Long-acting GM-CSF and method of use

Abstract

The present invention provides compositions comprising granulocyte-macrophage colony-stimulating factor (GM-CSF) peptides and GM-CSF peptides, which may be useful for the treatment of neurological disorders or conditions. [Solution] A composition comprising a granulocyte-macrophage colony-stimulating factor (GM-CSF) peptide and a GM-CSF peptide is provided, comprising a first polypeptide comprising granulocyte-macrophage colony-stimulating factor (GM-CSF) and a second polypeptide comprising a sequence that is at least 98% identical to a specific sequence.

Description

[Technical Field]

[0001] cross reference This application claims the rights of International Application PCT / CN2020 / 074834, filed on 12 February 2020, which is incorporated herein by reference in its entirety.

[0002] Parkinson's disease (PD) is a progressive neurodegenerative disease associated with a substantial morbidity, increased mortality, and particularly high economic burden. [Background technology]

[0003] Background of the Invention The progression of Parkinson's disease (PD) and other neurodegenerative conditions is associated with inflammation. Preclinically, GM-CSF therapy modulates congenital microglial immunity and regulatory T cells (T) that migrate from the periphery to the brain. reg ) increases, resulting in anti-inflammatory and neuroprotective responses. In one embodiment, a GM-CSF molecule for the treatment of neurodegenerative and / or inflammation-related conditions is provided herein. In some embodiments, the GM-CSF molecule provided herein is used for the treatment of one or more of the following conditions: Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), acute radiation syndrome, traumatic brain injury, cancer, and Crohn's disease (CD). [Overview of the project]

[0004] Due to the limited bioavailability and short half-life of GM-CSF, GM-CSF peptide therapeutics have required high doses and daily administration. Mild to moderate adverse events such as injection site reactions, elevated white blood cell counts, and bone pain have been experienced with daily GM-CSF treatment. In one embodiment, provided herein are long-acting GM-CSF molecules comprising GM-CSF attached to a scaffold to increase the half-life of GM-CSF. The exemplary scaffold includes an antibody-variable domain. Furthermore, the various long-acting GM-CSF molecules provided herein have increased the bioavailability of GM-CSF compared to GM-CSF peptide alone. The various long-acting GM-CSF molecules provided herein can be administered at frequencies ranging from once every 7 days to once every 1 month, for example, once every 2 weeks.

[0005] In one embodiment, the herein provides a composition comprising a first polypeptide containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and a second polypeptide containing a sequence that is at least 98% identical to SEQ ID NO: 2. In some embodiments, the GM-CSF contains a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16. In some embodiments, the GM-CSF contains a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 77. In some embodiments, the GM-CSF comprises human GM-CSF or mouse GM-CSF. In some embodiments, the first polypeptide includes a modified light chain of the antibody variable domain. In some embodiments, the modified light chain of the antibody variable domain includes a GM-CSF located between the first amino acid sequence of the antibody variable domain and the second amino acid sequence of the antibody variable domain. In some embodiments, the first amino acid sequence includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. In some embodiments, the first amino acid sequence includes SEQ ID NO: 14. In some embodiments, the second amino acid sequence includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15. In some embodiments, the second amino acid sequence includes SEQ ID NO: 15. In some embodiments, the GM-CSF is located within the complementarity-determining region (CDR) of the modified light chain. In some embodiments, the GM-CSF is located within the light chain CDR1, CDR2, or CDR3. In some embodiments, the GM-CSF is located within the light chain CDR3. In some embodiments, the modified light chain is modified from a variable light chain containing the sequence of SEQ ID NO: 17. In some embodiments, the first polypeptide further comprises a first linker peptide. In some embodiments, the first linker peptide contains SEQ ID NO: 10.In some embodiments, the first linker peptide includes the sequence of SEQ ID NO: 8. In some embodiments, the first linker peptide includes the sequence of SEQ ID NO: 11. In some embodiments, the first linker peptide includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 12. In some embodiments, the first polypeptide further includes a second linker peptide. In some embodiments, the second linker peptide includes the sequence of SEQ ID NO: 10. In some embodiments, the second linker peptide includes the sequence of SEQ ID NO: 9. In some embodiments, the second linker peptide includes the sequence of SEQ ID NO: 11. In some embodiments, the second linker peptide includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 13. In some embodiments, the first polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18. In some embodiments, the first polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6. In some embodiments, the first polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7. In some embodiments, the first polypeptide comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the second polypeptide comprises a heavy chain of the antibody variable region. In some embodiments, the second polypeptide comprises the sequence of SEQ ID NO: 2. In some embodiments, the second polypeptide further comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4.In some embodiments, the second polypeptide comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, the first polypeptide and the second polypeptide are connected via one or more disulfide bonds. In some embodiments, the first polypeptide and the second polypeptide form an antibody variable domain. In some embodiments, the antibody variable domain is about 10. -2 M, 10 -3 M, or 10 -4 The equilibrium dissociation constant (K D ) does not bind to the antigen at less than. In some embodiments, the antibody variable domain comprises a modified paritumab variable domain. In some embodiments, the modified paritumab variable domain comprises a heavy chain CDR1 comprising the sequence of SEQ ID NO: 19. In some embodiments, the modified paritumab variable domain comprises a heavy chain CDR2 comprising the sequence of SEQ ID NO: 20. In some embodiments, the modified paritumab variable domain comprises a heavy chain CDR3 comprising the sequence of SEQ ID NO: 21. In some embodiments, the modified paritumab variable domain comprises a light chain CDR1 comprising the sequence of SEQ ID NO: 22. In some embodiments, the modified paritumab variable domain comprises a light chain CDR2 comprising the sequence of SEQ ID NO: 23. In some embodiments, the modified paritumab variable domain comprises a light chain CDR3 comprising the sequence of SEQ ID NO: 24, 77 or 16. In some embodiments, the modified paritumab variable domain is about 10 -2 M, 10 -3 M, or 10 -4 The K of less than M DIt does not bind to respiratory syncytial virus (RSV). In some embodiments, the composition further comprises an Fc region comprising reduced effector function compared to human IgG1. In some embodiments, human IgG1 comprises the sequence of SEQ ID NO: 25. In some embodiments, reduced effector function comprises reduced antibody-dependent cytotoxicity (ADCC). In some embodiments, reduced effector function comprises reduced complement-dependent cytotoxicity (CDC). In some embodiments, the first polypeptide further comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3, and / or the first polypeptide comprises an Fc region comprising human IgG1 comprising E233P, L234V, L235A, ΔG236, A327G, A330S, P331S by Kabat numbering.

[0006] In one embodiment, the herein provides a composition comprising an antibody variable domain comprising a light chain sequence comprising a first polypeptide having a sequence at least about 90% identical to SEQ ID NO: 6, and a heavy chain sequence comprising a second polypeptide having a sequence at least about 90% identical to SEQ ID NO: 2. In some embodiments, the first polypeptide comprises a sequence at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6. In some embodiments, the second polypeptide comprises a sequence at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the composition comprises GM-CSF. In some embodiments, GM-CSF is human GM-CSF or mouse GM-CSF. In some embodiments, the GM-CSF contains a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 16. In some embodiments, the GM-CSF contains a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 77. In some embodiments, the light chain contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 7. In some embodiments, the light chain includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 5. In some embodiments, the heavy chain includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 4. In some embodiments, the heavy chain includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 1.In some embodiments, the composition further comprises an Fc region comprising reduced effector function compared to human IgG1. In some embodiments, human IgG1 comprises the sequence of SEQ ID NO: 25. In some embodiments, reduced effector function comprises reduced antibody-dependent cytotoxicity (ADCC). In some embodiments, reduced effector function comprises reduced complement-dependent cytotoxicity (CDC). In some embodiments, the heavy chain further comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3, and / or the heavy chain comprises an Fc region comprising human IgG1 comprising E233P, L234V, L235A, ΔG236, A327G, A330S, P331S by Kabat numbering.

[0007] In one embodiment, provided herein is Sequence ID No. 26 [ka] The composition comprises an antibody variable domain, comprising a light chain sequence containing a sequence that is at least about 90% identical to sequence number 2, wherein the light chain sequence contains X1, and X1 contains GM-CSF, and a heavy chain sequence containing a sequence that is at least about 90% identical to sequence number 2. In some embodiments, GM-CSF is human GM-CSF or mouse GM-CSF. In some embodiments, GM-CSF contains a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 16. In some embodiments, GM-CSF contains a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 77. In some embodiments, the light chain sequence includes a sequence that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 26. In some embodiments, the light chain sequence includes sequence number 27 [ka] comprising a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, wherein the light chain sequence comprises X2, and X2 comprises GM-CSF. In some embodiments, the heavy chain comprises a sequence that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the light chain comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 7. In some embodiments, the light chain comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5. In some embodiments, the heavy chain comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, the heavy chain comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1. In some embodiments, the composition further comprises an Fc region that comprises a reduced effector function as compared to human IgG1. In some embodiments, human IgG1 comprises the sequence of SEQ ID NO: 25. In some embodiments, the reduced effector function comprises a reduction in antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the reduced effector function comprises a reduction in complement-dependent cytotoxicity (CDC). In some embodiments, the heavy chain further comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3, and / or the heavy chain comprises an Fc region comprising human IgG1 comprising E233P, L234V, L235A, ΔG236, A327G, A330S, P331S according to Kabat numbering.

[0008] In one embodiment, provided herein is a composition comprising a sequence that is at least about 90% identical to Sequence ID No. 18. In some embodiments, the sequence is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No. 18. In some embodiments, the sequence is attached to an antibody domain. In some embodiments, the antibody domain is an antibody variable domain. In some embodiments, the sequence is located within the antibody domain. In some embodiments, the sequence is located within the CDR of the antibody variable domain. In some embodiments, the sequence is located within the CDR of a modified trastuzumab antibody variable domain. In some embodiments, the sequence is located within the CDR of a modified palivizumab antibody variable domain. In some embodiments, the composition includes a region that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 42, wherein the region includes X5, and X5 includes the sequence. In some embodiments, the composition further includes a region that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 31. In some embodiments, the composition includes a region that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 43, wherein the region includes X6, and X6 includes the sequence. In some embodiments, the composition further comprises a region that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the composition further comprises an Fc region that includes reduced effector function compared to human IgG1. In some embodiments, the human IgG1 includes the sequence of SEQ ID NO: 25. In some embodiments, the reduced effector function includes reduced antibody-dependent cytotoxicity (ADCC). In some embodiments, the reduced effector function includes reduced complement-dependent cytotoxicity (CDC).In some embodiments, the Fc region comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3, and / or the Fc region comprises a human IgG1 comprising E233P, L234V, L235A, ΔG236, A327G, A330S, P331S according to Kabat numbering.

[0009] In one aspect, provided herein is a composition comprising a first polypeptide comprising the sequences of SEQ ID NOs: 22, 23, and 16, and a second polypeptide comprising the sequences of SEQ ID NOs: 19 - 21. In one aspect, provided herein is a composition comprising a first polypeptide comprising the sequences of SEQ ID NOs: 22, 23, and 77, and a second polypeptide comprising the sequences of SEQ ID NOs: 19 - 21. In some embodiments, the first polypeptide is the light chain of an antibody variable domain. In some embodiments, the second polypeptide is the heavy chain of an antibody variable domain. In some embodiments, the first polypeptide comprises the sequence of SEQ ID NO: 24. In some embodiments, the composition further comprises an Fc region comprising a reduced effector function as compared to human IgG1. In some embodiments, the human IgG1 comprises the sequence of SEQ ID NO: 25. In some embodiments, the reduced effector function comprises a reduction in antibody - dependent cell - mediated cytotoxicity (ADCC). In some embodiments, the reduced effector function comprises a reduction in complement - dependent cytotoxicity (CDC). In some embodiments, the second polypeptide further comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3, and / or the second polypeptide comprises an Fc region comprising a human IgG1 comprising E233P, L234V, L235A, ΔG236, A327G, A330S, P331S according to Kabat numbering.

[0010] In one embodiment, the composition provided herein comprises a first polypeptide comprising the sequences of SEQ ID NOs: 37-39 and a second polypeptide comprising the sequences of SEQ ID NOs: 34, 35, and 16. In one embodiment, the composition provided herein comprises a first polypeptide comprising the sequences of SEQ ID NOs: 37-39 and a second polypeptide comprising the sequences of SEQ ID NOs: 34, 35, and 77. In some embodiments, the first polypeptide is the light chain of the antibody variable domain. In some embodiments, the second polypeptide is the heavy chain of the antibody variable domain. In some embodiments, the first polypeptide comprises the sequence of SEQ ID NO: 36. In some embodiments, the composition further comprises an Fc region that includes reduced effector function compared to human IgG1. In some embodiments, human IgG1 comprises the sequence of SEQ ID NO: 25. In some embodiments, the reduced effector function includes reduced antibody-dependent cytotoxicity (ADCC). In some embodiments, the reduced effector function includes reduced complement-dependent cytotoxicity (CDC). In some embodiments, the second polypeptide further comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3, and / or the second polypeptide comprises an Fc region containing human IgG1, including E233P, L234V, L235A, ΔG236, A327G, A330S, P331S by Kabat numbering.

[0011] In one embodiment, provided herein is a composition comprising a first polypeptide comprising the sequence of SEQ ID NO: 31 and a second polypeptide comprising granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the GM-CSF comprises a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16. In some embodiments, the GM-CSF comprises a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 77. In some embodiments, the GM-CSF comprises human GM-CSF or mouse GM-CSF. In some embodiments, the second polypeptide comprises a modified heavy chain of the antibody variable region. In some embodiments, the modified heavy chain of the antibody variable domain includes a GM-CSF located between the first amino acid sequence of the antibody variable region and the second amino acid sequence of the antibody variable region. In some embodiments, the first amino acid sequence includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32. In some embodiments, the first amino acid sequence includes the sequence of SEQ ID NO: 32. In some embodiments, the second amino acid sequence includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33. In some embodiments, the second amino acid sequence includes the sequence of SEQ ID NO: 33. In some embodiments, the GM-CSF is located within the complementarity-determining region (CDR) of the modified heavy chain. In some embodiments, GM-CSF is located within the heavy chain CDR1, CDR2, or CDR3; in some embodiments, GM-CSF is located within the heavy chain CDR3. In some embodiments, the modified heavy chain is modified from a variable heavy chain containing the sequence of SEQ ID NO: 44. In some embodiments, the second polypeptide further comprises the first linker peptide. In some embodiments, the first linker peptide contains the sequence of SEQ ID NO: 10.In some embodiments, the first linker peptide contains the sequence of SEQ ID NO: 8. In some embodiments, the first linker peptide contains the sequence of SEQ ID NO: 11. In some embodiments, the first linker peptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 12. In some embodiments, the second polypeptide further contains a second linker peptide. In some embodiments, the second linker peptide contains the sequence of SEQ ID NO: 10. In some embodiments, the second linker peptide contains the sequence of SEQ ID NO: 9. In some embodiments, the second linker peptide contains the sequence of SEQ ID NO: 11. In some embodiments, the second linker peptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 13. In some embodiments, the second polypeptide includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 18. In some embodiments, the second polypeptide includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 29. In some embodiments, the second polypeptide includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 4. In some embodiments, the second polypeptide comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28. In some embodiments, the first polypeptide comprises a light chain of the antibody variable region. In some embodiments, the first polypeptide comprises the sequence of SEQ ID NO: 31. In some embodiments, the first polypeptide further comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.In some embodiments, the second polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 30. In some embodiments, the first polypeptide and the second polypeptide are linked by one or more disulfide bonds. In some embodiments, the first polypeptide and the second polypeptide form an antibody variable domain. In some embodiments, the antibody variable domain is about 10. -2 M, 10 -3 M, or 10 -4 Equilibrium dissociation constant (K) less than M D ) does not bind to the antigen. In some embodiments, the antibody variable domain includes a modified trastuzumab variable domain. In some embodiments, the modified trastuzumab variable domain includes a heavy chain CDR1 containing the sequence of SEQ ID NO: 34. In some embodiments, the modified trastuzumab variable domain includes a heavy chain CDR2 containing the sequence of SEQ ID NO: 35. In some embodiments, the modified trastuzumab variable domain includes a heavy chain CDR3 containing the sequence of SEQ ID NO: 36, 77, or 16. In some embodiments, the modified trastuzumab variable domain includes a light chain CDR1 containing the sequence of SEQ ID NO: 37. In some embodiments, the modified trastuzumab variable domain includes a light chain CDR2 containing the sequence of SEQ ID NO: 38. In some embodiments, the modified trastuzumab variable domain includes a light chain CDR3 containing the sequence of SEQ ID NO: 39. In some embodiments, the modified trastuzumab variable domain includes about 10 -2 M, 10 -3 M, or 10 -4 Less than M K DIt does not bind to human epidermal growth factor receptor 2 (Her2). In some embodiments, the composition further comprises an Fc region comprising reduced effector function compared to human IgG1. In some embodiments, human IgG1 comprises the sequence of SEQ ID NO: 25. In some embodiments, reduced effector function comprises reduced antibody-dependent cytotoxicity (ADCC). In some embodiments, reduced effector function comprises reduced complement-dependent cytotoxicity (CDC). In some embodiments, the second polypeptide further comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3, and / or the second polypeptide comprises an Fc region comprising human IgG1 comprising E233P, L234V, L235A, ΔG236, A327G, A330S, P331S by Kabat numbering.

[0012] In one embodiment, the herein provides a composition comprising an antibody variable domain comprising a light chain sequence comprising a first polypeptide having a sequence at least about 90% identical to SEQ ID NO: 31, and a heavy chain sequence comprising a second polypeptide having a sequence at least about 90% identical to SEQ ID NO: 29. In some embodiments, the first polypeptide comprises a sequence at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31. In some embodiments, the second polypeptide comprises a sequence at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29. In some embodiments, the composition comprises GM-CSF. In some embodiments, GM-CSF is human GM-CSF or mouse GM-CSF. In some embodiments, the GM-CSF contains a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 16. In some embodiments, the GM-CSF contains a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 77. In some embodiments, the light chain contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 7. In some embodiments, the light chain includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 30. In some embodiments, the heavy chain includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 4. In some embodiments, the heavy chain includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 28.In some embodiments, the composition further comprises an Fc region comprising reduced effector function compared to human IgG1. In some embodiments, human IgG1 comprises the sequence of SEQ ID NO: 25. In some embodiments, reduced effector function comprises reduced antibody-dependent cytotoxicity (ADCC). In some embodiments, reduced effector function comprises reduced complement-dependent cytotoxicity (CDC). In some embodiments, the heavy chain further comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3, and / or the heavy chain comprises an Fc region comprising human IgG1 comprising E233P, L234V, L235A, ΔG236, A327G, A330S, P331S by Kabat numbering.

[0013] In one embodiment, provided herein is Sequence ID No. 42 [ka] A composition comprising an antibody variable domain comprising a heavy chain sequence comprising a sequence that is at least about 90% identical to sequence number 31, wherein the heavy chain sequence comprises X6, X6 comprises GM-CSF, and the light chain sequence comprises a sequence that is at least about 90% identical to sequence number 31. In some embodiments, GM-CSF is human GM-CSF or mouse GM-CSF. In some embodiments, GM-CSF comprises a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 16. In some embodiments, GM-CSF comprises a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 77. In some embodiments, the heavy chain sequence includes a sequence that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 42. In some embodiments, the heavy chain sequence includes sequence number 43. [ka] The heavy chain sequence includes a sequence that is at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 43, where the heavy chain sequence includes X6, and X6 includes GM-CSF. In some embodiments, the heavy chain sequence includes a sequence that is at least approximately 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 43. In some embodiments, the light chain includes a sequence that is at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 7. In some embodiments, the heavy chain includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 29. In some embodiments, the heavy chain includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 4. In some embodiments, the heavy chain includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 28. In some embodiments, the composition further includes an Fc region that exhibits reduced effector function compared to human IgG1. In some embodiments, human IgG1 includes the sequence of sequence number 25. In some embodiments, the reduction in effector function includes a decrease in antibody-dependent cytotoxicity (ADCC). In some embodiments, the reduction in effector function includes a decrease in complement-dependent cytotoxicity (CDC). In some embodiments, the heavy chain further comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3, and / or the heavy chain comprises an Fc region containing human IgG1, including E233P, L234V, L235A, ΔG236, A327G, A330S, P331S by Kabat numbering.

[0014] In one embodiment, this specification provides a method of use of the compositions described herein for the treatment of a neurological disorder or condition. Also provided herein is a method of treating a neurological disorder or condition, comprising the step of administering one of the compositions described herein to a subject of need. In some embodiments, the neurological disorder or condition includes Parkinson's disease. Also provided herein is a method of treating Alzheimer's disease, comprising the step of administering one of the compositions described herein to a subject of need. Also provided herein is a method of treating traumatic brain injury, comprising the step of administering one of the compositions described herein to a subject of need. Also provided herein is a method of treating amyotrophic lateral sclerosis (ALS), comprising the step of administering one of the compositions described herein to a subject of need. Also provided herein is a method of treating acute radiation syndrome, comprising the step of administering one of the compositions described herein to a subject of need. Also provided herein is a method of treating cancer, comprising the step of administering one of the compositions described herein to a subject of need. In some embodiments, the composition is administered once every approximately 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days during the treatment period. In some embodiments, the composition is administered once every approximately 14 days during the treatment period. In some embodiments, the composition is administered once every approximately 2 weeks during the treatment period. In some embodiments, the composition is administered once every approximately 3 weeks during the treatment period. In some embodiments, the composition is administered once every approximately 4 weeks during the treatment period. In some embodiments, the composition is administered once every approximately 1 month during the treatment period. In some embodiments, the treatment period ranges from approximately 8 weeks to approximately 2 years. [Brief explanation of the drawing]

[0015] The summary above, as well as the detailed description of the disclosure below, will be better understood when read in conjunction with the accompanying drawings. However, this disclosure is not limited to the exact examples shown and in accordance with common practice, and various features in the drawings are not to scale. In some cases, for clarity, the dimensions of various features have been arbitrarily enlarged or reduced. The drawings include the following figures: [Figure 1-1] Her-mGMCSF CDR treatment affects peripheral blood and spleen T cell populations. Quantification of CD8+ levels (Figure 1A), CD4+ levels (Figure 1B), and CD4+CD25+FoxP3+ regulatory T cell (Treg) levels (Figure 1C) in peripheral blood of mice treated with escalating doses of Her-mGMCSF CDR. Mean differences (±SEM, n=5) were determined, with p<0.05 compared to treatments of (a) 0 mg / kg, (b) 0.3 mg / kg, (c) 1.0 mg / kg, (d) 3.0 mg / kg, or (e) 10.0 mg / kg. Quantification of CD8+ levels (Figure 1D), CD4+ levels (Figure 1E), and CD4+CD25+FoxP3+Treg levels (Figure 1F) in peripheral blood of mice treated with escalating doses of rGM-CSF. [Figure 1-2]Her-mGMCSF CDR treatment affects peripheral blood and spleen T cell populations. Quantification of CD8+ levels (Figure 1G), CD4+ levels (Figure 1H), and CD4+CD25+FoxP3+Treg levels (Figure 1I) in spleens isolated from mice treated with escalating doses of Her-mGMCSF CDR. Mean differences (±SEM, n=5) were determined, and p<0.05 was observed compared to (a) 0 mg / kg treatment, (b) 0.3 mg / kg treatment, (c) 1.0 mg / kg treatment, and (d) 3.0 mg / kg treatment. Quantification of CD8+ levels (Figure 1J), CD4+ levels (Figure 1K), and CD4+CD25+FoxP3+Treg levels (Figure 1L) in spleens isolated from mice treated with escalating doses of rGM-CSF. The difference in mean values ​​(±SEM, n=5) was determined, and p<0.05 was observed compared to (a) 0 mg / kg, (b) 0.01 mg / kg, (c) 0.03 mg / kg, or (d) 0.10 mg / kg rGM-CSF treatment. Treatment with both Her-mGMCSF CDR and rGM-CSF resulted in a dose-dependent significant increase in splenic Treg levels. The results of linear regression analysis are shown in graphs (Figure 1I) and (Figure 1L). [Figure 2] Her-mGMCSF CDR treatment reduces neuroinflammatory responses observed after MPTP poisoning. Quantification of reactive microglia in the substantia nigra 2 days after MPTP poisoning. Mean differences (±SEM, n=5) were determined, and p<0.05 compared to (a) PBS or (b) MPTP treatment. [Figure 3] Her-mGMCSF CDR treatment preserves (spare) dopaminergic neurons after MPTP poisoning. Stereometric quantification of the total number of surviving dopaminergic (TH+ / Nissl+) and non-dopaminergic (TH- / Nissl+) neurons in the substantia nigra after MPTP poisoning. Mean differences (±SEM, n=7) were determined, and p<0.05 was observed compared to the groups treated with (a)PBS or (b)MPTP. The mean percentage of the remaining total number of neurons is shown for each treatment bar. [Figure 4]Her-mGMCSF CDR treatment reduces terminal striatal loss. Densitometry analysis of TH+ terminals in the striatum after MPTP poisoning. Treatment groups were normalized to PBS control density. The difference in mean values ​​(±SEM, n=7) was determined, and p<0.05 was observed compared to groups treated with (a) PBS or (b) MPTP. [Figure 5] Figures 5A-B show that Her-mGMCSF CDR treatment exhibits long-acting anti-inflammatory and immunomodulatory properties. Figure 5A: Quantification of reactive microglia (mac-1+) in the substantia nigra 2 days after MPTP poisoning. Mean differences (±SEM, n=5) were determined, and p<0.05 compared to (a) PBS or (b) MPTP treatment. Figure 5B: Stereometric quantification of the total number of surviving dopaminergic (TH+ / Nissl+) and non-dopaminergic (TH- / Nissl+) neurons in the substantia nigra 7 days after MPTP poisoning. Mean differences (±SEM, n=5) were determined, and p<0.05 compared to groups treated with (a) PBS, (b) MPTP, (c) -15 day Her-mGMCSF CDR+MPTP, and (d) -10 day Her-mGMCSF CDR+MPTP. [Figure 6] Figures 6A-B: Efficacy of long-acting GM-CSF molecules Syn hGMCSF CDRL3, NhGM Syn HC, and NhGM Syn LC (Figure 6A), and Her hGMCSF CDR (Figure 6B) in a TF-1 proliferation assay. [Figure 7] Schematic diagrams of the Fab domains of various long-acting GM-CSF molecules, where GM-CSF is located at the amino terminus or CDR of the IgG scaffold. [Figure 8A] Figures 8A-B: GM-CSF molecules Syn-hGMCSF CDR, Her-hGMCSF CDR, Syn-mGMCSF CDR, and Syn-mGMCSF NT (HC fusion) show increased half-lives compared to recombinant GM-CSF in rat and mouse plasma, respectively. Figure 8A shows the concentrations of Syn-hGMCSF CDR and Her-hGMCSF CDR in rat plasma over time. [Figure 8B]Figures 8A-B: GM-CSF molecules Syn-hGMCSF CDR, Her-hGMCSF CDR, Syn-mGMCSF CDR, and Syn-mGMCSF NT (HC fusion) show increased half-lives compared to recombinant GM-CSF in rat and mouse plasma, respectively. Figure 8B shows the concentrations of Syn-mGMCSF CDR and Syn-mGMCSF NT (N-terminal HC fusion) in mouse plasma over time. [Figure 9] Subchronic treatment using Syn mGMCSF CDR significantly increases the enlargement of Tregs in mice. [Figure 10] The long-acting GM-CSF, Her-hGMCSF CDR, increased Treg enlargement for up to 14 days. [Figure 11] Pharmacokinetic and pharmacodynamic studies in cynomolgus monkeys. Her-hGMCSF CDR increases circulating Treg in a dose-dependent manner. [Figure 12] Long-acting GM-CSF is actively transported to the mouse brain. [Modes for carrying out the invention]

[0016] Detailed description of the invention This specification describes GM-CSF molecules and compositions comprising GM-CSF molecules. Exemplary molecules include GM-CSF peptides attached to a scaffold that significantly increases the half-life of the GM-CSF peptide compared to that of GM-CSF alone. Such molecules may be referred to as long-acting GM-CSF molecules. Exemplary scaffolds for increasing the GM-CSF half-life include an antibody-variable domain, where GM-CSF is optionally attached to the antibody-variable domain via one or more linkers. In some cases, the antibody-variable domain reduces antigen binding or has no antigen binding. The reduction in antigen binding may be produced by modifying the complementarity-determining region (CDR) of the antibody-variable domain. Modifications may include insertion of a GM-CSF peptide and / or mutation, addition, or deletion of one or more CDR amino acids. The antibody scaffold may also include fragment crystallizable (FC) regions, which exhibit reduced effector functions such as decreased antibody-dependent cytotoxicity (ADCC) and / or reduced complement-dependent cytotoxicity (CDC) compared to antibody scaffolds containing wild-type IgG1 Fc regions.

[0017] The various GM-CSF molecules described herein may have been beneficial for Parkinson's disease (PD) and / or other neurodegenerative and neuroinflammatory diseases. REG To increase the number and / or function. For example, as shown in the examples herein, treatment (treatment) with long-acting GM-CSF, after a single injection, T REG A dose-dependent increase in the number of cells was observed, which resulted in increased cellular function in peripheral blood and spleen, more so than that observed with recombinant GM-CSF (RGM-CSF) alone. Furthermore, T14 isolated from mice treated with long-acting GM-CSF reg This showed increased antiproliferative effect, and T was isolated from mice treated with RGM-CSF. reg This method was able to suppress TRESP proliferation to a greater extent than other methods. Clinically, it was able to suppress the T of disease. reg Population modification has been tested in both ALS and PD. ALS patients have a high incidence of dysfunction that correlates with disease severity and survival. regThis indicates the T of disease. reg When isolated and stimulated exogenically, its inhibitory function is restored, suggesting a potential therapeutic target. The various GM-CSF molecules described herein also exhibit neuroprotective properties. As shown in the examples, a single dose of long-acting GM-CSF was neuroprotective in the MPTP mouse model.

[0018] Before describing the current methods and compositions, it should be understood that this disclosure is not limited to the specific methods or compositions described. The terminology used is for the sole purpose of describing specific embodiments and is not intended to limit them, for the scope of this disclosure is limited only by the appended claims. The examples are provided to those skilled in the art to disclose and describe how the compositions and methods of the present invention are made and used, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to indicate that the following experiments are all or only those that have been performed. Efforts have been made to ensure accuracy with respect to the figures used (e.g., quantities, temperatures, etc.), however, some degree of experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0019] Where a range of values ​​is provided, it is understood that each intervening value up to one-tenth of the lower limit unit is also specifically disclosed between the upper and lower limits of that range, unless the context clearly indicates otherwise. Each smaller range between any mentioned value or intervening value within the mentioned range and any other mentioned value or intervening value within that mentioned range is included in the invention. The upper and lower limits of these smaller ranges may be independently included in or excluded from that range, and each range in which either limit is included in the smaller range, or both limits are included in the smaller range, is also included in the invention and is below the limits specifically excluded within the mentioned range. If the scope mentioned includes one or both of the limits, the scope excluding one or both of these included limits is also included in the present invention.

[0020] When used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context explicitly indicates otherwise. For example, a reference to “cell” includes multiple such cells, and a reference to “peptide” includes one or more peptides and their equivalents, such as polypeptides known to those skilled in the art.

[0021] The terms “complementarity-determining region” and “CDR,” which are synonymous with “hypervariable region” or “HVR,” are known in the art to refer to discontinuous sequences of amino acids within an antibody variable region that confer antigen specificity and / or binding affinity. Generally, each heavy chain variable region has three CDRs (CDRH1, CDRH2, CDRH3), and each light chain variable region has three CDRs (CDRL1, CDRL2, CDRL3). In some embodiments, the antibody scaffolds in the GM-CSF molecules provided herein include one or more amino acid mutations, additions, and / or deletions in one or more CDRs such that the CDRs reduce or prevent antigen binding. Such modified antibody scaffolds are still considered to contain six CDRs (CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3) without requiring antigen binding, where the CDRs are positioned between the framework regions of the antibody (e.g., the heavy chain contains FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4, and the light chain contains FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4). In some embodiments, the CDRs contain GM-CSFs, where the GM-CSFs replace one or more amino acids of the CDRs. It is known in the art that "framework region" and "FR" refer to the non-CDR portions of the variable regions of the heavy and light chains. Generally, each full-length heavy chain variable region (FRH1, FRH2, FRH3, and FRH4) has four FRs, and each full-length light chain variable region (FRL1, FRL2, FRL3, and FRL4) has four FRs. The exact amino acid sequence boundaries of a given CDR or FR can be easily determined using one of many well-known schemes, including those described by: Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al.,(1997)JMB 273,927-948 ("Chothia" numbering scheme)) MacCallum et al.,J.Mol.Biol.262:732-745(1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J.Mol.Biol.262,732-745. ("Contact" numbering scheme)) Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol,2003 Jan;27(1):55-77 ("IMGT" numbering scheme)) Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun 8;309(3):657-70, ("Aho" numbering scheme); and Whitelegg NR and Rees AR, "WAM: an improved algorithm for modelling antibodies on the WEB," "Protein Eng. 2000 Dec;13(12):819-24 ("AbM" numbering scheme). In certain embodiments, the CDR of the antibodies described herein may be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof.

[0022] The sequence identity percentage (%) relative to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after the sequences have been aligned and gaps introduced, to achieve maximum percentage sequence identity if necessary, and without considering conservative substitutions as part of the sequence identity. Alignment for determining amino acid sequence identity percentage can be achieved in various known ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes of this invention, the amino acid sequence identity % value is generated using the NCBI sequence comparison computer program BLAST.

[0023] GM-CSF peptide In one embodiment, GM-CSF molecules comprising GM-CSF peptides such as human, bovine, rat, and / or mouse GM-CSF are provided herein. In some embodiments, the GM-CSF peptide comprises an amino acid sequence identical to or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 16. Long-acting GM-CSF molecules provided herein may comprise a GM-CSF peptide comprising an amino acid sequence identical to or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 16. The long-acting CSF molecules provided herein may include GM-CSF peptide variants containing amino acid sequences having approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid additions, deletions, or substitutions compared to GM-CSF containing SEQ ID NO: 16. In some embodiments, the GM-CSF peptide contains an amino acid sequence that is identical to or at least approximately 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 77. The long-acting CSF molecules provided herein may include GM-CSF peptide variants containing amino acid sequences having approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid additions, deletions, or substitutions compared to GM-CSF containing SEQ ID NO: 77. Such GM-CSF peptide variants may include those having one or more conserved amino acid substitutions. Conservative substitutions may include substitutions found in one of the following groups of conservative substitutions:Group 1: Alanine (Ala; A), Glycine (Gly; G), Serine (Ser; S), Threonine (Thr; T); Group 2: Aspartic acid (Asp; D), Glutamic acid (Glu; E); Group 3: Asparagine (Asn; N), Glutamine (Gln; Q); Group 4: Arginine (Arg; R), Lysine (Lys; K), Histidine (His; H); Group 5: Isoleucine (Ile; I), Leucine (Leu; L), Methionine (Met; M), Valine (Val; V); and Group 6: Phenylalanine (Phe; F), Tyrosine (Tyr; Y), Tryptophan (Trp; W). Furthermore, amino acids can be classified into conserved substitution groups based on similar functions, chemical structures, or compositions. For example, aliphatic groups may include Gly, Ala, Val, Leu, and Ile for substitution purposes. Other groups of amino acids that are considered conserved substitutions with each other may include: sulfur-containing: Met and Cys; acidic: Asp, Glu, and Asn; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, and Glu; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp.

[0024] Long-acting GM-CSF molecule In one embodiment, a GM-CSF molecule, sometimes referred to as a long-acting GM-CSF molecule, is provided herein, which is attached to a scaffold to increase the half-life of a GM-CSF peptide. Non-limiting examples of scaffolds include antibody variable domains. A scaffold may include variable domains of the heavy chain (VH) and / or light chain (VL), and / or one or more constant regions of a full-length antibody. Thus, as used herein, a scaffold having an antibody variable domain includes Fab, a full-length antibody, and any other antibody containing an antibody variable domain. The GM-CSF peptide does not need to be directly attached to the antibody and can be attached via one or more linker molecules. In some cases, the GM-CSF is located at the terminal of the heavy or light chain of the antibody. In some cases, the GM-CSF is located within and / or replaces one or more amino acids of the CDR of the antibody variable domain. In some cases, the GM-CSF is located between two amino acids of the CDR, between the antibody and the first amino acid of the CDR, between the antibody and the last amino acid of the CDR, and / or the GM-CSF replaces part or all of the CDR, and thus occupies the location where the CDR previously existed. In some cases, the GM-CSF molecule comprises a first antibody portion, a GM-CSF peptide, and a second antibody portion. For example, the first antibody portion comprises one or more framework regions and, where applicable, any other CDR at the N-terminus of the CDR where the GM-CSF is located, and the second antibody portion comprises one or more framework regions and / or Fc, and, where applicable, any other CDR at the C-terminus of the CDR where the GM-CSF peptide is located. Each of the first and second antibody parietals may independently have a length selected from at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50 amino acids. In the case of the palivizumab scaffold, the first antibody parietal may contain a sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14.In the case of a palivizumab scaffold, the first antibody moiety may contain a sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15. In the case of a trastuzumab scaffold, the first antibody moiety may contain a sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32. In the case of a trastuzumab scaffold, the second antibody moiety may contain a sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33. In some cases, being located within a CDR indicates that no amino acids are deleted in the CDR. In some cases, being located within a CDR indicates that at least 1, 2, 3, 4, 5, 6, 7, 8 or the entire CDR is replaced by a GM-CSF peptide. In some cases, the antibody CDR contains a GM-CSF peptide sequence. For example, the heavy chain or light chain CDR3 contains a GM-CSF peptide sequence.

[0025] In various GM-CSF molecules, the GM-CSF peptide is linked to a scaffold by one or more linkers. In some embodiments, the linker includes a sequence configured to form an alpha-helix. In some embodiments, the linker includes a sequence configured not to have a regular secondary structure (e.g., alpha-helix, 3-10 helix, beta chain, no beta turn). Non-limiting exemplary linkers may include one or more of SEQ ID NOs: 8-13.

[0026] The connections discussed herein may include peptide bonds, and therefore GM-CSF molecules may be produced from a gene construct containing DNA encoding a GM-CSF fusion molecule. As used herein, “placed within” and “inserted” may indicate the position of the GM-CSF peptide within a polypeptide containing both the GM-CSF peptide and the scaffold, and therefore do not necessarily indicate how the GM-CSF molecule is produced in such a manner. For example, “inserted” does not necessarily limit the molecule to one produced by modifying the DNA encoding the scaffold by inserting the DNA encoding the GM-CSF peptide, although the de novo synthesis of the DNA encoding the scaffold and the GM-CSF peptide may also, or alternatively, indicate this.

[0027] In various GM-CSF molecules, the GM-CSF peptide is attached to a scaffold containing an antibody-variable domain. The antibody-variable domain may comprise a first polypeptide and a second polypeptide, which may contain or otherwise attach to the GM-CSF. In some cases, the first polypeptide comprises the light chain of the antibody-variable domain, and the second polypeptide comprises the heavy chain-variable domain. In other cases, the first polypeptide comprises the heavy chain of the antibody-variable domain, and the second polypeptide comprises the light chain-variable domain. Non-limiting exemplary antibody-variable domains include trastuzumab or palivizumab-variable domains, which may be attached to and modified by the GM-CSF peptide. The trastuzumab or palivizumab-variable domain may include modifications that reduce antigen binding compared to unmodified trastuzumab or palivizumab (e.g., unmodified antibodies Herceptin and Synagis, respectively). In some cases, the GM-CSF peptide is attached to the amino terminus of the light or heavy chain. In other cases, the GM-CSF peptide is located within the light or heavy chain. For example, the GM-CSF peptide is located within the light or heavy chain CDR. As a further non-limiting example, the GM-CSF peptide is located within the light or heavy chain CDR3.

[0028] In some embodiments, the scaffold includes an antibody Fc region with reduced effector function compared to human IgG1 (SEQ ID NO: 25). Reduced effector function may include reduced antibody-dependent cytotoxicity (ADCC) and / or reduced complement-dependent cytotoxicity (CDC). In some cases, the scaffold includes an Fc sequence with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to SEQ ID NO: 3. In some cases, the scaffold includes an Fc region containing human IgG1, including E233P, L234V, L235A, ΔG236, A327G, A330S, and P331S according to Kabat numbering.

[0029] In some embodiments, the first polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6. In some cases, the first polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7. In some cases, the first polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the second polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. In some cases, the second polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4. In some cases, the second polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, a GM-CSF molecule is provided that includes an antibody-variable domain comprising a light chain sequence comprising a first polypeptide having a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6, and a heavy chain sequence comprising a second polypeptide having a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2.

[0030] In some embodiments, the first polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31. In some cases, the first polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 30. In some cases, the first polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7. In some embodiments, the second polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29. In some cases, the second polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28. In some cases, the second polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, a GM-CSF molecule is provided that includes an antibody-variable domain comprising a light chain sequence comprising a first polypeptide having a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31, and a heavy chain sequence comprising a second polypeptide having a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29.

[0031] In various GM-CSF molecules, the GM-CSF peptide is, as shown in SEQ ID NO: 26 [ka] The antibody is attached to a scaffold containing an antibody variable domain which contains a light chain sequence which contains a sequence which is at least approximately 90% identical to sequence number 16, where the light chain sequence contains X1, where X1 contains GM-CSF, and the heavy chain sequence contains a sequence which is at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 2. GM-CSF may be human, bovine, or mouse GM-CSF. GM-CSF may contain a sequence which is at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 16. GM-CSF may contain a sequence that is at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 77. GM-CSF may contain variants or homologs of GM-CSF. In some embodiments, the light chain sequence contains a sequence that is at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 26. In some embodiments, the light chain sequence contains a sequence that is at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 27 [ka] The light chain sequence includes a sequence that is at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 2, where the light chain sequence includes X2, and X2 includes GM-CSF. In some embodiments, the heavy chain sequence includes a sequence that is at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 2. In some embodiments, the light chain sequence includes a sequence that is at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 7. In some embodiments, the light chain sequence includes a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 5. In some embodiments, the heavy chain sequence includes a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 4. In some embodiments, the heavy chain sequence includes a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, the GM-CSF molecule includes an antibody Fc region with reduced effector function compared to human IgG1 (SEQ ID NO: 25). Reduced effector function may include reduced antibody-dependent cytotoxicity (ADCC) and / or reduced complement-dependent cytotoxicity (CDC). In some cases, the scaffold includes an Fc sequence with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 3. In some cases, the Fc region includes human IgG1 containing E233P, L234V, L235A, ΔG236, A327G, A330S, and P331S according to Kabat numbering.

[0032] In various GM-CSF molecules, the GM-CSF peptide is, as shown in SEQ ID NO: 42 [ka] The antibody is attached to a scaffold containing an antibody variable domain which includes a heavy chain sequence which includes a sequence which is at least approximately 90% identical to a sequence, where the heavy chain sequence includes X6, which includes GM-CSF, and the light chain sequence which includes a sequence which is at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 31. The GM-CSF may be human, bovine, or mouse GM-CSF. The GM-CSF may include a sequence which is at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 16. GM-CSF may contain a sequence that is at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 77. GM-CSF may contain variants or homologs of GM-CSF. In some embodiments, the heavy chain sequence contains a sequence that is at least approximately 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 42. In some embodiments, the heavy chain sequence contains a sequence that is at least approximately 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 43 [ka] The heavy chain includes a sequence that is at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 43, where the heavy chain includes X6, and X6 includes GM-CSF. In some embodiments, the heavy chain includes a sequence that is at least approximately 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 43. In some embodiments, the light chain includes a sequence that is at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 7. In some embodiments, the heavy chain sequence includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29. In some embodiments, the heavy chain includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, the heavy chain sequence includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28. In some embodiments, the GM-CSF molecule includes an antibody Fc region with reduced effector function compared to human IgG1 (SEQ ID NO: 25). Reduced effector function may include decreased antibody-dependent cytotoxicity (ADCC) and / or decreased complement-dependent cytotoxicity (CDC). The Fc region may contain sequences that are at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3. In some cases, the Fc region contains human IgG1 including E233P, L234V, L235A, ΔG236, A327G, A330S, and P331S according to Kabat numbering.

[0033] In one embodiment, the GM-CSF molecule provided herein comprises a first linker, a GM-CSF peptide, and a second linker. In non-limiting examples, the GM-CSF peptide may be human, bovine, rat, or mouse. For example, GM-CSF may contain a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 16. GM-CSF may contain a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 77. In some cases, the GM-CSF peptide contains a homolog or variant of GM-CSF. In some embodiments, the first linker contains a first peptide configured to form an alpha-helix. Alpha-helix formation can be predicted based on an analysis of the primary structure using readily available bioinformatics tools in the art. In some embodiments, the second linker comprises a second peptide configured to form an alpha-helix. In some embodiments, the GM-CSF molecule comprises first and second peptides configured to form a coiled coil. The coiled coil may be an antiparallel coiled coil. The first peptide may comprise a sequence with or without approximately 2, 3, or 4 amino acid substitutions or deletions from SEQ ID NO: 8, or with fewer than approximately 2, 3, or 4 amino acid substitutions or deletions. The second peptide may comprise a sequence with or without approximately 2, 3, or 4 amino acid substitutions or deletions from SEQ ID NO: 9, or with fewer than approximately 2, 3, or 4 amino acid substitutions or deletions. In some embodiments, the first linker comprises an amino acid sequence comprising at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (and up to about 30 amino acids), wherein the amino acid sequence does not contain regular secondary structures (e.g., alpha helix, beta chain, 310 helix, beta turn) and / or is a flexible linker.In some embodiments, the second linker comprises an amino acid sequence containing at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (and up to about 30 amino acids), where the amino acid sequence does not contain regular secondary structures (e.g., alpha helices, beta chains, 310 helices, beta turns) and / or is a flexible linker. The first linker may comprise a sequence with or without one or two amino acid substitutions from SEQ ID NO: 10, or with one or two amino acid substitutions. The first linker may comprise a sequence with or without amino acid substitutions from SEQ ID NO: 11, or with one or two amino acid substitutions. The second linker may comprise a sequence with or without amino acid substitutions from SEQ ID NO: 10, or with one or two amino acid substitutions. The second linker may comprise a sequence with or without amino acid substitutions from SEQ ID NO: 11, or with one or two amino acid substitutions. The first linker may comprise a sequence with or without amino acid substitutions from SEQ ID NO: 12, or with one, two, three, or four amino acid substitutions. The second linker may contain a sequence that has no amino acid substitutions from SEQ ID NO: 13, or has one, two, three, or four amino acid substitutions. In some embodiments, the GM-CSF molecule contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18.

[0034] In some embodiments, a GM-CSF molecule (referred to in some cases as a GM-CSF insert) comprising a first linker, a GM-CSF peptide, and a second linker is attached to an antibody variable domain. The GM-CSF insert may be positioned between a first sequence of the antibody variable domain (e.g., heavy chain or light chain framework 1) and a second sequence of the antibody variable domain (e.g., heavy chain or light chain framework 4). The first sequence of the antibody variable domain may include a sequence that does not have one, two, or three amino acid substitutions from SEQ ID NO: 14, or that has one, two, or three amino acid substitutions. The second sequence of the antibody variable domain may include a sequence that does not have one, two, or three amino acid substitutions from SEQ ID NO: 15, or that has one, two, or three amino acid substitutions. The first sequence of the antibody variable domain may include a sequence that does not have one, two, or three amino acid substitutions from SEQ ID NO: 32, or that has one, two, or three amino acid substitutions. The second sequence of the antibody variable domain contains a sequence that either lacks one, two, or three amino acid substitutions from SEQ ID NO: 33, or has one, two, or three amino acid substitutions.

[0035] In some embodiments, the GM-CSF molecule includes a region that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 42, wherein this region includes X5, and X5 includes the GM-CSF insert. In some embodiments, the GM-CSF molecule further includes a region that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31.

[0036] In some embodiments, the GM-CSF molecule includes a region that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26, wherein this region includes X1, where X1 includes the GM-CSF insert. In some embodiments, the GM-CSF molecule further includes a region that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2.

[0037] In one embodiment, the GM-CSF molecule provided herein comprises a first polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29, and a second polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31. In some embodiments, the first polypeptide contains a sequence that is at least about 95% identical to SEQ ID NO: 29, and the second polypeptide contains a sequence that is at least about 95% identical to SEQ ID NO: 31. In some embodiments, the first polypeptide contains a sequence that is at least about 96% identical to SEQ ID NO: 29, and the second polypeptide contains a sequence that is at least about 96% identical to SEQ ID NO: 31. In some embodiments, the first polypeptide includes a sequence that is at least about 97% identical to SEQ ID NO: 29, and the second polypeptide includes a sequence that is at least about 97% identical to SEQ ID NO: 31. In some embodiments, the first polypeptide includes a sequence that is at least about 98% identical to SEQ ID NO: 29, and the second polypeptide includes a sequence that is at least about 98% identical to SEQ ID NO: 31. In some embodiments, the first polypeptide includes a sequence that is at least about 99% identical to SEQ ID NO: 29, and the second polypeptide includes a sequence that is at least about 99% identical to SEQ ID NO: 31. In some embodiments, the first polypeptide includes SEQ ID NO: 29, and the second polypeptide includes SEQ ID NO: 31.

[0038] In one embodiment, the first polypeptide includes a sequence that is at least about 95% identical to SEQ ID NO: 28, and the second polypeptide includes a sequence that is at least about 95% identical to SEQ ID NO: 30. In some embodiments, the first polypeptide includes a sequence that is at least about 96% identical to SEQ ID NO: 28, and the second polypeptide includes a sequence that is at least about 96% identical to SEQ ID NO: 30. In some embodiments, the first polypeptide includes a sequence that is at least about 97% identical to SEQ ID NO: 28, and the second polypeptide includes a sequence that is at least about 97% identical to SEQ ID NO: 30. In some embodiments, the first polypeptide includes a sequence that is at least about 98% identical to SEQ ID NO: 28, and the second polypeptide includes a sequence that is at least about 98% identical to SEQ ID NO: 30. In some embodiments, the first polypeptide includes a sequence that is at least about 99% identical to SEQ ID NO: 28, and the second polypeptide includes a sequence that is at least about 99% identical to SEQ ID NO: 30. In some embodiments, the first polypeptide includes SEQ ID NO: 28, and the second polypeptide includes SEQ ID NO: 30.

[0039] In one embodiment, the first polypeptide includes a sequence that is at least about 95% identical to SEQ ID NO: 2, and the second polypeptide includes a sequence that is at least about 95% identical to SEQ ID NO: 6. In some embodiments, the first polypeptide includes a sequence that is at least about 96% identical to SEQ ID NO: 2, and the second polypeptide includes a sequence that is at least about 96% identical to SEQ ID NO: 6. In some embodiments, the first polypeptide includes a sequence that is at least about 97% identical to SEQ ID NO: 2, and the second polypeptide includes a sequence that is at least about 97% identical to SEQ ID NO: 6. In some embodiments, the first polypeptide includes a sequence that is at least about 98% identical to SEQ ID NO: 2, and the second polypeptide includes a sequence that is at least about 98% identical to SEQ ID NO: 6. In some embodiments, the first polypeptide includes a sequence that is at least about 99% identical to SEQ ID NO: 2, and the second polypeptide includes a sequence that is at least about 99% identical to SEQ ID NO: 6. In some embodiments, the first polypeptide includes SEQ ID NO: 2, and the second polypeptide includes SEQ ID NO: 6.

[0040] In one embodiment, the first polypeptide includes a sequence that is at least about 95% identical to SEQ ID NO: 1, and the second polypeptide includes a sequence that is at least about 95% identical to SEQ ID NO: 5. In some embodiments, the first polypeptide includes a sequence that is at least about 96% identical to SEQ ID NO: 1, and the second polypeptide includes a sequence that is at least about 96% identical to SEQ ID NO: 5. In some embodiments, the first polypeptide includes a sequence that is at least about 97% identical to SEQ ID NO: 1, and the second polypeptide includes a sequence that is at least about 97% identical to SEQ ID NO: 5. In some embodiments, the first polypeptide includes a sequence that is at least about 98% identical to SEQ ID NO: 1, and the second polypeptide includes a sequence that is at least about 98% identical to SEQ ID NO: 5. In some embodiments, the first polypeptide includes a sequence that is at least about 99% identical to SEQ ID NO: 1, and the second polypeptide includes a sequence that is at least about 99% identical to SEQ ID NO: 5. In some embodiments, the first polypeptide includes SEQ ID NO: 1, and the second polypeptide includes SEQ ID NO: 5.

[0041] In one embodiment, the GM-CSF molecule provided herein comprises a first polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 45, and a second polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 46. In one embodiment, the GM-CSF molecule provided herein comprises a first polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 47, and a second polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 48. In one embodiment, the GM-CSF molecule provided herein comprises a first polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 49, and a second polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 50. In one embodiment, the GM-CSF molecule provided herein comprises a first polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 51, and a second polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 52. In one embodiment, the GM-CSF molecule provided herein comprises a first polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 53, and a second polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 54.In one embodiment, the GM-CSF molecule provided herein comprises a first polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 55, and a second polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 56. In one embodiment, the GM-CSF molecule provided herein comprises a first polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57, and a second polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 58. In one embodiment, the GM-CSF molecule provided herein comprises a first polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 59, and a second polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 60. In one embodiment, the GM-CSF molecule provided herein comprises a first polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 61, and a second polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 62. In one embodiment, the GM-CSF molecule provided herein comprises a first polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 63, and a second polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 64.

[0042] In one embodiment, a GM-CSF molecule is provided herein that contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 67. In one embodiment, a GM-CSF molecule is provided herein that contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 68.

[0043] In one embodiment, a GM-CSF molecule is provided herein encoded by a first nucleic acid comprising a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 69, and a second nucleic acid comprising a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 70. In one embodiment, a GM-CSF molecule is provided herein encoded by a first nucleic acid comprising a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 71, and a second nucleic acid comprising a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 72. In one embodiment, a GM-CSF molecule is provided herein encoded by a first nucleic acid comprising a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 73, and a second nucleic acid comprising a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 74. In one embodiment, a GM-CSF molecule is provided herein encoded by a first nucleic acid comprising a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 75, and a second nucleic acid comprising a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 76.

[0044] In some embodiments, the GM-CSF molecule contains one or more sequences from SEQ ID NOs: 1-76. In some embodiments, the GM-CSF molecule contains sequences from Table 4. The antibody abbreviations include "HC" for the heavy chain, "VH" for the variable region of the heavy chain, "Fc" for the fragment crystallizable region, "LC" for the light chain, "VL" for the variable region of the light chain, "CDR3L" for the complementarity-determining region 3 of the light chain, "CDR3H" for the complementarity-determining region 3 of the heavy chain, and "FR" for the framework.

[0045] scaffold In one embodiment, a GM-CSF molecule comprising a GM-CSF peptide attached to a scaffold is provided herein. The scaffold can extend the half-life of the GM-CSF peptide. The half-life can be measured using experiments detailed in the examples provided herein or using methods readily available in the art. The half-life can be increased by at least about 10%, 20%, 30%, 40%, 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 100% compared to GM-CSF without a fusion, such as recombinant GM-CSF like sargramostim. Thus, a long-acting GM-CSF molecule with an increased half-life compared to the GM-CSF peptide alone is provided herein.

[0046] In some embodiments, the scaffold comprises one or more antibody moieties. The antibody moieties may comprise the entire antibody molecule, or any polypeptide comprising antibody fragments including, but not limited to, the entire antibody molecule, or heavy chains, light chains, variable domains, variable light chain regions (VL), variable heavy chain regions (VH), constant domains (e.g., CH1, CH2, CH3, and / or CL), complementarity-determining regions (CDR, e.g., CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3), framework regions (e.g., FRH1, FRH2, FRH3, FRH4, FRL1, FRL2, FRL3, and / or FRL4), antigen-binding fragments, single-domain antibodies, fragment antigen-binding (Fab) regions, Fab', F(ab')2, F(ab')3, Fab', fragment crystallizable (Fc) regions, single-chain variable fragments (scFv), di-scFv, single-domain antibodies, trifunctional antibodies, chemically bound F(ab')2, and any combination thereof. The antibody moiety may include heavy and light chains linked by a linker or disulfide bond. In some cases, the antibody moiety includes variable domains that have been modified or otherwise manipulated to reduce or eliminate antigen binding. Antigen binding may be reduced or eliminated by mutation, for example, by mutating the CDR3 of the light and / or heavy chains. Antibodies may originate from any type known to those skilled in the art, including but not limited to IgA, IgD, IgE, IgG, IgM, IgY, and IgW.

[0047] As used herein, the antibody variable domain is not limited to antibody fragments capable of binding to an antigen, but also includes antibody fragments derived from antibody fragments capable of binding to an antigen, where derivatization includes reducing or eliminating antigen binding. In some such cases, the amino acid length of the antibody-binding fragment derivatized to reduce or eliminate antigen binding may be the same as, or within about 10%–120% of, the amino acid length of the original antigen-binding fragment from which it was derived (i.e., the antigen-binding fragment that binds to the antigen). In some cases, the antibody variable domain is an antibody region comprising CDR1, CDR2, and CDR3 of the antibody heavy chain, and CDR1, CDR2, and CDR3 of the antibody light chain, wherein one or more of the CDRs are mutated or otherwise modified in terms of amino acid sequence identity to reduce or eliminate antigen binding compared to a non-mutated or modified antibody. In the case of a GM-CSF molecule where the GM-CSF peptide is located within a CDR, the antibody variable domain may include an antibody region comprising CDR1, CDR2, and CDR3 of the antibody heavy chain, and CDR1, CDR2, and CDR3 of the antibody light chain, where one or more of the CDRs have had their amino acid sequence identity mutated or otherwise altered to reduce or eliminate antigen binding. In some cases, the alteration is positioned within the CDR region by replacing, for example, one, two, three, four, five, or all of the CDR amino acids with the GM-CSF peptide.

[0048] The antibody moiety can be modified from a trastuzumab antibody containing a heavy chain variable region including SEQ ID NO: 44 and a light chain variable region including SEQ ID NO: 31. The antibody moiety can be modified from a palivizumab antibody containing a heavy chain variable region including SEQ ID NO: 65 and a light chain variable region including SEQ ID NO: 17. The antibody moiety can be modified by inserting a GM-CSF peptide into the heavy chain or light chain sequence. The GM-CSF peptide can replace one or more amino acids in the heavy chain or light chain sequence. The GM-CSF can be placed within a CDR of the heavy chain or light chain sequence. The CDR may be a CDR3. The antibody moiety can be attached to the GM-CSF peptide at the amino or carboxyl terminus of the heavy chain or light chain sequence. In some embodiments, the antibody moiety is modified to reduce antigen binding. For example, the palivizumab heavy chain CDR3 can be modified to replace the NWY with FGG.

[0049] In some embodiments, the antibody portion includes SEQ ID NO: 14. In some embodiments, the antibody portion includes SEQ ID NO: 15. The antibody portion may include SEQ ID NOs: 14 and 15. The GM-CSF peptide may be located between SEQ ID NOs: 14 and 15. In some embodiments, the antibody portion includes SEQ ID NO: 32. In some embodiments, the antibody portion includes SEQ ID NO: 33. The antibody portion may include SEQ ID NOs: 32 and 32. The GM-CSF peptide may be located between SEQ ID NOs: 32 and 33.

[0050] In some cases, the scaffold comprises "at least a portion" of the antibody or antibody fragment. In certain embodiments, "at least a portion" means that at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the antibody or antibody fragment is present in the composition with at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, a scaffold comprising at least a portion of the heavy chain having SEQ ID NO: 44 (120 amino acids) comprises at least about 96 amino acids (80%), 102 amino acids (85%), 108 amino acids (90%), or 114 amino acids (95%) of SEQ ID NO: 44 with at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. "At least a portion of" can be a continuous amino acid sequence, or the sum of two continuous amino acid sequences separated by the GM-CSF peptide. For example, a GM-CSF molecule containing at least a portion of the antibody variable domain may contain a first continuous amino acid sequence of the antibody variable domain, a GM-CSF peptide, and a second continuous amino acid sequence of the antibody variable domain, where the first and second continuous amino acid sequences of the antibody variable domain together constitute at least approximately 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the sequence length of the antibody variable domain. In some cases, the GM-CSF molecule comprises at least a portion of an antibody or antibody fragment selected from an antibody variable domain and / or antigen-binding fragment (e.g., a fragment containing CDR1, CDR2, and CDR3 of the antibody heavy chain and / or antibody light chain), wherein the molecule comprises a first antibody or antibody fragment region, a GM-CSF peptide, and a second antibody or antibody fragment region, where “at least a portion” means that the sum of the lengths of the first antibody or antibody fragment region and the second antibody or antibody fragment region is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the antibody variable domain and / or antigen-binding fragment.

[0051] The antibody moiety may contain an antibody sequence from a trastuzumab antibody. The antibody moiety is at least approximately 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least a portion of the trastuzumab antibody. For example, the antibody moiety is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from one or more of SEQ ID NOs. 30-35, 37-39, and 44. As another example, the antibody moiety contains at least approximately 10 consecutive amino acids of a sequence selected from one or more of SEQ ID NOs. 30-35, 37-39, and 44.

[0052] The antibody portion may contain an antibody sequence from an anti-Her2 antibody. The antibody portion may contain at least a portion of an anti-Her2 antibody. The antibody portion may contain an amino acid sequence that is identical to at least a portion of an anti-Her2 antibody, or identical by at least approximately 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 97% or more.

[0053] In some cases, the antibody moiety contains the palivizumab antibody sequence. The antibody moiety is at least approximately 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least a portion of the palivizumab antibody. For example, the antibody moiety is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from one or more of SEQ ID NOs. 14, 15, 17, 65, and 19-23. As another example, the antibody moiety contains at least approximately 10 consecutive amino acids of a sequence selected from one or more of 14, 15, 17, 65, and 19-23.

[0054] In some embodiments, the antibody scaffolds provided herein are not specific to the antigen or have reduced binding to the antigen. For example, the antibody scaffolds provided herein may not bind to the antigen or, as determined by assays described in the examples herein, for example, about 10M -2 , 10M -3 , or 10M -5It may bind to the antigen with weaker affinity than the original antibody. An exemplary antibody comprises six CDRs, one or more of which are modified CDRs, including the addition, substitution, or deletion of one or more amino acids that reduce or eliminate antigen binding. In some cases, the CDRs are modified by insertion of a therapeutic peptide, such as GM-CSF. In exemplary embodiments, GM-CSF is inserted into the heavy or light chain CDR3. The exemplary GM-CSF molecule provided herein comprises a GM-CSF peptide located within the heavy chain CDR3 of the antibody variable domain, where the antibody has no antigen binding or reduced antigen binding compared to an antibody without the GM-CSF fusion. GM-CSF may replace one, two, three, four, five, or all amino acids of the CDR. The antibody may comprise a trastuzumab antibody variable domain scaffold modified by insertion of GM-CSF into the CDR. In some cases, insertion of GM-CSF into the CDR3 of trastuzumab reduces antigen binding. Antibodies containing insertions may further contain one or more amino acid deletions, for example, if the insertion replaces one or more amino acids in the CDR. In some embodiments, the CDR sequence is mutated to reduce antigen binding. In some embodiments, the heavy chain CDR3 of palivizumab comprises SMITX(i)X(ii)X(iii)FDV (SEQ ID NO: 66), where X(i) is selected from F, A, G, and P; X(ii) is selected from G, A, S, T, and P; and X(iii) is selected from G, A, V, L, and P. In some embodiments, X(i) is F. In some embodiments, X(ii) is G. In some embodiments, X(ii) is A. In some embodiments, X(iii) is G. As an example, the heavy chain CDR3 of the antibody palivizumab is a mutation that replaces NWY with FGG, which reduces binding to RSV-F compared to unmutated palivizumab. As another example, the heavy chain CDR3 of the antibody trastuzumab is mutated to remove approximately one, two, three, four, five, or all of the CDR3 amino acids, thereby reducing binding to Her2 compared to non-mutated trastuzumab.In some cases, one or more amino acids of trastuzumab's CDR3 are replaced with a GM-CSF peptide or GM-CSF insert.

[0055] In some embodiments, the antibody moiety is not specific to mammalian targets. In some embodiments, the antibody is an antiviral antibody. In some embodiments, the antibody is an antibacterial antibody. In some embodiments, the antibody is an antiparasitic antibody. In some embodiments, the antibody is an antifungal antibody. In some embodiments, the antibody moiety is derived from an antibody vaccine.

[0056] In some embodiments, the antibody moiety includes, but is not limited to, antibody sequences from actoxumab, bezlotoxumab, CR6261, edbacomab, efungumab, exbivirumab, felbizumab, folavirumab, ibalizumab (TMB-355, TNX-355), ribivirumab, motabizumab, nevacumab, pagibaximab, palivizumab, panobacumab, rafibirumab, laxibacumab, regavirumab, sevilumab (MSL-109), suvizumab, tefivazumab, tubirumab, and urtoxazumab.

[0057] In some embodiments, the antibody moiety includes an antibody sequence from an antibody targeting Clostridium difficile, Orthomyxoviruses (influenza virus A, influenza virus B, influenza virus C, Isavirus, Togotovirus), Escherichia coli, Candida, Rabies, human immunodeficiency virus, Hepatitis, Staphylococcus, respiratory syncytial virus, Pseudomonas aeruginosa, Bacillus anthracis, cytomegalovirus, or Staphylococcus aureus.

[0058] The antibody portion may contain an antibody sequence from an antiviral antibody. The antiviral antibody may be directed against an epitope of a viral protein. The antiviral antibody may target one or more viruses, including but not limited to adenoviruses, herpesviruses, poxviruses, parvoviruses, reoviruses, picornaviruses, togaviruses, orthomyxoviruses, rhabdoviruses, retroviruses, and hepadnaviruses. The viral protein may be derived from respiratory syncytial virus. The viral protein may be the F protein of respiratory syncytial virus. The epitope may be at the A antigen site of the F protein. The antiviral antibody may be based on or derived from palivizumab. The antibody may be based on or derived from an antiviral vaccine. The antiviral antibodies may be based on or derived from exvivirumab, foravirumab, rivivirumab, rafivirumab, regavirumab, cevirumab, tubirumab, felbizumab, motavizumab, palivizumab, and / or suvizumab.

[0059] The antibody portion may include an antibody sequence derived from antiviral antibody G. The antibody portion may include at least a portion of antiviral antibody G. The antibody portion may include an amino acid sequence that is identical to at least a portion of antiviral antibody G by at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 97% or more. In some embodiments, the antibody portion includes the amino acid sequence of antiviral antibody M.

[0060] The antibody portion may include antibody sequences from exvivirumab, foravirumab, ribivirumab, rafibirumab, regavirumab, cevirumab, tubirumab, felbizumab, motavizumab, palivizumab, and / or subizumab antibodies. The antibody portion may include at least a portion of exvivirumab, foravirumab, ribivirumab, rafibirumab, regavirumab, cevirumab, tubirumab, felbizumab, motavizumab, palivizumab, and / or subizumab antibodies. The antibody portion is identical to at least a portion of exvivirumab, foravirumab, rivivirumab, rafibirumab, regavirumab, sevilumab, tubirumab, felbizumab, motavizumab, palivizumab, and / or suvizumab antibodies by at least approximately 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 97% or more.

[0061] The antibody portion may contain antibody sequences from antibacterial antibodies. Antibacterial antibodies may be directed against epitopes of bacterial proteins. Antimicrobial antibodies may target bacteria including, but not limited to, the following bacteria (Chemical Formula 9). Antibodies may be based on or derived from bacterial vaccines. Antiviral antibodies may be based on or derived from nevacumab, panobacumab, laxibacumab, edobacomab, pagibaximab, and / or tefibazumab. [ka]

[0062] The antibody portion may include an antibody sequence derived from antibacterial antibody G. The antibody portion may include at least a portion of antibacterial antibody G. The antibody portion may include an amino acid sequence that is identical to at least a portion of antibacterial antibody G, or identical to at least a portion of antibacterial antibody G by at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 97%, or more. In some embodiments, the antibody portion includes an amino acid sequence based on or derived from antibacterial antibody M.

[0063] The antibody moiety may contain antibody sequences from nevacumab, panobacumab, laxibacumab, edbacomab, pagibaximab, and / or tefibazumab antibodies. The antibody moiety may contain amino acid sequences that are identical to, or at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 97% or more identical to, at least a portion of, the nevacumab, panobacumab, laxibacumab, edbacomab, pagibaximab, and / or tefibazumab antibodies.

[0064] The antibody portion may contain an antibody sequence from an antiparasitic antibody. The antiparasitic antibody may be directed against an epitope of a parasitic protein. The antiparasitic antibody may target parasites or parasitic proteins, including but not limited to the following parasites (Chemical Formula 10). [ka] Parasites or parasitic proteins, including but not limited to these, may be targeted.

[0065] The antibody portion may include an antibody sequence derived from antiparasitic antibody G. The antibody portion may include at least a part of antiparasitic antibody G. The antibody portion may include an amino acid sequence that is identical to at least a part of antiparasitic antibody G, or that is identical to at least a part of antiparasitic antibody G by at least approximately 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 97% or more.

[0066] The antibody portion may contain antibody sequences from antifungal antibodies. Antimicrobial antibodies can be directed against epitopes of fungal proteins. Antifungal antibodies may target fungi or fungal proteins, including but not limited to the following fungi (Chemical Formula 11). [ka] Antifungal antibodies may be based on or derived from efungumab.

[0067] The antibody portion may include an antibody sequence derived from antifungal antibody G. The antibody portion may include at least a part of antifungal antibody G. The antibody portion may include an amino acid sequence that is identical to at least a part of antifungal antibody G, or that is identical to at least a part of antifungal antibody G by at least approximately 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 97% or more.

[0068] The antibody portion may include antibody sequences from anticancer antibodies. Examples of anticancer antibodies include, but are not limited to, absiximab, adalimumab, alemtuzumab, basiliximab, belimumab, bevacizumab, brentuximab, canakinumab, certolizumab, cetuximab, daclizumab, denosumab, eculizumab, efalizumab, gemtuzumab, golimumab, ibritumomab, infliximab, ipilimumab, muromonab-cd3, natalizumab, ofatumumab, omalizumab, palivizumab, panitumumab, ranibizumab, rituximab, tocilizumab, tocitumomab, and trastuzumab.

[0069] The antibody portion may include at least a portion of human antibodies. The antibody portion may include at least a portion of humanized antibodies. The antibody portion may include at least a portion of chimeric antibodies. The antibody portion may be based on or derived from human antibodies. The antibody portion may be based on or derived from humanized antibodies. The antibody portion may be based on or derived from chimeric antibodies. The antibody portion may be based on or derived from monoclonal antibodies. The antibody portion may be based on or derived from polyclonal antibodies. The antibody portion may include at least a portion of antibodies derived from mammals, birds, reptiles, amphibians, or combinations thereof. Mammals may be humans. Mammals may be non-human primates. Mammals may be dogs, cats, sheep, goats, cattle, rabbits, rats, or mice.

[0070] In one embodiment, the antibody variable domain provided herein includes: (a) a light chain comprising: (i) CDRL1 comprising a sequence that has no substitution or deletion from SEQ ID NO: 22, or has about one, two, or three amino acid substitutions or deletions from SEQ ID NO: 22; (ii) CDRL2 comprising a sequence that has no substitution or deletion from SEQ ID NO: 23, or has about one, two, or three amino acid substitutions or deletions from SEQ ID NO: 23; and (iii) a sequence that has no substitution or deletion from SEQ ID NO: 16 or 77, or has about one, two, or three amino acid substitutions or deletions from SEQ ID NO: 16 or 77. A light chain comprising CDRL3 containing a sequence with a deletion, and (b) a heavy chain comprising: (i) CDRH1 containing a sequence that does not have substitutions or deletions from SEQ ID NO: 19, or has about one, two, or three amino acid substitutions or deletions from SEQ ID NO: 19; (ii) CDRH2 containing a sequence that does not have substitutions or deletions from SEQ ID NO: 20, or has about one, two, or three amino acid substitutions or deletions from SEQ ID NO: 20; and (iii) CDRH3 containing a sequence that does not have substitutions or deletions from SEQ ID NO: 21, or has about one, two, or three amino acid substitutions or deletions from SEQ ID NO: 21. In some embodiments, CDRL3 comprises a sequence that does not have amino acid substitutions or deletions from SEQ ID NO: 24, or has about one, two, three, four, or five amino acid substitutions or deletions. In some embodiments, the antibody variable domain comprises a light chain comprising SEQ ID NOs: 22, 23, and 16, and a heavy chain comprising SEQ ID NOs: 19-21. In some embodiments, the antibody variable domain comprises a light chain containing SEQ ID NOs: 22, 23, and 77, and a heavy chain containing SEQ ID NOs: 19-21. In some embodiments, the antibody variable domain comprises a light chain containing SEQ ID NOs: 22, 23, and 24, and a heavy chain containing SEQ ID NOs: 19-21. Further antibodies are provided that contain an antibody variable domain and further comprise an Fc region having reduced effector function compared to human IgG (SEQ ID NO: 25). The reduced effector function may be a reduction in ADCC and / or CDC.The Fc region may contain a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 3. In some embodiments, the Fc region contains modified human IgG1 including E233P, L234V, L235A, ΔG236, A327G, A330S, and P331S by Kabat numbering.

[0071] In one embodiment, the antibody variable domain provided herein includes: (a) a light chain comprising: (i) CDRL1 comprising a sequence that has no substitution or deletion from SEQ ID NO: 37, or has about one, two, or three amino acid substitutions or deletions from SEQ ID NO: 37; (ii) CDRL2 comprising a sequence that has no substitution or deletion from SEQ ID NO: 38, or has about one, two, or three amino acid substitutions or deletions from SEQ ID NO: 38; and (iii) a sequence that has no substitution or deletion from SEQ ID NO: 39, or has about one, two, or three amino acid substitutions or deletions from SEQ ID NO: 39. A light chain comprising CDRL3 containing the sequence, and (b) a heavy chain comprising: (i) CDRH1 containing a sequence that does not have substitutions or deletions from SEQ ID NO: 34, or has about one, two, or three amino acid substitutions or deletions from SEQ ID NO: 34; (ii) CDRH2 containing a sequence that does not have substitutions or deletions from SEQ ID NO: 35, or has about one, two, or three amino acid substitutions or deletions from SEQ ID NO: 35; and (iii) CDRH3 containing a sequence that does not have substitutions or deletions from SEQ ID NO: 16, or has about one, two, or three amino acid substitutions or deletions from SEQ ID NO: 16. In some embodiments, CDRH3 comprises a sequence that does not have amino acid substitutions or deletions from SEQ ID NO: 36, or has about one, two, three, four, or five amino acid substitutions or deletions. In some embodiments, the antibody variable domain comprises a light chain comprising SEQ ID NOs: 37-39 and a heavy chain comprising SEQ ID NOs: 34, 35, and 16. In some embodiments, the antibody variable domain comprises a light chain comprising SEQ ID NOs: 37-39. and heavy chains including SEQ ID NOs. 34-36 are also provided. Further antibodies are provided that include an antibody variable domain and an Fc region containing reduced effector function compared to human IgG (SEQ ID NOs. 25). Reduced effector function may be reduced ADCC and / or CDC. The Fc region may contain a sequence that is at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs. 3.In some embodiments, the Fc region includes modified human IgG1, which includes E233P, L234V, L235A, ΔG236, A327G, A330S, and P331S according to Kabat numbering.

[0072] In one embodiment, the antibody variable domain provided herein includes: (a) a light chain comprising: (i) CDRL1 comprising a sequence that has no substitution or deletion from SEQ ID NO: 37, or has about one, two, or three amino acid substitutions or deletions from SEQ ID NO: 37; (ii) CDRL2 comprising a sequence that has no substitution or deletion from SEQ ID NO: 38, or has about one, two, or three amino acid substitutions or deletions from SEQ ID NO: 38; and (iii) a sequence that has no substitution or deletion from SEQ ID NO: 39, or has about one, two, or three amino acid substitutions or deletions from SEQ ID NO: 39. A light chain comprising CDRL3 containing the sequence, and (b) a heavy chain comprising: (i) CDRH1 containing a sequence that has no substitution or deletion from SEQ ID NO: 34, or has about one, two, or three amino acid substitutions or deletions from SEQ ID NO: 34; (ii) CDRH2 containing a sequence that has no substitution or deletion from SEQ ID NO: 35, or has about one, two, or three amino acid substitutions or deletions from SEQ ID NO: 35; and (iii) CDRH3 containing a sequence that has no substitution or deletion from SEQ ID NO: 77, or has about one, two, or three amino acid substitutions or deletions from SEQ ID NO: 77. In some embodiments, CDRH3 comprises a sequence that has no amino acid substitution or deletion from SEQ ID NO: 36, or has about one, two, three, four, or five amino acid substitutions or deletions. In some embodiments, the antibody variable domain comprises a light chain comprising SEQ ID NOs: 37-39 and a heavy chain comprising SEQ ID NOs: 34, 35, and 77. Further antibodies are provided that include an antibody-variable domain and further include an Fc region with reduced effector function compared to human IgG (SEQ ID NO: 25). The reduced effector function may be a reduction in ADCC and / or CDC. The Fc region may include a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3. In some embodiments, the Fc region includes modified human IgG1 including E233P, L234V, L235A, ΔG236, A327G, A330S, and P331S by Kabat numbering.

[0073] Degraded effect pedal functionality The antibody scaffolds provided herein may include the addition, deletion, and / or substitution of one or more amino acids to the wild-type IgG Fc region to reduce binding to effector molecules compared to wild-type IgG. The antibodies may exhibit reduced antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cell-mediated cytotoxicity. As an example, the scaffold includes an IgG1 Fc region containing one or more of the following mutations: E233P, L234V, L235A, ΔG236, A327G, A330S, and P331S. In some embodiments, the antibody includes a constant region containing an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to 25. In some embodiments, the antibody includes a CH1 domain containing an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 3. In some embodiments, the antibody includes an Fc region containing an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to 4.

[0074] Linker A GM-CSF molecule may contain a GM-CSF peptide linked to a scaffold via one or more linkers. In some cases, the linker molecule contains a linker peptide that includes a secondary structure. The secondary structure may be an alpha-helix. Exemplary alpha-helix peptides contain a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 8-9. In some embodiments, the linker molecule contains a linker peptide that is flexible and does not have a regular secondary structure. Exemplary linker peptides contain a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 10-11. In some embodiments, the first linker includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 12. In some embodiments, the second linker includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 13.

[0075] In some embodiments, the GM-CSF molecule comprises a GM-CSF peptide positioned within a scaffold such that the amino terminus and carboxyl terminus of the GM-CSF peptide are respectively connected to the scaffold. The GM-CSF peptide may be connected to the scaffold by a first linker at its amino terminus and by a second linker at its carboxyl terminus. As an example, a GM-CSF molecule comprising A1-L1-T-L2-A2 is provided, where A1 and A2 are the first and second parts of scaffold A3 (e.g., an antibody sequence), L1 is the first linker, L2 is the second linker, and T is the GM-CSF peptide. A3 may be the heavy or light chain of the antibody variable domain. In some cases, based on the primary sequence of L1, L1 is configured to form a helix. In some cases, based on the primary sequence of L2, L2 is configured to form a helix. In some cases, L1 is configured to form a first helix, L2 is configured to form a second helix, and the first and second helices are configured to form a coiled coil. The coils may be antiparallel. In some embodiments, L1 includes a sequence that has no amino acid substitutions or deletions from SEQ ID NO: 8, or has about one, two, three, or four amino acid substitutions or deletions. In some embodiments, L2 includes a sequence that has no amino acid substitutions or deletions from SEQ ID NO: 9, or has about one, two, three, or four amino acid substitutions or deletions. In some embodiments, L1 includes a flexible linker. L1 may include a sequence that has no amino acid substitutions or deletions from SEQ ID NO: 10, or has about one or two amino acid substitutions or deletions. L1 may include a sequence that has no amino acid substitutions or deletions from SEQ ID NO: 11, or has about one or two amino acid substitutions or deletions. In some embodiments, L2 includes a flexible linker. L2 may contain a sequence that has no amino acid substitutions or deletions from SEQ ID NO: 10, or may have approximately one or two amino acid substitutions or deletions.In some embodiments, L2 includes a sequence that has no amino acid substitutions or deletions from SEQ ID NO: 11, or has about one, two, three, or four amino acid substitutions or deletions. In some embodiments, L1 includes a sequence that has no amino acid substitutions or deletions from SEQ ID NO: 12, or has about one, two, three, or four amino acid substitutions or deletions. In some embodiments, L2 includes a sequence that has no amino acid substitutions or deletions from SEQ ID NO: 13, or has about one, two, three, or four amino acid substitutions or deletions. L1-T-L2 may include a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18.

[0076] Vectors, host cells, and recombination methods In one embodiment, the GM-CSF molecule provided herein comprises a polypeptide sequence. Such GM-CSF molecules disclosed herein (referred to in some cases as GM-CSF protein fusions) can be expressed and purified by known recombination and protein purification methods. In exemplary embodiments, a nucleic acid encoding a protein fusion is synthesized, amplified (e.g., by PCR), digested with restriction enzymes, and gel-purified. The digested nucleic acid can be inserted into a replicable vector. The replicable vector containing the digested protein fusion insert can be transformed or transduced into a host cell for further cloning (DNA amplification) or expression. The host cell may be a prokaryotic or eukaryotic cell. Furthermore, phage vectors containing replicons and regulatory sequences compatible with host microorganisms can be used as transformation vectors relevant to these hosts. For example, bacteriophages such as λGEM®-11 can be used in the production of recombinant vectors that can be used to transform susceptible host cells such as Escherichia coli LE392. The protein fusion can be expressed intracellularly (e.g., cytoplasmically) or extracellularly (e.g., secreted). For extracellular expression, the vector may contain a secretory signal that enables the transfer of the antibody protein outside the cell.

[0077] Suitable host cells for cloning or expressing vectors encoding protein fusions include prokaryotic and eukaryotic cells. The host cell may be a eukaryote. Examples of eukaryotic cells include, but are not limited to, human fetal kidney (HEK) cells (e.g., HEK 293F cells), Chinese hamster ovary (CHO) cells, fungi, yeast, invertebrate cells (e.g., plant and insect cells), and lymphocytes (e.g., YO, NSO, Sp20 cells). Other examples of suitable mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7), baby hamster kidney cells (BHK), mouse Sertoli cells, monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical tumor cells (HELA), canine kidney cells (MDCK), buffalo rat hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor cells (MMT 060562), TR1 cells, MRC5 cells, and FS4 cells. The host cell may be a prokaryotic cell (e.g., Escherichia coli (E. coli)).

[0078] Host cells can be transformed with a vector containing nucleotides encoding a GM-CSF protein fusion. Transformed host cells can be cultured in a culture medium. One or more agents may be added to the medium for promoter induction, selection of transformants, or amplification or expression of a gene encoding a desired sequence. Methods for transforming host cells are known in the art and may include electroporation, calcium chloride, or polyethylene glycol / DMSO. Host cells can be transfected or transduced with a vector containing nucleotides encoding a GM-CSF protein fusion. Transfected or transduced host cells can be cultured in a culture medium. One or more agents may be added to the medium for promoter induction, selection of transfected or transduced cells, or expression of a gene encoding a desired sequence.

[0079] The expressed GM-CSF protein fusion can be secreted into the periplasm of host cells and recovered from there, or transferred to culture medium. Recovery of the protein from the periplasm may involve disruption of host cells. Disruption of host cells may include osmotic shock, sonication, and / or lysis. Cell debris or whole cells may be removed using centrifugation or filtration. The GM-CSF protein fusion can be further purified, for example, by affinity resin chromatography. Alternatively, the GM-CSF protein fusion secreted into the culture medium can be isolated therein. To further purify the produced protein, cells may be removed from the culture, and the culture supernatant may be filtered and concentrated. The expressed polypeptide can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blotting assays.

[0080] GM-CSF protein fusion production can be carried out on a large scale by fermentation processes. Various large-scale supply batch fermentation procedures are available for recombinant protein production. Large-scale fermentation involves vessels with a capacity of at least 1,000 liters, e.g., approximately 1,000 to 100,000 liters. These fermenters use impellers to distribute oxygen and nutrients, particularly glucose (a preferred carbon / energy source). Small-scale fermentation generally refers to fermentation in vessels with a capacity of approximately 100 liters or less, which can range from approximately 1 liter to approximately 100 liters. In the fermentation process, induction of protein expression is typically initiated after cells have grown to a desired density, e.g., OD550 at approximately 180-220, under appropriate conditions, at which point the cells are in the early stationary phase. Various inducers can be used, depending on the vector construct used, as are known in the art and described herein. Cells may be grown for a short period before induction. Cells are usually induced for approximately 12-50 hours, but longer or shorter induction times may be used.

[0081] Various fermentation conditions can be modified to improve the production yield and quality of the GM-CSF protein fusions disclosed herein. For example, Dsb proteins (DsbA, DsbB, DsbC, DsbD, or DsbG) or FkpA (peptidyl prolyl cis,trans isomerase with chaperone activity) can be used to co-transform host prokaryotic cells in order to improve the proper assembly and folding of the secreted GM-CSF protein fusions. Chaperone proteins have been shown to promote the proper folding and solubility of heterologous proteins produced in bacterial host cells.

[0082] To minimize the proteolysis of expressed heterologous proteins (particularly those that are proteolytically sensitive), specific host strains lacking proteolytic enzymes may be used for this disclosure. For example, host cell lines may be modified to introduce genetic mutations in genes encoding known bacterial proteases such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Several Escherichia coli (E. coli) protease-deficient strains are available.

[0083] Standard protein purification methods known in the art may be used. The following procedure is a non-limiting example of a suitable purification procedure: fractionation on an immunoaffinity or ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica or on a cation exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, hydroxyl apatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, and gel filtration using, for example, Sephadex G-75.

[0084] GM-CSF protein fusions can be concentrated using commercially available protein concentration filters, such as Amicon or Millipore Pellicon® ultrafiltration units.

[0085] A protease inhibitor or a protease inhibitor cocktail may be included in any of the aforementioned steps to inhibit the proteolysis of the GM-CSF protein fusion.

[0086] In some cases, GM-CSF protein fusions may not be biologically active upon isolation. Biological activity can be restored using various methods to “refold” or convert the polypeptide to its tertiary structure and to generate disulfide bonds. Such methods involve exposing the solubilized polypeptide to a pH usually above 7 in the presence of a specific concentration of chaotrope. The selection of chaotrope is similar to that used for solubilizing inclusion bodies, but usually the chaotrope used is at a lower concentration and not necessarily the same as the chaotrope used for solubilization. In most cases, the refolding / oxidation solution also contains a reducing agent, or a reducing agent and its oxidized form in a specific ratio, to generate a specific redox potential and allow disulfide shuffling to occur in the formation of cysteine ​​crosslinks of the protein. Some commonly used redox pairs include cysteine / cystamine, glutathione (GSH) / dithiobis(GSH), cupric chloride, dithiothreitol (DTT) / dithiane(DTT), and 2-mercaptoethanol (bME) / dithio-b(ME). Often, cosolvents can be used to increase the efficiency of refolding, and common reagents used for this purpose include glycerol, polyethylene glycol of various molecular weights, and arginine.

[0087] composition Disclosed herein are compositions comprising a GM-CSF peptide, such as a GM-CSF molecule containing a GM-CSF peptide and a scaffold (e.g., a long-acting GM-CSF molecule). In the case of a scaffold containing a polypeptide sequence, the GM-CSF molecule may in some cases be referred to as a GM-CSF protein fusion.

[0088] The composition may further comprise one or more pharmaceutically acceptable salts, excipients, or vehicles. Pharmacologically acceptable salts, excipients, or vehicles for use in the present pharmaceutical composition include carriers, excipients, diluents, antioxidants, preservatives, colorants, fragrances and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, isotonic agents, cosolvents, wetting agents, complexing agents, buffers, antimicrobial agents, and surfactants.

[0089] Examples of suitable carriers include neutral buffered saline or saline mixed with serum albumin. Pharmaceutical compositions may include antioxidants such as ascorbic acid, proteins such as low molecular weight polypeptides, serum albumin, gelatin, or antibodies, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine, and lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin, chelating agents such as EDTA, sugar alcohols such as mannitol and sorbitol, salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, Pluronic®, or polyethylene glycol (PEG). Examples of suitable isotonic enhancers include alkali metal halides (preferably sodium chloride or potassium chloride), mannitol, and sorbitol. Suitable preservatives include benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, and sorbic acid. Hydrogen peroxide can also be used as a preservative. Suitable cosolvents include glycerin, propylene glycol, and PEG. Suitable complexing agents include caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin. Suitable surfactants or wetting agents include sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, and tyloxapearl. Buffers may be conventional buffers such as acetic acid, boric acid, citric acid, phosphoric acid, bicarbonate, or Tris-HCl. Acetate buffers may have a pH of approximately 4–5.5, and Tris buffers may have a pH of approximately 7–8.5. Additional pharmaceuticals are listed in Remington's Pharmaceutical Sciences, 18th edition, edited by Argennaro, Mack Publishing Company, 1990.

[0090] The composition may be in liquid form or in lyophilized or freeze-dried form and may contain one or more cryoprotectants, excipients, surfactants, high molecular weight structural additives, and / or fillers. In one embodiment, the cryoprotectant may be a non-reducing sugar such as sucrose, lactose, or trehalose. The amount of cryoprotectant generally included is such that the formulation obtained upon reconstitution is isotonic, although hypertonic or slightly hypotonic formulations may also be suitable. Furthermore, the amount of cryoprotectant should be sufficient to prevent unacceptable degradation and / or aggregation of proteins during lyophilization. In another embodiment, for example, nonionic surfactants and ionic surfactants, such as ionic surfactants such as polysorbate (e.g., polysorbate 20, polysorbate 80), poloxamer (e.g., poloxamer 188), poly(ethylene glycol)phenyl ether (e.g., Triton), sodium dodecyl sulfate (SDS), sodium lauryl sulfate, sodium octyl glycoside, lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine, lauryl-, myristyl-, linoleyl These surfactants include yl- or stearyl-sarcosine, linoleyl, myristyl- or cetyl-betaine, lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl- or isostearamidopropyl-betaine (e.g., lauroamidopropyl), myristamidopropyl-, palmidopropyl- or isostearamidopropyl-dimethylamine, methyl cocoyl-sodium or methylophenyl-taurate disodium, MONAQUAT® series (Mona Industries, Inc., Paterson, NJ), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.). An exemplary amount of surfactant that may be present in the formulation before lyophilization is about 0.001–0.5%.High molecular weight structural additives (e.g., fillers, binders) include, for example, acacia, albumin, alginic acid, calcium phosphate (dibasic), cellulose, carboxymethylcellulose, sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, dextran, dextrin, dextrate, sucrose, tyrose, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose, disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressed sugar, aluminum magnesium silicate, maltodextrin, polyethylene oxide, polymethacrylate, povidone, sodium alginate, tragacanth microcrystalline cellulose, starch, and zein. Exemplary concentrations of high molecular weight structural additives range from 0.1% to 10% by weight. In other embodiments, a bulking agent (e.g., mannitol, glycine) may be included.

[0091] Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions, including saline and buffer media. Parenteral vehicles include sodium chloride solutions, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate oil, or fixative oils. Intravenous vehicles include liquids and nutritional supplements, electrolyte supplements, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present. For general information, see Remington's Pharmaceutical Science, 16th edition, Mack Eds., 1980.

[0092] The compositions described herein may be formulated for controlled or sustained delivery in a manner that provides local concentrations of the product (e.g., bolus, depot effect) and / or increased stability or half-life in a specific local environment. The compositions include the GM-CSF molecule proteins, polypeptides, nucleic acids, or vectors disclosed herein, along with microparticle preparations of high molecular weight compounds such as polylactic acid and polyglycolic acid, as well as implantable delivery devices that provide controlled or sustained release of the activator, such as biodegradable matrices, injectable microspheres, microcapsule particles, microcapsules, bioerosive particle beads, liposomes, and depot injections. Techniques for formulating such sustained or controlled delivery means are known, and various polymers have been developed and used for controlled release and delivery of drugs. Such polymers are typically biodegradable and biocompatible. Polymeric hydrogels, including those formed by the complex formation of enantiomerized polymers or polypeptide segments, and hydrogels with temperature or pH-sensitive properties, may be desirable to provide a drug depot effect due to the mild, aqueous conditions involved in the capture of bioactive protein agents.

[0093] The pharmaceutical compositions disclosed herein may be administered to subjects by any suitable route, including parenteral (intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, subarachnoid, intravitreous, injectable, or local), topical, oral, and / or nasal administration.

[0094] Formulations suitable for intramuscular, subcutaneous, peritumoral, or intravenous injection may include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injection solutions or dispersions. Examples of suitable aqueous or non-aqueous carriers, diluents, excipients, or vehicles include water, ethanol, polyols (such as propylene glycol, polyethylene glycol, glycerol, and cremophor), suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Adequate fluidity is maintained, for example, by the use of coatings such as lecithin, maintaining the required particle size in the case of dispersions, and the use of surfactants. Formulations suitable for subcutaneous injection may also include optional additives such as preservatives, humectants, emulsifiers, and dispersants.

[0095] For intravenous injection, the activator may be optionally formulated in an aqueous solution, preferably in a physiologically suitable buffer such as Hanks' solution, Ringer's solution, or physiological saline buffer.

[0096] Parenteral injection may optionally include bolus injection or continuous infusion. Formulations for injection may optionally be provided in unit dosage forms, e.g., ampoules or multi-dose containers, with preservatives added. The pharmaceutical compositions described herein may be in forms suitable for parenteral injection as sterile suspensions, solutions or emulsions in oily or aqueous vehicles and may include formulation agents such as suspending agents, stabilizers and / or dispersants. Pharmaceutical formulations for parenteral administration may include aqueous solutions of the active agent in a water-soluble form. Furthermore, suspensions may optionally be prepared as suitable oily injection suspensions.

[0097] Alternatively, or further, the composition may be administered topically by implantation of a membrane, sponge, or other suitable material into which the GM-CSF molecules disclosed herein are absorbed or encapsulated. Where an implantable device is used, the device may be implanted in any suitable tissue or organ, and the delivery of the GM-CSF peptides, nucleic acids, or vectors disclosed herein may be carried out directly through the device via a bolus, via a series of doses, or via a catheter using a continuous infusion.

[0098] The pharmaceutical compositions containing the GM-CSF molecule disclosed herein may be formulated for inhalation, for example, as a dry powder. The inhalation solution may also be formulated with a liquefied propellant for aerosol delivery. In yet another formulation, the solution may be sprayed. For pulmonary delivery, the particle size should be suitable for delivery to the distal lungs. For example, the particle size may be 1 μm to 5 μm, however, larger particles may be used, for example, if each particle is quite porous.

[0099] Certain formulations containing the GM-CSF molecule disclosed herein may be administered orally. Formulations administered in this manner may be formulated with or without carriers conventionally used in the formulation of solid dosage forms such as tablets and capsules. For example, capsules may be designed to release the active portion of the formulation at a point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to enhance the absorption of selective binders. Diluents, fragrances, low-melting-point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may also be used.

[0100] Another preparation may contain an effective amount of GM-CSF molecules in a mixture with a non-toxic excipient suitable for tablet production. The solution can be prepared in unit dose form by dissolving the tablets in sterile water or another suitable vehicle. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose, or calcium phosphate; or binders such as starch, gelatin, or acacia; or lubricants such as magnesium stearate, stearic acid, or talc.

[0101] "Pharmacologically acceptable" can mean that it is approved or eligible for approval by a federal or state regulatory agency, or that it is listed in the United States Pharmacopeia or any other generally accepted pharmacopoeia for use in animals, including humans.

[0102] A "pharmaceutically acceptable salt" can refer to a salt of a compound that is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound.

[0103] "Pharmacologically acceptable excipients, carriers or adjuvants" may refer to excipients, carriers or adjuvants that can be administered to a subject together with at least one antibody of the present disclosure, which do not disrupt the pharmacological activity thereof and are non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the compound.

[0104] "Pharmacologically acceptable vehicle" may refer to a diluent, adjuvant, excipient, or carrier to which at least one of the antibodies of this disclosure is administered together.

[0105] therapeutic use In one embodiment, the GM-CSF molecules disclosed herein comprise GM-CSF peptides for treating, mitigating, inhibiting, and / or preventing one or more diseases and / or conditions. In some embodiments, the disease or condition is a neurological disease or condition. Non-limiting examples of neurological diseases or conditions include Parkinson's disease, Alzheimer's disease, or diseases or conditions characterized by neuroinflammation. In some embodiments, the disease or condition is an inflammatory disease or condition. Non-limiting examples of inflammatory diseases or conditions include Crohn's disease and colitis. In some embodiments, the disease or condition is an infectious disease. Infectious diseases may include cytomegalovirus infection. In some embodiments, the disease or condition includes Parkinson's disease (PD). In some embodiments, the disease or condition includes amyotrophic lateral sclerosis (ALS). In some embodiments, the disease or condition includes Alzheimer's disease (AD). In some embodiments, the disease or condition includes traumatic brain injury. In some embodiments, the disease or condition includes inflammatory bowel disease (IBD), including colitis and Crohn's disease (CD). In some embodiments, the disease or condition includes acute radiation syndrome. In some embodiments, the disease or condition includes cancer.

[0106] In some embodiments, the GM-CSF molecules disclosed herein are administered after induction chemotherapy in a subject. The GM-CSF molecules may shorten the time to neutrophil recovery and / or reduce the incidence of infection. In some cases, the subject includes acute myeloid leukemia.

[0107] In some embodiments, the GM-CSF molecules disclosed herein are administered to mobilize hematopoietic progenitor cells into the peripheral blood for collection by leukocyte apheresis transfusion. Mobilization may enable the collection of an increased number of transplantable progenitor cells compared to collection without mobilization. Transplanting an increased number of progenitor cells after chemotherapy, such as bone marrow depletion chemotherapy, may result in faster engraftment.

[0108] In some embodiments, the GM-CSF molecule disclosed herein is administered to accelerate bone marrow recovery in subjects with non-Hodgkin lymphoma (NHL). In some embodiments, the GM-CSF molecule disclosed herein is administered to accelerate bone marrow recovery in subjects with acute lymphoblastic leukemia (ALL). In some embodiments, the GM-CSF molecule disclosed herein is administered to accelerate bone marrow recovery in subjects with Hodgkin's disease. In some embodiments, the subjects have undergone autologous bone marrow transplantation (BMT). GM-CSF administration may accelerate bone marrow engraftment compared to bone marrow engraftment without GM-CSF administration. GM-CSF administration may reduce the median duration of antibiotic administration compared to periods without GM-CSF administration. Administration may reduce the median duration of infectious episodes compared to periods without GM-CSF administration. Administration may shorten the median length of hospital stay compared to periods without GM-CSF administration. Hematological responses to GM-CSF can be detected by complete blood count testing.

[0109] In some embodiments, the GM-CSF molecules disclosed herein are administered to accelerate bone marrow recovery in subjects receiving allogeneic BMT from HLA-matching-related donors.

[0110] In some embodiments, the GM-CSF molecules disclosed herein are administered to subjects who have undergone allogeneic or autologous bone marrow transplantation (BMT) with delayed or failed engraftment. Administration may prolong the survival of the subjects. Recombinant human GM-CSF is used in a variety of hematopoietic disorders, including reducing the severity of chemotherapy-induced neutropenia, accelerating hematopoietic recovery after bone marrow transplantation, and mobilizing blood progenitor cells for transplantation. However, recombinant GM-CSF has a short half-life in humans and is usually administered by daily injection for 15–21 days after chemotherapy. The need for daily administration also limits the appeal of GM-CSF for chemotherapy patients and for the treatment of radiation-exposed patients such as ARS patients. Therefore, methods for using long-acting GM-CSF molecules are provided herein for such conditions, including acute radiation syndrome.

[0111] Furthermore, preliminary preclinical results have shown that GM-CSF can protect and treat cells from radiation toxic damage or side effects. GM-CSF may also help accelerate the healing of radiation-damaged tissue.

[0112] To evaluate the efficacy of GM-CSF in humans, a retrospective analysis was conducted on human patients undergoing cancer treatment who acquired cellular damage after radiotherapy. In patients who received GM-CSF before radiotherapy, the treatment was found to improve their condition, and in some patients, damaged tissue healed faster than in the control group. Therefore, the method of the present invention comprises treating a subject who is receiving, undergoing, or has undergone radiotherapy with the GM-CSF molecule specified herein.

[0113] In some embodiments, the GM-CSF molecule disclosed herein is administered for acute radiation syndrome. The GM-CSF molecule may increase the survival rate of subjects exposed to myelosuppressive doses of radiation (hematopoietic syndrome of acute radiation syndrome, H-ARS). Myelosuppression occurs when radiation damages the bone marrow. Myelosuppression blocks the production of blood cells. The GM-CSF molecule may promote the recovery of myelocytes that develop into leukocytes. Therefore, GM-CSF may also be used to treat and / or prevent infection.

[0114] GM-CSF plays a crucial role in immunomodulation and hematopoiesis. While not tied to any specific treatment, experimental evidence suggests that GM-CSF, frequently upregulated in multiple types of human cancer, tags cancer cells that are targeted by the immune system. Activation of the GM-CSF receptor promotes the survival, proliferation, and differentiation of many different immune cell types, including neutrophils, macrophages, and various T cells, in addition to its direct stimulating effects on multiple immune functions. Preliminary preclinical results demonstrate that GM-CSF may stimulate the immune system in various ways to halt or delay the proliferation of tumor cells. GM-CSF may increase the number of immune cells found in the bone marrow or peripheral blood. GM-CSF may also induce extensive tumor destruction.

[0115] To evaluate the efficacy of GM-CSF for immuno-oncology, a retrospective analysis was conducted on human patients diagnosed with cancer. Patients who received GM-CSF were found to have improved condition with treatment. In some patients who received GM-CSF, tumor growth was delayed with treatment compared to the control group. Furthermore, in some patients who received GM-CSF, tumor size was reduced compared to the control group. These results suggest that GM-CSF can create a favorable environment for tumor antigen presentation. Therefore, the method of the present invention comprises treating a subject having cancer with the GM-CSF molecule of this specification.

[0116] In some embodiments, the methods described herein include the treatment of subjects suffering from dementia, such as Alzheimer's disease, vascular dementia, and cerebral amyloid vascular disease (CAA).

[0117] Preliminary preclinical results demonstrated that GM-CSF rapidly reduced cerebral amyloid deposition and completely reversed memory impairment in a transgenic mouse model of Alzheimer's disease (AD). A retrospective analysis was conducted on cognitive studies of human patients who underwent hematopoietic stem cell transplantation for cancer and acquired cognitive impairment due to chemotherapy or radiation (NCT01409915). In patients administered colony-stimulating factor (CSF) to stimulate bone marrow and restore immune system function, patients administered GM-CSF plus G-CSF showed significantly improved cognitive function compared to patients administered G-CSF alone. These findings, coupled with over 20 years of safety data obtained using recombinant human GM-CSF in elderly leukopenic patients, support the use of GM-CSF as a treatment to reverse cerebral amyloid pathology and cognitive impairment in AD.

[0118] A treatment method may include administering a composition comprising one or more GM-CSF molecules disclosed herein to a subject in need. This composition may further comprise a pharmaceutically acceptable carrier. The GM-CSF molecules may be substantially purified (e.g., substantially free of substances that limit their efficacy or produce undesirable side effects). The subject may be an animal, including but not limited to animals such as cattle, pigs, sheep, goats, rabbits, horses, chickens, cats, dogs, and mice. The subject may be a mammal. The subject may be a human. The subject may be a non-human primate. The subject may be a cattle. The subject may be a bird, reptile, or amphibian.

[0119] Provided herein are methods for treating (treating) a disease or condition in a subject of interest, the method comprising administering to the subject a composition comprising a GM-CSF molecule comprising a GM-CSF peptide attached to a scaffold comprising an antibody variable domain. In some embodiments, the disease or condition is a neurological disease or condition. In some embodiments, the neurological disease or condition includes Parkinson's disease. In some embodiments, the disease or condition includes amyotrophic lateral sclerosis (ALS). In some embodiments, the disease or condition includes Alzheimer's disease (AD). In some embodiments, the disease or condition includes traumatic brain injury. In some embodiments, the disease or condition includes inflammatory bowel disease (IBD), including colitis and / or Crohn's disease (CD). In some embodiments, the disease or condition includes acute radiation syndrome. In some embodiments, the disease or condition includes cancer. In some embodiments, the GM-CSF molecule is administered approximately every 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 days, weekly, bi-weekly, or monthly. The antibody variable domain may comprise a first polypeptide and a second polypeptide, where the first or second polypeptide comprises the GM-CSF peptide or is otherwise linked to the GM-CSF peptide. In some embodiments, the first polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6. In some cases, the first polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7. In some cases, the first polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5.In some embodiments, the second polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. In some cases, the second polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4. In some cases, the second polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, a GM-CSF molecule is provided that includes an antibody-variable domain comprising a light chain sequence comprising a first polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6, and a heavy chain sequence comprising a second polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the first polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31. In some cases, the first polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 30. In some cases, the first polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7. In some embodiments, the second polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29. In some cases, the second polypeptide contains a sequence that is at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 28.In some cases, the second polypeptide contains a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 4. In some embodiments, a GM-CSF molecule is provided that includes an antibody variable domain comprising a light chain sequence containing a first polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 31, and a heavy chain sequence containing a second polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 29.

[0120] Provided herein is a method for treating a disease or condition in a subject of interest, the method comprising administering to a subject a composition comprising a GM-CSF molecule comprising a GM-CSF peptide attached to a scaffold comprising an antibody-variable domain, the antibody-variable domain comprising SEQ ID NO: 26 [ka] The heavy chain sequence comprises a light chain sequence containing a sequence that is at least about 90% identical to sequence 2, wherein the light chain sequence contains X1, and X1 contains GM-CSF, and a second polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence 2. In some embodiments, the disease or condition is a neurological disease or condition. In some embodiments, the neurological disease or condition includes Parkinson's disease. In some embodiments, the neurological disease or condition includes amyotrophic lateral sclerosis (ALS). In some embodiments, the disease or condition includes Alzheimer's disease (AD). In some embodiments, the disease or condition includes traumatic brain injury. In some embodiments, the disease or condition includes inflammatory bowel disease (IBD), including colitis and / or Crohn's disease (CD). In some embodiments, the disease or condition includes acute radiation syndrome. In some embodiments, the disease or condition includes cancer. In some embodiments, the GM-CSF molecule is administered approximately every 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 days, weekly, bi-weekly, or monthly. The GM-CSF may be human, bovine, or mouse GM-CSF. GM-CSF may contain sequences that are at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 16. GM-CSF may contain sequences that are at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 77. GM-CSF may contain variants or homologs of GM-CSF.In some embodiments, the light chain sequence includes a sequence that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence 26. In some embodiments, the light chain sequence includes sequence 27. [ka] The light chain sequence includes a sequence that is at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 2, where the light chain sequence includes X2, and X2 includes GM-CSF. In some embodiments, the heavy chain sequence includes a sequence that is at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 2. In some embodiments, the light chain sequence includes a sequence that is at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 7. In some embodiments, the light chain sequence includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the heavy chain sequence includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, the heavy chain sequence includes a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, the GM-CSF molecule further includes an Fc region containing reduced effector function compared to human IgG1 (SEQ ID NO: 25). Reduced effector function may include decreased antibody-dependent cell-mediated cytotoxicity (ADCC) and decreased complement-dependent cell-mediated cytotoxicity (CDC). The Fc region may contain sequences that are at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3. In some cases, the Fc region contains human IgG1 including E233P, L234V, L235A, ΔG236, A327G, A330S, and P331S according to Kabat numbering.

[0121] Provided herein is a method for treating a disease or condition in a subject of interest, the method comprising administering to a subject a composition comprising a GM-CSF molecule comprising a GM-CSF peptide attached to a scaffold comprising an antibody-variable domain, wherein the antibody-variable domain is SEQ ID NO: 42

change

[0122] Provided herein are methods for treating a disease or condition in a subject of interest, the method comprising administering to a subject a composition comprising a GM-CSF molecule containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29, and a second polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31. In some embodiments, the first polypeptide contains a sequence that is at least about 95% identical to SEQ ID NO: 29, and the second polypeptide contains a sequence that is at least about 95% identical to SEQ ID NO: 31. In some embodiments, the first polypeptide contains a sequence that is at least about 96% identical to SEQ ID NO: 29, and the second polypeptide contains a sequence that is at least about 96% identical to SEQ ID NO: 31. In some embodiments, the first polypeptide comprises a sequence that is at least about 97% identical to SEQ ID NO: 29, and the second polypeptide comprises a sequence that is at least about 97% identical to SEQ ID NO: 31. In some embodiments, the first polypeptide comprises a sequence that is at least about 98% identical to SEQ ID NO: 29, and the second polypeptide comprises a sequence that is at least about 98% identical to SEQ ID NO: 31. In some embodiments, the first polypeptide comprises a sequence that is at least about 99% identical to SEQ ID NO: 29, and the second polypeptide comprises a sequence that is at least about 99% identical to SEQ ID NO: 31. In some embodiments, the first polypeptide comprises SEQ ID NO: 29, and the second polypeptide comprises SEQ ID NO: 31. In some embodiments, the disease or condition is a neurological disease or condition. In some embodiments, the neurological disease or condition includes Parkinson's disease. In some embodiments, the neurological disease or condition includes amyotrophic lateral sclerosis (ALS). In some embodiments, the disease or condition includes Alzheimer's disease (AD). In some embodiments, the disease or condition includes traumatic brain injury. In some embodiments, the disease or condition includes inflammatory bowel disease (IBD), including colitis and / or Crohn's disease (CD).In some embodiments, the disease or condition includes acute radiation syndrome. In some embodiments, the disease or condition includes cancer. In some embodiments, the GM-CSF molecule is administered approximately every 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 days, weekly, bi-weekly, or monthly.

[0123] Provided herein are methods for treating a disease or condition in a subject of interest, the method comprising administering to a subject a composition comprising a GM-CSF molecule containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28, and a second polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 30. In some embodiments, the first polypeptide contains a sequence that is at least about 95% identical to SEQ ID NO: 28, and the second polypeptide contains a sequence that is at least about 95% identical to SEQ ID NO: 30. In some embodiments, the first polypeptide contains a sequence that is at least about 96% identical to SEQ ID NO: 28, and the second polypeptide contains a sequence that is at least about 96% identical to SEQ ID NO: 30. In some embodiments, the first polypeptide contains a sequence that is at least about 97% identical to SEQ ID NO: 28, and the second polypeptide contains a sequence that is at least about 97% identical to SEQ ID NO: 30. In some embodiments, the first polypeptide contains a sequence that is at least about 98% identical to SEQ ID NO: 28, and the second polypeptide contains a sequence that is at least about 98% identical to SEQ ID NO: 30. In some embodiments, the first polypeptide contains a sequence that is at least about 99% identical to SEQ ID NO: 28, and the second polypeptide contains a sequence that is at least about 99% identical to SEQ ID NO: 30. In some embodiments, the first polypeptide contains SEQ ID NO: 28, and the second polypeptide contains SEQ ID NO: 30. In some embodiments, the disease or condition is a neurological disease or condition. In some embodiments, the neurological disease or condition includes Parkinson's disease. In some embodiments, the neurological disease or condition includes amyotrophic lateral sclerosis (ALS). In some embodiments, the disease or condition includes Alzheimer's disease (AD). In some embodiments, the disease or condition includes traumatic brain injury. In some embodiments, the disease or condition includes inflammatory bowel disease (IBD), including colitis and / or Crohn's disease (CD).In some embodiments, the disease or condition includes acute radiation syndrome. In some embodiments, the disease or condition includes cancer. In some embodiments, the GM-CSF molecule is administered approximately every 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 days, weekly, bi-weekly, or monthly.

[0124] Provided herein are methods for treating a disease or condition in a subject of interest, the method comprising administering to a subject a composition comprising a GM-CSF molecule containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, and a second polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6. In some embodiments, the first polypeptide contains a sequence that is at least about 95% identical to SEQ ID NO: 2, and the second polypeptide contains a sequence that is at least about 95% identical to SEQ ID NO: 6. In some embodiments, the first polypeptide contains a sequence that is at least about 96% identical to SEQ ID NO: 2, and the second polypeptide contains a sequence that is at least about 96% identical to SEQ ID NO: 6. In some embodiments, the first polypeptide contains a sequence that is at least about 97% identical to SEQ ID NO: 2, and the second polypeptide contains a sequence that is at least about 97% identical to SEQ ID NO: 6. In some embodiments, the first polypeptide contains a sequence that is at least about 98% identical to SEQ ID NO: 2, and the second polypeptide contains a sequence that is at least about 98% identical to SEQ ID NO: 6. In some embodiments, the first polypeptide contains a sequence that is at least about 99% identical to SEQ ID NO: 2, and the second polypeptide contains a sequence that is at least about 99% identical to SEQ ID NO: 6. In some embodiments, the first polypeptide contains SEQ ID NO: 2, and the second polypeptide contains SEQ ID NO: 6. In some embodiments, the disease or condition is a neurological disorder or condition. In some embodiments, the neurological disorder or condition includes Parkinson's disease. In some embodiments, the neurological disorder or condition includes amyotrophic lateral sclerosis (ALS). In some embodiments, the disease or condition includes Alzheimer's disease (AD). In some embodiments, the disease or condition includes traumatic brain injury. In some embodiments, the disease or condition includes inflammatory bowel disease (IBD), including colitis and / or Crohn's disease (CD). In some embodiments, the disease or condition includes acute radiation syndrome.In some embodiments, the disease or condition includes cancer. In some embodiments, the GM-CSF molecule is administered approximately every 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 days, weekly, bi-weekly, or monthly.

[0125] Provided herein are methods for treating a disease or condition in a subject of interest, the method comprising administering to a subject a composition comprising a GM-CSF molecule containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1, and a second polypeptide containing a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the first polypeptide contains a sequence that is at least about 95% identical to SEQ ID NO: 1, and the second polypeptide contains a sequence that is at least about 95% identical to SEQ ID NO: 5. In some embodiments, the first polypeptide contains a sequence that is at least about 96% identical to SEQ ID NO: 1, and the second polypeptide contains a sequence that is at least about 96% identical to SEQ ID NO: 5. In some embodiments, the first polypeptide comprises a sequence that is at least about 97% identical to SEQ ID NO: 1, and the second polypeptide comprises a sequence that is at least about 97% identical to SEQ ID NO: 5. In some embodiments, the first polypeptide comprises a sequence that is at least about 98% identical to SEQ ID NO: 1, and the second polypeptide comprises a sequence that is at least about 98% identical to SEQ ID NO: 5. In some embodiments, the first polypeptide comprises a sequence that is at least about 99% identical to SEQ ID NO: 1, and the second polypeptide comprises a sequence that is at least about 99% identical to SEQ ID NO: 5. In some embodiments, the first polypeptide comprises SEQ ID NO: 1, and the second polypeptide comprises SEQ ID NO: 5. In some embodiments, the disease or condition is a neurological disease or condition. In some embodiments, the neurological disease or condition includes Parkinson's disease. In some embodiments, the neurological disease or condition includes amyotrophic lateral sclerosis (ALS). In some embodiments, the disease or condition includes Alzheimer's disease (AD). In some embodiments, the disease or condition includes traumatic brain injury. In some embodiments, the disease or condition includes inflammatory bowel disease (IBD), including colitis and / or Crohn's disease (CD). In some embodiments, the disease or condition includes acute radiation syndrome.In some embodiments, the disease or condition includes cancer. In some embodiments, the GM-CSF molecule is administered approximately every 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 days, weekly, bi-weekly, or monthly.

[0126] Administration In one embodiment, the GM-CSF molecule containing the scaffold provided herein is long-acting and has a longer half-life than the GM-CSF peptide alone. Such a GM-CSF molecule is administered approximately every 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 days, or once a month. The GM-CSF molecule provided herein may be administered once every 2 weeks. The GM-CSF molecule provided herein may be administered once every 3 weeks. The GM-CSF molecule provided herein may be administered once every 4 weeks. The GM-CSF molecule provided herein may be administered approximately once a month. The amount of the composition described herein that is effective in treating, inhibiting, and / or preventing a disease or disorder may be determined by standard clinical techniques. Furthermore, in vitro assays may be optionally employed to help identify the optimal dose range. The exact dose employed in the formulation may also depend on the route of administration and the severity of the disease or disorder, and should be determined according to the physician's judgment and the circumstances of each patient. The effective dose may be extrapolated from dose-response curves derived from in vitro, animal model test systems, or clinical trials. In non-limiting embodiments, the GM-CSF molecule is administered to treat Parkinson's disease.

[0127] Pharmacological properties In one embodiment, a method for improving one or more pharmacological properties of GM-CSF is disclosed herein. GM-CSF includes at least human, bovine, rat, and mouse GM-CSF, as well as GM-CSF having at least about 90% identity to SEQ ID NO: 16 or 77. The method may include a step of producing a GM-CSF molecule, such as a GM-CSF molecule containing a GM-CSF peptide bound to a scaffold disclosed herein. Examples of pharmacological properties may include, but are not limited to, half-life, stability, solubility, immunogenicity, toxicity, bioavailability, absorption, release, distribution, metabolism, and elimination. Release may refer to the process of releasing GM-CSF from a pharmaceutical formulation. Absorption may refer to the process of a substance entering the bloodstream. Distribution may refer to the dispersion or propagation of a substance throughout the body's fluids and tissues. Metabolism (or biotransformation, or inactivation) may refer to the recognition by an organism of the presence of a foreign substance and its irreversible conversion to daughter metabolites of the parent compound. Discharge can refer to the removal of substances from the body.

[0128] The half-lives of GM-CSF molecules are at least approximately 5 hours to 1000 hours, at least approximately 10 hours to 1000 hours, at least approximately 15 hours to 1000 hours, at least approximately 20 hours to 1000 hours, at least approximately 25 hours to 1000 hours, at least approximately 30 hours to 1000 hours, at least approximately 40 hours to 1000 hours, at least approximately 50 hours to 1000 hours, at least approximately 60 hours to 1000 hours, at least approximately 70 hours to 1000 hours, at least approximately 80 hours to 1000 hours, at least approximately 90 hours to 1000 hours, and at least approximately 100 hours to 10 It could be 0 hours, at least about 125 hours to about 1000 hours, at least about 150 hours to about 1000 hours, at least about 175 hours to about 1000 hours, at least about 200 hours to about 1000 hours, at least about 225 hours to about 1000 hours, at least about 250 hours to about 1000 hours, at least about 275 hours to about 1000 hours, at least about 300 hours to about 1000 hours, at least about 350 hours to about 1000 hours, at least about 400 hours to about 1000 hours, at least about 450 hours to about 1000 hours, or at least about 500 hours to about 1000 hours. In some embodiments, the half-lives of the GM-CSF molecules provided herein are between about 100 hours and about 500 hours, between about 150 hours and about 500 hours, between about 200 hours and about 500 hours, between about 100 hours and about 500 hours, between about 150 hours and about 500 hours, between about 200 hours and about 500 hours, between about 100 hours and about 400 hours, between about 150 hours and about 400 hours, between about 200 hours and about 400 hours, or between about 100 hours and about 300 hours. For example, the half-lives are about 100, 125, 150, 175, 200, 225, 250, 275, or 300 hours. The half-lives may be measured in human, rat, or mouse blood, serum, and / or plasma. The half-lives may be measured using the methods provided in the examples herein. The half-life can be measured after administration of the GM-CSF molecule to the subject. Alternatively, the half-life can be measured after incubation of the GM-CSF molecule in a biological sample isolated from the subject.

[0129] The half-life of GM-CSF molecules constituting the scaffold may be at least approximately 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 times longer than the half-life of GM-CSF peptide alone. The half-life of GM-CSF molecules may be at least approximately 50 times longer than the half-life of GM-CSF peptide alone. The half-life of GM-CSF molecules may be at least approximately 100 times longer than the half-life of GM-CSF peptide alone. The half-life of GM-CSF molecules may be at least approximately 200 times longer than the half-life of GM-CSF peptide alone. The half-life of a GM-CSF molecule can be at least approximately 300 times longer than that of a GM-CSF peptide alone. The half-life of a GM-CSF molecule can be at least approximately 400 times longer than that of a GM-CSF peptide alone.

[0130] kit Further disclosed herein are kits comprising one or more GM-CSF molecules or compositions thereof provided herein. The GM-CSF molecules may be packaged in a manner that facilitates their use in carrying out the methods of this disclosure. For example, a kit comprises the GM-CSF molecules described herein, packaged in a container with a label affixed to the container or a package insert describing the use of the composition in carrying out the method. Suitable containers include, for example, bottles, vials, syringes, etc. Containers may be formed from a variety of materials, such as glass or plastic. Containers may have a sterile access port (for example, a container may be a vial with a stopper that can be pierced by an intravenous solution bag or a subcutaneous injection needle). A kit may also include a container containing the GM-CSF molecules. A kit may further include a package insert indicating that the GM-CSF molecules may be used to treat a particular condition. Alternatively, the kit may further include a second (or third) container containing a pharmaceutically acceptable buffer (e.g., water for bacteriostatic injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution). This may further include other materials desirable from a commercial and user perspective, including but not limited to other buffers, diluents, filters, needles, and syringes. The GM-CSF molecule may be packaged in unit dosage forms. The kit may further include a device suitable for administering the antibody fusion protein according to a specific route of administration. The kit may include a label describing the use of the GM-CSF molecule. [Examples]

[0131] The activity data provided in the following examples are generally obtained using molecules defined in the examples and illustrated by the provided sequence numbers. It should be understood that the activity of any molecule disclosed herein may be enhanced or diminished depending on conditions independent of the primary sequence, such as expression and purification conditions.

[0132] Example 1: Production of long-acting GM-CSF Expression constructThe gene encoding GM-CSF was synthesized by IDT (Coralville, IA) and amplified by polymerase chain reaction using PfuUltra II DNA polymerase (Agilent Technologies, CA). DNA fragments encoding the heavy and light chains of palivizumab and trastuzumab antibodies with reduced RSV-F binding, along with linkers, were also synthesized by IDT and amplified by PCR. The fusion gene fragments were assembled into a pFuse backbone (Invivogen, CA) using Gibson Assembly Master Mix (New England Biolabs, MA). The sequences of the resulting mammalian expression vectors were confirmed by DNA sequencing (GENEWIZ, CA). The constructs encoding the fusions in Table 1 were generated in this manner. Figure 7 provides schematic diagrams of the Fab domains of various long-acting GM-CSF molecules, where GM-CSF is positioned at the amino terminus or CDR of an IgG scaffold to generate an IgG fusion.

[0133] The heavy chain of the long-acting GM-CSF molecule contains a human IgG1 heavy chain constant region with mutations (E233P, L234V, L235A, ΔG236, A327G, A330S, P331S) that reduce complement-dependent and antibody-dependent cell-mediated cytotoxicity.

[0134] Expression and PurificationGenes containing the heavy and light chains of each GM-CSF molecule were co-expressed in HEK293F cells (Life Technologies, CA) by transient transfection according to Table 1. HEK293F cells were cultured in a shaker flask containing FreeStyle medium (Life Technologies, CA) and shaken at 125 rpm, 37°C, and 5% CO2. For transfection, 293F cells were grown to a density of 1 million cells per mL and transfected with the light chain, heavy chain plasmid, and 293fectin in a ratio of 1:2:6 according to the manufacturer's instructions. Expression medium was collected 5 days post-transfection to collect the secreted proteins. Fusion antibodies were purified by protein A chromatography (Thermo Fisher, IL) and analyzed by SDS-PAGE and mass spectrometry.

[0135] [Table 1]

[0136] Confirmation of Fc null caused by 7-point mutations To confirm that seven point mutations in the Fc of protein fusions can significantly reduce undesirable ADCC and CDC effects mediated by Fcγ receptor interaction, antibodies containing a therapeutic peptide (INVKCSLPQQCIKPCKDAGMRFGKCMNKKCRCYS, SEQ ID NO: 77) placed within the antibody heavy chain CDR3 having a mutated Fc (SEQ ID NO: 25, Fc null), or wild-type IgG1 Fc labeled with Alexa Fluor 488, were incubated with THP-1, a human monocyte cell line exhibiting high expression of the Fcγ receptor. The Fc null antibody fusion did not bind to THP-1 at concentrations up to 100 nM, while the antibody fusion with wild-type IgG1 Fc significantly bound to THP-1 at a low concentration of 1 nM.

[0137] Example 2: Characterization of long-acting GM-CSF molecules and in vitro activity In the TF-1 proliferation assay, the biological activity of the GM-CSF molecule generated in Example 1 was investigated. TF-1 human leukocytes (ATCC, CRL-2003) were cultured in RPM1640 medium of an ATCC preparation supplemented with 10% fetal bovine serum (FBS) at 37°C under 5% CO2. For the proliferation assay, cells were washed five times with PBS to remove FBS. The GM-CSF fusion or a commercially available GM-CSF standard (R&D systems) was added to cells in a 96-well plate at a density of 5,000 cells / well in 150 μL of RPM1640 medium containing 2% FBS. After 72 hours of incubation, 15 μL of AlamarBlue (Invitrogen, DAL1025) was added to each well, and after incubation at 37°C under 5% CO2 for 4 hours, the fluorescence signal at 565 / 595 nm was measured. The corresponding data is shown in Figures 6A-B and Table 2.

[0138] [Table 2] JPEG2026082829000019.jpg176170

[0139] Example 3: Pharmacokinetics (PK), pharmacodynamics (PD), and in vivo efficacy of long-acting GM-CSF in rats Pharmacokinetic assay in ratsLong-acting GM-CSF protein samples were administered intravenously to Sprague-Dawley rats at a single dose of 10 mg / kg, or intraperitoneally to mice at 3 mg / kg or 10 mg / kg. Blood samples were collected at various time points. Samples were stored in heparinized collection tubes, centrifuged, and stored at -80°C until further processing. After thawing and appropriately diluting the samples, the amount of antibody in each blood sample was quantified by ELISA. Pharmacokinetic parameters were determined by analyzing the data using a PK modeling program (WinNonlin, Certara, NJ). The experiments were performed using the molecules listed in Table 1, and the data are shown in Figures 8A-B and Table 3. Figure 8A shows the concentrations of Syn-hGMCSF CDR and Her-hGMCSF CDR in rat plasma over time. Figure 8B shows the concentrations of Syn-mGMCSF CDR and Syn-mGMCSF NT (N-terminal HC fusion) in mouse plasma over time. Syn-hGMCSF CDR and Her-hGMCSF CDR showed extended half-lives compared to recombinant GM-CSF (t 1 / 2 , t of Sarglamostim 1 / 2 (<0.5 hours versus approximately 200 hours). The hGMCSF CDR fusion showed a Cmax of 2,481 nM and an AUC∞(hr*nM) of 63,257.

[0140] T reg Assay Figure 9 shows that subchronic treatment with Syn-mGMCSF CDR (shown as Syn-GMCSF) is effective in mice. reg This shows a significant increase in proliferation. Figure 10 shows that a single long-acting GM-CSF Her-hGMCSF CDR significantly increases T reg This indicates that the expansion increased for up to 14 days.

[0141] Cynomolgus monkey PK / PD assaySamples of Her-hGMCSF CDR were administered intravenously (IV) or subcutaneously (SC) to cynomolgus monkeys (n=4, ~3kg, 2-5 years old) in single doses of 1 mg / kg (IV), 5-10 mg / kg (IV), or 5-10 mg / kg (SC). Plasma PK was collected at 13 time points: pre-administration, 10 minutes (30 minutes for SC), 1 hour, 3 hours, 10 hours, 24 hours, 48 ​​hours, 72 hours, 120 hours, 168 hours, 240 hours, 336 hours, and 504 hours. Blood samples were collected at 6 time points: pre-administration, 72 hours, 120 hours, 240 hours, 336 hours, and 504 hours for flow cytometry. There were no apparent negative clinical signs. As shown in Figure 11, Her-hGMCSF CDR was found to have circulating T reg It increases T in a dose-dependent manner. reg The increase peaked on day 8, rising to a 20-fold increase in the 5 mg / kg treatment group.

[0142] In vivo efficacy The assay is further described in Example 4.

[0143] Brain exposure in miceLong-acting GMCSFs (Her-hGMCSF CDR and Her-mGMCSF CDR) are actively transported to the mouse brain, as shown in Figure 12. Samples of long-acting GM-CSF protein were intravenously injected into CD1 mice (n=4, female, 12 weeks old) at a single dose of 10 mg / kg. Brain tissue was collected 3 hours after administration. Brain samples were pulverized with dry ice and transferred to homogenization tubes pre-loaded with 1.4 mm ceramic beads. The tissue was homogenized in RIPA buffer supplemented with a protease inhibitor. After centrifugation at 4°C and 17000 g for 30 minutes, the supernatant was collected and the protein concentration was measured. For ELISA assays, plates were coated with 2.5 μg / mL anti-human IgG Fc antibody at 37°C for 2 hours, then washed and blocked with 2% skim milk in 0.5% PBST for 1 hour. After adding standard proteins and diluted samples, the plates were incubated overnight at 4°C and washed with 0.5% PBST. Then, 1 μg / mL of anti-human or mouse GMCSF antibody was added to the wells and incubated at room temperature for 2 hours. After washing, 0.5 μg / mL of HRP-conjugated anti-mouse IgG antibody was added and incubated at room temperature for 1 hour. Preheated TMB substrate was added to the wells and incubated in the dark at room temperature for 10 minutes. Absorbance at 450 nM was measured using a microplate reader.

[0144] [Table 3]

[0145] Example 4: Neuroprotective and anti-inflammatory capabilities of long-acting GM-CSF Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons along the nigrostriatal axis. Immune dysfunction and neuroinflammation are associated with disease progression and neuronal loss. Innate immune dysfunction in PD is associated with the production of pro-inflammatory cytokines, microgliosis, reactive oxygen species levels, and increased neurotoxins, all of which have the ability to influence neuronal cell death. Similarly, dysfunction of the adaptive immune response in PD is associated with decreased CD4+ T cell levels, the presence of effector memory T cells, decreased regulatory T cell (Treg) levels, and Th1 and Th17 T effector T cells (T eff This includes an increased frequency of ). While not theoretically bound, these immune changes may contribute to the pathogenesis of the disease. Similar adaptive immune abnormalities have been observed in amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and Crohn's disease (CD), suggesting that this may be related to the suppression of inflammation. reg This suggests a decisive role.

[0146] Following treatment with long-acting GM-CSF in mice, complete blood analysis and blood chemistry profiles were determined by flow cytometry. The neuroprotective and anti-inflammatory capabilities of long-acting GM-CSF were evaluated in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease. Treatment with long-acting GM-CSF resulted in T reg This induced and enhanced the immunosuppressive function of Treg.

[0147] The use of long-acting GM-CSF compositions induced neuroprotective immunoconversion in the periphery. The observed phenotypic changes and neuroprotective responses were greater than those observed with recombinant GM-CSF (rGM-CSF), suggesting long-acting GM-CSF as a candidate for the treatment of PD and other neurodegenerative diseases.

[0148] method Animals, antibody GM-CSF treatment, and MPTP poisoning Male C57BL / 6 mice (6-8 weeks old) were obtained from Jackson Laboratories. After acclimatization, the mice were intraperitoneally (ip) injected with long-acting GM-CSF Her-mGMCSF CDR (SEQ ID NOs. 55, 56). In dose-response studies, mice were given single injections of doses ranging from 0 mg / kg to 30.0 mg / kg. For rGM-CSF (recombinant granulocyte-macrophage colony-stimulating factor) protein injections, mice were given either a single injection (1X) or five daily injections (5X) of 0.1 mg / kg prior to either sacrifice or MPTP poisoning. For neuroprotective experiments, mice were injected with either a vehicle (DPBS, 10 ml / kg body weight) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP-HCl) reconstituted in PBS obtained from Sigma-Aldrich, St. Louis, MO. Mice received four subcutaneous injections of MPTP-HCl (16 mg / kg, free MPTP base) at 2-hour intervals. MPTP safety measures were implemented according to the MPTP safety and handling protocol. Mice were sacrificed and their brains were collected for processing on days 2 and 7 of MPTP poisoning. All animals were cared for and maintained according to the National Institutes of Health Institutional guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Nebraska Medical Center.

[0149] Perfusion and immunohistochemistry Under local anesthesia (Fatal Plus, pentobarbital), mice were perfused with DPBS by cardiac puncture, followed by perfusion with 4% paraformaldehyde (Sigma-Aldrich) in DPBS. After perfusion, the entire brain was collected and processed to assess the survival of dopaminergic neurons in the substantia nigra and striatum. Frozen midbrain sections were cut into 30 μm sections and immunostained with anti-tyrosine hydroxylase (TH) (anti-TH, 1:2000, EMD Millipore), counterstained for Nissl substance. Striatal sections were also labeled with anti-TH (1:1000, EMD Millipore) to assess dopaminergic terminals. Midbrain sections were immunostained with anti-macrophage antigen complex-1 (Mac-1) (anti-CD11b, 1:1000, AbD Serotech) for microglia labeling. To visualize all antibody-labeled tissues, sections were incubated with streptavidin-horseradish peroxidase (HRP) solution (ABC Elite Vector Kit, Vector Laboratories), color was generated using a glucose oxidase chromogenic system, and then incubated with diaminobenzidine (DAB) chromogen (ACROS Organics) for visualization. Within the SN, the total number of Mac-1+ cells, TH+Nissl+ (dopaminergic neurons), and TH-Nissl+ (non-dopaminergic neurons) was estimated by stereochemistry using Stereo Investigator software under an optical fractionator module (MBF Bioscience). Densitometry analysis of dopaminergic neuron terminals in the striatum was determined using ImageJ software (National Institutes of Health).

[0150] CD4+ T cell isolation Five days after a single dose of Her-mGMCSF CDR or five doses of rGM-CSF, donor mice were sacrificed and single-cell suspensions were obtained from the spleen. For genomic studies, total CD4+ cells were isolated using the EasySep Mouse CD4+T Cell Isolation Kit (StemCell) according to the manufacturer's instructions. For proliferation assays, CD4+CD25+ Regulatory T Cell Isolation Kit II (StemCell) was used according to the manufacturer's instructions. reg CD4+CD25-conventional responder T cells (Tresp) were isolated from the spleen. The purity of the isolated cells was assessed by flow cytometry analysis and was determined to be >90% for all isolates.

[0151] Flow cytometry evaluation Five days after the procedure, whole blood and spleen were collected, and T cell and B cell profiles were determined by flow cytometry analysis. Antibodies against extracellular markers CD3, CD4, CD25, CD8, and CD19, and the intracellular marker FoxP3 were used to analyze whole blood (50 μl) and spleen cells (1 × 10⁶). 6 ) was fluorescently labeled. Mouse blood and splenocytes were labeled using PerCP-Cy5.5-anti-CD3 (eBioscience), PE-Cy7-anti-CD4 (eBioscience), PE-anti-CD25 (eBioscience), FITC-anti-CD8 (eBioscience), and PE-anti-CD19 (eBioscience). For intracellular staining, cells were permeabilized at 4°C for 45 minutes using FoxP3 / Transcription Factor Staining Buffer Set (eBioscience). Next, cells were labeled with APC-anti-FoxP3 (eBioscience) and then fixed. Samples were analyzed using an LSRII flow cytometer and FACSDiva software (BD Biosciences, San Jose, CA). All cell frequencies were determined from the total lymphocyte population.

[0152] Blood chemistry and peripheral blood evaluation At the time of slaughter, 250 μl of whole blood was collected in K2EDTA blood collection tubes for complete blood cell count (CBC) levels, or in heparinized blood collection tubes for blood chemistry and metabolite levels. After isolation, heparinized blood was centrifuged and plasma was collected. A complete metabolic panel was performed on a VetScan VS2 machine using the VetScan Chemistry Comprehensive Test cartridge (Abaxis). For CBC analysis, whole blood collected from K2EDTA tubes was immediately analyzed on a VetScan HM5 machine.

[0153] statistical analysis All values ​​are expressed as mean ± SEM. Differences in means between groups were analyzed using ANOVA followed by a Newman-Keuls post-hoc test (GraphPad Software, Inc., La Jolla, CA). Comparison of slopes and elevations in the CFSE inhibition assay was evaluated using linear regression. All line slopes are not significantly zero, and the values ​​from the linear regression analysis are shown in the corresponding graphs or in the figure legend.

[0154] result Long-acting GM-CSF treatment significantly increases CD4+CD25+FoxP3+ levels in the blood and spleen, enhancing immunosuppressive function. Flow cytometry analysis of lymphocyte populations in peripheral blood revealed that Her-mGMCSF CDR treatment did not affect CD8+ levels (Figure 1A). However, CD4+ levels were significantly reduced (Figure 1B). In contrast, only treatment with 30.0 mg / kg of Her-mGMCSF CDR significantly increased the frequency of CD4+CD25+Foxp3+ cells above control levels (Figure 1C). Treatment with increased doses of rGM-CSF did not alter CD8+ or CD4+ levels (Figures 1D and 1E), but resulted in a moderate, non-significant increase in CD4+CD25+FoxP3 levels (Figure 1F). Flow cytometry analysis of splenic lymphocyte populations after treatment with Her-mGMCSF CDR revealed slightly different results. Her-mGMCSF CDR treatment resulted in a significant decrease in CD8+ levels at 3.0 mg / kg, 10.0 mg / kg, and 30.0 mg / kg (Figure 1G), but no significant change in CD4+ levels (Figure 1H). A dose-dependent increase in CD4+CD25+FoxP3+ levels was also observed (R2=0.72, p=0.0337) (Figure 1I). Similar to blood observations, rGM-CSF treatment did not alter CD8 or CD4 levels (Figures 1J and 1K), but it did result in a dose-dependent increase in CD4+CD25+FoxP3+ levels (R2=0.78, p=0.0197) (Figure 1L).

[0155] Long-acting GM-CSF pretreatment reduces microgliosis and is neuroprotective in MPTP-toxic mice. T regFollowing induction evaluation, the ability of Her-mGMCSF CDR treatment to attenuate neuroinflammatory responses associated with MPTP poisoning was assessed by quantifying the level of reactive microglia. Two days after MPTP poisoning, the ventral midbrain was collected at the peak of inflammation after pretreatment with Her-mGMCSF CDR or rGM-CSF. Reactivity was assessed by the presence of Mac-1+ microglia with an amoeba-like morphology (Figure 2). MPTP poisoning significantly increased the number of reactive microglia from 4 ± 0.8 cells / mm2 to 70 ± 6.4 cells / mm2 compared to PBS control (Figure 2). Treatment with Her-mGMCSF CDR significantly reduced the number of reactive microglia to 47±4 cells / mm2 at 1.0 mg / kg, 49±5.5 cells / mm2 at 3.0 mg / kg, 46±6.8 cells / mm2 at 5.0 mg / kg, and 33±3.6 cells / mm2 at 10.0 mg / kg, compared to MPTP poisoning alone. Treatment with 0.1 mg / kg of rGM-CSF also reduced the neuroinflammatory response, with the cell count decreasing to 46±5.6 cells / mm2.

[0156] The neuroprotective capacity of Her-mGMCSF CDR pretreatment in an MPTP mouse model was evaluated (Figure 3). After MPTP poisoning, the number of dopaminergic neurons was significantly reduced from 9313±418 to 4873±211 compared to PBS controls (Figure 3). Pretreatment with a single dose of 0.1 mg / kg, 0.3 mg / kg, 1.0 mg / kg, or 3.0 mg / kg Her-mGMCSF CDR did not result in significant neuronal viability, producing 3891±248, 3631±364, 5104±488, and 5089±378 neurons after treatment. However, treatment with 5.0 mg / kg and 10.0 mg / kg Her-mGMCSF CDR resulted in significant neuroprotection compared to MPTP poisoning alone, preserving 81% and 76% of neurons, respectively. In contrast, treatment with five consecutive doses of 0.1 mg / kg rGM-CSF also significantly preserved the number of dopaminergic neurons, increasing survival rates from 45% to 78%, respectively. However, the use of a single injection of 0.1 mg / kg rGM-CSF did not provide neuroprotection, indicating that consecutive administration is necessary when using non-long-acting rGM-CSF. Striatal terminal survival was also assessed by immunohistochemistry of TH+ terminals. Digital image analysis showed that treatment with MPTP significantly reduced striatal terminal density compared to controls treated with PBS (Figure 4). Pretreatment with Her-mGMCSF CDR at 1.0 mg / kg, 20.0 mg / kg, and 25.0 mg / kg moderately avoided striatal terminal reduction; however, levels were still significantly lower than those of PBS controls. Treatment with Her-mGMCSF CDR and all other doses, including rGM-CSF, does not protect the striatal terminals and supports the concept of increased sensitivity of the striatal dopaminergic terminals.

[0157] The potential of a single injection of Her-mGMCSF CDR as a long-acting treatment was evaluated. Mice were poisoned with MPTP and sacrificed 2 or 7 days after poisoning, and inflammatory responses and dopaminergic neuron survival were assessed (Figures 5A and 5B). MPTP poisoning alone resulted in a significant increase in the number of reactive Mac-1+ microglia in the ventral midbrain (Figure 5A). Treatment with Her-mGMCSF CDR 15 days prior to MPTP poisoning did not reduce the observed microgliosis. Treatment with Her-mGMCSF CDR 10 or 5 days prior significantly reduced the number of reactive microglia from 113±7 cells / mm2 to 69±9 and 38±10 cells / mm2, respectively. Similarly, MPTP poisoning significantly reduced the number of TH+ / Nissl+ dopaminergic neurons from 8418±130 to 6252±292 (Figure 5B). However, only Her-mGMCSF CDR treatment five days prior to MPTP poisoning resulted in significant neuronal preservation, restoring neuronal counts to control levels. In summary, these findings suggest a potential Her-mGMCSF CDR treatment duration of up to 10 days.

[0158] These experiments showed that peripheral Her-mGMCSF CDR treatment was T reg This study demonstrates that it increased the frequency and activity of [specific neurotoxicity], attenuated neuroinflammatory responses, and selectively reduced MPTP-induced neurotoxicity.

[0159] Long-acting GM-CSF treatment results in an anti-inflammatory CD4+ T cell phenotype. The effects of treatment with 10 mg / kg Her-mGMCSF CDR on peripherally adapted immune populations, particularly CD4+ T cells, were investigated. Transcriptome analysis of CD4+ T cells treated with Her-mGMCSF CDR revealed significant dysregulation of numerous genes related to T cell differentiation, normalized compared to PBS-treated controls. In contrast, treatment with 0.1 mg / kg rGM-CSF resulted in only slight quantifiable changes in gene expression, normalized compared to PBS controls.

[0160] Use of long-acting GM-CSF for neuroinflammatory diseases GM-CSF treatment is known for its anti-inflammatory capacity and T reg Based on their ability to induce population changes, they have the potential to modify neuroinflammatory diseases. However, the impairment of clinical translation is due to their relatively short half-life and limited bioavailability. The example presented here characterizes the neuroprotective potential of the long-acting GM-CSF formulation, Her-mGMCSF CDR.

[0161] The procedure using HermGMCSF CDR is T reg This resulted in a dose-dependent increase in the number of cells, which could persist up to 10 days after a single injection, and also led to increased cellular function in peripheral blood and spleen compared to rGM-CSF treatment. reg The increase in numbers was also prolonged up to 14 days after injection, supporting the possibility of long-acting immunomodulation. Similarly, T isolated from Her-mGMCSF CDR-treated mice reg It showed increased antiproliferative effect, and T isolated from rGM-CSF treated animals reg This method significantly suppressed Tresp proliferation.

[0162] Daily administration of rGM-CSF has the potential to increase neuronal survival and reduce microgliosis in models of neurodegenerative diseases. Due to the long-acting nature of Her-mGMCSF CDR, nearly identical levels of neuroprotection were achieved in MPTP mouse models using a single-injection scheme. Treatment with Her-mGMCSF CDR significantly preserved dopaminergic cell bodies, along with striatal protrusions in MPTP-injured mice. Her-mGMCSF CDR treatment also resulted in a greater anti-inflammatory response than rGM-CSF treatment alone, as indicated by a reduction in microgliosis within the lesion site. The resulting reduction in the reactive microglial population may be due to the overall anti-inflammatory phenotype changes observed in the overall CD4+ T cell population after Her-mGMCSF CDR treatment. MPTP poisoning generally results in approximately 10% neuronal cell death from the neurotoxin itself. However, CD4+ T cells are necessary to maintain the inflammatory microenvironment in the brain for MPTP-induced lesions to progress and manifest. Following MPTP toxicity, CD4+ T cells cross the blood-brain barrier (BBB), interact with microglia, alter their phenotype, and promote the production of pro-inflammatory and neurotoxic mediators.

[0163] The experiments conducted in the examples demonstrate that the long-acting GM-CSF molecules described herein may result in reduced administration frequency and increased GM-CSF bioavailability. Her-mGMCSF CDR treatment is T reg The group was selectively induced by more than twice the number of people, T regIt enhanced immunosuppressive and antiproliferative cell function. Her-mGMCSF CDR treatment preserved dopaminergic neurons in the SN after MPTP toxicity and reduced the corresponding immune-mediated inflammatory response. The reduction in microgliosis and the resulting neuroprotection were associated with the anti-inflammatory and regulatory T cell phenotype induced by Her-mGMCSF CDR treatment. Along with its potent neuroprotective and anti-inflammatory profile, all the positive changes obtained were achieved using a single dose of Her-mGMCSF CDR rather than five consecutive doses, supporting the idea that long-acting GM-CSF treatment is attractive and clinically beneficial when it transitions to clinical practice for the treatment of PD.

[0164] List of abbreviations: AD - Alzheimer's disease, ALS - Amyotrophic lateral sclerosis, CFSE - 5(6)-carboxyfluorescein N-hydroxysuccinimidyl ester, DAB - 3,3'-diaminobenzidine, DPBS - Dulbecco's phosphate-buffered saline, GM - CSF - Granulocyte-macrophage colony-stimulating factor, HRP - Horseradish peroxidase, IPA - Ingenuity pathway analysis, MPTP - 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, PBS - Phosphate-buffered saline, PD - Parkinson's disease, rGM - CSF - Recombinant granulocyte-macrophage colony-stimulating factor, RT - PCR - Reverse transcription polymerase chain reaction, SEM - Standard error of mean, TBI - Traumatic brain injury, TH - Tyrosine hydroxylase, UPDRS - Integrated Parkinson's Disease Assessment Scale.

[0165] Example 5: Clinical trial for Parkinson's disease Randomized, double-blind, placebo-controlled, single / multiple dose escalation studies of long-acting GM-CSFs (e.g., Her-hGMCSF CDR, Syn-hGMCSF CDR) from Table 1 will be conducted in healthy subjects and patients with Parkinson's disease. Inclusion criteria : Indications: Idiopathic Parkinson's disease (moderate idiopathic PD - Hoehn and Yahr 1-3, with bradykinesia plus another PD sign (resting tremor or rigidity), undergoing PD medication versus washout). Healthy individuals vs. Parkinson's disease patients: Ph1a: healthy → Ph1b: PD patient or Ph1a / b: PD patient. The study involved a number of subjects per group (10), six dose-escalating cohorts, and random assignment to receive either long-acting GM-CSF or placebo. Treatment period (8-week induction period, 8-26 weeks of treatment), long-acting GM-CSF or placebo was administered every 2-4 weeks, and the patient was monitored during the observation period. Exclusion criteria Abnormal MRI, serious test abnormalities. endpoint : Safety and tolerability assessment: physical and neurological examination, clinical tests, vital signs, and adverse events. Pharmacokinetic parameters (serum and CSF): Maximum long-acting GM-CSF concentration, area under the curve, and half-life. Pharmacodynamics: T reg WBC, improved motor control and mobility, UPDRS score. Immunogenicity (ADA).

[0166] Example 6: Clinical trial of bone marrow transplantation The subjects are treated with placebo or a GM-CSF molecule as exemplified herein after autologous or allogeneic bone marrow or peripheral blood progenitor cell (PBPC) transplantation as exemplified herein. Pediatric and adult patients receive bi-weekly intravenous infusions of the GM-CSF molecule or placebo for 30 days.

[0167] Example 7: Treatment of ALS with long-acting GM-CSF in a mouse model Previous experiments have demonstrated that a SOD1 mouse model of ALS predicts treatment success in humans (Cleveland and Rothstein (2001), Nat Rev Neurosci, 2, 806-19). The primary endpoints of such analyses are both the onset of motor symptoms and mortality. For example, the onset of motor symptoms can be defined as the first day on which a mouse is unable to remain on a rotator rod (totatod) at a speed of 20 rpm for 7 minutes (Li, et al (2000), Science, 288, 335-9). Mortality is recorded as the day of death, or the day on which the defect is so severe that the mouse must be sacrificed (e.g., apathy and inability to right itself). Additional parameters are determined by measuring exercise intensity by grip strength tests, the number of motor neurons in the spinal cord, nerve thickness (e.g., sciatic nerve, phrenic nerve), and the presence of apoptotic staining of spinal motor neurons. The GM-CSF molecules in Table 1 are injected into the ventricles at a predetermined dose, e.g., 60 μg / kg body weight / day, via an osmotic pump. Alternatively, the GM-CSF molecule is administered via IV or IP injection at a dose of 60 ug / kg body weight per day or higher. Treatment is initiated at day 60 in the late presymptomatic stage of the SOD1 G93A mutant. In untreated familial ALS mice, motor impairment appears at 12-14 weeks of age, but paralysis is not observed before 20 weeks of age. The mean life expectancy is 140-170 days. Mice are monitored for effective treatment, including a life extension of more than 15% compared to the control group (Cleveland and Rothstein (2001), Nat. Rev. Neurosci., 2, 806-19). Both vehicle-treated animals and animals treated with zVADfmk (a potent caspase inhibitor that demonstrated efficacy in this model) are used as control groups for treatment. Each group consists of 10 animals.

[0168] Example 8: Long-acting GM-CSF for the treatment of ALS ALS patients will be treated with the GM-CSF molecules shown in Table 1, and improvements in motor function will be monitored.

[0169] Example 9: Treatment of acute radiation syndrome with long-acting GM-CSF The GM-CSF molecules shown in Table 1 were administered to mice after irradiation to demonstrate their effectiveness in increasing survival rates after radiation exposure.

[0170] Simply put, these studies will use Mus musculus / C57BL / 6 mice. LD50 / 30 is the radiation dose at which 50% of the exposed population is expected to die within 30 days. LD70 / 30 is the radiation dose at which 70% of the exposed population is expected to die within 30 days. A radiation dose equivalent to LD50 / 30 or LD70 / 30 will be administered as a single, uniform whole-body dose of gamma radiation. The effectiveness of GM-CSF molecules or a control (vehicle) in increasing 30-day survival will be tested with different doses of GM-CSF and different doses of radiation. Mice will be monitored for survival until day 30. Study endpoints will include 30-day overall survival, mean survival time (MST), and complete blood count (CBC) analysis. An increase in CBC may indicate accelerated hematopoietic recovery in GM-CSF-treated mice compared to untreated control mice. This experiment will be repeated to determine whether administering GM-CSF molecules before radiation exposure improves survival.

[0171] Experiments similar to those described herein will be conducted to determine whether administration of the molecules in Table 1 improves survival and promotes hematopoietic recovery in dogs and other animal species such as monkeys after lethal radiation exposure. Radiation doses corresponding to LD50 to LD70 doses can be determined. Survival can be tracked for 30–60 days post-irradiation. The frequency and / or dosage of protein administration may be adjusted to reflect differences in molecular clearance rates between species. Optimal doses and administration plans may be determined for each molecule.

[0172] Example 10-1: Treatment of radiotoxicity caused by long-acting GM-CSF Patients undergoing radiotherapy are treated with the GM-CSF molecules listed in Table 1 before, during, or after radiotherapy. Patients are monitored for a reduction in post-radiation cell damage.

[0173] Example 10-2: Long-acting GM-CSF for cancer treatment Patients diagnosed with cancer are treated with the GM-CSF molecules listed in Table 1. Patients are monitored for delayed tumor growth and reduction in tumor size.

[0174] Example 11: Treatment of Alzheimer's disease with long-acting GM-CSF in a mouse model Mice are treated with the GM-CSF molecules shown in Table 1. The treatment group is then evaluated to determine whether untreated mice exhibit the same level of impairment as the treated group.

[0175] Radial Arm Water Maze (RAWM) Test for Assessing Short-Term Memory The experiment will be conducted as generally described (Arendash et al. (2001); Ethell et al. (2006); Arendash et al. (2007)).

[0176] Simply put, aluminum inserts are placed in a circular pool, creating six swim arms that radiate outwards from a central circular swim area. Various 2D and 3D visual cues surround the pool. The number of errors before identifying which of the six swim arms contains the submerged escape platform is determined over an 8-day pre-treatment test and a 4-day post-treatment test, with five trials per day. During each trial (up to 60 seconds), the mouse swims to the wrong arm and returns to the starting arm for that trial, and the number of seconds it takes to find the underwater platform is recorded. If the mouse fails to find the platform within the 60-second trial, it is guided to the platform for a 30-second stay. Both the number of errors and escape wait times are considered indicators of working memory and are temporally similar to standard registration / recall tests of specific items clinically used in the assessment of AD patients.

[0177] Cognitive interference task The experiment should be carried out as described (Loewenstein et al. (2004)).

[0178] Following the completion of the RAWM test (4 days), all mice are further evaluated for 6 days on a novel cognitive interference task. This task involves setting up two radial-arm water mazes in two different rooms and three different sets of visual cues. In this task, the animals need to memorize sets of visual cues so that they can successfully solve the radial-arm water maze task by recalling the first set of cues after interference with another set of cues.

[0179] Example 12: Treatment of Alzheimer's disease with long-acting GM-CSF A clinical trial to evaluate the safety and efficacy of the GM-CSF molecules listed in Table 1 in the treatment of patients with mild cognitive impairment due to Alzheimer's disease.

[0180] The subjects will receive either the GM-CSF molecule or placebo listed in Table 1 for up to 24 weeks. Florbetapir will be administered for PET scans to examine baseline brain imaging pathology and changes in treatment.

[0181] Primary outcome measure: Change from baseline in standardized uptake ratio measured by PET using Florbetapir F18 (Amyvid) [Time frame: Baseline to 24 weeks].

[0182] Secondary outcome measures: number of patients experiencing treatment-induced adverse events (TEAEs) [timeframe: week 24]; change from baseline in CSF analysis [timeframe: before the first injection on day 1, and optionally at day 155, to serve as a baseline for necessary follow-up]; MRI to assess the appearance of amyloid-related imaging abnormalities (ARIAs) [timeframe: at screening and at days 43, 85, and 155]; measurement of anti-drug antibody levels [timeframe: days 1, 29, 57, 85, and 155].

[0183] Inclusion criteria: Men and women aged ≥40 and ≤80 years who have been diagnosed with mild cognitive impairment (MCI) due to Alzheimer's disease (AD) according to the National Institute of Aging Alzheimer's Association (NIA-AA) criteria (moderate or high probability), with a sporadic or familial inheritance pattern. Mild cognitive impairment (AD) is defined as follows: evidence of concern regarding a change in cognition compared to a person's previous level (a complaint of subjective memory loss for more than six months in the past year, and / or confirmed by an informant and / or clinician), and objective impairment of memory function as recorded by error scores in the delayed recall section of the Alzheimer's Disease Assessment Scale Cognitive Subscale (ADAS-cog) ≥1.5 standard deviations (SD), from the age-group mean; i.e., age 55-69 years: ≥6 errors, age 70-74 years: ≥7 errors, age 75+ years: ≥8 errors, but without clear impairment in activities of daily living (ADL), as assessed by the Alzheimer's Disease Collaborative Study (ADCS) in the opinion of the principal investigator, adapted ADL for MCI, and evidence of elevated cortical amyloid by positron emission tomography (PET) using florbetapir F18 (Amyvid) (positive scan), according to qualitative assessment as per the product label. A dedicated partner / caregiver information provider will be available to assist the patient with clinical trial procedures and administration of the investigational drug, and will be with the patient for at least 12 hours per week. We have the willingness and ability to provide signed informed consent.

[0184] Exclusion criteria: Pre-treatment for an anti-amyloid therapy currently under investigation. Contraindications to lumbar puncture, or contraindications to or inability to complete magnetic resonance imaging (MRI), or a history of or planned exposure to ionizing radiation that, together with the radiation resulting from the administration of the PET tracer used in this study, exceeds applicable institutional, local, or national recommendations for annual or lifetime exposure. Modified Huchinsky ischemia score > 4. Other neurological or psychiatric conditions that may impair cognition (other than AD), or evidence of potentially significant intracranial abnormalities unrelated to AD on computed tomography (CT) / MRI (e.g., evidence of stroke or lacune in areas important for cognition, infection, cancer, hydrocephalus, multiple sclerosis, etc.); or abnormal cerebrospinal fluid (CSF) unrelated to AD. MRI evidence of four microbleeds: A patient who may be prone to spontaneous amyloid-associated imaging abnormalities (ARIA-H) and / or susceptible to adverse effects of ARIA-H. Untreated or unstable medical conditions that, in the opinion of the principal investigator, may interfere with the evaluation of the clinical trial or may require immunostimulatory, immunosuppressive, or immunomodulatory treatment during the course of the trial; e.g., immunoglobulins, treatment vaccines, cytokines, anti-cytokine monoclonal antibodies; a history of asplenia, hypoplenia, or splenectomy (for whatever surgical reason). Current mood or anxiety disorders and / or psychiatric disorders and / or substance-related disorders that, according to the Diagnostic and Statistical Manual of Psychiatric Disorders, Edition IV, Text Revision (DSM-IV-TR or DSM-V), or that are considered suicidal or present with suicidal ideation as assessed by an investigator. Laboratory abnormalities indicating untreated medical or hematological conditions that may increase risk or interfere with study evaluation, including untreated hypothyroidism or hyperthyroidism, vitamin B12 deficiency, hyperleukocyte syndrome (including, but not limited to, chronic myeloid leukemia, Hodgkin lymphoma and non-Hodgkin lymphoma), monoclonal immunoglobulinemia, and thrombocythemia. Known renal impairment or serum creatinine >150 μmol / L. Known liver dysfunction (excluding Gilbert's syndrome) or serum alanine aminotransferase (ALT) levels greater than 3 times the upper limit of normal (ULN). Pregnant or breastfeeding women. Presence or history of drug hypersensitivity; or known hypersensitivity to salglamostim, yeast-derived products, other components of this product, or benzyl alcohol (contained in bacteriostatic water or infusion saline). Evidence of fluid retention (clinical or radiological), respiratory symptoms (e.g., dyspnea), cardiovascular symptoms, or electrocardiographic evidence of cardiac disease (e.g., supraventricular arrhythmia) justifying procedural intervention. A history of deep vein thrombosis (DVT) or pulmonary embolism, or a history of familial predisposition to DVT or pulmonary embolism. Women and female partners who may give birth and are not protected by a highly effective method of contraception (i.e., oral or depot contraceptives or intrauterine devices [IUDs] or surgically sterilized objects), and / or who do not want to or are unable to take a pregnancy test, or who are pregnant or breastfeeding. Recipients of the investigational drug within the past 60 days, or within five times the drug's elimination half-life, whichever is longer. A history of latex or yeast allergy. The following patients: If you are likely to be in non-compliance for more than 3 days during the test, have left the area, or have been separated from your designated caregiver / information provider, Unable to cooperate due to language problems or developmental disorders, To oversee or conduct all aspects of the research.

[0185] The above is merely an explanation of the principles of this disclosure. Those skilled in the art will understand that various configurations embodying the principles of the invention and falling within its spirit and scope can be devised, although these are not explicitly described or illustrated herein. Furthermore, all examples and conditional language enumerated herein are intended primarily to help the reader understand the principles of this disclosure and to understand the concepts to which the inventors have contributed to advancing the art, and are to be construed as not being limited to such specifically enumerated examples and conditions. Furthermore, all descriptions herein enumerating the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both their structural and functional equivalents. Moreover, such equivalents are intended to include both currently known equivalents and future-developed equivalents, i.e., both developed elements that perform the same function regardless of structure. Thus, the scope of this disclosure is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of this disclosure are embodied by the appended claims.

[0186] [Table 4] JPEG2026082829000022.jpg246170 JPEG2026082829000023.jpg239170 JPEG2026082829000024.jpg234170 JPEG2026082829000025.jpg243170 JPEG2026082829000026.jpg236170 JPEG2026082829000027.jpg243170 JPEG2026082829000028.jpg239170 JPEG2026082829000029.jpg242170 JPEG2026082829000030.jpg242170 JPEG2026082829000031.jpg238170 JPEG2026082829000032.jpg240170 JPEG2026082829000033.jpg119170

Claims

1. A composition comprising a first polypeptide containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and a second polypeptide containing a sequence that is at least 98% identical to SEQ ID NO:

2.

2. The composition according to claim 1, wherein the GM-CSF comprises a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 16 or 77.

3. The composition according to claim 1 or claim 2, wherein the GM-CSF comprises human GM-CSF or mouse GM-CSF.

4. The composition according to any one of claims 1 to 3, wherein the first polypeptide comprises a modified light chain in the antibody variable region.

5. The composition according to claim 4, wherein the modified light chain of the antibody variable domain includes a GM-CSF located between the first amino acid sequence of the antibody variable region and the second amino acid sequence of the antibody variable region.

6. The composition according to claim 5, wherein the first amino acid sequence is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

14.

7. The composition according to claim 6, wherein the first amino acid sequence comprises SEQ ID NO:

14.

8. The composition according to any one of claims 5-7, wherein the second amino acid sequence is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

15.

9. The composition according to claim 8, wherein the second amino acid sequence comprises SEQ ID NO:

15.

10. The composition according to any one of claims 4-9, wherein the GM-CSF is located within the complementarity determination region (CDR) of the modified light chain.

11. The composition according to claim 10, wherein the GM-CSF is located within the light chain CDR1, CDR2, or CDR3.

12. The composition according to claim 11, wherein the GM-CSF is located within the light chain CDR3.

13. The composition according to any one of claims 4-12, wherein the modified light chain is modified from a variable light chain containing the sequence of sequence number 17.

14. The composition according to any one of claims 1 to 13, wherein the first polypeptide further comprises a first linker peptide.

15. The composition according to claim 14, wherein the first linker peptide comprises the sequence of SEQ ID NO:

10.

16. The composition according to claim 14 or claim 15, wherein the first linker peptide comprises the sequence of SEQ ID NO:

8.

17. The composition according to any one of claims 14-16, wherein the first linker peptide comprises the sequence of SEQ ID NO:

11.

18. The composition according to any one of claims 14-17, wherein the first linker peptide comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

12.

19. The composition according to any one of claims 1 to 18, wherein the first polypeptide further comprises a second linker peptide.

20. The composition according to claim 19, wherein the second linker peptide comprises the sequence of SEQ ID NO:

10.

21. The composition according to claim 19 or claim 20, wherein the second linker peptide comprises the sequence of SEQ ID NO:

9.

22. The composition according to any one of claims 19-21, wherein the second linker peptide comprises the sequence of SEQ ID NO:

11.

23. The composition according to any one of claims 19-22, wherein the second linker peptide comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

13.

24. The composition according to any one of claims 1 to 23, wherein the first polypeptide comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

18.

25. The composition according to any one of claims 1 to 24, wherein the first polypeptide comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

6.

26. The composition according to any one of claims 1 to 25, wherein the first polypeptide comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

7.

27. The composition according to any one of claims 1 to 26, wherein the first polypeptide comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

5.

28. The composition according to any one of claims 1 to 27, wherein the second polypeptide comprises a heavy chain in the antibody variable region.

29. The composition according to any one of claims 1 to 28, wherein the second polypeptide comprises the sequence of Sequence ID No.

2.

30. The composition according to any one of claims 1 to 29, wherein the second polypeptide further comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

4.

31. The composition according to any one of claims 1 to 31, wherein the second polypeptide comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

1.

32. The composition according to any one of claims 1 to 31, wherein the first polypeptide and the second polypeptide are linked by one or more disulfide bonds.

33. The composition according to any one of claims 1 to 32, wherein the first polypeptide and the second polypeptide form an antibody variable domain.

34. The antibody variable domain is approximately 10 －２ M, 10 －３ M, or 10 －４ Equilibrium dissociation constant (K) less than M Ｄ The composition according to claim 33, which does not bind to the antigen.

35. The composition according to claim 33 or claim 34, wherein the antibody variable domain comprises a modified palivizumab variable domain.

36. The composition according to claim 35, wherein the modified palivizumab variable domain comprises a heavy chain CDR1 containing the sequence of SEQ ID NO:

19.

37. The composition according to claim 35 or claim 36, wherein the modified palivizumab variable domain comprises a heavy chain CDR2 containing the sequence of SEQ ID NO:

20.

38. The composition according to any one of claims 35-37, wherein the modified palivizumab variable domain comprises a heavy chain CDR3 containing the sequence of SEQ ID NO:

21.

39. The composition according to any one of claims 35-38, wherein the modified palivizumab variable domain comprises a light chain CDR1 containing the sequence of SEQ ID NO:

22.

40. The composition according to any one of claims 35-39, wherein the modified palivizumab variable domain comprises a light chain CDR2 containing the sequence of SEQ ID NO:

23.

41. The composition according to any one of claims 35-40, wherein the modified palivizumab variable domain comprises a light chain CDR3 having the sequence of SEQ ID NO: 24, 77, or 16.

42. The modified palivizumab variable domain is approximately 10 －２ M, 10 －３ M, or 10 －４ K less than M Ｄ The composition according to any one of claims 35-41, wherein it does not bind to respiratory syncytial virus (RSV).

43. The composition according to any one of claims 1 to 42, further comprising an Fc region that includes a decrease in effector function compared to human IgG1.

44. The composition according to claim 43, wherein the human IgG1 comprises the sequence of Sequence ID No.

25.

45. The composition according to claim 43 or claim 44, wherein the reduction in the effector function includes a reduction in antibody-dependent cell cytotoxicity (ADCC).

46. The composition according to any one of claims 43-45, wherein the reduction in the effector function includes a reduction in complement-dependent cytotoxicity (CDC).

47. The composition according to any one of claims 1 to 46, wherein the first polypeptide further comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3, and / or the first polypeptide comprises an Fc region comprising human IgG1 comprising E233P, L234V, L235A, ΔG236, A327G, A330S, P331S by Kabat numbering.

48. A composition comprising an antibody variable domain comprising a light chain sequence containing a first polypeptide having a sequence that is at least about 90% identical to SEQ ID NO: 6, and a heavy chain sequence containing a second polypeptide having a sequence that is at least about 90% identical to SEQ ID NO:

2.

49. The composition according to claim 48, wherein the first polypeptide comprises a sequence that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

6.

50. The composition according to claim 48 or claim 49, wherein the second polypeptide comprises a sequence that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

2.

51. A composition according to any one of claims 48-50, comprising GM-CSF.

52. The composition according to claim 51, wherein the GM-CSF is human GM-CSF or mouse GM-CSF.

53. The composition according to claim 51 or claim 52, wherein the GM-CSF comprises a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 16 or 77.

54. The composition according to any one of claims 48-53, wherein the light chain comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

7.

55. The composition according to any one of claims 48-54, wherein the light chain comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

5.

56. The composition according to any one of claims 48-55, wherein the heavy chain comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

4.

57. The composition according to any one of claims 48-56, wherein the heavy chain comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

1.

58. The composition according to any one of claims 48-57, further comprising an Fc region that includes a reduced effector function compared to human IgG1.

59. The composition according to claim 58, wherein the human IgG1 comprises the sequence of Sequence ID No.

25.

60. The composition according to claim 58 or claim 59, wherein the reduction in the effector function includes a reduction in antibody-dependent cytotoxicity (ADCC).

61. The composition according to any one of claims 58-60, wherein the reduction in the effector function includes a reduction in complement-dependent cytotoxicity (CDC).

62. The composition according to any one of claims 48-61, wherein the heavy chain further comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No. 3, and / or the heavy chain comprises an Fc region comprising human IgG1 comprising E233P, L234V, L235A, ΔG236, A327G, A330S, P331S by Kabat numbering.

63. Sequence ID 26 【Chemistry 1】 A composition comprising an antibody variable domain, comprising a light chain sequence having a sequence that is at least about 90% identical to sequence number 2, wherein the light chain sequence comprises X1, and X1 comprises GM-CSF, and a heavy chain sequence having a sequence that is at least about 90% identical to sequence number 2.

64. The composition according to claim 63, wherein the GM-CSF is human GM-CSF or mouse GM-CSF.

65. The composition according to claim 63 or claim 64, wherein the GM-CSF comprises a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 16 or 77.

66. The composition according to any one of claims 63-65, wherein the light chain sequence comprises a sequence that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 26.

67. The aforementioned light chain sequence is SEQ ID NO: 27 【Chemistry 2】 The composition according to any one of claims 63-66, comprising a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, wherein the light chain sequence comprises X2, and X2 comprises GM-CSF.

68. The composition according to any one of claims 63-67, wherein the heavy chain sequence comprises a sequence that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

2.

69. The composition according to any one of claims 63-68, wherein the light chain comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

7.

70. The composition according to any one of claims 63-69, wherein the light chain comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

5.

71. The composition according to any one of claims 63-70, wherein the heavy chain comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

4.

72. The composition according to any one of claims 63-71, wherein the heavy chain comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

1.

73. The composition according to any one of claims 63-72, further comprising an Fc region that includes a reduced effector function compared to human IgG1.

74. The composition according to claim 73, wherein the human IgG1 comprises the sequence of Sequence ID No.

25.

75. The composition according to claim 73 or claim 74, wherein the reduction in the effector function includes a reduction in antibody-dependent cell cytotoxicity (ADCC).

76. The composition according to any one of claims 73 to 75, wherein the reduction in the effector function includes a reduction in complement-dependent cytotoxicity (CDC).

77. The composition according to any one of claims 63-76, wherein the heavy chain further comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No. 3, and / or the heavy chain comprises an Fc region comprising human IgG1 comprising E233P, L234V, L235A, ΔG236, A327G, A330S, P331S by Kabat numbering.

78. A composition comprising a sequence that is at least about 90% identical to sequence number 18.

79. The composition according to claim 78, wherein the sequence is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 18.

80. The composition according to claim 78 or claim 79, wherein the sequence is connected to the antibody domain.

81. The composition according to claim 80, wherein the antibody domain is an antibody variable domain.

82. The composition according to claim 80 or claim 81, wherein the sequence is located within the antibody domain.

83. The composition according to claim 81, wherein the sequence is located within the CDR of the antibody variable domain.

84. The composition according to claim 83, wherein the sequence is located within the CDR of the modified trastuzumab antibody variable domain.

85. The composition according to claim 83, wherein the sequence is located within the CDR of the modified palivizumab antibody variable domain.

86. The composition according to any one of claims 78-83, comprising a region that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 42, wherein the region comprises X5, and X5 comprises the sequence.

87. The composition according to claim 86, further comprising regions that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

31.

88. The composition according to any one of claims 78-83, comprising a region that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 43, wherein the region comprises X6, and X6 comprises the sequence.

89. The composition according to claim 88, further comprising a region that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

2.

90. The composition according to any one of claims 78-89, further comprising an Fc region that includes a decrease in effector function compared to human IgG1.

91. The composition according to claim 90, wherein the human IgG1 comprises the sequence of Sequence ID No.

25.

92. The composition according to claim 90 or claim 91, wherein the reduction in the effector function includes a reduction in antibody-dependent cytotoxicity (ADCC).

93. The composition according to any one of claims 90-92, wherein the reduction in the effector function includes a reduction in complement-dependent cytotoxicity (CDC).

94. The composition according to any one of claims 90-93, wherein the Fc region comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 3, and / or the Fc region comprises human IgG1 comprising E233P, L234V, L235A, ΔG236, A327G, A330S, P331S by Kabat numbering.

95. (i) a first polypeptide comprising the sequences of SEQ ID NOs: 22, 23, and 16, and a second polypeptide comprising the sequences of SEQ ID NOs: 19-21, or (ii) a first polypeptide comprising the sequences of SEQ ID NOs: 22, 23, and 77, and a second polypeptide comprising the sequences of SEQ ID NOs: 19-21.

96. The composition according to claim 95, wherein the first polypeptide is the light chain of an antibody variable domain.

97. The composition according to claim 95 or claim 96, wherein the second polypeptide is the heavy chain of the antibody variable domain.

98. The composition according to any one of claims 95-97, wherein the first polypeptide comprises the sequence of Sequence ID No.

24.

99. The composition according to any one of claims 95-98, further comprising an Fc region that includes a decrease in effector function compared to human IgG1.

100. The composition according to claim 99, wherein the human IgG1 comprises the sequence of Sequence ID No.

25.

101. The composition according to claim 99 or claim 100, wherein the reduction in the effector function includes a reduction in antibody-dependent cell cytotoxicity (ADCC).

102. The composition according to any one of claims 99-101, wherein the reduction in the effector function includes a reduction in complement-dependent cytotoxicity (CDC).

103. The composition according to any one of claims 95-102, wherein the second polypeptide further comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No. 3, and / or the second polypeptide comprises an Fc region comprising human IgG1 comprising E233P, L234V, L235A, ΔG236, A327G, A330S, P331S by Kabat numbering.

104. (i) a first polypeptide comprising the sequence of SEQ ID NOs: 37-39 and a second polypeptide comprising the sequences of SEQ ID NOs: 34, 35, 16, or (ii) a first polypeptide comprising the sequence of SEQ ID NOs: 37-39 and a second polypeptide comprising the sequences of SEQ ID NOs: 34, 35, 77.

105. The composition according to claim 104, wherein the first polypeptide is the light chain of an antibody variable domain.

106. The composition according to claim 104 or claim 105, wherein the second polypeptide is the heavy chain of the antibody variable domain.

107. The composition according to any one of claims 104-106, wherein the first polypeptide comprises the sequence of sequence number 36.

108. The composition according to any one of claims 104-107, further comprising an Fc region that includes a reduced effector function compared to human IgG1.

109. The composition according to claim 108, wherein the human IgG1 comprises the sequence of Sequence ID No.

25.

110. The composition according to claim 108 or claim 109, wherein the reduction in the effector function includes a reduction in antibody-dependent cell cytotoxicity (ADCC).

111. The composition according to any one of claims 108-110, wherein the reduction in the effector function includes a reduction in complement-dependent cytotoxicity (CDC).

112. The composition according to any one of claims 104-111, wherein the second polypeptide further comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3, and / or the second polypeptide comprises an Fc region comprising human IgG1 comprising E233P, L234V, L235A, ΔG236, A327G, A330S, P331S by Kabat numbering.

113. A composition comprising a first polypeptide containing the sequence of SEQ ID NO: 31, and a second polypeptide containing granulocyte-macrophage colony-stimulating factor (GM-CSF).

114. The composition according to claim 113, wherein the GM-CSF comprises a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 16 or 77.

115. The composition according to claim 113 or claim 114, wherein the GM-CSF comprises human GM-CSF or mouse GM-CSF.

116. The composition according to any one of claims 113-115, wherein the second polypeptide comprises a modified heavy chain in the antibody variable region.

117. The composition according to claim 116, wherein the modified heavy chain of the antibody variable domain includes a GM-CSF located between the first amino acid sequence of the antibody variable region and the second amino acid sequence of the antibody variable region.

118. The composition according to claim 117, wherein the first amino acid sequence is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

32.

119. The composition according to claim 118, wherein the first amino acid sequence comprises the sequence of SEQ ID NO:

32.

120. The composition according to any one of claims 117-119, wherein the second amino acid sequence is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

33.

121. The composition according to claim 120, wherein the second amino acid sequence comprises the sequence of SEQ ID NO:

33.

122. The composition according to any one of claims 116-121, wherein the GM-CSF is located within the complementarity determination region (CDR) of the modified heavy chain.

123. The composition according to claim 122, wherein the GM-CSF is located within the heavy chain CDR1, CDR2, or CDR3.

124. The composition according to claim 123, wherein the GM-CSF is located within the heavy chain CDR3.

125. The composition according to any one of claims 116-124, wherein the modified heavy chain is modified from a variable heavy chain containing the sequence of sequence number 44.

126. The composition according to any one of claims 113-125, wherein the second polypeptide further comprises the first linker peptide.

127. The composition according to claim 126, wherein the first linker peptide comprises the sequence of SEQ ID NO:

10.

128. The composition according to claim 126 or claim 127, wherein the first linker peptide comprises the sequence of SEQ ID NO:

8.

129. The composition according to any one of claims 126-128, wherein the first linker peptide comprises the sequence of SEQ ID NO:

11.

130. The composition according to any one of claims 126-129, wherein the first linker peptide comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

12.

131. The composition according to any one of claims 113-130, wherein the second polypeptide further comprises a second linker peptide.

132. The composition according to claim 131, wherein the second linker peptide comprises the sequence of SEQ ID NO:

10.

133. The composition according to claim 131 or claim 132, wherein the second linker peptide comprises the sequence of SEQ ID NO:

9.

134. The composition according to any one of claims 131-133, wherein the second linker peptide comprises the sequence of SEQ ID NO:

11.

135. The composition according to any one of claims 131-134, wherein the second linker peptide comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

13.

136. The composition according to any one of claims 113-135, wherein the second polypeptide comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

18.

137. The composition according to any one of claims 113-136, wherein the second polypeptide comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

29.

138. The composition according to any one of claims 113-137, wherein the second polypeptide comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

4.

139. The composition according to any one of claims 113-138, wherein the second polypeptide comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

28.

140. The composition according to any one of claims 113-139, wherein the first polypeptide comprises a light chain in the antibody variable region.

141. The composition according to any one of claims 113-140, wherein the first polypeptide comprises the sequence of Sequence ID No.

31.

142. The composition according to any one of claims 113-141, wherein the first polypeptide further comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

7.

143. The composition according to any one of claims 113-142, wherein the second polypeptide comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

30.

144. The composition according to any one of claims 113-143, wherein the first polypeptide and the second polypeptide are linked by one or more disulfide bonds.

145. The composition according to any one of claims 113-144, wherein the first polypeptide and the second polypeptide form an antibody variable domain.

146. The antibody variable domain is about 10 －２ M, 10 －３ M, or less than 10 －４ M of the equilibrium dissociation constant (K Ｄ ), which does not bind to the antigen. The composition according to claim 145.

147. The composition according to claim 145 or claim 146, wherein the antibody variable domain comprises a modified trastuzumab variable domain.

148. The composition according to claim 147, wherein the modified trastuzumab variable domain comprises a heavy chain CDR1 containing the sequence of SEQ ID NO:

34.

149. The composition according to claim 147 or claim 148, wherein the modified trastuzumab variable domain comprises a heavy chain CDR2 containing the sequence of SEQ ID NO:

35.

150. The composition according to any one of claims 147-149, wherein the modified trastuzumab variable domain comprises a heavy chain CDR3 having the sequence of SEQ ID NO: 36, 77, or 16.

151. The composition according to any one of claims 147-150, wherein the modified trastuzumab variable domain comprises a light chain CDR1 containing the sequence of Sequence ID No.

37.

152. The composition according to any one of claims 147-151, wherein the modified trastuzumab variable domain comprises a light chain CDR2 containing the sequence of SEQ ID NO:

38.

153. The composition according to any one of claims 147-152, wherein the modified trastuzumab variable domain comprises a light chain CDR3 containing the sequence of SEQ ID NO:

39.

154. The modified trastuzumab variable domain is approximately 10 －２ M, 10 －３ M, or 10 －４ K less than M Ｄ The composition according to any one of claims 147-153, wherein it does not bind to human epidermal growth factor receptor 2 (Her2).

155. The composition according to any one of claims 113-154, further comprising an Fc region that includes a reduced effector function compared to human IgG1.

156. The composition according to claim 155, wherein the human IgG1 comprises the sequence of Sequence ID No.

25.

157. The composition according to claim 155 or claim 156, wherein the reduction in the effector function includes a reduction in antibody-dependent cell cytotoxicity (ADCC).

158. The composition according to any one of claims 155-157, wherein the reduction in the effector function includes a reduction in complement-dependent cytotoxicity (CDC).

159. The composition according to any one of claims 113-158, wherein the second polypeptide further comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3, and / or the second polypeptide comprises an Fc region comprising human IgG1 comprising E233P, L234V, L235A, ΔG236, A327G, A330S, P331S by Kabat numbering.

160. A composition comprising an antibody variable domain comprising a light chain sequence containing a first polypeptide having a sequence that is at least about 90% identical to SEQ ID NO: 31, and a heavy chain sequence containing a second polypeptide having a sequence that is at least about 90% identical to SEQ ID NO:

29.

161. The composition according to claim 160, wherein the first polypeptide comprises a sequence that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

31.

162. The composition according to claim 160 or claim 161, wherein the second polypeptide comprises a sequence that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

29.

163. A composition according to any one of claims 160-162, comprising GM-CSF.

164. The composition according to claim 163, wherein the GM-CSF is human GM-CSF or mouse GM-CSF.

165. The composition according to claim 163 or claim 164, wherein the GM-CSF comprises a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 16 or 77.

166. The composition according to any one of claims 160-165, wherein the light chain comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

7.

167. The composition according to any one of claims 160-166, wherein the light chain comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

30.

168. The composition according to any one of claims 160-167, wherein the heavy chain comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

4.

169. The composition according to any one of claims 160-168, wherein the heavy chain comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

28.

170. The composition according to any one of claims 160-169, further comprising an Fc region that includes a decrease in effector function compared to human IgG1.

171. The composition according to claim 170, wherein the human IgG1 comprises the sequence of Sequence ID No.

25.

172. The composition according to claim 170 or claim 171, wherein the reduction in the effector function includes a reduction in antibody-dependent cytotoxicity (ADCC).

173. The composition according to any one of claims 170-172, wherein the reduction in the effector function includes a reduction in complement-dependent cytotoxicity (CDC).

174. The composition according to any one of claims 160-173, wherein the heavy chain further comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No. 3, and / or the heavy chain comprises an Fc region comprising human IgG1 including E233P, L234V, L235A, ΔG236, A327G, A330S, P331S by Kabat numbering.

175. Sequence ID 42 【Transformation 3】 A composition comprising an antibody variable domain comprising a heavy chain sequence having at least about 90% identical sequence to sequence number 31, wherein the heavy chain sequence comprises X6, X6 comprises GM-CSF, and the light chain sequence comprises at least about 90% identical sequence to sequence number 31.

176. The composition according to claim 175, wherein the GM-CSF is human GM-CSF or mouse GM-CSF.

177. The composition according to claim 175 or claim 176, wherein the GM-CSF comprises a sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 16 or 77.

178. The composition according to any one of claims 175-177, wherein the heavy chain sequence comprises a sequence that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 42.

179. The aforementioned heavy chain sequence is sequence number 43 【Chemistry 4】 The composition according to any one of claims 175-178, comprising a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain sequence, wherein the heavy chain sequence comprises X6, and X6 comprises GM-CSF.

180. The composition according to any one of claims 175-179, wherein the heavy chain sequence comprises a sequence that is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 43.

181. The composition according to any one of claims 175-180, wherein the light chain comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

7.

182. The composition according to any one of claims 175-181, wherein the heavy chain comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

29.

183. The composition according to any one of claims 175-182, wherein the heavy chain comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

4.

184. The composition according to any one of claims 175-183, wherein the heavy chain comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No.

28.

185. The composition according to any one of claims 175-184, further comprising an Fc region that includes a decrease in effector function compared to human IgG1.

186. The composition according to claim 185, wherein the human IgG1 comprises the sequence of Sequence ID No.

25.

187. The composition according to claim 185 or claim 186, wherein the reduction in the effector function includes a reduction in antibody-dependent cell cytotoxicity (ADCC).

188. The composition according to any one of claims 185-187, wherein the reduction in the effector function includes a reduction in complement-dependent cytotoxicity (CDC).

189. The composition according to any one of claims 175-188, wherein the heavy chain further comprises a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No. 3, and / or the heavy chain comprises an Fc region comprising human IgG1 comprising E233P, L234V, L235A, ΔG236, A327G, A330S, P331S by Kabat numbering.

190. Use of the composition according to any one of claims 1-189 for the treatment of neurological disorders or conditions.

191. A method for treating a neurological disorder or condition, comprising the step of administering a composition comprising any one of claims 1 to 189 to a subject requiring treatment.

192. The use according to claim 190 or the method according to claim 191, wherein the neurological disorder or condition includes Parkinson's disease.

193. Use of the composition according to any one of claims 1-189 for the treatment of Alzheimer's disease and / or traumatic brain injury.

194. Use of the composition according to any one of claims 1-189 for the treatment of ALS.

195. Use of the composition according to any one of claims 1-189 for the treatment of acute radiation syndrome.

196. Use of the composition according to any one of claims 1-189 for the treatment of cancer.

197. The use or method according to any one of claims 190-196, wherein the composition is administered once every approximately 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days during the treatment period.

198. The use or method according to claim 196, wherein the composition is administered once every 14 days or so during the treatment period.

199. The use or method according to any one of claims 190-196, wherein the composition is administered once approximately every two weeks during the treatment period.

200. The use or method according to any one of claims 190-196, wherein the composition is administered once approximately every three weeks during the treatment period.

201. The use or method according to any one of claims 190-196, wherein the composition is administered once approximately every four weeks during the treatment period.

202. The use or method according to any one of claims 190-196, wherein the composition is administered once approximately every month during the treatment period.

203. The use or method according to any one of claims 197-202, wherein the treatment period includes a period of approximately 8 weeks to approximately 2 years.

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