Fully closed method and system for enrichment of natural killer cells

Abstract

This application provides a method and system for enriching natural killer (NK) cells in blood samples.

Description

Technical Field

[0001] This application generally relates to the field of biotechnology, and more specifically, to a fully enclosed method and system for enriching natural killer cells. Background Technology

[0002] Natural killer (NK) cells, as innate immune cells capable of directly killing malignant cells, are increasingly recognized as a promising immunotherapeutic tool and are currently undergoing various clinical trials. To obtain large quantities 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] Currently, conventional sources of NK cells mainly employ non-closed conditions, using Ficoll separation solution to separate materials such as peripheral blood, umbilical cord blood, and bone marrow. The proportion of NK cells in these materials is not high; for example, T cells (CD3+) in peripheral blood... + ) accounts for approximately 50%–84%, NK cells (CD3) - CD16 + and / or CD56 + The proportion is approximately 7% to 40% (see Zhu Lihua and Wang Jianzhong, “Investigation of Reference Values ​​for Blood Lymphocyte Immunophenotypes in Chinese People”, Chinese Journal of Medical Laboratory Science, 1998.21(4):223-226, and it has the following shortcomings:

[0004] 1. The operation is carried out in a biosafety cabinet, which is not a fully enclosed preparation process. It has high environmental requirements and poses risks of contamination and cross-contamination.

[0005] 2. Ficoll separation solution is generally a research reagent, and its application in clinical practice carries significant risks.

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

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

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

[0009] Therefore, there is a need in this field for methods and systems to address the aforementioned problems and shortcomings. Summary of the Invention

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

[0011] 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.

[0012] 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;

[0013] c) Collect NK cells after the treatment in step b); and

[0014] d) Optionally, NK cells may be cryopreserved;

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

[0016] In a second aspect, this application provides a fully enclosed system for enriching natural killer (NK) cells in blood samples, the system comprising:

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

[0018] A sedimentation generator supply device, the sedimentation generator comprising a bispecific antibody targeting erythrocyte surface antigen and T cell surface antigen and an erythrocyte sedimentation agent, the sedimentation generator supply device being fluidly connected to the sedimentation container and configured to supply the sedimentation generator to the sedimentation container;

[0019] A separation device (e.g., a slurry separator) configured to perform phase separation of a multiphase liquid in the settling container;

[0020] A cell processing device configured to perform one or more of the following processes on the enriched NK cells: cell washing, cell density adjustment, sampling for NK cell counting and / or NK cell property detection, and cryopreservation solution replacement; and

[0021] Optionally, a collection container (e.g., a collection bag) is provided for collecting a portion of the enriched NK cells treated by the cell processing device.

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

[0023] In some embodiments of the second aspect, the sedimentation generator supply device supplies the sedimentation generator to the sedimentation container via a pipe.

[0024] In some embodiments of the first and / or second aspects, 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:

[0025] 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).

[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 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).

[0027] The amino acid sequences of HCDR and LCDR are defined according to Kabat.

[0028] Preferably, the CD235a binding domain comprises a first light chain variable region as shown in SEQ ID No. 6 and a first heavy chain variable region as shown in SEQ ID No. 7, and / or the CD3 binding domain comprises a second light chain variable region as shown in SEQ ID No. 18 and a second heavy chain variable region 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 a 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 that forms a second heavy chain variable region-first linker-second light chain variable region through 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, and preferably the amino acid sequence of the second linker is as shown in SEQ ID No. 15. Optionally, the amino acid sequence of the fusion heavy chain is as shown in SEQ ID No. 20.

[0033] Optionally, the bispecific antibody is a tetravalent IgG4 molecule comprising a variable region from mice and a constant region from humans.

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

[0035] In some embodiments of the first aspect, the processing step b) includes cryopreservation solution replacement. In some embodiments of the second aspect, the cell processing apparatus includes a cryopreservation solution replacement module for replacing the components of the enriched NK cells with cryopreservation solution. In some specific embodiments, the cryopreservation solution contains one or more cryoprotectants. The cryoprotectants are, for example, permeable cryoprotectants (e.g., dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, acetamide, and / or methanol) and / or non-permeable cryoprotectants (e.g., polyvinylpyrrolidone, sucrose, polyethylene glycol, dextran, albumin, and / or hydroxyethyl starch). In some specific embodiments, the cryopreservation solution also contains one or more amino acids and / or vitamins. For example, the cryopreservation solution may contain dimethyl sulfoxide (e.g., final concentration ≤ 5%), dextran, sodium alginate, trehalose, human serum albumin, sorbitol, compound electrolytes, sodium chloride injection, glucose sodium chloride injection, dextran 40 glucose injection, compound amino acid injection, and compound vitamin B injection.

[0036] In some embodiments of the first and / or second aspects, the blood sample is a peripheral blood sample and / or umbilical cord blood sample derived from a donor. The donor may undergo screening, for example, by testing for pathogens (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 troponinovarian virus (HTLV), human parvovirus B19, human herpesvirus 6 / 7 / 8, adenovirus, JC virus) and / or genetic history. Attached Figure Description

[0037] Figure 1 A general flowchart of an exemplary method for enriching NK cells in biological samples according to this application is shown.

[0038] Figure 2 The flowcharts of methods for enriching NK cells in biological samples according to some specific embodiments of this application are shown. PPC3S is an exemplary bispecific antibody of this 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. Its design and preparation methods can be found in Examples 7 to 9.

[0039] Figures 3 to 7 This shows a comparison of NK cell recovery rate, total recovery rate, and pre-cryopreservation viability obtained using Stemcell cryopreservation solution and homemade 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 curves ( Figure 6 ) and the curve of change in NK cell proportion ( Figure 7 ).

[0040] Figures 8 to 10 The percentage of T cells in Example 4 compared to Comparative Example 1 is shown. Figure 8 ), NK cell percentage ( Figure 9 ), cell viability ( Figure 10 The differences between Example 4 and Comparative Example 1 are as follows: By comparing the data before and after separation in Example 4 and Comparative Example 1, it can be seen that the method in Example 4 can significantly remove T cells and other contaminating cells in peripheral blood, increase the NK cell ratio, and there is no significant difference in cell viability before and after separation; in Comparative Example 1, the ratio of T cells and NK cells after separation is comparable to that before separation.

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

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

[0043] Figure 27 and Figure 28 The flow cytometry plots of two batches of cell samples (referred to as batch 1 and batch 2, respectively) prepared by the method in Example 5 are shown on day 18 (D18) of culture.

[0044] Figure 29 A schematic diagram of the architecture of an exemplary bispecific antibody PPC3S of this application is shown. Detailed Implementation

[0045] definition

[0046] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art. For definitions and terminology in this field, those skilled in the art may refer 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 this field 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 this application are intended to be as accurate as possible in the specific embodiments, any numerical value inherently contains a certain degree of error due to the standard deviation present in their respective measurements. Furthermore, all ranges disclosed herein should be understood to encompass any and all subranges contained therein. For example, the stated range “1 to 10” should be considered to include any and all subranges between the minimum value 1 and the maximum value 10 (inclusive); that is, all subranges beginning with a minimum value of 1 or greater, such as 1 to 6.1, and subranges ending with a maximum value of 10 or less, such as 5.5 to 10. Additionally, any references marked “incorporated herein” should be understood to be incorporated herein in their entirety.

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

[0049] As used herein, the term "specific binding" is a well-known term in the art, and methods for measuring such specific binding of antibodies to antigens are also well-known in the art. For example, in some embodiments, "specific binding" means that an antibody binds to the intended target but does not bind significantly to other targets. Compared to binding to other epitopes, the antibody binds to the intended target epitope with significantly increased affinity and / or for a longer duration.

[0050] The term "natural killer cell" used in this article refers to NK cells, which are a type of lymphocyte that is not MHC-restricted, does not require prior sensitization, does not require antibody participation, and can directly and non-specifically kill tumor cells and virus-infected cells.

[0051] The term "mononuclear cell (MNC)" as used in this article refers to cells with a single nucleus found 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 meglumine solution, which utilizes the difference in specific gravity of blood cells to distribute blood cells of a certain specific gravity into different independent zones according to a corresponding density gradient, thereby achieving the purpose of blood cell separation.

[0053] The term "bispecific antibody" as used in this article refers to an antibody that simultaneously possesses the ability to bind to two antigenic epitopes. These two epitopes can be on different antigens or on the same antigen. Bispecific antibodies can have various structural configurations. For example, a bispecific antibody can consist of two Fc fragments and two antigen-binding moieties fused to them (similar to natural antibodies, except that the two arms bind to different antigenic targets or epitopes). The antigen-binding moieties can be in the form of a single-chain antibody (scfv) or a Fab fragment. Each of the two different binding moieties of the bispecific antibody binds to the N-terminus of an Fc fragment. The antigen-binding moieties of the two arms can be configured in four ways: scfv+Fab fragment, Fab fragment+scfv, scfv+scfv, and Fab fragment+Fab fragment. The Fc fragment can contain mutations that ensure heavy chain heteropolymerization. Knob-in-hole (KIH) technology is a strategy to address heavy chain heteropolymerization. Typically, KIH technology involves modifying the amino acid sequence of the CH3 region to form a structure that facilitates the pairing of heterologous half-antibodies, thus constructing a bispecific antibody while maintaining the structure of a normal antibody as much as possible.

[0054] As used in this article, the term "single-chain antibody (scFv)" refers to a single polypeptide chain consisting of VH and VL domains linked by peptide linkers. (scFv)2 contains two VH domains linked by peptide linkers and two VL domains, the two VL domains being combined with the two VH domains via disulfide bridges.

[0055] The term "CD235a" used in this article refers to glycoprotein A, a major, intrinsic membrane protein of red blood cells, expressed on the surface of mature red blood cells, with an average of 1 × 10⁶ cells per red blood cell surface. 5 ~1×10 6This molecule (Merry et al. Biochem J. (1986) 233:93-98; Loken et al. Blood (1987) 69:255-263). Furthermore, CD235a is highly expressed in kidney, bladder, and urethral tissues, but almost not expressed in lymphocytes (https: / / www.proteinatlas.org / ENSG00000170180-GYPA). CD235a is a component of the ankylosing sac complex, which is involved in the stability and shape of the erythrocyte membrane. Its N-terminus is a glycosylated fragment located outside the erythrocyte cell membrane, acting as a mononuclear blood group receptor. Amino acids 20-91 form the extracellular domain, 92-114 the transmembrane domain, and 115-150 the intracellular domain; see SEQ ID No. 1 for details. The CD235 family includes CD235b (blood group glycoprotein B, GYPB), which exhibits very high homology and high sequence identity in its extracellular N segment (see SEQ ID No. 2). Its function is similar to that of CD235a. In apheresis peripheral blood, CD235a is expressed in erythrocytes but not in lymphocytes, making it a highly selective target.

[0056] CD3, a component of the T cell receptor-CD3 (TCR-CD3) complex on the surface of T lymphocytes, plays a crucial role in the adaptive immune response. When antigen-presenting cells activate the T cell receptor, TCR-mediated signals are transmitted intracellularly via the δ, ε, γ, and ζ subunits of CD3, thereby activating downstream signaling pathways in T cells. In cell identification, CD3 is a well-known surface marker of T cells, including naive and memory CD8+ cells. + T cells, initial and memory CD4 + It is specifically expressed on T cells, γδT cells, T reg cells, etc. In conventional T cell positive or negative selection kits, CD3 antibody-conjugated magnetic beads and matching reagents are usually used for treatment.

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

[0058] 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.

[0059] 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;

[0060] c) Collect NK cells after the treatment in step b); and

[0061] d) Optionally, NK cells may be cryopreserved;

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

[0063] In a second aspect, this application provides a fully enclosed system for enriching natural killer (NK) cells in blood samples, the system comprising:

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

[0065] A sedimentation generator supply device, the sedimentation generator comprising a bispecific antibody targeting erythrocyte surface antigen and T cell surface antigen and an erythrocyte sedimentation agent, the sedimentation generator supply device being fluidly connected to the sedimentation container and configured to supply the sedimentation generator to the sedimentation container;

[0066] A separation device (e.g., a slurry separator) configured to perform phase separation of a multiphase liquid in the settling container;

[0067] A cell processing device configured to perform one or more of the following processes on the enriched NK cells: cell washing, cell density adjustment, sampling for NK cell counting and / or NK cell property detection, and cryopreservation solution replacement; and

[0068] Optionally, a collection container (e.g., a collection bag) is provided for collecting a portion of the enriched NK cells treated by the cell processing device.

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

[0070] In some embodiments of the second aspect, the sedimentation generator supply device supplies the sedimentation generator to the sedimentation container via a pipe.

[0071] In some embodiments of the first and / or second aspects, 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:

[0072] 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).

[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 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).

[0074] The amino acid sequences of HCDR and LCDR are defined according to Kabat.

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

[0076] Figure 29 This application demonstrates an exemplary construction scheme for the bispecific antibody. Figure 29The bispecific antibody comprises two light chains and two heavy chains, with an N-terminus of a CD235a-binding domain (containing variable regions of the light and heavy chains that bind to CD235a) and a C-terminus of a CD3a-binding domain (containing variable regions of the light and heavy chains 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 the CD235a-binding domain (the light chain variable region contains three CDR regions: LCDR1, LCDR2, and LCDR3). The heavy chain includes a heavy chain constant region (human IgG4 subtype), a heavy chain variable region of the CD235a-binding domain (the heavy chain variable region contains three CDR regions: HCDR1, HCDR2, and HCDR3), and a CD3-binding domain in the form of scFv (containing LCDR1, LCDR2, and LCDR3, as well as HCDR1, HCDR2, and HCDR3 regions).

[0077] It should be understood that Figure 29 The construction schemes for bispecific antibodies shown are merely exemplary and not limiting. Those skilled in the art will reasonably recognize other construction schemes for bispecific antibodies. For example, the bispecific antibody of this application can be obtained in the following ways (for example only, not limited to):

[0078] (i) Based on a full-length anti-CD235a antibody, an antibody fragment that binds to CD3 (e.g., a CD3-binding single-chain antibody scFv containing a heavy chain variable region and a light chain variable region) is linked to the Fc fragment of the anti-CD235a antibody.

[0079] (ii) Based on a full-length anti-CD3 antibody, an antibody fragment binding to CD235a (e.g., a CD235a-binding single-chain antibody scFv containing a heavy chain variable region and a light chain variable region) is linked to the Fc fragment of the anti-CD3 antibody; or

[0080] (iii) Replace one antigen-binding arm of an anti-CD235a antibody (or an anti-CD3 antibody) with 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 CD235a and CD3 respectively.

[0081] In some embodiments of the first and / or second aspects, 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 alternative embodiments, the heavy chain constant region may contain a mutation in its hinge region, for example, a mutation of serine (S) at the 10th amino acid position of 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 containing the S10P mutation in the hinge region may be as 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 and / or second aspects, 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 and / or second aspects, the CD3-binding domain is in the form of a single-chain antibody (scFv), for example, a structure forming a second heavy chain variable region-first adapter-second light chain variable region via a first linker (e.g., an N-terminal-second heavy chain variable region-first linker-second light chain variable region-C-terminal structure). The first linker may be, for example, a GS-type flexible linker, and preferably, the amino acid sequence of the first linker is as shown in SEQ ID No. 16. In some more specific embodiments, the amino acid sequence of the CD3-binding domain is as shown in SEQ ID No. 19.

[0084] In some embodiments of the first and / or second aspects, 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 with an amino acid sequence as 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 and / or second aspects, the fusion protein is a tetravalent IgG4 molecule comprising a variable region from mice and a constant region from humans.

[0086] Several studies in this field have investigated the druggability of antibodies from different sources of CD235a:

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

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

[0089] In patent WO2017 / 015141A1, the murine antibody against CD235a is named 10F7, with its light chain variable region sequence detailed in SEQ ID No. 6 and its heavy chain variable region sequence detailed in SEQ ID No. 7. In this patent, it is humanized as scFv. Through binding activity, functional studies, and stability tests, a combination with Glycophorin A binding activity was confirmed, and the sequence scFv-10F7-EPO was constructed, with its light chain variable region sequence detailed in SEQ ID No. 8 and its heavy chain variable region sequence 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 against CD235a: one of which is named 1C3 / 86, and 4.80 × 10⁻⁶ antibodies were found per red blood cell.5 The dissociation constant KD of one CD235a molecule is 23 × 10⁻⁶. -8 M, or 230 nM; another type, 10F7MN, was measured to have 4.66 × 10⁻⁶ mcg per red blood cell. 5 One CD235a molecule has a dissociation constant KD of 9.5 × 10⁻⁶. -8 M, which is 95nM.

[0091] However, none of these studies disclosed or taught the bispecific antibody of this application.

[0092] In some embodiments of the first and / or second aspects, the bispecific antibody of this application is added to a peripheral blood sample along with a settling agent, wherein the bispecific antibody is in the form of a concentration of about 5 mg / mL and is added to the blood sample at a volume of 5 mL to 25 mL per 100 mL of peripheral blood, for example at 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, or about 12.5 mL per 100 mL of blood. Add approximately 13 mL, 13.5 mL, 14 mL, 14.5 mL, 15 mL, 15.5 mL, 16 mL, 16.5 mL, 17 mL, 17.5 mL, 18 mL, 18.5 mL, 19 mL, 19.5 mL, 20 mL, 20.5 mL, 21 mL, 21.5 mL, 22 mL, 22.5 mL, 23 mL, 23.5 mL, 24 mL, 24.5 mL, or 25 mL per 100 mL of blood, preferably in a volume of approximately 5 mL, 15 mL, or 25 mL.

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

[0094] In some embodiments of the first aspect, the processing step b) includes cryopreservation solution replacement. In some embodiments of the second aspect, the cell processing apparatus includes a cryopreservation solution replacement module for replacing the components of the enriched NK cells with cryopreservation solution. In some specific embodiments, the cryopreservation solution contains one or more cryoprotectants. The cryoprotectants are, for example, permeable cryoprotectants (e.g., dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, acetamide, and / or methanol) and / or non-permeable cryoprotectants (e.g., polyvinylpyrrolidone, sucrose, polyethylene glycol, dextran, albumin, and / or hydroxyethyl starch). In some specific embodiments, the cryopreservation solution also contains one or more amino acids and / or vitamins. For example, the cryopreservation solution may contain dimethyl sulfoxide (e.g., final concentration ≤ 5%), dextran, sodium alginate, trehalose, human serum albumin, sorbitol, compound electrolytes, sodium chloride injection, glucose sodium chloride injection, dextran 40 glucose injection, compound amino acid injection, and compound vitamin B injection.

[0095] In some embodiments of the first and / or second aspects, the blood sample is a peripheral blood sample and / or umbilical cord blood sample derived from a donor. The donor may undergo screening, for example, by testing for pathogens (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 troponinovarian virus (HTLV), human parvovirus B19, human herpesvirus 6 / 7 / 8, adenovirus, JC virus) and / or genetic history.

[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 solution replacement module. The system and / or cell processing device may also include modules performing other functions, such as a temperature regulation module. One or more modules of the system and / or cell processing device may be combined into one; for example, the cryopreservation solution replacement module and the cell density adjustment module may be combined to simultaneously replace the existing solution in the system and adjust the cell density in the existing solution by introducing cryopreservation solution.

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

[0098] In some embodiments of the first aspect, the method further includes mixing the NK cells treated in step b) and aliquoting them (e.g., after mixing), for example, into cryopreservation containers (e.g., cryopreservation bags).

[0099] In some embodiments of the second aspect, the system further includes a cell mixing device (e.g., a shaker) and / or a dispensing device (e.g., a peristaltic pump), the cell mixing device being used to mix the components of the enriched NK cells treated by the cell processing device, and the dispensing device being used to dispense the components of the enriched NK cells (e.g., after mixing by the cell mixing device), for example, into cryopreservation containers (e.g., cryopreservation bags).

[0100] The inventors of this application have discovered that NK cell culture can achieve high NK purity (NK purity up to 95% or higher) and high NK cell viability density (8.5 × 10⁻⁶) by increasing the initial NK purity. 6 NK cells with high cell count / mL and high cytotoxic activity (up to 80% or more when the effector-to-target ratio is 10:1) are used to solve the global problem of low purity in NK cell culture.

[0101] This application utilizes hydroxyethyl starch and separating reagents (e.g., PPC3S mentioned above) to remove the vast majority of erythrocytes, T cells, and other contaminating cells. The cells are then concentrated, washed, and subjected to self-made cryopreservation solution replacement and density adjustment using a closed, automated system. Mononuclear cells are then dispensed via a shaker and peristaltic pump using a closed pipeline, and stored in a gas-phase liquid nitrogen tank after programmed cooling. In some embodiments, the method and system of this application can achieve one or more of the following technical effects:

[0102] 1. The materials used in the NK cell enrichment methods and systems of this application are abundant and widely applicable, and are not limited to single sources such as commonly used peripheral blood and umbilical cord blood.

[0103] 2. By using a fully enclosed preparation method, the risk of contamination and cross-contamination is reduced. A Class D environment is sufficient to meet environmental requirements, thereby reducing the cost of laboratory construction and maintenance.

[0104] 3. By using separation reagents, most red blood cells, T cells, and other contaminating cells in the material can be removed, while NK cells can be enriched within the material. Compared to flow cytometry and magnetic bead screening, the process described in this application is low-cost, time-efficient, and simple to operate.

[0105] 4. By using automated instruments, the risk of human error is reduced. Compared with similar products on the market, this instrument has a higher solution replacement rate and NK cell recovery rate, and avoids the cell accumulation problem caused by centrifugation, resulting in a high cell viability rate.

[0106] 5. The self-made cryopreservation solution of this application consists of pharmaceuticals or pharmaceutical excipients, and its components are safe and its cryopreservation effect is excellent.

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

[0108] In summary, the method of this application can overcome the shortcomings of non-fully enclosed methods (e.g., Ficoll separation method) and achieve the goal of obtaining mononuclear cells (MNCs) from peripheral blood, umbilical cord blood, bone marrow and other sources in a fully enclosed, automated manner with high recovery rate, batch-to-batch stability and high NK purity.

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

[0110] 1. The method described in this application may mainly include collection and transportation, sedimentation treatment, automated instrument processing, packaging, and cryopreservation, etc. For details, please refer to [link to relevant documentation]. Figure 1 and / or Figure 2 .

[0111] 2. All sampling, reagent addition, and cell transfer procedures in this application can be performed in a closed system using a sterile connector.

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

[0113] 4. During the collection and transportation process of this application, pathogen screening of the material supplier can be performed as needed, including HBV, HCV, syphilis, HIV, EBV, CMV, HTLV, human parvovirus B19, human herpesvirus 6 / 7 / 8, adenovirus, and JC virus. The material is transported via cold chain at 2–8°C after collection.

[0114] 5. In the sedimentation process of this application, the amount of separation reagent (e.g., PPC3S, for example, about 5 mg / ml) added is 5-25 ml / 100 ml of material, and the incubation time is 40-60 min. After addition, the final concentration of the hydroxyethyl starch solution is 1-2%, and the settling time is 30-60 min.

[0115] 6. In the automated instrument processing operation of this application, automated instruments can be used to concentrate, wash, and replace the cryopreservation solution for mononuclear cells, adjusting the density to 1.0–8.0 × 10⁻⁶. 6 Cells / ml. Cell transfer flow cytometry was 30–60 ml / min, washing and concentration volume was 2–5 mL, and replacement cycles were 2–5.

[0116] 7. In the automated instrument processing operation of this application, the cryopreservation solutions that can be used include DMSO, dextran, sodium alginate, trehalose, human serum 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 dispensing operation of this application, mononuclear cells can be uniformly dispensed into cryopreservation bags using a shaker and a peristaltic pump, with the shaker speed being 30-80 rpm.

[0118] It should be understood that the above detailed description is only intended to provide a clearer understanding of the contents of this application to those skilled in the art, and is not intended to limit in any way. Those skilled in the art can make various modifications and variations to the described embodiments.

[0119] Example

[0120] The present application will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present application.

[0121] Comparative Example 1: Isolation of mononuclear cells using Ficoll separation solution

[0122] 1. Collection and Transportation: Peripheral blood donors are screened for pathogens (Hepatitis B virus (HBV), Hepatitis C virus (HCV), Treponema pallidum, Human Immunodeficiency Virus (HIV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human T-cell troponinocyte virus (HTLV), Human parvovirus B19, Human herpesvirus 6 / 7 / 8, Adenovirus, JC virus) and genetic history. Materials are collected after passing the tests and transported via cold chain at 2–8℃.

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

[0124] 3. After centrifugation, discard the supernatant, collect the white membrane cells into a 225ml conical centrifuge flask, bring the volume up to 200ml with physiological saline, and centrifuge at 300g for 10min at room temperature.

[0125] 4. After centrifugation, discard the supernatant and adjust the density to 5.0 × 10⁻⁶. 6 The concentration of mononuclear cells was 1,000 per ml. After mixing, 5 ml was used for aseptic, mycoplasma, endotoxin, counting, and flow cytometry detection. The remaining mononuclear cells were aliquoted into cryovials.

[0126] 5. After the cryopreservation tubes are cooled to -80°C using a programmed method, they are placed in a gaseous liquid nitrogen tank for storage.

[0127] Example 1: Supplier Screening

[0128] Because materials such as peripheral blood and umbilical cord blood from donors may contain pathogens of blood-borne diseases, strict control over material donors and materials is essential to ensure the safety of PBMCs. Pathogen screening of material donors includes HBV, HCV, Treponema pallidum, HIV, EBV, CMV, HTLV, human parvovirus B19, HIV, human herpesvirus 6 / 7 / 8, adenovirus, and JC virus. Materials can only be collected after passing pathogen screening, and then subjected to tests for viable cell count, cell viability, flow cytometry, sterility, and mycoplasma. Only materials that pass all tests can be used for PBMC preparation.

[0129] Example 2: Settlement Treatment

[0130] In this embodiment, a separation reagent was used in combination with hydroxyethyl starch to remove most of the erythrocytes, T cells, and other impurity cells. The separation reagent used in Examples 2 to 5 was the bispecific antibody PPCS3, described above, which can specifically bind to CD235a molecules on erythrocytes and CD3 molecules on T cells, at a concentration of 5 mg / ml. The design and preparation of PPCS3 are described in Examples 7-9 below.

[0131] The settlement treatment steps are as follows:

[0132] Add equal volumes of peripheral blood to centrifuge tubes, and add different concentrations of separation reagent (5 mL / 100 mL peripheral blood, 15 mL / 100 mL peripheral blood, 25 mL / 100 mL peripheral blood). Mix well and incubate for 40 min. Add 6% hydroxyethyl starch solution (w / w), bringing the final concentration to 2%. After settling for 0.5 h, aspirate the supernatant plasma layer, resuspend to 45 mL, centrifuge (300 g, 10 min), discard the supernatant, and resuspend the cell pellet. Detect NK cell purity (CD3+) using flow cytometry. - CD56 + ) and the proportion of T cells (CD3+) as a major impurity + ) Specific experimental data are shown in Table 1 below.

[0133] Table 1. Experimental data on sedimentation treatment

[0134]

[0135] The results showed that, by using the separation reagent and hydroxyethyl starch in combination, the proportion of T cells in the mononuclear cells obtained in all three groups was ≤0.5%, and the purity of NK cells was >70%. Comparison with peripheral blood flow cytometry data showed that this procedure can remove the vast majority of T cells in peripheral blood and significantly increase the percentage of NK cells in mononuclear cells. Furthermore, compared with flow cytometry and magnetic bead screening, this method is low-cost, time-efficient, and simple to operate.

[0136] Example 3: Study on cryopreservation of cryopreservation solution

[0137] This embodiment uses a fully enclosed automated processing method to prepare PBMC samples from peripheral blood and studies the cryopreservation effect of a self-made cryopreservation solution prepared by the inventors. The self-made cryopreservation solution contains multiple components including DMSO, dextran, sodium alginate, trehalose, human serum albumin, sorbitol, compound electrolytes, sodium chloride injection, glucose sodium chloride injection, dextran 40 glucose injection, compound amino acid injection, and compound vitamin B injection. All components of the self-made cryopreservation solution are pharmaceuticals or pharmaceutical excipients, ensuring safety and excellent cryopreservation performance.

[0138] The self-made cryopreservation solution was compared with the commercial cryopreservation solution from Stemcell as follows. Steps 1-3 and / or 1-4 were performed in a fully enclosed system, which included a sedimentation container (a sedimentation bag in this embodiment), a sedimentation product supply device (used in this embodiment to provide separation reagents, sedimentation agent hydroxyethyl starch, and including components such as connectors to other devices and modules in the system), a separation device (a separator in this embodiment), and a cell processing device (including a fully enclosed automated concentration instrument and a shaker in this embodiment), etc.

[0139] 1. Settlement treatment:

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

[0141] 2) Adding separation reagent: Add separation reagent at a rate of 5 mL / 100 mL of material through a sterile tube, and incubate for 50 min after adding.

[0142] 3) Addition of hydroxyethyl starch: Add 6% hydroxyethyl starch solution, bringing the final concentration of the solution to 2%. Allow to settle for 0.5 hours. After settling, use a separator to transfer the supernatant from the settling bag to the sample bag, weigh it, and take a sample for counting and flow cytometry analysis. After mixing, divide the sample into two equal portions, labeled Sample Bag 1 and Sample Bag 2.

[0143] 2. Processing of Sample Bag 1:

[0144] Connected to a fully enclosed automated concentration instrument via sterile tubing, cell transfer flow cytometry was performed at 31 ml / min, with a washing and concentration volume of 3 mL and 3 replacement cycles. Stemcell cryopreservation solution was sampled, counted, and sent for flow cytometry analysis after replacement. The density was adjusted to 5.0 × 10⁻⁶ based on the count results. 6 Collect the samples at a rate of 1 / ml into a collection bag. Record the volume, count, and flow cytometry results before and after instrument processing.

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

[0146] 3. Processing of sample bag 2:

[0147] The cell transfer flow cytometry was connected to a fully enclosed automated concentration instrument via a sterile connector. The flow cytometry rate was 31 ml / min, the washing and concentration volume was 3 mL, and the number of replacements was 3. Samples of the self-made cryopreservation solution were collected and counted after replacement, and then sent for flow cytometry analysis. The density was adjusted to 5.0 × 10⁻⁶ based on the count results. 6 Collect the samples at a rate of 1 / ml into a collection bag. Record the volume, count, and flow cytometry results before and after instrument processing.

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

[0149] 4. After being frozen for one week, the samples were thawed and cultured separately.

[0150] Specific isolation and cryopreservation data are shown in Table 2 below, and resuscitation and culture data are shown in Table 3 below. Related images are available in [link to table]. Figures 3 to 7 .

[0151] Table 2. Separation and cryopreservation data

[0152]

[0153] Table 3. Resuscitation Culture Data

[0154]

[0155] The results showed that the NK cell recovery rate, total recovery rate, and proportion of mononuclear NK cells in the self-made cryopreservation solution group were superior to those in the Stemcell group. There were no significant differences in pre-cryopreservation viability and thawing culture data between the two groups. In terms of performance, the self-made cryopreservation solution was superior to the Stemcell cryopreservation solution, and since all components of the self-made cryopreservation solution were pharmaceuticals or pharmaceutical excipients, it was safer.

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

[0157] 1. Collection and Transportation: Peripheral blood donors are screened for pathogens (HBV, HCV, Treponema pallidum, HIV, EBV, CMV, HTLV, human parvovirus B19, human herpesvirus 6 / 7 / 8, adenovirus, JC virus) and genetic history. Materials are collected after passing the tests and transported via cold chain at 2–8℃.

[0158] Steps 2-4 are performed in the fully enclosed system described in Example 3:

[0159] 2. Settlement treatment

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

[0161] 2) Adding separation reagent: Add separation reagent at a rate of 5 mL / 100 mL of material through a sterile tube, and incubate for 50 min after adding.

[0162] 3) Addition of hydroxyethyl starch: Add 6% hydroxyethyl starch solution, bringing the final concentration of the solution to 2%. Allow the mixture to settle for 0.5 hours. After settling, use a separator to transfer the supernatant from the settling bag to the sample bag.

[0163] 3. Sample bags were connected to a fully enclosed automated concentration instrument via sterile tubing. Cell transfer flow cytometry was performed at 31 ml / min, with a washing and concentration volume of 3 mL and 3 replacement cycles. Samples were collected and counted after replacement with the self-made cryopreservation solution. The density was adjusted to 5.0 × 10⁻⁶ based on the counting results. 6 Collect each vial per ml into a collection bag.

[0164] 4. Mix the mononuclear cell suspension at 30 rpm on a shaker. Dispense the suspension into cryopreservation bags (20 ml per bag) using a peristaltic pump. Reserve 5 ml from each bag for aseptic, mycoplasma, endotoxin, counting, and flow cytometry analysis. After programmed cooling to -80°C, store the cryopreservation bags in a gas phase liquid nitrogen tank.

[0165] result

[0166] Four samples were separated using the separation methods provided in this embodiment and Comparative Example 1. The specific experimental data are shown in Table 4 below, and the detection results are shown in Table 5. Related images can be found... Figures 8 to 22 .

[0167] Table 4. Comparison of this embodiment and Comparative Example 1

[0168]

[0169] Table 5. Comparison of detection results between this embodiment and Comparative Example 1

[0170]

[0171] Therefore, the method provided in this application can remove the vast majority of T cells, with an NK cell recovery rate of >95%. The obtained mononuclear cells exhibit NK purity >45% and cell viability >95%. The entire separation process utilizes fully enclosed automated equipment, which reduces the risk of contamination and cross-contamination, demonstrating significant advantages over traditional methods.

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

[0173] 1. Collection and Transportation: Peripheral blood donors are screened for pathogens (HBV, HCV, Treponema pallidum, HIV, EBV, CMV, HTLV, human parvovirus B19, human herpesvirus 6 / 7 / 8, adenovirus, JC virus) and genetic history. Materials are collected after passing the tests and transported via cold chain at 2–8℃.

[0174] Steps 2-3 are performed in the fully enclosed system described in Example 3:

[0175] 2. Settlement treatment

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

[0177] 2) Adding separation reagent: Add separation reagent at a rate of 5 mL / 100 mL of material through a sterile tube, and incubate for 50 min after adding.

[0178] 3) Addition of hydroxyethyl starch: Add 6% hydroxyethyl starch solution, bringing the final concentration of the solution to 2%. Allow the mixture to settle for 0.5 hours. After settling, use a separator to transfer the supernatant from the settling bag to the sample bag.

[0179] 3. Sample bags were connected to a fully enclosed automated concentration instrument via sterile tubing. Cell transfer flow cytometry was performed at 31 ml / min, with a washing and concentration volume of 3 mL and 3 replacement cycles. Samples were taken and counted after replacement with the self-made cryopreservation solution. The density was adjusted to 6.0 × 10⁻⁶ based on the counting results. 6 Collect each vial per ml into a collection bag.

[0180] The mononuclear cell suspension was mixed on a shaker at 30 rpm. The suspension was then dispensed into cryopreservation bags (20 ml per bag) using a peristaltic pump. 5 ml was retained in each bag for aseptic, mycoplasma, endotoxin, counting, and flow cytometry analysis. The cryopreservation bags were then cooled to -80°C and stored in a gas-phase liquid nitrogen tank.

[0181] Two batches of PBMCs were prepared and cryopreserved in this manner. After being cryopreserved for a period of time, they were thawed 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. Culture Data

[0183]

[0184] Table 7. External Killing Data

[0185]

[0186] Example 6: Stability Study Data

[0187] PBMCs were stored long-term in a gaseous liquid nitrogen tank at a temperature ≤-150℃. To investigate whether PBMCs stored under these conditions could meet the post-resuscitation quality attributes and process requirements, their storage stability was studied. The quality attributes and processing capabilities of different PBMC samples stored at ≤-150℃ for 0, 12, and 24 months were examined.

[0188] Quality attributes: Quality characterization of PBMCs after resuscitation (appearance, total viable cell count, viable cell density, cell viability, cell phenotype, mycoplasma examination, sterility examination, bacterial endotoxins). Relevant data are shown in Table 8.

[0189] Processing capabilities: PBMC resuscitation culture, quality characterization of harvested cell suspensions (appearance, total viable cell count, cell viability, cell phenotype, mycoplasma examination, sterility examination, bacterial endotoxins, and cytotoxic 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] The data above shows that both batches of PBMC, when stored in a gas-phase liquid nitrogen tank at a temperature ≤-150℃ for 24 months, met the acceptance criteria for all quality attributes and process capabilities. Furthermore, there were no significant differences in any of the tested indicators between the 0-month and 24-month storage periods for the same batch of PBMC. Therefore, PBMC prepared using the method described in this application can be stably stored long-term in a gas-phase liquid nitrogen tank at a temperature ≤-150℃.

[0195] Example 7: Design of PPC3S molecules

[0196] This embodiment provides an exemplary bispecific antibody called PPC3S. This molecule is an artificially designed molecule that can crosslink red blood cells and T cells (CD3+) together to form a rosette-like complex. Then, through a cell sedimentation method, including the addition of hydroxyethyl starch and PPC3S polymer, most of the red blood cells and T cells are removed, making the treated PBMCs more suitable for culture as NK cells.

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

[0198] (1) The heavy chain constant region (CH1-hinge-CH2-CH3) is designed for the human IgG4 subtype, i.e., IGHG4 (UniProt accession number P01861-1, length 327aa), see SEQ ID No. 10 for details. The affinity of human IgG4 for CD16a is much lower than that of human IgG1 for CD16a (≤1 / 10), thus reducing the cross-linking of NK cells with T cells and erythrocytes, 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 for protein A, and high-purity PPC3S can be obtained through affinity chromatography to meet the requirements of commercial production;

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

[0200] (3) Regarding the variable region sequence of anti-CD235a, according to the NCBI database, the 10F7MN sequence (single chain antibody 10F7MN, partial [synthetic construct]-Protein-NCBI(nih.gov)) is the sequence of a whole-mouse single-chain antibody (scFv), as detailed in SEQ ID No. 12. Its light chain and heavy chain variable region sequences 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 the anti-CD235a human IgG4 monoclonal antibody 10F7MN;

[0201] (4) For the anti-CD3 domain, following the BiTE (i.e., tandem scFv) strategy, an anti-CD3 scFv is fused to the C-terminus of the 10F7MN of the IgG4 structure. The anti-CD3 scFv sequence is divided into four parts, from the N-terminus to the C-terminus: adapter 1, anti-CD3-VH, adapter 2, and anti-CD3-VL. Adapter 1 and adapter 2 are reproducible sequences composed of glycine and serine, which minimize the impact on target affinity while maintaining the conformation of the scFv. The sequences of adapter 1 and adapter 2 are detailed in SEQ ID No. 15 and SEQ ID No. 16. The variable region sequences of the anti-CD3 antibody, namely anti-CD3-VH (see SEQ ID No. 17) and anti-CD3-VL (see SEQ ID No. 18), are integrated into the scFv sequence, as detailed in SEQ ID No. 19.

[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 detailed in SEQ ID No. 20. The combination of SEQ ID No. 13 and SEQ ID No. 20 constitutes the PPC3S molecule.

[0203] Therefore, the PPC3S molecule in this embodiment 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. See the structural schematic diagram for details. Figure 29 .

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

[0205] The CDR sequence of the PPC3S molecule in this application

[0206]

[0207]

[0208] Example 8: Construction of expression vector and cell bank for 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 Biotechnology). The PHS10.0 expression vector has dual CMV promoters and open reading frames to ensure high expression of the PPC3S heavy and light chains in a 1:1 ratio and assembly into an IgG structure. After transformation of the recombinant plasmid into *E. coli*, recombinant plasmids containing the light and heavy chains of PPC3S were obtained through pressure selection and amplification using the ampicillin resistance gene (Amp). The amplified and purified recombinant plasmids were then electroporated into mammalian expression cells, and cells stably expressing PPC3S were obtained through pressure selection using the blast fungicide resistance gene (Bla).

[0210] The recombinant expression vector was transfected into host cells CHO-K1. Forty-eight hours post-transfection, CD CHO medium containing blastomycin and zeocin was added, and the cells were seeded into 96-well plates. The medium was changed twice weekly until cell confluence recovered to over 50%, which were then used as minipools. These minipools were transferred to new 96-well plates, and after medium change, expression was performed for approximately 24 hours. The expression level was detected and ranked using a human IgG HTR kit. The minipools with the highest expression levels were selected for mixed seeding and aliquoting to obtain 10 cell populations. Batch culture was performed on these cell populations, and the quality of the expression samples was assessed to confirm the optimal cell populations. The batch culture protocol is shown in Table 10.

[0211] Table 10. PPC3S Batch Feeding Culture Protocol

[0212]

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

[0214] Table 11. Expression levels of PPC3S in different cell populations during batch fed culture.

[0215]

[0216] Taking into account 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, purified by protein A in one step, and then subjected to protein quality analysis. The analysis results are detailed in Table 12.

[0217] Table 12. Quality analysis results of proteins expressed in the optimal cell populations of PPC3S batch fed 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 bank. Cells were expanded and passaged in CD CHO medium (containing 4 mM glutamine, 400 μg / ml genomicine, and 7 μg / ml cyprodinil) under the following conditions: 36.5℃, 6.0% CO2, and 110 rpm. After reaching the target cell count, cell viability was assessed, cells were collected by centrifugation, resuspended in cryopreservation buffer containing 10% DMSO, and then cryopreserved using a programmed cooling process. Cell samples were taken for testing; cell density and viability were measured after thawing. The 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 consists of cell culture and protein purification processes to obtain high-purity, low-impurity samples with controlled exogenous factors for the preparation of PBMCs under aseptic conditions. The cell culture process is shown in Table 14. After process confirmation, HSP6093-B7Z4-M003 cells were selected for the preparation of the PPC3S stock solution. Under these process conditions, the cell viability (VIA), viable cell density (VCD), and lactate (Lac) data during the cell culture stage are detailed in Table 15, while the expression level and quality data are detailed 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 from the cell culture process is a one-step affinity purification, it contains polymers, and the culture medium contains CHO-related HCP, HCD, exogenous viruses, etc., which need to be removed through a purification process. After harvesting the cell culture medium, it undergoes deep filtration, S / D incubation for virus inactivation, affinity chromatography, intermediate deep filtration, cation exchange chromatography, virus removal filtration, ultrafiltration and percolation, stock solution preparation, and dispensing to obtain the PPC3S stock solution. The stock solution consists of: 5 mg / ml PPC3S protein, 10 mM histidine-histidine hydrochloride, 9% sucrose, and pH 5.5.

[0232] The study employed a two-step process: S / D incubation for virus inactivation and virus removal filtration. Virus clearance was validated under reduced-scale and virus-added experimental conditions, and the results are shown in Table 17. The results indicate that the protein purification process can effectively remove exogenous viruses, with a removal efficiency (LRV) of no less than 9.4 logs, meeting the requirements for controlling exogenous factors.

[0233] Table 17. Virus Removal Verification Results of PPC3S

[0234]

[0235] Therefore, the test items and acceptable standards for PPC3S stock solution include, but are not limited to: (1) pH, which should be 5.5 ± 0.2; (2) SEC-HPLC purity, with the main peak not less than 90.0% and HMW not more than 10.0%; (3) NR-CE purity, with the main peak not less than 80.0% and LMW reported results; (4) bacterial endotoxins, not more than 1.0 EU / mg; and (5) sterility, with no bacterial growth. In non-GMP stages, microbial limits are used instead of sterility tests, with the total aerobic bacteria count not more than 3 cfu / 30 ml and the total mold and yeast count not more than 3 cfu / 30 ml. The PPC3S stock solution of this application meets the standards for these test items.

[0236] Sequence description of this application:

[0237]

[0238]

[0239]

[0240]

[0241]

[0242] The use of any and all embodiments or exemplary language (e.g., "for example," "such as") provided herein is intended only to better illustrate the application and does not constitute a limitation on the scope of the application, unless otherwise required. The language in the specification should not be construed as indicating that any unclaimed element is necessary for carrying out the application.

[0243] All publications and patent applications referenced in this specification are incorporated herein by reference, as if each individual publication or patent application were specifically and individually indicated to be incorporated herein by reference. Furthermore, any theories, mechanisms, proofs, or findings described herein are intended to further enhance the understanding of this application and are not intended to limit this application in any way to such theories, mechanisms, proofs, or findings. Although this application has been shown and described in detail in the accompanying drawings and the foregoing description, this 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) 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 enriched NK cell components, the processing steps comprising one or more of the following: cell washing, adjusting cell density, sampling for NK cell counting and / or NK cell property detection, and replacement of cryopreservation solution; and c) Collect NK cells after treatment in step b); At least steps a) through c) are performed in a fully enclosed manner. The bispecific antibody targets the CD235a molecule on erythrocytes and the CD3 molecule on T cells, and includes 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. The bispecific antibody further comprises a heavy chain constant region and a light chain constant region. The heavy chain constant region is described as being the human IgG4 subtype. The first light chain variable region of the CD235a binding domain is connected to 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 connected to the heavy chain constant region to form a heavy chain of the CD235a binding domain. The CD3 binding domain is in the form of a single-chain antibody. The CD3-bonded structural domain is a structure that forms a second heavy chain variable region - first joint - second light chain variable region through a first joint. The second heavy chain variable region of the CD3 binding domain is linked to the heavy chain constant region of the CD235a binding domain via a second linker to form a fusion heavy chain, and the fusion heavy chain combines with the light chain of the CD235a binding domain to form the bispecific antibody.

2. The method according to claim 1, wherein the method further comprises step d): cryopreserving NK cells.

3. The method according to claim 1 or 2, wherein at least steps a) to c) are performed in a fully enclosed automated manner.

4. The method according to any one of claims 1 to 3, wherein the CD235a binding domain comprises a first light chain variable region as shown in SEQ ID No. 6 and a first heavy chain variable region as shown in SEQ ID No. 7, and / or the CD3 binding domain comprises a second light chain variable region as shown in SEQ ID No. 18 and a second heavy chain variable region as shown in SEQ ID No.

17.

5. The method according to claim 1, wherein 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.

6. The method according to claim 1, wherein 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.

7. The method according to claim 1, wherein the first connector is a GS type flexible connector.

8. The method according to claim 7, wherein the amino acid sequence of the first linker is as shown in SEQ ID No.

16.

9. The method according to any one of claims 1-8, wherein the amino acid sequence of the CD3 binding domain is shown in SEQ ID No.

19.

10. The method according to claim 1, wherein the second connector is a GS type flexible connector.

11. The method of claim 10, wherein the amino acid sequence of the second linker is as shown in SEQ ID No.

15.

12. The method according to any one of claims 1-11, wherein the amino acid sequence of the fused heavy chain is as shown in SEQ ID No.

20.

13. The method according to any one of claims 1-12, wherein the bispecific antibody is a tetravalent IgG4 molecule comprising a variable region from mice and a constant region from humans.

14. The method according to any one of claims 1-13, wherein the erythrocyte sedimentation agent comprises hydroxyethyl starch, gelatin, dextran, polyvinylpyrrolidone and / or methylcellulose.

15. The method according to any one of claims 1-14, wherein the processing step in b) includes cryopreservation, wherein the cryopreservation contains one or more cryoprotectants.

16. The method of claim 15, wherein the cryoprotectant is a permeable cryoprotectant and / or a non-permeable cryoprotectant.

17. The method of claim 16, wherein: The penetrating cryoprotectant is dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, acetamide, and / or methanol; and / or The non-permeable cryoprotectant is polyvinylpyrrolidone, sucrose, polyethylene glycol, dextran, albumin, and / or hydroxyethyl starch.

18. The method according to any one of claims 15-17, wherein the cryopreservation solution further comprises one or more amino acids and / or vitamins.

19. The method according to claim 18, wherein the cryopreservation solution comprises dimethyl sulfoxide, dextran, sodium alginate, trehalose, human serum albumin, sorbitol, compound electrolyte, sodium chloride injection, glucose sodium chloride injection, dextran 40 glucose injection, compound amino acid injection, and compound vitamin B injection.

20. The method of claim 19, wherein the final concentration of dimethyl sulfoxide is ≤5%.

21. The method according to any one of claims 1-20, wherein the blood sample is a peripheral blood sample and / or umbilical cord blood sample derived from a donor.

22. The method of claim 21, wherein the donor is screened by pathogen detection and / or genetic history.

23. The method according to claim 22, wherein the pathogenic microorganism is hepatitis B virus, hepatitis C virus, Treponema pallidum, human immunodeficiency virus, Epstein-Barr virus, cytomegalovirus, human T-cell troponinophil, human parvovirus B19, human herpesvirus 6 / 7 / 8, adenovirus, or JC virus.

24. A fully enclosed system for enriching natural killer (NK) cells in a blood sample, the system comprising: A sedimentation container configured to receive the blood sample and allow the blood sample to undergo a sedimentation process therein; A sedimentation generator supply device, the sedimentation generator comprising a bispecific antibody targeting erythrocyte surface antigen and T cell surface antigen and an erythrocyte sedimentation agent, the sedimentation generator supply device being fluidly connected to the sedimentation container and configured to supply the sedimentation generator to the sedimentation container; A separation device configured to perform phase separation on a multiphase liquid in the settling container; as well as A cell processing device configured to perform one or more of the following processes on the enriched NK cells: cell washing, cell density adjustment, sampling for NK cell counting and / or detection of NK cell properties, and cryopreservation solution replacement. The bispecific antibody targets the CD235a molecule on erythrocytes and the CD3 molecule on T cells, and includes 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. The bispecific antibody further comprises a heavy chain constant region and a light chain constant region. The heavy chain constant region is described as being the human IgG4 subtype. The first light chain variable region of the CD235a binding domain is connected to 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 connected to the heavy chain constant region to form a heavy chain of the CD235a binding domain. The CD3 binding domain is in the form of a single-chain antibody. The CD3-bonded structural domain is a structure that forms a second heavy chain variable region - first joint - second light chain variable region through a first joint. The second heavy chain variable region of the CD3 binding domain is linked to the heavy chain constant region of the CD235a binding domain via a second linker to form a fusion heavy chain, and the fusion heavy chain combines with the light chain of the CD235a binding domain to form the bispecific antibody.

25. The system of claim 24, wherein the system further comprises a collection container for collecting a portion of the enriched NK cells treated by the cell processing device.

26. The system of claim 25, wherein the collection container is a collection bag.

27. The system according to any one of claims 24-26, wherein the settling container is a settling bag.

28. The system according to any one of claims 24-27, wherein the separating device is a slurry clamp.

29. The system according to any one of claims 24-28, wherein the system is a fully enclosed automated system.

30. The system according to any one of claims 24-29, wherein the sedimentation generator supply device supplies the sedimentation generator to the sedimentation container via a pipe.

31. The system according to any one of claims 24-30, wherein the CD235a binding domain comprises a first light chain variable region as shown in SEQ ID No. 6 and a first heavy chain variable region as shown in SEQ ID No. 7, and / or the CD3 binding domain comprises a second light chain variable region as shown in SEQ ID No. 18 and a second heavy chain variable region as shown in SEQ ID No.

17.

32. The system of claim 24, wherein 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.

33. The system of claim 24, wherein 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.

34. The system of claim 24, wherein the first connector is a GS type flexible connector.

35. The system of claim 34, wherein the amino acid sequence of the first linker is as shown in SEQ ID No.

16.

36. The system according to any one of claims 24-35, wherein the amino acid sequence of the CD3 binding domain is shown in SEQ ID No.

19.

37. The system of claim 24, wherein the second connector is a GS type flexible connector.

38. The system of claim 37, wherein the amino acid sequence of the second connector is as shown in SEQ ID No.

15.

39. The system according to any one of claims 24-38, wherein the amino acid sequence of the fused heavy chain is as shown in SEQ ID No.

20.

40. The system according to any one of claims 24-39, wherein the bispecific antibody is a tetravalent IgG4 molecule comprising a variable region from mice and a constant region from humans.

41. The system according to any one of claims 24-40, wherein the erythrocyte sedimentation agent comprises hydroxyethyl starch, gelatin, dextran, polyvinylpyrrolidone and / or methylcellulose.

42. The system according to any one of claims 24-41, wherein the cell processing device includes a cryopreservation solution replacement module for replacing the components of the enriched NK cells with cryopreservation solution, and wherein the cryopreservation solution contains one or more cryoprotectants.

43. The system of claim 42, wherein the cryoprotectant is a permeable cryoprotectant and / or a non-permeable cryoprotectant.

44. The system according to claim 43, wherein: The penetrating cryoprotectant is dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, acetamide, and / or methanol; and / or The non-permeable cryoprotectant is polyvinylpyrrolidone, sucrose, polyethylene glycol, dextran, albumin, and / or hydroxyethyl starch.

45. The system according to any one of claims 42-44, wherein the cryopreservation solution further comprises one or more amino acids and / or vitamins.

46. ​​The system according to claim 45, wherein the cryopreservation solution comprises dimethyl sulfoxide, dextran, sodium alginate, trehalose, human serum albumin, sorbitol, compound electrolyte, sodium chloride injection, glucose sodium chloride injection, dextran 40 glucose injection, compound amino acid injection, and compound vitamin B injection.

47. The system of claim 46, wherein the final concentration of the dimethyl sulfoxide is ≤5%.

48. The system according to any one of claims 24-47, wherein the blood sample is a peripheral blood sample and / or umbilical cord blood sample derived from a donor.

49. The system of claim 48, wherein the donor is screened by pathogen detection and / or genetic history.

50. The system according to claim 49, wherein the pathogenic microorganism is hepatitis B virus, hepatitis C virus, Treponema pallidum, human immunodeficiency virus, Epstein-Barr virus, cytomegalovirus, human T-cell troponinophil, human parvovirus B19, human herpesvirus 6 / 7 / 8, adenovirus, or JC virus.

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