FUNCTIONED ERYTHROID CELLS

专利摘要:
the present invention relates to preparations of functionalized erythroid cells and related compositions, reagents and methods for use in human and veterinary pharmaceutical applications.
公开号:BR112019016951A2
申请号:R112019016951-2
申请日:2018-02-16
公开日:2020-05-26
发明作者:Wickham Tom；F. Chen Tiffany；Zhang Xuqing；Hudak Carolyn；Mata-Fink Jordi；Elloul Sivan；Law Billy；Hoffman Lenka；Eric TEICHERT Kristian；Rabia Dastagir Shamael
申请人:Rubius Therapeutics, Inc.；
IPC主号:

专利说明:

Descriptive Report of the Invention Patent for FUNCTIONED ERYTHROID CELLS.
RELATED REQUESTS
[001] This application claims priority for the US patent with the serial ^{Q Q} 62 / 460,589, filed on February 17, 2017, and the US patent with the serial ^{Q Q} 62 / 542,142, filed on August 7 2017, the contents of each of which are incorporated herein by reference in their entirety.
SEQUENCE LISTING
[002] This application contains a listing of strings that has been deposited electronically in ASCII format and is incorporated by reference in its entirety. The ASCII copy, created on Thursday, February 15, 2018, is called R2081-7021WO_SL.txt and is 51,103 bytes in size.
BACKGROUND
[003] Erythroid cells, such as red blood cells, can be engineered to express a wide variety of exogenous therapeutic proteins in order to treat several different diseases, as described in WO2015 / 073587 (Rubius Therapeutics, Inc.). This manipulation may involve introducing a transgene into erythroid cell precursors, and then inducing the precursors to differentiate and express the transgene. However, some proteins are difficult to express, for example, because they require post-translational modification or because they compromise the growth or function of a host cell. There is a need for improved methods of producing cells comprising these proteins.
SUMMARY OF THE INVENTION
[004] New preparations of functionalized erythroid cells and related compositions, reagents and methods that have advantageous and surprising qualities for use in
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2/111 human and veterinary pharmaceutical applications. For example, the methods and compositions disclosed herein provide functionalized erythroid cells with yield, purity, stability, viability, immunogenicity, function, integrity and / or biological function optimized for use in therapeutic applications. The functionalized erythroid cells described herein are particularly well suited for delivering agents to the cell surface or for complex or difficult to express agents, for example, polypeptides, for example, multimeric polypeptides; large polypeptides; in vitro derivatized agents, for example, polypeptides; agents that may be toxic or that cannot be expressed efficiently in erythroid cells. The agent can also be a lipid, nucleic acid, sugar, drug, or small molecule.
[005] The methods and compositions described here provide optimized erythroid cells, derivatized with therapeutic or diagnostic agents, for use in a wide range of indications. Optimized reagents, intermediates and synthetic methods are also provided.
[006] In one aspect, the invention features a preparation, for example, pharmaceutical preparation, of erythroid cells, for example hematopoietic stem cells, reticulocytes or erythrocytes, comprising at least more than, or about 5,000, 10,000 , 50,000, 100,000, 200,000, 300,000, 400,000, 500,000 cell coupling reagents. For example, in some aspects, the disclosure discloses a preparation, for example, pharmaceutical preparation, of erythroid cells comprising at least 5,000, 10,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000 coupling reagents per cell. In some embodiments, a pharmaceutical preparation described herein comprises up to 10,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000 coupling reagents by
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3/111 cell. In one embodiment, at least 50, 60, 70, 80, 90, 95, 99 or 99.9% of the cells in the preparation have the recited level of the coupling reagent, for example, an alkaline coupling reagent (KR) per cell or an azide (AR) coupling reagent per cell. In one embodiment, at least about 1,2, 3, 5, 10,20, 30 or 40% of the cells in the preparation have the recited level of agent per cell. In one embodiment, the preparation comprises at least more than, or about, 10,000, 50,000, 100,000, 10 ⁶ , 10 ⁷ , 10 ⁸ or 10 ⁹ cells. In one embodiment, the preparation comprises at least more than, or about, 10 ¹⁰ , 10 ¹¹ or 10 ¹² cells. In one embodiment, the cell further comprises a polypeptide expressed from an exogenous nucleic acid, for example, inside the cell or on the cell surface.
[007] In another aspect, the invention features a preparation, for example, pharmaceutical preparation, of erythroid cells, for example hematopoietic stem cells, reticulocytes or erythrocytes, comprising at least more than, or about 1,000, 2,000, 3,000, 4,000, or 5,000 coupling reagents per cell. For example, in some aspects, the disclosure discloses a preparation, for example, pharmaceutical preparation, of erythroid cells comprising at least 1,000, 2,000, 3,000, 4,000, or 5,000 coupling reagents per cell.
[008] In another aspect, the invention features a preparation, for example, pharmaceutical preparation, of erythroid cells, for example, hematopoietic stem cells, reticulocytes or erythrocytes, comprising at least more than, or about 5,000, 10,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000 copies of an agent, for example, heterologous agent, coupled to the cell by a residual ligand. In some embodiments, the residual ligand comprises a click signature. For example, in some
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4/111 aspects, the disclosure presents a preparation, for example, pharmaceutical preparation, of erythroid cells comprising at least 5,000, 10,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000 copies of a heterologous agent, coupled to the cell by residual ligand comprising a click subscription.
[009] In one embodiment, at least about 50, 60, 70, 80, 90, 95, 99 or 99.9% of the cells in the preparation have the recited level of agent per cell. In one embodiment, at least about 50, 60, 70, 80, 90, 95, 99 or 99.9% of the cells in the preparation comprise a first agent and a second agent, for example, in which a cell is considered positive for an agent if the agent level is greater than that measured in 99% of similar unlabeled cells. In one embodiment, at least about 1,2, 3, 5, 10,20, 30 or 40% of the cells in the preparation have the recited level of agent per cell. In one embodiment, the preparation comprises at least more than, or about, 10,000, 50,000, 100,000, 10 ⁶ , 10 ⁷ , 10 ⁸ or 10 ⁹ cells. In one embodiment, the preparation comprises at least more than, or about, 10 ¹⁰ , 10 ¹¹ or 10 ¹² cells.
[0010] In one embodiment, erythroid cells are reticulocytes, for example, from expanded, differentiated and enucleated HSCs in vitro. In embodiments, erythroid cells comprise hematopoietic precursor cells, for example, CD34 + cells.
[0011] In one embodiment, erythroid cells are erythrocytes, for example, obtained from blood.
[0012] In one embodiment, erythroid cells are genetically modified, for example, the cells comprise a polypeptide expressed from an exogenous nucleic acid (for example, DNA or RNA, for example, mRNA).
[0013] In one embodiment, erythroid cells are encapsulated, for example, loaded hypotonically, with an exogenous protein.
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[0014] In one embodiment, the preparation is free or substantially free of free coupling reagent, unreacted coupling reagent, an organic solvent, a metal (e.g. copper), a catalyst, or unmarked cells or unmodified cells .
[0015] In one embodiment, the agent is an agent described in WO2015 / 15302; or WO2015 / 073587, each of which is incorporated herein by reference in its entirety.
[0016] In one embodiment, the agent comprises a peptide agent, for example, a polypeptide, an enzyme or an antibody. In one embodiment, the peptide agent comprises a cytokine, a receptor, a ligand, a hormone, a growth factor, a blood factor, a lysosomal storage enzyme, asparaginase or a fragment of any of the foregoing comprising an extracellular domain, domain of opposite binding or another biologically active domain, or a fragment or variant thereof. In one embodiment, the agent comprises an antigen, for example, a tumor antigen and antigen of the infectious and autoantigen disease.
[0017] In one embodiment, the agent comprises a lipid; nucleic acid, for example, RNA, DNA, siRNA; sugar; drug; or small molecule.
[0018] In one embodiment, the agent comprises a polypeptide of more than about 30, 50, 75, 100, 150, 200, 250, 300, 350 or 400 kilodaltons. In one embodiment, the agent comprises a polypeptide of about 1-2, 1-5, 2-5, 5-10, 10-20, 20-50, 50-100, 100-200 or 200500 kDa. In one embodiment, the agent comprises a polypeptide of more than 10, 15, 20, 25, 30, 40, 50, 100, 200, 300, 400, or 500 amino acids.
[0019] In one embodiment, the agent, for example, a polypeptide, comprises the post-translational modification, for example,
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6/111 a post-translation modification that is not made by erythroid cells, or made inefficiently by erythroid cells. In one embodiment, the polypeptide, for example, an antibody, comprises one or more disulfide bridges. In one embodiment, the agent, for example, a polypeptide, lacks a post-translational modification or comprises the modification at a lower level than the protein produced by a mammalian cell, for example, a CHO cell. In embodiments, the polypeptide (for example, an antibody) is subjected to deglycosylation. In modalities, deglycosylation leads to less ADCC induction and / or less interaction with gamma Fc receptors.
[0020] In some embodiments, the agent, for example, a polypeptide, does not comprise a post-translational modification that is normally present if the polypeptide is produced in a human cell, for example, a human erythroid cell. In some embodiments, the agent, for example, polypeptide, comprises a post-translational modification at a lower level than is normally present if the polypeptide is produced in a human cell, for example, at least 10%, 20%, 30% , 40%, 50%, 60%, 70%, 80%, 90% lower.
[0021] In some embodiments, a polypeptide agent comprises one or more non-canonical amino acids. The non-canon amino acid can be, for example, p-methoxyphenylalanine (pMpa); pacetylphenylalanine (pApa); p-benzoylphenylalanine (pBpa); pyophenylalanine (kite); p-azidophenylalanine (pAzpa); ppropargyloxyphenylalanine (pPpa); α-aminocaprylic acid; onitrobenzylcysteine (o-NBC); 1,5-dansylalanine; and o-nitrobenzylserine (oNBS).
[0022] In one embodiment, the agent, for example, a polypeptide, is toxic to, or compromises, the growth, function, life span, or development of an erythroid cell. In
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7/111 embodiments, an agent that is toxic to a cell is an agent (eg, enzyme) that produces a metabolite toxic to the cell.
[0023] In one embodiment, the agent comprises a multimeric polypeptide, for example, a dimer, for example, a homodimer or heterodimer, a trimer, for example, a homotrimer or heterotrimer, or a tetramer, for example, a homotetramer or heterotetramer, for example, an antibody or cell surface receptor, for example, a receptor for a disease vector, for example, a virus, a drug or a toxin.
[0024] In one embodiment, the agent comprises a polypeptide, for example, a multimeric polypeptide, comprising a plurality of cysteine bridges. In one embodiment, the agent comprises a polypeptide, for example, a multimeric polypeptide, comprising one or more cysteine bridges.
[0025] In one embodiment, the agent comprises a protein that is difficult to express. In embodiments, a protein that is difficult to express is a protein that comprises a post-translational modification that does not normally occur in erythroid cells. In embodiments, the post-translational modification includes divination by a protease that is not normally expressed in erythroid cells. In embodiments, the hard-to-express protein comprises an activated clotting factor, and the protease that activates the clotting factor is not normally expressed in erythroid cells. Examples of activated clotting factors that are activated by divage include Factor Va and Vila, Villa, IXa, Xa, Xia, XIIIa and thrombin.
[0026] In one embodiment, the agent comprises an antibody molecule, for example, a polypeptide comprising one or more of the following: a) variable region sufficient to bind the cognate antigen, for example, HC CDR1, HC CDR2 and HC CDR3, LC CDR1, LC CDR2 and LC CDR3; b) a heavy chain constant sequence
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8/111 comprising one or more of CH1, CH2 and CH3; c) a functional Fc region; and d) a modified or inactive Fc region, for example, a mutationally inactivated Fc region or an Fc region that has a state of glycosylation that impairs Fc activity, for example, a deglycosylated Fc region. In one embodiment, an antibody is a multispecific antibody, for example, a bispecific antibody. Examples of antibodies include, but are not limited to, Fab, Fab ', F (ab') 2, Fv fragments, scFv antibody fragments, disulfide-bound Fvs (sdFv), an Fd fragment consisting of the VH and CH1 domains, antibodies linear, single domain antibodies such as sdAb (VL or VH), camelid VHH domains, multispecific antibodies formed from antibody fragments, such as a divalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region, an epitope binding fragment isolated from an antibody, maxibodies, minibodies, nanobodies, intrabodies, diabodies, tribodies, tetribodies, v-NAR and bis-scFv. In modalities, CDRs are defined according to Kabat etal. (1991), Sequences of Proteins of Immunological Interest, 5- Ed. Public Health Service, National Institutes of Health, Bethesda, MD (Kabat numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (scheme numbering system), or a combination thereof.
[0027] In one embodiment, the agent comprises an antibody, for example, IgA, IgG, IgG1, IgG2, IgM, IgEor IgD.
[0028] In one embodiment, the agent comprises an anti-PDL1 antibody molecule, an anti-4-1BB antibody molecule or an anti-a4p7 antibody molecule. In one embodiment, the agent comprises an anti-PDL1 antibody, an anti-4-1BB antibody, anti-a4p7 antibody or protein A / G. In one embodiment, the agent comprises 4-1BBL, Factor Vila, Factor Xa, asparaginase, IL-10 or MOG peptide. In one embodiment, the agent understands
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9/111 asparaginase and cell asparaginase activity is about 1x1 O ^'12 - 1x1 O' ⁹ U / cell, 1x1 O ^'12 - 1x10 ¹¹ U / cell, 1x1 O' ¹¹ - 1x1 O ^'10 U / cell or 1x1 O ^'10 - 1x1 O' ⁹ U / cell.
[0029] In another aspect, the invention features a preparation, for example, pharmaceutical preparation, of erythroid cells, for example hematopoietic stem cells, reticulocytes or erythrocytes, comprising: as many as at least more than, or about 1,000 , 2,000, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, 200,000, 300,000, 400,000 or 500,000 copies of a first agent, for example, a heterologous agent, coupled to the cell by a residual ligand; and at least more than, or about 1,000, 2,000, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, 200,000, 300,000, 400,000 or 500,000 copies of a second agent, for example, a heterologous agent, coupled to the cell by a second residual binder. For example, in some aspects, the disclosure features a preparation, for example, pharmaceutical preparation, of erythroid cells, comprising at least 1,000 copies of a first heterologous agent, coupled to the cell by a residual linker having a click signature; and at least 1,000 copies of a second heterologous agent, coupled to the cell by a second residual ligand having a second click signature. In one embodiment, the first and second residual ligands have different structures. In one embodiment, the first and second residual ligands have the same structure. In one embodiment, the first and second residual ligands have the same structures, but have the opposite orientation. In modalities, the first click signature is different from the second click signature. In some embodiments, the cell comprises at least 2,000 copies of the first heterologous agent and at least 2,000 copies of the second heterologous agent. In some embodiments, the cell comprises at least 5,000 copies of the first heterologous agent and at least
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5,000 copies of the second heterologous agent. In some embodiments, the cell comprises at least 10,000 copies of the first heterologous agent and at least 10,000 copies of the second heterologous agent. In some embodiments, the cell comprises at least 50,000 copies of the first heterologous agent and at least 50,000 copies of the second heterologous agent. In some embodiments, the cell comprises at least 100,000 copies of the first heterologous agent and at least 100,000 copies of the second heterologous agent. [0030] In one embodiment, at least about 1,2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99 or 99.9% of the cells in the preparation have the recited level of agent per cell. In one embodiment, the preparation comprises at least more than, or about, 10,000, 50,000, 100,000, 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , or 10 ¹² cells. In one embodiment, the preparation comprises at least 10,000, 50,000, 100,000, 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ or 10 ¹² cells.
[0031] In one embodiment, erythroid cells still comprise one or more additional agents, for example, an Nth agent, where N is at least 3, 4, 5,6, 7, 8, 9,10, 50,100 or 200 agents , where for each additional agent the cell comprises as many as at least more than, or about 1,000, 2,000, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, 200,000, 300,000, 400,000 or 500,000 copies of the additional agent, for example, a heterologous agent, coupled to the cell by a residual ligand.
[0032] In one embodiment, a preparation, for example, pharmaceutical preparation, described herein is free or substantially free of coupling-free reagent, unreacted coupling reagent, an organic solvent, a metal (for example, copper) , a catalyst, or unlabeled cells or unmodified cells.
[0033] In modalities, in a cell or preparation, for example, pharmaceutical preparation, described here, less than 10%, 20%,
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30%, 40%, 50%, 60%, 70%, 80% or 90% of the coupling reagent in the cell is unreacted coupling reagent. In embodiments, in a cell or preparation, for example, pharmaceutical preparation, described herein, less than 15%, 14%, 13%, 12%, 11% or 10% of the coupling reagent in the cell is unreacted coupling reagent.
[0034] In embodiment, the agent comprises a peptide agent, for example, a polypeptide, a protein drug, an enzyme, an antibody (for example, a scFv), a cytokine, a cytokine receptor, a receptor molecule, a linker , a hormone, a growth factor, a blood factor, a lysosomal storage enzyme, asparaginase, an antigen (for example, a tumor antigen, an infectious or self-antigen disease antigen), or an immunostimulatory molecule (for example, a co-stimulatory molecule ). In embodiments, the antibody comprises a complete antibody, a fragment thereof, single chain antibody, humanized antibody; murine antibody; mouse-human chimeric monoclonal antibody, mouse primate, primata human, anti-idiotype antibody, antibody fragments, such as, for example, scFv, (scFv) 2, Fab, Fab 'and F (ab') 2, F ( ab1) 2, Fv, dAb and Fd, diacorpose an antibody related polypeptide.
[0035] In another aspect, the invention presents a method of producing a pharmaceutical preparation, product or intermediate comprising: a) coupling a first coupling reagent, for example, a GMP grade coupling reagent, to an erythroid cell, producing thus, a pharmaceutical preparation, product, or intermediate. In one embodiment, the method further comprises: b) contacting the cell with an agent coupled to a second coupling reagent, for example, a GMP grade coupling reagent, for example, under conditions suitable for the reaction of the first coupling reagent with the second coupling reagent. In
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12/111 embodiments, the method comprises coupling the second coupling reagent to the agent, for example, before or after step a). In modalities, the intermediate of step a) is stored before the contact of step b). In embodiments, the agent coupled to a second coupling reagent is stored before contacting step b).
[0036] In one embodiment, the method comprises providing a population of erythroid cells for coupling in a). In one embodiment, the method comprises reducing or minimizing entities in the preparation that react with the first coupling reagent. In one embodiment, the method comprises treating, for example, washing the cell to remove unbound material, for example, protein, from the cell, for example, capable of reacting with the first coupling reagent.
[0037] In one embodiment, a second agent is coupled to the cell, in which the method comprises: c) the coupling of a second coupling reagent, for example, a GMP grade coupling reagent with an erythroid cell and d) contact of the cell with a second agent coupled to a second coupling reagent, for example, a GMP grade coupling reagent, for example, under conditions suitable for reacting the first coupling reagent with the second coupling reagent.
[0038] In modalities, the method comprises:
a) coupling a first coupling reagent to the cell,
b) contacting the cell with a first agent coupled to a second coupling reagent that is capable of reacting with the first coupling reagent,
c) coupling a third coupling reagent to the cell and
d) contacting the cell with a second agent coupled to a fourth coupling reagent that is capable of reacting with the third
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13/111 coupling reagent.
[0039] In modalities, steps a), b), c) and d) are performed in one of the following orders:
a), then b), then c) and then d);
a), then c), then b) and then d);
a), then c), then d) and then b);
a), then c), then b) and d) simultaneously;
c), then d), then a) and then b);
c), then a), then b) and then d);
c), then a), then d) and then b);
c), then a), then b) and d) simultaneously;
a) and c) simultaneously, then b) and then d);
a) and c) simultaneously, after d) and then b); or
a) and c) simultaneously, then b) and d) simultaneously.
[0040] In one embodiment, the efficiency of the coupling reaction is greater than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97% or 98%.
[0041] In one embodiment, erythroid cells are reticulocytes, for example, from expanded, differentiated and enucleated HSCs in vitro.
[0042] In one embodiment, erythroid cells are erythrocytes, for example, obtained from blood.
[0043] In one embodiment, erythroid cells are genetically modified, for example, the cells comprise a polypeptide expressed from an exogenous nucleic acid (for example, DNA or RNA, for example, mRNA).
[0044] In one embodiment, erythroid cells are encapsulated, for example, hypotonically charged, with an exogenous protein, an agent that binds to a cellular protein, DNA or RNA.
[0045] In some embodiments, an enucleated cell described here is a reticulocyte, an erythrocyte or a platelet.
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[0046] In another aspect, the invention provides a method of administering an agent to an individual, for example, treating an individual, comprising administering a preparation, composition or cells described herein to the individual, thereby administering an agent to the individual. individual, for example, treatment of the individual. In one embodiment, the method comprises providing, for example, making, or obtaining from another entity, the preparation, composition or cells. In another aspect, the invention features a cell (e.g., enucleated erythroid cell) described herein, for use in the treatment of a disease or disorder, for example, a disease or disorder described herein. In another aspect, the invention provides a cell (e.g., enucleated erythroid cell) described herein, for the manufacture of a medicament for the treatment of a disease or disorder, for example, a disease or disorder described herein.
[0047] In one embodiment, the cells are allogeneic to the individual.
[0048] In one embodiment, the cells are autologous to the individual.
[0049] In one embodiment, an agent is attached to the cells. In one embodiment, the agent comprises a peptide agent, for example, a polypeptide, an enzyme or an antibody. In one embodiment, the agent comprises a cytokine, a receptor, a ligand, a hormone, a growth factor, a blood factor, a lysosomal storage enzyme, asparaginase or a fragment of any of the foregoing comprising an extracellular domain, binding domain contrary or other biologically active domain.
[0050] In one embodiment, less than 7, 6, 5, 4, 3, 2 or 1 days elapses between the coupling of an agent to the cells and administering the cells to the individual.
[0051] In one embodiment, the cells are autologous and less than 7, 6, 5, 4, 3, 2 or 1 days elapses between the removal of the cell from the
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15/111 individual and administer the cells to the individual.
[0052] In one embodiment, at least 1,2, 3, 4, 5, 6, 7, 14, 21 or 28 days elapse between removing an individual's cell and administering the cells to an individual (e.g. the same or a different individual).
[0053] In one embodiment, the method comprises evaluating the individual and responding to the evaluation, selecting an agent for coupling to the cell.
[0054] In modalities, a coupling step occurs in vivo or ex vivo. For example, in embodiments, a coupling agent is administered to an individual under conditions that allow the coupling reagent to attach to a cell, for example, an erythroid cell. In some embodiments, an agent coupled to a second coupling reagent is administered to an individual under conditions that allow the agent to attach to a cell, for example, an erythroid cell.
[0055] In another aspect, the invention provides a method of providing a preparation, composition or cells comprising: receiving from an entity the identity of an agent, for example, an agent suitable for treating an individual and coupling the agent to a cell by a method described herein, thereby providing a preparation, composition or cells.
[0056] In another aspect, the invention features a kit comprising one or more of the following: a) optionally, an erythroid cell; b) a first coupling reagent; c) a second coupling reagent; d) an agent; e) optionally, an erythroid cell coupled to a coupling reagent; f) an agent coupled to a coupling reagent; or g) a reagent to detect the presence of any of a-f.
[0057] In one embodiment, the kit comprises one or more of b, c,
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16/111 d, f eg.
[0058] In some respects, the invention features a kit comprising: a) an activated cell (e.g., erythroid cell), b) a first activated agent, c) a second activated agent and d) optionally a third or other activated agents.
[0059] In some respects, the invention features a method of manufacturing a functionalized cell (for example, a functionalized cell described here), comprising: a) receiving instructions from a third party (for example, a doctor, office or hospital) for provide a functionalized function; cell having one or more specified agents, b) contacting an activated cell with a different agent or agents (eg 2, 3, 4, 5, 10, 20 or more), making the cell functionalized and c) providing the functionalized cell to the third.
[0060] In some respects, the invention features a method of obtaining a functionalized cell (for example, a functionalized cell described here), comprising: a) transmitting instructions to a third party (for example, a laboratory) to provide a functionalized cell having one or more agents, b) receiving the functionalized cell from the third and c) administering the functionalized cell to an individual in need.
[0061] In some respects, the disclosure features a cell (for example, an erythroid cell and / or an enucleated cell, for example, an enucleated erythroid cell), comprising:
an agent (for example, an exogenous polypeptide) on the cell surface and a linker, for example, a residual ligand, ligand covalently to the agent's cell surface (for example, a polypeptide or carbohydrate on the cell surface), wherein the ligand residual comprises a click subscription.
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[0062] In some embodiments, the click signature was formed as the product of a click reaction. In some embodiments, the click signature has the structure of a click signature described here.
[0063] The disclosure provides in some respects a cell (e.g., an erythroid cell and / or an enucleated cell, e.g., an enucleated erythroid cell), comprising an exogenous polypeptide agent covalently attached to the cell surface by a residual ligand comprising a click signature, via an amino acid side chain of a protein comprised by the cell, for example, a protein on the cell surface, where the click signature was formed as the product of a click reaction or has the structure of a signature click, for example, a click subscription described here.
[0064] The disclosure provides, in some respects, a cell (for example, an erythroid cell and / or an enucleated cell, for example, an enucleated erythroid cell), comprising:
a plurality of exogenous polypeptide agents, each exogenous polypeptide agent of the plurality being covalently attached to the cell surface by a residual linker comprising a click signature, in which at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of exogenous polypeptide agents in the cell are linked via a side chain of amino acids to a protein comprised by the cell, for example, a protein on the cell surface , where the click signature was formed as the product of a click reaction or has the structure of a click signature, for example, a click signature described here.
[0065] In some ways, disclosure provides a cell
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18/111 enucleated erythroid, comprising a plurality of copies of an exogenous polypeptide agent covalently attached to the cell surface by a residual ligand comprising a click signature, where the click signature has been formed as the product of a click reaction or has the structure of a click subscription, where one or more of List 1 applies here. In some embodiments, the cell does not have a sortase transfer subscription. In some embodiments, the cell does not comprise a sortase transfer subscription covalently linked to the click signature.
[0066] In some respects, the disclosure provides an enucleated erythroid cell, comprising a plurality of copies of an exogenous polypeptide agent covalently attached to the cell surface by a residual ligand comprising a click signature, in which the click signature has been formed as the product of a click reaction or has the structure of a click signature, in which at least 50%, 60%, 70%, 80%, or 90% of the plurality of exogenous polypeptide agents have the same orientation in relation to cell surface. In some embodiments, the cell does not have a sortase transfer subscription. In some embodiments, the cell does not comprise a sortase transfer subscription covalently linked to the click signature. In some embodiments, the cell is not genetically modified, for example, it does not comprise a polypeptide that has been expressed from an exogenous nucleic acid. In other embodiments, the cell is genetically modified, for example, it comprises a polypeptide that has been expressed from an exogenous nucleic acid. In some embodiments, the cell does not comprise an unnatural amino acid. In some embodiments, the cell does not comprise a transmembrane protein with an unnatural amino acid.
[0067] In some embodiments, the exogenous polypeptide agent
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19/111 comprises a π-clip. In some embodiments, the exogenous polypeptide agent is covalently linked to the enucleated erythroid cell through the π-clip. In some embodiments, the exogenous polypeptide agent comprises an ncAA. In some embodiments, the exogenous polypeptide agent is covalently linked to the enucleated erythroid cell via ncAA. In some embodiments, the exogenous polypeptide agent comprises two or more cysteine residues. In some embodiments, the exogenous polypeptide agent is covalently linked to the enucleated erythroid cell through the two or more cysteine residues, for example, through a ThioLinker. [0068] In some embodiments, the exogenous polypeptide agent is a peptide ligand that binds to a binding partner.
[0069] In some embodiments, the cell further comprises a second agent, for example, a second exogenous polypeptide agent, for example, wherein the second exogenous polypeptide agent is covalently attached to the cell surface by a second residual linker comprising a click signature . In some embodiments, the cell further comprises a third agent, for example, a third exogenous polypeptide agent, for example, wherein the third exogenous polypeptide agent is covalently linked to the cell surface by a second residual linker comprising a click signature.
[0070] In some embodiments, the agent, for example, the exogenous polypeptide agent, is covalently attached to an endogenous molecule of the cell, for example, an endogenous polypeptide on the cell surface.
[0071] In some embodiments, at least 50%, 60%, 70%, 80% or 90% of exogenous polypeptide agents are covalently linked to the cell surface with a pre-selected orientation, for example, they are linked by the same portion or portions of agents
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20/111 exogenous polypeptides (for example, an N-terminus, a C-terminus, or a particular residue, for example, a particular ncAA, or a particular plurality of residues, for example, two cysteine residues). In some embodiments of the preparations described herein, at least 50%, 60%, 70%, 80% or 90% of the exogenous polypeptide agents in the preparation are covalently attached to an enucleated erythroid cell surface with a pre-selected orientation, for example, they are linked by same portion or portions of the exogenous polypeptide agents (for example, an N-terminus, a C-terminus, or a particular residue, for example, a particular ncAA, or a particular plurality of residues, for example, two cysteine residues).
[0072] In some embodiments, the agent, for example, the exogenous polypeptide agent, is covalently attached to an endogenous molecule of the cell, for example, an endogenous polypeptide on the cell surface. In some embodiments, the ligand binds the agent to an endogenous cell molecule, for example, an endogenous polypeptide or sugar on the cell surface. In some embodiments, the ligand binds the agent to an exogenous molecule in the cell, for example, a polypeptide or exogenous sugar on the cell surface.
[0073] In some embodiments, the click signature comprises a cyclic portion, for example, a heterocycle, such as triazole, for example, a disubstituted triazole, or a cicloaduto. In some embodiments, the click signature comprises a cycloalkene, such as cyclohexene, an alkyl sulfide, a dihydropyrazine, such as a 1,2-dihydropyrazine, a diazole or a sulfur-containing ring, such as a thiopyran.
[0074] In some modalities, the signature of clicks was formed or is capable of being formed by cycloaddition (for example, a 1,3-dipolar cycloaddition or hetero-Diels-Alder cycloaddition), opening
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21/111 nucleophilic ring (for example, tensioned heterocyclic electrophile openings, such as aziridines, epoxides, cyclic sulfates, aziridinium and episulfonium ions), non-aldolic carbonyl chemistry (eg, urea formation, thioureas, hydrazones, oxime ethers, amides or aromatic heterocycles) or an addition to a multiple carbo-carbon bond (for example, epoxidation, aziridination, dihydroxylation, sulfenyl halide addition, nitosyl halide addition or Michael addition).
[0075] In some respects, the disclosure presents a method of fabricating (for example, manufacturing) a cell (for example, an erythroid cell, for example, an enucleated erythroid cell), functionalized with an agent comprising:
(a) providing an activated cell comprising a cell bond, for example, covalently linked, to a first coupling portion, for example, a first click loop, (b) providing an activated agent comprising an agent (for example, an exogenous polypeptide ) attached, for example, covalently attached, to a second coupling portion capable of reacting with the first coupling portion, for example, a second click loop capable of reacting with the first click loop and (c) placing the activated cell in contact with the activated agent, thus producing a cell functionalized with the agent.
[0076] In embodiments, the method comprises contacting a cell with a first coupling reagent that comprises the first coupling portion, for example, the first click loop, thereby producing the activated cell. In embodiments, the method comprises contacting the agent with a second coupling reagent comprising the second coupling portion, for example, second click loop, thereby producing the activated agent. In modalities, the method comprises to synthesize the agent
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22/111 to contain the second coupling unit, for example, second click loop, for example, by incorporating a non-canonical amino acid. In embodiments, the method comprises preparing a cell comprising a first coupling portion, for example, by incorporating a non-canonical amino acid that comprises the first coupling portion or by incorporating a sugar (for example, in a carbohydrate), comprising the first coupling portion.
[0077] In modalities, the contact of the cell with the first coupling reagent occurs before or after the contact of the agent with the second coupling reagent. In embodiments, the contact of the cell with the first coupling reagent and the contact of the agent with the second coupling reagent occurs at the same time, for example, they start at the same time, end at the same time, or have a partial overlap.
[0078] In modalities, the first coupling reagent is impermeable to the membrane. In embodiments, the second coupling reagent is impermeable to the membrane. In embodiments, the first coupling reagent is permeable to the membrane. In embodiments, the second coupling reagent is permeable to the membrane.
[0079] In some respects, the disclosure features a cell, for example, an erythroid cell, for example, an enucleated erythroid cell, produced by a method described herein. In some embodiments, the method comprises (a) providing an activated cell comprising a cell covalently linked to a first click loop, (b) providing an activated agent comprising an agent (e.g., an exogenous polypeptide) covalently attached to a second click loop capable of reacting with the first click loop and (c) placing the activated cell in contact with the activated agent, thus producing a cell functionalized with the agent.
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[0080] In some aspects, the disclosure presents a method of treating a disease, comprising the administration of a functionalized cell described here to an individual in need. [0081] In some embodiments, the first coupling reagent comprises a first portion reactive to the substrate that reacts with one of the following portions in the agent:
a) a primary amine (-NH ₂ ), for example, at a lysine or N-terminus,
b) carboxyl (-COOH), for example, in an aspartic acid, glutamic acid, or C-terminus,
c) sulfhydryl (-SH), for example, in cysteine or
d) carbonyl (-CHO), for example, a ketone or aldehyde group, for example, in a glycoprotein, for example, an oxidized glycoprotein.
[0082] In some embodiments, the second coupling reagent comprises a first substrate-reactive portion that reacts with one of the following portions in the cell (for example, a portion of a protein or carbohydrate in the cell):
a) a primary amine (-NH ₂ ), for example, at a lysine or N-terminus,
b) carboxyl (-COOH), for example, in an aspartic acid, glutamic acid, or C-terminus,
c) sulfhydryl (-SH), for example, in cysteine or
d) carbonyl (-CHO), for example, a ketone or aldehyde group, for example, in a glycoprotein, for example, an oxidized glycoprotein.
[0083] In some embodiments, the first substrate-reactive portion and the second substrate-reactive portion react with the same type of portion, for example, the first substrate-reactive portion reacts with a first primary amine and the second portion
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24/111 reactive to the substrate reacts with a second primary amine. In some embodiments, the first substrate-reactive portion and the second substrate-reactive portion react with different types of molecules.
[0084] In some embodiments, the first or second coupling reagent comprises a sortase recognition site, for example, a GGG at the Nouum LPXTG terminal at the C terminal (SEQ ID NO: 1).
[0085] In some embodiments, the coupling reagent (for example, first or second coupling reagent) comprises a click handle. In embodiments, the click loop comprises an alkyne, for example, a tensioned alkyne, for example, a cyclooctyl, for example, the DBCO-sulfo-NHS ester. In some embodiments, the dedicated handle comprises an azide, for example, an estersulfo-NHS of 3-azidopropionic acid.
[0086] In some embodiments, a method described herein further comprises contacting the functionalized cell with a terminating reagent comprising only a coupling unit, for example, a click loop. In embodiments, the terminating reagent can react with an unreacted click loop in the cell, thereby reducing the number of unreacted click loops in the cell. In embodiments, the terminating reagent can react with unreacted click handle on the agent, thereby reducing the number of unreacted click handles on the agent. In embodiments, the method sequentially comprises, in any order, the contact of the functionalized cell with a terminating reagent that can react with unreacted click handles on the cell and the contact of the functionalized cell with a terminating reagent that can react with reactive handles unreacted click on the agent. In embodiments, the unreacted terminating reagent is removed by washing between the two
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25/111 contact steps. In embodiments, the terminating reagent has a lower molecular weight and / or less steric impedance than the agent. In embodiments, the terminating reagent comprises a detectable marker. In some embodiments, the terminating reagent with a detectable marker is placed in contact with an aliquot of a batch of functionalized cells. In some embodiments, if the amount of detectable marker that reacts with the functionalized cell is below a predetermined threshold, the batch is approved or released. In some embodiments, if the amount of detectable marker that reacts with the functionalized cell is above a predetermined threshold, the batch is suspended or subjected to further processing, for example, it is brought into contact with a terminating reagent. In embodiments, the terminating reagent comprises an azide or an alkaline group. In embodiments, since the terminating reagent reacts with an agent or cell, which is not substantially reactive, for example, no more reactive than the N-terminus of human wild type glycophorin Ade.
[0087] In some modalities, at least 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.5%, 99.8% or 99.9% of the Enucleated erythroid cells are labeled, for example, in which a cell is considered labeled if the level of agent is greater than that measured in 99% of similar unmarked cells.
[0088] In some embodiments, enucleated erythroid cells are marked with an average of (or an enucleated erythroid cell described here is marked with) 50-200,000 copies per cell of the agent, for example, 50-100,100-200, 200-500 , 500-1,000, 1,000-2,000, 2,0005,000, 5,000-10,000, 10,000-20,000, 20,000-50,000, 50,000-100,000 or 100,000-200,000 copies per cell of the agent, or with at least 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000 or 200,000 copies per cell of the agent, or up to 50, 100, 200, 500,
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11/26
1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, or 200,000 copies per cell of the agent.
[0089] In some embodiments, at least 10 ⁷ , 10 ⁸ or 10 ⁹ enucleated erythroid cells are labeled.
[0090] In some embodiments, the population of enucleated erythroid cells is marked with at least 1,2, 5, 10, 20, 50, 100, 200 or 500 ng of the agent, for example, exogenous polypeptide.
[0091] In some embodiments, the bound polypeptide agent comprises anti-PD-L1, anti-a4p7, anti-m41BBL, 4-1BBL, Vila Factor, Factor Xa, asparaginase or MOG peptide.
[0092] In some embodiments, at least 50.60, 70.80, 95.90, 95, 96, 97, 98 or 99% of the labeled erythroid cells bind to a ligand, for example, in a cytometry assay flow rate of Example 6. In some embodiments, at least 50, 60, 70, 80, 95, 90, 95, 96, 97, 98 or 99% of the enucleated erythroid cells comprise an agent that binds to a ligand, for example , in an Example 6 flow cytometry assay. In embodiments, the cell is considered to bind to the ligand by an Example 6 flow cytometry assay, if it has a signal greater than that measured in 99% of otherwise similarly lacking the agent.
[0093] In embodiments, the agent is attached (for example, adjacent to non-intervening atoms, or having one or more atoms between the agent and the endogenous polypeptide) of an amino acid of an endogenous polypeptide. In embodiments, the binder has a length of at least 5.10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nm. In embodiments, the binder has a length of about 30-100, 40-90, 50-80, or 60-70 nm. In embodiments, the ligand is of such length that the agent is outside the glycocalyx of the erythroid cell. In some embodiments, the linker comprises PEG, for example, PEG with a length of about 3-20, for example, 4
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13, for example, about 4, 5, 12 or 13. In some embodiments, the length of the PEG component is between about 30-60 Angstroms, for example, 30-40, 40-50 or 50-60 Angstroms. In some embodiments, the linker comprises PEG with a length of about 50-200, 200-400, 400-600, 600-800 or 8001000 Angstroms.
[0094] In some modalities, one or more of List 1 applies, where List 1 is:
a) the agent is linked (for example, via a residual linker) to an amino acid other than a glycine of an endogenous membrane protein, for example, it is linked to at least one non-glycine residue;
b) the agent is bound (for example, via a residual linker) to a site other than the N-terminus or C-terminus of an endogenous membrane protein;
c) the agent is bound (for example, via a residual linker) to a site other than the N-terminus or C-terminus of a membrane protein;
d) the agent is linked (for example, via a residual linker) to a full-length endogenous membrane protein;
e) the agent is linked to at least 10, 20, 50 or 100 polypeptides distinct from the sequence, for example, endogenous polypeptides;
f) the cell functionalized with an agent does not have a sortase transfer signature (i.e., a sequence that can be created by a sortase reaction) such as LPXTG (SEQ ID NO: 1).
g) the agent is not linked to a subscription transfer of sortase,
h) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the agents in the cell are not linked to a
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11/28 sortase transfer subscription,
i) the click subscription is not linked to a random transfer subscription,
j) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of click subscriptions in the cell are not linked to a transfer subscription of sortase,
k) the agent is not linked to an extracellular sortase transfer subscription;
l) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the agents in the cell are not linked to a sortase transfer subscription extracellular,
m) the click signature is not linked to an extracellular sort transfer subscription,
n) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of cell click subscriptions are not linked to a transfer subscription extracellular sortase,
o) the agent is not linked to an extracellular sortase transfer subscription that is within 1,2,3, 4,5, 10, 20, 50 or 100 amino acids of a transmembrane segment;
p) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the agents in the cell are not linked to a sortase transfer subscription extracellular which is within 1, 2, 3, 4, 5, 10, 20, 50 or 100 amino acids of a transmembrane segment,
q) the click signature is not linked to an extracellular sortase transfer signature that is within 1,2, 3, 4, 5, 10, 20, 50 or 100 amino acids of a transmembrane segment,
r) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of click subscriptions in the cell are not linked to a transfer subscription extracellular sortase that is
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29/111 within 1,2, 3, 4, 5, 10, 20, 50 or 100 amino acids of a transmembrane segment,
s) the agent is not linked to a sortase transfer subscription that is within 1, 2, 3, 4, 5, 10, 20, 50 or 100 amino acids of the agent,
t) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the agents in the cell are not linked to a sortase transfer subscription which is within 1,2, 3, 4, 5, 10, 20, 50 or 100 amino acids of the agent,
u) the click signature is not linked to a sort transfer subscription that is within 1,2, 3,4, 5, 10, 20, 50 or 100 amino acids of the click signature,
v) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of cell click subscriptions are not linked to a transfer subscription of sortase that is within 1,2, 3, 4, 5, 10,20, 50 or 100 amino acids from the click signature,
w) the polypeptide, for example, an endogenous polypeptide, to which the agent is attached does not have a sortase transfer signature at a position corresponding to the N or C terminus;
x) at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the agents in the cell are linked via an amino acid side chain of a protein comprised by the cell, for example, a protein on the cell surface, where in one embodiment the side chain is a side chain of lysine, cysteine, aspartic acid or glutamic acid, bound in such a way that by least one atom of a side chain of the amino acid is arranged between the agent and the main chain of the protein,
y) the agent is linked to a polypeptide (for example,
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30/111 endogenous protein) on the cell surface,
z) the functionalized cell has not been contacted with a sortase, aa) the functionalized cell does not comprise a sortase transfer signature that comprises a bond that has been formed extracellularly, bb) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the click signatures on the cell were produced by reacting the cell with a coupling reagent or cc) in which the cell is produced by a method that it does not understand the contact of the cell with an unnatural sugar, for example, a sugar comprising a click loop or a combination thereof.
[0095] In some embodiments, the cell comprises more than 1,000, 5,000, 5,000, 10,000, 50,000, 100,000, 200,000, 300,000, 400,000 or 500,000 copies of the agent.
[0096] In some ways:
i) the agent has an elimination rate in which at least 20% of the agent remains in the individual's circulatory system for
1,2, 3, 4, 5, 6 or 7 days, ii) the cell population has an elimination rate in which at least 20% of the agent remains in the individual's circulatory system for 1,2, 3, 4, 5 , 6 or 7 days, iii) the cell population has an elimination rate in which at least 20% of functional erythroid cells remain in the individual's circulatory system for 1,2, 3, 4, 5, 6 or 7 days ;
iv) the agent has an elimination rate in which at least 20% of the agent that is in the individual's circulatory system after 1 day remains in the circulatory system after another 1,2, 3, 4, 5, 6, 7, 14 or 21 days;
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31/111 iv) the cell population has an elimination rate in which at least 20% of the agent that is in the individual's circulatory system after 1 day remains in the circulatory system after another 1, 2, 3, 4, 5, 6, 7, 14 or 21 days;
iv) the cell population has an elimination rate in which at least 20% of the cell population that is in the individual's circulatory system after 1 day remains in the circulatory system after another 1,2, 3, 4, 5, 6, 7 , 14 or 21 days;
[0097] In some modalities, at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98% or 99% of the erythroid cells of the population are enucleated.
[0098] In some embodiments, cells are not hypotonic charged cells.
[0099] In some embodiments, the enucleated erythroid cell has one or more of the following characteristics:
a) an osmotic fragility with less than 50% cell lysis at 0.3%, 0.35%, 0.4%, 0.45% or 0.5% NaCI;
b) a cell volume of about 10-200 µL or a cell diameter between about 1 micron and about 20 microns, between about 2 microns and about 20 microns, between about 3 microns and about 20 microns, between about of about 4 microns and about 20 microns, between about 5 microns and about 20 microns, between about 6 microns and about 20 microns, between about 5 microns and about 15 microns, or between about 10 microns and about 30 microns;
c) greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of fetal hemoglobin; or at least about 20, 25 or 30 pg / hemoglobin cell; or
d) the phosphatidylserine content of the external part is less than 30%, 25%, 20%, 15%, 10% or 5%, as measured by staining with Annexin V.
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[0100] In some embodiments, the residual ligand or signature dykes is within 1,2, 5,10, 20 or 50 atoms of an amino acid (for example, a canonical amino acid) of a polypeptide in the cell. In some embodiments, the residual ligand or click signature is not within 1, 2, 5, 10, 20 or 50 atoms of a carbohydrate portion of the cell. In some embodiments, the agent is linked to a polypeptide that is not glycosylated. In some embodiments, the agent is attached to an amino acid side chain, N-terminus or C-terminus of a polypeptide that is glycosylated.
[0101] In some embodiments, the residual ligand is not within 1,2, 5, 10, 20 or 50 atoms of a portion of mannose in the cell. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of residual binders or cell click signatures are not within 1,2, 5, 10, 20 or 50 atoms of a portion of mannose in the cell. In some embodiments, the residual ligand is linked to at least two (for example, 3, 4, 5 or more) types of sugar in the cell. For example, a first residual linker can be within 1, 2, 5, 10, 20 or 50 atoms of a first portion of sugar in the cell and a second residual linker can be within 1, 2, 5, 10, 20 or 50 atoms of a second portion of sugar in the cell.
[0102] In some embodiments, the cell is not genetically modified.
[0103] In some embodiments, the cell does not comprise a non-canonical amino acid. In some embodiments, less than 1%, 0.1%, 0.01%, 0.001% or 0.0001% of amino acids in the cell are non-canonical amino acids.
[0104] Disclosure includes all combinations of any one or more of the previous aspects and / or modalities, as well as combinations with any one or more of the modalities
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33/111 presented in the detailed description and examples.
[0105] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references (for example, reference numbers from the sequence database) mentioned herein are incorporated by reference in their entirety. For example, all GenBank, Unigene, NCBI and Entrez strings referred to herein, for example, in any Table here, are incorporated by reference. Unless otherwise specified, the sequence access numbers specified here, including in any Table here, refer to the current database entries as of Monday, August 7, 2017. When a gene or protein does reference to a plurality of sequence access numbers, all variants of the sequence are covered.
BRIEF DESCRIPTION OF THE FIGURES
[0106] FIG. 1 is a diagram showing a method of clicking an agent in a cell. An activated agent can be produced, for example, by contacting an agent with a first coupling reagent having a first reactive substrate portion and a first coupling portion that is a click loop and allowing the first reactive substrate portion to react with the agent. An activated cell can be made, for example, by contacting a cell with a second coupling reagent having a second reactive substrate portion and a second coupling portion, which is a click loop, and allowing the second reactive portion of the substrate reacts with the cell. The activated agent and the activated cell are then combined under conditions that allow the first click loop to react with the second click loop, producing a residual binder that comprises a click signature.
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[0107] FIGS. 2A-2C are flow cytometry images of erythroid cells that have been coupled to an exemplary agent (an anti-a4p7 antibody) using the coupling reagents described herein.
[0108] FIG. 3 is a graph showing the percentage of tumor cells bound by erythroid cells functionalized with anti-PD-L1 antibody or control cells functionalized with isotype control antibody.
[0109] FIG. 4 is a time course showing tumor size in mice treated with 41BBL-ECR and an untreated control.
[0110] FIG. 5 is a graph showing serum asparagine levels over time in C57BL / 6 mice treated with RCTasparaginase.
[0111] FIG. 6 is a graph showing serum asparagine levels over time in RAG1 - / - mice treated with RCTasparaginase.
[0112] FIG. 7A and 7B are graphs showing fluorescence over time in mice treated with Cy5-labeled RCT-asparaginase. FIG. 7A, high dose of ASNase mRBCs; FIG. 7B, low dose of ASNase mRBCs.
[0113] FIG. 8A and 8B are graphs showing that the targeted marking of the HIS6 41BBL mouse, using a ThioLinker click loop, results in increased functional activity of the coupled cells. FIG. 8A, IL-2 secretion; FIG. 8B, interferon Y secretion [0114] FIGS. 9A and 9B show the site specific incorporation of a non-canonical amino acid, exo (BCN) -lysine, in the 41BBL mouse, to create a clickable 41BBL mouse. FIG. 9A, chemical structure of exo (BCN) -lysine; FIG. 9B, western blot showing that the
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35/111 mouse 41 BBL clicked was produced at exo (BCN) -lysine concentrations of 1 mM or higher.
DETAILED DESCRIPTION OF THE INVENTION
[0115] Here are described methods and compositions that include functionalization of erythroid cells using selective, biocompatible reactions to couple the cells with an agent (for example, a peptide agent) of interest. In some embodiments, the reactions are cycloaddition reactions (for example, a Huisgen 1,3-dipolar cycloaddition reaction) using reagents that are water-soluble and impermeable to the membrane.
Definitions
[0116] A click signature, as the term is used here, refers to a plurality of atoms arranged between and covalently linking entity A and entity B, in which the click signature is formed as the product of a reaction of click that links entity A and entity B. In one embodiment, the click signature has the structure of a click signature that is formed as the product of a click reaction that links entity A and entity B, but does not limits to a click signature made by any specific process, a click signature formed by a click reaction, but can be formed or provided by another process. In one embodiment, the click signature is an alkaline / azide click signature, for example, the click signature comprises a triazole.
[0117] A click reaction, as the term is used here, refers to a range of reactions used to covalently link a first and a second portion, for convenient production of linked products. It usually has one or more of the following characteristics: it is fast, it is specific, it has high performance, it is efficient, it is spontaneous, it does not significantly alter the
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36/111 biocompatibility of linked entities, has a high reaction rate, produces a stable product, favors the production of a single reaction product, has high atom savings, is chemoselective, is modular, is stereoselective, is insensitive to oxygen, is insensitive to water, is of high purity, generates only harmless or relatively non-toxic by-products that can be removed by non-chromatographic methods (for example, crystallization or distillation), do not need a solvent or can be carried out in a solvent that is benign or physiologically compatible , for example, water, stable under physiological conditions. Examples include an alkaline / azide reaction, a diene / dienophile reaction or a thiol / alkene reaction. Other reactions can be used. In some modalities, the click reaction is fast, specific and of high performance. For example, in modalities, a quick click reaction has a second constant order of forward speed of 10-200 M ' ¹ s' ¹ , 1-20 M' ¹ s ^_1 , or at least 1, 2, 3, 5, 10, 20, 50, 60, 100, 200, 500, 1E3, 2E3, 5E3, 1E4, 2E4, 5E4, 1E5, 2E5, 5E5 or 1E6 M ' ¹ s' ¹ , for example, at 20 ° C in PBS. In some embodiments, a specific click reaction is one in which, when an unmodified cell (for example, a human red blood cell isolated from the peripheral circulation) is contacted with an agent that has a click handle, less than 10%, 5 %, 4% 3%, 2% or 1% of cells are detectably bound to the agent, for example, after a reaction time of 1 hour at 20 ° C in PBS, for example, in an Example 21 assay. In some modalities, a high yield click reaction is one that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% yield, for example, for a time 1 hour reaction time at 20 ° C in PBS. [0118] A click loop, as the term is used here, refers to a chemical portion that is capable of reacting with a second click loop in a click reaction to produce a
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37/111 click subscription. In embodiments, a click loop is comprised of a coupling reagent, and the coupling reagent can further comprise a reactive portion of substrate. [0119] As used herein, a sortase transfer signature is a sequence that can be created by a sortase reaction that links a first sortase recognition motif with a second sortase recognition motif, where the transfer transfer signature sortase comprises the amino acid sequence of the first sortase recognition motif and the second amino acid sequence motif, minus any amino acids (e.g., Gly-Gly) removed during the sortase reaction. For example, an LPXTGG sortase-mediated reaction (SEQ ID NO: 2) with (G) _n can produce an LPXT (G) _n sortase transfer signature (SEQ ID NO: 1).
Cells
[0120] The present invention features compositions comprising functionalized cells and methods of using them. In embodiments, the cells comprise erythroid cells. In modalities, the cell is different from platelets, platelet precursor or platelet parent. In embodiments, cells are nucleated or enucleated. In embodiments, the cells are eukaryotic cells, for example, mammalian cells, for example, human cells. In embodiments, the cells comprise T cells (for example, CD4 T cells or CD8 T cells), B cells, natural killer cells, natural killer T cells, Myeloid Cells, Dendritic Cells, Platelets or Neutrophils. In embodiments, the cells comprise cells derived from the endoderm, cells derived from the ectoderm, or cells derived from the mesoderm. In modalities, the cells comprise stem cells, mesenchymal stem cells, neural stem cells, cardiomyocytes, cells for
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38/111 allogeneic transplantation, cells for xenogenic transplantation or pancreatic beta cells.
Erythroid cells
[0121] The present invention features compositions comprising functionalized erythroid cells and methods of using them. Erythroid cells, as used here, are cells of the erythrocyte series including hematopoietic stem cells (HSCs) and precursors of nucleated and enucleated red blood cells, enucleated red blood cells and any intermediates between HSCs and enucleated red blood cells. In one embodiment, an erythroid cell is a proeritroblast, basophilic erythroblast, polychromatophilic erythroblast, orthochromatic erythroblast, reticulocyte and erythrocyte. In one embodiment, the erythroid cell is a cord blood stem cell, a CD34 + cell, a hematopoietic stem cell (HSC), a spleen colony forming cell (CFU-S), a common myeloid progenitor cell (CPM ), a blastocyte colony-forming cell, a rupture erythroid-forming unit (BFU-E), a megakaryocyte erythrocyte (MEP) progenitor cell, an erythroid colony-forming unit (CFU-E), a reticulocyte, an erythrocyte, a cell- induced pluripotent trunk (IPSC), a mesenchymal stem cell (MSC), a polychromatic normoblast, an orthochromatic normoblast, or a combination thereof.
[0122] In modalities, erythroid cells are, or are derived from, immortal or immortalized cells. For example, immortalized erythroblast cells can be generated by retroviral transduction of CD34 + hematopoietic progenitor cells to express Oct4, Sox2, Klf4, cMyc and suppress TP53 (for example, as described in Huang etal. (2013) Mol Ther, epub print of 3 de September).
[0123] In modalities, erythroid cells can be destined for autologous use or provide a source for allogeneic transfusion. In
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39/111 some modalities, erythroid cells are placed in culture. In one embodiment, an erythroid cell is an enucleated red blood cell.
[0124] In modalities, an erythroid cell is obtained from a biological sample, for example, erythrocytes from human blood, or HSC from the bone marrow.
[0125] In embodiments, an erythroid cell is obtained from an ex-vivo or in vitro process, for example, by which precursor cells, for example, hematopoietic stem cells (for example, human hematopoietic stem cells isolated from bone marrow, cytokine-stimulated peripheral blood or umbilical cord blood) are expanded and / or differentiated and enucleated ex vivo to produce, for example, reticulocytes. Ex vivo methods of making enucleated erythroid cells (e.g., reticulocytes) from stem cells are described, for example, in Migliaccio and Palis (2011) Drug Discov Today Dis Meeh. 8 (1-2): e3-e8; WO2015 / 073587 and WO2015 / 153102, each of which is incorporated by reference in its entirety. Erythroid cells, for example reticulocytes, prepared using this process can be functionalized according to the methods described herein.
[0126] In one embodiment, an enucleated cell is a cell that has lost its nucleus through differentiation from a precursor cell, for example, a hematopoietic stem cell (for example, a CD34 cell), a common myeloid progenitor (CMP) , a megakaryocyte erythrocyte progenitor cell (MEP), a rupture-forming erythroid unit (BFU-E), a colony-forming erythroid unit (CFU-E), a basal erythroblast, a late basophilic erythroblast, a polychromatic erythroblast or orthochromatic erythroblast, or an induced pluripotent cell, in a mature red blood cell or reticulocyte. In one embodiment, a cell
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Enucleated 40/111 is a cell that has lost its nucleus through in vitro differentiation from a precursor cell, for example, a hematopoietic stem cell (for example, a CD34 cell), a common myeloid progenitor (CMP), a progenitor cell erythrocyte megakaryocyte (MEP), a rupture-forming erythroid unit (BFU-E), a colony-forming erythroid unit (CFU-E), a basal erythroblast, a late basophilic erythroblast, a polychromatic erythroblast or an orthochromatic erythroblast, an induced pluripotent cell, into a mature red blood cell or reticulocyte. [0127] An erythroid cell used in the functionalization methods described here may not be modified or may be modified, for example, genetically modified (for example, modified by genetic engineering to express an exogenous protein); it can be encapsulated, for example, hypotonically charged, with an exogenous protein. An erythroid cell used in the treatment methods described here can be autologous, allogeneic or xenogenic.
[0128] Examples of cells for use in the preparations, compounds, methods and kits described herein are described herein. In one embodiment, the cell, for example, erythroid cells, for example, erythrocytes, comprises one or more of the following properties: a) it is obtained from blood, an in vitro culture, or has been differentiated from a cell type more primitive in vitro, for example, a hematopoietic stem cell; b) was loaded hypothetically with an agent; or c) is a genetically modified erythroid cell, for example, expressing an exogenous agent, for example, a polypeptide.
[0129] In one embodiment, the cell is differentiated from a more primitive cell type in vitro, for example, a hematopoietic stem cell.
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[0130] In one embodiment, the cell is a genetically modified erythroid cell, for example, expressing an exogenous agent, for example, a polypeptide.
[0131] In one embodiment, the cell is an erythrocyte obtained from the blood.
[0132] In one embodiment, the cell was loaded hypothetically with an agent.
[0133] In one embodiment, the cell is from an in vitro culture.
[0134] In one embodiment, the cell is a genetically modified erythroid cell, for example, expressing an exogenous agent, for example, a polypeptide.
Physical characteristics of enucleated erythroid cells
[0135] In some embodiments, the enucleated erythroid cells described herein have one or more (for example, 2, 3, 4 or more) physical characteristics described here, for example, osmotic fragility, cell size, hemoglobin concentration or content of phosphatidylserine. Although not wanting to be limited by theory, in some embodiments an enucleated erythroid cell described here has physical characteristics that resemble an untreated wild type erythroid cell. In contrast, a hypotonically charged erythroid cell has aberrant physical characteristics, such as increased osmotic fragility, altered cell size, reduced hemoglobin concentration or increased levels of phosphatidylserine on the outside of the cell membrane.
[0136] In some embodiments, the erythroid cell is in a composition that lacks a stabilizer.
Osmotic fragility
[0137] In some embodiments, the enucleated erythroid cell exhibits substantially the same fragility as the osmotic membrane as an isolated erythroid cell, not in enucleated culture. In some
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42/111 modalities, the population of enucleated erythroid cells has an osmotic fragility of less than 50% of cell lysis to 0.3%, 0.35%, 0.4%, 0.45% or 0.5 NaCl %. Osmotic fragility is determined, in some embodiments, using the method of Example 59 of WO2015 / 073587.
Cell size
[0138] In some embodiments, the enucleated erythroid cell is approximately the diameter or volume like a wild, untreated erythroid cell.
[0139] In some embodiments, the population of erythroid cells has an average diameter of about 4, 5, 6, 7 or 8 microns and, optionally, the population standard deviation is less than 1, 2 or 3 microns. In some embodiments, the one or more erythroid cells have a diameter of about 4-8, 5-7, or about 6 microns. In some embodiments, the diameter of the erythroid cell is less than about 1 micron, greater than about 20 microns, between about 1 micron and about 20 microns, between about 2 microns and about 20 microns, between about 3 microns and about 20 microns, between about 4 microns and about 20 microns, between about 5 microns and about 20 microns, between about 6 microns and about 20 microns, between about 5 microns and about 15 microns or between about 10 microns and about 30 microns. The cell diameter is measured, in some modalities, using an Advia 120 hematology system.
[0140] In some modalities, the volume of the average corpuscular volume of the erythroid cell is greater than 10 f L, 20 f L, 30 f L, 40 f L, 50 f L, 60 fL, 70 f L, 80 f L , 90 fL, 100 f L, 110 fL, 120 fL, 130 fL, 140 fL, 150 fL or greater than 150 fL. In one embodiment, the mean corpuscular volume of the erythroid cell is less than 30 fL, 40 fL, 50 fL, 60 fL, 70 fL, 80 fL, 90 fL, 100 fL, 110 fL, 120 fL, 130 fL, 140 fL, 150 fL, 160 fL, 170 fL, 180 fL, 190 fL, 200 fL or less than 200 fL. In one embodiment, the volume
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43/111 average corpuscular cell of erythroid cells is between 80-100, 100-200, 200-300, 300-400 or 400-500 fentoliters (fL). In some embodiments, a population of erythroid cells has an average corpuscular volume established in this paragraph and the population standard deviation is less than 50, 40, 30, 20, 10, 5 or 2 fL. The average corpuscular volume is measured, in some modalities, using a hematological analysis instrument, for example, a Coulter counter.
Hemoglobin concentration
[0141] In some embodiments, the enucleated erythroid cell has a hemoglobin content similar to an untreated wild-type erythroid cell. In some embodiments, erythroid cells comprise fetal hemoglobin greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or greater than 10%. In some embodiments, erythroid cells comprise at least about 20, 22, 24, 26, 28 or 30 pg and, optionally, up to about 30 pg, of total hemoglobin. Hemoglobin levels are determined, in some embodiments, using the Drabkin reagent method of Example 33 of WO2015 / 073587.
Phosphatidylserine content
[0142] In some embodiments, the enucleated erythroid cell has approximately the same phosphatidylserine content on the outside of its cell membrane as a wild, untreated erythroid cell. Phosphatidylserine is predominantly on the inner part of the cell membrane of untreated wild-type erythroid cells, and the hypotonic charge can cause phosphatidylserine to be distributed outside, where it can trigger an immune response. In some embodiments, the erythroid cell population comprises less than about 30, 25, 20, 15, 10, 9, 8, 6, 5, 4, 3, 2 or 1% of cells that are positive for Annexin V staining Exposure to phosphatidylserine is assessed, in some modalities,
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44/111 by annexin-V-FITC staining, which preferentially binds PS and FITC fluorescence measurement by flow cytometry, for example, using the method of Example 54 of WO2015 / 073587.
Other features
[0143] In some embodiments, the erythroid cell population comprises at least about 50%, 60%, 70%, 80%, 90% or 95% (and, optionally, up to 90 or 100%) of cells that are positive for GPA. The presence of GPA is detected, in some modalities, using FACS.
[0144] In some embodiments, enucleated erythroid cells have a half-life of at least 30, 45 or 90 days in an individual.
[0145] In some embodiments, a cell population comprising erythroid cells comprises less than about 10, 5, 4, 3, 2 or 1% echinocytes.
[0146] In some embodiments, an erythroid cell is enucleated. In some embodiments, a cell, for example, an erythroid cell, contains a nucleus that is non-functional, for example, it has been inactivated.
Universal donor erythroid cells
[0147] In some embodiments, the erythroid cells described here are autologous or allogeneic to the individual to whom the cells will be administered. For example, erythroid cells allogenic to the individual include one or more blood type specific erythroid cells (for example, the cells may be the same blood type as the individual) or one or more universal donor erythroid cells. In some embodiments, the enucleated erythroid cells described herein have reduced immunogenicity compared to a reference cell, for example, have reduced levels of one or more blood group antigens.
[0148] When allogeneic cells are used for transfusion, one
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45/111 ABO compatible blood group can be chosen to prevent an acute intravascular hemolytic transfusion reaction. ABO blood types are defined based on the presence or absence of blood type A and B antigens, monosaccharide carbohydrate structures that are found at the ends of the oligosaccharide chains associated with glycoproteins and glycolipids on the erythrocyte surface (reviewed in Liu etal., Nat. Biotech. 25: 454-464 (2007)). Since group O erythrocytes contain neither A nor B antigens, they can be safely transfused into recipients of any ABO blood group, eg recipient group A, B, AB, or O. Group erythrocytes O are considered universal and can be used in all blood transfusions. Thus, in some embodiments, an erythroid cell described here is type O. In contrast, group A erythroid cells can be administered to group A and AB receptors, group B erythroid cells can be administered to group B receptors. and AB and AB group erythroid cells can be administered to AB receptors.
[0149] In some cases, it may be beneficial to convert a non-group O erythroid cell to a universal blood type. Enzymatic elimination of immunodominant monosaccharides on the surface of group A and group B erythrocytes can be used to generate a population of group O-like erythroid cells (see, for example, Liu etal, Nat Biotech 25: 454-464 (2007) ). Group B erythroid cells can be converted using a-galactosidase derived from green coffee beans. Alternatively or additionally, the enzymatic activities of aN-acetylgalactosaminidase and agalactosidase derived from E. meningosepticum bacteria can be used to remove immunodominant antigens A and B, respectively (Liu et al., Nat. Biotech. 25: 454-464 (2007)) , if present in erythroid cells. In one example, packed erythroid cells and
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46/111 isolated as described herein, are incubated in 200 mM glycine (pH 6.8) and 3 mM NaCI in the presence of aN-acetylgalactosaminidase and agalactosidase (about 300 pg / ml packed erythroid cells) for 60 minutes at 26 ° Ç. After treatment, erythroid cells are washed with 3-4 washes in saline with centrifugation and ABO typed according to standard blood bank techniques. [0150] Although the ABO blood group system is the most important in transfusion and transplantation, in some modalities it may be useful to associate other blood groups between the erythroid cells to be administered and the recipient, or to select or make erythroid cells that are universal for one or more other (for example, smaller) blood groups. A second blood group is the Rh system, where an individual can be Rh + or Rh-. Thus, in some embodiments, an erythroid cell described here is Rh-. In some embodiments, the erythroid cell is of the O and Rh- type.
[0151] In some embodiments, an erythroid cell described here is negative for one or more smaller blood group antigens, for example, Le (ab-) (for Lewis antigen system), Fy (ab-) (for Duffy system ), Jk (ab-) (for the Kidd system), MN- (for the MNS system), Kk- (for the Kell system), Lu (ab-) (for the Lutheran system) and H-negative antigen (phenotype Bombay), or any combination thereof. In some embodiments, the erythroid cell is also Type O and / or Rh-. Smaller blood groups are described, for example, in Agarwal et al Blood group phenotype frequencies in blood donors from a tertiary care hospital in north India Blood Res. 2013 Mar; 48 (1): 51-54 and Mitra et al Blood groups systems Indian J Anaesth. 2014 set-out; 58 (5): 524-528, each of which is incorporated herein by reference in its entirety.
Compositions of erythroid cells
[0152] In some embodiments, the population of erythroid cells
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47/111 comprises at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% (and optionally up to about 80, 90 or 100%) cells enucleated erythroids. In some embodiments, the erythroid cell population contains less than 1% live nucleated cells, for example, it does not contain detectable live nucleated cells. Enucleation is measured, in some modalities, by FACS using a nuclear dye. In some modalities, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80% (and, optionally, up to about 70, 80, 90 or 100%) of erythroid cells in the population comprises one or more (e.g., 2, 3, 4 or more) of the agents. The presence of the agent is measured, in some embodiments, by FACS using a labeled protein (eg, antibody) that binds to the agent. In some modalities, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80% (and, optionally, up to about 70, 80, 90 or 100%) of erythroid cells in the population are enucleated and comprise one or more agents. In some embodiments, the erythroid cell population comprises about 1x10 ⁹ - 2x10 ⁹ , 2x10 ⁹ - 5x10 ⁹ , 5x10 ⁹ - 1x10 ¹⁰ , 1x10 ¹⁰ - 2x10 ¹⁰ , 2x10 ¹⁰ - 5x10 ¹⁰ , 5x10 ¹⁰ - 1x10 ¹¹ , 1x10 ¹¹ - 2x10 ¹¹ , 2x10 ¹¹ - 5x10 ¹¹ , 5x10 ¹¹ - 1x10 ¹² , 1x10 ¹² - 2x10 ¹² , 2x10 ¹² - 5x10 ¹² or 5x10 ¹² - 1x10 ¹³ cells.
Clickable shape cells
[0153] In some embodiments, the cell comprises a conjugating agent without requiring a chemical reaction step of the cell with the conjugating agent. For example, the cell can be contacted with a molecule that comprises a metabolite (for example, an amino acid or sugar) and a coupling portion (for example, a click handle). The cell is then left to incorporate the metabolite, for example, into proteins or carbohydrates, for example, on the cell surface. The metabolite can be, for example, a non-canonical amino acid. In modalities, the metabolite is incorporated using a
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48/111 RNAt / amino-acyl-RNAt-synthetase pair that directs the metabolite in a specific position, for example, one encoded by an Amber terminating codon. The resulting activated cell comprises the coupling portion (for example, click handle). The activated cell can then be contacted with an activated agent, for example, an activated agent described herein.
[0154] A variety of molecules comprising a metabolite and a coupling portion can be used. For example, in modalities, the molecule is chosen from:
NH
H ₂ N
HOOC
HC02H
SCO-Lysine, for example, for use in an alkaline azide cycloaddition reaction (SPAAC) promoted by tension;
A ^{H 9}
JX ... N
Y OH NH
Cyclopropene lysine, for example, for use in a Diels-Alder cicloaddition reaction promoted by electron-induced inverse voltage (SPIEDAC, from the strain-promoted inverse-electrondemand Diels-Alder cycloaddition);
Q>
)-The
NH
HOOC
TCO * A-Lysine, for example, for use in a SPIEDAC reaction;
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49/111 exo
H'
O ^ OH
Ό
O ^ 'N
H
Exo-BCN-Lysine, for example, for use in a SPAAC reaction;
β ...... 'I ^μ o .COOH
HCI
NBO-Lysine, for example, for use in a SPIEDAC reaction;
OH
Ν '’
H
YsfNH ₂ rac-BCN-Lysine, for example, for use in a SPAAC reaction;
NH h ₂ n
HOOC
TCO-Lysine, for example, for use in a SPIEDAC reaction;
I endo j
H ^X O
NH ₂ H
Endo-BCN-Lysine, for example, for use in a SPAAC reaction nh ₂ o ho ₂ c
HCI
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50/111
PrK-HCL-Sal, for example, for use in a SPAAC or SPIEDAC reaction;
COOH
N ₃ ^s > NH ₂
N3-Lysine, for example, for use in a SPAAC reaction or
SPIEDAC
p-acetylphenylalanine, for example, for use with a site-specific oxime bond (for example, mediated by DBCO-amine), followed by SPAAC p-azidomethylphenylalanine, for example, for use in a reaction
SPAAC or SPIEDAC; or
HO and SeH
NH _; .
seleno-cysteine, for example, for reaction with maleimide.
Coupling Reagents
[0155] Compositions of erythroid cells functionalized with an agent are described herein, in which the cells and the agent are
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51/111 linked through a set of bispecific coupling reagents. In some embodiments, a set of bispecific coupling reagents comprises a first coupling reagent and a second coupling reagent. The first coupling reagent comprises a coupling portion, for example, an alkaline portion, which reacts specifically with the coupling portion, for example, an azide, in the second coupling reagent. The coupling portions are not self-reacting. In embodiments, the coupling portion is a click handle. A click handle can include an azide or an alkaline.
[0156] Each coupling reagent also comprises a reactive portion of substrate, suitable, for example, for binding (for example, covalently) to a substrate of interest, for example, an erythroid cell, or an agent for binding to an erythroid cell , for example, a polypeptide, lipid, nucleic acid, sugar, drug, small molecule. In embodiments, the reactive portion of the substrate is able to react non-enzymatically with a substrate. In modalities, the reactive portion of the substrate is able to react with a different substrate in a sortase reaction.
[0157] In embodiments, a coupling reagent is capable of covalently linking a first entity (eg, a cell) with a second entity (eg, an exogenous polypeptide). [0158] The first coupling reagent can be linked through its reactive portion of the substrate to a first substrate, for example, an erythroid cell. The second coupling reagent can be linked through its reactive portion of the substrate to a second substrate, for example, a polypeptide or drug. The substrates thus derived can then be bonded together.
[0159] The bonding of the two substrates typically results in a residual binder between the first and the second substrate. For example, in
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52/111 In the case of coupling reagents comprising an azide and an alkali, a residual binder comprising a triazole (for example, a 1,2,3-triazole) can be formed. Exemplary coupling reagents include alkaline coupling reagents (KR) and azide coupling reagents (AR).
[0160] In some embodiments, the coupling reagent comprises an alkaline coupling reagent. In some embodiments, the alkaline coupling reagent comprises a propargyl moiety or a cyclooctyl moiety. Exemplary coupling reagents include (DBCO) -sulfo-NHS-diarylcyclooctine ester, (DBCO) -PEG-NHS-diarylcyclooctine ester, (DBCO) C6-NHS-diarylcyclooctine ester, (DBCO) NHS- diarylcyclooctin ester, (DBCO) -drylcyclooctine amine, (DBCO) -drylcyclooctin acid, (DBCO) -drylcyclo-octin sulphate chameleid, (DBCO) -drylcyclo-octine sulphamimide, bis-sulfone-PEG- diarylcyclooctine (DBCO), propargyl-NHS ester, propargyl-maleimide, alkaline-PEG-NHS ester, alkino-PEG-maleimide or its derivatives.
[0161] In some embodiments, the coupling reagent comprises an azide coupling reagent. In some embodiments, the azide coupling reagent comprises an azide-alkyl moiety, azide-aryl moiety, or an azidaheteroaryl moiety. Exemplary azide coupling reagents include sulfo-NHS ester of 3-azidopropionic acid, NHS ester of azidoacetic acid, azido-PEG-NHS ester, azidopropylamine, azido-PEGamine, azido-PEG-maleimide, bis-sulfone-PEG-azide, or their derivative. [0162] The coupling reagents may also comprise an alkene moiety, for example, a transcycloalkene moiety, an oxanorbornadiene moiety, or a tetrazine moiety. Additional coupling reagents can be found at Click Chemistry Tools (https://clickchemistrytools.com/), Lahann, J (ed) (2009) Click
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53/111
Chemistry for Biotechnology and Materials Science, McKay etal, Click chemistry in complex mixtures: bioorthogonal bioconjugation Chem Biol, September 2014 18; 21 (9): 1075-101, Becer et al. Click chemistry beyond metal-catalyzed cycloaddition Angew Chem Int Ed Engl. 2009; 48 (27): 4900-8., And Hein et al. Click chemistry, a powerful tool for pharmaceutical sciences Pharm Res. October 2008; 25 (10): 221630, each of which is incorporated herein by reference in its entirety.
[0163] In embodiments, the coupling reagent comprises a tetrazine moiety, for example, by reaction with an alkene radical. For example, in embodiments, the tetrazine is a 1,2,4,5-tetrazine and the alkene is a tensioned alkene. In embodiments, the alkene coupling reagent comprises a trans-cyclooctene, (E) -Cyclo-oct4-enol, (E) -Cyclo-oct-4-enyl 2,5-dioxo-1-pyrrolidinyl carbonate, ester succinimidyl of 5-Norbornene-2-acetic acid, 5-Norbornene-2endo-acetic acid, succinimidyl ester TCO PEG4, TCO-amine, or TCOPEG3-maleimide. In embodiments, the tetrazine coupling reagent comprises (4- (1,2,4,5-Tetrazin-3-yl) phenyl) methanamine or 2,5Dioxo-1-pyrrolidinyl 5- [4- (1,2,4 , 5-tetrazin-3-yl) benzylamino] -5oxopentanoate, 5- [4- (1,2,4,5-Tetrazin-3-yl) benzylamino] -5oxopentanoic acid. In modalities, tetrazine and alkene react in a Diels-Alder cycloaddition to obtain a stable covalent bond. In embodiments, a catalyst is not necessary. In modalities, the only by-product is dinitrogen. In modalities, the reaction of at least an order of magnitude faster than azide-cyclooctin based on click chemistry. Without wishing to be bound by theory, tetrazine / alkene reactions can be used with low concentrations of reagent (eg, the agent).
[0164] In some embodiments, the coupling portions of each coupling reagent react via a cycloaddition of azide
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54/111 alcino Huisgen. In some embodiments, a Huisgen azload alkaline cycloaddition comprises a copper (I) catalyzed azide alkali cycloaddition or a stress-promoted azide alkali cycloaddition. [0165] In some embodiments, the coupling portions of each coupling reagent react to form a heteroaryl, for example, a triazole. In some embodiments, triazole comprises a 1,2,3-triazole, for example, a 1,4-disubstituted 1,2,3-triazole or a 1,5-disubstituted 1,2,3-triazole.
[0166] In some embodiments, the coupling reagent comprises a reactive portion of substrate to bind the coupling reagent to an agent (for example, an agent described herein). In some embodiments, the reactive portion of the substrate reacts with a carbonyl group, an ester, a carboxylic acid, an amine or a sulfhydryl group. In some embodiments, the reactive portion of the substrate comprises a succinimide (for example, NHS-ter), a maleimide, an amine, a hydrazine, an alkoxyamine, a carboxylic acid, an aldehyde, a ketone, a disulfide, an acyl halide, an isothiocyanate or a derivative thereof.
[0167] In some embodiments, the reactive portion of the substrate comprises a binder. In some embodiments, the binder comprises a portion of polyethylene glycol (PEG). In some embodiments, the linker is a linear chain. In some embodiments, the linker is a branched chain.
[0168] In some embodiments, the coupling reagent comprises an alkali and reacts with an amine. In some embodiments, the coupling agent comprises a cyclooctin and reacts with an amine. In some embodiments, the coupling agent is diarylcyclooctyl ester (DBCO) -sulfo-NHS or diarylcyclooctyl ester (DBCO) -PEG5-NHS.
[0169] In some embodiments, the coupling agent
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55/111 comprises an azide and reacts with an amine. In some embodiments, the coupling agent is sulfo-NHS ester of 3azidopropionic acid or azido-PEG4-NHS ester.
[0170] In one embodiment, the coupling reagent is soluble in water. In one embodiment, the coupling reagent is impermeable to the membrane, for example, it has sufficient charge to make it impermeable to the membrane. In one embodiment, the coupling reagent is charged, for example, positively charged or negatively charged. In one embodiment, the coupling reagent comprises a cationic portion or an anionic portion, for example, an SO3 portion.
[0171] In some embodiments, the coupling agent comprises a detection agent, for example, useful for the detection of the functionalized erythroid cell. Exemplary detection agents can include a fluorescent molecule (for example, a cyanine dye, for example, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 or Cy7.5), a metal chelate, a contrast agent, a radionuclide, a positron emission tomography (PET) imaging agent, an infrared imaging agent, an infrared imaging agent, a computer aided tomography imaging agent (CAT), a computerized tomography imaging agent photon emission (for example, DIBO-DFO, where DFO chelates Zirconium-89), an X-ray imaging agent or a magnetic resonance imaging (MRI) agent.
[0172] In one embodiment, a coupling reagent is a GMP grade material.
[0173] In some embodiments, the click reaction is a cycloaddition (for example, a 1,3-dipolar cycloaddition or hetero-Diels-Alder cycloaddition), nucleophilic ring opening (for example, tensioned heterocyclic electrophile openings, such as like aziridines,
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56/111 epoxides, cyclic sulfates, aziridinium and episulfonium ions), carbonyl chemistry of the non-aldolic type (eg formation of ureas, thioureas, hydrazones, oxime ethers, amides or aromatic heterocycles) or an addition to a multiple bond of carbo no-carbo no (for example, epoxidation, aziridination, dihydroxylation, addition of sulfenyl halide, addition of nitosyl halide or addition of Michael). Examples of these types of click reactions are described in more detail in Hein et al., Pharm. October 2008; 25 (10): 2216-2230, which is incorporated herein by reference in its entirety. In modalities, the click reaction is a metal-free cycloaddition reaction [3 + 2], Diels-Alder reaction, or radical thiol-alkene reaction. Examples of these types of click reactions are described in more detail in Becer et al., Angew. Chem. Int. Ed. 2009, 48, 4900-4908, which is incorporated herein by reference in its entirety.
[0174] In one embodiment, the click signature is an alkaline / azide click signature (for example, the alkaline is a cyclooctin, activated alkaline or electron deficient alkaline), for example, the click signature comprises a triazole, for example, 1,2; 3-triazole and / or a disubstituted triazole. In one embodiment, the click signature is a diene / dienophile click signature (for example, where the dienophile comprises an alken portion), for example, the click signature comprises a cycloalkene, for example, a disubstituted alkene. In embodiments, the click signature is a tetrazine / alkene click signature, for example, the click signature comprises a dihydropyrazine, for example, a 1,2-dihydropyrazine. In modalities, the click signature is a tetrazole / alkene click signature, for example, the click signature comprises a diazole. In modalities, the click signature is a dithioester / diene click signature, for example, the click signature comprises a ring containing sulfur, for example, a tetra
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57/111 hydrothiophene, for example, a disubstituted tetrahydrothiophene. In embodiments, the click signature is a dithioester / diene signature, for example, the click signature comprises a ring containing sulfur, for example, a thiopyran. In modalities, the click signature is a thiol / alkene click signature, for example, the click signature comprises an alkyl sulfide.
[0175] In modalities, the click reaction does not require a catalyst. In modalities, the click signature does not require copper ions, for example, it takes place at substantially the same rate in the absence of copper ions, as in the presence of copper ions, for example, under conditions described in Tornoe, CW et al (2002 ). Peptidotriazoles on Solid Phase: [1,2,3] -Triazoles by Regiospecific Copper (l) -Catalyzed 1,3-Dipolar Cycloadditions of Terminal Alkynes to Azides. In modalities, the click signature proceeds efficiently at a temperature of about 10-40, 20-40, 20-30, 2025, 30-40 or 35-40 or about 37 ^Q C. In modalities, the click reaction proceeds efficiently at a temperature below 50, 45, 40, 35, 30, 25 or 20 ^Q C.
[0176] In modalities, the activation barrier for a click reaction is 24-30, 25-29 or 26-28 kcal / mol, for example, about 27.8 kcal / mol or 26 kcal / mol. In modalities, the activation barrier for a click reaction is the same as or not less than 50%, 40%, 30%, 20%, or 10%, different from the activation barrier of a Huisgen cycloaddition reaction catalyzed by Cu between an azide and a terminal alkene, for example, as described in Hein etal. Click chemistry, a powerful tool for pharmaceutical sciences Pharm Res. 2008 Oct; 25 (10): 2216-30.
[0177] In modalities, the click reaction is exergonic, for example, it has an AG ° of between -10 and -100, -20 and -90, -30 and -70, -40 and 70, -50 and -60 , or about - 61 kcal / mol. In modalities, AG ° for a
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58/111 click reaction is equal to or less than 50%, 40%, 30%, 20% or 10%, different from AG ° of a Huisgen cycloaddition reaction catalyzed by Cu between an azide and a terminal alkene.
[0178] In modalities, the click reaction has AG ° between -30 and -140, -40 and -130, -50 and -120, -60 and -110, -70 and -100, -80 and -90 , or about 84 kJ / mol.
[0179] An example of a cycloaddition reaction is the Huisgen 1,3-dipolar cycloaddition of a dipolarophile with a 1,3-dipolar component that produces (hetero) five-membered cycles. Examples of dipolarophils are alkenes, alkynes and molecules that have related functional groups of heteroatom, such as carbonyls and nitriles. Specifically, another example is the cycloaddition 2 + 3 of alkyl azides and acetylenes. Other cycloaddition reactions include DielsAlder reactions from a conjugated diene and a dienophile (such as, an alkaline or alkene). Examples of cycloaddition reactions are described, for example, in Pat. US 9,517,291, which is incorporated herein by reference in its entirety.
[0180] Other examples of click reactions include a H-Si hydrosylation reaction and simple non-activated vinyl compounds, formation of urethane from alcohols and isocyanates, Menshutkin reactions of tertiary amines with alkyl iodides or alkyl trifluoromethanesulfonates, additions Michael, for example, very efficient maleimide-thiol, radical addition reactions of transferring atoms between --SO2CI and an olefin (R ¹ , R ² —C = C — R ³ , R ⁴ ), metathesis, Staudinger reaction of phosphines with alkyl azides, oxidative coupling of thiols, nucleophilic substitution, especially of small tensioned rings such as epoxy and aziridine compounds, carbonyl chemistry such as urea formation and addition reactions to carbo no-carbo double bonds as di-idroxylation. Therefore, the attached functionality can be chosen from acetylene bonding,
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59/111 an azido group, a nitrile group, acetylenic group, amine group, phosphine group. The chemical click reaction can result in the addition of a functional group selected from amine, primary amine, hydroxyl, sulfonate, benzotriazole, bromide, chloride, chloroformate, trimethylsilane, phosphonium bromide or bio-responsive functional group, including polypeptides, proteins and nucleic acids.
[0181] Thus, suitable coupling reagents can comprise, for example, an amine, sulfate, thiol, hydroxyl, azide, alkaline, alkene, carboxyl groups, aldehyde groups, sulfone groups, vinyl sulfone groups, isocyanate groups, acid anhydride groups, epoxide groups, aziridine groups, episulfide groups, groups, such as -CO ₂ N (COCH ₂ ) 2, -CO ₂ N (COCH ₂ ) _2j -CO ₂ H, -CHO, -CHOCH _2j N.dbd.C.dbd .O, -SO ₂ CH.dbd.CH ₂ , -N (COCH) ₂ , -SS- (C ₅ H ₄ N) and groups of the following structures where X is halogen and R is hydrogen or C 1 to C ₄ alkyl :
[0182] In some embodiments, a click reaction forms very energy-efficient carbon-heteroatom bonds, in particular a ring-opening nucleophilic reaction or a cycloaddition reaction. One type of reaction that is widely represented in click chemistry is the aforementioned cyclo-loading of alkaline azide catalyzed with Cu (I). Examples of click reactions are also described, for example, in Pat. US 9,453,843, which is incorporated herein by reference in its entirety.
[0183] Click chemistry can generate substances quickly and reliably by joining small modular units (see, for example, Kolb et al. (2001) Angewandte Chemie Intl. Ed. 40: 2004-2011; Evans
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60/111 (2007) Australian J. Chem. 60: 384-395; Carlmark et al. (2009) Chem. Soc. Rev. 38: 352-362; each incorporated herein by reference in its entirety). Examples of click chemistry are described, for example, in 8,912,323, which is incorporated herein by reference in its entirety. Agents, for example, Exogenous Polypeptide Agents
[0184] The present invention features compositions of erythroid cells functionalized with an agent, and methods, preparations and kits comprising them. Exemplary agents for use in the invention are described herein.
[0185] In one embodiment, the agent is an agent described in WO2015 / 15302; or WO2015 / 073587, each of which is incorporated herein by reference in its entirety.
[0186] In one embodiment, the agent comprises a peptide agent, for example, a polypeptide, an enzyme or an antibody. [0187] In one embodiment, the agent comprises an exogenous polypeptide, for example, a polypeptide that is not produced by a wild-type cell of that type or is present at a lower level in a wild-type cell than in a cell containing the exogenous polypeptide. In some embodiments, an exogenous polypeptide is a polypeptide conjugated to the cell surface by chemical or enzymatic means. In some embodiments, an exogenous polypeptide is a polypeptide encoded by a nucleic acid that has been introduced into the cell, which acid, optionally, is not retained by the cell.
[0188] In one embodiment, the agent comprises a cytokine, a receptor, a ligand, a hormone, a growth factor, a blood factor, a lysosomal storage enzyme, asparaginase or a fragment of any of the foregoing comprising an extracellular domain , backlink domain, or other biologically active domain.
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[0189] In one embodiment, the agent comprises an antigen, for example, a tumor antigen and antigen of infectious disease and autoantigen.
[0190] In one embodiment, the agent comprises a lipid, nucleic acid, for example, RNA, DNA, siRNA, sugar, drug or small molecule.
[0191] In one embodiment, the agent comprises a polypeptide of more than about 30, 50, 75, 100, 150, 200, 250, 300, 350 or 400 kilodaltons.
[0192] In one embodiment, the agent, for example, a polypeptide, comprises the post-translational modification, for example, a post-translational modification that is not made by erythroid cells, or done inefficiently by erythroid cells.
[0193] In one embodiment, the agent, for example, a polypeptide, is toxic to, or compromises, the growth, function, life span, or development of an erythroid cell.
[0194] In one embodiment, the agent comprises a multimeric polypeptide, for example, a dimer, for example, a homodimer or heterodimer, a trimer, for example, a homotrimer or heterotrimer, or a tetramer, for example, a homotetramer or heterotetramer, for example, an antibody or cell surface receptor, for example, a receptor for a disease vector, for example, a virus, a drug or a toxin.
[0195] In one embodiment, the agent comprises a polypeptide, for example, a multimeric polypeptide, comprising a plurality of cysteine bridges. In one embodiment, the agent comprises a polypeptide, for example, a multimeric polypeptide, comprising one or more cysteine bridges.
[0196] In one embodiment, the agent comprises a protein that is difficult to express. For example, in modalities, the agent
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62/111 polypeptide has an amino acid sequence that, if expressed in a genetically erythroid cell, would reach a copy number of less than 1,000, 500, 200, or 100 copies of the protein per cell. In embodiments, the polypeptide agent has an amino acid sequence that, if expressed genetically in an erythroid cell, has ineffective translation or transcription, for example, reaching a protein or mRNA level of less than 50%, 40%, 30% , 20%, 10%, 5%, 2%, 1% of a reference value, for example, expression of a control protein, such as ADA as described in WO2015 / 073587. In one embodiment, the polypeptide agent comprises an isoform, for example, a splice variant, which is expressed genetically in an erythroid cell, would not be the most abundant isoform, for example, it would be present at a level of at least 10 %, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% below the level of the most abundant isoform.
[0197] In one embodiment, the agent comprises an antibody molecule, for example, a polypeptide comprising one or more of the following: a) variable region sufficient to bind the cognate antigen, for example, HC CDR1, HC CDR2 and HC CDR3, LC CDR1, LC CDR2 and LC CDR3; b) a heavy chain constant sequence comprising one or more of CH1, CH2 and CH3; c) a functional Fc region; and d) a modified or inactive Fc region, for example, a mutationally inactivated Fc region or an Fc region that has a state of glycosylation that impairs Fc activity, for example, a deglycosylated Fc region.
[0198] In one embodiment, the agent comprises an antibody, for example, IgA, IgG, IgG1, IgG2, IgM, IgEor IgD.
[0199] In one embodiment, the agent comprises an anti-PDL1 antibody, an anti-4-1BB antibody, anti-a4p7 antibody or protein A / G.
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[0200] In some embodiments, the peptide is deglycosylated. For example, a glycosylated precursor can be treated with a deglycosylating enzyme (for example, EndoS) to produce an agent.
[0201] In some embodiments, the agent comprises a polypeptide selected from or derived from one or more of the following classes, including but not limited to: an enzyme, a protease, a nuclease, a glycosidase, a lipase, a DNAse, an antigen , an antibody-like molecule (for example, a nanobody, scFv, duibody, or multispecific antibody), an antibody linker, a growth factor, a transporter, a cytokine, a chemokine, a growth factor receptor, a receptor cytokine, a chemokine receptor, an enzyme recognition sequence, a transpeptidase recognition sequence, a protease recognition sequence, a cleavable domain, an intein, a DNA-binding protein, an RNA-binding protein, a complement regulatory molecule, a complement cascade molecule, a coagulation cascade molecule, a chelator, a complement regulatory domain, an SCR domain, a CCP domain, an im unoglobulin or an immunoglobulin-like domain, an armadillo repeat, a leucine lock, an effector of death domain, a cadherin repeat, an EF hand, a phosphotyrosine binding domain, a plecstrin homology domain, a SCR 2 homology, a zinc finger domain, a cyclic peptide, a cell penetrating peptide, a chaperone molecule, an integrin, a collagen, a carrier protein (eg albumin), a peptide binding toxin (eg a peptide that binds to a bacterial toxin, a parasite, a fungus or the environment), a myelination molecule, a prion protein binding molecule, a differentiation molecule (CD), an immunomodulatory molecule, a co-stimulatory molecule , an activator
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64/111 of a co-stimulatory molecule, an inhibitor of a co-stimulatory molecule, a co-inhibiting molecule, an inhibitor of a co-inhibiting molecule or an activator of a co-inhibiting molecule), a cancer antigen or cancer cell marker, an antigen presenting molecule , a pro-apoptotic molecule, a targeting fraction, an Fc receptor binding molecule, a tumor-depriving enzyme, a DNA damage inhibitor, a cell cycle inhibitor or an epitope marker. Specific examples of agents, for example, polypeptides, are found, for example, in WO2015 / 073587, W02015 / 153102 and WO2016 / 183482, each of which is incorporated by reference in its entirety.
[0202] In some embodiments, an agent comprises one or more non-canonical amino acids. Non-canonical amino acids include, for example, p-methoxyphenylalanine (pMpa); p-acetylphenylalanine (pApa); pbenzoylphenylalanine (pBpa); p-iodophenylalanine (kite); p-azidophenylalanine (pAzpa); p-propargyloxyphenylalanine (pPpa); α-aminocaprylic acid; onitrobenzylcysteine (o-NBC); 1,5-dansylalanine; and o-nitrobenzylserine (oNBS), and others described, for example, in Pat. US 9,624,485, which is incorporated herein by reference in its entirety.
[0203] In some embodiments, the agent is different from a polypeptide. For example, the agent can be a carbohydrate, a small molecule, a lipid, a nucleic acid, a therapeutic agent, a natural or synthetic compound, or combinations thereof.
[0204] An exemplary exogenous polypeptide, for example, a polypeptide agent from Table 1 or a variant thereof, includes:
a) a naturally occurring form of the polypeptide;
b) the polypeptide having a sequence appearing in a database, for example, GenBank database, on August 7, 2017;
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c) a polypeptide having a sequence that does not differ by more than 1, 2, 3, 4, 5 or 10 amino acid residues from a sequence of a) or b);
d) a polypeptide having a sequence that differs from no more than 1,2, 3, 4, 5 or 10% of its amino acid residues from a sequence of a) or b);
e) a polypeptide having a sequence that does not differ substantially from a sequence of a) or b); or
f) a polypeptide with a sequence of c), d) or e) that does not differ substantially in a biological activity, for example, an enzymatic activity (for example, specificity or renewal) or binding activity (for example, specificity or affinity ligation) from a protein with the sequence of a) or b).
[0205] In embodiments, the polypeptide comprises a polypeptide or a fragment thereof, for example, all or a fragment of a polypeptide from a), b), c), d), e) or f) of the previous paragraph.
[0206] In embodiments, the agent comprises a polypeptide from Table 1, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity, or a functional fragment thereof.
Table 1. Amino acid sequences of exemplary agents
Agent Sequence 4-1BBL ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFA QLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKA GVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAQQQGFRQLGHQLQGLQFGQLQQ Anti-CD20 Heavy chain of chimeric rituximab:QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
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LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK (SEQ ID NO: 4)Rituximab chimeric light chain: QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKP WIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQW TSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 5) TRAIL TRAIL soluble variant DR4-1MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQM QDKYSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLV RKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRRRSN TLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKG FYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSAR NSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEAS FFGAFLVG (SEQ ID NO: 6)Soluble TRAIL variant DR4-2 MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQM QDKYSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLV RKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSN TLSSPNSKNEKALGRKINSWESSRRGHSFLSNLHLRNGELVIHEKG FYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSAR NSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEAS FFGAFLVG (SEQ ID NO: 7)Soluble TRAIL variant DR4-3 MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQM QDKYSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLV RKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRRRSN TLSSPNSKNEKALGIKINSWESSRRGHSFLSNLHLRNGELVIHEKG FYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTDYPDPILLMKSAR NSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEAS
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WED 11
FFGAFLVG (SEQ ID NO: 8)TRAIL soluble variant DR5-1MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQM QDKYSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLV RKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSN TLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKG FYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSAR NSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMHHEAS FFGAFLVG (SEQ ID NO: 9)Soluble TRAIL variant DR5-2 MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQM QDKYSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLV RKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSN TLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKG FYYIYSQTYFRFQERIKENTKNDKQMVQYIYKYTSYPDPILLMKSAR NSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMHHEAS FFGAFLVG (SEQ ID NO: 10) Anti-PD-L1 scFv VQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGL EWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAED TAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGG SIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPK LLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYL YHPATFGQGTKVEIK (SEQ ID NO: 11) PAL MKTLSQAQSKTSSQQFSFTGNSSANVIIGNQKLTINDVARVARNGT LVSLTNNTDILQGIQASCDYINNAVESGEPIYGVTSGFGGMANVAIS REQASELQTNLVWFLKTGAGNKLPLADVRAAMLLRANSHMRGAS GIRLELIKRMEIFLNAGVTPYVYEFGSIGASGDLVPLSYITGSLIGLD PSFKVDFNGKEMDAPTALRQLNLSPLTLLPKEGLAMMNGTSVMTG IAANCVYDTQILTAIAMGVHALDIQALNGTNQSFHPFIHNSKPHPGQ LWAADQMISLLANSQLVRDELDGKHDYRDHELIQDRYSLRCLPQY LGPIVDGISQIAKQIEIEINSVTDNPLIDVDNQASYHGGNFLGQYVG MGMDHLRYYIGLLAKHLDVQIALLASPEFSNGLPPSLLGNRERKVN MGLKGLQICGNSIMPLLTFYGNSIADRFPTHAEQFNQNINSQGYTS ATLARRSVDIFQNYVAIALMFGVQAVDLRTYKKTGHYDARACLSPA TERLYSAVRHVVGQKPTSDRPYIWNDNEQGLDEHIARISADIAAGG VIVQAVQDILPCLH (SEQ ID NO: 12)
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Y vbAsparaginase MADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEV KKLANVKGEQFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVI THGTDTVEESAYFLHLTVKSDKPVVFVAAMRPATAISADGPMNLLE AVRVAGDKQSRGRGVMVVLNDRIGSARYITKTNASTLDTFKANEE GYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDD PEYLYDAAIQHG VKGIVYAG MG AGS VS VRGIAG MRKAM EKG VV VI RSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQ EYFHTY (SEQ ID NO: 13) Anti-a4b7 Variable heavy chain region:QVQLVQSGAEVKKPGASVKVSCKGSGYTFTSYWMHWVRQAPGQ RLEWIGEIDPSESNTNYNQKFKGRVTLTVDISASTAYMELSSLRSE DTAVYYCARGGYDGWDYAIDYW GQQTLVTVDWMTQSPLSLPVTPGEPASISCRSSQSLAKSYGNTYLSWYLQKP GQSPQLLIYGISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVY YCLQGTHQPYTFGQGTKVEIK (SEQ ID NO: 15) IL10Human SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLD NLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAH VNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKNY Coagulation factor X ANSFLEEMKKGHLERECMEETCSYEEAREVFEDSDKTNEFWNKY KDGDQCETSPCQNQGKCKDGLGEYTCTCLEGFEGKNCELFTRKL CSLDNGDCDQFCHEEQNSVVCSCARGYTLADNGKACIPTGPYPC GKQTLERRKRSVAQATSSSGEAPDSITWKPYDAADLDPTENPFDL LDFNQTQPERGDNNLTRIVGGQECKDGECPWQALLINEENEGFC GGTILSEFYILTAAHCLYQAKRFKVRVGDRNTEQEEGGEAVHEVEV VIKHNRFTKETYDFDIAVLRLKTPITFRMNVAPACLPERDWAESTLM TQKTGIVSGFGRTHEKGRQSTRLKMLEVPYVDRNSCKLSSSFHTQ NMFCAGYDTKQEDACQGDSGGPHVTRFKDTYFVTGIVSWGEGCA RKGKYGIYTKVTAFLKWIDRSMKTRGLPKAKSHAPEVITSSPLK (SEQ ID NO: 17)
[0207] In some embodiments, an exogenous polypeptide described herein is at least 200, 300, 400, 500, 600, 700 or 800 amino acids in length. In some embodiments, the exogenous polypeptide is between 200-300, 300-400, 400-500, 500-600, 600-700 or
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700-800 amino acids in length.
[0208] In some embodiments, an erythroid cell, for example, an enucleated erythroid cell, comprises at least 1,000, 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 50,000, 100,000, 200,000, or 500,000 copies of an exogenous polypeptide described here , for example, from Table 1.
[0209] In modalities, the agent comprises one or more post-translation modifications. Post-translation modifications include cleavage (for example, proteolytic cleavage), cyclization, glycosylation, phosphorylation, conjugation to a cofactor (for example, lipylation, flavin fraction (for example, FMN or FAD), heme C binding, phospopantetainylation or base formation retinylidene Schiff), diphenamide formation, ethanolamine phosphoglycerol fixation, hypusin formation, acylation (eg, O-acylation, N-acylation or Sacylation), formylation, acetylation, alkylation (for example, methylation or ethylation), amidation, butyrylation, gamma-carboxylation, malonylation, hydroxylation, iodination, addition of nucleotides, such as ADP ribosylation, oxidation, phosphate ester (linked to O) or phosphoramidate (linked to N), (for example, phosphorylation or adenylation), for propylation, pyroglutamate formation, S-glutathylation, S-nitrosylation, succinylation, sulfation, ISGylation, SUMOylation, ubiquitination, Nidilation or a chemical modification of an amino acid (eg, citrullination, deamidation, el imination or carbamylation), formation of a disulfide bridge, racemization (for example, of proline, serine, alanine or methionine) or any combination thereof. In modalities, glycosylation includes the addition of a glycosyl group for arginine, asparagine, cysteine, hydroxylisin, serine, threonine, tyrosine or tryptophan, resulting in a glycoprotein. In embodiments, glycosylation comprises, for example, O-linked glycosylation or N-linked glycosylation.
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[0210] In embodiments, the cell comprises a plurality of agents, for example, at least 10, 20, 50, 100, 200, 500, or 1,000 different agents. In some embodiments, the plurality of agents comprises a plurality of vaccine antigens. In some embodiments, the plurality of agents have sequence similarity to each other, but can vary between each other by at least 1,2, 5, 10, 20, 50, or 100 amino acid positions. In some embodiments, each agent in the plurality has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with each other in the plurality. In some embodiments, each agent in plurality has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with at least 1,2, 5, 10, 20, 50 or 100 other agents in the plurality. Geometry of coupling reagents in agents
[0211] A coupling agent can be attached to a variety of different positions in an agent. A coupling agent may comprise a reactive portion of substrate suitable, for example, for attachment (for example, covalently) to a substrate, such as an agent (for example, polypeptide). A coupling agent may further comprise a coupling portion, for example, a click coupling portion suitable, for example, for attachment (e.g., covalently) to a second coupling agent. A suitable reactive portion of substrate can be chosen to direct the binding of the coupling agent to the agent (e.g., polypeptide).
[0212] For example, in some embodiments, the reaction portion of the substrate is able to react with an NH ₂ group, for example, in a side chain of lysine or at the N-terminus of the agent. An example of a substrate reaction portion capable of reacting with an NH ₂ group is an NHS ester, Imidoester, Pentafluorophenyl ester or Hydroxymethyl phosphine. In some embodiments, the reaction portion of the
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71/111 substrate is able to react with a carboxyl of the agent, for example, in a side chain of aspartic acid or glutamic acid or the C-terminus of the agent. An example of a substrate reaction portion capable of reacting with a carboxyl is a carbodiimide. In some embodiments, the reaction portion of the substrate is capable of reacting with a sulfhydryl of the agent, for example, in a cysteine side chain. An example of a substrate reaction unit capable of reacting with a sulfhydryl is Maleimide, Haloacetyl (e.g., Bromo- or sludge), Pyridyl-disulfide, Thiosulfonate or Vinylsulfone. An agent having a disulfide bridge can be placed under reducing conditions to convert the disulfide bridge to sulfhydryl. In some embodiments, the reaction portion of the substrate is capable of reacting with a carbonyl of the agent. For example, a ketone or aldehyde group can be created in glycoproteins, for example, by oxidizing the post-translational modifications of the polysaccharides, for example, with sodium metaperiodate. An example of a substrate reaction portion capable of reacting with a carbonyl group is a hydrazide or alkoxyamine.
[0213] A portion of an agent can be attached to a pre-selected portion in a cell. For example, in some embodiments, an NH ₂ group in the agent is attached to an NH ₂ , carboxyl, sulfhydryl or carbonyl group in the cell. In embodiments, a carboxyl group in the agent is attached to an NH ₂ , carboxyl, sulfhydryl or carbonyl group in the cell. In embodiments, a sulfhydryl group in the agent is attached to an NH ₂ , carboxyl, sulfhydryl or carbonyl group in the cell. In embodiments, a carbonyl group in the agent is attached to an NH ₂ , carboxyl, sulfhydryl or carbonyl group in the cell.
Clickable format agents
[0214] In some embodiments, the agent is a polypeptide with a coupling agent attached to a predetermined amino acid.
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Briefly, the polypeptide agent can be produced in a first cell (a factory cell, such that a non-canonical amino acid (ncAA) comprising a first coupling reagent is present at one or more amino acid positions of the polypeptide agent. then be coupled to a second cell that has a second coupling reagent on the cell surface, thereby coupling the polypeptide agent to the second cell. This technology can use the ncAA site specific incorporation, such that the orientation of the agent in the cell surface is controlled.
[0215] For example, ncAAs can be introduced into the polypeptide agent by genetically incorporating an Amber termination codon (TAG) at the site of interest into a nucleic acid encoding the polypeptide agent, for example, as described in Nikic etal. Nature Protocols 2015, which is incorporated herein by reference in its entirety. The Amber termination codon can be incorporated, for example, at the N-terminus, C-terminus, or inside the protein. This plasmid encoding the polypeptide agent can be co-transfected into a factory cell (e.g., HEK293or a CHO cell) with another plasmid encoding an RNAt / aminoacyl-RNAt synthase pair that is orthogonal to the host's translation machinery. In the presence of ncAA, there is a reading of the Amber codon and incorporation of ncAA. For example, M. maze / 7RNAt RNAt-aminoacyl pyrrolysin synthetase (PyIRS / tRNAPyl) can be used with the Y306A and Y384F mutations and the ncAAs: cyclooctyl-lysine (SCO), Endo Biciclo [6.1.0] nonin-lysine (endoBCN), Exo Biciclo [6.1.0] nonine-lysine (exoBCN) or Rac Biciclo [6.1.0] nonine-lysine (racBCN). Other suitable ncAAs are described in the section here entitled Clickable shape cells. The plasmid can also encode a sequence that directs the secretion of the protein. Transient transfection or a stable cell line can be used.
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[0216] Once the clickable-shaped protein is produced, it can react with a cell that has a second coupling agent. For example, the cell can be an erythroid cell having a click linker, for example, a click linker comprising an azide. Geometry of coupling reagents in cells
[0217] A coupling agent can be attached to a variety of different positions in a cell. A coupling agent may comprise a reactive portion of substrate suitable, for example, for attachment (e.g., covalently) to a substrate such as a cell (for example, a polypeptide or carbohydrate portion in the cell). A coupling agent may further comprise a coupling portion, for example, a click coupling portion suitable, for example, for attachment (e.g., covalently) to a second coupling agent. A suitable reactive substrate portion can be chosen to direct the binding of the coupling agent to the cell.
[0218] Suitable substrate reaction portions are described here, for example, in the section entitled Geometry of coupling reagents in agents.
[0219] A portion in a cell can be linked to a pre-selected portion in an agent. For example, in some embodiments, an NH ₂ group in the cell is attached to an NH ₂ , carboxyl, sulfhydryl or carbonyl group of the agent. In embodiments, a carboxyl group in the cell is attached to an NH ₂ , carboxyl, sulfhydryl or carbonyl group of the agent. In embodiments, a sulfhydryl group on the cell is linked to an NH ₂ , carboxyl, sulfhydryl or carbonyl group of the agent. In embodiments, a carbonyl group in the cell is attached to an NH ₂ , carboxyl, sulfhydryl or carbonyl group of the agent.
Sortase and click
[0220] In some embodiments, a transpeptidase reaction, such as
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74/111 as a sortase reaction is used to attach a binding element to an agent or a cell.
[0221] For example, in some embodiments, a polypeptide agent comprises a transpeptidase recognition sequence, for example, a sortase recognition sequence. The polypeptide agent can be brought into contact with a sortase and a coupling reagent that has a compatible sortase recognition sequence, thus sorting the coupling reagent into the polypeptide agent. The polypeptide agent can then react with a cell with a second coupling agent. The second coupling agent can be, for example, in an NH ₂ group, carboxyl, sulfhydryl or carbonyl of the cell.
[0222] In some embodiments, a cell (for example, a polypeptide or carbohydrate in the cell) comprises a transpeptidase recognition sequence, for example, a sortase recognition sequence. For example, the cell can genetically express a transmembrane protein comprising a sortase recognition sequence on the cell's surface. The cell can be brought into contact with a sortase and a coupling reagent that has a compatible sortase recognition sequence, thus sorting the coupling reagent in the cell. The cell can then be reacted with a polypeptide agent having a second coupling agent. The second coupling agent can be, for example, in an NH ₂ , carboxyl, sulfhydryl or carbonyl group of the polypeptide agent.
[0223] In some embodiments, the polypeptide agent does not comprise a transpeptidase recognition sequence, for example, a sortase recognition sequence. In embodiments, the cell does not comprise an exogenous transpeptidase recognition sequence, for example, an
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75/111 sortase recognition sequence. In modalities, the method does not include a sortagging step. In modalities, the functionalized erythroid cell does not include a sortase transfer signature.
[0224] A sortase can conjugate two reasons for the recognition of sortase enzymatically. The first sortase recognition motive can be a sortase donor motif and the second sortase recognition motif can be a sortase acceptor motif.
[0225] Reasons for sortase recognition include LPXTA (SEQ ID NO: 18) and LPXTG (SEQ ID NO: 1), where X is any amino acid residue. An example of a sortase recognition motif is LPXTG (SEQ ID NO: 1), where X can be any amino acid residue (natural or non-canonical), for example, any of the 20 standard amino acids most commonly found in proteins found in living organisms. In some examples, the reason for recognition is LPXTG (SEQ ID NO: 19) or LPXT, where XD, E, A, N, Q, K or R. In other examples, X is selected from K, E, N, Q or A in an LPXTG motif (SEQ ID NO: 20) or LPXT, which are recognizable by a sortase A. In other examples, X is selected from K, S, E, L, A, or N in an LPXTG motif ( SEQ ID NO: 21) or LPXT, which are recognizable by a class C of sortase. Exemplary reasons for sortase recognition include, but are not limited to, LPKTG (SEQ ID NO: 22), LPITG (SEQ ID NO: 23), LPDTA (SEQ ID NO: 24), SPKTG (SEQ ID NO: 25), LAETG (SEQ ID NO: 26), LAATG (SEQ ID NO: 27), LAHTG (SEQ ID NO: 28), LASTG (SEQ ID NO: 29), LPLTG (SEQ ID NO: 30), LSRTG (SEQ ID NO : 31), LPETG (SEQ ID NO: 32), VPDTG (SEQ ID NO: 33), IPQTG (SEQ ID NO: 34), YPRRG (SEQ ID NO: 35), LPMTG (SEQ ID NO: 36), LAFTG (SEQ ID NO: 37), LPQTS (SEQ ID NO: 38), LPXT, LAXT, LPXA, LGXT, IPXT, NPXT, NPQS (SEQ ID NO: 39), LPST (SEQ ID NO: 40), NSKT (SEQ ID
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NO: 41), NPQT (SEQ ID NO: 42), NAKT (SEQ ID NO: 43), LPIT (SEQ ID NO: 44), LAET (SEQ ID NO: 45), LPXAG (SEQ ID NO: 46), LPNAG (SEQ ID NO: 47), LPXTA (SEQ ID NO: 18), LPNTA (SEQ ID NO: 48), LGXTG (SEQ ID NO: 49), LGATG (SEQ ID NO: 50), IPXTG (SEQ ID NO : 51), IPNTG (SEQ ID NO: 52), IPETG (SEQ ID NO: 53), NPXTX, NP [Q / K] - [T / s] - [N / G / s], NPQTN (SEQ ID NO : 54), NPKTG (SEQ ID NO: 55), NSKTA (SEQ ID NO: 56), NPQTG (SEQ ID NO: 57), NAKTN (SEQ ID NO: 58), NPQSS (SEQ ID NO: 59), NA - [E / A / S / H] -TG (SEQ ID NO: 60), LAXTG (SEQ ID NO: 61), QVPTGV (SEQ ID NO: 62), LPXTX, LP [Q / K] T [A / S] T (SEQ ID NO: 63), or LPXT [A / S].
[0226] Sortase acceptor motifs include oligoglycines or oligoalanines, such as a 1-5-glycine fragment or a 1-5-alanine fragment. In some examples, oligoglycine consists of 3 or 5 glycine residues. In other examples, oligoalanine consists of 3 or 5 alanine residues.
[0227] A sortase transfer subscription can be created by a sortase reaction. For example, an LPXTGG sortase-mediated reaction (SEQ ID NO: 2) with (G) _n can produce an LPXT (G) _n sortase transfer signature (SEQ ID NO: 1). In modalities, the sortase transfer subscription comprises a sequence of a sortase recognition motive described here, for example, in this section. In embodiments, the sortase transfer signature further comprises one or more alanine or glycine amino acids, for example, at the C-terminus of the sortase recognition motif sequence.
[0228] A variety of sortases are described, for example, in WQ2014 / 183071 (for example, on pages 33-37 of the same document), the order of which is incorporated herein by reference in its entirety.
Second agents added by various methods
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[0229] In some embodiments, a cell (e.g., enucleated erythroid cell) described herein comprises (in addition to its first agent) a second agent, for example, an exogenous polypeptide agent. In some embodiments, the second agent is conjugated to the cell, for example, using click chemistry. In some embodiments, the second agent comprises a protein expressed from an exogenous nucleic acid (for example, DNA or RNA) introduced into the cell or its precursor. In some embodiments, the second agent comprises a sortagged protein in the cell. In some embodiments, the second agent is loaded hypothetically into the cell. In some embodiments, the second agent is not covalently linked to a click signature or a residual binder.
Non-conjugation methods for adding an agent to a cell [0230] Although in many embodiments, an agent is conjugated to a cell, for example, using click chemistry, it is understood that any agent described here can also be added to a cell using a variety of methods. Accordingly, in some respects, the present disclosure provides a cell (for example, an erythroid cell, for example, an enucleated erythroid cell) comprising an agent, for example, exogenous polypeptide agent, described herein, for example, a polypeptide from Table 1 , or a fragment or its variant. The cell can be prepared, for example, by introducing a nucleic acid that encodes the protein in the cell or in a precursor of the cell, by sortagging, by hypotonic charge or by chemical conjugation.
Treatment methods with compositions described herein, for example, erythroid cells
[0231] Methods of administration of modified erythroid cells are described, for example, in documents
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W02015 / 153102 and WO2015 / 073587, each of which is incorporated by reference in its entirety.
[0232] In embodiments, the enucleated erythroid cells described herein are administered to an individual, for example, a mammal, for example, a human. Exemplary mammals that can be treated include, without limitation, humans, domestic animals (eg dogs, cats and the like), farm animals (eg cows, sheep, pigs, horses and the like) and laboratory animals (eg monkeys, rats, mice, rabbits, guinea pigs and the like). The methods described herein are applicable to both human therapy and veterinary applications.
[0233] In some embodiments, the erythroid cells described herein are administered to an individual for the treatment or prevention of inflammation and diseases associated with inflammation, including sepsis, autoimmune disease, cancer and microbial infections. In some embodiments, the erythroid cells described herein are administered to an individual with an autoimmune disease, for example, multiple sclerosis, type 1 diabetes, rheumatoid arthritis, membranous nephritis or any of the diseases listed in Table F of W02015 / 153102, which is incorporated by reference here in its entirety.
[0234] In some respects, the present invention provides a method of treating a disease or condition described herein, comprising administering to an individual in need of a composition described herein, for example, an erythroid cell described herein. In some embodiments, the disease or condition is a cancer, for example, a cancer as described herein. In some respects, the disclosure provides a use of an erythroid cell described herein to treat a disease or condition described herein, for example, a cancer. In some ways, disclosure provides a use of
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79/111 an erythroid cell described herein for the manufacture of a medicament for the treatment of a disease or condition described herein, for example,
a cancer. Cancers exemplary are described in WO2015 / 073587, which is incorporated here by reference in your wholeness. [0235] In some modalities, the cells erythroid are
administered intravenously, for example, by intravenous infusion. [0236] All references cited herein are hereby incorporated by reference in their entirety.
EXAMPLES
[0237] In order for the invention described here to be more fully understood, the following examples are presented. The examples described in this application are offered to illustrate the compositions, preparations and methods provided herein and are not to be construed as limiting their scope.
Example 1: Preparation of an erythroid cell comprising a click loop
[0238] A population of erythroid cells was prepared for labeling with a coupling reagent. The erythrocytes obtained from whole blood were filtered and concentrated to a density of 3.31 x 10 ⁹ cells / ml. The cells were washed twice with phosphate buffered saline (PBS, 2 x 1 ml) and the residual volume was removed by pipette. PH 8 PBS (100 µL) was then added to the cells.
[0239] Stock solutions of the following coupling reagents were prepared at 1 mM (DBCO-sulfo-NHS ester, DBCO-PEG5-NHS ester, 3-azidopropionic acid sulfo-NHS ester and azido-PEG4-NHS ester), and each solution was added to a sample of cells to a final concentration of coupling reagent of 0.1 mM or 0.04 mM. The reaction was incubated at room temperature for
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[0240] The labeling reaction was stopped by adding 1 mL of PBSA (PBS with 0.1% BSA) to each reaction, and allowing each reaction to incubate for 5 minutes at room temperature. The cells were pelleted by centrifugation (5 minutes at 2,500 rpm), and the supernatant was removed by aspiration. The cell pellet was then washed with PBS (1 ml) and pelleted again (5 minutes at 2500 rpm), and the supernatant was removed by aspiration.
[0241] In order to detect the level of labeling, a reagent stock solution comprising Cy5-biotin-azide or Cy5-DBCOa 100 nM was prepared. The detection reagent (100 µL) was added to each reaction, and the reactions were incubated at room temperature for 30 minutes. The labeling efficiency was determined by flow cytometry and is summarized below in Table 2.
Table 2. Efficiency of conjugation of click reactions
Sample # Click chemistry reagent Reagentdetection Conjugation Efficiency (%) 1 DBCO-Sulfo-NHS ester 0.1 mM Biotin Cy5 Azide 88.5 2 0.04 mM DBCO-Sulfo-NHS ester Biotin Cy5 Azide 38.3 3 Sulfo-DBCO-NHS ester 0.1 mM Cy5 azide 74.4 4 0.04 mM Sulfo-DBCO-NHS Ester Cy5 azide 55.1 5 DBCO-PEG4-NHS ester 0.1 mM Cy5 azide 97.4 6 0.04 mM DBCO-PEG4-NHS ester Cy5 azide 96.1 7 DBCO-PEG5-NHS ester 0.1 mM Biotin Cy5 Azide 83.1 8 0.04 mM DBCO-PEG5-NHS ester Biotin Cy5 Azide 29.7 9 DBCO-PEG13-NHS ester 0.1 mM Cy5 azide 98.0 10 0.04 mM DBCO-PEG13-NHS ester Cy5 azide 95.5
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11 Sulfo-NHS ester of 0.1 mM 3azidopropionic acid Cy5 DBCO 99.8 12 Sulfo-NHS ester of 0.04 mM 3azidopropionic acid Cy5 DBCO 97.9 13 0.1 mM 6azidohexanoic acid Sulfo-NHS ester Cy5 DBCO 98.9 14 0.04 mM 6azidohexanoic acid Sulfo-NHS ester Cy5 DBCO 99.3 15 0.1 mM Azido-PEG4-NHS Ester Cy5 DBCO 99.1 16 0.04 mM Azido-PEG4-NHS Ester Cy5 DBCO 67.0 17 0.1 mM TCO-PEG4-NHS ester Methyltetrazine fromCy5 1.4 18 0.04 mM TCO-PEG4-NHS ester Methyltetrazine fromCy5 3.0 19 0.1 mM TCO-PEG12-NHS ester Methyltetrazine fromCy5 96.7 20 0.04 mM TCO-PEG12-NHS ester Methyltetrazine fromCy5 80.5 21 Methyltetrazine-Sulfo-NHS ester0.1 mM Cy5-TCO 97.6 22 Methyltetrazine-Sulfo-NHS ester0.04 mM Cy5-TCO 95.1 23 Methyl-tetrazine-PEG4-NHS ester0.1 mM Cy5-TCO 97.5 24 Methyl-tetrazine-PEG4-NHS ester0.04 mM Cy5-TCO 98.7 25 Control (N / A) Biotin Cy5 Azide 0.74 26 Control (N / A) Cy5 DBCO 0.43 Example 2: Preparation of an agenl comprising you a handle of
click
[0242] Proteins of interest for coupling to erythroid cells were prepared by staining with a coupling reagent. Exemplary proteins for labeling include antibodies (for example, an anti-PDL1 (mouse anti-mouse PDL1 antibody) and anti-a4p7 (a mouse anti-mouse α4β7 antibody)), 4-1 BB ligand and A / G protein. The proteins were typically desalted, exchanged with PBS buffer and concentrated to> 1 mg / ml before labeling, and the pH was adjusted to pH 7-8. Antibodies were
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[0243] Coupling reagent stock solutions were prepared as described in Example 1. For protein solutions with a concentration greater than 5 mg / mL, the coupling reagent was added to a 10-fold molar excess over the protein concentration . For protein solutions with a concentration below 5 mg / mL, the coupling reagent was added in a molar excess of 20 to 50 times over the protein concentration. The protein labeling reaction was incubated at room temperature for 30 minutes, with gentle shaking every 10 minutes.
[0244] In order to detect the level of labeling, a detection reagent solution comprising Cy5-biotin-azide or WS-DBCO-biotin was prepared and added to each reaction and the reactions were incubated at room temperature for 30 minutes . The labeling efficiency was determined by Western blotting, using an antibiotin antibody for detection.
[0245] In an alternative detection method for degree of labeling, the Nanodrop UV-Vis program was used to read approximately 1-3pL of labeled protein at the absorbance of 280 nm and 309 nm with a baseline correction at 750 nm.
Example 3: Production of enucleated erythroid cells comprising an agent covalently attached to the cell surface by a residual ligand comprising a click signature
[0246] The proteins were coupled to erythroid cells according to the general procedure described below. Erythroid cells marked with sulfo-NHS ester of 3-azidopropionic acid (Sample #
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83/111 in Table 2) as described in Example 1 were incubated with 1.53 molar equivalents of an anti-a4p7 antibody (mouse) labeled with DBCO-sulfo-NHS ester or DBCO-PEG5-NHS ester, and the reaction was incubated at room temperature for up to 12 hours at room temperature or 4 ° C. The cells were then washed with PBS or PBSA, and stained with an anti-mouse antibody bound to a detection agent. The cells were then analyzed using flow cytometry to determine the protein staining efficiency. As shown in FIGS. 2A-2C, protein labeling efficiency was determined to be 98.4% for anti-α4β7 antibody bound to DBCO-sulfo-NHS ester (FIG. 2B) and 94.4% for anti-a4p7 antibody bound to DBCO-PEG5-NHS ester (FIG. 2C).
[0247] In another experiment, proteins were coupled to erythroid cells in small reaction volumes using high concentrations of protein. To perform the reaction, erythroid cells were marked with Sulfo-NHS ester of 3-azidopropionic acid (AS). An anti-a4p7 antibody (mouse) was labeled with DBCOsulfo-NHS ester (DS) or DBCO-PEG5-NHS ester (DP). The labeled cells were incubated with 1-20E6 molar equivalents per cell of the anti-a4p7 antibody in an undiluted volume in a volume of 5-10 µl, for 1 hour at 23 ° C. The cells were then washed with phosphate-buffered saline with 0.1% bovine serum albumin and stained with a fluorophore-bound anti-mouse antibody (fluorescein isothiocyanate). The cells were then analyzed by flow cytometry to determine the click efficiency of the protein. As shown in Table 3, the protein click efficiency was determined to be in the range of 98.9-99.8%. A cell is considered positive for fluorescence if its fluorescence is greater than 99% of similar unlabeled cells. This experience indicates that it is possible to score a very high percentage of
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84/111 cells with a protein, for example, using a small reaction volume and a high amount of protein. This experiment also indicates that it is possible to attach a multimer to the cell, since the antibody has two heavy chains and two light chains. This example also indicates that it is possible to attach large portions to a cell, since the antibody has a molecular weight of about 150 kDa.
Table 3. Efficiency of anti-a4p7 labeling cells
Number of cells marked with AS AS concentration Click reaction volume molar excess DBCO: protein ReagentDBCO Percentage of cells positive for fluorescence 1E7 0.1mM 5ul 10 DS 99.8% 1E7 0.1mM 10ul 10 DP 98.9% 1E7 0.1mM 10ul 50 DS 99.6% 1E8 0.1mM 10ul 10 DP 99.7% 1E8 0.1mM 10ul 50 DS 99.7%
Example 4: Coupling of an antibody agent to erythroid cells via a residual ligand comprising a click signature
[0248] It is sometimes desirable to conjugate a protein that has multiple subunits and / or post-translational modifications to a cell. This example describes the conjugation of an antibody to a cell surface.
[0249] An anti-PD-L1 antibody (aPD-L1) (rat IgG2b) was coupled to erythroid cells under different reaction conditions described in Table 4. To carry out the reaction, the erythroid cells were marked with Sulfo-NHS ester from 0.1 mM 3-azidopropionic acid (AS) (resuspended in DMSO) to a 0.1 mM AS label concentration. aPD-L1 was marked with an excess of 10x DBCO-Sulfo-NHS ester (DS) (20 μΙ_ of 10 mM DS). The labeled cells were incubated with the concentration, volume and quantity of labeled aPD-L1 indicated in Table 4 for 1 hour at 25 ° C. The cells were
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85/111 then washed with PBS, and stained with an anti-mouse kappa light chain antibody attached to a fluorophore (PE). The cells were then analyzed using flow cytometry to determine the protein staining efficiency. As shown in Table 4, the protein labeling efficiency was determined to be in the range of 99.799.9%. A cell is considered positive for fluorescence if its fluorescence is greater than 99% of similar unlabeled cells. The number of aPD-L1 molecules present per cell was determined and found to be 7000-15000 aPD-L1 molecules per cell. The amount of protein (in ng) clicked per cell was also determined and is shown in Table 4. In general, the addition of higher concentrations of protein resulted in a higher percentage of cells being modified and a greater number of conjugated molecules per cell . This experiment demonstrates the production of a population of cells having a very high labeling efficiency with a protein, for example, reaching an average number of molecules per cell, in the range of 7000 to 14000 molecules per cell. This experiment also demonstrates that a multimer can be attached to a cell, as the antibody has two heavy chains and two light chains. Experience also indicates that large populations of cells, for example, having 1E8 or 1E9 erythroid cells, can be labeled, with a high percentage of labeled cells and a desired amount of protein per cell reached.
Table 4. Efficiency of aPD-L1 labeling cells
Number of cells Protein Volume Protein Concentration (mg / mL) % of cells added Molecules / Cell Protein (ng) 1E7 10ul 4.5 99.9 6,955 2.88 1E7 10ul 2 99.8 7.014 2.91 1E7 10ul 0.5 99.7 6,728 2.78 1E8 50ul 4.5 99.9 13,892 57.61
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1E8 50ul 2 99.8 10,934 45.30 1E8 50ul 0.5 99.7 9,797 40.55 1E9 100ul 4.5 99.9 14,916 618.61 1E9 100ul 2 99.8 12,126 502.40 1E9 100ul 0.5 99.7 7,476 309.43 Example 5: Coupling an agent protein equal to
erythroid cells through a residual ligand that comprises a click signature
[0250] Ligand 41 BB (m41BBL) was coupled to erythroid cells. To carry out the reaction, erythroid cells were marked with Sulfo-NHS Ester of 3-azidopropionic acid (AS) and m41 BBL was marked with DBCO-sulfo-NHS ester (DS). The labeled cells were incubated with 1-5 mg / ml of labeled m41BBL, in a volume of 10-30 µl, for 1 hour at 23 ° C. The cells were then washed with phosphate buffered saline with 0.1% bovine serum albumin, and stained with fluorescent detection reagent (anti-m41BBL-PE). The cells were then analyzed by flow cytometry to determine the click efficiency of the protein. As shown in Table 5, the protein click efficiency was determined to be in the range of 1.14-99.9%. A cell is considered positive for fluorescence if its fluorescence is greater than 99% of similar unlabeled cells. This experiment demonstrates the production of a population of cells that have a very high labeling efficiency with a protein ligand.
Table 5. Efficiency of m41BBL marking cells
Number of cells m41 BBL concentration (mg / mL) Molecules m41 BBL / cell Percentage of cells positive for fluorescence 1E7 1,024 44.158 88.7% 1E8 1,024 40,926 86.5% 1E8 5 94,874 99.9% Example 6: A protein ligand coupled to heaven erythroid ulcers have
bonding activity
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[0251] It is often desirable to produce a cell population with a high percentage of labeled cells and a high level of clicked protein per cell. At the same time, it is usually desirable to avoid excessive labeling, for example, to destroy the functionality of a protein by conjugating ligands to many sites in the protein. Consequently, erythroid cells functionalized with clicked proteins were tested for their ability to bind to a physiological ligand.
[0252] The 41 BB ligand (m41BBL) was coupled to erythroid cells as described in Example 5. The cells were then contacted with 41 BB (the 41BBL cognate binding partner), an antibody labeled with Phycoerythrin that binds to 41 BB , and an allophicocyanin-labeled antibody (APC) that binds to 41 BB. The binding of 41 BB to cells indicates that not only 41 BBL is present in cells, but that its binding site is functional and oriented to allow binding. The binding of the anti-41 BBL antibody confirms that 41 BBL is present in the cells. The cells were analyzed by flow cytometry to determine the protein staining efficiency. As shown in Table 6, the 41 BB binding was determined to be in the range of 71.4-97.0%. A cell is considered positive for fluorescence if its fluorescence is greater than 99% of similar unlabeled cells. This experiment demonstrates the production of cells with very high labeling efficiency yet without excessive labeling, for example, without destroying the ligand binding site.
Table 6. Connection of erythroid-m41BBL cell to 41 BB
Number of cells m41BBL degree of marking with DS Percentage of cells positive for fluorescence 1E7 -2.64 71.4% 1E8 -2.64 72.4% 1E8 -1.74 97.0%
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Example 7: An antibody coupled to erythroid cells has binding activity
[0253] It is often desirable to produce a cell population with a high percentage of labeled cells and a high level of clicked protein per cell, without excessive labeling. Consequently, a clicked antibody was tested for the ability to bind to its antigen.
[0254] Anti-PD-L1 has been coupled to erythroid cells. To carry out the reaction, erythroid cells were marked with the Sulfo-NHS ester of 3-azidopropionic acid (AS) and the PD-L1 was marked with the DBCO-sulfo-NHS ester (DS). The labeled cells were incubated with 2 mg / ml of labeled anti-PD-L1, in a volume of 20 µL, for 1 hour at 20 ° C. The cells were then placed in contact with the recombinant mouse PD-L1 with an Fc chemical marker. This can be detected with the anti-Fc antibody. The binding of mouse erythroid cells that are clicked with aPD-L1 to the recombinant protein has been demonstrated by a complete population shift so that all cells are doubly positive for rat antibody (indicating the presence of the antibody) as well as Fc marker (indicating binding to the recombinant protein).
[0255] Additionally, the ability of aPD-L1-labeled erythroid cells to bind to murine tumor cell lines expressing PD-L1 was evaluated. Initially, PD-L1 expression was evaluated in three cell lines: CT-26, B16F.10 and A20 with or without IFNg stimulation for 24 h (which induces PD-L1 expression in tumor cells). PD-L1 expression was assessed by staining with the aPD-L1 antibody compared to the isotype control antibody. The binding of erythroid cells that are clicked with APD-L1 to cells that express PD-L1 was assessed by incubation of cancer cells that were previously marked with Far red
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89/111 cell tracking and erythroid cells at 4 ° C for 2 hours. The cell suspension was stained with anti-Kappa chain antibody and evaluated on a flow cytometer. A population that is doubly positive for Far Red and kappa would indicate that erythroid cells bind to tumor cells. This experiment assesses conditions with or without IFNg pre-incubation and compares the erythroid cells clicked with PD-L1 with the cells that are clicked with an isotype control antibody. The percentage of tumor cells that bound to mouse APD-L1 in erythroid cells varied between 60-99%, depending on the level of expression of PD-L1 in the tumor cell line (FIG. 3). The percentage of tumor cells that bind to mouse erythroid cells clicked on isotype control did not correlate with the level of expression of PD-L1, as expected. Overall, the binding of aD-L1 mouse erythroid cells to tumor cells was significantly increased with increased expression of PD-L1. The binding of erythroid cells that express aPD-L1 to recombinant PD-L1 and to tumor cells that express PDL1, indicates that not only is anti-PD-L1 present in cells, but that its binding site is functional and oriented towards allow connection.
Example 8: Quantification of unreacted coupling reagent in cells and proteins
[0256] In some embodiments, it is desirable to have none, or very low levels, of unreacted click ligand present in the cell and the protein to which it is attached. Although not intended to be limited by theory, in some embodiments, lower levels of unreacted click ligands are associated with lower immunogenicity of the functionalized cell. This example describes the quantification of the unreacted click ligand present in the cell and the protein.
[0257] The cells were labeled by reaction with Sulfo-NHS Ester of 3-azidopropionic acid (AS), and the m41BBL protein was labeled
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90/111 through reaction with DBCO-sulfo-NHS ester (DS). The cells and proteins were then combined to conjugate the protein to the cell surface. The unreacted residual ligand was then detected by adding fluorescence-labeled ligand, in which the addition of Cy5 DBCO reacted with and identified the residual ligand in the cell, and the addition of Cy5 Azide biotin reacted and identified the residual ligand in the protein. The experiment indicated low levels of unreacted ligand in proteins, specifically 11.2%. This Example demonstrates the ability to detect previously unreacted binding sites on the manufactured cell and protein, for example, as a quality control test. It also demonstrates the ability to reduce the number of unreacted ligand sites in the manufactured cell and protein, making them react with a ligand, for example, a ligand with low stereochemical impedance.
Example 9: Conjugating exogenous polypeptide agents in various types of cells
[0258] A mixture of mouse spleen cells was obtained by mechanical disruption and lysis of red blood cells. The remaining cells were labeled with Sulfo-NHS Ester of 0.1 mM 3-azidopropionic acid (AS). Purified E. coii asparaginase was labeled with DBCO. The labeled cells and proteins were then combined to allow conjugation. Surface asparaginase was detected with a rabbit anti-asparaginase antibody and a secondary anti-rabbit antibody labeled with Alexa Fluor 657. The identity of each cell type was detected using the markers described in Table 7. The cells were analyzed by cytometry flow. The experiment indicated significant labeling of all cell types tested as indicated in Table 7, using a blocking strategy designed to distinguish between different cell types in the population.
Table 7. Efficiency of marking different cell types
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Bookmarks Cell type Percentage Clicked by Protein Detection CD3 + CD4 + CD4 T cells 99.1% CD3 + CD8 + CD8 T cells 99.2% CD3- NK1.1 + Natural Killer Cells 99.4% CD3 + NK1.1 + Natural Killer T Cells 99.3% CD11b + Myeloid Cells 89.4% CD11c + Dendritic Cells 88.5% CD19 + CD61- B cells 95.0% CD61 + CD19-CD11b- Platelets 96.7% Ly6G ++ Neutrophils 84.1%
[0259] The experiment also demonstrates the conjugation of an enzyme to the surface of several cells, including immune cells, nucleated cells and enucleated cells.
Example 10: Erythroid cells conjugated to an exogenous polypeptide agent retard tumor growth in vivo [0260] A MC38 mouse model system for colon cancer was used to test the effects of functionalized erythroid cells on tumor growth. Without being limited by theory, 41BBL is thought to slow tumor growth, eliciting diverse effector immune responses in both innate and adaptive immune arms. The most potent responses stimulate CD8 + cytotoxic T cells to proliferate and increase their effector potential by increasing the production of interferon gamma and expression of multiple granzymes. In contrast, published preclinical data using multiple 4-1 BB agonists showed little or no activity from the single antitumor agent in MC38 or other models (Chen, etal, 2014; Kudo-Saito, etal, 2006; Kocak, etal, Cane Res 2006; Tirapu, et al, Int J Cancer 2004; John, et al, Canc Res 2012). These
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92/111 differences in activity suggest that cell-cell binding of effector cells 4-1 BB (for example, T cells, NK cells) using cell presentation by erythroid cells expressing 41BB-L is more effective in stimulating antitumor responses than approaches antibody-based agonists.
[0261] The cells were conjugated to 41BBL as described in Example 5. In this study, 94.7% of the cells were labeled with m41BB-L. The number of labeled molecules per cell was quantified using flow cytometry. For dosing in animals, there was an average of 1.1e9m4-1BB-L mRBCs administered per dose, with an average of 36,200 m4-1 BB-L molecules per cell, corresponding to 0.084 mg / kg of m4-1BB-L per dose.
[0262] Fourteen 6-8 week old female C57 / B6 mice were inoculated sc on the left flank with 5 x 10 ⁵ MC38 cells. The animals' weight and condition were recorded daily, and tumors were measured 3 times a week.
[0263] Tumors were measured three times a week, measuring each tumor in 2 dimensions. Tumor volumes were calculated using the standard formula: (L x W ² ) / 2. The mean tumor weight and the standard error of the mean were calculated for each group at each time point.
[0264] The observed antitumor activity of m4-1BB-L mRBC compared to untreated controls is shown in FIG. 4 and demonstrates a reduction in tumor growth in mice treated with m4-1BB-L mRBC. The distributions of tumor volume showed statistically significant differences between groups from day 5 of the study and ^Q 9 to ^Q day (P <0.05, t-test).
[0265] Body weight was recorded daily. Changes in body weight were calculated for each mouse relative to the body weight recorded on day 1. The treatment was well tolerated,
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93/111 as indicated by the overall increase in body weight for most mice. The mice that showed some decrease in body weight, did not lose more than 5% of the total body weight throughout the study.
[0266] These data support a significant advantage in the efficacy and potency of the cellular presentation of 4-1BB-L via erythroid cells through agonist antibody approaches. Significant antitumor activity was observed with m4-1BB-L mRBC in model MC38 that was not previously observed with a 4-1 BB agonist antibody administered at a level 10 times higher than that of m41BB-L in the same model (Chen, et al , 2014). The increased potency and mRBC activity m4-1BB-L compared to agonist antibody approaches is consistent with the 4-1BBL cell presentation and a corresponding 4-1 BB receptor multimerization that is necessary to induce potent 4-1 pathway signaling. BB (Bremer, 2013) and that would normally occur within the immune synapse.
Example 11: Erythroid cells comprising three exogenous polypeptide agents covalently linked to the cell surface by a residual ligand comprising a click signature
[0267] To test the ability to label cells with a plurality of agents, erythroid cells were functionalized with three different agents. The three agents (Factor Vila, Fator Xa and Cy5) were marked with DBCO-sulfo-NHS ester (DS) or purchased as marked with DBCO (i.e., Cy5). The cells were marked with Sulfo-NHS Ester of 3-azidopropionic acid (AS). The labeled cells were combined with the labeled agents to allow conjugation. The presence of the three agents in the cells was detected by flow cytometry. The experiment indicated that 33.6% of the cells were positive for all three agents.
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Example 12: Agents linked to sugars
[0268] The agents can bind directly to proteins on the cell surface, for example, as described above. However, the agents can also be linked to sugars on the cell surface, for example, to glycan chains on glycoproteins, as described in this Example. Cells that have glycans on the cell surface are marked with a bifunctional ligand having a click loop and a group that reacts with a glycan.
[0269] In one approach, the click loop is added to the glycan using an adaptation of a commercially available kit designed to mark glycans in antibodies. In this adaptation, the cells, instead of the antibody, are first treated with comendoglycosidase to hydrolyze glycans after the GlcNAc nucleus. Then, the cells are washed and treated with a modified galactosyltransferase, GaIT (Y289L) and GaINAzide. GaIT binds a residue of GaINAzide to the exposed GlcNAc, resulting in an available azide for a chemical reaction of azide-alkaline promoted by a biocompatible strain with an alkaline modified protein.
[0270] In another approach, portions of sugar within glycans are first modified to create aldehydes and ketones through moderate periodate-mediated oxidation of vicinal diols using the methodology described in de Bank et al Biotechnol Bioeng 81: 800-808, 2003 An alkoxide or azide-containing click loop can then be added to the resulting aldehyde and ketone groups using alkaline hydrazide or ethyl ethyl hydrazide, respectively. The agent can then be labeled with the corresponding azide or alkali as described in the other examples above. The labeled agent is then incubated with the labeled cells to allow conjugation. The binding of the agent to the cell can be detected by flow cytometry, for example, as described above.
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[0271] The bond may also involve a sugar in the agent. For example, an agent (for example, an exogenous protein or antibody), which has a sugar can be labeled with a bifunctional ligand having a click loop and a group that reacts with a glycan, for example, one of the previously described ligands. A cell can be labeled with a second ligand, for example, a ligand that reacts with a protein or sugar on the cell's surface. The labeled cell and the labeled agent are then mixed to allow conjugation. Binding of the agent to the cell can be detected by flow cytometry, for example, as described above.
Example 13: Measurement of the immunogenicity of functionalized erythroid cells
[0272] In some embodiments, functionalized erythroid cells have low immunogenicity. Immunogenicity can be tested by measuring antibodies generated against a protein expressed in an erythroid or another cell. A standard method for measuring these antibodies is using a direct ELISA. Immunogenicity against a protein or agent attached to the erythroid cell can be measured by administering the functionalized erythroid cell to an animal or patient and then taking samples of plasma or serum over a period of days or weeks. Serial dilutions of samples are prepared and then incubated for 10-120 minutes in ELISA plate wells that have been pre-coated with the protein or agent used to functionalize the erythroid cell so that any antibodies generated against the protein or agent can connect. The plates are then washed and incubated with enzyme-labeled polyclonal antibodies (for example, horseradish peroxidase) that bind to any antibodies that have bound to the protein or agent. The wells are then washed and the level of enzyme activity remaining in the well is measured to assess the level of antibodies raised against the protein or
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96/111 agent used to functionalize the erythroid cell.
Example 14: Functionalizing erythroid cells with a cytotoxic agent from erythroid cells
[0273] This example describes how a cell can be functionalized with a toxic agent, for example, an agent that expresses itself in the erythroid cell would be toxic to it, for example, an agent that reduces the growth rate, viability, cell life or function (cytotoxic erythroid cell). Cytotoxic agents include, for example, enzymatic proteins that degrade amino acids and hinder cell growth and expansion, enzymes that are involved in the modification or degradation of key metabolic molecules or molecular intermediates, or proteins or molecules such as ricin that interfere with processes critical cell phones.
[0274] An example of a cytotoxic agent for erythroid cells is asparaginase, which is used clinically to deprive cancer cells. The overexpression of asparaginase in matured erythroid cells interferes with cell growth and cell maturation.
[0275] Cells, for example, B6.129S7-Rag1 ^tm1Mom IJ murine red blood cells (Rag1 knockout mice), are scored in vivo by injecting the mice with 2 mg of Sulfo-NHS ester of 3-azidopropionic acid and then collecting the cells 2 days later.
[0276] An erythroid cell cytotoxic agent, for example, asparaginase, is marked with 5x and 2.5x molar excess of DBCO-sulfo-NHS ester for 30 minutes at 25 ° C. The 5x molar excess of DBCO-sulfo-NHS ester leads to ~ 2 markers per asparaginase monomer and 2.5x molar excess of DBCO-sulfo-NHS ester leads to ~ 1.2 markers per asparaginase monomer.
[0277] The labeled cells are then combined with 2
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QUA 11 different concentrations of DBCO labeled with asparaginase in different degrees for 60 minutes to allow conjugation to occur and reach the labeled cells in different degrees. The presence of the agent on the cell surface can be detected using an asparaginase activity assay, using flow cytometry with an asparaginase antibody.
Table 8. Asparaginase activity of labeled cells
Cell labeling reaction Activityasparaginase frommarked cells Higher degree of marking 1e9 cells + 1.6 mg asparaginase modified with ~ 2 markers / tetramer 2.16e-10 units / cell Lower degree of marking 1e9 cells + 0.2 mg asparaginase modified with ~ 1.2 markers / tetramer 4.32e-11 units / cell
[0278] The relative ability of asparaginase-conjugated versus non-asparaginase-conjugated red blood cells to eliminate serum asparaginase over time can be tested in mice. The mice were injected with control red blood cells, RBC labeled with high or low amounts of asparaginase together, or with low, medium or high amounts of unconjugated recombinant asparaginase as shown in Table 9. The asparaginase-conjugated and control RBCs were additionally labeled with a fluorescent marker, Cy5, to determine the pharmacokinetics of RBCs after injection. Blood samples are collected at various times after injection to determine asparagine levels and levels of marked RBCs.
Table 9. Configuration for injection of cells marked with asparaginase in mice.
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Recipient mouse Number of animals description Number of cells injected Total units of Asparaginase injected Group 1 C57BL / 6 3 Unmarked asparaginase cell 1e9 - Group 2 C57BL / 6 3 Cells labeled with the highest dose of asparaginase 1e9 0.22 Group 3 C57BL / 6 3 Cells labeled with the lowest dose of asparaginase 1e9 0.043 Group 4 C57BL / 6 3 2.8 ug of recombinant asparaginase - 0.19 Group 5 C57BL / 6 3 0.39 u _g of recombinant asparaginase - 0.025 Group 6 C57BL / 6 3 15 u _g of recombinant asparaginase - 0.97 Group 7 B6,129S7- R _ag1 tm1Mom | J 3 Unmarked asparaginase cell 1e9 - Group 8 B6,129S7- R _ag1 tm1Mom | J 3 Cells labeled with the highest dose of asparaginase 1e9 0.22 Group 9 B6,129S7- R _ag1 tm1Mom | J 3 Cells labeled with the lowest dose of asparaginase 1e9 0.043 Group 10 B6,129S7- R _ag1 tm1Mom | J 3 2.83 ug of recombinant asparaginase - 0.19 Group 11 B6,129S7- R _ag1 tm1Mom | J 3 0.385 u _g of recombinant asparaginase - 0.025 [0279] When 0.3S 1, 2.8 or 15 micrograms of asparaginase
recombinant cells were injected into the C557BL / 6 mice injected into the mice, the serum asparagine was reduced to close to zero
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99/111 hours after the injection (FIG. 5). However, for the 0.39 µg group, serum asparagine levels rose to almost normal levels within 1 day after injection, while the 2.8 and 15 µg dose groups increased close to or above normal levels in the day 4. In contrast, asparagine levels were reduced to close to zero at 6 hours of 8 days in C57BL / 6 mice treated with low or high amounts of asparaginase coupled with red blood cells with levels starting to increase again on day 11 (FIG . 5). Similar results were obtained in the Rag1 mice (FIG. 6); however, in contrast to the C57BL / 6 mice, asparagine levels remained close to zero until day 29 (FIG. 6). The pharmacokinetics of ASNas-conjugated cells is also measured by detecting the percentage of Cy5 positive cells at various times after injection using flow cytometry. One day after the injection, the elimination of the remaining ASNase-conjugated cells was similar for the cells labeled with Cy5 (FIGS. 7A and 7B). Approximately 20% of RBCs present in the circulation on day 1 remained in the blood on day 18. For cells with a higher level of ASNase, the initial rapid elimination of cells was greater than for cells with lower levels of ASNase. Despite this rapid initial elimination, the cells remaining in the circulation after the first day remained detectable in the circulation at least until the 22nd. This experiment demonstrates that the conjugation of asparaginase in red blood cells dramatically improves the circulation time of asparaginase in the blood. This increased exposure is associated with a dramatic improvement in the ability to reduce serum asparagine levels over days and weeks with a single dose.
[0280] Furthermore, the pharmacokinetic profile for asparaginase-labeled RBCs also did not change with repeated dosing,
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100/111 indicating a lack of immunogenic response to asparaginase-labeled RBCs for all three levels of asparaginase, as detectable by this assay.
Example 15: Coupling of an enzymatic agent to erythroid cells through a residual ligand that comprises a click signature
[0281] Factor Xa (FXa) has been coupled to erythroid cells. To perform the reaction, 1e9 - 1.36e10 erythroid cells / mL were marked with Sulfo-NHS Ester of 3-azidopropionic acid (AS) and FXa was marked with DBCO-sulfo-NHS ester (DS). The labeled cells were incubated with 0.5 -1.7 mg / mL of labeled FXa, in a volume of 10 pL -3 mL, for 1 hour at 23 ° C. The cells were then washed with phosphate-buffered saline with 0.1% bovine serum albumin, and stained with fluorescent detection reagent (anti-FXaPE). The cells were then analyzed by flow cytometry to determine the click efficiency of the protein. The protein click efficiency was determined to be in the 99% range. This experiment demonstrates the production of a population of cells that have a very high labeling efficiency with an enzyme.
[0282] The number of Factor Xa protein molecules per cell was quantified by the antibody's binding capacity being 1,000 -250,000 molecules per cell. The number of proteins per cell can be adjusted, for example, using the protein concentration, the number of cells and reaction volume, for example, as described in Example 4.
[0283] Factor Xa activity was quantified by TGA activity in up to 14,000 active molecules per cell.
Example 16: Coupling of protein agents to human erythroid cells through a residual ligand comprising a click signature
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[0284] FactorXa (FXa) and asparaginase (ASNase) have been coupled to human erythroid cells. To carry out the reaction, erythroid cells were marked with Sulfo-NHS ester of 3azidopropionic acid (AS) and FXa and ASNase was marked with DBCOPEG5-NHS ester (DP) and DBCO-sulfo-NHS ester (DS), respectively. The labeled cells were incubated with 1.03 mg / ml of labeled FXa and 4.535 mg / ml of ASNase in a volume of 10 pL, for 1 hour at 23 ° C. The cells were then washed with phosphate-buffered saline with 0.1% bovine serum albumin, and stained with fluorescent detection reagent (anti-FX-PE and anti-ASNaseAlexaFluor 488). The cells were then analyzed by flow cytometry to determine the click efficiency of the protein. The protein click efficiency was determined in the range of 99.9% for FXa and 71.1% for ASNase. This experiment demonstrates the production of a population of human erythroid cells having a very high labeling efficiency with different proteins.
Example 17: Coupling of a peptide agent to erythroid cells through a residual ligand comprising a click signature
[0285] The 35-55 peptide from biotinylated oligodendrocyte myelin glycoprotein (MOG) was coupled to erythroid cells. To carry out the reaction, erythroid cells were marked with different amounts (high and low) of Sulfo-NHS ester of acid
3-azidopropionic (AS) and the MOG peptide was labeled with DBCO-sulfo-NHS ester (DS). The labeled cells were incubated with 2.7 mg / ml of labeled MOG-DBCO, in a volume of 10 pL, for 1 hour at 23 ° C. The cells were then washed with phosphate buffered saline with 0.1% bovine serum albumin, and stained with fluorescent detection reagent (anti-biotin-PE). The cells were then analyzed by flow cytometry to determine
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102/111 the click efficiency of the peptide. The click efficiency of the peptide was determined in the range of 67.4% to 91.8% for MOG. This experiment demonstrates the production of a population of cells with an adjustable degree of peptide staining efficiency. This experiment also demonstrates that a short peptide (about 20 amino acids) can be efficiently clicked on erythroid cells.
Example 18: Coupling of a cytokine agent to erythroid cells via a residual ligand comprising a click signature
[0286] Human IL10 (hlL10) was coupled to erythroid cells. To carry out the reaction, erythroid cells were marked with SulfoNHS Ester of 3-azidopropionic acid (AS) and hLL10 was marked with DBCO-sulfo-NHS ester (DS). The labeled cells were incubated with 0.24 to 0.79 mg / ml hlL10 labeled in a volume of 2 pl_ for 13 hours at 23 ° C or 16 hours at 4 ^Q C. The cells were then washed with brine phosphate buffered with 0.1% bovine serum albumin, and stained with fluorescent detection reagent (antihlL10-PE). The cells were then analyzed by flow cytometry to determine the click efficiency of the protein. As shown in Table 10, the protein click efficiency was determined to be in the range of 76.7-100%. A cell is considered positive for fluorescence if its fluorescence is greater than 99% of similar unlabeled cells. This experiment demonstrates the production of a population of cells that have a very high labeling efficiency, for example more than 200,000 molecules per cell.
Table 10. Efficiency of hlL10 labeling cells
Number of cells hlL10 concentration(mg / mL) Click reaction time (hours) HlL10 molecules / cell Percentage of cells positive for fluorescence 4E7 0.79 1 69,964 76.7% 4E7 0.79 3 128,766 99.9% 4E7 0.24 16 158,572 99.9% 4E7 0.583 1 208,519 100%
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Example 19: A cytokine coupled to erythroid cells has binding activity
[0287] Erythroid cells functionalized with clicked proteins were tested for their ability to bind to a physiological ligand. As discussed above, it is often desirable to have a high percentage of labeled cells and a high level of clicked protein per cell, while preserving the protein's binding activity.
[0288] Human IL10 (hlL10) was coupled to erythroid cells as described in Example 18. The cells were then contacted with the fusion of the human IL10 alpha Fc receptor (the cognate binding partner of hlL10), an antibody labeled with allophicocyanin (APC) that binds to Fc was used to detect the interaction of hlL10 clicked on cells and their binding to the human IL10 alpha receptor. The binding of the hlL10 alpha receptor to cells indicates that not only is hlL10 present in erythroid cells, but that its binding site is functional and oriented to allow binding. The cells were analyzed by flow cytometry to determine the protein staining efficiency. As shown in Table 11, the hlL10 binding was determined to be in the range of 90.0-95.0%. A cell is considered positive for fluorescence if its fluorescence is greater than 99% of similar unlabeled cells. This experiment demonstrates the production of cells with very high labeling efficiency yet without excess labeling, for example, without destroying the ligand binding site.
Table 11. Connection of the erythroid-hlL10 cell to the alpha hlL10 receptor
Number of cells hlL10 degree of DS marking Percentage of cells positive for fluorescence 5E6 -0.63 93.3% 5E6 -1.39 95.0% 1E7 -0.43 90.0%
Example 20: Coupling a cytokine in combination with
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[0289] Human IL10 (hlL10) in combination with an anti-a4p7 Fab was coupled to erythroid cells. To perform the reaction, erythroid cells were labeled with Sulfo-NHS Ester of 3azidopropionic acid (AS), and anti-hlL10 α4β7 Fab was individually labeled with DBCO-sulfo-NHS ester (DS). The labeled cells were incubated with 0.583 mg / mL of labeled hLL10, in a volume of 30 µl, and with 2.434 mg / mL of labeled anti-a4p7 Fab, in a volume of 19.35 pL, for 2 hours at 23 ° C . The cells were then washed with phosphate-buffered saline with 0.1% bovine serum albumin, and stained with fluorescent detection reagents (anti hlL10-BV421 and anti-kappa light chain mouse-PE). The cells were then analyzed by flow cytometry to determine the protein's click efficiency. The click efficiency of the double protein was determined to be approximately 98.3%. A cell is considered positive for fluorescence if its fluorescence is greater than 99% of similar unlabeled cells. This experiment demonstrates the production of a population of cells that have a very high labeling efficiency with a cytokine and a targeting portion.
Example 21: Click specificity
[0290] This example demonstrates the specificity of click markup. Three samples were analyzed. First, untreated murine red blood cells (without a click loop) were mixed with a protein agent (anti-a4b7 Fab) with a DBCO click loop. Second, the murine red blood cells with an azide click loop were mixed with an anti-a4b7 Fab that did not have a click loop. Third, murine red blood cells having an azide click handle were mixed with Fabanti-a4b7 having a DBCO compatible click handle. The reactions were allowed to proceed
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105/111 for 1 hour at room temperature in PBS. The cells were then tested for the presence of the anti-a4b7 Fab, contacting them with a PE-conjugated anti-mouse Kappa IgG antibody (phycoerythrin) and flow cytometry. A cell was considered positive for fluorescence if the signal was greater than 99% of the untreated, otherwise similar cells in the presence of the detection reagent, but in the absence of anti-a4b7 Fab protein. As expected, the first and second samples showed low fluorescence (1.81% and 1.53% of the fluorescent cells, respectively), while the third sample was highly fluorescent (95.2% of the cells showed fluorescence). This example demonstrates that click markup is highly specific.
Example 22: Conjugating a click loop to an agent's N terminal
[0291] It is often desirable to avoid disrupting the protein's biology, for example, by polarizing the N-terminal labeling of a protein of interest, for example, to better preserve the functionality of a C-terminal domain. interest for coupling to erythroid cells by staining with a coupling reagent. To better preserve the functionality of human IL10, the chemistry of the NHS ester was influenced by the N-terminal staining. The proteins were saline and the buffer was exchanged for PBS and then concentrated to> 1 mg / mL before staining. The pH is maintained at neutral pH, approximately 7-7.4.
[0292] Coupling reagent stock solutions were prepared as described in Example 1. The coupling reagent was added at a 1 to 1 molar ratio to the protein concentration. The protein labeling reaction was incubated at room temperature for 1 hour to 1 hour and 30 minutes, with gentle shaking every 10 minutes. In order to detect the level of
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106/111 labeling, the Nanodrop UV-Vis program was used to read approximately 1-3 pL of labeled protein at absorbance of 280 nm and 309 nm with a baseline correction at 750 nm. The degree of labeling ranged from 0.63 to 1.39 DBCO click loop molecules per protein.
Example 23: Conjugation of a click loop with an agent, comprising a free cysteine residue, for example, located on a π-clip, using maleimide chemistry
[0293] It may be desirable to better preserve protein biology, for example, by introducing a free cysteine to a protein of interest for the specific chemical conjugation of the maleimide site. Proteins of interest (e.g., mouse 41BBL) were prepared for coupling to erythroid cells by staining with a coupling reagent. To better preserve protein functionality, a free cysteine was introduced into the recombinant 41BBL protein in the sequence of four amino acids (FCPF, SEQ ID NO: 64) known as π-clamp for site-specific maleimide chemical conjugation. Although a π-clamp is used in this example, it is contemplated that any free cysteine residue can be used for site specific conjugation, for example, as described herein. The proteins were desalted, exchanged with PBS buffer and concentrated to> 1 mg / ml before labeling. The proteins were reduced with 1 mM DTT, incubated for 1 hour at room temperature, and desalted on a NAP5 column.
[0294] The maleimide coupling reagent was added in a 5-fold molar excess over the protein concentration. The protein labeling reaction was incubated at room temperature for 4 hours and desalted using a NAP10 column. In order to detect the level of labeling, the Nanodrop UV-Vis program was used to read approximately 1 -3 μΙ_ of labeled protein in absorbance
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107/111 of 280 nm and 309 nm with a baseline correction at 750 nm. The degree of labeling ranged from 6 to 7.6 DBCO click loop molecules per protein.
Example 24: Conjugation of a click loop for an agent containing two cysteine residues, using the ThioLinker chemistry [0295] It may be desirable to introduce a click loop at a specific site on a protein of interest and / or an orientation particular. Proteins of interest for coupling to erythroid cells were prepared by staining with a coupling reagent. In one method, ThioLinker chemistry was introduced for the recombinant anti-CTLA-4 Fab protein for specific click loop site conjugation via disulfide bond bridge. The Fabs were desalted, the buffer was exchanged for PBS and concentrated to> 1 mg / ml before staining. Fabs were then reduced with 5 mM DTT, incubated for 1 hour at 37 ° C and then exchanged with buffer in PBS. The ThioLinker coupling reagent was added in a 15-fold molar excess over the protein concentration to mark the reduced disulfide bond. The protein labeling reaction was incubated at 4 ° C for 16-18 hours and desalted using a Zeba column. In order to detect the level of labeling, the Nanodrop UV-Vis program was used to read approximately 1-3 pL of labeled protein at absorbance of 280 nm and 309 nm with a baseline correction at 750 nm. The degree of labeling was 8.3 DBCO click loop molecules per protein. ThioLinker-oriented click loop marking on anti-CTLA4 Fab was compared with other click reagents and has been shown to result in substantial cell marking. ThioLinker-labeled anti-CTLA4 was also found to significantly increase functional binding to recombinant CTLA4.
[0296] In another method, ThioLinker chemistry was used to
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108/111 specifically locate a recombinant protein (41BBL of mouse HIS6) through the HIS6 purification marker. The proteins were typically desalted, exchanged with PBS buffer and concentrated to> 1 mg / ml before labeling. The ThioLinker binding reagent was then added in a 20-fold molar excess relative to the protein concentration to mark the protein's HIS6 marker (not reduced). The protein labeling reaction was incubated at room temperature for 3 hours and the buffer was exchanged for PBS using a Zeba desalination column. The ThioLinker tagging of 41BBL from HIS6 mouse clicked on AS-labeled RBC resulted in increased functional capacity of 41 BBL clickable cells to activate immune cells, as shown by the production of IL-2 (FIG. 8A) and interferon-γ (IFN- γ) FIG 8B), respectively. It was observed that the targeted binding of 41 HIS6 mouse BBL resulted in substantially greater secretion of IL-2 and IFN-γ compared to random binding, although the latter induced significantly more cytokine secretion than the click CTL control (labeled RBCs) with AS only).
Example 25: Production of an agent that comprises a non-canonical amino acid (ncAA)
[0297] It may be desirable to introduce a coupling reagent to a specific site of interest within a protein of interest, for example, incorporating non-canonical amino acids (ncAA) into a protein of interest in a site-specific manner. In this example, proteins of interest were produced by co-transfecting Expi293 cells with a plasmid containing an tRNA / amino-acyl-tRNA synthase pair and a plasmid encoding the protein of interest (mlg-mouse 41 BBL) with a termination codon amber (TAG) embedded in a site of interest. The cells were plated and the plasmids were transfected at 1.5 pg / ml of each plasmid
Petition 870190079090, of 8/15/2019, p. 130/195
109/111 using the ExpiFectamine kit on day 1. Different concentrations (2 mM, 1.3 mM, 1 mM, 250 mM) of ncAA (Exo (BCN) -Lys; FIG. 9A) were added to the transfected cells for specific incorporation site in place of the Amber termination codon on the same day. On day 3 after transfection, the medium was harvested to obtain secreted proteins. The secreted proteins were confirmed to have site specific ncAA incorporation by incubation with Azide-Biotin Cy5 for 30 min at room temperature and then Western blot analysis. The proteins were detected with an anti-41BBL antibody and a secondary HRP antibody, and the click handle loop incorporation was detected with an anti-biotin-HRP antibody.
Table 12. Clicked proteins
Protein Marking chemistry Ovalbumin DBCO-Sulfo-NHS ester Erwinase (Erwinia asparaginase) Ester DBCO-Sulfo-NHS Uricase Ester DBCO-Sulfo-NHS Lysozyme DBCO-Sulfo-NHS ester Anti-CTLA4 Fab ThioLinker-DBCO Anti-CD3 Fab ThioLinker-DBCO Anti-PDL1 Fab ThioLinker-DBCO
[0298] As shown in FIG. 9B, the specific incorporation of the ncAA exo (BCN) -lysine site resulted in the production of clickable murine 41BBL.
Example 26: Coupling of urate oxidase to erythroid cells via a click-signed ligand
[0299] Recombinant Candida utilis oxidase urate-Hise was expressed and purified from E. coli and labeled with DBCOsulfo-NHS ester giving urate oxidase at a ratio of approximately 1 label per monomer. Murine red blood cells (RBCs) were labeled with Sulfo-NHS ester of acid 6
Petition 870190079090, of 8/15/2019, p. 131/195
110/111 azidohexanoic. Coupling reactions were carried out in which 1e9-labeled murine RBCs were incubated in the presence of 0 uM DBCO-labeled urate oxidase or 75 uM for 2 hours at room temperature. The degree of conjugation of urate oxidase was evaluated by staining the murine RBCs for Hise-urate oxidase with anti-Hise antibody labeled with DyLight 488, followed by flow cytometry analysis. 100% of RBCs incubated with 75 µM urate oxidase were labeled with the enzyme; in contrast, only 0.16% of the negative control cells treated with 0 uM urate oxidase showed positive fluorescence in this assay. The erythrocytes coupled with urate oxidase were able to efficiently deplete uric acid, showing urate oxidase activity of about 4.6e12 units / cell.
Example 27: Erythroid cells comprising exogenous polypeptide agent are active In vivo
[0300] Enucleated erythroid cells were conjugated to antiPD-L1 on their surface and tested for the ability to infiltrate tumors in mice.
[0301] The mice were inoculated with B16F10SC cells. The tumors were allowed to grow up to 400 cubic mm before dosing. Murine RBCs were conjugated to PD-L1 anti-murine antibody (Fab) fragments and isotype control. Conjugated murine RBCs were labeled with CTFR according to the manufacturer's protocol. The cells were infused into the animals. One day after the infusion, the tumors were collected. The tumors were sectioned and stained with anti-CD31 to visualize the tumor vasculature and DAPI to visualize the nuclei. Stained sections were scanned and photographs were taken. Using the Halo software, tumor areas and vascular areas were identified. The total cell count of red cells marked in these two areas was taken both
Petition 870190079090, of 8/15/2019, p. 132/195
111/111 for isotype control and anti-PD-L1. The ratio between the RBCs found in the tumor and the RBCs found in the vessels was calculated. The relationship between RBC in the vessels and RBC in the tumor is 1 (average measurement in tumors of 8 mice) for the RBC combined with the isotype control, indicating similar amounts in the tumor and in the vasculature. The ratio between RBC in the vessel and RBC in the tumor is 1.7 for the mice treated with anti-PD-L1, indicating the enrichment of RBC in the tumor in the anti-PD-L1 group compared to the isotype control mice. The difference in the ratio between the 2 groups was statistically significant with P <0.01 (Student's T test).
[0302] While not wishing to be bound by theory, tumors that express higher levels of PD-L1 may respond better to RCTs comprising anti-PD-L1 than tumors that express lower levels of PD-L1. B16F10 cells expressed about 300,000 copies per cell of PD-L1 when stimulated with IFN-gamma at 10 ng / µL. In contrast, CT26 cells expressed about 150,000 copies per cell of PD-L1 and A20 cells expressed about 100,000 copies per cell of PD-L1 under the same conditions. The PD-L1 copy number was measured using a Quantum Simply Cellular kit (Bangs Laboratories). Erythroid cells comprising an anti-PD-L1 antibody on their surface showed greater binding to B16F10 cells treated with IFN-gamma and CT26 than to A20 cells, consistent with higher levels of PD-L1 expression in tumor cells leading to greater binding from erythroid cells to tumor cells.

权利要求:
Claims (27)
[1]
1. Enucleated erythroid cell, characterized by the fact that it comprises an exogenous polypeptide agent covalently linked to the cell surface by a residual ligand comprising a click signature, via an amino acid side chain of a protein on the cell surface, in which the click signature was formed as the product of a click reaction or has the structure of a click signature.
[2]
2. Enucleated erythroid cell, characterized by the fact that it comprises:
a plurality of exogenous polypeptide agents, each exogenous polypeptide agent of the plurality being covalently linked to the cell surface by a residual linker comprising a click signature, in which at least 10% of exogenous polypeptide agents in the cell are linked via a chain side of amino acids to a protein on the cell surface, where the click signature was formed as the product of a click reaction or has the structure of a click signature.
[3]
3. Enucleated erythroid cell according to claim 2, characterized by the fact that at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99 % of exogenous polypeptide agents in the cell are linked via a side chain of amino acids to a protein on the cell surface.
[4]
4. Enucleated erythroid cell, according to any of the preceding claims, characterized by the fact that it is not genetically manipulated.
[5]
5. Enucleated erythroid cell according to any of the preceding claims, characterized by the fact that it does not comprise an unnatural amino acid.
Petition 870190079090, of 8/15/2019, p. 134/195
2/5
[6]
6. Enucleated erythroid cell according to any of the preceding claims, characterized by the fact that the exogenous polypeptide agent comprises a π-clip.
[7]
7. Enucleated erythroid cell according to claim 6, characterized by the fact that the exogenous polypeptide agent is covalently linked to the enucleated erythroid cell through the π-clip.
[8]
8. Enucleated erythroid cell according to any of the preceding claims, characterized by the fact that the exogenous polypeptide agent comprises a non-canonical amino acid (ncAA).
[9]
9. Enucleated erythroid cell according to claim 8, characterized by the fact that the exogenous polypeptide agent is covalently linked to the enucleated erythroid cell via ncAA.
[10]
10. Enucleated erythroid cell, according to any of the preceding claims, characterized by the fact that it lacks a sortase transfer subscription.
[11]
11. Enucleated erythroid cell according to any of the preceding claims, characterized by the fact that the exogenous polypeptide agent is selected from an alpha4beta7 antibody, IL-10, an enzyme, such as asparaginase, or a coagulation factor, such as Factor Xa.
[12]
12. Enucleated erythroid cell according to any of the preceding claims, characterized by the fact that it comprises at least about 5,000, 10,000, 50,000, 100,000 or 200,000 copies of the exogenous polypeptide agent, or between 5,000, 10,000, 10,000-50,000, 50,000-100,000 or 100,000-200,000 copies of the exogenous polypeptide agent.
[13]
13. Enucleated erythroid cell, according to any
Petition 870190079090, of 8/15/2019, p. 135/195
3/5 one of the preceding claims, characterized by the fact that the exogenous poHpeptide agent is a peptide linker that binds to a binding partner.
[14]
14. Enucleated erythroid cell according to any of the preceding claims, characterized by the fact that it has an elimination rate in which at least 20% of the agent remains in the individual's circulatory system for 1, 2, 3, 4, 5, 6 or 7 days.
[15]
15. Enucleated erythroid cell according to any of the preceding claims, characterized by the fact that less than 10% of the coupling reagent in the cell is unreacted coupling reagent.
[16]
16. Enucleated erythroid cell according to any of the preceding claims, characterized by the fact that it further comprises a second exogenous polypeptide agent, for example, in which the second exogenous polypeptide agent is covalently linked to the cell surface by a second residual ligand comprising a dike signature.
[17]
17. Enucleated erythroid cell according to claim 16, characterized by the fact that it further comprises a third exogenous polypeptide agent, for example, in which the second exogenous polypeptide agent is covalently linked to the cell surface by a second residual ligand comprising a signature click.
[18]
18. Enucleated erythroid cell according to any of the preceding claims, characterized by the fact that at least 50%, 60%, 70%, 80%, or 90% of the exogenous polypeptide agents are covalently attached to the surface of the cells with a pre-selected orientation, for example, are linked by the same portion of exogenous polypeptide agents (for example, a
Petition 870190079090, of 8/15/2019, p. 136/195
4/5 N-terminus, a C-terminus, or a particular residue, for example, a cysteine).
[19]
19. Enucleated erythroid cell according to any of the preceding claims, characterized by the fact that the exogenous polypeptide agent is covalently linked to an endogenous molecule of the cell, for example, an endogenous polypeptide on the cell surface.
[20]
20. Preparation, characterized by the fact that it comprises at least 10 ¹⁰ , 10 ¹¹ or 10 ¹² enucleated erythroid cells, as defined in any of the preceding claims.
[21]
21. Preparation according to claim 20, characterized in that at least 70% of the erythroid cells enucleated in the preparation comprise the agent.
[22]
22. Preparation according to claim 20 or 21, characterized in that at least 70% of the erythroid cells enucleated in the preparation comprise an exogenous polypeptide agent that binds to a ligand, for example, in a cytometry assay flow of Example 6.
[23]
23. Preparation according to any one of claims 20 to 22, characterized in that at least 50%, 60%, 70%, 80% or 90% of the exogenous polypeptide agents in the preparation are covalently attached to a surface of enucleated erythroid cells with a pre-selected orientation, for example, are linked by the same portion of exogenous polypeptide agents (for example, an N-terminus, a C-terminus, or a particular residue, for example, a cysteine).
[24]
24. Method of producing an enucleated erythroid cell functionalized with an agent, characterized by the fact that it comprises:
Petition 870190079090, of 8/15/2019, p. 137/195
5/5 (a) providing an activated cell comprising a cell covalently linked to a first click loop, (b) providing an activated agent comprising an agent (e.g., an exogenous polypeptide) covalently linked to a second click loop capable of react with the first click loop, and (c) put the activated cell in contact with the activated agent, thus producing a cell functionalized with the agent.
[25]
25. Enucleated erythroid cell, characterized by the fact that it is produced by the method, as defined in claim 24.
[26]
26. Method of administering an agent to a human individual, characterized in that it comprises administering an effective amount of cells, as defined in any one of claims 1 to 19 to the human individual, thereby administering the agent to the human individual.
[27]
27. Cell according to any of claims 1 to 19, characterized by the fact that it is for use as a medicine.

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法律状态:
2021-10-19| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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