Fully enclosed method and system for enriching natural killer cells

Abstract

Provided are a method and system for enriching natural killer (NK) cells in a blood sample.

Description

Closed method and system for enriching natural killer cells

[0001] Related applications

[0002] This application claims priority to and the benefit of Chinese Patent Application No. CN 202411288100.9, filed September 13, 2024, entitled “Closed method and system for enriching natural killer cells,” which is incorporated by reference herein in its entirety for all purposes. TECHNICAL FIELD

[0003] The present application relates generally to the field of biotechnology, and more specifically, the present application relates to a closed method and system for enriching natural killer cells, and the like. BACKGROUND

[0004] Natural killer (NK) cells, as a kind of natural immune cells with the ability to directly kill malignant cells, are increasingly recognized as a promising immunotherapy tool, and various clinical trials are currently underway. In order to obtain a large number of NK cells with high purity and high cytotoxicity, various materials including peripheral blood mononuclear cells (PBMCs), umbilical cord blood (UCB), induced pluripotent stem cells (iPSCs), and NK cell lines have been used as sources to generate NK cells for immunotherapy.

[0005] Currently, the conventional NK sources mainly use Ficoll separation solution to separate peripheral blood, umbilical cord blood, bone marrow, and other materials under non-fully closed conditions, and the proportion of NK in the materials is not high. For example, the proportion of T cells (CD3 + ) in peripheral blood is about 50% to 84%, and the proportion of NK cells (CD3 - , CD16 + , and / or CD56 + ) is about 7% to 40% (see Zhu, Lihua, and Wang, Jianzhong. “Investigation of reference values of immunophenotypes of lymphocytes in Chinese blood.” Zhonghua Yi Xue Za Zhi 21.4 (1998): 223-226), and there are the following disadvantages:

[0006] 1. The operation is carried out in a biological safety cabinet, and the preparation is not fully closed, which requires a high environment and has the risk of pollution and cross-contamination.

[0007] 2. Ficoll separation solution is generally a research reagent, and its application in clinical practice has a high risk.

[0008] 3. It relies heavily on personnel operation, and the batch-to-batch stability is not stable.

[0009] 4. The recovery rate of mononuclear cells is low.

[0010] 5. The ratio of T cells in the obtained mononuclear cells is high, which affects the purity of the subsequent NK cell culture.

[0011] Therefore, there is a need in the art for methods and systems that address the above problems and deficiencies. SUMMARY

[0012] In a first aspect, the present application provides a method of enriching natural killer (NK) cells in a blood sample, the method comprising:

[0013] a) adding to the blood sample a bispecific antibody targeting an erythrocyte surface antigen and a T cell surface antigen and an erythrocyte sedimentation agent, and after a sedimentation process, isolating an upper fraction to obtain an NK cell-enriched fraction;

[0014] b) processing the NK cell-enriched fraction, the processing step comprising one or more of the following: cell washing, adjusting cell density, sampling for counting and / or detecting NK cell properties, cryopreservation medium exchange;

[0015] c) collecting the NK cells after step b); and

[0016] d) optionally, cryopreserving the NK cells;

[0017] wherein at least steps a) to c) are performed in a fully closed manner, preferably in a fully closed automated manner.

[0018] In a second aspect, the present application provides a fully closed system for enriching natural killer (NK) cells in a blood sample, the system comprising:

[0019] a sedimentation container (e.g., a sedimentation bag) configured to receive the blood sample and allow a sedimentation process to occur therein;

[0020] a sedimentation agent providing device, the sedimentation agent comprising a bispecific antibody targeting an erythrocyte surface antigen and a T cell surface antigen and an erythrocyte sedimentation agent, the sedimentation agent providing device being in fluid connection with the sedimentation container and configured to provide the sedimentation agent to the sedimentation container;

[0021] a separation device (e.g., a plasma separator clamp) configured to perform a phase separation of the multi-phase liquid in the sedimentation container;

[0022] a cell processing device configured to perform one or more of the following on the NK cell-enriched fraction: cell washing, adjusting cell density, sampling for counting and / or detecting NK cell properties, cryopreservation medium exchange; and

[0023] Optionally, a collection container (e.g. a collection bag) for collecting the NK cell enriched fraction treated by the cell treatment device,

[0024] wherein the system is preferably a fully closed automated system.

[0025] In some embodiments of the second aspect, the sedimentation providing device provides the sedimentation providing means to the sedimentation container by a pipe.

[0026] In some embodiments of the first and / or second aspect, the bispecific antibody targets a CD235a molecule on red blood cells and a CD3 molecule on T cells and comprises a CD235a binding domain and a CD3 binding domain, wherein:

[0027] the CD235a binding domain comprises a first light chain variable region comprising LCDR1 of the amino acid sequence RASSNVKYMY (SEQ ID No. 22), LCDR2 of the amino acid sequence YTSNLAS (SEQ ID No. 23), and LCDR3 of the amino acid sequence QQFTSSPYT (SEQ ID No. 24), and a first heavy chain variable region comprising HCDR1 of the amino acid sequence SYFMH (SEQ ID No. 25), HCDR2 of the amino acid sequence MIRPNGGTTDYNEKFKN (SEQ ID No. 26), and HCDR3 of the amino acid sequence WEGSYYALDY (SEQ ID No. 27), and

[0028] the CD3 binding domain comprises a second light chain variable region comprising LCDR1 of the amino acid sequence RASSSVSYMN (SEQ ID No. 28), LCDR2 of the amino acid sequence DTSKVAS (SEQ ID No. 29), and LCDR3 of the amino acid sequence QQWSSNPLT (SEQ ID No. 30), and a second heavy chain variable region comprising HCDR1 of the amino acid sequence RYTMH (SEQ ID No. 31), HCDR2 of the amino acid sequence YINPSRGYTNYNQKFKD (SEQ ID No. 32), and HCDR3 of the amino acid sequence YYDDHYCLDY (SEQ ID No. 33),

[0029] wherein the amino acid sequences of the HCDRs and LCDRs are according to the definition of Kabat;

[0030] Preferably, the CD235a binding domain comprises a first light chain variable region having an amino acid sequence as set forth in SEQ ID No. 6 and a first heavy chain variable region having an amino acid sequence as set forth in SEQ ID No. 7, and / or the CD3 binding domain comprises a second light chain variable region having an amino acid sequence as set forth in SEQ ID No. 18 and a second heavy chain variable region having an amino acid sequence as set forth in SEQ ID No. 17.

[0031] In some embodiments of the first and / or second aspect, the bispecific antibody further comprises a heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is of human IgG4 subtype, more preferably, the heavy chain constant region has an amino acid sequence as set forth in SEQ ID No. 10 or SEQ ID No. 21; and / or the light chain constant region has an amino acid sequence as set forth in SEQ ID No. 11.

[0032] Optionally, the first light chain variable region of the CD235a binding domain is associated with the light chain constant region to form a light chain of the CD235a binding domain, and the first heavy chain variable region of the CD235a binding domain is associated with the heavy chain constant region to form a heavy chain of the CD235a binding domain; preferably, the light chain of the CD235a binding domain has an amino acid sequence as set forth in SEQ ID No. 13 and / or the heavy chain of the CD235a binding domain has an amino acid sequence as set forth in SEQ ID No. 14.

[0033] Optionally, the CD3 binding domain is in a single chain antibody (scFv) format, for example, in a structure of second heavy chain variable region-first linker-second light chain variable region formed by a first linker; for example, the first linker is a GS type flexible linker, preferably, the first linker has an amino acid sequence as set forth in SEQ ID No. 16; optionally, the CD3 binding domain has an amino acid sequence as set forth in SEQ ID No. 19.

[0034] Optionally, the CD3 binding domain is associated with the heavy chain constant region of the CD235a binding domain to form a fusion heavy chain and the fusion heavy chain is combined with the light chain of the CD235a binding domain to form the bispecific antibody, preferably, the CD3 binding domain is associated with the CD235a binding domain through a second linker; for example, the second linker is a GS type flexible linker, preferably, the second linker has an amino acid sequence as set forth in SEQ ID No. 15; optionally, the fusion heavy chain has an amino acid sequence as set forth in SEQ ID No. 20.

[0035] Optionally, the bispecific antibody is a tetravalent IgG4 molecule comprising variable regions from a mouse and constant regions from a human.

[0036] In some embodiments of the first and / or second aspects, wherein the erythrocyte sedimentation agent comprises hydroxyethyl starch, gelatin, dextran, polyvinylpyrrolidone, and / or methylcellulose.

[0037] In some embodiments of the first aspect, the processing step in b) comprises a cryopreservation solution exchange. In some embodiments of the second aspect, the cell processing device comprises a cryopreservation solution exchange module for performing a cryopreservation solution exchange on the NK cell-enriched fraction. In some particular embodiments, the cryopreservation solution comprises one or more cryoprotective agents. The cryoprotective agents are, for example, permeating cryoprotective agents (e.g., dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, acetamide, and / or methanol) and / or non-permeating cryoprotective agents (e.g., polyvinylpyrrolidone, sucrose, polyethylene glycol, dextran, albumin, and / or hydroxyethyl starch). In some particular embodiments, the cryopreservation solution further comprises one or more amino acids and / or vitamins. For example, the cryopreservation solution can comprise dimethyl sulfoxide (e.g., at a final concentration of < 5%), dextran, sodium alginate, trehalose, human blood albumin, sorbitol, compound electrolyte, sodium chloride injection, glucose sodium chloride injection, dextran 40 glucose injection, compound amino acid injection, compound vitamin b injection.

[0038] In some embodiments of the first and / or second aspects, the blood sample is a peripheral blood sample and / or a cord blood sample derived from a donor. The donor can undergo screening, for example, by pathogenic microorganism (e.g., hepatitis B virus (HBV), hepatitis C virus (HCV), Treponema pallidum, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human T-cell lymphotropic virus (HTLV), human parvovirus B19, human herpes virus types 6 / 7 / 8, adenovirus, JC virus) detection and / or genetic disease history. BRIEF DESCRIPTION OF DRAWINGS

[0039] FIG. 1 shows a general flow chart of an exemplary method of enriching NK cells from a biological sample according to the present application.

[0040] FIG. 2 shows a flow chart of a method of enriching NK cells from a biological sample in some particular embodiments of the present application, wherein PPC3S is an exemplary bispecific antibody according to the present application, which is a tetravalent bispecific antibody capable of simultaneously specifically binding to CD235a molecules on red blood cells and CD3 molecules on T cells, and the design and preparation method thereof can be found in Examples 7 to 9.

[0041] Figures 3-7 show the comparison of NK cell recovery, total recovery and pre-freeze viability of NK cells obtained using Stemcell cryopreservation solution and self-made cryopreservation solution, respectively, in Example 3 (Figure 3), cell proliferation change curve (Figure 4), cell culture viability change curve (Figure 5), NKT and T cell ratio change curve (Figure 6), and NK cell ratio change curve (Figure 7).

[0042] Figures 8-10 show the difference between T cell percentage (Figure 8), NK cell percentage (Figure 9), and cell viability (Figure 10) of Example 4 compared to Comparative Example 1. By comparing the data before and after separation of Example 4 and Comparative Example 1, it can be seen that the method of Example 4 can greatly remove T cells and other impurities in peripheral blood, increase the ratio of NK cells, and the cell viability before and after separation has no significant difference; the ratio of T cells and NK cells after separation of Comparative Example 1 is similar to that before separation.

[0043] Figures 11-22 show the flow cytometry graphs of four samples prepared by the method of Example 4 (indicated as A1, A2, A3 and A4, respectively) and four samples prepared by the method of Comparative Example 1 (indicated as B1, B2, B3 and B4, respectively).

[0044] Figures 23-26 show the cell growth change curve (Figure 23), cell viability change curve (Figure 24), NK purity change curve (Figure 25), and data of killing Daudi tumor cells in vitro (Figure 26) of the sample prepared by the method in Example 5.

[0045] Figures 27 and 28 show the flow cytometry graphs of two batches of cell samples prepared by the method in Example 5 (indicated as Batch 1 and Batch 2, respectively) on day 18 (D18) of culture.

[0046] Figure 29 shows a schematic diagram of the framework of an exemplary bispecific antibody PPC3S of the present application. DETAILED DESCRIPTION

[0047] DEFINITIONS

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present application, the phrase "professional" specifically refers to Current Protocols in Molecular Biology (Ausubel) for definitions and terminology of the art. Abbreviations for amino acid residues are the standard three letter and / or one letter codes used in the art to designate one of the 20 commonly occurring L-amino acids.

[0049] Notwithstanding that the numerical ranges and parameters setting forth the broadest scope of the application are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a range of "1 to 10" is to be understood to include any and all subranges between (and including) the minimum value of 1 and the maximum value of 10, that is, all subranges beginning with a minimum of 1 or more, e.g. 1 to 6.1, and ending with a maximum of 10 or less, e.g. 5.5 to 10. Additionally, any reference to a "consisting of" is to be construed as a "consisting of, or consisting essentially of, or consisting of. Further, any reference to a "consisting of" is to be construed as a "consisting of, or consisting essentially of, or consisting of.

[0050] The terms "comprising" and "including," as used herein, are to be construed as being inclusive and open ended, and not exclusive. Specifically, when used in the specification and in the claims, the terms "comprising" and "including" and variations thereof mean "comprising," but also "consisting essentially of" and "consisting of."

[0051] The term "specifically binds," as used herein, is a term of art well known in the field and methods of determining such specific binding of an antibody to an antigen are well known in the art. For example, in some embodiments, "specifically binds" means that an antibody binds to an intended target, but does not significantly bind to other targets. The antibody binds to the intended target epitope with significantly increased affinity and / or for a longer duration as compared to binding to other epitopes.

[0052] The term "natural killer cell," as used herein, is a type of lymphocyte that can kill tumor cells and virus-infected cells without prior sensitization and without the participation of antibody. It can kill in an MHC unrestricted manner.

[0053] The term "mononuclear cell," as used herein, refers to a cell having a single nucleus in peripheral blood, umbilical cord blood, bone marrow, etc., and includes lymphocytes and monocytes.

[0054] The term "Ficoll solution," as used herein, refers to a solution of Ficoll-Hypaque, which is used to separate blood cells by density gradient. The solution is used to separate blood cells by density gradient.

[0055] The term "bispecific antibody" as used herein refers to an antibody that has the ability to bind to two antigen epitopes simultaneously. The two antigen epitopes can be on different antigens or on the same antigen. Bispecific antibodies can have various structural configurations. For example, a bispecific antibody can be composed of two Fc fragments and two antigen binding moieties fused to each of them respectively (similar to a natural antibody, except that the two arms bind to different antigen targets or epitopes), which can be in the form of single chain antibody (scFv) or Fab fragment. The two different binding moieties of the bispecific antibody are each bound to the N-terminus of one Fc fragment, and the configuration of the antigen binding moieties of the two arms can have four combinations: scFv + Fab fragment, Fab fragment + scFv, scFv + scFv, Fab fragment + Fab fragment. The Fc fragment can contain mutations that can ensure heterodimerization of the heavy chains, and the KIH technology (knob-in-hole, KIH) is a strategy to solve the heterodimerization of the heavy chains. Generally, the KIH technology refers to the formation of a structure that is conducive to the pairing of hetero-hybrid antibodies by modifying the amino acid sequence of the CH3 region, which can form a bispecific antibody while maintaining the structure of a normal antibody as much as possible.

[0056] The term "single chain antibody (scFv)" as used herein refers to a single polypeptide chain in which a VH domain and a VL domain are connected via a peptide linker. (scFv)2 contains two VH domains and two VL domains connected by a peptide linker, and the two VL domains are combined with the two VH domains via a disulfide bridge.

[0057] The term "CD235a" as used herein, i.e., Glycophorin A, is a major, intrinsic membrane protein of red blood cells, expressed on the surface of mature red blood cells, with an average of 1 x 10 5 ~1 x 10 6CD235a (Glycophorin A, GPA) is a sialoglycoprotein that is a major component of the red blood cell membrane. It is a single-pass transmembrane protein with a large extracellular N-terminal domain and a short cytoplasmic C-terminal domain. The N-terminal domain is heavily glycosylated and is the major sialic acid-containing protein on the red blood cell surface. The C-terminal domain is involved in the formation of the ankyrin-1 complex, which is important for the stability and shape of the red blood cell. The N-terminal domain is a single-nucleated blood group receptor. The extracellular domain is from amino acid 20 to 91, the transmembrane domain is from amino acid 92 to 114, and the intracellular domain is from amino acid 115 to 150. See SEQ ID No. 1. CD235a is highly homologous to CD235b (Glycophorin B, GYPB), which has a very high sequence identity in the extracellular N-terminal domain. See SEQ ID No. 2. CD235a and CD235b have similar functions. CD235a is expressed on red blood cells and not on lymphocytes in peripheral blood, making it a very selective target for depletion.

[0058] CD3 is a component of the T cell receptor-CD3 (TCR-CD3) complex on the surface of T lymphocytes and plays an important role in the adaptive immune response. When an antigen-presenting cell activates the T cell receptor, the TCR-mediated signal is transmitted to the intracellular through the δ, ε, γ, ζ subunits of CD3, thereby activating the downstream signaling pathway of T cells. In cell identification, CD3 is a well-known surface marker of T cells and is specifically expressed on T cells (including naive and memory CD8 + T cells, naive and memory CD4 + T cells, γδ T cells, T reg, etc.). In a conventional T cell positive or negative selection kit, magnetic beads coupled with CD3 antibody and matching reagents are usually selected for processing.

[0059] In a first aspect, the present application provides a method for enriching natural killer (NK) cells in a blood sample, the method comprising:

[0060] a) adding a bispecific antibody targeting a red blood cell surface antigen and a T cell surface antigen and a red blood cell sedimentation agent to the blood sample, after the sedimentation process, separating the upper part to obtain a NK cell-enriched component;

[0061] b) processing the NK cell-enriched component, the processing step comprising one or more of the following: cell washing, adjusting cell density, sampling for counting and / or detecting NK cell properties, freezing medium replacement;

[0062] c) collecting the NK cells after the treatment of step b); and

[0063] d) optionally, cryopreserving the NK cells;

[0064] wherein at least steps a) to c) are performed in a fully closed manner, preferably in a fully closed automated manner.

[0065] In a second aspect, the present application provides a fully closed system for enriching natural killer (NK) cells in a blood sample, the system comprising:

[0066] a sedimentation container (e.g., a sedimentation bag) configured to receive the blood sample and allow a sedimentation process to occur therein;

[0067] a sedimentation inducer comprising a bispecific antibody targeting a red blood cell surface antigen and a T cell surface antigen and a red blood cell sedimentation agent, the sedimentation inducer being in fluid connection with the sedimentation container and configured to provide the sedimentation inducer to the sedimentation container;

[0068] a separation device (e.g., a plasmapheresis clamp) configured to perform a phase separation of the multi-phase liquid in the sedimentation container;

[0069] a cell processing device configured to perform one or more of the following on the NK cell enriched fraction: cell washing, adjusting cell density, sampling for counting and / or detecting NK cell properties, cryopreservation medium replacement; and

[0070] optionally, a collection container (e.g., a collection bag) for collecting the NK cell enriched fraction treated by the cell processing device,

[0071] wherein the system is preferably a fully closed automated system.

[0072] In some embodiments of the second aspect, the sedimentation inducer providing device provides the sedimentation inducer to the sedimentation container through a tap.

[0073] In some embodiments of the first and / or second aspect, the bispecific antibody targets a CD235a molecule on a red blood cell and a CD3 molecule on a T cell and comprises a CD235a binding domain and a CD3 binding domain, wherein:

[0074] the CD235a binding domain comprises a first light chain variable region comprising a LCDR1 of the amino acid sequence RASSNVKYMY (SEQ ID No. 22), a LCDR2 of the amino acid sequence YTSNLAS (SEQ ID No. 23), and a LCDR3 of the amino acid sequence QQFTSSPYT (SEQ ID No. 24), and a first heavy chain variable region comprising a HCDR1 of the amino acid sequence SYFMH (SEQ ID No. 25), a HCDR2 of the amino acid sequence MIRPNGGTTDYNEKFKN (SEQ ID No. 26), and a HCDR3 of the amino acid sequence WEGSYYALDY (SEQ ID No. 27), and

[0075] the CD3 binding domain comprises a second light chain variable region comprising a LCDR1 of the amino acid sequence RASSSVSYMN (SEQ ID No. 28), a LCDR2 of the amino acid sequence DTSKVAS (SEQ ID No. 29), and a LCDR3 of the amino acid sequence QQWSSNPLT (SEQ ID No. 30), and a second heavy chain variable region comprising a HCDR1 of the amino acid sequence RYTMH (SEQ ID No. 31), a HCDR2 of the amino acid sequence YINPSRGYTNYNQKFKD (SEQ ID No. 32), and a HCDR3 of the amino acid sequence YYDDHYCLDY (SEQ ID No. 33),

[0076] wherein the amino acid sequences of the HCDRs and LCDRs are according to the definition of Kabat;

[0077] Preferably, the CD235a binding domain comprises a first light chain variable region of the amino acid sequence as set forth in SEQ ID No. 6 and a first heavy chain variable region of the amino acid sequence as set forth in SEQ ID No. 7, and / or the CD3 binding domain comprises a second light chain variable region of the amino acid sequence as set forth in SEQ ID No. 18 and a second heavy chain variable region of the amino acid sequence as set forth in SEQ ID No. 17.

[0078] Figure 29 shows one exemplary construction scheme of the bispecific antibody of the present application. In Figure 29, the bispecific antibody comprises two light chains and two heavy chains, and the N-terminal is a CD235a binding domain (comprising a light chain variable region and a heavy chain variable region that bind to CD235a), and the C-terminal is a CD3a binding domain (comprising a light chain variable region and a heavy chain variable region that bind to CD3). Specifically, the light chain of this exemplary bispecific antibody comprises a light chain constant region (human IgG4 subtype) and a light chain variable region of the CD235a binding domain (which comprises three CDR regions of LCDR1, LCDR2 and LCDR3), and the heavy chain comprises a heavy chain constant region (human IgG4 subtype), a heavy chain variable region of the CD235a binding domain (which comprises three CDR regions of HCDR1, HCDR2 and HCDR3), and a CD3 binding domain in the form of scFv (comprising LCDR1, LCDR2 and LCDR3, and HCDR1, HCDR2 and HCDR3 regions).

[0079] It should be understood that the construction scheme of the bispecific antibody shown in Figure 29 is merely exemplary, but not limiting. Those skilled in the art can reasonably know other construction schemes of the bispecific antibody. For example, the bispecific antibody of the present application can be obtained by the following ways (only for example, but not limited thereto):

[0080] (i) on the basis of an anti-CD235a full-length antibody, an antibody fragment (for example, a single-chain antibody scFv comprising a heavy chain variable region and a light chain variable region that binds to CD3) that binds to CD3 is connected to the Fc fragment of the anti-CD235a antibody;

[0081] (ii) on the basis of an anti-CD3 full-length antibody, an antibody fragment (for example, a single-chain antibody scFv comprising a heavy chain variable region and a light chain variable region that binds to CD235a) that binds to CD235a is connected to the Fc fragment of the anti-CD3 antibody; or

[0082] (iii) one antigen binding arm of an anti-CD235a antibody (or an anti-CD3 antibody) is replaced by a set of heavy chain variable regions and light chain variable regions of an anti-CD3 antibody (or an anti-CD235a antibody), so that the two arms of the parent antibody can bind to CD235a and CD3, respectively.

[0083] In some embodiments of the first and / or second aspect, the bispecific antibody further comprises a heavy chain constant region and a light chain constant region. In some specific embodiments, the heavy chain constant region is of human IgG4 subtype. In some more specific embodiments, the amino acid sequence of the heavy chain constant region is set forth in SEQ ID No. 10. In some alternative embodiments, the heavy chain constant region can comprise a mutation in its hinge region, for example, a mutation of serine (S) at the 10th amino acid position in the hinge region to proline (P), the amino acid position being numbered according to the EU numbering system. For example, the amino acid sequence of the heavy chain constant region comprising the hinge region S10P mutation can be set forth in SEQ ID No. 21. In some specific embodiments, the amino acid sequence of the light chain constant region is set forth in SEQ ID No. 11.

[0084] In some embodiments of the first and / or second aspect, the first light chain variable region of the CD235a binding domain is associated with the light chain constant region (e.g., the first light chain variable region is connected to the N-terminus of the light chain constant region) to form the light chain of the CD235a binding domain, and the first heavy chain variable region of the CD235a binding domain is associated with the heavy chain constant region (e.g., the first heavy chain variable region is connected to the N-terminus of the heavy chain constant region) to form the heavy chain of the CD235a binding domain. In some specific embodiments, the amino acid sequence of the light chain of the CD235a binding domain is set forth in SEQ ID No. 13 and / or the amino acid sequence of the heavy chain of the CD235a binding domain is set forth in SEQ ID No. 14;

[0085] In some embodiments of the first and / or second aspect, the CD3 binding domain is in the form of a single chain antibody (scFv), for example, in the structure of second heavy chain variable region-first linker-second light chain variable region formed by the first linker (e.g., in the structure of N-terminal-second heavy chain variable region-first linker-second light chain variable region-C-terminal). The first linker may, for example, be a GS type flexible linker, preferably, the amino acid sequence of the first linker is set forth in SEQ ID No. 16. In some more specific embodiments, the amino acid sequence of the CD3 binding domain is set forth in SEQ ID No. 19.

[0086] In some embodiments of the first and / or second aspect, the CD3 binding domain is associated with the heavy chain constant region of the CD235a binding domain (e.g., the CD3 binding domain is connected to the C-terminus of the heavy chain constant region) to form a fusion heavy chain and the fusion heavy chain is combined with the light chain of the CD235a binding domain to form the bispecific antibody. For example, the CD3 binding domain can be associated with the CD235a binding domain via a second linker. The second linker can be, for example, a GS-type flexible linker, preferably the amino acid sequence of the second linker is set forth in SEQ ID No. 15. In some more specific embodiments, the amino acid sequence of the fusion heavy chain is set forth in SEQ ID No. 20.

[0087] In some embodiments of the first and / or second aspect, the fusion protein is a tetravalent IgG4 molecule comprising variable regions from mouse and constant regions from human.

[0088] There are some studies on the drugability of antibodies of different sources of CD235a in the art:

[0089] In patent US2022 / 0153859A1, the humanized antibody of CD235a is named 10F7-M10, the sequence of the light chain variable region is set forth in SEQ ID No. 3, the sequence of the heavy chain variable region is set forth in SEQ ID No. 4, and the dissociation constant KD of the antibody to CD235a is 140nM.

[0090] In patent US9879090B2, the VHH antibody of CD235a is named IH5, which is a single domain antibody, the sequence of the heavy chain variable region is set forth in SEQ ID No. 5, and the dissociation constant KD of the antibody to CD235a is 33.7nM.

[0091] In patent WO2017 / 015141A1, the murine antibody of CD235a is named 10F7, the sequence of the light chain variable region is set forth in SEQ ID No. 6, and the sequence of the heavy chain variable region is set forth in SEQ ID No. 7. In this patent, it is humanized into scFv, and through the combination of binding activity, functional research, and stability, it is confirmed that there is a combination of Glycophorin A binding activity, and the sequence scFv-10F7-EPO is constructed, the sequence of the light chain variable region is set forth in SEQ ID No. 8, and the sequence of the heavy chain variable region is set forth in SEQ ID No. 9.

[0092] In the literature (Catimel et al. J. Immunol. Methods (1993) 165(2): 183-192), there are two antibodies of CD235a: one of which is named 1C3 / 86, and it is measured that there are 4.80x105 a CD235a molecule with a dissociation constant KD of 23 x 10 -8 M, i.e. 230 nM; another named 10F7MN was measured to have 4.66 x 10 5 a CD235a molecule with a dissociation constant KD of 9.5 x 10 -8 M, i.e. 95 nM.

[0093] However, none of these studies disclose or teach the bispecific antibodies of the present application.

[0094] In some embodiments of the first and / or second aspect, the bispecific antibody of the present application is added to a peripheral blood sample with a sedimentation agent, wherein the bispecific antibody is in a form of a concentration of about 5 mg / mL and is added to the blood sample in a volume of 5 mL to 25 mL per 100 mL of peripheral blood, for example in a volume of about 5 mL, about 5.5 mL, about 6 mL, about 6.5 mL, about 7 mL, about 7.5 mL, about 8 mL, about 8.5 mL, about 9 mL, about 9.5 mL, about 10 mL, about 10.5 mL, about 11 mL, about 11.5 mL, about 12 mL, about 12.5 mL, about 13 mL, about 13.5 mL, about 14 mL, about 14.5 mL, about 15 mL, about 15.5 mL, about 16 mL, about 16.5 mL, about 17 mL, about 17.5 mL, about 18 mL, about 18.5 mL, about 19 mL, about 19.5 mL, about 20 mL, about 20.5 mL, about 21 mL, about 21.5 mL, about 22 mL, about 22.5 mL, about 23 mL, about 23.5 mL, about 24 mL, about 24.5 mL, or about 25 mL per 100 mL of blood, preferably in a volume of about 5 mL, about 15 mL, or about 25 mL per 100 mL of blood.

[0095] In some embodiments of the first and / or second aspect, the red blood cell sedimentation agent comprises hydroxyethyl starch, gelatin, dextran, polyvinylpyrrolidone, and / or methylcellulose, and combinations, analogs, or derivatives thereof. The red blood cell sedimentation agent can be added in a final concentration of 0.5-4%, for example in a final concentration of 0.5-1%, 1-2%, or 2-4%. For example, the sedimentation agent can be hydroxyethyl starch in a final concentration of about 2%.

[0096] In some embodiments of the first aspect, the processing step in b) comprises a cryopreservation solution exchange. In some embodiments of the second aspect, the cell processing device comprises a cryopreservation solution exchange module for performing a cryopreservation solution exchange on the NK cell-enriched component. In some specific embodiments, the cryopreservation solution comprises one or more cryoprotective agents. The cryoprotective agents are, for example, permeating cryoprotective agents (e.g., dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, acetamide, and / or methanol) and / or non-permeating cryoprotective agents (e.g., polyvinylpyrrolidone, sucrose, polyethylene glycol, dextran, albumin, and / or hydroxyethyl starch). In some specific embodiments, the cryopreservation solution further comprises one or more amino acids and / or vitamins. For example, the cryopreservation solution can comprise dimethyl sulfoxide (e.g., at a final concentration of < 5%), dextran, sodium alginate, trehalose, human blood albumin, sorbitol, compound electrolytes, sodium chloride injection, dextrose sodium chloride injection, dextran 40 dextrose injection, compound amino acid injection, compound vitamin b injection.

[0097] In some embodiments of the first aspect and / or the second aspect, the blood sample is a peripheral blood sample and / or a cord blood sample derived from a donor. The donor can undergo screening, for example, by pathogenic microorganism (e.g., hepatitis B virus (HBV), hepatitis C virus (HCV), Treponema pallidum, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human T-cell lymphotropic virus (HTLV), human parvovirus B19, human herpes virus types 6 / 7 / 8, adenovirus, JC virus) testing and / or genetic history.

[0098] In some embodiments of the second aspect, the cell processing device can comprise one or more of a cell washing module, a cell density adjustment module, a sampling module, and / or a cryopreservation solution exchange module. The system and / or cell processing device can further comprise modules that perform other functions, such as a temperature regulation module, etc. One or more modules in the system and / or cell processing device can be combined, for example, a cryopreservation solution exchange module and a cell density adjustment module can be combined such that the introduction of a cryopreservation solution simultaneously replaces the existing solution in the system and adjusts the cell density in the existing solution.

[0099] In some embodiments of the second aspect, the cell processing device performs one or more of the following on the NK cell-enriched component: liquid volume concentration, sodium chloride injection perfusion washing, cryopreservation solution exchange, cryopreservation solution resuspension, and / or sampling for counting.

[0100] In some embodiments of the first aspect, the method further comprises mixing the NK cells after being treated by step b) and aliquoting (e.g., after mixing), for example, into cryogenic containers (e.g., cryogenic bags).

[0101] In some embodiments of the second aspect, the system further comprises a cell mixing device (e.g., a shaker) for mixing the enriched NK cell component treated by the cell processing device and / or an aliquoting device (e.g., a peristaltic pump) for aliquoting the enriched NK cell component (e.g., after being mixed by the cell mixing device), for example, into cryogenic containers (e.g., cryogenic bags).

[0102] The inventors of the present application found that the culture of NK cells can be improved by increasing the initial purity of NK cells, obtaining NK cells with high purity (up to 95% or more), high viable cell density (8.5 x 10 6 The present application can solve the world problem of low purity of NK cells by obtaining NK cells with high purity (up to 95% or more), high viable cell density (8.5 x 10

[0103] The present application can remove most of the red blood cells, T cells and other impurity cells by using hydroxyethyl starch and separation reagents (e.g., PPC3S described above). Further, the cells can be concentrated, washed, and replaced with self-made cryopreservation solution by using a closed and automated instrument, and the density can be adjusted. The single nuclear cells can be aliquoted by using a shaker and a peristaltic pump through a closed pipeline, and stored in a gas phase liquid nitrogen tank after programmed cooling. In some embodiments, the method and system of the present application can achieve one or more of the following technical effects:

[0104] 1. The materials used in the NK cell enrichment method and system of the present application are abundant and widely applicable, and are not limited to a single source such as peripheral blood and cord blood.

[0105] 2. The preparation is fully closed, which reduces the risk of contamination and cross-contamination, and can meet the environmental requirements in a D-level environment, thereby reducing the construction and maintenance costs of the laboratory.

[0106] 3. The separation reagents can remove most of the red blood cells, T cells and other impurity cells in the material, and enrich the NK cells in the material. Compared with flow cytometry and magnetic bead selection, the process of the present application is low in cost, short in time and simple in operation.

[0107] 4. The use of an automated instrument reduces the risk of human error. Compared with similar products on the market, the solution replacement rate and NK cell recovery rate of this instrument are high, and the problem of cell accumulation caused by centrifugation is avoided, and the cell viability after processing is high.

[0108] 5. The self-made cryopreservation solution of the present application, which is composed of safe components of pharmaceutical products or pharmaceutical excipients and has excellent cryopreservation effect.

[0109] 6. The single nuclear cells are sub-packaged into cryopreservation bags using a peristaltic pump and a shaker, and the sub-packaging is performed in a sterile manner.

[0110] In summary, the method of the present application can make up for the shortcomings of non-fully-closed methods (e.g., Ficoll separation method), and achieve the goal of obtaining peripheral blood, umbilical cord blood, bone marrow and other multi-source mononuclear cells (MNC) in a fully-closed, automated, high-recovery, batch-stable and high-NK-purity manner.

[0111] In some embodiments, the present subject matter is further characterized by one or more of the following:

[0112] 1. The method of the present application can mainly include collection and transportation, sedimentation treatment, automated instrument treatment, sub-packaging, cryopreservation and the like, and specific details can be found in FIG. 1 and / or FIG. 2.

[0113] 2. In the method of the present application, all sampling, reagent addition and cell transfer can be performed in a sterile manner using a sterile pipe connection machine.

[0114] 3. In the collection and transportation operation of the present application, the material source can be peripheral blood, umbilical cord blood, bone marrow, iPSC and the like.

[0115] 4. In the collection and transportation operation of the present application, the material donor can be subjected to pathogenic microorganism screening as needed, including HBV, HCV, syphilis, HIV, EBV, CMV, HTLV, human parvovirus B19, human herpes virus 6 / 7 / 8, adenovirus, JC virus. After collection, the sample is transported at 2-8°C in a cold chain.

[0116] 5. In the sedimentation treatment operation of the present application, the addition amount of separation reagent (e.g., PPC3S, e.g., about 5 mg / ml) is 5-25 ml / 100 ml of c material, and the incubation time is 40-60 min. After addition, the final concentration of hydroxyethyl starch solution is 1-2%, and the standing sedimentation time is 30-60 min.

[0117] 6. In the automated instrument treatment operation of the present application, an automated instrument can be used to concentrate, wash and replace the cryopreservation solution of the single nuclear cells, and the density is adjusted to 1.0-8.0 x 10 6 cells / ml. The cell transfer flow is 30-60 ml / min, the washing concentration volume is 2-5 mL, and the replacement number is 2-5.

[0118] 7. The automated instrument processing operation of the present application can use a freezing solution containing DMSO, dextran, sodium alginate, trehalose, human blood albumin, sorbitol, compound electrolyte, sodium chloride injection, glucose sodium chloride injection, dextran 40 glucose injection, compound amino acid injection, and compound vitamin B injection. The DMSO can be ≤5%.

[0119] 8. In the aliquoting operation of the present application, single nucleated cells can be uniformly aliquoted into freezing bags by using a shaker and a peristaltic pump. The shaker speed is 30-80 rpm.

[0120] It should be understood that the foregoing detailed description is only intended to give a clearer understanding of the present application to those skilled in the art and is not intended to limit the present application in any way. Those skilled in the art can make various modifications and changes to the described embodiments.

[0121] Examples

[0122] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application.

[0123] Comparative Example 1: Ficoll separation solution for separating single nucleated cells

[0124] 1. Collection and transportation: pathogenic microorganisms (hepatitis B virus (HBV), hepatitis C virus (HCV), Treponema pallidum, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human T cell lymphotropic virus (HTLV), human parvovirus B19, human herpes viruses 6 / 7 / 8, adenovirus, JC virus), genetic history screening are performed on peripheral blood donors. After testing, the material is collected and transported at 2-8°C in a cold chain.

[0125] 2. In a biological safety cabinet, the peripheral blood is transferred to a 225 ml conical bottom centrifuge bottle, diluted with an equal volume of physiological saline, and then 20 ml of the diluted peripheral blood is slowly added to 20 ml of Ficoll separation solution. Centrifuge at 600g for 15 min at room temperature.

[0126] 3. After centrifugation, the supernatant is discarded, and the white membrane layer cells are collected into a 225 ml conical bottom centrifuge bottle, and the volume is adjusted to 200 ml with physiological saline, and centrifuged at 300g for 10 min at room temperature.

[0127] 4. After centrifugation, the supernatant is discarded, and the density is adjusted to 5.0 x 10 6 6 ml is taken for sterile, mycoplasma, endotoxin, counting, and flow cytometry detection, and the remaining single nucleated cells are aliquoted into freezing tubes.

[0128] 5. The cryotubes were stored in a tank of gaseous phase liquid nitrogen after being programmed to -80°C.

[0129] Example 1: Donor screening

[0130] In order to ensure the safety of PBMC, the material donor or material must be strictly controlled, because the peripheral blood, umbilical cord blood and other materials from the donor may contain pathogens of blood-borne diseases. The pathogenic microorganism screening of the material donor includes HBV, HCV, Treponema pallidum, HIV, EBV, CMV, HTLV, human parvovirus B19, HIV, human herpes virus 6 / 7 / 8, adenovirus, JC virus. After passing the pathogenic microorganism screening, the material collection can be carried out, and the material is subjected to viable cell count, cell viability, flow detection, sterility test, mycoplasma test. After all tests are qualified, it can be used for PBMC preparation.

[0131] Example 2: sedimentation treatment

[0132] In this embodiment, the separation reagent is combined with hydroxyethyl starch to remove most of the red blood cells, T cells and other impurity cells. The separation reagent used in examples 2 to 5 is the bispecific antibody PPCS3 described above, which can specifically bind to CD235a molecules on red blood cells and CD3 molecules on T cells, and the concentration is 5 mg / ml. The design and preparation of PPCS3 are described below in examples 7-9.

[0133] The sedimentation treatment steps are as follows:

[0134] The same amount of peripheral blood was added to the centrifuge tube, and different concentrations of separation reagent were added (5 mL / 100 m peripheral blood, 15 mL / 100 m peripheral blood, 25 mL / 100 m peripheral blood). Mix well, incubate for 40 min. Add a hydroxyethyl starch solution with a mass concentration of 6%, and the final concentration of the hydroxyethyl starch solution is 2% after addition. After standing and sedimentation for 0.5 h, the upper plasma layer was aspirated, resuspended to 45 mL, centrifuged (300 g, 10 min), the supernatant was discarded, and the cell pellet was resuspended. The purity of NK cells (CD3 - CD56 + ) and the proportion of main impurity T cells (CD3 + ) were detected by flow cytometry. The specific experimental data are shown in Table 1 below.

[0135] Table 1. Sedimentation treatment experimental data

[0136] The results show that by using the separation reagent and hydroxyethyl starch in combination, the proportion of T cells in the mononuclear cells obtained in the three groups is all ≤0.5%, and the purity of NK is all >70%. Compared with the peripheral blood flow data, it can be known that this operation can remove most of the T cells in the peripheral blood, and greatly improve the percentage of NK cells in the mononuclear cells. Compared with flow cytometry and magnetic bead screening, this method has low cost, short time consumption and simple operation.

[0137] Example 3: Cryopreservation solution freezing study

[0138] In this example, PBMC samples are prepared from peripheral blood in a fully closed and automated processing mode, and the freezing effect of the self-made cryopreservation solution prepared by the inventors of the present application is studied. The self-made cryopreservation solution contains DMSO, dextran, sodium alginate, trehalose, human blood albumin, sorbitol, compound electrolyte, sodium chloride injection, glucose sodium chloride injection, dextran 40 glucose injection, compound amino acid injection, and compound vitamin B injection, etc. The components of the self-made cryopreservation solution are all drugs or pharmaceutical excipients, which are safe and have excellent freezing effect.

[0139] The self-made cryopreservation solution and the commercial cryopreservation solution from Stemcell are compared as follows. Steps 1-3 and / or steps 1-4 below are carried out in a fully closed system, which includes a sedimentation container (in this example, a sedimentation bag), a sedimentation agent providing device (in this example, for providing the separation reagent, the sedimentation agent hydroxyethyl starch, and containing connecting pipes and other components with other devices and modules of the system), a separation device (in this example, a split-clamp), a cell processing device (in this example, including a fully closed and automated concentration instrument, a shaker), and other devices and modules.

[0140] 1. Sedimentation treatment:

[0141] 1) Bag transfer: transfer the peripheral blood into the sedimentation bag. Calculate the material volume by the mass difference before and after the transfer. Take about 0.5 ml for cell counting, flow cytometry detection (CD3, CD56), KIR detection.

[0142] 2) Separation reagent addition: add the separation reagent at 5 mL / 100 ml of material through a sterile connecting pipe, and incubate for 50 min after addition.

[0143] 3) Hydroxyethyl starch addition: add 6% hydroxyethyl starch solution, and the final concentration of the hydroxyethyl starch solution after addition is 2%, and incubate for 0.5 h after addition. After sedimentation, use the split-clamp to transfer the supernatant from the incubation bag to the sample bag, weigh and take samples for counting and flow cytometry detection. After mixing, divide into two equal parts, labeled as sample bag 1 and sample bag 2.

[0144] 2. Sample bag 1 treatment:

[0145] Connect to the fully enclosed automated concentration instrument through a sterile connector, the cell transfer flow rate is 31 ml / min, the washing concentration volume is 3 mL, and the replacement number is 3. After the homemade freezing solution is replaced, sample counting and flow cytometry detection are performed. After adjusting the density to 5.0 x 10 6 The volume, counting and flow cytometry detection results before and after instrument processing are recorded.

[0146] Mix the mononuclear cell suspension on a shaker at 30 rpm, and then divide the mononuclear cell suspension into freezing bags through a peristaltic pump, 20 ml per bag. After the freezing bags are programmed to -80°C, they are stored in a gas-phase liquid nitrogen tank.

[0147] 3. Sample bag 2 processing:

[0148] Connect to the fully enclosed automated concentration instrument through a sterile connector, the cell transfer flow rate is 31 ml / min, the washing concentration volume is 3 mL, and the replacement number is 3. After the homemade freezing solution is replaced, sample counting and flow cytometry detection are performed. After adjusting the density to 5.0 x 10 6 The volume, counting and flow cytometry detection results before and after instrument processing are recorded.

[0149] Mix the mononuclear cell suspension on a shaker at 30 rpm, and then divide the mononuclear cell suspension into freezing bags through a peristaltic pump, 20 ml per bag. After the freezing bags are programmed to -80°C, they are stored in a gas-phase liquid nitrogen tank.

[0150] 4. After freezing for one week, recover and culture separately.

[0151] The specific separation and freezing data are shown in Table 2 below, and the recovery and culture data are shown in Table 3 below. The relevant pictures are shown in Figures 3 to 7.

[0152] Table 2. Separation and freezing data

[0153] Table 3. Recovery and culture data

[0154] The results show that the NK recovery rate, total recovery rate, and mononuclear NK cell ratio of the homemade freezing solution group are better than those of the Stemcell group, and there is no significant difference in the pre-freezing viability and recovery and culture data between the two groups. In terms of performance, the homemade freezing solution is better than the Stemcell freezing solution, and the components of the homemade freezing solution are all drugs or pharmaceutical excipients, which are safer.

[0155] Example 4: Peripheral blood-derived mononuclear cell separation

[0156] 1. Collection and transportation: Peripheral blood donors are screened for pathogenic microorganisms (HBV, HCV, Treponema pallidum, HIV, EBV, CMV, HTLV, human parvovirus B19, human herpes virus 6 / 7 / 8, adenovirus, JC virus), genetic history. After passing the test, collect the material and transport it at 2-8°C in the cold chain.

[0157] The following steps 2-4 are carried out in the fully closed system described in Example 3:

[0158] 2. Sedimentation treatment

[0159] 1) Bag transfer: Transfer the peripheral blood to the sedimentation bag. Calculate the volume of the material by the difference in mass of the sedimentation bag before and after transfer. Take about 0.5 ml for cell counting, flow detection (CD3, CD56), KIR detection.

[0160] 2) Separation reagent addition: Add separation reagent at 5 mL / 100 ml of material through a sterile adapter. Incubate for 50 min after addition.

[0161] 3) Hydroxyethyl starch addition: Add 6% hydroxyethyl starch solution, and the final concentration of the hydroxyethyl starch solution after addition is 2%. Incubate and sediment for 0.5 h after addition. After sedimentation, use a plasma splitter to transfer the supernatant from the incubation bag to the sample bag.

[0162] 3. The sample bag is connected to the fully closed automatic concentration instrument through a sterile adapter, the cell transfer flow rate is 31 ml / min, the washing and concentration volume is 3 mL, and the replacement number is 3. After replacement with self-made cryopreservation solution, take a sample for counting. After adjusting the density to 5.0 x 10 6 / ml according to the counting result, collect it into the collection bag.

[0163] 4. Shake the mononuclear cell suspension at 30 rpm, and use a peristaltic pump to divide the mononuclear cell suspension into cryopreservation bags, each containing 20 ml. The collection bag retains 5 ml for sterile, mycoplasma, endotoxin, counting, and flow detection. After the cryopreservation bag is programmed to -80°C, it is stored in a gas-phase liquid nitrogen tank.

[0164] Results

[0165] According to the separation method provided in this example and Comparative Example 1, four samples were separated, and the specific experimental data obtained are shown in Table 4, and the detection results are shown in Table 5. The related pictures are shown in Figures 8 to 22.

[0166] Table 4. Comparison of this example and Comparative Example 1

[0167] Table 5. Comparison of detection results of this example and Comparative Example 1

[0168] Therefore, the method provided by the application can remove most of the T cells, and the recovery rate of NK cells is greater than 95%. In the obtained mononuclear cells, the purity of NK is greater than 45%, and the cell survival rate is greater than 95%. The whole separation process uses a fully closed automatic instrument, which can reduce the risk of pollution and cross contamination, and has obvious advantages over the traditional method.

[0169] Example 5: Peripheral blood-derived mononuclear cell separation

[0170] 1. Collection and transportation: screening of peripheral blood donors for pathogenic microorganisms (HBV, HCV, Treponema pallidum, HIV, EBV, CMV, HTLV, human parvovirus B19, human herpes virus 6 / 7 / 8, adenovirus, JC virus), genetic history. After passing the test, collect the material and transport it at 2-8 DEG C in the cold chain.

[0171] The following steps 2-3 are carried out in the fully closed system described in Example 3:

[0172] 2. Sedimentation treatment

[0173] 1) Transfer bag: transfer the peripheral blood to the sedimentation bag. Calculate the material volume by the difference in mass of the sedimentation bag before and after transfer. Take about 0.5 ml for cell counting, flow detection (CD3, CD56), KIR detection.

[0174] 2) Separation reagent addition: add separation reagent at 5 mL / 100 ml of material through a sterile connection pipe, and incubate for 50 min after addition.

[0175] 3) Hydroxyethyl starch addition: add 6% hydroxyethyl starch solution, and the final concentration of the hydroxyethyl starch solution after addition is 2%, and the hydroxyethyl starch solution is added. After standing for 0.5 h. After sedimentation, use a slurry clamp to transfer the supernatant from the standing bag to the sample bag.

[0176] 3. The sample bag is connected to the fully closed automatic concentration instrument through a sterile connection pipe, the cell transfer flow rate is 31 ml / min, the washing and concentration volume is 3 mL, and the replacement number is 3. After replacement with the self-made cryopreservation solution, take a sample for counting. After adjusting the density to 6.0 x 10 6 / ml according to the counting result, collect it into the collection bag.

[0177] Mix the mononuclear cell suspension on a shaker at 30 rpm, and divide the mononuclear cell suspension into cryopreservation bags through a peristaltic pump, 20 ml per bag. The collection bag retains 5 ml for sterile, mycoplasma, endotoxin, counting, and flow detection. The cryopreservation bag is programmed to be cooled to -80 DEG C and then stored in a gas-phase liquid nitrogen tank.

[0178] Prepare 2 batches of PBMC in this way, and after a period of cryopreservation, culture them. The cell culture data are shown in Table 6, and the data of killing Daudi tumor cells in vitro are shown in Table 7.

[0179] Table 6. Culture data

[0180] Table 7. In vitro killing data

[0181] Example 6: Stability study data

[0182] PBMCs were stored in a gas phase liquid nitrogen tank at a temperature of ≤-150°C for a long time. To investigate whether PBMCs stored for a long time under this condition can meet the quality attributes after recovery and satisfy the process culture requirements, a study was conducted on the storage stability of PBMCs. The quality attributes and process capacity of PBMCs stored at a temperature of ≤-150°C for 0, 12, and 24 months were investigated.

[0183] Quality attributes: quality characterization of PBMCs after recovery (appearance, total number of viable cells, viable cell density, cell viability, cell phenotype, mycoplasma test, sterility test, bacterial endotoxin). Relevant data are shown in Table 8.

[0184] Process capacity: quality characterization of PBMCs after recovery culture (appearance, total number of viable cells, cell viability, cell phenotype, mycoplasma test, sterility test, bacterial endotoxin, killing activity). Relevant data are shown in Table 9.

[0185] Table 8. Stability quality attribute data

[0186] Table 9. Stability process capacity data

[0187] From the above data, it can be seen that for two batches of PBMCs, stored in a gas phase liquid nitrogen tank at a temperature of ≤-150°C for 24 months, the results of each investigation of the quality attributes and process capacity meet the acceptance criteria. There is no significant difference between the investigation indexes of PBMCs stored for 0 months and 24 months. The PBMCs prepared by the method of the present application can be stored stably in a gas phase liquid nitrogen tank at a temperature of ≤-150°C for a long time.

[0188] Example 7: Design of PPC3S molecule

[0189] This example provides an exemplary bispecific antibody, referred to as PPC3S, which is an artificially designed molecule that can cross-link red blood cells and T cells (CD3+) together to form a rosette-like complex, and then through a cell sedimentation method, including adding hydroxyethyl starch, PPC3S multimers, removing most of the red blood cells and T cells, the treated PBMCs are more suitable for culture into NK cells.

[0190] PPC3S is a multivalent structure targeting both CD235a and CD3, and is based on the framework of IgG, in which:

[0191] (1) The heavy chain constant region (CH1-hinge-CH2-CH3) part is designed as human IgG4 subtype, i.e. IGHG4 (UniProt accession number P01861-1, length 327 aa), as shown in SEQ ID No. 10. The affinity of human IgG4 to CD16a is much lower than that of human IgG1 to CD16a (≤1 / 10), so it can reduce the cross-linking of NK cells with T cells, red blood cells, thereby reducing the killing and loss of NK cells, and improving the yield of NK cells. At the same time, IgG4 has high affinity to protein A, and high-purity PPC3S can be obtained by affinity chromatography to meet the requirements of commercial production;

[0192] (2) To match the heavy chain constant region (CH1-hinge-CH2-CH3) of human IgG4, the light chain constant region of human IgG4, i.e. IGKC (UniProt accession number P01834, length 107 aa) is selected. IGHG4 and IGKC can be matched, as exemplified by antibodies such as Nivolumab, Lambrolizumab, Gemtuzumab, in which IGHG4 is matched with IGKC.

[0193] (3) For the variable region sequence of anti-CD235a, according to the sequence of 10F7MN single chain antibody (single chain antibody 10F7MN, partial [synthetic construct]-Protein-NCBI (nih.gov)) in NCBI database, which is a full mouse-derived single chain antibody (scFv), as shown in SEQ ID No. 12, the light chain and heavy chain variable region sequences thereof are completely identical to SEQ ID No. 6 and SEQ ID No. 7, respectively. Therefore, the inventors fused SEQ ID No. 6 to the N-terminus of IGKC (SEQ ID No. 11) to obtain SEQ ID No. 13, and fused SEQ ID No. 7 to the N-terminus of IGHG4 (SEQ ID No. 10) to obtain SEQ ID No. 14. SEQ ID No. 13 and SEQ ID No. 14 can be considered as the light chain and heavy chain of anti-CD235a human IgG4 monoclonal antibody 10F7MN;

[0194] (4) For the domain of anti-CD3, the strategy of BiTE (i.e. tandem scFv) is referred, i.e. at the C-terminal of 10F7MN of IgG4 structure, the scFv of anti-CD3 is fused, and the sequence of the scFv of anti-CD3 is divided into 4 parts, from N-terminal to C-terminal, in turn: linker 1, anti-CD3-VH, linker 2, anti-CD3-VL. Among them, linker 1 and linker 2 are repeatable sequences composed of glycine and serine, which can reduce the influence on the affinity of the target as much as possible while ensuring the conformation of the scFv. The sequences of linker 1 and linker 2 are shown in SEQ ID No. 15 and SEQ ID No. 16. The variable region sequences of anti-CD3 antibody, i.e. anti-CD3-VH (shown in SEQ ID No. 17) and anti-CD3-VL (shown in SEQ ID No. 18) are integrated into the scFv sequence, shown in SEQ ID No. 19.

[0195] (5) Finally, the scFv of anti-CD3 is integrated into the C-terminal of the heavy chain of human IgG4 monoclonal antibody 10F7MN, and the sequence of the obtained fusion heavy chain is shown in SEQ ID No. 20. The combination of SEQ ID No. 13 and SEQ ID No. 20 is the PPC3S molecule.

[0196] Therefore, the PPC3S molecule of the present embodiment is a dual-target (CD235a, CD3) tetravalent (2 Fv binding CD235a, 2 scFv binding CD3) IgG4 molecule containing constant region from human and variable region from mouse, and the structural schematic diagram is shown in Figure 29.

[0197] More specifically, the CDR sequence information in the CD235a binding domain and the CD3 binding domain of the PPC3S molecule of the present application is shown in the following table.

[0198] CDR sequence of the PPC3S molecule of the present application

[0199] Example 8: Construction of expression vector and cell bank of PPC3S molecule

[0200] The DNA sequences of the light chain and the heavy chain of PPC3S were artificially synthesized (Genscript), and cloned into PHS10.0 expression vector (Haoyang Biotech). The PHS10.0 expression vector has a double CMV promoter and an open reading frame to ensure that the heavy chain and the light chain of PPC3S are highly expressed in a 1:1 ratio and assembled into an IgG structure. After the recombinant plasmid is transformed into E. coli, the recombinant plasmid with the light chain and the heavy chain of PPC3S can be obtained by ampicillin resistance gene (Amp), pressure screening and amplification. After amplification and purification, the recombinant plasmid is electroporated into mammalian expression cells, and the cells stably expressing PPC3S can be obtained by blasticidin resistance gene (Bla) and pressure screening.

[0201] The recombinant expression vector was transfected into the host cell CHO-K1, and 48 hours after transfection, CD CHO medium containing blasticidin and zeocin was added and plated into a 96-well plate. The culture medium was changed twice a week until the cell confluence recovered to more than 50%, as a minipool. The minipool was transferred to a new 96-well plate, and after changing the medium, the expression was detected for about 24 hours. The expression amount was detected by HTRF using a human IgG HTR kit, and the minipools with the highest expression amount were selected for mixed inoculation and placement to obtain 10 cell populations. Batch fed culture was performed on the cell populations, and the expression sample quality was detected to confirm the preferred cell population. The batch fed culture scheme is shown in Table 10.

[0202] Table 10. PPC3S batch fed culture scheme

[0203] The expression amount results of each cell population are shown in Table 11.

[0204] Table 11. PPC3S batch fed culture of each cell population

[0205] Considering the expression level and cell growth of the cell population, the D14 supernatant of HSP6093-B7Z4-M001, HSP6093-B7Z4-M003 and HSP6093-B7Z4-M007 was collected, and the protein quality analysis was performed after one-step purification by protein A. The analysis results are shown in Table 12.

[0206] Table 12. Quality analysis results of the expressed protein of the preferred cell population of PPC3S batch fed culture

[0207] Based on the above quality analysis data, HSP6093-B7Z4-M003 and HSP6093-B7Z4-M007 were selected for the original cell bank library, and the cells were amplified and subcultured in CD CHO medium (containing 4 mM glutamine, 400 μg / ml zeocin and 7 μg / ml blasticidin), and the culture conditions were maintained at 36.5°C, 6.0% CO2, and 110 rpm. After culturing to the target cell number, the cell viability was detected, the cells were collected by centrifugation, resuspended with a cryopreservation solution containing 10% DMSO, and then programmed cooling and cryopreservation. The cells were sampled and detected, and the cell density and viability were detected after recovery. The detection results are shown in Table 13.

[0208] Table 13. PCB viable cell density and viability of PPC3S

[0209] Example 9: Preparation of PPC3S molecules

[0210] The preparation process of PPC3S is divided into cell culture process and protein purification process to obtain high-purity, low-impurity, and exogenous factor controlled samples for the preparation of PBMC under sterile conditions. The cell culture process is shown in Table 14. After process confirmation, the cells of HSP6093-B7Z4-M003 are preferred for the preparation of the stock solution of PPC3S. Under the process conditions, the detection data of cell viability (VIA), viable cell density (VCD), lactic acid (Lac), etc. during the cell culture stage are shown in Table 15, and the expression amount and quality data are shown in Table 16.

[0211] Table 14. Cell culture process of PPC3S

[0212] Table 15. Cell culture monitoring data of PPC3S

[0213] Table 16. Expression amount and quality data of PPC3S

[0214] Because the sample obtained by the cell culture process is obtained by one-step affinity purification, the sample contains multimers, and the culture solution contains CHO-related HCP, HCD, exogenous viruses, etc. which need to be removed by purification process. After the cell culture solution is harvested, the following steps are performed: deep filtration, S / D virus inactivation, affinity chromatography, intermediate product deep filtration, cation exchange chromatography, virus removal filtration, ultrafiltration and diafiltration, stock solution preparation and subpackaging, to obtain the stock solution of PPC3S. The components of the stock solution include: 5 mg / ml PPC3S protein, 10 mM histidine-histidine hydrochloride, 9% sucrose, pH 5.5.

[0215] Among them, the two-step process of S / D incubation virus inactivation and virus removal filtration is selected, and under the experimental conditions of scale-down and adding virus, the virus removal verification is carried out, and the detection results are shown in Table 17. The results show that the protein purification process can effectively remove the exogenous virus, and the removal efficiency LRV is not less than 9.4 logs, which meets the requirements of exogenous factor control.

[0216] Table 17. Virus removal verification results of PPC3S

[0217] Therefore, the detection items and acceptable standards of the PPC3S stock solution include but are not limited to: (1) pH, which should be 5.5±0.2; (2) SEC-HPLC purity, the main peak should be not less than 90.0%, and the HMW should be not higher than 10.0%; (3) NR-CE purity, the main peak should be not less than 80.0%, and the LMW report result; (4) bacterial endotoxin, which should be not higher than 1.0 EU / mg; (5) sterility, which should be sterile growth. In the non-GMP stage, microbial limit is used instead of sterility test, the total number of aerobic bacteria should be not higher than 3 cfu / 30 ml, and the total number of mold and yeast should be not higher than 3 cfu / 30 ml. The PPC3S stock solution of the present application meets the standards for these detection items.

[0218] Sequence description of the present application:

[0219] The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0220] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. In addition, any theory, mechanism, proof, or finding stated herein is intended to further enhance the understanding of the application, and is not intended to limit the application to a certain theory, mechanism, proof, or finding. Although the application has been shown and described in detail in the accompanying drawings and foregoing description, the application should be considered as illustrative and not restrictive.

Claims

1. A method for enriching natural killer (NK) cells in a blood sample, the method comprising: a) Add a bispecific antibody targeting erythrocyte surface antigen and T cell surface antigen and an erythrocyte sedimentation agent to the blood sample. After sedimentation, separate the upper part to obtain the NK cell-enriched component. b) Processing the components of the enriched NK cells, the processing steps including one or more of the following: cell washing, adjusting cell density, sampling for NK cell counting and / or detection of NK cell properties, and replacement of cryopreservation solution; c) Collect NK cells after the treatment in step b); and d) Optionally, NK cells may be cryopreserved; At least steps a) through c) are performed in a fully enclosed manner, preferably in a fully enclosed automated manner.

2. The method according to claim 1, wherein the bispecific antibody targets CD235a molecules on erythrocytes and CD3 molecules on T cells, and comprises a CD235a binding domain and a CD3 binding domain, wherein: The CD235a binding domain comprises a first light chain variable region and a first heavy chain variable region. The first light chain variable region comprises LCDR1 with the amino acid sequence RASSNVKYMY (SEQ ID No. 22), LCDR2 with the amino acid sequence YTSNLAS (SEQ ID No. 23), and LCDR3 with the amino acid sequence QQFTSSPYT (SEQ ID No. 24). The first heavy chain variable region comprises HCDR1 with the amino acid sequence SYFMH (SEQ ID No. 25), HCDR2 with the amino acid sequence MIRPNGGTTDYNEKFKN (SEQ ID No. 26), and HCDR3 with the amino acid sequence WEGSYYALDY (SEQ ID No. 27). The CD3 binding domain comprises a second light chain variable region and a second heavy chain variable region. The second light chain variable region comprises LCDR1 with the amino acid sequence RASSSVSYMN (SEQ ID No. 28), LCDR2 with the amino acid sequence DTSKVAS (SEQ ID No. 29), and LCDR3 with the amino acid sequence QQWSSNPLT (SEQ ID No. 30). The second heavy chain variable region comprises HCDR1 with the amino acid sequence RYTMH (SEQ ID No. 31), HCDR2 with the amino acid sequence YINPSRGYTNYNQKFKD (SEQ ID No. 32), and HCDR3 with the amino acid sequence YYDDHYCLDY (SEQ ID No. 33). The amino acid sequences of HCDR and LCDR are defined according to Kabat. Preferably, the CD235a binding domain comprises a first light chain variable region having an amino acid sequence as set forth in SEQ ID No. 6 and a first heavy chain variable region having an amino acid sequence as set forth in SEQ ID No. 7, and / or the CD3 binding domain comprises a second light chain variable region having an amino acid sequence as set forth in SEQ ID No. 18 and a second heavy chain variable region having an amino acid sequence as set forth in SEQ ID No.

17.

3. The method of claim 2, wherein the bispecific antibody further comprises a heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is of human IgG4 subtype, more preferably, the heavy chain constant region has an amino acid sequence as set forth in SEQ ID No. 10 or SEQ ID No. 21; and / or the light chain constant region has an amino acid sequence as set forth in SEQ ID No.

11. Optionally, wherein the first light chain variable region of the CD235a binding domain is associated with the light chain constant region to form a light chain of the CD235a binding domain, and the first heavy chain variable region of the CD235a binding domain is associated with the heavy chain constant region to form a heavy chain of the CD235a binding domain; preferably, the light chain of the CD235a binding domain has an amino acid sequence as set forth in SEQ ID No. 13 and / or the heavy chain of the CD235a binding domain has an amino acid sequence as set forth in SEQ ID No.

14. Optionally, wherein the CD3 binding domain is in a single chain antibody (scFv) format, for example, in a structure of second heavy chain variable region-first linker-second light chain variable region formed by a first linker; for example, the first linker is a GS type flexible linker, preferably, the first linker has an amino acid sequence as set forth in SEQ ID No. 16; optionally, the CD3 binding domain has an amino acid sequence as set forth in SEQ ID No.

19. Optionally, wherein the CD3 binding domain is associated with the heavy chain constant region of the CD235a binding domain to form a fusion heavy chain and the fusion heavy chain is combined with the light chain of the CD235a binding domain to form the bispecific antibody, preferably, the CD3 binding domain is associated with the CD235a binding domain through a second linker; for example, the second linker is a GS type flexible linker, preferably, the second linker has an amino acid sequence as set forth in SEQ ID No. 15; optionally, the fusion heavy chain has an amino acid sequence as set forth in SEQ ID No.

20. Optionally, the bispecific antibody is a tetravalent IgG4 molecule comprising variable regions from mice and constant regions from humans.

4. The method of any one of claims 1-3, wherein the erythrocyte sedimentation agent comprises hydroxyethyl starch, gelatin, dextran, polyvinylpyrrolidone, and / or methylcellulose.

5. The method of any one of claims 1-4, wherein the processing step in b) comprises a cryopreservation medium exchange, wherein the cryopreservation medium comprises one or more cryoprotectants, such as permeating cryoprotectants (e.g., dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, acetamide, and / or methanol) and / or non-permeating cryoprotectants (e.g., polyvinylpyrrolidone, sucrose, polyethylene glycol, dextran, albumin, and / or hydroxyethyl starch); Optionally, the cryopreservation medium further comprises one or more amino acids and / or vitamins; Preferably, the cryopreservation medium comprises dimethyl sulfoxide (e.g., at a final concentration of < 5%), dextran, sodium alginate, trehalose, human blood albumin, sorbitol, compound electrolytes, sodium chloride injection, dextrose sodium chloride injection, dextran 40 dextrose injection, compound amino acid injection, compound vitamin b injection.

6. The method of any one of claims 1-5, wherein the blood sample is a peripheral blood sample and / or a cord blood sample derived from a donor, optionally the donor is screened for a history of pathogenic microorganisms (e.g., hepatitis B virus (HBV), hepatitis C virus (HCV), Treponema pallidum, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human T-cell lymphotropic virus (HTLV), human parvovirus B19, human herpes viruses 6 / 7 / 8, adenovirus, JC virus) and / or genetic diseases.

7. A closed system for enriching natural killer (NK) cells in a blood sample, the system comprising: a sedimentation container (e.g., a sedimentation bag) configured to receive the blood sample and allow a sedimentation process to occur therein; a sedimentation agent providing device comprising a bispecific antibody targeting red blood cell surface antigens and T cell surface antigens and a red blood cell sedimentation agent, the sedimentation agent providing device being in fluid connection with the sedimentation container and configured to provide the sedimentation agent to the sedimentation container; a separation device (e.g., a plasmapheresis clamp) configured to perform a phase separation of the multi-phase liquid in the sedimentation container; a cell processing device configured to perform one or more of the following on the NK cell-enriched fraction: cell washing, adjusting cell density, sampling for counting and / or detecting NK cell properties, cryopreservation medium exchange; and optionally, a collection container (e.g., a collection bag) for collecting the NK cell-enriched fraction processed by the cell processing device, wherein the system is preferably a closed automated system.

8. The system of claim 7, wherein the sedimentation agent providing device provides the sedimentation agent to the sedimentation container through a dip tube.

9. The system according to any one of claims 7-8, wherein the bispecific antibody targets a CD235a molecule on a red blood cell and a CD3 molecule on a T cell, and comprises a CD235a binding domain and a CD3 binding domain, wherein: the CD235a binding domain comprises a first light chain variable region comprising a LCDR1 of the amino acid sequence RASSNVKYMY (SEQ ID No. 22), a LCDR2 of the amino acid sequence YTSNLAS (SEQ ID No. 23), and a LCDR3 of the amino acid sequence QQFTSSPYT (SEQ ID No. 24), and a first heavy chain variable region comprising a HCDR1 of the amino acid sequence SYFMH (SEQ ID No. 25), a HCDR2 of the amino acid sequence MIRPNGGTTDYNEKFKN (SEQ ID No. 26), and a HCDR3 of the amino acid sequence WEGSYYALDY (SEQ ID No. 27), and the CD3 binding domain comprises a second light chain variable region comprising a LCDR1 of the amino acid sequence RASSSVSYMN (SEQ ID No. 28), a LCDR2 of the amino acid sequence DTSKVAS (SEQ ID No. 29), and a LCDR3 of the amino acid sequence QQWSSNPLT (SEQ ID No. 30), and a second heavy chain variable region comprising a HCDR1 of the amino acid sequence RYTMH (SEQ ID No. 31), a HCDR2 of the amino acid sequence YINPSRGYTNYNQKFKD (SEQ ID No. 32), and a HCDR3 of the amino acid sequence YYDDHYCLDY (SEQ ID No. 33), wherein the amino acid sequences of the HCDRs and LCDRs are according to the definition of Kabat; preferably, the CD235a binding domain comprises a first light chain variable region of the amino acid sequence as set forth in SEQ ID No. 6 and a first heavy chain variable region of the amino acid sequence as set forth in SEQ ID No. 7, and / or the CD3 binding domain comprises a second light chain variable region of the amino acid sequence as set forth in SEQ ID No. 18 and a second heavy chain variable region of the amino acid sequence as set forth in SEQ ID No.

17.

10. The system according to claim 9, wherein the bispecific antibody further comprises a heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is of human IgG4 subtype, more preferably, the amino acid sequence of the heavy chain constant region is as set forth in SEQ ID No. 10 or SEQ ID No. 21; and / or the amino acid sequence of the light chain constant region is as set forth in SEQ ID No.

11. Optionally, a first light chain variable region of the CD235a binding domain is associated with the light chain constant region to form a light chain of the CD235a binding domain, and a first heavy chain variable region of the CD235a binding domain is associated with the heavy chain constant region to form a heavy chain of the CD235a binding domain; preferably, the amino acid sequence of the light chain of the CD235a binding domain is set forth in SEQ ID No. 13 and / or the amino acid sequence of the heavy chain of the CD235a binding domain is set forth in SEQ ID No. 14; Optionally, the CD3 binding domain is in the form of a single chain antibody (scFv), for example, in the structure of second heavy chain variable region-first linker-second light chain variable region formed by a first linker; for example, the first linker is a flexible linker of GS type, preferably, the amino acid sequence of the first linker is set forth in SEQ ID No. 16; optionally, the amino acid sequence of the CD3 binding domain is set forth in SEQ ID No. 19; Optionally, the CD3 binding domain is associated with the heavy chain constant region of the CD235a binding domain to form a fusion heavy chain and the fusion heavy chain is combined with the light chain of the CD235a binding domain to form the bispecific antibody, preferably, the CD3 binding domain is associated with the CD235a binding domain through a second linker; for example, the second linker is a flexible linker of GS type, preferably, the amino acid sequence of the second linker is set forth in SEQ ID No. 15; optionally, the amino acid sequence of the fusion heavy chain is set forth in SEQ ID No. 20; Optionally, the bispecific antibody is a tetravalent IgG4 molecule comprising variable regions from mice and constant regions from humans.

11. The system of any one of claims 7-10, wherein the erythrocyte sedimentation agent comprises hydroxyethyl starch, gelatin, dextran, polyvinylpyrrolidone, and / or methylcellulose.

12. The system of any one of claims 7-11, wherein the cell processing device comprises a cryopreservation solution exchange module for performing a cryopreservation solution exchange on the NK cell-enriched component, and wherein the cryopreservation solution comprises one or more cryoprotective agents, such as a permeating cryoprotective agent (e.g., dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, acetamide, and / or methanol) and / or a non-permeating cryoprotective agent (e.g., polyvinylpyrrolidone, sucrose, polyethylene glycol, dextran, albumin, and / or hydroxyethyl starch); Optionally, the cryopreservation solution further comprises one or more amino acids and / or vitamins; Preferably, the cryopreservation solution comprises dimethyl sulfoxide (e.g., at a final concentration of < 5%), dextran, sodium alginate, trehalose, human blood albumin, sorbitol, compound electrolyte, sodium chloride injection, glucose sodium chloride injection, dextran 40 glucose injection, compound amino acid injection, compound vitamin b injection.

13. The system of any one of claims 7-12, wherein the blood sample is a peripheral blood sample and / or a cord blood sample derived from a donor, optionally the donor is screened for a history of a pathogenic microorganism (e.g., hepatitis B virus (HBV), hepatitis C virus (HCV), Treponema pallidum, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human T-lymphotropic virus (HTLV), human parvovirus B19, human herpes viruses 6 / 7 / 8, adenovirus, JC virus) and / or a genetic disease.

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