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  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
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  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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  • C12N5/0602—Vertebrate cells
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  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01076—L-Iduronidase (3.2.1.76)
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  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
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  • C12N2510/00—Genetically modified cells

Definitions

• a number of neurodegenerative and other central nervous system (CNS) disorders are theoretically treatable by proteins such as enzymes, cytokines and antibodies.
• CNS disorders are theoretically treatable by proteins such as enzymes, cytokines and antibodies.
• BBB blood-brain barrier
• BECs brain endothelial cells
• RMT Receptor-mediated transcytosis
• the RMT approach typically employs intravenous or subcutaneous administration of a fusion protein that links the therapeutic protein to a molecule that binds one of the endogenous receptors expressed on the surface of the BECs. Following receptor binding, the RMT process requires internalization of the receptor-fusion protein complex and exocytosis of the fusion protein on the abluminal side of the cell.
• Candidate target receptors for RMT include the transferrin receptor, insulin receptor, insulinlike growth factor- 1 receptor (IGF1R), low-density lipoprotein (LDL) receptor related proteins 1 and 2 (LRP-1 and LRP-2), diptheria toxin receptor and TMEM30A. Results obtained in pre- clinical and clinical studies indicate that a variety of factors can impact the amount and/or activity of therapeutic protein that crosses the BBB, including plasma half-life of the fusion protein and its binding affinity for the RMT target receptor.
• BBB-transporting fusion proteins that comprise a cargo moiety, e.g., a molecule with an activity useful for treating or diagnosing a central nervous system (CNS) condition or disorder of interest, located C-terminal to separate domains that bind to human serum albumin (HSA) and to the extracellular portion of human IGF1R (hIGFIR).
• a cargo moiety e.g., a molecule with an activity useful for treating or diagnosing a central nervous system (CNS) condition or disorder of interest, located C-terminal to separate domains that bind to human serum albumin (HSA) and to the extracellular portion of human IGF1R (hIGFIR).
• HSA human serum albumin
• hIGFIR extracellular portion of human IGF1R
• a linker moiety is disposed in between the two binding domains and / or between the hIGFl -binding domain and the cargo moiety.
• one or both of the HSA-binding and hIGFIR-binding domains has a molecular weight of less than about any of 75 kDa, 50 kDa or 25 kDa.
• the amino acid sequence of each of the HSA-binding and hIGFIR-binding domains is from a single chain Fab (scFab), a single chain Fv (scFv) or a single domain antibody (sdAb).
• the cargo moiety (molecule) has a molecular weight of about 1 kD to about 200 kD, or about 2 kD to about 100 kD.
• the cargo moiety (molecule) consists essentially of, or consists of the amino acid sequence of a polypeptide, e.g., a cytokine, an enzyme, or an antibody.
• the linker moiety is a linker peptide that is less than 50 amino acids in length.
• the present disclosure features a BBB -transporting fusion protein which comprises a primary structure defined by formula I: AB-L1-RB-L2-C or by formula II: RB-L1- AB-L2-C, wherein in each formula AB comprises an HSA-binding domain, LI comprises a first linker amino acid sequence, RB comprises a hIGFIR-binding domain, L2 comprises a second linker amino acid sequence, and C comprises the amino acid sequence of a cargo polypeptide.
• the fusion protein binds via AB to domain 1 (DI) or domain 2 (DII) of HSA and does not substantially inhibit binding of human FcRn (h-FcRn) to HSA.
• the fusion protein binds via the AB domain to HSA with a KD affinity of less than about 1 nM to about 100 nM within a pH range of about 5.0 to about 7.4 as determined by surface plasmon resonance at 25° C.
• the fusion protein also binds via AB to at least one mammalian serum albumin ortholog at 25° C within a pH range of about 5.5 to about 7.4.
• the albumin ortholog is from mouse, rat, hamster, rabbit, guinea pig, pig, cat, dog, or a non-human primate (e.g., cynomolgus or rhesus monkey).
• AB comprises first, second and third amino acid sequences corresponding to the three complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region of an anti -HSA antibody (e.g., a conventional antibody with two heavy chains and two light chains, an scFab, an scFv, a sdAb).
• an anti -HSA antibody e.g., a conventional antibody with two heavy chains and two light chains, an scFab, an scFv, a sdAb.
• the CDR1, CDR2 and CDR3 amino acid sequences in AB are: GRTFIAYA (SEQ ID NO: 1) or a conservatively substituted variant thereof; ITNFAGGTT (SEQ ID NO:2) or a conservatively substituted variant thereof; and AADRSAQTMRQVRPVLPY (SEQ ID N0:3) or a conservatively substituted variant thereof.
• AB consists essentially of, or consists of: QVQLVESGGGLVQAGGSLRLSCVASGRTFIAYAMGWFRQAPGKEREFVAAITNFAGGT TYYADSVKGRFTISRDNAKTTVYLQMNSLKPEDTALYYCAADRSAQTMRQVRPVLPY WGQGTQVTVSS (SEQ ID NO:4), or a conservatively substituted variant thereof.
• AB consists essentially of, or consists of: QVQLVESGGGLVQPGGSLRLSCAASGRTFIAYAMGWFRQAPGKEREFVAAITNFAGGT TYYADSVKGRFTISRDNAKTTVYLQMNSLRAEDTAVYYCAADRSAQTMRQVRPVLPY WGQGTLVTVSS (SEQ ID NO:5), or a conservatively substituted variant thereof.
• AB consists essentially of, or consists of, an amino acid sequence of the heavy chain variable region of an antibody that cross-competes with a sdAb consisting of SEQ ID NO:4 or SEQ ID NO: 5 for binding to HSA.
• the fusion protein binds via the RB domain to hIGFIR expressed on the surface of human brain endothelial cells. In some embodiments, the fusion protein does not substantially bind to the human insulin receptor (h-IR). In some embodiments, the fusion protein does not substantially inhibit binding of insulin, insulin growth factor 1 (IGF1) or insulin growth factor 2 (IGF2) to hIGFIR. In an embodiment, the fusion protein binds via RB to an epitope in the hIGFIR extracellular domain which comprises FENFLHNSIFVPR (SEQ II) NO:6).
• the fusion protein binds via RB to hIGFIR with a KD affinity of about 0.5 nM to about 50 nM within a pH range of about 5.0 to about 7.4, as determined by surface plasmon resonance at 25° C.
• the fusion protein also binds via RB to at least one mammalian IGF1R ortholog at 25° C and within a pH range of about 5.0 to about 7.4.
• the IGF1R ortholog is from mouse, rat, hamster, rabbit, guinea pig, dog, cat, or a non-human primate (e.g., cynomolgus or rhesus monkey).
• RB comprises first, second and third amino acid sequences corresponding to the three complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region of an anti -hIGFIR antibody (e.g., a conventional antibody with two heavy chains and two light chains, an scFab, an scFv, a sdAb).
• an anti -hIGFIR antibody e.g., a conventional antibody with two heavy chains and two light chains, an scFab, an scFv, a sdAb.
• the CDR1, CDR2 and CDR3 amino acid sequences in RB are GRTIDNYA (SEQ ID NO:7) or a conservatively substituted variant thereof; IDWGDGGX, where X is A or T (SEQ ID NO:8) or a conservatively substituted variant thereof; and AMARQSRVNLDVARYDY (SEQ ID N0:9) or a conservatively substituted variant thereof.
• the CDR2 sequence in RB is IDWGDGGA (SEQ ID NO: 10).
• RB consists essentially of, or consists of: QVKLEESGGGLVQAGGSLRLSCAASGRTIDNYAMAWSRQAPGKDREFVATIDWGDGG ARYANSVKGRFTISRDNAKGTMYLQMNNLEPEDTAVYSCAMARQSRVNLDVARYDY WGQGTQVTVSS (SEQ ID NO: 11) or a conservatively substituted variant thereof.
• RB consists essentially of, or consists of: QVQLVESGGGLVQPGGSLRLSCAASGRTIDNYAMAWVRQAPGKGLEWVATIDWGDGG TRYANSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCAMARQSRVNLDVARYDY WGQGTLVTVSS (SEQ ID NO: 12) or a conservatively substituted variant thereof.
• RB consists essentially of, or consists of, an amino acid sequence of the heavy chain variable region of an antibody that cross-competes with a sdAb consisting of SEQ ID NO: 11 or SEQ ID NO: 12 for binding to hIGFIR.
• the cargo moiety in the fusion protein comprises, consists essentially of, or consists of the amino acid sequence for an enzyme that is deficient in a lysosomal storage disorder (LSD), e.g., alpha-L-iduronidase (IDUA), iduronate-2-sulfatase (IDS), arylsulfatase B (ARSB), N-sulfoglucosamine sulfohydrolase (SGSH), glucosylceramidase (GBA), alpha-galactosidase A (GLA), and alpha- 1,4-glucosidase (GAA).
• LSD lysosomal storage disorder
• IDUA alpha-L-iduronidase
• IDS iduronate-2-sulfatase
• ARSB arylsulfatase B
• SGSH N-sulfoglucosamine sulfohydrolase
• GAA glucosylcer
• the cargo moiety comprises, consists essentially of, or consists of the amino acid sequence for an antibody or antigen-binding fragment thereof that binds to a target protein in the brain, e.g., beta- seer etase 1 (BACE1), an immunotherapy target (e.g., programmed death receptor 1 (PD-1)).
• BACE1 beta- seer etase 1
• PD-1 programmed death receptor 1
• each of LI and L2 is 3 to about 35 amino acids in length, 4 to about 30 amino acids in length or 5 to about 20 amino acids in length.
• each of LI and L2 consists essentially of, or consists of, (GGGGS) n (SEQ ID NO: 13), where n is equal to 3, 4 or 5.
• each of LI and L2 consists essentially of, or consists of, (GGGGS)4 (SEQ ID NO: 14).
• the cargo moiety in the BBB- transporting fusion protein is an IDUA protein.
• the BBB -transporting IDUA fusion protein comprises, consists essentially of, or consists of the amino acid sequence shown in FIG. 6 (SEQ ID NO:38).
• the present disclosure provides a polynucleotide (e.g., an isolated polynucleotide) which comprises a nucleotide sequence that encodes a BBB -transporting fusion protein described herein.
• the nucleotide sequence is operably linked to a promoter sequence and a polyA signal sequence.
• the promoter sequence is identical to, or substantially identical to, one of the promoter sequences in FIG. 4 (SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34).
• the polyA signal sequence is identical to, or substantially identical to, one of the polyA signal sequences shown in FIG. 5 (SEQ ID NO:35, SEQ ID NO:36 or SEQ ID NO:37).
• the BBB -transporting fusion protein consists of the amino acid sequence shown in FIG. 6 and the polynucleotide comprises, consists essentially or, or consists of the nucleotide sequence shown in FIG. 7.
• the present disclosure provides a mammalian cell (e.g., a mouse cell, a Chinese hamster ovary (CHO) cell, a monkey cell, a human cell (e.g., an RPE cell) that is genetically modified to express and secrete a BBB-transporting fusion protein described herein.
• the mammalian cell is genetically modified by transfection with a polynucleotide described herein (e.g., an expression vector) which encodes the BBB-transporting fusion protein.
• the cargo moiety in the fusion protein is an IDUA protein.
• the mammalian cell is derived from an ARPE-19 cell by transfection with an expression vector comprising the nucleotide sequence shown in FIG. 7.
• the present disclosure also provides a composition comprising a plurality of genetically modified cells described herein and a method of manufacturing the composition.
• the composition comprises a cell culture media or a storage medium.
• the composition comprises a polymer solution in which the cells are suspended, e.g., a polymer solution described herein, e.g., comprising alginate and a cell-binding substance, e.g., as defined herein.
• the method of manufacturing the composition comprises culturing a plurality of a genetically modified cell described herein until a desired number of cultured cells has been produced, and combining the desired number of cultured cells with a cell culture media, a storage medium or a polymer solution.
• the present disclosure features a device comprising at least one cellcontaining compartment which comprises a genetically modified mammalian cell as described herein or a plurality of such cells.
• the device is configured to shield the cell(s) from the recipient’s immune system and mitigate the foreign body response (FBR) (as defined herein) to the implanted device.
• FBR foreign body response
• the surface of the device comprises a compound or polymer that mitigates the FBR (as defined herein) to the device (e.g., an afibrotic compound or afibrotic polymer).
• an afibrotic polymer comprises a biocompatible, zwitterionic polymer, e.g., as described in WO 2017/218507, WO 2018/140834, or Liu et al., Zwitterionically modified alginates mitigate cellular overgrowth for cell encapsulation, Nature Communications (2019)10:5262.
• the compound is a compound of Formula (III): or a pharmaceutically acceptable salt thereof, wherein the variables A, L 1 , M, L 2 , P, L 3 , and Z, as well as related subvariables, are defined herein.
• the compound of Formula (III) or a pharmaceutically acceptable salt thereof e.g., Formulas (Ill-a), (Ill-b), (III-b-i), (Ill-b- ii), (in-c), (in-d), (Ill-e), (Ill-f), (IV), (IV-a), (V), (V-a), (V-b), (V-c), (V-d), (Vl-a), (VLb), (VI- c), (Vl-d), or (VI-e)) is a compound described herein, including for example, one of the compounds shown in Table 6 herein.
• the compound of Formula (III) is a compound selected from Compound 100, Compound 101, Compound 102 or Compound 122 shown in Table 6.
• a device of the disclosure is a two-compartment hydrogel capsule (e.g., a microcapsule (less than 1 mm in diameter) or a millicapsule (at least 1 mm in diameter)) in which a cell-containing compartment (e.g., the inner compartment) comprising a plurality of live genetically modified cells described herein (and optionally one or more cell binding substances) is surrounded by a barrier compartment comprising an afibrotic polymer (e.g., the outer compartment, e.g., hydrogel layer).
• the afibrotic polymer comprises an afibrotic compound.
• the afibrotic compound is a compound of Formula (III).
• the present disclosure features a preparation (e.g., a composition) comprising a plurality (at least any of 3, 6, 12, 25, 50 or more) of a cell -containing device described herein, e.g, a preparation of hydrogel capsules encapsulating genetically modified ARPE-19 cells.
• a preparation e.g., a composition
• the preparation is a pharmaceutically acceptable composition.
• the present disclosure features a method of making or manufacturing a device comprising a genetically modified cell described herein.
• the method comprises providing the genetically modified cell, or a plurality of such cells, and disposing the cell(s) in an enclosing component, e.g., a cell-containing compartment of the device as described herein.
• the enclosing component comprises a flexible polymer (e.g., PL A, PLG, PEG, CMC, or a polysaccharide, e.g., alginate).
• the enclosing component comprises an inflexible polymer or metal housing.
• the surface of the device is chemically modified, e.g., with a compound of Formula (III) as described herein.
• a device described herein, or a plurality of the device is combined with a pharmaceutically acceptable excipient to prepare a device preparation or a composition which may be administered to a subject (e.g., into the intraperitoneal cavity) in need of treatment with the BBB -transporting fusion protein produced by the device.
• the genetically modified cells are derived from a human cell (e.g., an RPE cell, an ARPE-19 cell) and the device preparation or composition is capable of continuously delivering an effective amount of the BBB- transporting fusion protein to the subject for a sustained time period, e.g., at least any of 3 months, 6 months, one year, two years or longer.
• the present disclosure features a method of evaluating a composition, device or device preparation described herein.
• the method comprises providing the composition, device or device preparation and evaluating a functional parameter of the composition, device or device preparation.
• the functional parameter is the amount of the BBB-transporting fusion protein produced by the cells in the composition, device or device preparation in vitro (e.g., when placed in a suitable culture medium) and/or in vivo (e.g., after implant into a subject, e.g., a non-human subject or a human subject.
• the present disclosure features a method of treating a subject in need of therapy with a BBB-transporting fusion protein described herein.
• the method comprises administering to the subject an implantable element (e.g., a device or device preparation) comprising a genetically modified cell that expresses and secrets the fusion protein, or a plurality of such cells.
• the administering step comprises placing into the subject a pharmaceutically acceptable preparation comprising a plurality of devices, each of which has the ability to produce the BBB-transporting fusion protein.
• the implantable element is administered to, placed in, or injected in the peritoneal cavity (e.g., the lesser sac), the omentum, or the subcutaneous fat of a subject.
• the method further comprises measuring the amount of the BBB-transporting fusion protein present in a tissue sample removed from the subject, e.g., in plasma separated from a blood sample or in a tissue sample obtained from the CNS or from an organ of interest.
• the tissue sample is removed from the patient at 15, 30, 60 or 120 days after administration, implantation, or placement of the device or device preparation.
• the subject is a human.
• the subject is a human patient diagnosed as having a neuronopathic MPS disease and the fusion protein comprises the glycosaminoglycan-metabolizing enzyme that is deficient in the neuronopathic MPS disease.
• the implantable element produces the BBB -transporting fusion protein in an amount sufficient to reduce one or more symptoms of the neuronopathic MPS disease.
• the treatment results in a reduction in heparan sulfate levels in the brain and optionally in one or more other organs or tissues outside the CNS, e.g., liver, spleen, kidney, lung and heart.
• the patient has been diagnosed with Mucopolysaccharidosis type I (MPS I) and the BBB-transporting fusion protein comprises a human IDUA protein.
• MPS I Mucopolysaccharidosis type I
• FIGS. 1A-1J show the amino acid sequences of the wild-type, human precursor polypeptides for exemplary LSD enzymes that may be included as the cargo polypeptide in BBB- transporting fusion proteins described herein: GAA (FIG. 1A, SEQ ID NO: 15); GBA (FIG. IB, SEQ ID NO: 16); GLA; (FIG. 1C, SEQ ID NO:17); GNS (FIG. ID, SEQ ID NO: 18); GUSB (FIG. IE, SEQ ID NO: 19); HGSNAT (FIG. IF, SEQ ID NO:20); IDS (FIG. 1G, SEQ ID NO:21); IDUA (FIG. 1H, SEQ ID NO:22); NAGLU (FIG. II, SEQ ID NO:23); and SGSH (FIG. 1 J, SEQ ID NO:24).
• GAA FIG. 1A, SEQ ID NO: 15
• GBA FIG. IB, SEQ ID NO: 16
• GLA (
• FIGS. 2A-2B show exemplary amino acid sequences of the precursor forms of human proteins bound by BBB-transporting fusion proteins described herein, with FIG. 2A (SEQ ID NO:25) showing the amino acid sequence of a precursor human IGF1R monomer (UniProtKB - P08069), with the signal peptide italicized, the furin cleavage site shown in lower case italics, and underlined bold font indicating amino acids that are points of contact in putative binding sites for certain hIGFIR-binding domains described herein and FIG. 2B (SEQ ID NO:95) showing the amino acid sequence of precursor human serum albumin, with the signal peptide underlined.
• FIG. 2A SEQ ID NO:25
• FIG. 2B shows the amino acid sequence of a precursor human IGF1R monomer (UniProtKB - P08069), with the signal peptide italicized, the furin cleavage site shown in lower case italics, and underlined
• FIGS. 3A-3F show the amino acid sequences of exemplary BBB-transporting IDUA fusion proteins of the disclosure, with the fusion proteins in FIG. 3A (SEQ ID NO:26), FIG. 3B (SEQ ID NO:27) and FIG. 3C (SEQ ID NO:28) comprising a parental anti-HSA and one of three different parental anti-IGFIR sdAb sequences; the fusion proteins in FIG. 3D (SEQ ID NO:29), FIG. 3E (SEQ ID NO:30), and FIG. 3F (SEQ ID NO:31) comprising humanized variants of the anti-HSA and anti-IGFIR sdAb sequences shown in FIGs. 3 A, 3B and 3C; and each of the fusion proteins comprising two linker sequences with 4 or 5 repeats of GGGGS (SEQ ID NO:92.
• FIGS. 4A-4C show exemplary promoter sequences that are useful in an expression construct for BBB-transporting fusion proteins of the disclosure: pCAG promoter sequence (FIG. 4A, SEQ ID NO:32); EFla promoter sequence (FIG. 4B, SEQ ID NO:33); and EFS promoter sequence (FIG. 4C, SEQ ID NO:34).
• FIGS. 5A-5C show exemplary polyA signal sequences that are useful in an expression construct for BBB-transporting fusion proteins of the disclosure: rBG poly A signal sequence (FIG. 5A, SEQ ID NO:35); SV40 late poly A signal sequence (FIG. 5B, SEQ ID NO:36) and BGH poly A signal sequence (FIG. 5C, SEQ ID NO:37).
• FIG. 6 shows the amino acid sequence (SEQ ID NO:38) for an exemplary BBB- transporting IDUA fusion protein, with underlining indicating a VHH consensus signal peptide, bold font indicating the anti-HSA sdAb, italics indicating the anti-IGFIR sdAb, bold italics font indicating the wild-type, human mature IDUA amino acid sequence and dash-dot underlining indicating the flexible linker sequences.
• FIG. 7 shows the nucleotide sequence (SEQ ID NO:39) of an exemplary transcription unit useful for expressing the IDUA fusion protein described in Figure 6, with wavy underlining indicating the EFl A promoter sequence, straight underlining indicating an exemplary coding sequence for the VHH consensus signal peptide, bold font indicating an exemplary coding sequence for the anti-HSA sdAb, italics indicating an exemplary coding sequence for the anti- IGFIR sdAb, bold italics indicating an exemplary coding sequence for the wild-type, human mature IDUA amino acid sequence, dash-dot underline indicating a coding sequence for the flexible inker sequences, dotted underline indicating the stop codon and underlined italics font indicating the rBG poly A signal sequence.
• FIGS. 8A-8B illustrate IDUA activity in single IDUA fusion proteins containing an exemplary anti-IGFIR sdAb fused to hIDUA via an amino acid linker
• FIG. 8 A showing in vitro IDUA activity in culture media of cells expressing one of six different fusion constructs based on the sdAb orientation and linker length
• FIG. 8B comparing in vivo hIDUA activity in liver and plasma samples from MPS-1 mice implanted with encapsulated cells expressing either wildtype hIDUA (light grey bars) or the fusion construct that had produced the highest in vitro hIDUA activity in FIG. 8A (IGFlrR-hlDUA, dark grey bars).
• FIG. 9A is a graph showing the in vitro IDUA activity in conditioned culture media of cells expressing one of six different double IDUA fusion proteins containing various orientations of hIDUA, an exemplary anti -IGF 1R sdAb (IGF1R5) and an exemplary anti-HSA sdAb (R28).
• FIG. 9B is a graph comparing the in vitro IDUA activity in conditioned culture media of cells expressing an exemplary anti-IGFIR-hlDUA fusion enzyme (IGFlR4-hIDUA, light grey bar) with cells expressing an exemplary anti-HSA-anti-IGFIR-hlDUA fusion enzyme (R28- IGFlR5-hIDUA, dark grey bar).
• IGFlR4-hIDUA an exemplary anti-IGFIR-hlDUA fusion enzyme
• R28- IGFlR5-hIDUA dark grey bar
• FIG. 10 is a graph comparing in vivo hIDUA activity in plasma and systemic (non-brain) tissues of MPS-1 mice implanted with encapsulated cells expressing either wild-type hIDUA (light grey bars) or an exemplary anti-HSA-anti-IGFIR-hlDUA fusion enzyme (R28-IGFlR5-hIDUA, dark grey bars).
• FIG. 11 is a graph comparing heparan sulfate levels in brain tissue samples from untreated MPS-1 mice (light grey bars) and MPS-1 mice implanted with encapsulated cells expressing an exemplary anti-HSA-anti-IGFIR-hlDUA fusion enzyme (R28-IGFlR5-hIDUA, dark grey bars).
• FIGS. 12A and 12B are amino acid sequences (SEQ ID NO: 93 and SEQ ID NO: 94) of exemplary BBB -transporting IDS fusion proteins of the disclosure.
• FIG. 13 is a chart illustrating a comparison of enzyme activity levels generated with exemplary constructs described herein, as outlined in Example 5.
• FIG. 14 is a graph comparing heparan sulfate levels in liver, spleen, kidney, lung and heart tissue samples from untreated MPS-1 mice (grey bars) and MPS-1 mice implanted with encapsulated cells expressing an exemplary anti-HSA-anti-IGFIR-hlDUA fusion enzyme (R28- IGFlR5-hIDUA, black bars).
• FIG. 15 is a graph comparing heparan sulfate levels in brain tissue samples from untreated MPS-1 mice (solid black bar) and MPS-1 mice implanted with encapsulated cells expressing an exemplary hIDUA fusion enzyme grey bars).
• the present disclosure features BBB -transporting fusion proteins that comprise HSA- and hIGFIR-binding domains and a cargo moiety, mammalian cells (e.g., human RPE cells) genetically modified to express and secrete these fusion proteins, as well as compositions and devices comprising the genetically modified cells.
• the devices comprise a cell-containing compartment which includes a cell binding substance as well as the genetically modified cells.
• the devices are configured to mitigate the FBR when placed inside a subject, e.g., a human subject.
• the fusion proteins, genetically modified cells, compositions, and devices are useful for the treatment of a CNS condition or disorder such as a lysosomal storage disease. Various embodiments will be described below.
• HGSNAT heparan-alpha-glucosaminide N-acetyltransferase protein
• “About” or “approximately” when used herein to modify a numerically defined parameter means that the recited numerical value is within an acceptable functional range for the defined parameter as determined by one of ordinary skill in the art, which will depend in part on how the numerical value is measured or determined, e.g., the limitations of the measurement system, including the acceptable error range for that measurement system. For example, “about” can mean a range of 20% above and below the recited numerical value.
• a hydrogel capsule defined as having a diameter of about 1.5 millimeters (mm) and encapsulating about 5 million (M) cells may have a diameter of 1.2 to 1.8 mm and may encapsulate 4 M to 6 M cells.
• a preparation of about 100 devices includes preparations having 80 to 120 devices.
• the term “about” means that the modified parameter may vary by as much as 15%, 10% or 5% above and below the stated numerical value for that parameter.
• “Acquire” or “acquiring” as used herein, refer to obtaining possession of a value, e.g., a numerical value, or image, or a physical entity (e.g., a sample), by “directly acquiring” or “indirectly acquiring” the value or physical entity.
• “Directly acquiring” means performing a process (e.g., performing an analytical method or protocol) to obtain the value or physical entity.
• “Indirectly acquiring” refers to receiving the value or physical entity from another party or source (e.g., a third-party laboratory that directly acquired the physical entity or value).
• Directly acquiring a value or physical entity includes performing a process that includes a physical change in a physical substance or the use of a machine or device. Examples of directly acquiring a value include obtaining a sample from a human subject.
• Directly acquiring a value includes performing a process that uses a machine or device, e.g., using a fluorescence microscope to acquire fluorescence microscopy
• administering refers to implanting, absorbing, ingesting, injecting, placing, or otherwise introducing into a subject, an entity described herein (e.g., a device or a preparation of devices), or providing such an entity to a subject for administration.
• an entity described herein e.g., a device or a preparation of devices
• Afibrotic means a compound or material that mitigates the foreign body response (FBR).
• FBR foreign body response
• the amount of FBR in a biological tissue that is induced by implant into that tissue of a device e.g., hydrogel capsule
• an afibrotic compound e.g., a hydrogel capsule comprising a polymer covalently modified with a compound listed in Table 6
• the FBR induced by implantation of an afibrotic-null reference device i.e., a device that lacks any afibrotic compound, but is of substantially the same composition (e.g., same cell type(s)) and structure (e.g., size, shape, no. of compartments).
• the degree of the FBR is assessed by the immunological response in the tissue containing the implanted device (e.g., hydrogel capsule), which may include, for example, protein adsorption, macrophages, multinucleated foreign body giant cells, fibroblasts, and angiogenesis, using assays known in the art, e.g., as described in WO 2017/075630, or using one or more of the assays / methods described Vegas, A., et al., Nature Biotechnol (supra), (e.g., subcutaneous cathepsin measurement of implanted capsules, Masson’s tri chrome (MT), hematoxylin or eosin staining of tissue sections, quantification of collagen density, cellular staining and confocal microscopy for macrophages (CD68 or F4/80), myofibroblasts (alpha-muscle actin, SMA) or general cellular deposition, quantification of 79 RNA sequences of known inflammation
• the FBR is assessed by measuring the levels in the tissue containing the implant of one or more biomarkers of immune response, e.g., cathepsin, TNF-a, IL-13, IL-6, G-CSF, GM- CSF, IL-4, CCL2, or CCL4.
• biomarkers of immune response e.g., cathepsin, TNF-a, IL-13, IL-6, G-CSF, GM- CSF, IL-4, CCL2, or CCL4.
• the FBR induced by a device of the invention is at least about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% lower than the FBR induced by an FBR-null reference device, e.g., a device that is substantially identical to the test or claimed device except for lacking the means for mitigating the FBR (e.g., a hydrogel capsule that does not comprise an afibrotic compound but is otherwise substantially identical to the claimed capsule.
• the FBR (e.g., level of a biomarker(s)) is measured after about 30 minutes, about 1 hour, about 6 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 1 week, about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, or longer.
• “Acid alpha-glucosidase protein”, “acid maltase protein”, “alpha-1, 4-glucosidase protein” and “GAA protein” may be used interchangeably herein and refer to a protein comprising the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) GAA gene or any fragment, mutant, variant or derivative thereof that has GAA enzyme activity that is within 80- 120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature GAA protein, as measured by any art-recognized GAA activity assay.
• the GAA enzyme catalyzes the hydrolysis of alpha(l,4) and alpha(l,6) linkages in glycogen, yielding free glucose and shortened glycogen polymers. GAA enzymatic activity can be measured using any art-recognized assay.
• the wild-type human GAA gene encodes a 952 amino acid precursor pro-polypeptide, of which the N- terminal 27 amino acids constitute a signal peptide, and amino acids 28-69 constitute a pro-peptide (UniProtKB - Pl 0253).
• the GAA amino acid sequence in a BBB- transporting fusion protein described herein comprises amino acids 70-952 of the human precursor GAA sequence shown in FIG. 1 A.
• the human GAA amino acid sequence in a BBB -transporting fusion protein consists essentially of amino acids 28-952 of the sequence shown in FIG. 1 A.
• Alpha-galactosidase A protein may be used interchangeably herein and refer to a protein comprising the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) GLA gene or any fragment, mutant, variant or derivative thereof that has GLA enzyme activity that is within 80-120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature GLA protein, as measured by any art-recognized GLA activity assay.
• the GLA enzyme hydrolyzes the terminal alpha-D-galactose residues in glycosphingolipids, particularly in globotriaosylceramide (Gbs).
• GLA enzymatic activity can be measured using any art-recognized assay.
• the wild-type human GLA gene encodes a 429-amino acid polypeptide, of which the N-terminal 31 amino acids constitute a signal peptide (UniProtKB - P06280).
• the human GLA amino acid sequence in a BBB-transporting fusion protein described herein consists essentially of amino acids 32-429 of human precursor GLA amino acid sequence shown in FIG. 1C.
• Alpha-L-iduronidase protein and “IDUA protein” may be used interchangeably herein and refer to a protein comprising the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) IDUA gene or any fragment, mutant, variant or derivative thereof that has IDUA enzyme activity that is within 80-120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature IDUA protein, as measured by any art-recognized IDUA activity assay (e.g., hydrolysis of the substrate 4-methylumbelliferyl-a-L-iduronide (4MU- iduronide), see, e.g., Ou, L. et al., Mol Genet Metab.
• IDUA activity assay e.g., hydrolysis of the substrate 4-methylumbelliferyl-a-L-iduronide (4MU- iduronide), see, e.g., Ou, L. et al., Mol Genet Metab.
• IDUA protein hydrolyzes nonreducing terminal alpha-L-iduronic acid residues in glycosaminoglycans (GAGs) (e.g., dermatan sulfate and heparan sulfate).
• GAGs glycosaminoglycans
• the wild-type human IDUA gene encodes a 653 amino acid precursor protein, of which the N-terminal 26 or 27 amino acids constitute a signal peptide (GenBAnk Accession No. AAA81589.1, GenBAnk Accession No. AAA51698.1; UniProtKB - P35475).
• the mature human IDUA amino acid sequence in a BBB-transporting fusion protein described herein comprises amino acid 26, 27 or 28 to amino acid 653 of the precursor human IDUA amino acid sequence shown in FIG. 1H.
• the mature human IDUA amino acid sequence in a BBB-transporting fusion protein consists essentially of amino acids 27-653 of the amino acid sequence shown in FIG. 1H.
• Alpha-N-acetyl-glucosaminidase protein N-acetyl-alpha-glucosaminidase
• NAGLU protein may be used interchangeably herein to refer to a protein that comprises the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) NAGLU gene or any fragment, mutant, variant or derivative thereof that has enzyme activity that is within 80-120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature NAGLU protein, as measured by any art-recognized NAGLU assay.
• NAGLU catalyzes the hydrolysis of terminal non-reducing N-acetyl-D-glucosamine residues in N-acetyl-alpha-D-glucosaminides.
• the wild-type human NAGLU gene encodes a 743 amino acid precursor polypeptide, of which the N-terminal 23 amino acids constitute a signal peptide (UniProtKB - P54802).
• the mature human NAGLU amino acid sequence in a BBB-transporting fusion protein described herein consists essentially of amino acids 24-743 of the amino acid sequence shown in FIG. II.
• Beta-glucuronidase protein and “GUSB protein” may be used interchangeably herein to refer to a protein that comprises the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) GUSB gene or any fragment, mutant, variant or derivative thereof that has enzyme activity that is within 80-120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature GUSB protein, as measured by any art-recognized GUSB assay.
• GUSB catalyzes the hydrolysis of beta-D-glucuronoside into an alcohol and D-glucuronate.
• the wild-type human GUSB gene encodes a 651 amino acid precursor polypeptide, of which the N- terminal 22 amino acids constitute a signal peptide (UniProtKB PO8236).
• the mature human GUSB amino acid sequence in a BBB -transporting fusion protein described herein consists essentially of amino acids 23-651 of the sequence shown in FIG. IE.
• Beta-glucosidase protein may be used interchangeably herein to refer to a protein that comprises the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) GBA gene or any fragment, mutant, variant or derivative thereof that has enzyme activity that is within 80-120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature GBA protein, as measured by any art-recognized GBA assay.
• GBA within the lysosomal compartment, catalyzes the breakdown of the glycolipid glucosylceramide (GlcCer) to ceramide and glucose.
• the wild-type human GBA gene encodes a 536 amino acid precursor polypeptide, of which the N-terminal 39 amino acids constitute a signal peptide (UniProtKB P04062.3).
• the mature human GBA amino acid sequence in a BBB-transporting fusion protein described herein consists essentially of amino acids 40-536 of the sequence shown in FIG. IB.
• Cell refers to an engineered cell (e.g., a genetically modified cell), or a cell that is not engineered.
• a cell is an immortalized cell, or an engineered cell derived from an immortalized cell.
• the cell is a live cell, e.g., is viable as measured by any technique described herein or known in the art.
• Cell-binding peptide means a linear or cyclic peptide that comprises an amino acid sequence that is derived from the cell binding domain of a ligand for a cell-adhesion molecule (CAM) (e.g., that mediates cell-matrix junctions or cell-cell junctions).
• CAM cell-adhesion molecule
• the CBP is any of the CBPs described in international patent publication W02020069429.
• the CBP is a linear peptide comprising RGD (SEQ ID NO:87) and is less than 10 amino acids in length.
• the CBP is a linear peptide that consists essentially of GRGD (SEQ ID NO:88) or GRGDSP (SEQ ID NO:89).
• CBP-polymer means a polymer comprising at least one cell-binding peptide molecule covalently attached to the polymer via a linker.
• the polymer in a CBP-polymer is a synthetic or naturally-occurring polysaccharide, e.g., an alginate, e.g., a sodium alginate.
• the linker is an amino acid linker (i.e., consists essentially of a single amino acid, or a peptide of several identical or different amino acids), which is joined via a peptide bond to the N-terminus or C-terminus of the CBP.
• the CBP-polymer is any of the CBP-alginates defined in W02020069429.
• Cell-binding substance means any chemical, biological, or other type of substance (e.g., a small organic compound, a peptide, a polypeptide) that is capable of mimicking at least one activity of a ligand for a cell-adhesion molecule (CAM) or other cellsurface molecule that mediates cell-matrix junctions or cell-cell junctions or other receptor- mediated signaling.
• CAM cell-adhesion molecule
• the CBS when present in a polymer composition encapsulating live cells, the CBS is capable of forming a transient or permanent bond or contact with one or more of the cells.
• the CBS facilitates interactions between two or more live cells encapsulated in the polymer composition.
• the presence of a CBS in a polymer composition encapsulating a plurality of cells is correlated with one or both of increased cell productivity (e.g., expression of a therapeutic agent) and increased cell viability when the encapsulated cells are implanted into a test subject, e.g., a mouse.
• the CBS is physically attached to one or more polymer molecules in the polymer composition.
• the CBS is a cell-binding peptide, as defined herein or in W02020069429.
• Constantly modified variants refers to a variant of a reference peptide or polypeptide that is identical to the reference molecule, except for having one or more conservative amino acid substitutions in its amino acid sequence.
• a conservatively modified variant consists of an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the reference amino acid sequence.
• a conservatively modified variant of an HSA-binding domain sequence and / or IGF 1R binding domain sequence does not include substitutions of any amino acids in the CDRs.
• a conservative amino acid substitution refers to substitution of an amino acid with an amino acid having similar characteristics (e.g., charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) and which has minimal impact on the biological activity of the resulting substituted peptide or polypeptide.
• Conservative substitution tables of functionally similar amino acids are well known in the art, and exemplary substitutions grouped by functional features are set forth in Table 1 below. Table 1. Exemplary conservative amino acid substitution groups.
• Consists essentially of and variations such as “consist essentially of’ or “consisting essentially of’ as used throughout the specification and claims, indicate the inclusion of any recited elements or group of elements, and the optional inclusion of other elements, of similar or different nature than the recited elements, that do not materially change the basic or novel properties of the specified molecule, composition, device, or method.
• an HSA-binding domain or an IGFIR-binding domain that consists essentially of a recited amino acid sequence may also include one or more amino acids, including substitutions in the recited amino acid sequence, of one or more amino acid residues, which do not materially affect the relevant biological activity of the HSA-binding domain or an IGFIR-binding domain, respectively.
• “Derived from”, as used herein with respect to a cell or cells, refers to cells obtained from tissue, cell lines, or cells, which optionally are then cultured, passaged, immortalized, differentiated and/or induced, etc. to produce the derived cell(s).
• Device refers to any implantable object (e.g., a particle, a hydrogel capsule, an implant, a medical device), which contains an engineered cell or cells (e.g., live cells) capable of expressing and secreting a fusion protein following implant of the device, and has a configuration that supports the viability of the cells by allowing cell nutrients to enter the device.
• an engineered cell or cells e.g., live cells
• Effective amount refers to an amount of any of the following: genetically-modified cells secreting a BBB -transporting fusion protein, a device preparation producing the fusion protein, number of genetically-modified cells in a device, amount of a CBS and/or afibrotic compound in a device that is sufficient to elicit a desired biological response.
• the term “effective amount” refers to the amount of a component of the device (e.g., number of cells in the device, the density of an afibrotic compound disposed on the surface and/or in a barrier compartment of the device, the density of a CBS in the cell -containing compartment.
• the desired biological response is an increase in levels of the cargo molecule (e.g., cargo polypeptide) in a tissue sample removed from a subject treated with (e.g., implanted with) the genetically modified cells, a device or a device preparation containing such cells.
• the effective amount may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the secreted BBB -transporting fusion protein, composition or device, the condition being treated, the mode of administration, and the age and health of the subject.
• An effective amount encompasses therapeutic and prophylactic treatment.
• an effective amount of a compound of Formula (III) disposed on or in a device is an amount that reduces the FBR to the implanted device compared to a reference device, e.g., reduces fibrosis or amount of fibrotic tissue on or near the implanted device.
• an effective amount of a CBS disposed with engineered cells in a cellcontaining compartment is an amount that enhances the viability of the cells (e.g., number of live cells) compared to a reference device and/or increases the production of the BBB-transporting fusion protein by the cells (e.g., increased levels of the fusion protein in plasma of a subject implanted with the device) compared to a reference device.
• An effective amount of a device, composition or component e.g., afibrotic compound, CBS, engineered cells
• “’’Engineered cell” or “genetically-modified cell,” as used herein, is a mammalian cell (e.g., a human cell, e.g., an RPE cell, a cell derived from a cell line, (e.g., ARPE-19 or other cell line), a stem cell, a cell differentiated from an iPSC) having a non-naturally occurring alteration, and typically comprises an exogenous nucleotide sequence (e.g., a vector or an altered chromosomal sequence), encoding a BBB-transporting fusion protein described herein.
• a mammalian cell e.g., a human cell, e.g., an RPE cell, a cell derived from a cell line, (e.g., ARPE-19 or other cell line), a stem cell, a cell differentiated from an iPSC) having a non-naturally occurring alteration, and typically comprises an exogenous nucleotide sequence (e.
• the exogenous nucleotide sequence is chromosomal (e.g., the exogenous sequence is disposed in endogenous chromosomal sequence) or is extra chromosomal (e.g., a non-integrated expression vector).
• the exogenous nucleotide sequence in a genetically modified cell comprises a codon optimized coding sequence for one, two or all three of the AB, BBB or C domains that achieves higher expression of the fusion protein than a naturally-occurring coding sequence for each of these domains.
• the codon optimized sequence may be generated using a commercially available algorithm, e.g., GeneOptimizer (ThermoFisher Scientific), OptimumGeneTM (GenScript, Piscataway, NJ USA), GeneGPS® (ATUM, Newark, CA USA), or Java Codon Adaptation Tool (JCat, www.jcat.de, Grote, A. et al., Nucleic Acids Research, Vol 33, Issue suppl_2, pp. W526-W531 (2005).
• the cell is also genetically modified to reduce or eliminate expression of one or more proteins naturally expressed by the parental cell.
• a genetically modified cell e.g., modified RPE cell, a modified ARPE-19 cell
• a genetically modified cell is cultured from a population of stably -transfected cells, or from a monoclonal cell line.
• nucleotide sequence is a nucleotide sequence that does not occur naturally in a subject cell.
• exogenous polypeptide is a polypeptide that does not occur naturally in a subject cell, e.g., engineered cell.
• Reference to an amino acid position of a specific sequence means the position of said amino acid in a reference amino acid sequence, e.g., sequence of a full- length mature (after signal peptide cleavage) wild-type protein (unless otherwise stated), and does not exclude the presence of variations, e.g., deletions, insertions and/or substitutions at other positions in the reference amino acid sequence.
• “Expression vector”, as used herein, refers to a recombinant polynucleotide comprising one or more expression constructs encoding one or more proteins to be expressed. Each expression construct contains expression control sequences operatively linked to one or more nucleotide sequences to be expressed.
• An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
• the vector may comprise additional sequence elements used for the expression of and/or the integration of the expression cassette(s) into the genome of a mammalian cell.
• Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.
• the expression vectors suitable for use in engineering mammalian cells to express any of the fusion proteins described herein may also contain a nucleotide sequence encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker are genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, nourseothricin, or zeocin.
• Fabry disease “GLA deficiency”, “alpha-galactosidase A deficiency”, and “Anderson- Fabry disease”, may be used interchangeably herein, to refer to an X-linked, LSD caused by deficient activity of the enzyme alpha-galactosidase A (GLA or GALA), which leads to damaging accumulation of the glycosphingolipid globotriaosylceramide (Gb3) in various tissues and organs. More than 370 different mutations in the human GLA gene have been identified in people with Fabry disease, many of which are unique to single families. Mutations that eliminate GLA activity lead to the severe, classic form of Fabry disease, which typically begins in childhood.
• Fabry patient refers to an individual who has been diagnosed with or suspected of having Fabry disease.
• a Fabry patient may be diagnosed using any method known in the art.
• a human Fabry disease patient has a GLA gene mutation associated with deficient GLA enzymatic activity and / or Fabry disease.
• Gaucher disease refers to an autosomal recessive LSD caused by deficient activity of beta-glucocerebrosidase, which leads to intracellular accumulation of the glycolipid glucocerebroside throughout the body.
• Gaucher disease type 2 also known as acute neuronopathic Gaucher disease, occurs in newborns and infants, who typically die within the first three years of life.
• the type 3 form also known as chronic neuronopathic Gaucher disease, typically occurs during the first decade of life, with CNS complications that develop and progress slower than in Type 2 patients.
• Gaucher patient refers to an individual who has been diagnosed with or suspected of having Gaucher disease.
• a human Gaucher patient has a GBA gene mutation associated with deficient GBA enzymatic activity and/or Gaucher disease type 3.
• Heparan-alpha-glucosaminide N-acetyltransf erase protein may be used interchangeably herein to a protein that comprises the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) gene or any fragment, mutant, variant or derivative thereof that has HGSNAT enzyme activity that is within 80-120%, 85-115%, 90- 110% or 95-105% of the corresponding wild-type mammalian mature HGSNAT protein, as measured by any art-recognized HGSNAT assay.
• HGSNAT catalyzes acetylation of the terminal glucosamine residues of intralysosomal heparan or heparan sulfate, converting it into a substrate for hydrolysis by NAGLU.
• the wild-type human HGSNAT gene encodes a 663 amino acid polypeptide, which includes a predicted signal sequence that is not cleaved upon translocation into the endoplasmic reticulum (UniProtKB - Q68CP4).
• the HGSNAT amino acid sequence in a BBB-transporting fusion protein described herein consists essentially of amino acids 1-663 of the human HGSNAT sequence shown in FIG. IF.
• High molecular weight alginate or “HMW-Alg”, as used herein, means an alginate with an approximate molecular weight of 150 kDa - 250 kDa.
• Iduronate-2-sulfatase protein may be used interchangeably herein to refer to a protein that comprises the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) IDS gene or any fragment, mutant, variant or derivative thereof that has IDS enzyme activity that is within 80- 120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature IDS protein, as measured by any art-recognized IDS assay.
• IDS hydrolyzes the 2-sulfate groups of the L-iduronate 2-sulfate units of dermatan sulfate, heparan sulfate and heparan.
• the wild-type human IDS gene encodes a 550 amino acid precursor pro-polypeptide, of which the N-terminal 25 amino acids constitute a signal peptide, and the remaining amino acids constitute a pro-polypeptide that is processed into the mature polypeptide by removal of the pro-peptide of amino acids 26-33 and then cleavage into two chains formed by amino acids 34-455 and amino acids 456-550. (UniProtKB - P22304).
• the IDS amino acid sequence in a BBB- transporting fusion protein described herein comprises amino acids 34-550 of the human precursor IDS sequence shown in FIG. 1G.
• the human IDS amino acid sequence in a BBB -transporting fusion protein consists essentially of amino acids 26-550 of the sequence shown in FIG. 1G.
• Low molecular weight alginate or “LMS-Alg” as used herein, means an alginate with an approximate molecular weight of ⁇ 75 kDa.
• LSD Lysosomal Storage Disorder
• GAGs glycosaminoglycans
• CS chondroitin sulfate
• DS dermatan sulfate
• HS heparan sulfate
• KS keratan sulfate
• LSD is a mucopolysaccharidoses (MPS) or a sphingolipidoses (SP).
• Medium molecular weight alginate or “MMW-Alg” as used herein, means an alginate with an approximate molecular weight of 75 kDa to 150 kDa.
• “Mucopolysaccharidoses” and “MPS”, as used herein, refer to a condition caused by deficiency of an enzyme involved in metabolism of glycosaminoglycans that leads to accumulation of glycosaminoglycan fragments in lysosomes and can result in bone, soft tissue, and CNS manifestations, particularly in neuronopathic forms of MPS.
• the neuronal damage in neuronopathic MPS is related to the storage of undegraded heparan sulfate (HS), and secondary toxic products such as GM2 and GM3 gangliosides, inflammatory cytokines, and reactive oxygen species.
• Neuronopathic MPS includes MPS-1 Hurler (MPS-1H), MPS-2, MPS-3(A-D) and MPS- 7.
• “Mucopolysaccharidosis type I” and “MPS 1” may be used interchangeably herein to refer to an LSD caused by deficient IDUA enzymatic activity and consequent accumulation of GAGs (primarily DS and HS) within lysosomes in multiple organs and tissues.
• MPS 1 patient refers to an individual (e.g., a human) who has been diagnosed with or suspected of having MPS 1 disease, e.g., severe MPS 1 or attenuated MPS 1. The patient may be diagnosed using any method known in the art, including clinical, biochemical and genetic methods for diagnosing MPS 1.
• a human MPS 1 patient has a mutation in the IDUA gene that is associated with deficient IDUA enzymatic activity and/or MPS 1 disease.
• MPS 2 patient refers to an individual (e.g., a human) who has been diagnosed with or suspected of having MPS 2 disease, e.g., severe early-onset MPS 2 (symptoms become apparent within 2-4 years of age) or mild, late-onset MPS 2.
• the patient may be diagnosed using any method known in the art, including clinical, biochemical and genetic methods for diagnosing MPS 2.
• a human MPS 2 patient has a mutation in the IDS gene that is associated with deficient IDS activity and/or MPS 2 disease .
• “Mucopolysaccharidosis type IIIA”, “MPS 3 A” and “Sanfilippo syndrome type A” may be used interchangeably herein to refer to an autosomal recessive LSD caused by deficient SGSH activity and consequent accumulation of heparan sulfate in the CNS.
• MPS 3 A is characterized by severe CNS degeneration. More than 80 different mutations in the SGSH gene have been identified in MPS 3A patients.
• MPS 3A patient refers to an individual (e.g., a human) who has been diagnosed with or suspected of having MPS3A disease. The patient may be diagnosed using any method known in the art, including clinical, biochemical and genetic methods for diagnosing MPS 3A.
• a human MPS 3A patient has a SGSH gene mutation associated with deficient SGSH enzymatic activity and/or MPS 3 A disease.
• “Mucopolysaccharidosis type IIIB”, “MPS 3B” and “Sanfilippo syndrome type B” may be used interchangeably herein to refer to an autosomal recessive LSD caused by deficient NAGLU enzyme activity and consequent accumulation of HS in the CNS. More than 100 different mutations in the human NAGLU gene have been associated with the MPS 3B phenotype.
• MPS 3B patient refers to an individual (e.g., a human) who has been diagnosed with or suspected of having MPS 3B disease. The patient may be diagnosed using any method known in the art, including clinical, biochemical and genetic methods for diagnosing MPS 3B.
• a human MPS 3B patient has a NAGLU gene mutation associated with deficient NAGLU enzymatic activity and/or MPS 3B disease.
• “Mucopolysaccharidosis type IIIC”, “MPS 3C” and “Sanfilippo syndrome type C” may be used interchangeably herein to refer to an autosomal recessive LSD caused by deficient HGSNAT enzyme activity and consequent accumulation of heparan sulfate in the CNS.
• MPS 3C disease onset is typically before age 10 years and is characterized by progressive CNS degeneration. More than 50 different mutations in the HGSNAT gene have been identified in MPS 3C patients.
• MPS 3C patient refers to an individual (e.g., a human) who has been diagnosed with or suspected of having MPS 3C disease. The patient may be diagnosed using any method known in the art, including clinical, biochemical and genetic methods for diagnosing MPS 3C.
• a human MPS 3C patient has an HGSNAT gene mutation associated with deficient HGSNAT enzymatic activity and/or MPS 3C disease.
• “Mucopolysaccharidosis type IIID”, “MPS 3D”, and “Sanfilippo syndrome type D” may be used interchangeably herein to refer to an autosomal recessive LSD caused by deficient GNS enzyme activity and consequent accumulation of HS in the CNS.
• MPS 3D disease onset is typically between ages 2 and 6 years and is characterized by severe neurological degeneration in most patients between 6 and 10 years of age.
• MPS 3D patient refers to an individual (e.g., a human) who has been diagnosed with or suspected of having MPS 3D disease. The patient may be diagnosed using any method known in the art, including clinical, biochemical and genetic methods for diagnosing MPS-3A.
• a human MPS 3D patient has a GNS gene mutation associated with deficient GNS enzymatic activity and/or MPS 3D disease.
• “Mucopolysaccharidosis type VII”, “MPS7” and Sly syndrome” may be used interchangeably herein to refer to an autosomal recessive LSD caused by deficient GUSB enzyme activity and consequent accumulation of CS, DS and HS in the lysosome of multiple tissues. At least 49 different mutations in the GUSB gene have been identified in MPS 7 patients.
• MPS 7 patient refers to an individual (e.g., a human) who has been diagnosed with or suspected of having MPS 7 disease. The patient may be diagnosed using any method known in the art, including clinical, biochemical and genetic methods for diagnosing MPS 7.
• a human MPS 7 patient has a GUSB gene mutation associated with deficient GUSB enzymatic activity and/or MPS 7 disease.
• N-acetylgalactosamine-6-sulfatase protein Glucosamine N-acetyl-6-sulfatase protein
• GNS protein may be used interchangeably herein to a protein that comprises the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) GNS gene or any fragment, mutant, variant or derivative thereof that has GNS enzyme activity that is within 80- 120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature GNS protein, as measured by any art-recognized GNS enzymatic assay.
• GNS catalyzes the hydrolysis of the 6-sulfate groups of the N-acetyl-D-glucosamine 6-sulfate units of heparan sulfate and keratan sulfate.
• the wild-type human GNS gene encodes a 552 amino acid precursor polypeptide, of which the N-terminal 36 amino acids constitute a signal peptide (UniProtKB - P15586).
• the mature human GNS amino acid sequence in a BBB-transporting fusion protein described herein consists essentially of amino acids 37-552 of the human precursor GNS sequence shown in FIG. ID.
• N-sulfoglucosamine sulfohydrolase SGSH
• Sulfamidase heparan-N-sulfatase
• SGSH wild-type mammalian (e.g., human) SGSH gene or any fragment, mutant, variant or derivative thereof that has enzyme activity that is within 80-120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature SGSH protein, as measured by any art-recognized SGSH assay.
• the wild-type human SGSH gene encodes a 502 amino acid precursor polypeptide, of which the N-terminal 20 amino acids constitute a signal peptide (UniProtKB - P51688).
• the mature human SGSH amino acid sequence in a BBB- transporting fusion protein described herein consists essentially of amino acids 21-502 of the sequence shown in FIG. 1 J.
• “Peptide”, as used herein, is a polypeptide of less than 50 amino acids, typically, less than 25 amino acids.
• PolyA signal refers to any continuous sequence that terminates transcription of a coding sequence into RNA and directs addition of a polyA tail onto the RNA.
• Examples of polyA signals are the rabbit binding globulin (rBG) polyA signal, the SV40 late poly A signal, the SV50 polyA signal, the bovine growth hormone (BGH) poly A signal, the human growth hormone (HGH) polyA signal and synthetic polyA signals known in the art.
• Polymer composition is a composition (e.g., a solution, mixture) comprising one or more polymers.
• polymers includes homopolymers, heteropolymers, co-polymers, block polymers, block co-polymers and can be both natural and synthetic. Homopolymers contain one type of building block, or monomer, whereas co-polymers contain more than one type of monomer.
• Polypeptide is a polymer comprising amino acid residues linked through peptide bonds and having at least two, and in some embodiments, at least 10, 50, 75, 100, 150 or 200 amino acid residues.
• PD Pore disease
• Acid alpha-glucosidase deficiency and “glycogen storage disease type II” may be used interchangeably herein to refer to an autosomal recessive LSD caused by deficient GAA enzymatic activity and consequent excessive accumulation of lysosomal glycogen primarily in the heart, skeletal, smooth muscles, and the nervous system.
• PD is broadly classified into infantile PD (IPD) and late-onset PD (LOPD). More than 580 mutations in the GAA gene have been identified in PD patients.
• PD patient refers to an individual (e.g., a human) who has been diagnosed with or suspected of having PD.
• the patient may be diagnosed using any method known in the art, including clinical, biochemical and genetic methods for diagnosing PD.
• a human PD patient has a GAA gene mutation associated with deficient GAA enzymatic activity and/or PD.
• the patient has been diagnosed with LOPD.
• Prevention refers to a treatment that comprises administering or applying a BBB-transporting fusion protein described herein, e.g., administering a composition of devices encapsulating modified cells expressing the fusion protein (e.g., as described herein), prior to the onset of one or more symptoms of a CNS condition or disease to preclude the physical manifestation of the symptom(s).
• prevention require that signs or symptoms of the CNS condition / disease have not yet developed or have not yet been observed.
• treatment comprises prevention and in other embodiments it does not.
• Promoter sequence refers to a nucleotide sequence that is capable of driving expression in a mammalian cell, e.g., a human cell, e.g., an RPE cell, e.g., an ARPE-19 cell.
• the promoter sequence is from a strong mammalian promoter, e.g., a human promoter sequence.
• strong promoters for use in expression cassettes described herein include the EFl A promoter, CAG promoter, PGK (phosphoglycerate kinase) promoter and the ACTB (human beta-actin) promoter.
• a promoter sequence useful for driving expression of a ST protein described herein may be from a medium-strength promoter, e.g., the EFS promoter sequence, which is a shortened form of the EFl A promoter sequence.
• RPE cell refers to a cell having one or more of the following characteristics: a) it comprises a retinal pigment epithelial cell (RPE) (e.g., cultured using the ARPE-19 cell line (ATCC® CRL-2302TM)) or a cell derived or modified therefrom, e.g., by stably transfecting cells cultured from the ARPE-19 cell line with an exogenous sequence that encodes a BBB -transporting fusion protein), a cell derived from a primary cell culture of RPE cells, a cell isolated directly (without long term culturing, e.g., less than 5 or 10 passages or rounds of cell division since isolation) from naturally occurring RPE cells, e.g., from a human or other mammal, a cell derived from a transformed, an immortalized, or a long term (e.g., more than 5 or 10 passages or rounds of cell division) RPE cell culture; b) a cell that has been obtained from a retinal pigment epit
• an RPE described herein is genetically modified, e.g., to have a new property, e.g., the cell is modified to express and secrete a fusion protein described herein.
• the cell is also genetically modified to reduce or eliminate expression of one or more proteins naturally expressed by the parental cell.
• an RPE cell is not genetically modified.
• Sequence identity when used herein to refer to two nucleotide sequences or two amino acid sequences, means the two sequences are the same within a specified region, or have the same nucleotides or amino acids at a specified percentage of nucleotide or amino acid positions within the specified when the two sequences are compared and aligned for maximum correspondence over a comparison window or designated region. Sequence identity may be determined using standard techniques known in the art including, but not limited to, any of the algorithms described in US Patent Application Publication No. 2017/02334455 Al. In an embodiment, the specified percentage of identical nucleotide or amino acid positions is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.
• Spherical as used herein, mean a device (e.g., a hydrogel capsule or other particle) having a curved surface that forms a sphere (e.g., a completely round ball) or sphere-like shape, which may have waves and undulations, e.g., on the surface.
• Spheres and sphere-like objects can be mathematically defined by rotation of circles, ellipses, or a combination around each of the three perpendicular axes, a, b, and c. For a sphere, the three axes are the same length.
• a sphere-like shape is an ellipsoid (for its averaged surface) with semi -principal axes within 10%, or 5%, or 2.5% of each other.
• the diameter of a sphere or sphere-like shape is the average diameter, such as the average of the semi-principal axes.
• Sphingolipidoses and “SP”, as used herein, refer to a deficiency of an enzyme involved in the metabolism of sphingolipids that causes sphingolipid accumulation in lysosomes and can lead to manifestations in various organs and tissues, including in visceral and neurological systems. SP diseases include Gaucher disease and Fabry disease.
• Subject refers to a human or non-human animal.
• the subject is a human (i.e., a male or female) of any age group, e.g., a pediatric human subject (e.g., infant, child, adolescent) or adult human subject (e.g., young adult, middle-aged adult, or senior adult)).
• the subject is a non-human animal, for example, a mammal (e.g., a mouse, a dog, a primate (e.g., a cynomolgus monkey or a rhesus monkey).
• the subject is a commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog) or a bird (e.g., a commercially relevant bird such as a chicken, duck, goose, or turkey).
• the animal is a mammal.
• the animal may be a male or female and at any stage of development.
• a non-human animal may be a transgenic animal.
• Treatment,” “treat,” and “treating” as used herein refers to one or more of reducing, reversing, alleviating, delaying the onset of, or inhibiting the progress of one or more of a symptom, manifestation, or underlying cause, of a CNS condition or disease.
• treating comprises increasing the activity of a therapeutic protein in the CNS.
• treating comprises reducing, reversing, alleviating, delaying the onset of, or inhibiting the progress of a symptom associated with the condition or disease.
• “treatment,” “treat,” and “treating” require that signs or symptoms associated with the CNS condition / disease have developed or have been observed.
• treatment may be administered in the absence of signs or symptoms of disease, e.g., in preventive treatment.
• treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.
• treatment comprises prevention and in other embodiments it does not.
• Wild-type refers to the natural form, including sequence, of a polynucleotide, polypeptide or protein in a species. A wild-type form is distinguished from a mutant form of a polynucleotide, polypeptide or protein arising from genetic mutation(s).
• C 1 -C 6 alkyl is intended to encompass, C 1 , C 2 , C 3 , C 4 , c 5 , C 6 , C 1 - C 6 , C 1 -C 5 , C 1 -C 4 , C 1 -C 3 , C 1 -C 2 , C 2 -C 6 , C 2 -C 5 , C 2 -C 4 , C 2 -C 3 , C 3 -C 6 , C 3 -C 5 , C 3 -C 4 , C 4 -C 6 , c 4 -c 5 , and c 5 -C 6 alkyl.
• alkyl refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 24 carbon atoms (“C 1 -C 2 4 alkyl”).
• an alkyl group has 1 to 12 carbon atoms (“C 1 -C 1 2 alkyl”), 1 to 10 carbon atoms (“C 1 -C 1 2 alkyl”), 1 to 8 carbon atoms (“C 1 -C 8 alkyl”), 1 to 6 carbon atoms (“C 1 -C 6 alkyl”), 1 to 5 carbon atoms (“C 1 -c 5 alkyl”), 1 to 4 carbon atoms (“C 1 -C 4 alkyl”), 1 to 3 carbon atoms (“C 1 -C 3 alkyl”), 1 to 2 carbon atoms (“C 1 -C 2 alkyl”), or 1 carbon atom (“C 1 alkyl”).
• an alkyl group has 2 to 6 carbon atoms (“C 2 -C 6 alkyl”).
• C 1 -C 6 alkyl groups include methyl (C 1 ), ethyl (C 2 ), n-propyl (C 3 ), isopropyl (C 3 ), n-butyl (C 4 ), tert-butyl (C 4 ), sec-butyl (C 4 ), iso-butyl (C 4 ), n-pentyl (c 5 ), 3-pentanyl (c 5 ), amyl (c 5 ), neopentyl (c 5 ), 3-methyl-2-butanyl (c 5 ), tertiary amyl (c 5 ), and n-hexyl (C 6 ).
• alkyl groups include n-heptyl (C 7 ), n-octyl (C 8 ) and the like.
• Each instance of an alkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents, e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
• alkenyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 24 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds (“C 2 -C 2 4 alkenyl”).
• an alkenyl group has 2 to 10 carbon atoms (“C 2 - C 1 0 alkenyl”), 2 to 8 carbon atoms (“C 2 -C8 alkenyl”), 2 to 6 carbon atoms (“C 2 -C 6 alkenyl”), 2 to 5 carbon atoms (“C 2 -c 5 alkenyl”), 2 to 4 carbon atoms (“C 2 -C 4 alkenyl”), 2 to 3 carbon atoms (“C 2 -C 3 alkenyl”), or 2 carbon atoms (“C 2 alkenyl”).
• the one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl).
• Examples of C 2 -C 4 alkenyl groups include ethenyl (C 2 ), 1-propenyl (C 3 ), 2-propenyl (C 3 ), 1-butenyl (C 4 ), 2-butenyl (C 4 ), butadienyl (C 4 ), and the like.
• Examples of C 2 -C 6 alkenyl groups include the aforementioned C 2 -4 alkenyl groups as well as pentenyl (c 5 ), pentadienyl (c 5 ), hexenyl (C 6 ), and the like.
• Each instance of an alkenyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
• alkynyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 24 carbon atoms, one or more carbon-carbon triple bonds (“C 2 -C 2 4 alkenyl”).
• an alkynyl group has 2 to 10 carbon atoms (“C 2 -C 1 0 alkynyl”), 2 to 8 carbon atoms (“C 2 -C8 alkynyl”), 2 to 6 carbon atoms (“C 2 -C 6 alkynyl”), 2 to 5 carbon atoms (“C 2 -c 5 alkynyl”), 2 to 4 carbon atoms (“C 2 -C 4 alkynyl”), 2 to 3 carbon atoms (“C 2 - C 3 alkynyl”), or 2 carbon atoms (“C 2 alkynyl”).
• the one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl).
• C 2 -C 4 alkynyl groups include ethynyl (C 2 ), 1-propynyl (C 3 ), 2-propynyl (C 3 ), 1-butynyl (C 4 ), 2-butynyl (C 4 ), and the like.
• Each instance of an alkynyl group may be independently optionally substituted, /. ⁇ ?., unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
• heteroalkyl refers to a non-cyclic stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized.
• the heteroatom(s) O, N, P, S, and Si may be placed at any position of the heteroalkyl group.
• heteroalkyl Up to two or three heteroatoms may be consecutive, such as, for example, -CH 2 -NH-OCH3 and -CH 2 -O- Si(CH3)3.
• heteroalkyl is recited, followed by recitations of specific heteroalkyl groups, such as -CH 2 O, -NR C R D , or the like, it will be understood that the terms heteroalkyl and -CH 2 O or -NR C R D are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term “heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as -CH 2 O, -NR C R D , or the like.
• Each instance of a heteroalkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted heteroalkyl”) or substituted (a “substituted heteroalkyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
• alkylene alkenylene, alkynylene, or heteroalkylene, alone or as part of another substituent, mean, unless otherwise stated, a divalent radical derived from an alkyl, alkenyl, alkynyl, or heteroalkyl, respectively.
• alkylene, alkenylene, alkynylene, or heteroalkylene group may be described as, e.g., a C 1 -C 6 -membered alkylene, C 2 -C 6 -membered alkenylene, C 2 -C 6 -membered alkynylene, or C 1 -C 6 -membered heteroalkylene, wherein the term “membered” refers to the non-hydrogen atoms within the moiety.
• heteroatoms can also occupy either or both chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like).
• aryl refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 it electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C 6 - C 1 4 aryl”).
• an aryl group has six ring carbon atoms (“C 6 aryl”; e.g., phenyl).
• an aryl group has ten ring carbon atoms (“C 1 o aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C 1 4 aryl”; e.g., anthracyl).
• An aryl group may be described as, e.g., a C 6 -C 1 o-membered aryl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety.
• Aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Each instance of an aryl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents.
• heteroaryl refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g, having 6 or 10 it electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-10 membered heteroaryl”).
• heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits.
• Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings.
• Heteroaryl also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl/heteroaryl) ring system.
• Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom e.g, indolyl, quinolinyl, carbazolyl, and the like
• the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5- indolyl).
• a heteroaryl group may be described as, e.g., a 6-10-membered heteroaryl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety.
• a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”).
• a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”).
• a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”).
• the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
• the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
• the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.
• Each instance of a heteroaryl group may be independently optionally substituted, /. ⁇ ?., unsubstituted (an “unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more substituents.
• Exemplary 5-membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl.
• Exemplary 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl.
• Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl.
• Exemplary 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl.
• Exemplary 6- membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl.
• Exemplary 6-membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl.
• Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively.
• Exemplary 7-membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl.
• Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotri azolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadi azolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl.
• Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.
• Other exemplary heteroaryl groups include heme and heme derivatives.
• arylene and “heteroarylene,” alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively.
• cycloalkyl refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C 3 -C 1 0 cycloalkyl”) and zero heteroatoms in the non- aromatic ring system.
• a cycloalkyl group has 3 to 8 ring carbon atoms (“C 3 - C 8 cycloalkyl”), 3 to 6 ring carbon atoms (“C 3 -C 6 cycloalkyl”), or 5 to 10 ring carbon atoms (“c 5 - C 1 0 cycloalkyl”).
• a cycloalkyl group may be described as, e.g., a C 4 -C?-membered cycloalkyl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety.
• Exemplary C 3 -C 6 cycloalkyl groups include, without limitation, cyclopropyl (C 3 ), cyclopropenyl (C 3 ), cyclobutyl (C 4 ), cyclobutenyl (C 4 ), cyclopentyl (c 5 ), cyclopentenyl (c 5 ), cyclohexyl (C 6 ), cyclohexenyl (C 6 ), cyclohexadienyl (C 6 ), and the like.
• Exemplary C 3 -C 8 cycloalkyl groups include, without limitation, the aforementioned C 3 -C 6 cycloalkyl groups as well as cycloheptyl (C 7 ), cycloheptenyl (C 7 ), cycloheptadienyl (C 7 ), cycloheptatrienyl (C 7 ), cyclooctyl (C 8 ), cyclooctenyl (C 8 ), cubanyl (C 8 ), bicyclo[l.
• C 3 -C 1 0 cycloalkyl groups include, without limitation, the aforementioned C 3 -C 8 cycloalkyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C 1 0), cyclodecenyl (C 1 0), octahydro- I //-in deny!
• the cycloalkyl group is either monocyclic (“monocyclic cycloalkyl”) or contain a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic cycloalkyl”) and can be saturated or can be partially unsaturated.
• Cycloalkyl also includes ring systems wherein the cycloalkyl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is on the cycloalkyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the cycloalkyl ring system.
• Each instance of a cycloalkyl group may be independently optionally substituted, /. ⁇ ?., unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents.
• Heterocyclyl refers to a radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-10 membered heterocyclyl”).
• the point of attachment can be a carbon or nitrogen atom, as valency permits.
• a heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”), and can be saturated or can be partially unsaturated.
• Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings.
• Heterocyclyl also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more cycloalkyl groups wherein the point of attachment is either on the cycloalkyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system.
• a heterocyclyl group may be described as, e.g., a 3-7-membered heterocyclyl, wherein the term “membered” refers to the non-hydrogen ring atoms, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon, within the moiety.
• Each instance of heterocyclyl may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents.
• the heterocyclyl group is unsubstituted 3-10 membered heterocyclyl.
• the heterocyclyl group is substituted 3-10 membered heterocyclyl.
• a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“5-10 membered heterocyclyl”).
• a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”).
• a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”).
• the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
• the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
• the 5-6 membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen, and sulfur.
• Exemplary 3-membered heterocyclyl groups containing one heteroatom include, without limitation, azirdinyl, oxiranyl, thiorenyl.
• Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl.
• Exemplary 5- membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2, 5-dione.
• Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one.
• Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl.
• Exemplary 6-membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl, piperazinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl.
• Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, dioxanyl.
• Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, triazinanyl or thiomorpholinyl-l,l-dioxide.
• Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl.
• Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl.
• Exemplary 5-membered heterocyclyl groups fused to a G> aryl ring include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like.
• Exemplary 6-membered heterocyclyl groups fused to an aryl ring include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.
• Amino refers to the radical -NR 70 R 71 , wherein R 70 and R 71 are each independently hydrogen, C 1 -C 8 alkyl, C 3 -C 1 0 cycloalkyl, C 4 -C 1 0 heterocyclyl, C 6 -C 1 o aryl, and c 5 -C 1 0 heteroaryl. In some embodiments, amino refers to NH2.
• cyano refers to the radical -CN.
• halo or “halogen,” independently or as part of another substituent, mean, unless otherwise stated, a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom.
• hydroxy refers to the radical -OH.
• Alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are optionally substituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” heteroalkyl, “substituted” or “unsubstituted” cycloalkyl, “substituted” or “unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl group).
• substituted means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.
• a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position.
• substituted is contemplated to include substitution with all permissible substituents of organic compounds, such as any of the substituents described herein that result in the formation of a stable compound.
• the present disclosure contemplates any and all such combinations to arrive at a stable compound.
• heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.
• Two or more substituents may optionally be joined to form aryl, heteroaryl, cycloalkyl, or heterocyclyl groups.
• Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic base structure.
• the ring-forming substituents are attached to adjacent members of the base structure.
• two ring-forming substituents attached to adjacent members of a cyclic base structure create a fused ring structure.
• the ring-forming substituents are attached to a single member of the base structure.
• two ring-forming substituents attached to a single member of a cyclic base structure create a spirocyclic structure.
• the ring-forming substituents are attached to non-adjacent members of the base structure.
• Compounds of Formula (III) and pharmaceutically acceptable salts thereof described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers.
• the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer.
• Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high-pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses.
• HPLC high-pressure liquid chromatography
• a pure enantiomeric compound is substantially free from other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess).
• an “S” form of the compound is substantially free from the “R” form of the compound and is, thus, in enantiomeric excess of the “R” form.
• enantiomerically pure or “pure enantiomer” denotes that the compound comprises more than 75% by weight, more than 80% by weight, more than 85% by weight, more than 90% by weight, more than 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 99% by weight, more than 99.5% by weight, or more than 99.9% by weight, of the enantiomer.
• the weights are based upon total weight of all enantiomers or stereoisomers of the compound.
• H may be in any isotopic form, including 'H, 2 H (D or deuterium), and 3 H (T or tritium); C may be in any isotopic form, including 12 C, 13 C, and 14 C; O may be in any isotopic form, including 16 O and 18 O; and the like.
• pharmaceutically acceptable salt is meant to include salts of the active compounds that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein.
• base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent.
• pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt.
• acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
• Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like.
• inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
• salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galacturonic acids and the like (see, e.g., Berge et al, Journal of Pharmaceutical Science 66: 1-19 (1977)).
• Certain specific compounds used in the devices of the present disclosure e.g., a particle, a hydrogel capsule
• These salts may be prepared by methods known to those skilled in the art.
• Other pharmaceutically acceptable carriers known to those of skill in the art are suitable for use in the present disclosure.
• Devices of the present disclosure may contain a compound of Formula (III) in a prodrug form.
• Prodrugs are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds useful for preparing devices in the present disclosure. Additionally, prodrugs can be converted to useful compounds of Formula (III) by chemical or biochemical methods in an ex vivo environment.
• Certain compounds of Formula (III) described herein can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure. Certain compounds of Formula (III) described herein may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present disclosure and are intended to be within the scope of the present disclosure.
• solvate refers to forms of the compound that are associated with a solvent, usually by a solvolysis reaction. This physical association may include hydrogen bonding.
• Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like.
• the compounds described herein may be prepared, e.g, in crystalline form, and may be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include both stoichiometric solvates and non-stoichiometric solvates.
• hydrate refers to a compound which is associated with water.
• the number of the water molecules contained in a hydrate of a compound is in a definite ratio to the number of the compound molecules in the hydrate. Therefore, a hydrate of a compound may be represented, for example, by the general formula R x H2O, wherein R is the compound and wherein x is a number greater than 0.
• tautomer refers to compounds that are interchangeable forms of a compound structure, and that vary in the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium through the movement of it electrons and an atom (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Tautomeric forms may be relevant to the attainment of the optimal chemical reactivity and biological activity of a compound of interest.
• connection refers to a connection to an entity, e.g., a polymer (e.g., hydrogel -forming polymer such as alginate) or surface of an implantable device, e.g., a particle, a hydrogel capsule.
• entity e.g., a polymer (e.g., hydrogel -forming polymer such as alginate) or surface of an implantable device, e.g., a particle, a hydrogel capsule.
• the connection represented by “ ” may refer to direct attachment to the entity, e.g., a polymer or an implantable element, may refer to linkage to the entity through an attachment group.
• An “attachment group,” as described herein, refers to a moiety for linkage of a compound of Formula (III) to an entity (e.g., a polymer or an implantable element (e.g., a device) as described herein), and may comprise any attachment chemistry known in the art.
• an attachment group comprises an amine, ketone, ester, amide, alkyl. In some embodiments, an attachment group is a cross-linker. In some embodiments, the ⁇ attachment group is -C(O)(C 1 -C 6 1 , and R 1 is as described herein ⁇ . In some embodiments, the attachment group is -C(O)(C 1 -C 6 -alkylene)-, wherein alkylene is substituted with 1-2 alkyl groups (e.g., 1-2 methyl groups). In some embodiments, the attachment group is -C(O)C(CH3)2-.
• the attachment group is -C(O)(methylene)-, wherein alkylene is substituted with 1- 2 alkyl groups (e.g., 1-2 methyl groups).
• the attachment group is - C(0)CH(CH3)-.
• the attachment group is -C(O)C(CH3)-.
• BBB -transporting fusion proteins of the disclosure comprise an HSA binding domain (AB) and an IGF1R binding domain (RB) located upstream of a cargo moiety (C) (e.g., amino acid sequence of a therapeutic protein).
• a first linker moiety e.g., a linker peptide
• a second linker moiety e.g., a linker peptide
• HSA Mature human serum albumin
• albumin The long half-life of albumin in blood is mainly driven by two characteristics: (i) the large size (65 kDa) of albumin limits its glomerular filtration and (ii) albumin binds to FcRn at low pH (pH 6), which protects albumin from degradation in the lysosomes after passive endocytosis in endothelial and epithelial cells, by recycling from early endosome back to the extracellular environment.
• the AB domain confers upon the BBB-transporting fusion protein a serum half-life in man (expressed as tl/2-beta) that is more than any of 6 hours, 12 hours, 24 hours, 72 hours, one week, two weeks or up to the half-life of HSA in man (estimated to be around 19 days).
• the AB domain is specific for serum albumin, i.e., it does not substantially bind to any non-albumin proteins.
• the fusion protein does not substantially inhibit binding of FcRn to HSA, the binding site of which is in Dili. In an embodiment, less than about 20%, 15%, 10%, 5% or 1% inhibition of FcRn binding to HSA occurs in the presence of a fusion protein of the disclosure, which may be determined by any competition binding assay known in the art, e.g., the SPR competition binding assay described in WO 2019/204925 at [0095] or in Example 3 of US 2019/0367596A1.
• the potential for interference with FcRn binding can be reduced by deriving the AB domain from an anti-HSA antibody that does not binds to DHL
• the fusion protein binds via the A 13 domain to DI of HSA.
• the fusion protein cross-competes via AB for HSA binding with a sdAb that consists of the amino acid sequence for a single domain antibody (sdAb) described in WO20 19/204925, i.e., Rl l, R28, M75 or XI 79.
• AB (as part of the fusion protein) also binds to (e.g., cross-reacts with) serum albumin from one, two, three, four or more other mammalian species, e.g., any combination of two, three, four or more of mouse, rat, guinea pig, hamster, rabbit, cat, dog, pig, sheep, horse, cow and monkey (e.g., rhesus and / or cynomolgus).
• the fusion protein binds via the AB domain to serum albumin from at least mouse, rat, monkey (rhesus or cynomolgus) and human.
• the fusion protein binds via the AB domain to serum albumin from at least mouse, dog and human.
• the AB domain (as part of the fusion protein) binds to HSA, and optionally to at least one other mammalian serum albumin, with a desired affinity within a pH range of between about 5.0 or about 5.5 up to about 7.4.
• the desired affinity is a dissociation constant (KD) ranging from any of about 0.1 nM to about 1,000 nM, about 0.5 nM to about 500 nM, about 1 nM to about 250 nM, about 5 nM to about 50 nM, about 10 nM to about 25 nM, or about 0.5 nM to about 1 nM.
• KD dissociation constant
• the affinity is determined by surface plasmon resonance (SPR) at 25° C and a pH range of about 5.5 to about 7.4.
• the affinity of the fusion protein for serum albumin from mouse, rat and monkey is similar to the affinity for HSA, e.g., within 70% to 130%, 80% to 120%, or 90% to 110% of the KD for HSA.
• the AB domain of a BBB-transporting fusion protein of the disclosure comprises the amino acid sequence of a heavy chain variable region (HCVR) of an anti-HSA antibody.
• the AB domain has a molecular weight of less than about any of 75 kDa, 50 kDa or 25 kDa.
• the AB domain may be derived from any anti-HSA antibody molecule known in the art, including conventional 4-chain antibodies, antigen-binding fragments, Fab, Fab’, F(ab’)z, Fv (double-chain and single-chain (scFv)), minibody, diabody and sdAb.
• AB consists essentially of, or consists of, the HCVR amino acid sequence of a sdAb.
• the AB domain comprises the set of three CDR amino acid sequences in one of the anti-HSA sdAbs described in Table I of WO 2006/122787.
• AB consists essentially of, or consists of, the amino acid sequence in one of the anti-HSA sdAbs described in Table II of WO 2006/22787 (e.g., Alb-1), or in one of the humanized variants of Alb- 1 described in Table III of WO 2006/22787 (e.g., Alb-8).
• AB consists essentially of, or consists of, any of the Alb-23 sequences described in WO 2012/175400 (e.g., Alb-23D).
• a BBB-transporting fusion protein of the present disclosure crosscompetes for binding to HSA with any of the anti-HSA sdAbs described in WO 2006/22787 (e.g., Alb-8) or in WO 2012/175400 (e.g,, Alb-23D).
• the BBB-transporting fusion protein does not comprise the amino acid sequence for Alb-1.
• the BBB- transporting fusion protein does not comprise the amino acid sequence for Alb-8.
• the AB domain consists essentially of, or consists of, the amino acid sequence of an albumin binding domain (ABD) described in Table 7 of WO 2019/246003, e g., LAEAI ⁇ VLANRELDI ⁇ YGVSDYYI ⁇ NLINNAI ⁇ TVEGVI ⁇ ALIDEILAALP (SEQ ID NO:40), which is described in WO 2019/246003 as an ABD having a KD to HSA of about 1.2 nM.
• the AB domain comprises the three CDRs of the anti-HSA P367 antibody described in Table 14 of WO 2019/2460003.
• AB consists essentially of, or consists of, the amino acid sequence of the anti-HSA P367 antibody or its humanized variant P494, each of which is listed in Table 14 of WO 2019/2460003.
• a BBB-transporting fusion protein of the present disclosure cross-competes for binding to HSA with the P494 sdAb described in WO 2019/2460003.
• the AB domain comprises a set of three heavy chain CDR amino acid sequences in one of the anti-HSA sdAbs described in Table 5 of WO 2021/119551.
• AB consists essentially of, or consists of, the VH amino acid sequence in one of the anti-HSA sdAbs described in Table 5 of WO 2021/119551.
• a BBB-transporting fusion protein of the present disclosure cross-competes for binding to HSA with one or more of the sdAbs described in Table 5 of WO 2021/119551.
• the AB domain comprises a set of the three CDR amino acid sequences that are in the T0235002C06 sdAb described in Table B of US 20190367597A1.
• AB consists essentially of, or consists of, the amino acid sequence of T0235002C06 described in Table B of US 2019/0367597A1.
• a BBB -transporting fusion protein of the present disclosure cross-competes for binding to HSA with the T0235002C06 sdAb described in Table B of US 20190367597A1.
• the AB domain comprises a set of the three CDR amino acid sequences that are in the T0235005D04 sdAb described in Table B of US 20190367596A1.
• AB consists essentially of, or consists of, the amino acid sequence of T0235005D04 described in Table B of US 2019/0367596A1.
• a BBB-transporting fusion protein of the present disclosure cross-competes for binding to HSA with the T0235002D04 sdAb described in Table B of US 20190367596A1.
• the AB domain comprises a set of the three CDR amino acid sequences that are in the T0235005G01 or T023500043 sdAbs described in Table B of US 20190367598A1.
• AB consists essentially of, or consists of, the amino acid sequence of T0235005G01 or T023500043 described in Table B of US 20190367598A1.
• a BBB-transporting fusion protein of the present disclosure cross-competes for binding to HSA with the T0235005G01 or T023500043 sdAbs described in Table B of US 20190367598A1.
• the AB domain comprises a set of the three CDR amino acid sequences that are in the R28, R11, M75 or M79 sdAbs described in WO 2019/204925. These CDR sequences are set forth in Table 2A below.
• Table 2 A Exemplary CDR amino acid sequences for the AB domain.
• AB consists essentially of, or consists of, the amino acid sequence of the R28, R11, M75 or M79 sdAbs described in WO 2019/204925. In an embodiment, AB consists essentially of, or consists of, the amino acid sequence of one of the humanized variants of R28, Rl l, M75 or M79 described in WO 2019/204925. The amino acid sequences of the parental and humanized variants of R28, Rl l, M75 and M79 are shown in the SEQUENCES Table on pages 46-48 of WO 2019/204925. In an embodiment, AB consists essentially of, or consists of, an amino acid sequence selected from the parental and humanized sequences shown in Table 2B herein below. In an embodiment, AB consists essentially of, or consists of, the parental or humanized amino acid sequence of R28 shown in Table 2B below. Table 2B: Exemplary amino acid sequences for the AB domain.
• the RB domain of the BBB -transporting fusion proteins of the disclosure confers upon the fusion protein the capability of crossing the BBB via transcytosis mediated by the IGF1R, also known as CD221, IGFIR, IGFR, and JTK13.
• IGF1R also known as CD221, IGFIR, IGFR, and JTK13.
• Human IGF1R is synthesized as a monomeric 1367- amino acid pre-proreceptor with a 30-amino acid signal sequence (UniProtKB - P08069).
• the proreceptor is glycosylated, dimerized, and transported to the Golgi apparatus, where furin cleavage yields alpha and beta subunits that form a disulfide-linked tetramer (beta-alpha-alpha-beta), which is transported to the plasma membrane.
• the fully mature cell membrane-bound IGFIR consists of two 130- to 135-kDa alpha subunits and two 90- to 95-kDa beta subunits, with several alpha-alpha and alpha-beta disulfide bonds.
• the alpha subunits are entirely extracellular and form the ligand-binding domain.
• the beta subunits contain an extracellular domain, a transmembrane domain and an intracellular domain. Ligand binding induces transautophosphorylation and phosphorylation of a wide variety of downstream signaling molecules.
• RB domain to confer hIGFIR mediated transcytosis of a fusion protein described herein may be assessed by any method known in the art.
• in vitro assays may evaluate internalization of a fusion protein into IGF1-R expressing cells such as MCF-7 cells or primary human microvascular brain endothelial cells (HMBEC) as described in [0317]-[0315] of EP3725806A1 .
• HMBEC primary human microvascular brain endothelial cells
• a similar assay that may be used when RB cross-reacts with rat IGF1-R employs immortalized rat brain endothelial cells (svARBEC) as described in US 10,100,117.
• svARBEC immortalized rat brain endothelial cells
• known in vitro and in vivo BBB models may be used to evaluate BBB-tran sporting ability ef fusion proteins that bind to human and rat IGFIR, e.g., as described in Examples 10 and 12
• the RB domain is specific for IGF1R, i.e., it does not substantially bind to the insulin receptor or any other non-IGFIR proteins.
• the fusion protein containing RB should not induce signaling through IGFIR or IR, and also should not inhibit signaling through IGFIR or IR that is induced by insulin, IGF-1 or IGF-2.
• the signaling impact of a BBB- transporting fusion protein described herein can be assessed by analyzing phosphorylation of the IGFIR and IR and / or of the receptor-stimulated downstream kinase Akt.
• This assessment may be performed using any method known in the art, e.g., as described in Example 14 of US 10,100,117 or in paragraphs [0325]-[0327] of EP3725806A1.
• Any impact of the fusion protein on IGF1 signaling through the IGFIR may be assessed using any method known in the art, e.g., by an MCF-7 cell line proliferation assay as described in [0320]-[0324] of EP3725806A1.
• the potential for interference with ligand binding can be reduced by deriving the RB domain from an anti-hlGFIR antibody molecule that is known not to interfere with ligand binding or selecting an antibody that does not binds to the alpha subunit.
• the fusion protein cross-competes via the RB domain for hIGFIR binding with the IGFIR- 5 sdAb described in US 10,100,117, the IGF1R-3 sdAb described in US 10,106,614 or the IGFIR- 5 sdAb described in US 10, 112,998.
• the fusion protein cross-competes via the RB domain for hIGFIR binding with any of the 996, 1226 and 1564 antibodies described in EP3725806A1.
• the epitope for the RB domain has three binding sites: binding site 1 includes one or more of R650, Y775, P776, F778, E779, S791, and L798; binding site 2 includes one or more of L641, H808, E809 and L813; and binding site 3 includes one or more ofV397, W434, D435, Y460 and C 4 88.
• RB (as part of the fusion protein) also binds to (e.g., cross-reacts with) IGF1R from one, two, three, four or more other mammalian species, e.g., any combination of two, three, four or more of mouse, rat, guinea pig, hamster, rabbit, cat, dog, pig, sheep, horse, cow and monkey (e.g., rhesus and / or cynomolgus).
• the fusion protein binds via the RB domain to IGF 1R from at least mouse, rat, monkey (rhesus or cynomolgus) and human.
• the fusion protein binds via the RB domain to IGF1R from at least mouse, dog and human.
• the RB domain (as part of the fusion protein) binds to hIGFIR, and optionally to at least one other mammalian IGF1R, with a desired affinity within a pH range of between about 5.0 or about 5.5 up to about 7.4.
• the desired affinity is a dissociation constant (KD) ranging from: (i) about 0.1 nM to about 1,000 nM; (ii) about 0.2 nM to about any one of 500 nM, 250 nM, 100 nM, 50 nM , 25 nM or 10 nM; (iii) about 0.5 nM to about any one of 250 nM, 100 nM, 50 nM, 25 nM, 10 nM or 5 nM; or (iv) about 1 nM to about any one of 100 nM, 50 nM, 25 nM, 10 nM or 5 nM.
• KD dissociation constant
• the desired affinity is a dissociation constant (KD) of 1 nM to 10 nM.
• the affinity is determined by surface plasmon resonance (SPR) at 25° C and a pH range of about 5.5 to about 7.4.
• the affinity of the fusion protein for IGF1R from mouse, rat and monkey is similar to the affinity for hIGFIR, e.g., a KD within 70% to 130%, 80% to 120%, or 90% to 110% of the K D for hIGFIR.
• An exemplary SPR assay for measuring KD of a BBB-containing fusion protein is described in US 10,100,117.
• the RB domain of a BBB-transporting fusion protein of the disclosure comprises the amino acid sequence of a heavy chain variable region (HCVR) of an anti-hlGFIR antibody.
• the RB domain has a molecular weight of less than about any of 75 kDa, 50 kDa or 25 kDa.
• the RB domain may be derived from any anti-IGFIR antibody molecule known in the art, including conventional 4-chain antibodies, antigen-binding fragments, Fab, Fab’, F(ab’)?, Fv (double-chain and single-chain (scFv)), minibody, diabody and sdAb.
• RB consists essentially of, or consists of, the HCVR amino acid sequence of a sdAb.
• the RB domain comprises a set of the three CDR amino acid sequences in a sdAb selected from the group consisting of: the IGF1R-5 sdAb described in US 10,100,117, the IGFlR-3 sdAb described in US 10,106,614 and the IGF 1R-5 sdAb described in US 10,112,998.
• a sdAb selected from the group consisting of: the IGF1R-5 sdAb described in US 10,100,117, the IGFlR-3 sdAb described in US 10,106,614 and the IGF 1R-5 sdAb described in US 10,112,998.
• RB consists essentially of, or consists of, the parental amino acid sequence of IGF1R-5, IGF1R-3 or IGF1R-4, or a humanized variant of one of these sdAbs. In an embodiment, RB consists essentially of, or consists of a sequence selected from the group consisting of:
• X9 is A or S, Xiois A or T
• Xu is A or S
• X12 is G or N
• Xu is M or L
• X>.4 is N or R
• X15 is E or R
• Xi6 is P or A
• Xj? is S or Y
• Xis is Q or L (SEQ ID NO:64);
• Xiois F or W Xu is G or S; X>.?.is V or Y, Xu is D or G, Xu is N or S; Xis is A or S; Xu is L or V; Xu is K or R; Xis is A or S; and X !9 is L or Q (SEQ ID NO: 66).
• RB consists essentially of, or consists of, the amino acid sequence of any of the humanized variants of IGF1R-5, IGF1R-3 or IGF1R-5 described in US 10,100,117, US 10,106,614 and US 10,112,998, respectively.
• RB consists essentially of, or consists of, an amino acid sequence selected from the parental or humanized sequences shown in Table 3B herein below.
• RB consists essentially of, or consists of, the parental or humanized amino acid sequence of IGF1R-5 shown in Table 3B below.
• Table 3B Exemplary amino acid sequences for the RB domain. Generation ofHSA and hIGFIR Binding Domains
• the AB and RB domains may be derived from any antibody or antigen binding fragment thereof that has the desired properties described herein.
• the antibody or antigen binding fragment may be already known in the art or identified by via any approach known in the art.
• one or both of the AB and RB domains are derived from a singledomain Ab.
• sdAbs of camelid origin lack light chains and thus their antigen binding sites consist of one domain, termed VHH.
• sdAb have also been observed in shark and are termed VNAR .
• Other sdAb may be engineered based on human Ig heavy and light chain sequences.
• sdAb includes sdAbs directly isolated from VH, VHH, VL, or VNAR reservoir of any origin through phage display or other technologies, recombinantly produced sdAbs, as well as those sdAb generated through further modification of such sdAbs by humanization, affinity maturation, stabilization, solubilization, or other methods of antibody engineering. Also encompassed by the present disclosure are homologues, derivatives, or fragments that retain the antigen-binding function and specificity of the parental sdAb.
• the skilled person may employ any approach known in the art, e.g., substantially as described in WO 2019/204925 and US 10,100,117, respectively, to generate and screen a phage-displayed VHH library from the heavy-chain-only antibody repertoire of llama or other camelid immunized with a desired antigen.
• the IGF1R antigen used to immunize the camelid is a fragment of precursor hIGFIR, e.g., comprises amino acids 1 to 932 of the sequence shown in Figure 1.
• the antigenic hIGFIR fragment does not contain the signal peptide.
• AB or RB domains derived from sdAbs may include the parental framework regions; alternatively, the parental CDRs may be grafted onto VNAR, VHH, VH or VL framework regions of other sdAbs or onto the framework regions of other types of antibody fragments or antibody -like molecules (Fv, scFv, Fab) of any source (e.g., human) or proteins of similar size and nature onto which CDRs can be grafted (for example, see Nicaise, M. et al, Protein Sci 13: 1882-91 2004).
• Fv, scFv, Fab antibody fragments or antibody -like molecules
• the amino acid sequence in one or both of the AB and RB domains is a humanized version (humanized variant) of the parental variable region.
• Humanization of an antibody or antibody fragment comprises replacing an amino acid in the sequence with its human counterpart, as found in the human consensus sequence, without loss of antigen-binding ability or specificity; this approach reduces immunogenicity of the antibody or fragment thereof when introduced into human subjects.
• the parental sequence may be humanized using any suitable method known in the art, for example, but not limited to CDR grafting and veneering.
• one or more than one of the CDR defined herein may be fused or grafted to a human variable region (VH, or VL), to another human antibody (IgA, IgD, IgE, IgG, and IgM), to antibody fragment framework regions (Fv, scFv, Fab), or to proteins of similar size and nature onto which CDR can be grafted.
• VH human variable region
• IgA human antibody
• IgD IgD
• IgE IgE
• IgG IgG
• IgM antibody fragment framework regions
• Fv, scFv, Fab antibody fragment framework regions
• Veneering also referred to in the art as “variable region resurfacing”, involves humanizing solvent-exposed positions of the antibody or antibody fragment; thus, buried non-humanized residues, which may be important for CDR conformation, are preserved while the potential for immunological reaction against solvent-exposed regions is minimized. Veneering is known in the art and is described in at least the following: U.S. Pat. Nos. 5,869,619, 5,766,886, and 5,821,123, and European Patent No. 519596.
• the BBB -transporting fusion protein may contain one or more linkers, e.g., between the AB and RB domains and / or between the RB domain and the cargo moiety (e.g., therapeutic polypeptide).
• Each linker should be of sufficient length to allow the linked polypeptides to individually fold into 3-dimensional structures having the desired functional activity, e.g,, binding or therapeutic. Also, each linker should not be cleavable by any proteases or other enzymes present in the serum.
• the linker is a peptide linker comprising at least two, three or four amino acids and less than about 30 amino acids, e.g., less than about any of 25, 20, 15 or 10 amino acids.
• Peptide linkers are known in the art and nonlimiting examples are described herein.
• the peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence.
• a sequence derived from the hinge region of heavy chain only antibodies may be used as the linker. See, for example, WO 1996/34103.
• Suitable linker peptides typically include G and/or S residues in various formats, with exemplary linkers including GGGG (SEQ ID NO:71), TGGGG (SEQ ID NO:72), GGSSGGSGSSSGSGGSGSSG (SEQ ID NO:73), (GGSS)n (SEQ ID NO:74), (GGGGS)n (SEQ ID NO: 13), (SGGGG)n (SEQ ID NO:75) and GGGG(SGGGG)n (SEQ ID NO:76), wherein "n” in each case is generally a number between 1 and 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10, provided that the maximum length of about 40 amino acids is not exceeded.
• Another exemplary peptide linker is SKPTCPPPELLGGPSVFIFPPK (SEQ ID NO:77).
• the fusion protein comprises two peptide linkers LI and L2, with peptide linker LI located between AB and RB and L2 located between RB and the cargo polypeptide.
• the length of each of LI and L2 is between about 15 and 30 amino acids, or between about 20 and 25 amino acids.
• LI and L2 may have the same or different amino acid sequences.
• each LI and L2 independently consists essentially of, or consists of: (GGGGS)4 (SEQ ID NO: 14) or (GGGGS)s (SEQ ID NO:78).
• each of LI and L2 consists essentially of, or consists of: (GGGGSfi.
• the cargo moiety may be any therapeutic or diagnostic molecule that may be joined or linked to the hIGFIR binding domain.
• the therapeutic molecule is a polypeptide, e.g., a cytokine, an enzyme, a growth factor, or an antibody or antigen binding fragment thereof.
• the polypeptide has activity useful for treating a neurological disorder in a mammal, e.g., a human.
• the neurological disorder is selected from the group consisting of: LSDs with neurological manifestations (nLSDs), Alzheimer’s disease (AD), ataxias (e.g., hereditary ataxias such as Friedreich’s ataxia), Huntington’s disease, stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman’s syndrome Liddle syndrome, Parkinson’s disease.
• LSDs with neurological manifestations nLSDs
• AD Alzheimer’s disease
• ataxias e.g., hereditary ataxias such as Friedreich’s ataxia
• Huntington’s disease e.g., hereditary ataxias such as Friedreich’s ataxia
• Huntington’s disease e.g., hereditary ataxias such as Friedreich’s ataxia
• stroke stroke
• dementia muscular dystrophy
• MS multiple sclerosis
• ALS amyotrophic lateral
• the therapeutic molecule is an enzyme.
• the enzyme is deficient in an LSD, e.g., any of the deficient enzymes listed in Table 1 of Edelman, M.J. and Maegawa, G.H.B., Frontiers in Molecular Biosciences, Volume 7, Article 559804 (12 November 2020).
• the enzyme is IDUA, IDS, SGSH, GLA or GAA.
• the LSD is MPS-I and the enzyme is IDUA.
• the enzyme is IDS. In an embodiment, the enzyme is not IDS.
• the therapeutic molecule is an antibody or antigen-binding fragment thereof that specifically binds to a target protein (e.g., antigen) in the brain.
• the target protein is a Tau protein (e.g., cis P-tau), Abeta, BACE1 or a splice isoform thereof, human epidermal growth factor receptor (2) (HER2), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), interferon gamma, interleukin-1 receptor (IL-1R), interleukin-6 (IL-6), interleukin 6 receptor (IL6R), interleukin- 12, interleukin-23,
• HER2
• the therapeutic molecule is a cytokine or growth factor, e.g., an immunomodulatory cytokine, e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-beta, nerve growth factor, glial-cell line-derived neurotrophic factor (GNDF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), fibroblast growth factor-2 (FGF2), epidermal growth factor (EGF), or transforming growth factor (TGF)-beta 2.
• an immunomodulatory cytokine e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-beta, nerve growth factor, glial-cell line-derived neurotrophic factor (GNDF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), fibroblast growth factor-2 (FGF2), epidermal growth factor (EGF), or
• any of the BBB-transporting fusion proteins described above may be expressed by a mammalian cell(s) genetically modified to express and secrete the fusion protein.
• the genetically modified cell(s) may be derived from a variety of different mammalian cell types (e.g., human cells), including epithelial cells, endothelial cells, fibroblast cells, mesenchymal stem cells, keratinocyte cells and stem cells, e.g., embryonic stem cells or induced pluripotent stem cells.
• Exemplary cell types include the cell types recited in WO 2017/075631.
• the cells are derived from a cell-line shown in Table 4 below.
• any of the genetically modified mammalian cells described herein is derived from an RPE cell, e.g., an ARPE-19 cell.
• a genetically modified ARPE-19 cell comprises any of the expression cassettes, transposons and polynucleotides described herein.
• Cells may be genetically modified to express and secrete a desired BBB-transporting fusion protein using any of a variety of genetic engineering techniques known in the art.
• a cell may be transfected with an expression vector comprising an exogenous nucleotide sequence(s) encoding the desired fusion protein operably linked to control elements necessary or useful for gene expression, e.g., promoters, ribosomal binding sites, enhancers, poly A signal and the like.
• the exogenous nucleotide sequence is part of a transcription unit that is stably integrated into the genome of the parental cell.
• the exogenous sequence includes a nucleotide sequence encoding a secretory signal sequence for the fusion protein.
• the signal sequence is from a naturally secreted protein.
• the signal sequence is MELGLSWVVLAALLQGVQA (SEQ ID NO:79).
• the signal sequence consists essentially of an amino acid sequence shown in Table 5 below. Table 5: Exemplary secretory signal peptide sequences
• the genetically modified mammalian cells for use in devices, compositions and methods described herein, e.g., as a plurality of cells in a hydrogel capsule, may be in various stages of the cell cycle.
• at least one cell in the plurality of genetically modified cells is undergoing cell division.
• Cell division may be measured using any known method in the art, e.g., as described in DeFazio A et al (1987) J Histochem Cytochem 35:571-577 and Dolbeare F et al (1983) Proc Natl Acad Sci USA 80:5573-5577, each of which is incorporated by reference in its entirety.
• At least 1, 2, 3, 4, 5, 10, or 20% of the cells are undergoing cell division, e.g., as determined by 5-ethynyl-2’deoxyuridine (EdU) assay or 5-bromo-2’ -deoxyuridine (BrdU) assay.
• cell proliferation is visualized or quantified by microscopy (e.g., fluorescence microscopy (e.g., time-lapse or evaluation of spindle formation) or flow cytometry.
• none of the cells in the plurality of genetically modified cells are undergoing cell division and are quiescent.
• less than 1, 2, 3, 4, 5, 10, or 20% of the cells are undergoing cell division, 5-ethynyl-2’deoxyuridine (EdU) assay, 5-bromo-2’- deoxyuridine (BrdU) assay, microscopy (e.g., fluorescence microscopy (e.g., time-lapse or evaluation of spindle formation), or flow cytometry.
• EdU 5-ethynyl-2’deoxyuridine
• BadU 5-bromo-2’- deoxyuridine
• At least 50%, 60%, 70%, 80%, 90% or more of the genetically modified cells in the plurality are viable.
• Cell viability may be measured using any known method in the art, e.g., as described in Riss, T. et al (2013) “Cell Viability Assays” in Assay Guidance Manual (Sittapalam, G.S. et al, eds).
• cell viability may be measured or quantified by an ATP assay, 5-ethynyl -2’ deoxyuridine (EdU) assay, 5-bromo-2’-deoxyuridine (BrdU) assay.
• cell viability is visualized or quantified by microscopy (e.g., fluorescence microscopy (e.g., time-lapse or evaluation of spindle formation) or flow cytometry.
• microscopy e.g., fluorescence microscopy (e.g., time-lapse or evaluation of spindle formation) or flow cytometry.
• at least 80% of the engineered cells in the plurality are viable, e.g., as determined by an ATP assay, a 5-ethynyl-2’deoxyuridine (EdU) assay, a 5-bromo-2’ -deoxyuridine (BrdU) assay, microscopy (e.g., fluorescence microscopy (e.g., time-lapse or evaluation of spindle formation), or flow cytometry.
• EdU 5-ethynyl-2’deoxyuridine
• BadU 5-bromo-2’ -deoxyuridine
• a genetically modified cell described herein or a plurality of such cells may be incorporated into an implantable device for use in providing a therapeutic or diagnostic cargo to a subject with a CNS disease or condition, e.g., to a patient with an LSD, e.g., MPS-I.
• a CNS disease or condition e.g., to a patient with an LSD, e.g., MPS-I.
• An implantable device of the present disclosure comprises at least one barrier that prevents immune cells from contacting cells contained inside the device. At least a portion of the barrier needs to be sufficiently porous to allow proteins (e.g., the fusion protein) expressed and secreted by the cells to exit the device.
• proteins e.g., the fusion protein
• the device e.g., particle
• the device can have any configuration and shape appropriate for supporting the viability and productivity of the contained cells after implant into the intended target location.
• device shapes may be cylinders, rectangles, disks, ovoids, stellates, or spherical.
• the device can be comprised of a mesh-like or nested structure.
• a device is capable of preventing materials over a certain size from passing through a pore or opening.
• a device e.g., particle
• a device is capable of preventing materials greater than 50 kD, 75 kD, 100 kD, 125 kD, 150 kD, 175 kD, 200 kD, 250 kD, 300 kD, 400 kD, 500 kD, 750 kD, or 1,000 kD from passing through.
• the device is a macroencapsulation device.
• macrodevices are described in: WO 2019/068059, WO 2019/169089, US Patent Numbers 9,526,880, 9,724,430 and 8,278,106; European Patent No. EP742818B1, and Sang, S. and Roy, S . , Biotechnol. Bioeng. 113 (7) : 1381 - 1402 (2016).
• the device is a macrodevice having one or more cell-containing compartments.
• a device with two or more cell-containing compartments may be configured to produce two or more proteins, e.g., cells expressing the fusion protein would be placed in one compartment and cells expressing a different protein (e.g., a therapeutic protein that can alleviate one or more symptoms of the targeted CNS disease or condition) would be placed in a separate compartment.
• WO 2018/232027 describes a device with multiple cell-containing compartments formed in a micro-fabricated body and covered by a porous membrane.
• the device is configured as a thin, flexible strand as described in US Patent No. 10,493,107.
• This strand comprises a substrate, an inner polymeric coating surrounding the substrate and an outer hydrogel coating surrounding the inner polymeric coating.
• the proteinexpressing cells are positioned in the outer coating.
• a device e.g., particle
• LLD largest linear dimension
• mm millimeter
• mm millimeter
• a device can be as large as 10 mm in diameter or size.
• a device or particle described herein is in a size range of 0.5 mm to 10 mm, 1 mm to 10 mm, 1 mm to 8 mm, 1 mm to 6 mm, 1 mm to 5 mm, 1 mm to 4 mm, 1 mm to 3 mm, 1 mm to 2 mm, 1 mm to 1.5 mm, 1.5 mm to 8 mm, 1.5 mm to 6 mm, 1.5 mm to 5 mm, 1.5 mm to 4 mm,
• a device of the disclosure (e.g., particle, capsule) comprises at least one pore or opening, e.g., to allow for the free flow of materials.
• the mean pore size of a device is between about 0.1 ⁇ m to about 10 ⁇ m.
• the mean pore size may be between 0.1 ⁇ m to 10 ⁇ m, 0.1 ⁇ m to 5 ⁇ m, 0.1 ⁇ m to 2 ⁇ m, 0.15 ⁇ m to 10 ⁇ m, 0.15 ⁇ m to 5 ⁇ m, 0.15 ⁇ m to 2 ⁇ m, 0.2 ⁇ m to 10 ⁇ m, 0.2 ⁇ m to 5 ⁇ m, 0.25 ⁇ m to 10 ⁇ m, 0.25 ⁇ m to 5 ⁇ m, 0.5 ⁇ m to 10 ⁇ m, 0.75 ⁇ m to 10 ⁇ m, 1 ⁇ m to 10 ⁇ m, 1 ⁇ m to 5 ⁇ m, 1 ⁇ m to 2 ⁇ m, 2 ⁇ m to 10 ⁇ m, 2 ⁇ m to 5 ⁇ m, or 5 ⁇ m to 10 ⁇ m.
• the mean pore size of a device is between about 0.1 ⁇ m to 10 ⁇ m. In some embodiments, the mean pore size of a device is between about 0.1 ⁇ m to 5 ⁇ m. In some embodiments, the mean pore size of a device is between about 0.1 ⁇ m to 1 ⁇ m.
• the device comprises a semi-permeable, biocompatible membrane surrounding the genetically modified cells that are encapsulated in a polymer composition (e.g., an alginate hydrogel).
• a polymer composition e.g., an alginate hydrogel.
• the membrane pore size is selected to allow oxygen and other molecules important to cell survival and function to move through the semi-permeable membrane while preventing immune cells from traversing through the pores.
• the semi-permeable membrane has a molecular weight cutoff of less than 1000 kD or between 50-700 kD, 70-300 kD, or between 70-150 kD, or between 70 and 130 kD.
• the device may contain a cell -containing compartment that is surrounded with a barrier compartment formed from a cell-free biocompatible material, such as the core-shell microcapsules described in Ma, M et al., Adv. Healthc Mater 2(5) : 667 -672 (2012).
• a barrier compartment formed from a cell-free biocompatible material, such as the core-shell microcapsules described in Ma, M et al., Adv. Healthc Mater 2(5) : 667 -672 (2012).
• a barrier compartment could be used with or without the semi-permeable membrane.
• Cells in the cell-containing compartment(s) of a device of the disclosure may be encapsulated in a polymer composition.
• the polymer composition may comprise one or more hydrogel-forming polymers.
• the device e.g., macrodevice, particle, hydrogel capsule
• the device may comprise or be formed from materials such as metals, metallic alloys, ceramics, polymers, fibers, inert materials, and combinations thereof.
• a device may be completely made up of one type of material, or may comprise other materials within the cell-containing compartment and any other compartments.
• the device comprises a metal or a metallic alloy.
• one or more of the compartments in the device comprises a metal or a metallic alloy.
• Exemplary metallic or metallic alloys include comprising titanium and titanium group alloys (e.g., nitinol, nickel titanium alloys, thermo-memory alloy materials), platinum, platinum group alloys, stainless steel, tantalum, palladium, zirconium, niobium, molybdenum, nickel-chrome, chromium molybdenum alloys, or certain cobalt alloys (e.g., cobalt-chromium and cobalt-chromium -nickel alloys, e.g., ELGILOY® and PHYNOX®).
• a metallic material may be stainless steel grade 316 (SS 316L) (comprised of Fe, ⁇ 0.3% C, 16-18.5% Cr, 10-14% Ni, 2-3% Mo, ⁇ 2% Mn, ⁇ 1% Si, ⁇ 0.45% P, and ⁇ 0.03% S).
• the amount of metal e.g., by % weight, actual weight
• the device comprises a ceramic.
• one or more of the compartments in the device comprises a ceramic.
• Exemplary ceramic materials include oxides, carbides, or nitrides of the transition elements, such as titanium oxides, hafnium oxides, iridium oxides, chromium oxides, aluminum oxides, and zirconium oxides. Silicon based materials, such as silica, may also be used.
• the amount of ceramic (e.g., by % weight, actual weight) can be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, e.g., w/w; less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less.
• the device has two hydrogel compartments, in which the inner, cellcontaining compartment is completely surrounded by the second, outer (e.g., barrier) compartment.
• the inner boundary of the second compartment forms an interface with the outer boundary of the first compartment.
• the thickness of the second (outer) compartment means the average distance between the outer boundary of the second compartment and the interface between the two compartments, e.g., the average of the distances measured at each of the thinnest and thickest points visually observed in the outer compartment.
• the thinnest and thickest distances for the outer compartment are between 25 and 110 micrometers ( ⁇ m) and between 270 and 480 ⁇ m, respectively.
• the thickness of the outer compartment is greater than about 10 nanometers (nm), preferably 100 nm or greater and can be as large as 1 millimeter (mm).
• the thickness (e.g., average distance) of the outer compartment in a hydrogel capsule device described herein may be 10 nm to 1 mm, 100 nm to 1mm, 500 nm to 1 millimeter, 1 micrometer ( ⁇ m) to 1 mm, 1 ⁇ m to 1 mm, 1 ⁇ m to 500 ⁇ m, 1 ⁇ m to 250 ⁇ m, 1 ⁇ m to 1 mm, 5 ⁇ m to 500 ⁇ m, 5 ⁇ m to 250 ⁇ m, 10 ⁇ m to 1 mm, 10 ⁇ m to 500 ⁇ m, or 10 ⁇ m to 250 ⁇ m.
• the thickness (e.g., average distance) of the outer compartment is 100 nm to 1 mm, between 1 ⁇ m and 1 mm, between 1 ⁇ m and 500 ⁇ m or between 5 ⁇ m and 1 mm. In some embodiments, the thickness (e.g., average distance) of the outer compartment is between about 50 ⁇ m and about 100 ⁇ m. In some embodiments (e.g., the device is about 1.5 mm in diameter), the thickness of the outer compartment (e.g., average distance) is between about 180 ⁇ m and 260 ⁇ m or between about 310 ⁇ m and 440 ⁇ m.
• the mean pore size of the cell-containing inner compartment and the outer compartment is substantially the same.
• the mean pore size of the inner compartment and the second compartment differ by about 1.5%, 2%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more.
• the mean pore size of the device e.g., mean pore size of the first compartment and/or mean pore size of the second compartment
• the polymer composition in the cell-containing compartment(s) comprises a polysaccharide or other hydrogel-forming polymer (e.g., alginate, hyaluronate or chondroitin).
• a polysaccharide or other hydrogel-forming polymer e.g., alginate, hyaluronate or chondroitin.
• the polymer is an alginate, which is a polysaccharide made up of P-D-mannuronic acid (M) and a-L-guluronic acid (G).
• the alginate has a low molecular weight (e.g., approximate molecular weight of ⁇ 75 kD) and G:M ratio > 1.5, (ii) a medium molecular weight alginate, e.g., has approximate molecular weight of 75-150 kDa and G:M ratio > 1.5, (iii) a high molecular weight alginate, e.g., has an approximate MW of 150 kDa - 250 kDa and G:M ratio > 1.5, (iv) or a blend of two or more of these alginates.
• a low molecular weight e.g., approximate molecular weight of ⁇ 75 kD
• G:M ratio > 1.5 e.g., a medium molecular weight alginate, e.g., has approximate molecular weight of 75-150 kDa and G:M ratio > 1.5
• a high molecular weight alginate e.g., has an approximate MW of 150 k
• the cell-containing compartment(s) further comprises at least one cell-binding substance (CBS), e.g., a cell-binding peptide (CBP) or cell-binding polypeptide (CBPP) described in W02020069429.
• CBS cell-binding substance
• CBP cell-binding peptide
• CBPP cell-binding polypeptide
• the cell-containing compartment(s) comprises an alginate covalently modified with a linker-cell-binding peptide moiety, e.g., GRGD or GRGDSP.
• a linker-cell-binding peptide moiety e.g., GRGD or GRGDSP.
• the cell-binding peptide density in the cell-containing compartment(s) (% nitrogen as determined by combustion analysis, e.g., as described in WO2020198695) to be at least 0.05%, 0.1%, 0.2% or 0.3% but less than 4%, 3%, 2% or 1%.
• the total density of the linker-CBP in a cell containing compartment is about 0.1 to about 1.0 micromoles of the CBP per g of CBP-polymer (e.g., a MMW-alginate covalently modified with GRGD (SEQ ID NO:88) or GRGDSP (SEQ ID NO:89) in solution as determined by a quantitative peptide conjugation assay, e.g., an assay described in W02020198695.
• the linker-CBP is GRGDSP and the alginate has a molecular weight of 75 kDa to 150 kDa and a G:M ratio of greater than or equal to 1.5.
• the cell -containing compartment also comprises an unmodified alginate with a molecular weight of 75 kDa to 150 kDa and a G:M ratio of greater than or equal to 1.5.
• the device may form part of a plurality of substantially the same devices in a preparation (e.g., composition).
• the devices e.g., particles, hydrogel capsules
• the devices in the preparation have a mean diameter or size between about 0.5 mm to about 8 mm.
• the mean diameter or size of devices in the preparation is between about 0.5 mm to about 4 mm or between about 0.5 mm to about 2 mm.
• the devices in the preparation are two-compartment hydrogel capsules and have a mean diameter or size of about 0.7 mm to about 1.3 mm or about 1.2 mm to about 1.8 mm.
• the surface of the device comprises a compound capable of mitigating the FBR upon implant into a subject, an afibrotic compound as described herein below.
• the afibrotic compound may covalently modify a polymer disposed throughout the barrier compartment and optionally throughout the cell-containing compartment.
• one or more compartments in a device comprises an afibrotic polymer, e.g., an afibrotic compound of Formula (III) covalently attached to a polymer.
• an afibrotic polymer e.g., an afibrotic compound of Formula (III) covalently attached to a polymer.
• some or all the monomers in the afibrotic polymer are modified with the same compound of Formula (III).
• some or all the monomers in the afibrotic polymer are modified with different compounds of Formula (III).
• the afibrotic polymer is present only in the outer, barrier compartment.
• One or more compartments in a device may comprise an unmodified polymer that is the same or different than the polymer in any afibrotic polymer that is present in the device.
• the first compartment, second compartment or all compartments in the device comprise the unmodified polymer.
• Each of the modified and unmodified polymers in the device may be a linear, branched, or cross-linked polymer, or a polymer of selected molecular weight ranges, degree of polymerization, viscosity or melt flow rate.
• Branched polymers can include one or more of the following types: star polymers, comb polymers, brush polymers, dendronized polymers, ladders, and dendrimers.
• a polymer may be a thermoresponsive polymer, e.g., gel (e.g., becomes a solid or liquid upon exposure to heat or a certain temperature) or a photocrosslinkable polymer.
• Exemplary polymers include polystyrene, polyethylene, polypropylene, polyacetylene, poly(vinyl chloride) (PVC), polyolefin copolymers, poly(urethane)s, polyacrylates and polymethacrylates, polyacrylamides and polymethacrylamides, poly(methyl methacrylate), poly(2-hydroxyethyl methacrylate), polyesters, poly siloxanes, poly dimethyl siloxane (PDMS), poly ethers, poly(orthoester), poly(carbonates), poly(hydroxyalkanoate)s, polyfluorocarbons, PEEK®, Teflon® (polytetrafluoroethylene, PTFE), PEEK, silicones, epoxy resins, Kevlar®, Dacron® (a condensation polymer obtained from ethylene glycol and terephthalic acid), polyethylene glycol, nylon, polyalkenes, phenolic resins, natural and synthetic elastomers, adhesives and sealants, polyo
• the amount of a polymer (e.g., by % weight of the device, actual weight of the polymer) can be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, e.g., w/w; less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less.
• one or more of the modified and unmodified polymers in the device comprises a polyethylene.
• exemplary polyethylenes include ultra-low-density polyethylene (ULDPE) (e.g., with polymers with densities ranging from 0.890 to 0.905 g/cm 3 , containing comonomer); very -low-density polyethylene (VLDPE) (e.g., with polymers with densities ranging from 0.905 to 0.915 g/cm 3 , containing comonomer); linear low-density polyethylene (LLDPE) (e.g., with polymers with densities ranging from 0.915 to 0.935 g/cm 3 , contains comonomer); low- density polyethylene (LDPE) (e.g., with polymers with densities ranging from about 0.915 to 0.935 g/m 3 ); medium density polyethylene (MDPE) (e.g., with polymers with densities ranging from ULDPE
• one or more of the modified and unmodified polymers in the device comprises a polypropylene.
• exemplary polypropylenes include homopolymers, random copolymers (homophasic copolymers), and impact copolymers (heterophasic copolymers), e.g., as described in McKeen, Handbook of Polymer Applications in Medicine and Medical Devices, 3- Plastics Used in Medical Devices, (2014):21-53.
• one or more of the modified and unmodified polymers in the device comprises a polypropylene.
• exemplary polystyrenes include general purpose or crystal (PS or GPPS), high impact (HIPS), and syndiotactic (SPS) polystyrene.
• one or more of the modified and unmodified polymers comprises a comprises a thermoplastic elastomer (TPE).
• TPEs include (i) TPA-polyamide TPE, comprising a block copolymer of alternating hard and soft segments with amide chemical linkages in the hard blocks and ether and/or ester linkages in the soft blocks; (ii) TPC -co-poly ester TPE, consisting of a block copolymer of alternating hard segments and soft segments, the chemical linkages in the main chain being ester and/or ether; (iii) TPO-olefinic TPE, consisting of a blend of a polyolefin and a conventional rubber, the rubber phase in the blend having little or no crosslinking; (iv) TPS-styrenic TPE, consisting of at least a triblock copolymer of styrene and a specific diene, where the two end blocks (hard blocks) are polystyrene and
• the unmodified polymer is an unmodified alginate.
• the alginate is a high guluronic acid (G) alginate, and comprises greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more guluronic acid (G).
• the alginate is a high mannuronic acid (M) alginate, and comprises greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more mannuronic acid (M).
• the ratio of M:G is about 1.
• the ratio of M:G is less than 1.
• the ratio of M:G is greater than 1.
• the unmodified alginate has a molecular weight of 150 kDa - 250 kDa and a G:M ratio of > 1.5.
• the afibrotic polymer comprises an alginate chemically modified with a Compound of Formula (HI).
• the alginate in the afibrotic polymer may be the same or different than any unmodified alginate that is present in the device.
• the density of the Compound of Formula (III) in the afibrotic alginate e.g., amount of conjugation
• the density of the Compound of Formula (III) in the afibrotic alginate is between about 4.0% and about 8.0%, between about 5.0% and about 7.0 %, or between about 6.0% and about 7.0 % nitrogen (e.g., as determined by combustion analysis for percent nitrogen).
• the amount of Compound 101 produces an increase in % N (as compared with the unmodified alginate) of about 0.5% to 2% 2% to 4% N, about 4% to 6% N, about 6% to 8%, or about 8% to 10% N), where % N is determined by combustion analysis and corresponds to the amount of Compound 101 in the modified alginate.
• the density (e.g., concentration) of the Compound of Formula (III) (e.g., Compound 101) in the afibrotic alginate is defined as the % w/w, e.g., % of weight of amine / weight of afibrotic alginate in solution (e.g., saline) as determined by a suitable quantitative amine conjugation assay (e.g.
• the density of a Compound of Formula (III) is between about 1.0 % w/w and about 3.0 % w/w, between about 1.3 % w/w and about 2.5 % w/w or between about 1.5 % w/w and 2.2 % w/w.
• the amount of modified and unmodified alginates (e.g., by % weight of the device, actual weight of the alginate) can be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, e.g., w/w; less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less.
• the alginate in an afibrotic polymer can be chemically modified with a compound of Formula (III) using any suitable method known in the art.
• the alginate carboxylic acid moiety can be activated for coupling to one or more amine-functionalized compounds to achieve an alginate modified with a compound of Formula (III).
• the alginate polymer may be dissolved in water (30 mL/gram polymer) and treated with an activating agent (e.g., 2-chloro-4,6- dimethoxy-l,3,5-triazine (0.5 eq)) and a base (e.g., N-methylmorpholine (1 eq)).
• an activating agent e.g., 2-chloro-4,6- dimethoxy-l,3,5-triazine (0.5 eq)
• a base e.g., N-methylmorpholine (1 eq)
• the reaction may be warmed to 55°C for 16h, then cooled to room temperature and gently concentrated via rotary evaporation, then the residue may be dissolved, e.g., in water.
• the mixture may then be filtered, e.g., through a bed of cyano-modified silica gel (Silicycle) and the filter cake washed with water.
• the resulting solution may then be dialyzed (10,000 MWCO membrane) against water for 24 hours, e.g., replacing the water twice.
• the resulting solution can be concentrated, e.g., via lyophilization, to afford the desired chemically modified alginate.
• the alginate in an afibrotic polymer can be chemically modified with a compound of Formula (III) using any suitable method known in the art, e.g., as described in any of WO 2021/119522, WO 2019/195055, WO 2018/067615, WO 2017/075631, WO 2016/019391 and WO 2012/167223.
• the device comprises at least one cell -containing compartment, and in some embodiments contains two, three, four or more cell -containing compartments.
• each cell-containing compartment comprises a plurality of cells (e.g., live cells) and the cells in at least one of the compartments are capable of expressing and secreting a BBB- transporting fusion protein when the device is implanted into a subject.
• all the cells in a cell-containing compartment are derived from a single parental cell-type or a mixture of at least two different parental cell types. In an embodiment, all of the cells in a cell -containing compartment are derived from the same parental cell type, but a first plurality of the derived cells are engineered to express the BBB-transporting fusion protein, and a second plurality of the derived cells are engineered to express a different therapeutic protein. In devices with two or more cell -containing compartments, the cells and the protein(s) produced thereby may be the same or different in each cell-containing compartment. In some embodiments, all of the cell -containing compartments are surrounded by a single barrier compartment. In some embodiments, the barrier compartment is substantially cell-free.
• cells to be incorporated into a device described herein are prepared in the form of a cell suspension prior to being encapsulated within the device.
• the cells in the suspension may take the form of single cells (e.g., from a monolayer cell culture), or provided in another form, e.g., disposed on a microcarrier (e.g., a bead or matrix) or as a three- dimensional aggregate of cells (e.g., a cell cluster or spheroid).
• the cell suspension can comprise multiple cell clusters (e.g., as spheroids) or microcarriers.
• a device may comprise one or more exogenous agents that are not expressed by the cells, and may include, e.g., a nucleic acid (e.g., an RNA or DNA molecule), a protein (e.g., a hormone, an enzyme (e.g., glucose oxidase, kinase, phosphatase, oxygenase, hydrogenase, reductase) antibody, antibody fragment, antigen, or epitope)), an active or inactive fragment of a protein or polypeptide, a small molecule, or drug.
• the device is configured to release such an exogenous agent.
• Afibrotic e.g., FBR-mitigating
• the devices described herein comprise at least one compound of Formula (III): or a pharmaceutically acceptable salt thereof, wherein:
• L 2 is a bond
• M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted by one or more R 3 ;
• P is absent, cycloalkyl, heterocyclyl, or heteroaryl, each of which is optionally substituted by one or more R 4 ;
• Z is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, -0R A , -C(0)R A , -C(0)0R A , -C(0)N(R C )(R D ), -N(R C )C(0)R A , -N(R C )(R D ), cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substituted by one or more R 5 ; each R A , R B , R C , R D , R E , R F , and R G is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, azido, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each alkyl
• the compound of Formula (III) is a compound of Formula (Ill-a):
• L 2 is a bond
• M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted by one or more R 3 ;
• P is heteroaryl optionally substituted by one or more R 4 ;
• Z is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted by one or more R 5 ; each R A , R B , R C , R D , R E , R F , and R G is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, azido, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substituted with one or more R 6 ; or R c and R D , taken together with the nitrogen atom to which they are attached, form a ring (e.g., a 5-7 membered ring), optionally substituted with one or more R 6 ; each R 1
• A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O) -, -N(R c )C(O)-, -N(R c )C(O)(C 1 -C 6 -alkylene)-, -N(R c )C(O)(C 1 -C 6 -alkenylene)-, or -N(R C )-.
• A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, — C(O)O— , — C(O) ⁇ , -OC(O) -, or -N(R C )-.
• A is alkyl, alkenyl, alkynyl, heteroalkyl, -O-, -C(O)O-, -C(O)-,-OC(O) -, or -N(R C )-.
• A is alkyl, -O-, — C(O)O— , -C(O)-, -OC(O), or -N(R C )-.
• A is -N(R c )C(O)-, -N(R c )C(O)(C 1 -C 6 -alkylene)-, or -N(R c )C(O)(C 1 -C 6 -alkenylene)-.
• A is -N(R C )-.
• A is -N(R C ) -, and R c an R D is independently hydrogen or alkyl.
• A is -NH-. In some embodiments, A is -N(R c )C(O)(C 1 -C 6 -alkylene)-, wherein alkylene is substituted with R 1 . In some embodiments, A is -N(R c )C(O)(C 1 -C 6 -alkylene)-, and R 1 is alkyl (e.g., methyl). In some embodiments, A is -NHC(O)C(CH 3 )2-. In some embodiments, A is -N(R c )C(O)(methylene)-, and R 1 is alkyl (e.g., methyl).
• A is -NHC(O)CH(CH 3 )-. In some embodiments, A is -NHC(O)C(CH 3 )-.
• L 1 is a bond, alkyl, or heteroalkyl. In some embodiments, L 1 is a bond or alkyl. In some embodiments, L 1 is a bond. In some embodiments, L 1 is alkyl. In some embodiments, L 1 is C 1 -C 6 alkyl. In some embodiments, L 1 is -CH 2 -, -CH(CH 3 )-, -CH 2 CH 2 CH 2 , or -CH 2 CH 2 -. In some embodiments, L 1 is -CH 2 -or -CH 2 CH 2 -.
• L 3 is a bond, alkyl, or heteroalkyl. In some embodiments, L 3 is a bond. In some embodiments, L 3 is alkyl. In some embodiments, L 3 is C 1 -C 1 2 alkyl. In some embodiments, L 3 is C 1 -C 6 alkyl. In some embodiments, L 3 is -CH 2 -. In some embodiments, L 3 is heteroalkyl. In some embodiments, L 3 is C 1 -C 1 2 heteroalkyl, optionally substituted with one or more R 2 (e.g., oxo).
• R 2 e.g., oxo
• L 3 is C 1 -C 6 heteroalkyl, optionally substituted with one or more R 2 (e.g., oxo). In some embodiments, L 3 is -C(O)OCH 2 -, -CH 2 (OCH 2 CH 2 )2-, -CH 2 (OCH 2 CH 2 )3-, CH 2 CH 2 O-, or -CH 2 O-. In some embodiments, L 3 is -CH 2 O-.
• M is absent, alkyl, heteroalkyl, aryl, or heteroaryl. In some embodiments, for Formulas (III) and (Ill-a), M is absent, alkyl, heteroalkyl, aryl, or heteroaryl. In some embodiments, M is heteroalkyl, aryl, or heteroaryl. In some embodiments, M is absent. In some embodiments, M is alkyl (e.g., C 1 -C 6 alkyl). In some embodiments, M is -CH 2 -. In some embodiments, M is heteroalkyl (e.g., C 1 -C 6 heteroalkyl).
• M is (-OCH 2 CH 2 -Jz, wherein z is an integer selected from 1 to 10. In some embodiments, z is an integer selected from 1 to 5. In some embodiments, M is -(OCH 2 ) 2 -, (-OCH 2 CH 2 -)2, (-OCH 2 CH 2 -)3, (-OCH 2 CH 2 -) 4 , or
• M is -OCH 2 CH 2 -, (-OCH 2 CH 2 -)2, (-OCH 2 CH 2 -) 3 , or (-OCH 2 CH 2 -)4.
• M is (-OCH 2 -h.
• M is aryl.
• M is phenyl.
• M is unsubstituted phenyl.
• M is .
• M is .
• M is phenyl substituted with 1-4 R 3 (e.g., 1 R 3 ).
• R 3 is CF3.
• P is absent, heterocyclyl, or heteroaryl. In some embodiments, for Formulas (III) and (Ill-a), P is absent, heterocyclyl, or heteroaryl. In some embodiments, P is absent. In some embodiments, for Formulas (III) and (Ill-a), P is a tricyclic, bicyclic, or monocyclic heteroaryl. In some embodiments, P is a monocyclic heteroaryl. In some embodiments, P is a nitrogen-containing heteroaryl. In some embodiments, P is a monocyclic, nitrogen-containing heteroaryl. In some embodiments, P is a 5-membered heteroaryl.
• P is a 5-membered nitrogen-containing heteroaryl. In some embodiments, P is tetrazolyl, imidazolyl, pyrazolyl, or triazolyl, or pyrrolyl. In some embodiments, P is imidazolyl. In some embodiments, P is 1,2, 3 -triazolyl. In some embodiments, P is
• P is . In some embodiments, P is
• P is heterocyclyl. In some embodiments, P is heterocyclyl. In some embodiments, P is a 5-membered heterocyclyl. In some embodiments, P is imidazolidinonyl. In some embodiments, P is . In some embodiments, P is thiomorpholinyl-l,l-dioxidyl. In some embodiments, P is
• Z is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl. In some embodiments, for Formulas (III) and (Ill-a), Z is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl. In some embodiments, Z is heterocyclyl. In some embodiments, Z is monocyclic or bicyclic heterocyclyl, 5-membered heterocyclyl, or 6-membered heterocyclyl. In some embodiments, Z is a 6-membered oxygen-containing heterocyclyl. In some embodiments, Z is tetrahydropyranyl. In some embodiments, Z is In some embodiments, Z is a 4-membered oxygen-containing heterocyclyl.
• Z is In some embodiments, Z is a bicyclic oxygen-containing heterocyclyl. In some embodiments,
• Z is a bicyclic oxygen-containing heterocyclyl. In some embodiments, Z is phthalic anhydridyl. In some embodiments, Z is a sulfur-containing heterocyclyl some embodiments, Z is a
• Z is a
• Z is thiomorpholinyl- 1,1 -di oxidyl. In some embodiments, some embodiments, Z is a nitrogen-containing heterocyclyl. In some embodiments, Z is a 6-membered nitrogen- containing heterocyclyl. In some embodiments, Z is
• Z is a bicyclic heterocyclyl. In some embodiments, Z is a bicyclic heterocyclyl In some embodiments, Z is a bicyclic nitrogen-containing heterocyclyl, optionally substituted with one or more R 5 . In some embodiments, Z is 2-oxa-7-azaspiro[3.5]nonanyl
• Z is l-oxa-3,8-diazaspiro[4.5]decan-
• Z is aryl. In some embodiments, Z is monocyclic aryl. In some embodiments, Z is phenyl. In some embodiments, Z is monosubstituted phenyl (e.g., with 1 R 5 ). In some embodiments, Z is monosubstituted phenyl, wherein the 1 R 5 is a nitrogen-containing group. In some embodiments, Z is monosubstituted phenyl, wherein the 1 R 5 is NH2. In some embodiments, Z is monosubstituted phenyl, wherein the 1 R 5 is an oxygencontaining group.
• Z is monosubstituted phenyl, wherein the 1 R 5 is an oxygen-containing heteroalkyl. In some embodiments, Z is monosubstituted phenyl, wherein the 1 R 5 is OCH3. In some embodiments, Z is monosubstituted phenyl, wherein the 1 R 5 is in the ortho position. In some embodiments, Z is monosubstituted phenyl, wherein the 1 R 5 is in the meta position. In some embodiments, Z is monosubstituted phenyl, wherein the 1 R 5 is in the para position.
• Z is alkyl. In some embodiments, Z is C 1 -C 1 2 alkyl. In some embodiments, Z is C 1 -C 1 0 alkyl. In some embodiments, Z is C 1 -C 8 alkyl. In some embodiments, Z is C 1 -C 8 alkyl substituted with 1-5 R 5 . In some embodiments, Z is C 1 -C 8 alkyl substituted with 1 R 5 .
• Z is C 1 -C 8 alkyl substituted with 1 R 5 , wherein R 5 is alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 ,-OC(O)R B1 , or -N(R C1 )(R D1 ).
• Z is C 1 -C 8 alkyl substituted with 1 R 5 , wherein R 5 is -OR A1 or -C(O)OR A1 .
• Z is C 1 -C 8 alkyl substituted with 1 R 5 , wherein R 5 is -OR A1 or -C(O)OH.
• Z is -CH3.
• Z is heteroalkyl.
• Z is C 1 -C 1 2 heteroalkyl.
• I n some embodiments, Z is C 1 -C 1 0 heteroalkyl.
• Z is C 1 -C 8 heteroalkyl.
• I n some embodiments, Z is C 1 -C 6 heteroalkyl.
• Z is a nitrogen-containing heteroalkyl optionally substituted with one or more R 5 .
• 1 n some embodiments, Z is a nitrogen and sulfur-containing heteroalkyl substituted with 1-5 R 5 .
• Z is N-methyl-2-(methylsulfonyl)ethan-l -aminyl.
• Z is -OR A or -C(O)OR A . In some embodiments, Z is -OR A (e.g., -OH or -OCH3). In some embodiments, Z is -OCH3. In some embodiments, Z is -C(O)OR A (e.g., -C(O)OH).
• Z is hydrogen
• L 2 is a bond and P and L 3 are independently absent.
• L 2 is a bond
• P is heteroaryl
• L 3 is a bond
• Z is hydrogen
• P is heteroaryl
• L 3 is heteroalkyl
• Z is alkyl.
• the compound of Formula (III) is a compound of Formula (Ill-b): or a pharmaceutically acceptable salt thereof, wherein Ring M 1 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1-5 R 3 ; Ring Z 1 is cycloalkyl, heterocyclyl’ aryl or heteroaryl, optionally substituted with 1-5 R 5 ; each of R 2a , R 2b , R 2c , and R 2d is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, nitro, amino, cycloalkyl, heterocyclyl, aryl, or heteroaryl, or each of R 2a and R 2b or R 2c and R 2d is taken together to form an oxo group; X is absent, N(R 10 )(R n ), O, or S; R c is hydrogen, alkyl, alkeny
• each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally and independently substituted with halogen, oxo, cyano, cycloalkyl, or heterocyclyl.
• the compound of Formula (III-b) is a compound of Formula (III-b- or a pharmaceutically acceptable salt thereof, wherein Ring M 2 is aryl or heteroaryl optionally substituted with one or more R 3 ; Ring Z 2 is cycloalkyl, heterocyclyl, aryl’ or heteroaryl; each of R 2a , R 2b , R 2C , and R 2d is independently hydrogen, alkyl, or heteroalkyl, or each of R 2a and R 2b or R 2C and R 2d is taken together to form an oxo group; X is absent, O, or S; each R 3 and R 5 is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 , wherein each alkyl and heteroalkyl is optionally substituted with halogen; or two R 5 are taken together to form a 5-6 membered ring fuse
• the compound of Formula (III-b-i) is a compound of Formula (III- b-ii): or a pharmaceutically acceptable salt thereof, wherein Ring Z 2 is cycloalkyl, heterocyclyl, aryl or heteroaryl; each of R 2c and R 2d is independently hydrogen, alkyl, or heteroalkyl, or R 2c and R and taken together to form an oxo group; each R 3 and R 5 is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(0)0R A1 , or -C(0)R B1 , wherein each alkyl and heteroalkyl is optionally substituted with halogen; each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; each of p and q is independently 0, 1, 2, 3, 4, 5, or 6; and “ refers to a connection to an attachment group or a polymer described herein.
• the compound of Formula (III) is a compound of Formula (III-c): or a pharmaceutically acceptable salt thereof, wherein Ring Z 2 is cycloalkyl, heterocyclyl’ aryl or heteroaryl; each of R 2c and R 2d is independently hydrogen, alkyl, or heteroalkyl, or R 2c and R 2d is taken together to form an oxo group; each R 3 and R 5 is independently alkyl, heteroalkyl, halogen, oxo, -OR, -C(0)0R, or -C(0)R B1 , wherein each alkyl and heteroalkyl is optionally substituted with halogen; each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; m is 1, 2, 3, 4, 5, or 6; each of p and q is independently 0, 1, 2, 3, 4, 5, or 6; and “ refers to a connection to an attachment group or a polymer described herein.
• the compound of Formula (III) is a compound of Formula (Ill-d): or a pharmaceutically acceptable salt thereof, wherein Ring Z 2 is cycloalkyl, heterocyclyl’ aryl or heteroaryl; X is absent, O, or S; each of R 2a , R 2b , R 2c , and R 2d is independently hydrogen, alkyl, or heteroalkyl, or each of R 2a and R 2b or R 2c and R 2d is taken together to form an oxo group; each R 5 is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 , wherein each alkyl and heteroalkyl is optionally substituted with halogen; each R A1 and R is independently hydrogen, alkyl, or heteroalkyl; each of m and n is independently 1, 2, 3, 4, 5, or 6; p is 0, 1, 2, 3, 4, 5,
• the compound of Formula (III) is a compound of Formula (Ill-e): or a pharmaceutically acceptable salt thereof, wherein Ring Z 2 is cycloalkyl, heterocyclyl, aryl or heteroaryl; X is absent, O, or S; each of R 2a , R 2b , R 2c , and R 2d is independently hydrogen, alkyl, or heteroalkyl, or each of R 2a and R 2b or R 2c and R 2d is taken together to form an oxo group; each R 5 is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or-C(O)R B1 ; each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; each of m and n is independently 1, 2, 3, 4, 5, or 6; p is 0, 1, 2, 3, 4, 5, or 6; and refers to a connection to an attachment group or a polymer described
• the compound of Formula (III) is a compound of Formula (Ill-f): or a pharmaceutically acceptable salt thereof, wherein M is alkyl optionally substituted with one or more R 3 ; Ring P is heteroaryl optionally substituted with one or more R 4 ; L 3 is alkyl or heteroalkyl optionally substituted with one or more R 2 ; Z is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with one or more R 5 ; each of R 2a and R 2b is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b is taken together to form an oxo group; each R 2 , R 3 , R 4 , and R 5 is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 ; each R A1
• the compound of Formula (III) is a compound of Formula (IV): or a pharmaceutically acceptable salt thereof, wherein M is a bond, alkyl or aryl, wherein alkyl and aryl is optionally substituted with one or more R 3 ; L 3 is alkyl or heteroalkyl optionally substituted with one or more R 2 ; Z is hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl or -OR, wherein alkyl, , cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substituted with one or more R 5 ; R A is hydrogen; each of R 2a and R 2b is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b is taken together to form an oxo group; each R 2 , R 3 , and R 5 is independently alkyl, heteroalkyl, halogen, oxo, -OR A1
• the compound of Formula (IV) is a compound of Formula (IV-a): (IV-a), or a pharmaceutically acceptable salt thereof, wherein L 3 is alkyl or heteroalkyl, each of which is optionally substituted with one or more R 2 ; Z is hydrogen, alkyl, heteroalkyl or -OR A , heteroalkyl are optionally substituted with one or more R 5 ; each of R 2a and R 2b is independently hydrogen, alkyl, or heteroalkyl, or each of R 2a and R 2b is taken together to form an oxo group’ each R 2 , R 3 , and R 5 is independently heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 ; R A is hydrogen; each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; n is independently 1, 2, 3, 4, 5, or 6; and “ refers to a connection to an attachment group or a polymer described herein.
• the compound of Formula (III) is a compound of Formula (V): or a pharmaceutically acceptable salt thereof, wherein Z 1 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1-5 R 5; each of R 2a , R 2b , R 2c , and R 2d is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, nitro, amino, cycloalkyl, heterocyclyl, aryl, or heteroaryl; or each of R 2a and R 2b or R 2c and R 2d is taken together to form an oxo group; R c is hydrogen, alkyl, alkenyl, alkynyl, or heteroalkyl, wherein each of alkyl, alkenyl, alkynyl, or heteroalkyl is optionally substitute
• the compound of Formula (V) is a compound of Formula (V-a): (V-a), or a pharmaceutically acceptable salt thereof, wherein Ring Z 1 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1-5 R 5 ; each of R 2a , R 2b , R 2c , and R 2d is independently hydrogen, alkyl, heteroalkyl, halo; or R 2a and R 2b or R 2c and R 2d are taken together to form an oxo group; R c is hydrogen, alkyl, alkenyl, alkynyl, or heteroalkyl, wherein each of alkyl, alkenyl, alkynyl, or heteroalkyl is optionally substituted with 1-6 R 6 ; each of R 3 , R 5 , and R 6 is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)
• the compound of Formula (V) is a compound of Formula (V-b): (V-b), or a pharmaceutically acceptable salt thereof, wherein Ring Z 1 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1-5 R 5 ;
• R c is hydrogen, alkyl, -N(R C )C(O)R B , -N(R c )C(O)(C 1 -C 6 -alkyl), or -N(R c )C(O)(C 1 -C 6 -alkenyl), wherein each of alkyl and alkenyl is optionally substituted with 1-6 R 6 ;
• each of R 2a , R 2b , R 2c , and R 2d is independently hydrogen or alkyl; or R 2a and R 2b or R 2c and R 2d are taken together to form an oxo group;
• the compound of Formula (V) is a compound of Formula (V-c): or a pharmaceutically acceptable salt thereof, wherein R c is hydrogen, alkyl, -N(R c )C(O)R B , -N(R c )C(O)(C 1 -C 6 -alkyl), or -N(R c )C(O)(C 1 -C 6 -alkenyl), wherein each of alkyl and alkenyl is optionally substituted with 1-6 R 6 ; each of R 2a , R 2b , R 2c , and R 2d is independently hydrogen or alkyl; or R 2a and R 2b or R 2c and R 2d are taken together to form an oxo group; each of R 3 , R 5 , and R 6 is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)
• the compound of Formula (V) is a compound of Formula (V-d): or a pharmaceutically acceptable salt thereof, wherein X is C(R’)(R”), N(R’), or S(O) X ; each of R’ and R” is independently hydrogen, alkyl, or halogen; R c is hydrogen, alkyl, -N(R c )C(O)R B , -N(R c )C(O)(C 1 -C 6 -alkyl), or -N(R c )C(O)(C 1 -C 6 -alkenyl), wherein each of alkyl and alkenyl is optionally substituted with 1-6 R 6 ; each of R 2a , R 2b , R 2c , and R 2d is independently hydrogen or alkyl; or R 2a and R 2b or R 2c and R 2d are taken together to form an oxo group; each of R 3 , R 5 , and R
• X is S(O) X . In some embodiments, x is 2. In some embodiments, X is S(O) 2 .
• each of R 2a , R 2b , R 2c , and R 2d is independently hydrogen.
• R c is hydrogen, -C(O)(C 1 -C 6 -alkyl), or -C(O)(C 1 -C 6 -alkenyl). In some embodiments, each of alkyl and alkenyl is substituted with 1 R 6 (e.g., -CFF). In some embodiments, R c is hydrogen.
• n is 1. In some embodiments, q is 2, 3, 4, or 5. In some embodiments, q is 3. In some embodiments, m is 1. In some embodiments, p is 0. In some embodiments, R 12 is halo (e.g., Cl).
• the compound is a compound of Formula (III).
• L 2 is a bond and P and L 3 are independently absent.
• the compound is a compound of Formula (Ill-a).
• L 2 is a bond, P is heteroaryl, L 3 is a bond, and Z is hydrogen.
• P is heteroaryl, L 3 is heteroalkyl, and Z is alkyl.
• L 2 is a bond and P and L 3 are independently absent.
• L 2 is a bond, P is heteroaryl, L 3 is a bond, and Z is hydrogen.
• P is heteroaryl, L 3 is heteroalkyl, and Z is alkyl.
• the compound is a compound of Formula (Ill-b).
• P is absent
• L 1 is -NHCH 2
• L 2 is a bond
• M is aryl (e.g., phenyl)
• L 3 is -CH 2 O
• Z is heterocyclyl (e.g., a nitrogen-containing heterocyclyl, e.g., thiomorpholinyl-l,l-dioxide).
• the compound of Formula (I-b) is Compound 116.
• P is absent, L 1 is -NHCH 2 , L 2 is a bond, M is absent, L 3 is a bond, and Z is heterocyclyl (e.g., an oxygen-containing heterocyclyl, e.g., tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl).
• the compound of Formula (Ill-b) is Compound 105.
• the compound is a compound of Formula (III-b-i).
• each of R 2a and R 2b is independently hydrogen or CH3, each of R 2C and R 2d is independently hydrogen, m is 1 or 2, n is 1, X is O, p is 0, M 2 is phenyl optionally substituted with one or more R 3 , R 3 is -CF3, and Z 2 is heterocyclyl (e.g., an oxygen-containing heterocyclyl, e.g., tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl).
• the compound of Formula (III-b-i) is Compound 100, Compound 106, Compound 107, Compound 108, Compound 109, or Compound 111.
• the compound is a compound of Formula (III-b-ii).
• each of R 2a , R 2b , R 2c , and R 2d is independently hydrogen, q is 0, p is 0, m is 1, and Z 2 is heterocyclyl (e.g., an oxygen-containing heterocyclyl, e.g., tetrahydropyranyl).
• the compound of Formula (III-b-ii) is Compound 100.
• the compound is a compound of Formula (III-c).
• each of R 2c and R 2d is independently hydrogen, m is 1, p is 1, q is 0, R 5 is -CH3, and Z is heterocyclyl (e.g., a nitrogen-containing heterocyclyl, e.g., piperazinyl).
• the compound of Formula (I-c) is Compound 113.
• the compound is a compound of Formula (Ill-d).
• each of R 2a , R 2b , R 2c , and R 2d is independently hydrogen, m is 1, n is 3, X is O, p is 0, and Z is heterocyclyl (e.g., an oxygen-containing heterocyclyl, e.g., tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl).
• the compound of Formula (Ill-d) is Compound 110 or Compound 114.
• the compound is a compound of Formula (Ill-f).
• each of R 2a and R 2b is independently hydrogen, n is 1, M is -CH 2 -, P is a nitrogen-containing heteroaryl (e.g., imidazolyl), L 3 is -C(O)OCH 2 -, and Z is CH3.
• the compound of Formula (III-f) is Compound 115.
• the compound is a compound of Formula (IV-a).
• each of R 2a and R 2b is independently hydrogen, n is 1, q is 0, L 3 is -CH 2 (OCH 2 CH 2 )2, and Z is -OCH3.
• the compound of Formula (IV-a) is Compound 112.
• each of R 2a and R 2b is independently hydrogen, n is 1, L 3 is a bond or -CH 2 , and Z is hydrogen or -OH.
• the compound of Formula (IV-a) is Compound 103 or Compound 104.
• the compound is a compound of Formula (V).
• each of R 2a , R 2b , R 2c , and R 2d is independently hydrogen
• m is l
• n is 2
• q is 3
• p is 0,
• R c is hydrogen
• Z 1 is heteroalkyl optionally substituted with R 5 (e.g., -N(CH3)(CH 2 CH 2 )S(O)2CH3).
• the compound of Formula (V) is Compound 120.
• the compound is a compound of Formula (V-b).
• each of R 2a , R 2b , R 2c , and R 2d is independently hydrogen, m is 0, n is 2, q is 3, p is 0, and Z 2 is aryl (e.g., phenyl) substituted with 1 R 5 (e.g., -NH2).
• the compound of Formula (Ill-b) is Compound 102.
• the compound is a compound of Formula (V-b).
• each of R 2a , R 2b , R 2c , and R 2d is independently hydrogen
• m is 1
• n is 2
• q is 3
• p is 0,
• R c is hydrogen
• Z 2 is heterocyclyl (e.g., a nitrogen-containing heterocyclyl, e.g., a nitrogen-containing spiro heterocyclyl, e.g., 2-oxa-7-azaspiro[3.5]nonanyl).
• the compound of Formula (V-b) is Compound 121.
• the compound is a compound of Formula (V-d).
• each of R 2a , R 2b , R 2c , and R 2d is independently hydrogen, m is 1, n is 2, q is 1, 2, 3, or 4, p is 0, and X is S(O)2.
• each of R 2a and R 2b is independently hydrogen, m is 1, n is 2, q is 1, 2, 3, or 4, p is 0, and X is S(O)2.
• the compound of Formula (V-d) is Compound 101, Compound 117, Compound 118, or Compound 119.
• the compound is a compound of Formula (Ill-b), (Ill-d), or (Ill-e). In some embodiments, the compound is a compound of Formula (Ill-b), (Ill-d), or (IV). In some embodiments, the compound is a compound of Formula (Ill-b), (Ill-d), or (Ill-f). In some embodiments, the compound is a compound of Formula (Ill-b), (Ill-d), or (V).
• the compound of Formula (I) is not a compound disclosed in WO2012/112982, WO2012/167223, WO2014/153126, W02016/019391, WO 2017/075630, US2012-0213708, US 2016-0030359 or US 2016-0030360.
• the compound of Formula (III) comprises a compound shown in Table 6 herein below, or a pharmaceutically acceptable salt thereof.
• the exterior surface and / or one or more compartments within a device described herein comprises a small molecule compound shown in Table 6, or a pharmaceutically acceptable salt thereof.
• Table 6 Exemplary afibrotic (FBR-mitigating) compounds
• Conjugation of any of the compounds in Table 6 to a polymer may be performed as described in Example 2 of WO 2019/195055 or in or any other suitable chemical reaction.
• the compound is a compound of Formula (III or a pharmaceutically acceptable salt thereof and is selected from: pharmaceutically acceptable salt thereof.
• the device described herein comprises the compound of pharmaceutically acceptable salt of either compound.
• a compound of Formula (III) e.g., Compound 101 in Table 6
• an alginate e.g., an alginate with approximate MW ⁇ 75 kDa, G:M ratio > 1.5
• an alginate e.g., an alginate with approximate MW ⁇ 75 kDa, G:M ratio > 1.5
• a conjugation density of at least 2.0 % and less than 9.0 %, or 3.0 % to 8.0 %, 4.0-7.0, 5.0 to 7.0, or 6.0 to 7.0 or about 6.8 as determined by combustion analysis for percent nitrogen as described in WO 2020/069429.
• the conjugation density of Compound 101 in the modified alginate is determined by quantitative free amine analysis, e.g., as described in WO2020198695, wherein the determined conjugation density is 1.0 % w/w to 3.0 % w/w, 1.3 % w/w to 2.8 % w/w, 1.3 % w/w to 2.6 % w/w, 1.5 % w/w to 2.4 % w/w, 1.5 % w/w to 2.2 % w/w, or 1.7 % w/w to 2.2 % w/w.
• a device, device preparation or device composition may be configured for implantation, or is implanted or disposed, into or onto any site or part of the body.
• the implantable device or device preparation is configured for implantation into the peritoneal cavity (e.g., the lesser sac, also known as the omental bursa or bursalis omentum).
• a device, device preparation or device composition may be implanted in the peritoneal cavity (e.g., the omentum, e.g., the lesser sac) or disposed on a surface within the peritoneal cavity (e.g., omentum, e.g., lesser sac) via injection or catheter. Additional considerations for implantation or disposition of a device, device preparation or device composition into the omentum (e.g., the lesser sac) are provided in M. Pellicciaro et al. (2017) CellR4 5(3):e2410.
• ARPE-19 cells for use in manufacturing a device described herein may be generated and cultured using methods known in the art.
• stably-transfected ARPE-19 cells may be cultured in vitro substantially as described in W02020198695.
• Alginate solutions for making afibrotic, two-compartment hydrogel capsules may be obtained using procedures known in the art, e.g., substantially as described in WO2020198695.
• Two-compartment hydrogel capsules encapsulating in the inner compartment genetically modified mammalian cells and an afibrotic alginate in the outer layer (“shielded capsules”) may be generated using procedures known in the art, e.g., substantially as described in W02020198696.
• Described herein are methods for preventing or treating a CNS disease or condition in a subject (e.g., a lysosomal storage disease, e.g., MPS-1) through administration or implantation of a pharmaceutical composition or genetically modified cells described herein.
• the pharmaceutical composition comprises a BBB -transporting fusion protein and is formulated for intravenous or subcutaneous administration.
• the pharmaceutical composition comprises a plurality of cells that are genetically modified to express the BBB- transporting fusion protein.
• the cells are encapsulated in hydrogel capsules described herein.
• the cells are encapsulated in a macrodevice described herein.
• the methods described herein directly or indirectly reduce or alleviate at least one symptom of the CNS disease or condition, or prevent or slow the onset of the disease.
• the method comprises administering (e.g., implanting) an effective amount of a composition of two-compartment alginate hydrogel capsules which comprise in the inner compartment genetically modified RPE cells and a cell-binding polymer described herein and comprise a Compound of Formula (III), e.g., Compound 101, on the outer capsule surface and optionally within the outer compartment.
• a composition of two-compartment alginate hydrogel capsules which comprise in the inner compartment genetically modified RPE cells and a cell-binding polymer described herein and comprise a Compound of Formula (III), e.g., Compound 101, on the outer capsule surface and optionally within the outer compartment.
• the present disclosure provides a method of treating a human patient with MPS-1 by administering to the patient a BBB -transporting IDUA fusion protein described herein.
• the administering step comprises implanting cells engineered to express and secrete the BBB-transporting IDUA fusion protein, which cells may be encapsulated in a macrodevice or in two-compartment alginate hydrogel capsules described herein.
• encapsulated cells expressing the BBB-transporting IDUA fusion protein are implanted into the peritoneal cavity (e.g., the lesser sac, also known as the omental bursa or bursalis omentum).
• the BBB-transporting IDUA fusion protein expressed and secreted by the implanted cells comprises SEQ ID NO:28 or SEQ ID NO:29.
• the therapeutic efficacy of treatment with the BBB-transporting IDUA fusion protein may be assessed using one or more efficacy measurements that have been used for approved and experimental MPS1 therapies.
• efficacy measurements typically include: reduction in urinary glycosaminoglycan (e.g., heparan sulfate) levels; reduction in liver volume; stable forced vital capacity; increase in 6-minute walk distance; improvement in the apnea/hypopnea index; increase in shoulder flexion; improvement in the Child Health Assessment Questionnaire/Health Assessment Questionnaire disability index.
• the efficacy measurement is taken at a desired timepoint after implant of the cells expressing the GGG-transporting IDUA fusion protein and is compared to the baseline level prior to implant.
• the desired timepoint is any one or more of 15 days, 30 days, 60 days, 120 days, one year or longer.
• treatment of a subject with a BBB-transporting IDUA fusion protein described herein results in reduced heparan sulfate levels in the subject’s brain and optionally in one or more non-CNS organs and tissues, e.g., liver, spleen, kidney, heart and lung.
• heparan sulfate levels are reduced by at least 10%, 25%, 50% or more at the desired timepoint.
• a fusion protein which comprises an N-terminal to C-terminal structure defined by formula I: AB-L1-RB-L2-C or by formula II: RB-L1-AB-L2-C, wherein in each of formula I and II:
• HSA human serum albumin
• LI which may be present or absent, comprises a first linker amino acid sequence
• RB comprises a domain that binds to the extracellular domain of human IGF1R (hIGFIR);
• L2 which may be present or absent, comprises a second linker amino acid sequence that is the same or different than the first linker amino acid sequence
• C is a cargo moiety.
• RB has a molecular weight of less than about any of 75 kDa, 50 kDa or 25 kDa.
• AB comprises first, second and third amino acid sequences corresponding to the three complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region of an anti- HSA antibody or (ii) AB comprises the CDR1, CDR2 and CDR3 sequences of the Rl l sdAb, R28 sdAb, M75 sdAb or M79 sdAb set forth in Table 2A.
• AB comprises, consists essentially of, or consists of the amino acid sequence from a single domain antibody (sdAb), optionally wherein the amino acid sequence is selected from the group consisting of the Rl l, R28, M75 and M79 amino acid sequences disclosed in Table 2B above, the Alb-1 and Alb-8 amino acid sequences described in Table II and Table III of WO 2006/22787, and the Alb-23 amino acid sequences described in WO 2012/175400.
• sdAb single domain antibody
• the fusion protein of any one of the above embodiments, wherein AB comprises, consists essentially of, or consists of the amino acid sequence from a sdAb.
• fusion protein of any one of embodiments 1 to 10 wherein AB consists essentially of, or consists of, an amino acid sequence of the heavy chain variable region of an antibody that cross-competes with a sdAb consisting of SEQ ID NO:4 or SEQ ID NO:5 for binding to HSA. 16.
• a sdAb consisting of SEQ ID NO:4 or SEQ ID NO:5 for binding to HSA. 16.
• the fusion protein of any one of the above embodiments, wherein the fusion protein binds via AB to domain 1 (DI) or domain 2 (DII) of HSA and does not substantially inhibit binding of human FcRn to HSA.
• fusion protein of any one of the above embodiments wherein the fusion protein binds via the AB domain to HSA with a dissociation constant (KD) affinity of less than about 0.1 nM to about 1,000 nM within a pH range of about 5.0 to about 7.4 as determined by surface plasmon resonance at 25° C.
• KD dissociation constant
• the fusion protein of any one of the above embodiments, wherein the fusion protein binds via the RB domain to hIGFIR expressed on the surface of human brain endothelial cells. 2. The fusion protein of any one of the above embodiments, wherein the fusion protein does not substantially bind to the human insulin receptor (h-IR). 3. The fusion protein of any one of the above embodiments, wherein the fusion protein does not substantially inhibit binding of insulin, insulin growth factor 1 (IGF1) or insulin growth factor 2 (IGF2) to hIGFIR. 4.
• IGF1 insulin growth factor 1
• IGF2 insulin growth factor 2
• fusion protein of any one of the above embodiments wherein the fusion protein binds via RB to at least one mammalian IGF1R ortholog at 25° C and within a pH range of about 5.0 to about 7.4.
• fusion protein of any one of the above embodiments wherein the fusion protein binds via RB to two or more mammalian IGF1R proteins selected from the group consisting of mouse IGF1R, rat IGF1R, hamster IGF1R, rabbit IGF1R, guinea pig IGF1R, dog IGF1R, cat IGF1R and a non-human primate IGF1R, optionally wherein the non-human primate IGF1R is cynomolgus IGF1R or rhesus monkey IGF1R.
• mammalian IGF1R proteins selected from the group consisting of mouse IGF1R, rat IGF1R, hamster IGF1R, rabbit IGF1R, guinea pig IGF1R, dog IGF1R, cat IGF1R and a non-human primate IGF1R, optionally wherein the non-human primate IGF1R is cynomolgus IGF1R or r
• RB comprises a set of first, second and third amino acid sequences corresponding to the three complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region of an anti- hlGFlR antibody or (ii) RB comprises a set of first, second and third amino acid sequences selected from the CDR1, CDR2 and CDR3 amino acid sequence of the IGF1R-5 sdAb, the IGF1R-3 sdAb and the IGF1R-4 sdAb set forth in Table 3 A above.
• RB CDR1 sequence is GRTIDNYA (SEQ ID NO:7) or a conservatively substituted variant thereof
• the RB CDR2 sequence is IDWGDGGX, where X is A or T (SEQ ID NO:8) or a conservatively substituted variant thereof
• the B3 CDR3 sequence is AMARQSRVNLDVARYDY (SEQ ID NO:9) or a conservatively substituted variant thereof.
• the RB CDR2 sequence is IDWGDGGA (SEQ ID NOTO).
• RB consists essentially of, or consists of: QVKLEESGGGLVQAGGSLRLSCAASGRTIDNYAMAWSRQAPGKDREFVATIDW GDGGARYANSVKGRFTISRDNAKGTMYLQMNNLEPEDTAVYSCAMARQSRVN LDVARYDYWGQGTQVTVSS (SEQ ID NO: 11) or a conservatively substituted variant thereof.
• RB consists essentially of, or consists of: QVQLVESGGGLVQPGGSLRLSCAASGRTIDNYAMAWVRQAPGKGLEWVATID WGDGGTRYANSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCAMARQSRV NLDVARYDYWGQGTLVTVSS (SEQ ID NO: 12) or a conservatively substituted variant thereof.
• RB consists essentially of, or consists of, an amino acid sequence of the heavy chain variable region of an antibody that (i) cross-competes with a sdAb consisting of SEQ ID NO: 11 or SEQ ID NO: 12 for binding to hIGFIR or (ii) cross-competes with any of the 996, 1226 and 1564 antibodies described in EP3725806A1.
• each of LI and L2 is a linker peptide that is less than 50 amino acids in length, optionally wherein the length of each of LI and L2 is between about 15 and 30 amino acids, or between about 20 and 25 amino acids.
• each of LI and L2 consists essentially of, or consists of: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 14).
• the cargo moiety is a cargo polypeptide selected from the group consisting of: an enzyme, a growth factor, a cytokine, or an antibody or antigen binding fragment thereof.
• the cargo polypeptide is an acid alpha-glucosidase protein (GAA), an alpha-galactosidase A (GLA) protein, an alpha-L-iduronidase (IDUA) protein, an alpha-N-acetyl-glucosaminidase (NAGLU) protein, a beta-glucoronidase (GUSB) protein, a beta-glucosidase (GBA) protein, an iduronate-2-sulfatase (IDS) protein, an heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT) protein, an N-acetylgalactosamine-6-sulfatase (GNS) protein, or an N- sulfoglucosamine sulfohydrolase (SGSH) protein, optionally wherein the cargo polypeptide is not an IDS protein.
• GAA acid alpha-glucosidase protein
• the fusion protein of any one of embodiments 46 to 48 which has an IDUA enzymatic activity that is within 80-120% of the corresponding enzymatic activity of wild-type human IDUA protein.
• the fusion protein of embodiment 50 which has an IDS enzymatic activity that is within 80-120% of the corresponding enzymatic activity of wild-type human IDS protein.
• a polynucleotide which comprises a first nucleotide sequence that encodes the fusion protein of any one of the above embodiments.
• polynucleotide of embodiment 56 wherein the first nucleotide sequence is operably linked to a nucleotide sequence encoding a secretory signal sequence for the fusion protein, optionally wherein the secretory signal sequence consists essentially of, or consists of, (i) MELGLSWVVLAALLQGVQA (SEQ ID NO:79) or (ii) one of the amino acid sequences set forth in Table 5.
• the polynucleotide of embodiment 56 which comprises the nucleotide sequence shown in FIG. 7.
• the polynucleotide of any one of embodiments 56 to 64 which is one strand in an isolated double-stranded DNA molecule.
• a genetically modified mammalian cell which is transiently or stably transfected with the polynucleotide of any one of embodiments 56 to 65.
• the genetically modified mammalian cell of embodiment 66 wherein the polynucleotide is inserted into at least one location in the genome of the mammalian cell.
• the genetically modified mammalian cell of embodiment 66 or 67 wherein the cell is derived from a human cell.
• the genetically modified mammalian cell of embodiment 68 which is derived from an RPE cell, optionally an ARPE-19 cell.
• the genetically modified mammalian cell of embodiment 68 which is derived from an induced pluripotent stem cell (iPSC) or a mesenchymal stem cell.
• iPSC induced pluripotent stem cell
• a composition comprising a plurality of genetically modified cells, wherein each cell in the plurality is a genetically modified cell as defined by any one of embodiments 68 to 70.
• the composition of embodiment 71, wherein the plurality of genetically modified cells is obtained from a culture of a monoclonal cell line.
• An implantable device comprising at least one cell-containing compartment which comprises the genetically modified cell of any one of embodiments 66 to 70 or the composition of embodiment 71 or 72 and further comprises at least one means for mitigating the foreign body response (FBR) when the device is implanted into the subject.
• FBR foreign body response
• the cell-containing compartment comprises a polymer composition, wherein the polymer composition comprises an alginate covalently modified with a peptide, wherein the peptide consists essentially of or consists of GRGDSP (SEQ ID NO:89), GGRGDSP (SEQ ID NO:90) or GGGRGDSP (SEQ ID NO:91).
• the polymer composition comprises an alginate covalently modified with a peptide, wherein the peptide consists essentially of or consists of GRGDSP (SEQ ID NO:89), GGRGDSP (SEQ ID NO:90) or GGGRGDSP (SEQ ID NO:91).
• the polymer composition comprises an alginate covalently modified with a peptide, wherein the peptide consists essentially of or consists of GRGDSP, and wherein the barrier compartment comprises an alginate chemically modified with or a pharmaceutically acceptable salt thereof.
• the implantable device of any one of embodiments 73 to 76 which is a spherical, two- compartment hydrogel capsule of about 0.75 mm to about 2 mm in diameter.
• each device in the preparation is a device of any one of embodiments 73 to 77.
• a hydrogel capsule comprising:
• an inner compartment which comprises a plurality of the genetically modified cell of any one of embodiments 66 to 70 encapsulated in a first polymer composition, wherein the first polymer composition comprises a hydrogel -forming polymer; and (b) a barrier compartment surrounding the inner compartment and comprising a second polymer composition, wherein the second polymer composition comprises an alginate covalently modified with at least one compound of Formula (III) or a pharmaceutically acceptable salt thereof.
• a capsule composition comprising a plurality of the hydrogel capsule of any one of embodiments 79 to 82 in a pharmaceutically acceptable carrier.
• a pharmaceutical composition comprising the fusion protein of any one of embodiments 1 to 55 and a pharmaceutically acceptable carrier.
• a method of preventing or treating a disease or condition in the central nervous system (CNS) of a subject which comprises:
• a method of treating a human subject diagnosed with Mucopolysaccharidosis type 1 (MPS-1) disease comprising:
• the disposing step comprises placing the capsule composition into the greater sac of the peritoneal cavity.
• the capsule composition produces a BBB-transporting IDUA fusion protein which comprises, consists essentially or consists of SEQ ID NO:28 or SEQ ID NO:29.
• Example 1 Effect of fusing an exemplary IGF1R binding domain to hIDUA on in vitro hIDUA activity.
• DNA expression vectors were engineered to encode six different hIDUA fusion enzymes containing a BBB-penetrant sdAb (IGF1R4) fused to the N-terminus or C-terminus of the hIDUA open reading frame via a variable length G4S linker unit repeated 3, 4 or 5 times.
• ARPE-19 cells were transfected with these expression vectors and polyclonal colonies stably expressing a hIDUA fusion enzyme were generated for each of the six different transfections.
• the expression vectors and transfected cells were named based upon the orientation of the encoding sequences for the composite modules with respect to one another. For example, hIDUA-(G4S)3-IGFlR4 cells express a fusion enzyme in which IGF1R4 is fused to the C-terminus of hIDUA via a G4S linker repeated 3 times.
• the in vitro IDUA activity of the hIDUA fusion proteins secreted from the six polyclonal colonies was assessed by seeding cells from each polyclonal colony at approximately 400,000 cells in 2 ml fresh medium per well of a 6-well tissue culture plate. Twenty to twenty-four hours later, the conditioned cell culture medium was collected and assayed for IDUA protein concentration using an IDUA activity assay, and compared to a known standard (laronidase), substantially as described in Ou, L., et al., (2014). Standardization of a-L-iduronidase enzyme assay with Michaelis-Menten kinetics. Molecular Genetics and Metabolism, 777(2), 113-115.
• the expression vector for this fusion was used to perform a new transfection of ARPE-19 cells, from which a higher hIDUA activity polyclonal pool was identified.
• Cells from this pool were encapsulated at 50 million cell/ml in the inner compartment of shielded capsules, essentially as described in WO2020198696.
• a dose of 0.5 ml capsules was implanted in the IP space of each of five MPS-1 mice. At 28 days post-administration, the mice were euthanized, and the amount of hIDUA activity in liver and plasma tissue samples was assessed.
• FIG. 8B shows the measured hIDUA activity levels compared with typical hIDUA levels observed in substantially similar experiments with implanted capsules encapsulating ARPE-19 cells genetically modified to express and secrete wild-type hIDUA. This comparison indicates that implanting MPS-1 mice with cells producing the IGFlR4-hIDUA fusion did not result in higher liver and plasma hIDUA activity compared to implanting cells producing wild-type hIDUA.
• Example 2 Effect of fusing exemplary HSA and IGF1R binding domains to hIDUA on in vitro hIDUA activity.
• DNA expression vectors were engineered to encode six different hIDUA fusion enzymes containing different orientations of open reading frames for a BBB-penetrant sdAb (IGF1R5), an anti-HSA sdAb (R28) and hIDUA with a (G4S)4 amino acid linker located between each of the open reading frames.
• IGF1R5 BBB-penetrant sdAb
• R28 anti-HSA sdAb
• G4S G4S
• the different orientations evaluated were: (i) 2 fusions in which the sdAbs were both fused to the N-terminus of hIDUA (N-terminal fusions); (ii) 2 fusions in which the sdAbs flanked hIDUA (flanking fusions); and (iii) 2 fusions in which the sdAbs were both fused to the C-terminus of hIDUA (C-terminal fusions).
• ARPE-19 cells were transfected with these expression vectors and polyclonal colonies stably expressing a hIDUA fusion enzyme were generated for each of the six different transfections.
• IGFlR5-R28-hIDUA cells express a fusion enzyme in which IGF1R5 is N-terminal to R28, which is itself N-terminal to hIDUA.
• the in vitro hIDUA activity of the hIDUA fusion proteins secreted from the six polyclonal colonies was assessed as described in Example 1, and the results are shown in Figure 9A.
• the polyclonal pools expressing the N-terminal double fusion enzymes (IGFlR5-R28-hIDUA and R28-IGFlR5-hIDUA) exhibited higher hIDUA activity levels than the cell lines expressing the flanking fusions or the C-terminal fusions, results that are consistent with the hIDUA activity results obtained in Example 1, which showed higher hIDUA activity generated for the single N- terminal fusions than the C-terminal fusions.
• the orientation of the anti-HSA and anti-IGFIR sdAbs in the N-terminal fusions had a significant effect on hIDUA activity, with the fusion containing R28 upstream of IGF1R5 generating about 50% higher hIDUA activity than the fusion with IGF1R-5 upstream of R28.
• the difference in activity between each of these six cell lines is statistically significant.
• the in vitro hIDUA activity in conditioned media from culturing cells from the topperforming double fusion polyclonal pool was compared to the in vitro hIDUA activity in conditioned media from culturing cells from the IGF1R4-(G4S)4- hlDUA polyclonal pool used for the MPS-1 mice experiment described in Example 1.
• FIG. 9B there was essentially no difference in the amount of hIDUA activity in the single N- terminal and double N-terminal fusion cultures.
• Example 3 Effect of HSA and IGF1R binding domains on in vivo hIDUA activity.
• FIG. 10 shows the measured hIDUA activity levels compared with typical hIDUA levels observed in substantially similar experiments with implanted shielded capsules encapsulating ARPE-19 cells genetically modified to express and secrete wild-type hIDUA.
• Mice implanted with cells expressing the R28-IGFlR5-hIDUA fusion enzyme had substantially higher hIDUA activity in tissues (except for kidney) and plasma than mice implanted with cells expressing the wild-type hIDUA enzyme.
• Example 4 Effect of implanting MPS-1 mice with shielded capsules producing an exemplary BBB -transporting fusion protein on brain heparan sulfate levels.
• Shielded capsules encapsulating cells from the polyclonal pool secreting the R28-(G4S)4- IGFlR5-(G4S)4-hIDUA fusion enzyme were implanted into the IP space of each of six MPS-1 mice.
• a group of six, untreated MPS-1 mice were observed as a control.
• both groups of mice were euthanized, and the amount of heparan sulfate in two distinct brain regions (hippocampus and frontal lobe) was assessed.
• tissue homogenates of each brain region were obtained, and tissue homogenates (10 ul) were combined with an equal volume of a heparinase cocktail (a mixture of heparinase I, heparinase II and heparinase III in a heparinase reaction buffer).
• a heparinase cocktail a mixture of heparinase I, heparinase II and heparinase III in a heparinase reaction buffer.
• the individual heparinases and heparinase reaction buffer were from New England Biolabs, catalogue numbers P0735S, P0736S, P0737S and B0735S).
• the reaction was incubated at 37° C for 3 days, and the resulting heparan sulfate disaccharides were quantified via LC/MS.
• MPS-1 mice implanted with cells secreting the hIDUA BBB- transporting fusion protein had a statistically significant reduction in heparan sulfate in both brain tissues compared to the control mice.
• Example 5 Double IDS fusions with different orientations of exemplary HSA and IGF1R binding domains.
• DNA expression vectors were engineered to encode two different hIDS fusion enzymes containing different orientations of open reading frames for a BBB-penetrant sdAb (IGF1R5), an anti-HSA sdAb (R28) and hIDS with a (G4S)4 amino acid linker located between each of the open reading frames.
• the different orientations evaluated were R28-(G4S)4-IGFlR5-(G4S)4-hIDS (e.g., a formula I fusion) and IGFlR5-(G4S)4-R28-(G4S)4-hIDS (e.g., a formula II fusion).
• ARPE-19 cells were transfected with one of the two expression vectors and polyclonal colonies stably expressing a hIDS fusion enzyme were generated for each of the two transfections.
• the in vitro IDS activity of the hIDS fusion proteins secreted from each polyclonal pool was assessed by seeding cells from each cell line at approximately 400,000 cells in 2 ml fresh medium per well of a 6-well tissue culture plate. Twenty to twenty-four hours later, the conditioned cell culture medium was collected and assayed for IDS protein concentration using a 2-step enzymatic IDS activity assay and compared to a known standard (idursulfase). In brief, 3ul of the cell culture medium was added to 15.6ul of water and 20ul buffer containing 20 mM lead(II) acetate in 0.2 M sodium acetate (pH 5.0), in a black 96-well plate.
• the substrate (4MU-a-iduronide 2-sulfate) is hydrolyzed by Iduronate 2-Sulfatase (IDS) to product 4MU-iduronide.
• Iduronate 2-Sulfatase activity is quenched by addition of excess phosphate, and a-L-iduronidase catalyzed the cleavage of the non-fluorescent product of the first reaction (4MU-iduronide) into a fluorescent product (4-MU). Fluorescence intensity was measured on a Biotek Cytation 3 at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively, in endpoint mode.
• Enzyme activity levels were compared to a standard curve generated with idursulfase, and the results are shown in FIG. 13.
• the polyclonal pools expressing the R28-(G4S)4-IGFlR5-(G4S)4-hIDS (formula I) double fusion enzyme exhibited higher hIDS activity levels than the cell lines expressing the IGFlR5-(G4S)4-R28-(G4S)4-hIDS (formula II) double fusion, results that are consistent with the hIDUA activity results obtained in Example 3, which showed higher hIDUA activity generated for the formula I fusion (AB-L1-RB- L2-C) than the formula II fusion (RB-L1-AB-L2-C).
• Example 6 Effect of implanting MPS-1 mice with shielded capsules producing an exemplary BBB -transporting fusion protein on heparan sulfate in systemic tissues.
• Shielded capsules encapsulating cells from the polyclonal pool secreting the R28-(G4S)4- IGFlR5-(G4S)4-hIDUA fusion enzyme were implanted into the IP space of each of six MPS-1 mice.
• a group of six, untreated MPS-1 mice were observed as a control.
• both groups of mice were euthanized, and the amount of heparan sulfate in various organs (liver, spleen, kidney, lung and heart) was assessed.
• organ tissue homogenates were obtained, and the tissue homogenates (10 ul) were combined with an equal volume of a heparinase cocktail (a mixture of heparinase I, heparinase II and heparinase III in a heparinase reaction buffer).
• a heparinase cocktail a mixture of heparinase I, heparinase II and heparinase III in a heparinase reaction buffer.
• the individual heparinases and heparinase reaction buffer were from New England Biolabs, catalogue numbers P0735S, P0736S, P0737S and B0735S.
• the reaction was incubated at 37° C for 3 days, and the resulting heparan sulfate disaccharides were quantified via LC/MS.
• MPS-1 mice treated with the dual fusion enzyme exhibited statistically significant reduction in heparan sulfate levels relative to untreated mice (grey bars) in each of the liver, spleen, kidney, lung and heart tissue samples.
• Example 7 Assessment of immunogenicity potential of an exemplary BBB -transporting fusion protein.
• R28-(G4S)4-IGFlR5-(G4S)4-hIDUA fusion enzyme (2) R28-H5-(G4S)4-IGF lR5-H2-(G4S)4-hIDUA fusion enzyme, in which the R28- H5 component is a humanized R28 variant with the amino acid sequence (SEQ ID NO:5), and the IGF1R5-H2 component is a humanized IGF1R5 variant with the amino acid of (SEQ ID NO: 12); (3) native human IDUA protein alone (i.e., not fused to any other molecule); and (4) the individual parental and humanized sdAbs present in the two fusion enzymes.
• EpiVax evaluated the amino acid sequences of the fusion constructs and of the individual comprising molecules for predicted T-cell epitopes using their proprietary EpiMatrix algorithm. Each molecule was scored for total predicted T-cell epitope content and ranked against the EpiMatrix Protein Immunogenicity Scale where the molecules could be compared directly to proteins of known immunogenicity. As shown in the table immediately below, each of the fusion enzymes had a lower predicted immunogenicity score than native hIDUA alone.
• Example 8 Comparison of exemplary humanized and camelid BBB-transporting fusion proteins in vivo.
• tissue homogenates were obtained, and tissue homogenates (10 ul) were combined with an equal volume of a heparinase cocktail (a mixture of heparinase I, heparinase II and heparinase III in a heparinase reaction buffer.
• the individual heparinases and heparinase reaction buffer were from New England Biolabs, catalogue numbers P0735S, P0736S, P0737S and B0735S.

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