Fcrn / HSA-binding molecules and methods of use

Abstract

Provided herein are binding molecules comprising a human neonatal Fc receptor (FcRn) binding molecule and at least one HSA-binding domain linked to the FcRn-binding molecule. Polynucleotides, vectors, host cells, and methods of production are also provided herein. Methods of treating an antibody-mediated disorder with an FcRn / HSA-binding molecule are further provided.

Description

FCRN / HSA-BINDING MOLECULES AND METHODS OF USE CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional PatentApplication No.63 / 612,866, filed on December 20, 2023, the contents of which are incorporated herein by reference in their entirety. SUBMISSION OF SEQUENCE LISTING XML

[0002] The content of the following submission of Sequence Listing XML is incorporatedherein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 404373-T2301WO_SL.xml, date created: December 19, 2024, size: 62,594 bytes). FIELD

[0003] The present disclosure relates to human neonatal Fc receptor (FcRn) / HSA-bindingmolecules and methods of using the same. BACKGROUND

[0004] Immunoglobulin gamma (IgG) antibodies play a key role in the pathology of manydisorders, such as autoimmune diseases, inflammatory diseases, and disorders in which the pathology is characterized by over-expression of IgG antibodies.

[0005] The half-life of IgG in the serum is prolonged relative to the serum half-life of otherplasma proteins due, in part, to the binding of the Fc region of IgG to the Fc receptor, FcRn. FcRn binds to IgG and protects the IgG from transport to degradative lysosomes by recycling it back to the extracellular compartment. This recycling is facilitated by the pH-dependent binding of IgG to FcRn, where the IgG / FcRn interaction is stronger at acidic endosomal pH than at extracellular physiological pH.

[0006] When the serum concentration of IgG reaches a level that exceeds available FcRnmolecules, unbound IgG is not protected from lysosomal degradation and will consequently have a reduced serum half-life. Thus, inhibition of IgG binding to FcRn reduces the serum half-life of IgG by preventing endosomal recycling of IgG. Agents that antagonize the binding of IgG to FcRn,such as FcRn-binding molecules, are useful for regulating, treating, or preventing antibody- mediated disorders, such as autoimmune diseases or inflammatory diseases.

[0007] Efgartigimod is a modified human immunoglobulin (Ig) gamma (IgG) 1-derived Fcof the za allotype that binds with nanomolar affinity to human FcRn. Efgartigimod encompasses the IgG1 Fc-region and has been engineered using ABDEG technology to increase its affinity for FcRn at both physiological and acidic pH. The increased affinity for FcRn of efgartigimod at both acidic and physiological pH results in a blockage of FcRn-mediated recycling of IgGs. Efgartigimod has been approved as a weekly intravenous injection for use in the treatment of generalized myasthenia gravis in the U.S. and other countries and is under development for the treatment of several other antibody-mediated disorders.

[0008] FcRn also binds to and recycles serum albumin, a modulator of serum cholesterollevels. Efgartigimod advantageously does not negatively impact serum albumin levels in human subjects. However, it has recently been shown that anti-FcRn antibodies can cause a reduction in serum albumin levels and a concomitant increase in serum cholesterol levels in human subjects, both of which are undesirable.

[0009] Accordingly, there is a need in the art for improved agents that antagonize FcRnbinding to IgG with a longer half-life, lower dose, less frequent administration, better maintenance of albumin levels, and / or reduction or elimination of FcRn degradation, for use in the treatment of antibody-mediated disorders. SUMMARY

[0010] The instant disclosure is broadly directed to neonatal Fc receptor (FcRn) bindingmolecules linked to an antigen-binding domain which specifically bind to human serum albumin (FcRn / HSA-binding molecules) and methods of use thereof. It has been shown in this application that, unexpectedly, FcRn / HSA-binding molecules with relatively low affinity for HSA have favorable PD / PK profiles and do not alter HSA serum concentrations when administered in vivo.

[0011] In an aspect, provided herein is an FcRn / HSA-binding molecule comprising anFcRn-binding molecule and an HSA-binding domain, wherein the HSA-binding domain comprises a VHH comprising the CDR1, CDR2, and CDR3 amino acid sequences of a VHH comprising an amino acid sequence selected from SEQ ID NOs: 23-26.

[0012] In some embodiments, the FcRn-binding molecule is a variant IgG Fc region.

[0013] In some embodiments, the variant IgG Fc region comprises a first Fc domain and asecond Fc domain which form a dimer, wherein the first Fc domain and / or the second Fc domain comprise amino acids Y, P, and Y at EU positions 252, 308, and 434, respectively.

[0014] In some embodiments, the variant IgG Fc region comprises a first Fc domain and asecond Fc domain which form a dimer, wherein the first Fc domain and / or the second Fc domain comprise amino acids Y, T, E, K, and F at EU positions 252, 254, 256, 433, and 434, respectively. In some embodiments, the first Fc domain and / or the second Fc domain comprise amino acids Y, T, E, K, F, and Y at EU positions 252, 254, 256, 433, 434, and 436, respectively. In some embodiments, both the first Fc domain and the second Fc domain comprise amino acids Y, T, E, K, and F at EU positions 252, 254, 256, 433, and 434, respectively. In some embodiments, both the first Fc domain and the second Fc domain comprise amino acids Y, T, E, K, F, and Y at EU positions 252, 254, 256, 433, 434, and 436, respectively.

[0015] In some embodiments, the first Fc domain and / or the second Fc domain is an IgG1Fc domain. In some embodiments, the first Fc domain and / or the second Fc domain is a human IgG Fc domain. In some embodiments, the first Fc domain and / or the second Fc domain is a human IgG1 Fc domain. In some embodiments, both the first Fc domain and the second Fc domain are IgG1 Fc domains. In some embodiments, both the first Fc domain and the second Fc domain are human IgG Fc domains. In some embodiments, both the first Fc domain and the second Fc domain are human IgG1 Fc domains.

[0016] In some embodiments, the HSA-binding domain is fused to the C-terminus of thefirst Fc domain or the second Fc domain. In some embodiments, the HSA-binding domain is fused to the N-terminus of the first Fc domain or the second Fc domain. In some embodiments, the HSA- binding domain is fused to the first Fc domain or the second Fc domain via a linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is a peptide linker, optionally, a GS linker, optionally from 8 to 40 amino acids in length, optionally 20 or 30 amino acids in length. In some embodiments, the HSA-binding domain is fused to the first Fc domain or the second Fc domain via an IgG hinge region or portion thereof.

[0017] In some embodiments, the first Fc domain and / or the second Fc domain comprisean amino acid sequence independently selected from an amino acid sequence set forth in SEQ ID NO: 1, 2, or 3. In some embodiments, the first Fc domain and / or the second Fc domain comprise the amino acid sequence of SEQ ID NO: 2. In some embodiments, both the first Fc domain and the second Fc domain comprise an amino acid sequence independently selected from an aminoacid sequence set forth in SEQ ID NO: 1, 2, or 3. In some embodiments, both the first Fc domain and the second Fc domain comprise the amino acid sequence of SEQ ID NO: 2. In some embodiments, the amino acid sequence of each of the first Fc domain and the second Fc domain consists of an amino acid sequence independently selected from an amino acid sequence set forth in SEQ ID NO: 1, 2, or 3. In some embodiments, the amino acid sequence of the first Fc domain or the amino acid sequence of the second Fc domain consists of SEQ ID NO: 2. In some embodiments, the amino acid sequences of both the first Fc domain and the second Fc domain consist of SEQ ID NO: 2.

[0018] In some embodiments, the variant Fc region comprises one or more mutations ofamino acid residues forming the interface of the CH3 domain of the Fc domains.

[0019] In some embodiments, the amino acid sequence of the first Fc domain furthercomprises amino acid W at EU position 366. In some embodiments, the amino acid sequence of the first Fc domain comprises an amino acid sequence selected from an amino acid sequence set forth in SEQ ID NO: 4, 5, or 6. In some embodiments, the amino acid sequence of the first Fc domain comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the amino acid sequence of the first Fc domain consists of an amino acid sequence selected from an amino acid sequence set forth in SEQ ID NO: 4, 5, or 6. In some embodiments, the amino acid sequence of the first Fc domain consists of the amino acid sequence of SEQ ID NO: 5.

[0020] In some embodiments, the amino acid sequence of the second Fc domain furthercomprises amino acids S, A, and V at EU positions 366, 368, and 407, respectively. In some embodiments, the amino acid sequence of the second Fc domain comprises an amino acid sequence selected from an amino acid sequence set forth in SEQ ID NO: 7, 8, or 9. In some embodiments, the amino acid sequence of the second Fc domain comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the amino acid sequence of the second Fc domain consists of an amino acid sequence selected from an amino acid sequence set forth in SEQ ID NO: 7, 8, or 9. In some embodiments, the amino acid sequence of the second Fc domain consists of the amino acid sequence of SEQ ID NO: 8.

[0021] In some embodiments, the HSA-binding domain comprises a VHH comprising anamino acid sequence selected from the group consisting of: a) an amino acid sequence comprising SEQ ID NO: 14 (CDR1), SEQ ID NO: 15 (CDR2), and SEQ ID NO: 13 (CDR3);b) an amino acid sequence comprising SEQ ID NO: 14 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 16 (CDR3); c) an amino acid sequence comprising SEQ ID NO: 14 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 17 (CDR3); and d) an amino acid sequence comprising SEQ ID NO: 18 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 19 (CDR3).

[0022] In some embodiments, the HSA-binding domain comprises a VHH comprising anamino acid sequence comprising SEQ ID NO: 18 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 19 (CDR3).

[0023] In some embodiments, the HSA-binding domain comprises a VHH comprising anamino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from an amino acid sequence set forth in SEQ ID NOs: 23-26. In some embodiments, the HSA-binding domain comprises a VHH comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the HSA-binding domain comprises an amino acid sequence selected from an amino acid sequence set forth in SEQ ID NOs: 23-26. In some embodiments, the HSA-binding domain comprises an amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the FcRn / HSA-binding molecule further comprises one or more additional amino acids at the C-terminal end of the HSA-binding domain. In some embodiments, the one or more additional amino acids are selected from the group consisting of: A, AG, GG, PP, and AA.

[0024] In some embodiments, the FcRn / HSA-binding molecule further comprises anantigen-binding domain.

[0025] In another aspect, provided herein is an FcRn / HSA-binding molecule comprising afirst heavy chain, wherein the first heavy chain comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 42-45. In some embodiments, the first heavy chain comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 45. In some embodiments, the first heavy chain comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 42-45. In some embodiments, the first heavy chain comprises or consists of an amino acid sequence of SEQ ID NO: 45. In someembodiments, the first heavy chain further comprises one or more amino acids added at the C- terminus of the first heavy chain, optionally selected from A, AG, GG, and PP.

[0026] In some embodiments, the FcRn / HSA-binding molecule further comprises a secondheavy chain, wherein the second heavy chain consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the second heavy chain consists of the amino acid sequence of SEQ ID NO: 8.

[0027] In another aspect, provided herein is an HSA-binding domain comprising CDR1,CDR2, and CDR3 amino acid sequences of a VHH comprising an amino acid sequence selected from SEQ ID NOs: 23-26.

[0028] In some embodiments, the HSA-binding domain comprises an amino acid sequenceselected from the group consisting of: a) an amino acid sequence comprising SEQ ID NO: 14 (CDR1), SEQ ID NO: 15 (CDR2), and SEQ ID NO: 13 (CDR3); b) an amino acid sequence comprising SEQ ID NO: 14 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 16 (CDR3); c) an amino acid sequence comprising SEQ ID NO: 14 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 17 (CDR3); and d) an amino acid sequence comprising SEQ ID NO: 18 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 19 (CDR3).

[0029] In some embodiments, the HSA-binding domain comprises an amino acid sequencecomprising SEQ ID NO: 18 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 19 (CDR3).

[0030] In some embodiments, the HSA-binding domain comprises or consists of an aminoacid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in SEQ ID NOs: 23-26. In some embodiments, the HSA-binding domain comprises or consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the HSA-binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NOs: 23-26. In some embodiments, the HSA-binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 26.

[0031] In some embodiments, the HSA-binding domain is an sdAb. In some embodiments,the sdAb is a VHH. In some embodiments, the HSA-binding domain further comprises one ormore additional amino acids at the C-terminal end of the VHH. In some embodiments, the one or more additional amino acids are selected from the group consisting of A, AG, GG, PP, and AA.

[0032] Also provided is an isolated polynucleotide or polynucleotides encoding anyFcRn / HSA-binding molecule described herein or any HSA-binding domain described herein.

[0033] Also provided is an expression vector comprising any isolated polynucleotide orpolynucleotides described herein.

[0034] Also provided is a host cell comprising any isolated polynucleotide orpolynucleotides described herein, or any expression vector described herein.

[0035] A method for producing an FcRn / HSA-binding molecule or an HSA-bindingdomain is also provided, the method comprising culturing a host cell as described herein under conditions which permit the expression of the FcRn / HSA-binding molecule or HSA-binding domain.

[0036] Also provided is a pharmaceutical composition comprising an FcRn / HSA-bindingmolecule as described herein or an HSA-binding domain as described herein and at least one pharmaceutically acceptable carrier.

[0037] Also provided is an FcRn / HSA-binding molecule as described herein, or an HSA-binding domain as described herein, or a pharmaceutical composition thereof for use as a medicament.

[0038] Also provided is a method of reducing serum IgG in a subject in need thereofcomprising administering to the subject a therapeutically effective amount of an FcRn / HSA- binding molecule as described herein, or an HSA-binding domain as described herein, or a pharmaceutical composition thereof.

[0039] Also provided is a method of treating an antibody-mediated disorder in a subject inneed thereof comprising administering to the subject a therapeutically effective amount of an FcRn / HSA-binding molecule as described herein, or an HSA-binding domain as described herein, or a pharmaceutical composition thereof. In some embodiments, the antibody-mediated disorder is an IgG-mediated disorder. In some embodiments, the antibody-mediated disorder is an autoimmune disease.

[0040] Also provided is an FcRn / HSA-binding molecule as described herein, or an HSA-binding domain as described herein, or a pharmaceutical composition thereof, for use in the treatment of an antibody-mediated disorder.

[0041] Also provided is an FcRn / HSA-binding molecule as described herein, or an HSA-binding domain as described herein, for the manufacture of a medicament for treating an antibody- mediated disorder. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG. 1 shows the effect of one-armed (OA) Fc-ABDEG molecules with an HSA-binding VHH (Alb23 and variants thereof) fused at the C-terminus of one Fc domain on FcRn degradation in HEK FcRn WT GFP+ cells in the presence or absence of HSA. Bars represent mean ± SEM of individual experiments (indicated with n) each performed in two technical replicates.

[0043] FIGs. 2A-2B show the pharmacodynamic (PD) effect of OA-Fc-ABDEG-Alb23variants in AlbuMus Rag1KO mice. Normalized levels (% pre-dose) of the tracer human IgG (FIG.2A) and total preloaded human IgG (FIG.2B) are shown after a single IV injection of OA- Fc-ABDEG-3Rab (control with irrelevant rabies virus-binding VHH) and OA-Fc-ABDEG-Alb23 variants. Changes in tracer hIgG and total hIgG concentrations were plotted over time (days post- injection) as % to pre-dose on day 0, -2h. The datapoints show the mean ± SEM of 3-5 mice per group.

[0044] FIG. 2C shows pharmacokinetic (PK) profiles in AlbuMus Rag1KO mice after asingle IV injection of OA-Fc-ABDEG-3Rab (control) and OA-Fc-ABDEG-Alb23 variants. Serum concentrations of the test items were plotted as an average per group over time. The datapoints show the mean ± SD of 3-5 mice per group. Values below low limit of quantification (LLOQ) are excluded from the graph.

[0045] FIG. 2D shows levels of HSA (% to pre-dose) in AlbuMus Rag1KO mice after asingle IV injection of OA-Fc-ABDEG-3Rab (control) and OA-Fc-ABDEG-Alb23 variants. Albumin (HSA) levels were plotted over time (days post-injection) as % relative to pre-dose (day 0 -2h) averaged per group. The datapoints show the mean ± SEM of 3-5 mice per group.

[0046] FIGs. 3A-3B show the PD effect of OA-Fc-ABDEG-Alb23 variants in AlbuMusRag1KO mice. Normalized levels (% pre-dose) of the tracer human IgG (FIG. 3A) and total preloaded human IgG (FIG.3B) are shown after a single IP injection of PBS (control), OA-Fc- ABDEG-3Rab (control), efgartigimod, and OA-Fc-ABDEG-Alb23 variants. Changes in tracer hIgG and total hIgG concentrations were plotted over time (days post-injection) as % to pre-dose on day 0, -2h. The datapoints show the mean ± SEM of 4-5 mice per group.

[0047] FIG. 3C shows PK profiles in AlbuMus Rag1KO mice after a single IP injectionof OA-Fc-ABDEG-3Rab (control), efgartigimod, and OA-Fc-ABDEG-Alb23 variants. Serum concentrations of the test items were plotted as an average per group over time. The datapoints show the mean ± SD of 4-5 mice per group. Values below low limit of quantification (LLOQ) are excluded from the graph.

[0048] FIG. 3D shows levels of HSA (% to pre-dose) in AlbuMus Rag1KO mice after asingle IP injection of PBS, OA-Fc-ABDEG-3Rab (control), efgartigimod, and OA-Fc-ABDEG- Alb23 variants. Albumin (HSA) levels were plotted over time (days post-injection) as % relative to pre-dose (day 0 -2h) averaged per group. The datapoints show the mean ± SEM of 4-5 mice per group.

[0049] FIGs. 4A-4B show the PD effect of OA-Fc-ABDEG-Alb23 variants in Tg32 SCIDmice. Normalized levels (% pre-dose) of the tracer human IgG (FIG. 4A) and total preloaded human IgG (FIG. 4B) are shown after a single IV injection of PBS (control), efgartigimod, and OA-Fc-ABDEG-Alb23 variants. Changes in tracer hIgG and total hIgG concentrations were plotted over time (days post-injection) as % to pre-dose on day 0, -1h. The datapoints show the mean ± SEM of 3-4 mice per group.

[0050] FIG. 4C shows PK profiles in Tg32 SCID mice after a single IV injection ofefgartigimod and OA-Fc-ABDEG-Alb23 variants. Serum concentrations of the test items were plotted as an average per group over time. The datapoints show the mean ± SD of 3-4 mice per group. Values below low limit of quantification (LLOQ) are excluded from the graph.

[0051] FIG.5 shows the effect of OA-Fc-ABDEG-Alb23 with a 20GS linker, and OA-Fc-ABDEG-Alb23-F32A / L102A and OA-Fc-ABDEG-Alb23-M34A / F33A / S101A with 20, 25, and 30 GS linkers on FcRn degradation in the presence or absence of HSA. Bars represent mean ± SEM of individual experiments (indicated with n) each performed in two technical replicates.

[0052] FIGs. 6A-6B show the PD / PK effect of OA-Fc-ABDEG-Alb23-F32A, OA-Fc-ABDEG-Alb23-F32A / L102A, and OA-Fc-ABDEG-Alb23-M34A / G33A / S101A, each with a 20GS linker or a 30GS linker. OA-Fc-ABDEG-3Rab is included as a control. FIG.6A shows PK profiles after a single IP injection in AlbuMus Rag1KO mice. Serum concentrations of the test items were plotted as an average per group over time. The datapoints show the mean ± SD of 3-4 animals per group. Values below low limit of quantification (LLOQ) are excluded from the graph. FIG. 6B shows normalized levels (% pre-dose) of total preloaded human IgG in AlbuMusRag1KO after a single IP injection of test items. The datapoints show the mean ± SEM of 3-4 animals per group.

[0053] FIG. 7A shows normalized HSA levels (% pre-dose) after 4 IP injections onceevery week of PBS (placebo), OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A, TA-Fc- ABDEG-Alb23, anti-FcRn mAb1, or anti-FcRn mAb2 in AlbuMus Rag1KO mice. Albumin (HSA) levels were plotted over time (days post-injection) as % relative to pre-dose (day -6), averaged per group. The datapoints show the mean ± SEM of 3-5 mice per group, per timepoint. Dotted lines show injections of test items on day 0, 7, 14, and 21.

[0054] FIG. 7B shows the effect of OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A,TA-Fc-ABDEG-Alb23, anti-FcRn mAb1, or anti-FcRn mAb2 on FcRn degradation in the presence or absence of HSA. Bars represent mean ± SEM of individual experiments (indicated with n) each performed in two technical replicates.

[0055] FIGs. 8A-8B show PD profiles in cynomolgus monkeys treated with one IV doseof an Fc-ABDEG molecule equipped with one anti-HSA VHH with M34A / G33A / S101A mutations (Alb23-TM) fused at the C-terminus of an Fc domain via a 20GS linker (ABDEG-20GS- Alb23-TM). % Cynomolgus total serum IgG relative to pre-dose were plotted over time. The datapoints show the mean ± SD of 3 individual monkeys (n=3) dosed with 10 mg / kg (FIG. 8A) and 5 individual monkeys (n=5) dosed with 60 mg / kg (FIG. 8B) of ABDEG-20GS-Alb23-TM. PD profiles in cynomolgus monkeys of equimolar doses of efgartigimod (model simulation) plotted for comparison. Time points when presence of ADA was detected and with a steep concentration decline in PK curves are excluded from the graphs.

[0056] FIGs. 9A-9B show PK profiles in cynomolgus monkeys treated with one IV doseof ABDEG-20GS-Alb23-TM. ABDEG-20GS-Alb23-TM concentration values were plotted over time. The datapoints show the mean ± SD of 3 individual monkeys (n=3) dosed with 10 mg / kg (FIG. 9A) and 5 individual monkeys (n=5) dosed with 60 mg / kg (FIG. 9B) of ABDEG-20GS- Alb23-TM. PK profiles in cynomolgus monkeys of equimolar doses of efgartigimod (model simulation) plotted for comparison. Time points when presence of ADA was detected and with a steep concentration decline in PK curves are excluded from the graphs.

[0057] FIG. 10 shows serum albumin levels in cynomolgus monkeys after single orrepeated administration of ABDEG-20GS-Alb23-TM. The dotted lines indicate administration of ABDEG-20GS-Alb23-TM. The data points show the mean ± SD of individual monkeys per treatment group. The light grey shaded area shows min and max levels of albumin measured atpre-dose in the monkeys used in this study. The dark grey shaded area shows a normal range of serum albumin in cynomolgus monkeys according to Park et al. (Lab Anim Res.2016 Jun; 32(2): 79–86).

[0058] FIG. 11 shows a reduced binding of pre-existing ADA to OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A with C-terminal extensions (A, AG, PP, or GG). Plotted are absorbance values at OD450.

[0059] FIG. 12A shows FcRn occupancy by efgartigimod, TA-Alb23-Fc-ABDEG, andOA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A with and without C-terminal extensions (A, AG, PP, or GG). Presented are means ± SD of 2-9 independent experiments each performed in technical duplicates.

[0060] FIG. 12B shows the effect of OA-Fc-ABDEG-20GS-Alb23, OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A with and without C-terminal extensions, and anti-FcRn mAb1 on FcRn degradation in the presence or absence of HSA. Bars represent mean ± SD of at least three independent experiments each performed in two technical replicates. DETAILED DESCRIPTION

[0061] The present disclosure provides engineered FcRn-binding molecules linked to anHSA-binding domain (FcRn / HSA-binding molecules). In an aspect, an FcRn / HSA-binding molecule is provided comprising an FcRn-binding molecule linked to an HSA-binding domain at the C-terminus or at the N-terminus, or at a position other than the C-terminus or N-terminus. In some embodiments, the FcRn / HSA-binding molecule comprises an FcRn-binding molecule and only one HSA-binding domain. Nucleic acids encoding such FcRn / HSA-binding molecules, vectors, host cells, methods of manufacture, and methods for their use in treating antibody- mediated disorders are also provided herein. Definitions

[0062] As used herein, the term “FcRn” refers to a neonatal Fc receptor. Exemplary FcRnmolecules include human FcRn encoded by the FCGRT gene as set forth in RefSeq NM 004107. The amino acid sequence of the corresponding protein is set forth in RefSeq NP_004098.

[0063] As used herein, the term “FcRn-binding molecule” refers to any agent thatspecifically binds to FcRn. As used herein, the term “FcRn antagonist” refers to any agent that specifically binds to FcRn and inhibits the binding of immunoglobulin to FcRn (e.g., humanFcRn). In an embodiment, the FcRn antagonist comprises an Fc region (e.g., a variant Fc region disclosed herein) that specifically binds to FcRn through the Fc region and inhibits the binding of immunoglobulin to FcRn. In an embodiment, the FcRn antagonist is not a full-length IgG antibody. In an embodiment, the FcRn antagonist comprises an antigen-binding domain that binds a target antigen and a variant Fc region. In an embodiment, the term “FcRn antagonist” refers to an antibody or antigen-binding fragment thereof that specifically binds to FcRn via its antigen binding domain and / or via its Fc region and inhibits the binding of the Fc region of immunoglobulin (e.g., IgG autoantibodies) to FcRn. As used herein, the term “FcRn / HSA-binding molecule” refers to any agent that specifically binds to FcRn and specifically binds to HSA.

[0064] As used herein, the term “affinity” or “binding affinity” refers to the strength of thebinding interaction between two molecules. As used herein, the term “equilibrium dissociation constant” or “KD” refers to the propensity of bound complex of two molecules to dissociate into two free molecules. Thus, as the binding affinity increases, the KD decreases.

[0065] As used herein, the term “specifically binds” refers to the ability of any moleculeto preferentially bind with a given target. For example, a molecule that specifically binds to a given target can bind to other molecules, generally with lower affinity as determined by, e.g., immunoassays, BIAcoreTM8K+ instrument (Sapidyne Instruments, Boise, Id.), or other assays known in the art. In a specific embodiment, molecules that specifically bind to a given target bind to the antigen with a KD that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or less than the KD when the molecules bind non-specifically to another target.

[0066] As used herein, the term “operably linked” refers to a linkage of polynucleotidesequence elements in a functional relationship. For example, a polynucleotide sequence is operably linked when it is placed into a functional relationship with another polynucleotide sequence. In some embodiments, a transcription regulatory polynucleotide sequence, e.g., a promoter, enhancer, or other expression control element is operably linked to a polynucleotide sequence that encodes a protein if it affects the transcription of the polynucleotide sequence that encodes the protein. Operably linked elements may be contiguous or non-contiguous.

[0067] As used herein, the term “linked” refers to a physical linkage (e.g., directly orindirectly linked) between amino acid sequences (e.g., different segments, regions, fragments, or domains). Linked regions, fragments, domains, and segments of the FcRn / HSA-binding molecules of the disclosure may be contiguous or non-contiguous (e.g., linked to one another through alinker). In some embodiments, linkages are covalent. In some embodiments, linkages are non- covalent.

[0068] As used herein, the term “covalently linked” refers to the linkage of two moleculesor chemical moieties by a covalent bond. In some embodiments, the covalent bond is a peptide bond or a disulfide bond. As used herein, the term “fused” refers to the linkage of two peptides by a peptide bond or a peptide linker. In some embodiments, two proteins are directly and contiguously fused together by a peptide bond. In some embodiments, two proteins are indirectly and non-contiguously fused through a peptide linker. In some embodiments, one protein is fused to a peptide linker by a peptide bond at a first position, and a second protein is fused to a peptide linker by a peptide bond at a second position. As used herein, the term “non-covalently linked” refers to the linkage of two molecules or chemical moieties by a non-covalent interaction or bond. In some embodiments, non-covalent interactions or bonds include hydrogen bonds, electrostatic bonds or interactions, halogen bonds, pi stacking, and van der Waals interactions.

[0069] The determination of “percent identity” between two sequences (e.g., amino acidsequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin S & Altschul SF, (1990) PNAS 87: 2264-2268, modified as in Karlin S & Altschul SF, (1993) PNAS 90: 5873-5877, each of which is herein incorporated by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215: 403, which is herein incorporated by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., at score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecule described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., at score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389-3402, which is herein incorporated by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules. Id. When utilizing BLAST, Gapped BLAST, and PSI BLAST programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm utilizedfor the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS 4:11-17, which is herein incorporated by reference in its entirety. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0070] The percent identity between two sequences can be determined using techniquessimilar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0071] As used herein, the terms “antibody” and “antibodies” include full-lengthantibodies, antigen-binding fragments of full-length antibodies, and molecules comprising antibody CDRs, VH domains (VH), or VL domains (VL). Examples of antibodies include monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multi- specific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain-antibody heavy chain pair, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single-domain antibodies (sdAb), monovalent antibodies, single chain antibodies or single-chain Fvs (scFv), camelid antibodies, affibody molecules, VHH, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g., anti- anti-Id antibodies), and antigen-binding fragments of any of the above. Antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or species (e.g., mouse IgG2a or IgG2b) of immunoglobulin molecule.

[0072] As used herein, the term “antigen-binding domain” refers to any polypeptide thatspecifically binds to an antigen. Examples of antigen-binding domains include polypeptides derived from antibodies, such as Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv), single-chain Fvs (scFv), CDRs, VH domains (VH), VL domains (VL), single-domain antibodies (sdAb), VHH, camelid antibodies, and antigen-binding fragments of any of the above. The term also encompasses synthetic antigen-binding proteins or antibody mimetic proteins such as, for example, anticalins and DARPins.

[0073] As used herein, the term “HSA-binding domain” refers to any polypeptide thatspecifically binds to human serum albumin (HSA). Examples of HSA-binding domains includepolypeptides derived from antibodies, such as Fab fragments, F(ab')2fragments, disulfide-linked Fvs (sdFv), single-chain Fvs (scFv), CDRs, VH domains (VH), VL domains (VL), single-domain antibodies (sdAb), VHH, camelid antibodies, and HSA-binding fragments of any of the above. The term also encompasses synthetic antigen-binding proteins or antibody mimetic proteins such as, for example, anticalins and DARPins.

[0074] In some embodiments, the HSA-binding domain or antigen-binding domain is aVHH. In some embodiments, the VHH has one or more additional amino acids at its C-terminal end. In some embodiments, the one or more additional amino acids are selected from the group consisting of A, AG, GG, and PP.

[0075] As used herein, the term “Fc region” refers to the portion of an immunoglobulinformed by the Fc domains of its two heavy chains. The Fc region can be a wild-type Fc region (native Fc region) or a variant Fc region. A native Fc region is homodimeric. The Fc region can be derived from any native immunoglobulin. In some embodiments, the Fc region is formed from an IgA, IgD, IgE, or IgG heavy chain constant region. In some embodiments, the Fc region is formed from an IgG heavy chain constant region. In some embodiments, the IgG heavy chain is an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the Fc region is formed from an IgG1 heavy chain constant region. In some embodiments, the IgG1 heavy chainconstant region comprises a G1m1(a), G1m2(x), G1m3(f), or G1m17(z) allotype. See, e.g., Jefferisand Lefranc (2009) mAbs 1(4): 332-338, and de Taeye et al., (2020) Front Immunol. 11: 740, incorporated herein by reference in their entirety.

[0076] As used herein, the term “variant Fc region” refers to a variant of an Fc region withone or more alteration(s) relative to a native Fc region. Alterations can include amino acid substitutions, additions and / or deletions, linkage of additional moieties, and / or alteration of the native glycans. The term encompasses heterodimeric Fc regions where each of the constituent Fc domains is different. The term also encompasses single chain Fc regions where the constituent Fc domains are linked together by a linker moiety.

[0077] As used herein, the term “Fc domain” refers to the portion of a singleimmunoglobulin heavy chain comprising both the CH2 and CH3 domains of the antibody. In some embodiments, the Fc domain comprises at least a portion of a hinge (e.g., upper, middle, and / or lower hinge region) region, a CH2 domain, and a CH3 domain. In some embodiments, the Fc domain does not include the hinge region.

[0078] As used herein, the term “hinge region” refers to the portion of a heavy chainmolecule that joins the CH1 domain to the CH2 domain. In some embodiments, the hinge region is at most 70 amino acid residues in length. In some embodiments, this hinge region comprises approximately 11-17 amino acid residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. In some embodiments, the hinge region is 12 amino acid residues in length. In some embodiments, the hinge region is 15 amino acid residues in length. In some embodiments, the hinge region is 62 amino acid residues in length. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains. The FcRn / HSA- binding molecules of the instant disclosure can include all or any portion of a hinge region. In some embodiments, the hinge region is from an IgG1 antibody. In some embodiments, the hinge region comprises the amino acid sequence of EPKSCDKTHTCPPCP (SEQ ID NO: 37).

[0079] As used herein, the term “FcRn-binding fragment” refers to a portion of an FcRn-binding molecule, e.g., a portion of an Fc region, that is sufficient to confer FcRn binding.

[0080] As used herein, the term “VHH” refers to a portion of a VHH that is sufficient toconfer antigen (e.g., HSA) binding. In some embodiments, a VHH comprises CDR1, CDR2, and CDR3 of a VHH. In some embodiments, a VHH is a full-length VHH.

[0081] As used herein, the terms, “one-armed,” “one armed,” “one-arm,” “one arm,” or“OA” refers to an FcRn / HSA-binding molecule comprising an FcRn-binding molecule linked to only one HSA-binding domain. In some embodiments, “one-armed,” “one armed,” “one-arm,” “one arm,” or “OA” refers to an FcRn / HSA-binding molecule comprising an Fc region comprising the Fc domains of two heavy chains, wherein one of the Fc domains of the two heavy chains is linked to an HSA-binding domain and the other Fc domain of the two heavy chains is not linked to an HSA-binding domain. In some embodiments, the HSA-binding domain is linked to the C- terminus of one of the Fc domains of the two heavy chains. In some embodiments, the HSA- binding domain is linked to the N-terminus of one of the Fc domains of the two heavy chains. In some embodiments, the HSA-binding domain is linked to a position other than the N-terminus or the C-terminus of one of the Fc domains of the two heavy chains. The linkage can be covalent or non-covalent. In some embodiments, the HSA-binding domain is fused to the C-terminus of one of the Fc domains of the two heavy chains. In some embodiments, the HSA-binding domain is fused to the N-terminus of one of the Fc domains of the two heavy chains. In some embodiments, the HSA-binding domain is fused to a position other than the N-terminus or the C-terminus of one of the Fc domains of the two heavy chains.

[0082] As used herein, the term “EU position” refers to the amino acid position in the EUnumbering convention for the Fc region described in Edelman, GM et al. Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., in “Sequences of Proteins of Immunological Interest,” U.S. Dept. Health and Human Services, 5thedition, 1991.

[0083] As used herein, the term, “antibody-mediated disorder” refers to any disorderwherein the symptoms of the disorder are caused by abnormal levels of one or more antibodies in a subject. As used herein, the term “autoantibody-mediated disorder” refers to any disease or disorder in which the underlying pathology is caused, at least in part, by pathogenic IgG autoantibodies.

[0084] As used herein, the term “treat,” “treating,” and “treatment” refer to therapeutic orpreventative measures described herein. The methods of “treatment” employ administration of a polypeptide to a subject having a disease or disorder, or predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. In some embodiments, the methods of “treatment” employ administration of a polypeptide to a subject having a disease or disorder, or predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate the disease or disorder or recurring disease or disorder.

[0085] As used herein, the term “effective amount” in the context of the administration ofa therapy to a subject refers to the amount of a therapy that achieves a desired prophylactic or therapeutic effect.

[0086] As used herein, the term “dose” or “dosing” refers to an amount of an agentadministered to a subject in a single administration.

[0087] As used herein, the term, “equivalent dose” refers to a dose of a first and a secondtherapeutic agent wherein the number of molecules of the first and second agents is about the same. In some embodiments, an equivalent dose is an equimolar dose. As used herein, the term “equimolar dose” refers to a dose of a first and a second therapeutic agent wherein the number of moles of the first and second agent is the same. In some embodiments, the first agent is a FcRn / HSA-binding molecule and the second agent is efgartigimod.

[0088] As used herein, the terms “pharmacodynamics,” and “PD,” refer to the biologicaleffect of a therapeutic agent on an organism. In some embodiments, the biological effect is modulation of the amount of circulating IgG in an organism administered a therapeutic agent. Insome embodiments, the biological effect is modulation of the amount of circulating albumin in an organism administered a therapeutic agent. In some embodiments, the therapeutic agent is an FcRn / HSA-binding molecule.

[0089] As used herein, the terms “pharmacokinetics,” and “PK,” refer to the effect of anorganism on a therapeutic agent administered to the organism. In some embodiments, the effect is metabolization and / or clearance of the therapeutic agent. In some embodiments, PK refers to the rate of metabolization and / or clearance of the therapeutic agent. In some embodiments, the therapeutic agent is an FcRn / HSA-binding molecule.

[0090] As used herein, the term “subject” or “patient” or “participant” includes any humanor non-human animal. In an embodiment, the subject or patient or participant is a human or non- human mammal. In an embodiment, the subject or patient or participant is a human.

[0091] As used herein, the term “about” or “approximately” when referring to ameasurable value, such as a dosage, encompasses variations of ±5% of a given value or range, as are appropriate to perform the methods disclosed herein. FcRn / HSA-Binding Molecules

[0092] In an aspect, the disclosure provides FcRn / HSA-binding molecules or fragmentsthereof. In some embodiments, the FcRn / HSA-binding molecules disclosed herein comprise an FcRn-binding molecule and an HSA-binding domain. The FcRn-binding molecule may be any FcRn-binding molecule described herein. Similarly, the HSA-binding domain may be any HSA- binding domain described herein. In some embodiments, the FcRn / HSA-binding molecules disclosed herein comprise one or more FcRn-binding molecules in combination with one or more HSA-binding domains. In some embodiments, the FcRn / HSA-binding molecules disclosed herein comprise one or more Fc regions, or FcRn-binding fragments thereof, in combination with one or more HSA-binding domains.

[0093] In some embodiments, the FcRn / HSA-binding molecule comprises only one HSA-binding domain (e.g., one-armed FcRn / HSA-binding molecule).

[0094] In some embodiments, the HSA-binding domain is linked or fused to the C-terminus of the FcRn-binding molecule. In some embodiments, the HSA-binding domain is linked or fused to the N-terminus of the FcRn-binding molecule.

[0095] In some embodiments, the FcRn-binding molecule is an Fc region, e.g., a variantFc region. In some embodiments, HSA-binding domain is linked or fused to the C-terminus of oneof the Fc domains of the variant Fc region. In some embodiments, the HSA-binding domain is linked or fused to the N-terminus of the one of the Fc domains of the variant Fc region.

[0096] In some embodiments, the HSA-binding domain may be linked or fused directly tothe N-terminus or the C-terminus of an FcRn-binding molecule. In some embodiments, the HSA- binding domain is linked or fused to the N-terminus or the C-terminus of an FcRn-binding molecule via a linker. The linker may be any suitable linker, including those described herein. In some embodiments, the linker is a non-cleavable linker. FcRn-Binding Molecules

[0097] FcRn-binding molecules disclosed herein include any molecule that binds to FcRn,including, but not limited to, any anti-FcRn antibody, any anti-FcRn-binding region, or any Fc domain or Fc region.

[0098] In some embodiments, the FcRn-binding molecules are FcRn antagonists whichinclude any molecule that binds to and inhibits FcRn, including, but not limited to, any anti-FcRn antibody, any anti-FcRn-binding region, or any Fc domain or Fc region.

[0099] In some embodiments, the FcRn-binding molecules disclosed herein comprise two,three, or four FcRn-binding regions, such as an Fc region.

[0100] In some embodiments, the FcRn-binding molecules disclosed herein comprise oneor more Fc regions, or FcRn-binding fragment thereof, in combination with one or more antigen- binding domains (e.g., an sdAb, a Fab fragment, an scFv, or an antibody mimetic).

[0101] Any Fc region can be altered to produce a variant Fc region as disclosed herein. Ingeneral, an Fc region, or FcRn-binding fragment thereof, is from a human immunoglobulin. It is understood, however, that the Fc region may be derived from an immunoglobulin of any other mammalian species, including for example, a camelid species, a rodent (e.g., a mouse, rat, rabbit, guinea pig) or non-human primate (e.g., chimpanzee, macaque) species. Moreover, the Fc region or FcRn-binding portion thereof may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4. In an embodiment, the Fc region is an IgG Fc region (e.g., a human IgG region). In an embodiment, the Fc region is an IgG1 Fc region (e.g., a human IgG1 region). In an embodiment, the Fc region is a chimeric Fc region comprising portions of several different Fc regions. Suitable examples of chimeric Fc regions are set forth in US 2011 / 0243966A1, which is incorporated herein byreference in its entirety. A variety of Fc region gene sequences (e.g., human constant region gene sequences) are available in the form of publicly accessible deposits.

[0102] An Fc region can be further truncated or internally deleted to produce a minimalFcRn-binding fragment thereof. The ability of an Fc-region fragment to bind to FcRn can be determined using any art recognized binding assay e.g., ELISA.

[0103] To enhance the manufacturability of FcRn-binding molecules, and FcRn / HSA-binding molecules containing the same, as disclosed herein, it is preferable that the constituent Fc regions do not comprise any non-disulfide bonded cysteine residues. Accordingly, in an embodiment, the Fc regions do not comprise a free cysteine residue.

[0104] In some embodiments, any Fc variant, or FcRn-binding fragment thereof, thatspecifically binds to FcRn with increased affinity and reduced pH dependence relative to the native Fc region can be used herein. In an embodiment, the variant Fc region comprises amino acid alterations, substitutions, insertions, and / or deletions that confer the desired characteristics. In some embodiments, the FcRn-binding molecule comprises a variant Fc region, or FcRn-binding fragment thereof, which binds to FcRn with a higher affinity at pH 5.5 as compared to a corresponding wild-type Fc region. In some embodiments, the FcRn-binding molecule comprises a variant Fc region, or FcRn-binding fragment thereof, which binds to FcRn with a higher affinity at pH 6.0 and / or at pH 7.4 as compared to a corresponding wild-type Fc region. In some embodiments, the FcRn-binding molecule comprises a variant Fc region, or FcRn-binding fragment thereof, which binds to FcRn with a higher affinity at both acidic and neutral pH as compared to a corresponding wild-type Fc region.

[0105] In some embodiments, the variant Fc region is derived from the Fc region of anynative immunoglobulin. In some embodiments, the native immunoglobulin is a human immunoglobulin. In some embodiments, the immunoglobulin is IgA, IgD, IgE, or IgG. In some embodiments, the immunoglobulin is IgG. In some embodiments, the immunoglobulin is human IgA, human IgD, human IgE, or human IgG. In some embodiments, the immunoglobulin is human IgG. In some embodiments, the IgG is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the human IgG is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the variant Fc region varies from the human IgG1 Fc region. In some embodiments, the human IgG1 Fc region comprises a G1m1(a), G1m2(x), G1m3(f), or G1m17(z) allotype.

[0106] In some embodiments, the FcRn-binding molecule is an FcRn antagonist.

[0107] In some embodiments, the variant Fc region, or FcRn-binding fragment thereofcomprises or consists of at least one Fc domain. In some embodiments, the variant Fc region comprises or consists of two Fc domains. In some embodiments, the Fc domains are the same. In some embodiments, the Fc domains are different. In certain embodiments, at least one of the variant Fc domains or FcRn-binding fragments described herein comprises at least one amino acid or at least two amino acids selected from the following: 237M; 238A; 239K; 248I; 250A; 250F; 250I; 250M; 250Q; 250S; 250V; 250W; 250Y; 252F; 252W; 252Y; 254T; 255E; 256D; 256E; 256Q; 257A; 257G; 257I; 257L; 257M; 257N; 257S; 257T; 257V; 258H; 265A; 270F; 286A; 286E; 289H; 297A; 298G; 303A; 305A; 307A; 307D; 307F; 307G; 307H; 307I; 307K; 307L; 307M; 307N; 307P; 307Q; 307R; 307S; 307V; 307W; 307Y; 308A; 308F; 308I; 308L; 308M; 308P; 308Q; 308T; 309A; 309D; 309E; 309P; 309R; 311A; 311H; 311I; 312A; 312H; 314K; 314R; 315A; 315H; 317A; 325G; 332V; 334L; 360H; 376A; 378V; 380A; 382A; 384A; 385D; 385H; 386P; 387E; 389A; 389S; 424A; 428A; 428D; 428F; 428G; 428H; 428I; 428K; 428L; 428N; 428P; 428Q; 428S; 428T; 428V; 428W; 428Y; 433K; 434A; 434F; 434H; 434S; 434W; 434Y; 436H; 436I and 436F, wherein the positions are defined in accordance with EU numbering. EU numbering refers to the convention for the Fc region described in Edelman, G.M. et al., Proc. Natl. Acad. Sci. USA, 63: 78-85 (1969); and Kabat et al., in “Sequences of Proteins of Immunological Interest”, U.S. Dept. Health and Human Services, 5thedition, 1991. In some embodiments, at least one of the variant Fc domains or FcRn-binding fragments described herein comprises 2, 3, 4 or 5 amino acids selected from the following: 237M; 238A; 239K; 248I; 250A; 250F; 250I; 250M; 250Q; 250S; 250V; 250W; 250Y; 252F; 252W; 252Y; 254T; 255E; 256D; 256E; 256Q; 257A; 257G; 257I; 257L; 257M; 257N; 257S; 257T; 257V; 258H; 265A; 270F; 286A; 286E; 289H; 297A; 298G; 303A; 305A; 307A; 307D; 307F; 307G; 307H; 307I; 307K; 307L; 307M; 307N; 307P; 307Q; 307R; 307S; 307V; 307W; 307Y; 308A; 308F; 308I; 308L; 308M; 308P; 308Q; 308T; 309A; 309D; 309E; 309P; 309R; 311A; 311H; 311I; 312A; 312H; 314K; 314R; 315A; 315H; 317A; 325G; 332V; 334L; 360H; 376A; 378V; 380A; 382A; 384A; 385D; 385H; 386P; 387E; 389A; 389S; 424A; 428A; 428D; 428F; 428G; 428H; 428I; 428K; 428L; 428N; 428P; 428Q; 428S; 428T; 428V; 428W; 428Y; 433K; 434A; 434F; 434H; 434S; 434W; 434Y; 436H; 436I and 436F, wherein the positions are defined in accordance with EU numbering and wherein any combinations are contemplated.

[0108] In certain embodiments, at least one of the variant Fc domains or FcRn-bindingfragments described herein comprises at least one non-naturally occurring amino acid or at leasttwo non-naturally occurring amino acids selected from the following: 234, 235, 236, 239, 240, 241, 243, 244, 245, 247, 252, 254, 256, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 326, 327, 328, 329, 330, 332, 333, and 334 as numbered by the EU index as set forth in Kabat. Optionally, at least one of the variant Fc domains may comprise a non-naturally occurring amino acid residue at additional and / or alternative positions known to one skilled in the art (see, e.g., U.S. Pat. Nos.5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO 01 / 58957; WO 02 / 06919; WO 04 / 016750; WO 04 / 029207; WO 04 / 035752 and WO 05 / 040217, the contents of which are incorporated by reference herein in their entirety).

[0109] In certain embodiments, at least one of the variant Fc domains comprises at leastone non-naturally occurring amino acid or comprises at least two non-naturally occurring amino acids selected from the group consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R, 243W, 243L, 243Y, 243R, 243Q, 244H, 245A, 247V, 247G, 252Y, 254T, 256E, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 269H, 269Y, 269F, 269R, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 313F, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, and 332A as numbered by the EU index as set forth in Kabat. Optionally, at least one of the variant Fc domains may comprise additional and / or alternative non-naturally occurring amino acid residues known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO 01 / 58957; WO 02 / 06919; WO 04 / 016750; WO 04 / 029207; WO 04 / 035752 and WO 05 / 040217, the contents of which are incorporated by reference herein in their entirety).

[0110] Other known Fc domain variants that may be used in the compositions disclosedherein include without limitations those disclosed in Ghetie et al., 1997, Nat. Biotech.15:637-40; Duncan et al., 1988, Nature 332:563-564; Lund et al., 1991, J. Immunol, 147:2657-2662; Lund et al., 1992, Mol. Immunol, 29:53-59; Alegre et al., 1994, Transplantation 57: 1537-1543; Hutchins et al., 1995, Proc Natl. Acad Sci USA, 92: 11980-11984; Jefferis et al., 1995, Immunol Lett., 44:111-117; Lund et al., 1995, Faseb J., 9: 115-119; Jefferis et al., 1996, Immunol Lett., 54: 101-104; Lund et al., 1996, J. Immunol, 157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et al., 2000, J. Immunol, 164:4178-4184; Reddy et al., 2000, J. Immunol, 164: 1925- 1933; Xu et al., 2000, Cell Immunol, 200: 16-26; Idusogie et al., 2001, J. Immunol, 166:2571- 2575; Shields et al., 2001, J Biol. Chem., 276:6591-6604; Jefferis et al., 2002, Immunol Lett., 82:57-65; Presta et al., 2002, Biochem Soc Trans., 30:487-490); U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,528,624; 6,194,551; 6,737,056; 6,821,505; 6,277,375; U.S. Patent Publication Nos. 2004 / 0002587 and PCT Publications WO 94 / 29351; WO 99 / 58572; WO 00 / 42072; WO 02 / 060919; WO 04 / 029207; WO 04 / 099249; WO 04 / 063351, the contents of which are incorporated by reference herein in their entirety.

[0111] In an embodiment, the variant Fc region, or FcRn-binding fragment thereofcomprises or consists of two Fc domains. In an embodiment, the variant Fc region, or FcRn- binding fragment thereof, comprises at least one Fc domain comprising amino acids Y, T, E, K, and F at EU positions 252, 254, 256, 433, and 434, respectively. In an embodiment, the variant Fc region, or FcRn-binding fragment thereof, comprises at least one Fc domain comprising amino acids Y, T, E, K, F, and Y at EU positions 252, 254, 256, 433, 434, and 436, respectively. In an embodiment, the variant Fc region, or FcRn-binding fragment thereof, comprises one Fc domain comprising amino acids Y, T, E, K, and F at EU positions 252, 254, 256, 433, and 434, respectively, and a second Fc domain comprising amino acid K and F at EU positions 433 and 434, respectively. In an embodiment, the variant Fc region, or FcRn-binding fragment thereof, comprises one Fc domain comprising amino acids Y, T, E, K, F, and Y at EU positions 252, 254, 256, 433, 434, and 436, respectively, and a second Fc domain comprising amino acid K and F at EU positions 433 and 434, respectively. In an embodiment, the variant Fc region, or FcRn-binding fragment thereof consists of two Fc domains, both of which comprise amino acids Y, T, E, K, and F at EU positions 252, 254, 256, 433, and 434, respectively. In an embodiment, the variant Fc region, or FcRn-binding fragment thereof, consists of two Fc domains, both of which comprise amino acids Y, T, E, K, F, and Y at EU positions 252, 254, 256, 433, 434, and 436, respectively.

[0112] In certain embodiments, at least one of the variant Fc domains or FcRn-bindingfragments described herein comprises a combination of amino acids selected from the following:

[0113] (i) Q and L at EU positions 250 and 428, respectively;

[0114] (ii) P and A at EU positions 308 and 434, respectively;

[0115] (iii) P and Y at EU positions 308 and 434, respectively; or

[0116] (iv) Y, E and Y at EU positions 252, 286 and 434, respectively.

[0117] In certain embodiments, at least one of the variant Fc domains or FcRn-bindingfragments described herein comprises at least one amino acid substitution selected from: G237M; P238A; S239K; K248I; T250A; T250F; T250I; T250M; T250Q; T250S; T250V; T250W; T250Y; M252F; M252W; M252Y; S254T; R255E; T256D; T256E; T256Q; P257A; P257G; P257I; P257L; P257M; P257N; P257S; P257T; P257V; E258H; D265A; D270F; N286A; N286E; T289H; N297A; S298G; V303A; V305A; T307A; T307D; T307F; T307G; T307H; T307I; T307K; T307L; T307M; T307N; T307P; T307Q; T307R; T307S; T307V; T307W; T307Y; V308A; V308F; V308I; V308L; V308M; V308P; V308Q; V308T; V309A; V309D; V309E; V309P; V309R; Q311A; Q311H; Q311I; D312A; D312H; L314K; L314R; N315A; N315H; K317A; N325G; I332V; K334L; K360H; D376A; A378V; E380A; E382A; N384A; G385D; G385H; Q386P; P387E; N389A; N389S; S424A; M428A; M428D; M428F; M428G; M428H; M428I; M428K; M428L; M428N; M428P; M428Q; M428S; M428T; M428V; M428W; M428Y; H433K; N434A; N434F; N434H; N434S; N434W; N434Y; Y436H; Y436I and Y436F, wherein the positions are defined in accordance with EU numbering. In some embodiments, at least one of the variant Fc domains or FcRn-binding fragments described herein comprises 2, 3, 4 or 5 amino acid substitutions selected from the following: G237M; P238A; S239K; K248I; T250A; T250F; T250I; T250M; T250Q; T250S; T250V; T250W; T250Y; M252F; M252W; M252Y; S254T; R255E; T256D; T256E; T256Q; P257A; P257G; P257I; P257L; P257M; P257N; P257S; P257T; P257V; E258H; D265A; D270F; N286A; N286E; T289H; N297A; S298G; V303A; V305A; T307A; T307D; T307F; T307G; T307H; T307I; T307K; T307L; T307M; T307N; T307P; T307Q; T307R; T307S; T307V; T307W; T307Y; V308A; V308F; V308I; V308L; V308M; V308P; V308Q; V308T; V309A; V309D; V309E; V309P; V309R; Q311A; Q311H; Q311I; D312A; D312H; L314K; L314R; N315A; N315H; K317A; N325G; I332V; K334L; K360H; D376A; A378V; E380A; E382A; N384A; G385D; G385H; Q386P; P387E; N389A; N389S; S424A; M428A; M428D; M428F; M428G; M428H; M428I; M428K; M428L; M428N; M428P; M428Q; M428S; M428T; M428V; M428W; M428Y; H433K; N434A; N434F; N434H; N434S; N434W; N434Y; Y436H; Y436I and Y436F, wherein the positions are defined in accordance with EU numbering, and wherein any combinations of substitutions are contemplated.

[0118] In certain embodiments, at least one of the variant Fc domains or FcRn-bindingfragments described herein comprises a combination of amino acid substitutions selected from the following:

[0119] (i) M252Y, S254T, T256E, H433K and N434F;

[0120] (ii) T250Q and M428L;

[0121] (iii) V308P and N434A;

[0122] (iv) V308P and N434Y;

[0123] (v) M252Y, V308P, and N434Y; or

[0124] (vi) M252Y, N286E and N434Y.

[0125] In an embodiment, one, two, or more mutations (e.g., amino acid substitutions) areintroduced into the hinge region of a polypeptide described herein, such that the number of cysteine residues in the hinge region is altered (e.g., increased or decreased) as described in, e.g., U.S. Patent No. 5,677,425, herein incorporated by reference in its entirety. The number of cysteine residues in the hinge region may be altered to, e.g., facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the polypeptide.

[0126] In an embodiment, one, two, or more amino acid mutations (e.g., substitutions,insertions, or deletions) are introduced into an Fc region, Fc domain, or FcRn-binding fragment thereof to alter (e.g., decrease or increase) half-life of the polypeptide in vivo. See, e.g., International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375 and 6,165,745, all of which are herein incorporated by reference in their entireties, for examples of mutations that will alter (e.g., decrease or increase) the half-life of an antibody in vivo. In certain embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into a Fc region, Fc domain, or FcRn-binding fragment thereof to decrease the half-life of the polypeptide in vivo. In other embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into a Fc region, Fc domain, or FcRn-binding fragment thereof to increase the half- life of the antibody in vivo. In an embodiment, the Fc region or Fc domain may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or the third constant (CH3) domain (residues 341-447 of human IgG1), numbered according to the EU numbering system. In an embodiment, the constant region of the IgG1 of a polypeptide described herein comprises a methionine (M) to tyrosine (Y) substitution in position 252, a serine (S) to threonine (T) substitution in position 254, and a threonine (T) toglutamic acid (E) substitution in position 256, numbered according to the EU numbering system. See U.S. Patent No.7,658,921, which is herein incorporated by reference in its entirety. This type of mutant Fc domain, referred to “as "YTE mutant" has been shown to display fourfold increased half-life as compared to wild-type versions of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281: 23514-24, which is herein incorporated by reference in its entirety). In an embodiment, the polypeptide comprises an IgG constant region comprising one, two, three, or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385- 389, and 428-436, numbered according to the EU numbering system.

[0127] In an embodiment, one, two, or more mutations (e.g., amino acid substitutions) areintroduced into a Fc region, Fc domain, or FcRn-binding fragment thereof (e.g., a CH2 domain (residues 231-340 of human IgG1) and / or a CH3 domain (residues 341-447 of human IgG1, numbered according to the EU numbering system) and / or a hinge region (residues 216-230, numbered according to the EU numbering system)) of a polypeptide described herein, to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region, Fc domain, or FcRn-binding fragment thereof that decrease or increase the affinity of an antibody for an Fc receptor and techniques for introducing such mutations into the Fc receptor or fragment thereof are known to one of skill in the art. Examples of mutations in the Fc region, Fc domain, or FcRn-binding fragment thereof that can be made to alter the affinity of the variant Fc region, or FcRn-binding fragment thereof for an Fc receptor are described in, e.g., Smith P et al., (2012) PNAS 109: 6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, all of which are herein incorporated by reference in their entireties.

[0128] In an embodiment, one, two, or more amino acid substitutions are introduced intoa Fc region, Fc domain, or FcRn-binding fragment thereof to alter the effector function(s) of the polypeptide. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 239, 243, 267, 292, 297, 300, 318, 320, 322, 328, 330, 332, and 396, numbered according to the EU numbering system, can be replaced with a different amino acid residue such that the polypeptide has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent polypeptide. The effector ligand to which affinity is altered can be, for example, an Fc receptor. This approach is described in further detail in U.S. Patent Nos.5,624,821 and 5,648,260, each of which is herein incorporated by reference in its entirety. In an embodiment, one or more amino acid substitutions may be introduced into the Fc region or Fc domain of a polypeptidedescribed herein to remove potential glycosylation sites on the Fc region or Fc domain, which may reduce Fc receptor binding (see, e.g., Shields RL et al., (2001) J Biol Chem 276: 6591-604, which is herein incorporated by reference in its entirety). In an embodiment, one or more of the following mutations in the constant region of a polypeptide described herein may be made: an N297A substitution; an N297Q substitution; an L234A substitution; an L234F substitution; an L235A substitution; an L235F substitution; an L235V substitution; an L237A substitution; an S239D substitution; an E233P substitution; an L234V substitution; an L235A substitution; a C236 deletion; a P238A substitution; an S239D substitution; an F243L substitution; a D265A substitution; an S267E substitution; an L328F substitution; an R292P substitution; a Y300L substitution; an A327Q substitution; a P329A substitution; an A330L substitution; an I332E substitution; or a P396L substitution, numbered according to the EU numbering system.

[0129] In an embodiment, a mutation selected from the group consisting of D265A,P329A, and a combination thereof, numbered according to the EU numbering system, may be made in the constant region of a polypeptide described herein. In an embodiment, a mutation selected from the group consisting of L235A, L237A, and a combination thereof, numbered according to the EU numbering system, may be made in the constant region of a polypeptide described herein. In an embodiment, a mutation selected from the group consisting of S267E, L328F, and a combination thereof, numbered according to the EU numbering system, may be made in the constant region of a polypeptide described herein. In an embodiment, a mutation selected from the group consisting of S239D, I332E, optionally A330L, and a combination thereof, numbered according to the EU numbering system, may be made in the constant region of a polypeptide described herein. In an embodiment, a mutation selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, and a combination thereof, numbered according to the EU numbering system, may be made in the constant region of a polypeptide described herein. In an embodiment, a mutation selected from the group consisting of S267E, L328F, and a combination thereof, numbered according to the EU numbering system, may be made in the constant region of a polypeptide described herein.

[0130] In an embodiment, an Fc region, Fc domain, or FcRn-binding fragment thereofdescribed herein comprises the constant region of an IgG1 with an N297Q or N297A amino acid substitution, numbered according to the EU numbering system. In an embodiment, an Fc region, Fc domain, or FcRn-binding fragment thereof described herein comprises the constant region of an IgG1 with a mutation selected from the group consisting of D265A, P329A, and a combinationthereof, numbered according to the EU numbering system. In an embodiment, an Fc region, Fc domain, or FcRn-binding fragment thereof described herein comprises the constant region of an IgG1 with a mutation selected from the group consisting of L234A, L235A, and a combination thereof, numbered according to the EU numbering system. In another embodiment, an Fc region, Fc domain, or FcRn-binding fragment thereof described herein comprises the constant region of an IgG1 with a mutation selected from the group consisting of L234F, L235F, N297A, and a combination thereof, numbered according to the EU numbering system. In an embodiment, amino acid residues in the constant region of an Fc region, Fc domain, or FcRn-binding fragment thereof described herein in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, numbered according to the EU numbering system, are not L, L, and D, respectively. This approach is described in detail in International Publication No. WO 14 / 108483, which is herein incorporated by reference in its entirety. In an embodiment, the amino acids corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A; or A, A, and A, respectively, numbered according to the EU numbering system.

[0131] In an embodiment, the amino acids at positions 433, 434, and 436 of the heavychain constant region, according to the EU numbering system, are K, F, and Y, respectively. In an embodiment, the amino acids at positions 252, 254, and 256 of the heavy chain constant region, according to the EU numbering system, are Y, T, and E, respectively. In an embodiment, the amino acids at positions 428 and 434 of the heavy chain constant region, according to the EU numbering system, are L and S, respectively. In an embodiment, the amino acid at positions 309, 311, and 434 of the heavy chain constant region, according to the EU numbering system, are D, H, and S, respectively.

[0132] In an embodiment, the polypeptide does not have amino acids Y, T, E, K, and F atEU positions 252, 254, 256, 433, and 434, respectively.

[0133] In an embodiment, one or more amino acids selected from amino acid residues 329,331, and 322 in the constant region of a polypeptide described herein, numbered according to the EU numbering system, can be replaced with a different amino acid residue such that the antibody has altered C1q binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No.6,194,551 (Idusogie et al.), which is herein incorporated by reference in its entirety. In an embodiment, one or more amino acid residues within amino acid positions 231 to 238 in the N-terminal region of the CH2 domain of a polypeptide described herein are altered to thereby alter the ability of the antibody to fixcomplement, numbered according to the EU numbering system. This approach is described further in International Publication No. WO 94 / 29351, which is herein incorporated by reference in its entirety. In an embodiment, the Fc region or Fc domain of a polypeptide described herein is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the polypeptide for an Fc receptor by mutating one or more amino acids (e.g., introducing amino acid substitutions) at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439, numbered according to the EU numbering system. This approach is described further in International Publication No. WO 00 / 42072, which is herein incorporated by reference in its entirety.

[0134] In an embodiment, any of the constant region mutations or modifications describedherein can be introduced into one or both heavy chain constant regions of a polypeptide described herein having two heavy chain constant regions. In an embodiment, any of the constant region mutations or modifications described herein can be introduced into the heavy chain constant region of a polypeptide described herein having one heavy chain constant region.

[0135] In an embodiment, the instant disclosure provides a polypeptide comprising one,two or three binding sites for human FcRn, that specifically binds to FcRn and functions as an antagonist.

[0136] In an embodiment, the amino acid sequence of the Fc domains of the variant Fcregion comprises or consists of the amino acid sequence of SEQ ID NO: 1. In an embodiment, the amino acid sequence of the Fc domains of the variant Fc region comprises or consists of the amino acid sequence of SEQ ID NO: 2. In an embodiment, the amino acid sequence of the Fc domains of the variant Fc region comprises or consists of the amino acid sequence of SEQ ID NO: 3.

[0137] In an embodiment, the FcRn-binding molecule comprises a variant Fc region,wherein the variant Fc region comprises two Fc domains, wherein the amino acid sequence of each of the Fc domains is independently selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0138] In certain embodiments, the variant Fc region is a heterodimer, where theconstituent Fc domains are different from each other. Methods of producing Fc heterodimers are known in the art (see, e.g., US 8,216,805, which is incorporated by reference herein in its entirety).In an embodiment, the FcRn-binding molecule consists of a variant Fc region, wherein the variant Fc region consists of two Fc domains which form a heterodimer, wherein the amino acid sequence of each of the Fc domains is independently selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In an embodiment, the FcRn-binding molecule consists of or comprises a variant Fc region, wherein the variant Fc region consists of or comprises two Fc domains which form a heterodimer, wherein the amino acid sequence of the first Fc domain consists of or comprises the amino acid sequence of SEQ ID NO: 1, and the amino acid sequence of the second Fc domain consists of or comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. In an embodiment, the FcRn-binding molecule consists of or comprises a variant Fc region, wherein the variant Fc region consists of or comprises two Fc domains which form a heterodimer, wherein the amino acid sequence of the first Fc domain consists of or comprises the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of the second Fc domain consists of or comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. In an embodiment, the FcRn-binding molecule consists of or comprises a variant Fc region, wherein the variant Fc region consists of or comprises two Fc domains which form a heterodimer, wherein the amino acid sequence of the first Fc domain consists of or comprises the amino acid sequence of SEQ ID NO: 3, and the amino acid sequence of the second Fc domain consists of or comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0139] In an embodiment, the FcRn-binding molecule consists of or comprises a variantFc region, wherein the variant Fc region consists of or comprises two Fc domains which form a homodimer, wherein the amino acid sequence of each of the Fc domains consists of or comprises the amino acid sequence of SEQ ID NO: 1.

[0140] In an embodiment, the FcRn-binding molecule consists of or comprises a variantFc region, wherein the variant Fc region consists of or comprises two Fc domains which form a homodimer, wherein the amino acid sequence of each of the Fc domains consists of or comprises the amino acid sequence of SEQ ID NO: 2.

[0141] In an embodiment, the FcRn-binding molecule consists of or comprises a variantFc region, wherein the variant Fc region consists of or comprises two Fc domains which form a homodimer, wherein the amino acid sequence of each of the Fc domains consists of or comprises the amino acid sequence of SEQ ID NO: 3.

[0142] In an embodiment, the FcRn-binding molecule comprises or consists of a variantFc region, wherein the variant Fc region comprises or consists of efgartigimod (CAS Registry No.1821402-21-4). The term “efgartigimod” as used herein is interchangeable with “efgartigimod alfa” and “ARGX-113.” In some embodiments, efgartigimod is efgartigimod alfa-fcab.

[0143] In an embodiment, the variant Fc region is modified to promote heterodimerization.Such modifications are known in the art and any suitable means to promote heterodimerization may be used to generate the FcRn / HSA-binding molecules described herein. In some embodiments, the variant Fc region comprises one or more mutations of amino acid residues forming the interface of the CH3 domain of the Fc domains. In some embodiments, the variant Fcregion comprises knob-into-hole mutations (see, e.g., Intl. Publ. WO 2006 / 028936, incorporatedby reference in its entirety). The mispairing of Ig heavy chains is reduced in this technology by mutating selected amino acids forming the interface of the CH3 domains in IgG. At positions within the CH3 domain at which the two heavy chains interact directly, one or more amino acids with a small side chain (hole) is / are introduced into the sequence of one heavy chain and one or more amino acids with a large side chain (knob) into the counterpart interacting residue location(s) on the other heavy chain. The Fc domains of an Fc region can be composed of immunoglobulin chains of the same subclass (e.g., IgG1 or IgG3) or different subclasses (e.g., IgG1 and IgG3, or IgG3 and IgG4).

[0144] In some embodiments, the variant Fc region comprises or consists of two Fcdomains in which one of the Fc domains comprises amino acid W at EU position 366. In some embodiments, the variant Fc region comprises or consists of two Fc domains in which one of the Fc domains comprises amino acid S, A, and V at EU positions 366, 368, and 407, respectively. In some embodiments, the variant Fc region comprises or consists of two Fc domains in which one Fc domain comprises amino acid W at EU position 366, and the other Fc domain comprises amino acid S, A, and V at EU positions 366, 368, and 407, respectively.

[0145] In some embodiments, the variant Fc region comprises or consists of two Fcdomains in which one Fc domain comprises amino acids E and D at EU positions 370 and 409, respectively, and the other Fc domain comprises amino acid K at EU positions 357 and 399. In some embodiments, the variant Fc region comprises or consists of two Fc domains in which one Fc domain comprises amino acids H and A at EU positions 364 and 405, respectively, and the other Fc domain comprises amino acids T and F at EU positions 349 and 394, respectively. In some embodiments, the variant Fc region comprises or consists of two Fc domains in which one Fc domain comprises amino acids V, Y, A, and V at EU positions 350, 351, 405, and 407, respectively, and the other Fc domain comprises amino acids V, L, L, and W at EU positions 350,366, 392, and 394, respectively. In some embodiments, the variant Fc region comprises or consists of two Fc domains in which one Fc domain comprises amino acids D, M, and A at EU positions 360, 399, and 407, respectively, and the other Fc domain comprises amino acids R, R, V, and V at EU positions 345, 347, 366, and 409, respectively. In some embodiments, the variant Fc region comprises or consists of two Fc domains in which one Fc domain comprises amino acid D at EU positions 409 and 392, and the other Fc domain comprises amino acid K at EU positions 399 and 356. In some embodiments, the variant Fc region comprises or consists of two Fc domains in which one Fc domain comprises amino acids E, W, and C at EU positions 360, 409, and 349, respectively, and the other Fc domain comprises amino acids R, V, T, and C at EU positions 347, 399, 405, and 354, respectively. In some embodiments, the variant Fc region comprises or consists of two Fc domains in which one Fc domain comprises amino acids E and W at EU positions 370 and 409, respectively, and the other Fc domain comprises amino acids N, V, and T at EU positions 357, 399, and 405, respectively.

[0146] In an embodiment, the FcRn-binding molecule consists of a variant Fc region,wherein the variant Fc region comprises or consists of two Fc domains which form a heterodimer, wherein the amino acid sequence of the first Fc domain is selected from an amino acid sequence comprising or consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 and / or the amino acid sequence of the second Fc domain is selected from an amino acid sequence comprising or consisting of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. In an embodiment, the FcRn- binding molecule consists of a variant Fc region, wherein the variant Fc region comprises or consists of two Fc domains which form a heterodimer, wherein the amino acid sequence of the first Fc domain is selected from an amino acid sequence comprising or consisting of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 and / or the amino acid sequence of the second Fc domain is selected from an amino acid sequence comprising or consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. In an embodiment, the FcRn-binding molecule consists of a variant Fc region, wherein the variant Fc region comprises or consists of two Fc domains which form a heterodimer, wherein the amino acid sequence of the first Fc domain comprises or consists of SEQ ID NO: 4 and the amino acid sequence of the second Fc domain comprises or consists of SEQ ID NO: 7. In an embodiment, the FcRn-binding molecule consists of a variant Fc region, wherein the variant Fc region comprises or consists of two Fc domains which form a heterodimer, wherein the amino acid sequence of the first Fc domain comprises or consists of SEQ ID NO: 5 and the amino acid sequence of the second Fc domain comprises or consists of SEQ ID NO: 8. In an embodiment, theFcRn-binding molecule consists of a variant Fc region, wherein the variant Fc region comprises or consists of two Fc domains which form a heterodimer, wherein the amino acid sequence of the first Fc domain comprises or consists of SEQ ID NO: 6 and the amino acid sequence of the second Fc domain comprises or consists of SEQ ID NO: 9. In some embodiments, the FcRn-binding molecule is an FcRn antagonist. Table 1. Amino acid sequences of Fc domains SEQ ID NO: Amino Acid Sequence 1 CPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPE E S E S Y E EDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS E SAmino Acid Sequence SEQ ID NO:

[0147] In some embodiments, the FcRn-binding molecule comprises or consists of avariant Fc region, wherein the variant Fc region comprises or consists of a dimer of a first Fc domain and a second Fc domain, wherein the amino acid sequence of the first and second Fc domain comprises or consists of the amino acid sequence of SEQ ID NO: 10.

[0148] In some embodiments, the variant Fc region comprises a first Fc domain comprisingamino acids Y, T, E, K, and F at EU positions 252, 254, 256, 433, and 434, respectively, and a second Fc domain comprising amino acids K and F at EU positions 433 and 434, respectively. In some embodiments, the first Fc domain comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first Fc domain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the first Fc domain comprises the amino acid sequence of SEQ ID NO: 3.

[0149] In some embodiments, the variant Fc region comprises a first Fc domain comprisingamino acids Y, T, E, W, K, and F at EU positions 252, 254, 256, 366, 433, and 434, respectively, and a second Fc domain comprising amino acids S, A, V, K, and F at EU positions 366, 368, 407, 433, and 434, respectively. In some embodiments, the first Fc domain comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the first Fc domain comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the first Fc domain comprises the amino acid sequence of SEQ ID NO: 6.

[0150] In some embodiments, the variant Fc region comprises a first Fc domain comprisingamino acids Y, T, E, S, A, V, K, and F at EU positions 252, 254, 256, 366, 368, 407, 433, and 434, respectively, and a second Fc domain comprising amino acids W, K, and F at EU positions 366, 433, and 434, respectively. In some embodiments, the first Fc domain comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the first Fc domain comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first Fc domain comprises the amino acid sequence of SEQ ID NO: 9. HSA-Binding Domains

[0151] In an aspect, HSA-binding domains are provided by the present disclosure.

[0152] In some embodiments, the HSA-binding domain is selected from a Fab fragment,an scFv, an sdAb, a VH, a VL, HSA, and HSA-binding fragments thereof. In some embodiments, the HSA-binding domain is an sdAb, such as a VHH. In some embodiments, the HSA-binding domain is a synthetic antigen-binding protein or antibody mimetic protein including, but not limited to, an anticalin or a DARPin. In some embodiments, HSA comprises an amino acid sequence at least 95% identical to the amino acid sequence provided in GenBank Accession No.: AAA98797.1. In some embodiments, HSA comprises the amino acid sequence provided in GenBank Accession No.: AAA98797.1.

[0153] In some embodiments, the HSA-binding domain further comprises one or moreamino acids added at its C-terminus. In some embodiments, the HSA-binding domain further comprises one or more amino acids added at the C-terminus selected from A, AG, GG, PP, and AA. In some embodiments, the C-terminus of VHH is the amino acid sequence VTVSS (SEQ ID NO: 38). In some embodiments, the C-terminus of VHH consists of the amino acid sequence VTVSS (SEQ ID NO: 38).

[0154] In some embodiments, the HSA-binding domain is a VHH comprising the CDR1,CDR2, and CDR3 amino acid sequences of a VHH comprising an amino acid sequence selected from SEQ ID NOs: 22-34.

[0155] In some embodiments, the HSA-binding domain is a VHH comprising or consistingof a combination of CDR1, CDR2, and CDR3 wherein 1, 2, 3, 4, or 5 amino acids differ in at least one of the amino acid sequences selected from SEQ ID NOs: 11, 12 and 13; 14, 15, and 13; 14, 12, and 16; 14, 12, and 17; 18, 12, and 19; 14, 12, and 13; 20, 12, and 13; 21, 12, and 13; 11, 15, and 13; 11, 12, and 16; 11, 12, and 19; 11, 12, and 17; and 21, 12, and 19.

[0156] In some embodiments, the HSA-binding domain is a VHH comprising orconsisting of a combination of CDR1, CDR2, and CDR3 selected from SEQ ID NOs: 11, 12 and 13; 14, 15, and 13; 14, 12, and 16; 14, 12, and 17; 18, 12, and 19; 14, 12, and 13; 20, 12, and 13; 21, 12, and 13; 11, 15, and 13; 11, 12, and 16; 11, 12, and 19; 11, 12, and 17; and 21, 12, and 19.

[0157] In some embodiments, the HSA-binding domain is a VHH comprising or consistingof an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 22-34. In some embodiments, the HSA-binding domain is a VHH comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 22-34. Linkers

[0158] The HSA-binding domain may be linked to the N-terminus or the C-terminus of anFcRn-binding molecule (e.g., an Fc domain). Alternatively, the HSA-binding domain may to linked at a position other than the N-terminus or the C-terminus an FcRn-binding molecule (e.g., an Fc domain). Preferably, the HSA-binding domain is linked to the C-terminus of an FcRn- binding molecule (e.g., an Fc domain).

[0159] In some embodiments, the HSA-binding domain may be non-covalently linked tothe FcRn-binding molecule. In some embodiments, the HSA-binding domain may be covalently linked to the FcRn-binding molecule.

[0160] In some embodiments, the HSA-binding domain may be linked (e.g., fused) directlyto the N-terminus or the C-terminus of an FcRn-binding molecule. In some embodiments, the HSA-binding domain is linked to the N-terminus or the C-terminus of an FcRn-binding molecule via a linker. In some embodiments, the linker is a non-cleavable linker.

[0161] In some embodiments, the HSA-binding domain may be linked (e.g., fused) directlyto the N-terminus or the C-terminus of an Fc domain. In some embodiments, the HSA-binding domain is linked to the N-terminus or the C-terminus of an Fc domain via a linker. In some embodiments, the linker is a non-cleavable linker. As used herein, the term “non-cleavable linker” refers to a linker that is not readily cleaved by one or more of a given enzyme, chemical agent, or photo-irradiation. In some embodiments, the enzyme is a protease.

[0162] In some embodiments, the linker is a synthetic compound linker such as, forexample, a chemical cross-linking agent. Non-limiting examples of suitable cross-linking agents that are available on the market include N-hydroxysuccinimide (NHS), disuccinimidylsuberate(DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethyleneglycol bis(succinimidylsuccinate) (EGS), ethyleneglycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2- (succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2- (sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0163] As described above, Fc domains disclosed herein may comprise a portion of a hingeregion. As such, the HSA-binding domain may be linked to the N-terminus of an Fc domain via this hinge region. In some embodiments, one or more amino acids are included between the C- terminus of the HSA-binding domain and the N-terminus of the Fc domain. In some embodiments, the one or more amino acids included between the C-terminus of the HSA-binding domain and the N-terminus of the Fc domain are amino acids of a natural hinge region. In some embodiments, the C-terminus of the HSA-binding domain is fused to the N-terminus of the Fc domain via a hinge region or a portion thereof. In some embodiments, the hinge region is and IgG hinge region, such as a human IgG hinge region.

[0164] In some embodiments, the linker is a peptide linker. Examples of peptide linkersare well known and those of skill in the art could select a suitable peptide linker for use in fusing an HSA-binding domain to an FcRn-binding molecule, e.g., an Fc domain.

[0165] Peptide linkers may be of any length. In some embodiments, the length and aminoacid composition of the linker peptide sequence can be optimized to vary the orientation and / or proximity of the polypeptide domains to one another to achieve a desired activity of the FcRn / HSA-binding molecule. In some embodiments, the peptide linker is between about 1 and about 100 amino acids in length, between about 8 and about 40 amino acids in length, or between about 15 amino acids and about 25 amino acids in length. In some embodiments, the peptide linker is between 1 and 100 amino acids in length, between 8 and 40 amino acids in length, or between 15 and 25 amino acids in length. In some embodiments, the peptide linker is about 8 amino acid in length, about 9 amino acids in length, about 10 amino acids in length, about 11 amino acids in length, about 12 amino acids in length, about 13 amino acids in length, about 14 amino acids in length, about 15 amino acids in length, about 16 amino acids in length, about 17 amino acids in length, about 18 amino acids in length, about 19 amino acids in length, about 20 amino acids in length, about 21 amino acids in length, about 22 amino acids in length, about 23 amino acids in length, about 24 amino acids in length, about 25 amino acids in length, about 26 amino acids inlength, about 27 amino acids in length, about 28 amino acids in length, about 29 amino acids in length, about 30 amino acids in length, about 31 amino acids in length, about 32 amino acids in length, about 33 amino acids in length, about 34 amino acids in length, about 35 amino acids in length, about 36 amino acids in length, about 37 amino acids in length, about 38 amino acids in length, about 39 amino acids in length, or about 40 amino acids in length. In some embodiments, the peptide linker is 8 amino acids in length, 9 amino acids in length, 10 amino acids in length, 11 amino acids in length, 12 amino acids in length, 13 amino acids in length, 14 amino acids in length, 15 amino acids in length, 16 amino acids in length, 17 amino acids in length, 18 amino acids in length, 19 amino acids in length, 20 amino acids in length, 21 amino acids in length, 22 amino acids in length, 23 amino acids in length, 24 amino acids in length, 25 amino acids in length, 26 amino acids in length, 27 amino acids in length, 28 amino acids in length, 29 amino acids in length, 30 amino acids in length, 31 amino acids in length, 32 amino acids in length, 33 amino acids in length, 34 amino acids in length, 35 amino acids in length, 36 amino acids in length, 37 amino acids in length, 38 amino acids in length, 39 amino acids in length, or 40 amino acids in length.

[0166] In some embodiments, the peptide linker contains only glycine and / or serineresidues (e.g., glycine-serine linker or GS linker). Examples of such peptide linkers include: Gly(x) Ser, where x is 0 to 6; or Ser Gly(x), where x is 0 to 6; (Gly Gly Gly Gly Ser)n, wherein n is an integer of one or more; and (Ser Gly Gly Gly Gly)n, wherein n is an integer of one or more. In some embodiments, the peptide linker includes an amino acid sequence selected from the group consisting of: (GGGGS)n and (SGGGG)n, where n is 1 to 8. In some embodiments, the linker peptides are modified such that the amino acid sequence GSG (that occurs at the junction of traditional Gly / Ser linker peptide repeats) is not present. For example, in some embodiments, the peptide linker includes an amino acid sequence selected from the group consisting of: (GGGXX)nGGGGS and GGGGS(XGGGS)n, where X is any amino acid that can be inserted into the sequence and not result in a polypeptide including the sequence GSG, and n is 0 to 4. In some embodiments, the sequence of a linker peptide is (GGGX1X2)nGGGGS and X1 is P and X2 is S and n is 0 to 4. In some other embodiments, the sequence of a linker peptide is (GGGX1X2)nGGGGS and X1 is G and X2 is Q and n is 0 to 4. In some other embodiments, the sequence of a linker peptide is (GGGX1X2)nGGGGS and X1 is G and X2 is A and n is 0 to 4. In yet other embodiments, the sequence of a linker peptide is GGGGS(XGGGS)n, and X is P and n is 0 to 4. In some embodiments, a linker peptide of the disclosure comprises or consists of the amino acid sequence (GGGGA)2GGGGS. In some embodiments, a linker peptide comprises orconsists of the amino acid sequence (GGGGQ)2GGGGS. In another embodiment, a linker peptide comprises or consists of the amino acid sequence (GGGPS)2GGGGS. In another embodiment, a linker peptide comprises or consists of the amino acid sequence GGGGS(PGGGS)2. In yet a further embodiment, a linker peptide comprises or consists of the amino acid sequence GSGGS or SGGSGS. In some embodiments, a linker peptide comprises or consists of the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 39), GGGGSGGGGS (SEQ ID NO: 40), or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 41).

[0167] In some embodiments, the peptide linker is a GS linker of about 20 or about 30amino acids in length. In some embodiments, the peptide linker is a GS linker of 20 or 30 amino acids in length. Heavy Chain Molecules

[0168] In some embodiments, FcRn / HSA-binding molecules can comprise a first heavychain described herein. In some embodiments, the first heavy chain comprises an Fc domain and an HSA-binding domain joined by a linker. In some embodiments, FcRn / HSA-binding molecules can further comprise second heavy chain described herein. In some embodiments, the second heavy chain comprises an Fc domain.

[0169] In some embodiments, the first and second heavy chains have the same Fc domain.In some embodiments, the first and second heavy chains have different Fc domains. In some embodiments, the first heavy chain comprises an Fc domain and an HSA-binding domain, while the second heavy chain comprises an Fc domain but does not comprise an HSA-binding domain. In some embodiments, the first heavy chain comprises an Fc domain and an HSA-binding domain, while the second heavy chain comprises an Fc domain but does not comprise an HSA-binding domain or a linker. In some embodiments, the first heavy chain comprises an Fc domain, an HSA- binding domain, and a linker, while the second heavy chain comprises an Fc domain but does not comprise an HSA-binding domain or a linker.

[0170] In some embodiments, the HSA-binding domain is fused to the N-terminus of theFc domain. In some embodiments, the HSA-binding domain is fused to the C-terminus of the Fc domain. In some embodiments, the HSA-binding domain is fused to the N-terminus of the Fc domain by a peptide linker. In some embodiments, the HSA-binding domain is fused to the C- terminus of the Fc domain by a peptide linker.

[0171] In some embodiments, the Fc domain comprises an amino acid sequence at least70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1-9. In some embodiments, the Fc domain consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1-9.

[0172] In some embodiments, the Fc domain comprises the amino acid sequence of anyone of SEQ ID NOs: 1-9. In some embodiments, the Fc domain consists of the amino acid sequence of any one of SEQ ID NOs: 1-9.

[0173] In some embodiments, the Fc domain comprises the amino acid sequence of SEQID NO: 10. In some embodiments, the Fc domain consists of the amino acid sequence of SEQ ID NO: 10.

[0174] In some embodiments, the first and second heavy chains comprise the same Fcdomain. In some embodiments, both the first and second heavy chains comprise an Fc domain comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, both the first and second heavy chains comprise an Fc domain consisting of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, both the first and second heavy chains comprise an Fc domain comprising the amino acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, both the first and second heavy chains comprise an Fc domain consisting of the amino acid sequence of any one of SEQ ID NOs: 1-3.

[0175] In some embodiments, the first and second heavy chains comprise different Fcdomains. In some embodiments, the first heavy chain comprises an Fc domain comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 4-6 and the second heavy chain comprises an Fc domain comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 7-9. In some embodiments, the first heavy chain comprises an Fc domain consisting of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 4-6 and the second heavy chain comprises an Fc domain consisting of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 7-9. In someembodiments, the first heavy chain comprises an Fc domain comprising the amino acid sequence of any one of SEQ ID NOs: 4-6 and the second heavy chain comprises an Fc domain comprising the amino acid sequence of any one of SEQ ID NOs: 7-9. In some embodiments, when the first heavy chain comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof, the second heavy chain comprises an Fc domain comprising SEQ ID NO: 7 or a variant thereof. In some embodiments, when the first heavy chain comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 5 or a variant thereof, the second heavy chain comprises an Fc domain comprising SEQ ID NO: 8 or a variant thereof. In some embodiments, when the first heavy chain comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof, the second heavy chain comprises an Fc domain comprising SEQ ID NO: 9 or a variant thereof.

[0176] In some embodiments, the first and second heavy chains further comprise a peptidelinker. In some embodiments, the first and second heavy chains further comprise the same peptide linker. In some embodiments, the first and second heavy chains further comprise different peptide linkers. In some embodiments, the first heavy chain comprises an Fc domain, a peptide linker, and an HSA-binding domain and the second heavy chain comprises an Fc domain but no peptide linker or HSA-binding domain. The peptide linkers encoded by the first and heavy chains can be any described herein. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 39, 40, or 41.

[0177] In some embodiments, the FcRn / HSA-binding molecule comprises an amino acidsequence selected from Table 3 or a variant thereof.:O N D)I8Q8E3 2 3 4 5 6 7 8 94S4 4 4 4 4 4 4 40512(O W1032T-373404L T G Q L T G Q L T G Q L T G Q L T G Q L T G Q L T G Q L T G Q L T G Q T Y G L T Y G L T Y G L T Y G L T Y G L T Y G L T Y G L T Y G L T Y G L D V G Y D V G Y D V G Y D V G Y D V G Y D V G Y D V G Y D V G Y D V G Y K Q G L K Q G L K Q G L K Q G L K Q G L K Q G L K Q G L K Q G L K Q G L P P P T P P P T P P P T P P P T P P P T P P P T P P P T P P P T P P P T K E S N K E S N K E S N K E S N K E S N K E S N K E S N K E S N K E S N P R L K P R L K P R L K P R L K P R L K P R L K P R L K P R L K P R L K P P S S P P S S P P S S P P S S P P S S P P S S P P S S P P S S P P S S F Q L N F Q L N F Q L N F Q L N F Q L N F Q L N F Q L N F Q L N F Q L N L G S D L G S D L G S D L G S D L G S D L G S D L G S D L G S D L G S D F K K R F K K R F K K R F K K R F K K R F K K R F K K R F K K R F K K R V A Q S V A Q S V A Q S V A Q S V A Q S V A Q S V A Q S V A Q S V A Q S S K T I S K T I S K T I S K T I S K T I S K T I S K T I S K T I S K T I P S Y T P S Y T P S Y T P S Y T P S Y T P S Y T P S Y T P S Y T P S Y T G I H F G I H F G I H F G I H F G I H F G I H F G I H F G I H F G I H F G T F R G T F R G T F R G T F R G T F R G T F R G T F R G T F R G T F R L K K G L K K G L K K G L K K G L K K G L K K G L K K G L K K G L K K G L E L K L E L K L E L K L E L K L E L K L E L K L E L K L E L K L E L K E I A V E I A V E I A V E I A V E I A V E I A V E I A V E I A V E I A V P P E S P P E S P P E S P P E S P P E S P P E S P P E S P P E S P P E S A A H D A A H D A A H D A A H D A A H D A A H D A A H D A A H D A A H D P P M A P P M A P P M A P P M A P P M A P P M A P P M A P P M A P P M A e C L V Y C L V Y C L V Y C L V Y C L V Y C L V Y C L V Y C L V Y C L V Y c P A S A P A S L P A S L P A S L P A S L P A S L P A S L P A S A P A S L n P K C T P K C T P K C T P K C T P K C T P K C T P K C T P K C T P K C T e C N S D C N S D C N S D C N S D C N S D C N S D C N S D C N S D C N S D u T S F S T S F S T S F S T S F S T S F S T S F S T S F S T S F S T S F S q H V V G H V V G H V V G H V V G H V V G H V V G H V V G H V V G H V V G e T K N S T K N S T K N S T K N S T K N S T K N S T K N S T K N S T K N S K C G G K C G G K C G G K C G G K C G G K C G G K C G G K C G G K C G G 3 S D K Q S D K Q S D K Q S D K Q S D K Q S D K Q S D K Q S D K Q S D K Q S elba en TolC1#2#3#4#5#6#7#8#9#)883412(O W1032T-373404Q T F Q T F Q T F Q T S A E T G E T G E T G E T G S E K S E K S E K S E K G S R Y A R Y A R Y A R Y P V P N A P N A P N A P N Q T K N C K N C K N C K N V V T E S T E S T E S T E L L K P L K P L K P L K P G T A Q R A Q R A Q R A Q G G N G L N G L N G L N G G Q H N S H N S H N S H N S S V S G S V S G S V S G S V S E S E E G S E E G S E E G S E E L R V W P V V W P V V W P V V W L S G E Q T G E Q T G E Q T G E Q L D V V V D V V V D V V V D V V S V A L L V A L L V A L L V A E G Y I G T Y I G T Y I G T Y I S G W D G G W D G G W D G G W D G I N S G Q N S G Q N S G Q N S G T F P S S F P S S F P S S F P G C K Y E S K Y E S K Y E S K Y G Y V F L R V F L R V F L R V F S Y E G L S E G L S E G L S E G G V P K Q L P K Q A P K Q L P K G A D V V A D V V S D V V A D V G T E L E G E L E G E L E G E L G D3H C S G H C S G H C S G H C S E4S W G I S W G I S W G I S W G P V L G T V L G T V L G T V L G R D S G C D S G C D S G C D S G L V V G Y V V G Y V V G Y V V G S V Q S Y V Q S Y V Q S Y V Q S N V N G V V N G V V N G V V N G M C K G A C K G A C K G A C K G Q T T G T T T G T T T G T T T G L V L G D V L G D V L G D V L G Y E E S E E E S E E E S E E E S L P D G P P D G P P D G P P D G T E R G R E R G R E R G R E R G N R S G L R S G L R S G L R S G K T P G S T P G S T P G S T P G S I P S N I P S N I P S N I P S N Y L G M Y L G M Y L G M Y L G D L T G Q L T G Q L T G Q L T G R T Y G L T Y G L T Y G L T Y G S D V G Y D V G Y D V G Y D V G I K Q G L K Q G L K Q G L K Q G T P P P T P P P T P P P T P P P F K E S N K E S N K E S N K E S R P R L K P R L K P R L K P R L G P P S S P P S S P P S S P P S K F Q L N F Q L N F Q L N F Q L V L G S D L G S D L G S D L G S S F K K R F K K R F K K R F K K D V A Q S V A Q S V A Q S V A Q A S K T I S K T I S K T I S K T Y P S Y T P S Y T P S Y T P S Y L G I H F G I H F G I H F G I H T G T F R G T F R G T F R G T F D L K K G L K K G L K K G L K K S L E L K L E L K L E L K L E L G E I A V E I A V E I A V E I A S P P E S P P E S P P E S P P E G A A H D A A H D A A H D A A H S P P M A P P M A P P M A P P M I C L V Y C L V Y C L V Y C L V S P A S L P A S L P A S L P A S S P K C T P K C T P K C T P K C V C N S D C N S D C N S D C N S W T S F S T S F S T S F S T S F E H V V G H V V G H V V G H V V P T K N S T K N S T K N S T K N G K C G G K C G G K C G G K C G K D K Q S D K Q S D K Q S D K Q G 0 111213# # #1#

[0178] In some embodiments, the FcRn / HSA-binding molecule comprises or consists ofan amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 42-54. In some embodiments, the FcRn / HSA-binding molecule comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 42-54.

[0179] In some embodiments, the FcRn / HSA-binding molecule comprises the amino acidsequence of any one of SEQ ID NOs: 42-54 or a variant thereof and one or more amino acids added at the C-terminus. In some embodiments, the FcRn / HSA-binding molecule comprises the amino acid sequence of any one of SEQ ID NOs: 42-54 or a variant thereof and one or more amino acids added at the C-terminus selected from A, AG, GG, PP, and AA.

[0180] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises or consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 42-54 and the second heavy chain of the FcRn / HSA-binding molecule does not comprise an HSA-binding domain. In some embodiments, the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain, but does not comprise an HSA-binding domain. Optionally, the second heavy chain of the FcRn / HSA-binding molecule comprises or consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8.

[0181] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises or consists of the amino acid sequence of any one of SEQ ID NOs: 42-54 and the second heavy chain of the FcRn / HSA-binding molecule does not comprise an HSA-binding domain. In some embodiments, the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain, but does not comprise an HSA-binding domain. Optionally, the second heavy chain of the FcRn / HSA-binding molecule comprises or consists of the amino acid sequence of SEQ ID NO: 8.

[0182] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 42 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the firstheavy chain of the FcRn / HSA-binding molecule consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 42 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0183] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises the amino acid sequence of SEQ ID NO: 42 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the first heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 42 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0184] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 43 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the first heavy chain the FcRn / HSA-binding molecule consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 43 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0185] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises the amino acid sequence of SEQ ID NO: 43 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the first heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 43 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0186] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 44 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the firstheavy chain of the FcRn / HSA-binding molecule consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 44 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0187] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises the amino acid sequence of SEQ ID NO: 44 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the first heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 44 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0188] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 45 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the first heavy chain of the FcRn / HSA-binding molecule consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 45 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0189] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises the amino acid sequence of SEQ ID NO: 45 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the first heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 45 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0190] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 46 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the firstheavy chain of the FcRn / HSA-binding molecule consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 46 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0191] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises the amino acid sequence of SEQ ID NO: 46 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the first heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 46 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0192] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 47 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the first heavy chain of the FcRn / HSA-binding molecule consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 47 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0193] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises the amino acid sequence of SEQ ID NO: 47 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the first heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 47 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0194] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 48 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the firstheavy chain of the FcRn / HSA-binding molecule consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 48 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0195] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises the amino acid sequence of SEQ ID NO: 48 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the first heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 48 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0196] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 49 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the first heavy chain of the FcRn / HSA-binding molecule consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 49 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0197] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises the amino acid sequence of SEQ ID NO: 49 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the first heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 49 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0198] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 50 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the firstheavy chain of the FcRn / HSA-binding molecule consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 50 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0199] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises the amino acid sequence of SEQ ID NO: 50 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the first heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 50 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0200] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 51 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the first heavy chain of the FcRn / HSA-binding molecule consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 51 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0201] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises the amino acid sequence of SEQ ID NO: 51 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the first heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 51 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0202] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 52 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the firstheavy chain of the FcRn / HSA-binding molecule consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 52 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0203] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises the amino acid sequence of SEQ ID NO: 52 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the first heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 52 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0204] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 53 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the first heavy chain of the FcRn / HSA-binding molecule consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 53 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0205] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises the amino acid sequence of SEQ ID NO: 53 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the first heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 53 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0206] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 54 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the firstheavy chain of the FcRn / HSA-binding molecule consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 54 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0207] In some embodiments, the first heavy chain of the FcRn / HSA-binding moleculecomprises the amino acid sequence of SEQ ID NO: 54 and the second heavy chain of the FcRn / HSA-binding molecule comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 8, but does not comprise an HSA-binding domain. In some embodiments, the first heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 54 and the second heavy chain of the FcRn / HSA-binding molecule consists of the amino acid sequence of SEQ ID NO: 8.

[0208] In some embodiments, the FcRn / HSA-binding molecule comprises the amino acidsequence of any one of SEQ ID NOs: 42-54 or a variant thereof as described herein and one or more amino acids added at the C-terminus. In some embodiments, the FcRn / HSA-binding molecule comprises the amino acid sequence of any one of SEQ ID NOs: 42-54 or a variant thereof as described herein and one or more amino acids added at the C-terminus selected from A, AG, GG, PP, and AA. Polynucleotides, Vectors, and Methods of Production

[0209] The disclosure also provides polynucleotides encoding the FcRn / HSA-bindingmolecules disclosed herein or fragments thereof. In some embodiments, the polynucleotide encodes an HSA-binding domain of the disclosure. In some embodiments, the polynucleotide encodes an FcRn-binding molecule of the disclosure. In some embodiments, the polynucleotide encodes an Fc region of the disclosure. In some embodiments, the polynucleotide encodes an Fc domain of the disclosure. In some embodiments, the polynucleotide encodes one or more of an HSA-binding domain, an FcRn-binding molecule, and a linker. In some embodiments, the polynucleotide encodes an HSA-binding domain and an FcRn-binding molecule, and optionally a linker. In some embodiments, the polynucleotide encodes one or more of an HSA-binding domain, an Fc region, and a linker. In some embodiments, the polynucleotide encodes an HSA-binding domain and an Fc region, and optionally a linker. In some embodiments, the polynucleotide encodes an FcRn / HSA-binding molecule comprising one or more HSA-binding domains and an Fc region. In some embodiments, the polynucleotide encodes one or more of an HSA-bindingdomain, an Fc domain, and a linker. In some embodiments, the polynucleotide encodes an HSA- binding domain and an Fc domain, and optionally a linker. In some embodiments, the polynucleotide encodes an FcRn / HSA-binding molecule comprising one or more HSA-binding domains and one or more Fc domains. In some embodiments, the polynucleotide encodes one or more heavy chains of the disclosure.

[0210] As used herein, an “isolated” polynucleotide or nucleic acid molecule is one whichis separated from other nucleic acid molecules which are present in the natural source (e.g., in a mouse or a human) of the nucleic acid molecule. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the language “substantially free” includes preparations of polynucleotide or nucleic acid molecules having less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (in particular, less than about 10%) of other material, e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors and / or other chemicals. In an embodiment, a nucleic acid molecule(s) encoding a polypeptide described herein is isolated or purified.

[0211] In an aspect, provided herein are polynucleotides comprising a nucleotide sequenceencoding an FcRn-binding molecule or FcRn / HSA-binding molecule described herein. In another aspect, provided herein are polynucleotides comprising a nucleotide sequence encoding an HSA- binding domain described herein. In another aspect, provided herein are polynucleotides comprising a nucleotide sequence encoding an FcRn / HSA-binding molecule described herein.

[0212] The polynucleotides can comprise nucleotide sequences encoding an sdAb (e.g., aVHH), a Fab fragment, an scFv, a VH, or a VL comprising FRs and CDRs of HSA-binding domains described herein. The polynucleotides can also comprise nucleotide sequences encoding an antibody mimetic as described herein. In some embodiments, the polynucleotides can comprise nucleotide sequences encoding a VHH comprising FR and CDRs of HSA-binding domains described herein. In some embodiments, the polynucleotides can comprise nucleotide sequences encoding a heavy chain comprising VHH FRs and CDRs of HSA-binding domains described herein and / or an Fc domain as described herein.

[0213] In some embodiments, the polynucleotides can comprise nucleotide sequencesencoding a first heavy chain described herein. In some embodiments, the first heavy chain comprises an Fc domain and an HSA-binding domain joined by a linker. In some embodiments,polynucleotides can comprise nucleotide sequences encoding a second heavy chain described herein. In some embodiments, the second heavy chain comprises an Fc domain and an antigen- binding domain (e.g., another HSA-binding domain) joined by a linker. In some embodiments, the first and second heavy chains are the same. In some embodiments, the first and second heavy chains are different.

[0214] In some embodiments, the first and second heavy chains have the same Fc domain.In some embodiments, the first and second heavy chains have different Fc domains. In some embodiments, the first and second heavy chains both comprise an antigen-binding domain. In some embodiments, the antigen-binding domains on the first and second heavy chains are the same. In some embodiments, the antigen-binding domains on the first and second heavy chains are different. In some embodiments, the second heavy chain comprises an Fc domain but does not comprise an antigen-binding domain, while the first heavy chain comprises an Fc domain and an HSA-binding domain. In some embodiments, the second heavy chain comprises an Fc domain but does not comprise an antigen-binding domain or a linker, while the first heavy chain comprises an Fc domain and an HSA-binding domain. In some embodiments, the second heavy chain comprises an Fc domain but does not comprise an antigen-binding domain or a linker, while the first heavy chain comprises an Fc domain and an HSA-binding domain and a linker.

[0215] In some embodiments, the polynucleotides comprise or consist of a nucleotidesequence that encodes an Fc domain comprising or consisting of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1-9. In some embodiments, the polynucleotides comprise or consist of a nucleotide sequence that encodes an Fc domain comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1-9. In some embodiments, the polynucleotides comprise or consist of a nucleotide sequence that encodes an Fc domain comprising or consisting of the amino acid sequence of SEQ ID NO: 10.

[0216] In some embodiments, the polynucleotides comprise nucleotide sequences thatencode two or more Fc domains. In some embodiments, the polynucleotides comprise nucleotide sequences that encode two Fc domains. In some embodiments, the polynucleotides comprise a first nucleotide sequence that encodes a first Fc domain and a second nucleotide sequence that encodes a second Fc domain. In some embodiments, the first nucleotide sequence and the second nucleotide sequence are comprised in distinct nucleic acid molecules. In some embodiments, the firstnucleotide sequence and the second nucleotide sequence are comprised in the same nucleic acid molecule.

[0217] In some embodiments, the first and second nucleotide sequence encode the sameFc domain. In some embodiments, both the first and second nucleotide sequence encode an Fc domain comprising or consisting of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, both the first and second nucleotide sequence encode an Fc domain comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, both the first and second nucleotide sequence encode an Fc domain comprising or consisting of the amino acid sequence of SEQ ID NO: 10.

[0218] In some embodiments, the first and second nucleotide sequence encode differentFc domains. In some embodiments, the first nucleotide sequence encodes an Fc domain comprising or consisting of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 4-6 and the second nucleotide sequence encodes an Fc domain comprising or consisting of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 7-9. In some embodiments, the first nucleotide sequence encodes an Fc domain comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 4-6 and the second nucleotide sequence encodes an Fc domain comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 7-9. In some embodiments, when the first nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 4 or a variant thereof, the second nucleotide sequence encodes SEQ ID NO: 7 or a variant thereof. In some embodiments, when the first nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 5 or a variant thereof, the second nucleotide sequence encodes SEQ ID NO: 8 or a variant thereof. In some embodiments, when the first nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 6 or a variant thereof, the second nucleotide sequence encodes SEQ ID NO: 9 or a variant thereof.

[0219] In some embodiments, the first and / or second nucleotide sequence also encode anHSA-binding domain. In some embodiments, the first and second nucleotide sequence encode the same HSA-binding domain. In some embodiments, the first and second nucleotide sequence encode different HSA-binding domains. In some embodiments, the first nucleotide sequence encodes an Fc domain and an HSA-binding domain and the second nucleotide sequence encodesan Fc domain but no HSA-binding domain. The HSA-binding domains encoded by the first and / or second nucleotide sequences can be any described herein.

[0220] In some embodiments, the first and / or second nucleotide sequence also encode apeptide linker. In some embodiments, the first and second nucleotide sequence encode the same peptide linker. In some embodiments, the first and second nucleotide sequence encode different peptide linkers. In some embodiments, the first nucleotide sequence encodes an Fc domain, a peptide linker, and an HSA-binding domain and the second nucleotide sequence encodes an Fc domain but no peptide linker or HSA-binding domain. In some embodiments, the first nucleotide sequence encodes an HSA-binding domain, a peptide linker, and an Fc domain and the second nucleotide sequence encodes an Fc domain but no peptide linker or HSA-binding domain. The peptide linkers encoded by the first and / or second nucleotide sequences can be any described herein. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 39, 40, or 41.

[0221] In some embodiments, the polynucleotide comprises a nucleotide sequence thatencodes a protein comprising or consisting of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 42-54.

[0222] In some embodiments, the polynucleotide comprises a nucleotide sequence thatencodes a protein comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 42-54.

[0223] In some embodiments, the first nucleotide sequence encodes a protein comprisingor consisting of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 42-54 and the second nucleotide sequence encodes an Fc domain, but does not encode an antigen-binding domain. Optionally, the second nucleotide sequence encodes a protein comprising or consisting of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8.

[0224] In some embodiments, the first nucleotide sequence encodes a protein comprisingor consisting of the amino acid sequence of any one of SEQ ID NOs: 42-54 and the second nucleotide sequence encodes an Fc domain, but does not encode an antigen-binding domain. Optionally, the second nucleotide sequence encodes a protein comprising or consisting of the amino acid sequence of SEQ ID NO: 8.

[0225] Also provided herein are polynucleotides encoding a polypeptide as provided abovethat are optimized, e.g., by codon / RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA instability elements. Methods to generate optimized nucleic acids for recombinant expression by introducing codon changes and / or eliminating inhibitory regions in the mRNA can be carried out by adapting the optimization methods described in, e.g., U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly, all of which are herein incorporated by reference in their entireties. For example, potential splice sites and instability elements (e.g., A / T or A / U rich elements) within the RNA can be mutated without altering the amino acids encoded by the nucleic acid sequences to increase stability of the RNA for recombinant expression. The alterations utilize the degeneracy of the genetic code, e.g., using an alternative codon for an identical amino acid. In an embodiment, it can be desirable to alter one or more codons to encode a conservative mutation, e.g., a similar amino acid with similar chemical structure and properties and / or function as the original amino acid.

[0226] The polynucleotides can be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. Nucleotide sequences encoding proteins described herein, and modified versions of these antibodies can be determined using methods well known in the art, i.e., nucleotide codons known to encode particular amino acids are assembled in such a way to generate a nucleic acid that encodes the protein. Such a polynucleotide encoding the protein can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et al., (1994) BioTechniques 17: 242-6, herein incorporated by reference in its entirety), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing, and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

[0227] Alternatively, a polynucleotide encoding a protein described herein can begenerated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the polypeptide of interest. Such PCR amplification methods can be used to obtain nucleic acids comprising the sequence encoding the polypeptide. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning.

[0228] If a clone containing a nucleic acid encoding a particular polypeptide is notavailable, but the sequence of the polypeptide is known, a nucleic acid encoding the polypeptide can be chemically synthesized or obtained from a suitable source (e.g., a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from any tissue or cells expressing the polypeptide described herein) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the polypeptide. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[0229] DNA encoding proteins described herein can be readily isolated and sequencedusing conventional procedures. Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS System™ (Lonza)), or myeloma cells that do not otherwise produce the proteins described herein.

[0230] Also provided are polynucleotides that hybridize under high stringency,intermediate or lower stringency hybridization conditions to polynucleotides that encode a protein described herein.

[0231] Hybridization conditions have been described in the art and are known to one ofskill in the art. For example, hybridization under stringent conditions can involve hybridization to filter-bound DNA in 6x sodium chloride / sodium citrate (SSC) at about 45° C followed by one or more washes in 0.2xSSC / 0.1% SDS at about 50-65° C; hybridization under highly stringent conditions can involve hybridization to filter-bound nucleic acid in 6xSSC at about 45° C followed by one or more washes in 0.1xSSC / 0.2% SDS at about 68° C. Hybridization under other stringenthybridization conditions is known to those of skill in the art and has been described, see, e.g.,Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3, which is herein incorporated by reference in its entirety.

[0232] In an aspect, provided herein are cells (e.g., host cells) expressing (e.g.,recombinantly) a protein described herein, and related polynucleotides and expression vectors. Provided herein are vectors (e.g., expression vectors) comprising polynucleotides comprising nucleotide sequences encoding a protein described herein for recombinant expression in host cells,preferably in mammalian cells (e.g., CHO cells). Also provided herein are host cells comprising such vectors for recombinantly expressing proteins described herein. In an aspect, provided herein are methods for producing a protein described herein, comprising expressing the polypeptide from a host cell.

[0233] Recombinant expression of a protein described herein generally involvesconstruction of an expression vector containing a polynucleotide that encodes the polypeptide. Once a polynucleotide encoding a polypeptide described herein has been obtained, the vector for the production of the polypeptide can be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing a polypeptide encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing polypeptide coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors comprising a nucleotide sequence encoding containing a polypeptide described herein, operably linked to a promoter. Such vectors can, for example, include a nucleotide sequence encoding afirst heavy chain of the disclosure (see, e.g., International Publication Nos. WO 86 / 05807 and WO89 / 01036; and U.S. Patent No. 5,122,464, which are herein incorporated by reference in their entireties), and a second heavy chain of the disclosure can be cloned into such a vector for expression of the first heavy chain, the second heavy chain, or both the first and second heavy chains.

[0234] In an embodiment, a vector comprises a polynucleotide encoding an sdAb, Fabfragment, scFv, VHH, VH, VL, heavy chain, and / or light chain of a polypeptide described herein.

[0235] An expression vector can be transferred to a cell (e.g., host cell) by conventionaltechniques and the resulting cells can then be cultured by conventional techniques to produce a polypeptide described herein or a fragment thereof. Thus, provided herein are host cells containing a polynucleotide encoding containing a polypeptide described herein or fragments thereof, or a heavy or light chain thereof, or fragment thereof, or a single chain antibody described herein, operably linked to a promoter for expression of such sequences in the host cell.

[0236] In an embodiment, a host cell comprises a polynucleotide comprising one of thefirst nucleotide sequences and one of the second nucleotide sequences described above. In another embodiment, a host cell comprises a first polynucleotide comprising one of the first nucleotidesequences described above, and a second polynucleotide comprising one of the first nucleotide sequences described above. In another embodiment, a host cell comprises a first vector comprising one of the first nucleotide sequences and one of the second nucleotide sequences described above. In another embodiment, a host cell comprises a first vector comprising one of the first nucleotide sequences and one of the second nucleotide sequences described above, and a second vector comprising a second polynucleotide comprising one of the first nucleotide sequences described above.

[0237] In some embodiments, an FcRn / HSA-binding molecule expressed by a first hostcell is associated with an FcRn / HSA-binding molecule expressed by a second host cell to form a two-armed FcRn / HSA-binding molecule. In some embodiments, an FcRn / HSA-binding molecule expressed by a first host cell is associated with an FcRn-binding molecule expressed by a second host cell to form a one-armed FcRn / HSA-binding molecule. In some embodiments, provided herein are populations of host cells comprising such first host cells and such second host cells.

[0238] In some embodiments, provided herein is a population of vectors comprising a firstvector comprising a polynucleotide encoding an FcRn / HSA-binding molecule, and a second vector comprising a polynucleotide encoding an FcRn / HSA-binding molecule. In some embodiments, provided herein is a population of vectors comprising a first vector comprising a polynucleotide encoding an FcRn / HSA-binding molecule, and a second vector comprising a polynucleotide encoding an FcRn-binding molecule. In some embodiments, provided herein is a population of vectors comprising a first vector comprising a polynucleotide encoding an FcRn / HSA-binding molecule and a polynucleotide encoding an FcRn / HSA-binding molecule. In some embodiments, provided herein is a population of vectors comprising a first vector comprising a polynucleotide encoding two FcRn / HSA-binding molecules.

[0239] A variety of host-expression vector systems can be utilized to express polypeptidesdescribed herein (see, e.g., U.S. Patent No. 5,807,715, which is herein incorporated by referencein its entirety). Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express a polypeptide described herein in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with, e.g., recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing FcRn / HSA-binding molecule coding sequences; yeast (e.g., Saccharomyces and Pichia) transformed with, e.g., recombinant yeastexpression vectors containing FcRn / HSA binding molecule coding sequences; insect cell systems infected with, e.g., recombinant virus expression vectors (e.g., baculovirus) containing FcRn / HSA-binding molecule coding sequences; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with, e.g., recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with, e.g., recombinant plasmid expression vectors (e.g., Ti plasmid) containing FcRn / HSA-binding molecule coding sequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, NIH 3T3, HEK- 293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB / 20, and BMT10 cells) harboring, e.g., recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In an embodiment, cells for expressing FcRn / HSA- binding molecules described herein are Chinese hamster ovary (CHO) cells, for example CHO cells from the CHO GS System™ (Lonza). In an embodiment, the heavy chain and / or light chain produced by a CHO cell may have an N-terminal glutamine or glutamate residue replaced by pyroglutamate. In an embodiment, cells for expressing polypeptides described herein are human cells, e.g., human cell lines. In an embodiment, a mammalian expression vector is pOptiVEC™ or pcDNA3.3. In an embodiment, bacterial cells such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells), are used for the expression of a recombinant polypeptide. For example, mammalian cells such as CHO cells, in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus, are an effective expression system for antibodies (Foecking MK & Hofstetter H (1986) Gene 45: 101-5; and Cockett MI et al., (1990) Biotechnology 8(7): 662-7, each of which is herein incorporated by reference in its entirety). In an embodiment, polypeptides described herein are produced by CHO cells or NS0 cells. In an embodiment, the expression of nucleotide sequences encoding polypeptides described herein which comprise two, three, or four binding sites for human FcRn is regulated by a constitutive promoter, inducible promoter, or tissue specific promoter.

[0240] In bacterial systems, a number of expression vectors can be advantageouslyselected depending upon the use intended for the molecule being expressed. For example, when a large quantity of such a polypeptide is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified can be desirable. Such vectors include, but are not limitedto, the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791- 1794), in which the coding sequence can be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye S & Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24: 5503-5509); and the like, all of which are herein incorporated by reference in their entireties. For example, pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0241] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV),for example, can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

[0242] In mammalian host cells, a number of viral-based expression systems can beutilized. In cases where an adenovirus is used as an expression vector, the coding sequence of interest can be ligated to an adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressingthe molecule in infected hosts (see, e.g., Logan J & Shenk T (1984) PNAS 81(12): 3655-9, whichis herein incorporated by reference in its entirety). Specific initiation signals can also be required for efficient translation of inserted coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (see, e.g., Bitter G et al., (1987)Methods Enzymol.153: 516-544, which is herein incorporated by reference in its entirety).

[0243] In addition, a host cell strain can be chosen which modulates the expression of theinserted sequences or modifies and processes the gene product in the specific fashion desired. Suchmodifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells. In an embodiment, proteins described herein are produced in mammalian cells, such as CHO cells.

[0244] In an embodiment, a polypeptide described herein comprises a portion of anantibody with reduced fucose content or no fucose content. Such proteins can be produced using techniques known to one skilled in the art. For example, the proteins can be expressed in cells deficient in or lacking the ability to fucosylate. In an example, cell lines with a knockout of both alleles of α1,6-fucosyltransferase can be used to produce antibodies with reduced fucose content. The Potelligent® system (Lonza) is an example of such a system that can be used to produce antibodies with reduced fucose content.

[0245] For long-term, high-yield production of recombinant proteins, stable expressioncells can be generated. For example, cell lines which stably express a protein described herein can be engineered. In an embodiment, a cell provided herein stably expresses an HSA-binding domain, an FcRn / HSA-binding molecule, or an FcRn-binding molecule which associate to form a one- armed or two-armed polypeptide described herein.

[0246] In certain aspects, rather than using expression vectors which contain viral originsof replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA / polynucleotide, engineered cells can be allowed to grow for one to two days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and growto form foci, which in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express a polypeptide comprising two, three, or four binding sites for human FcRn described herein or a fragment thereof. Such engineered cell lines can be particularly useful in the screening and evaluation of compositions that interact directly or indirectly with the polypeptide.

[0247] A number of selection systems can be used, including but not limited to the herpessimplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1): 223-32), hypoxanthineguanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12): 2026-2034) and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3): 817-23) genes in tk-, hgprt- or aprt-cells, respectively, all of which are herein incorporated by reference in their entireties. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler M et al., (1980) PNAS 77(6): 3567-70; O’Hare K et al., (1981) PNAS 78: 1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo, which confers resistance to the aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan RC (1993) Science 260: 926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62: 191-217; Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5): 211-5); and hygro, which confers resistance to hygromycin (Santerre RF et al., (1984) Gene 30(1-3): 147- 56), all of which are herein incorporated by reference in their entireties. Methods commonly known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clone and such methods are described, for example, in Ausubel FM et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli NC et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbère-Garapin F et al., (1981) J Mol Biol 150: 1-14, all of which are herein incorporated by reference in their entireties.

[0248] The expression levels of a polypeptide can be increased by vector amplification (fora review, see, Bebbington CR & Hentschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, p. 163-188. In DNA Cloning, Vol III, A Practical Approach. D. M. Glover (Ed.) (Academic Press, New York, 1987), which is herein incorporated by reference in its entirety). When a marker in the vector system is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the numberof copies of the marker gene. Since the amplified region is associated with the gene of interest, production of the polypeptide will also increase (Crouse GF et al., (1983) Mol Cell Biol 3: 257- 66, which is herein incorporated by reference in its entirety).

[0249] The host cell can be co-transfected with two or more expression vectors describedherein. The two vectors can contain identical selectable markers which enable equal expression of polypeptides, such as a first heavy chain and a second heavy chain polypeptide. The host cells can be co-transfected with different amounts of the two or more expression vectors. For example, host cells can be transfected with any one of the following ratios of a first expression vector and a second expression vector: about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

[0250] Alternatively, a single vector can be used which encodes, and is capable ofexpressing, both polypeptides. The coding sequences can comprise cDNA or genomic DNA. The expression vector can be monocistronic or multicistronic. A multicistronic nucleic acid construct can encode 2, 3, 4, 5, 6, 7, 8, 9, 10, or more genes / nucleotide sequences, or in the range of 2-5, 5- 10, or 10-20 genes / nucleotide sequences. For example, a bicistronic nucleic acid construct can comprise, in the following order, a promoter, a first gene and a second gene. In such an expression vector, the transcription of both genes can be driven by the promoter, whereas the translation of the mRNA from the first gene can be by a cap-dependent scanning mechanism, and the translation of the mRNA from the second gene can be by a cap-independent mechanism, e.g., by an IRES.

[0251] Once a polypeptide described herein has been produced by recombinant expression,it can be purified by any method known in the art for purification of a protein, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the polypeptides described herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0252] In an embodiment, a polypeptide described herein is isolated or purified. In anembodiment, an isolated polypeptide is one that is substantially free of other polypeptides with different antigenic specificities than the isolated polypeptide. For example, in certain embodiments, a preparation of a protein described herein is substantially free of cellular material and / or chemical precursors. The language “substantially free of cellular material” includes preparations of a polypeptide in which the polypeptide is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. Thus, a polypeptide that is substantially free of cellular material includes preparations of polypeptide having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous protein (also referred to herein as a “contaminating protein”) and / or variants of a polypeptide, for example, different post-translational modified forms of a polypeptide or other different versions of a polypeptide (e.g., polypeptide fragments). When the polypeptide is recombinantly produced, it is also generally substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When the polypeptide is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals, which are involved in the synthesis of the protein. Accordingly, such preparations of the protein have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the molecule of interest. In an embodiment, polypeptides described herein are isolated or purified.

[0253] A polypeptide described herein can be produced by any method known in the artfor the synthesis of proteins, for example, by chemical synthesis or by recombinant expression techniques. The methods described herein employ, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described, forexample, in the references cited herein and are fully explained in the literature. See, e.g., ManiatisT et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates); Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press, all of which are herein incorporated by reference in their entireties.

[0254] In an embodiment, a polypeptide described herein is prepared, expressed, created,or isolated by any means that involves creation, e.g., via synthesis, genetic engineering of DNAsequences. In an embodiment, such a polypeptide comprises sequences (e.g., DNA sequences or amino acid sequences) that do not naturally exist within the antibody germline repertoire of an animal or mammal (e.g., human) in vivo. Pharmaceutical Compositions

[0255] In an aspect, the instant disclosure provides pharmaceutical compositionscomprising an FcRn / HSA-binding molecule as disclosed herein for use in methods of treating an antibody-mediated disorder (e.g., an autoantibody-mediated disorder). In certain embodiments, these compositions comprise an FcRn / HSA-binding molecule comprising an FcRn-binding molecule and an HSA-binding domain. In some embodiments, the FcRn-binding molecule is an FcRn antagonist. In some embodiments, the FcRn antagonist comprises or consists of a variant Fc region, or FcRn-binding fragment thereof that inhibits the binding of an Fc region of immunoglobulin to FcRn. In general, these FcRn antagonists inhibit the binding of Fc-containing agents (e.g., antibodies and immunoadhesins) to FcRn in vivo, which results in an increased rate of degradation of the Fc-containing agents and, concomitantly, a reduced serum level of these agents.

[0256] The formulations disclosed herein include bulk drug compositions useful in themanufacture of pharmaceutical compositions (e.g., compositions that are suitable for administration to a subject or patient) which can be used in the preparation of unit dosage forms. In an embodiment, a composition of the invention is a pharmaceutical composition. Such compositions comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic agents (e.g., an FcRn / HSA-binding molecule) of the invention (or other prophylactic or therapeutic agent), and a pharmaceutically acceptable carrier.

[0257] In some embodiments the pharmaceutical compositions are formulated foradministration to a subject via any suitable route of administration including, but not limited to, intramuscular, intravenous, intradermal, intraperitoneal, subcutaneous, epidural, nasal, oral, rectal, topical, inhalation, buccal (e.g., sublingual), and transdermal administration. In an embodiment, the pharmaceutical compositions are formulated to be suitable for intravenous administration to a subject. In an embodiment, the pharmaceutical compositions are formulated to be suitable for subcutaneous administration to a subject.Methods of Treatment

[0258] The disclosure also provides methods for treating an antibody-mediated disorder(e.g., an autoantibody-mediated disorder) in a subject comprising administering to the subject a therapeutically effective amount of an FcRn / HSA-binding molecule according to the disclosure or a pharmaceutical composition comprising the same. In some embodiments, the antibody-mediated disorder is an IgG-mediated disorder. In some embodiments, the antibody-mediated disorder is an autoimmune disease.

[0259] In an embodiment, the FcRn / HSA-binding molecule antagonizes FcRn binding toan antibody Fc region. In an embodiment, the FcRn / HSA-binding molecule does not antagonize FcRn binding to albumin.

[0260] The disclosure provides methods of reducing serum IgG in a subject comprisingadministering to the subject a therapeutically effective amount of an FcRn / HSA-binding molecule according to the disclosure or a pharmaceutical composition comprising the same. In an embodiment, at least one of the IgG subtypes is reduced in a subject following administration of the FcRn / HSA-binding molecule. In some embodiments, IgG1, IgG2, IgG3, IgG4, or any combination thereof is reduced. In some embodiments, the administration of the FcRn / HSA- binding molecule is a single administration (e.g., a single therapeutic administration) of the FcRn / HSA-binding molecule. In an embodiment, the level of serum IgG is decreased in the subject following administration of the FcRn / HSA-binding molecule compared to a baseline level of serum IgG.

[0261] In an embodiment, a total serum IgG reduction of at least about 40% compared tobaseline serum IgG level is obtained. In an embodiment, a total serum IgG reduction of at least about 45% compared to baseline serum IgG level is obtained. In an embodiment, a total serum IgG reduction of at least about 50% compared to baseline serum IgG level is obtained. In an embodiment, a total serum IgG reduction of at least about 55% compared to baseline serum IgG level is obtained. In an embodiment, a total serum IgG reduction of at least about 60% compared to baseline serum IgG level is obtained. In an embodiment, a total serum IgG reduction of at least about 65%, about 70%, about 75%, or about 80% compared to baseline serum IgG level is obtained. In an embodiment, a total serum IgG reduction of at least about 65% compared to baseline serum IgG level is obtained. In an embodiment, a total serum IgG reduction of at least about 70% compared to baseline serum IgG level is obtained. In an embodiment, a total serum IgG reduction of at least about 75% compared to baseline serum IgG level is obtained. In anembodiment, a total serum IgG reduction of at least about 80% compared to baseline serum IgG level is obtained.

[0262] In an embodiment, the level of serum IgG is decreased in the subject followingadministration of the FcRn / HSA-binding molecule compared to a baseline level of serum IgG. In an embodiment, a total serum IgG reduction of about 40% compared to baseline serum IgG level is obtained. In an embodiment, a total serum IgG reduction of about 45% compared to baseline serum IgG level is obtained. In an embodiment, a total serum IgG reduction of at about 50% compared to baseline serum IgG level is obtained. In an embodiment, a total serum IgG reduction of about 55% compared to baseline serum IgG level is obtained. In an embodiment, a total serum IgG reduction of about 60% compared to baseline serum IgG level is obtained. In an embodiment, a total serum IgG reduction of about 65%, about 70%, about 75%, or about 80% compared to baseline serum IgG level is obtained. In an embodiment, a total serum IgG reduction of about 65% compared to baseline serum IgG level is obtained. In an embodiment, a total serum IgG reduction of about 70% compared to baseline serum IgG level is obtained. In an embodiment, a total serum IgG reduction of about 75% compared to baseline serum IgG level is obtained. In an embodiment, a total serum IgG reduction of about 80% compared to baseline serum IgG level is obtained.

[0263] In an embodiment, the level of FcRn is not decreased in the subject followingadministration of the FcRn / HSA-binding molecule compared to a baseline level of FcRn. In an embodiment, an FcRn reduction of less than about 1%, 2%, 3%, 4%, or 5% compared to baseline FcRn level is observed. In an embodiment, an FcRn reduction of less than about 10% compared to baseline FcRn level is observed.

[0264] In an embodiment, the level of albumin is not decreased in the subject followingadministration of the FcRn / HSA-binding molecule compared to a baseline level of albumin. In an embodiment, an albumin reduction of less than about 1%, 2%, 3%, 4%, or 5% compared to baseline albumin level is observed. In an embodiment, an albumin reduction of less than about 10% compared to baseline albumin level is observed.

[0265] In an embodiment, the total IgG, FcRn / HSA-binding molecule, FcRn, or albuminin a serum sample of the patient is analyzed using a bioanalytical method. In an embodiment, the total IgG, FcRn / HSA-binding molecule, FcRn, or albumin in a serum sample of the patient is analyzed using ELISA or automated diagnostic analyzer (IVD). In an embodiment, the total IgG, FcRn / HSA-binding molecule, FcRn, or albumin in a serum sample of the patient is analyzed using ELISA. In an embodiment, the total IgG, FcRn / HSA-binding molecule, FcRn, or albumin in aserum sample of the patient is analyzed using automated diagnostic analyzer (IVD). In an embodiment, the total FcRn in a blood sample of the patient is analyzed using a bioanalytical method, preferably flow cytometry, microscopy, or an immunoblot.

[0266] In some embodiments, the reduction of total serum IgG is measured by area underthe percentage of reduction curve (AUEC). In some embodiments, the reduction of total serum IgG is measured by clearance of total serum IgG (CL).

[0267] In some embodiments, the FcRn / HSA-binding molecule is administeredintravenously or subcutaneously. EXAMPLES

[0268] The following examples are offered by way of illustration, and not by way oflimitation. Example 1: Identification of Alb23 VHH variants with reduced affinity to albumin

[0269] The MHC class I-related receptor, FcRn, plays a central role in regulating the serumlevels of IgG (Ghetie et al., (1996) Immunology Today 18(12): 592-8) and albumin (Chaudhury et al., (2003) Journal of Experimental Medicine 197(3): 315-22) and is ubiquitously expressed e.g., in endothelial, epithelial, and hematopoietic such as monocytes, macrophages, dendritic cells, and B cells. The Fc portion of IgG binds with high affinity to FcRn at an acidic pH (<6.5) but not at a physiological pH (7.4) (Rodewald R., (1976) Journal of Cell Biology 71(2): 666-9). A mutated, human IgG1-derived antibody (MST-HN) binds with higher affinity and reduced pH dependence to FcRn and competes effectively with wild-type IgGs for FcRn-mediated transport resulting in a rapid decrease of IgG levels in mice (Vaccaro et al., (2005) Nature Biotechnology 23(10): 1283- 8). In humans, such FcRn blockers (or “ABDEGs,” for antibodies that enhance IgG degradation) may be desirable in multiple therapeutic situations, e.g., clearance of autoreactive antibodies in autoimmune diseases such as systemic lupus erythematosus, myasthenia gravis, and immune thrombocytopenic purpura (ITP) or other antibody-mediated diseases.

[0270] Efgartigimod is a human IgG1 Fc-fragment that utilizes the ABDEG Fc engineeringtechnology. Its presumed in vivo mechanism of action is the constitutive blockage of FcRn- mediated IgG recycling leading to IgG degradation. The effectiveness of efgartigimod depends in large part on its pharmacokinetic properties. For this reason, it is desired to explore methods tofurther improve the half-life of the efgartigimod molecule which could allow the use of lower dose and / or less frequent administrations. An effective means of improving the pharmacokinetic properties is by binding to long-lived plasma proteins. Albumin is the most abundant protein in plasma, has a half-life of 19 days in humans, and could represent an optimal carrier for therapeutic peptides / proteins.

[0271] Based on previous data demonstrating that VHHs with low affinity for albuminhave favorable PD / PK profiles when fused to Fc-ABDEG, Alb23 variants with alanine substitutions in two or three amino acid positions within the CDRs were developed to identify variants with reduced affinity for albumin. Alb23 VHH variant sequences are provided below in Tables S1 and S2.)883Q.7 D4 712(1O W1032T-373404C D L S L S D D D D D D D S S S S S S S D D D D D S S S S S WS S WG WS G S S A stnairsG QG L G Q aecG L P Y G Y P L G L Y vn3eQL 2 S S S S S S S S S S S S S T QT P QL u V V N V T b 1 l R M M M M A M M AM M M M MqeL N L K s G K G S L N G K A D G G G G A G foC F AG G G G G A S N S S A S A S A F F F F F F F F G G G S S A S S S S S S S S H G N G D G N s H S D S R S D ecVtE R LSESLIT E R Snen aiH LIL T L F LIF R T u qeSlAAa0A2A1A1raH Q Q QF t95010 01A A 01vV V R E G V G R E K V E G Rtn L / 1S A AA4A0A1 232D nerAG / L / 3 / A2333395010101S / AblAC ai a:1rap 233A 2 A 222F3F3bl33F3 G M L3 G S L 3FAG3AA00 / 23232 2323232G :2l 9a5 1S Vbl323 3Ablblblblblblbl3 Se3 t 3 L / G / elAbl 2b2b A l43A A A A A A2beln2 ner 2A32A baAl lb olblbl23bl23T A A M AaaT C ApA F A F)883412(152OW K10V3S2DT- A37Y L34T04DS GS G SISS V W EP G K GP A Q R V WS M GAS C FS C AS C FS C FS C FS C F C F C F C F C27Y Y Y Y Y Y S Y S Y S Y S R Y RF Y RF Y RF Y R Y F F Y RF Y RF Y RF Y RF Y RF Y TF V TF V TF V TF V TF V T V T V T V T V T V A F F F F F G G A G A G A A A A A A A S T S T S T S T GS T GS T GS T GS T GS T GS T A DE A DE A DE A DE A DE A D A D A D A D A D A P A P A P A P A P A EP A EP A EP A EP A EP CS R CS R CS R CS R CS R C R C R C R C R C R L LS L LS L LS L LS L L S S L L S S L L S S L L S S L L S S L LS RL N RL N RL N RL N RL N RL N RL N RL N R N R N S M M M M M M M M L M L M QS Q S QS S S S S S S G G G G QG QG Q Q Q Q Q L L L L L L G L G L G L G GP G G G G L YL P Y G G G G G L P YL P YL P YL P YL P YL P Y P Y P Y Q Q Q Q Q Q Q QL QL L T T T T T Q V V T T T T T L N L N VL N VL N VL N VL N VL N V N V N V N G KS G KS G KS G K G K G K G K L G K L G K L G K G G G G S G S S S S S S N N N N N G N G N G N G G G G G G G G G G G N G N S D S D S D D D D D D D D R R R S R S R S S S S S E E E E E E R E R E R R R L LSILST LILST LILST LILSLSLSLSELSELST LIT LIT LIT LIT LIT LIF F F F F T Q F F F F F R QR QR QR Q Q Q Q Q Q V V V V V R G V R R R R R E G E G E G E G E E G V E G V E G V E G V E G / A2A A A A AA0A01A2A11332 3 439 0 0 0 0G3 / 3FG M5 1LG1S1L13L / AA S / 2A313 3 3 3 3 3 3 3A b2l23bl43021b2lb2lb2lb2lb2lb2lb2lbl33A F A M S A A A A A A A A G

[0272] One Alb23 VHH variant was fused at the C-terminus of an Fc-ABDEG via a 20GSlinker to generate the one-armed (OA) Fc-ABDEG molecules described herein except where specified. The generated OA-Fc-ABDEG-Alb23-based molecules were tested for their albumin binding at pH 5.5 and pH 7.4. Human, cynomolgus monkey, and mouse serum albumin proteins were immobilized on CM5 chips and the OA-Fc-ABDEG-Alb23-based molecules were injected in-solution in a five step 2-fold dilution series (62.5 nM, 125 nM, 250 nM, 500 nM, and 1000 nM) in either 1XHBS-EP+ buffer (pH 7.4) or McIlvaine buffer (pH 5.5) for 2 minutes at 30 µL / min. Buffer only and OA-Fc-ABDEG-Alb23 were included as controls. Data are presented below in Table S3. Table S3: Binding of OA-Fc-ABDEG-Alb23 variants to human serum albumin, cynomolgus serum albumin, and mouse serum albumin at pH 7.4 and pH 5.5 Human SA Cyno SA Mouse SA pH74 pH55 pH74 pH55 pH74 pH55 x

[0273] All double and triple mutant constructs showed lower RUmax values than theirrespective single mutant counterparts. In general, lower binding RUmax values were observed for mouse serum albumin (MSA) relative to human serum albumin (HSA) and cynomolgus serum albumin (CSA).

[0274] The F32A Alb23 VHH was fused to the C-terminus of an Fc-ABDEG via a 30GSlinker with a single alanine added to the C-terminus of the VHH (referred to interchangeably herein as “OA-Fc-ABDEG-30GS-Alb23-F32A-A” or “OA-Fc-ABDEG-30GS-Alb23-SM”). The M34A / G33A / S101A Alb 23 VHH was fused to the C-terminus of an Fc-ABDEG via a 20GS linker with a single alanine added to the C-terminus of the VHH (referred to interchangeably herein as “OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A-A” or “OA-Fc-ABDEG-20GS-Alb23- TM”). Binding affinities of these constructs to HSA, MSA, rat serum albumin (RSA), and bovine serum albumin (BSA) were compared to OA-Fc-ABDEG-20GS-Alb23. Kinetic parameters and / or affinity values were determined using the Langmuir 1:1 binding model or steady state model of OA-Fc-ABDEG-Alb23 variants binding to serum albumin at pH 7.4 and 5.5. Results are shown in Table S4.Table S4: Albumin binding affinity of OA-Fc-ABDEG-Alb23 variants at pH 7.4 and pH 5.5 Name CaptureAnalyte pH KD KD Rmax Conc. Conc. (µM) pH (RU) estconcentration tested (60 µM) N / A = Not applicableExample 2: FcRn degradation induced by OA-Fc-ABDEG-Alb23 variants

[0275] Previous studies have shown that anti-FcRn antibodies decrease serum albuminwhen administered to patients. It is believed that this effect is mediated, at least in part, by degradation of FcRn induced by these anti-FcRn antibodies.

[0276] Thus, the effect of OA-Fc-ABDEG-Alb23 variants on FcRn degradation wasexplored using a cell-based assay in the presence or absence of albumin. Briefly, HEK FcRn WT GFP+ cells / well were seeded on a 96-well microplate overnight at 37°C in growth medium (DMEM + 10% FBS + P / S + L-glutamine). OA-Fc-ABDEG-Alb23 variant molecules (12,500 nM) were pre-incubated with 50,000 nM human serum albumin (HSA) in a 1:4 ratio in treatment medium (DMEM + 1% BSA + P / S + L-glutamine) for 30 minutes at 37°C, 5% CO2prior to adding to the cells. After incubation of the OA-Fc-ABDEG-Alb23 variant / HSA mix or OA-Fc-ABDEG- Alb23 variant with the cells, plates were then placed on ice and cells harvested by trypsin. Harvested cells were transferred to FACS plate and centrifuged. LD stain (1:800) was added in FACS buffer and incubated for 15 minutes at 4°C. Cells were washed, centrifuged, and resuspended in FACS buffer. GFP signal was measured and compared to untreated controls.

[0277] As shown in FIG. 1, OA-Fc-ABDEG-Alb23 molecules containing F32A / G100A,F32A / L102A, F32A / L59A, and M34A / G33A / S101A mutations did not induce FcRn degradation in the absence or presence of HSA, whereas the parental OA-Fc-ABDEG-Alb23 molecule did induce FcRn degradation in the presence of HSA. Not all mutations had an impact on FcRn degradation. Of note, OA-Fc-ABDEG-Alb23 molecules containing an M34A mutation in combination with an L59A, G100A, or L102A mutation (M34A / L59A, M34A / G100A, M34A / L102A) were indistinguishable from OA-Fc-ABDEG-Alb23 (data not shown), although OA-Fc-ABDEG-Alb23 molecules containing an F32A mutation in combination with an L59A, G100A, or L102A mutation (F32A / L59A, F32A / G100A, F32A / L102A) did not induce FcRn degradation, as described above and shown in FIG.1. Example 3: Pharmacokinetics / Pharmacodynamics of OA-Fc-ABDEG-Alb23 variants in AlbuMus Rag1KO mice

[0278] A comparison of PK and PD of OA-Fc-ABDEG-Alb23 variant molecules to OA-Fc-ABDEG-Alb23 in the AlbuMus Rag1KO mouse model was conducted as described below.

[0279] Methods

[0280] Briefly, a total of 30 AlbuMus Rag1KO mice were randomly assigned into 6groups. AlbuMus Rag1KO mice are double-humanized for serum albumin / neonatal Fc receptor, Rag1 knock-out mouse model (hFcRn+ / +, hAlb+ / +, Rag1- / -). Fcgrt and hAlb are knocked-in and expressed under the endogenous mouse promoter. Knock out of Rag1 produces immunodeficiency such that the mice do not develop ADA. Mice were single-dosed IP with human IgG (tracer IgG) prior to administration of test items. On day 0, mice were injected IV according to the designated group and doses in Table S5.

[0281] All animals were pre-weighed before dosing and dosed according to their bodyweights. Blood samples were collected before dosing of the test article (pre-dose, D0-2h) and aftertreatment for PD, PK, and albumin read-outs for 7 days (see Table S5) (for all timepoints). Bloodsamples were processed to serum and added to a 96-well plate (polypropylene) per group. Table S5: Groups and dosing regimen Group N Test Article Dose, Administration k t Blood sampling times 2, 2, 2, 2, 2, 2,

[0282] Tracer IgG serum levels were determined using a sandwich ELISA. A NuncMaxiSorp F-bottom plate (Thermo Fisher Scientific, Cat. 44-24004-21) was coated overnight (4°C) with a specific antigen for the tracer IgG and non-specific binding sites were blocked with 1% casein-PBS (Bio-Rad, Cat. #1610783). Study serum samples were diluted 1 / 100 and 1 / 4000 (depending on time post-injection) and incubated on the immunoplate together with a fresh 11-point hIgG1 (Evitria ##32101.1 – SEC) calibration curve and 3 quality control (QC) samples (HQC, MQC, LQC) for 1 hour. IgG tracer was bound and detected for 1 hour by a goat anti-hIgG, Fc-specific (abcam #98595). The plate was developed by adding 3,3',5,5'-tetramethylbenzidine (TMB) substrate for approximately 7 minutes. The enzymatic reaction was stopped with sulfuric acid (H2SO4) and optical density values at 450 nm were recorded using a Tecan plate reader. For data analysis, the obtained values were back-calculated on a 11-point calibrator curve in GraphPad Prism (log(agonist) vs. response -- Variable slope (four parameters), Least squares fit). Tracer IgG concentrations were plotted in absolute values measured (µg / mL) and percentage to pre-dose (2h prior to test article injection, D0-2h).

[0283] Total human IgG serum levels were determined using a sandwich ELISA. A NuncMaxiSorp F-bottom plate (Thermo Fisher Scientific, Cat. 44-24004-21) was coated overnight (4°C) with mouse anti-human lambda light chain (Sigma-Aldrich L6522, clone HP-6054) and non- specific binding sites are blocked with 1% casein-PBS (Bio-Rad, Cat. #1610783). Study serum samples were diluted 1 / 5000 and 1 / 1000 (depending on time post-injection) and incubated on the immunoplate together with a fresh 11-point hIgG (IVIg, CSL Behring (Privigen Lot. P100071321)) calibration curve and 3 quality control (QC) samples (HQC, MQC, LQC) for 1 hour. hIgG were bound and detected for 1 hour by a goat anti-human IgG, Fc-specific (abcam #98595). The plate was developed by adding 3,3',5,5'-tetramethylbenzidine (TMB) substrate for approximately 9 minutes. The enzymatic reaction was stopped with sulfuric acid (H2SO4) and optical density values at 450 nm were recorded using a Tecan plate reader. For data analysis, the obtained values were back-calculated on a 11-point calibrator curve in GraphPad Prism (log(agonist) vs. response -- Variable slope (four parameters), Least squares fit). hIgG were plotted in absolute values measured (µg / mL) and percentage to pre-dose (2h prior to test article injection, D0-2h).

[0284] Concentrations of OA-Fc-ABDEG-based molecules, comprising OA-Fc-ABDEG-3Rab (control), OA-Fc-ABDEG-Alb23 (WT), and OA-Fc-ABDEG-Alb23 variants: OA-Fc- ABDEG-Alb23-F32A / G100A, OA-Fc-ABDEG-Alb23-F32A / L102A, OA-Fc-ABDEG-Alb23- F32A / L59A, and OA-Fc-ABDEG-Alb23-M34A / G33A / S101A were determined using a sandwich ELISA method. Briefly, Nunc MaxiSorp F-bottom plates (Thermo Fisher Scientific, Cat. 44- 24004-21) were coated overnight (4°C) with neutravidin (Thermo Fisher Scientific, Lot VI312512) and nonspecific binding sites were blocked with 1% casein-PBS (G Biosciences, Part#097B, Lot# 210104). anti-ABDEG-biotin (10x molar excess) was captured for 1 hour. Next,100% study serum samples were diluted to the concentration range of quantitation or at least the minimum required dilution (MRD). The calibration curve and quality control (QC) samples (HQC, MQC and LQC) with OA-Fc-ABDEG-based molecules were spiked in 100% AlbuMus serum and pre-incubated for 30 min at room temperature before applying MRD 100. Samples were incubated on the immunoplate together with a fresh calibration curve and two sets of QC samples (HQC, MQC and LQC) for 2 hours. OA-Fc-ABDEG-based molecules were detected by the subsequent addition of goat anti-Human IgG - Fc (HRP) (Abcam #ab98595, Lot GR3345397-1) for 1 hour. Plates were developed by adding TMB substrate for approximately 20 minutes. The enzymatic reaction was stopped with 0.5M H2SO4 and optical density values at 450 nm were recorded using a Tecan plate reader. For data analysis, the obtained values were back-calculated on a 11-point calibrator curve in GraphPad Prism (Nonlinear regression; Asymmetric Sigmoidal, 5PL, X is concentration). Concentrations were plotted in absolute values measured (µg / mL) for each tested molecule.

[0285] Albumin levels in serum samples were assessed by using a sandwich ELISA.Ninety-six-well ELISA plates were coated with 1.0 µg / mL of Goat anti-Human Albumin (Sigma, A1151) and incubated O / N at 4 °C. Non-specific binding sites were blocked with 1xPBS with 1% (w / v) casein for 1h at room temperature. Study serum samples were diluted 1 / 1,000,000 and incubated on the immunoplate together with a fresh 11-point HSA (Sigma-A3782) calibration curve and 3 quality control (QC) samples (HQC, MQC, LQC) for 1 hour. Bound HSA was detected using an HRP-conjugated polyclonal goat anti-HSA antibody (Bethyl, A80-129P). ELISAs were developed by adding 100 µL of TMB substrate and the enzymatic reaction was stopped with H2SO4. Optical density values at 450 nm were recorded using a Tecan plate reader. For data analysis, the obtained values were back-calculated on an 11-point calibrator curve in GraphPad Prism (log(agonist) vs. response -- Variable slope (four parameters), Least squares fit). Albumin concentrations were plotted in absolute values measured (µg / mL) and percentage to pre-dose (2h prior to test article injection, D0-2h).

[0286] Results

[0287] To assess the efficacy of the tested OA-Fc-ABDEG-based molecules to depleteIgG, the PD effect over time was measured. The measured tracer IgG concentrations (FIG. 2A) and total hIgG (FIG.2B) were plotted as percentage to pre-dose at 2h prior to test article injection (D0-2h) per treatment group. A clear PD effect was observed for all test articles starting at day 1.

[0288] To evaluate the PK profile of the test items after a single IV administration, theirlevels in mouse serum were determined post-dose according to the bleeding scheme in Table S5. The obtained values were plotted in µg / mL (FIG.2C). Values below the low limit of quantification (LLOQ) were excluded from FIG.2C. OA-Fc-ABDEG-Alb23 showed the best PK profile among the test items. No clear differences were observed in PK profile between the OA-Fc-ABDEG- Alb23 variants (OA-Fc-ABDEG-Alb23-F32A / G100A, OA-Fc-ABDEG-Alb23-F32A / L102A, OA-Fc-ABDEG-Alb23-F32A / L59A, and OA-Fc-ABDEG-Alb23-M34A / G33A / S101A). The control molecule, OA-Fc-ABDEG-3Rab, containing an irrelevant VHH which binds to rabies virus, showed the worst PK profile. Overall, no clear correlation was found between measured PD effect and serum half-life. For all tested molecules, concentrations were quantifiable in the serum during the entire study (7 days).

[0289] To evaluate a potential impact of OA-Fc-ABDEG-based molecules on albuminlevels after the administration to AlbuMus Rag1KO mice, total human serum albumin levels were measured throughout the study at baseline (pre-dose) and post-dose according to the scheme in Table S5. Measured albumin concentrations were plotted as percentage to pre-dose (day 0, -2h) prior test item administration (FIG. 2D). No meaningful decreases in albumin levels were observed in mice treated with OA-Fc-ABDEG-3Rab, OA-Fc-ABDEG-Alb23, OA-Fc-ABDEG- Alb23-F23A / G100A, OA-Fc-ABDEG-Alb23-F32A / L102A, and OA-Fc-ABDEG-Alb23- F32A / L59A during the course of the study. A transient albumin decrease was observed in mice treated with OA-Fc-ABDEG-Alb23-M34A / G33A / S101A. Albumin levels returned to baseline by day 4 in these mice.

[0290] A further study was conducted to evaluate these OA-Fc-ABDEG-based moleculesin comparison to efgartigimod.

[0291] Methods

[0292] Briefly, a total of 40 AlbuMus Rag1KO mice were randomly assigned into 8groups. Mice were dosed IP with human IgG (tracer IgG) prior to administration of test items and again on day 7 and day 14 after administration of the test items. On day 0, mice were injected IP according to the designated group and doses in Table S6.

[0293] All animals were pre-weighed before dosing and dosed according to their bodyweights.25 mg / kg dose for OA-Fc-ABDEG-based molecules and 20 mg / kg dose for efgartigimod were selected based on MW of the test items to have equimolar doses thereof. Blood samples were collected before dosing of the test article (pre-dose, D0-2h) and after treatment for PD, PK, andalbumin read-outs for 21 days (see Table S6) (for all timepoints). Blood samples collected on day7 and day 14 were collected 1 hour prior to and 1 hour after administration of tracer IgG. Blood samples were processed to serum and added to a 96-well plate (polypropylene) per group. Table S6: Groups and dosing regimen Group N Test Article Dose, Administration mg / kg route Blood sampling times 4, , 8,

[0294] All PD, PK, and albumin measurements were conducted as described above.

[0295] Results

[0296] To assess the efficacy of the tested OA-Fc-ABDEG-based molecules to depleteIgG, the PD effect over time was measured. The measured tracer IgG concentrations (FIG. 3A) and total hIgG (FIG.3B) were plotted as percentage to pre-dose at 2h prior to test article injection (D0-2h) per treatment group. A clear PD effect was observed for all test articles starting at day 1 as compared to PBS control. No clear difference between the OA-Fc-ABDEG-based articles and efgartigimod was observed when tracer IgG was measured (FIG. 3A). However, all OA-Fc- ABDEG-based articles tended to decrease total hIgG serum levels more than efgartigimod (FIG. 3B).

[0297] To evaluate the PK profile of the test items after a single IP administration, theirlevels in mouse serum were determined post-dose according to the bleeding scheme in Table S6. The obtained values were plotted in nM (FIG.3C). Values below the low limit of quantification (LLOQ) were excluded from FIG.3C. Consistent with the earlier results, OA-Fc-ABDEG-Alb23 showed the best PK profile among the test items. No clear differences were observed in PK profile between the OA-Fc-ABDEG-Alb23 variants (OA-Fc-ABDEG-Alb23-F32A / G100A, OA-Fc- ABDEG-Alb23-F32A / L102A, OA-Fc-ABDEG-Alb23-F32A / L59A, and OA-Fc-ABDEG-Alb23- M34A / G33A / S101A). The control molecule, OA-Fc-ABDEG-3Rab, and efgartigimod showed the worst PK profiles. Overall, no clear correlation was found between measured PD effect and serum half-life. Concentrations of OA-Fc-ABDEG-Alb23 and variants thereof were quantifiable in the serum during the entire study (21 days).

[0298] No decreases in albumin levels were observed in mice treated with the test articlescompared to PBS during the course of the study (FIG.3D).

[0299] In conclusion, this experiment shows that incorporation of one albumin-bindingVHH can substantially improve PK of Fc-ABDEG and allow Fc-ABDEG to mediate an efficient IgG depletion without depleting serum albumin levels. Example 4: Pharmacokinetics / Pharmacodynamics of OA-Fc-ABDEG-Alb23 variants in Tg32 SCID mice

[0300] The current experiment was conducted to compare PK and PD of one-armed Fc-ABDEG-based molecules to efgartigimod in the Tg32-SCID mouse model instead of the AlbuMus Rag1KO mouse model (hFcRn + / +, hSA + / +, Rag1 - / -) used in Example 3.

[0301] Methods

[0302] Briefly, a total of 36 Tg32 SCID mice were randomly assigned into 9 groups. Micewere dosed IP with human IgG (tracer IgG) and IVIg prior to administration of test items. On day 0, mice were injected IV according to the designated group and doses in Table S7.25 mg / kg dose for OA-Fc-ABDEG-based molecules and 20 mg / kg dose for efgartigimod were selected based on MW of the test items to have equimolar doses thereof. All animals were pre-weighed before dosing and dosed according to their body weights. Blood samples were collected before dosing of the test article (pre-dose, D0-1h) and after treatment for PD and PK read-outs for 21 days (Table S7).

[0303] Blood samples were processed to serum and added to a 96-well plate(polypropylene) and stored at -80°C.Table S7: Groups and dosing regimen Group N Test Article Dose, Administration mg / kg route Blood sampling times , 6,

[0304] PK and PD measurements were conducted as described in Example 3.

[0305] Results

[0306] To assess the PD effects of the OA-Fc-ABDEG-Alb23-based molecules, the levelsof preloaded hIgG and tracer IgG were measured during the course of the study. The measured tracer IgG concentrations (FIG.4A) and total hIgG (FIG.4B) were plotted as percentage to pre- dose at 1h prior to test article injection (D0-1h) per treatment group. A clear PD effect was observed for all test articles starting at day 1 as compared to PBS control. No clear difference between the OA-Fc-ABDEG-based articles and efgartigimod was observed when either tracer IgG or total IgG was measured. This is in contrast to data obtained from AlbuMus mice, where all OA- Fc-ABDEG-based articles tended to decrease total hIgG serum levels more than efgartigimod (FIG.3B).

[0307] To evaluate the PK profile of the test items after a single IV administration, theirlevels in mouse serum were determined post-dose according to the bleeding scheme in Table S7. The obtained values were plotted in nM (FIG.4C). Values below the low limit of quantification (LLOQ) were excluded from FIG.4C. No clear differences were observed in PK profile between OA-Fc-ABDEG-Alb23 and the OA-Fc-ABDEG-Alb23 variants (OA-Fc-ABDEG-Alb23- F32A / G100A, OA-Fc-ABDEG-Alb23-F32A / L102A, OA-Fc-ABDEG-Alb23-F32A / L59A, and OA-Fc-ABDEG-Alb23-M34A / G33A / S101A). This is in contrast to data obtained from AlbuMus mice, where OA-Fc-ABDEG-Alb23 showed the best PK profile among the test items. Similar to data seen in AlbuMus mice, efgartigimod showed the worst PK profile. Concentrations of OA-Fc- ABDEG-Alb23 and variants thereof were quantifiable in the serum during the entire study (21 days).

[0308] In conclusion, this experiment shows that incorporation of one albumin-bindingVHH can substantially improve PK of Fc-ABDEG and allow Fc-ABDEG to mediate an efficient IgG depletion. Example 5: Optimization of linker length

[0309] The goal of this study was to explore optimal linker lengths in OA-Fc-ABDEG-Alb23-based molecules.

[0310] FcRn degradation was measured as described in previous Examples. Briefly, HEKFcRn WT GFP+ cells / well were seeded on a 96-well microplate overnight at 37°C in growth medium (DMEM + 10% FBS + P / S + L-glutamine). OA-Fc-ABDEG-Alb23-based molecules (OA-Fc-ABDEG-Alb23, OA-Fc-ABDEG-Alb23-F32A / L102A, and OA-Fc-ABDEG-Alb23- M34A / G53A / S101A) (12,500 nM) were pre-incubated with 50,000 nM HSA in treatment medium (DMEM + 1% BSA + P / S + L-glutamine) for 30 minutes at 37°C, 5% CO2prior to adding to the cells. After incubation of the OA-Fc-ABDEG-Alb23-based molecule / HSA mix or OA-Fc- ABDEG-Alb23-based molecule with the cells, plates were then placed on ice and cells harvested by trypsin. Harvested cells were transferred to FACS plate and centrifuged. LD stain (1:800) was added in FACS buffer and incubated for 15 minutes at 4°C. Cells were washed, centrifuged, and resuspended in FACS buffer. GFP signal was measured and compared to untreated controls.

[0311] Results are shown in FIG. 5. As shown previously in Example 2, OA-Fc-ABDEG-Alb23 (20GS linker) induces FcRn degradation in the presence of HSA, which is ameliorated in the variants OA-Fc-ABDEG-Alb23-F32A / L102A and OA-Fc-ABDEG-Alb23-M34A / G53A / S101A (see FIG. 1). Inclusion of longer linker lengths (25GS and 30GS) had littleto no impact on FcRn degradation in these two variant molecules.

[0312] The effect of linker length on PD and PK was studied in AlbuMus Rag1KO mice.26 AlbuMus Rag1KO mice were assigned to one of seven groups as shown below in Table S8. Mice were preloaded intraperitoneally with IVIg and tracer hIgG1 on day -3 (due to low levels of endogenous mIgG in mouse model). OA-Fc-ABDEG-Alb23-based molecules or OA-Fc-ABDEG- 3Rab (containing an irrelevant rabies virus-binding VHH as a control) were administered on day 0. Blood samples were drawn on Day 0, 2 hours prior to administration of the test articles and again 1 hour after administration of the test articles, and on Days 1, 2, 4, 7, 11, and 14 post- administration. Table S8: Groups and dosing regimen Group Test article Dose Route, (mg / kg) # animals frequency e

[0313] Results are shown in FIGs. 6A-6B. A slight improvement in PK (FIG. 6A) wasseen with OA-Fc-ABDEG-30GS-Alb23-F32A when compared to OA-Fc-ABDEG-20GS-Alb32- F32A, supporting the benefit of using a 30GS linker over a 20GS linker. No difference in PK was seen with OA-Fc-ABDEG-Alb23-F32A / L102A or OA-Fc-ABDEG-Alb23-M34A / G33A / S101A, regardless of linker length. A slight improvement in PD, as measured by percent reduction in total serum IgG, was seen in molecules including the longer 30GS linker as compared to the 20GS linker (FIG.6B).Example 6: Impact of repeated administration of OA-Fc-ABDEG-20GS-Alb23- M34A / G33A / S101A on albumin in AlbuMus Rag1KO mouse model

[0314] The impact of OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A on serumalbumin levels was further explored by measuring serum albumin levels following repeated weekly injections to AlbuMus Rag1KO mice.

[0315] Briefly, AlbuMus Rag1KO mice were randomly assigned into 5 groups (3-5 miceper group). The mice were dosed intraperitoneally (IP) according to the designated group and doses in Table S9. The doses were selected based on MW of the test items to have equimolar doses thereof. All animals were pre-weighed before dosing and dosed according to their body weights. Blood samples were collected before dosing of the test article and after treatment for albumin read- outs (Table S9).

[0316] Blood samples were processed to serum and added to a 96-well plate(polypropylene) and stored at -80°C. Table S9: Groups and dosing regimen Group N Test Article Dose, Administration Blood sampling times , ,

[0317] PBS was included as a negative control and TA-Fc-ABDEG-Alb23, anti-FcRnmAb1 and anti-FcRn mAb2 were included as comparators since all three were previously shown to decrease serum albumin levels and cause FcRn degradation. TA-Fc-ABDEG-Alb23 is an Fc- ABDEG molecule with one Alb23 VHH fused at the C-terminus of each Fc domain (“two- armed”). Anti-FcRn mAb1 comprises the light chain sequence of SEQ ID NO: 57 and the heavychain sequence of SEQ ID NO: 58. Anti-FcRn mAb2 comprises the light chain sequence of SEQ ID NO: 59 and the heavy chain sequence of SEQ ID NO: 60.

[0318] SEQ ID NO: 57:SYVLTQSPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSAS NSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQ ANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRS YSCQVTHEGSTVEKTVAPTECS

[0319] SEQ ID NO: 58:QLLLQESGPGLVKPSETLSLTCTVSGGSLSSSFSYWVWIRQPPGKGLEWIGTIYYSGNTYYNPS LKSRLTISVDTSKNHFSLKLSSVTAADTAVYYCARRAGILTGYLDSWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG

[0320] SEQ ID NO: 59:QSALTQPASVSGSPGQSITISCTGTGSDVGSYNLVSWYQQHPGKAPKLMIYGDSERPSGVSNRF SGSKSGNTASLTISGLQAEDEADYYCSSYAGSGIYVFGTGTKVTVLGQPKAAPSVTLFPPSSEE LQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSH KSYSCQVTHEGSTVEKTVAPTECS

[0321] SEQ ID NO: 60:EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMGWVRQAPGKGLEWVSSIGASGSQTRYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLAIGDSYWGQGTMVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0322] Results are shown in FIG. 7A. Values are presented as a percentage of predose (D-6) values averaged per group. As expected, administration of TA-Fc-ABDEG-Alb23, anti-FcRn mAb1, and anti-FcRn mAb2 resulted in significant decreases in serum albumin. In contrast,repeated administration of OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A not only failed to decrease serum albumin, but actually increased serum albumin levels over time.

[0323] To explore if the effect on albumin levels is consistent with the effect of thesemolecules on FcRn degradation, HEK FcRn WT GFP+ cells were incubated with each of the test articles listed in Table S9 in the presence (50,000 nM) or absence of HSA and FcRn degradation was measured as previously described above. Results are shown in FIG.7B. Incubation of HEK FcRn WT GFP+ cells with anti-FcRn mAbs (1 & 2) resulted in significant FcRn degradation in the presence and absence of HSA. As previously shown, FcRn degradation is seen upon incubation of HEK FcRn WT GFP+ cells with TA-Fc-ABDEG-Alb23 in the presence of HSA but not in the absence of HSA. In contrast, incubation of HEK FcRn WT GFP+ cells with OA-Fc-ABDEG- 20GS-Alb23-M34A / G33A / S101A in the presence of HSA did not impact FcRn degradation to the same extent as seen with the other molecules. Example 7: PD / PK of OA-Fc-ABDEG-20GS-Alb23-M34 / G33A / S101A in cynomolgus monkeys

[0324] Based on previous in vitro and in vivo data showing reduced FcRn degradation andimproved PK for OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A (ABDEG-20GS-Alb23- TM) without compromising PD, this study sought to measure PD / PK of OA-Fc-ABDEG-20GS- Alb23-M34A / G33A / S101A in cynomolgus monkeys.

[0325] Briefly, cynomolgus monkeys were randomly assigned to one of three groups anddosed according to the schedule described in Table S10. Table S10: Groups and dosing regimen Grou N Test Article Dose, Administration Blood sam lin times , ,

[0326] To evaluate pharmacodynamic effects of OA-Fc-ABDEG-20GS-Alb23-TM after asingle IV administration at 10 and 60 mg / kg doses, the levels of total circulating IgG in serum samples were determined at baseline (pre-dose) and post-dose according to the bleeding schemein Table S10. The obtained IgG concentrations were plotted as percentage to pre-dose (D0-2h) prior to of OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A administration (FIGs. 8A-8B). OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A demonstrated a pronounced PD effect on the clearance of total circulating IgG with an average observed maximum total IgG reduction of 48% and 60%, defined as IgG Cmin, in individual monkeys in the dose groups of 10 and 60 mg / kg, respectively (FIGs.8A-8B, Table S11). Table S11: Pharmacodynamic properties of OA-Fc-ABDEG-30GS-Alb23-F32A in cynomolgus monkeys Test Article Dose, µmol / kg Dose, mg / kg Cmin (% d0) HED (mg / kg)OA-HEL-ABDEG 0.1 10 36 1.7larOA-HEL-ABDEG was not possible due to non-linear PK / PD, thus two values are shown which bracket the target dose of 0.15 µmol / kg.

[0327] The potential role of ADA developed in the monkeys after OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A administration cannot be excluded and should be taken into account when interpreting PD effects on total circulating IgG. Time points when presence of ADA was detected coupled with a steep concentration decline in PK curves are excluded from results.

[0328] Overall, the low dose OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A (10mg / kg) reduced IgG better than equimolar dosed efgartigimod, and comparable to near-equimolar dosed OA-HEL-ABDEG (HEL (anti-hen egg lysozyme) Fab fragment fused to N-terminus of Fc- ABDEG). At a higher dose of OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A (60 mg / kg), the IgG reduction was comparable to efgartigimod and to OA-HEL-ABDEG.

[0329] To evaluate the PK profile of OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101Aafter a single IV administration at 10 and 60 mg / kg doses, OA-Fc-ABDEG-Alb23 levels were determined in serum post-dose samples according to the bleeding scheme in Table S10. The obtained OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A concentrations were plotted over time during the course of the study (FIGs. 9A-9B). Sampling times with steep concentration decline of OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A levels and when ADA presence was detected were excluded from data. PK profiles in cynomolgus monkeys treated with equimolardoses of efgartigimod (model simulation) are plotted for comparison. Values for Cmaxand AUC(0-7) for OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A, efgartigimod (model simulation) and nearly equimolar doses of OA-HEL-ABDEG (experimental data) and TA-Fc-ABDEG-Alb23 (experimental data) are summarized in Table S12. Table S12: Pharmacokinetic properties of OA-Fc-ABDEG-30GS-Alb23-M34A / G33A / S101A in cynomolgus monkeys Test Article Dose, µmol / kg Dose, mg / kg Cmax(µmol / L) AUC0-7, day*µmol / L

[0330] propert es o O - G- 0GS- b 3- 3 / G33 / S 0 were mprovedover equimolar doses of both efgartigimod and OA-HEL-ABDEG, and were similar, although slightly lower than PK of equimolar TA-Fc-ABDEG-Alb23.

[0331] Safety of OA-ABDEG-20GS-Alb23-M34A / G33A / S101A was evaluated bymeasuring serum albumin levels in response to single or repeated administration to cynomolgus monkeys. On day 1, group 1 (n=3) received a 10 mg / kg dose of OA-ABDEG-20GS-Alb23- M34A / G33A / S101A; group 2 (n=2) and group 3 (n=3) received a 60 mg / kg dose of OA-ABDEG- 20GS-Alb23-M34A / G33A / S101A. After a 4-week follow-up period, the monkeys in groups 1 and 2 were additionally dosed four times, once every week on day 29, day 36, day 43, and day 50 with 60 mg / kg of OA-ABDEG-20GS-Alb23-M34A / G33A / S101A. Serum albumin was analyzed with a BCG albumin assay. Results are shown in FIG. 10. Albumin levels were not significantly affected by single or repeated administration of OA-ABDEG-20GS-Alb23-M34A / G33A / S101A, staying within the minimum and maximum albumin serum levels seen in these monkeys prior to administration of OA-ABDEG-20GS-Alb23-M34A / G33A / S101A (predose). The serum albumin levels obtained during the study are also generally within the normal range of serum albumin in cynomolgus monkeys according to Park et al. (Lab Anim Res.2016 Jun; 32(2):79-86).Example 8: Optimization of OA-Fc-ABEDEG-20GS-Alb23-M34A / G33A / S101A to reduce binding of pre-existing antibodies

[0332] The C-terminus of VHHs is known to interact with pre-existing antibodies inhuman subjects. Since the C-terminus of the Alb23-M34A / G33A / S101A VHH is exposed in the OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A molecule, the goal of this study was to explore modifications to reduce ADA reactivity while maintaining functionality.

[0333] OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A was modified by adding oneof the following to the C-terminus of the molecule: A, AG, PP, or GG (OA-Alb23- M34A / G33A / S101A-A, OA-Alb23-M34A / G33A / S101A-AG, OA-Alb23-M34A / G33A / S101A- PP, or OA-Alb23-M34A / G33A / S101A-GG, respectively). Binding of each molecule to pre- existing ADA was tested along with PBS as a negative control. Serum from 40 humans positive for pre-existing ADA to ABDEG was used. OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A with or without different C-terminal extensions or PBS (blank, no coating) were coated on a 96- well plate, the plate was blocked with 1% PBS-casein, and the serum was applied. Binding of pre- existing ADA to the test articles was detected with HRP-conjugated anti-human Fab IgG. Results are presented in FIG. 11 showing that all C-terminal extensions reduced binding to pre-existing ADA.

[0334] Effect of the additional C-terminal extensions on FcRn occupancy was measuredusing an FcRn occupancy assay. Efgartigimod and TA-Alb23-Fc-ABDEG (one Alb23 VHH linked to the N-terminus of each Fc domain) were included for comparison. Briefly, U937 cells were incubated with a titration series of the test items in the presence of 2,500 nM HSA. Free FcRn was detected with a fluorescently labelled anti-FcRn Fab fragment recognizing IgG binding site on FcRn. Detected levels of free FcRn were normalized to the FcRn levels in cells treated with the assay buffer (placebo, 100%) and results are presented in FIG.12A and in Table S13. Table S13: FcRn occupancy after incubation of with ABDEG-Alb23-SM molecules in the presence of albumin in U937 cells Name Mean IC50, nM SD n totalOA-Fc-ABDEG-20GS-Alb23- 3.8 2.2 2 M34A / G33A / S101A-AGon was measuredusing an FcRn degradation assay as described in detail above. Briefly, HEK FcRn WT GFP+ cells were incubated with indicated concentrations of the test molecules (12.5 µM OA-Fc-ABDEG- 20GS-Alb23 and variants thereof, and 5 nM anti-FcRn mAb1) in the absence of HSA or in the presence of 50,000 nM HSA and levels of FcRn were measured and presented as a percentage of untreated control values. Results are presented in FIG. 12B showing no difference in FcRn degradation between OA-Fc-ABDEG-20GS-Alb23-M33A / G33A / S101A and any of the C- terminal extensions: OA-Fc-ABDEG-20GS-Alb23-M33A / G33A / S101A-A, OA-Fc-ABDEG- 20GS-Alb23-M33A / G33A / S101A-AG, OA-Fc-ABDEG-20GS-Alb23-M33A / G33A / S101A-PP, and OA-Fc-ABDEG-20GS-Alb23-M33A / G33A / S101A-GG.

[0336] Based upon the in vitro data for the C-terminal extensions of OA-Fc-ABDEG-20GS-Alb23-M34A / G33A / S101A, these molecules have similar functional properties to OA-Fc- ABDEG-20GS-Alb23-M34A / G33A / S101A but have the advantage of reduced binding to ADA. ***

[0337] The invention is not to be limited in scope by the specific embodiments describedherein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims

Claims 1. An FcRn / HSA-binding molecule comprising an FcRn-binding molecule and an HSA- binding domain, wherein the HSA-binding domain comprises a VHH comprising the CDR1, CDR2, and CDR3 amino acid sequences of a VHH comprising an amino acid sequence selected from SEQ ID NOs: 23-26.

2. The FcRn / HSA-binding molecule of claim 1, wherein the FcRn-binding molecule is a variant IgG Fc region.

3. The FcRn / HSA-binding molecule of claim 2, wherein the variant IgG Fc region comprises a first Fc domain and a second Fc domain which form a dimer, wherein the first Fc domain and / or the second Fc domain comprise amino acids Y, P, and Y at EU positions 252, 308, and 434, respectively.

4. The FcRn / HSA-binding molecule of claim 2, wherein the variant IgG Fc region comprises a first Fc domain and a second Fc domain which form a dimer, wherein the first Fc domain and / or the second Fc domain comprise amino acids Y, T, E, K, and F at EU positions 252, 254, 256, 433, and 434, respectively.

5. The FcRn / HSA-binding molecule of claim 4, wherein the first Fc domain and / or the second Fc domain comprise amino acids Y, T, E, K, F, and Y at EU positions 252, 254, 256, 433, 434, and 436, respectively.

6. The FcRn / HSA-binding molecule of claim 4 or 5, wherein both the first Fc domain and the second Fc domain comprise amino acids Y, T, E, K, and F at EU positions 252, 254, 256, 433, and 434, respectively.

7. The FcRn / HSA-binding molecule of any one of claims 4-6, wherein both the first Fc domain and the second Fc domain comprise amino acids Y, T, E, K, F, and Y at EU positions 252, 254, 256, 433, 434, and 436, respectively.

8. The FcRn / HSA-binding molecule of any one of claims 3-7, wherein the first Fc domain and / or the second Fc domain is an IgG1 Fc domain.

9. The FcRn / HSA-binding molecule of any one of claims 3-7, wherein the first Fc domain and / or the second Fc domain is a human IgG Fc domain.

10. The FcRn / HSA-binding molecule of any one of claims 3-9, wherein the first Fc domain and / or the second Fc domain is a human IgG1 Fc domain.

11. The FcRn / HSA-binding molecule of any one of claims 3-8, wherein both the first Fc domain and the second Fc domain are IgG1 Fc domains.

12. The FcRn / HSA-binding molecule of any one of claims 3-7, wherein both the first Fc domain and the second Fc domain are human IgG Fc domains.

13. The FcRn / HSA-binding molecule of any one of claims 3-12, wherein both the first Fc domain and the second Fc domain are human IgG1 Fc domains.

14. The FcRn / HSA-binding molecule of any one of claims 3-13, wherein the HSA-binding domain is fused to the C-terminus of the first Fc domain or the second Fc domain.

15. The FcRn / HSA-binding molecule of any one of claims 3-13, wherein the HSA-binding domain is fused to the N-terminus of the first Fc domain or the second Fc domain.

16. The FcRn / HSA-binding molecule of any one of claims 3-15, wherein the HSA-binding domain is fused to the first Fc domain or the second Fc domain via a linker.

17. The FcRn / HSA-binding molecule of claim 16, wherein the linker is a non-cleavable linker.

18. The FcRn / HSA-binding molecule of claim 16 or 17, wherein the linker is a peptide linker.

19. The FcRn / HSA-binding molecule of claim 18, wherein the peptide linker is a GS linker, optionally from 8 to 40 amino acids in length, optionally 20 or 30 amino acids in length.

20. The FcRn / HSA-binding molecule of any one of claims 3-18, wherein the HSA-binding domain is fused to the first Fc domain or the second Fc domain via an IgG hinge region or portion thereof.

21. The FcRn / HSA-binding molecule of any one of claims 4-20, wherein the first Fc domain and / or the second Fc domain comprise an amino acid sequence independently selected from an amino acid sequence set forth in SEQ ID NO: 1, 2, or 3.

22. The FcRn / HSA-binding molecule of claim 21, wherein the first Fc domain and / or the second Fc domain comprise the amino acid sequence of SEQ ID NO:

2.

23. The FcRn / HSA-binding molecule of claim 21, wherein both the first Fc domain and the second Fc domain comprise an amino acid sequence independently selected from an amino acid sequence set forth in SEQ ID NO: 1, 2, or 3.

24. The FcRn / HSA-binding molecule of claim 23, wherein both the first Fc domain and the second Fc domain comprise the amino acid sequence of SEQ ID NO:

2.

25. The FcRn / HSA-binding molecule of any one of claims 4-21, wherein the amino acid sequence of each of the first Fc domain and the second Fc domain consists of an amino acid sequence independently selected from an amino acid sequence set forth in SEQ ID NO: 1, 2, or 3.

26. The FcRn / HSA-binding molecule of claim 25, wherein the amino acid sequence of the first Fc domain or the amino acid sequence of the second Fc domain consists of SEQ ID NO:

2.

27. The FcRn / HSA-binding molecule of claim 26, wherein the amino acid sequences of both the first Fc domain and the second Fc domain consist of SEQ ID NO: 2.

28. The FcRn / HSA-binding molecule of any one of claims 3-20, wherein the variant Fc region comprises one or more mutations of amino acid residues forming the interface of the CH3 domain of the Fc domains.

29. The FcRn / HSA-binding molecule of any one of claims 3-20 and 28, wherein the amino acid sequence of the first Fc domain further comprises amino acid W at EU position 366.

30. The FcRn / HSA-binding molecule of claim 29, wherein the amino acid sequence of the first Fc domain comprises an amino acid sequence selected from an amino acid sequence set forth in SEQ ID NO: 4, 5, or 6.

31. The FcRn / HSA-binding molecule of claim 30, wherein the amino acid sequence of the first Fc domain comprises the amino acid sequence of SEQ ID NO:

5.

32. The FcRn / HSA-binding molecule of claim 30, wherein the amino acid sequence of the first Fc domain consists of an amino acid sequence selected from an amino acid sequence set forth in SEQ ID NO: 4, 5, or 6.

33. The FcRn / HSA-binding molecule of claim 32, wherein the amino acid sequence of the first Fc domain consists of the amino acid sequence of SEQ ID NO:

5.

34. The FcRn / HSA-binding molecule of any one of claims 3-20 and 28-33, wherein the amino acid sequence of the second Fc domain further comprises amino acids S, A, and V at EU positions 366, 368, and 407, respectively.

35. The FcRn / HSA-binding molecule of claim 34, wherein the amino acid sequence of the second Fc domain comprises an amino acid sequence selected from an amino acid sequence set forth in SEQ ID NO: 7, 8, or 9.

36. The FcRn / HSA-binding molecule of claim 35, wherein the amino acid sequence of the second Fc domain comprises the amino acid sequence of SEQ ID NO:

8.

37. The FcRn / HSA-binding molecule of claim 35, wherein the amino acid sequence of the second Fc domain consists of an amino acid sequence selected from an amino acid sequence set forth in SEQ ID NO: 7, 8, or 9.

38. The FcRn / HSA-binding molecule of claim 37, wherein the amino acid sequence of the second Fc domain consists of the amino acid sequence of SEQ ID NO:

8.

39. The FcRn / HSA-binding molecule of any one of claims 1-38, wherein the HSA-binding domain comprises a VHH comprising an amino acid sequence selected from the group consisting of: a) an amino acid sequence comprising SEQ ID NO: 14 (CDR1), SEQ ID NO: 15 (CDR2), and SEQ ID NO: 13 (CDR3); b) an amino acid sequence comprising SEQ ID NO: 14 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 16 (CDR3); c) an amino acid sequence comprising SEQ ID NO: 14 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 17 (CDR3); and d) an amino acid sequence comprising SEQ ID NO: 18 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 19 (CDR3).

40. The FcRn / HSA-binding molecule of any one of claims 1-39, wherein the HSA-binding domain comprises a VHH comprising an amino acid sequence comprising SEQ ID NO: 18 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 19 (CDR3).

41. The FcRn / HSA-binding molecule of any one of claims 1-39, wherein the HSA-binding domain comprises a VHH comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from an amino acid sequence set forth in SEQ ID NOs: 23-26.

42. The FcRn / HSA-binding molecule of any one of claims 1-41, wherein the HSA-binding domain comprises a VHH comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in SEQ ID NO:

26.

43. The FcRn / HSA-binding molecule of any one of claims 1-41, wherein the HSA-binding domain comprises an amino acid sequence selected from an amino acid sequence set forth in SEQ ID NOs: 23-26.

44. The FcRn / HSA-binding molecule of any one of claims 1-43, wherein the HSA-binding domain comprises an amino acid sequence set forth in SEQ ID NO:

26.

45. The FcRn / HSA-binding molecule of any one of claims 1-44, further comprising one or more additional amino acids at the C-terminal end of the HSA-binding domain.

46. The FcRn / HSA-binding molecule of claim 45, wherein the one or more additional amino acids are selected from the group consisting of: a) A; b) AG; c) GG; d) PP; and e) AA.

47. The FcRn / HSA-binding molecule of any one of claims 1-46, further comprising an antigen- binding domain.

48. An FcRn / HSA-binding molecule comprising a first heavy chain, wherein the first heavy chain comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 42-45.

49. The FcRn / HSA-binding molecule of claim 48, wherein the first heavy chain comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

45.

50. The FcRn / HSA-binding molecule of claim 48, wherein the first heavy chain comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 42-45.

51. The FcRn / HSA-binding molecule of any one of claims 48-50, wherein the first heavy chain comprises or consists of an amino acid sequence of SEQ ID NO:

45.

52. The FcRn / HSA-binding molecule of any one of claims 48-51, wherein the first heavy chain further comprises one or more amino acids added at the C-terminus of the first heavy chain, optionally selected from A, AG, GG, and PP.

53. The FcRn / HSA-binding molecule of any one of claims 48-52, further comprising a second heavy chain, wherein the second heavy chain consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

8.

54. The FcRn / HSA-binding molecule of claim 53, wherein the second heavy chain consists of the amino acid sequence of SEQ ID NO:

8.

55. An HSA-binding domain comprising CDR1, CDR2, and CDR3 amino acid sequences of a VHH comprising an amino acid sequence selected from SEQ ID NOs: 23-26.

56. The HSA-binding domain of claim 55, comprising an amino acid sequence selected from the group consisting of: a) an amino acid sequence comprising SEQ ID NO: 14 (CDR1), SEQ ID NO: 15 (CDR2), and SEQ ID NO: 13 (CDR3); b) an amino acid sequence comprising SEQ ID NO: 14 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 16 (CDR3);c) an amino acid sequence comprising SEQ ID NO: 14 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 17 (CDR3); and d) an amino acid sequence comprising SEQ ID NO: 18 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 19 (CDR3).

57. The HSA-binding domain of claim 55 or 56, wherein the HSA-binding domain comprises an amino acid sequence comprising SEQ ID NO: 18 (CDR1), SEQ ID NO: 12 (CDR2), and SEQ ID NO: 19 (CDR3).

58. The HSA-binding domain of claim 55 or 56, wherein the HSA-binding domain comprises or consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in SEQ ID NOs: 23-26.

59. The HSA-binding domain of any one of claims 55-58, wherein the HSA-binding domain comprises or consists of an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence set forth in SEQ ID NO:

26.

60. The HSA-binding domain of any one of claims 55-58, wherein the HSA-binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NOs: 23-26.

61. The HSA-binding domain of any one of claims 55-60, wherein the HSA-binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:

26.

62. The HSA-binding domain of any one of claims 55-61, wherein the HSA-binding domain is an sdAb.

63. The HSA-binding domain of claim 62, wherein the sdAb is a VHH.

64. The HSA-binding domain of claim 63, further comprising one or more additional amino acids at the C-terminal end of the VHH.

65. The HSA-binding domain of claim 64, wherein the one or more additional amino acids are selected from the group consisting of: a) A; b) AG; c) GG; d) PP; and e) AA.

66. An isolated polynucleotide or polynucleotides encoding the FcRn / HSA-binding molecule of any one of claims 1-54 or the HSA-binding domain of any one of claims 55-65.

67. An expression vector comprising the isolated polynucleotide or polynucleotides of claim 66.

68. A host cell comprising the isolated polynucleotide or polynucleotides of claim 66, or the expression vector of claim 67.

69. A method for producing an FcRn / HSA-binding molecule or an HSA-binding domain, comprising culturing the host cell of claim 68 under conditions which permit the expression of the FcRn / HSA-binding molecule or HSA-binding domain.

70. A pharmaceutical composition comprising an FcRn / HSA-binding molecule of any one of claims 1-54 or an HSA-binding domain of any one of claims 55-65 and at least one pharmaceutically acceptable carrier.

71. An FcRn / HSA-binding molecule of any one of claims 1-54, or an HSA-binding domain of any one of claims 55-65, or a pharmaceutical composition of claim 70 for use as a medicament.

72. A method of reducing serum IgG in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an FcRn / HSA-binding molecule of any one ofclaims 1-54, or an HSA-binding domain of any one of claims 55-65, or a pharmaceutical composition of claim 70.

73. A method of treating an antibody-mediated disorder in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an FcRn / HSA-binding molecule of any one of claims 1-54, or an HSA-binding domain of any one of claims 55-65, or a pharmaceutical composition of claim 70.

74. The method of claim 73, wherein the antibody-mediated disorder is an IgG-mediated disorder.

75. The method of claim 73 or 74, wherein the antibody-mediated disorder is an autoimmune disease.

76. An FcRn / HSA-binding molecule of any one of claims 1-54, or an HSA-binding domain of any one of claims 55-65, or a pharmaceutical composition of claim 70, for use in the treatment of an antibody-mediated disorder.

77. An FcRn / HSA-binding molecule of any one of claims 1-54, or an HSA-binding domain of any one of claims 55-65, for the manufacture of a medicament for treating an antibody-mediated disorder.

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