Compositions and methods for crossing the blood-brain barrier

Abstract

The present disclosure relates to compositions and methods for the preparation, use, and / or formulation of active agents conjugated to ligands to increase passage across the blood-brain barrier.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 394,849, filed August 3, 2022, and U.S. Provisional Application No. 63 / 471,167, filed June 5, 2023, the entire contents of each of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy, created on August 2, 2023, is named V2071-3006PCT_SL.xml and is 4,777,611 bytes in size.

[0003] The present disclosure relates to compositions and methods for the preparation, use, and / or formulation of active agents conjugated to ligands to increase passage across the blood-brain barrier. [Background technology]

[0004] Administering active agents, such as therapeutic and diagnostic agents, to the adult central nervous system (CNS) remains a significant challenge. Engineered compositions comprising the active agent fused or conjugated to a ligand capable of binding to a receptor on cells present at the blood-brain barrier offer an attractive solution to the limitations of CNS delivery.

[0005] Attempts to provide compositions with improved ability to cross the blood-brain barrier have met with limited success. Thus, there is a need for improved methods of producing and delivering active agents of interest to target cells or tissues, such as CNS cells or tissues. Summary of the Invention

[0006] The present disclosure relates, at least in part, to compositions and methods for the production and use of compositions comprising a ligand capable of binding to a receptor present on cells of the blood-brain barrier. In some embodiments, the ligand is fused or attached, e.g., covalently or non-covalently, to an active agent, e.g., a therapeutic or diagnostic agent. The compositions may be useful for delivering an active agent, e.g., a therapeutic or diagnostic agent described herein, to cells or tissues, e.g., CNS cells or tissues, for the treatment of a disorder, e.g., a neurological or neurodegenerative disorder, a muscular or neuromuscular disorder, or a neuro-oncological disorder.

[0007] Thus, in one aspect, the present disclosure provides a composition, e.g., a fusion or conjugated molecule, comprising: (i) a ligand that binds to a glycosylphosphatidylinositol (GPI)-anchored protein, e.g., alkaline phosphatase (ALPL); and (ii) an active agent, e.g., a therapeutic or diagnostic agent, wherein the ligand is fused or attached, e.g., covalently or non-covalently, to the active agent.

[0008] In another aspect, the disclosure provides multispecific antibody molecules comprising a first binding domain that binds to ALPL (e.g., an anti-ALPL binding domain) and a second binding domain that binds to a therapeutic target.

[0009] In yet another aspect, the disclosure provides a method of making a composition described herein, comprising: (i) providing a ligand that binds to a GPI-anchored protein, e.g., ALPL, and an active agent; and (ii) incubating the ligand and the active agent under conditions suitable for fusing or conjugating the ligand to the active agent, thereby producing the composition.

[0010] In yet another aspect, the present disclosure provides a method for delivering an active agent, e.g., a therapeutic or diagnostic agent, to a cell or tissue (e.g., a CNS cell or tissue), comprising administering to a subject an effective amount of a composition comprising (i) a ligand that binds to a glycosylphosphatidylinositol (GPI)-anchored protein, e.g., alkaline phosphatase (ALPL), and (ii) an active agent, as described herein.

[0011] In yet another aspect, the disclosure provides a method for increasing central nervous system transduction (e.g., increasing blood-brain barrier penetration) in a subject, the method comprising administering to the subject an effective amount of a composition comprising (i) a ligand that binds to a glycosylphosphatidylinositol (GPI)-anchored protein, e.g., alkaline phosphatase (ALPL), and (ii) an active agent, as described herein.

[0012] In yet another aspect, the disclosure provides a method of treating a subject having or diagnosed with a genetic disorder, e.g., a monogenic or polygenic disorder, comprising administering to the subject an effective amount of a composition comprising (i) a ligand that binds to a glycosylphosphatidylinositol (GPI)-anchored protein, e.g., alkaline phosphatase (ALPL), and (ii) an active agent, as described herein.

[0013] In yet another aspect, the disclosure provides a method of treating a subject having or diagnosed with a neurological disorder, e.g., a neurodegenerative disorder, comprising administering an effective amount of a composition comprising (i) a ligand that binds to a glycosylphosphatidylinositol (GPI)-anchored protein, e.g., alkaline phosphatase (ALPL), and (ii) an active agent, as described herein.

[0014] In yet another aspect, the present disclosure provides a method of treating a subject having or diagnosed with a neuro-oncology disorder, the method comprising administering an effective amount of a composition comprising (i) a ligand that binds to a glycosylphosphatidylinositol (GPI)-anchored protein, e.g., alkaline phosphatase (ALPL), and (ii) an active agent, as described herein.

[0015] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the embodiments recited below.

[0016] Enumeration of Embodiments 1. (i) a ligand that binds to a glycosylphosphatidylinositol (GPI)-anchored protein, such as alkaline phosphatase (ALPL); (ii) an active agent, e.g., a therapeutic or diagnostic agent; a composition, e.g., a fusion or conjugate molecule, comprising: the ligand is fused or attached, e.g., covalently or non-covalently, to the active agent; Optionally, the ligand binds to the GPI-anchored protein, e.g., ALPL, with a K of at least about 10-250 nM, 10-150 nM (e.g., at least 10 nM, 15 nM, 20 nM, 30 nM, 32 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 175 nM, 200 nM, 215 nM, or 250 nM), as measured, e.g., by an SPR assay, e.g., as described in Example 8. D can be combined with The composition.

[0017] 2. The ligand binds to the GPI-anchored protein, e.g., ALPL, and optionally the following (a), (b), (c), and (d) are measured by an SPR assay, e.g., as described in Example 8 or 13, and the following K D : (a) at least about 10-250 nM; (b) at least about 10-150 nM (e.g., at least 10 nM, 15 nM, 20 nM, 30 nM, 32 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM) (e.g., the ligand is a viral particle or a peptide); (c) at least about 10-55 nM, 15-30 nM, 20-30 nM, 25-50 nM, or 30-50 nM (e.g., at least 10 nM, 15 nM, 20 nM, 30 nM, 32 nM, 50 nM, or 55 nM) (e.g., the ligand is a viral particle (e.g., an AAV viral particle) or a peptide); or (c) at least about 150-250 nM, 150-225 nM, 175-250 nM, 175-225 nM, 200-225 nM, 200-250 nM (e.g., 150 nM, 175 nM, 200 nM, 215 nM, or 250 nM) (e.g., the ligand is an antibody molecule); can be combined with 10. The composition of claim 1.

[0018] 3. The ligand can bind to the GPI-anchored protein, e.g., ALPL, in a pH-dependent manner, e.g., as measured by an assay, e.g., an SPR or Biacore assay, e.g., as described in Example 8 or 13, and optionally, the ligand can bind to ALP at physiological pH (e.g., at least about pH 6.5-8.0, 7.0-8.0, 6.5-7.5, 7.0-7.5, 7.0, 7.1, 7.2, 7.3, or 7.4). 3. The composition of claim 1 or 2, wherein the composition binds to L and / or does not substantially bind to ALPL at an acidic pH (e.g., at a pH of at least about 1.0-5.7, 1.0-5.5, 2.0-5.7, 2.5-5.5, 2.5-5.7, 3.0-5.7, 3.0-5.5, 3.5-5.7, 3.5-5.5, 4.0-5.7, 4.0-5.5, 4.5-5.7, 4.5-5.5, 5.0-5.7, 5.5-5.7, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5).

[0019] 4. The composition of any one of embodiments 1 to 3, wherein the ligand is or comprises a peptide, a protein, an antibody molecule, a nucleic acid molecule (e.g., an aptamer), or a small molecule.

[0020] 5. The composition of any one of embodiments 1 to 3, wherein the ligand comprises a linear or cyclic peptide. 6. The composition of any one of embodiments 1 to 5, wherein the active agent is or comprises a therapeutic agent selected from a protein (e.g., an enzyme), an antibody molecule, a nucleic acid molecule (e.g., an RNAi agent), or a small molecule.

[0021] 7. The composition of any one of embodiments 1 to 5, wherein the active agent is or comprises a ribonucleic acid complex (e.g., a Cas9 / gRNA complex), a plasmid, a closed-end DNA, a circ-RNA, or an mRNA.

[0022] 8. The composition of any one of embodiments 1 to 5, wherein the active agent is or comprises a diagnostic and imaging agent (e.g., a protein or small molecule compound bound to a detectable moiety).

[0023] 9. The composition of any one of embodiments 1-8, wherein the ligand is covalently bound to the active agent. 10. The composition of any one of embodiments 1 to 9, wherein the ligand is conjugated to the active agent.

[0024] 11. The composition of any one of embodiments 1 to 8, wherein the ligand is fused to the active agent, for example as part of a fusion peptide or protein. 12. The composition of any one of embodiments 1 to 11, wherein the ligand is not a component of a viral particle, such as an adeno-associated virus (AAV) particle.

[0025] 13. The composition of any one of embodiments 1-12, wherein the ligand is not a component of a capsid protein, e.g., an AAV capsid protein. 14. The composition of embodiment 13, wherein the ligand is not a component of the AAV9 capsid or a variant thereof.

[0026] 15. The composition of any one of embodiments 1 to 14, wherein the GPI-anchored protein is conserved in at least two to three species, e.g., at least three species (e.g., mouse, NHP (e.g., Macaca fascicularis), and / or human).

[0027] 16. The composition of embodiment 15, wherein the GPI-anchored proteins of the at least two species are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to each other.

[0028] 17. The composition of any one of embodiments 1 to 16, wherein the GPI-anchored protein is present on the surface of a cell within the blood-brain barrier. 18. The composition of any one of embodiments 1-17, wherein the GPI-anchored protein is ALPL, CD59, LY6E, CA4, GPC5, NTM, HYAL2, LSAMP, BST2, EMP2, ALPL, CPM, NCAM1, EFNA1, PIBF1, SEC24B, PRNP, TFPI, OPCML, CD109, DPM3, CNTN4, PIGN, HBP1, CNTN2, CD55, NEGR1, EFNA5, RECK, NRN1, CNTN1, GPAA1, PGAP1, PIGF, PIGK, MDGA2, DPM1, SVIP, NTNG1, CNTN5, GPC6, PIGG, TMEM8A, THY1, GPIHBP1, PIGT, PIGL, ZFAND2B, PLAUR, DPM2, or GPC1.

[0029] 19. The composition of any one of embodiments 1 to 18, wherein the GPI-anchored protein is ALPL. 20. The composition of any one of embodiments 1-19, wherein the ligand binds to human, cynomolgus monkey, or mouse ALPL.

[0030] 21. The composition of any one of embodiments 1-20, wherein the ligand is fused or conjugated to a therapeutic or diagnostic agent. 22. The composition of any one of embodiments 1-21, wherein the ligand is covalently bound to the active agent, for example, directly or indirectly via a linker.

[0031] 23. The composition of embodiment 22, wherein the ligand is covalently attached to the active agent via a linker. 24. The composition of any one of embodiments 1 to 23, wherein the ligand is conjugated to the active agent, for example, directly or indirectly via a linker.

[0032] 25. The composition of embodiment 24, wherein the ligand is conjugated to the active agent via a linker. 26. The composition of any one of embodiments 22-25, wherein the linker is a cleavable linker or a non-cleavable linker.

[0033] 27. The composition of embodiment 26, wherein the cleavable linker is a pH-sensitive linker or an enzyme-sensitive linker. 28. The composition of embodiment 27, wherein the pH-sensitive linker comprises a hydrazine / hydrazone linker or a disulfide linker.

[0034] 29. The composition of embodiment 28, wherein the enzyme-sensitive linker comprises a peptide-based linker, such as a peptide linker that is sensitive to a protease (e.g., a lysosomal protease), or a beta-glucuronide linker.

[0035] 30. The composition of embodiment 26, wherein the non-cleavable linker is a linker comprising a thioether group or a maleimidocaproyl group. 31. The composition of any one of embodiments 1 to 23, wherein the ligand is fused to the active agent, e.g., directly or indirectly via a linker, e.g., as part of a fusion peptide or protein.

[0036] 32. The composition of any one of embodiments 1 to 31, wherein the ligand and the active agent are post-translationally fused or conjugated, for example, using click chemistry. 33. The composition of any one of embodiments 1-32, wherein the ligand and the active agent are fused or linked via chemically induced dimerization.

[0037] 34. The composition of any one of embodiments 1-33, wherein the ligand is N-terminal to the active agent. 35. The composition of any one of embodiments 1-33, wherein the ligand is C-terminal to the active agent.

[0038] 36. The composition of any one of embodiments 1-33, wherein the ligand is fused or conjugated at or near the C-terminus of the active agent, and the active agent is a therapeutic protein, enzyme, or antibody molecule.

[0039] 37. The composition of embodiment 36, wherein the ligand is fused or conjugated within 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acids from the C-terminus of the therapeutic protein, enzyme, or antibody molecule.

[0040] 38. The ligand is or comprises a protein or peptide comprising an amino acid sequence having the following formula: [N1]-[N2]-[N3]; (i) optionally, [N1] includes X1, X2, and X3, and at least one of X1, X2, or X3 is G; (ii) [N2] comprises the amino acid sequence of SPH, and optionally, S comprises a modification, e.g., a phosphate group; (ii) [N3] comprises X4, X5, and X6, and at least one of X4, X5, or X6 is a basic amino acid, e.g., K or R; 37. The composition of any one of embodiments 1 to 36.

[0041] 39. The composition of embodiment 38, wherein X4, X5, or both of [N3] are K. 40. The composition of embodiment 38 or 39, wherein X4, X5, or X6 of [N3] is R.

[0042] 41. (a) Position X4 of [N3] is independently selected from K, S, A, V, T, G, F, W, V, N, or R; (b) position X5 of [N3] is independently selected from S, K, T, F, I, L, Y, H, M, or R; and / or (c) position X6 of [N3] is independently selected from G, A, R, M, I, N, T, Y, D, P, V, L, E, W, N, Q, K, or S; Optionally, the protein or peptide comprises an amino acid modification, e.g., a conservative substitution, of any of the aforementioned amino acids in (a)-(c); 41. The composition of any one of embodiments 38 to 40.

[0043] 42. The composition of any one of embodiments 38 to 41, wherein [N3] comprises SK, KA, KS, AR, RM, VK, AS, SR, VK, KR, KK, KN, VR, RS, RK, KT, TS, KF, FG, KI, IG, KL, LG, TT, TY, KY, YG, KD, KP, TR, RG, VR, GA, SL, SS, FL, WK, SA, RA, LR, KW, RR, GK, TK, NK, AK, KV, KG, KH, KM, TG, SE, SV, SW, SN, HG, SQ, LW, MG, MA, or SG.

[0044] 43. The composition of any one of embodiments 38 to 42, wherein [N3] is SKA, KSG, ARM, VKS, ASR, VKI, KKN, VRM, RKA, KTS, KFG, KIG, KLG, KTT, KTY, KYG, SKD, SKP, TRG, VRG, KRG, GAR, KSA, KSR, SKL, SRA, SKR, SLR, SRG, SSR, FLR, SKW, SKS, WKA, VRR, SKV, SKT, SKG, GKA, TKA, NKA, SKL, SKN, AKA, KTG, KSL, KSE, KSV, KSW, KSN, KHG, KSQ, KSK, KLW, WKG, KMG, KMA, or RSG.

[0045] 44. [N2]-[N3] are SPHSK (SEQ ID NO: 4701), SPHKS (SEQ ID NO: 4704), SPHAR (SEQ ID NO: 4705), SPHVK (SEQ ID NO: 4706), SPHAS (SEQ ID NO: 4707), SPHKK (SEQ ID NO: 4708), SPHVR (SEQ ID NO: 4709), SPHRK (SEQ ID NO: 4710), SPHKT (SEQ ID NO: 4711), SPHKF (SEQ ID NO: 4712), SPHKI (SEQ ID NO: 4713), SPHKL (SEQ ID NO: 4714), SPHKY (SEQ ID NO: 4715), SPHTR (SEQ ID NO: 4716), SPHKR (SEQ ID NO: 4717), SPHKL (SEQ ID NO: 4718), SPHKY (SEQ ID NO: 4719), SPHTR (SEQ ID NO: 4720), SPHKR (SEQ ID NO: 4721), SPHKKL (SEQ ID NO: 4722), SPHKKL (SEQ ID NO: 4723), SPHKY (SEQ ID NO: 4724), SPHTR (SEQ ID NO: 4725), SPHKR (SEQ ID NO: 4726), SPHKR (SEQ ID NO: 4727), SPHKR (SEQ ID NO: 4728), SPHKR (SEQ ID NO: 4729), SPHKR (SEQ ID NO: 4730), SPHKR (SEQ ID NO: 4731), SPHKR (SEQ ID NO: 4732), SPHKR (SEQ ID NO: 4733), SPHKR (SEQ ID NO: 47 4717), SPHGA (SEQ ID NO: 4718), SPHSR (SEQ ID NO: 4719), SPHSL (SEQ ID NO: 4720), SPHSS (SEQ ID NO: 4721), SPHFL (SEQ ID NO: 4722), SPHWK (SEQ ID NO: 4723), SPHGK (SEQ ID NO: 4724), SPHTK (SEQ ID NO: 4725), SPHNK (SEQ ID NO: 4726), SPHAK (SEQ ID NO: 4727), SPHKH (SEQ ID NO: 4728), SPHKM (SEQ ID NO: 4729), or SPHRS (SEQ ID NO: 4730).

[0046] 45.[N2]-[N3] (i) SPHSKA (SEQ ID NO: 941), SPHKSG (SEQ ID NO: 946), SPHARM (SEQ ID NO: 947), SPHVKS (SEQ ID NO: 948), SPHASR (SEQ ID NO: 949), SPHVKI (SEQ ID NO: 950), SPHKKN (SEQ ID NO: 954), SPHVRM (SEQ ID NO: 955), SPHRKA (SEQ ID NO: 956), SPHKFG (SEQ ID NO: 957), SPHKIG (SEQ ID NO: 958), SPHKLG (SEQ ID NO: 959), SPHKTS (SEQ ID NO: 963), SPHKTT (SEQ ID NO: 964), SPHKTY (SEQ ID NO: 965), Sequence number 965), SPHKYG (SEQ ID NO: 966), SPHSKD (SEQ ID NO: 967), SPHSKP (SEQ ID NO: 968), SPHTRG (SEQ ID NO: 972), SPHVRG (SEQ ID NO: 973), SPHKRG (SEQ ID NO: 974), SPHGAR (SEQ ID NO: 975), SPHKSA (SEQ ID NO: 977), SPHKSR (SEQ ID NO: 951), SPHSKL (SEQ ID NO: 960), SPHSRA (SEQ ID NO: 969), SPHSKR (SEQ ID NO: 978), SPHSLR (SEQ ID NO: 952), SPHSRG (SEQ ID NO: 961), SPH SSR (SEQ ID NO: 970), SPHFLR (SEQ ID NO: 979), SPHSKW (SEQ ID NO: 953), SPHSKS (SEQ ID NO: 962), SPHWKA (SEQ ID NO: 971), SPHVRR (SEQ ID NO: 980), SPHSKT (SEQ ID NO: 4731), SPHSKG (SEQ ID NO: 4732), SPHGKA (SEQ ID NO: 4733), SPHNKA (SEQ ID NO: 4734), SPHSKN (SEQ ID NO: 4735), SPHAKA (SEQ ID NO: 4736), SPHSKV (SEQ ID NO: 4737), SPHKTG (SEQ ID NO: 4738), SPHTKA (SEQ ID NO: 4739), SPHKSL (SEQ ID NO: 4740), SPHKSE (SEQ ID NO: 4741), SPHKSV (SEQ ID NO: 4742), SPHKSW (SEQ ID NO: 4743), SPHKSN (SEQ ID NO: 4744), SPHKHG (SEQ ID NO: 4745), SPHKSQ (SEQ ID NO: 4746), SPHKSK (SEQ ID NO: 4747), SPHKLW (SEQ ID NO: 4748), SPHWKG (SEQ ID NO: 4749), SPHKMG (SEQ ID NO: 4750), SPHKMA (SEQ ID NO: 4751), or SPHRSG (SEQ ID NO: 976), (ii) an amino acid sequence comprising a portion of any of the amino acid sequences in (i), e.g., 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids, of any of them; (iii) an amino acid sequence that contains one, two, or three, but not more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or (iv) an amino acid sequence containing one, two, or three, but not more than four, different amino acids from any one of the amino acid sequences in (i); 45. The composition of any one of embodiments 38 to 44, which is or comprises:

[0047] 46. ​​The composition of any one of embodiments 38 to 45, wherein [N1] comprises X1, X2, and X3, and at least one of X1, X2, or X3 is G. 47. (a) Position X1 of [N1] is independently selected from G, V, R, D, E, M, T, I, S, A, N, L, K, H, P, W, or C; (b) position X2 of [N1] is independently selected from S, V, L, N, D, H, R, P, G, T, I, A, E, Y, M, or Q; and / or (c) position X3 of [N1] is independently selected from G, C, L, D, E, Y, H, V, A, N, P, or S; Optionally, the protein or peptide comprises an amino acid modification, e.g., a conservative substitution, of any of the aforementioned amino acids in (a)-(c); 47. The composition of any one of embodiments 38 to 46.

[0048] 48. The composition of any one of embodiments 38 to 47, wherein [N1] comprises GS, SG, GH, HD, GQ, QD, VS, CS, GR, RG, QS, SH, MS, RN, TS, IS, GP, ES, SS, GN, AS, NS, LS, GG, KS, GT, PS, RS, GI, WS, DS, ID, GL, DA, DG, ME, EN, KN, KE, AI, NG, PG, TG, SV, IG, LG, AG, EG, SA, YD, HE, HG, RD, ND, PD, MG, QV, DD, HN, HP, GY, GM, GD, or HS.

[0049] 49. The composition of any one of embodiments 38-48, wherein [N1] is or comprises GSG, GHD, GQD, VSG, CSG, GRG, CSH, GQS, GSH, RVG, GSC, GLL, GDD, GHE, GNY, MSG, RNG, TSG, ISG, GPG, ESG, SSG, GNG, ASG, NSG, LSG, GGG, KSG, HSG, GTG, PSG, GSV, RSG, GIG, WSG, DSG, IDG, GLG, DAG, DGG, MEG, ENG, GSA, KNG, KEG, AIG, GYD, GHG, GRD, GND, GPD, GMG, GQV, GHN, GHP, or GHS.

[0050] 50.[N1]-[N2] (i) SGSPH (SEQ ID NO: 4752), HDSPH (SEQ ID NO: 4703), QDSPH (SEQ ID NO: 4753), RGSPH (SEQ ID NO: 4754), SHSPH (SEQ ID NO: 4755), QSSPH (SEQ ID NO: 4756), DDSPH (SEQ ID NO: 4757), HESPH (SEQ ID NO: 4758), NYSPH (SEQ ID NO: 4759), VGSPH (SEQ ID NO: 4760), SCSPH (SEQ ID NO: 4761), LLSPH (SEQ ID NO: 4762), NGSPH (SEQ ID NO: 4763), PGSPH (SEQ ID NO: 4764), GGSPH (SEQ ID NO: 4765), TGSPH (SEQ ID NO: 4766), SVSPH (SEQ ID NO: No. 4767), IGSPH (SEQ ID NO: 4768), DGSPH (SEQ ID NO: 4769), LGSPH (SEQ ID NO: 4770), AGSPH (SEQ ID NO: 4771), EGSPH (SEQ ID NO: 4772), SASPH (SEQ ID NO: 4773), YDSPH (SEQ ID NO: 4774), HGSPH (SEQ ID NO: 4775), RDSPH (SEQ ID NO: 4776), NDSPH (SEQ ID NO: 4777), PDSPH (SEQ ID NO: 4778), MGSPH (SEQ ID NO: 4779), QVSPH (SEQ ID NO: 4780), HNSPH (SEQ ID NO: 4781), HPSPH (SEQ ID NO: 4782), or HSSPH (SEQ ID NO: 4783), (ii) an amino acid sequence comprising a portion of any of the amino acid sequences in (i), e.g., 2, 3, or 4 amino acids, e.g., consecutive amino acids, of any of them; (iii) an amino acid sequence that contains one, two, or three, but not more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or (iv) an amino acid sequence containing one, two, or three, but not more than four, different amino acids from any one of the amino acid sequences in (i); 50. The composition of any one of embodiments 38-49, comprising:

[0051] 51.[N1]-[N2] (i) GSGSPH (SEQ ID NO: 4695), GHDSPH (SEQ ID NO: 4784), GQDSPH (SEQ ID NO: 4785), VSGSPH (SEQ ID NO: 4786), CSGSPH (SEQ ID NO: 4787), GRGSPH (SEQ ID NO: 4788), CSHSPH (SEQ ID NO: 4789), GQSSPH (SEQ ID NO: 4790), GSHSPH (SEQ ID NO: 4791), GDDSPH (SEQ ID NO: 4792), GHESPH (SEQ ID NO: 4793), GNYSPH (SEQ ID NO: 4794), RVGSPH (SEQ ID NO: 4795), GSCSPH (SEQ ID NO: 4796) ), GLLSPH (SEQ ID NO: 4797), MSGSPH (SEQ ID NO: 4798), RNGSPH (SEQ ID NO: 4799), TSGSPH (SEQ ID NO: 4800), ISGSPH (SEQ ID NO: 4801), GPGSPH (SEQ ID NO: 4802), ESGSPH (SEQ ID NO: 4803), SSGSPH (SEQ ID NO: 4804), GNGSPH (SEQ ID NO: 4805), ASGSPH (SEQ ID NO: 4806), NSGSPH (SEQ ID NO: 4807), LSGSPH (SEQ ID NO: 4808), GGGSPH (SEQ ID NO: 4809), KSGSPH (SEQ ID NO: 4810), HSGSPH (SEQ ID NO: 4811), GTGSPH (SEQ ID NO: 4812), PSGSPH (SEQ ID NO: 4813), GSVSPH (SEQ ID NO: 4814), RSGSPH (SEQ ID NO: 4815), GIGSPH (SEQ ID NO: 4816), WSGSPH (SEQ ID NO: 4817), DSGSPH (SEQ ID NO: 4818), IDGSPH (SEQ ID NO: 4819), GLGSPH (SEQ ID NO: 4820), DAGSPH (SEQ ID NO: 4821), DGGSPH (SEQ ID NO: 4822), MEGSPH (SEQ ID NO: 4823), ENGSPH (SEQ ID NO: 4824), GS ASPH (SEQ ID NO: 4825), KNGSPH (SEQ ID NO: 4826), KEGSPH (SEQ ID NO: 4827), AIGSPH (SEQ ID NO: 4828), GYDSPH (SEQ ID NO: 4829), GHGSPH (SEQ ID NO: 4830), GRDSPH (SEQ ID NO: 4831), GNDSPH (SEQ ID NO: 4832), GPDSPH (SEQ ID NO: 4833), GMGSPH (SEQ ID NO: 4834), GQVSPH (SEQ ID NO: 4835), GHNSPH (SEQ ID NO: 4836), GHPSPH (SEQ ID NO: 4837), or GHSSPH (SEQ ID NO: 4838), (ii) an amino acid sequence comprising a portion of any of the amino acid sequences in (i), e.g., 2, 3, 4, or 5 amino acids, e.g., consecutive amino acids, of any of them; (iii) an amino acid sequence that contains one, two, or three, but not more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or (iv) an amino acid sequence containing one, two, or three, but not more than four, different amino acids from any one of the amino acid sequences in (i); 51. The composition of any one of embodiments 38 to 50, which is or comprises:

[0052] 52.[N1]-[N2]-[N3] (i) SGSPHSK (SEQ ID NO: 4839), HDSPHKS (SEQ ID NO: 4840), SGSPHAR (SEQ ID NO: 4841), SGSPHVK (SEQ ID NO: 4842), QDSPHKS (SEQ ID NO: 4843), SGSPHKK (SEQ ID NO: 4844), SGSPHVR (SEQ ID NO: 4845), SGSPHAS (SEQ ID NO: 4846), SGSPHRK (SEQ ID NO: 4847), SGSPHKT (SEQ ID NO: 4848), SHSPHKS (SEQ ID NO: 4849), QSSPHRS (SEQ ID NO: 4850), RGSPHAS (SEQ ID NO: 4851), RGSPHSK (SEQ ID NO: No. 4852), SGSPHKF (SEQ ID NO: 4853), SGSPHKI (SEQ ID NO: 4854), SGSPHKL (SEQ ID NO: 4855), SGSPHKY (SEQ ID NO: 4856), SGSPHTR (SEQ ID NO: 4857), SHSPHKR (SEQ ID NO: 4858), SGSPHGA (SEQ ID NO: 4859), HDSPHKR (SEQ ID NO: 4860), DDSPHKS (SEQ ID NO: 4861), HESPHKS (SEQ ID NO: 4862), NYSPHKI (SEQ ID NO: 4863), SGSPHSR (SEQ ID NO: 4864), SGSPHSL (SEQ ID NO: 4865), SGSPHSS (SEQ ID NO: 4866), VGSPHSK (SEQ ID NO: 4867), SCSPHRK (SEQ ID NO: 4868), SGSPHFL (SEQ ID NO: 4869), LLSPHWK (SEQ ID NO: 4870), NGSPHSK (SEQ ID NO: 4871), PGSPHSK (SEQ ID NO: 4872), GGSPHSK (SEQ ID NO: 4873), TGSPHSK (SEQ ID NO: 4874), SVSPHGK (SEQ ID NO: 4875), SGSPHTK (SEQ ID NO: 4876), IGSPHSK (SEQ ID NO: 4877), DGSPHSK (SEQ ID NO: 4878), SGSPHNK (SEQ ID NO: 4879), LGS PHSK (SEQ ID NO: 4880), AGSPHSK (SEQ ID NO: 4881), EGSPHSK (SEQ ID NO: 4882), SASPHSK (SEQ ID NO: 4883), SGSPHAK (SEQ ID NO: 4884), HDSPHKI (SEQ ID NO: 4885), YDSPHKS (SEQ ID NO: 4886), HDSPHKT (SEQ ID NO: 4887), RGSPHKR (SEQ ID NO: 4888), HGSPHSK (SEQ ID NO: 4889), RDSPHKS (SEQ ID NO: 4890), NDSPHKS (SEQ ID NO: 4891), QDSPHKI (SEQ ID NO: 4892), PDSPHKI (SEQ ID NO: 4893),PDSPHKS (SEQ ID NO: 4894), MGSPHSK (SEQ ID NO: 4895), HDSPHKH (SEQ ID NO: 4896), QVSPHKS (SEQ ID NO: 4897), HNSPHKS (SEQ ID NO: 4898), NGSPHKR (SEQ ID NO: 4899), HDSPHKY (SEQ ID NO: 4900), NDSPHKI (SEQ ID NO: 4901), HDSPHKL (SEQ ID NO: 4902), HPSPHWK (SEQ ID NO: 4903), HDSPHKM (SEQ ID NO: 4904), or HSSPHRS (SEQ ID NO: 4905), (ii) an amino acid sequence comprising any part of the amino acid sequences in (i), e.g., 2, 3, 4, 5, or 6 amino acids, e.g., consecutive amino acids, of any of them; (iii) an amino acid sequence that contains one, two, or three, but not more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or (iv) an amino acid sequence containing one, two, or three, but not more than four, different amino acids from any one of the amino acid sequences in (i); 52. The composition of any one of embodiments 38-51, comprising:

[0053] 53.[N1]-[N2]-[N3] (i) GSGSPHSKA (SEQ ID NO: 4697), GHDSPHKSG (SEQ ID NO: 4698), GSGSPHARM (SEQ ID NO: 4906), GSGSPHVKS (SEQ ID NO: 4907), GQDSPHKSG (SEQ ID NO: 4908), GSGSPHASR (SEQ ID NO: 4909), GSGSPHVKI (SEQ ID NO: 4910), GSGSPHKKN (SEQ ID NO: 4911), GSGSPHVRM (SEQ ID NO: 4912), VSGSPHSKA (SEQ ID NO: 4913), CSGSPHSKA (SEQ ID NO: 4914), GSGSPHRKA (SEQ ID NO: 4915), CS GSPHKTS (SEQ ID NO: 4916), CSHSPHKSG (SEQ ID NO: 4917), GQSSPHRSG (SEQ ID NO: 4918), GRGSPHASR (SEQ ID NO: 4919), GRGSPHSKA (SEQ ID NO: 4920), GSGSPHKFG (SEQ ID NO: 4921), GSGSPHKIG (SEQ ID NO: 4922), GSGSPHKLG (SEQ ID NO: 4923), GSGSPHKTS (SEQ ID NO: 4924), GSGSPHKTT (SEQ ID NO: 4925), GSGSPHKTY (SEQ ID NO: 4926), GSGSPHKYG (SEQ ID NO: 4927), GSGSPHSK D (SEQ ID NO: 4928), GSGSPHSKP (SEQ ID NO: 4929), GSGSPHTRG (SEQ ID NO: 4930), GSGSPHVRG (SEQ ID NO: 4931), GSHSPHKRG (SEQ ID NO: 4932), GSHSPHKSG (SEQ ID NO: 4933), VSGSPHASR (SEQ ID NO: 4934), VSGSPHGAR (SEQ ID NO: 4935), VSGSPHKFG (SEQ ID NO: 4936), GHDSPHKRG (SEQ ID NO: 4937), GDDSPHKSG (SEQ ID NO: 4938), GHESPHKSA (SEQ ID NO: 4939), GHDSPHKSA (SEQ ID NO: 4940), GNYSPHKIG (SEQ ID NO: 4941), GHDSPHKSR (SEQ ID NO: 4942), GSGSPHSKL (SEQ ID NO: 4943), GSGSPHSRA (SEQ ID NO: 4944), GSGSPHSKR (SEQ ID NO: 4945), GSGSPHSLR (SEQ ID NO: 4946), GSGSPHSRG (SEQ ID NO: 4947), GSGSPHSSR (SEQ ID NO: 4948), RVGSPHSKA (SEQ ID NO: 4949), GSCSPHRKA (SEQ ID NO: 4950), GSGSPHFLR (SEQ ID NO: 4951), GSGSPHSKW (SEQ ID NO: 4952),GSGSPHSKS (SEQ ID NO: 4953), GLLSPHWKA (SEQ ID NO: 4954), GSGSPHVRR (SEQ ID NO: 4955), GSGSPHSKV (SEQ ID NO: 4956), MSGSPHSKA (SEQ ID NO: 4957), RNGSPHSKA (SEQ ID NO: 4958), TSGSPHSKA (SEQ ID NO: 4959), ISGSPHSKA (SEQ ID NO: 4960), GPGSPHSKA (SEQ ID NO: 4961), GSGSPHSKT (SEQ ID NO: 4962), ESGSPHSKA (SEQ ID NO: 4963), SSGSPHSKA (SEQ ID NO: 4964), GNGSPHSKA ( SEQ ID NO: 4965), ASGSPHSKA (SEQ ID NO: 4966), NSGSPHSKA (SEQ ID NO: 4967), LSGSPHSKA (SEQ ID NO: 4968), GGGSPHSKA (SEQ ID NO: 4969), KSGSPHSKA (SEQ ID NO: 4970), GGGSPHSKS (SEQ ID NO: 4971), GSGSPHSKG (SEQ ID NO: 4972), HSGSPHSKA (SEQ ID NO: 4973), GTGSPHSKA (SEQ ID NO: 4974), PSGSPHSKA (SEQ ID NO: 4975), GSVSPHGKA (SEQ ID NO: 4976), RSGSPHSKA (SEQ ID NO: 4977), GSGSPHTKA (SEQ ID NO: 4978), GIGSPHSKA (SEQ ID NO: 4979), WSGSPHSKA (SEQ ID NO: 4980), DSGSPHSKA (SEQ ID NO: 4981), IDGSPHSKA (SEQ ID NO: 4982), GSGSPHNKA (SEQ ID NO: 4983), GLGSPHSKS (SEQ ID NO: 4984), DAGSPHSKA (SEQ ID NO: 4985), DGGSPHSKA (SEQ ID NO: 4986), MEGSPHSKA (SEQ ID NO: 4987), ENGSPHSKA (SEQ ID NO: 4988), GSASPHSKA (SEQ ID NO: 4989), GNGSPHSKS ( SEQ ID NO: 4990), KNGSPHSKA (SEQ ID NO: 4991), KEGSPHSKA (SEQ ID NO: 4992), AIGSPHSKA (SEQ ID NO: 4993), GSGSPHSKN (SEQ ID NO: 4994), GSGSPHAKA (SEQ ID NO: 4995), GHDSPHKIG (SEQ ID NO: 4996), GYDSPHKSG (SEQ ID NO: 4997), GHESPHKSG (SEQ ID NO: 4998), GHDSPHKTG (SEQ ID NO: 4999), GRGSPHKRG (SEQ ID NO: 5000), GQDSPHKSG (SEQ ID NO: 4908), GHDSPHKSL (SEQ ID NO: 5001),GHGSPHSKA (SEQ ID NO: 5002), GHDSPHKSE (SEQ ID NO: 5003), VSGSPHSKA (SEQ ID NO: 4913), GRDSPHKSG (SEQ ID NO: 5004), GNDSPHKSV (SEQ ID NO: 5005), GQDSPHKIG (SEQ ID NO: 5006), GHDSPHKSV (SEQ ID NO: 5007), GPDSPHKIG (SEQ ID NO: 5008), GPDSPHKSG (SEQ ID NO: 5009), GHDSPHKSW (SEQ ID NO: 5010), GHDSPHKSN (SEQ ID NO: 5011), GMGSPHSKT (SEQ ID NO: 5012), GHDSPHKHG (SEQ ID NO: 5013), GQ VSPHKSG (SEQ ID NO: 5014), GDDSPHKSV (SEQ ID NO: 5015), GHNSPHKSG (SEQ ID NO: 5016), GNGSPHKRG (SEQ ID NO: 5017), GHDSPHKYG (SEQ ID NO: 5018), GHDSPHKSQ (SEQ ID NO: 5019), GNDSPHKIG (SEQ ID NO: 5020), GHDSPHKSK (SEQ ID NO: 5021), GHDSPHKLW (SEQ ID NO: 5022), GHPSPHWKG (SEQ ID NO: 5023), GHDSPHKMG (SEQ ID NO: 5024), GHDSPHKMA (SEQ ID NO: 5025), or GHSSPHRSG (SEQ ID NO: 5026), (ii) an amino acid sequence comprising any part of the amino acid sequences in (i), e.g., 2, 3, 4, 5, 6, 7, or 8 amino acids, e.g., consecutive amino acids, of any of them; (iii) an amino acid sequence that contains one, two, or three, but not more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or (iv) an amino acid sequence containing one, two, or three, but not more than four, different amino acids from any one of the amino acid sequences in (i); 53. The composition of any one of embodiments 38 to 52, which is or comprises:

[0054] 54. The composition of any one of embodiments 38 to 53, wherein [N3] comprises SK, KA, KS, or SG. 55. The composition of any one of embodiments 38-54, wherein [N3] is or comprises SKA, KSG, or KYG.

[0055] 56. The composition of any one of embodiments 38 to 55, wherein [N2]-[N3] comprises SPHSK (sequence number 4701), SPHKS (sequence number 4704), or SPHKY (sequence number 4715).

[0056] 57. The composition according to any one of embodiments 38 to 56, wherein [N2]-[N3] is or comprises SPHSKA (SEQ ID NO: 941). 58. The composition of any one of embodiments 38 to 56, wherein [N2]-[N3] is or comprises SPHKSG (sequence number 946).

[0057] 59. The composition of any one of embodiments 38 to 56, wherein [N2]-[N3] is or comprises SPHKYG (sequence number 966). 60. The composition of any one of embodiments 38-59, wherein [N1] comprises GS, SG, GH, or HD.

[0058] 61. The composition of any one of embodiments 38 to 60, wherein [N1] is or comprises GSG. 62. The composition according to any one of embodiments 38 to 60, wherein [N1] is or comprises GHD.

[0059] 63. The composition of any one of embodiments 38 to 57, 60, or 61, wherein [N1]-[N2]-[N3] comprises SGSPHSK (sequence number 4839). 64. The composition of any one of embodiments 38-56, 58, 60, or 62, wherein [N1]-[N2]-[N3] comprises HDSPHKS (sequence number 4840).

[0060] 65. The composition of any one of embodiments 38 to 56, or 59 to 61, wherein [N1]-[N2]-[N3] comprises SGSPHKYG (sequence number 5027). 66. The composition of any one of embodiments 38 to 57, 60, 61, or 63, wherein [N1]-[N2]-[N3] is or comprises GSGSPHSKA (sequence number 4697).

[0061] 67. The composition of any one of embodiments 38 to 56, 58, 60, 62, or 54, wherein [N1]-[N2]-[N3] is or comprises GHDSPHKSG (sequence number 4698).

[0062] 68. The composition of any one of embodiments 38-56, or 59-61, or 65, wherein [N1]-[N2]-[N3] is or comprises GSGSPHKYG (SEQ ID NO: 4927).

[0063] 69. [N4] further includes X7, X8, X9, and X10; (a) position X7 is independently selected from Q, W, K, R, G, L, V, S, P, H, K, I, M, A, E, or F; (b) position X8 is independently selected from N, Y, C, K, T, H, R, D, V, S, P, G, W, E, F, A, I, M, Q, or L; (c) position X9 is independently selected from Q, G, K, H, R, T, L, D, A, P, I, F, V, M, W, Y, S, E, N, or Y; (d) position X10 is independently selected from Q, H, L, R, W, K, A, P, E, M, I, S, G, N, Y, C, V, T, D, or V; Optionally, the protein includes an amino acid modification, e.g., a conservative substitution, of any of the aforementioned amino acids in (a)-(d). 69. The composition of any one of embodiments 38 to 68.

[0064] 70.(a) The X7 position of [N4] is Q or R, (b) the X8 position of [N4] is N or R; (c) The X9 position of [N4] is Q or R; (d) The X10 position of [N4] is Q, L, or R; 70. The composition of embodiment 69.

[0065] 71. [N4] (i) QNQQ (SEQ ID NO: 5028), WNQQ (SEQ ID NO: 5029), QYYV (SEQ ID NO: 5030), RRQQ (SEQ ID NO: 5031), GCGQ (SEQ ID NO: 5032), LRQQ (SEQ ID NO: 5033), RNQQ (SEQ ID NO: 5034), VNQQ (SEQ ID NO: 5035), FRLQ (SEQ ID NO: 5036), FNQQ (SEQ ID NO: 5037), LLQQ (SEQ ID NO: 5038), SNQQ (SEQ ID NO: 5039), RLQQ (SEQ ID NO: 5040), LNQQ (SEQ ID NO: 5041), QRKL (SEQ ID NO: 5042), LRRQ (SEQ ID NO: 5043), QRLR ( SEQ ID NO: 5044), QRRL (SEQ ID NO: 5045), RRLQ (SEQ ID NO: 5046), RLRQ (SEQ ID NO: 5047), SKRQ (SEQ ID NO: 5048), QLYR (SEQ ID NO: 5049), QLTV (SEQ ID NO: 5050), QNKQ (SEQ ID NO: 5051), KNQQ (SEQ ID NO: 5052), QKQQ (SEQ ID NO: 5053), QTQQ (SEQ ID NO: 5054), QNHQ (SEQ ID NO: 5055), QHQQ (SEQ ID NO: 5056), QNQH (SEQ ID NO: 5057), QHRQ (SEQ ID NO: 5058), LTQQ (SEQ ID NO: 5059), QNQW (SEQ ID NO: 5060 ), QNTH (SEQ ID NO: 5061), RRRQ (SEQ ID NO: 5062), QYQQ (SEQ ID NO: 5063), QNDQ (SEQ ID NO: 5064), QNRH (SEQ ID NO: 5065), RDQQ (SEQ ID NO: 5066), PNLQ (SEQ ID NO: 5067), HVRQ (SEQ ID NO: 5068), PNQH (SEQ ID NO: 5069), HNQQ (SEQ ID NO: 5070), QSQQ (SEQ ID NO: 5071), QPAK (SEQ ID NO: 5072), QNLA (SEQ ID NO: 5073), QNQL (SEQ ID NO: 5074), QGQQ (SEQ ID NO: 5075), LNRQ (SEQ ID NO: 5076), QNPP (SEQ ID NO: Sequence number 5077), QNLQ (SEQ ID NO: 5078), QDQE (SEQ ID NO: 5079), QDQQ (SEQ ID NO: 5080), HWQQ (SEQ ID NO: 5081), PNQQ (SEQ ID NO: 5082), PEQQ (SEQ ID NO: 5083), QRTM (SEQ ID NO: 5084), LHQH (SEQ ID NO: 5085), QHRI (SEQ ID NO: 5086), QYIH (SEQ ID NO: 5087), QKFE (SEQ ID NO: 5088), QFPS (SEQ ID NO: 5089), QNPL (SEQ ID NO: 5090), QAIK (SEQ ID NO: 5091), QNRQ (SEQ ID NO: 5092), QYQH (SEQ ID NO: 5093),QNPQ (SEQ ID NO: 5094), QHQL (SEQ ID NO: 5095), QSPP (SEQ ID NO: 5096), QAKL (SEQ ID NO: 5097), KSQQ (SEQ ID NO: 5098), QDRP (SEQ ID NO: 5099), QNLG (SEQ ID NO: 5100), QAFH (SEQ ID NO: 5101), QNAQ (SEQ ID NO: 5102), HNQL (SEQ ID NO: 5103), QKLN (SEQ ID NO: 5104), QNVQ (SEQ ID NO: 5105), QAQQ (SEQ ID NO: 5106), QTPP (SEQ ID NO: 5107), QPPA (SEQ ID NO: 5108), QERP (SEQ ID NO: 5109), QDLQ (SEQ ID NO: 5110), No. 5110), QAMH (SEQ ID NO: 5111), QHPS (SEQ ID NO: 5112), PGLQ (SEQ ID NO: 5113), QGIR (SEQ ID NO: 5114), QAPA (SEQ ID NO: 5115), QIPP (SEQ ID NO: 5116), QTQL (SEQ ID NO: 5117), QAPS (SEQ ID NO: 5118), QNTY (SEQ ID NO: 5119), QDKQ (SEQ ID NO: 5120), QNHL (SEQ ID NO: 5121), QIGM (SEQ ID NO: 5122), LNKQ (SEQ ID NO: 5123), PNQL (SEQ ID NO: 5124), QLQQ (SEQ ID NO: 5125), QRMS (SEQ ID NO: 5126), QGIL (SEQ ID NO: 5127), QDRQ (SEQ ID NO: 5128), RDWQ (SEQ ID NO: 5129), QERS (SEQ ID NO: 5130), QNYQ (SEQ ID NO: 5131), QRTC (SEQ ID NO: 5132), QIGH (SEQ ID NO: 5133), QGAI (SEQ ID NO: 5134), QVPP (SEQ ID NO: 5135), QVQQ (SEQ ID NO: 5136), LMRQ (SEQ ID NO: 5137), QYSV (SEQ ID NO: 5138), QAIT (SEQ ID NO: 5139), QKTL (SEQ ID NO: 5140), QLHH (SEQ ID NO: 5141), QNII (SEQ ID NO: 5142), QGHH (SEQ ID NO: 514 ...GIL (SEQ ID NO: 5127), QDRQ (SEQ ID NO: 5128), RDWQ (SEQ ID NO: 5129), QERS (SEQ ID NO: 5130), QNYQ (SEQ ID NO: 5131), QRTC (SEQ ID NO: 5132), QIGH (SEQ ID NO: 51 No. 5143), QSKV (SEQ ID NO: 5144), QLPS (SEQ ID NO: 5145), IGKQ (SEQ ID NO: 5146), QAIH (SEQ ID NO: 5147), QHGL (SEQ ID NO: 5148), QFMC (SEQ ID NO: 5149), QNQM (SEQ ID NO: 5150), QHLQ (SEQ ID NO: 5151), QPAR (SEQ ID NO: 5152), QSLQ (SEQ ID NO: 5153), QSQL (SEQ ID NO: 5154), HSQQ (SEQ ID NO: 5155), QMPS (SEQ ID NO: 5156), QGSL (SEQ ID NO: 5157), QVPA (SEQ ID NO: 5158), HYQQ (SEQ ID NO: 5159),QVPS (SEQ ID NO: 5160), RGEQ (SEQ ID NO: 5161), PGQQ (SEQ ID NO: 5162), LEQQ (SEQ ID NO: 5163), QNQS (SEQ ID NO: 5164), QKVI (SEQ ID NO: 5165), QNND (SEQ ID NO: 5166), QSVH (SEQ ID NO: 5167), QPLG (SEQ ID NO: 5168), HNQE (SEQ ID NO: 5169), QIQQ (SEQ ID NO: 5170), QVRN (SEQ ID NO: 5171), PSNQ (SEQ ID NO: 5172), QVGH (SEQ ID NO: 5173), QRDI (SEQ ID NO: 5174), QMPN (SEQ ID NO: 5175), RGLQ (SEQ ID NO: 5176), No. 5176), PSLQ (SEQ ID NO: 5177), QRDQ (SEQ ID NO: 5178), QAKG (SEQ ID NO: 5179), QSAH (SEQ ID NO: 5180), QSTM (SEQ ID NO: 5181), QREM (SEQ ID NO: 5182), QYRA (SEQ ID NO: 5183), QRQQ (SEQ ID NO: 5184), QWQQ (SEQ ID NO: 5185), QRMN (SEQ ID NO: 5186), GDSQ (SEQ ID NO: 5187), QKIS (SEQ ID NO: 5188), PSMQ (SEQ ID NO: 5189), SPRQ (SEQ ID NO: 5190), MEQQ (SEQ ID NO: 5191), QYQN (SEQ ID NO: 5192), QIRQ (SEQ ID NO: 5193), QSVQ (SEQ ID NO: 5194), RSQQ (SEQ ID NO: 5195), QNKL (SEQ ID NO: 5196), QIQH (SEQ ID NO: 5197), PRQQ (SEQ ID NO: 5198), HTQQ (SEQ ID NO: 5199), QRQH (SEQ ID NO: 5200), RNQE (SEQ ID NO: 5201), QSKQ (SEQ ID NO: 5202), QNQP (SEQ ID NO: 5203), QSPQ (SEQ ID NO: 5204), QTRQ (SEQ ID NO: 5205), QNLH (SEQ ID NO: 5206), QNQE (SEQ ID NO: 5207), LNQP (SEQ ID NO: 5208), QNQD (SEQ ID NO: 5209), QNQH (SEQ ID NO: 5210), QNQH (SEQ ID NO: 5211), QNQH (SEQ ID NO: 5212), QNQH (SEQ ID NO: 5213), QNQH (SEQ ID NO: 5214), QNQH (SEQ ID NO: 5215), QNQH (SEQ ID NO: 5216), QNQH (SEQ ID NO: 5217), QNQH (SEQ ID NO: 5218), QNQH (SEQ ID NO: 5219), QNQH (SEQ ID NO: 5220), QNQH (SEQ ID NO: 5221), QNQH (SEQ ID NO: 5222), QNQH (SEQ ID NO: 5223), QNQH (SEQ ID NO: 5224), QNQH (SEQ ID No. 5209), QNLL (SEQ ID NO: 5210), QLVI (SEQ ID NO: 5211), RTQE (SEQ ID NO: 5212), QTHQ (SEQ ID NO: 5213), QDQH (SEQ ID NO: 5214), QSQH (SEQ ID NO: 5215), VRQQ (SEQ ID NO: 5216), AWQQ (SEQ ID NO: 5217), QSVP (SEQ ID NO: 5218), QNIQ (SEQ ID NO: 5219), LDQQ (SEQ ID NO: 5220), PDQQ (SEQ ID NO: 5221), ESQQ (SEQ ID NO: 5222), QRQL (SEQ ID NO: 5223), QIIV (SEQ ID NO: 5224), QKQS (SEQ ID NO: 5225),QSHQ (SEQ ID NO: 5226), QFVV (SEQ ID NO: 5227), QSQP (SEQ ID NO: 5228), QNEQ (SEQ ID NO: 5229), INQQ (SEQ ID NO: 5230), RNRQ (SEQ ID NO: 5231), RDQK (SEQ ID NO: 5232), QWKR (SEQ ID NO: 5233), ENRQ (SEQ ID NO: 5234), QTQP (SEQ ID NO: 5235), QKQL (SEQ ID NO: 5236), RNQL (SEQ ID NO: 5237), ISIQ (SEQ ID NO: 5238), QTVC (SEQ ID NO: 5239), QQIM (SEQ ID NO: 5240), LNHQ (SEQ ID NO: 5241), QNQA (SEQ ID NO: 5242), QMIH (SEQ ID NO: 5243), RNHQ (SEQ ID NO: 5244), or QKMN (SEQ ID NO: 5245), (ii) an amino acid sequence containing any part of the amino acid sequence in (i), for example, any two or three amino acids thereof, for example, consecutive amino acids; (iii) an amino acid sequence that contains one, two, or three, but not more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or (iv) an amino acid sequence containing one, two, or three, but not more than four, different amino acids from any one of the amino acid sequences in (i); 71. The composition of embodiment 69 or 70, which is or comprises:

[0066] 72.[N1]-[N2]-[N3]-[N4] (i) any amino acid sequence of SEQ ID NOs: 1800 to 2241; (ii) an amino acid sequence comprising any part of the amino acid sequences in (i), e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids, e.g., consecutive amino acids, of any of them; (iii) an amino acid sequence that contains one, two, or three, but not more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or (iv) The composition of any one of embodiments 69 to 71, which is or comprises an amino acid sequence that contains 1, 2, or 3, but not more than 4, different amino acids relative to any one of the amino acid sequences in (i).

[0067] 73. The composition of any one of embodiments 69 to 72, wherein [N1]-[N2]-[N3]-[N4] is or comprises GSGSPHSKAQNQQ (sequence number 1801).

[0068] 74. The composition of any one of embodiments 69 to 72, wherein [N1]-[N2]-[N3]-[N4] is or comprises GHDSPHKSGQNQQ (sequence number 1800).

[0069] 75. The composition of any one of embodiments 69 to 72, wherein [N1]-[N2]-[N3]-[N4] is or comprises GSGSPHKYGQNQQT (sequence number 910).

[0070] 76. [N0] further includes XA, XB, and XC; (a) position XA is independently selected from T, S, Y, M, A, C, I, R, L, D, F, V, Q, N, H, E, or G; (b) the XB positions are independently selected from I, M, P, E, N, D, S, A, T, G, Q, F, V, L, C, H, R, W, or L; (c) the XC positions are independently selected from N, M, E, G, Y, W, T, I, Q, F, V, A, L, I, P, K, R, H, S, D, or S; Optionally, the protein or peptide comprises an amino acid modification, e.g., a conservative substitution, of any of the aforementioned amino acids in (a)-(c); 76. The composition of any one of embodiments 38 to 75.

[0071] 77.[N0] is TIN, SMN, TIM, YLS, GLS, MPE, MEG, MEY, AEW, CEW, ANN, IPE, ADM, IEY, ADY, IET, MEW, CEY, RIN, MEI, LEY, ADW, IEI, DIM, FEQ, MEF, CDQ, LPE, IEN, MES, AEI, VEY, IIN, TSN, IEV, MEM, AEV, MDA, V EW, AEQ, LEW, MEL, MET, MEA, IES, MEV, CEI, ATN, MDG, QEV, ADQ, NMN, IEM, ISN, TGN, QQQ, HDW, IEG, TI I, TFP, TEK, EIN, TVN, TFN, SIN, TER, TSY, ELH, AIN, SVN, TDN, TFH, TVH, TEN, TSS, TID, TCN, NIN, TEH, AEM, AIK, TDK, TFK, SDQ, TEI, NTN, TET, SIK, TEL, TEA, TAN, TIY, TFS, TES, TTN, TED, TNN, EVH, TIS, T VR, TDR, TIK, NHI, TIP, ESD, TDL, TVP, TVI, AEH, NCL, TVK, NAD, TIT, NCV, TIR, NAL, VIN, TIQ, TEF, TR The composition of embodiment 76 is or comprises E, QGE, SEK, NVN, GGE, EFV, SDK, TEQ, EVQ, TEY, NCW, TDV, SDI, NSI, NSL, EVV, TEP, SEL, TWQ, TEV, AVN, GVL, TLN, TEG, TRD, NAI, AEN, AET, ETA, NNL, or any dipeptide thereof.

[0072] 78. [N0]-[N1]-[N2]-[N3]-[N4] (i) any one of the amino acid sequences of SEQ ID NOs: 2242 to 2886; (ii) an amino acid sequence comprising any part of the amino acid sequences in (i), e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, e.g., consecutive amino acids, of any of them; (iii) an amino acid sequence that contains one, two, or three, but not more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the amino acid sequences in (i); or (iv) an amino acid sequence containing one, two, or three, but not more than four, different amino acids from any one of the amino acid sequences in (i); 78. The composition of embodiment 76 or 77, which is or comprises:

[0073] 79. The composition of any one of embodiments 76 to 78, wherein [N0]-[N1]-[N2]-[N3]-[N4] is or comprises TINGSGSPHSKAQNQQ (sequence number 2242).

[0074] 80. The composition of any one of embodiments 76 to 78, wherein [N0]-[N1]-[N2]-[N3]-[N4] is or comprises TINGHDSPHKSGQNQQ (sequence number 2243).

[0075] 81. The composition of any one of embodiments 76 to 78, wherein [N0]-[N1]-[N2]-[N3]-[N4] is or comprises TINGSGSPHKYGQNQQT (sequence number 5246).

[0076] 82. The composition of any one of embodiments 38-81, wherein [N3] is present immediately after [N2]. 83. The composition of any one of embodiments 38-82, comprising, from N-terminus to C-terminus, [N2]-[N3].

[0077] 84. The composition of any one of embodiments 38-83, comprising, from N-terminus to C-terminus, [N1]-[N2]-[N3]. 85. The composition of any one of embodiments 76-84, comprising, from N-terminus to C-terminus, [N0]-[N1]-[N2]-[N3].

[0078] 86. The composition of any one of embodiments 69-85, comprising, from N-terminus to C-terminus, [N1]-[N2]-[N3]-[N4]. 87. The composition of any one of embodiments 76-86, comprising, from N-terminus to C-terminus, [N0]-[N1]-[N2]-[N3]-[N4].

[0079] 88. The composition according to any one of embodiments 1 to 87, wherein the ligand comprises at least 1 to 5, for example at least 1, 2, 3, 4, or 5, proteins or peptides according to any one of embodiments 35 to 84.

[0080] 89. The composition of embodiment 88, wherein the at least 1 to 5, for example at least 1, 2, 3, 4, or 5, proteins or peptides comprise the same amino acid sequence. 90. The composition of embodiment 88, wherein the at least 1 to 5, for example at least 1, 2, 3, 4, or 5, proteins or peptides comprise different amino acid sequences.

[0081] 91. The composition of any one of embodiments 88 to 90, wherein the at least 1 to 5, e.g., at least 1, 2, 3, 4, or 5, peptides are present in a tandem (e.g., connected directly or indirectly via a linker) or multimeric configuration.

[0082] 92. The composition of any one of embodiments 38-91, wherein the protein or peptide comprises an amino acid sequence of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, or 35 amino acids in length.

[0083] 93. The composition of embodiment 92, wherein the protein or peptide further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all of the amino acids TLKFSVAGPSNMAVQG (SEQ ID NO: 4694), and optionally at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all of the amino acids LKFSVAGPSNMAVQG (SEQ ID NO: 21) are C-terminal to [N4].

[0084] 94. The composition of any one of embodiments 5 to 93, wherein the peptide comprises the amino acid sequence of SPH, and the S comprises a modification, for example, a phosphate group. 95. The composition of any one of embodiments 5 to 94, wherein the peptide comprises the amino acid sequence of SPHSKA (SEQ ID NO: 941), and optionally the S at position 1 numbered according to SEQ ID NO: 941 comprises a modification, for example a phosphate group.

[0085] 96. The composition of any one of embodiments 2 to 94, wherein the peptide comprises the amino acid sequence of SPHK (sequence number 6398), and optionally, S comprises a modification, for example, a phosphate group.

[0086] 97. The composition of any one of embodiments 2 to 94 or 96, wherein the peptide comprises the amino acid sequence HDSPHK (SEQ ID NO: 2), and optionally, S comprises a modification, for example, a phosphate group.

[0087] 98. The composition of any one of embodiments 38-97, wherein the modification comprises a phosphate group. 99. The composition of any one of embodiments 38 to 98, wherein the peptide further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids N-terminal to the amino acid sequence of SPH.

[0088] 100. The composition of any one of embodiments 96-99, wherein the peptide further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids N-terminal to the amino acid sequence of HDSPHK (SEQ ID NO: 2).

[0089] 101. The composition of any one of embodiments 96-100, wherein the peptide further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids C-terminal to the amino acid sequence of HDSPHK (SEQ ID NO: 2).

[0090] 102. The composition of any one of embodiments 94, 95, or 98, wherein the peptide further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids N-terminal to the amino acid sequence of SPHSKA (SEQ ID NO: 941).

[0091] 103. The composition of any one of embodiments 94, 95, 98, or 102, wherein the peptide further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids C-terminal to the amino acid sequence of SPHSKA (SEQ ID NO: 941).

[0092] 104. The composition of any one of embodiments 76 to 94, wherein the peptide further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids N-terminal to the amino acid sequence [N0]-[N2]-[N3]-[N4].

[0093] 105. The composition of any one of embodiments 76-94, or 104, wherein the peptide further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acids C-terminal to the amino acid sequence of [N0]-[N2]-[N3]-[N4].

[0094] 106. The peptide has the following amino acid sequence: (i) GHDSPHKS (SEQ ID NO: 4487) (optionally, S at position 4 of SEQ ID NO: 4487 contains a modification, e.g., a phosphate group); (ii) NGHDSPHKSG (SEQ ID NO: 4489) (optionally, S at position 5 of SEQ ID NO: 4489 contains a modification, e.g., a phosphate group); (iii) INGHDSPHKSGQ (SEQ ID NO: 4490) (optionally, S at position 6 of SEQ ID NO: 4490 contains a modification, e.g., a phosphate group); (iv) TINGHDSPHKSGQN (SEQ ID NO: 4491) (optionally, S at position 7 of SEQ ID NO: 4491 contains a modification, e.g., a phosphate group); (v) KTINGHDSPHKSGQNQ (SEQ ID NO: 4492) (optionally, S at position 8 of SEQ ID NO: 4492 contains a modification, e.g., a phosphate group); (vi) LYYLSKTINGHDSPHKSGQNQQTLKF (SEQ ID NO: 4518) (optionally, S at position 13 of SEQ ID NO: 4518 contains a modification, e.g., a phosphate group); (vii) RLMNPLIDQYLYYLSKTINGHDSPHKSGQNQQTLKFSVAGPSNMAV (SEQ ID NO: 4519) (optionally, S at position 23 of SEQ ID NO: 4519 includes a modification, e.g., a phosphate group); (viii) GSPHSKAQ (SEQ ID NO: 4493) (optionally, S at position 2 of SEQ ID NO: 4493 contains a modification, e.g., a phosphate group); (ix) SGSPHSKAQN (SEQ ID NO: 4494) (optionally, S at position 3 of SEQ ID NO: 4494 contains a modification, e.g., a phosphate group); (x) GSGSPHSKAQNQ (SEQ ID NO: 4495) (optionally, S at position 4 of SEQ ID NO: 4495 contains a modification, e.g., a phosphate group); (xi) NGSGSPHSKAQNQQ (SEQ ID NO: 4496) (optionally, S at position 5 of SEQ ID NO: 4496 contains a modification, e.g., a phosphate group), or (xii) INGSGSPHSKAQNQQT (SEQ ID NO: 4497) (optionally, S at position 6 of SEQ ID NO: 4497 ​​contains a modification, e.g., a phosphate group). 106. The composition of any one of embodiments 5-105, comprising:

[0095] 107. The composition of any one of embodiments 5 to 94 or 96 to 106, wherein the peptide comprises the amino acid sequence NGHDpSPHKSG (sequence number 4515). 108. The composition of any one of embodiments 5 to 94 or 96 to 107, wherein the peptide comprises the amino acid sequence KTINGHDpSPHKSGQNQ (sequence number 4516).

[0096] 109. The composition of any one of embodiments 5 to 94 or 96 to 107, wherein the peptide comprises the amino acid sequence YLSKTINGHDpSPHKSGQNQQTLKFS (sequence number 4517).

[0097] 110. The composition of embodiments 1 to 109, wherein the ligand is a conjugate comprising at least 2 to 5, for example at least 2, 3, 4, or 5, proteins or peptides described in any one of embodiments 38 to 109, and the conjugate comprises a chemical bond, for example a succinimidyl ester or biotin.

[0098] 111. The composition of any one of embodiments 1 to 110, wherein the ligand is a fusion protein comprising at least 2 to 5, for example at least 2, 3, 4, or 5, proteins or peptides of any one of embodiments 3 to 108, and each protein or peptide of the fusion protein is connected directly or via a linker.

[0099] 112. The composition of any one of embodiments 1 to 111, wherein the peptide or protein was identified using phage display. 113. The composition according to any one of embodiments 1 to 37, wherein the ligand is or comprises an aptamer.

[0100] 114. The composition of embodiment 113, wherein the aptamer binds to human, mouse, or NHP ALPL. 115. The composition of embodiment 113 or 114, wherein the aptamer is or comprises DNA, RNA, modified DNA, modified RNA, or a combination thereof.

[0101] 116. The composition of any one of embodiments 114 to 115, wherein the aptamer is fused or conjugated to a therapeutic agent selected from a protein (e.g., an enzyme), an antibody molecule, a nucleic acid molecule (e.g., an RNAi agent), or a small molecule.

[0102] 117. The composition according to any one of embodiments 1 to 37, wherein the ligand is or comprises an antibody molecule that binds to a GPI-anchored protein, such as ALPL. 118. The composition of embodiment 117, wherein the antibody molecule comprises a complete antibody or an antigen-binding fragment.

[0103] 119. The composition of embodiment 117 or 118, wherein the antigen-binding fragment is a Fab or Fab fragment, a F(ab)2 fragment, an Fv fragment, a dAb fragment, a single-chain antibody (scFv) or scFv fragment, a variable region of an antibody, a diabody, a VHH, a camelid antibody, a single-domain antibody, or a nanobody.

[0104] 120. The composition of any one of embodiments 117 to 119, wherein the antibody molecule is a monospecific antibody, a multispecific antibody, such as a bispecific or biparatopic antibody. 121. The composition according to any one of embodiments 117 to 120, wherein the antibody molecule is a human antibody, a humanized antibody, a chimeric antibody, a phage-displayed antibody, a recombinant antibody, or a murine antibody.

[0105] 122. The composition of any one of embodiments 117 to 121, wherein the antibody molecule comprises a half-life extender. 123. The composition of any one of embodiments 117-122, wherein the variable domain of the antibody molecule binds to ALPL, for example human ALPL.

[0106] 124. The composition of any one of embodiments 117 to 123, wherein the antibody molecule is an antibody as provided in Table 40 (e.g., Ab9), AF2910-SP, AF2909, NBP2-67295, LS-B3666, MA524845, 2F4, or a variant thereof.

[0107] 125. The composition of any one of embodiments 117-124, wherein the antibody molecule binds to the same or substantially the same epitope as any one of the antibodies as provided in Table 40 (e.g., Ab9), AF2910-SP, AF2909, NBP2-67295, LS-B3666, MA524845, or 2F4.

[0108] 126. The composition of any one of embodiments 117-125, wherein the antibody molecule competes for binding with any one of the antibodies provided in Table 40 (e.g., Ab9), AF2910-SP, AF2909, NBP2-67295, LS-B3666, MA524845, or 2F4.

[0109] 127. The composition of any one of embodiments 117 to 126, further comprising a therapeutic antibody molecule, for example a multispecific antibody comprising a first binding domain that binds to ALPL (e.g., an anti-ALPL binding domain) and a second binding domain that binds to a therapeutic target.

[0110] 128. A multispecific antibody molecule comprising a first binding domain that binds to ALPL (e.g., an anti-ALPL binding domain) and a second binding domain that binds to a therapeutic target. 129. The multispecific antibody molecule of embodiment 128, wherein the first and / or second binding domain is a full-length antibody or an antigen-binding fragment (e.g., a Fab, F(ab')2, Fv, single-chain Fv (scFv), single-domain antibody, half-arm antibody, diabody (dAb), bivalent antibody, bispecific antibody, or fragment thereof, single-domain variant thereof, or camelid antibody).

[0111] 130. (i) the anti-ALPL binding domain is a Fab and the second binding domain is an scFv; (ii) the anti-ALPL binding domain is a Fab and the second binding domain is a Fab; or (iii) the anti-ALPL binding domain is an scFv and the second binding domain is an scFv; or (iv) the anti-ALPL binding domain is an scFv and the second binding domain is a Fab; 130. A multispecific antibody molecule according to embodiment 128 or 129.

[0112] 131. The multispecific antibody molecule of any one of embodiments 128 to 130, wherein the multispecific antibody molecule comprises an immunoglobulin constant region (e.g., an Fc region). 132. The multispecific antibody molecule of embodiment 131, wherein the immunoglobulin constant region (e.g., Fc region) is linked (e.g., covalently linked) to the first and / or second binding domain.

[0113] 133. The multispecific antibody molecule of any one of embodiments 128 to 132, wherein the first and / or second binding domain comprises a light chain constant region selected from the kappa or lambda light chain constant region, or a fragment thereof.

[0114] 134. The multispecific antibody molecule of any one of embodiments 128 to 133, wherein the first binding domain and the second binding domain comprise a common light chain variable region. 135. The multispecific antibody molecule of any one of embodiments 128 to 134, comprising a dimerization domain, e.g., an interface of the first and second immunoglobulin chain constant regions (e.g., Fc regions).

[0115] 136. The multispecific antibody molecule of embodiment 135, wherein the dimerization domain has been engineered, e.g., mutated, to increase or decrease dimerization, e.g., compared to an unengineered interface.

[0116] 137. The multispecific antibody molecule of embodiment 136, wherein dimerization of the immunoglobulin chain constant regions (e.g., Fc regions) is enhanced by providing one or more of paired depression-protrusions ("knobs-in-a-hole"), electrostatic interactions, or chain exchange at the Fc interface of the first and second Fc regions, e.g., such that a greater ratio of heteromultimers:homomultimers is formed compared to an unengineered interface.

[0117] 138. The multispecific antibody molecule of any one of embodiments 135 to 137, wherein the immunoglobulin chain constant region (e.g., Fc region) comprises an amino acid substitution at one or more of positions 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409 of an antibody, e.g., in the Fc region of a human IgG1.

[0118] 139. The multispecific antibody molecule of any one of embodiments 135 to 138, wherein the immunoglobulin chain constant region (e.g., Fc region) comprises an amino acid substitution selected from T366S, L368A, or Y407V (e.g., corresponding to a depression or hole), or T366W (e.g., corresponding to a protrusion or knob), or a combination thereof.

[0119] 140. The anti-ALPL binding domain comprises a first polypeptide and a second polypeptide, and the second binding domain comprises a third polypeptide and a fourth polypeptide; (i) the first polypeptide comprises, e.g., from N-terminus to C-terminus, a first heavy chain variable region (VH), a first heavy chain constant region 1 (CH1), and a first Fc region that promotes association between the first polypeptide and a third polypeptide, the first Fc region comprising a first heavy chain constant region 2 (CH2) and a first heavy chain constant region 3 (CH3); (ii) the second polypeptide comprises, e.g., from N-terminus to C-terminus, a first light chain variable region (VL) and a first light chain constant region (CL); (iii) the third polypeptide comprises, e.g., from N-terminus to C-terminus, a second heavy chain variable region (VH), a second heavy chain constant region 1 (CH1), and a second Fc region that promotes association between the first polypeptide and the third polypeptide, the second Fc region comprising a second heavy chain constant region 2 (CH2) and a second heavy chain constant region 3 (CH3); (iv) the fourth polypeptide comprises, for example, from the N-terminus to the C-terminus, a second light chain variable region (VL) and a second light chain constant region (CL); A multispecific antibody molecule according to any one of embodiments 128 to 139.

[0120] 141. (i) the anti-ALPL binding domain (e.g., anti-ALPL Fab or scFv) is located N-terminal to the second binding domain (e.g., Fab or scFv) that binds to the therapeutic target; or (i) the second binding domain (e.g., a Fab or scFv) that binds to a therapeutic target is located N-terminal to the anti-ALPL binding domain (e.g., an anti-ALPL Fab or scFv); Optionally, the Fc region is located between the anti-ALPL binding domain and the second binding domain that binds to a therapeutic target. A multispecific antibody molecule according to any one of embodiments 128 to 139.

[0121] 142. The Fc region of the first and / or second binding domain comprises: (i) has reduced, e.g., eliminated, affinity for an Fc receptor, e.g., as compared to a reference, wherein the reference is a wild-type Fc receptor; (ii) containing a mutation at one, two, or all of positions I253 (e.g., I253A), H310 (e.g., H310A or H310Q), and / or H435 (e.g., H435A or H435Q), numbered according to the EU index of Kabat; (iii) has a reduced effector function (e.g., reduced ADCC) when compared to a reference, wherein the reference is a wild-type Fc receptor; (iv) containing a mutation at one, two, three, four, or all of positions L235 (e.g., L235V), F243 (e.g., F243L), R292 (e.g., R292P), Y300 (e.g., Y300L), and P396 (e.g., P396L), numbered according to the EU index of Kabat; 142. A multispecific antibody molecule according to any one of embodiments 128 to 141.

[0122] 143. The therapeutic target is: (i) CNS-related targets, e.g., antigens associated with neurological or neurodegenerative disorders, e.g., β-amyloid, APOE, tau, SOD1, TDP-43, huntingtin (HTT), and / or synuclein; (ii) a muscle- or neuromuscular-related target, e.g., an antigen associated with a myopathy or neuromuscular disorder, or (iii) a neuro-oncology-associated target, e.g., an antigen associated with a neuro-oncology disorder, e.g., HER2, or EGFR (e.g., EGFRvIII). 143. The multispecific antibody molecule of any one of embodiments 128 to 142, comprising:

[0123] 144. The composition of any one of embodiments 1-37, wherein the ligand is or comprises a first Fc polypeptide. 145. The composition of embodiment 144, wherein the first Fc polypeptide is fused or conjugated to an active agent comprising a second Fc polypeptide.

[0124] 146. The composition of embodiment 145, wherein the first Fc polypeptide and the second Fc polypeptide form a dimer. 147. The composition of embodiment 145 or 146, wherein the second Fc polypeptide is fused or conjugated (e.g., directly or indirectly via a linker) to a therapeutic protein or variant thereof (e.g., an enzyme).

[0125] 148. The composition of any one of embodiments 145-147, wherein the second Fc polypeptide is covalently linked to the therapeutic protein or variant thereof. 149. The composition of any one of embodiments 145-148, wherein the second Fc polypeptide is indirectly connected to the therapeutic protein or variant thereof via a linker.

[0126] 150. The composition of embodiment 149, wherein the linker is a peptide linker (e.g., a flexible peptide linker (e.g., a glycine-serine linker) or a protease-sensitive peptide linker), a cleavable linker (e.g., a pH-sensitive linker or an enzyme-sensitive linker), or a non-cleavable linker (e.g., a linker comprising a thioether group or a maleimidocaproyl group).

[0127] 151. The composition of embodiment 149 or 150, wherein the linker is a glycine-serine linker, such as a G4S linker (SEQ ID NO: 6407) or a (G4S)2 linker (SEQ ID NO: 6408).

[0128] 152. The composition of any one of embodiments 147-151, wherein the therapeutic protein is present at the N-terminus of the second Fc polypeptide. 153. The composition of any one of embodiments 147-151, wherein the therapeutic protein is present at the C-terminus of the second Fc polypeptide.

[0129] 154. The composition of any one of embodiments 147-153, wherein the therapeutic protein or functional variant thereof is associated with (e.g., aberrant expression in) a neurological or neurodegenerative disorder, a myopathic or neuromuscular disorder, or a neuro-oncological disorder.

[0130] 155. The composition of any one of embodiments 147-154, wherein the therapeutic protein or functional variant thereof is selected from apolipoprotein E (APOE) (e.g., ApoE2, ApoE3, and / or ApoE4), human motor neuron survival (SMN) 1 or SMN2, glucocerebrosidase (GBA1), aromatic L-amino acid decarboxylase (AADC), aspartoacylase (ASPA), tripeptidyl peptidase I (CLN2), beta-galactosidase (GLB1), N-sulfoglucosamine sulfohydrolase (SGSH), N-acetyl-alpha-glucosaminidase (NAGLU), iduronate 2-sulfatase (IDS), intracellular cholesterol transporter (NPC1), or gigaxonin (GAN).

[0131] 156. The composition of any one of embodiments 144-155, wherein the first Fc polypeptide is fused or conjugated to a second therapeutic protein or variant thereof, e.g., an enzyme, and optionally the therapeutic protein or variant thereof is fused or conjugated to the N-terminus or C-terminus of the first Fc polypeptide.

[0132] 157. The composition of any one of embodiments 145-156, wherein the first Fc polypeptide and the second Fc polypeptide comprise a dimerization domain, e.g., an interface between the first and second Fc polypeptides.

[0133] 158. The composition of embodiment 157, wherein the dimerization domain has been engineered, e.g., mutated, to increase or decrease dimerization, e.g., compared to an unengineered interface.

[0134] 159. The composition of embodiment 158, wherein dimerization between the first Fc polypeptide and the second Fc polypeptide is enhanced by providing one or more of paired depression-protrusions ("knobs-in-holes"), electrostatic interactions, or strand exchange at the Fc interface of the first and second Fc polypeptides, e.g., so that a greater ratio of heteromultimers:homomultimers is formed compared to an unengineered interface.

[0135] 160. The composition of any one of embodiments 145-159, wherein the first Fc polypeptide comprises an amino acid substitution selected from T366S, L368A, or Y407V (e.g., corresponding to a depression or hole) (or a combination thereof).

[0136] 161. The composition of any one of embodiments 145-160, wherein said second Fc polypeptide comprises the amino acid substitution T366W (e.g., corresponding to a protrusion or knob). 162. The composition of any one of embodiments 145-161, wherein the first Fc polypeptide comprises an amino acid substitution selected from T366S, L368A, or Y407V (e.g., corresponding to a depression or hole) (or a combination thereof), and the second Fc polypeptide comprises the amino acid substitution T366W (e.g., corresponding to a protrusion or knob).

[0137] 163. The composition of any one of embodiments 145-162, wherein the second Fc polypeptide comprises an amino acid substitution selected from T366S, L368A, or Y407V (e.g., corresponding to a depression or hole) (or a combination thereof).

[0138] 164. The composition of any one of embodiments 145-159, or 163, wherein the first Fc polypeptide comprises the amino acid substitution T366W (e.g., corresponding to a protrusion or knob).

[0139] 165. The composition of any one of embodiments 145-159, 163, or 164, wherein the second Fc polypeptide comprises an amino acid substitution selected from T366S, L368A, or Y407V (e.g., corresponding to a depression or hole) (or a combination thereof), and the first Fc polypeptide comprises the amino acid substitution T366W (e.g., corresponding to a protrusion or knob).

[0140] 166. The first Fc polypeptide, the second Fc polypeptide, or both, (i) has reduced, e.g., eliminated, affinity for an Fc receptor, e.g., as compared to a reference, wherein the reference is a wild-type Fc receptor; (ii) containing a mutation at one, two, or all of positions I253 (e.g., I253A), H310 (e.g., H310A or H310Q), and / or H435 (e.g., H435A or H435Q), numbered according to the EU index of Kabat; (iii) has a reduced effector function (e.g., reduced ADCC) when compared to a reference, wherein the reference is a wild-type Fc receptor; (iv) containing a mutation at one, two, three, four, or all of positions L235 (e.g., L235V), F243 (e.g., F243L), R292 (e.g., R292P), Y300 (e.g., Y300L), and P396 (e.g., P396L), numbered according to the EU index of Kabat; 166. The composition of any one of embodiments 145 to 165.

[0141] 167. The composition of any one of embodiments 145 to 166, wherein the first Fc polypeptide, the second Fc polypeptide, or both, comprise a half-life extender or amino acid modification that increases serum half-life (e.g., (i) Leu at position 428 and Ser at position 434, or (ii) Ser or Ala at position 434, according to EU numbering).

[0142] 168. The composition of any one of embodiments 144-167, wherein the first Fc polypeptide comprises a protein or peptide of any one of embodiments 35-84.

[0143] 169. The composition of any one of embodiments 168, wherein the protein or peptide is present in the CH3 domain of the first Fc polypeptide. 170. The composition of embodiment 169, wherein the CH3 domain is modified from the CH3 domain of human IgG1, IgG2, IgG3, or IgG4.

[0144] 171. The composition of embodiment 169 or 170, wherein the CH3 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 substitutions at a set of amino acid positions comprising 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421 according to EU numbering.

[0145] 172. The composition of any one of embodiments 168-171, wherein the protein or peptide is present at or near the C-terminus of the Fc polypeptide (e.g., within 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acids of the C-terminus of the therapeutic protein, enzyme, or antibody).

[0146] 173. The composition of any one of embodiments 145-172, wherein the first Fc polypeptide, the second Fc polypeptide, or both the first Fc polypeptide and the second Fc polypeptide do not comprise immunoglobulin heavy and / or light chain variable region sequences or antigen-binding portions thereof.

[0147] 174. The composition of embodiments 1-11 or 15-37, wherein the ligand is a component of a viral particle, such as an AAV particle or a lentivirus. 175. The composition of any one of embodiments 1-11, 15-37, or 174, wherein the ligand is a capsid protein, e.g., a component of an AAV capsid protein.

[0148] 176. The composition of any one of embodiments 1-11, 15-37, 174, or 175, wherein the ligand is a component of the AAV9 capsid or a variant thereof. 177. The composition of any one of embodiments 1-11, 15-37, or 174-176, wherein the ligand is an AAV9 capsid variant comprising a modification, e.g., a substitution, insertion, and / or deletion, in loop IV of AAV9.

[0149] 178. The composition of any one of embodiments 1 to 11, 15 to 37, or 174 to 176, wherein the ligand is an AAV9 capsid variant comprising an amino acid sequence described in any one of embodiments 35 to 84.

[0150] 179. The composition of any one of embodiments 1 to 11, 15 to 37, or 174, wherein the ligand is a lentiviral particle and at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the surface of the lentiviral particle comprises at least 1 to 5, such as at least 1, 2, 3, 4, or 5, proteins or peptides, such as ALPL-binding peptides, or peptides or proteins described in any one of embodiments 38 to 109.

[0151] 180. The composition of any preceding embodiment, wherein the ligand is a small molecule. 181. The composition of embodiment 180, wherein the small molecule is an inhibitor of ALPL, for example, a small molecule that interferes with the dimerization of ALPL.

[0152] 182. The composition of embodiment 180 or 181, wherein the small molecule is an arylsulfonamide, a phosphonate derivative, a pyrazole, a triazole, or an imidazole, and optionally, the small molecule is 2,5-dimethoxy-N-(quinolin-3-yl)benzenesulfonamide (tissue-nonspecific alkaline phosphatase inhibitor (TNAPi)), or 5-((5-chloro-2-methoxyphenyl)sulfonamido)nicotinamide (SBI-425).

[0153] 183. The composition of any one of the preceding embodiments, wherein binding to ALPL results in increased cell transduction, e.g., compared to a reference sequence of SEQ ID NO: 138, e.g., when measured by a transduction assay or a binding / internalization assay as described (e.g., as described in Example 8).

[0154] 184. The composition of any one of the preceding embodiments, wherein binding to ALPL results in increased passage through the blood-brain barrier, e.g., compared to a reference sequence of SEQ ID NO: 138, e.g., when measured by a transduction assay or a binding / internalization assay as described (e.g., as described in Example 8).

[0155] 185. The composition of any one of embodiments 1-127 or 144-184, wherein the therapeutic or diagnostic agent is an antibody molecule or an Fc polypeptide. 186. The composition of embodiment 185, wherein the antibody molecule comprises a complete antibody or an antigen-binding fragment.

[0156] 187. The composition of embodiment 186, wherein the antigen-binding fragment is a Fab or Fab fragment, a F(ab)2 fragment, an Fv fragment, a dAb fragment, a single-chain antibody (scFv) or scFv fragment, a variable region of an antibody, a diabody, a VHH, a camelid antibody, a single-domain antibody, or a nanobody.

[0157] 188. The composition of any one of embodiments 185 to 187, wherein the antibody molecule is a monospecific antibody, a multispecific antibody, such as a bispecific or biparatopic antibody. 189. The composition according to any one of embodiments 185 to 188, wherein the antibody molecule is a human antibody, a humanized antibody, a chimeric antibody, a phage-displayed antibody, a recombinant antibody, or a murine antibody.

[0158] 190. The composition of any one of embodiments 185 to 189, wherein the antibody molecule is an antibody-drug conjugate. 191. The composition of embodiment 190, wherein the antibody molecule is conjugated to a cytotoxic or cytostatic agent, such as a chemotherapeutic or antineoplastic agent.

[0159] 192. The composition of any one of embodiments 185-189, wherein the antibody molecule is conjugated to a radioisotope, such as an α-, β-, or γ-emitter, or a β- and γ-emitter.

[0160] 193. The composition of any one of embodiments 185-192, wherein the antibody molecule comprises an Fc region comprising an amino acid modification that increases serum half-life. 194. The composition of embodiment 193, wherein the amino acid modifications that increase serum half-life include (i) Leu at position 428 and Ser at position 434, or (ii) Ser or Ala at position 434, according to EU numbering.

[0161] 195. The Fc region of the antibody molecule (i) has reduced, e.g., eliminated, affinity for an Fc receptor, e.g., as compared to a reference, wherein the reference is a wild-type Fc receptor; (ii) containing a mutation at one, two, or all of positions I253 (e.g., I253A), H310 (e.g., H310A or H310Q), and / or H435 (e.g., H435A or H435Q), numbered according to the EU index of Kabat; (iii) has a reduced effector function (e.g., reduced ADCC) when compared to a reference, wherein the reference is a wild-type Fc receptor; (iv) containing a mutation at one, two, three, four, or all of positions L235 (e.g., L235V), F243 (e.g., F243L), R292 (e.g., R292P), Y300 (e.g., Y300L), and P396 (e.g., P396L), numbered according to the EU index of Kabat; 195. The composition of embodiment 193 or 194.

[0162] 196. The antibody molecule, (i) CNS-related targets, e.g., antigens associated with neurological or neurodegenerative disorders, e.g., β-amyloid, APOE, tau, SOD1, TDP-43, huntingtin (HTT), and / or synuclein; (ii) a muscle- or neuromuscular-related target, e.g., an antigen associated with a myopathy or neuromuscular disorder, or (iii) a neuro-oncology-associated target, e.g., an antigen associated with a neuro-oncology disorder, e.g., HER2, or EGFR (e.g., EGFRvIII). binds to 196. The composition of any one of embodiments 185 to 195.

[0163] 197. The composition of any one of embodiments 1-127 or 144-118565, wherein the ligand is present in or bound to a carrier, such as an exosome, microvesicle, or lipid nanoparticle (LNP).

[0164] 198. The composition of embodiment 197, wherein the carrier is an exosome or LNP. 199. The composition of embodiment 197 or 198, wherein the ligand is present on the surface of the carrier.

[0165] 200. The composition according to any one of embodiments 197 to 199, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the surface of the carrier comprises at least 1 to 5, for example at least 1, 2, 3, 4, or 5, proteins or peptides according to any one of embodiments 35 to 84.

[0166] 201. The composition of any one of embodiments 197-200, wherein the carrier comprises a therapeutic agent. 202. The composition of any one of embodiments 197 to 201, wherein the carrier comprises an RNAi agent, mRNA, a ribonucleoprotein complex (e.g., a Cas9 / gRNA complex), or a circRNA.

[0167] 203. The composition according to any one of embodiments 197 to 202, wherein the ligand is conjugated to the surface of the carrier by post-insertion. 204. The composition of any one of embodiments 197 to 202, wherein the ligand is conjugated to the surface of the carrier via a covalent bond (e.g., using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry or a thiol-maleimide coupling reaction).

[0168] 205. The composition of any one of embodiments 1-127 or 144-184, wherein the ligand is attached to the RNAi agent directly or via a linker. 206. The composition of embodiment 205, wherein the RNAi agent is a dsRNA, siRNA, shRNA, pre-miRNA, pri-miRNA, miRNA, stRNA, lncRNA, piRNA, antisense oligonucleotide agent (ASO), or snoRNA.

[0169] 207. The composition of embodiment 205 or 206, wherein the RNAi agent is an siRNA or an ASO. 208. The composition of embodiment 206 or 207, wherein the siRNA or ASO comprises at least one modified nucleotide.

[0170] 209. The composition of any one of embodiments 206-208, wherein no more than five of the nucleotides in the sense strand of the siRNA and no more than five of the nucleotides in the antisense strand of the siRNA are unmodified nucleotides.

[0171] 210. The composition of any one of embodiments 206-209, wherein all of the nucleotides of the sense strand of the siRNA and all of the nucleotides of the antisense strand of the siRNA are modified.

[0172] 211. The composition of any one of embodiments 206-208, wherein no more than five of the nucleotides of the ASO are unmodified nucleotides. 212. The composition of any one of embodiments 206-208 or 211, wherein all of the nucleotides of the ASO are modified.

[0173] 213. The composition of any one of embodiments 208 to 312, wherein the modified nucleotide is selected from the group consisting of deoxynucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, non-locked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural base containing nucleotides, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimetics, glycol modified nucleotides, and 2-O-(N-methylacetamido) modified nucleotides, and combinations thereof.

[0174] 214. The composition of any one of embodiments 205 to 213, wherein the RNAi agent modulates, e.g., inhibits, the expression of a CNS-related gene, mRNA, and / or protein.

[0175] 215. The composition of embodiment 214, wherein the CNS-related gene is selected from SOD1, MAPT, APOE, HTT, C9ORF72, TDP-43, APP, BACE, SNCA, ATXN1, ATXN3, ATXN7, SCN1A-SCN5A, SCN8A-SCN11A, SMN, or a combination thereof.

[0176] 216. The composition according to any one of embodiments 205 to 215, wherein the ligand comprises a protein or peptide according to any one of embodiments 35 to 84. 217. The composition according to embodiments 205 to 216, wherein the ligand comprises at least 1 to 5, such as at least 1, 2, 3, 4, or 5, proteins or peptides according to any one of embodiments 35 to 84.

[0177] 218. The composition of embodiment 216 or 217, wherein the at least 1 to 5, e.g., at least 1, 2, 3, 4, or 5, peptides are present in a tandem (e.g., connected directly or indirectly via a linker) or multimeric configuration.

[0178] 219. The composition of any one of embodiments 216-218, wherein the protein or peptide comprises an amino acid sequence of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, or 35 amino acids in length.

[0179] 220. The composition of embodiment 219, wherein the protein or peptide further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all of the amino acids LKFSVAGPSNMAVQG (SEQ ID NO: 21).

[0180] 221. The composition of any one of embodiments 205-221, wherein the ligand is covalently bound to the RNAi agent, for example directly or indirectly via a linker.

[0181] 222. The composition of any one of embodiments 205-221, wherein the ligand is conjugated to the RNAi agent, for example directly or indirectly via a linker.

[0182] 223. The composition of any one of embodiments 205-222, wherein the ligand is conjugated to the RNAi agent via a linker, e.g., a crosslinker. 224. The composition of embodiment 223, wherein the cross-linking agent comprises succinimidyl-4-(N-maleimidomethyl) and / or a saturated or unsaturated hydrocarbon chain (e.g., cyclohexane-1-carboxylate).

[0183] 225. The composition of embodiment 223 or 224, wherein the cross-linking agent comprises succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate. 226. The composition of any one of embodiments 205 to 224, wherein the ligand is conjugated to the RNAi agent via a linker comprising an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide bond, a product of a click reaction, or a carbamate.

[0184] 227. The composition of any one of embodiments 205 to 226, wherein the ligand is conjugated, for example directly or indirectly via a linker, to the N-terminus of at least one strand of the RNAi agent.

[0185] 228. The composition of any one of embodiments 205 to 226, wherein the ligand is conjugated, for example directly or indirectly via a linker, to the C-terminus of at least one strand of the RNAi agent.

[0186] 229. The composition of any one of embodiments 205 to 226, wherein the ligand is conjugated, for example directly or indirectly via a linker, to an internal nucleotide of at least one strand of the RNAi agent.

[0187] 230. The composition of any one of embodiments 227-229, wherein the at least one strand of the RNAi agent is the sense strand. 231. The composition of any one of embodiments 205-230, further comprising a lipophilic moiety.

[0188] 232. The composition of embodiment 231, wherein the lipophilic moiety is an aliphatic, alicyclic, or polycyclic compound. 233. The composition of embodiment 231 or 232, wherein the lipophilic moiety is selected from the group consisting of lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine.

[0189] 234. The composition of any one of embodiments 231-233, wherein the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0190] 235. The composition of embodiment 234, wherein the lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain, such as a saturated or unsaturated C16 hydrocarbon chain. 236. The composition of any one of embodiments 231-235, wherein the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotide(s) at an internal position(s) of the iRNA agent, e.g., the siRNA or ASO.

[0191] 237. The composition of embodiment 236, wherein the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl, or an acyclic moiety based on a serinol or diethanolamine backbone.

[0192] 238. The composition of any one of embodiments 231 to 237, wherein the lipophilic moiety is conjugated to the RNAi agent, e.g., the siRNA or ASO, via a linker containing an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide bond, a product of a click reaction, or a carbamate.

[0193] 239. The composition of any one of embodiments 231-238, wherein the lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage. 240. The composition of any one of embodiments 231 to 239, wherein the lipophilic moiety is conjugated via a biologically cleavable linker selected from the group consisting of functionalized monosaccharides or oligosaccharides of DNA, RNA, disulfides, amides; galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0194] 241. The composition of any one of embodiments 231 to 240, wherein the lipophilic moiety is conjugated, for example directly or indirectly via a linker, to the N-terminus of at least one strand of the RNAi agent.

[0195] 242. The composition of any one of embodiments 231 to 241, wherein the lipophilic moiety is conjugated, for example directly or indirectly via a linker, to the C-terminus of at least one strand of the RNAi agent.

[0196] 243. The composition of any one of embodiments 231 to 242, wherein the lipophilic moiety is conjugated, for example directly or indirectly via a linker, to an internal nucleotide of at least one strand of the RNAi agent.

[0197] 244. The composition of any one of embodiments 241-243, wherein the at least one strand of the RNAi agent is the sense strand. 245. The composition of any one of embodiments 231-244, wherein the ligand and the lipophilic moiety are present on the same strand, e.g., the sense strand.

[0198] 246. The composition of any one of embodiments 231-244, wherein the ligand and the lipophilic moiety are present on different chains. 247. The composition of embodiments 206-246, wherein the 3' end of the sense strand of the siRNA agent is protected via an end cap that is an amine-bearing cyclic group, and the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

[0199] 248. The composition of any one of embodiments 205-247, further comprising an N-acetylgalactosamine (GalNAc) conjugate. 249. The composition of embodiment 248, wherein the GalNAc conjugate is attached via a monovalent linker, or a bivalent, trivalent, or tetravalent branched linker.

[0200] 250. The composition of any one of embodiments 1-127 or 144-184, wherein the active agent is a diagnostic agent. 251. The composition of embodiment 250, wherein the diagnostic agent is or comprises an imaging agent (e.g., a protein or small molecule compound bound to a detectable moiety).

[0201] 252. The composition of embodiment 251, wherein the imaging agent comprises a PET or MRI ligand, or an antibody molecule conjugated to a detectable moiety. 253. The composition of embodiment 252, wherein the detectable moiety is or comprises a radiolabel, a fluorophore, a chromophore, or an affinity tag.

[0202] 254. The composition of embodiment 253, wherein the radiolabel is or comprises tc99m, iodine-123, a spin label, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0203] 255. A vector comprising a polynucleotide encoding a ligand according to any one of embodiments 1 to 127 or 144 to 184. 256. A cell comprising a composition according to any one of embodiments 1 to 127 or 144 to 254, a multispecific antibody molecule according to any one of embodiments 128 to 143, or a vector according to embodiment 255, wherein the cell is optionally a mammalian cell, a cell of the central nervous system, and / or a cell present at the blood-brain barrier.

[0204] 257. A method of making a composition according to any one of embodiments 1-127 or 144-254, comprising: (i) providing a ligand that binds to the GPI-anchored protein, e.g., ALPL, and the active agent; and (ii) incubating the ligand and the active agent under conditions suitable for fusing or binding the ligand to the active agent; Including, thereby producing said composition. The method.

[0205] 258. A pharmaceutical composition comprising a composition according to any one of embodiments 1 to 127 or 144 to 254 or a multispecific antibody molecule according to any one of embodiments 128 to 143, and a pharmaceutically acceptable excipient.

[0206] 259. A method for delivering an active agent, such as a therapeutic or diagnostic agent, to a cell or tissue (e.g., a CNS cell or tissue), comprising administering a composition according to any one of embodiments 1 to 127 or 144 to 254, a multispecific antibody molecule according to any one of embodiments 128 to 143, or a pharmaceutical composition according to embodiment 258.

[0207] 260. The method of embodiment 259, wherein the cell is a cell of a brain region or a spinal cord region, optionally a cell of the frontal cortex, sensory cortex, motor cortex, caudate nucleus, cerebellar cortex, cerebral cortex, brainstem, hippocampus, or thalamus.

[0208] 261. The method of embodiment 259 or 260, wherein the cell or tissue is in a subject. 262. A method for increasing transduction of the central nervous system in a subject (e.g., increasing passage through the blood-brain barrier), comprising administering a composition according to any one of embodiments 1 to 127 or 144 to 254, a multispecific antibody molecule according to any one of embodiments 128 to 143, or a pharmaceutical composition according to embodiment 258.

[0209] 263. The method of embodiment 261 or 262, wherein the subject has, has been diagnosed with, or is at risk of having a genetic disorder, such as a monogenic or polygenic disorder.

[0210] 264. The method of any one of embodiments 261-263, wherein the subject has, has been diagnosed with, or is at risk of having a neurological disorder, e.g., a neurodegenerative disorder.

[0211] 265. The method of any one of embodiments 261-264, wherein the subject has, has been diagnosed with, or is at risk of having a neuro-oncology disorder. 266. The method of any one of embodiments 261-265, wherein the subject has, has been diagnosed with, or is at risk of having a myopathy or neuromuscular disorder.

[0212] 267. A method for treating a subject having or diagnosed as having a genetic disorder, such as a monogenic or polygenic disorder, comprising administering to the subject a composition according to any one of embodiments 1 to 127 or 144 to 254, a multispecific antibody molecule according to any one of embodiments 128 to 143, or a pharmaceutical composition according to embodiment 258.

[0213] 268. A method for treating a subject having or diagnosed as having a neurological disorder, such as a neurodegenerative disorder, comprising administering to the subject an effective amount of a composition according to any one of embodiments 1 to 127 or 144 to 254, a multispecific antibody molecule according to any one of embodiments 128 to 143, or a pharmaceutical composition according to embodiment 258.

[0214] 269. A method for treating a subject having or diagnosed as having a myopathy or neuromuscular disorder, comprising administering to the subject an effective amount of the composition of any one of embodiments 1 to 127 or 144 to 254, the multispecific antibody molecule of any one of embodiments 128 to 143, or the pharmaceutical composition of embodiment 258.

[0215] 270. A method for treating a subject having or diagnosed as having a neuro-oncology disorder, comprising administering to the subject an effective amount of the composition of any one of embodiments 1 to 127 or 144 to 254, the multispecific antibody molecule of any one of embodiments 128 to 143, or the pharmaceutical composition of embodiment 258.

[0216] 271. The method of any one of embodiments 263-270, wherein the genetic, neurological, neurodegenerative, myopathic, neuromuscular, or neuro-oncological disorder is Huntington's disease, amyotrophic lateral sclerosis (ALS), Gaucher disease, dementia with Lewy bodies, Parkinson's disease, spinal muscular atrophy, Alzheimer's disease, leukodystrophy (e.g., Alexander disease, autosomal dominant leukodystrophy with autonomic neuropathy (ADLD), Canavan disease, cerebrotendinous xanthomatosis (CTX), metachromatic leukodystrophy (MLD), Pelizaeus-Merzbacher disease, or Refsum disease), or cancer (e.g., HER2 / neu-positive cancer or glioblastoma).

[0217] 272. The method of any one of embodiments 266-271, wherein treating comprises preventing the progression of said disease or disorder in said subject. 273. The method of embodiments 261-272, wherein the subject is a human.

[0218] 274. The method of any one of embodiments 261-273, wherein the composition is administered to the subject intravenously, via intracisternal injection (ICM), intracerebrally, intrathecally, intracerebroventricularly, via intraparenchymal administration, intraarterially, or intramuscularly.

[0219] 275. The method of any one of embodiments 261 to 274, wherein the composition is administered to the subject via focused ultrasound (FUS), for example in combination with intravenous administration of microbubbles (FUS-MB), or via MRI-guided FUS in combination with intravenous administration.

[0220] 276. The method of any one of embodiments 261-275, wherein the composition is administered intravenously to the subject. 277. The method of any one of embodiments 261-276, wherein the composition is administered to the subject via intracisternal injection (ICM).

[0221] 278. The method of any one of embodiments 261-277, wherein the composition is administered to the subject intra-arterially. 279. The method of any one of embodiments 274-278, wherein administration of the composition results in a decrease in the presence, level, and / or activity of a gene, mRNA, protein, or a combination thereof.

[0222] 280. The method of any one of embodiments 274-278, wherein administration of the composition results in an increase in the presence, level, and / or activity of a gene, mRNA, protein, or a combination thereof.

[0223] 281. A composition according to any one of embodiments 1 to 127 or 144 to 254, a multispecific antibody molecule according to any one of embodiments 128 to 143, or a pharmaceutical composition according to embodiment 258, for use in a method for delivering a payload to a cell or tissue.

[0224] 282. A composition according to any one of embodiments 1 to 127 or 144 to 254, a multispecific antibody molecule according to any one of embodiments 128 to 143, or a pharmaceutical composition according to embodiment 258 for use in a method for treating a genetic, neurological, neurodegenerative, myopathic, neuromuscular, or neuro-oncological disorder.

[0225] 283. A composition according to any one of embodiments 1 to 127 or 144 to 254, a multispecific antibody molecule according to any one of embodiments 128 to 143, or a pharmaceutical composition according to embodiment 258, for use in the manufacture of a medicament.

[0226] 284. A composition according to any one of embodiments 1 to 127 or 144 to 254, a multispecific antibody molecule according to any one of embodiments 128 to 143, or a pharmaceutical composition according to embodiment 258, for use in a method for increasing transduction of the central nervous system in a subject (e.g., increasing passage through the blood-brain barrier).

[0227] 285. Use of a composition according to any one of embodiments 1 to 127 or 144 to 254, a multispecific antibody molecule according to any one of embodiments 128 to 143, or a pharmaceutical composition according to embodiment 258 in the manufacture of a medicament.

[0228] 286. Use of a composition according to any one of embodiments 1 to 127 or 144 to 254, a multispecific antibody molecule according to any one of embodiments 128 to 143, or a pharmaceutical composition according to embodiment 258 in the manufacture of a medicament for treating a genetic, neurological, neurodegenerative, myopathic, neuromuscular, or neuro-oncological disorder.

[0229] 287. Use of a composition according to any one of embodiments 1 to 127 or 144 to 254, a multispecific antibody molecule according to any one of embodiments 128 to 143, or a pharmaceutical composition according to embodiment 258 in the manufacture of a medicament for increasing central nervous system transduction (e.g., increasing passage through the blood-brain barrier) in a subject.

[0230] The details of one or more embodiments of the present disclosure are set forth in the accompanying description below. Other features, objects, and advantages of the present disclosure will become apparent from the specification. As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Certain terms are defined in the definitions section and throughout.

[0231] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0232] [Figure 1A] 1 is a violin diagram showing expression levels of payloads on the Y-axis in various cell types shown on the X-axis, including, from left to right, microglia, astrocytes, endothelial cell subset 1, vascular smooth muscle cells, pericytes, endothelial cell subset 2, committed oligodendrocytes, macrophages, vascular and leptomeningeal cells, committed oligodendrocyte subset 2, and mature oligodendrocytes. Relates to data shown in Example 7 and Table 22. [Figure 1B] Violin diagram showing the expression levels of ALPL on the Y-axis in various cell types indicated on the X-axis, including, from left to right, microglia, astrocytes, endothelial cell subset 1, vascular smooth muscle cells, pericytes, endothelial cell subset 2, committed oligodendrocytes, macrophages, vascular and leptomeningeal cells, committed oligodendrocyte subset 2, and mature oligodendrocytes. [Figure 2A] 1 is a graph showing the binding of TTM-002 to ALPL at increasing concentrations of AAV over time by surface plasmon resonance (SPR). [Figure 2B] 1 is a graph showing the binding of AAV9 to ALPL over time by SPR at increasing concentrations of AAV. [Figure 2C] 1 is a graph showing the binding of ALPL to TTM-002 over time by surface plasmon resonance (SPR) at increasing concentrations of ALPL. [Figure 2D] 1 is a graph showing the binding of ALPL to AAV9 over time by SPR at increasing concentrations of ALPL. [Figure 3] (A) is a graph showing the binding of ALPL to TTM-002 at increasing concentrations of ALPL over time by surface plasmon resonance (SPR) at pH 7.4, where the left half of the graph shows association and the right half of the graph shows dissociation. (B) is a graph showing the binding of ALPL to TTM-002 at increasing concentrations of ALPL over time by surface plasmon resonance (SPR) at pH 5.5, where the left half of the graph shows association and the right half of the graph shows dissociation. [Figure 4]Graph showing luciferase activity (RLU) as a measure of TTM-002 (right side of graph) or AAV9 (left side of graph) at 24 hours and 48 hours post-transfection with ALPL-targeting siRNA1, 2, or both siRNA1 and 2, or a non-ALPL control siRNA that did not knock down ALPL. [Figure 5A]

[0023] Figure 1 is a series of graphs demonstrating the effect of TNAPi, a small molecule inhibitor of the ALPL receptor, on TTM-002 transduction in ALPL-expressing HeLa cells in vitro.

[0024] Figure 1 is a graph showing luciferase activity as a measure of transduction of TTM-002 capsid variants or AAV9 capsid variants in the presence of increasing concentrations of TNAPi inhibitor. The concentrations tested, from left to right on the x-axis, include 0 nM (no inhibitor control), 24 nM, 48 nM, 95 nM, 190 nM, and 380 nM. [Figure 5B]

[0023] Figure 1 is a series of graphs demonstrating the effect of TNAPi, a small molecule inhibitor of the ALPL receptor, on TTM-002 transduction in ALPL-expressing HeLa cells in vitro.

[0024] Figure 1 is a graph showing luciferase activity as a measure of transduction of TTM-002 capsid variants in the presence of increasing concentrations of TNAPi inhibitor or DMSO vehicle control. Concentrations tested, from left to right on the x-axis, include 0 nM, 0.019 nM, 0.19 nM, 1.9 nM, 19 nM, and 190 nM. [Figure 5C]

[0023] Figure 1 is a series of graphs demonstrating the effect of TNAPi, a small molecule inhibitor of the ALPL receptor, on TTM-002 transduction in ALPL-expressing HeLa cells in vitro.

[0024] Figure 2 is a graph showing the IC50 of TNAPi inhibitors for TTM-002 capsid variants compared to vehicle control. [Figure 6A]

[0023] Figure 1 is a series of graphs demonstrating the effect of SBI-425, a small molecule inhibitor of the ALPL receptor, on TTM-002 transduction in vitro in HeLa cells expressing ALPL.

[0024] Figure 1 shows luciferase activity as a measure of transduction of TTM-002 capsid variants in the presence of increasing concentrations of the SBI-425 inhibitor or a DMSO vehicle control.

[0025] Concentrations tested, from left to right on the x-axis, include 0 nM, 0.00019 nM, 0.0019 nM, 0.019 nM, 0.19 nM, 1.9 nM, or 19.0 nM. [Figure 6B]

[0023] Figure 1 is a series of graphs demonstrating the effect of SBI-425, a small molecule inhibitor of the ALPL receptor, on TTM-002 transduction in vitro in HeLa cells expressing ALPL.

[0024] Figure 1 shows luciferase activity as a measure of transduction of AAV9 capsid variants in the presence of increasing concentrations of the SBI-425 inhibitor or DMSO vehicle control.

[0025] Concentrations tested, from left to right on the x-axis, include 0 nM, 0.00019 nM, 0.0019 nM, 0.019 nM, 0.19 nM, 1.9 nM, or 19.0 nM. [Figure 6C]

[0023] Figure 1 is a series of graphs demonstrating the effect of SBI-425, a small molecule inhibitor of the ALPL receptor, on TTM-002 transduction in ALPL-expressing HeLa cells in vitro.

[0024] Figure 1 is a graph showing the IC50 of the SNBI-425 inhibitor for TTM-002 capsid variants compared to vehicle control. [Figure 7] A is the effect of increasing concentrations of ALPL GSGSKTINGHDSPHKSGQNQ (SEQ ID NO: 4503) (left graph) or

[0233] [ka]

[0234] A is a series of graphs demonstrating the binding of ALPL to GSGSNGHDSPHKSG (SEQ ID NO: 4500) (left graph) or GSGSNGHDSPHKSG (right graph) over time (seconds) by SPR at increasing concentrations of ALPL.

[0235] [ka]

[0236] (Right graph) over time (seconds) by SPR. [Figure 8] A is the result of increasing concentrations of the following peptide: GSGSKTINGHDSPHKSGQNQ (SEQ ID NO: 4503) (left graph) or

[0237] [ka]

[0238] A is a series of graphs demonstrating the binding of GSGSNGHDSPHKSG (SEQ ID NO: 4500) (left graph) or (right graph) to ALPL over time (seconds) by SPR at increasing concentrations of the following peptides:

[0239] [ka]

[0240] 1 is a series of graphs demonstrating the binding of (right graph) to ALPL over time (seconds) by SPR. [Figure 9A] GSGSKTINGHDSPHKSGQNQ (SEQ ID NO: 4503) (left graph) or

[0241] [ka]

[0242] (Right graph) is a series of graphs showing the binding of ALPL over time by BioLayer Interferometry (BLI) / Octet. [Figure 9B]GSGSNGHDSPHKSG (SEQ ID NO: 4500) (left graph) or

[0243] [ka]

[0244] (Right graph) is a series of graphs showing the binding of ALPL over time by BioLayer Interferometry (BLI) / Octet. [Figure 10] A is increasing concentrations of GSGSNGHDSPHKSG (SEQ ID NO: 4500) or

[0245] [ka]

[0246] A is a graph showing the binding (OD450) of ALPL to increasing concentrations of GSGSKTINGHDSPHKSGQNQ (SEQ ID NO: 4503) or (μg / mL) by ELISA.

[0247] [ka]

[0248] 1 is a graph showing the binding (OD450) of ALPL to (μg / mL) by ELISA. [Figure 11A] Graph showing antibody concentrations in the upper chamber measured in μg / ml before the transcytosis assay. Antibodies from left to right on the X-axis include PT3 (non-ALPL binding control), MOPC (isotype control), Ab9 (ALPL binding antibody), and Ab22 (ALPL binding antibody). [Figure 11B] Graph showing antibody concentration (pg / ml) in the lower chamber for antibodies indicated on the X-axis. [Figure 11C]1 is a graph showing the percentage of antibody detected in the lower chamber relative to loading, for antibodies indicated on the X-axis. The left portion of the graph (labeled "MDCK ALPL single clone") shows the percentage in single-clone ALPL-expressing MDCK cells generated in Example 8, and the right portion of the graph (labeled "MDCK") shows the percentage in MDCK cells that do not express ALPL. [Figure 12] 1 is a graph showing luciferase activity (RLU) in cells preincubated with antibodies against ALPL listed on the X-axis and described in Table 40, and subsequently transduced with AAV particles containing the TTM-002 capsid variant and encoding a GFP luciferase transgene. The low luciferase activity measured indicates that the antibody was able to compete with the TTM-002 capsid variant for binding to ALPL. DETAILED DESCRIPTION OF THE INVENTION

[0249] Described herein are compositions, e.g., fused or conjugated molecules, comprising, among other things, a ligand that binds to a glycosylphosphatidylinositol (GPI)-anchored protein, e.g., alkaline phosphatase (ALPL), and an active agent, e.g., a therapeutic or diagnostic agent. In some embodiments, the ligand is fused or conjugated, e.g., covalently or non-covalently, to the active agent. In some embodiments, the GPI-anchored protein is conserved in at least two to three species, e.g., at least three species (e.g., mouse, NHP (e.g., Macaca fascicularis), and / or human). In some embodiments, the GPI-anchored protein is present on the surface of cells at the blood-brain barrier. In some embodiments, the GPI-anchored protein is ALPL, e.g., human or mouse ALPL.

[0250] In some embodiments, the ligand to be used in the compositions described herein is a ligand capable of binding to ALPL. In some embodiments, the ligand of the present disclosure is or includes a peptide, a protein, an antibody molecule, a nucleic acid molecule (e.g., an aptamer), or a small molecule. In some embodiments, the active agent described herein is a therapeutic agent (e.g., a protein (e.g., an enzyme), an antibody molecule, a nucleic acid molecule (e.g., an RNAi agent), or a small molecule). In some embodiments, the active agent described herein is a diagnostic agent.

[0251] Without wishing to be bound by theory, in some embodiments, it is believed that fusion or attachment, e.g., covalently (e.g., directly or via a linker) or non-covalently, of a ligand capable of binding to ALPL to an active agent increases passage of the blood-brain barrier by the active agent compared to an active agent that is not fused or attached to a ligand capable of binding to ALPL. Without wishing to be bound by theory, in some embodiments, it is believed that fusion or attachment, e.g., covalently (e.g., directly or via a linker) or non-covalently, of a peptide comprising an amino acid sequence provided herein, e.g., Tables 1, 2A, 2B, 2C, 13-19 (e.g., SEQ ID NOs: 2, 941, or 943), to an active agent, e.g., a therapeutic or diagnostic agent, can enhance passage of the blood-brain barrier and biodistribution within the CNS of the active agent compared to the active agent alone.

[0252] Ligand Disclosed herein are ligands capable of binding to proteins present on cells, e.g., cells present at the blood-brain barrier. In some embodiments, the ligand binds to a GPI-anchored protein. In some embodiments, the GPI-anchored protein is conserved in at least two to three species, e.g., at least three species (e.g., mouse, NHP (e.g., Macaca fascicularis), and / or human). In some embodiments, the GPI-anchored protein is alkaline phosphatase issue-nonspecific isozyme (NM_000478.4, which is incorporated herein by reference) (ALPL).

[0253] ALPL is part of a family of membrane-bound glycoproteins that hydrolyze monophosphate esters at high pH (see, e.g., Weiss et al., Isolation and characterization of a cDNA encoding a human liver / bone / kidney-type alkaline phosphatase. Proc. Nat. Acad. Sci., 83:7182-7186 (1986), the contents of which are incorporated herein by reference in their entirety). ALPL is highly conserved among humans, mice, and cynomolgus monkeys (Macaca fascicularis) when compared by sequence alignment (e.g., as shown in Table 24). In addition, in humans, ALPL is expressed in endothelial cells and neurons, and at lower levels in astrocytes. In humans, the highest level of ALPL expression is in endothelial cells. In mice, ALPL is more highly expressed in astrocytes, oligodendrocyte precursor cells (OPCs), and to a lesser extent in endothelial cells. Without wishing to be bound by theory, in some embodiments, the high conservation of the ALPL receptor protein across species is believed to predict the cross-species compatibility of the AAV capsid variants described herein.

[0254] In some embodiments, the ligand binds to ALPL comprising an amino acid sequence encoded by an amino acid or nucleotide sequence provided in Table 32, or a sequence at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical thereto. In some embodiments, the ligand binds to human ALPL protein, e.g., a human ALPL protein comprising the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical thereto. In some embodiments, the ALPL is mouse ALPL, e.g., a mouse ALPL comprising the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical thereto.

[0255] [Table 1-1]

[0256] [Table 1-2]

[0257] [Table 1-3]

[0258] [Table 1-4]

[0259] [Table 1-5]

[0260] [Table 1-6]

[0261] [Table 1-7]

[0262] [Table 1-8]

[0263] In some embodiments, the GPI-anchored protein described herein is CD59, LY6E, CA4, GPC5, NTM, HYAL2, LSAMP, BST2, EMP2, ALPL, CPM, NCAM1, EFNA1, PIBF1, SEC24B, PRNP, TFPI, OPCML, CD109, DPM3, CNTN4, PIGN, HBP1, CNTN2, CD55, NEGR1, EFNA5, RECK, NRN1, CNTN1, GPAA1, PGAP1, PIGF, PIGK, MDGA2, DPM1, SVIP, NTNG1, CNTN5, GPC6, PIGG, TMEM8A, THY1, GPIHBP1, PIGT, PIGL, ZFAND2B, PLAUR, DPM2, or GPC1.

[0264] In some embodiments, the ligand is or comprises a peptide, a protein, an antibody molecule, a nucleic acid molecule (eg, an aptamer), or a small molecule. In some embodiments, the ligand is not a component of a viral particle, e.g., an AAV viral particle. In some embodiments, the ligand is not a component of a capsid protein, e.g., an AAV capsid protein.

[0265] In some embodiments, the ligand is covalently attached, e.g., directly or indirectly via a linker, to an active agent (e.g., a therapeutic or diagnostic agent) described herein. In some embodiments, the ligand is conjugated, e.g., directly or indirectly via a linker, to an active agent (e.g., a therapeutic or diagnostic agent) described herein. In some embodiments, the ligand is fused to the active agent, e.g., as part of a fusion peptide or protein.

[0266] In some embodiments, the ligand is directly conjugated to the active agent described herein.In some embodiments, direct conjugation includes, but is not limited to, forming a covalent bond between the reactive group on the ligand and the corresponding group or acceptor on the active agent; modifying the ligand or active agent (for example, genetic modification) so that it is conjugated to a reactive group (for example, sulfhydryl group or carboxyl group) that forms a covalent bond with other molecules under appropriate conditions and conjugates.For example, desired active groups can be introduced into the ligand, the active agent, or both, and disulfide bonds can be formed.

[0267] In some embodiments, the ligand is non-covalently bound or fused, e.g., conjugated, to the active agent, e.g., by hydrophobic bonds, electrostatic interactions, and / or ionic bonds.

[0268] In some embodiments, the ligand is conjugated to the ligand via a linker. In some embodiments, the linker is a cleavable linker (e.g., an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker). In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is an enzyme-sensitive linker or a pH-sensitive linker. In some embodiments, the pH-sensitive linker comprises a hydrazine / hydrazone linker or a disulfide linker. In some embodiments, the enzyme-sensitive linker comprises a peptide-based linker, e.g., a peptide linker that is sensitive to a protease (e.g., a lysosomal protease), or a beta-glucuronide linker. In some embodiments, the non-cleavable linker is a linker comprising a thioether group or a maleimidocaproyl group. In some embodiments, the linker is a chemical linker. In some embodiments, the linker is a peptide linker, e.g., a flexible polypeptide. In some embodiments, the linker is a glycine-serine linker. In some embodiments, the linker is a crosslinker selected from, for example, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, or SVSB (succinimidyl (4-vinylsulfone) benzoate).

[0269] In some embodiments, ligands may be conjugated to active agents described herein using bifunctional protein binding agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate H), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0270] In some embodiments, the ligand and active agent are fused or conjugated post-translationally, for example, using click chemistry, hi some embodiments, the ligand and active agent are fused or conjugated via chemically induced dimerization.

[0271] In some embodiments, the ligand may be conjugated to the active agent described herein using the methods described in: Shadish JA and DeForest CA, Site-Selective Protein Modification: From Functionalized Proteins to Functional Biomaterials. Matter 2020 2:50-70; Fu et al. Antibody drug conjugate: the “biological missile” for targeted cancer therapy. Signal Transduction and Targeted Therapy 2022 7:93; and Drago et al. Unlocking the potential of antibody-drug conjugates for cancer therapy. Nat Rev Clin Oncol 2021 18:327-344; Eyford et al. A Nanomule Peptide Carrier Delivers siRNA Across the Intact Blood-Brain Barrier to Attenuate Ischemic Stroke. Front Mol Biosci 2021 8:611367; A microfluidic method for synthesis of transferrin-lipid nanoparticles loaded with siRNA LOR-1284 for therapy of acute myeloid leukemia. Nanoscale 2014 6(16):9742-9751; or US20220125823A1; all of which are incorporated by reference in their entirety.

[0272] In some embodiments, the ligand is N-terminal to the active agent. In some embodiments, the ligand is C-terminal to the active agent. In some embodiments, the ligand is fused or conjugated at or near the C-terminus of the active agent, which is a therapeutic protein, enzyme, or antibody molecule. In some embodiments, the ligand is fused or conjugated within 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acids of the C-terminus of the therapeutic protein, enzyme, or antibody.

[0273] In some embodiments, binding to ALPL results in one or both of increased cell signaling and / or transcytosis, hi some embodiments, binding to ALPL results in increased passage across the blood-brain barrier, for example, compared to a reference sequence of SEQ ID NO: 138.

[0274] peptide Disclosed herein is a ligand comprising a peptide or protein for binding to a protein on a cell, for example, a cell present at the blood-brain barrier.In some embodiments, the protein is a GPI-anchored protein.In some embodiments, the protein is ALPL, for example, human or mouse ALPL.In some embodiments, the peptide is an isolated, for example, recombinant peptide.In some embodiments, the nucleic acid encoding the peptide is an isolated, for example, recombinant nucleic acid.

[0275] The present disclosure also provides peptides and related AAV particles, including AAV capsid variants and peptides, for enhanced or improved transduction of target cells or tissues (e.g., cells or tissues of the CNS). In some embodiments, the peptides can increase the distribution of AAV particles to cells, regions, or tissues of the CNS. Cells of the CNS can be, but are not limited to, brain support cells, such as neurons (e.g., excitatory, inhibitory, motor, sensory, autonomic, sympathetic, parasympathetic, Purkinje, Betz, etc.), glial cells (e.g., microglia, astrocytes, oligodendrocytes), and / or immune cells (e.g., T cells). Tissues of the CNS can be, but are not limited to, the cortex (e.g., frontal, parietal, occipital, and / or temporal regions), thalamus, hypothalamus, striatum, nucleus pallidum, caudate nucleus, hippocampus, entorhinal cortex, basal ganglia, or deep cerebellar nuclei. In some embodiments, the peptide may increase the distribution of AAV particles to the CNS (e.g., the cortex) after intravenous administration.

[0276] In some embodiments, the peptides of the ligands described herein can vary in length. In some embodiments, the peptides are about 3 to about 20 amino acids in length. By way of non-limiting example, the peptides may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 3-5, 3-8, 3-10, 3-12, 3-15, 3-18, 3-20, 5-10, 5-15, 5-20, 10-12, 10-15, 10-20, 12-20, or 15-20 amino acids in length. In some embodiments, the peptides comprise about 6-12 amino acids in length, e.g., about 9 amino acids in length. In some embodiments, the peptides comprise about 5-10 amino acids in length, e.g., about 7 amino acids in length. In some embodiments, the peptides comprise about 7-11 amino acids in length, e.g., about 8 amino acids in length. In some embodiments, the peptide comprises about 4-9 amino acids in length, for example, about 6 amino acids in length.

[0277] In some embodiments, the ligands described herein comprise proteins or peptides comprising a sequence set forth in Table 1 (e.g., comprising the amino acid sequence of any one of SEQ ID NOS: 200-940, 1800-2241, 2242-2886, or 2887-3076). In some embodiments, the peptides may comprise a sequence set forth in Table 2A, 2B, or 2C. In some embodiments, the peptides may comprise a sequence set forth in Table 13 or 14. In some embodiments, the peptides may comprise a sequence set forth in Table 15. In some embodiments, the peptides may comprise a sequence set forth in Table 16. In some embodiments, the peptides may comprise a sequence set forth in Table 17. In some embodiments, the peptides may comprise a sequence set forth in Table 18. In some embodiments, the peptides may comprise a sequence set forth in Table 19. In some embodiments, the peptides are isolated, e.g., recombinant.

[0278] [Table 2-1]

[0279] [Table 2-2]

[0280] [Table 2-3]

[0281] [Table 2-4]

[0282] [Table 2-5]

[0283] [Table 2-6]

[0284]

Table 2-7

[0285]

Table 2-8

[0286]

Table 2-9

[0287]

Table 2-10

[0288]

Table 2-11

[0289]

Table 2-12

[0290]

Table 2-13

[0291]

Table 2-14

[0292]

Table 2-15

[0293]

Table 2-16

[0294]

Table 2-17

[0295]

Table 2-18

[0296]

Table 2-19

[0297]

Table 2-20

[0298]

Table 2-21

[0299]

Table 2-22

[0300]

Table 2-23

[0301]

Table 2-24

[0302]

Table 2-25

[0303]

Table 2-26

[0304]

Table 2-27

[0305]

Table 2-28

[0306]

Table 2-29

[0307]

Table 2-30

[0308]

Table 2-31

[0309]

Table 2-32

[0310]

Table 2-33

[0311]

Table 2-34

[0312]

Table 2-35

[0313]

Table 3

[0314]

Table 4

[0315] [Table 5]

[0316] In some embodiments, the ligands described herein include proteins or peptides comprising an amino acid sequence having the formula [N1]-[N2]-[N3], where [N2] comprises the amino acid sequence of SPH, and [N3] comprises X4, X5, and X6, wherein at least one of X4, X5, or X6 is a basic amino acid, e.g., K or R. In some embodiments, the X4 position of [N2] is K. In some embodiments, the X5 position of [N2] is K.

[0317] In some embodiments, [N1] comprises X1, X2, and X3, and at least one of X1, X2, or X3 is G. In some embodiments, the X1 position of [N1] is independently selected from G, V, R, D, E, M, T, I, S, A, N, L, K, H, P, W, or C. In some embodiments, the X2 position of [N1] is independently selected from S, V, L, N, D, H, R, P, G, T, I, A, E, Y, M, or Q. In some embodiments, the X3 position of [N1] is independently selected from G, C, L, D, E, Y, H, V, A, N, P, or S. In some embodiments, [N1] comprises GS, SG, GH, HD, GQ, QD, VS, CS, GR, RG, QS, SH, MS, RN, TS, IS, GP, ES, SS, GN, AS, NS, LS, GG, KS, GT, PS, RS, GI, WS, DS, ID, GL, DA, DG, ME, EN, KN, KE, AI, NG, PG, TG, SV, IG, LG, AG, EG, SA, YD, HE, HG, RD, ND, PD, MG, QV, DD, HN, HP, GY, GM, GD, or HS. In some embodiments, [N1] comprises GS, SG, GH, or HD. In some embodiments, [N1] is or comprises GSG, GHD, GQD, VSG, CSG, GRG, CSH, GQS, GSH, RVG, GSC, GLL, GDD, GHE, GNY, MSG, RNG, TSG, ISG, GPG, ESG, SSG, GNG, ASG, NSG, LSG, GGG, KSG, HSG, GTG, PSG, GSV, RSG, GIG, WSG, DSG, IDG, GLG, DAG, DGG, MEG, ENG, GSA, KNG, KEG, AIG, GYD, GHG, GRD, GND, GPD, GMG, GQV, GHN, GHP, or GHS. In some embodiments, [N1] is or comprises GSG. In some embodiments, [N1] is or comprises GHD. In some embodiments, [N1]-[N2] is selected from the group consisting of SGSPH (SEQ ID NO: 4752), HDSPH (SEQ ID NO: 4703), QDSPH (SEQ ID NO: 4753), RGSPH (SEQ ID NO: 4754), SHSPH (SEQ ID NO: 4755), QSSPH (SEQ ID NO: 4756), DDSPH (SEQ ID NO: 4757), HESPH (SEQ ID NO: 4758),NYSPH (SEQ ID NO: 4759), VGSPH (SEQ ID NO: 4760), SCSPH (SEQ ID NO: 4761), LLSPH (SEQ ID NO: 4762), NGSPH (SEQ ID NO: 4763), PGSPH (SEQ ID NO: 4764), GGSPH (SEQ ID NO: 4765), TGSPH (SEQ ID NO: 4766), SVSPH (SEQ ID NO: 4767), IGSPH (SEQ ID NO: 4768), DGSPH (SEQ ID NO: 4769), LGSPH (SEQ ID NO: 4770), AGSPH (SEQ ID NO: 4771), EGSPH (SEQ ID NO: 4772), SASPH (SEQ ID NO: 4773), YDSPH (SEQ ID NO: 4774), HGSPH (SEQ ID NO: 4775), RDSPH (SEQ ID NO: 4776), NDSPH (SEQ ID NO: 4777), PDSPH (SEQ ID NO: 4778). 778), MGSPH (SEQ ID NO: 4779), QVSPH (SEQ ID NO: 4780), HNSPH (SEQ ID NO: 4781), HPSPH (SEQ ID NO: 4782), or HSSPH (SEQ ID NO: 4783), an amino acid sequence comprising any portion of any of these aforementioned amino acid sequences (e.g., any 2, 3, or 4 amino acids, e.g., contiguous amino acids), an amino acid sequence that includes one, two, or three, but not more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforementioned amino acid sequences, or an amino acid sequence that includes one, two, or three, but not more than four different amino acids, relative to any one of the aforementioned amino acid sequences. In some embodiments, [N1]-[N2] is selected from the group consisting of GSGSPH (SEQ ID NO: 4695), GHDSPH (SEQ ID NO: 4784), GQDSPH (SEQ ID NO: 4785), VSGSPH (SEQ ID NO: 4786), CSGSPH (SEQ ID NO: 4787), GRGSPH (SEQ ID NO: 4788), CSHSPH (SEQ ID NO: 4789), GQSSPH (SEQ ID NO: 4790), GSHSPH (SEQ ID NO: 4791), GDDSPH (SEQ ID NO: 4792), GHESPH (SEQ ID NO: 4793), GNYSPH (SEQ ID NO: 4794), RVGSPH (SEQ ID NO: 4795), GSCSPH (SEQ ID NO: 4796), GLLSPH (SEQ ID NO: 4797), MSGSPH (SEQ ID NO: 4798), RNGSPH (SEQ ID NO: 4799), TSGSPH (SEQ ID NO: 4800), ISGSPH (SEQ ID NO: 4801), GPGSPH (SEQ ID NO: 4802),ESGSPH (SEQ ID NO: 4803), SSGSPH (SEQ ID NO: 4804), GNGSPH (SEQ ID NO: 4805), ASGSPH (SEQ ID NO: 4806), NSGSPH (SEQ ID NO: 4807), LSGSPH (SEQ ID NO: 4808), GGGSPH (SEQ ID NO: 4809), KSGSPH (SEQ ID NO: 4810), HSGSPH (SEQ ID NO: 4811), GTGSPH (SEQ ID NO: 4812), PSGSPH (SEQ ID NO: 4813), GSVSPH (SEQ ID NO: 4814), RSGSPH (SEQ ID NO: No. 4815), GIGSPH (SEQ ID NO: 4816), WSGSPH (SEQ ID NO: 4817), DSGSPH (SEQ ID NO: 4818), IDGSPH (SEQ ID NO: 4819), GLGSPH (SEQ ID NO: 4820), DAGSPH (SEQ ID NO: 4821), DGGSPH (SEQ ID NO: 4822), MEGSPH (SEQ ID NO: 4823), ENGSPH (SEQ ID NO: 4824), GSASPH (SEQ ID NO: 4825), KNGSPH (SEQ ID NO: 4826), KEGSPH (SEQ ID NO: 4827), AIG SPH (SEQ ID NO: 4828), GYDSPH (SEQ ID NO: 4829), GHGSPH (SEQ ID NO: 4830), GRDSPH (SEQ ID NO: 4831), GNDSPH (SEQ ID NO: 4832), GPDSPH (SEQ ID NO: 4833), GMGSPH (SEQ ID NO: 4834), GQVSPH (SEQ ID NO: 4835), GHNSPH (SEQ ID NO: 4836), GHPSPH (SEQ ID NO: 4837), or GHSSPH (SEQ ID NO: 4838), an amino acid sequence comprising any portion of any of these aforesaid amino acid sequences (e.g., any 2, 3, 4, or 5 amino acids, e.g., contiguous amino acids), an amino acid sequence that includes one, two, or three, but not more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences, or an amino acid sequence that includes one, two, or three, but not more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [N1]-[N2] is or comprises GSGSPH (SEQ ID NO: 4695). In some embodiments, [N1]-[N2] is or comprises GHDSPH (SEQ ID NO: 4784).

[0318] In some embodiments, X4, X5, or both of [N3] are K. In some embodiments, X4, X5, or X6 of [N3] are R. In some embodiments, the X4 position of [N3] is independently selected from A, K, V, S, T, G, F, W, V, N, or R. In some embodiments, the X5 position of [N3] is independently selected from S, K, T, F, I, L, Y, H, M, or R. In some embodiments, the X6 position of [N3] is independently selected from G, R, A, M, I, N, T, Y, D, P, V, L, E, W, N, Q, K, or S. In some embodiments, [N3] comprises SK, KA, KS, AR, RM, VK, AS, SR, VK, KR, KK, KN, VR, RS, RK, KT, TS, KF, FG, KI, IG, KL, LG, TT, TY, KY, YG, KD, KP, TR, RG, VR, GA, SL, SS, FL, WK, SA, RA, LR, KW, RR, GK, TK, NK, AK, KV, KG, KH, KM, TG, SE, SV, SW, SN, HG, SQ, LW, MG, MA, or SG. In some embodiments, [N3] comprises SK, KA, KS, or SG. In some embodiments, [N3] is or includes SKA, KSG, ARM, VKS, ASR, VKI, KKN, VRM, RKA, KTS, KFG, KIG, KLG, KTT, KTY, KYG, SKD, SKP, TRG, VRG, KRG, GAR, KSA, KSR, SKL, SRA, SKR, SLR, SRG, SSR, FLR, SKW, SKS, WKA, VRR, SKV, SKT, SKG, GKA, TKA, NKA, SKL, SKN, AKA, KTG, KSL, KSE, KSV, KSW, KSN, KHG, KSQ, KSK, KLW, WKG, KMG, KMA, or RSG. In some embodiments, [N3] is or includes SKA. In some embodiments, [N3] is or includes KSG.In some embodiments, [N2]-[N3] is selected from the group consisting of SPHSK (SEQ ID NO: 4701), SPHKS (SEQ ID NO: 4704), SPHAR (SEQ ID NO: 4705), SPHVK (SEQ ID NO: 4706), SPHAS (SEQ ID NO: 4707), SPHKK (SEQ ID NO: 4708), SPHVR (SEQ ID NO: 4709), SPHRK (SEQ ID NO: 4710), SPHKT (SEQ ID NO: 4711), SPHKF (SEQ ID NO: 4712), SPHKI (SEQ ID NO: 4713), SPHKL (SEQ ID NO: 4714), SPHKY (SEQ ID NO: 4715), SPHTR (SEQ ID NO: 4716), SPHKL (SEQ ID NO: 4717), SPHKY (SEQ ID NO: 4718), SPHKR (SEQ ID NO: 4719), SPHKT (SEQ ID NO: 4720), SPHKF (SEQ ID NO: 4721), SPHKI (SEQ ID NO: 4722), SPHKL (SEQ ID NO: 4723), SPHKY (SEQ ID NO: 4724), SPHTR (SEQ ID NO: 4725), SPHKR (SEQ ID NO: 4726), SPHKT (SEQ ID NO: 4727), SPHKT (SEQ ID NO: 4728), SPHKT (SEQ ID NO: 4729), SPHKT (SEQ ID NO: 4730), SPHKT (SEQ ID NO: 4731), SPHKT (SEQ ID NO: 4732), SPHKT (SEQ ID NO: 4733), SPHKT (SEQ ID NO In some embodiments, [N2]-[N3] comprises (SEQ ID NO: 4701) or SPHKS (SEQ ID NO: 4704).In some embodiments, [N2]-[N3] is selected from the group consisting of SPHSKA (SEQ ID NO: 941), SPHKSG (SEQ ID NO: 946), SPHARM (SEQ ID NO: 947), SPHVKS (SEQ ID NO: 948), SPHASR (SEQ ID NO: 949), SPHVKI (SEQ ID NO: 950), SPHKKN (SEQ ID NO: 954), SPHVRM (SEQ ID NO: 955), SPHRKA (SEQ ID NO: 956), SPHKFG (SEQ ID NO: 957), SPHKIG (SEQ ID NO: 958), SPHKLG (SEQ ID NO: 959), SPHKTS (SEQ ID NO: 963), SPHKTT (SEQ ID NO: 964), SPHKT (SEQ ID NO: 965), SPHKT (SEQ ID NO: 966), SPHKT (SEQ ID NO: 967), SPHKT (SEQ ID NO: 968), SPHKT (SEQ ID NO: 969), SPHKT (SEQ ID NO: 970), SPHKT (SEQ ID NO: 971), SPHKT (SEQ ID NO: 972), SPHKT (SEQ ID NO: 973), SPHKT (SEQ ID NO: 974), SPHKT (SEQ ID NO: 975), SPHKT (SEQ ID NO: 976), SPHKT (SEQ ID NO: 977), SPHKT (SEQ ID NO: 978), SPHKT (SEQ ID NO: 979), SPHKT (SEQ ID NO: 980), SPHKT (SEQ ID NO: 981), SPHKT (SEQ ID NO: 982), SPHKT (SEQ ID NO: 983 64), SPHKTY (SEQ ID NO: 965), SPHKYG (SEQ ID NO: 966), SPHSKD (SEQ ID NO: 967), SPHSKP (SEQ ID NO: 968), SPHTRG (SEQ ID NO: 972), SPHVRG (SEQ ID NO: 973), SPHKRG (SEQ ID NO: 974), SPHGAR (SEQ ID NO: 975), SPHKSA (SEQ ID NO: 977), SPHKSR (SEQ ID NO: 951), SPHSKL (SEQ ID NO: 960), SPHSRA (SEQ ID NO: 969), SPHSKR (SEQ ID NO: 978), SPHSLR (SEQ ID NO: 952), SPHSRG (SEQ ID NO: 961), SPHSSR (SEQ ID NO: 970), SPHFLR (SEQ ID NO: 979), SPHSKW (SEQ ID NO: 953), SPHSKS (SEQ ID NO: 962), SPHWKA (SEQ ID NO: 971), SPHVRR (SEQ ID NO: 980), SPHSKT (SEQ ID NO: 4731), SPHSKG (SEQ ID NO: 4732), SPHGKA (SEQ ID NO: 4733), SPHNKA (SEQ ID NO: 4734), SPHSKN (SEQ ID NO: 4735), SPHAKA (SEQ ID NO: 4736), SPHSKV (SEQ ID NO: 4737), SPHKTG (SEQ ID NO: 4738), SPHTKA (SEQ ID NO: 4739), SPHKSL (SEQ ID NO: 4740), SPHKSE (SEQ ID NO: 4741), SPHKSV (SEQ ID NO: 4742), SPHKSW (SEQ ID NO: 4743), SPHKSN (SEQ ID NO: 4744), SPHKHG (SEQ ID NO: 4745), SPHKSQ (SEQ ID NO: 4746), SPHKSK (SEQ ID NO: 4747), SPHKLW (SEQ ID NO: 4748), SPHWKG (SEQ ID NO: 4749), SPHKMG (SEQ ID NO: 4750), SPHKMA (SEQ ID NO: 4751), or SPHRSG (SEQ ID NO: 976).In some embodiments, [N2]-[N3] is or comprises SPHSKA (SEQ ID NO: 941). In some embodiments, [N2]-[N3] is or comprises SPHKSG (SEQ ID NO: 946).

[0319] In some embodiments, [N1]-[N2]-[N3] is selected from the group consisting of SGSPHSK (SEQ ID NO: 4839), HDSPHKS (SEQ ID NO: 4840), SGSPHAR (SEQ ID NO: 4841), SGSPHVK (SEQ ID NO: 4842), QDSPHKS (SEQ ID NO: 4843), SGSPHKK (SEQ ID NO: 4844), SGSPHVR (SEQ ID NO: 4845), SGSPHAS (SEQ ID NO: 4846), SGSPHRK (SEQ ID NO: 4847), SGSPHKT (SEQ ID NO: 4848), SHSPHKS (SEQ ID NO: 4849), QSSPHRS (SEQ ID NO: 4850), RGSPHA S (SEQ ID NO: 4851), RGSPHSK (SEQ ID NO: 4852), SGSPHKF (SEQ ID NO: 4853), SGSPHKI (SEQ ID NO: 4854), SGSPHKL (SEQ ID NO: 4855), SGSPHKY (SEQ ID NO: 4856), SGSPHTR (SEQ ID NO: 4857), SHSPHKR (SEQ ID NO: 4858), SGSPHGA (SEQ ID NO: 4859), HDSPHKR (SEQ ID NO: 4860), DDSPHKS (SEQ ID NO: 4861), HESPHKS (SEQ ID NO: 4862), NYSPHKI (SEQ ID NO: 4863), SGSPHSR (SEQ ID NO: 4864), SGSP HSL (SEQ ID NO: 4865), SGSPHSS (SEQ ID NO: 4866), VGSPHSK (SEQ ID NO: 4867), SCSPHRK (SEQ ID NO: 4868), SGSPHFL (SEQ ID NO: 4869), LLSPHWK (SEQ ID NO: 4870), NGSPHSK (SEQ ID NO: 4871), PGSPHSK (SEQ ID NO: 4872), GGSPHSK (SEQ ID NO: 4873), TGSPHSK (SEQ ID NO: 4874), SVSPHGK (SEQ ID NO: 4875), SGSPHTK (SEQ ID NO: 4876), IGSPHSK (SEQ ID NO: 4877), DGSPHSK (SEQ ID NO: 4878), SG SPHNK (SEQ ID NO: 4879), LGSPHSK (SEQ ID NO: 4880), AGSPHSK (SEQ ID NO: 4881), EGSPHSK (SEQ ID NO: 4882), SASPHSK (SEQ ID NO: 4883), SGSPHAK (SEQ ID NO: 4884), HDSPHKI (SEQ ID NO: 4885), YDSPHKS (SEQ ID NO: 4886), HDSPHKT (SEQ ID NO: 4887), RGSPHKR (SEQ ID NO: 4888), HGSPHSK (SEQ ID NO: 4889), RDSPHKS (SEQ ID NO: 4890), NDSPHKS (SEQ ID NO: 4891), QDSPHKI (SEQ ID NO: 4892),PDSPHKI (SEQ ID NO: 4893), PDSPHKS (SEQ ID NO: 4894), MGSPHSK (SEQ ID NO: 4895), HDSPHKH (SEQ ID NO: 4896), QVSPHKS (SEQ ID NO: 4897), HNSPHKS (SEQ ID NO: 4898), NGSPHKR (SEQ ID NO: 4899), HDSPHKY (SEQ ID NO: 4900), NDSPHKI (SEQ ID NO: 4901), HDSPHKL (SEQ ID NO: 4902), HPSPHWK (SEQ ID NO: 4903), HDSPHKM (SEQ ID NO: 4904), or HSSPHRS (SEQ ID NO: 4905). In some embodiments, [N1]-[N2]-[N3] is selected from the group consisting of GSGSPHSKA (SEQ ID NO: 4697), GHDSPHKSG (SEQ ID NO: 4698), GSGSPHARM (SEQ ID NO: 4906), GSGSPHVKS (SEQ ID NO: 4907), GQDSPHKSG (SEQ ID NO: 4908), GSGSPHASR (SEQ ID NO: 4909), GSGSPHVKI (SEQ ID NO: 4910), GSGSPHKKN (SEQ ID NO: 4911), GSGSPHVRM (SEQ ID NO: 4912), VSGSPHSKA (SEQ ID NO: 4913), CSGSPHSKA (SEQ ID NO: 4914), GSGSPHRKA (SEQ ID NO: 4915), CSGSPHKTS (SEQ ID NO: 4916), CSHSPHKSG (SEQ ID NO: 4917), GQSSPHRSG (SEQ ID NO: 4918), GRGSPHASR (SEQ ID NO: 4919), GRGSPHSKA (SEQ ID NO: 4920), GSGSPH KFG (SEQ ID NO: 4921), GSGSPHKIG (SEQ ID NO: 4922), GSGSPHKLG (SEQ ID NO: 4923), GSGSPHKTS (SEQ ID NO: 4924), GSGSPHKTT (SEQ ID NO: 4925), GSGSPHKTY (SEQ ID NO: 4926), GSGSPHKYG (SEQ ID NO: 4927), GSGSPHSKD (SEQ ID NO: 4928), GSGSPHSKP (SEQ ID NO: 4929), GSGSPHTRG (SEQ ID NO: No. 4930), GSGSPHVRG (SEQ ID NO: 4931), GSHSPHKRG (SEQ ID NO: 4932), GSHSPHKSG (SEQ ID NO: 4933), VSGSPHASR (SEQ ID NO: 4934), VSGSPHGAR (SEQ ID NO: 4935), VSGSPHKFG (SEQ ID NO: 4936), GHDSPHKRG (SEQ ID NO: 4937), GDDSPHKSG (SEQ ID NO: 4938), GHESPHKSA (SEQ ID NO: 4939),GHDSPHKSA (SEQ ID NO: 4940), GNYSPHKIG (SEQ ID NO: 4941), GHDSPHKSR (SEQ ID NO: 4942), GSGSPHSKL (SEQ ID NO: 4943), GSGSPHSRA (SEQ ID NO: 4944), GSGSPHSKR (SEQ ID NO: 4945), GSGSPHSLR (SEQ ID NO: 4946), GSGSPHSRG (SEQ ID NO: 4947), GSGSPHSSR (SEQ ID NO: 4948), RVGSPHSKA (SEQ ID NO: 4949), GSCSPHRKA (SEQ ID NO: 4950), GSGSPHFLR (SEQ ID NO: 4951), GSGSPHSKW ( SEQ ID NO: 4952), GSGSPHSKS (SEQ ID NO: 4953), GLLSPHWKA (SEQ ID NO: 4954), GSGSPHVRR (SEQ ID NO: 4955), GSGSPHSKV (SEQ ID NO: 4956), MSGSPHSKA (SEQ ID NO: 4957), RNGSPHSKA (SEQ ID NO: 4958), TSGSPHSKA (SEQ ID NO: 4959), ISGSPHSKA (SEQ ID NO: 4960), GPGSPHSKA (SEQ ID NO: 4961), GSGSPHSKT (SEQ ID NO: 4962), ESGSPHSKA (SEQ ID NO: 4963), SSGSPHSKA (SEQ ID NO: 4964), GNGSPHSKA (SEQ ID NO: 4965), ASGSPHSKA (SEQ ID NO: 4966), NSGSPHSKA (SEQ ID NO: 4967), LSGSPHSKA (SEQ ID NO: 4968), GGGSPHSKA (SEQ ID NO: 4969), KSGSPHSKA (SEQ ID NO: 4970), GGGSPHSKS (SEQ ID NO: 4971), GSGSPHSKG (SEQ ID NO: 4972), HSGSPHSKA (SEQ ID NO: 4973), GTGSPHSKA (SEQ ID NO: 4974), PSGSPHSKA (SEQ ID NO: 4975), GSVSPHGKA (SEQ ID NO: 4976), RSGSPHSKA ( SEQ ID NO: 4977), GSGSPHTKA (SEQ ID NO: 4978), GIGSPHSKA (SEQ ID NO: 4979), WSGSPHSKA (SEQ ID NO: 4980), DSGSPHSKA (SEQ ID NO: 4981), IDGSPHSKA (SEQ ID NO: 4982), GSGSPHNKA (SEQ ID NO: 4983), GLGSPHSKS (SEQ ID NO: 4984), DAGSPHSKA (SEQ ID NO: 4985), DGGSPHSKA (SEQ ID NO: 4986), MEGSPHSKA (SEQ ID NO: 4987), ENGSPHSKA (SEQ ID NO: 4988), GSASPHSKA (SEQ ID NO: 4989),GNGSPHSKS (SEQ ID NO: 4990), KNGSPHSKA (SEQ ID NO: 4991), KEGSPHSKA (SEQ ID NO: 4992), AIGSPHSKA (SEQ ID NO: 4993), GSGSPHSKN (SEQ ID NO: 4994), GSGSPHAKA (SEQ ID NO: 4995), GHDSPHKIG (SEQ ID NO: 4996), GYDSPHKSG (SEQ ID NO: 4997), GHESPHKSG (SEQ ID NO: 4998), GHDSPHKTG (SEQ ID NO: 4999), GRGSPHKRG (SEQ ID NO: 5000), GQDSPHKSG (SEQ ID NO: 4908), GHDSPHKS L (SEQ ID NO: 5001), GHGSPHSKA (SEQ ID NO: 5002), GHDSPHKSE (SEQ ID NO: 5003), VSGSPHSKA (SEQ ID NO: 4913), GRDSPHKSG (SEQ ID NO: 5004), GNDSPHKSV (SEQ ID NO: 5005), GQDSPHKIG (SEQ ID NO: 5006), GHDSPHKSV (SEQ ID NO: 5007), GPDSPHKIG (SEQ ID NO: 5008), GPDSPHKSG (SEQ ID NO: 5009), GHDSPHKSW (SEQ ID NO: 5010), GHDSPHKSN (SEQ ID NO: 5011), GMGSPHSKT (SEQ ID NO: 501 2), GHDSPHKHG (SEQ ID NO: 5013), GQVSPHKSG (SEQ ID NO: 5014), GDDSPHKSV (SEQ ID NO: 5015), GHNSPHKSG (SEQ ID NO: 5016), GNGSPHKRG (SEQ ID NO: 5017), GHDSPHKYG (SEQ ID NO: 5018), GHDSPHKSQ (SEQ ID NO: 5019), GNDSPHKIG (SEQ ID NO: 5020), GHDSPHKSK (SEQ ID NO: 5021), GHDSPHKLW (SEQ ID NO: 5022), GHPSPHWKG (SEQ ID NO: 5023), GHDSPHKMG (SEQ ID NO: 5024), GHDSP HKMA (SEQ ID NO: 5025), or GHSSPHRSG (SEQ ID NO: 5026), an amino acid sequence comprising any portion of any of these aforementioned amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, or 8 amino acids, e.g., contiguous amino acids), an amino acid sequence that includes one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, to any of the aforementioned amino acid sequences, or one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, to any one of the aforementioned amino acid sequences.[N1]-[N2]-[N3] is or comprises an amino acid sequence containing no more than four different amino acids. In some embodiments, [N1]-[N2]-[N3] is or comprises GSGSPHSKA (SEQ ID NO: 4697). In some embodiments, [N1]-[N2]-[N3] is or comprises GHDSPHKSG (SEQ ID NO: 4698).

[0320] In some embodiments, the ligand comprising a protein or peptide comprising an amino acid sequence having the formula [N1]-[N2]-[N3] further comprises an [N4] comprising X7, X8, X9, and X10. In some embodiments, the X7 position of [N4] is independently selected from W, Q, K, R, G, L, V, S, P, H, K, I, M, A, E, or F. In some embodiments, the X8 position of [N4] is independently selected from N, Y, C, K, T, H, R, D, V, S, P, G, W, E, F, A, I, M, Q, or L. In some embodiments, the X9 position of [N4] is independently selected from Q, G, K, H, R, T, L, D, A, P, I, F, V, M, W, Y, S, E, N, or Y. In some embodiments, position X10 of [N4] is independently selected from Q, H, L, R, W, K, A, P, E, M, I, S, G, N, Y, C, V, T, D, or V. In some embodiments, [N4] is selected from QNQQ (SEQ ID NO: 5028), WNQQ (SEQ ID NO: 5029), QYYV (SEQ ID NO: 5030), RRQQ (SEQ ID NO: 5031), QNQQ (SEQ ID NO: 5028), GCGQ (SEQ ID NO: 5032), LRQQ (SEQ ID NO: 5033), RNQQ (SEQ ID NO: 5034), VNQQ (SEQ ID NO: 5035), FRLQ (SEQ ID NO: 5036), FNQQ (SEQ ID NO: 5037), LLQQ (SEQ ID NO: 5038), SNQQ (SEQ ID NO: 5039), RLQQ (SEQ ID NO: 5040), LNQQ (SEQ ID NO: 5041), QRKL (SEQ ID NO: 5042), LRRQ (SEQ ID NO: 5043), QRLR (SEQ ID NO: 5044), QRRL (SEQ ID NO: 5045), RRLQ (SEQ ID NO: 5046), RLRQ (SEQ ID NO: 5047), SKRQ (SEQ ID NO: 5048), QL YR (SEQ ID NO: 5049), QLTV (SEQ ID NO: 5050), QNKQ (SEQ ID NO: 5051), KNQQ (SEQ ID NO: 5052), QKQQ (SEQ ID NO: 5053), QTQQ (SEQ ID NO: 5054), QNHQ (SEQ ID NO: 5055), QHQQ (SEQ ID NO: 5056), QNQH (SEQ ID NO: 5057), QHRQ (SEQ ID NO: 5058), LTQQ (SEQ ID NO: 5059), QNQW (SEQ ID NO: 5060), QNTH (SEQ ID NO: 5061), RRRQ (SEQ ID NO: 5062), QYQQ (SEQ ID NO: 5063), QNDQ (SEQ ID NO: 5064), QNRH (SEQ ID NO: 5065), RDQQ (SEQ ID NO: 5066),PNLQ (SEQ ID NO: 5067), HVRQ (SEQ ID NO: 5068), PNQH (SEQ ID NO: 5069), HNQQ (SEQ ID NO: 5070), QSQQ (SEQ ID NO: 5071), QPAK (SEQ ID NO: 5072), QNLA (SEQ ID NO: 5073), QNQL (SEQ ID NO: 5074), QGQQ (SEQ ID NO: 5075), LNRQ (SEQ ID NO: 5076), QNPP (SEQ ID NO: 5077), QNLQ (SEQ ID NO: 5078), QDQE (SEQ ID NO: 5079), QDQQ (SEQ ID NO: 5080), HWQQ (SEQ ID NO: 5081), PNQQ (SEQ ID NO: 5082), PEQQ (SEQ ID NO: 5083), No. 5083), QRTM (SEQ ID NO: 5084), LHQH (SEQ ID NO: 5085), QHRI (SEQ ID NO: 5086), QYIH (SEQ ID NO: 5087), QKFE (SEQ ID NO: 5088), QFPS (SEQ ID NO: 5089), QNPL (SEQ ID NO: 5090), QAIK (SEQ ID NO: 5091), QNRQ (SEQ ID NO: 5092), QYQH (SEQ ID NO: 5093), QNPQ (SEQ ID NO: 5094), QHQL (SEQ ID NO: 5095), QSPP (SEQ ID NO: 5096), QAKL (SEQ ID NO: 5097), KSQQ (SEQ ID NO: 5098), QDRP (SEQ ID NO: 5099), QNLG (SEQ ID NO: 5100), QAFH (SEQ ID NO: 5101), QNAQ (SEQ ID NO: 5102), HNQL (SEQ ID NO: 5103), QKLN (SEQ ID NO: 5104), QNVQ (SEQ ID NO: 5105), QAQQ (SEQ ID NO: 5106), QTPP (SEQ ID NO: 5107), QPPA (SEQ ID NO: 5108), QERP (SEQ ID NO: 5109), QDLQ (SEQ ID NO: 5110), QAMH (SEQ ID NO: 5111), QHPS (SEQ ID NO: 5112), PGLQ (SEQ ID NO: 5113), QGIR (SEQ ID NO: 5114), QAPA (SEQ ID NO: 5115), QIPP (SEQ ID NO: 5116), No. 5116), QTQL (SEQ ID NO: 5117), QAPS (SEQ ID NO: 5118), QNTY (SEQ ID NO: 5119), QDKQ (SEQ ID NO: 5120), QNHL (SEQ ID NO: 5121), QIGM (SEQ ID NO: 5122), LNKQ (SEQ ID NO: 5123), PNQL (SEQ ID NO: 5124), QLQQ (SEQ ID NO: 5125), QRMS (SEQ ID NO: 5126), QGIL (SEQ ID NO: 5127), QDRQ (SEQ ID NO: 5128), RDWQ (SEQ ID NO: 5129), QERS (SEQ ID NO: 5130), QNYQ (SEQ ID NO: 5131), QRTC (SEQ ID NO: 5132),QIGH (SEQ ID NO: 5133), QGAI (SEQ ID NO: 5134), QVPP (SEQ ID NO: 5135), QVQQ (SEQ ID NO: 5136), LMRQ (SEQ ID NO: 5137), QYSV (SEQ ID NO: 5138), QAIT (SEQ ID NO: 5139), QKTL (SEQ ID NO: 5140), QLHH (SEQ ID NO: 5141), QNII (SEQ ID NO: 5142), QGHH (SEQ ID NO: 5143), QSKV (SEQ ID NO: 5144), QLPS (SEQ ID NO: 5145), IGKQ (SEQ ID NO: 5146), QAIH (SEQ ID NO: 5147), QHGL (SEQ ID NO: 5148), QFMC (SEQ ID NO: 5149), No. 5149), QNQM (SEQ ID NO: 5150), QHLQ (SEQ ID NO: 5151), QPAR (SEQ ID NO: 5152), QSLQ (SEQ ID NO: 5153), QSQL (SEQ ID NO: 5154), HSQQ (SEQ ID NO: 5155), QMPS (SEQ ID NO: 5156), QGSL (SEQ ID NO: 5157), QVPA (SEQ ID NO: 5158), HYQQ (SEQ ID NO: 5159), QVPS (SEQ ID NO: 5160), RGEQ (SEQ ID NO: 5161), PGQQ (SEQ ID NO: 5162), LEQQ (SEQ ID NO: 5163), QNQS (SEQ ID NO: 5164), QKVI (SEQ ID NO: 5165), QNND (SEQ ID NO: 5166), QSVH (SEQ ID NO: 5167), QPLG (SEQ ID NO: 5168), HNQE (SEQ ID NO: 5169), QIQQ (SEQ ID NO: 5170), QVRN (SEQ ID NO: 5171), PSNQ (SEQ ID NO: 5172), QVGH (SEQ ID NO: 5173), QRDI (SEQ ID NO: 5174), QMPN (SEQ ID NO: 5175), RGLQ (SEQ ID NO: 5176), PSLQ (SEQ ID NO: 5177), QRDQ (SEQ ID NO: 5178), QAKG (SEQ ID NO: 5179), QSAH (SEQ ID NO: 5180), QSTM (SEQ ID NO: 5181), QREM (SEQ ID NO: 5182), No. 5182), QYRA (SEQ ID NO: 5183), QRQQ (SEQ ID NO: 5184), QWQQ (SEQ ID NO: 5185), QRMN (SEQ ID NO: 5186), GDSQ (SEQ ID NO: 5187), QKIS (SEQ ID NO: 5188), PSMQ (SEQ ID NO: 5189), SPRQ (SEQ ID NO: 5190), MEQQ (SEQ ID NO: 5191), QYQN (SEQ ID NO: 5192), QIRQ (SEQ ID NO: 5193), QSVQ (SEQ ID NO: 5194), RSQQ (SEQ ID NO: 5195), QNKL (SEQ ID NO: 5196), QIQH (SEQ ID NO: 5197), PRQQ (SEQ ID NO: 5198),HTQQ (SEQ ID NO: 5199), QRQH (SEQ ID NO: 5200), RNQE (SEQ ID NO: 5201), QSKQ (SEQ ID NO: 5202), QNQP (SEQ ID NO: 5203), QSPQ (SEQ ID NO: 5204), QTRQ (SEQ ID NO: 5205), QNLH (SEQ ID NO: 5206), QNQE (SEQ ID NO: 5207), LNQP (SEQ ID NO: 5208), QNQD (SEQ ID NO: 5209), QNLL (SEQ ID NO: 5210), QLVI (SEQ ID NO: 5211), No. 5211), RTQE (SEQ ID NO: 5212), QTHQ (SEQ ID NO: 5213), QDQH (SEQ ID NO: 5214), QSQH (SEQ ID NO: 5215), VRQQ (SEQ ID NO: 5216), AWQQ (SEQ ID NO: 5217), QSVP (SEQ ID NO: 5218), QNIQ (SEQ ID NO: 5219), LDQQ (SEQ ID NO: 5220), PDQQ (SEQ ID NO: 5221), ESQQ (SEQ ID NO: 5222), QRQL (SEQ ID NO: 5223), Q IIV (SEQ ID NO: 5224), QKQS (SEQ ID NO: 5225), QSHQ (SEQ ID NO: 5226), QFVV (SEQ ID NO: 5227), QSQP (SEQ ID NO: 5228), QNEQ (SEQ ID NO: 5229), INQQ (SEQ ID NO: 5230), RNRQ (SEQ ID NO: 5231), RDQK (SEQ ID NO: 5232), QWKR (SEQ ID NO: 5233), ENRQ (SEQ ID NO: 5234), QTQP (SEQ ID NO: 5235), QKQL (SEQ ID NO: 5236), RNQL (SEQ ID NO: 5237), ISIQ (SEQ ID NO: 5238), QTVC (SEQ ID NO: 5239), QQIM (SEQ ID NO: 5240), LNHQ (SEQ ID NO: 5241), QNQA (SEQ ID NO: 5242), QMIH (SEQ ID NO: 5243), RNHQ (SEQ ID NO: 5244), or QKMN (SEQ ID NO: 5245), or any dipeptide or tripeptide thereof. In some embodiments, [N1]-[N2]-[N3]-[N4] is any of the amino acid sequences of SEQ ID NOs: 1800-2241, an amino acid sequence comprising any portion of any of these aforementioned amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids, e.g., contiguous amino acids), an amino acid sequence that includes 1, 2, or 3, but not more than 4, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforementioned amino acid sequences,or an amino acid sequence that contains 1, 2, or 3, but not more than 4, different amino acids from any one of the above amino acid sequences. In some embodiments, [N1]-[N2]-[N3]-[N4] is or comprises GSGSPHSKAQNQQ (SEQ ID NO: 1801). In some embodiments, [N1]-[N2]-[N3]-[N4] is or comprises GHDSPHKSGQNQQ (SEQ ID NO: 1800).

[0321] In some embodiments, the ligand comprising a protein or peptide comprising an amino acid sequence having the formula [N1]-[N2]-[N3] further comprises [N0] comprising XA XB and XC. In some embodiments, XA of [N0] is independently selected from T, S, Y, M, A, C, I, R, L, D, F, V, Q, N, H, E, or G. In some embodiments, XB of [N0] is independently selected from I, M, P, E, N, D, S, A, T, G, Q, F, V, L, C, H, R, W, or L. In some embodiments, XC of [N0] is independently selected from N, M, E, G, Y, W, T, I, Q, F, V, A, L, I, P, K, R, H, S, D, or S. In some embodiments, [N0] is TIN, SMN, TIM, YLS, GLS, MPE, MEG, MEY, AEW, CEW, ANN, IPE, ADM, IEY, ADY, IE T, MEW, CEY, RIN, MEI, LEY, ADW, IEI, DIM, FEQ, MEF, CDQ, LPE, IEN, MES, AEI, VEY, IIN, TSN, IEV, MEM , AEV, MDA, VEW, AEQ, LEW, MEL, MET, MEA, IES, MEV, CEI, ATN, MDG, QEV, ADQ, NMN, IEM, ISN, TGN, QQQ, HDW, IEG, TII, TFP, TEK, EIN, TVN, TFN, SIN, TER, TSY, ELH, AIN, SVN, TDN, TFH, TVH, TEN, TSS, TID, T CN, NIN, TEH, AEM, AIK, TDK, TFK, SDQ, TEI, NTN, TET, SIK, TEL, TEA, TAN, TIY, TFS, TES, TTN, TED, TN N, EVH, TIS, TVR, TDR, TIK, NHI, TIP, ESD, TDL, TVP, TVI, AEH, NCL, TVK, NAD, TIT, NCV, TIR, NAL, VIN , TIQ, TEF, TRE, QGE, SEK, NVN, GGE, EFV, SDK, TEQ, EVQ, TEY, NCW, TDV, SDI, NSI, NSL, EVV, TEP, SEL, TWQ, TEV, AVN, GVL, TLN, TEG, TRD, NAI, AEN, AET, ETA, NNL, or any dipeptide thereof.In some embodiments, [N0]-[N1]-[N2]-[N3]-[N4] is or comprises the amino acid sequence of any one of SEQ ID NOs: 2242-2886, an amino acid sequence comprising any portion of any of these aforementioned amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, e.g., contiguous amino acids), an amino acid sequence comprising one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforementioned amino acid sequences, or an amino acid sequence comprising one, two, or three, but not more than four different amino acids, relative to any one of the aforementioned amino acid sequences. In some embodiments, [N0]-[N1]-[N2]-[N3]-[N4] is or comprises TINGSGSPHSKAQNQQ (SEQ ID NO: 2242). In some embodiments, [N0]-[N1]-[N2]-[N3]-[N4] is or includes TINGHDSPHKSGQNQQ (sequence number 2243).

[0322] In some embodiments, [N3] immediately follows [N2]. In some embodiments, the peptide comprises, from N-terminus to C-terminus, [N2]-[N3]. In some embodiments, the peptide comprises, from N-terminus to C-terminus, [N1]-[N2]-[N3]. In some embodiments, the peptide comprises, from N-terminus to C-terminus, [N1]-[N2]-[N3]-[N4]. In some embodiments, the peptide comprises, from N-terminus to C-terminus, [N0]-[N1]-[N2]-[N3]. In some embodiments, the peptide comprises, from N-terminus to C-terminus, [N0]-[N1]-[N2]-[N3]-[N4].

[0323] In some embodiments, the ligand comprises a protein or peptide comprising an amino acid sequence having the formula [A][B] (SEQ ID NO: 6410), wherein [A] comprises the amino acid sequence of GSGSPH (SEQ ID NO: 4695), and [B] comprises X1, X2, X3, X4, X5, X6, and X7. In some embodiments, position X1 of [B] is independently selected from S, C, F, or V. In some embodiments, position X2 of [B] is independently selected from K, L, R, I, E, Y, V, or S. In some embodiments, X3 of [B] is independently selected from A, R, L, G, I, Y, S, F, or W. In some embodiments, X4 of [B] is independently selected from W, Q, R, G, L, V, S, or F. In some embodiments, position X5 of [B] is independently selected from N, Y, R, C, K, or L. In some embodiments, position X6 of [B] is independently selected from Q, G, K, R, T, L, or Y. In some embodiments, position X7 of [B] is independently selected from Q, L, R, or V. In some embodiments, [B] is selected from SLLWNQQ (SEQ ID NO: 5247), SKAQYYV (SEQ ID NO: 5248), SKLRRQQ (SEQ ID NO: 5249), SIWQNQQ (SEQ ID NO: 5250), SKAGCGQ (SEQ ID NO: 5251), SRAQNQQ (SEQ ID NO: 5252), SKRLRQQ (SEQ ID NO: 5253), SLRRNQQ (SEQ ID NO: 5254), SRGRNQQ (SEQ ID NO: 5255), SEIVNQQ (SEQ ID NO: 5256), SSRRNQQ (SEQ ID NO: 5257), CLLQNQQ (SEQ ID NO: 5258), SKAFRLQ (SEQ ID NO: 5259), CLAQNQQ (SEQ ID NO: 5260), FLRQNQQ (SEQ ID NO: 5261), SLRFNQQ (SEQ ID NO: 5262), SYLRNQQ (SEQ ID NO: 5263), CSLQNQQ (SEQ ID NO: 5264), VLWQNQQ (SEQ ID NO: 5265), SKWLLQQ (SEQ ID NO: 5266), SLWSNQQ (SEQ ID NO: 5267), SKRRLQQ (SEQ ID NO: 5268), SVYLNQQ (SEQ ID NO: 5269), SLWLNQQ (SEQ ID NO: 5270), SKAQRKL (SEQ ID NO: 5271), SKALRRQ (SEQ ID NO: 5272), SKAQRLR (SEQ ID NO: 5273), SKAQNQQ (SEQ ID NO: 5274), SKAQRRL (SEQ ID NO: 5275),SKARRQQ (SEQ ID NO: 5276), SKARRLQ (SEQ ID NO: 5277), SKSRRQQ (SEQ ID NO: 5278), SKALRQ (SEQ ID NO: 5279), SKASKRQ (SEQ ID NO: 5280), VRRQNQQ (SEQ ID NO: 5281), SKAQLYR (SEQ ID NO: 5282), SLFRNQQ (SEQ ID NO: 5283), SKAQLTV (SEQ ID NO: 5284), or any dipeptide, tripeptide, tetrapeptide, pentapeptide, or hexapeptide thereof. In some embodiments, [A][B] is selected from the group consisting of GSGSPHSLLWNQQ (SEQ ID NO: 5285), GSGSPHSKAQYYV (SEQ ID NO: 2060), GSGSPHSKLRRQQ (SEQ ID NO: 2061), GSGSPHSIWQNQQ (SEQ ID NO: 5286), GSGSPHSKAGCGQ (SEQ ID NO: 2062), GSGSPHSRAQNQQ (SEQ ID NO: 2063), GSGSPHSKRLRQQ (SEQ ID NO: 2064), GSG SPHSLRRNQQ (SEQ ID NO: 2065), GSGSPHSRGRNQQ (SEQ ID NO: 2066), GSGSPHSEIVNQQ (SEQ ID NO: 5287), GSGSPHSSRRNQQ (SEQ ID NO: 2067), GSGSPHCLLQNQQ (SEQ ID NO: 5288), GSGSPHSKAFRLQ (SEQ ID NO: 2068), GSGSPHCLAQNQQ (SEQ ID NO: 5289), GSGSPHFLRQNQQ (SEQ ID NO: 2070), GS GSPHSLRFNQQ (SEQ ID NO: 2071), GSGSPHSYLRNQQ (SEQ ID NO: 5290), GSGSPHCSLQNQQ (SEQ ID NO: 5291), GSGSPHVLWQNQQ (SEQ ID NO: 5292), GSGSPHSKWLLQQ (SEQ ID NO: 2072), GSGSPHSLWSNQQ (SEQ ID NO: 5293), GSGSPHSKRRLQQ (SEQ ID NO: 2073), GSGSPHSVYLNQQ (SEQ ID NO: 5294), G SGSPHSLWLNQQ (SEQ ID NO: 5295), GSGSPHSKAQRKL (SEQ ID NO: 2074), GSGSPHSKALRRQ (SEQ ID NO: 2075), GSGSPHSKAQRLR (SEQ ID NO: 2076), GSGSPHSKAQNQQ (SEQ ID NO: 1801), GSGSPHSKAQRRL (SEQ ID NO: 2077), GSGSPHSKARRQQ (SEQ ID NO: 2078), GSGSPHSKARRLQ (SEQ ID NO: 2079),GSGSPHSKSRRQQ (SEQ ID NO: 2080), GSGSPHSKARLRQ (SEQ ID NO: 2082), GSGSPHSKASKRQ (SEQ ID NO: 2083), GSGSPHVRRQNQQ (SEQ ID NO: 2084), GSGSPHSKAQLYR (SEQ ID NO: 2085), GSGSPHSLFRNQQ (SEQ ID NO: 5296), GSGSPHSKAQLTV (SEQ ID NO: 2086), or any portion thereof, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids, e.g., consecutive amino acids, of any of them. In some embodiments, [B] is immediately followed by [A]. In some embodiments, the peptide comprises, from the N-terminus to the C-terminus, [A][B].

[0324] In some embodiments, the ligand comprises a protein or peptide comprising an amino acid sequence having the formula [A][B] (SEQ ID NO: 6411), wherein [A] comprises X1, X2, X3, X4, X5, and X6, and [B] comprises SPHKSG (SEQ ID NO: 946). In some embodiments, position X1 of [A] is independently selected from T, M, A, C, I, R, L, D, F, V, Q, N, or H. In some embodiments, position X2 of [A] is independently selected from I, P, E, N, D, S, A, T, M, or Q. In some embodiments, position X3 of [A] is independently selected from N, E, G, Y, W, M, T, I, K, Q, F, S, V, A, or L. In some embodiments, position X4 of [A] is independently selected from G, D, R, or E. In some embodiments, position X5 of [A] is independently selected from H, Q, N, or D. In some embodiments, position X6 of [A] is independently selected from D or R. In some embodiments, [A] is selected from the group consisting of TINGHD (SEQ ID NO: 5297), MPEGHD (SEQ ID NO: 5298), MEGGHD (SEQ ID NO: 5299), MEYGHD (SEQ ID NO: 5300), AEWGHD (SEQ ID NO: 5301), CEWGHD (SEQ ID NO: 5302), ANNGQD (SEQ ID NO: 5303), IPEGHD (SEQ ID NO: 5304), ADMGHD (SEQ ID NO: 5305), IEYGHD (SEQ ID NO: 5306), ADYGHD (SEQ ID NO: 5307), IETGHD (SEQ ID NO: 5308), MEWGHD (SEQ ID NO: 5309), CEYGHD (SEQ ID NO: 5310), RINGHD (SEQ ID NO: 5311), MEIGHD (SEQ ID NO: 5312), LEYGHD (SEQ ID NO: 5313), ADWGHD (SEQ ID NO: 5314), IEIGHD (SEQ ID NO: 5315), TIKDND (SEQ ID NO: 5316), DIMGHD (SEQ ID NO: 5317), FEQGHD (SEQ ID NO: 5318), MEFGHD (SEQ ID NO: 5319), CDQGHD (SEQ ID NO: 5320), LPEGHD (SEQ ID NO: 5321), IENGHD (SEQ ID NO: 5322), MESGHD (SEQ ID NO: 5323), AEIGHD (SEQ ID NO: 5324), VEYGHD (SEQ ID NO: 5325), TSNGDD (SEQ ID NO: 5326), IEVGHD (SEQ ID NO: 5327), MEMGHD (SEQ ID NO: 5328), AEVGHD (SEQ ID NO: 5329),MDAGHD (SEQ ID NO: 5330), VEWGHD (SEQ ID NO: 5331), AEQGHD (SEQ ID NO: 5332), LEWGHD (SEQ ID NO: 5333), MELGHD (SEQ ID NO: 5334), METGHD (SEQ ID NO: 5335), MEAGHD (SEQ ID NO: 5336), TINRQR (SEQ ID NO: 5337), IESGHD (SEQ ID NO: 5338), TAKDHD (SEQ ID NO: 5339), MEVGHD (SEQ ID NO: 5340), CEIGHD (SEQ ID NO: 5341), ATNGHD (SEQ ID NO: 5342), MDGGHD ( SEQ ID NO: 5343), QEVGHD (SEQ ID NO: 5344), ADQGHD (SEQ ID NO: 5345), NMNGHD (SEQ ID NO: 5346), TPWEHD (SEQ ID NO: 5347), IEMGHD (SEQ ID NO: 5348), TANEHD (SEQ ID NO: 5349), QQQGHD (SEQ ID NO: 5350), TPQDHD (SEQ ID NO: 5351), HDWGHD (SEQ ID NO: 5352), IEGGHD (SEQ ID NO: 5353), or any dipeptide, tripeptide, tetrapeptide, or pentapeptide thereof. In some embodiments, [A][B] is selected from the group consisting of TINGHDSPHKR (SEQ ID NO: 5354), MPEGHDSPHKS (SEQ ID NO: 5355), MEGGHDSPHKS (SEQ ID NO: 5356), MEYGHDSPHKS (SEQ ID NO: 5357), AEWGHDSPHKS (SEQ ID NO: 5358), CEWGHDSPHKS (SEQ ID NO: 5359), ANNGQDSPHKS (SEQ ID NO: 5360), IPEGHDSPHKS (SEQ ID NO: 5361), ADMGHDSPHKS (SEQ ID NO: 5362), IEYGHDSPHKS (SEQ ID NO: 5363), ADYGHDSPHKS (SEQ ID NO: 5364), I ETGHDSPHKS (SEQ ID NO: 5365), MEWGHDSPHKS (SEQ ID NO: 5366), CEYGHDSPHKS (SEQ ID NO: 5367), RINGHDSPHKS (SEQ ID NO: 5368), MEIGHDSPHKS (SEQ ID NO: 5369), LEYGHDSPHKS (SEQ ID NO: 5370), ADWGHDSPHKS (SEQ ID NO: 5371), IEIGHDSPHKS (SEQ ID NO: 5372), TIKDNDSPHKS (SEQ ID NO: 5373), DIMGHDSPHKS (SEQ ID NO: 5374), FEQGHDSPHKS (SEQ ID NO: 5375), MEFGHDSPHKS (SEQ ID NO: 5376),CDQGHDSPHKS (SEQ ID NO: 5377), LPEGHDSPHKS (SEQ ID NO: 5378), IENGHDSPHKS (SEQ ID NO: 5379), MESGHDSPHKS (SEQ ID NO: 5380), AEIGHSDPHKS (SEQ ID NO: 5381), VEYGHDSPHKS (SEQ ID NO: 5382), TSNGDDSPHKS (SEQ ID NO: 5383), IEVGHDSPHKS (SEQ ID NO: 5384), MEMGHDSPHKS (SEQ ID NO: 5385), AEVGHDSPHKS (SEQ ID NO: 5386), No. 5386), MDAGHDSPHKS (SEQ ID NO: 5387), VEWGHDSPHKS (SEQ ID NO: 5388), AEQGHDSPHKS (SEQ ID NO: 5389), LEWGHDSPHKS (SEQ ID NO: 5390), MELGHDSPHKS (SEQ ID NO: 5391), METGHDSPHKS (SEQ ID NO: 5392), MEAGHDSPHKS (SEQ ID NO: 5393), TINRQRSPHKS (SEQ ID NO: 5394), IESGHDSPHKS (SEQ ID NO: 5395), TAKDHD SPHKS (SEQ ID NO: 5396), MEVGHDSPHKS (SEQ ID NO: 5397), CEIGHDSPHKS (SEQ ID NO: 5398), ATNGHDSPHKS (SEQ ID NO: 5399), MDGGHDSPHKS (SEQ ID NO: 5400), QEVGHDSPHKS (SEQ ID NO: 5401), ADQGHDSPHKS (SEQ ID NO: 5402), NMNGHDSPHKS (SEQ ID NO: 5403), TPWEHDSPHKS (SEQ ID NO: 5404), IEMGHDSPHKS (SEQ ID NO: 5405) ), TANEHDSPHKS (SEQ ID NO: 5406), TINGHDSPHKS (SEQ ID NO: 5407), QQQGHDSPHKS (SEQ ID NO: 5408), TPQDHDSPHKS (SEQ ID NO: 5409), HDWGHDSPHKS (SEQ ID NO: 5410), IEGGHDSPHKS (SEQ ID NO: 5411), or any portion thereof, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids, e.g., consecutive amino acids, of any of them. In some embodiments, [B] is immediately followed by [A]. In some embodiments, the peptide comprises, from the N-terminus to the C-terminus, [A][B].

[0325] In some embodiments, the ligands described herein include proteins or peptides comprising an amino acid sequence comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 consecutive amino acids from any one of the sequences provided in Tables 1, 2A, 2B, 2C, 13-19. In some embodiments, the peptide comprises an amino acid sequence comprising at least 3, 4, or 5 consecutive amino acids from any one of SEQ ID NOs: 945-980 or 985-986. In some embodiments, the peptide comprises an amino acid sequence comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 consecutive amino acids from any one of SEQ ID NOs: 2, 200, 201, 941, 943, 204, 208, 404, or 903-909. In some embodiments, the peptide comprises a modification. In some embodiments, the peptide comprises a phosphate group. In some embodiments, the peptide includes a modification, eg, a phosphate group, at a serine residue.

[0326] In some embodiments, three consecutive amino acids comprise SPH. In some embodiments, four consecutive amino acids comprise SPHS (SEQ ID NO: 4700). In some embodiments, five consecutive amino acids comprise SPHSK (SEQ ID NO: 4701). In some embodiments, six consecutive amino acids comprise SPHSKA (SEQ ID NO: 941). In some embodiments, the peptide comprises a modification. In some embodiments, the peptide comprises a phosphate group. In some embodiments, the peptide comprises a modification, e.g., a phosphate group, at a serine residue. In some embodiments, the peptide comprises a modification, e.g., a phosphate group, at a serine residue located at position 1 numbered according to SEQ ID NO: 941.

[0327] In some embodiments, three consecutive amino acids comprise HDS. In some embodiments, four consecutive amino acids comprise HDSP (SEQ ID NO: 4702). In some embodiments, five consecutive amino acids comprise HDSPH (SEQ ID NO: 4703). In some embodiments, six consecutive amino acids comprise HDSPHK (SEQ ID NO: 2). In some embodiments, seven consecutive amino acids comprise HDSPHKS (SEQ ID NO: 4840). In some embodiments, eight consecutive amino acids comprise HDSPHKSG (SEQ ID NO: 943).

[0328] In some embodiments, three consecutive amino acids comprise HDS. In some embodiments, four consecutive amino acids comprise HDSP (SEQ ID NO: 4702). In some embodiments, five consecutive amino acids comprise HDSPH (SEQ ID NO: 4703). In some embodiments, six consecutive amino acids comprise HDSPHK (SEQ ID NO: 2). In some embodiments, the peptide comprises a modification. In some embodiments, the peptide comprises a phosphate group. In some embodiments, the peptide comprises a modification, e.g., a phosphate group, at a serine residue. In some embodiments, the peptide comprises a modification, e.g., a phosphate group, at a serine residue located at position 3 numbered according to SEQ ID NO: 2.

[0329] In some embodiments, three consecutive amino acids comprise SPH. In some embodiments, four consecutive amino acids comprise SPHK (SEQ ID NO: 6398). In some embodiments, five consecutive amino acids comprise SPHKY (SEQ ID NO: 4715). In some embodiments, six consecutive amino acids comprise SPHKYG (SEQ ID NO: 966).

[0330] In some embodiments, the ligands described herein include proteins or peptides that include an amino acid sequence that includes at least one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of the sequences provided in Tables 1, 2A, 2B, 9, 13-19. In some embodiments, the peptide includes an amino acid sequence that includes at least one, two, or three, but not more than four, different amino acids relative to the amino acid sequence of any one of the sequences provided in Tables 1, 2A, 2B, 13-19. In some embodiments, the peptide includes an amino acid sequence that includes at least one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of SEQ ID NOs: 945-980 or 985-986. In some embodiments, the peptide includes an amino acid sequence that includes at least one, two, or three, but not more than four, different amino acids relative to the amino acid sequence of any one of SEQ ID NOs: 945-980 or 985-986. In some embodiments, the peptide comprises an amino acid sequence that includes at least one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of SEQ ID NOs: 2, 200, 201, 941, 943, 204, 208, 404, or 903-909. In some embodiments, the peptide comprises an amino acid sequence that includes at least one, two, or three, but not more than four, different amino acids relative to the amino acid sequence of any one of SEQ ID NOs: 2, 200, 201, 941, 943, 204, 208, 404, or 903-909. In some embodiments, the peptide comprises an amino acid sequence that includes at least one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 3589. In some embodiments, the peptide comprises an amino acid sequence that includes at least 1, 2, or 3, but no more than 4, different amino acids relative to the amino acid sequence of SEQ ID NO:3589.In some embodiments, the peptide comprises an amino acid sequence that includes at least one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SEQ ID NO: 1754. In some embodiments, the peptide comprises an amino acid sequence that includes at least one, two, or three, but not more than four, different amino acids relative to the amino acid sequence of SEQ ID NO: 1754.

[0331] In some embodiments, the ligands described herein include proteins or peptides that include an amino acid sequence that includes at least one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SPHSKA (SEQ ID NO: 941). In some embodiments, the peptides include an amino acid sequence that includes at least one, two, or three, but not more than four, different amino acids relative to the amino acid sequence of SPHSKA (SEQ ID NO: 941).

[0332] In some embodiments, the ligands described herein include proteins or peptides that include an amino acid sequence that contains at least one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of HDSPHKSG (SEQ ID NO: 943). In some embodiments, the peptides include an amino acid sequence that includes at least one, two, or three, but not more than four, different amino acids relative to the amino acid sequence of HDSPHKSG (SEQ ID NO: 943).

[0333] In some embodiments, the ligands described herein include proteins or peptides that include an amino acid sequence that contains at least one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of HDSPHK (SEQ ID NO: 2). In some embodiments, the peptides include an amino acid sequence that contains at least one, two, or three, but not more than four, different amino acids relative to the amino acid sequence of HDSPHK (SEQ ID NO: 2).

[0334] In some embodiments, the ligands described herein include proteins or peptides that include an amino acid sequence that includes at least one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SPHKYG (SEQ ID NO: 966). In some embodiments, the peptides include an amino acid sequence that includes at least one, two, or three, but not more than four, different amino acids relative to the amino acid sequence of SPHKYG (SEQ ID NO: 966).

[0335] In some embodiments, the ligands described herein comprise proteins or peptides comprising the amino acid sequence of any of the sequences provided in Tables 1, 2A, 2B, 13-19. In some embodiments, the peptide comprises the amino acid sequence of any of SEQ ID NOs: 945-980 or 985-986. In some embodiments, the peptide comprises the amino acid sequence of any of SEQ ID NOs: 200, 201, 941, 943, 204, 208, 404, or 903-909. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 941. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 943. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3589. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1754.

[0336] In some embodiments, the ligands described herein include proteins or peptides comprising an amino acid sequence encoded by a nucleotide sequence described herein, e.g., a nucleotide sequence in Table 2A. In some embodiments, the peptide comprises an amino acid sequence encoded by a nucleotide sequence that includes at least 1, 2, 3, 4, 5, 6, or 7 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 942. In some embodiments, the peptide comprises an amino acid sequence encoded by a nucleotide sequence that includes at least 1, 2, 3, 4, 5, 6, or 7, but not more than 10, different nucleotides relative to the nucleotide sequence of SEQ ID NO: 942. In some embodiments, the peptide comprises an amino acid sequence encoded by a nucleotide sequence that includes the nucleotide sequence of SEQ ID NO: 942, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In some embodiments, the peptide comprises an amino acid sequence encoded by a nucleotide sequence that includes at least 1, 2, 3, 4, 5, 6, or 7 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 944. In some embodiments, the peptide comprises an amino acid sequence encoded by a nucleotide sequence that includes at least 1, 2, 3, 4, 5, 6, or 7, but not more than 10, different nucleotides relative to the nucleotide sequence of SEQ ID NO: 944. In some embodiments, the peptide comprises an amino acid sequence encoded by a nucleotide sequence that is substantially identical thereto (e.g., has at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity).

[0337] In some embodiments, the ligands described herein include proteins or peptides that include a modification. In some embodiments, the peptide includes a phosphate group. In some embodiments, the peptide includes a modification, e.g., a phosphate group, at a serine residue. In some embodiments, the peptide includes a modification, e.g., a phosphate group, at a serine residue located at position 3 numbered according to SEQ ID NO: 2. In some embodiments, the peptide includes a modification, e.g., a phosphate group, at a serine residue located at position 1 numbered according to SEQ ID NO: 941. In some embodiments, the peptide includes a modification, e.g., a phosphate group, at a serine residue located in the amino acid sequence of SPH.

[0338] In some embodiments, the nucleotide sequence encoding a peptide of a ligand described herein comprises a nucleotide sequence described herein, e.g., as set forth in Table 2A. In some embodiments, the nucleotide sequence encoding a peptide described herein is codon-optimized. In some embodiments, the nucleotide sequence encoding a peptide described herein is isolated, e.g., recombinant.

[0339] In some embodiments, a nucleotide sequence encoding a peptide of a ligand described herein comprises the nucleotide sequence of SEQ ID NO:942, or a nucleotide sequence that includes at least 1, 2, 3, 4, 5, 6, or 7 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO:942. In some embodiments, a nucleotide sequence encoding a peptide described herein comprises a nucleotide sequence that includes at least 1, 2, 3, 4, 5, 6, or 7, but not more than 10, different nucleotides relative to the nucleotide sequence of SEQ ID NO:942. In some embodiments, a nucleic acid sequence encoding a peptide described herein comprises a nucleotide sequence that includes the nucleotide sequence of SEQ ID NO:942, or a nucleotide sequence that is substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity).

[0340] In some embodiments, a nucleic acid encoding a peptide of a ligand described herein comprises the nucleotide sequence of SEQ ID NO:944, or a nucleotide sequence that includes at least 1, 2, 3, 4, 5, 6, or 7 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO:944. In some embodiments, a nucleotide sequence encoding a peptide described herein comprises a nucleotide sequence that includes at least 1, 2, 3, 4, 5, 6, or 7, but not more than 10, different nucleotides relative to the nucleotide sequence of SEQ ID NO:944. In some embodiments, a nucleic acid encoding a peptide described herein comprises a nucleotide sequence that includes the nucleotide sequence of SEQ ID NO:944, or a nucleotide sequence that is substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity).

[0341] The present disclosure also provides nucleic acids or polynucleotides encoding any of the peptides described herein, as well as ligands, compositions, AAV capsid variants, AAV particles, vectors, and cells comprising them.

[0342] antibody molecule In some embodiments, a ligand described herein is or comprises an antibody molecule, hi other embodiments, an active agent described herein, e.g., a therapeutic or diagnostic agent, is or comprises an antibody molecule.

[0343] As used herein, the term "antibody molecule" refers to a protein that comprises at least one immunoglobulin variable domain sequence, e.g., an immunoglobulin chain or fragment thereof. The term "antibody molecule" includes, for example, monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region). In certain embodiments, an antibody molecule comprises a full-length antibody or a full-length immunoglobulin chain. In certain embodiments, an antibody molecule comprises an antigen-binding or functional fragment of a full-length antibody or a full-length immunoglobulin chain.

[0344] In certain embodiments, the antibody molecule is a monospecific antibody molecule, binding to a single epitope, for example, a monospecific antibody molecule having multiple immunoglobulin variable domain sequences, each of which binds to the same epitope.

[0345] In some embodiments, the antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In some embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In some embodiments, the multispecific antibody molecule comprises a third, fourth, or fifth immunoglobulin variable domain. In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0346] In some embodiments, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody does not have specificity for more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In some embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In some embodiments, a bispecific antibody molecule comprises heavy and light chain variable domain sequences that have binding specificity for a first epitope and heavy and light chain variable domain sequences that have binding specificity for a second epitope. In certain embodiments, a bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In certain embodiments, a bispecific antibody molecule comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In certain embodiments, a bispecific antibody molecule comprises an scFv, or fragment thereof, having binding specificity for a first epitope and an scFv, or fragment thereof, having binding specificity for a second epitope.

[0347] In some embodiments, an antibody molecule comprises at least one immunoglobulin variable domain sequence. Antibody molecules can include, for example, full-length mature antibodies and antigen-binding fragments of antibodies. For example, an antibody molecule can comprise a heavy (H) chain variable domain sequence (abbreviated herein as VH) and a light (L) chain variable domain sequence (abbreviated herein as VL). In another example, an antibody can comprise two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequences, thereby forming two antigen-binding sites, such as Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single-chain antibodies (e.g., scFv), single variable domain antibodies, diabodies (DAbs) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which can be produced by modification of whole antibodies or de novo synthesized antibodies using recombinant DNA technology. These functional antibody fragments retain the ability to selectively bind to their respective antigens or receptors. Antibodies and antibody fragments can be derived from any antibody class, including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Antibody molecules can be monoclonal or polyclonal. The encoded antibody can also be human, humanized, CDR-grafted, or in vitro generated. The antibody can have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. The antibody can also have a light chain selected from, for example, kappa or lambda.

[0348] Examples of antigen-binding fragments include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a diabody (dAb) fragment consisting of a VH domain; (vi) a camel or camelized variable domain; (vii) a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426, and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and (viii) a single-domain antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology, 23:1126-1136, 2005).

[0349] The term "antibody" includes intact molecules as well as functional fragments thereof. The constant region of an antibody can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function).

[0350] In some embodiments, the antibody molecule may be a single-domain antibody. Single-domain antibodies may include antibodies in which the complementarity-determining regions are part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, antibodies naturally devoid of light chains, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. The single-domain antibody may be any known or future single-domain antibody in the art. The single-domain antibody may be derived from any species, including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit, and cow. According to another aspect of the present invention, the single-domain antibody is a naturally occurring single-domain antibody known as a heavy-chain antibody devoid of light chains. Such single-domain antibodies are disclosed, for example, in WO9404678. For clarity, this variable domain derived from a heavy-chain antibody naturally devoid of light chains is referred to herein as a VHH or nanobody to distinguish it from the conventional VH of four-chain immunoglobulins. Such VHH molecules may be derived from antibodies produced in Camelidae species, such as camel, llama, dromedary, alpaca, and guanaco. Non-Camelidae species may also produce heavy chain antibodies that are naturally devoid of light chains, and such VHHs are within the scope of the present invention.

[0351] In some embodiments, the VH and VL regions of an antibody molecule can be subdivided into regions of hypervariability, termed "complementarity-determining regions" (CDRs), interspersed with regions that are highly conserved, termed "framework regions" (FR or FW).

[0352] The extent of framework regions and CDRs has been precisely defined in several ways (see Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and the AbM definitions used by Oxford Molecular's AbM antibody modeling software. See generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg).

[0353] "Complementarity determining region" and "CDR" as used herein refer to the sequences of amino acids in an antibody variable region that confer antigen specificity and binding affinity. Generally, each heavy chain variable region has three CDRs (HCDR1, HCDR2, HCDR3), and each light chain variable region has three CDRs (LCDR1, LCDR2, LCDR3).

[0354] The precise amino acid sequence boundaries of a given CDR can be determined using any of several well-known schemes, including those described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (Kabat numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (Chothia numbering scheme). In some embodiments, CDRs defined according to the Chothia numbering scheme are also referred to as hypervariable loops.

[0355] For example, in Kabat, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). In Chothia, the CDR amino acids of the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues of the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.

[0356] In some embodiments, the antigen-binding domain of an antibody molecule of the present disclosure is the portion of the antibody molecule that contains determinants that form an interface that binds to a therapeutic protein or its epitope. For proteins (or protein mimetics), the antigen-binding site typically includes one or more loops (of at least four amino acids or amino acid analogs) that form an interface that binds to the therapeutic protein. Typically, the antigen-binding site of an antibody molecule includes at least one or two CDRs and / or hypervariable loops, and more typically includes at least three, four, five, or six CDRs and / or hypervariable loops.

[0357] Antibody molecules may be monoclonal or polyclonal. In some embodiments, a monoclonal antibody or monoclonal antibody composition refers to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Monoclonal antibodies can be produced by hybridoma technology or by methods that do not use hybridoma technology (e.g., recombinant methods).

[0358] In some embodiments, the sequences of antibody molecules to be included in the encoded payloads described herein can be generated by recombinant libraries, e.g., by phage display or by combinatorial methods.

[0359] Phage display and combinatorial methods for generating antibodies are known in the art (e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. WO 92 / 18619; Dower et al. WO 91 / 17271; Winter et al. WO 92 / 20791; Markland et al. WO 92 / 15679; Breitling et al. WO 93 / 01288; McCafferty et al. WO 92 / 01047; Garrard et al. WO 92 / 09690; Ladner et al. WO 90 / 02809; Fuchs et al. (1991) Bio / Technology 9:1370-1372; Hay et al. al.(1992)Hum Antibod Hybridomas 3:81-85;Huse et al.(1989)Science 246:1275-1281;Griftth et al.(1993)EMBO J 12:725-734;Hawkins et al.(1992)J Mol Biol 226:889-896;Clackson et al. al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio / Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. al.(1991)PNAS 88:7978-7982, the entire contents of which are incorporated herein by reference).

[0360] In some embodiments, the sequences of antibody molecules to be included in the encoded payloads described herein can be generated from antibody molecules designed using VERSITOPE™ Antibody Generation or BIOATLA®, e.g., in US20130303399, US20130281303, WO2012009026, WO2016033331, WO2016036916, and US8859467, the contents of which are incorporated by reference in their entireties. In some embodiments, the sequences of antibody molecules included in the encoded payloads described herein can be derived from antibody molecules designed and / or produced using the methods described in, e.g., WO2017189959 and WO2020223276, the contents of which are incorporated by reference in their entireties.

[0361] In some embodiments, the antibody molecule comprises the amino acid sequence of a fully human antibody (e.g., an antibody generated in a mouse genetically engineered to produce antibodies from human immunoglobulin sequences) or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), or camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Methods for producing rodent antibodies are known in the art.

[0362] Human monoclonal antibodies may also be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with an antigen of interest are used to produce hybridomas secreting human mAbs with specific affinity for epitopes derived from human proteins (e.g., (e.g., Wood et al. International Application WO 91 / 00906; Kucherlapati et al. PCT Publication WO 91 / 10741; Lonberg et al. International Application WO 92 / 03918; Kay et al. International Application 92 / 03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, LL et al. 1994 Nature Genet. 7:13-21; Morrison, SL et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326).

[0363] In some embodiments, the antibody comprises a variable region or portion thereof, e.g., a CDR, comprising the amino acid sequence of an antibody generated in a non-human organism, e.g., a rat or a mouse. Antibody molecules, including chimeric, CDR-grafted, and humanized antibodies, are within the scope of the present invention. Antibody molecules comprising the sequence of an antibody generated in a non-human organism, e.g., a rat or a mouse, and then modified, e.g., in the variable framework or constant region, to reduce antigenicity in humans are within the scope of the present invention.

[0364] A substantially human protein is one that does not provoke a substantially neutralizing antibody response, such as a human anti-mouse antibody (HAMA) response. HAMA can be a problem in many situations, for example, when antibody molecules are administered repeatedly, such as in the treatment of chronic or recurring disease states. HAMA responses can potentially render repeated antibody administration ineffective due to increased antibody clearance from serum (see, e.g., Saleh et al., Cancer Immunol. Immunother, 32:180-190 (1990)) and potential allergenicity (see, e.g., LoBuglio et al., Hybridoma, 5:5117-5123 (1986)).

[0365] Chimeric antibodies can be produced by recombinant DNA techniques known in the art (Robinson et al., International Patent Publication No. PCT / US86 / 02269; Akira, et al., European Patent Application No. 184,187; Taniguchi, M., European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neberger et al., International Application No. WO 86 / 01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application No. 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

[0366] A humanized antibody or CDR-grafted antibody will have at least one or two, but generally all three, recipient CDRs (of the immunoglobulin heavy and / or light chain) replaced with donor CDRs. The antibody may have at least a portion of the non-human CDRs replaced, or only some CDRs replaced with non-human CDRs. Preferably, the donor will be a rodent antibody, e.g., a rat or mouse antibody, and the recipient will be a human framework or human consensus framework. Typically, the immunoglobulin providing the CDRs is referred to as the donor, and the immunoglobulin providing the framework is referred to as the acceptor. In some embodiments, the donor immunoglobulin is non-human (e.g., rodent). The acceptor framework is a naturally occurring (e.g., human) framework or consensus framework, or a sequence about 85% or more, preferably 90%, 95%, 99% or more identical thereto.

[0367] In some embodiments, a consensus sequence refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of related sequences (see, e.g., Winnaker, From Genes to Clones (Verlaggsgesellschft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. In some embodiments, a consensus framework refers to the framework region in a consensus immunoglobulin sequence.

[0368] Antibodies can be humanized by methods known in the art (see, e.g., Morrison, SL, 1985, Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; and Queen et al., US Pat. Nos. 5,585,089, 5,693,761, and 5,693,762, the contents of all of which are incorporated herein by reference).

[0369] Humanized antibodies or CDR-grafted antibodies may be produced by CDR grafting or CDR replacement, and one, two, or all CDRs of an immunoglobulin chain may be replaced. See, for example, U.S. Patent 5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; Winter, U.S. Patent 5,225,539 (the contents of all of which are expressly incorporated herein by reference). Winter describes a CDR-grafting method (UK Patent Application GB2188638A filed March 26, 1987; Winter, U.S. Patent 5,225,539) (the contents of which are expressly incorporated herein by reference) that can be used to prepare the humanized antibodies of the present invention.

[0370] In some embodiments, the antibody comprises a humanized antibody sequence in which specific amino acids have been substituted, deleted, or added. Criteria for selecting amino acids from a donor are described in US 5,585,089, e.g., columns 12-16 of US 5,585,089, e.g., columns 12-16 of US 5,585,089, the contents of which are incorporated herein by reference. Other techniques for humanizing antibodies are described in EP 519596 A1, published December 23, 1992, by Padlan et al.

[0371] In some embodiments, the antibody molecule may be a single-chain antibody. Single-chain antibodies (scFV) may be engineered (see, e.g., Colcher, D. et al. (1999) Ann NY Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). Single-chain antibodies may be dimerized or multimerized to generate multivalent antibodies with specificities for different epitopes of the same target protein.

[0372] In yet other embodiments, the antibody molecule has a heavy chain constant region selected from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE heavy chain constant regions; particularly, e.g., IgG1, IgG2, IgG3, and IgG4 (e.g., human) heavy chain constant regions. In another embodiment, the antibody molecule has a light chain constant region selected from, e.g., kappa or lambda (e.g., human) light chain constant regions. The constant region can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, and / or complement function). In some embodiments, the antibody has effector function and can fix complement. In other embodiments, the antibody neither recruits effector cells nor fixes complement. In other embodiments, the antibody has reduced or no ability to bind to Fc receptors. For example, the antibody is an isotype or subtype, fragment or other variant that does not support binding to an Fc receptor, eg, the Fc receptor binding region thereof has been mutated or deleted.

[0373] Methods for altering antibody constant regions are known in the art. Antibodies with altered function, e.g., altered affinity for effector ligands such as FcR on cells or the C1 component of complement, can be generated by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see, e.g., EP 388,151 A1, U.S. Pat. No. 5,624,821, and U.S. Pat. No. 5,648,260, the contents of all of which are incorporated herein by reference). Similar types of alterations may be described that, when applied to immunoglobulins of mice or other species, reduce or eliminate these functions.

[0374] Antibody molecules can be derivatized or linked to another functional molecule (e.g., another peptide or protein). As used herein, a "derivatized" antibody molecule is one that has been modified. Methods of derivatization include, but are not limited to, the addition of a fluorescent moiety, a radionucleotide, a toxin, an enzyme, or an affinity ligand, such as biotin. Thus, the antibody molecules of the present invention are intended to include derivatized and other modified forms of antibodies described herein, such as immunoadhesion molecules. For example, an antibody molecule can be functionally linked (by chemical conjugation, genetic fusion, noncovalent bonding, or other methods) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (e.g., a streptavidin core region or a polyhistidine tag).

[0375] One type of derivatized antibody molecule is produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include heterobifunctional (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate), which have two distinct reactive groups separated by an appropriate spacer. Such linkers are available from Pierce Chemical Company, Rockford, Ill.

[0376] Useful detection agents with which the antibody molecules of the present invention can be derivatized (or labeled) include fluorescent compounds, various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, fluorescent metal atoms such as europium (Eu) and other lanthanides, and radioactive materials (described below). Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and the like. Antibodies may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, β-galactosidase, acetylcholinesterase, glucose oxidase, and the like. When an antibody is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, in the presence of the detectable agent horseradish peroxidase, addition of hydrogen peroxide and diaminobenzidine produces a detectable colored reaction product. Antibody molecules may also be derivatized with prosthetic groups (e.g., streptavidin / biotin and avidin / biotin). For example, antibodies may be derivatized with biotin and detected through indirect measurement of avidin or streptavidin binding. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of suitable luminescent materials include luminol; and examples of bioluminescent materials include luciferase, luciferin, and aequorin.

[0377] Labeled antibody molecules can be used diagnostically and / or experimentally in a number of contexts, for example, (i) to isolate a given antigen by standard techniques such as affinity chromatography or immunoprecipitation, (ii) to detect a given antigen (e.g., in a cell lysate or cell supernatant) to assess protein abundance and expression patterns, and (iii) to monitor protein levels in tissues as part of a clinical testing procedure, for example, to determine the effectiveness of a given treatment regimen.

[0378] The antibody molecule may be conjugated to another molecular entity, typically a label or a therapeutic (e.g., cytotoxic or cytostatic) agent or moiety. Radioisotopes can be used for diagnostic or therapeutic applications. Radioisotopes that can be bound to the antibodies described herein include, but are not limited to, α, β, or γ emitters, or β and γ emitters. Such radioisotopes include iodine ( 131 I or 125 I), yttrium ( 90 Y), lutetium ( 177 Lu), actinium ( 225 Ac), praseodymium, astatine ( 211 At), rhenium ( 186 Re), Bismuth ( 212 Bi or 213 Bi), Indium ( 111 In), technetium ( 99 mTc), phosphorus ( 32 P), rhodium ( 188 Rh), sulfur ( 35 S), carbon ( 14 C), tritium ( 3 H), chromium ( 51 Cr), chlorine ( 36 Cl), cobalt ( 57 Co or 58 Co), iron ( 59 Fe), Selenium ( 75 Se), or gallium ( 67 Radioisotopes useful as therapeutic agents include, but are not limited to, yttrium (90 Y), lutetium ( 177 Lu), actinium ( 225 Ac), praseodymium, astatine ( 211 At), rhenium ( 186 Re), Bismuth ( 212 Bi or 213 Bi), and rhodium ( 188 Radioisotopes useful as labels, for example for diagnostic use, include iodine ( 131 I or 125 I), indium ( 111 In), technetium ( 99 mTc), phosphorus ( 32 P), carbon ( 14 C), and tritium ( 3 H), or one or more of the therapeutic isotopes listed above.

[0379] The present invention provides radiolabeled antibody molecules and methods for labeling the same. In one embodiment, a method for labeling an antibody molecule is disclosed. The method comprises contacting the antibody molecule with a chelating agent, thereby producing a conjugated antibody. The conjugated antibody can be fused to a radioisotope, e.g., 111 indium, 90 yttrium, and 177 It is radiolabeled with lutetium, thereby producing a labeled antibody molecule.

[0380] As discussed above, the antibody molecule can be conjugated to a therapeutic agent. Therapeutically active radioisotopes have already been mentioned. Examples of other therapeutic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat. Nos. 5,475,092, 5,585,499, 5,846,545), and analogs or homologs thereof. Therapeutic agents include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decylcarbazine), alkylating agents (e.g., mechlorethamine, thioepa, chlorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclosporine, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamin). Anti-inflammatory drugs include, but are not limited to, platinum(II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine, vinblastine, taxol, and maytansinoids).

[0381] In some embodiments, the ligand described herein is or comprises an antibody molecule that binds to a GPI-anchored protein. In some embodiments, the antibody molecule binds to ALPL, for example, human or mouse ALPL. In some embodiments, the antibody molecule is F2910-SP, AF2909, NBP2-67295, LS-B3666, MA524845, 2F4, or a variant thereof. In some embodiments, the antibody molecule is an antibody provided in Table 40 or a variant thereof, for example, Ab9 in Table 40.

[0382] multispecific antibodies In some embodiments, the antibody molecule is a multispecific antibody, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In some embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In some embodiments, the multispecific antibody molecule comprises a third, fourth, or fifth immunoglobulin variable domain. In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule. In some embodiments, the antibody molecules described herein are multispecific antibody molecules.

[0383] In some embodiments, a multispecific antibody is a bispecific antibody. A bispecific antibody does not have specificity for more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In some embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In some embodiments, a bispecific antibody molecule comprises heavy and light chain variable domain sequences that have binding specificity for a first epitope and heavy and light chain variable domain sequences that have binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises an scFv, or fragment thereof, having binding specificity for a first epitope and an scFv, or fragment thereof, having binding specificity for a second epitope. In some embodiments, the antibody molecules described herein are bispecific antibody molecules.

[0384] In some embodiments, the sequences of antibody molecules can be generated from bispecific or heterodimeric antibody molecules generated using protocols known in the art, such as the "knobs-in-holes" approach described, for example, in U.S. Pat. No. 5,731,168; electrostatic steering Fc pairing, for example, as described in WO 09 / 089004, WO 06 / 106905, and WO 2010 / 129304; SEED (Strand Exchange Engineered) antibody pairing, for example, as described in WO 07 / 110205. Fab arm exchange, as described, for example, in WO 08 / 119353, WO 2011 / 131746, and WO 2013 / 060867; biantibody conjugates, e.g., by crosslinking antibodies using heterobifunctional reagents with amine-reactive groups and sulfhydryl-reactive groups to generate bispecific structures, as described, for example, in US 4,433,059; bispecific antibody determinants generated by recombining half antibodies (heavy-light chain pairs or Fab) from different antibodies through cycles of reduction and oxidation of the disulfide bond between the two heavy chains, as described, for example, in US 4,444,878; trifunctional antibodies, e.g., three Fab' fragments crosslinked via sulfhydryl-reactive groups, as described, for example, in US 5,273,743; biosynthetic binding proteins, such as pairs of scFvs cross-linked through their C-terminal tails, preferably via disulfide or amine-reactive chemical cross-linking; bifunctional antibodies, such as Fab fragments with different binding specificities dimerized via leucine zippers (e.g., c-fos and c-jun) replacing the constant domains, as described, for example, in US Pat. No. 5,582,996; bispecific and oligospecific monovalent and oligovalent receptors, such as those described, for example, in US Pat. No. 5,591,828, for example, the VH-CH1 regions of two antibodies (two Fab fragments) linked via a polypeptide spacer between the CH1 region of one antibody and the VH region of the other antibody, typically with the associated light chain; bispecific DNA-antibody conjugates, such as those described, for example, in US Pat. No. 5,635,602.Cross-linking of antibodies or Fab fragments via double-stranded sections of DNA; bispecific fusion proteins, such as described in US Pat. No. 5,637,481, for example, an expression construct comprising two scFvs and a complete constant region with a hydrophilic helical peptide linker between them; multivalent and multispecific binding proteins, such as described in US Pat. No. 5,837,242, for example, a polypeptide dimer having a first domain with an Ig heavy chain variable region binding region and a second domain with an Ig light chain variable region binding region, commonly referred to as a diabody (higher order structures creating bispecific, trispecific or tetraspecific molecules are also disclosed); minibody constructs, such as described in US Pat. No. 5,837,821, having linked VL and VH chains further connected to antibody hinge and CH3 regions by a peptide spacer, which can be dimerized to form bispecific / multivalent molecules; These include, but are not limited to, VH and VL domains linked by a linker (e.g., 5 or 10 amino acids) or no linker at all in either orientation, which can dimerize to form bispecific diabodies; trimers and tetramers, as described, for example, in US 5,844,094; consecutive VH domains (or VL domains of family members) connected at the C-terminus by a peptide bond to a crosslinking group and further associated with a VL domain to form a series of FVs (or scFvs), as described, for example, in US 5,864,019; and single-chain binding polypeptides, as described, for example, in US 5,869,620, in which both VH and VL domains linked via peptide linkers are joined into multivalent structures via non-covalent or chemical crosslinking to form, for example, homobivalent, heterobivalent, trivalent, and tetravalent structures using both scFv or diabody-type formats. Additional exemplary multispecific and bispecific molecules and methods for making them can be found in, e.g., US5910573, US5932448, US5959083, US5989830, US6005079, US6239259, US6294353, US6333396, US6476198, US6511663, US6670453, US6743896, US6809185, US6833441, US7129330, US7183076,US7521056、US7527787、US7534866、US7612181、US2002 / 004587A1、US2002 / 076406A1、US2002 / 103345A1、US2003 / 207346A1、US2003 / 211078A1、US2004 / 219643A1、US2004 / 220388A1、US2004 / 242847A1、US2005 / 003403A1、US2005 / 004352A1、US2005 / 069552A1、US2005 / 079170A1、US2005 / 100543A1、US2005 / 136049A1、US2005 / 136051A1、US2005 / 163782A1、US2005 / 266425A1、US2006 / 083747A1、US2006 / 120960A1、US2006 / 204493A1、US2006 / 263367A1、US2007 / 004909A1、US2007 / 087381A1、US2007 / 128150A1、US2007 / 141049A1、US2007 / 154901A1、US2007 / 274985A1、US2008 / 050370A1、US2008 / 069820A1、US2008 / 152645A1、US2008 / 171855A1、US2008 / 241884A1、US2008 / 254512A1、US2008 / 260738A1、US2009 / 130106A1、US2009 / 148905A1、US2009 / 155275A1、US2009 / 162359A1、US2009 / 162360A1、US2009 / 175851A1、US2009 / 175867A1、US2009 / 232811A1、US2009 / 234105A1、US2009 / 263392A1、US2009 / 274649A1、EP346087A2、WO00 / 06605A2、WO02 / 072635A2、WO04 / 081051A1、WO06 / 020258A2、WO2007 / 044887A2、WO2007 / 095338A2、WO2007 / 137760A2、WO2008 / 119353A1、WO2009 / 021754A2、WO2009 / 068630A1、WO91 / 03493A1、WO93 / 23537A1、WO94 / 09131A1、WO94 / 12625A2、WO95 / 09917A1、WO96 / 37621A2、WO99 / 64460A1, the contents of which are incorporated herein by reference in their entirety.

[0385] In some embodiments, the ligands described herein include multispecific, e.g., bispecific, antibody molecules that include a first binding domain that binds to ALPL (e.g., an anti-ALPL binding domain) and a second binding domain that binds to a therapeutic target.

[0386] Fc polypeptide In some embodiments, the ligand described herein comprises an Fc polypeptide. In some embodiments, the ligand is or comprises a first Fc polypeptide. In some embodiments, the ligand is a first Fc polypeptide and the active agent is a second Fc polypeptide.

[0387] In some embodiments, the first Fc polypeptide and the second Fc polypeptide form dimers. In some embodiments, the first Fc polypeptide and the second Fc polypeptide comprise a dimerization domain, e.g., the interface between the first and second Fc polypeptides. In some embodiments, the dimerization domain has been engineered, e.g., mutated, to increase or decrease dimerization, e.g., compared to an unengineered interface. In some embodiments, dimerization between the first Fc polypeptide and the second Fc polypeptide is enhanced by providing one or more of paired knobs-and-protrusions ("knobs-in-holes"), electrostatic interactions, or strand exchange at the Fc interface of the first and second Fc polypeptides, e.g., such that a greater ratio of heteromultimers to homomultimers is formed, compared to an unengineered interface. In some embodiments, the first Fc polypeptide comprises an amino acid substitution selected from T366S, L368A, or Y407V (e.g., corresponding to a knob or hole) (or a combination thereof). In some embodiments, the second Fc polypeptide comprises the amino acid substitution T366W (e.g., corresponding to a protrusion or knob). In some embodiments, the first Fc polypeptide comprises an amino acid substitution selected from T366S, L368A, or Y407V (e.g., corresponding to a depression or hole) (or a combination thereof), and the second Fc polypeptide comprises the amino acid substitution T366W (e.g., corresponding to a protrusion or knob). In some embodiments, the second Fc polypeptide comprises an amino acid substitution selected from T366S, L368A, or Y407V (e.g., corresponding to a depression or hole) (or a combination thereof). In some embodiments, the first Fc polypeptide comprises the amino acid substitution T366W (e.g., corresponding to a protrusion or knob). In some embodiments, the second Fc polypeptide comprises an amino acid substitution selected from T366S, L368A, or Y407V (e.g., corresponding to a depression or hole) (or a combination thereof), and the first Fc polypeptide comprises the amino acid substitution T366W (e.g., corresponding to a protrusion or knob).

[0388] In some embodiments, the first Fc polypeptide, the second Fc polypeptide, or both (i) have reduced affinity, e.g., eliminated affinity, for an Fc receptor, e.g., as compared to a reference, wherein the reference is a wild-type Fc receptor; and (ii) have a nucleotide sequence at one of positions I253 (e.g., I253A), H310 (e.g., H310A or H310Q), and / or H435 (e.g., H435A or H435Q), numbered according to the EU index of Kabat. (iii) has a reduced effector function (e.g., reduced ADCC) when compared to a reference, wherein the reference is a wild-type Fc receptor; and (iv) comprises mutations at one, two, or all of positions L235 (e.g., L235V), F243 (e.g., F243L), R292 (e.g., R292P), Y300 (e.g., Y300L), and P396 (e.g., P396L), numbered according to the EU index of Kabat. In some embodiments, the first Fc polypeptide, the second Fc polypeptide, or both, comprise a half-life extender or amino acid modification that increases serum half-life (e.g., (i) Leu at position 428 and Ser at position 434, or (ii) Ser or Ala at position 434, according to EU numbering).

[0389] In some embodiments, the ligand comprises a first Fc polypeptide, wherein the first Fc polypeptide comprises a protein or peptide sequence provided herein, e.g., as set forth in any of Tables 1, 2A, 2B, 13-19. In some embodiments, the protein or peptide is present in a CH3 domain of the first Fc polypeptide. In some embodiments, the CH3 domain is modified from the CH3 domain of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the CH3 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 substitutions at a set of amino acid positions including 380, 384, 386, 387, 388, 389, 390, 413, 415, 416, and 421, according to EU numbering. The protein or peptide is present at or near the C-terminus of the Fc polypeptide (e.g., within 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acids of the C-terminus of the therapeutic protein, enzyme, or antibody). The first Fc polypeptide, the second Fc polypeptide, or both the first and second Fc polypeptides do not comprise immunoglobulin heavy and / or light chain variable region sequences or antigen-binding portions thereof.

[0390] The second Fc polypeptide is fused or conjugated (eg, directly or indirectly via a linker) to a therapeutic protein or variant thereof (eg, an enzyme). Other Exemplary Ligands In some embodiments, the ligands described herein comprise nucleic acid molecules. In some embodiments, the ligands described herein comprise aptamers. In some embodiments, the aptamers bind to GPI-anchored proteins. In some embodiments, the aptamers bind to ALPL, e.g., human or mouse ALPL. In some embodiments, the aptamers are or comprise DNA, RNA, modified DNA, modified RNA, or combinations thereof. In some embodiments, the aptamers are fused or conjugated to a therapeutic agent selected from a protein (e.g., an enzyme), an antibody molecule, a nucleic acid molecule (e.g., an RNAi agent), or a small molecule.

[0391] In some embodiments, the ligand described herein is or comprises a small molecule. In some embodiments, the small molecule is an inhibitor of ALPL, e.g., a small molecule that interferes with ALPL dimerization. In some embodiments, the small molecule is an arylsulfonamide, a phosphonate derivative, a pyrazole, a triazole, or an imidazole. In some embodiments, the small molecule is 5-((5-chloro-2-methoxyphenyl)sulfonamido)nicotinamide (SBI-425). In some embodiments, the small molecule is 2,5-dimethoxy-N-(quinolin-3-yl)benzenesulfonamide (tissue-nonspecific alkaline phosphatase inhibitor (TNAPi)).

[0392] In some embodiments, the ligand described herein is present on or attached to a carrier, such as an exosome, microvesicle, or lipid nanoparticle (LNP). In some embodiments, the carrier is an exosome or LNP. In some embodiments, the peptide is present on the surface of the carrier. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the surface of the carrier comprises at least 1 to 5, e.g., at least 1, 2, 3, 4, or 5, proteins or peptides comprising an amino acid sequence provided herein, e.g., as shown in any one of Tables 1, 2A, 2B, or 13-19. In some embodiments, the ligand is conjugated to the surface of the carrier by post-insertion. In some embodiments, the ligand is conjugated to the surface of the carrier via a covalent bond (e.g., using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry or thiol-maleimide coupling reaction). In some embodiments, the carrier is conjugated to a therapeutic agent. In some embodiments, the carrier comprises an RNAi agent, an mRNA, a ribonucleoprotein complex (e.g., a Cas9 / gRNA complex), or a circRNA.

[0393] AAV serotypes and capsids In some embodiments, the ligand described herein is a component of a viral particle, e.g., an AAV particle or a lentivirus. In some embodiments, the ligand is a component of a capsid protein, e.g., an AAV capsid protein described herein.

[0394] In some embodiments, AAV particles may comprise capsid proteins or variants thereof of any natural or recombinant AAV serotype. AAV serotypes may differ in characteristics such as, but not limited to, packaging, tropism, transduction, and immunogenicity profiles. Without wishing to be bound by theory, it is believed that in some embodiments, AAV capsid proteins, e.g., AAV capsid variants, can, for example, modulate, e.g., direct, the tropism of AAV particles to specific tissues.

[0395] In some embodiments, the AAV comprises a small, non-enveloped, icosahedral-capsid virus of the Parvoviridae family, characterized by a single-stranded DNA viral genome. The Parvoviridae family of viruses consists of two subfamilies: the Parvovirinae subfamily, which infect vertebrates, and the Densovirinae subfamily, which infect invertebrates. The Parvoviridae family includes the Dependovirus genus, which includes AAVs that can replicate in vertebrate hosts, including, but not limited to, humans, primates, bovine, canine, equine, and ovine species.

[0396] In some embodiments, AAVs are used as biological tools due to their relatively simple structure, their ability to infect a wide range of cells (including quiescent and dividing cells) without integrating into the host genome and without replication, and their relatively benign immunogenic profile. The viral genome can be engineered to contain the minimal components for assembly of functional recombinant viruses or viral particles loaded with a desired payload or engineered to express or deliver a desired payload to specific tissues.

[0397] In some embodiments, the AAV is a naturally occurring (e.g., wild-type) AAV or a recombinant AAV. In some embodiments, the wild-type AAV vector genome is a linear single-stranded DNA (ssDNA) molecule approximately 5,000 nucleotides (nt) in length. In some embodiments, inverted terminal repeats (ITRs) cap the viral genome at both the 5' and 3' ends and provide origins of replication for the viral genome. In some embodiments, the AAV viral genome typically contains two ITR sequences. These ITRs have a characteristic T-shaped hairpin structure defined by self-complementary regions (145 nt in wild-type AAV) at the 5' and 3' ends of the ssDNA that form an energetically stable double-stranded region. The double-stranded hairpin structure has multiple functions, including, but not limited to, acting as an origin of DNA replication by serving as a primer for the endogenous DNA polymerase complex of the host viral replicating cell.

[0398] In some embodiments, the wild-type AAV viral genome further comprises nucleotide sequences for two open reading frames: one for four nonstructural Rep proteins (Rep78, Rep68, Rep52, and Rep40, encoded by the Rep genes) and one for three capsid, or structural, proteins (VP1, VP2, and VP3, encoded by the capsid or Cap genes). The Rep proteins are used for replication and packaging, while the capsid proteins assemble to create the protein shell of AAV or AAV capsid polypeptides, e.g., AAV capsid variants. Alternative splicing and alternative start codons and promoters result in the production of four different Rep proteins from one open reading frame and the production of three capsid proteins from one open reading frame. While variations vary depending on the AAV serotype, as a non-limiting example, for AAV9 / hu.14 (SEQ ID NO: 123 of US Pat. No. 7,906,111 , the entire contents of which are incorporated herein by reference), VP1 refers to amino acids 1-736, VP2 refers to amino acids 138-736, and VP3 refers to amino acids 203-736. In some embodiments, with respect to any one of the amino acid sequences of SEQ ID NO: 981 or 982, VP1 includes amino acids 1-742, VP2 includes amino acids 138-742, and VP3 includes amino acids 203-742. In other words, VP1 is the full-length capsid sequence, while VP2 and VP3 are shorter components. As a result, variations in the sequence of the VP3 region will result in variations in both VP1 and VP2, but the percent difference compared to the parent sequence will be greatest for VP3, since it is the shortest of the three sequences. While described herein with respect to amino acid sequences, the nucleic acid sequences encoding these proteins can be similarly described. Together, the three capsid proteins assemble to create the AAV capsid protein, which, without wishing to be bound by theory, typically comprises a 1:1:10 molar ratio of VP1:VP2:VP3.

[0399] The AAV vectors of the present disclosure may be recombinantly produced and may be based on adeno-associated virus (AAV) reference sequences. In addition to single-stranded AAV viral genomes (e.g., ssAAV), the present disclosure also provides self-complementary AAV (scAAV) viral genomes. The scAAV vector genome comprises DNA strands that anneal together to form double-stranded DNA. By omitting second-strand synthesis, scAAV allows for rapid expression in transduced cells. In some embodiments, the AAV particles of the present disclosure are scAAV. In some embodiments, the AAV particles of the present disclosure are ssAAV.

[0400] Methods for producing and / or modifying AAV particles have been disclosed in the art, such as pseudotyped AAV vectors (PCT Patent Publication Nos. WO200028004, WO200123001, WO2004112727, WO2005005610, and WO2005072364, the contents of each of which are incorporated herein by reference in their entirety).

[0401] As described herein, the AAV particles of the present disclosure, comprising an AAV capsid variant and a viral genome, have enhanced tropism for a cell type or tissue, e.g., a CNS cell type, region, or tissue.

[0402] In some embodiments, the AAV capsid variants described herein enable blood-brain barrier penetration after intravenous administration. In some embodiments, the AAV capsid variants enable blood-brain barrier penetration after intravenous administration, focused ultrasound (FUS), or MRI-guided FUS combined with intravenous administration, for example, intravenous administration of microbubbles (FUS-MB). In some embodiments, the AAV capsid variants enable increased distribution to brain regions. In some embodiments, the brain regions include the frontal cortex, sensory cortex, motor cortex, caudate cortex, dentate nucleus, cerebellar cortex, cerebral cortex, brainstem, hippocampus, thalamus, nucleus pallidum, or a combination thereof. In some embodiments, the AAV capsid variants enable preferential transduction in brain regions compared to transduction in dorsal root ganglia (DRG). In some embodiments, the AAV capsid variants enable transduction in non-neuronal cells, such as glial cells (e.g., astrocytes, oligodendrocytes, or a combination thereof).

[0403] In some embodiments, the AAV capsid variant allows for increased distribution to spinal cord regions, including the cervical spinal cord region, the thoracic spinal cord region, and / or the lumbar spinal cord region.

[0404] In some embodiments, the AAV capsid variant is suitable for intramuscular administration and / or transduction of muscle fibers. In some embodiments, the AAV capsid variant allows for increased distribution to muscle regions. In some embodiments, the muscle region includes myocardium, quadriceps, diaphragm muscle regions, or combinations thereof. In some embodiments, the muscle region includes myocardium regions, such as atrial muscle regions or ventricular muscle regions.

[0405] In some embodiments, the start codon for translation of the AAV VP1 capsid protein, e.g., capsid variants described herein, can be CTG, TTG, or GTG, as described in U.S. Pat. No. 8,163,543, the contents of which are incorporated herein by reference in their entirety.

[0406] This disclosure refers to structural capsid proteins (including VP1, VP2, and VP3) encoded by capsid (Cap) genes. These capsid proteins form the outer protein structural shell (e.g., capsid) of viral vectors, such as AAV. VP capsid proteins synthesized from Cap polynucleotides generally contain a methionine (Met1) as the first amino acid in the peptide sequence associated with the initiation codon (AUG or ATG) in the corresponding Cap nucleotide sequence. However, the first methionine (Met1) residue, or generally any first amino acid (AA1), is typically cleaved after or during polypeptide synthesis by a protein-processing enzyme, such as Met-aminopeptidase. This "Met / AA clipping" process often correlates with the corresponding acetylation of a second amino acid (e.g., alanine, valine, serine, threonine, etc.) in the polypeptide sequence. Met clipping occurs most commonly in the VP1 and VP3 capsid proteins, but can also occur in the VP2 capsid protein.

[0407] If Met / AA-clipping is incomplete, a mixture of one or more (1, 2, or 3) VP capsid proteins that make up the viral capsid may be produced, some of which may contain the Met1 / AA1 amino acid (Met+ / AA+) and some of which may lack the Met1 / AA1 amino acid as a result of Met / AA-clipping (Met- / AA-). For further discussion of Met / AA-clipping in capsid proteins, see Jin, et al. Direct Liquid Chromatography / Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins. Hum Gene Ther Methods. 2017, Oct. 28(5):255-267; Hwang, et al. N-Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals. Science. 2010, February 19. 327(5968):973-977, the contents of each of which are incorporated herein by reference in their entirety.

[0408] According to the present disclosure, reference to a capsid protein, e.g., an AAV capsid variant, is not limited to either clipped (Met- / AA-) or non-clipped (Met+ / AA+) and, in context, may refer to an individual capsid protein, a viral capsid composed of a mixture of capsid proteins, and / or a polynucleotide sequence (or fragment thereof) that encodes, describes, produces, or results in a capsid protein of the present disclosure. Direct reference to a capsid protein or capsid polypeptide (such as VP1, VP2, or VP2) can also include VP capsid proteins, including those lacking the Met1 / AA1 amino acids (Met+ / AA+), as well as the corresponding VP capsid protein lacking the Met1 / AA1 amino acids as a result of Met / AA clipping (Met- / AA-).

[0409] Further, in accordance with the present disclosure, reference to specific SEQ ID NOs (either protein or nucleic acid) that each comprise or encode one or more capsid proteins that include Met1 / AA1 amino acids (Met+ / AA+) should be understood to teach VP capsid proteins that lack Met1 / AA1 amino acids, since sequences that simply lack the first-described amino acids (whether Met1 / AA1 or not) are readily apparent upon review of the sequences.

[0410] As a non-limiting example, reference to a VP1 polypeptide sequence that is 736 amino acids in length and that includes the "Met1" amino acid (Met+) encoded by an AUG / ATG start codon may also be understood to teach a VP1 polypeptide sequence that is 735 amino acids in length and that does not include the "Met1" amino acid (Met-) of the 736 amino acid Met+ sequence. As a second non-limiting example, reference to a VP1 polypeptide sequence that is 736 amino acids in length and that includes the "AA1" amino acid (AA1+) encoded by an optional NNN start codon may also be understood to teach a VP1 polypeptide sequence that is 735 amino acids in length and that does not include the "AA1" amino acid (AA1-) of the 736 amino acid AA1+ sequence.

[0411] Reference to a viral capsid formed from VP capsid proteins (e.g., reference to a particular AAV capsid serotype) can incorporate VP capsid proteins that contain Met1 / AA1 amino acids (Met+ / AA1+), corresponding VP capsid proteins that lack Met1 / AA1 amino acids as a result of Met / AA1 clipping (Met- / AA1-), and combinations thereof (Met+ / AA1+ and Met- / AA1-).

[0412] As non-limiting examples, AAV capsid serotypes can include VP1(Met+ / AA1+), VP1(Met- / AA1-), or a combination of VP1(Met+ / AA1+) and VP1(Met- / AA1-). AAV capsid serotypes can also include VP3(Met+ / AA1+), VP3(Met- / AA1-), or a combination of VP3(Met+ / AA1+) and VP3(Met- / AA1-), as well as similar optional combinations of VP2(Met+ / AA1) and VP2(Met- / AA1-).

[0413] AAV capsid variants In some embodiments, an AAV capsid variant disclosed herein comprises a modification of loop IV of AAV9, e.g., at positions 449-460, e.g., at positions 454 and / or 455, numbered relative to SEQ ID NO: 138, 981, or 982. In some embodiments, loop (e.g., loop IV) is used interchangeably herein with the term variable region (e.g., variable region IV) or VR (e.g., VR-IV). In some embodiments, loop IV comprises positions 449-475, numbered according to SEQ ID NO: 138 (e.g., amino acids KTINGSGQNQQTLKFSVAGPSNMAVQG (SEQ ID NO: 6404)). In some embodiments, loop IV comprises positions 449-460, numbered according to SEQ ID NO: 138 (e.g., amino acids KTINGSGQNQQT (SEQ ID NO: 6405)).

[0414] The AAV particles and payloads of the present disclosure can be delivered to one or more target cells, tissues, organs, or organisms. In some embodiments, the AAV particles of the present disclosure exhibit enhanced tropism for target cell types, tissues, or organs. As a non-limiting example, the AAV particles may have enhanced tropism for cells and tissues of the central nervous system or peripheral nervous system (CNS and PNS, respectively). In some embodiments, the AAV particles of the present disclosure may additionally or alternatively have decreased tropism for cell types, tissues, or organs.

[0415] As shown in the examples below, certain AAV capsid variants described herein exhibit multiple advantages over wild-type AAV9, including (i) increased penetration through the blood-brain barrier after intravenous administration, (ii) broader distribution throughout multiple brain regions, e.g., the frontal cortex, sensory cortex, motor cortex, nucleus, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus, and / or hippocampus, and / or (iii) increased expression of the payload in multiple brain regions. Without wishing to be bound by theory, it is believed that these advantages may be due, in part, to dissemination of the AAV capsid variants through the cerebral vasculature. In some embodiments, the AAV capsids described herein enhance delivery of the payload to multiple brain regions, including, e.g., the frontal cortex, sensory cortex, motor cortex, nucleus, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus, and / or hippocampus.

[0416] In some embodiments, the AAV particles described herein comprise an AAV capsid variant, e.g., an AAV capsid variant described herein (e.g., an AAV capsid variant comprising a peptide described herein). In some embodiments, the AAV capsid variant comprises a peptide described in any of Tables 1, 2A, 2B, 13-19.

[0417] In some embodiments, the AAV capsid variants described herein comprise an amino acid sequence having the formula [N1]-[N2]-[N3], wherein [N2] comprises the amino acid sequence of SPH, and [N3] comprises X4, X5, and X6, wherein at least one of X4, X5, or X6 is a basic amino acid, e.g., K or R. In some embodiments, the X4 position of [N2] is K. In some embodiments, the X5 position of [N2] is K.

[0418] In some embodiments, [N1] comprises X1, X2, and X3, and at least one of X1, X2, or X3 is G. In some embodiments, the X1 position of [N1] is independently selected from G, V, R, D, E, M, T, I, S, A, N, L, K, H, P, W, or C. In some embodiments, the X2 position of [N1] is independently selected from S, V, L, N, D, H, R, P, G, T, I, A, E, Y, M, or Q. In some embodiments, the X3 position of [N1] is independently selected from G, C, L, D, E, Y, H, V, A, N, P, or S. In some embodiments, [N1] comprises GS, SG, GH, HD, GQ, QD, VS, CS, GR, RG, QS, SH, MS, RN, TS, IS, GP, ES, SS, GN, AS, NS, LS, GG, KS, GT, PS, RS, GI, WS, DS, ID, GL, DA, DG, ME, EN, KN, KE, AI, NG, PG, TG, SV, IG, LG, AG, EG, SA, YD, HE, HG, RD, ND, PD, MG, QV, DD, HN, HP, GY, GM, GD, or HS. In some embodiments, [N1] comprises GS, SG, GH, or HD. In some embodiments, [N1] is or comprises GSG, GHD, GQD, VSG, CSG, CSH, GQS, GRG, GSH, RVG, GSC, GLL, GDD, GHE, GNY, MSG, RNG, TSG, ISG, GPG, ESG, SSG, GNG, ASG, NSG, LSG, GGG, KSG, HSG, GTG, PSG, GSV, RSG, GIG, WSG, DSG, IDG, GLG, DAG, DGG, MEG, ENG, GSA, KNG, KEG, AIG, GYD, GHG, GRD, GND, GPD, GMG, GQV, GHN, GHP, or GHS. In some embodiments, [N1] is or comprises GSG. In some embodiments, [N1] is or comprises GHD. In some embodiments, [N1]-[N2] is selected from the group consisting of SGSPH (SEQ ID NO: 4752), HDSPH (SEQ ID NO: 4703), QDSPH (SEQ ID NO: 4753), RGSPH (SEQ ID NO: 4754), SHSPH (SEQ ID NO: 4755), QSSPH (SEQ ID NO: 4756), DDSPH (SEQ ID NO: 4757), HESPH (SEQ ID NO: 4758),NYSPH (SEQ ID NO: 4759), VGSPH (SEQ ID NO: 4760), SCSPH (SEQ ID NO: 4761), LLSPH (SEQ ID NO: 4762), NGSPH (SEQ ID NO: 4763), PGSPH (SEQ ID NO: 4764), GGSPH (SEQ ID NO: 4765), TGSPH (SEQ ID NO: 4766), SVSPH (SEQ ID NO: 4767), IGSPH (SEQ ID NO: 4768), DGSPH (SEQ ID NO: 4769), LGSPH (SEQ ID NO: 4770), AGSPH (SEQ ID NO: 4771), EGSPH (SEQ ID NO: 4772), SASPH (SEQ ID NO: 4773), YDSPH (SEQ ID NO: 4774), HGSPH (SEQ ID NO: 4775), RDSPH (SEQ ID NO: 4776), NDSPH (SEQ ID NO: 4777), PDSPH (SEQ ID NO: 4778). 778), MGSPH (SEQ ID NO: 4779), QVSPH (SEQ ID NO: 4780), HNSPH (SEQ ID NO: 4781), HPSPH (SEQ ID NO: 4782), or HSSPH (SEQ ID NO: 4783), an amino acid sequence comprising any portion of any of these aforementioned amino acid sequences (e.g., any 2, 3, or 4 amino acids, e.g., contiguous amino acids), an amino acid sequence that includes one, two, or three, but not more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforementioned amino acid sequences, or an amino acid sequence that includes one, two, or three, but not more than four different amino acids, relative to any one of the aforementioned amino acid sequences. In some embodiments, [N1]-[N2] is selected from the group consisting of GSGSPH (SEQ ID NO: 4695), GHDSPH (SEQ ID NO: 4784), GQDSPH (SEQ ID NO: 4785), VSGSPH (SEQ ID NO: 4786), CSGSPH (SEQ ID NO: 4787), GRGSPH (SEQ ID NO: 4788), CSHSPH (SEQ ID NO: 4789), GQSSPH (SEQ ID NO: 4790), GSHSPH (SEQ ID NO: 4791), GDDSPH (SEQ ID NO: 4792), GHESPH (SEQ ID NO: 4793), GNYSPH (SEQ ID NO: 4794), RVGSPH (SEQ ID NO: 4795), GSCSPH (SEQ ID NO: 4796), GLLSPH (SEQ ID NO: 4797), MSGSPH (SEQ ID NO: 4798), RNGSPH (SEQ ID NO: 4799), TSGSPH (SEQ ID NO: 4800), ISGSPH (SEQ ID NO: 4801), GPGSPH (SEQ ID NO: 4802),ESGSPH (SEQ ID NO: 4803), SSGSPH (SEQ ID NO: 4804), GNGSPH (SEQ ID NO: 4805), ASGSPH (SEQ ID NO: 4806), NSGSPH (SEQ ID NO: 4807), LSGSPH (SEQ ID NO: 4808), GGGSPH (SEQ ID NO: 4809), KSGSPH (SEQ ID NO: 4810), HSGSPH (SEQ ID NO: 4811), GTGSPH (SEQ ID NO: 4812), PSGSPH (SEQ ID NO: 4813), GSVSPH (SEQ ID NO: 4814), RSGSPH (SEQ ID NO: No. 4815), GIGSPH (SEQ ID NO: 4816), WSGSPH (SEQ ID NO: 4817), DSGSPH (SEQ ID NO: 4818), IDGSPH (SEQ ID NO: 4819), GLGSPH (SEQ ID NO: 4820), DAGSPH (SEQ ID NO: 4821), DGGSPH (SEQ ID NO: 4822), MEGSPH (SEQ ID NO: 4823), ENGSPH (SEQ ID NO: 4824), GSASPH (SEQ ID NO: 4825), KNGSPH (SEQ ID NO: 4826), KEGSPH (SEQ ID NO: 4827), AIG SPH (SEQ ID NO: 4828), GYDSPH (SEQ ID NO: 4829), GHGSPH (SEQ ID NO: 4830), GRDSPH (SEQ ID NO: 4831), GNDSPH (SEQ ID NO: 4832), GPDSPH (SEQ ID NO: 4833), GMGSPH (SEQ ID NO: 4834), GQVSPH (SEQ ID NO: 4835), GHNSPH (SEQ ID NO: 4836), GHPSPH (SEQ ID NO: 4837), or GHSSPH (SEQ ID NO: 4838), an amino acid sequence comprising any portion of any of these aforesaid amino acid sequences (e.g., any 2, 3, 4, or 5 amino acids, e.g., contiguous amino acids), an amino acid sequence that includes one, two, or three, but not more than four modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforesaid amino acid sequences, or an amino acid sequence that includes one, two, or three, but not more than four different amino acids, relative to any one of the aforesaid amino acid sequences. In some embodiments, [N1]-[N2] is or comprises GSGSPH (SEQ ID NO: 4695). In some embodiments, [N1]-[N2] is or comprises GHDSPH (SEQ ID NO: 4784).

[0419] In some embodiments, X4, X5, or both of [N3] are K. In some embodiments, X4, X5, or X6 of [N3] are R. In some embodiments, the X4 position of [N3] is independently selected from A, K, V, S, T, G, F, W, V, N, or R. In some embodiments, the X5 position of [N3] is independently selected from S, K, T, F, I, L, Y, H, M, or R. In some embodiments, the X6 position of [N3] is independently selected from G, R, A, M, I, N, T, Y, D, P, V, L, E, W, N, Q, K, or S. In some embodiments, [N3] comprises SK, KA, KS, AR, RM, VK, AS, SR, VK, KR, KK, KN, VR, RS, RK, KT, TS, KF, FG, KI, IG, KL, LG, TT, TY, KY, YG, KD, KP, TR, RG, VR, GA, SL, SS, FL, WK, SA, RA, LR, KW, RR, GK, TK, NK, AK, KV, KG, KH, KM, TG, SE, SV, SW, SN, HG, SQ, LW, MG, MA, or SG. In some embodiments, [N3] comprises SK, KA, KS, or SG. In some embodiments, [N3] is or includes SKA, KSG, ARM, VKS, ASR, VKI, KKN, VRM, RKA, KTS, KFG, KIG, KLG, KTT, KTY, KYG, SKD, SKP, TRG, VRG, KRG, GAR, KSA, KSR, SKL, SRA, SKR, SLR, SRG, SSR, FLR, SKW, SKS, WKA, VRR, SKV, SKT, SKG, GKA, TKA, NKA, SKL, SKN, AKA, KTG, KSL, KSE, KSV, KSW, KSN, KHG, KSQ, KSK, KLW, WKG, KMG, KMA, or RSG. In some embodiments, [N3] is or includes SKA. In some embodiments, [N3] is or includes KSG.In some embodiments, [N2]-[N3] is selected from the group consisting of SPHSK (SEQ ID NO: 4701), SPHKS (SEQ ID NO: 4704), SPHAR (SEQ ID NO: 4705), SPHVK (SEQ ID NO: 4706), SPHAS (SEQ ID NO: 4707), SPHKK (SEQ ID NO: 4708), SPHVR (SEQ ID NO: 4709), SPHRK (SEQ ID NO: 4710), SPHKT (SEQ ID NO: 4711), SPHKF (SEQ ID NO: 4712), SPHKI (SEQ ID NO: 4713), SPHKL (SEQ ID NO: 4714), SPHKY (SEQ ID NO: 4715), SPHTR (SEQ ID NO: 4716), SPHKL (SEQ ID NO: 4717), SPHKY (SEQ ID NO: 4718), SPHKR (SEQ ID NO: 4719), SPHKT (SEQ ID NO: 4720), SPHKF (SEQ ID NO: 4721), SPHKI (SEQ ID NO: 4722), SPHKL (SEQ ID NO: 4723), SPHKY (SEQ ID NO: 4724), SPHTR (SEQ ID NO: 4725), SPHKR (SEQ ID NO: 4726), SPHKT (SEQ ID NO: 4727), SPHKT (SEQ ID NO: 4728), SPHKT (SEQ ID NO: 4729), SPHKT (SEQ ID NO: 4730), SPHKT (SEQ ID NO: 4731), SPHKT (SEQ ID NO: 4732), SPHKT (SEQ ID NO: 4733), SPHKT (SEQ ID NO In some embodiments, [N2]-[N3] comprise SPHSK (SEQ ID NO: 4701) or SPHKS (SEQ ID NO: 4704).In some embodiments, [N2]-[N3] is selected from the group consisting of SPHSKA (SEQ ID NO: 941), SPHKSG (SEQ ID NO: 946), SPHARM (SEQ ID NO: 947), SPHVKS (SEQ ID NO: 948), SPHASR (SEQ ID NO: 949), SPHVKI (SEQ ID NO: 950), SPHKKN (SEQ ID NO: 954), SPHVRM (SEQ ID NO: 955), SPHRKA (SEQ ID NO: 956), SPHKFG (SEQ ID NO: 957), SPHKIG (SEQ ID NO: 958), SPHKLG (SEQ ID NO: 959), SPHKTS (SEQ ID NO: 963), SPHKTT (SEQ ID NO: 964), SPHKT (SEQ ID NO: 965), SPHKT (SEQ ID NO: 966), SPHKT (SEQ ID NO: 967), SPHKT (SEQ ID NO: 968), SPHKT (SEQ ID NO: 969), SPHKT (SEQ ID NO: 970), SPHKT (SEQ ID NO: 971), SPHKT (SEQ ID NO: 972), SPHKT (SEQ ID NO: 973), SPHKT (SEQ ID NO: 974), SPHKT (SEQ ID NO: 975), SPHKT (SEQ ID NO: 976), SPHKT (SEQ ID NO: 977), SPHKT (SEQ ID NO: 978), SPHKT (SEQ ID NO: 979), SPHKT (SEQ ID NO: 980), SPHKT (SEQ ID NO: 981), SPHKT (SEQ ID NO: 982), SPHKT (SEQ ID NO: 983 64), SPHKTY (SEQ ID NO: 965), SPHKYG (SEQ ID NO: 966), SPHSKD (SEQ ID NO: 967), SPHSKP (SEQ ID NO: 968), SPHTRG (SEQ ID NO: 972), SPHVRG (SEQ ID NO: 973), SPHKRG (SEQ ID NO: 974), SPHGAR (SEQ ID NO: 975), SPHKSA (SEQ ID NO: 977), SPHKSR (SEQ ID NO: 951), SPHSKL (SEQ ID NO: 960), SPHSRA (SEQ ID NO: 969), SPHSKR (SEQ ID NO: 978), SPHSLR (SEQ ID NO: 952), SPHSRG (SEQ ID NO: 961), SPHSSR (SEQ ID NO: 970), SPHFLR (SEQ ID NO: 979), SPHSKW (SEQ ID NO: 953), SPHSKS (SEQ ID NO: 962), SPHWKA (SEQ ID NO: 971), SPHVRR (SEQ ID NO: 980), SPHSKT (SEQ ID NO: 4731), SPHSKG (SEQ ID NO: 4732), SPHGKA (SEQ ID NO: 4733), SPHNKA (SEQ ID NO: 4734), SPHSKN (SEQ ID NO: 4735), SPHAKA (SEQ ID NO: 4736), SPHSKV (SEQ ID NO: 4737), SPHKTG (SEQ ID NO: 4738), SPHTKA (SEQ ID NO: 4739), SPHKSL (SEQ ID NO: 4740), SPHKSE (SEQ ID NO: 4741), SPHKSV (SEQ ID NO: 4742), SPHKSW (SEQ ID NO: 4743), SPHKSN (SEQ ID NO: 4744), SPHKHG (SEQ ID NO: 4745), SPHKSQ (SEQ ID NO: 4746), SPHKSK (SEQ ID NO: 4747), SPHKLW (SEQ ID NO: 4748), SPHWKG (SEQ ID NO: 4749), SPHKMG (SEQ ID NO: 4750), SPHKMA (SEQ ID NO: 4751), or SPHRSG (SEQ ID NO: 976).In some embodiments, [N2]-[N3] is or comprises SPHSKA (SEQ ID NO: 941). In some embodiments, [N2]-[N3] is or comprises SPHKSG (SEQ ID NO: 946).

[0420] In some embodiments, [N1]-[N2]-[N3] is selected from the group consisting of SGSPHSK (SEQ ID NO: 4839), HDSPHKS (SEQ ID NO: 4840), SGSPHAR (SEQ ID NO: 4841), SGSPHVK (SEQ ID NO: 4842), QDSPHKS (SEQ ID NO: 4843), SGSPHKK (SEQ ID NO: 4844), SGSPHVR (SEQ ID NO: 4845), SGSPHAS (SEQ ID NO: 4846), SGSPHRK (SEQ ID NO: 4847), SGSPHKT (SEQ ID NO: 4848), SHSPHKS (SEQ ID NO: 4849), QSSPHRS (SEQ ID NO: 4850), RGSPHA S (SEQ ID NO: 4851), RGSPHSK (SEQ ID NO: 4852), SGSPHKF (SEQ ID NO: 4853), SGSPHKI (SEQ ID NO: 4854), SGSPHKL (SEQ ID NO: 4855), SGSPHKY (SEQ ID NO: 4856), SGSPHTR (SEQ ID NO: 4857), SHSPHKR (SEQ ID NO: 4858), SGSPHGA (SEQ ID NO: 4859), HDSPHKR (SEQ ID NO: 4860), DDSPHKS (SEQ ID NO: 4861), HESPHKS (SEQ ID NO: 4862), NYSPHKI (SEQ ID NO: 4863), SGSPHSR (SEQ ID NO: 4864), SGSP HSL (SEQ ID NO: 4865), SGSPHSS (SEQ ID NO: 4866), VGSPHSK (SEQ ID NO: 4867), SCSPHRK (SEQ ID NO: 4868), SGSPHFL (SEQ ID NO: 4869), LLSPHWK (SEQ ID NO: 4870), NGSPHSK (SEQ ID NO: 4871), PGSPHSK (SEQ ID NO: 4872), GGSPHSK (SEQ ID NO: 4873), TGSPHSK (SEQ ID NO: 4874), SVSPHGK (SEQ ID NO: 4875), SGSPHTK (SEQ ID NO: 4876), IGSPHSK (SEQ ID NO: 4877), DGSPHSK (SEQ ID NO: 4878), SG SPHNK (SEQ ID NO: 4879), LGSPHSK (SEQ ID NO: 4880), AGSPHSK (SEQ ID NO: 4881), EGSPHSK (SEQ ID NO: 4882), SASPHSK (SEQ ID NO: 4883), SGSPHAK (SEQ ID NO: 4884), HDSPHKI (SEQ ID NO: 4885), YDSPHKS (SEQ ID NO: 4886), HDSPHKT (SEQ ID NO: 4887), RGSPHKR (SEQ ID NO: 4888), HGSPHSK (SEQ ID NO: 4889), RDSPHKS (SEQ ID NO: 4890), NDSPHKS (SEQ ID NO: 4891), QDSPHKI (SEQ ID NO: 4892),PDSPHKI (SEQ ID NO: 4893), PDSPHKS (SEQ ID NO: 4894), MGSPHSK (SEQ ID NO: 4895), HDSPHKH (SEQ ID NO: 4896), QVSPHKS (SEQ ID NO: 4897), HNSPHKS (SEQ ID NO: 4898), NGSPHKR (SEQ ID NO: 4899), HDSPHKY (SEQ ID NO: 4900), NDSPHKI (SEQ ID NO: 4901), HDSPHKL (SEQ ID NO: 4902), HPSPHWK (SEQ ID NO: 4903), HDSPHKM (SEQ ID NO: 4904), or HSSPHRS (SEQ ID NO: 4905). In some embodiments, [N1]-[N2]-[N3] is selected from the group consisting of GSGSPHSKA (SEQ ID NO: 4697), GHDSPHKSG (SEQ ID NO: 4698), GSGSPHARM (SEQ ID NO: 4906), GSGSPHVKS (SEQ ID NO: 4907), GQDSPHKSG (SEQ ID NO: 4908), GSGSPHASR (SEQ ID NO: 4909), GSGSPHVKI (SEQ ID NO: 4910), GSGSPHKKN (SEQ ID NO: 4911), GSGSPHVRM (SEQ ID NO: 4912), VSGSPHSKA (SEQ ID NO: 4913), CSGSPHSKA (SEQ ID NO: 4914), GSGSPHRKA (SEQ ID NO: 4915), CSGSPHKTS (SEQ ID NO: 4916), CSHSPHKSG (SEQ ID NO: 4917), GQSSPHRSG (SEQ ID NO: 4918), GRGSPHASR (SEQ ID NO: 4919), GRGSPHSKA (SEQ ID NO: 4920), GSGSPH KFG (SEQ ID NO: 4921), GSGSPHKIG (SEQ ID NO: 4922), GSGSPHKLG (SEQ ID NO: 4923), GSGSPHKTS (SEQ ID NO: 4924), GSGSPHKTT (SEQ ID NO: 4925), GSGSPHKTY (SEQ ID NO: 4926), GSGSPHKYG (SEQ ID NO: 4927), GSGSPHSKD (SEQ ID NO: 4928), GSGSPHSKP (SEQ ID NO: 4929), GSGSPHTRG (SEQ ID NO: No. 4930), GSGSPHVRG (SEQ ID NO: 4931), GSHSPHKRG (SEQ ID NO: 4932), GSHSPHKSG (SEQ ID NO: 4933), VSGSPHASR (SEQ ID NO: 4934), VSGSPHGAR (SEQ ID NO: 4935), VSGSPHKFG (SEQ ID NO: 4936), GHDSPHKRG (SEQ ID NO: 4937), GDDSPHKSG (SEQ ID NO: 4938), GHESPHKSA (SEQ ID NO: 4939),GHDSPHKSA (SEQ ID NO: 4940), GNYSPHKIG (SEQ ID NO: 4941), GHDSPHKSR (SEQ ID NO: 4942), GSGSPHSKL (SEQ ID NO: 4943), GSGSPHSRA (SEQ ID NO: 4944), GSGSPHSKR (SEQ ID NO: 4945), GSGSPHSLR (SEQ ID NO: 4946), GSGSPHSRG (SEQ ID NO: 4947), GSGSPHSSR (SEQ ID NO: 4948), RVGSPHSKA (SEQ ID NO: 4949), GSCSPHRKA (SEQ ID NO: 4950), GSGSPHFLR (SEQ ID NO: 4951), GSGSPHSKW ( SEQ ID NO: 4952), GSGSPHSKS (SEQ ID NO: 4953), GLLSPHWKA (SEQ ID NO: 4954), GSGSPHVRR (SEQ ID NO: 4955), GSGSPHSKV (SEQ ID NO: 4956), MSGSPHSKA (SEQ ID NO: 4957), RNGSPHSKA (SEQ ID NO: 4958), TSGSPHSKA (SEQ ID NO: 4959), ISGSPHSKA (SEQ ID NO: 4960), GPGSPHSKA (SEQ ID NO: 4961), GSGSPHSKT (SEQ ID NO: 4962), ESGSPHSKA (SEQ ID NO: 4963), SSGSPHSKA (SEQ ID NO: 4964), GNGSPHSKA (SEQ ID NO: 4965), ASGSPHSKA (SEQ ID NO: 4966), NSGSPHSKA (SEQ ID NO: 4967), LSGSPHSKA (SEQ ID NO: 4968), GGGSPHSKA (SEQ ID NO: 4969), KSGSPHSKA (SEQ ID NO: 4970), GGGSPHSKS (SEQ ID NO: 4971), GSGSPHSKG (SEQ ID NO: 4972), HSGSPHSKA (SEQ ID NO: 4973), GTGSPHSKA (SEQ ID NO: 4974), PSGSPHSKA (SEQ ID NO: 4975), GSVSPHGKA (SEQ ID NO: 4976), RSGSPHSKA ( SEQ ID NO: 4977), GSGSPHTKA (SEQ ID NO: 4978), GIGSPHSKA (SEQ ID NO: 4979), WSGSPHSKA (SEQ ID NO: 4980), DSGSPHSKA (SEQ ID NO: 4981), IDGSPHSKA (SEQ ID NO: 4982), GSGSPHNKA (SEQ ID NO: 4983), GLGSPHSKS (SEQ ID NO: 4984), DAGSPHSKA (SEQ ID NO: 4985), DGGSPHSKA (SEQ ID NO: 4986), MEGSPHSKA (SEQ ID NO: 4987), ENGSPHSKA (SEQ ID NO: 4988), GSASPHSKA (SEQ ID NO: 4989),GNGSPHSKS (SEQ ID NO: 4990), KNGSPHSKA (SEQ ID NO: 4991), KEGSPHSKA (SEQ ID NO: 4992), AIGSPHSKA (SEQ ID NO: 4993), GSGSPHSKN (SEQ ID NO: 4994), GSGSPHAKA (SEQ ID NO: 4995), GHDSPHKIG (SEQ ID NO: 4996), GYDSPHKSG (SEQ ID NO: 4997), GHESPHKSG (SEQ ID NO: 4998), GHDSPHKTG (SEQ ID NO: 4999), GRGSPHKRG (SEQ ID NO: 5000), GQDSPHKSG (SEQ ID NO: 4908), GHDSPHKS L (SEQ ID NO: 5001), GHGSPHSKA (SEQ ID NO: 5002), GHDSPHKSE (SEQ ID NO: 5003), VSGSPHSKA (SEQ ID NO: 4913), GRDSPHKSG (SEQ ID NO: 5004), GNDSPHKSV (SEQ ID NO: 5005), GQDSPHKIG (SEQ ID NO: 5006), GHDSPHKSV (SEQ ID NO: 5007), GPDSPHKIG (SEQ ID NO: 5008), GPDSPHKSG (SEQ ID NO: 5009), GHDSPHKSW (SEQ ID NO: 5010), GHDSPHKSN (SEQ ID NO: 5011), GMGSPHSKT (SEQ ID NO: 501 2), GHDSPHKHG (SEQ ID NO: 5013), GQVSPHKSG (SEQ ID NO: 5014), GDDSPHKSV (SEQ ID NO: 5015), GHNSPHKSG (SEQ ID NO: 5016), GNGSPHKRG (SEQ ID NO: 5017), GHDSPHKYG (SEQ ID NO: 5018), GHDSPHKSQ (SEQ ID NO: 5019), GNDSPHKIG (SEQ ID NO: 5020), GHDSPHKSK (SEQ ID NO: 5021), GHDSPHKLW (SEQ ID NO: 5022), GHPSPHWKG (SEQ ID NO: 5023), GHDSPHKMG (SEQ ID NO: 5024), GHDSP HKMA (SEQ ID NO: 5025), or GHSSPHRSG (SEQ ID NO: 5026), an amino acid sequence comprising any portion of any of these aforementioned amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, or 8 amino acids, e.g., contiguous amino acids), an amino acid sequence that includes one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, to any of the aforementioned amino acid sequences, or one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, to any one of the aforementioned amino acid sequences.An amino acid sequence containing no more than four different amino acids. In some embodiments, [N1]-[N2]-[N3] is or comprises GSGSPHSKA (SEQ ID NO: 4697). In some embodiments, [N1]-[N2]-[N3] is or comprises GHDSPHKSG (SEQ ID NO: 4698).

[0421] In some embodiments, the AAV capsid variant comprising an amino acid sequence having the formula [N1]-[N2]-[N3] further comprises [N4], wherein [N4] comprises X7, X8, X9, and X10. In some embodiments, position X7 of [N4] is independently selected from W, Q, K, R, G, L, V, S, P, H, K, I, M, A, E, or F. In some embodiments, position X8 of [N4] is independently selected from N, Y, C, K, T, H, R, D, V, S, P, G, W, E, F, A, I, M, Q, or L. In some embodiments, position X9 of [N4] is independently selected from Q, G, K, H, R, T, L, D, A, P, I, F, V, M, W, Y, S, E, N, or Y. In some embodiments, the X10 position of [N4] is independently selected from Q, H, L, R, W, K, A, P, E, M, I, S, G, N, Y, C, V, T, D, or V. In some embodiments, [N4] is selected from the group consisting of QNQQ (SEQ ID NO: 5028), WNQQ (SEQ ID NO: 5029), QYYV (SEQ ID NO: 5030), RRQQ (SEQ ID NO: 5031), GCGQ (SEQ ID NO: 5032), LRQQ (SEQ ID NO: 5033), RNQQ (SEQ ID NO: 5034), VNQQ (SEQ ID NO: 5035), FRLQ (SEQ ID NO: 5036), FNQQ (SEQ ID NO: 5037), LLQQ (SEQ ID NO: 5038), SNQQ (SEQ ID NO: 5039), RLQQ (SEQ ID NO: 5040), LNQQ (SEQ ID NO: 5041), QRKL (SEQ ID NO: 5042), LRRQ (SEQ ID NO: 5043), QRLR (SEQ ID NO: 5044), QRRL (SEQ ID NO: 5045), RRLQ (SEQ ID NO: 5046), RLRQ (SEQ ID NO: 5047), RLRQ (SEQ ID NO: 5048), RLRQ (SEQ ID NO: 5049), RLRQ (SEQ ID NO: 5050), RLRQ (SEQ ID NO: 5051), RLRQ (SEQ ID NO: 5052), RLRQ (SEQ ID NO: 5053), RLRQ (SEQ ID NO: 5054), RLRQ (SEQ ID NO: 5055), RLRQ (SEQ ID NO: 5056), RLRQ (SEQ ID NO: 5057), RLRQ (SEQ ID NO: 5058), RLRQ (SEQ ID NO: 5059), RLRQ ( Sequence number 5047), SKRQ (SEQ ID NO: 5048), QLYR (SEQ ID NO: 5049), QLTV (SEQ ID NO: 5050), QNKQ (SEQ ID NO: 5051), KNQQ (SEQ ID NO: 5052), QKQQ (SEQ ID NO: 5053), QTQQ (SEQ ID NO: 5054), QNHQ (SEQ ID NO: 5055), QHQQ (SEQ ID NO: 5056), QNQH (SEQ ID NO: 5057), QHRQ (SEQ ID NO: 5058), LTQQ (SEQ ID NO: 5059), QNQW (SEQ ID NO: 5060), QNTH (SEQ ID NO: 5061), RRRQ (SEQ ID NO: 5062), QYQQ (SEQ ID NO: 5063), QNDQ (SEQ ID NO: 5064), QNRH (SEQ ID NO: 5065), RDQQ (SEQ ID NO: 5066), PNLQ (SEQ ID NO: 5067),HVRQ (SEQ ID NO: 5068), PNQH (SEQ ID NO: 5069), HNQQ (SEQ ID NO: 5070), QSQQ (SEQ ID NO: 5071), QPAK (SEQ ID NO: 5072), QNLA (SEQ ID NO: 5073), QNQL (SEQ ID NO: 5074), QGQQ (SEQ ID NO: 5075), LNRQ (SEQ ID NO: 5076), QNPP (SEQ ID NO: 5077), QNLQ (SEQ ID NO: 5078), QDQE (SEQ ID NO: 5079), QDQQ (SEQ ID NO: 5080), HWQQ (SEQ ID NO: 5081), PNQQ (SEQ ID NO: 5082), PEQQ (SEQ ID NO: 5083), QRTM (SEQ ID NO: 5084), No. 5084), LHQH (SEQ ID NO: 5085), QHRI (SEQ ID NO: 5086), QYIH (SEQ ID NO: 5087), QKFE (SEQ ID NO: 5088), QFPS (SEQ ID NO: 5089), QNPL (SEQ ID NO: 5090), QAIK (SEQ ID NO: 5091), QNRQ (SEQ ID NO: 5092), QYQH (SEQ ID NO: 5093), QNPQ (SEQ ID NO: 5094), QHQL (SEQ ID NO: 5095), QSPP (SEQ ID NO: 5096), QAKL (SEQ ID NO: 5097), KSQQ (SEQ ID NO: 5098), QDRP (SEQ ID NO: 5099), QNLG (SEQ ID NO: 5100), QAFH (SEQ ID NO: 5101), QNAQ (SEQ ID NO: 5102), HNQL (SEQ ID NO: 5103), QKLN (SEQ ID NO: 5104), QNVQ (SEQ ID NO: 5105), QAQQ (SEQ ID NO: 5106), QTPP (SEQ ID NO: 5107), QPPA (SEQ ID NO: 5108), QERP (SEQ ID NO: 5109), QDLQ (SEQ ID NO: 5110), QAMH (SEQ ID NO: 5111), QHPS (SEQ ID NO: 5112), PGLQ (SEQ ID NO: 5113), QGIR (SEQ ID NO: 5114), QAPA (SEQ ID NO: 5115), QIPP (SEQ ID NO: 5116), QTQL (SEQ ID NO: 5117), QTQL (SEQ ID NO: 5118), QTQL (SEQ ID NO: 5119), QTQL (SEQ ID NO: 5120), QTQL (SEQ ID NO: 5121), QTQL (SEQ ID NO: 5122), QTQL (SEQ ID NO: 5123), QTQL (SEQ ID NO: 5124), QTQL (SEQ ID NO: 5125), QTQL (SEQ ID NO: 5126), QTQL (SEQ ID NO: 5127), QTQL (SEQ ID NO: 5128), QTQL (SEQ ID NO: 5129), QTQL (SEQ ID NO: 5130), QTQL (SEQ ID NO: 5131), QTQL (SEQ ID NO: 5132), QTQL (SEQ ID NO: 5 No. 5117), QAPS (SEQ ID NO: 5118), QNTY (SEQ ID NO: 5119), QDKQ (SEQ ID NO: 5120), QNHL (SEQ ID NO: 5121), QIGM (SEQ ID NO: 5122), LNKQ (SEQ ID NO: 5123), PNQL (SEQ ID NO: 5124), QLQQ (SEQ ID NO: 5125), QRMS (SEQ ID NO: 5126), QGIL (SEQ ID NO: 5127), QDRQ (SEQ ID NO: 5128), RDWQ (SEQ ID NO: 5129), QERS (SEQ ID NO: 5130), QNYQ (SEQ ID NO: 5131), QRTC (SEQ ID NO: 5132), QIGH (SEQ ID NO: 5133),QGAI (SEQ ID NO: 5134), QVPP (SEQ ID NO: 5135), QVQQ (SEQ ID NO: 5136), LMRQ (SEQ ID NO: 5137), QYSV (SEQ ID NO: 5138), QAIT (SEQ ID NO: 5139), QKTL (SEQ ID NO: 5140), QLHH (SEQ ID NO: 5141), QNII (SEQ ID NO: 5142), QGHH (SEQ ID NO: 5143), QSKV (SEQ ID NO: 5144), QLPS (SEQ ID NO: 5145), IGKQ (SEQ ID NO: 5146), QAIH (SEQ ID NO: 5147), QHGL (SEQ ID NO: 5148), QFMC (SEQ ID NO: 5149), QNQM (SEQ ID NO: 5150), No. 5150), QHLQ (SEQ ID NO: 5151), QPAR (SEQ ID NO: 5152), QSLQ (SEQ ID NO: 5153), QSQL (SEQ ID NO: 5154), HSQQ (SEQ ID NO: 5155), QMPS (SEQ ID NO: 5156), QGSL (SEQ ID NO: 5157), QVPA (SEQ ID NO: 5158), HYQQ (SEQ ID NO: 5159), QVPS (SEQ ID NO: 5160), RGEQ (SEQ ID NO: 5161), PGQQ (SEQ ID NO: 5162), LEQQ (SEQ ID NO: 5163), QNQS (SEQ ID NO: 5164), QKVI (SEQ ID NO: 5165), QNND (SEQ ID NO: 5166), QSVH (SEQ ID NO: 5167), QPLG (SEQ ID NO: 5168), HNQE (SEQ ID NO: 5169), QIQQ (SEQ ID NO: 5170), QVRN (SEQ ID NO: 5171), PSNQ (SEQ ID NO: 5172), QVGH (SEQ ID NO: 5173), QRDI (SEQ ID NO: 5174), QMPN (SEQ ID NO: 5175), RGLQ (SEQ ID NO: 5176), PSLQ (SEQ ID NO: 5177), QRDQ (SEQ ID NO: 5178), QAKG (SEQ ID NO: 5179), QSAH (SEQ ID NO: 5180), QSTM (SEQ ID NO: 5181), QREM (SEQ ID NO: 5182), QYRA (SEQ ID NO: 5183), QYRA (SEQ ID NO: 5184), QYRA (SEQ ID NO: 5185), QYRA (SEQ ID NO: 5186), QYRA (SEQ ID NO: 5187), QYRA (SEQ ID NO: 5188), QYRA (SEQ ID NO: 5189), QYRA (SEQ ID NO: 5190), QYRA (SEQ ID NO: 5191), QYRA (SEQ ID NO: 5192), QYRA (SEQ ID NO: 5193), QYRA (SEQ ID NO: 5194), QYRA (SEQ ID NO: 5195), QYRA (SEQ ID NO: 5196), QYRA (SEQ ID NO: 5197), QYRA (SEQ ID NO: 5198), QYRA (SEQ ID NO: 5199), QYRA (SEQ ID NO: 5199 No. 5183), QRQQ (SEQ ID NO: 5184), QWQQ (SEQ ID NO: 5185), QRMN (SEQ ID NO: 5186), GDSQ (SEQ ID NO: 5187), QKIS (SEQ ID NO: 5188), PSMQ (SEQ ID NO: 5189), SPRQ (SEQ ID NO: 5190), MEQQ (SEQ ID NO: 5191), QYQN (SEQ ID NO: 5192), QIRQ (SEQ ID NO: 5193), QSVQ (SEQ ID NO: 5194), RSQQ (SEQ ID NO: 5195), QNKL (SEQ ID NO: 5196), QIQH (SEQ ID NO: 5197), PRQQ (SEQ ID NO: 5198), HTQQ (SEQ ID NO: 5199),QRQH (SEQ ID NO: 5200), RNQE (SEQ ID NO: 5201), QSKQ (SEQ ID NO: 5202), QNQP (SEQ ID NO: 5203), QSPQ (SEQ ID NO: 5204), QTRQ (SEQ ID NO: 5205), QNLH (SEQ ID NO: 5206), QNQE (SEQ ID NO: 5207), LNQP (SEQ ID NO: 5208), QNQD (SEQ ID NO: 5209), QNLL (SEQ ID NO: 5210), QLVI (SEQ ID NO: 5211), R TQE (SEQ ID NO: 5212), QTHQ (SEQ ID NO: 5213), QDQH (SEQ ID NO: 5214), QSQH (SEQ ID NO: 5215), VRQQ (SEQ ID NO: 5216), AWQQ (SEQ ID NO: 5217), QSVP (SEQ ID NO: 5218), QNIQ (SEQ ID NO: 5219), LDQQ (SEQ ID NO: 5220), PDQQ (SEQ ID NO: 5221), ESQQ (SEQ ID NO: 5222), QRQL (SEQ ID NO: 5223), QI IV (SEQ ID NO: 5224), QKQS (SEQ ID NO: 5225), QSHQ (SEQ ID NO: 5226), QFVV (SEQ ID NO: 5227), QSQP (SEQ ID NO: 5228), QNEQ (SEQ ID NO: 5229), INQQ (SEQ ID NO: 5230), RNRQ (SEQ ID NO: 5231), RDQK (SEQ ID NO: 5232), QWKR (SEQ ID NO: 5233), ENRQ (SEQ ID NO: 5234), QTQP (SEQ ID NO: 5235), QKQ L (SEQ ID NO: 5236), RNQL (SEQ ID NO: 5237), ISIQ (SEQ ID NO: 5238), QTVC (SEQ ID NO: 5239), QQIM (SEQ ID NO: 5240), LNHQ (SEQ ID NO: 5241), QNQA (SEQ ID NO: 5242), QMIH (SEQ ID NO: 5243), RNHQ (SEQ ID NO: 5244), or QKMN (SEQ ID NO: 5245), or any dipeptide or tripeptide thereof. In some embodiments, [N1]-[N2]-[N3]-[N4] is any of the amino acid sequences of SEQ ID NOs: 1800-2241, an amino acid sequence comprising any portion of any of these aforementioned amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids, e.g., contiguous amino acids), an amino acid sequence that includes one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforementioned amino acid sequences, or one, two,or an amino acid sequence containing three, but not more than four, different amino acids. In some embodiments, [N1]-[N2]-[N3]-[N4] is or comprises GSGSPHSKAQNQQ (SEQ ID NO: 1801). In some embodiments, [N1]-[N2]-[N3]-[N4] is or comprises GHDSPHKSGQNQQ (SEQ ID NO: 1800).

[0422] In some embodiments, the AAV capsid variant comprising an amino acid sequence having the formula [N1]-[N2]-[N3] further comprises [N0], wherein [N0] comprises XA XB and XC. In some embodiments, XA of [N0] is independently selected from T, S, Y, M, A, C, I, R, L, D, F, V, Q, N, H, E, or G. In some embodiments, XB of [N0] is independently selected from I, M, P, E, N, D, S, A, T, G, Q, F, V, L, C, H, R, W, or L. In some embodiments, XC of [N0] is independently selected from N, M, E, G, Y, W, T, I, Q, F, V, A, L, I, P, K, R, H, S, D, or S. In some embodiments, [N0] is TIN, SMN, TIM, YLS, GLS, MPE, MEG, MEY, AEW, CEW, ANN, IPE, ADM, IEY, ADY, I ET, MEW, CEY, RIN, MEI, LEY, ADW, IEI, DIM, FEQ, MEF, CDQ, LPE, IEN, MES, AEI, VEY, IIN, TSN, IEV, MEM, AEV, MDA, VEW, AEQ, LEW, MEL, MET, MEA, IES, MEV, CEI, ATN, MDG, QEV, ADQ, NMN, IEM, ISN, TGN, QQQ, HDW, IEG, TII, TFP, TEK, EIN, TVN, TFN, SIN, TER, TSY, ELH, AIN, SVN, TDN, TFH, TVH, TEN, TSS , TID, TCN, NIN, TEH, AEM, AIK, TDK, TFK, SDQ, TEI, NTN, TET, SIK, TEL, TEA, TAN, TIY, TFS, TES, TT N, TED, TNN, EVH, TIS, TVR, TDR, TIK, NHI, TIP, ESD, TDL, TVP, TVI, AEH, NCL, TVK, NAD, TIT, NCV, TI R, NAL, VIN, TIQ, TEF, TRE, QGE, SEK, NVN, GGE, EFV, SDK, TEQ, EVQ, TEY, NCW, TDV, SDI, NSI, NSL, EVV, TEP, SEL, TWQ, TEV, AVN, GVL, TLN, TEG, TRD, NAI, AEN, AET, ETA, NNL, or any dipeptide thereof.In some embodiments, [N0]-[N1]-[N2]-[N3]-[N4] is or comprises the amino acid sequence of any one of SEQ ID NOs: 2242-2886, an amino acid sequence comprising any portion of any of these aforementioned amino acid sequences (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, e.g., contiguous amino acids), an amino acid sequence comprising one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to any of the aforementioned amino acid sequences, or an amino acid sequence comprising one, two, or three, but not more than four different amino acids, relative to any one of the aforementioned amino acid sequences. In some embodiments, [N0]-[N1]-[N2]-[N3]-[N4] is or comprises TINGSGSPHSKAQNQQ (SEQ ID NO: 2242). In some embodiments, [N0]-[N1]-[N2]-[N3]-[N4] is or includes TINGHDSPHKSGQNQQ (sequence number 2243).

[0423] In some embodiments, [N1]-[N2]-[N3] are present in loop IV of the AAV capsid variant. In some embodiments, [N0] and [N4] are present in loop IV of the AAV capsid variant. In some embodiments, [N0]-[N1]-[N2]-[N3]-[N4] are present in loop IV of the AAV capsid variant.

[0424] In some embodiments, [N0] is located immediately after position 449 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, [N0] is located immediately after position 449 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 981 or 982. In some embodiments, [N0] replaces positions 450, 451, and 452 (e.g., amino acids T450, I451, and N452) relative to a reference sequence numbered according to SEQ ID NO: 138, 981, or 982, and [N0] is located immediately after position 449, and [N0] replaces positions 450-452 (e.g., T450, I451, and N452) relative to a reference sequence numbered according to SEQ ID NO: 138, 981, or 982. In some embodiments, [N1] is located immediately after position 452 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138, 981, or 982. In some embodiments, [N1] replaces positions 453-455 (e.g., G453, S454, and G455) relative to a reference sequence numbered according to SEQ ID NO: 138, 981, or 982. In some embodiments, [N1] is located immediately after position 452, and [N1] replaces positions 453-455 (e.g., G453, S454, and G455) relative to a reference sequence numbered according to SEQ ID NO: 138, 981, or 982. In some embodiments, [N2] is located immediately after position 455 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138, 981, or 982. In some embodiments, [N2]-[N3] are located immediately after position 455 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138, 981, or 982. In some embodiments, [N1]-[N2]-[N3] is located immediately after position 452, as numbered relative to SEQ ID NO: 138, 981, or 982. In some embodiments, [N1]-[N2]-[N3] replaces positions 453-455 (e.g., G453, S454, and G455) relative to a reference sequence numbered according to SEQ ID NO: 138, 981, or 982.In some embodiments, [N1] occurs immediately after position 452, and [N1]-[N2]-[N3] replace positions 453-455 (e.g., G453, S454, and G455) relative to a reference sequence numbered according to SEQ ID NO: 138, 981, or 982. In some embodiments, [N4] occurs immediately after position 455 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, [N4] replaces positions 456-459 (e.g., Q456, N457, Q458, and Q459) relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, [N4] occurs immediately after position 455, and [N4] replaces positions 456-459 (e.g., Q456, N457, Q458, and Q459) relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, [N2]-[N3]-[N4] replace positions 456-459 (e.g., Q456, N457, Q458, and Q459) relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, [N2]-[N3]-[N4] occurs immediately after position 455, and [N2]-[N3]-[N4] replace positions 456-459 (e.g., Q456, N457, Q458, and Q459) relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, [N1]-[N2]-[N3]-[N4] replaces positions 453-459 (e.g., G453, S454, G455, Q456, N457, Q458, and Q459) relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, [N1]-[N2]-[N3]-[N4] is located immediately after position 452, and [N1]-[N2]-[N3]-[N4] replaces positions 453-459 (e.g., G453, S454, G455, Q456, N457, Q458, and Q459) relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.In some embodiments, [N0]-[N1]-[N2]-[N3]-[N4] replace positions 450-456 (e.g., T450, I451, N452, G453, S454, G455, Q456, N457, Q458, and Q459) relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, [N0]-[N1]-[N2]-[N3]-[N4] is located immediately after position 449, and [N0]-[N1]-[N2]-[N3]-[N4] replaces positions 450-456 (e.g., T450, I451, N452, G453, S454, G455, Q456, N457, Q458, and Q459) relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0425] In some embodiments, [N3] occurs immediately after [N2]. In some embodiments, the AAV capsid variant comprises, from N- to C-terminus, [N2]-[N3]. In some embodiments, the AAV capsid variant comprises, from N- to C-terminus, [N1]-[N2]-[N3]. In some embodiments, the AAV capsid variant comprises, from N- to C-terminus, [N1]-[N2]-[N3]-[N4]. In some embodiments, the AAV capsid variant comprises, from N- to C-terminus, [N0]-[N1]-[N2]-[N3]. In some embodiments, the AAV capsid variant comprises, from N- to C-terminus, [N0]-[N1]-[N2]-[N3]-[N4].

[0426] In some embodiments, the AAV capsid variants described herein comprise an amino acid sequence comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 16, or 17 contiguous amino acids from any one of the sequences provided in Tables 1, 2A, 2B, 13-19. In some embodiments, the AAV capsid variants comprise an amino acid sequence comprising at least 3, 4, or 5 contiguous amino acids from any one of SEQ ID NOs: 945-980 or 985-986. In some embodiments, the AAV capsid variants comprise an amino acid sequence comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 contiguous amino acids from any one of SEQ ID NOs: 2, 200, 201, 941, 943, 204, 208, 404, or 903-909. In some embodiments, the amino acid sequence is present in loop IV. In some embodiments, the amino acid sequence is located immediately after position 448, 452, 453, or 455 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138, 981, or 982. In some embodiments, the amino acid sequence is located immediately after position 455 numbered according to SEQ ID NO: 982. In some embodiments, the amino acid sequence is located immediately after position 455 numbered according to SEQ ID NO: 138. In some embodiments, the amino acid sequence is located immediately after position 453 numbered according to SEQ ID NO: 981. In some embodiments, the amino acid sequence is located immediately after position 453 numbered according to SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces one, two, three, four, five, six, seven, eight, nine, ten, eleven, or all of positions 499 (e.g., K499), 450 (e.g., T450), 451 (e.g., I451), 452 (e.g., N452), 453 (e.g., G453), 454 (e.g., S454), 455 (e.g., G455), 456 (e.g., Q456), 457 (e.g., N457), 458 (e.g., Q458), 459 (e.g., Q459), and 460 (e.g., T460), numbered according to SEQ ID NO: 138.In some embodiments, the AAV capsid variant comprises one or more amino acid substitutions at positions 499 (e.g., K499), 450 (e.g., T450), 451 (e.g., I451), 452 (e.g., N452), 453 (e.g., G453), 454 (e.g., S454), 455 (e.g., G455), 456 (e.g., Q456), 457 (e.g., N457), 458 (e.g., Q458), 459 (e.g., Q459), and / or 460 (e.g., T460), numbered according to SEQ ID NO: 138.

[0427] In some embodiments, three consecutive amino acids comprise SPH. In some embodiments, four consecutive amino acids comprise SPHS (SEQ ID NO: 4700). In some embodiments, five consecutive amino acids comprise SPHSK (SEQ ID NO: 4701). In some embodiments, six consecutive amino acids comprise SPHSKA (SEQ ID NO: 941).

[0428] In some embodiments, three consecutive amino acids comprise HDS. In some embodiments, four consecutive amino acids comprise HDSP (SEQ ID NO: 4702). In some embodiments, five consecutive amino acids comprise HDSPH (SEQ ID NO: 4703). In some embodiments, six consecutive amino acids comprise HDSPHK (SEQ ID NO: 2). In some embodiments, seven consecutive amino acids comprise HDSPHKS (SEQ ID NO: 4840). In some embodiments, eight consecutive amino acids comprise HDSPHKSG (SEQ ID NO: 943).

[0429] In some embodiments, three consecutive amino acids comprise HDS. In some embodiments, four consecutive amino acids comprise HDSP (SEQ ID NO: 4702). In some embodiments, five consecutive amino acids comprise HDSPH (SEQ ID NO: 4703). In some embodiments, six consecutive amino acids comprise HDSPHK (SEQ ID NO: 2).

[0430] In some embodiments, the AAV capsid variants described herein comprise an amino acid sequence that includes at least one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of the sequences provided in Tables 1, 2A, 2B, 13-19. In some embodiments, the AAV capsid variants comprise an amino acid sequence that includes at least one, two, or three, but not more than four, different amino acids relative to the amino acid sequence of any one of the sequences provided in Tables 1, 2A, 2B, 13-19. In some embodiments, the AAV capsid variants comprise an amino acid sequence that includes at least one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of any one of SEQ ID NOs: 945-980 or 985-986. In some embodiments, the AAV capsid variant comprises an amino acid sequence that contains at least one, two, or three, but not more than four, different amino acids from the amino acid sequence of any one of SEQ ID NOs: 945-980 or 985-986. In some embodiments, the AAV capsid variant comprises an amino acid sequence that contains at least one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, from the amino acid sequence of any one of SEQ ID NOs: 2, 200, 201, 941, 943, 204, 208, 404, or 903-909. In some embodiments, the AAV capsid variant comprises an amino acid sequence that contains at least one, two, or three, but not more than four, different amino acids from the amino acid sequence of any one of SEQ ID NOs: 2, 200, 201, 941, 943, 204, 208, 404, or 903-909. In some embodiments, the amino acid sequence is present in loop IV. In some embodiments, the amino acid sequence is located immediately after position 448, 452, 453, or 455 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138, 981, or 982. In some embodiments, the amino acid sequence is located immediately after position 455 numbered according to SEQ ID NO: 982.In some embodiments, the amino acid sequence is located immediately after position 455, numbered according to SEQ ID NO: 138. In some embodiments, the amino acid sequence is located immediately after position 453, numbered according to SEQ ID NO: 981. In some embodiments, the amino acid sequence is located immediately after position 453, numbered according to SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces one, two, three, four, five, six, seven, eight, nine, ten, eleven, or all of positions 499 (e.g., K499), 450 (e.g., T450), 451 (e.g., I451), 452 (e.g., N452), 453 (e.g., G453), 454 (e.g., S454), 455 (e.g., G455), 456 (e.g., Q456), 457 (e.g., N457), 458 (e.g., Q458), 459 (e.g., Q459), and 460 (e.g., T460), numbered according to SEQ ID NO: 138.

[0431] In some embodiments, the AAV capsid variant comprises an amino acid sequence that includes at least one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of SPHSKA (SEQ ID NO: 941). In some embodiments, the AAV capsid variant comprises an amino acid sequence that includes at least one, two, or three, but not more than four, different amino acids from the amino acid sequence of SPHSKA (SEQ ID NO: 941).

[0432] In some embodiments, the AAV capsid variant comprises an amino acid sequence that contains at least one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of HDSPHKSG (SEQ ID NO: 943). In some embodiments, the AAV capsid variant comprises an amino acid sequence that contains at least one, two, or three, but not more than four, different amino acids relative to the amino acid sequence of HDSPHKSG (SEQ ID NO: 2).

[0433] In some embodiments, the AAV capsid variant comprises an amino acid sequence that contains at least one, two, or three, but not more than four, modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the amino acid sequence of HDSPHK (SEQ ID NO: 2). In some embodiments, the AAV capsid variant comprises an amino acid sequence that contains at least one, two, or three, but not more than four, different amino acids relative to the amino acid sequence of HDSPHK (SEQ ID NO: 2).

[0434] In some embodiments, the AAV capsid variant comprises the amino acid sequence of any of the sequences provided in Tables 1, 2A, 2B, 13-19. In some embodiments, the peptide comprises the amino acid sequence of any of SEQ ID NOs: 945-980 or 985-986. In some embodiments, the AAV capsid variant comprises the amino acid sequence of any of SEQ ID NOs: 200, 201, 941, 943, 204, 208, 404, or 903-909. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 941. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 943. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 3589. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 1754. In some embodiments, the amino acid sequence is present in loop IV. In some embodiments, the amino acid sequence is located immediately after position 448 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 449-460 (e.g., K449, T450, 1451, N452, G453, S454, G455, Q456, N457, Q458, Q459, and T460) numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence is located immediately after position 448 and replaces positions 449-460 (e.g., K449, T450, 1451, N452, G453, S454, G455, Q456, N457, Q458, Q459, and T460) numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence is located immediately after position 449 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 450-460 (e.g., T450, I451, N452, G453, S454, G455, Q456, N457, Q458, Q459, and T460) numbered relative to SEQ ID NO: 138.In some embodiments, the amino acid sequence occurs immediately after position 449 and replaces positions 450-460, numbered relative to SEQ ID NO: 138 (e.g., T450, I451, N452, G453, S454, G455, Q456, N457, Q458, Q459, and T460). In some embodiments, the amino acid sequence occurs immediately after position 450 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 451-460, numbered relative to SEQ ID NO: 138 (e.g., I451, N452, G453, S454, G455, Q456, N457, Q458, Q459, and T460). In some embodiments, the amino acid sequence occurs immediately after position 450 and replaces positions 451-460, numbered relative to SEQ ID NO: 138 (e.g., I451, N452, G453, S454, G455, Q456, N457, Q458, Q459, and T460). In some embodiments, the amino acid sequence occurs immediately after position 451 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 452-460, numbered relative to SEQ ID NO: 138 (e.g., N452, G453, S454, G455, Q456, N457, Q458, Q459, and T460). In some embodiments, the amino acid sequence occurs immediately after position 451 and replaces positions 452-460, numbered relative to SEQ ID NO: 138 (e.g., N452, G453, S454, G455, Q456, N457, Q458, Q459, and T460). In some embodiments, the amino acid sequence occurs immediately after position 452 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 453-460, numbered relative to SEQ ID NO: 138 (e.g., G453, S454, G455, Q456, N457, Q458, Q459, and T460). In some embodiments, the amino acid sequence immediately follows position 452 and replaces positions 453-460 (e.g., G453, S454, G455, Q456, N457, Q458, Q459, and T460), numbered relative to SEQ ID NO: 138.In some embodiments, the amino acid sequence is located immediately after position 453 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 454 and 455 (e.g., S454 and G455) numbered according to SEQ ID NO: 138. In some embodiments, the amino acid sequence is located immediately after position 453 and replaces positions 454 and 455 (e.g., S454 and G455) numbered according to SEQ ID NO: 138. In some embodiments, the amino acid sequence replaces positions 454-460 (e.g., S454, G455, Q456, N457, Q458, Q459, and T460) numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence is located immediately after position 453 and replaces positions 454-460, numbered relative to SEQ ID NO: 138 (e.g., S454, G455, Q456, N457, Q458, Q459, and T460). In some embodiments, the amino acid sequence is located immediately after position 454 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence is located immediately after position 454 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 981. In some embodiments, the amino acid sequence replaces positions 455-460, numbered relative to SEQ ID NO: 138 (e.g., G455, Q456, N457, Q458, Q459, and T460). In some embodiments, the amino acid sequence is located immediately after position 454 and replaces positions 455-460, numbered relative to SEQ ID NO: 138 (e.g., positions G455, Q456, N457, Q458, Q459, and T460). In some embodiments, the amino acid sequence is located immediately after position 455 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence is located immediately after position 455 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 982. In some embodiments, the amino acid sequence replaces positions 456-460, numbered relative to SEQ ID NO: 138 (e.g., Q456, N457, Q458, Q459, and T460).In some embodiments, the amino acid sequence immediately follows position 455 and replaces positions 456-460 (e.g., Q456, N457, Q458, Q459, and T460), numbered relative to SEQ ID NO: 138.

[0435] In some embodiments, an AAV capsid variant (e.g., an AAV capsid variant described herein) comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 942 or 944, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In some embodiments, an AAV capsid variant described herein comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 942 or 944, or a nucleotide sequence that includes at least 1, 2, 3, 4, 5, 6, or 7 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 942 or 944. In some embodiments, the AAV capsid variant comprises an amino acid sequence encoded by a nucleotide sequence that contains at least 1, 2, 3, 4, 5, 6, or 7, but not more than 10, different nucleotides relative to the nucleotide sequence of SEQ ID NO: 942 or 944.

[0436] In some embodiments, a nucleotide sequence encoding an AAV capsid variant (e.g., an AAV capsid variant described herein) comprises the nucleotide sequence of SEQ ID NO:942, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In some embodiments, a nucleic acid sequence encoding an AAV capsid variant comprises a nucleotide sequence that includes at least 1, 2, 3, 4, 5, 6, or 7 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO:942. In some embodiments, a nucleotide sequence encoding an AAV capsid variant described herein comprises a nucleotide sequence that includes at least 1, 2, 3, 4, 5, 6, or 7, but not more than 10, different nucleotides relative to the nucleotide sequence of SEQ ID NO:942.

[0437] In some embodiments, a nucleotide sequence encoding an AAV capsid variant (e.g., an AAV capsid variant described herein) comprises the nucleotide sequence of SEQ ID NO:944, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In some embodiments, a nucleic acid sequence encoding an AAV capsid variant comprises a nucleotide sequence that includes at least 1, 2, 3, 4, 5, 6, or 7 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO:944. In some embodiments, a nucleotide sequence encoding an AAV capsid variant described herein comprises a nucleotide sequence that includes at least 1, 2, 3, 4, 5, 6, or 7, but not more than 10, different nucleotides relative to the nucleotide sequence of SEQ ID NO:944.

[0438] In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SPHSKA (SEQ ID NO:941), which amino acid sequence occurs immediately after position 455 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, an AAV capsid variant described herein comprises the amino acid sequence of SPHSKA (SEQ ID NO:941), which amino acid sequence occurs immediately after position 455 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO:981.

[0439] In some embodiments, the AAV capsid variants described herein comprise the amino acid sequence of HDSPHKSG (SEQ ID NO:943), which amino acid sequence occurs immediately after position 453 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variants described herein comprise the amino acid sequence of HDSPHKSG (SEQ ID NO:943), which amino acid sequence occurs immediately after position 453 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO:982.

[0440] In some embodiments, the AAV capsid variants described herein comprise the amino acid sequence of HDSPHK (SEQ ID NO:2), which amino acid sequence occurs immediately after position 453 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variants described herein comprise the amino acid sequence of HDSPHK (SEQ ID NO:2), which amino acid sequence occurs immediately after position 453 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO:982.

[0441] In some embodiments, the AAV capsid variants described herein comprise (i) the amino acid sequence HDSPHKSG immediately following position 453 (SEQ ID NO: 943), and (ii) a deletion of amino acids SG at positions 454 and 455, wherein (i) and (ii) are numbered according to SEQ ID NO: 138.

[0442] In some embodiments, the AAV capsid variants described herein comprise (i) the amino acid sequence of HDSPHSKA (SEQ ID NO: 4486) located immediately after position 453, and (ii) a deletion of amino acids SG at positions 454 and 455, wherein (i) and (ii) are numbered according to SEQ ID NO: 138.

[0443] In some embodiments, the AAV capsid variants described herein comprise an amino acid other than S at position 454 and / or an amino acid other than G at position 455, numbered according to SEQ ID NO: 138. In some embodiments, the AAV capsid variants comprise an amino acid H at position 454 and an amino acid D at position 455, numbered according to SEQ ID NO: 138. In some embodiments, the AAV capsid variants further comprise the amino acid sequence of SPHKSG (SEQ ID NO: 946). In some embodiments, the AAV capsid variants comprise (i) an amino acid H at position 454 and an amino acid D at position 455, and (ii) the amino acid sequence of SPHKSG (SEQ ID NO: 946), where the amino acid sequence of SPHKSG (SEQ ID NO: 946) immediately follows position 455, and (i) and (ii) are numbered according to SEQ ID NO: 138.

[0444] In some embodiments, the AAV capsid variants described herein comprise an amino acid other than S at position 454 and / or an amino acid other than G at position 455, numbered according to SEQ ID NO: 138. In some embodiments, the AAV capsid variants comprise an amino acid H at position 454 and an amino acid D at position 455, numbered according to SEQ ID NO: 138. In some embodiments, the AAV capsid variants further comprise the amino acid sequence of SPHSKA (SEQ ID NO: 941). In some embodiments, the AAV capsid variants comprise (i) an amino acid H at position 454 and an amino acid D at position 455, and (ii) the amino acid sequence of SPHSKA (SEQ ID NO: 941), where the amino acid sequence of SPHSKA (SEQ ID NO: 941) immediately follows position 455, and (i) and (ii) are numbered according to SEQ ID NO: 138.

[0445] In some embodiments, the AAV capsid variants described herein comprise a modification, e.g., a substitution, relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variants comprise a modification, e.g., a substitution, at positions S454 and / or G455, numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variants comprise a S454H substitution and / or a G455D substitution, numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variants comprise a S454H substitution and a G455D substitution, numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variants further comprise the amino acid sequence of SPHKSG (SEQ ID NO: 946). In some embodiments, the AAV capsid variant comprises (i) a S454H substitution and a G455D substitution, and (ii) the amino acid sequence of SPHKSG (SEQ ID NO: 946), which occurs immediately after position 455, and (i) and (ii) are numbered according to SEQ ID NO: 138.

[0446] In some embodiments, the AAV capsid variants described herein comprise a modification, e.g., a substitution, relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variants comprise a modification, e.g., a substitution, at positions S454 and / or G455, numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variants comprise a S454H substitution and / or a G455D substitution, numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variants comprise a S454H substitution and a G455D substitution, numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid variants further comprise the amino acid sequence of SPHSKA (SEQ ID NO: 941). In some embodiments, the AAV capsid variant comprises (i) a S454H substitution and a G455D substitution, and (ii) the amino acid sequence of SPHSKA (SEQ ID NO: 941), which occurs immediately after position 455, and (i) and (ii) are numbered according to SEQ ID NO: 138.

[0447] In some embodiments, the AAV capsid variant further comprises one, two, or all of an amino acid other than T at position 450 (e.g., S, Y, or G), an amino acid other than I at position 451 (e.g., M or L), and / or an amino acid other than N at position 452 (e.g., S) relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an S at position 450 and an M at position 451 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a Y at position 450, an L at position 451, and an S at position 452 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a G at position 450, an L at position 451, and an S at position 452 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0448] In some embodiments, the AAV capsid variant further comprises one, two, three, four, or all of an amino acid other than Q at position 456 (e.g., R or L), an amino acid other than N (e.g., H, K, or R) at position 457, an amino acid other than Q (e.g., R or T) at position 458, an amino acid other than Q (H) at position 459, and / or an amino acid other than T (N or S) at position 460 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an R at position 456 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an L at position 456 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an H at position 457 and an R at position 458 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a K at position 457 and an N at position 460 relative to the reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a T at position 458, an H at position 459, and an S at position 460 relative to the reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an R at position 456, an R at position 457, and an R at position 458 relative to the reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0449] In some embodiments, the AAV capsid variants described herein comprise an amino acid other than I at position 451, an amino acid other than N at position 452, and an amino acid other than G at position 453, numbered according to SEQ ID NO: 138 or 981. In some embodiments, the AAV capsid variants comprise an E at position 451, an R at position 452, and a V at position 453, numbered according to SEQ ID NO: 138 or 981. In some embodiments, the AAV capsid variants comprise the substitutions I451E, N452R, and G453V, numbered according to SEQ ID NO: 138 or 981.

[0450] In some embodiments, the AAV capsid variant comprises the amino acid sequence of SPHSKA (SEQ ID NO:941), the amino acid sequence occurring immediately after position 455, the AAV capsid variant comprising an E at position 451, an R at position 452, and a V at position 453, numbered according to the amino acid sequence of SEQ ID NO:138 or 981. In some embodiments, the AAV capsid variant comprises the substitutions I451E, N452R, and G453V, and further comprises the amino acid sequence of SPHSKA (SEQ ID NO:941), the amino acid sequence occurring immediately after position 455, all numbered according to SEQ ID NO:138 or 981. In some embodiments, the AAV capsid variant comprises the amino acid sequence of ERVSGSPHSKA (SEQ ID NO:6399), the amino acid sequence occurring immediately after position 449, replacing positions 450-455, numbered according to SEQ ID NO:138. In some embodiments, the AAV capsid variant comprises the amino acid sequence KTERVSGSPHSKAQNQQT (SEQ ID NO: 3589), which occurs immediately after position 448 and replaces positions 449-460, numbered according to SEQ ID NO: 138.

[0451] In some embodiments, an AAV capsid variant described herein comprises an amino acid other than T at position 450, an amino acid other than I at position 451, and an amino acid other than N at position 452, numbered according to SEQ ID NO: 138 or 982. In some embodiments, an AAV capsid variant comprises an A at position 450, an E at position 451, and an I at position 452, numbered according to SEQ ID NO: 138 or 982. In some embodiments, an AAV capsid variant comprises the substitutions T450A, I451E, and N452I, numbered according to SEQ ID NO: 138 or 982.

[0452] In some embodiments, an AAV capsid variant comprises the amino acid sequence of SPHKSG (SEQ ID NO:946) occurring immediately after position 455, and further comprising an A at position 450, an E at position 451, an I at position 452, an H at position 454, and a D at position 455, all numbered according to SEQ ID NO: 138 or 982. In some embodiments, an AAV capsid variant comprises the substitutions T450A, I451E, N452I, S454H, and G455D, and further comprising the amino acid sequence SPHKSG (SEQ ID NO:946) occurring immediately after position 455, all numbered according to SEQ ID NO: 138 or 982. In some embodiments, an AAV capsid variant comprises the amino acid sequence of AEIGHDSPHKSG (SEQ ID NO:6400), which amino acid sequence occurs immediately after position 449 and replaces positions 450-455, numbered according to SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises the amino acid sequence KAEIGHDSPHKSGQNQQT (SEQ ID NO: 1754), which amino acid sequence occurs immediately after position 448 and replaces positions 449-460, numbered according to SEQ ID NO: 138.

[0453] In some embodiments, the AAV capsid variant further comprises a substitution at position K449, e.g., a K449R substitution, numbered according to SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an amino acid other than K (e.g., R) at position 449 relative to the reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an R at position 449 relative to the reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a modification, e.g., an insertion, substitution, and / or deletion in loops I, II, VI, and / or VIII.

[0454] In some embodiments, the AAV capsid variant further comprises an amino acid sequence that includes at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 30, 20, or 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises an amino acid sequence that includes at least one, two, or three, but not more than 30, 20, or 10 amino acids, that differ from the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence having at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.

[0455] In some embodiments, the AAV capsid variant is (a) a VP1 protein comprising the amino acid sequence of SEQ ID NO: 138, 981, or 982; (b) a VP2 protein comprising the amino acid sequence of positions 138 to 736 of SEQ ID NO: 138 or positions 138 to 742 of SEQ ID NO: 981 or 982; (c) a VP3 protein comprising the amino acid sequence of positions 203 to 736 of SEQ ID NO: 138 or positions 203 to 742 of SEQ ID NO: 981 or 982; or (d) a VP4 protein comprising at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, (c) or (d) (6, 97, 98, or 99%); an amino acid sequence that contains at least 1, 2, or 3 but not more than 30, 20, or 10 different amino acids to any of the amino acid sequences in (a)-(c); or an amino acid sequence that contains at least 1, 2, or 3 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 30, 20, or 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, to any of the amino acid sequences in (a)-(c).

[0456] In some embodiments, the AAV capsid variant further comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 137, or a sequence having at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, the AAV capsid variant further comprises an amino acid sequence encoded by a nucleotide sequence that includes at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 30, 20, or 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 137. In some embodiments, the AAV capsid variant further comprises an amino acid sequence encoded by a nucleotide sequence that includes at least one, two, or three, but not more than 30, 20, or 10, different nucleotides relative to the amino acid sequence of SEQ ID NO: 137.

[0457] In some embodiments, the nucleotide sequence encoding the AAV capsid variant further comprises the nucleotide sequence of SEQ ID NO: 137, or a sequence having at least 70% (e.g., at least about 80, 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, the nucleotide sequence encoding the AAV capsid variant further comprises a nucleotide sequence that includes at least one, two, or three modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, but not more than 30, 20, or 10 modifications, e.g., substitutions (e.g., conservative substitutions), insertions, or deletions, relative to the nucleotide sequence of SEQ ID NO: 137. In some embodiments, the nucleotide sequence encoding the AAV capsid variant further comprises a nucleotide sequence that includes at least one, two, or three, but not more than 30, 20, or 10, different nucleotides relative to the amino acid sequence of SEQ ID NO: 137.

[0458] In some embodiments, the AAV capsid variants of the present disclosure comprise an amino acid sequence described herein, e.g., the amino acid sequence of an AAV capsid variant described in Tables 3 and 4, e.g., TTM-001 or TTM-002.

[0459] In some embodiments, the AAV capsid variants described herein comprise VP1, VP2, and / or VP3 proteins comprising an amino acid sequence described herein, e.g., the amino acid sequence of an AAV capsid variant described in Tables 3 and 4, e.g., TTM-001 or TTM-002.

[0460] In some embodiments, the AAV capsid variants described herein comprise an amino acid sequence encoded by a nucleotide sequence described herein, e.g., the nucleotide sequence of an AAV capsid variant of TTM-001 or TTM-002, e.g., as described in Tables 3 and 5.

[0461] In some embodiments, a polynucleotide or nucleic acid encoding an AAV capsid variant of the present disclosure comp...

Claims

1. (i) A ligand that binds to alkaline phosphatase (ALPL), comprising a peptide containing the amino acid sequence of HDSPHK (SEQ ID NO: 2), wherein the S comprises a phosphate group, (ii) Activators and Includes, A composition in which the ligand is fused to or bound to the activator.

2. The composition according to claim 1, wherein the ligand is fused directly to the activator or indirectly to the activator via a linker.

3. The composition according to claim 1, wherein the peptide comprises the amino acid sequence of GHDSPHKS (SEQ ID NO: 4487), and the S at position 4 of SEQ ID NO: 4487 comprises a phosphate group.

4. The composition according to claim 1, wherein the peptide comprises the amino acid sequence of NGHDSPHKSG (SEQ ID NO: 4489), and the S at position 5 of SEQ ID NO: 4489 comprises a phosphate group.

5. The composition according to claim 1, wherein the peptide comprises the amino acid sequence of INGHDSPHKSGQ (SEQ ID NO: 4490), and the S at position 6 of SEQ ID NO: 4490 comprises a phosphate group.

6. The composition according to claim 1, wherein the peptide comprises the amino acid sequence of TINGHDSPHKSGQN (SEQ ID NO: 4491), and the S at position 7 of SEQ ID NO: 4491 comprises a phosphate group.

7. The composition according to claim 1, wherein the peptide comprises the amino acid sequence of KTINGHDSPHKSGQNQ (SEQ ID NO: 4492), and the S at position 8 of SEQ ID NO: 4492 comprises a phosphate group.

8. The composition according to claim 1, wherein the peptide comprises the amino acid sequence LYYLSKTINGHDSPHKSGQNQQTLKF (SEQ ID NO: 4518), and the S at position 13 of SEQ ID NO: 4518 comprises a phosphate group.

9. The composition according to claim 1, wherein the peptide comprises the amino acid sequence RLMNPLIDQYLYYLSKTINGHDSPHKSGQNQQTLKFSVAGPSNMAV (SEQ ID NO: 4519), and the S at position 23 of SEQ ID NO: 4519 comprises a phosphate group.

10. The composition according to claim 1, wherein the peptide comprises the amino acid sequence NGHDpSPHKSG (SEQ ID NO: 4515), KTINGHDpSPHKSGQNQ (SEQ ID NO: 4516), or YLSKTINGHDpSPHKSGQNQQTLKFS (SEQ ID NO: 4517).

11. The composition according to claim 1, wherein the activator comprises a therapeutic agent selected from proteins, antibody molecules, nucleic acid molecules, and small molecules.

12. The composition according to claim 11, wherein the therapeutic agent is an antibody molecule comprising a complete antibody or an antigen-binding fragment.

13. The composition according to claim 12, wherein the antigen-binding fragment is Fab, Fab fragment, F(ab)2 fragment, Fv fragment, dAb fragment, single-chain antibody (scFv), scFv fragment, variable region of an antibody, diabody, VHH, camelid antibody, single-domain antibody, or nanobody.

14. The composition according to claim 12, wherein the antibody molecule is a monospecific antibody, a multispecific antibody, a bispecific antibody, or a biparatope antibody.

15. The composition according to claim 11, wherein the therapeutic agent is an antibody molecule that binds to an antigen associated with nerve damage, neurodegenerative damage, muscle damage, neuromuscular damage, or neurotumor damage.

16. The composition according to claim 11, wherein the therapeutic agent is an RNAi agent, and the RNAi agent is dsRNA, siRNA, shRNA, pre-miRNA, prim-miRNA, miRNA, stRNA, lncRNA, piRNA, antisense oligonucleotide agent (ASO), or snoRNA.

17. The composition according to claim 1, wherein the activator is a ribonucleic acid complex, a plasmid, closed-end DNA, circ-RNA, or mRNA.

18. The composition according to claim 11, wherein the therapeutic agent is a protein or a functional variant thereof, and the protein or functional variant thereof is related to neuropathy, neurodegenerative disorders, muscle disorders, neuromuscular disorders, or neurotumor disorders.

19. The composition according to claim 1, wherein the activator is a diagnostic agent.

20. The composition according to claim 19, wherein the diagnostic agent comprises an imaging agent.

21. A cell comprising the composition according to any one of claims 1 to 20.

22. The cell according to claim 21, which is a mammalian cell.

23. The cell according to claim 21, which is a cell of the central nervous system and / or a cell present in the blood-brain barrier.

24. A pharmaceutical composition comprising the composition according to any one of claims 1 to 20 and a pharmaceutically acceptable excipient.

25. A composition according to any one of claims 1 to 20, for use in a method of delivering an activator to cells or tissues.

26. The aforementioned cells, (i) Cells from brain or spinal cord regions, optionally selected, including cells from the prefrontal cortex, sensory cortex, motor cortex, caudate nucleus, cerebellar cortex, cerebral cortex, brainstem, hippocampus, or thalamus, and / or (ii) Individuals who have, have been diagnosed with, or are at risk of having, a genetic disorder, neurological disorder, neurodegenerative disorder, neurotumor disorder, muscle disorder, or neuromuscular disorder The composition according to claim 25.

27. ​​The composition according to claim 25, wherein the composition is formulated for intravenous administration, intracisional injection (ICM), intracerebral administration, intrathecal administration, intraventricular administration, intraparenchymal administration, intraarterial administration, or intramuscular administration.

28. The composition according to any one of claims 1 to 20, for use in a method for increasing transduction of the central nervous system in a subject or for increasing passage across the blood-brain barrier.

29. The composition according to claim 28, wherein the subject has, has been diagnosed with, or is at risk of having, a genetic disorder, neurological disorder, neurodegenerative disorder, neurotumor disorder, muscle disorder, or neuromuscular disorder.

30. The composition according to claim 28, wherein the composition is formulated for intravenous administration, intracisional injection (ICM), intracerebral administration, intrathecal administration, intraventricular administration, intraparenchymal administration, intraarterial administration, or intramuscular administration.

31. Use of the composition according to any one of claims 1 to 20 in the manufacture of a pharmaceutical product for delivering an active agent to cells or tissues.

32. The cell is (i) Cells from brain or spinal cord regions, optionally selected, including cells from the prefrontal cortex, sensory cortex, motor cortex, caudate nucleus, cerebellar cortex, cerebral cortex, brainstem, hippocampus, or thalamus, and / or (ii) Individuals who have, have been diagnosed with, or are at risk of having, a genetic disorder, neurological disorder, neurodegenerative disorder, neurotumor disorder, muscle disorder, or neuromuscular disorder The use described in claim 31.

33. The use according to claim 31, wherein the composition is formulated for intravenous administration, intracisional injection (ICM), intracerebral administration, intrathecal administration, intraventricular administration, intraparenchymal administration, intraarterial administration, or intramuscular administration.

34. Use of the composition according to any one of claims 1 to 20 in the manufacture of a pharmaceutical product for increasing CNS transduction or increasing passage across the blood-brain barrier.

35. The use according to claim 34, wherein the subject has, has been diagnosed with, or is at risk of having, a genetic disorder, neurological disorder, neurodegenerative disorder, neurotumor disorder, muscle disorder, or neuromuscular disorder.

36. The use according to claim 34, wherein the composition is formulated for intravenous administration, intracisional injection (ICM), intracerebral administration, intrathecal administration, intraventricular administration, intraparenchymal administration, intraarterial administration, or intramuscular administration.