Full-closed method and system for enriching natural killer cells

Abstract

The invention provides a method and a system for enriching natural killer (NK) cells in a blood sample.

Description

Technical Field

[0001] The present application generally relates to the field of biotechnology, and in particular, to a fully enclosed method and system for enriching natural killer cells. Background Art

[0002] Natural killer (NK) cells, as innate immune cells with the ability to directly kill malignant cells, are increasingly considered a promising immunotherapy tool, and various clinical trials are currently underway. In order to obtain a large number of highly pure and cytotoxic NK cells, 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 for generating NK cells for immunotherapy.

[0003] At present, 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. The proportion of NK in the materials is not high. For example, T cells (CD3 + ) account for about 50% to 84%, NK cells (CD3 - 、CD16 + and / or CD56 + ) accounted for about 7% to 40% (see Zhu Lihua and Wang Jianzhong, "Survey on Reference Values ​​of Blood Lymphocyte Immunophenotypes of Chinese People", "Chinese Journal of Medical Laboratory Science", 1998.21(4): 223-226, and there are the following deficiencies:

[0004] 1. The operation is carried out in a biological safety cabinet, which is not a fully enclosed preparation. It has high requirements on the environment and has the risk of pollution and cross contamination.

[0005] 2. Ficoll separation fluid is generally a scientific research reagent and has great risks in clinical use.

[0006] 3. It is highly dependent on human operation and unstable between batches.

[0007] 4. The recovery rate of mononuclear cells is low.

[0008] 5. The proportion of T cells in the obtained mononuclear cells is high, which affects the purity of subsequent NK cell culture.

[0009] Therefore, there is a need in the art for methods and systems that solve the above problems and deficiencies. Summary of the invention

[0010] In a first aspect, the present application provides a method for enriching natural killer (NK) cells in a blood sample, the method comprising:

[0011] a) adding a bispecific antibody targeting an erythrocyte surface antigen and a T cell surface antigen and an erythrocyte sedimentation agent to the blood sample, and after the sedimentation process, separating the upper layer to obtain a component enriched in NK cells;

[0012] b) processing the NK cell-enriched fraction, wherein the processing steps include one or more of the following: cell washing, adjusting cell density, sampling for NK cell counting and / or detecting NK cell properties, and cryopreservation fluid replacement;

[0013] c) collecting the NK cells treated in step b); and

[0014] d) optionally, cryopreserving the NK cells;

[0015] At least steps a) to c) are performed in a fully closed manner, preferably in a fully closed automated manner.

[0016] 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:

[0017] a sedimentation container (e.g., a sedimentation bag) configured to receive the blood sample and allow a sedimentation process of the blood sample to occur therein;

[0018] A sedimentation product providing device, wherein the sedimentation product comprises a bispecific antibody targeting an erythrocyte surface antigen and a T cell surface antigen and an erythrocyte sedimentation agent, wherein the sedimentation product providing device is fluidically connected to the sedimentation container and is configured to provide the sedimentation product to the sedimentation container;

[0019] a separation device (e.g., a slurry separator), the separation device being configured to phase separate the multiphase liquid in the settling vessel;

[0020] A cell processing device, wherein the cell processing device is configured to perform one or more of the following treatments on the NK cell-enriched portion: cell washing, adjusting cell density, sampling for counting NK cells and / or detecting NK cell properties, and cryopreservation fluid replacement; and

[0021] Optionally, a collection container (e.g., a collection bag) for collecting the NK cell-enriched portion processed by the cell processing device,

[0022] The system is preferably a fully closed automated system.

[0023] In some embodiments of the second aspect, the sedimentation product providing device provides the sedimentation product to the sedimentation container through a pipe.

[0024] In some embodiments of the first aspect and / or the second aspect, the bispecific antibody targets the CD235a molecule on erythrocytes and the CD3 molecule on T cells, and comprises a CD235a binding domain and a CD3 binding domain, wherein:

[0025] The CD235a binding domain comprises a first light chain variable region and a first heavy chain variable region, wherein the first light chain variable region comprises LCDR1 with an amino acid sequence of RASSNVKYMY (SEQ ID No. 22), LCDR2 with an amino acid sequence of YTSNLAS (SEQ ID No. 23), and LCDR3 with an amino acid sequence of QQFTSSPYT (SEQ ID No. 24), the first heavy chain variable region comprises HCDR1 with an amino acid sequence of SYFMH (SEQ ID No. 25), HCDR2 with an amino acid sequence of MIRPNGGTTDYNEKFKN (SEQ ID No. 26), and HCDR3 with an amino acid sequence of WEGSYYALDY (SEQ ID No. 27), and

[0026] 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 a LCDR1 with an amino acid sequence of RASSSVSYMN (SEQ ID No.28), a LCDR2 with an amino acid sequence of DTSKVAS (SEQ ID No.29), and a LCDR3 with an amino acid sequence of QQWSSNPLT (SEQ ID No.30), the second heavy chain variable region comprises a HCDR1 with an amino acid sequence of RYTMH (SEQ ID No.31), a HCDR2 with an amino acid sequence of YINPSRGYTNYNQKFKD (SEQ ID No.32), and a HCDR3 with an amino acid sequence of YYDDHYCLDY (SEQ ID No.33),

[0027] Among them, the amino acid sequences of HCDR and LCDR are based on the Kabat definition;

[0028] Preferably, the CD235a binding domain comprises a first light chain variable region with an amino acid sequence as shown in SEQ ID No.6 and a first heavy chain variable region with an amino acid sequence as shown in SEQ ID No.7, and / or the CD3 binding domain comprises a second light chain variable region with an amino acid sequence as shown in SEQ ID No.18 and a second heavy chain variable region with an amino acid sequence as shown in SEQ ID No.17.

[0029] In some embodiments of the first aspect and / or the 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 amino acid sequence of the heavy chain constant region is as shown in SEQ ID No.10 or SEQ ID No.21; and / or the amino acid sequence of the light chain constant region is as shown in SEQ ID No.11;

[0030] Optionally, the first light chain variable region of the CD235a binding domain is associated with 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 to form the heavy chain of the CD235a binding domain; preferably, the amino acid sequence of the light chain of the CD235a binding domain is as shown in SEQ ID No.13 and / or the amino acid sequence of the heavy chain of the CD235a binding domain is as shown in SEQ ID No.14;

[0031] Optionally, the CD3 binding domain is in the form of a single-chain antibody (scFv), for example, a structure of a second heavy chain variable region-a first linker-a second light chain variable region formed by a first linker; for example, the first linker is a GS-type flexible linker, preferably, the amino acid sequence of the first linker is as shown in SEQ ID No.16; Optionally, the amino acid sequence of the CD3 binding domain is as shown in SEQ ID No.19;

[0032] 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 via a second linker; for example, the second linker is a GS-type flexible linker, preferably the amino acid sequence of the second linker is shown in SEQ ID No.15; optionally, the amino acid sequence of the fusion heavy chain is shown in SEQ ID No.20;

[0033] Optionally, the bispecific antibody is a tetravalent IgG4 molecule comprising variable regions from mouse and constant regions from human.

[0034] In some embodiments of the first aspect and / or the second aspect, the erythrocyte sedimentation agent comprises hydroxyethyl starch, gelatin, dextran, polyvinyl pyrrolidone and / or methylcellulose.

[0035] In some embodiments of the first aspect, the processing step in b) includes cryopreservation fluid replacement. In some embodiments of the second aspect, the cell processing device includes a cryopreservation fluid replacement module for replacing the cryopreservation fluid of the NK cell-enriched component. In some specific embodiments, the cryopreservation fluid comprises one or more cryoprotectants. The cryoprotectant is, for example, a permeable cryoprotectant (e.g., dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, acetamide and / or methanol) and / or a non-permeable cryoprotectant (e.g., polyvinyl pyrrolidone, sucrose, polyethylene glycol, dextran, albumin and / or hydroxyethyl starch). In some specific embodiments, the cryopreservation fluid also comprises one or more amino acids and / or vitamins. For example, the cryopreservation fluid may comprise dimethyl sulfoxide (e.g., final concentration ≤5%), dextran, sodium alginate, trehalose, human albumin, sorbitol, compound electrolytes, sodium chloride injection, glucose sodium chloride injection, dextran 40 glucose injection, compound amino acid injection, compound vitamin b injection.

[0036] In some embodiments of the first aspect and / or the second aspect, the blood sample is a peripheral blood sample and / or an umbilical cord blood sample derived from a donor. The donor can undergo screening, for example, by 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 virus (HTLV), human parvovirus B19, human herpes virus 6 / 7 / 8 type, adenovirus, JC virus) detection and / or genetic history for screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 A general flow chart of an exemplary method of enriching NK cells in a biological sample of the present application is shown.

[0038] Figure 2 A flow chart of a method for enriching NK cells in a biological sample in some specific embodiments of the present application is shown, wherein PPC3S is an exemplary bispecific antibody of the present application, which is a tetravalent bispecific antibody that can specifically bind to CD235a molecules on red blood cells and CD3 molecules on T cells at the same time. The design and preparation methods thereof can be found in Examples 7 to 9.

[0039] Figures 3 to 7 The comparison of NK cell recovery rate, total recovery rate and pre-cryopreservation viability obtained by using Stemcell cryopreservation solution and homemade cryopreservation solution in Example 3 is shown ( Figure 3 ), cell proliferation 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 ).

[0040] Figures 8 to 10 It shows that compared with Comparative Example 1, the percentage of T cells in Example 4 ( Figure 8 ), NK cell percentage ( Fig. 9 ), cell viability ( Fig.10 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 significantly remove T cells and other miscellaneous cells in peripheral blood, increase the NK ratio, and there is no significant difference in cell viability before and after separation; the ratio of T cells and NK cells after separation in Comparative Example 1 is equivalent to that before separation.

[0041] Figure 11-Figure 22 Flow cytometry graphs of four samples (denoted as A1, A2, A3 and A4, respectively) prepared by the method of Example 4 and four samples (denoted as B1, B2, B3 and B4, respectively) prepared by the method of Comparative Example 1 are shown respectively.

[0042] Figure 23 to Figure 26 The cell growth change curves of the samples prepared by the method in Example 5 are shown respectively ( Fig.23 ), cell viability change curve ( Fig.24 ), NK purity change curve ( Fig.25 ) and in vitro killing data of Daudi tumor cells ( Fig.26 ).

[0043] Fig. 27 and Fig.28 Flow cytometric graphs of two batches of cell samples (respectively indicated as batch 1 and batch 2) prepared by the method in Example 5 on the 18th day (D18) of culture are shown respectively.

[0044] Fig.29 A schematic diagram of the framework of an exemplary bispecific antibody PPC3S of the present application is shown. DETAILED DESCRIPTION

[0045] definition

[0046] Unless otherwise defined, all scientific and technical terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terms in the art, professionals can refer specifically to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0047] Although the numerical ranges and parameter approximations shown in the broad scope of the present application, the numerical values ​​shown in the specific examples are recorded as accurately as possible. However, any numerical value is bound to contain a certain error, which is caused by the standard deviation in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, the range of "1 to 10" recorded should be considered to include any and all sub-ranges between the minimum value 1 and the maximum value 10 (including the endpoints); that is, all sub-ranges starting with a minimum value of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum value of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0048] The terms "include" and "comprising" used herein should be interpreted as inclusive and open-ended, rather than exclusive. Specifically, when used in the specification and claims, the terms "include" and "comprising" and their variations mean including the specified features, steps or components. These terms should not be interpreted to exclude the presence of other features, steps or components.

[0049] The term "specific binding" as used herein is a term well known in the art, and methods for determining such specific binding of an antibody to an antigen are also well known in the art. For example, in some embodiments, "specific binding" refers to an antibody binding to an intended target but not significantly binding to other targets. An antibody binds to an intended target epitope with significantly increased affinity and / or for a longer duration than binding to other epitopes.

[0050] The term "natural killer cells" used in this article, namely NK cells (natural killer cells), are a type of lymphocytes that are not MHC-restricted, do not require prior sensitization, do not require the participation of antibodies, and can directly kill tumor cells and virus-infected cells in a non-specific manner.

[0051] The term "mononuclear cell (MNC)" used herein refers to cells with a single nucleus in peripheral blood, umbilical cord blood, bone marrow, etc., including lymphocytes and monocytes.

[0052] The term "Ficoll separation solution" used in this article refers to a polysucrose-diatrizoate solution, which utilizes the difference in specific gravity of each blood cell to distribute blood cells of a certain specific gravity in different independent bands according to the corresponding density gradient, thereby achieving the purpose of blood cell separation.

[0053] The term "bispecific antibody" used herein refers to an antibody that has the ability to bind to two antigen epitopes at the same time. The two antigen epitopes can be on different antigens or on the same antigen. Bispecific antibodies can have a variety of structural configurations. For example, a bispecific antibody can be composed of two Fc fragments and two antigen binding parts fused thereto respectively (similar to natural antibodies, the difference is that the two arms bind to different antigen targets or epitopes), and the antigen binding part can be in the form of a single-chain antibody (scFv) or a Fab fragment. The two different binding parts of the bispecific antibody each bind to the N-terminus of an Fc fragment, and the antigen binding part configuration 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 heavy chain heteropolymerization, and KIH technology (knob-in-hole, KIH) is a strategy to solve heavy chain heteropolymerization. Generally, KIH technology refers to the formation of a structure that is conducive to the mutual pairing of heterogeneous half antibodies by modifying the amino acid sequence of the CH3 region, which can maintain the structure of normal antibodies as much as possible while forming a bispecific antibody.

[0054] The term "single-chain antibody (scFv)" as used herein refers to a single polypeptide chain consisting of a VH domain and a VL domain connected by a peptide linker. (scFv) 2 It comprises two VH domains connected by a peptide linker and two VL domains, wherein the two VL domains are combined with the two VH domains via disulfide bridges.

[0055] The term "CD235a" as used herein refers to glycophorin A, which is a major, intrinsic membrane protein of erythrocytes and is expressed on the surface of mature erythrocytes. On average, there are 1×10 5 ~1×10 6This molecule (Merry et al. Biochem J. (1986) 233: 93-98; Loken et al. Blood (1987) 69: 255-263). In addition, CD235a is highly expressed in kidney, bladder, and urethral tissues, and is almost not expressed in lymphocytes (https: / / www.proteinatlas.org / ENSG00000170180-GYPA). CD235a is a component of the ankyrin 1 complex, which is related to the stability and shape of the erythrocyte membrane. Its N-terminus is a glycosylated fragment, which is outside the erythrocyte cell membrane and is a mononuclear blood group receptor. Its 20th to 91st amino acids are the extracellular domain, 92th to 114th amino acids are the transmembrane region, and 115th to 150th amino acids are the intracellular domain, see SEQ ID No. 1 for details. In the CD235 family, there is a very high homology CD235b (glycophorin B, GYPB), and the extracellular N segment sequence is very consistent, see SEQ ID No. 2 for details, and its function is similar to CD235a. In apheresis peripheral blood, CD235a is expressed in red blood cells, but not in lymphocytes, and is a very selective target.

[0056] 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 acquired immune response. When antigen presenting cells activate T cell receptors, TCR-mediated signals are transmitted to the cell through the δ, ε, γ, and ζ subunits of CD3, thereby activating the downstream signaling pathways of T cells. In cell identification, CD3 is a well-known surface marker of T cells in the art. + T cells, naive and memory CD4 + In conventional T cell positive or negative selection kits, CD3 antibody-coupled magnetic beads and supporting reagents are usually used for processing.

[0057] In a first aspect, the present application provides a method for enriching natural killer (NK) cells in a blood sample, the method comprising:

[0058] a) adding a bispecific antibody targeting an erythrocyte surface antigen and a T cell surface antigen and an erythrocyte sedimentation agent to the blood sample, and after the sedimentation process, separating the upper layer to obtain a component enriched in NK cells;

[0059] b) processing the NK cell-enriched fraction, wherein the processing steps include one or more of the following: cell washing, adjusting cell density, sampling for NK cell counting and / or detecting NK cell properties, and cryopreservation fluid replacement;

[0060] c) collecting the NK cells treated in step b); and

[0061] d) optionally, cryopreserving the NK cells;

[0062] At least steps a) to c) are performed in a fully closed manner, preferably in a fully closed automated manner.

[0063] 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:

[0064] a sedimentation container (e.g., a sedimentation bag) configured to receive the blood sample and allow a sedimentation process of the blood sample to occur therein;

[0065] A sedimentation product providing device, wherein the sedimentation product comprises a bispecific antibody targeting an erythrocyte surface antigen and a T cell surface antigen and an erythrocyte sedimentation agent, wherein the sedimentation product providing device is fluidically connected to the sedimentation container and is configured to provide the sedimentation product to the sedimentation container;

[0066] a separation device (e.g., a slurry separator), the separation device being configured to phase separate the multiphase liquid in the settling vessel;

[0067] A cell processing device, wherein the cell processing device is configured to perform one or more of the following treatments on the NK cell-enriched portion: cell washing, adjusting cell density, sampling for counting NK cells and / or detecting NK cell properties, and cryopreservation fluid replacement; and

[0068] Optionally, a collection container (e.g., a collection bag) for collecting the NK cell-enriched portion processed by the cell processing device,

[0069] The system is preferably a fully closed automated system.

[0070] In some embodiments of the second aspect, the sedimentation product providing device provides the sedimentation product to the sedimentation container through a pipe.

[0071] In some embodiments of the first aspect and / or the second aspect, the bispecific antibody targets the CD235a molecule on erythrocytes and the CD3 molecule on T cells, and comprises a CD235a binding domain and a CD3 binding domain, wherein:

[0072] The CD235a binding domain comprises a first light chain variable region and a first heavy chain variable region, wherein the first light chain variable region comprises LCDR1 with an amino acid sequence of RASSNVKYMY (SEQ ID No. 22), LCDR2 with an amino acid sequence of YTSNLAS (SEQ ID No. 23), and LCDR3 with an amino acid sequence of QQFTSSPYT (SEQ ID No. 24), the first heavy chain variable region comprises HCDR1 with an amino acid sequence of SYFMH (SEQ ID No. 25), HCDR2 with an amino acid sequence of MIRPNGGTTDYNEKFKN (SEQ ID No. 26), and HCDR3 with an amino acid sequence of WEGSYYALDY (SEQ ID No. 27), and

[0073] 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 a LCDR1 with an amino acid sequence of RASSSVSYMN (SEQ ID No.28), a LCDR2 with an amino acid sequence of DTSKVAS (SEQ ID No.29), and a LCDR3 with an amino acid sequence of QQWSSNPLT (SEQ ID No.30), the second heavy chain variable region comprises a HCDR1 with an amino acid sequence of RYTMH (SEQ ID No.31), a HCDR2 with an amino acid sequence of YINPSRGYTNYNQKFKD (SEQ ID No.32), and a HCDR3 with an amino acid sequence of YYDDHYCLDY (SEQ ID No.33),

[0074] Among them, the amino acid sequences of HCDR and LCDR are based on the Kabat definition;

[0075] Preferably, the CD235a binding domain comprises a first light chain variable region with an amino acid sequence as shown in SEQ ID No.6 and a first heavy chain variable region with an amino acid sequence as shown in SEQ ID No.7, and / or the CD3 binding domain comprises a second light chain variable region with an amino acid sequence as shown in SEQ ID No.18 and a second heavy chain variable region with an amino acid sequence as shown in SEQ ID No.17.

[0076] Fig.29 An exemplary construction scheme of the bispecific antibody of the present application is shown. Fig.29In the embodiment, the bispecific antibody includes two light chains and two heavy chains, and the N-terminus is a CD235a binding domain (comprising a light chain variable region and a heavy chain variable region that bind to CD235a), and the C-terminus 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 includes a light chain constant region (human IgG4 subtype) and a light chain variable region of a CD235a binding domain (the light chain variable region includes three CDR regions of LCDR1, LCDR2, and LCDR3), and the heavy chain includes a heavy chain constant region (human IgG4 subtype), a heavy chain variable region of a CD235a binding domain (the heavy chain variable region includes 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).

[0077] It should be understood that Fig.29 The construction scheme of the bispecific antibody shown in is merely exemplary and non-restrictive. Those skilled in the art can reasonably know other construction schemes of bispecific antibodies. For example, the bispecific antibody of the present application can be obtained by the following methods (for example only, not limited thereto):

[0078] (i) based on an anti-CD235a full-length antibody, linking an antibody fragment that binds to CD3 (e.g., a single-chain antibody scFv that binds to CD3 and includes a heavy chain variable region and a light chain variable region) to the Fc fragment of the anti-CD235a antibody;

[0079] (ii) based on an anti-CD3 full-length antibody, linking an antibody fragment that binds to CD235a (e.g., a single-chain antibody scFv that binds to CD235a and comprises a heavy chain variable region and a light chain variable region) to the Fc fragment of the anti-CD3 antibody; or

[0080] (iii) replacing one antigen-binding arm of an anti-CD235a antibody (or anti-CD3 antibody) with a set of heavy chain variable regions and light chain variable regions of an anti-CD3 antibody (or anti-CD235a antibody), thereby enabling the two arms of the parent antibody to bind to CD235a and CD3, respectively.

[0081] In some embodiments of the first aspect and / or the 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 a human IgG4 subtype. In some more specific embodiments, the amino acid sequence of the heavy chain constant region is shown in SEQ ID No.10. In some optional embodiments, the heavy chain constant region may include a mutation in its hinge region, such as a mutation of serine (S) at the 10th amino acid position in the hinge region to proline (P), and the amino acid position is numbered according to the EU numbering system. For example, the amino acid sequence of the heavy chain constant region comprising a hinge region S10P mutation can be shown in SEQ ID No.21. In some specific embodiments, the amino acid sequence of the light chain constant region is shown in SEQ ID No.11.

[0082] In some embodiments of the first aspect and / or the 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 as shown in SEQ ID No. 13 and / or the amino acid sequence of the heavy chain of the CD235a binding domain is as shown in SEQ ID No. 14;

[0083] In some embodiments of the first aspect and / or the second aspect, the CD3 binding domain is in the form of a single-chain antibody (scFv), for example, a structure of a second heavy chain variable region-first linker-second light chain variable region formed by a first linker (for example, a structure of N-terminus-second heavy chain variable region-first linker-second light chain variable region-C-terminus). The first linker may be, for example, a GS-type flexible linker, and preferably, the amino acid sequence of the first linker is shown in SEQ ID No. 16. In some more specific embodiments, the amino acid sequence of the CD3 binding domain is shown in SEQ ID No. 19.

[0084] In some embodiments of the first aspect and / or the 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, and preferably the amino acid sequence of the second linker is shown in SEQ ID No. 15. In some more specific embodiments, the amino acid sequence of the fusion heavy chain is shown in SEQ ID No. 20.

[0085] In some embodiments of the first aspect and / or the second aspect, the fusion protein is a tetravalent IgG4 molecule comprising a variable region from a mouse and a constant region from a human.

[0086] There are some studies in the field on the drugability of antibodies derived from different sources of CD235a:

[0087] In patent US2022 / 0153859A1, the humanized antibody for CD235a is named 10F7-M10, the light chain variable region sequence is shown in SEQ ID No. 3, and the heavy chain variable region sequence is shown in SEQ ID No. 4. Its dissociation constant KD for CD235a is 140nM.

[0088] In patent US9879090B2, the VHH antibody of CD235a is named IH5, which is a single-domain antibody. The heavy chain variable region sequence is shown in SEQ ID No. 5. Its dissociation constant KD for CD235a is 33.7 nM.

[0089] In patent WO2017 / 015141A1, the mouse antibody of CD235a is named 10F7, and its light chain variable region sequence is detailed in SEQ ID No.6, and its heavy chain variable region sequence is detailed in SEQ ID No.7. In this patent, it is humanized into scFv, and through binding activity, functional studies, and stability, a combination of Glycophorin A binding activity is confirmed, and the sequence scFv-10F7-EPO is constructed, and its light chain variable region sequence is detailed in SEQ ID No.8, and its heavy chain variable region sequence is detailed in SEQ ID No.9.

[0090] In the literature (Catimel et al. J. Immunol. Methods (1993) 165 (2): 183-192), there are two antibodies for CD235a: one of which is called 1C3 / 86, which is measured to have 4.80×105 CD235a molecules, with a dissociation constant KD of 23×10 -8 M, which is 230nM; another one is called 10F7MN, which is 4.66×10 5 CD235a molecules, with a dissociation constant KD of 9.5×10 -8 M, that is 95nM.

[0091] However, none of these studies discloses or teaches the bispecific antibodies of the present application.

[0092] In some embodiments of the first aspect and / or the second aspect, the bispecific antibody of the present application and a sedimentation agent are added to a peripheral blood sample, wherein the bispecific antibody is in 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, 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 per 100 mL of blood. 14mL, about 14.5mL, about 15mL, about 15.5mL, about 16mL, about 16.5mL, about 17mL, about 17.5mL, about 18mL, about 18.5mL, about 19mL, about 19.5mL, about 20mL, about 20.5mL, about 21mL, about 21.5mL, about 22mL, about 22.5mL, about 23mL, about 23.5mL, about 24mL, about 24.5mL or about 25mL is added, preferably in a volume of about 5mL, about 15mL or about 25mL per 100mL of blood.

[0093] In some embodiments of the first aspect and / or the second aspect, the erythrocyte sedimentation agent comprises hydroxyethyl starch, gelatin, dextran, polyvinyl pyrrolidone and / or methylcellulose, and combinations, analogs or derivatives thereof. The erythrocyte sedimentation agent can be added at a final concentration of 0.5%-4%, for example, at a final concentration of 0.5%-1%, 1%-2% or 2%-4%. For example, the sedimentation agent can be hydroxyethyl starch at a final concentration of about 2%.

[0094] In some embodiments of the first aspect, the processing step in b) includes cryopreservation fluid replacement. In some embodiments of the second aspect, the cell processing device includes a cryopreservation fluid replacement module for replacing the cryopreservation fluid of the NK cell-enriched component. In some specific embodiments, the cryopreservation fluid comprises one or more cryoprotectants. The cryoprotectant is, for example, a permeable cryoprotectant (e.g., dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, acetamide and / or methanol) and / or a non-permeable cryoprotectant (e.g., polyvinyl pyrrolidone, sucrose, polyethylene glycol, dextran, albumin and / or hydroxyethyl starch). In some specific embodiments, the cryopreservation fluid also comprises one or more amino acids and / or vitamins. For example, the cryopreservation fluid may comprise dimethyl sulfoxide (e.g., final concentration ≤5%), dextran, sodium alginate, trehalose, human albumin, sorbitol, compound electrolytes, sodium chloride injection, glucose sodium chloride injection, dextran 40 glucose injection, compound amino acid injection, compound vitamin b injection.

[0095] In some embodiments of the first aspect and / or the second aspect, the blood sample is a peripheral blood sample and / or an umbilical cord blood sample derived from a donor. The donor can undergo screening, for example, by 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 virus (HTLV), human parvovirus B19, human herpes virus 6 / 7 / 8 type, adenovirus, JC virus) detection and / or genetic history for screening.

[0096] In some embodiments of the second aspect, the cell processing device may include one or more of a cell washing module, a cell density adjustment module, a sampling module, and / or a cryopreservation fluid replacement module. The system and / or cell processing device may also include modules that perform other functions, such as a temperature adjustment module, etc. One or more modules in the system and / or cell processing device may be combined into one, such as a cryopreservation fluid replacement module and a cell density adjustment module, so that the original solution in the system is replaced and the cell density in the original solution is adjusted by introducing the cryopreservation fluid.

[0097] In some embodiments of the second aspect, the cell processing device performs one or more of the following operations on the NK cell-enriched component: liquid volume concentration, sodium chloride injection perfusion washing, freezing fluid replacement, freezing fluid resuspension and / or sampling for counting.

[0098] In some embodiments of the first aspect, the method further comprises mixing the NK cells treated in step b) and aliquoting (eg, after mixing), for example, into a freezing container (eg, a freezing bag).

[0099] In some embodiments of the second aspect, the system further comprises a cell mixing device (e.g., a shaker) and / or a filling device (e.g., a peristaltic pump), wherein the cell mixing device is used to mix the NK cell-enriched components processed by the cell processing device, and the filling device is used to fill the NK cell-enriched components (e.g., after mixing by the cell mixing device), for example, into a freezing container (e.g., a freezing bag).

[0100] The inventors of the present application have found that NK cell culture can improve the initial NK purity to obtain high NK purity (NK purity up to 95% or more), high NK cell viable cell density (8.5×10 6 / mL or more) and NK cells with high killing activity (up to 80% or more when the effector-target ratio is 10:1) to solve the world's difficult problem of low purity of NK cell culture.

[0101] The present application uses hydroxyethyl starch and separation reagents (e.g., PPC3S described above) to remove most red blood cells, T cells, and other miscellaneous cells. The cells are then concentrated, washed, replaced with homemade cryopreservation fluid, and density adjusted using a closed, automated instrument. The mononuclear cells are packaged using a shaker and a peristaltic pump through a closed pipeline, and stored in a gas phase liquid nitrogen tank after program cooling. In some embodiments, the method and system of the present application can achieve one or more of the following technical effects:

[0102] 1. The material sources that can be used for the NK cell enrichment method and system of the present application are abundant and have wide applicability, and are not limited to a single source such as commonly used peripheral blood and umbilical cord blood.

[0103] 2. Through fully enclosed preparation, the risk of pollution and cross-contamination is reduced. Class D environment can meet environmental requirements and reduce laboratory construction and maintenance costs.

[0104] 3. Through the separation reagent, most of the red blood cells, T cells and other miscellaneous cells in the material can be removed, and the NK cells in the material can be enriched. Compared with flow cytometry and magnetic bead screening, the process of this application is low in cost, short in time and simple in operation.

[0105] 4. Reduce the risk of human operating errors by using automated instruments. Compared with similar products on the market, this instrument has a high solution replacement rate and NK cell recovery rate, avoids the cell accumulation problem caused by centrifugation, and has a high cell viability.

[0106] 5. The homemade cryopreservation solution of the present application has components of medicines or pharmaceutical excipients, has safe ingredients and excellent cryopreservation effect.

[0107] 6. Use a peristaltic pump and shaker to dispense the mononuclear cells into cryopreservation bags, ensuring uniformity of the dispensing.

[0108] In summary, the method of the present application can make up for the shortcomings of non-fully enclosed methods (e.g., Ficoll separation method) and achieve the goal of obtaining mononuclear cells (MNC) from multiple sources such as peripheral blood, umbilical cord blood, and bone marrow with fully enclosed, automated, high recovery rate, batch-to-batch stability, and high NK purity.

[0109] In some embodiments, the subject matter of the present application is further characterized by one or more of the following:

[0110] 1. The method of this application may mainly include collection and transportation, sedimentation treatment, automated instrument processing, packaging, freezing and other parts. For details, please refer to Figure 1 and / or Figure 2 .

[0111] 2. All sampling, reagent addition, and cell transfer in the present application method can be performed in a closed manner after being docked with a sterile pipe connection machine.

[0112] 3. In the collection and transportation operation of this application, the source of materials can be peripheral blood, umbilical cord blood, bone marrow, iPSC, etc.

[0113] 4. During the collection and transportation operation of this application, the material donors can be screened for pathogenic microorganisms as needed, including HBV, HCV, syphilis, HIV, EBV, CMV, HTLV, human parvovirus B19, human herpes virus type 6 / 7 / 8, adenovirus, and JC virus. After collection, the material must be transported in a cold chain at 2-8°C.

[0114] 5. In the sedimentation treatment operation of the present application, the amount of separation reagent (e.g., PPC3S, e.g., about 5 mg / ml) added is 5-25 ml / 100 mlc material, the incubation time is 40-60 min. The final concentration of hydroxyethyl starch solution after addition is 1-2%, and the static sedimentation time is 30-60 min.

[0115] 6. In the automated instrument processing operation of the present application, the automated instrument can be used to concentrate, wash, replace the cryopreservation solution, and adjust the density to 1.0-8.0×10 6 The cell transfer flow rate is 30-60 ml / min, the washing and concentration volume is 2-5 ml, and the number of replacements is 2-5.

[0116] 7. In the automated instrument processing operation of the present application, the cryopreservation solution that can be used includes DMSO, dextran, sodium alginate, trehalose, human albumin, sorbitol, compound electrolytes, sodium chloride injection, glucose sodium chloride injection, dextran 40 glucose injection, compound amino acid injection, and compound vitamin B injection. Among them, DMSO can be ≤5%.

[0117] 8. In the packaging operation of the present application, the mononuclear cells can be evenly packaged into cryopreservation bags using a shaker and a peristaltic pump, and the shaker speed is 30 to 80 rpm.

[0118] It should be understood that the above detailed description is only for the purpose of enabling those skilled in the art to more clearly understand the content of the present application, and is not intended to be limiting in any aspect. Those skilled in the art can make various modifications and changes to the embodiments.

[0119] Example

[0120] The present application is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application.

[0121] Comparative Example 1: Separation of mononuclear cells using Ficoll separation medium

[0122] 1. Collection and transportation: Peripheral blood donors are screened for pathogenic microorganisms (hepatitis B virus (HBV), hepatitis C virus (HCV), syphilis spirochetes, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human T-lymphotropic virus (HTLV), human parvovirus B19, human herpes virus type 6 / 7 / 8, adenovirus, JC virus) and genetic disease history. After the test is qualified, the materials are collected and transported at 2-8℃ cold chain.

[0123] 2. In a biosafety cabinet, transfer the peripheral blood to a 225 ml conical bottom centrifuge bottle, dilute the peripheral blood with an equal volume of saline, and then slowly add 20 ml of the diluted peripheral blood to 20 ml of Ficoll separation solution. Centrifuge at 600 g for 15 min at room temperature.

[0124] 3. After centrifugation, discard the supernatant, collect the buffy coat cells into a 225 ml conical bottom centrifuge bottle, make up to 200 ml with physiological saline, and centrifuge at 300 g for 10 min at room temperature.

[0125] 4. After centrifugation, discard the supernatant and adjust the density to 5.0×10 6 / ml, after mixing, take 5ml for sterility, mycoplasma, endotoxin, counting, and flow cytometry detection, and distribute the remaining mononuclear cells into cryopreservation tubes.

[0126] 5. The cryotubes are cooled to -80°C and then stored in a gas phase liquid nitrogen tank.

[0127] Example 1: Donor Screening

[0128] Since peripheral blood, umbilical cord blood and other materials from donors may contain pathogens of blood-borne diseases, in order to ensure the safety of PBMC, the material donors or materials must be strictly controlled. Pathogenic microorganism screening of material donors includes HBV, HCV, Treponema pallidum, HIV, EBV, CMV, HTLV, human parvovirus B19, HIV, human herpes virus 6 / 7 / 8, adenovirus, and JC virus. Material collection can only be carried out after the pathogen screening is qualified, and the materials are tested for viable cell count, cell viability, flow cytometry, sterility test, and mycoplasma test. They can only be used for PBMC preparation after all tests are qualified.

[0129] Example 2: Sedimentation treatment

[0130] In this example, a separation agent is used in combination with hydroxyethyl starch to remove most of the red blood cells, T cells and other impurity cells. The separation agent used in Examples 2 to 5 is the bispecific antibody PPCS3 described above that can specifically bind to the CD235a molecule on red blood cells and the CD3 molecule on T cells, with a concentration of 5 mg / ml. The design and preparation of PPCS3 are shown in Examples 7 to 9 below.

[0131] The sedimentation treatment steps are as follows:

[0132] Add an equal amount of peripheral blood to a centrifuge tube, and add different concentrations of separation reagent (5mL / 100mL peripheral blood, 15mL / 100mL peripheral blood, 25mL / 100mL peripheral blood). Mix well and incubate for 40 minutes. Add 6% hydroxyethyl starch solution during incubation. The final concentration of hydroxyethyl starch solution after addition is 2%. After settling for 0.5 hours, aspirate the upper plasma layer, resuspend to 45mL, centrifuge (300g, 10min), discard the supernatant, and resuspend the cell pellet. Detect NK purity (CD3 - CD56 + ) and the main impurity item T cell ratio (CD3 + ). Specific experimental data are shown in Table 1 below.

[0133] Table 1. Sedimentation treatment experimental data

[0134]

[0135] The results showed that by using the separation reagent and hydroxyethyl starch together, the proportion of T cells in mononuclear cells obtained in the three groups was ≤0.5%, and the NK purity was >70%. Compared with the peripheral blood flow cytometry data, this operation can remove most of the T cells in the peripheral blood and greatly increase the percentage of NK cells in mononuclear cells. Compared with flow cytometry and magnetic bead screening, this method is low-cost, time-consuming, and simple to operate.

[0136] Example 3: Cryopreservation study of cryopreservation solution

[0137] This example prepares PBMC samples from peripheral blood in a fully enclosed automated processing mode, and studies the freezing effect of a homemade freezing solution prepared by the inventor of the present application, the homemade freezing solution comprising DMSO, dextran, sodium alginate, trehalose, human albumin, sorbitol, compound electrolytes, sodium chloride injection, glucose sodium chloride injection, dextran 40 glucose injection, compound amino acid injection, compound vitamin B injection, etc. The components of the homemade freezing solution are all medicines or pharmaceutical excipients, the ingredients are safe and the freezing effect is excellent.

[0138] The self-made cryopreservation solution was compared with the commercial cryopreservation solution from Stemcell as follows. Steps 1-3 and / or steps 1-4 are performed in a fully enclosed system, which includes a sedimentation container (a sedimentation bag in this embodiment), a sedimentation product providing device (in this embodiment, it is used to provide separation reagents and sedimentation agent hydroxyethyl starch, and includes components such as connecting pipes with other devices and modules of the system), a separation device (in this embodiment, a pulping clamp), a cell processing device (in this embodiment, including a fully enclosed automated concentration instrument, a shaker), and other devices and modules.

[0139] 1. Sedimentation treatment:

[0140] 1) Bag transfer: transfer peripheral blood to sedimentation bag. Calculate the volume of material by the difference in mass of sedimentation bag before and after transfer. Take about 0.5 ml for cell counting, flow cytometry (CD3, CD56), and KIR detection.

[0141] 2) Adding separation reagent: Add separation reagent at 5 mL / 100 ml of material through a sterile pipette and incubate for 50 min.

[0142] 3) Addition of hydroxyethyl starch: Add 6% hydroxyethyl starch solution, the final concentration of hydroxyethyl starch solution is 2% after addition, and let it stand for 0.5h. After settling, use a slurry separator to transfer the supernatant from the standing bag to the sample bag, weigh it and take samples for counting and flow detection. After mixing, divide it into 2 parts, marked as sample bag 1 and sample bag 2.

[0143] 2. Processing of sample bag 1:

[0144] Connect to the fully enclosed automated concentration instrument through a sterile pipe, the cell transfer flow is 31ml / min, the washing concentration volume is 3mL, and the number of replacements is 3. After the Stemcell cryopreservation solution is replaced, the sample is counted and sent for flow testing. The density is adjusted to 5.0×10 according to the counting results. 6 The volume, count and flow cytometry results before and after instrument processing were recorded.

[0145] The mononuclear cell suspension was mixed at 30 rpm on a shaker, and the mononuclear cell suspension was dispensed into cryopreservation bags by a peristaltic pump, 20 ml per bag. The cryopreservation bags were programmed to cool to -80°C and then stored in a gas phase liquid nitrogen tank.

[0146] 3. Processing of sample bag 2:

[0147] Connect to the fully enclosed automated concentration instrument through a sterile pipe, the cell transfer flow is 31ml / min, the washing concentration volume is 3mL, and the number of replacements is 3. After the homemade cryopreservation solution is replaced, the sample is counted and sent for flow cytometry. The density is adjusted to 5.0×10 according to the counting results. 6 The volume, count and flow cytometry results before and after instrument processing were recorded.

[0148] The mononuclear cell suspension was mixed at 30 rpm on a shaker, and the mononuclear cell suspension was dispensed into cryopreservation bags by a peristaltic pump, 20 ml per bag. The cryopreservation bags were programmed to cool to -80°C and then stored in a gas phase liquid nitrogen tank.

[0149] 4. After one week of cryopreservation, the cells were revived and cultured separately.

[0150] See Table 2 for specific separation and freezing data and Table 3 for recovery and culture data. Figures 3 to 7 .

[0151] Table 2. Isolation and cryopreservation data

[0152]

[0153] Table 3. Recovery culture data

[0154]

[0155] The results showed that the NK recovery rate, total recovery rate, and mononuclear NK cell ratio of the homemade cryopreservation solution group were better than those of the Stemcell group, and there was no significant difference in the pre-cryopreservation viability and resuscitation culture data between the two groups. In terms of performance, the homemade cryopreservation solution is better than the Stemcell cryopreservation solution, and the components of the homemade cryopreservation solution are all drugs or pharmaceutical excipients, which are safer.

[0156] Example 4: Isolation of mononuclear cells from peripheral blood

[0157] 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) and genetic disease history. After the test is qualified, the materials are collected and transported at a cold chain of 2-8℃.

[0158] The following steps 2-4 are carried out in the fully closed system described in Example 3:

[0159] 2. Sedimentation treatment

[0160] 1) Bag transfer: transfer peripheral blood to sedimentation bag. Calculate the volume of material by the difference in mass of sedimentation bag before and after transfer. Take about 0.5 ml for cell counting, flow cytometry (CD3, CD56), and KIR detection.

[0161] 2) Adding separation reagent: Add separation reagent at 5 mL / 100 ml of material through a sterile pipette and incubate for 50 min.

[0162] 3) Addition of hydroxyethyl starch: Add 6% hydroxyethyl starch solution, the final concentration of hydroxyethyl starch solution is 2% after addition, and let it stand for 0.5 hours after addition. After settling, use a slurry separator to transfer the supernatant from the standing bag to the sample bag.

[0163] 3. The sample bag is connected to the fully enclosed automated concentration instrument through a sterile pipe. The cell transfer flow is 31 ml / min, the washing concentration volume is 3 mL, and the number of replacements is 3. After replacement with the homemade freezing solution, the sample is counted. The density is adjusted to 5.0×10 6 After collecting into the collection bag.

[0164] 4. Mix the mononuclear cell suspension on a shaker at 30 rpm, and dispense the mononuclear cell suspension into cryopreservation bags using a peristaltic pump, 20 ml per bag. 5 ml is reserved in the collection bag for sterility, mycoplasma, endotoxin, counting, and flow cytometry. The cryopreservation bag is cooled to -80°C and then stored in a gas phase liquid nitrogen tank.

[0165] result

[0166] According to the separation method provided in this embodiment and comparative example 1, 4 samples were separated respectively, and the specific experimental data obtained were shown in Table 4 and the test results were shown in Table 5. Figures 8 to 22 .

[0167] Table 4. Comparison of this embodiment and comparative example 1

[0168]

[0169] Table 5. Comparison of the test results of this embodiment and comparative example 1

[0170]

[0171] It can be seen that the method provided by the present application can remove most of the T cells, and the NK cell recovery rate is greater than 95%. Among the mononuclear cells obtained, the NK purity is greater than 45%, and the cell viability is greater than 95%. The entire separation process uses a fully enclosed automated instrument, which can reduce the risk of contamination and cross contamination, and has obvious advantages over traditional methods.

[0172] Example 5: Isolation of mononuclear cells from peripheral blood

[0173] 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) and genetic disease history. After the test is qualified, the materials are collected and transported at a cold chain of 2-8℃.

[0174] The following steps 2-3 are carried out in the fully closed system described in Example 3:

[0175] 2. Sedimentation treatment

[0176] 1) Bag transfer: transfer peripheral blood to sedimentation bag. Calculate the volume of material by the difference in mass of sedimentation bag before and after transfer. Take about 0.5 ml for cell counting, flow cytometry (CD3, CD56), and KIR detection.

[0177] 2) Adding separation reagent: Add separation reagent at 5 mL / 100 ml of material through a sterile pipette and incubate for 50 min.

[0178] 3) Addition of hydroxyethyl starch: Add 6% hydroxyethyl starch solution, the final concentration of hydroxyethyl starch solution is 2% after addition, and let it stand for 0.5 hours after addition. After settling, use a slurry separator to transfer the supernatant from the standing bag to the sample bag.

[0179] 3. The sample bag is connected to the fully enclosed automated concentration instrument through a sterile pipe. The cell transfer flow is 31 ml / min, the washing concentration volume is 3 mL, and the number of replacements is 3. After replacement with the homemade freezing solution, the sample is counted. The density is adjusted to 6.0×10 6 After collecting into the collection bag.

[0180] Mix the mononuclear cell suspension on a shaker at 30 rpm, and dispense the mononuclear cell suspension into cryopreservation bags using a peristaltic pump, 20 ml per bag. Keep 5 ml in the collection bag for sterility, mycoplasma, endotoxin, counting, and flow cytometry. The cryopreservation bag is cooled to -80°C and then stored in a gas phase liquid nitrogen tank.

[0181] Two batches of PBMCs were prepared and cryopreserved in this manner. After a period of cryopreservation, they were revived and cultured. The cell culture data are shown in Table 6 below, and the data on in vitro killing of Daudi tumor cells are shown in Table 7 below.

[0182] Table 6. Cultivation data

[0183]

[0184] Table 7. In vitro killing data

[0185]

[0186] Example 6: Stability Study Data

[0187] PBMCs are stored in gas phase liquid nitrogen tanks at a temperature of ≤-150°C for a long time. In order to explore whether PBMCs stored for a long time under this condition can meet the quality attributes after resuscitation and meet the process culture requirements, a study was conducted on their storage stability. The quality attributes and process capabilities of PBMCs stored at different PBMC samples at a temperature of ≤-150°C for 0, 12, and 24 months were investigated.

[0188] Quality attributes: Quality characterization of PBMC after resuscitation (appearance, total number of viable cells, viable cell density, cell viability, cell phenotype, mycoplasma test, sterility test, bacterial endotoxin). See Table 8 for relevant data.

[0189] Process capability: PBMC recovery culture, quality characterization of harvested cell suspension (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.

[0190] Table 8. Stability quality attribute data

[0191]

[0192] Table 9. Stability process capability data

[0193]

[0194] From the above data, we can see that: 2 batches of PBMCs were stored in a gas phase liquid nitrogen tank at a temperature of ≤-150°C for up to 24 months, and the results of the quality attributes and process capabilities all met the acceptance criteria. And there was no significant difference in the various inspection indicators when the same batch of PBMCs were stored for 0 months and 24 months. The PBMCs prepared by the method of this application can be stably stored in a gas phase liquid nitrogen tank at a temperature of ≤-150°C for a long time.

[0195] Example 7: Design of PPC3S molecule

[0196] This embodiment provides an exemplary bispecific antibody, called PPC3S, which is an artificially designed molecule that can cross-link red blood cells and T cells (CD3+) to form a rosette-like complex, and then remove most of the red blood cells and T cells through a cell sedimentation method, including adding hydroxyethyl starch and PPC3S polymers, so that the treated PBMCs are more suitable for culturing into NK cells.

[0197] PPC3S is a multivalent structure that targets both CD235a and CD3 and is based on the IgG framework. In the PPC3S molecule:

[0198] (1) The heavy chain constant region (CH1-hinge-CH2-CH3) is designed as a human IgG4 subtype, i.e., IGHG4 (UniProt accession number is P01861-1, length is 327aa), see SEQ ID No. 10 for details. 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 and red blood cells, thereby reducing the killing and loss of NK cells and increasing the yield of NK cells. At the same time, IgG4 has a high affinity with protein A, and high-purity PPC3S can be obtained by affinity chromatography to meet the requirements of commercial production;

[0199] (2) To match the heavy chain constant region of human IgG4 (CH1-hinge-CH2-CH3), the light chain constant region of human IgG4, i.e., IGKC (UniProt accession number P01834, length 107aa), was selected. IGHG4 and IGKC can match, as exemplified by antibodies such as Nivolumab, Lambrolizumab, and Gemtuzumab, in which IGHG4 matches IGKC.

[0200] (3) Regarding the variable region sequence of anti-CD235a, according to the 10F7MN sequence in the NCBI database (single chain antibody 10F7MN, partial [synthetic construct] -Protein-NCBI (nih.gov)), it is the sequence of a single-chain antibody (scFv) of full mouse origin, see SEQ ID No.12 for details, and its light chain and heavy chain variable region sequences are completely consistent with 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 the human IgG4 monoclonal antibody 10F7MN against CD235a;

[0201] (4) For the anti-CD3 domain, the BiTE (i.e., tandem scFv) strategy was used as a reference, that is, the anti-CD3 scFv was fused to the C-terminus of the 10F7MN of the IgG4 structure. The anti-CD3 scFv sequence was divided into four parts, from the N-terminus to the C-terminus: linker 1, anti-CD3-VH, linker 2, and anti-CD3-VL. Among them, linker 1 and linker 2 are repeatable sequences composed of glycine and serine, which can minimize the impact on target affinity while ensuring the conformation of 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 the anti-CD3 antibody, i.e., anti-CD3-VH (see SEQ ID No. 17 for details) and anti-CD3-VL (see SEQ ID No. 18 for details), were integrated into the scFv sequence, see SEQ ID No. 19 for details.

[0202] (5) Finally, the anti-CD3 scFv was integrated into the C-terminus of the heavy chain of the human IgG4 monoclonal antibody 10F7MN, and the resulting fusion heavy chain sequence is shown in SEQ ID No. 20. The combination of SEQ ID No. 13 and SEQ ID No. 20 is the PPC3S molecule.

[0203] Therefore, the PPC3S molecule of this example is a dual-target (CD235a, CD3) tetravalent (2 Fvs bind CD235a, 2 scFvs bind CD3) IgG4 molecule containing a constant region from humans and a variable region from mice. The structural schematic diagram is shown in Fig.29 .

[0204] More specifically, the CDR sequence information in the CD235a binding domain and CD3 binding domain of the PPC3S molecule of the present application is shown in the following table.

[0205] CDR sequences of the PPC3S molecule of this application

[0206]

[0207]

[0208] Example 8: Construction of expression vector and cell bank of PPC3S molecule

[0209] The DNA sequences of the light and heavy chains of PPC3S were synthesized artificially (GenScript) and cloned into the PHS10.0 expression vector (Haoyang Bio). The PHS10.0 expression vector has a dual CMV promoter and an open reading frame to ensure that the heavy and light chains of PPC3S are highly expressed at a ratio of 1:1 and assembled into an IgG structure. After the recombinant plasmid is transformed into Escherichia coli, the recombinant plasmid with the light and heavy chains of PPC3S can be obtained by pressure screening and amplification through the ampicillin resistance gene (Amp). After the amplified and purified recombinant plasmid is electroporated into mammalian expression cells, it can be pressure screened through the blasticidin resistance gene (Bla) to obtain cells that stably express PPC3S.

[0210] The recombinant expression vector was transfected into the host cell CHO-K1. 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 expressed for about 24 hours after the medium was changed. The expression level was detected and sorted using the human IgG HTR kit by HTRF. The minipool with the highest expression level was selected for mixed inoculation and separation to obtain 10 cell groups. The cell group was subjected to batch feeding culture, and the quality of the expression sample was tested to confirm the preferred cell group. The batch feeding culture scheme is shown in Table 10.

[0211] Table 10. PPC3S fed-batch culture scheme

[0212]

[0213] The expression results of each cell population are shown in Table 11.

[0214] Table 11. Expression levels of each cell population in PPC3S fed-batch culture

[0215]

[0216] Taking into account the expression level and cell growth of the cell population, the D14 supernatants of HSP6093-B7Z4-M001, HSP6093-B7Z4-M003 and HSP6093-B7Z4-M007 were collected, purified by protein A in one step, and then the protein quality was analyzed. The analysis results are shown in Table 12.

[0217] Table 12. Quality analysis results of proteins expressed by the preferred cell population for PPC3S fed-batch culture

[0218]

[0219] Based on the above quality analysis data, HSP6093-B7Z4-M003 and HSP6093-B7Z4-M007 were selected for the construction of the original cell library. The cells were amplified and passaged in CD CHO medium (containing 4 mM glutamine, 400 μg / ml zeocin and 7 μg / ml blasticidin). The culture conditions were maintained at 36.5°C and 6.0% CO 2 , speed 110rpm. After culturing to the target cell number, the cell viability was detected, the cells were collected by centrifugation, resuspended in a freezing solution containing 10% DMSO, and then programmed to cool and freeze. The cells were sampled for testing, and the cell density and viability were tested after recovery. The test results are detailed in Table 13.

[0220] Table 13. PCB viable cell density and viability of PPC3S

[0221]

[0222] Example 9: Preparation of PPC3S molecules

[0223] The preparation process of PPC3S is divided into a cell culture process and a protein purification process to obtain a sample with high purity, low impurities and controlled exogenous factors for the preparation of PBMC under sterile conditions. Its cell culture process is shown in Table 14. After process confirmation, HSP6093-B7Z4-M003 cells are preferably used for the preparation of the stock solution of PPC3S. Under this process condition, the cell viability (VIA), viable cell density (VCD), lactic acid (Lac) and other test data in the cell culture stage are shown in Table 15, and the expression and quality data are shown in Table 16.

[0224] Table 14. Cell culture process of PPC3S

[0225]

[0226]

[0227] Table 15. Cell culture monitoring data of PPC3S

[0228]

[0229] Table 16. Expression and quality data of PPC3S

[0230]

[0231] Because the sample obtained by the cell culture process is obtained by one-step affinity purification, the sample contains polymers, and the culture medium contains CHO-related HCP, HCD, exogenous viruses, etc., which need to be removed through a fine purification process. After the cell culture medium is harvested, it undergoes deep filtration, S / D incubation virus inactivation, affinity chromatography, intermediate deep filtration, cation exchange chromatography, virus removal filtration, ultrafiltration and diafiltration, stock solution preparation and packaging to obtain the PPC3S stock solution. The components of the stock solution include: 5mg / ml PPC3S protein, 10mM histidine-histidine hydrochloride, 9% sucrose, pH 5.5.

[0232] Among them, the two-step process of S / D incubation virus inactivation and virus removal filtration was selected, and virus removal verification was carried out under the experimental conditions of reduced scale and virus addition, and the test results are shown in Table 17. The results show that the protein purification process can effectively remove exogenous viruses, and the removal efficiency LRV is not less than 9.4logs, which meets the requirements for the control of exogenous factors.

[0233] Table 17. Virus clearance validation results of PPC3S

[0234]

[0235] Therefore, the detection items and acceptable standards for the PPC3S stock solution include but are not limited to: pH, which should be 5.5±0.2; (2) SEC-HPLC purity, the main peak should not be less than 90.0%, and the HMW should not be higher than 10.0%; (3) NR-CE purity, the main peak should not be less than 80.0%, and the LMW report result; (4) Bacterial endotoxin, which should not be higher than 1.0EU / mg; (5) Sterility, which should be sterile growth. In the non-GMP stage, microbial limits are used instead of sterility tests. The total aerobic bacteria count should not be higher than 3cfu / 30ml, and the total mold and yeast count should not be higher than 3cfu / 30ml. The PPC3S stock solution of this application meets the standards for these detection items.

[0236] Sequence description of this application:

[0237]

[0238]

[0239]

[0240]

[0241]

[0242] The use of any and all examples or exemplary language (e.g., "for example," "such as") provided herein is intended only to better illustrate the present application and does not limit the scope of the present application unless otherwise required. Language in the specification should not be construed as indicating that any non-claimed element is essential to the practice of the present application.

[0243] All publications and patent applications cited in this specification are incorporated herein by reference, as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In addition, any theory, mechanism, proof or discovery described herein is intended to further enhance the understanding of the application, and is not intended to limit the application to such theory, mechanism, proof or discovery in any way. Although the application has been shown and described in detail in the accompanying drawings and the preceding description, the application should be considered illustrative rather than restrictive.

Claims

1. A method for enriching natural killer (NK) cells in a blood sample, the method comprising: a) adding a bispecific antibody targeting an erythrocyte surface antigen and a T cell surface antigen and an erythrocyte sedimentation agent to the blood sample, and after the sedimentation process, separating the upper layer to obtain a component enriched in NK cells; b) processing the NK cell-enriched fraction, wherein the processing steps include one or more of the following: cell washing, adjusting cell density, sampling for NK cell counting and / or detecting NK cell properties, and cryopreservation fluid replacement; c) collecting the NK cells treated in step b); and d) optionally, cryopreserving the NK cells; At least steps a) to c) are performed in a fully closed manner, preferably in a fully closed automated manner.

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

17.

3. The method according to 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 amino acid sequence of the heavy chain constant region is as shown in SEQ ID No.10 or SEQ ID No.21; and / or the amino acid sequence of the light chain constant region is as shown in SEQ ID No.11; Optionally, the first light chain variable region of the CD235a binding domain is associated with 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 to form the heavy chain of the CD235a binding domain; preferably, the amino acid sequence of the light chain of the CD235a binding domain is as shown in SEQ ID No.13 and / or the amino acid sequence of the heavy chain of the CD235a binding domain is as shown in SEQ ID No.14; Optionally, the CD3 binding domain is in the form of a single-chain antibody (scFv), for example, a structure of a second heavy chain variable region-a first linker-a second light chain variable region formed by a first linker; for example, the first linker is a GS-type flexible linker, preferably, the amino acid sequence of the first linker is as shown in SEQ ID No.16; Optionally, the amino acid sequence of the CD3 binding domain is as shown 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 via a second linker; for example, the second linker is a GS-type flexible linker, preferably the amino acid sequence of the second linker is shown in SEQ ID No.15; optionally, the amino acid sequence of the fusion heavy chain is shown in SEQ ID No.20; Optionally, the bispecific antibody is a tetravalent IgG4 molecule comprising variable regions from mouse and constant regions from human.

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

5. The method according to any one of claims 1 to 4, wherein the treatment step in b) comprises replacement with a cryopreservation solution, wherein the cryopreservation solution 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., polyvinyl pyrrolidone, 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 freezing solution comprises dimethyl sulfoxide (for example, final concentration ≤5%), dextran, sodium alginate, trehalose, human albumin, sorbitol, compound electrolytes, sodium chloride injection, glucose sodium chloride injection, dextran 40 glucose injection, compound amino acid injection, and compound vitamin B injection.

6. The method according to any one of claims 1-5, wherein the blood sample is a peripheral blood sample and / or an umbilical cord blood sample derived from a donor, and optionally the donor is screened 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-lymphotropic virus (HTLV), human parvovirus B19, human herpes virus type 6 / 7 / 8, adenovirus, JC virus) detection and / or genetic disease history.

7. A fully 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 of the blood sample to occur therein; A sedimentation product providing device, wherein the sedimentation product comprises a bispecific antibody targeting an erythrocyte surface antigen and a T cell surface antigen and an erythrocyte sedimentation agent, wherein the sedimentation product providing device is fluidically connected to the sedimentation container and is configured to provide the sedimentation product to the sedimentation container; a separation device (e.g., a slurry separator), the separation device being configured to phase separate the multiphase liquid in the settling vessel; A cell processing device, wherein the cell processing device is configured to perform one or more of the following treatments on the NK cell-enriched portion: cell washing, adjusting cell density, sampling for counting NK cells and / or detecting NK cell properties, and cryopreservation fluid replacement; and Optionally, a collection container (e.g., a collection bag) for collecting the NK cell-enriched portion processed by the cell processing device, The system is preferably a fully closed automated system.

8. The system according to claim 7, wherein the sedimentation product supply device supplies the sedimentation product to the sedimentation container through a pipe.

9. The system according to any one of claims 7-8, wherein the bispecific antibody targets the CD235a molecule on erythrocytes and the CD3 molecule 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, wherein the first light chain variable region comprises LCDR1 with an amino acid sequence of RASSNVKYMY (SEQ ID No. 22), LCDR2 with an amino acid sequence of YTSNLAS (SEQ ID No. 23), and LCDR3 with an amino acid sequence of QQFTSSPYT (SEQ ID No. 24), the first heavy chain variable region comprises HCDR1 with an amino acid sequence of SYFMH (SEQ ID No. 25), HCDR2 with an amino acid sequence of MIRPNGGTTDYNEKFKN (SEQ ID No. 26), and HCDR3 with an amino acid sequence of WEGSYYALDY (SEQ ID No. 27), and 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 a LCDR1 with an amino acid sequence of RASSSVSYMN (SEQ ID No.28), a LCDR2 with an amino acid sequence of DTSKVAS (SEQ ID No.29), and a LCDR3 with an amino acid sequence of QQWSSNPLT (SEQ ID No.30), the second heavy chain variable region comprises a HCDR1 with an amino acid sequence of RYTMH (SEQ ID No.31), a HCDR2 with an amino acid sequence of YINPSRGYTNYNQKFKD (SEQ ID No.32), and a HCDR3 with an amino acid sequence of YYDDHYCLDY (SEQ ID No.33), Among them, the amino acid sequences of HCDR and LCDR are based on the Kabat definition; Preferably, the CD235a binding domain comprises a first light chain variable region with an amino acid sequence as shown in SEQ ID No.6 and a first heavy chain variable region with an amino acid sequence as shown in SEQ ID No.7, and / or the CD3 binding domain comprises a second light chain variable region with an amino acid sequence as shown in SEQ ID No.18 and a second heavy chain variable region with an amino acid sequence as shown 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 shown in SEQ ID No.10 or SEQ ID No.21; and / or the amino acid sequence of the light chain constant region is as shown in SEQ ID No.11; Optionally, the first light chain variable region of the CD235a binding domain is associated with 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 to form the heavy chain of the CD235a binding domain; preferably, the amino acid sequence of the light chain of the CD235a binding domain is as shown in SEQ ID No.13 and / or the amino acid sequence of the heavy chain of the CD235a binding domain is as shown in SEQ ID No.14; Optionally, the CD3 binding domain is in the form of a single-chain antibody (scFv), for example, a structure of a second heavy chain variable region-a first linker-a second light chain variable region formed by a first linker; for example, the first linker is a GS-type flexible linker, preferably, the amino acid sequence of the first linker is as shown in SEQ ID No.16; Optionally, the amino acid sequence of the CD3 binding domain is as shown 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 via a second linker; for example, the second linker is a GS-type flexible linker, preferably the amino acid sequence of the second linker is shown in SEQ ID No.15; optionally, the amino acid sequence of the fusion heavy chain is shown in SEQ ID No.20; Optionally, the bispecific antibody is a tetravalent IgG4 molecule comprising variable regions from mouse and constant regions from human.

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

12. The system according to any one of claims 7-11, wherein the cell processing device comprises a cryopreservation fluid replacement module for performing cryopreservation fluid replacement on the NK cell-enriched component, and wherein the cryopreservation fluid 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., polyvinyl pyrrolidone, 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 freezing solution comprises dimethyl sulfoxide (for example, final concentration ≤5%), dextran, sodium alginate, trehalose, human albumin, sorbitol, compound electrolytes, sodium chloride injection, glucose sodium chloride injection, dextran 40 glucose injection, compound amino acid injection, and compound vitamin B injection.

13. The system according to any one of claims 7-12, wherein the blood sample is a peripheral blood sample and / or an umbilical cord blood sample derived from a donor, and optionally the donor is screened 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-lymphotropic virus (HTLV), human parvovirus B19, human herpes virus type 6 / 7 / 8, adenovirus, JC virus) detection and / or genetic disease history.

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