48 (atggacatgagagtgcctgcacagctgctgggcctgctgctgctgtggctgagaggcgccagatgc). The leader sequence may contain the amino acid sequence of SEQ ID NO: 49 (MAWALLLLTLLTQGTGSWA), which is encoded by SEQ ID NO: 50 (atggcctggg ctctgctgctcctcaccctc ctcactcagg gcacagggtc ctgggcc). The present invention contemplates molecular protein sequences without a leader sequence.

[0091] A variety of protein purification methods can be employed to purify the protein, including but not limited to antibodies or binding domains, and such methods are known in the art.

[0092] The molecules of the present invention can be biosynthesized, purified and formulated for administration by well-known methods. For example, appropriate host cells such as HEK 293 or CHO are transiently or stably transfected with an expression system for secreting an antibody or binding domain using a predetermined HC:LC or HCVR:LC vector ratio (if two vectors are used) or a single vector system encoding both the heavy and light chains. Vectors suitable for expressing and secreting antibodies or binding domains from these commonly used host cells are well-known. After expression and secretion of the antibody or binding domain, the culture medium is clarified to remove cells and the clarified medium is purified using any of a number of common techniques. For example, the medium can be applied to a Protein A or G column that has been equilibrated with a buffer such as phosphate buffered saline (pH 7.4). The column is washed to remove non-specifically bound components. For example, the bound antibody or binding domain is eluted by 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, for example, by SDS-PAGE and then pooled. Further purification is optional depending on the intended use. The antibody or binding domain can be concentrated and / or sterile filtered using common techniques. Other 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 chromatographic steps is typically greater than 95%. The product can be frozen at -70°C or lyophilized.

[0093] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. For stable transfection of mammalian cells, it is known that depending on the expression vector and transfection technique used, only a small fraction of cells integrate foreign DNA into their genome. To identify and select these integrants, a gene encoding a selectable marker (e.g., for antibiotic resistance) is typically introduced into the host cell along with the gene of interest. Additional selectable markers include those that confer resistance to drugs such as G418, hygromycin and methotrexate. Among other methods, cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive while other cells will die).

[0094] The polynucleotides encoding the amino acid sequences of the molecules of the present invention can be of any length suitable for the desired use or function and may contain one or more additional sequences, such as regulatory sequences, and / or can be part of a longer nucleic acid, such as a vector. Those skilled in the art will understand that due to the degeneracy of the genetic code, each polypeptide sequence disclosed herein is encoded by a large number of other nucleic acid sequences. Mutations can also be introduced into the nucleic acid without significantly altering the biological activity of the polypeptide it encodes. For example, nucleotide substitutions can be made to effect amino acid substitutions at non-essential amino acid residues.

[0095] Transformed cells can be cultured under conditions that promote polypeptide expression, 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.

[0096] In certain embodiments, a vector containing a nucleic acid molecule as described herein is provided. In certain embodiments, the present invention encompasses host cells containing 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 containing at least one molecule as described herein is provided.

[0097] Glutaminyl and asparaginyl residues are often deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under weakly acidic conditions. Either form of these residues is within the scope of the present invention.

[0098] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of serine or threonine residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, pp. 79-86), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0099] Another type of covalent modification of molecules included within the scope of the present invention includes altering the glycosylation pattern of the protein. As is known in the art, the glycosylation pattern can depend on the sequence of the protein (e.g., the presence or absence of specific glycosylated amino acid residues discussed below) or the host cell or organism in which the protein is produced. Specific expression systems are discussed below.

[0100] Glycosylation of polypeptides is typically 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 other than proline) are the recognition sequences for the 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 hydroxy amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0101] Immunoglobulins are produced by B cells and plasma cells and are important in the humoral immune response against bacteria, viruses, fungi, parasites, cellular antigens, chemicals, and synthetic substances. Immunoglobulins can be classified as IgG, IgM, IgA, IgD, or IgE according to their heavy chain constant regions. Immunoglobulins are mainly expressed on B cells and then secreted and circulate in the blood (see, for example, Hoffman et al., Clin J Am Soc Nephrol. [Clinical Journal of the American Society of Nephrology] January 7, 2016; 11(1):137-154). As used herein, the phrase "circulate in the blood" means that the immunoglobulin has been secreted and is circulating in the blood.

[0102] IgM serves as the first line of defense and provides short-term protection. IgA is also known as the secretory antibody and is secreted through mucous membranes. IgD and IgE account for relatively small percentages of serum antibodies, but they still play roles in the innate immune system and against parasitic infections, respectively.

[0103] IgG is the most common immunoglobulin in the body, accounting for approximately 75%-80% of the antibodies found in plasma. IgG is able to activate the complement system and also has the longest lifespan of the immunoglobulins. The long half-life of IgG is due to a recycling pathway involving the neonatal fragment crystallizable receptor (FcRn). Thus, the mode of inhibiting FcRn is thought to consume IgG via lysosomal degradation (see, for example, Hans-Hartmut et al., J Allergy Clin Immunol. [Journal of Allergy and Clinical Immunology] September 2020; 146(3):479-491). One such antibody-based FcRn inhibitor is called ABDEG (see Challa et al., MAbs. [Monoclonal Antibodies] September 1, 2013; 5(5):655-659). Efgartimgimod (a human IgG1-derived Fc fragment modified using ABDEG technology) has shown reduced IgG in humans (Ulrichts et al., J Clin Invest. [Journal of Clinical Investigation], 2018; 128(10):4372-438).

[0104] The multispecific molecules of the present invention are intended for the treatment of 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 syndrome, multiple sclerosis, pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, acquired epidermolysis bullosa, pemphigoid gestationis, mucous membrane pemphigoid, lichen sclerosus, antiphospholipid syndrome, relapsing polychondritis, autoimmune anemia, idiopathic thrombocytopenic purpura, autoimmune Graves' disease, dilated cardiomyopathy, vasculitis, Goodpasture syndrome, idiopathic membranous nephropathy, rheumatoid arthritis, and systemic lupus erythematosus (see, for example, Wang L et al., J. Internal Medicine [Journal of Internal Medicine], 2015, 278, 369-395; Ludwig RJ et al., Front Immunol. [Frontiers in Immunology] 2017, 8, 603; and Pruss H, 2021, Nat. Rev. Immunol [Nature Reviews Immunology], 21(12), 798-813).

[0105] The multispecific molecules of the present invention, or pharmaceutical compositions comprising the same, can be administered by parenteral routes, non-limiting examples of which are subcutaneous administration and intravenous injection. Intramuscular, intraarterial, intralesional, and intraperitoneal bolus injections are other possible routes of administration. The multispecific molecules can also be administered by infusion (e.g., intravenous or subcutaneous infusion). The multispecific molecules of the present invention can be administered to a patient in a single dose or multiple doses with a pharmaceutically acceptable carrier, diluent, or excipient. Optionally, the composition further comprises one or more bioactive agents. The pharmaceutical compositions of the present invention can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 22nd edition (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.

[0106] As used interchangeably herein, "treatment" (and / or "treating" and / or "treat") is intended to refer to all processes in which there may be a slowing, interruption, arrest, control, stoppage, or reversal of the progression of the disorders described herein, but does not necessarily mean the complete elimination of all symptoms of the disorder. Treatment includes administering the multispecific molecules of the present invention for treating a disease or disorder of a human who would benefit from the activity of the multispecific molecules of the present invention, and includes: (a) inhibiting further progression of the disease; and (b) alleviating the disease, i.e., causing the disease or disorder to regress or reducing its symptoms or complications. As used herein, "therapy" or "therapeutic" refers to treating a patient suffering from at least one autoantibody-induced disease.

[0107] A therapeutically effective amount (or dose) of the multispecific molecules of the invention can be administered. As used herein, an "effective amount" refers to the amount of the multispecific molecules of the invention or a pharmaceutical composition comprising such multispecific molecules that will elicit a biological or medical response or a desired therapeutic effect on a tissue, system, animal, mammal, or human being that is being sought by a researcher, physician, or other clinician. The effective amount of the multispecific molecules can vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the antibody to elicit the desired response in the individual. An effective amount is also an amount in which the therapeutically beneficial effects of the antibody outweigh any toxic or detrimental effects. Such benefits include improvement in the signs or symptoms of cancer. The effective amount of the multispecific molecules of the invention can be administered as a single dose or multiple doses. When determining the effective amount for a patient, the attending physician will consider a number of factors, including but not limited to: the size of the patient (e.g., weight or mass), body surface area, age, and general health; the specific disease or disorder involved; the degree or extent or severity of the disease or disorder; the response of the individual patient; the specific compound administered; the mode of administration; the bioavailability characteristics of the formulation administered; the dosing regimen selected; the use of concomitant medications; and other relevant circumstances known to the physician.

[0108] Examples

[0109] Example 1: Antibody binding as determined by surface plasmon resonance

[0110] Antibodies that bind to ASGR1 in a pH / Ca2+-dependent manner were screened by employing surface plasmon resonance. Antibodies were screened under conditions of pH 7.4 and 2 mM CaCl2 or pH 6.0 and 2 μM CaCl2 to analyze binding to ASGR1. The K D value was determined by immobilizing the receptor on the chip and using the antibody as the analyte.

[0111] The binding of anti-ASGR1 antibodies to ASGR1 in a pH / Ca2+-dependent or -independent manner was evaluated 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.

[0112] The BIAcore 3000 was used to determine the equilibrium binding affinity of the interaction between murine ASGR1 and anti-ASGR1 antibodies. The murine ASGR1 protein was obtained from R&D-systems (Catalog number 2755-AS / CF) and immobilized on a CM5 chip using amine coupling chemistry to a density of approximately 1500 RU. On each CM5 chip, a reference flow cell coupled only with coupling buffer was used. The antibodies were injected over the immobilized ASGR1 at a concentration range of 1000 nM - 0.2 nM (two-fold serial dilution). To determine pH / Ca-sensitive binding, experiments were conducted in phosphate-buffered saline (PBS) with 0.01% (v / v) Tween 20 and 0.05% azide at pH 7.4 / 2 mM calcium chloride or pH 6.0 / 2 μM calcium chloride. The chip was regenerated between each injection cycle using 0.15 M NaCl, 0.1 M glycine pH 1.5 buffer. The equilibrium dissociation constant was determined using BIAevaluation with a 1:1 interaction model.

[0113] Anti-ASGR1 antibodies that did not exhibit differential binding at pH 7.4 / 2 mM calcium chloride or pH 6.0 / 2 μM calcium chloride were designated as non-catabolic antibodies (non-CAT mAb), while antibodies that did not exhibit detectable binding at pH 6.0 / 2 μM calcium chloride were designated as catabolic antibodies (CAT mAb).

[0114] These data demonstrate that CAT mAb and non-CAT mAb have 20 nM and 6 nM affinity for ASGR1 at pH 7.4 and 2 mM CaCl2, respectively (Table 1). The non-CAT mAb exhibited a 5.5 nM binding affinity for ASGR1 under low pH / Ca2+ conditions, while the non-CAT mAb had no detectable binding under these assay conditions (Table 1).

[0115] Table 1. Equilibrium dissociation constants for the interaction between murine ASGR1 and antibodies at pH 7.4 and 2 mM CaCl2 or pH 6.0 and 2 μM CaCl2 Figure 1A

[0116]

[0117] Example 2: In vivo pharmacokinetics

[0118] The pharmacokinetic parameters of the antibodies observed in homozygous huFcRn Tg32 mice are highly correlated with those observed in non-human primates and humans (see, for example, Avery et al., mAbs [Monoclonal Antibodies] 2016, 8(6), 1064-1078). To determine the in vivo pharmacokinetics of the anti-ASGR1 antibodies, pharmacokinetic experiments were conducted in 8-12-week-old male or female C57BL / 6 mice carrying the human FcRn gene (homozygous huFcRn Tg32 mice; Jackson laboratory, stock number 014565) and wild-type C57BL / 6 mice. The CAT ASGR1 or non-CAT ASGR1 antibodies were administered to the mice via intravenous injection into the caudal vein in a manner of injecting 150 μl of the corresponding antibody buffer per animal. The dose ranged from 0.3–30 mg / kg. At the indicated time points, 50 μl of whole blood was collected (n = 3 mice / group / time point) using a sparse serial sampling protocol via submandibular vein puncture using a SARSTEDT serum separator tube. The whole blood was allowed to clot at room temperature for 20 minutes and then centrifuged at 11,500 rpm for 15 minutes, and the resulting serum was stored at -70 °C until further analysis. Non-compartmental analysis was performed on the concentration-time curve of the administered antibody. The AUClast was calculated for each individual animal, and the mean values are presented in Table 2 along with the standard deviation in parentheses.

[0119] The administered non-CAT-WT antibody did not exhibit dose-proportional exposure, indicating significant TMDD (target-mediated drug disposition) clearance ( Figure 1A ). The rapid decline to undetectable antibody concentrations 24 hours after administration of 0.3 mg / kg and 3 mg / kg of the non-CAT-WT antibody indicates that ASGR1 rapidly binds the antibody and degrades the bound antibody. At a dose of 10 mg / kg, the clearance curve was biphasic, with the clearance rate decreasing slightly after 48 hours post-administration. For the antibody administered at 30 mg / kg, a typical four-phase TMDD-mediated clearance ( Figure 1B ) was observed. In contrast, the CAT antibody showed a modified clearance curve when compared to the non-CAT antibody. At all dose levels, the CAT antibody exhibited an enhanced rapid decline in concentration within 24 hours post-administration ( Table 2. Serum exposure of the administered antibody)。After 24 hours of administration, the clearance rate of the CAT antibody decreased significantly, indicating that ASGR1-mediated clearance was minimal during this period. The CAT antibody designed to overcome TMDD was effective at low dose levels (Table 2). At doses of 0.3 mg / kg and 3 mg / kg, the exposure of the CAT antibody was 84.3-fold and 58-fold in excess of that of the non-CAT antibody, respectively. At a dose of 10 mg / kg, both the CAT and non-CAT antibodies had similar exposure (Table 2). However, at a dose of 30 mg / kg, the CAT antibody had a reduced exposure and was 0.4-fold that of the non-CAT antibody. The clearance properties of both the CAT and non-CAT antibodies suggest that the moiety targeting ASGR1 can be used to deplete soluble antigens by cross-linking the soluble antigen with an anti-ASGR1 antibody.

[0120] Figure 2 。

[0121]

[0122] These data indicate that at higher administration doses, the CAT antibody has a faster in vivo clearance compared to the non-CAT antibody.

[0123] Additional studies demonstrated the simultaneous binding of the multispecific molecules of the present invention to human IgG and ASGR1. The 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. The experiment was repeated twice, and both experiments are presented in Table 3. Equilibrium dissociation constants for the interaction between murine ASGR1 and an anti-ASGR1 antibody or a bispecific scFv molecule of the invention at pH 7.4 and 2 mM CaCl2 IVIg binding to the complexed ASGR1:bispecific scFv indicates that the bispecific scFv of the present invention can bind to both ASGR1 and IVIg simultaneously.

[0124] Example 3: Form and manufacturability of multispecific molecules

[0125] Based on the in vivo clearance of catabolic and non-catabolic anti-ASGR1 antibodies, the anti-ASGR1 moiety can be used as a vehicle to deplete soluble antigens. To target autoantibody-mediated diseases, it is necessary to deplete circulating human Ig (see, for example, Howard et al., Neurology 2019, 92(23). Ig (such as IgG) can be cross-linked with ASGR1 to facilitate Ig clearance).

[0126] In designing the form of the bispecific scFv molecules of the present invention that crosslink ASGR1 and human IgG, two key design principles were considered. First, in order to prevent the bispecific scFv molecules of the present invention from forming IgG complexes that activate the immune system and thus cause allergic reactions or other immune-mediated side effects (see, e.g., Mayadas et al., Circulation 120(20), November 17, 2009: 2012-2024), the portion targeting IgG binding should be monovalent. Second, the bispecific scFv molecules of the present invention should lack human IgG fragments that can cause self-binding. For example, if targeting the Fc fragment to deplete IgG, the bispecific scFv molecules of the present invention should lack the Fc fragment. Similarly, if targeting the Fc fragment to deplete IgG, the bispecific scFv molecules of the present invention should also lack the Fc fragment.

[0127] To generate bispecific scFv, the antibody sequences were reformatted into scFv in which the antibody heavy and light chains were fused via a 3xG4S linker and subsequently had a 6xHis at the C-terminus. Gene fragments of the scFv were synthesized and cloned into a mammalian stable expression (pTT5-derived vector carrying a puromycin selection marker). The secreted scFv protein was directly captured from the conditioned medium by affinity chromatography using Ni Sepharose Excel resin (GE Healthcare Life Sciences). Additional polishing was accomplished using CHT™ Ceramic Hydroxyapatite Type I 40um 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 buffer was exchanged into the final formulation: 25 mM citrate, 75 mM arginine, 4% sucrose, pH 7.0 by dialysis. The final product quality 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 scFv were selected for further in vitro and in vivo experiments. Bispecific scFv molecules that bind ASGR1 in a non-catabolic manner and bind Ig in a non-catabolic manner (non-CAT bispecific scFv molecules) contain the amino acid sequence given by SEQ ID NO: 37. Bispecific scFv molecules that bind ASGR1 in a catabolic manner and bind Ig in a non-catabolic manner (CAT bispecific scFv molecules) contain the amino acid sequence given by SEQ ID NO: 46.

[0128] Example 4: Bispecific scFv Molecules That Bind Both ASGR1 and Human IgG

[0129] To demonstrate the simultaneous binding of the bispecific scFv molecules of the invention to human IgG and ASGR1, ASGR1 was immobilized on an SPR chip, and subsequently 100 nM of the bispecific scFv molecules of the invention (containing a binding domain that binds ASGR1 in a catabolic manner and a binding domain that binds IgG in a non-catabolic manner) or an anti-ASGR1 antibody (containing a binding domain that binds ASGR1 in a catabolic manner) and 100 nM IVIg (intravenous immunoglobulin) were co-administered. IVIg consists of a pool of immunoglobulins from a large cohort of healthy human volunteers. K was determined by immobilizing the receptor on the chip and using the antibody as the analyte. DValue

[0130] The binding of anti-ASGR1 antibodies to ASGR1 in a pH / Ca2+-dependent or -independent manner was evaluated by BIAcore. The equilibrium binding affinities of the interactions between the following were determined using a BIAcore 3000: (i) murine or human ASGR1 and anti-ASGR1 antibodies; and (ii) murine or human ASGR1 and the bispecific scFv molecules of the present invention. Murine and human ASGR1 proteins were obtained from R&D-systems (catalog numbers 2755-AS / CF and 4394-AS / CF, respectively) and immobilized on a CM5 chip using amine coupling chemistry to a density of approximately 1500 RU. On each CM5 chip, a reference flow cell coupled only to the coupling buffer was used. Antibodies were injected over the immobilized ASGR1 at a concentration range of 1000 nM - 0.2 nM (two-fold serial dilutions). To determine pH / Ca-sensitive binding, experiments were performed in phosphate-buffered saline (PBS) with 0.01% (v / v) Tween 20 and 0.05% azide at pH 7.4 / 2 mM calcium chloride or pH 6.0 / 2 μM calcium chloride. The chip was regenerated between each injection cycle using 0.15 M NaCl, 0.1 M glycine pH 1.5 buffer. The equilibrium dissociation constant was determined using BIAevaluation with a 1:1 interaction model.

[0131] These data demonstrate that the bispecific scFv molecules of the present invention exhibit dose-dependent binding to immobilized ASGR1, and the binding of IVIg to the complexed ASGR1:bispecific scFv molecule of the present invention indicates that the bispecific scFv molecules of the present invention can bind both ASGR1 and IVIg simultaneously. The anti-huIgG component of the bispecific scFv molecules of the present invention binds to the IgG1, IgG2, and IgG4 subclasses of human IgG. Due to the conversion from the mAb form to the scFv, the affinity of the bispecific scFv molecules of the present invention for ASGR1 in the scFv is reduced (Table 3). These data demonstrate that in the bispecific scFv form, the bispecific scFv molecules of the present invention (bispecific scFv) can bind both ASGR1 and human IgG. The binding of one target to the bispecific scFv does not inhibit the binding to the other target.

[0132] Figure 3 Figure 4

[0133]

[0134] Example 5: In vivo clearance

[0135] 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). Blocking a portion of FcRn - mediated IgG recycling, such as anti - FcRn antibodies or ABDEG, takes approximately four days to deplete approximately 70% - 80% of human IgG administered in mice, and ABDEG takes 2 - 3 weeks in humans to clear approximately 50% - 70% of IgG (Vaccaro C et al., Nat. Biotechnol. 23, 1283 - 1288 (2005); Getman KE and 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., Neurology, 2019, 92(23)).

[0136] 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 the bispecific scFv molecule of the present invention that binds to ASGR1 in a pH - and calcium - sensitive manner.

[0137] To analyze the clearance of human IgG, IVIG (Sigma, catalog number 56834) was administered to mice by intravenous injection, and 72 hours later, the CAT bispecific scFv molecule of the present invention (which binds to ASGR1 in a catabolic manner and binds to IgG in a non - catabolic manner) (1.67 μM) or PBS was administered to the mice. At the indicated time points, 50 μl of whole blood was collected via submandibular vein puncture using a SARSTEDT serum separator tube using a sparse continuous sampling protocol (n = 3 mice / group / time point). The whole blood was allowed to clot at room temperature for 20 minutes, then centrifuged at 11,500 rpm for 15 minutes, and the resulting serum was stored at - 80 °C until further analysis.

[0138] For cynomolgus monkey analysis, an intravenous injection dose of the bispecific scFv molecule of the invention was administered to female naive cynomolgus monkeys via the saphenous vein, and blood was collected into tubes without anticoagulant (serum separator tubes) via the femoral vein at the indicated time points. The blood was allowed to clot at ambient temperature before centrifugation to obtain serum. Centrifugation was started within one hour after collection. The serum was placed in polypropylene tubes and kept on dry ice before storage at -80°C.

[0139] To quantify huIVIG, a mouse anti-human IgG F(ab’)2 specific antibody (Jackson ImmunoResearch Labs, catalog number 209-005-097) was used as the capture and detection reagent in an ELISA-based assay.

[0140] These data demonstrate that within three hours after administration of the CAT bispecific scFv molecule of the invention, the human IgG concentration in mice decreased by approximately 70% ( Figure 6 ). A similar enhanced clearance of the bispecific scFv molecule of the invention was observed in cynomolgus monkeys, in which the bispecific scFv molecule of the invention depleted 72% of human IgG within 12 hours after administration of the bispecific scFv molecule of the invention ( Table 4. Octet-based binding characterization of the generated molecules to murine IgG2a at neutral pH and 2 mM CaCl2 and acidic pH and 2 μM CaCl2 ).

[0141] Example 6: Generation of CAT and non-CAT bispecific scFv molecules

[0142] As shown in Figure 1, compared to a non-catabolic anti-ASGR1 antibody, an anti-ASGR1 antibody that binds to the receptor in a catabolic manner has a faster in vivo clearance. This rapid clearance indicates that the catabolic anti-ASGR1 accumulates faster into lysosomes. To analyze whether the rapid clearance of the catabolic anti-ASGR1 moiety would result in a faster clearance of human IgG if the bispecific scFv molecule of the invention binds to ASGR1 in a catabolic manner (and binds to Ig in a non-catabolic manner), the bispecific scFv molecules of the invention that bind to ASGR1 in catabolic and non-catabolic manners were tested in vivo. The bispecific scFv molecule that binds to ASGR1 in a non-catabolic manner contains the amino acid sequence given by SEQ ID NO:37. The bispecific scFv molecule that binds to ASGR1 in a catabolic manner contains the amino acid sequence given by SEQ ID NO:46.

[0143] Human IgG (IVIg) was labeled with Na125I (Perkin Elmer, catalog number NEZ033L), and radio-based pharmacokinetics were evaluated. 1.67 uM of human IVIg was injected into mice via the tail vein, and the radioactivity of the animals was immediately measured in a dose calibrator (Capintec, catalog number CRC-15R) to obtain the initial (T = 0) whole body activity. Then, 1.67 μM of the bispecific scFv molecule of the present invention was injected into the mice via the tail vein. At the indicated times, the whole body activity of the animals was read, and then blood samples were collected by pricking the tail vein and collected in capillary tubes. All capillary tubes were weighed before collection and then after collection to determine the exact blood weight / volume collected. The radioactivity of the serum samples was analyzed by a gamma counter.

[0144] As shown in Figure 5, the bispecific scFv molecules of the present invention that bind to ASGR1 in a catabolic or non-catabolic manner showed similar efficacy in depleting serum IgG and catabolizing serum IgG in vivo, indicating that the pH / Ca2+-dependent binding to ASGR1 had no significant effect on the clearance of exogenously administered human IgG in mice by the bispecific scFv molecules of the present invention.

[0145] Example 7: Bispecific scFv Molecule Induces IgG Hepatic Catabolism

[0146] ASGR1 is mainly expressed on both the cell membrane and cytoplasm in hepatocytes, especially on the limiting membrane of endosomes. Ligands targeting ASGR1 are expected to accumulate and catabolize in the liver.

[0147] To analyze the catabolism of exogenously administered IVIg in mice after administration of the targeting molecule, IVIg was labeled with I-125 and In-111 with non-residual and residual radioactivity, respectively. When IgG degrades, iodine (a non-residual dye) is secreted from the cell when cleaved from IgG and then undergoes renal clearance. However, indium is a residual dye, and thus it remains in the cell even after cleavage from IgG. Therefore, similar levels of I-125 and In-111 indicate a lack of catabolism, while different levels indicate that IgG is being catabolized in the liver.

[0148] Radioactively labeled IVIg was administered to mice via tail vein injection, and the radioactivity of the animals was measured in a dose calibrator (Capintec, catalog number CRC-15R) to obtain the initial (T = 0) whole body activity. After 72 hours, the CAT bispecific scFv molecule of the present invention (which binds to ASGR1 in a catabolic manner and binds to Ig in a non-catabolic manner) or PBS was administered to the animals. At the designated time points, the whole body activity of the animals was read, and then blood samples were collected by pricking the tail vein and collected in capillary tubes. All capillary tubes were weighed before collection and then after collection to determine the exact blood weight / volume collected. The radioactivity of the serum samples was analyzed by a gamma counter. The animals were perfused, and then the organs were harvested, weighed, and their radioactivity was measured.

[0149] Table 5. Octet-based binding characterization of the generated molecules to murine IgG2a at neutral pH and 2 mM CaCl2 and acidic pH and 2 μM CaCl2 These data shown in demonstrate that, compared to the PBS control group, three hours after administration of the targeting molecule, most of the circulating human IgG was localized to the liver, as indicated by the radioactivity of the residual label In-111. Since the radioactivity signal of the non-residual label I-125 was approximately 1% ID / g at all measurement time points, it was indicated that the accumulated human IgG was rapidly catabolized by hepatic lysosomes. Bringing an antigen (such as Ig) to the liver for clearance from the circulation is considered to reduce toxicity compared to antigen clearance by different mechanisms.

[0150] Example 8: Catabolic IgG Molecule

[0151] Molecules were generated that bind to ASGR1 in a non-catabolic manner and bind to IgG in a catabolic manner. This binding characteristic to ASGR1 and IgG will enable continuous recycling of the molecule by binding to ASGR1 while dissociating IgG in the endosomal compartment. Mouse IgG2a was selected as the antigen to be able to test the generated molecules in an in vivo disease model expressing autoantibodies of the IgG2a subclass.

[0152] Rabbits were immunized with mouse IgG2a, and B cell spleens of the immunized animals were harvested. The harvested cells were sorted using a FACS-based multiplex assay by analyzing the binding to mouse IgG2a, IgG2b, IgG2c, IgG1, and an irrelevant antigen. Clones that specifically bind to mouse IgG2a were identified by analyzing the binding at pH 6.0 and 2 μM CaCl2 and pH 7.4 and 2 mM CaCl2, and their catabolic binding was further screened. The heavy and light chain sequences of the antibody were extracted for the conjugates showing the desired catabolic binding properties, the conjugates were converted to the Fab-scFc form (anti-mIgG2a), and the binding characteristics to mouse IgG2a were analyzed for their catabolic binding affinity using BIAcore- and Octet-based assays.

[0153] For the Octet-based assay, the streptavidin biosensor was loaded with biotinylated mouse IgG2a and the association and dissociation of the conjugate were carried out under the above acidic and neutral pH conditions. The biosensor was regenerated with 10 mM glycine buffer at pH 1.5.

[0154] Similarly, for the BIAcore-based assay, cross-linked anti-mouse IgG (H+L) was amine-coupled to the CM5 sensor chip and mouse IgG2a was captured as a ligand, while the generated conjugate was injected as an analyte at neutral pH and 2 mM CaCl2 or acidic pH and 2 μM CaCl2. The analyte was injected in 3-fold serial dilutions, with the highest concentration ranging from 900 nM. The CM5 chip was regenerated with 10 mM glycine buffer at pH 1.5. Analysis of the binding was performed in BIAevaluation using a 1:1 Langmuir fit.

[0155] The Octet data for molecule 099 (containing HC with SEQ ID NO:53 and LC with SEQ ID NO:54) are shown in Table 4. Molecule 099 showed tight binding to mouse IgG2a at neutral pH and 2 mM CaCl2, while showing reduced affinity (about 3-fold) for mouse IgG2a at acidic pH and 2 μM CaCl2. The weaker the affinity for the antigen at acidic pH, the higher the efficiency of antigen release in endosomes.

[0156] Molecule 099 was further engineered by introducing histidine residues in the CDR regions to enhance pH-dependent binding. Two resulting clones (465, which contains HC with SEQ ID NO:55 and LC with SEQ ID NO:56; and 463, which contains HC with SEQ ID NO:57 and LC with SEQ ID NO:58) demonstrated further reduced binding to mouse IgG2a at pH 6.0 and 2 μM CaCl2, while maintaining tight binding at neutral pH and 2 mM CaCl2 (Table 5). The resulting sensorgrams (BIAcore data) showed catabolic binding characteristics similar to those of mouse IgG2a, as determined by the Octet assay.

[0157] ​ ​

[0158]

[0159] ​ ​ 。

[0160]

[0161] These data demonstrate that the test molecules bind to IgG in a catabolic manner. The binding of these molecules to murine IgG2a is reduced at pH 6.0 and 2 μM CaCl2, while remaining tightly bound at neutral pH and 2 mM CaCl2.

[0162] The conjugate was converted to scFv form by converting the conjugate to scFv and fusing the conjugate with an anti-mouse / human ASGR1 scFv (e.g., SEQ ID NO: 75). One or more additional cysteines were introduced to improve the stability of the bispecific scFv molecule. The binding of these bispecific scFvs to mouse / human ASGR1 and murine IgG2a was analyzed by flow cytometry, and it was determined that these bispecific scFvs remained bound to ASGR1 and IgG2a when converted from Fab to scFv form at neutral pH.

[0163] Sequence

[0164] Non-catabolic anti-ASGR1 antibody HCDR1 (SEQ ID NO: 1)

[0165] DYNMA

[0166] Non-catabolic anti-ASGR1 antibody HCDR2 (SEQ ID NO: 2)

[0167] TIIYDGGSTYYRHSVKG

[0168] Non-catabolic anti-ASGR1 antibody HCDR3 (SEQ ID NO: 3)

[0169] QTYFGSRDYFDY

[0170] Non-catabolic anti-ASGR1 antibody LCDR1 (SEQ ID NO: 4)

[0171] LTSEDIYNNLA

[0172] Non-catabolic anti-ASGR1 antibody LCDR2 (SEQ ID NO: 5)

[0173] YASNFQD

[0174] Non-catabolic anti-ASGR1 antibody LCDR3 (SEQ ID NO: 6)

[0175] LQDSEYPP

[0176] Non-catabolic anti-ASGR1 antibody HCVR (SEQ ID NO: 7)

[0177] EVQLVESGGGLVQPGRSLKLSCAASGFTFSDYNMAWVRQAPKKGLEWVATIIYDGGSTYYRHSVKGRFTISRDNAKSTHSLQMDSLRSEDTATYYCARQTYFGSRDYFDYWGQGVMVTVSS

[0178] Non-catabolic anti-ASGR1 antibody LCVR (SEQ ID NO:8)

[0179] DIQMTQSPTSLSASLGETVSIECLTSEDIYNNLAWYQQKPGKSPQLLISYASNFQ DGVPSRFSGSGSGTQYSLKINSLESEDAATYFCLQDSEYPPTFGGGTKLELKR

[0180] Non-catabolic anti-ASGR1 antibody HC (SEQ ID NO:9)

[0181] EVQLVESGGGLVQPGRSLKLSCAASGFTFSDYNMAWVRQAPKKGLEWVATIIYDGGSTYYRHSVKGRFTISRDNAKSTHSLQMDSLRSEDTATYYCARQTYFGSRDYFDYWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0182] Non-catabolic anti-ASGR1 antibody LC (SEQ ID NO:10)

[0183] DIQMTQSPTSLSASLGETVSIECLTSEDIYNNLAWYQQKPGKSPQLLISYASNFQDGVPSRFSGSGSGTQYSLKINSLESEDAATYFCLQDSEYPPTFGGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0184] Catabolic anti-ASGR1 antibody HCDR1 (SEQ ID NO:11)

[0185] SYGMH

[0186] Catabolic anti-ASGR1 antibody HCDR2 (SEQ ID NO:12)

[0187] VIWYDGSNKYYADSVKG

[0188] Catabolic anti-ASGR1 antibody HCDR3 (SEQ ID NO:13)

[0189] DSSPYGMDV

[0190] Catabolic anti-ASGR1 antibody LCDR1 (SEQ ID NO:14)

[0191] RASQGISSWLA

[0192] Catabolic anti-ASGR1 antibody LCDR2 (SEQ ID NO:15)

[0193] GASSLQS

[0194] Catabolic anti-ASGR1 antibody LCDR3 (SEQ ID NO:16)

[0195] QQSDSFPRT

[0196] Catabolic anti-ASGR1 antibody HCVR (SEQ ID NO:17)

[0197] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSSPYGMDVWGQGTTVTVSS

[0198] Catabolic anti-ASGR1 antibody LCVR (SEQ ID NO:18)

[0199] DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASS LQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYCQQSDSFPRTFGQGTKVEIKR

[0200] Catabolic anti-ASGR1 antibody HC (SEQ ID NO:19)

[0201] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSSPYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPCVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0202] Catabolic anti-ASGR1 antibody LC (SEQ ID NO:20)

[0203] DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYCQQSDSFPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0204] Anti-IgG HCDR1 (SEQ ID NO:21)

[0205] DTYIH

[0206] Anti-IgG HCDR2 (SEQ ID NO:22)

[0207] RIDPANGNTKYDPKFQD

[0208] Anti-IgG HCDR3 (SEQ ID NO:23)

[0209] NYGSNYDPMDY

[0210] Anti-IgG LCDR1 (SEQ ID NO:24)

[0211] RASQNIDTNIH

[0212] Anti-IgG LCDR2 (SEQ ID NO:25)

[0213] YASESIS

[0214] Anti-IgG LCDR3 (SEQ ID NO:26)

[0215] QQSDTWPWT

[0216] Non-catabolic anti-ASGR1 HCDR1 (SEQ ID NO:27)

[0217] DYSVH

[0218] Non-catabolic anti-ASGR1 HCDR2 (SEQ ID NO:28)

[0219] IMWTGGSTAYNSALKS

[0220] Non-catabolic anti-ASGR1 HCDR3 (SEQ ID NO:29)

[0221] DGDYGPDY

[0222] Non-catabolic anti-ASGR1 LCDR1 (SEQ ID NO:30)

[0223] QASQDIGNWLS

[0224] Non-catabolic anti-ASGR1 LCDR2 (SEQ ID NO:31)

[0225] GATSLAD

[0226] Non-catabolic anti-ASGR1 LCDR3 (SEQ ID NO:32)

[0227] LQAYSAPPWT

[0228] Anti-IgG HCVR (SEQ ID NO:33)

[0229] ELQLQQSGAELVRPGASVKLSCTTSGFNVKDTYIHWVRQRPEQGLEWIGRIDPANGNTKYDPKFQDRATITTDTSSITAYLQLSSLTSEDTAVYYCARNYGSNYDPMDYWGQGTSLTVSS

[0230] Anti-IgG LCVR (SEQ ID NO:34)

[0231] DILLTQSPAILSVSPGERVSFSCRASQNIDTNIHWYQRRTNDSPRLLIKYASESIS GIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSDTWPWTFGGGTKLEIKR

[0232] Non-catabolic anti-ASGR1 HCVR (SEQ ID NO:35)

[0233] EVQLKESGPGLVQPSQTLSLTCTVSGFSLTDYSVHWVRQSPGKGLEWMGIMWTGGSTAYNSALKSRLSISRDTSKSQVFLKMNSLQTEDTAIYYCTRDGDYGPDYWGQGVMVTVSS

[0234] Non-catabolic anti-ASGR1 LCVR (SEQ ID NO:36)

[0235] DIQMTQSPASLSASLEEIVTITCQASQDIGNWLSWYQQKPGKSPQLLIYGATSL ADGVPSRFSGSRSGTQYSLKISRLQVEDIGIYYCLQAYSAPPWTFGGGTKLELKR

[0236] Non-catabolic bispecific scFv (SEQ ID NO:37)

[0237] ELQLQQSGAELVRPGASVKLSCTTSGFNVKDTYIHWVRQRPEQGLEWIGRIDPANGNTKYDPKFQDRATITTDTSSITAYLQLSSLTSEDTAVYYCARNYGSNYDPMDYWGQGTSLTVSSGGGGSGGGGSGGGGSDILLTQSPAILSVSPGERVSFSCRASQNIDTNIHWYQRRTNDSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSDTWPWTFGGGTKLEIKRSGGGGSEVQLKESGPGLVQPSQTLSLTCTVSGFSLTDYSVHWVRQSPGKGLEWMGIMWTGGSTAYNSALKSRLSISRDTSKSQVFLKMNSLQTEDTAIYYCTRDGDYGPDYWGQGVMVTVSSGGGGSGGGGSGGGGSDIQMTQSPASLSASLEEIVTITCQASQDIGNWLSWYQQKPGKSPQLLIYGATSLADGVPSRFSGSRSGTQYSLKISRLQVEDIGIYYCLQAYSAPPWTFGGGTKLELKRHHHHHH

[0238] Catabolic anti-ASGR1 HCDR1 (SEQ ID NO:38)

[0239] SYGMH

[0240] Catabolic anti-ASGR1 HCDR2 (SEQ ID NO:39)

[0241] VIWYDGSNKYYADSVKG

[0242] Catabolic anti-ASGR1 HCDR3 (SEQ ID NO:40)

[0243] DSSPYGMDV

[0244] Catabolic anti-ASGR1 LCDR1 (SEQ ID NO:41)

[0245] RASQGISSWLA

[0246] Catabolic anti-ASGR1 LCDR2 (SEQ ID NO:42)

[0247] GASSLQS

[0248] Catabolic anti-ASGR1 LCDR3 (SEQ ID NO:43)

[0249] QQSDSFPRT

[0250] Catabolic anti-ASGR1 HCVR (SEQ ID NO:44)

[0251] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSSPYGMDVWGQGTTVTVSS

[0252] Catabolic anti-ASGR1 LCVR (SEQ ID NO:45)

[0253] DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASS LQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYCQQSDSFPRTFGQGTKVEIKR

[0254] Catabolic bispecific scFv (SEQ ID NO:46)

[0255] ELQLQQSGAELVRPGASVKLSCTTSGFNVKDTYIHWVRQRPEQGLEWIGRIDPANGNTKYDPKFQDRATITTDTSSITAYLQLSSLTSEDTAVYYCARNYGSNYDPMDYWGQGTSLTVSSGGGGSGGGGSGGGGSDILLTQSPAILSVSPGERVSFSCRASQNIDTNIHWYQRRTNDSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSDTWPWTFGGGTKLEIKRSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDSSPYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASSLQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYCQQSDSFPRTFGQGTKVEIKRHHHHHH

[0256] Leader sequence (SEQ ID NO:47)

[0257] MDMRVPAQLLGLLLLWLRGARC

[0258] Nucleic acid sequence encoding the leader sequence of SEQ ID NO:47 (SEQ ID NO:48)

[0259] ATGGACATGAGAGTGCCTGCACAGCTGCTGGGCCTGCTGCTGCTGTGGCTG AGAGGCGCCAGATGC

[0260] Leader sequence (SEQ ID NO:49)

[0261] MAWALLLLTLLTQGTGSWA

[0262] Nucleic acid sequence encoding the leader sequence of SEQ ID NO:49 (SEQ ID NO:50)

[0263] ATGGCCTGGGCTCTGCTGCTCCTCACCCTCCTCACTCAGGGCACAGGGTCC TGGGCC

[0264] Anti-Ig HC (SEQ ID NO:51)

[0265] ELQLQQSGAELVRPGASVKLSCTTSGFNVKDTYIHWVRQRPEQGLEWIGRIDPANGNTKYDPKFQDRATITTDTSSITAYLQLSSLTSEDTAVYYCARNYGSNYDPMDYWGQGTSLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0266] Anti-Ig LC (SEQ ID NO:52)

[0267] DILLTQSPAILSVSPGERVSFSCRASQNIDTNIHWYQRRTNDSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSDTWPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0268] Catabolic anti-mIgG2a 099Fab-scFc HC (SEQ ID NO:53)

[0269] EQLEESGGDLVKPGASLTLTCTASGFSFTSDYYMCWVRQAPGKGLEWIACIGAGDIHTTYYANWAKGRFTISKTSSTTVTLQMTTLTAADTATYFCARDTYNIGGYTGDFDLWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0270] Catabolic anti-mIgG2a 099 Fab-scFc LC (SEQ ID NO:54)

[0271] ALVMTQPPASVSAAVGGTVTINCQASESISTWLAWYQQKPGQPPKLLIYYASTLASGVPSRFKGSGSGTQFTLTISGVECDDAATYYCAGHKSYSSDDFAFGGGTEVVVKGTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0272] Catabolic anti-mIgG2a 465Fab-scFc HC (SEQ ID NO:55)

[0273] EQLEESGGDLVKPGASLTLTCTASGFSFTSDYYMCWVRQAPGKGLEWIACHGAGDIHTTYYANWAKGRFTISKTSSTTVTLQMTTLTAADTATYFCARDTYNIGGYTGDFDLWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0274] Catabolic anti-mIgG2a 465Fab-scFc LC (SEQ ID NO:56)

[0275] ALVMTQPPASVSAAVGGTVTINCQASESISTWLAWYQQKPGQPPKLLIYYASTLASGVPSRFKGSGSGTQFTLTISGVECDDAATYYCAGHKSYSSDDFAFGGGTEVVVKGTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0276] Catabolic anti-mIgG2a 463Fab-scFc HC (SEQ ID NO:57)

[0277] EQLEESGGDLVKPGASLTLTCTASGFSFTSDYYHCWVRQAPGKGLEWIACIGAGDIHTTYYANWAKGRFTISKTSSTTVTLQMTTLTAADTATYFCARDTYNIGGYTGDFDLWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0278] Catabolic anti-mIgG2a 463Fab-scFc LC (SEQ ID NO:58)

[0279] ALVMTQPPASVSAAVGGTVTINCQASESISTWLAWYQQKPGQPPKLLIYYASTLASGVPSRFKGSGSGTQFTLTISGVECDDAATYYCAGHKSYSSDDFAFGGGTEVVVKGTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0280] Catabolic anti-mIgG2a 099 HCDR1 (SEQ ID NO:59)

[0281] SDYYMC

[0282] Catabolic anti-mIgG2a 099 HCDR2 (SEQ ID NO:60)

[0283] CIGAGDIHTTYYANWAKG

[0284] Catabolic anti-mIgG2a 099 HCDR3 (SEQ ID NO:61)

[0285] DTYNIGGYTGDFDL

[0286] Catabolic anti-mIgG2a 099, 465 and 463 LCDR1 (SEQ ID NO:62)

[0287] QASESISTWLA

[0288] Catabolic anti-mIgG2a 099, 465 and 463 LCDR2 (SEQ ID NO:63)

[0289] YASTLAS

[0290] Catabolic anti-mIgG2a 099, 465 and 463 LCDR3 (SEQ ID NO:64)

[0291] AGHKSYSSDDFA

[0292] Catabolic anti-mIgG2a 465 HCDR1 (SEQ ID NO:65)

[0293] SDYYMC

[0294] Catabolic anti-mIgG2a 465 HCDR2 (SEQ ID NO:66)

[0295] CHGAGDIHTTYYANWAKG

[0296] Catabolic anti-mIgG2a 465 HCDR3 (SEQ ID NO:67)

[0297] DTYNIGGYTGDFDL

[0298] Catabolic anti-mIgG2a 463 HCDR1 (SEQ ID NO:68)

[0299] SDYYHC

[0300] Catabolic anti-mIgG2a 463 HCDR2 (SEQ ID NO:69)

[0301] CIGAGDIHTTYYANWAKG

[0302] Catabolic anti-mIgG2a 463 HCDR3 (SEQ ID NO:70)

[0303] DTYNIGGYTGDFDL

[0304] Catabolic anti-mIgG2a 099 HCVR (SEQ ID NO:71)

[0305] EQLEESGGDLVKPGASLTLTCTASGFSFTSDYYMCWVRQAPGKGLEWIACIGAGDIHTTYYANWAKGRFTISKTSSTTVTLQMTTLTAADTATYFCARDTYNIGGYTGDFDLWGPGTLVTVSS

[0306] Catabolic anti-mIgG2a 099, 463 and 465 LCVR (SEQ ID NO:72)

[0307] ALVMTQPPASVSAAVGGTVTINCQASESISTWLAWYQQKPGQPPKLLIYYASTLASGVPSRFKGSGSGTQFTLTISGVECDDAATYYCAGHKSYSSDDFAFGGGTEVVVKG

[0308] Catabolic anti-mIgG2a 465 HCVR (SEQ ID NO:73)

[0309] EQLEESGGDLVKPGASLTLTCTASGFSFTSDYYMCWVRQAPGKGLEWIACHGAGDIHTTYYANWAKGRFTISKTSSTTVTLQMTTLTAADTATYFCARDTYNIGGYTGDFDLWGPGTLVTVSS

[0310] Catabolic anti-mIgG2a 463 HCVR (SEQ ID NO:74)

[0311] EQLEESGGDLVKPGASLTLTCTASGFSFTSDYYHCWVRQAPGKGLEWIACIGAGDIHTTYYANWAKGRFTISKTSSTTVTLQMTTLTAADTATYFCARDTYNIGGYTGDFDLWGPGTLVTVSS

[0312] Catabolic anti-mIgG2a non-catabolic ASGR1 bispecific scFv (SEQ ID NO:75)

[0313] EQLEESGGDLVKPGASLTLTCTASGFSFTSDYYMCWVRQAPGKGLEWIACIGAGDIHTTYYANWAKGRFTISKTSSTTVTLQMTTLTAADTATYFCARDTYNIGGYTGDFDLWGPGTLVTVSSSGGGGSGGGGSGGGGSALVMTQPPASVSAAVGGTVTINCQASESISTWLAWYQQKPGQPPKLLIYYASTLASGVPSRFKGSGSGTQFTLTISGVECDDAATYYCAGHKSYSSDDFAFGGGTEVVVKGSGGGGSEVQLKESGPGLVQPSQTLSLTCTVSGFSLTDYSVHWVRQSPGKGLEWMGIMWTGGSTAYNSALKSRLSISRDTSKSQVFLKMNSLQTEDTAIYYCTRDGDYGPDYWGQGVMVTVSSGGGGSGGGGSGGGGSDIQMTQSPASLSASLEEIVTITCQASQDIGNWLSWYQQKPGKSPQLLIYGATSLADGVPSRFSGSRSGTQYSLKISRLQVEDIGIYYCLQAYSAPPWTFGGGTKLELKRHHHHHH

[0314] (Gly3Ser)3 (SEQ ID NO:76)

[0315] GGGSGGGSGGGS

[0316] (Gly4Ser)3(SEQ ID NO:77)

[0317] GGGGSGGGGSGGGGS

[0318] (Gly3Ser)4(SEQ ID NO:78)

[0319] GGGSGGGSGGGSGGGS

[0320] (Gly4Ser)4(SEQ ID NO:79)

[0321] GGGGSGGGGSGGGGSGGGGS

[0322] (Gly3Ser)5(SEQ ID NO:80)

[0323] GGGSGGGSGGGSGGGSGGGS

[0324] (Gly4Ser)5(SEQ ID NO:81)

[0325] GGGGSGGGGSGGGGSGGGGSGGGGS

[0326] (Gly3Ser)6(SEQ ID NO:82)

[0327] GGGSGGGSGGGSGGGSGGGSGGGS

[0328] (Gly4Ser)6(SEQ ID NO:83)

[0329] GGGGS GGGGSGGGGSGGGGSGGGGSGGGGS

[0330] GSADDAKKDAAKKDAAKKDDAKKDDAGS(SEQ ID NO:84)

[0331] GSADDAKKDAAKKDAAKKDDAKKDDAKKDAGS(SEQ ID NO:85)

[0332] (Gly3Gln)2(SEQ ID NO:86)

[0333] GGGQGGGQ

[0334] (Gly4Gln)2(SEQ ID NO:87)

[0335] GGGGQGGGGQ

[0336] (Gly3Gln)3(SEQ ID NO:88)

[0337] GGGQGGGQGGGQ

[0338] (Gly4Gln)3(SEQ ID NO:89)

[0339] GGGGQGGGGQGGGGQ

[0340] (Gly3Gln)4(SEQ ID NO:90)

[0341] GGGQGGGQGGGQGGGQ

[0342] (Gly4Gln)4(SEQ ID NO:91)

[0343] GGGGQGGGGQGGGGQGGGGQ

[0344] (Gly3Gln)5(SEQ ID NO:92)

[0345] GGGQGGGQGGGQGGGQGGGQ

[0346] (Gly4Gln)5(SEQ ID NO:93)

[0347] GGGGQGGGGQGGGGQGGGGQGGGGQ

[0348] (Gly3Gln)6(SEQ ID NO:94)

[0349] GGGQGGGQGGGQGGGQGGGQGGGQ

[0350] (Gly4Gln)6(SEQ ID NO:95)

[0351] GGGGQGGGGQGGGGQGGGGQGGGGQGGGGQ

[0352] (Gly3Ser)2(SEQ ID NO:96)

[0353] GGGSGGGS

[0354] (Gly4Ser)2(SEQ ID NO:97)

[0355] GGGGSGGGGS

[0356] SGGGGSSGGGGS (SEQ ID NO:98).

Claims

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

2. The multispecific molecule according to 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. The multispecific molecule according to claim 1 or claim 2, wherein the first binding domain and / or the second binding domain is an scFv.

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

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

6. The multispecific molecule according to any one of claims 1, 2 and 5, wherein the first binding domain and the second binding domain are each an scFab.

7. The multispecific molecule according to claim 1 or claim 2, wherein the first binding domain and / or the second binding domain is a Fab.

8. The multispecific molecule according to any one of claims 1, 2 and 7, wherein the first binding domain and the second binding domain are each a Fab.

9. The multispecific molecule according to claim 1 or claim 2, wherein the first binding domain is an scFv and the second binding domain is a Fab.

10. The multispecific molecule according to claim 1 or claim 2, wherein the first binding domain is an scFv and the second binding domain is an scFab.

11. The multispecific molecule according to claim 1 or claim 2, wherein the first binding domain is a Fab and the second binding domain is an scFv.

12. The multispecific molecule according to claim 1 or claim 2, wherein the first binding domain is an scFab and the second binding domain is an scFv.

13. The multispecific molecule according to claim 1 or claim 2, wherein the first binding domain is a Fab and the second binding domain is an scFab.

14. The multispecific molecule according to claim 1 or claim 2, wherein the first binding domain is an scFab and the second binding domain is a Fab.

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

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

17. The multispecific molecule according to claim 15, wherein the linker is an SG4S linker.

18. The multispecific molecule according to claim 15, wherein the linker comprises a sequence 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:6285), (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).

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

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

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

22. The multispecific molecule according to any one of claims 1-20, wherein the immunoglobulin circulates in the blood.

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

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

25. The multispecific molecule according to claim 24, wherein the multispecific molecule depletes at least 70% of the immunoglobulins.

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

27. The multispecific molecule according to any one of claims 24-26, wherein the immunoglobulins are depleted within less than 72 hours.

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

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

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

31. The multispecific molecule according to any one of claims 1-30, which is for use in therapy.

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

33. The multispecific molecule according to any one of claims 1-30, which is for use in the manufacture of a medicament for the treatment of an autoantibody-induced disease.

34. The multispecific molecule according to 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 disorders associated with streptococcal infection, neuromyotonia, Morvan syndrome, multiple sclerosis, pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, acquired epidermolysis bullosa, pemphigoid gestationis, mucous membrane pemphigoid, lichen sclerosus, antiphospholipid syndrome, relapsing polychondritis, autoimmune anemia, idiopathic thrombocytopenic purpura, autoimmune Graves' disease, dilated cardiomyopathy, vasculitis, Goodpasture syndrome, idiopathic membranous nephropathy, rheumatoid arthritis and systemic lupus erythematosus.

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

36. The method according to claim 35, wherein the autoimmune antibody-induced disease is selected from the group consisting of: myasthenia gravis, Guillain-Barré syndrome, epilepsy, autoimmune limbic encephalitis, spinal cord injury, pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections, neuromyotonia, Morvan 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 thrombocytopenic purpura, autoimmune Graves' disease, dilated cardiomyopathy, vasculitis, Goodpasture syndrome, idiopathic membranous nephropathy, rheumatoid arthritis, and systemic lupus erythematosus.

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