WO2020106710A1 - Veterinary device for adoptive cell therapy - Google Patents

Abstract

The present technology provides methods, devices, and kits for preparing a stabilized protein-infused nucleated blood cell suspension with greater ease of use, portability, and sterility. The suspension can be prepared by first adding a quantity of whole blood to a bag having a top end and a bottom end. The blood can be added through a first port on the bag. The first port on the bag is coupled to a first end of a first adapter. The quantity of blood is added to a second end of the adapter.

Description

VETERINARY DEVICE FOR ADOPTIVE CELL THERAPY

CROSS-REFERENCE TO RELATED APPLICATION

{0001] This application claims priority to U.S. provisional application no. 62/769,973, filed November 20, 2018, the entire contents of which are incorporated herein by reference.

FIELD OF USE

{0002] The following disclosure is directed generally to the therapeutic utilization of protein-infused nucleated blood cells, including kits and devices for creating, enriching, incubating, and stabilizing a concentration of protein-infused nucleated blood cells, and methods for separating and infusing the cells with exogenous proteins.

BACKGROUND

[0003] Preparation of stabilized cells obtained from primary blood sources and treated with exogenous proteins is important for a variety therapeutic applications, including adoptive cell therapy. Improved methods are needed for efficient, sterile administration without need for significant manipulation of the blood samples.

SUMMARY

[0004] The present technology overcomes the drawbacks of previous systems by providing methods, devices, and kits for preparing a stabilized protein-infused nucleated blood cell suspension with greater ease of use, portability, and sterility than the existing art. In some embodiments the suspension can be prepared by first adding a quantity of whole blood to a bag having a top end and a bottom end. In some embodiments, the blood can be added through a first port on the bag. In some embodiments, the first port on the bag is coupled to a first end of a first adapter. In some embodiments, the quantity of blood is added to a second end of the adapter. In some embodiments, the bag further contains an erythrocyte sedimentation solution. In some embodiments, the whole blood and the erythrocyte sedimentation solution are then mixed in the bag and placed in an upright position to allow the erythrocytes from the whole blood to form an erythrocyte layer at the bottom end of the bag and a plasma and leukocyte layer on top of the erythrocyte layer. In some embodiments, the erythrocyte layer is then removed. In some embodiments, the erythrocyte layer is removed through a second port at the bottom end of the bag leaving the plasma and leukocyte layer in the bag. In some embodiments, the second port on the bag is operatively connected to a conduit that optionally comprises a roller clamp and/or syringe for removal of the erythrocyte layer through the second port. In some embodiments, a vial containing a composition comprising a stabilizing agent is then coupled to the bag. In some embodiments, the stabilizing agent is a recombinant protein. In some embodiments, the protein stabilizes or increases the viability of cells in the blood cell suspension. In some embodiments, a vial containing a recombinant protein is coupled to the bag via the second end of the first adapter coupled to the first port on the bag. In some embodiments, a vial containing a recombinant protein is coupled to the bag via a second adapter coupled to a third port on the bag. By mixing the composition having the recombinant protein with the plasma and leukocyte layer in the bag, a stabilized protein-infused nucleated blood cell suspension is generated. Exposure of the stabilizing agent to lymphoid cells can result in a transient increase of cell survival proteins.

[0005] In certain embodiments of the methods, the recombinant protein composition is a lyophilized composition. In some embodiments, all items other than the recombinant protein may be stored at about 2°C to about 28°C prior to use, and the recombinant protein may be stored at about -20°C to about -80°C prior to use. In certain embodiments of the methods, the recombinant protein composition is mixed with the plasma and leukocyte layer in the bag by compressing the bag so that the plasma and leukocyte layer enters the vial through the first or second adapter to resuspend the lyophilized composition in the vial. In such embodiments, the vial is then inverted to allow the resuspended protein to flow back into the bag. In certain embodiments of the methods, the composition comprising the recombinant protein is a liquid composition. In certain embodiments of the methods, the protein-infused nucleated blood cell suspension is further incubated for about 1 hour to about 5 hours with the recombinant protein. In certain embodiments of the methods, the protein-infused nucleated blood cell suspension is further incubated for about 1 hour to about 5 hours with the recombinant protein at about 10- 30 °C. In certain embodiments of the methods, the protein-infused nucleated blood cell suspension is stored at about 2°C to about 8°C following incubation with the recombinant protein. In some embodiments, the protein-infused nucleated blood cell suspension is stable for about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days when stored at about 2°C to about 8°C compared to a blood sample that has not been infused with the recombinant protein. [0006] In certain embodiments of the methods, the protein-infused nucleated blood cell suspension is extracted from the bag. In one embodiment, the protein-infused nucleated blood cell suspension is extracted through a fourth port on the bag.

[0007] In some embodiments of the method, the first or second adapter is a luer lock adapter. In some embodiments, the luer lock adapter is a single female luer lock adapter or a double female luer lock adapter. In other embodiments, the luer lock adapter is a single male luer lock adapter or a double male luer lock adapter. In some embodiments, the first or second adapter on the bag is a Y -shaped adapter having a first branch, a second branch and a connector. In one embodiment of the method, the vial is coupled to the first branch of the Y-shaped adapter and the protein-infused nucleated blood cell suspension is extracted through the second branch of the Y-shaped adapter. In some embodiments, the quantity of whole blood added to the bag is about 10-60 mL. In some embodiments, the quantity of whole blood added to the bag is collected from a mammalian subject. Mammalian subjects include, but are not limited to, veterinary subjects, including dogs, cats, and horses. In certain embodiments, the mammalian subject is a human.

[0008] In some embodiments, an anticoagulant is added to and/or mixed with the quantity of whole blood prior to adding the whole blood to the bag. In some embodiments, the anticoagulant is pre-loaded into a syringe for collection of the whole blood sample. In some embodiments, a syringe containing an anticoagulant is used to extract the whole blood. In one embodiment, the syringe may be anywhere from an about 1 mL to an about 60 mL syringe. In some embodiments, the anticoagulant is pre-loaded into a syringe for collection of the whole blood sample. In some embodiments, the anticoagulant comprises heparin. In some embodiments, the anticoagulant comprises about 200 to about 1500 USP units of heparin sodium. In some embodiments, the anticoagulant comprises about 1000 USP units of heparin sodium. In some embodiments, the anticoagulant may be Heparin Sodium Injection, USP.

[0009] In some embodiments, the erythrocyte sedimentation solution comprises hetastarch (hydroxyethyl starch) and sodium chloride. In some embodiments, the erythrocyte sedimentation solution comprises about 0.5% by weight to about 10% by weight hetastarch. In some embodiments, the erythrocyte sedimentation solution comprises about 1.2% by weight hetastarch or 0.6 g hetastarch in 50 mL of erythrocyte sedimentation solution. In some embodiments, erythrocyte sedimentation solution comprises about 0.5% by weight to about 5% by weight sodium chloride. In some embodiments, erythrocyte sedimentation solution comprises about 0.9% by weight or 0.45 g sodium chloride in 50 mL of erythrocyte sedimentation solution. In some embodiments, erythrocyte sedimentation solution comprises about 1.2% by weight hetastarch and about 0.9% by weight sodium chloride.

[0010] In some embodiments, the recombinant protein comprises a protein transduction domain for uptake of the protein by the nucleated blood cells. In some embodiments, the recombinant protein comprises a polypeptide that promotes the survival and/or proliferation of the nucleated blood cells. In some embodiments, the recombinant protein comprises a MYC polypeptide. In some embodiments, the recombinant protein is a fusion protein. In some embodiments, the recombinant protein is a fusion protein comprising a MYC polypeptide fused to a protein transduction domain. In some embodiments, the recombinant protein is a polypeptide that promotes cell survival. In some embodiments, the recombinant protein is a MYC polypeptide fused to an HIV-TAT protein transduction domain. In some embodiments, the recombinant protein is TAT -MYC. In some embodiments, about 0.5 mg to about 5 mg of the recombinant protein is added to the nucleated blood cells. In some embodiments, about 1.25 mg of the recombinant protein is added to the nucleated blood cells. In other embodiments, about 1.50 mg of the recombinant protein is added to the nucleated blood cells.

[0011] Also provided, in certain embodiments, is a device for making a protein-infused nucleated blood cell suspension. In some embodiments, the device includes a bag having a top end and a bottom end, a first port at the top end, a second port on the bottom end, a third port on the top end, and an erythrocyte sedimentation solution in the bag. In some embodiments, the device includes a conduit that optionally includes a roller clamp, wherein the conduit operatively connects the second port. In some embodiments, a syringe is operatively coupled to the conduit connected to the second port. In some embodiments, a first adapter is operatively coupled to the first port and optionally, a second adapter is operatively coupled to the third port. In some embodiments, the first or second adapter is a luer lock adapter. In some embodiments, the first or second adapter is a Y-shaped adapter, having a first branch, a second branch, and a connector. In some embodiments, a spike adapter is coupled to the second branch of the Y-shaped adapter. In some embodiments, the erythrocyte sedimentation solution of the device comprises about 0.5-5% by weight hetastarch. In some embodiments, the erythrocyte sedimentation solution of the device comprises about 1.2% by weight hetastarch. In some embodiments, erythrocyte sedimentation solution of the device comprises about 0.5-5% by weight sodium chloride. In some embodiments, erythrocyte sedimentation solution of the device comprises about 0.9% by weight sodium chloride. In some embodiments, the erythrocyte sedimentation solution of the device comprises about 1.2% by weight hetastarch and about 0.9% by weight sodium chloride in about 50 mL of erythrocyte sedimentation solution.

[0012] Also provided, in certain embodiments, is a kit that includes the device disclosed above and a vial containing a composition comprising an isolated protein. In some embodiments, the vial contains a composition comprising a recombinant protein. In some embodiments, the composition comprising a recombinant protein is a lyophilized composition. In some embodiments, the recombinant protein comprises a protein transduction domain for uptake of the protein by the nucleated blood cells. In some embodiments, the recombinant protein comprises a polypeptide that promotes the survival and/or proliferation of the nucleated blood cells. In some embodiments, the recombinant protein comprises a MYC polypeptide. In some embodiments, the recombinant protein is a fusion protein. In some embodiments, the recombinant protein is a fusion protein comprising a MYC polypeptide fused to a protein transduction domain. In some embodiments, the recombinant protein is a polypeptide that promotes cell survival. In some embodiments, the recombinant protein is a MYC polypeptide fused to an HIV-TAT protein transduction domain. In some embodiments, the recombinant protein is TAT -MYC. In some embodiments, the vial contains about 0.5 mg to about 5 mg of the recombinant protein. In some embodiments, the vial contains about 1.25 mg of the recombinant protein. In some embodiments, the kit further includes one or more devices for the collection of a whole blood sample from a subject, including, but not limited to, a hemostat, a collection syringe, an anticoagulant and a vial adapter. In some embodiments, the anticoagulant is heparin. In certain aspects of the disclosure, any item in the kit other than the stabilizing agent may be stored at about 2°C to about 28°C prior to use and the vial of stabilizing agent is stored at about -20°C to about -80°C prior to use.

[0013] The kit, device, and methods of the present technology overcome shortcomings of existing art in the field of use by providing a simplified, sterile apparatus and method with desired mobility, affordability, and ease of use.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 illustrates an exemplary view of the kit, including the device, a vial of recombinant proteins, a hemostat, a syringe, and a vial adapter. [0015] FIG. 2 shows an embodiment of the device with a Y-shaped adapter having a first branch, a second branch, and a connector.

[0016] FIG. 3 illustrates an exemplary method step where a sample of blood is transferred to the bag.

[0017] FIG. 4 illustrates an exemplary method step where blood components are separated into different layers for extraction.

[0018] FIG. 5 illustrates an exemplary method step of removal of the red blood cell layer from the bag.

{0019] FIG. 6 illustrates an exemplary method step where the recombinant protein vial being attached to the bag.

[0020] FIG. 7 shows an exemplary graph depicting the total number of nucleated cells over four twenty-four-hour time intervals (four days).

[0021] FIG. 8 shows an exemplary graph depicting cell viability using the method of the present invention over four twenty-four-hour time intervals (four days).

[0022] FIG. 9 depicts an exemplary view of the kit packaged with the assembly bag visible in conjunction other components.

[0023] FIG. 10 depicts an exemplary view of the kit packaged with the roller clamp and conduit visible.

DETAILED DESCRIPTION

Definitions

[0024J The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. As used herein, the singular forms "a",“an” and“the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0025] As used herein, the term“about” means that a value can vary +/- 20%, +/- 15%, +/- 10% or +/- 5% and remain within the scope of the present disclosure. [0026] As used herein, the term“administration” of an agent to a subj ect includes any route of introducing or delivering the agent to a subject to perform its intended function. Administration can be carried out by any suitable route, including intravenously, intramuscularly, intraperitoneally, or subcutaneously. Administration includes self administration and the administration by another.

[0027] The terms“polypeptide,”“peptide,” and“protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non- naturally occurring amino acid, e.g., an amino acid analog. The terms encompass amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0028] As used herein, the term“effective amount” or“therapeutically effective amount” refers to a quantity of an agent sufficient to achieve a desired therapeutic effect. In the context of therapeutic applications, the amount of a therapeutic peptide administered to the subject can depend on the type and severity of the infection and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It can also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.

[0029] The terms“lyophilized,”“lyophilization” and the like as used herein refer to a process by which the material (e.g., nanoparticles) to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment. An excipient can be included in pre-lyophilized formulations to enhance stability of the lyophilized product upon storage. The lyophilized sample can further contain additional excipients.

[0030] As used herein, the term“nucleated blood cell” refer to any blood cell that has a nucleus. Generally, nucleated blood cells exclude mature erythrocytes, which are anucleated.

[0031] As used herein, the term immune cell refers to any cell that plays a role in the immune response. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, dendritic cells, eosinophils, neutrophils, mast cells, basophils, and granulocytes. [0032] The term “lymphocyte” refers to all immature, mature, undifferentiated and differentiated white lymphocyte populations including tissue specific and specialized varieties. It encompasses, by way of non-limiting example, B cells, T cells, NKT cells, and NK cells.

[0033] As used herein "adoptive cell therapeutic composition" refers to any composition comprising cells suitable for adoptive cell transfer. In exemplary embodiments, the adoptive cell therapeutic composition comprises a cell type selected from a group consisting of a tumor infiltrating lymphocyte (TIL), TCR (i.e. heterologous T-cell receptor) modified lymphocytes and CAR (i.e. chimeric antigen receptor) modified lymphocytes. In another embodiment, the adoptive cell therapeutic composition comprises a cell type selected from a group consisting of T-cells, CD8+ cells, CD4+ cells, NK-cells, delta-gamma T-cells, regulatory T-cells and peripheral blood mononuclear cells. In another embodiment, TILs, T-cells, CD8+ cells, CD4+ cells, NK-cells, delta-gamma T-cells, regulatory T-cells or peripheral blood mononuclear cells form the adoptive cell therapeutic composition. In one embodiment, the adoptive cell therapeutic composition comprises T cells.

[0034] As used herein, “protein-infused” means incorporating or containing proteins delivered to desired cells through the means or methods of the present methods, device, or kit.

{0035] As used herein,“recombinant protein” means any protein encoded by a gene with recombinant DNA that has been reproduced in a system that supports expression of the recombinant DNA.

[0036] The terms“MYC” and“MYC gene” are synonyms. They refer to a nucleic acid sequence that encodes a MYC polypeptide. A MYC gene comprises a nucleotide sequence of at least 120 nucleotides that is at least 60% to 100% identical or homologous, e.g., at least 60, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other percent from about 70% to about 100% identical to sequences of NCBI Accession Number NM— 002467. In some embodiments, the MYC gene is a proto-oncogene. In certain instances, a MYC gene is found on chromosome 8, at 8q24.21. In certain instances, a MYC gene begins at 128,816,862 bp from pter and ends at 128,822,856 bp from pter. In certain instances, a MYC gene is about 6 kb. In certain instances, a MYC gene encodes at least eight separate mRNA sequences— 5 alternatively spliced variants and 3 unspliced variants. [0037] The terms “MYC protein,” “MYC polypeptide,” and “MYC sequence” are synonyms and refer to the polymer of amino acid residues disclosed in NCBI Accession Number UniProtKB/Swiss-Prot:P01106.1 (MYC isoform 1) or NP_002458.2 (UniProtKB/Swiss-Prot:P01106.2; MYC isoform 2), and functional homologs, analogs or fragments thereof. The sequence of or UniProtKB/Swiss-Prot:P01106.1 is:

MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLP TPPL SP SRRSGLC SP S YVAVTPF SLRGDNDGGGGSF ST ADQLEM VTELLGGDMVNQ S FICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCS TSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQ GSPEPL VLHEETPPTT S SD SEEEQEDEEEID VV S VEKRQ APGKRSESGSP S AGGHSKPP HSPLVLKRCH V STHQHNY AAPP STRKD YP AAKRVKLD S VRVLRQISNNRKCT SPRS S DTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSV Q AEEQKLISEEDLLRKRREQLKHKLEQLRN SC A (SEQ ID NO: 2)

[0038] The sequence of NP 002458.2 (UniProtKB/Swiss-Prot: P01106.2) is:

MDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQ PP AP SEDIWKKFELLPTPPL SP SRRS GLC SP S Y V A VTPF SLRGDNDGGGGSF S T ADQLE MVTELLGGDMVNQ SFICDPDDETFIKNIIIQDCMW SGF S AAAKL VSEKL AS YQ AARK DSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAF SPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKR SESGSP S AGGHSKPPHSPL VLKRCHVSTHQHNY AAPP STRKD YP AAKRVKLD SVRVL RQI SNNRKC T SPRS SDTEEN VKRRTHNVLERQRRNELKRSFF ALRD QIPELENNEK AP K V VILKK AT A YIL S VQ AEEQKLI SEEDLLRKRREQLKHKLEQLRN S C A (SEQ ID NO: 3)

[0039] In some embodiments, the MYC polypeptide is a complete MYC polypeptide sequence. In some embodiments, the MYC polypeptide is a partial MYC polypeptide sequence. In some embodiments, the MYC polypeptide comprises at least 400 consecutive amino acids of SEQ ID NO: 2. In some embodiments, the MYC polypeptide comprises at least 400 consecutive amino acids of SEQ ID NO: 2 and retains at least one MYC activity. In some embodiments, the MYC polypeptide comprises at least 400, at least 410, at least 420, or at least 430 consecutive amino acids of SEQ ID NO: 2. In some embodiments, the MYC polypeptide comprises at least 400, at least 410, at least 420, or at least 430 consecutive amino acids of SEQ ID NO: 2 and retains at least one MYC activity. In some embodiments, the MYC polypeptide is c-MYC. In some embodiments, the MYC polypeptide sequence comprises the sequence shown below:

MDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQ PP AP SEDIWKKFELLPTPPL SP SRRS GLC SP S Y V A VTPF SLRGDNDGGGGSF S T ADQLE MVTELLGGDMVNQ SFICDPDDETFIKNIIIQDCMW SGF S AAAKL VSEKL AS YQAARK DSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAF SPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKR SESGSP S AGGHSKPPHSPL VLKRCHVSTHQHNY AAPP STRKDYP AAKRVKLD SVRVL RQI SNNRKC T SPRS SDTEEN VKRRTHNVLERQRRNELKRSFF ALRD QIPELENNEK AP K V VILKK AT A YIL S VQ AEEQKLI SEEDLLRKRREQLKHKLEQLR (SEQ ID NO: 4).

[0040] In some embodiments, the MYC polypeptide sequence comprises the sequence shown below:

PLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPT PPLSP SRRSGLC SP S YVAVTPF SLRGDNDGGGGSF ST ADQLEMVTELLGGDMVNQ SFI CDPDDETFIKNIIIQDCMW SGF S AAAKL V SEKL AS Y Q AARKD SGSPNP ARGHS VC STS SLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGS PEPL VLHEETPPTT S SD SEEEQEDEEEID VV S VEKRQ APGKRSESGSP S AGGHSKPPHS PL VLKRCH V STHQHN Y AAPP S TRKDYP AAKRVKLD SVRVLRQI SNNRKC T SPRS SDT EENVKRRTHNVLERQRRNELKRSFF ALRDQIPELENNEK APK VVILKK AT AYILS VQ AEEQKLI SEEDLLRKRREQLKHKLEQLR (SEQ ID NO: 5).

[0041 j In some embodiments, a MYC polypeptide comprises an amino acid sequence that is at least 40% to 100% identical, e.g ., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or any other percent from about 40% to about 100% identical to the sequence of NCBI Accession Number NP002458.2 or UniProtKB/Swiss-Prot Accession Number P01106.1. In some embodiments, MYC polypeptide refers to a polymer of 439 amino acids, a MYC polypeptide that has not undergone any post-translational modifications. In some embodiments, MYC polypeptide refers to a polymer of 439 amino acids that has undergone post-translational modifications. In some embodiments, the MYC polypeptide is 48,804 kDa. In some embodiments, the MYC polypeptide contains a basic Helix-Loop-Helix Leucine Zipper (bHLH/LZ) domain. In some embodiments, the bHLH/LZ domain comprises the sequence of:

ELKRSFF ALRDQIPELENNEK APK V VILKK AT A YIL S VQ AEEQKLI SEEDLLRKRREQL KHKLEQLR (SEQ ID NO: 6). In some embodiments, the MYC polypeptide is a transcription factor ( e.g ., Transcription Factor 64). In some embodiments, the MYC polypeptide contains an E-box DNA binding domain. In some embodiments, the MYC polypeptide binds to a sequence comprising CACGTG. In some embodiments, the MYC polypeptide promotes one or more of cell survival and/or proliferation. In some embodiments, a MYC polypeptide includes one or more of those described above, and includes one or more post-translational modifications (e.g., acetylation). In some embodiments, the MYC polypeptides comprise one or more additional amino acid residues at the N-terminus or C-terminus of the polypeptide. In some embodiments, the MYC polypeptides are fusion proteins. In some embodiments, the MYC polypeptides are linked to one or more additional peptides at the N-terminus or C-terminus of the polypeptide.

[0042] Proteins suitable for use in the methods described herein also includes functional variants, including proteins having between 1 to 15 amino acid changes, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions, deletions, or additions, compared to the amino acid sequence of any protein described herein. In other embodiments, the altered amino acid sequence is at least 75% identical, e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein inhibitor described herein. Such sequence-variant proteins are suitable for the methods described herein as long as the altered amino acid sequence retains sufficient biological activity to be functional in the compositions and methods described herein. Where amino acid substitutions are made, the substitutions can be conservative amino acid substitutions. Among the common, naturally occurring amino acids, for example, a“conservative amino acid substitution” is illustrated by a substitution among amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine. The BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff et ah, (1992), Proc. Natl Acad. Sci. USA, 89: 10915- 10919). Accordingly, the BLOSUM62 substitution frequencies are used to define conservative amino acid substitutions that, in some embodiments, are introduced into the amino acid sequences described or disclosed herein. Although it is possible to design amino acid substitutions based solely upon chemical properties (as discussed above), the language “conservative amino acid substitution” preferably refers to a substitution represented by a BLOSUM62 value of greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. According to this system, preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).

[00431 The term“MYC activity” or“MYC biological activity” or“biologically active MYC” includes one or more of enhancing or inducing cell survival, cell proliferation, and/or antibody production. By way of example and not by way of limitation, MYC activity includes enhancement of expansion of anti-CD3 and anti-CD28 activated T-cells and/or increased proliferation of long-term self-renewing hematopoietic stem cells. MYC activity also includes entry into the nucleus of a cell, binding to a nucleic acid sequence (e.g., binding an E-box sequence), and/or inducing expression of MYC target genes.

[0044] The terms“patient,”“subject,”“individual,” and the like are used interchangeably herein, and refer to an animal, typically a mammal. In one embodiment, the patient, subject, or individual is a mammal. In one embodiment, the patient, subject or individual is a human. In some embodiments the patient, subject or individual is an animal, such as, but not limited to, domesticated animals, such as equine, bovine, murine, ovine, canine, and feline.

[0045j The terms“protein transduction domain (PTD)” or“transporter peptide sequence” (also known as cell permeable proteins (CPP) or membrane translocating sequences (MTS)) are used interchangeably herein to refer to small peptides that are able to ferry much larger molecules into cells independent of classical endocytosis. In some embodiments, a nuclear localization signal can be found within the protein transduction domain, which mediates further translocation of the molecules into the cell nucleus.

[0046] The terms“treating” or“treatment” as used herein covers the treatment of a disease in a subject, such as a human, and includes: (i) inhibiting a disease, i.e., arresting its development; (ii) relieving a disease, i.e., causing regression of the disease; (iii) slowing progression of the disease; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease. With respect to a melanoma,“treating” or“treatment” also encompasses regression of a tumor, slowing tumor growth, inhibiting metastasis of a melanoma tumor, inhibiting relapse or recurrent melanoma and/or maintaining remission.

[0047] It is also to be appreciated that the various modes of treatment or prevention of medical diseases and conditions as described are intended to mean“substantial,” which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved. The treatment can be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition. j0048| The term“therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0049] As used herein,“spike adapter” means any male adapter that permits connection to a non-vented port or female adapter wherein the spike element helps maintain sterility and securely fitted coupling between the connections.

|0050) As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as“up to,”“at least,” “greater than,”“less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

|005l | Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

Kit and Device [0052] A kit, device, and method for preparing a protein-infused nucleated blood cell suspension is provided herein. Referring to FIG. 1, an exemplary kit used to create a protein- infused nucleated blood cell suspension in accordance with the principles of the present disclosure is described. The kit illustratively includes device 10 for making a protein-infused nucleated blood suspension. The device incorporates bag 100 having top end 102 and bottom end 104. Bag 100 further has a first port 106 at top end 102, a second port 108 at bottom end 104, and third port 110 at top end 102. In one embodiment, bag 100 contains an erythrocyte sedimentation solution.

[0053] Device 10 further includes conduit 112 which optionally includes roller clamp 141. Conduit 112 operatively connects to second port 108. Extraction syringe 116 can be operatively coupled to conduit 112. First adapter 118 can be operatively coupled to first port 106, and, optionally, second adapter 120 can be operatively coupled to third port 110. First adapter 118 or second adapter 120 can also be a luer lock adapter 122.

[0054] In addition to device 10, kit 1 further can include vial 124 containing a recombinant protein composition. In some embodiments, the recombinant protein composition can be lyophilized. The recombinant protein can be protein that stabilizes the cells in a blood cell sample, such as a TAT-MYC protein, or any recombinant protein or combination of one or more recombinant proteins. Kit 1 further can include one or more devices for the collection of a whole blood sample from a subject. In some embodiments, kit 1 includes a hemostat 126, a blood collection syringe 127, an anticoagulant, and a vial adapter 129. In one embodiment, the anticoagulant is heparin. In certain embodiments, 1000 USP units of heparin sodium can be used as the anticoagulant.

[0055] Referring to FIG. 2, in some alternative embodiments, first adapter 118 or second adapter 120 can be Y-shaped adapter 130. Y-shaped adapter 130 can have a first branch 132, a second branch 134, and connector 136 to operatively connect to one of the bag ports. Device 10 further can comprise spike adapter 137 coupled to second branch 134 of Y-shaped adapter

130

[0056] The erythrocyte sedimentation solution added to bag 100 can include hetastarch and/or sodium chloride. In some embodiments, the erythrocyte sedimentation solution includes about 1.2% by weight hetastarch. In some embodiments, the erythrocyte sedimentation solution includes about 0.9% by weight sodium chloride. [0057] Referring to FIGS. 9 and 10, in some embodiments, kit 1 can be enclosed in packaging. The first layer of packaging shown in FIG. 9, comprises blood collection syringe 127, anticoagulant vial 128, vial adapter 129, luer lock adapter 122, and bag 100. The second, or lower, layer of packaging, represented in FIG. 10, houses roller clamp 141, packaged underneath where bag 100 is depicted in FIG. 10.

Methods for Preparing Protein-Infused Nucleated Blood Cell Suspension

Blood Collection

[0058] Methods of preparing a protein infused nucleated blood cell suspension are also provided. Blood can first be collected from a subject. In one alternative method, an appropriately sized needle can be attached to an about 30 cc blood collection syringe 127 to withdraw about 0.5 mL of anticoagulant from anticoagulant vial 128. The first needle on blood collection syringe 127 can then be replaced with a new needle appropriately sized for venipuncture. From about 10 to about 60 mL of blood can be withdrawn from the patient into blood collection syringe 127 containing anticoagulant.

[0059] The source of the blood sample can be from any from any animal, typically a mammal. In some embodiments, the blood sample is obtained from a mammal. In some embodiments, the blood sample is obtained from a veterinary subject, such as a domesticated animal, such as, but not limited to, a horse, cow, camel, llama, alpaca, pig, sheep, goat, donkey, mule, dog, and cat. In some embodiments, the blood sample is obtained from a human.

Transferring the Blood to the Bag Assembly

[0060] Referring to FIG. 1, one method includes adding this quantity of whole blood to bag 100 of device 10, where bag 100 has top end 102 and bottom end 104. The blood can be added through first port 106 on bag 100 and first port 106 is coupled to first end 117 of first adapter 118, and the quantity of blood is added to second end 119 of first adapter 118. Bag 100 further can have an erythrocyte sedimentation solution.

[0061 ] Referring to FIG. 3, in one alternative of the method, luer lock adapter 122 can be sanitized prior to adding blood through first port 106. The whole blood and the erythrocyte sedimentation solution can be mixed in bag 100. Once the quantity of collected blood is added to bag 100, blood collection syringe 127 can be removed from first port 106 and discarded. Separation of Blood Components

[0062] Referring to FIG. 4, bag 100 can be placed in an upright position to allow erythrocytes from the whole blood to form erythrocyte layer 138 and plasma and leukocyte layer 140. In one alternative of the method, bag 100 can be suspended at about room temperature (e.g., about 20°C ± about 5°C) for about 60±15 minutes. Sufficient separation has occurred when a visible interface between erythrocyte layer 138 and plasma and leukocyte layer 140 is observed. If a visible interface is not apparent, then allow for another about 30 minutes to elapse for more separation to occur. If there is still no visible layer between erythrocyte layer 138 and plasma and leukocyte layer 140, then this cycle can be repeated every about 30 minutes until a visible interface is observed.

Removal of the Erythrocyte Cell Laver

[0063] Referring to FIG. 5, erythrocyte layer 138 can be removed through second port 108 at bottom end 104 of bag 100, leaving plasma and leukocyte layer 140 in bag 100. Referring to FIG. 5, erythrocyte layer 138 can be removed by moving roller clamp 141 from roller clamp closed position 142 to roller clamp open position 144. Erythrocyte layer 138 then flows through second port 108, enters conduit 112, and travels to extraction syringe 116. After removal of erythrocyte layer 138 through conduit 112, roller clamp 141 can be moved back to roller clamp closed position 142. Extraction syringe 116 can then be capped and discarded.

Attaching the Vial of Recombinant Protein to the Bag

[0064] Referring to FIG. 6, protein vial 124 can be coupled to second end 119 of first adapter 118 coupled to first port 106 on bag 100. Alternatively, protein vial 124 can be coupled to second adapter 120 coupled to third port 110 on bag 100. Protein vial 124 contains a composition of recombinant proteins 125. In one alternative of the method, a cap can be removed from recombinant protein vial 124 and wiped with an alcohol swab, and vial adapter 129 can be firmly inserted onto protein vial 124. In another alternative method, first port 106 or third port 110 have a luer lock adapter 122 that connects to protein vial 124.

Transferring the Recombinant Protein to the Bag Assembly and Mixing

[0065] The composition remaining in the bag, including plasma and leukocyte layer 140 and recombinant proteins 125 can be mixed to generate a protein-infused nucleated blood cell suspension. In one alternative of the method, bag 100 can be inverted so that recombinant protein vial 124 is below bag 100. Bag 100 can be squeezed to allow liquid, including plasma and leukocyte layer 140, to transfer into recombinant protein vial 124, reanimating recombinant proteins 125. This inverting and squeezing process can be continued until about half the volume of recombinant protein vial 124 is filled. The assembly can then be inverted again so that recombinant protein vial 124 is above bag 100, at which point bag 100 can be squeezed to transfer air from bag 100 into recombinant protein vial 124. This squeezing process should be continued until any liquid in recombinant protein vial 124, including recombinant proteins 125, is transferred to bag 100. When this is completed, recombinant protein vial 124 can be removed from the respective adapter.

[0066] In one alternative of the method, bag 100 can then be mixed to ensure thorough distribution of recombinant proteins 125 throughout the composition and hung about an hour at about room temperature or about 20°C ± 5°C. In another alternative, the protein-infused nucleated blood cell suspension can be incubated for about an hour at about 10-30°C. In another alternative of the method, bag 100 including the protein-infused nucleated blood cell suspension can then be stored for up to four days from about 2°C to about 8°C. In yet another alternative, the protein-infused nucleated blood cell suspension can be stable for about 3, 4, 5, 6, 7, 8, 9, 10 or more days when stored at about 2-8°C when compared to a blood sample that has not been infused with recombinant proteins 125.

Alternative Method:

Blood Collection

[0067] In one alternative of the method, a user may first attach an appropriately sized needle to an about 1 mL syringe and withdraw a measured amount of anticoagulant and air from a vial containing anticoagulant to the 1 mL syringe, after which they may then discard the needle. The user may then connect a double female luer lock adapter to an about 20 mL syringe and transfer the anticoagulant from the about 1 mL syringe to the about 20 mL syringe. The user may then attach an appropriately sized needle to the 20 mL syringe and safely obtain a measured amount of blood from a subject, rotating the syringe several times to ensure adequate mixing of blood and anticoagulant. Blood Processing

[0068] After blood collection, a vial of stabilizing agent may be removed from frozen storage and placed in conditions adequate to thaw the vial of stabilizing agent. The tubing with the roller clamp may be connected to a luer lock adapter at the bottom of the assembly bag with the roller clamp in the closed position. An about 60 mL syringe may then be connected to the tubing. The vial of erythrocyte sedimentation solution can then be attached to an about 20 mm Vial Adapter. Using the about 60 mL syringe, a measured amount of erythrocyte sedimentation solution and air can be withdrawn from the vial of erythrocyte sedimentation solution and transferred to the bag assembly via a top luer lock adapter. The syringe may then be removed and discard. Then, a user may inject the 20 mL blood-filled syringe from the previous steps into the injection port of the bag assembly, slowly transferring the blood into the bag assembly, after which the about 20 mL syringe may be removed and discarded. The bag may then be gently kneaded to mix and create a homogenous suspension. The bag should then be suspended, undisturbed, at around room temperature (about 20°C ± about 5°C) for anywhere from about 45 to about 75 minutes. The red blood cells should sediment to the bottom of the bag during this time. When a visible interface between the plasma/leukocyte layer and red blood cell layer is observed sedimentation is complete. If a visible interface is not apparent after the first about 45 to about 75 minutes, then another 30 minutes should be allowed for separation to occur. This process may be repeated in 30-minute cycles, for up to a total sedimentation time of about 4 hours, until a visible interface is observed. After sedimentation, the roller clamp on the tubing at the bottom of the assembly bag may be slowly opened, and, using, for example, an about 60 mL syringe attached to tubing, the red blood cell layer may be slowly withdrawn from the assembly bag. The roller clamp on the tubing should then be closed removed from the luer lock valve. The tubing and the syringe filled with the red blood cell layer should then be discarded.

Cell Stabilization

[0069] After the vial of stabilizing agent has completely thawed, an about 13 mm vial adapter may be attached to the vial of stabilizing agent. An about 3 mL syringe may be attached to an adapter on the vial of the stabilizing agent and used to withdraw a measured amount of stabilizing agent and air from the vial. The stabilizing agent and air should then be transferred to the bag assembly via the luer lock adapter. To help ensure thorough distribution of the stabilizing agent and the plasma or leukocyte layer, the assembly bag should be gently kneaded or compressed. The bag may then be left, undisturbed, at or around room temperature (20°C ± 5°C) for about 60 minutes. After these steps are complete, the bag of preserved cells can be stored at about 2°C to about 8°C for a number of days, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

Additional Embodiments

[0070] In one alternative method, recombinant proteins 125 is a lyophilized composition. In another alternative, when mixing recombinant proteins 125 and plasma and leukocyte layer 140, bag 100 is compressed so that plasma and leukocyte layer 140 enters recombinant protein vial 124 through first adapter 118 or second adapter 120 to resuspend the lyophilized composition in recombinant protein vial 124. Recombinant protein vial 124 can then be inverted to allow resuspended recombinant proteins 125 to flow back into bag 100. In another alternative method, recombinant proteins 125 can be a liquid composition.

[0071] After any of the previously mentioned methods, the protein-infused nucleated blood cell suspension can be extracted from the bag. In one alternative method, the protein-infused nucleated blood cell suspension can be extracted through a fourth port 200 on the bag. Further, in any of the above-mentioned methods, first adapter 118 or second adapter 120 can be Y- shaped adapter 130 having a first branch 132, a second branch 134 and a connector 136. In one alternative method, recombinant protein vial 124 can be coupled to first branch 132 of Y- shaped adapter 130, and the protein-infused blood cell suspension can be extracted through second branch 134 of Y-shaped adapter 130.

[0072] In any of the above-disclosed methods, the quantity of whole blood added to the bag can be from about 10 mL to about 60 mL. Further, in any of the above-disclosed methods, the quantity of whole blood can be collected from a mammalian subject, and further, the mammalian subject can be a veterinary subject. In some alternatives of the above-disclosed methods, the mammalian subject is a human.

[0073] As mentioned above, the quantity of whole blood extracted from the subject can be collected using a syringe containing an anticoagulant. In some embodiments, the syringe is pre filled with a composition containing the anticoagulant. In some embodiments, the anticoagulant comprises heparin. In some embodiments, the anticoagulant comprises about 200 to 1500 USP units of heparin sodium. In some embodiments, the anticoagulant comprises about 1000 USP units of heparin sodium.

[0074] In some embodiments, the erythrocyte sedimentation solution includes hetastarch (hydroxyethyl starch) and sodium chloride. In some embodiments, the erythrocyte sedimentation solution comprises about 0.5% by weight to about 10% by weight hetastarch. In some embodiments, the erythrocyte sedimentation solution comprises about 1.2% by weight hetastarch. In some embodiments, erythrocyte sedimentation solution comprises about 0.5% by weight to about 5% by weight sodium chloride. In some embodiments, erythrocyte sedimentation solution comprises about 0.9% by weight sodium chloride. In some embodiments, erythrocyte sedimentation solution comprises about 1.2% by weight hetastarch and about 0.9% by weight sodium chloride.

[0075] In some embodiments of the disclosed methods, the stabilizing agent is a recombinant protein. In some embodiments, the recombinant protein 125 comprises a protein transduction domain for uptake of the protein by the nucleated blood cells. In some embodiments, the recombinant protein comprises a polypeptide that promotes the survival and/or proliferation of the nucleated blood cells. In some embodiments, the recombinant protein comprises a MYC polypeptide. In some embodiments, the recombinant protein is a fusion protein. In some embodiments, the recombinant protein is a fusion protein comprising a MYC polypeptide fused to a protein transduction domain. In some embodiments, the recombinant protein is a polypeptide that promotes cell survival. In some embodiments, the recombinant protein is a MYC polypeptide fused to an HIV-TAT protein transduction domain. In some embodiments, the recombinant protein is TAT-MYC. In some embodiments, about 1.25 mg of the recombinant protein is added to the cell suspension. In some embodiments of the disclosed methods, the stabilizing agent further comprises one or additional therapeutic recombinant proteins. In some embodiments of the disclosed methods, the stabilizing agent further comprises one or additional agents to stabilize the cells.

MYC fusion proteins

[0076] In some embodiments, the recombinant protein added to the cell suspension is MYC fusion polypeptide comprising a protein transduction domain (PTD), a MYC

polypeptide that promotes one or more of cell survival or proliferation, and optionally a protein tag domain, e.g ., one or more amino acid sequences that facilitate detection and/or purification of the fusion protein, e.g. a V5 epitope tag and/or 6-histidine tag. In some embodiments, a cell contacted with MYC polypeptide exhibits increased survival time (e.g., as compared to an identical or similar cell of the same type that was not contacted with MYC), and/or increased proliferation (e.g, as compared to an identical or similar cell of the same type that was not contacted with MYC).

[0077] In some embodiments, the MYC fusion protein comprises one or more linker sequences. The linker sequences can be employed to link the protein transduction domain, MYC polypeptide sequence, V5 epitope tag and/or 6-histidine tag of the fusion protein. In some embodiments, the linker comprises one or more amino acids. In some embodiments, the amino acid sequence of the linker comprises KGELNSKLE. In some embodiments, the linker comprises the amino acid sequence of RTG.

[0078] Peptide transport using a PTD fused to the protein provides an alternative for delivery of small molecules, proteins, or nucleic acids across the cell membrane to an intracellular compartment of a cell. One non-limiting example and well-characterized protein transduction domain (PTD) is a TAT-derived peptide. Frankel et al, (see, e.g, U.S. Pat. No. 5,804,604, U.S. Pat. No. 5,747,641, U.S. Pat. No. 5,674,980, U.S. Pat. No. 5,670,617, and U.S. Pat. No. 5,652,122) demonstrated transport of a cargo protein (b-galactosidase or horseradish peroxidase) into a cell by conjugating a peptide containing amino acids 48-57 of TAT to the cargo protein. In some embodiments, TAT comprises an amino acid sequence of MRKKRRQRRR (SEQ ID NO: 7).

[0079] Another non-limiting example of a PTD is penetratin. Penetratin can transport hydrophilic macromolecules across the cell membrane (Derossi et al, Trends Cell Biol., 8:84-87 (1998) incorporated herein by reference in its entirety). Penetratin is a 16 amino acid peptide that corresponds to amino acids 43-58 of the homeodomain of Antennapedia, a Drosophila transcription factor which is internalized by cells in culture.

[0080] Yet another non-limiting example of a PTD is VP22. VP22, a tegument protein from Herpes simplex virus type 1 (HSV-1), has the ability to transport proteins and nucleic acids across a cell membrane (Elliot et al, Cell 88:223-233, 1997, incorporated herein by reference in its entirety). Residues 267-300 of VP22 are necessary but cannot be sufficient for transport. Because the region responsible for transport function has not been identified, the entire VP22 protein is commonly used to transport cargo proteins and nucleic acids across the cell membrane (Schwarze et al, Trends Pharmacol Sci, 21 :45-48, 2000). WO 2020/106710 Attorney DockpcT/uS2019/062200⁰²

[0081] In some embodiments, the PTD-MYC fusion polypeptide includes a protein transduction domain. By way of example, but not by way of limitation, in some

embodiments, the protein transduction domain comprises the protein transduction domain of one or more of TAT, penetratin, VP22, vpr, EPTD, R9, R15, VP16, and Antennapedia. In some embodiments, the protein transduction domain comprises the protein transduction domain of one or more of TAT, penetratin, VP22, vpr, and EPTD. In some embodiments, the protein transduction domain comprises the protein transduction domain of at least one of TAT, penetratin, VP22, vpr, EPTD, R9, R15, VP 16, and Antennapedia. In some

embodiments, the protein transduction domain comprises a synthetic protein transduction domain ( e.g ., polyarginine or PTD-5). In particular embodiments, the protein transduction domain comprises a TAT protein transduction domain. In some embodiments, the protein transduction domain is covalently linked to the MYC polypeptide. In some embodiments, the protein transduction domain is linked to the MYC polypeptide via a peptide bond. In some embodiments, the protein transduction domain is linked to the MYC polypeptide via a linker sequence. In some embodiments, the linker comprises a short amino acid sequence. By way of example, but not by way of limitation, in some embodiments, the linker sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length.

[0082] The MYC fusion protein of the present technology can be arranged in any desired order. For example, in some embodiments, the MYC fusion protein can be arranged in order of a) the protein transduction domain linked in frame to the MYC polypeptide, b) the MYC polypeptide linked in frame to the V5 domain, and c) the V5 domain linked in frame to the 6- histidine epitope tag. In some embodiments, the MYC fusion protein has an order of components of a) the MYC polypeptide linked in frame to the protein transduction domain, b) the protein transduction domain linked in frame to the V5 domain, and c) the V5 domain linked in frame to the 6-histidine epitope tag. In some embodiments, additional amino acid sequences can be included between each of the sequences. In some embodiments, additional amino acids can be included at the start and/or end of the polypeptide sequences.

[0083] In some embodiments, the protein transduction domain is a TAT protein transduction domain. In some embodiments, the protein transduction domain is TAT [48-57] . In some embodiments, the protein transduction domain is TAT [57-48] .

[0084] In some embodiments, the MYC fusion protein comprises a protein tag domain that comprises one or more amino acid sequences that facilitate purification of the fusion protein. In some embodiments, the protein tag domain comprises one or more of a 22

4826 3233 4252.1 WO 2020/106710 Attorney DockpcT/uS2019/062200⁰² polyhistidine tag, and an epitope tag. By way of example, but not by way of limitation, exemplary tags include one or more of a V5, a histidine-tag (e.g, a 6-histidine tag), HA (hemagglutinin) tags, FLAG tag, CBP (calmodulin binding peptide), CYD (covalent yet dissociable NorpD peptide), Strepll, or HPC (heavy chain of protein C). In some

embodiments, the protein tag domain comprises about 10 to 20 amino acids in length. In some embodiments, the protein tag domain comprises 2 to 40 amino acids in length, for example 6-20 amino acids in length. In some embodiments, two of the above listed tags (for example, V5 and the HIS-tag) are used together to form the protein tag domain.

[0085] In some embodiments, the histidine tag is a 6-histidine tag. In some embodiments, the histidine tag comprises the sequence HHHHHH (SEQ ID NO:8). In some embodiments, the fusion peptide disclosed herein comprises a V5 epitope tag. In some embodiments, the V5 tag comprises the amino acid sequence of: GKPIPNPLLGLDST (SEQ ID NO:9). In some embodiments, the V5 tag comprises the amino acid sequence of IPNPLLGLD (SEQ ID NO: 10).

[0086] The protein tags can be added to the fusion protein disclosed herein by any suitable method. By way of example, but not by way of limitation, in some embodiments, a TAT-MYC polypeptide sequence is cloned into an expression vector encoding one or more protein tags, e.g. , a polyHis-tag and/or a V5 tag. In some embodiments, a polyhistidine tag and/or a V5 tag is added by PCR (i.e., the PCR primers comprise a polyhistidine sequence and/ or V5 sequence).

[0087] PTD-MYC fusion polypeptides (e.g, TAT-MYC fusion polypeptide) disclosed herein can be constructed by methods well known in the art. By way of example, but not by way of limitation, a nucleotide sequence encoding a TAT-MYC fusion polypeptide can be generated by PCR. In some embodiments, a forward primer for a human MYC sequence comprises an in-frame N-terminal 9-amino-acid sequence of the TAT protein transduction domain (e.g, RKKRRQRRR). In some embodiments, a reverse primer for a human MYC sequence is designed to remove the stop codon. In some embodiments, the PCR product is cloned into any suitable expression vector. In some embodiments, the expression vector comprises a polyhistidine tag and a V5 tag.

[0088] In some embodiments, a fusion peptide disclosed herein comprises (a) TAT, and (b) c-MYC. In some embodiments, a fusion peptide disclosed herein comprises (a) TAT[48-57], and (b) c-MYC. In some embodiments, a fusion peptide disclosed herein comprises (a)

TAT [57-48], and (b) c-MYC. 23

4826 3233 4252.1 WO 2020/106710 Attorney DockpcT/uS2019/062200⁰²

[0089] In some embodiments, a fusion peptide disclosed herein comprises (a) TAT, (b) c- MYC, (c) linker(s), (d) V5 tag, and (e) 6-histidine tag. In some embodiments, a fusion peptide disclosed herein comprises (a) TAT [48-57], (b) c-MYC , (c) linker(s), (d) V5 tag, and (e) 6-histidine tag. In some embodiments, a fusion peptide disclosed herein comprises (a)

TAT [57-48], (b) c-MYC , (c) linker(s), (d) V5 tag, and (e) 6-histidine tag.

[0090] In some embodiments, the PTD-MYC fusion polypeptide comprises SEQ ID NO:

1; in some embodiments, the PTD-MYC fusion polypeptide is SEQ ID NO: 1.

MRKKRRQRRRPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSE

DIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTEL

LGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSP

NPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSD

SLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGS

P S AGGHSKPPHSPL VLKRCHVSTHQHNY AAPP STRKD YP AAKRVKLD S VRVLRQISN

NRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVIL

KKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRKGELNSKLEGKPIPNPLLG

LD STRT GHHHHHH (SEQ ID NO: 1).

[00911 The fusion protein can be modified during or after synthesis to include one or more functional groups. By way of example but not by way of limitation, the protein can be modified to include one or more of an acetyl, phosphate, acetate, amide, alkyl, and/or methyl group. This list is not intended to be exhaustive and is exemplary only. In some

embodiments, the protein includes at least one acetyl group.

[0092] A PTD-MYC fusion polypeptide can be generated by any suitable method known the art, e.g. by recombinant protein expression in a cell, such as a bacterial cell, an insect cell, or mammalian cell. In some embodiments, a PTD-MYC fusion polypeptide is recombinantly produced by microbial fermentation.

[0093] While various illustrative embodiments of the invention are described above, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the invention. The appended claims are intended to cover all such changes and modifications that fall within the true scope of the invention.

[0094] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the disclosure. All the various embodiments of the present disclosure will not be described 24

4826 3233 4252.1 WO 2020/106710 Attorney DockpcT/uS2019/062200⁰² herein. Many modifications and variations of the disclosure can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0095] It is to be understood that the present disclosure is not limited to particular uses, methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

EXAMPLES

|0096] A stabilized protein-infused nucleated cell suspension was prepared according to the methods described herein using the exemplary device illustrated in FIG. 1 and canine whole blood samples. Data for this experiment is provided in FIGS. 7 and 8. Referring to FIG. 7, a graph depicting the total nucleated cells is shown. Here, data presented in the graph are relative to T = 0 and are the average of n = 3 dogs. In experimental data, the kit, device, and methods provided herein stabilized cells derived from canine whole blood during storage from about 2°C to about 8°C for a number of days, including tests demonstrated over about four days. The number of live total nucleated cells preserved cell suspension for over four days. In contrast, the number of live total nucleated cells of control whole blood from the same dogs steadily declined during storage at about 2°C to about 8°C.

|0097) Referring to FIG. 8, a graph depicting cell viability is shown, with elapsed time depicted on the x-axis and cell viability percentage depicted on the y-axis. Data presented in this graph are the average of n = 3 dogs. Similar to FIG. 7, the cell viability of the preserved nucleated cell suspension was stable over four days. In contrast, the cell viability of control whole blood from the same dogs steadily declined during storage at about 2°C to about 8°C.

25

4826 3233 4252.1

Claims

WHAT IS CLAIMED IS:

1. A method for preparing a protein-infused nucleated blood cell suspension comprising:

(i) adding a quantity of whole blood to a bag comprising a top end and a bottom end, wherein the blood is added through a first port on the bag, wherein the first port on the bag is coupled to a first end of a first adapter, and the quantity of blood is added to a second end of the adapter, and wherein the bag contains a solution comprising an erythrocyte sedimentation solution;

(ii) mixing the whole blood and the erythrocyte sedimentation solution in the bag;

(iii) placing the bag in an upright position to allow the erythrocytes from the whole blood to form an erythrocyte layer at the bottom end of the bag and a plasma and leukocyte layer on top of the erythrocyte layer;

(iv) removing the erythrocyte layer through a second port at the bottom end of the bag leaving the plasma and leukocyte layer in the bag;

(v) coupling a vial to the second end of the first adapter coupled to the first port on the bag or coupling a vial to a second adapter coupled to a third port on the bag, wherein the vial contains a composition comprising a stabilizing agent; and

(vi) mixing the composition comprising the stabilizing agent with the plasma and leukocyte layer in the bag to generate the protein-infused nucleated blood cell suspension.

2. The method of claim 1, wherein the stabilizing agent comprises a recombinant protein.

3. The method of claim 2, wherein the composition comprising the recombinant protein is a lyophilized composition.

4. The method of claim 2, wherein mixing the composition comprising the recombinant protein with the plasma and leukocyte layer in the bag comprises compressing the bag so that the plasma and leukocyte layer enters the vial through the first or second adapter to resuspend the lyophilized composition in the vial; and then inverting the vial to allow the resuspended protein flow back into the bag.

5. The method of claim 2, wherein the composition comprising the recombinant protein is a liquid composition.

6. The method of any of claims 2-5, further comprising incubating the protein-infused nucleated blood cell suspension for about 1 hour at about 10-30 °C.

7. The method of any of claims 2-6, further comprising storing the protein-infused nucleated blood cell suspension at about 2-8 °C.

8. The method of any of claims 2-7, wherein the protein-infused nucleated blood cell suspension is stable for about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days when stored at about 2-8 °C compared to a blood sample that has not been infused with the recombinant protein.

9. The method of any of claims 2-8, wherein the second port is operatively connected to a conduit that optionally comprises a roller clamp and/or syringe for removal of the erythrocyte layer.

10. The method of any of claims 2-9, further comprising extracting the protein-infused nucleated blood cell suspension from the bag.

11. The method of claim 10, wherein the protein-infused nucleated blood cell suspension is extracted through a fourth port on the bag.

12. The method of any of claims 2-11, wherein the first or second adapter is a luer lock adapter.

13. The method of any of claims 2-11, wherein the first or second adapter on the bag is a Y-shaped adapter comprising a first branch, a second branch and a connector.

14. The method of claim 13, wherein the vial is coupled to the first branch of the Y- shaped adapter.

15. The method of claim 13 or claim 14, wherein the protein-infused nucleated blood cell suspension is extracted through the second branch of the Y-shaped adapter.

16. The method of any of claims 1-15, wherein the quantity of whole blood added to the bag is about 10 mL to about 60 mL.

17. The method of any of claims 1-16, further comprising collecting the quantity of whole blood from a mammalian subject.

18. The method of claim 17, wherein the mammalian subject is a veterinary subject.

19. The method of claim 18, wherein the veterinary subject is a dog, cat, or horse.

20. The method of claim 17, wherein the mammalian subject is a human.

21. The method of any of claims 1-20, wherein an anticoagulant is added to the quantity of whole blood prior to adding the whole blood to the bag.

22. The method of any of claims 1-21, wherein the quantity of whole blood is collected using a syringe containing an anticoagulant.

23. The method of either claim 21 or claim 22, wherein the anticoagulant comprises heparin.

24. The method of any of claims 1-23, wherein the erythrocyte sedimentation solution comprises hetastarch and sodium chloride.

25. The method of claim 24, wherein the erythrocyte sedimentation solution comprises about 1.2% by weight hetastarch.

26. The method of claim 24 or claim 25, wherein the erythrocyte sedimentation solution comprises about 0.9% by weight sodium chloride.

27. The method of any of claims 2-26, wherein the recombinant protein comprises a MYC polypeptide.

28. The method of any of claims 2-27, wherein the recombinant protein comprises a protein transduction domain.

29. The method of claim 27, wherein the protein transduction domain is an HIV-TAT protein transduction domain.

30. The method of any of claims 2-29, wherein the recombinant protein is TAT-MYC.

31. A device for making a protein-infused nucleated blood cell suspension comprising: a bag comprising a top end and a bottom end, the bag further comprising a first port at the top end, a second port on the bottom end, and a third port on the top end, wherein the bag contains an erythrocyte sedimentation solution; a conduit that optionally comprises a roller clamp, wherein the conduit operatively connects the second port; a syringe operatively coupled to the conduit; a first adapter operatively coupled to the first port and optionally, a second adapter operatively coupled to the third port.

32. The device of claim 31, wherein the first or second adapter is a luer lock adapter.

33. The device of claim 31, wherein the first or second adapter is a Y-shaped adapter, comprising a first branch, a second branch, and a connector.

34. The device of claim 33, further comprising a spike adapter coupled to the second branch of the Y-shaped adapter.

35. The device of any of claims 31-34, wherein the erythrocyte sedimentation solution comprises hetastarch and sodium chloride.

36. The device of claim 35, wherein the erythrocyte sedimentation solution comprises about 1.2% by weight or 0.6 g hetastarch in 50 mL of erythrocyte sedimentation solution.

37. The device of claim 35 or claim 36, wherein the erythrocyte sedimentation solution comprises about 0.9% by weight or 0.45 g sodium chloride in 50 mL of erythrocyte sedimentation solution.

38. A kit comprising the device of any of claims 31-37; and a vial containing a composition comprising a stabilizing agent.

39. The kit of claim 38, wherein the stabilizing agent comprises a recombinant protein.

40. The kit of claim 39, wherein the composition comprising the recombinant protein is a lyophilized composition.

41. The kit of claim 38 or claim 40, wherein the recombinant protein comprises a MYC polypeptide.

42. The kit of any of claims 38-41, wherein the recombinant protein comprises a protein transduction domain.

43. The kit of claim 42, wherein the protein transduction domain is an HIV-TAT protein transduction domain.

44. The kit of any of claims 38-43, wherein the recombinant protein is TAT -MYC.

45. The kit of any of claims 38-41, further comprising one or more devices for the collection of a whole blood sample from a subject.

46. The kit of claim 45, wherein the one or more devices for the collection of a whole blood sample comprises a hemostat, a collection syringe, an anticoagulant and a vial adapter.

47. The kit of claim 46, wherein the anticoagulant comprises heparin.

48. The kit of any of claims 38-47, wherein any item in the kit other than the stabilizing agent is stored at about 2°C to about 28°C prior to use and the vial of stabilizing agent is stored at about -20°C to about -80°C prior to use.