Fusion proteins binding cd235a and cd3 and methods of making and uses thereof

Abstract

The present application provides a fusion protein (e.g., bispecific antibody format) that binds CD235a and CD3, and methods of making and related uses thereof.

Description

Technical Field

[0001] This application generally relates to the field of biotechnology, and more specifically, to a fusion protein combining CD235a and CD3, its preparation method, and its uses. Background Technology

[0002] Natural killer (NK) cells are off-the-shelf cell therapy products derived from healthy allogeneic donors. These sources include peripheral blood mononuclear cells (PBMCs), umbilical cord blood (UCB), induced pluripotent stem cells (iPSCs), and NK cell lines (such as NK92). Among these, PBMCs, derived from donor-collected peripheral blood, yield NK cells that are widely available and possess good functional activity, making them a preferred key raw material. Combined with mature PBMC-derived NK cell culture and expansion processes, inexpensive and readily available off-the-shelf NK cell therapy products can be obtained.

[0003] Processing donor blood into PBMCs suitable for NK cell culture and expansion presents the following challenges: the NK cell percentage in the material is low, and its T cell (CD3+) content is low. + ) accounts for approximately 50% to 84%, NK cells (CD3) - CD16 + and / or CD56 + The proportion of T cells is approximately 7% to 40%. If the proportion of T cells in the obtained mononuclear cells is high, the proportion of T cells will increase and will seriously affect the purity of subsequent NK cell culture because the proliferative activity of T cells is higher than that of NK cells during the culture process. Furthermore, T cells may pose a safety risk to clinical use.

[0004] Therefore, there is a need in the art for improved methods to increase the proportion of NK cells in biological raw materials, particularly isolation tools and methods to increase the proportion of NK cells in PBMCs. Summary of the Invention

[0005] In a first aspect, this application provides a fusion protein (e.g., in the form of a bispecific antibody) that binds CD235a and CD3, said fusion protein comprising a CD235a-binding domain and a CD3-binding domain, wherein:

[0006] 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).

[0007] 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).

[0008] The amino acid sequences of HCDR and LCDR are defined according to Kabat.

[0009] In some embodiments of the first aspect, the fusion protein is capable of specifically binding to CD235a and CD3, for example, as a bispecific antibody that specifically binds to CD235a and CD3.

[0010] In some embodiments of the first aspect, 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.

[0011] In some embodiments of the first aspect, 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.

[0012] In some implementations, the fusion protein exists in monomeric or polymeric form.

[0013] In some embodiments of the first aspect, the fusion protein 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 as in SEQ ID No. 10 or SEQ ID No. 21. In some embodiments, the amino acid sequence of the light chain constant region is shown in SEQ ID No. 11.

[0014] In some embodiments of the first aspect, the first light chain variable region of the CD235a binding domain is associated with the light chain constant region to form a light chain of the CD235a binding domain, and the first heavy chain variable region of the CD235a binding domain is associated with the heavy chain constant region to form a heavy chain of the CD235a binding domain. 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.

[0015] In some embodiments of the first aspect, 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 linker-second light chain variable region via a first linker. For example, the first linker is 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 specific embodiments, the amino acid sequence of the CD3-binding domain is as shown in SEQ ID No. 19.

[0016] In some embodiments of the first aspect, 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 fusion protein. In some specific embodiments, 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 with the amino acid sequence shown in SEQ ID No. 15. In some specific embodiments, the amino acid sequence of the fusion heavy chain is shown in SEQ ID No. 20.

[0017] In some embodiments of the first aspect, the fusion protein is a tetravalent IgG4 molecule comprising a variable region from mice and a constant region from humans.

[0018] In a second aspect, this application provides a polynucleotide that encodes the fusion protein described in the first aspect above.

[0019] In a third aspect, this application provides an expression vector comprising the polynucleotides described in the second aspect above. In some embodiments, the expression vector is a recombinant plasmid.

[0020] In a fourth aspect, this application provides a host cell comprising the polynucleotide described in the second aspect or the expression vector described in the third aspect above. In some embodiments, the host cell is a mammalian cell, preferably a CHO-K1 cell.

[0021] In a fifth aspect, this application provides a method for preparing the fusion protein described in the first aspect above, the method comprising:

[0022] Cultivating the host cells described in the fourth aspect above, and

[0023] Harvest the cell culture medium containing cultured host cells, and extract and purify the fusion protein from the cell culture medium.

[0024] In some embodiments of the fifth aspect, the method further includes separating the extracted and purified fusion protein into monomers and polymers (e.g., by size exclusion chromatography (SEC)). In some specific embodiments, such as those shown by size exclusion chromatography, the monomer of the fusion protein has an SEC purity of about 99.3% at 280 nm and a retention time of about 7.29 min. In some specific embodiments, such as those shown by size exclusion chromatography, the polymer of the fusion protein has no peak with a retention time of about 7.29 min, wherein the content is about 68.2% at a retention time of about 5.78 min at 280 nm and about 31.8% at a retention time of about 6.30 min.

[0025] In a sixth aspect, this application provides a composition comprising the fusion protein described in the first aspect above. The composition can be used, for example, to enrich / isolate natural killer (NK) cells in peripheral blood mononuclear cell (PBMC) samples. In some embodiments, the composition comprises about 5 mg / ml of the fusion protein. The composition may also comprise at least one additional component, such as a buffer and / or an osmotic regulator. The buffer is, for example, histidine-histidine hydrochloride. The osmotic regulator is, for example, sucrose. In some embodiments, the composition comprises about 5 mg / ml of the fusion protein, about 10 mM histidine-histidine hydrochloride, about 9% sucrose, and has a pH of about 5.5.

[0026] In a seventh aspect, this application provides a method for enriching / isolating natural killer (NK) cells in a peripheral blood mononuclear cell (PBMC) sample, the method comprising:

[0027] The fusion protein and sedimentation agent described in the first aspect above are added to a peripheral blood sample to cause T cells and erythrocytes in the peripheral blood sample to settle; and

[0028] Obtain the upper layer of a peripheral blood sample containing NK cells.

[0029] In some embodiments of the seventh aspect, the settling agent may include hydroxyethyl starch, gelatin, dextran, polyvinylpyrrolidone, methylcellulose, and combinations thereof, analogs, or derivatives thereof. In some specific embodiments, the settling agent may be hydroxyethyl starch. The settling agent may 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 settling agent may be hydroxyethyl starch at a final concentration of about 2%.

[0030] In some embodiments of the seventh aspect, the fusion protein may be added to a peripheral blood sample in monomeric form along with a settling agent. In some specific embodiments, the monomeric fusion protein is added to the peripheral blood sample at a concentration of about 5 mg / mL and at a volume of 5 mL to 25 mL per 100 mL of peripheral blood, preferably at a volume of about 5 mL, about 15 mL, or about 25 mL per 100 mL of peripheral blood.

[0031] In some embodiments of the seventh aspect, the fusion protein is added to a peripheral blood sample in a mixture of monomers and polymers with a settling agent. In some specific embodiments, the mass ratio of the monomer to the polymer of the fusion protein is from 1:4 to 4:1, for example, about 1:4, about 1:3.5, about 1:3, about 1:2.5, about 1:2, about 1:1.5, about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, or about 4:1. In some specific embodiments, 0.82-3.25 mg of the monomer and 0.84-3.35 mg of the polymer are added per 1.2 mL of peripheral blood. In some more specific embodiments, about 0.94 mg of the monomer and about 1.09 mg of the polymer are added per 1.2 mL of peripheral blood. Attached Figure Description

[0032] Figure 1 A schematic diagram of the structure of the exemplary fusion protein PPC3S of this application is shown.

[0033] Figure 2 A schematic diagram of the structure of the recombinant expression vector HSP6093-LC-HC-10.0 used for recombinant expression of PPC3S in this application is shown.

[0034] Figure 3 A process flow diagram for purifying PPC3S of this application from cell culture is shown, wherein HSP6093DS in the diagram is PPC3S of this application.

[0035] Figure 4 The results show the SEC purity confirmation of the monomer (PPC3S-U2) of PPC3S in this application after separation by SEC-HPLC.

[0036] Figure 5 The results show the SEC purity confirmation of the polymer (PPC3S-U3) after separation by SEC-HPLC of PPC3S in this application.

[0037] Figures 6 to 9 The flow cytometry results of experiments using PPC3S monomer molecules followed by hydroxyethyl starch to precipitate cell cross-linked complexes are shown. The sample numbers in the experimental group are A1 to A4, and the control group uses solvent instead of PPC3S, with other operation steps being the same. The sample numbers in the control group are B1 to B4.

[0038] Figures 10 to 11 The results of the additive characteristic analysis of PPC3S-U2 and PPC3S-U3 are shown. The addition amounts of PPC3S-U2 and PPC3S-U3 were used as process parameters, and T% and NK% were used as key quality attributes. Polynomial fitting was performed on the experimental results in Table 11. The fitting results are shown below. Figure 10 For the optimal theoretical process, please refer to [link / reference]. Figure 11 .

[0039] Figure 12 The results of the validation experiments show the process design space of PPC3S molecules for processing PBMCs.

[0040] Figure 13 The sedimentation results of using both monomers and polymers of PPC3S molecules in peripheral blood lymphocyte samples from three donors are shown. Detailed Implementation

[0041] definition

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] As used in this article, "fusion protein" refers to the intentional linking of two or more gene segments encoding a functional protein together, thereby expressing the protein. This protein product, obtained by artificially linking the coding regions of two or more genes end-to-end and then expressing the gene under the control of a regulatory sequence, is called a fusion protein.

[0047] As used herein, the term "linker" or "peptide linker" refers to a short peptide, ranging in length from 3 to 76 amino acids, used to connect two functional proteins. Peptide linkers provide flexibility to the functional proteins in a fusion protein, enabling them to perform their respective functions.

[0048] 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.

[0049] 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.

[0050] Allogeneic peripheral blood mononuclear cells (PBMCs) derived from healthy donor peripheral blood refer to human peripheral blood mononuclear cells collected from healthy donor peripheral blood and serve as raw materials for allogeneic natural killer cells. In some embodiments, this application screens PBMCs for HLA and KIR. In some embodiments, this application screens PBMCs for CD16a variants (176V, 176F). In some embodiments, this application removes a majority (≥99%) of the T cells from the PBMCs during isolation.

[0051] As used herein, the term "monoclonal antibody" refers to an immunoglobulin with a defined amino acid sequence, expressed by a polyclonal or monoclonal host cell, such as a mammalian cell (e.g., CHO cells, 293 cells), and specifically targeting a specific target. In some embodiments, the host cell, such as a mammalian cell, is genetically engineered to highly express the specific target protein under selection pressure. In some embodiments, the specific target of the immunoglobulin may be human CD235a and CD3. In some embodiments, the immunoglobulin is chimeric, i.e., it comprises an immunoglobulin motif derived from humans and an immunoglobulin motif derived from mice, for example, comprising a constant region derived from humans and a variable region derived from mice. In some embodiments, the Fc of the immunoglobulin is the human G4 subtype (IgG4).

[0052] 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.

[0053] The term "bispecific antibody" or "biantibody" as used in this article refers to an antibody that simultaneously binds 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 a 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; KIH (knob-in-hole) technology is one strategy to address heavy chain heteropolymerization. Typically, KIH technology refers to modifying the amino acid sequence of the CH3 region to form a structure that facilitates the pairing of heterologous half-antibodies, thereby creating bispecific antibodies while maintaining the structure of normal antibodies as much as possible.

[0054] 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 6 This molecule has a glycosylated segment at its N-terminus, located outside the erythrocyte cell membrane, and is a mononuclear blood group receptor.

[0055] In a first aspect, this application provides a fusion protein (e.g., in the form of a bispecific antibody) that binds CD235a and CD3, said fusion protein comprising a CD235a-binding domain and a CD3-binding domain, wherein:

[0056] 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).

[0057] 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).

[0058] The amino acid sequences of HCDR and LCDR are defined according to Kabat.

[0059] Figure 1 This application illustrates an exemplary construction scheme for the fusion protein. Figure 1 In this embodiment, the fusion protein is in the form of a bispecific antibody. The bispecific antibody comprises two light chains and two heavy chains, with an N-terminus containing a CD235a-binding domain (including a variable region of the light chain and a variable region of the heavy chain that binds to CD235a) and a C-terminus containing a CD3a-binding domain (including a variable region of the light chain and a variable region of the heavy chain that binds 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 includes 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 includes three CDR regions: HCDR1, HCDR2, and HCDR3), and a CD3-binding domain in the form of an scFv (including LCDR1, LCDR2, and LCDR3, as well as HCDR1, HCDR2, and HCDR3 regions).

[0060] It should be understood that Figure 1 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):

[0061] (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.

[0062] (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

[0063] (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.

[0064] In some embodiments of the first aspect, 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. In some embodiments, 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.

[0065] In some implementations, the fusion protein exists in monomeric or polymeric form.

[0066] In some embodiments of the first aspect, the fusion protein 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.

[0067] In some embodiments of the first 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;

[0068] In some embodiments of the first aspect, 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.

[0069] In some embodiments of the first 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 fusion protein. 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 the amino acid sequence 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.

[0070] In some embodiments of the first aspect, the fusion protein is a tetravalent IgG4 molecule comprising a variable region from mice and a constant region from humans.

[0071] The inventors have designed and prepared the fusion protein described above, and through NK cell culture process research, have found that this fusion protein can improve the purity of NK cells in PBMCs. Furthermore, by combining it with a suitable culture process, PBMCs can be cultured into a high-purity NK cell product. In some specific embodiments, the inventors have discovered that an exemplary fusion protein of this application (i.e., PPC3S), as a human IgG4-based antibody, can target red blood cells (CD235a...) + ) and T cells (CD3) + The cells cross-link together to form a rosette-like complex. Subsequently, a settling agent, such as hydroxyethyl starch, can be added to accelerate the settling of erythrocytes and their cross-linked T cells, while NK cells are mostly diffused within the plasma layer. Then, the PBMCs, from which erythrocytes and T cells have been removed, can be transferred to sterile containers using various methods, aliquoted into key raw materials for NK cells, and frozen at deep cryogenic temperatures (≤-150℃).

[0072] The inventors also discovered an advantage of this application: the fusion protein (e.g., PPC3S) is a molecule with a clearly defined target gene, which can be constructed into a cell library suitable for GMP production and used for large-scale production, meeting the requirements for sterile raw materials necessary for NK cell culture. Furthermore, based on the molecule's mechanism of action and experimental data, the monomer and high molecular weight polymer (HMW, SEC-HPLC) of the fusion protein (e.g., PPC3S) each have advantages in separation effect and separation rate, and both have practical value, capable of replacing some of the flocculants (such as hydroxyethyl starch).

[0073] Specifically, the PPC3S monoclonal antibody of this application is a full-length IgG4 antibody that is expressed in mammalian cells and prepared through a purification process, targeting human CD235a and CD3, and containing a variable region from mice and a constant region from humans.

[0074] CD235a, or blood group glycoprotein A (GYPA), is 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 6The CD235a molecule (Merry et al. Biochem J. (1986) 233: 93-98; Loken et al. Blood (1987) 69: 255-263) is highly expressed in the kidney, bladder, and urethra, 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 and is a mononuclear blood group receptor. Amino acids 20-91 form the extracellular domain, amino acids 92-114 form the transmembrane domain, and amino acids 115-150 form 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.

[0075] 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.

[0076] Based on the design principles of product design, the inventors have selected CD235a and CD3 as targets to target red blood cells (RBCs) and T cells (CD3+). + They cross-link together to form a large cell complex and then settle, thus achieving the purpose of separation.

[0077] Several studies in this field have investigated the druggability of antibodies from different sources of CD235a:

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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 was found to have 4.80 × 10⁻⁶ antibodies 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, measured at 4.66 × 10⁻⁶ per red blood cell. 5 One CD235a molecule has a dissociation constant KD of 9.5 × 10⁻⁶. -8 M, which is 95 nM.

[0082] However, none of these studies disclosed or taught the fusion protein of this application.

[0083] In a second aspect, this application provides a polynucleotide that encodes the fusion protein described in the first aspect above.

[0084] In a third aspect, this application provides an expression vector comprising the polynucleotides described in the second aspect above. In some embodiments, the expression vector is a recombinant plasmid.

[0085] In a fourth aspect, this application provides a host cell comprising the polynucleotide described in the second aspect or the expression vector described in the third aspect above. In some embodiments, the host cell is a mammalian cell, preferably a CHO-K1 cell.

[0086] In a fifth aspect, this application provides a method for preparing the fusion protein described in the first aspect above, the method comprising:

[0087] Cultivating the host cells described in the fourth aspect above, and

[0088] Harvest the cell culture medium containing cultured host cells, and extract and purify the fusion protein from the cell culture medium.

[0089] In some embodiments, the host cells are cultured into more than one cell population. In some specific embodiments, the more than one cell population is subjected to batch fed-batch culture. In some more specific embodiments, cell populations are selected for protein quality analysis based on their expression levels and cell growth, and based on the protein quality analysis results, cell populations are selected for original cell bank construction and expansion culture.

[0090] In some implementations, the extraction and purification of the fusion protein includes 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.

[0091] In some embodiments of the fifth aspect, the method further includes separating the extracted and purified fusion protein into monomers and polymers (e.g., by size exclusion chromatography (SEC)). In some specific embodiments, the monomer has an SEC purity of approximately 99.3% at 280 nm and a retention time of approximately 7.29 min. In some specific embodiments, the polymer has no peak at a retention time of approximately 7.29 min, with a content of approximately 68.2% at a retention time of approximately 5.78 min at 280 nm and approximately 31.8% at a retention time of approximately 6.30 min.

[0092] In some implementations, the size exclusion chromatography (SEC) purity of the obtained fusion protein is greater than or equal to 45%, for example, greater than or equal to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0093] In a sixth aspect, this application provides a composition comprising the fusion protein described in the first aspect above, for example, the composition for enriching / isolating natural killer (NK) cells in peripheral blood mononuclear cell (PBMC) samples. In some embodiments, the fusion protein is present in the composition at a concentration of 0.5 mg / ml to 50 mg / ml. In some embodiments, the fusion protein is administered at 0.5 mg / ml, 1 mg / ml, 1.5 mg / ml, 2 mg / ml, 2.5 mg / ml, 3 mg / ml, 3.5 mg / ml, 4 mg / ml, 4.5 mg / ml, 5 mg / ml, 5.5 mg / ml, 6 mg / ml, 6.5 mg / ml, 7 mg / ml, 7.5 mg / ml, 8 mg / ml, 8.5 mg / ml, 9 mg / ml、9.5 mg / ml、10 mg / ml、10.5 mg / ml、11 mg / ml、11.5 mg / ml、12 mg / ml、12.5 mg / ml、13.5 mg / ml、14.5 mg / ml、15 mg / ml、15.5 mg / ml、16mg / ml、16.5 mg / ml、17 mg / ml、17.5 mg / ml、18 mg / ml, 18.5 mg / ml, 19 mg / ml, 20 The composition is present in concentrations of 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, or 50 mg / ml. In some embodiments, the fusion protein may be present in the composition at a concentration greater than 50 mg / ml. The composition may also contain at least one additional component, such as a buffer and / or an osmolarity regulator. Buffers may include, for example, weak acids or salts thereof, weak bases or salts thereof, and other buffers or combinations thereof well known in the art, such as borates, citric acid, citrates, phosphoric acid, phosphates, tris(hydroxymethyl)aminomethane (Tris), etc. Osmolarity regulators may include, for example, salts (e.g., sodium salts, potassium salts, magnesium salts, etc.), sugars (e.g., glucose, sucrose, etc.), amino acids (e.g., glycine, histidine, etc.), and other osmolarity regulators or combinations thereof well known in the art. For example, the composition comprises the fusion protein and a histidine-histidine hydrochloride buffer solution. In some embodiments, the composition also contains sucrose to improve the stability of the fusion protein. In some embodiments, the composition comprises about 5 mg / ml of the fusion protein, about 10 mM histidine-histidine hydrochloride, and about 9% sucrose. In some embodiments, the composition has a pH of about 5.5.

[0094] In a seventh aspect, this application provides a method for enriching / isolating natural killer (NK) cells in a peripheral blood mononuclear cell (PBMC) sample, the method comprising:

[0095] The fusion protein and sedimentation agent described in the first aspect above are added to a peripheral blood sample to cause T cells and erythrocytes in the peripheral blood sample to settle; and

[0096] Obtain the upper layer of a peripheral blood sample containing NK cells.

[0097] In some embodiments of the seventh aspect, the settling agent may include hydroxyethyl starch, gelatin, dextran, polyvinylpyrrolidone, methylcellulose, and combinations thereof, analogues, or derivatives thereof. In some specific embodiments, the settling agent may be hydroxyethyl starch.

[0098] The settling 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 settling agent can be hydroxyethyl starch at a final concentration of about 2%.

[0099] In some embodiments of the seventh aspect, the fusion protein is added to a peripheral blood sample in monomeric form along with a settling agent, wherein the monomer is added to the peripheral blood sample at a concentration of about 5 mg / mL and 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, about 12.5 mL, or about 13 mL per 100 mL of peripheral blood. Add approximately 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 peripheral blood, preferably in volumes of approximately 5 mL, 15 mL, or 25 mL.

[0100] In some embodiments of the seventh aspect, the fusion protein is added to peripheral blood in a mixture of monomers and polymers with a settling agent, wherein the mass ratio of the monomer to the polymer is 1:4 to 4:1, for example, about 1:4, about 1:3.5, about 1:3, about 1:2.5, about 1:2, about 1:1.5, about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, or about 4:1. In some specific embodiments, 0.82-3.25 mg of the monomer and 0.84-3.35 mg of the polymer are added per 1.2 mL of peripheral blood. In some more specific embodiments, about 0.94 mg of the monomer and about 1.09 mg of the polymer are added per 1.2 mL of peripheral blood.

[0101] In some embodiments of the seventh aspect, a polymer of the fusion protein is added to a peripheral blood sample, wherein the polymer may be in the form of about 5 mg / mL and added to the peripheral blood sample in volumes of 5 mL to 25 mL per 100 mL of peripheral blood, for example, at amounts of about 5 mL, about 5.5 mL, about 6 mL, about 6.5 mL, about 7 mL, about 7.5 mL, about 8 mL, about 8.5 mL, about 9 mL, about 9.5 mL, about 10 mL, about 10.5 mL, about 11 mL, about 11.5 mL, about 12 mL, about 12.5 mL, or about 13 mL per 100 mL of peripheral blood. Add approximately 13.5 mL, approximately 14 mL, approximately 14.5 mL, approximately 15 mL, approximately 15.5 mL, approximately 16 mL, approximately 16.5 mL, approximately 17 mL, approximately 17.5 mL, approximately 18 mL, approximately 18.5 mL, approximately 19 mL, approximately 19.5 mL, approximately 20 mL, approximately 20.5 mL, approximately 21 mL, approximately 21.5 mL, approximately 22 mL, approximately 22.5 mL, approximately 23 mL, approximately 23.5 mL, approximately 24 mL, approximately 24.5 mL, or approximately 25 mL, preferably at a volume of approximately 5 mL, approximately 15 mL, or approximately 25 mL per 100 mL of peripheral blood.

[0102] In some embodiments of this application, this application achieves one or more of the following beneficial technical effects, including but not limited to:

[0103] 1. Since magnetic beads are not used, the method for enriching / isolating natural killer (NK) cells in this application eliminates the need for a magnetic bead removal step and the control of magnetic bead residue;

[0104] 2. The NK% and double-negative % results obtained by treating peripheral blood samples with the fusion protein of this application are superior to commercially available products, and the T% is comparable, with good robustness in multiple donors; and / or

[0105] 3. The mixed use of monomers and polymers of the fusion protein in this application can promote cell sedimentation, improve processing efficiency, and reduce the purity requirements of the fusion protein (e.g., PPC3S).

[0106] 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.

[0107] Example

[0108] 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.

[0109] In general, the design of an exemplary fusion protein PPC3S of this application is detailed in Example 1, and the construction process of the expression vector and expression cell library for PPC3S is detailed in Example 2;

[0110] The cell resuscitation, expansion, and expression processes, as well as the preparation process for fusion protein purification, are detailed in Example 3. The PPC3S molecules were preserved in a histidine-histidine hydrochloride buffer solution, with sucrose added to improve stability. Quality control parameters for the purified molecules included, but were not limited to: pH, binding activity, purity (SEC-HPLC), purity (non-reducing CE), bacterial endotoxins, microbial limits, and sterility.

[0111] The effect of PPC3S on PBMCs achieved the goals of molecular design, with the content of NK cells and T cells in the treated PBMCs meeting the control requirements (<2.0%). For the combined effect with hydroxyethyl starch and its validation in peripheral blood from different donor sources, please refer to Example 4.

[0112] The combined effect of PPC3S monomers and polymers, and the comparison with the combined effect of commercially available reagents and hydroxyethyl starch, are detailed in Example 5.

[0113] Example 1 - Design of Fusion Protein

[0114] This embodiment provides an exemplary fusion protein (hereinafter referred to as "PPC3S"), which is an artificially designed molecule capable of binding red blood cells to T cells (CD3+). + They cross-link together to form a rosette-like complex, and then through cell sedimentation methods, including the addition of hydroxyethyl starch and PPC3S polymers, most of the red blood cells and T cells are removed, making the treated PBMCs more suitable for culture as NK cells.

[0115] PPC3S has a multivalent structure, dually targets CD235a and CD3, and is based on the IgG framework. Within the PPC3S molecule:

[0116] (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;

[0117] (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.

[0118] (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), see 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;

[0119] (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 with adapter 1 is divided into four parts, from the N-terminus to the C-terminus: adapter 1, anti-CD3-VH, adapter 2, and anti-CD3-VL. Among them, adapter 1 and adapter 2 are reproducible sequences composed of glycine and serine. This sequence can minimize the impact on target affinity while ensuring 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.

[0120] (5) Finally, the anti-CD3 scFv with adapter 1 was integrated into the C-terminus of the heavy chain of 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.

[0121] 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 1 .

[0122] 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.

[0123] The CDR sequence of the PPC3S molecule in this application

[0124]

[0125] Example 2 - Construction of expression vector and cell bank for PPC3S molecule

[0126] 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). The recombinant expression vector was named HSP6093-LC-HC-10.0. Figure 2 Its structure is schematically depicted.

[0127] 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 1.

[0128] Table 1. PPC3S Batch Feeding Culture Protocol

[0129]

[0130] The expression levels of each cell population are shown in Table 2.

[0131] Table 2. Expression levels of PPC3S in different cell populations during batch fed culture.

[0132]

[0133] 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 3.

[0134] Table 3. Quality analysis results of proteins expressed in the optimal cell populations of PPC3S batch fed culture.

[0135]

[0136] 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 a rotation speed of 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 4.

[0137] Table 4. PCB viable cell density and viability of PPC3S

[0138]

[0139] Example 3 - Preparation of PPC3S molecules

[0140] 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 5. 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 6, while the expression level and quality data are detailed in Table 7.

[0141] Table 5. Cell culture process of PPC3S

[0142]

[0143] Table 6. Cell culture monitoring data of PPC3S

[0144]

[0145] Table 7. Expression and quality data of PPC3S

[0146]

[0147] Because the sample obtained from cell culture is a one-step affinity purification, it contains polymers, and the culture medium contains CHO-related HCPs, HCDs, exogenous viruses, etc., which need to be removed through a purification process. The protein purification process flow is as follows: Figure 3As shown, after harvesting the cell culture medium, the solution underwent deep filtration, S / D incubation for virus inactivation, affinity chromatography, intermediate deep filtration, cation exchange chromatography, virus-free filtration, ultrafiltration and diafiltration, stock solution preparation, and dispensing to obtain the PPC3S stock solution. The stock solution consisted of: 5 mg / ml PPC3S protein, 10 mM histidine-histidine hydrochloride, 9% sucrose, and pH 5.5.

[0148] 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 8. 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.

[0149] Table 8. Virus Removal Verification Results of PPC3S

[0150]

[0151] 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, which should not exceed 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 exceeding 3 cfu / 30 ml and the total mold and yeast count not exceeding 3 cfu / 30 ml. The PPC3S stock solution of this application meets the standards for these test items.

[0152] Example 4 - Separation effect of PPC3S monomer

[0153] PPC3S can separate red blood cells (RBCs) from T cells (CD3+). + The cells cross-link together to form a rosette-like complex. Hydroxyethyl starch can accelerate the settling of erythrocytes and their cross-linked T cells, while NK cells are mostly diffused within the plasma layer. By using PPC3S in combination with hydroxyethyl starch, PPC3S cross-links erythrocytes and T cells together, followed by hydroxyethyl starch sedimentation, removing most of the erythrocytes and T cells. The experimental steps are as follows:

[0154] Using typical batches of PPC3S (SEC purity 98.8%, NR-CE purity 90.9%, considered predominantly monomeric), equal volumes of peripheral blood were added to centrifuge tubes, along with different concentrations of PPC3S (5 mL / 100 mL peripheral blood, 15 mL / 100 mL peripheral blood, and 25 mL / 100 mL peripheral blood). The mixture was thoroughly mixed and incubated for 40 min. Then, 6% hydroxyethyl starch solution was added, bringing the final concentration to 2%. After settling for 0.5 h, the supernatant plasma layer was aspirated, resuspended to 45 mL, centrifuged (300 g, 10 min), and the supernatant was discarded. The cell pellet was resuspended, and NK cell purity (CD3+) was determined by flow cytometry. - CD56 + ) and T cell ratio (CD3) + For detailed experimental data, please refer to Table 9.

[0155] Table 9. Effects of the classic method of isolating T cells using PPC3S monomers and hydroxyethyl starch.

[0156]

[0157] The results showed that by using PPC3S monomer molecules and hydroxyethyl starch in combination, the proportion of T cells in mononuclear cells obtained in all three groups was ≤0.5%, and the purity of NK cells was >70%.

[0158] Based on the results of the above experiments, PPC3S monomer molecules were added at a rate of 5 mL / 100 mL peripheral blood, and peripheral blood from four different donors (numbered 1, 2, 3, and 4) was processed to verify the effect of the combined use of PPC3S monomer molecules and hydroxyethyl starch. In the experimental group, PPC3S monomer molecules were used followed by hydroxyethyl starch to settle the cell cross-linking complex; samples A1 to A4 were used. In the control group, the solvent was used instead of PPC3S, with all other procedures identical; samples B1 to B4 were used. The separation results are summarized in Table 10 (percentages based on flow cytometry results, rounded to one decimal place). Detailed flow cytometry results can be found in [link to table]. Figure 6 (A1 / B1) Figure 7 (A2 / B2) Figure 8 (A3 / B3) Figure 9 (A4 / B4)

[0159] Table 10. Separation effect of PPC3S monomer molecules combined with hydroxyethyl starch on peripheral blood cells from different donors

[0160]

[0161] The above verification results demonstrate that the use of PPC3S monomer molecules in combination with hydroxyethyl starch can remove most of the T cells in peripheral blood, significantly increase the percentage of NK cells in mononuclear cells, and compared with flow cytometry and magnetic bead screening, this method is low in cost, short in time, and simple to operate.

[0162] Example 5 - Separation effect of using a mixture of PPC3S monomers and polymers

[0163] The PPC3S obtained by one-step affinity purification was separated into monomers and polymers (HMWs) by SEC-HPLC. SEC detection results for the samples are detailed below. Figure 4 , Figure 5 Among them, the monomer was named PPC3S-U2, with an SEC purity of 99.3% (280 nm) and a retention time of 7.29 min; the polymer was named PPC3S-U3, with no peak at a retention time of 7.29 min, a retention time of 5.78 min with a content of 68.2% (280 nm), and a retention time of 6.30 min with a content of 31.8% (280 nm), indicating that they are polymers of different degrees of PPC3S.

[0164] 5.1 Selection of Design Space

[0165] A single donor peripheral blood cell suspension was mixed thoroughly and then divided into multiple equal portions for experimental use. The isolated PPC3S-U2 and PPC3S-U3 were added to the peripheral blood in different proportions. Following the experimental steps of Example 4, T cells (CD3+) were isolated from the peripheral blood. + ), and detect NK cells (CD3) - CD56 + The purity of the cells was determined. The experimental protocol and flow cytometry results are shown in Table 11. The experimental groups are numbered A to I, with group E serving as the center point and repeated twice. Peripheral blood cells treated with solvent served as the control group, numbered K0.

[0166] The experimental steps are as follows:

[0167] Peripheral blood lymphocytes collected from apheresis (live cell density approximately 1.2 × 10⁻⁶) 8Cells / ml (red blood cells to white blood cells ratio approximately 14:1) were divided into ten 15ml centrifuge tubes, with 1.2 ml added to each tube. Different volumes of PPC3S-U2, PPC3S-U3, and 25 μl of a commercially available control sample were added according to the experimental design. After addition, the PPC3S sample adhering to the tube wall was flushed off with 2 ml of PBS (pH 7.4), and incubated at room temperature for 20 minutes. Each group was then diluted to 10 ml with PBS (pH 7.4), and 3.5 ml of lymphocyte separation medium was added. The cells were centrifuged at 1200g for 20 minutes. The white blood cell layer was collected from each group, resuspended in PBS (pH 7.4) to 15 ml, centrifuged at 1000g for 10 minutes, the supernatant was discarded, and the cells were resuspended in PBS (pH 7.4) to 1 ml for flow cytometry analysis.

[0168] Table 11. Preliminary screening study scheme and separation effect of using a mixture of PPC3S monomers and polymers.

[0169]

[0170] Analysis of PPC3S-U2 and PPC3S-U3 Addition Characteristics: Using the addition amounts of PPC3S-U2 and PPC3S-U3 as process parameters and T% and NK% as key quality attributes, polynomial fitting was performed on the experimental results in Table 11. The fitting results are detailed in [link to table]. Figure 10 For details on the optimal theoretical process, please refer to [link / reference]. Figure 11 The fitting coefficients of the T% and NK% models were 0.69 and 0.95, respectively, both of which can be used as process design spaces, and key data points were selected for process validation. Specifically, when the amount of monomeric PPC3S-U2 added was 0.94 mg / part of peripheral blood and the amount of polymeric PPC3S-U3 added was 1.09 mg / part of peripheral blood, the mixed use of the PPC3S monomer and polymer achieved the maximum effect, namely, NK cell purity (29.4%) and T cell content (0.18%).

[0171] 5.2 Confirmation of Design Space

[0172] The design space was validated through experiments, and its robustness was confirmed using peripheral blood lymphocyte samples from three donors. The criteria for parameter selection are detailed in [link to relevant documentation]. Figure 12 The white area represents the region with the preferred quality requirements for the design space (T% < 1.0%, NK% > 30%). The parameters and experimental group list are shown in Table 12, and the experimental results are summarized in Table 13. The experimental operation steps are the same as described in this embodiment, and flow cytometry was performed (results not shown). Furthermore, the settling effect 20 minutes after treatment (fastest 4, slowest 1) was ranked, as detailed in [link to table]. Figure 13 E represents the control group of commercially available sedimentation reagents.

[0173] Table 12. Experimental Study Protocol for Confirming the Separation Effect of PPC3S Monomer and Polymer Mixtures

[0174]

[0175] Table 13. Summary of experimental results confirming the separation effect of PPC3S monomers and polymers in combination.

[0176]

[0177] 5.3 Confirmation and summary analysis of experimental results

[0178] According to the confirmatory experimental design in Table 12, the separation effects of groups A, B, C, and D varied slightly among peripheral blood lymphocytes from different donors. Compared to the K0 (starting sample), the proportion of T% was significantly reduced, from 57.1%–65.0% to below 2.5%, with the optimal effect reaching 0.5%. Furthermore, each group also reduced the relative proportion of double-negative cells. In the K0 (starting sample), the double-negative percentage was close to or higher than the NK percentage. After treatment with groups A, B, C, and D, the double-negative percentage was lower than the NK percentage, confirming that the experimental objective was achieved. The selection of the proportion is usually based on the red-to-white ratio of peripheral blood lymphocytes from different donors. The proportions in groups B and C showed better applicability, and small-scale trials should be conducted to confirm their applicability before large-scale preparation.

[0179] In addition, based on the molecular principles of use, the sedimentation and hemolysis of each group were compared. The sedimentation effect remained basically consistent, C>B>D>A>E (commercially available sedimentation reagent), meaning that the higher the proportion of PPC3S-U3 polymer added, the faster the sedimentation. This also implies that in the process of preparing PPC3S, appropriately retaining some polymer is beneficial to accelerate sedimentation and improve experimental efficiency. Considering both separation effect and sedimentation rate, although group A had a slower sedimentation, the separation effect could ensure T%<2.0%. When considering the combined use of hydroxyethyl starch, the monomer ratio should be as high as possible, and the polymer can be appropriately retained (e.g., the preparation process can obtain PPC3S with 10% polymer and 90% monomer) to promote the sedimentation rate.

[0180] Therefore, this application discloses a fusion protein (e.g., PPC3S molecule) that combines CD235a and CD3, and discloses a method for using its monomers and a mixture of its polymers and monomers, which can effectively remove T cells, increase the proportion of NK cells, and accelerate cell sedimentation.

[0181] Sequence description of this application:

[0182]

[0183]

[0184]

[0185]

[0186] The use of any and all embodiments or exemplary language (e.g., “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.

[0187] 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 fusion protein that binds CD235a and CD3, said fusion protein being in the form of a bispecific antibody, said fusion protein comprising 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 fusion protein also includes a heavy chain constant region and a light chain constant region; The heavy chain constant region is the human IgG4 subtype; The first light chain variable region of the CD235a binding structural domain is connected to the light chain constant region to form the light chain of the CD235a binding structural domain, and the first heavy chain variable region of the CD235a binding structural domain is connected to the heavy chain constant region to form the heavy chain of the CD235a binding structural domain. The CD3 binding domain is in the form of a single-chain antibody (scFv), which forms a structure of second heavy chain variable region-first linker-second light chain variable region through a first linker; 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 fusion protein.

2. The fusion protein according to claim 1, wherein 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.

3. The fusion protein according to claim 1, wherein the CD3 binding domain comprises a second light chain variable region with the amino acid sequence as shown in SEQ ID No. 18 and a second heavy chain variable region with the amino acid sequence as shown in SEQ ID No.

17.

4. The fusion protein 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.

5. The fusion protein according to claim 1, wherein the amino acid sequence of the constant region of the light chain is shown in SEQ ID No.

11.

6. The fusion protein according to claim 1, wherein the first connector is a GS-type flexible connector.

7. The fusion protein according to claim 1, wherein the second connector is a GS-type flexible connector.

8. The fusion protein according to claim 6, wherein the amino acid sequence of the first linker is as shown in SEQ ID No.

16.

9. The fusion protein according to claim 7, wherein the amino acid sequence of the second linker is as shown in SEQ ID No.

15.

10. The fusion protein according to claim 1, wherein the amino acid sequence of the heavy chain of the CD235a binding domain is shown in SEQ ID No.

14.

11. The fusion protein according to claim 1, wherein the amino acid sequence of the CD3 binding domain is shown in SEQ ID No.

19.

12. The fusion protein of claim 1, wherein the fusion protein is a tetravalent IgG4 molecule comprising a variable region from a mouse and a constant region from a human.

13. The fusion protein according to claim 1, wherein the amino acid sequence of the light chain of the fusion protein is the amino acid sequence shown in SEQ ID No.

13.

14. The fusion protein according to claim 1, wherein the amino acid sequence of the heavy chain of the fusion protein is the amino acid sequence shown in SEQ ID No.

20.

15. The fusion protein of claim 1, wherein the fusion protein exists in monomeric or polymeric form.

16. A polynucleotide encoding a fusion protein according to any one of claims 1-15.

17. An expression vector comprising the polynucleotide of claim 16.

18. The expression vector according to claim 17, wherein the expression vector is a recombinant plasmid.

19. A host cell comprising the polynucleotide of claim 16 or the expression vector of any one of claims 17 to 18.

20. The host cell of claim 19, wherein the host cell is a mammalian cell.

21. The host cell according to claim 19, wherein the host cell is a CHO-K1 cell.

22. A method for preparing the fusion protein according to any one of claims 1-15, the method comprising: Cultivate the host cells as described in claim 19; as well as Harvest the cell culture medium containing cultured host cells, and extract and purify the fusion protein from the cell culture medium.

23. The method of claim 22, wherein the method further comprises separating the extracted and purified fusion protein into monomers and polymers.

24. The method of claim 23, wherein the separation is performed by size exclusion chromatography (SEC).

25. The method of claim 23, wherein the monomer has a SEC purity of 99.3% at 280 nm and a retention time of 7.29 min, and the polymer has no peak at a retention time of 7.29 min, the polymer has a content of 68.2% at a retention time of 5.78 min at 280 nm and a content of 31.8% at a retention time of 6.30 min.

26. A composition comprising the fusion protein of any one of claims 1-15, the composition being used to enrich / isolate natural killer (NK) cells in peripheral blood mononuclear cell (PBMC) samples.

27. The composition of claim 26, wherein the composition comprises 5 mg / ml of the fusion protein.

28. The composition of claim 26, wherein the composition further comprises a buffer and / or an osmotic pressure regulator.

29. The composition of claim 28, wherein the buffer is histidine-histidine hydrochloride.

30. The composition according to claim 28, wherein the osmotic pressure regulator is sucrose.

31. The composition of claim 26, wherein the composition comprises 5 mg / ml of the fusion protein, 10 mM histidine-histidine hydrochloride, 9% sucrose and has a pH of 5.

5.

32. A method for enriching / isolating natural killer (NK) cells in a peripheral blood mononuclear cell (PBMC) sample, the method comprising: The fusion protein of any one of claims 1-15 and the sedimentation agent are added to a peripheral blood sample to cause T cells and erythrocytes in the peripheral blood sample to settle; as well as Obtain the upper layer of a peripheral blood sample containing NK cells.

33. The method according to claim 32, wherein the settling agent is hydroxyethyl starch, gelatin, dextran, polyvinylpyrrolidone and / or methylcellulose.

34. The method according to claim 32, wherein the settling agent is hydroxyethyl starch; the settling agent is added at a final concentration of 0.5%-4%.

35. The method of claim 34, wherein the flocculant is added at a final concentration of 0.5%-1%, 1%-2%, or 2%-4%.

36. The method of claim 35, wherein the settling agent is 2% final concentration of hydroxyethyl starch.

37. The method according to claim 32, wherein: The fusion protein, in monomeric form, is added to the peripheral blood sample along with the settling agent, wherein the monomeric fusion protein is prepared as a solution at a concentration of 5 mg / mL and added to the peripheral blood sample at a volume of 5 mL to 25 mL per 100 mL of peripheral blood.

38. The method of claim 37, wherein the monomer is added in the form of a solution of fusion protein in the form of 5 mL, 15 mL, or 25 mL per 100 mL of peripheral blood.

39. The method according to claim 32, wherein: The fusion protein, in a mixture of monomers and polymers, is added to the peripheral blood sample along with the settling agent, wherein the mass ratio of the monomer to the polymer is 1:4 to 4:

1.

40. The method according to claim 39, wherein the mass ratio of the monomer to the polymer is 1:4, 1:3.5, 1:3, 1:2.5, 1:2, 1:1.5, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1 or 4:

1.

41. The method of claim 39, wherein 0.82-3.25 mg of the monomer and 0.84-3.35 mg of the polymer are added per 1.2 mL of peripheral blood.

42. The method of claim 39, wherein 0.94 mg of the monomer and 1.09 mg of the polymer are added per 1.2 mL of peripheral blood.

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