Fusion protein binding to CD235a and CD3, preparation method therefor, and use thereof

Abstract

Provided are a fusion protein (for example, in the form of a bispecific antibody) binding to CD235a and CD3, a preparation method therefor, and a related use thereof.

Description

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

[0001] Related applications

[0002] This application claims priority to and the benefit of Chinese Patent Application No. CN 202411288089.6, filed September 13, 2024, entitled “Fusion proteins binding cd235a and cd3 and methods of making and uses thereof,” which is incorporated by reference herein in its entirety for all purposes. TECHNICAL FIELD

[0003] The present application relates generally to the field of biotechnology. In particular, the present application relates to a fusion protein binding CD235a and CD3 and methods of making and uses thereof. BACKGROUND

[0004] Natural killer (NK) cells are a type of off-the-shelf cell therapy product suitable for healthy allogeneic donor sources, including peripheral blood mononuclear cells (PBMCs), umbilical cord blood (UCB), induced pluripotent stem cells (iPSCs), and NK cell lines (such as NK92). Among them, PBMCs are derived from donor apheresis peripheral blood, and NK cells derived therefrom have the characteristics of wide source and good functional activity, thus becoming the preferred key raw material. In combination with a mature PBMC-derived NK cell culture and expansion process, an off-the-shelf NK cell therapy product can be obtained at a low cost and good use.

[0005] Donor apheresis blood is processed into PBMCs suitable for culturing and expanding NK cells, which faces the following challenges: the NK proportion in the material is not high, the T cell (CD3 + ) proportion is about 50% to 84%, and the NK cell (CD3 - , CD16 + , and / or CD56 + ) proportion is about 7% to 40%. If the proportion of T cells in the mononuclear cells obtained is high, the proportion of T cells will increase and seriously affect the purity of subsequent NK cell culture due to the higher proliferation activity of T cells than NK cells during the culture process, and T cells will cause clinical use safety risks.

[0006] Therefore, there is a need in the art for improved methods of increasing the proportion of NK cells in biological raw materials, particularly separation tools and methods for increasing the proportion of NK cells in PBMCs.

[0007] SUMMARY

[0008] In a first aspect, the present application provides a fusion protein (e.g. in the form of a bispecific antibody) binding CD235a and CD3, comprising a CD235a binding domain and a CD3 binding domain, wherein:

[0009] the CD235a binding domain comprises a first light chain variable region comprising LCDR1 of amino acid sequence RASSNVKYMY (SEQ ID No. 22), LCDR2 of amino acid sequence YTSNLAS (SEQ ID No. 23), and LCDR3 of amino acid sequence QQFTSSPYT (SEQ ID No. 24), and a first heavy chain variable region comprising HCDR1 of amino acid sequence SYFMH (SEQ ID No. 25), HCDR2 of amino acid sequence MIRPNGGTTDYNEKFKN (SEQ ID No. 26), and HCDR3 of amino acid sequence WEGSYYALDY (SEQ ID No. 27), and

[0010] the CD3 binding domain comprises a second light chain variable region comprising LCDR1 of amino acid sequence RASSSVSYMN (SEQ ID No. 28), LCDR2 of amino acid sequence DTSKVAS (SEQ ID No. 29), and LCDR3 of amino acid sequence QQWSSNPLT (SEQ ID No. 30), and a second heavy chain variable region comprising HCDR1 of amino acid sequence RYTMH (SEQ ID No. 31), HCDR2 of amino acid sequence YINPSRGYTNYNQKFKD (SEQ ID No. 32), and HCDR3 of amino acid sequence YYDDHYCLDY (SEQ ID No. 33),

[0011] wherein the amino acid sequences of HCDRs and LCDRs are according to the definition of Kabat.

[0012] In some embodiments of the first aspect, the fusion protein is capable of specifically binding to CD235a and CD3, e.g. is a bispecific antibody specifically binding to CD235a and CD3.

[0013] In some embodiments of the first aspect, the CD235a binding domain comprises a first light chain variable region of amino acid sequence as set forth in SEQ ID No. 6 and a first heavy chain variable region of amino acid sequence as set forth in SEQ ID No. 7.

[0014] In some embodiments of the first aspect, the CD3 binding domain comprises a second light chain variable region having an amino acid sequence as set forth in SEQ ID No. 18 and a second heavy chain variable region having an amino acid sequence as set forth in SEQ ID No. 17.

[0015] In some embodiments, the fusion protein exists in a monomeric form or a multimeric form.

[0016] 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 of human IgG4 subtype. In some more specific embodiments, the amino acid sequence of the heavy chain constant region is set forth in SEQ ID No. 10 or SEQ ID No. 21. In some embodiments, the amino acid sequence of the light chain constant region is set forth in SEQ ID No. 11.

[0017] 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 set forth in SEQ ID No. 13 and / or the amino acid sequence of the heavy chain of the CD235a binding domain is set forth in SEQ ID No. 14.

[0018] In some embodiments of the first aspect, wherein the CD3 binding domain is in a single chain antibody (scFv) format, for example, in a structure of second heavy chain variable region-first linker-second light chain variable region formed by a first linker. For example, the first linker is a GS type flexible linker, preferably, the amino acid sequence of the first linker is set forth in SEQ ID No. 16. In some specific embodiments, the amino acid sequence of the CD3 binding domain is set forth in SEQ ID No. 19.

[0019] 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 through a second linker. For example, the second linker is a GS type flexible linker, preferably, the amino acid sequence of the second linker is set forth in SEQ ID No. 15. In some specific embodiments, the amino acid sequence of the fusion heavy chain is set forth in SEQ ID No. 20.

[0020] In some embodiments of the first aspect, the fusion protein is a tetravalent IgG4 molecule comprising variable regions from mouse and constant regions from human.

[0021] In a second aspect, the present application provides a polynucleotide encoding the fusion protein described in the first aspect above.

[0022] In a third aspect, the present application provides an expression vector comprising the polynucleotide described in the second aspect above. In some embodiments, the expression vector is a recombinant plasmid.

[0023] In a fourth aspect, the present application provides a host cell comprising the polynucleotide described in the second aspect above 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.

[0024] In a fifth aspect, the present application provides a method of preparing the fusion protein described in the first aspect above, the method comprising:

[0025] culturing the host cell described in the fourth aspect above, and

[0026] harvesting a cell culture broth comprising the cultured host cell, and extracting and purifying the fusion protein from the cell culture broth.

[0027] In some embodiments of the fifth aspect, the method further comprises separation of monomer and multimer of the fusion protein after extraction and purification (e.g., separation by size exclusion chromatography (SEC)). In some specific embodiments, the monomer of the fusion protein has a SEC purity of about 99.3% at 280 nm with a retention time of about 7.29 min, as shown in size exclusion chromatography. In some specific embodiments, the multimer of the fusion protein has a peak with no retention time of about 7.29 min, wherein the content at a retention time of about 5.78 min is about 68.2% and the content at a retention time of about 6.30 min is about 31.8% at 280 nm, as shown in size exclusion chromatography.

[0028] In a sixth aspect, the present 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 a peripheral blood mononuclear cell (PBMC) sample. In some embodiments, the composition comprises about 5 mg / ml of the fusion protein. The composition can further comprise at least one additional component, such as a buffer and / or an osmotic pressure regulator. The buffer is, for example, histidine-histidine hydrochloride. The osmotic pressure 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.

[0029] In a seventh aspect, the present application provides a method of enriching / isolating natural killer (NK) cells in a peripheral blood mononuclear cell (PBMC) sample, the method comprising:

[0030] adding the fusion protein described in the first aspect above and a sedimentation agent to a peripheral blood sample to sediment T cells and red blood cells in the peripheral blood sample; and

[0031] obtaining the supernatant of the peripheral blood sample comprising NK cells.

[0032] In some embodiments of the seventh aspect, the sedimentation agent can comprise hydroxyethyl starch, gelatin, dextran, polyvinylpyrrolidone, methylcellulose, and combinations, analogs or derivatives thereof. In some embodiments, the sedimentation agent can be hydroxyethyl starch. The sedimentation agent can be added at a final concentration of 0.5-4%, for example, at a final concentration of 0.5-1%, 1-2% or 2-4%. For example, the sedimentation agent can be hydroxyethyl starch at a final concentration of about 2%.

[0033] In some embodiments of the seventh aspect, the fusion protein can be added to the peripheral blood sample in monomeric form with the sedimentation agent. In some embodiments, the monomer of the fusion protein is in a concentration of about 5 mg / mL and is added to the peripheral blood sample in a volume of 5 mL to 25 mL per 100 mL of peripheral blood, preferably in a volume of about 5 mL, about 15 mL or about 25 mL per 100 mL of peripheral blood.

[0034] In some embodiments of the seventh aspect, the fusion protein is added to the peripheral blood sample in a mixture of monomers and multimers with a sedimentation agent. In some specific embodiments, the mass ratio of monomers to multimers 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 monomers and 0.84-3.35 mg of the multimers are added per 1.2 mL of peripheral blood. In some more specific embodiments, about 0.94 mg of the monomers and about 1.09 mg of the multimers are added per 1.2 mL of peripheral blood. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 shows a structural schematic of an exemplary fusion protein PPC3S of the present application.

[0036] Figure 2 shows a structural schematic of a recombinant expression vector HSP6093-LC-HC-10.0 used for recombinantly expressing PPC3S of the present application.

[0037] Figure 3 shows a process flow diagram for purifying PPC3S of the present application from cell culture, in which HSP6093DS in the figure is PPC3S of the present application.

[0038] Figure 4 shows the SEC purity confirmation results of the monomer (PPC3S-U2) of PPC3S of the present application after SEC-HPLC separation.

[0039] Figure 5 shows the SEC purity confirmation results of the multimer (PPC3S-U3) of PPC3S of the present application after SEC-HPLC separation.

[0040] Figures 6 to 9 respectively show the flow cytometry detection results of experiments in which PPC3S monomer molecules were used and then hydroxyethyl starch was used to sediment the cell crosslinking complexes, in which the sample numbers of the experimental groups are A1 to A4 respectively, and in the control groups, the PPC3S was replaced by a solvent, and the other operation steps were the same, and the sample numbers of the control groups are B1 to B4 respectively.

[0041] Figures 10 to 11 show the results of PPC3S-U2 and PPC3S-U3 addition property analysis, in which the PPC3S-U2 and PPC3S-U3 addition amount is used as a process parameter, and T% and NK% are used as key quality attributes, the experimental results of Table 11 are fitted by a polynomial, and the fitting results are shown in Figure 10, and the theoretical process optimum point is shown in Figure 11.

[0042] Figure 12 shows the results of the confirmation experiment of the process design space of the PPC3S molecule for treating PBMCs.

[0043] Figure 13 shows sedimentation results for PPC3S molecules, both monomeric and polymeric, used in combination on peripheral blood lymphocyte samples from 3 donors. DETAILED DESCRIPTION

[0044] DEFINITIONS

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application belongs. For the purposes of the present application, the phrase "phenyl" means a monovalent aromatic group derived from a benzene ring, namely a -C6H5 group. As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and derivatives. Unless otherwise specified, amino acid residues are referred to herein by either their commonly accepted three letter designations or by their commonly accepted one letter designations. The abbreviations for the standard L-amino acids are those used by the scientific community for the 20 standard amino acids commonly found in proteins.

[0046] Notwithstanding that the numerical ranges and parameters setting forth the broadest scope of the application are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a range of "1 to 10" should be considered to include any and all subranges between (and including) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more and ending with a maximum value of 10 or less, e.g., 5 to 10. Additionally, any reference to a "consisting of" is to be construed as a "consisting of" of the listed elements, but not excluding additional elements. Any reference to a "consisting essentially of" is to be construed as a "consisting essentially of" of the listed elements, but not excluding additional elements that do not materially affect the basic and novel characteristics of the composition and / or method. Any reference to an "including" is to be construed as an "including but not limited to" of the listed elements or the listed steps of the method. Any reference to a "comprising" is to be construed as a "comprising but not limited to" of the listed elements or the listed steps of the method.

[0047] The terms "comprising" and "including," as used herein, are to be construed as being inclusive and open ended, and not exclusive. Specifically, when used in the specification and in the claims, the terms "comprising" and "including" and variations thereof mean "comprising but not limited to." These terms are not to be construed as excluding the presence of other features, steps or components.

[0048] The term "specifically binds," as used herein, is a term of art well known in the field and methods of determining such specific binding of an antibody to an antigen are well known in the art. For example, in some embodiments, "specifically binds" means that an antibody binds to an intended target, but does not significantly bind to other targets. The antibody binds to the intended target epitope with significantly increased affinity and / or for a significantly longer duration than it binds to other epitopes.

[0049] The term "fusion protein" as used herein refers to the purposeful joining of two or more genes encoding functional proteins, which are then expressed. The resulting protein product of the gene expression controlled by regulatory sequences after the joining of the coding regions of two or more genes under artificial conditions is the fusion protein.

[0050] The term "linker" or "peptide linker" as used herein in the context of the present application refers to a short peptide used to connect two functional proteins, which can be from 3 amino acids up to 76 amino acids in length. The peptide linker can provide a certain flexibility to each functional protein in the fusion protein, allowing it to perform its respective function.

[0051] The term "natural killer cell" or NK cell as used herein refers to a type of lymphocyte that can non-specifically directly kill tumor cells and virus-infected cells without MHC restriction, without prior sensitization, and without the involvement of antibodies.

[0052] The term "mononuclear cell" or MNC as used herein refers to a cell with a single nucleus in peripheral blood, umbilical cord blood, bone marrow, etc., including lymphocytes and monocytes.

[0053] Allogeneic healthy donor peripheral blood-derived PBMC refers to human peripheral blood mononuclear cells collected from the peripheral blood of an allogeneic healthy donor, which is the raw material for allogeneic donor-derived natural killer cells. In some embodiments, the HLA and KIR of the PBMC are screened in the present application. In some embodiments, the variants (176V, 176F) of CD16a of the PBMC are screened in the present application. In some embodiments, most (≥99%) of the T cells are removed while isolating the PBMC.

[0054] The term "monoclonal antibody" as used herein refers to an immunoglobulin with a specific target, whose amino acid sequence is clear, expressed by polyclonal or monoclonal host cells such as mammalian cells (e.g., CHO cells, 293 cells). In some embodiments, the host cells, such as mammalian cells, are genetically engineered to highly express a specific protein of interest under screening pressure. In some embodiments, the specific target of the immunoglobulin can be human CD235a and CD3. In some embodiments, the immunoglobulin is human-mouse chimeric, i.e., contains an immunoglobulin portion from humans and an immunoglobulin portion from mice, such as containing a constant region from humans and a variable region from mice. In some embodiments, the Fc of the immunoglobulin is human G4 subtype (IgG4).

[0055] The term "single chain antibody (scFv)" as used herein refers to a single polypeptide chain of a VH domain and a VL domain connected via a peptide linker. (scFv)2 comprises two VH domains and two VL domains connected via a peptide linker, the two VL domains are combined with the two VH domains via disulfide bridges.

[0056] The term "bispecific antibody" or "diabody" as used herein refers to an antibody that has the ability to bind to two antigen epitopes simultaneously. The two antigen epitopes can be on different antigens or on the same antigen. Bispecific antibodies can have various structural configurations. For example, a bispecific antibody can be composed of two Fc fragments and two antigen binding moieties fused to each of them respectively (similar to a natural antibody, except that the two arms bind to different antigen targets or epitopes), the antigen binding moieties can be in the form of single chain antibodies (scfv) or Fab fragments. The two different binding moieties of the bispecific antibody are each combined with the N-terminus of one Fc fragment, and the configuration of the antigen binding moieties of the two arms can have four combinations: scfv+Fab fragment, Fab fragment+scfv, scfv+scfv, Fab fragment+Fab fragment. The Fc fragment can contain mutations that can ensure heterodimerization of the heavy chains, and the KIH technology (knob-in-hole, KIH) is a strategy to solve the heterodimerization of the heavy chains. Generally, the KIH technology refers to the formation of a structure that is conducive to the pairing of hetero-hybrid antibodies by modifying the amino acid sequence of the CH3 region, which can form a bispecific antibody while maintaining the structure of a normal antibody as much as possible.

[0057] The term "CD235a" as used herein, i.e., Glycophorin A, is a major, intrinsic membrane protein of red blood cells, expressed on the surface of mature red blood cells, with an average of 1 x 10 5 ~1 x 10 6 molecules on the surface of each red blood cell. Its N-terminus is a glycosylated fragment, and it is a single nuclear blood group receptor outside the red blood cell membrane.

[0058] In a first aspect, the present application provides a fusion protein (e.g., in the form of a bispecific antibody) that binds CD235a and CD3, the fusion protein comprising a CD235a binding domain and a CD3 binding domain, wherein:

[0059] the CD235a binding domain comprises a first light chain variable region comprising a LCDR1 of the amino acid sequence RASSNVKYMY (SEQ ID No. 22), a LCDR2 of the amino acid sequence YTSNLAS (SEQ ID No. 23), and a LCDR3 of the amino acid sequence QQFTSSPYT (SEQ ID No. 24), and a first heavy chain variable region comprising a HCDR1 of the amino acid sequence SYFMH (SEQ ID No. 25), a HCDR2 of the amino acid sequence MIRPNGGTTDYNEKFKN (SEQ ID No. 26), and a HCDR3 of the amino acid sequence WEGSYYALDY (SEQ ID No. 27), and

[0060] the CD3 binding domain comprises a second light chain variable region comprising a LCDR1 of the amino acid sequence RASSSVSYMN (SEQ ID No. 28), a LCDR2 of the amino acid sequence DTSKVAS (SEQ ID No. 29), and a LCDR3 of the amino acid sequence QQWSSNPLT (SEQ ID No. 30), and a second heavy chain variable region comprising a HCDR1 of the amino acid sequence RYTMH (SEQ ID No. 31), a HCDR2 of the amino acid sequence YINPSRGYTNYNQKFKD (SEQ ID No. 32), and a HCDR3 of the amino acid sequence YYDDHYCLDY (SEQ ID No. 33),

[0061] wherein the amino acid sequences of the HCDRs and LCDRs are according to the definition of Kabat.

[0062] Figure 1 shows an exemplary construction of the fusion protein of the present application. In Figure 1, the fusion protein is in the form of a bispecific antibody, which includes two light chains and two heavy chains, and the N-terminal is a CD235a binding domain (comprising light chain variable region and heavy chain variable region binding to CD235a), and the C-terminal is a CD3a binding domain (comprising light chain variable region and heavy chain variable region binding 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 (which includes three CDR regions of LCDR1, LCDR2 and LCDR3), and the heavy chain includes a heavy chain constant region (human IgG4 subtype), a heavy chain variable region of the CD235a binding domain (which includes three CDR regions of HCDR1, HCDR2 and HCDR3), and a CD3 binding domain in the form of scFv (comprising LCDR1, LCDR2 and LCDR3, and HCDR1, HCDR2 and HCDR3 regions).

[0063] It should be understood that the construction of the bispecific antibody shown in Figure 1 is merely exemplary, but not limiting. Those skilled in the art can reasonably know other constructions of the bispecific antibody. For example, the bispecific antibody of the present application can be obtained by the following ways (only for example, but not limited thereto):

[0064] (i) on the basis of an anti-CD235a full-length antibody, an antibody fragment binding to CD3 (for example, a single-chain antibody scFv comprising heavy chain variable region and light chain variable region binding to CD3) is connected to the Fc fragment of the anti-CD235a antibody;

[0065] (ii) on the basis of an anti-CD3 full-length antibody, an antibody fragment binding to CD235a (for example, a single-chain antibody scFv comprising heavy chain variable region and light chain variable region binding to CD235a) is connected to the Fc fragment of the anti-CD3 antibody; or

[0066] (iii) one antigen binding arm of an anti-CD235a antibody (or an anti-CD3 antibody) is replaced by a set of heavy chain variable region and light chain variable region of an anti-CD3 antibody (or an anti-CD235a antibody), so that the two arms of the parent antibody can bind to CD235a and CD3, respectively.

[0067] In some embodiments of the first aspect, the CD235a binding domain comprises a first light chain variable region having an amino acid sequence as set forth in SEQ ID No. 6 and a first heavy chain variable region having an amino acid sequence as set forth in SEQ ID No. 7. In some embodiments, the CD3 binding domain comprises a second light chain variable region having an amino acid sequence as set forth in SEQ ID No. 18 and a second heavy chain variable region having an amino acid sequence as set forth in SEQ ID No. 17.

[0068] In some embodiments, the fusion protein exists in a monomeric form or a multimeric form.

[0069] 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 of human IgG4 subtype. In some more specific embodiments, the amino acid sequence of the heavy chain constant region is as set forth in SEQ ID No. 10. In some alternative embodiments, the heavy chain constant region can comprise a mutation in its hinge region, for example, a mutation of serine (S) at the 10th amino acid position in the hinge region to proline (P), the amino acid position being numbered according to the EU numbering system. For example, the amino acid sequence of a heavy chain constant region comprising a hinge region S10P mutation can be as set forth in SEQ ID No. 21. In some specific embodiments, the amino acid sequence of the light chain constant region is as set forth in SEQ ID No. 11.

[0070] In some embodiments of the first aspect, the first light chain variable region of the CD235a binding domain is associated with (e.g., linked to the N-terminus of) 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 (e.g., linked to the N-terminus of) 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 set forth in SEQ ID No. 13 and / or the amino acid sequence of the heavy chain of the CD235a binding domain is as set forth in SEQ ID No. 14;

[0071] In some embodiments of the first aspect, the CD3 binding domain is in the form of a single chain antibody (scFv), for example, in the structure of second heavy chain variable region-first linker-second light chain variable region formed by the first linker (e.g., in the structure of N-terminal-second heavy chain variable region-first linker-second light chain variable region-C-terminal). The first linker can be, for example, a GS type flexible linker, preferably the amino acid sequence of the first linker is set forth in SEQ ID No. 16. In some more specific embodiments, the amino acid sequence of the CD3 binding domain is set forth in SEQ ID No. 19.

[0072] 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-terminal 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 through a second linker. The second linker can be, for example, a GS type flexible linker, preferably the amino acid sequence of the second linker is set forth in SEQ ID No. 15. In some more specific embodiments, the amino acid sequence of the fusion heavy chain is set forth in SEQ ID No. 20.

[0073] In some embodiments of the first aspect, the fusion protein is a tetravalent IgG4 molecule comprising variable regions from mouse and constant regions from human.

[0074] The inventors have designed and prepared the fusion proteins described above, and through the study of NK cell culture processes, found that the purity of NK cells in PBMC can be improved by the fusion proteins, and by coordinating suitable culture processes, PBMC can be cultured into high-purity NK cell products. In some specific embodiments, the inventors have found that an exemplary fusion protein of the present application (i.e., PPC3S) can cross-link red blood cells (CD235a + ) and T cells (CD3 + ) together to form a rosette-like complex as a human IgG4-based antibody. Subsequently, by adding a sedimentation agent such as hydroxyethyl starch, the red blood cells and the T cells cross-linked therewith can be accelerated to sink, while the NK cells are mostly dispersed in the plasma layer. Then the PBMC from which the red blood cells and T cells have been removed can be transferred to a sterile container by various means, and packaged as a key raw material of NK cells, and stored in deep low temperature (≤-150°C) conditions.

[0075] The inventors have also found that one advantage of the present application is that the fusion protein of the present application (e.g. PPC3S) is a well-defined molecule of the gene of interest, which can be constructed into a cell bank suitable for GMP production and used for large-scale production, meeting the requirements of sterile raw materials necessary for NK cell culture. In addition, based on the mechanism of action of the molecule 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 are both of practical value and can replace a part of the role of the sedimentation agent (such as hydroxyethyl starch).

[0076] In particular, the PPC3S monoclonal antibody of the present application is an IgG4 full-length antibody expressed by mammalian cells and prepared by a purification process, which targets CD235a and CD3 of human origin, and contains variable regions from mice and constant regions from humans.

[0077] CD235a, i.e. glycophorin 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 x 10 5 ~ 1 x 10 6 CD235a molecules (Merry et al. Biochem J. (1986) 233:93-98; Loken et al. Blood (1987) 69:255-263) on the surface of each red blood cell, and in addition, CD235a is highly expressed in kidney, bladder, urethral tissue, and almost not expressed in lymphocytes (https: / / www.proteinatlas.org / ENSG00000170180-GYPA). CD235a is a component of the ankyrin 1 complex, which is related to the stability and shape of the red blood cell membrane. Its N-terminal glycosylated fragment is a single nuclear blood group receptor outside the red blood cell membrane. Its 20th-91st amino acid is the extracellular domain, 92nd-114th amino acid is the transmembrane region, and 115th-150th amino acid is the intracellular domain, as shown in SEQ ID No. 1. CD235b (glycophorin B, GYPB) in the CD235 family has very high homology, and the extracellular N segment sequence has very high consistency, as shown in SEQ ID No. 2, and its function is similar to that of CD235a. In the peripheral blood of apheresis, CD235a is expressed in red blood cells and not expressed in lymphocytes, and is a very selective target.

[0078] CD3, a component of the T cell receptor-CD3 (TCR-CD3) complex on the surface of T lymphocytes, plays an important role in the acquired immune response. When the T cell receptor is activated by antigen-presenting cells, the TCR-mediated signal is transmitted through the delta, epsilon, gamma, and zeta subunits of CD3 to the intracellular, thereby activating the downstream signaling pathway of T cells. In cell identification, CD3 is a well-known surface marker of T cells in the art, which is specifically expressed on T cells (including naive and memory CD8 + T cells, naive and memory CD4 + T cells, gamma delta T cells, T reg, etc.). In a conventional T cell positive or negative selection kit, CD3 antibody-coupled magnetic beads and matching reagents are usually selected for processing.

[0079] Based on the design principle of product design, the inventors have selected CD235a and CD3 as target points to cross-link red blood cells (RBCs) with T cells (CD3 + ) to form a large cell complex and settle down, thereby achieving the purpose of separation.

[0080] There are some studies on the drugability of antibodies of different sources of CD235a in the art:

[0081] In patent US2022 / 0153859A1, the humanized antibody of CD235a is named 10F7-M10, the sequence of its light chain variable region is shown in SEQ ID No. 3, and the sequence of its heavy chain variable region is shown in SEQ ID No. 4. Its dissociation constant KD for CD235a is 140nM.

[0082] In patent US9879090B2, the VHH antibody of CD235a is named IH5, which is a single-domain antibody. The sequence of its heavy chain variable region is shown in SEQ ID No. 5. Its dissociation constant KD for CD235a is 33.7nM.

[0083] In patent WO2017 / 015141A1, the murine antibody of CD235a is named 10F7. The sequence of its light chain variable region is shown in SEQ ID No. 6, and the sequence of its heavy chain variable region is shown in SEQ ID No. 7. In this patent, it is humanized into scFv, and through the combination of binding activity, functional research, and stability, it is confirmed that there is a combination of Glycophorin A binding activity. The sequence of the light chain variable region of scFv-10F7-EPO is shown in SEQ ID No. 8, and the sequence of the heavy chain variable region is shown in SEQ ID No. 9.

[0084] In the document (Catimel et al. J. Immunol. Methods (1993) 165(2): 183-192), there are two antibodies for CD235a: one of them is named 1C3 / 86, and it was measured that there are 4.80 x 1010 molecules of CD235a on each red blood cell, and its dissociation constant KD is 23 x 1010 M, i.e. 230 nM; the other is named 10F7MN, and it was measured that there are 4.66 x 1010 molecules of CD235a on each red blood cell, and its dissociation constant KD is 9.5 x 1010 M, i.e. 95 nM. 5 -8 5 -8

[0085] However, none of these studies discloses or teaches the fusion protein of the present application.

[0086] In a second aspect, the present application provides a polynucleotide encoding the fusion protein described in the first aspect above.

[0087] In a third aspect, the present application provides an expression vector comprising the polynucleotide described in the second aspect above. In some embodiments, the expression vector is a recombinant plasmid.

[0088] In a fourth aspect, the present application provides a host cell comprising the polynucleotide described in the second aspect above 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.

[0089] In a fifth aspect, the present application provides a method for preparing the fusion protein described in the first aspect above, the method comprising:

[0090] culturing the host cell described in the fourth aspect above, and

[0091] harvesting a cell culture fluid comprising the cultured host cell, and extracting and purifying the fusion protein from the cell culture fluid.

[0092] In some embodiments, the host cell is cultured into more than one cell population. In some specific embodiments, the more than one cell population is subjected to batch fed culture. In some more specific embodiments, the cell populations are selected for protein quality analysis based on their expression levels and cell growth, and based on the results of the protein quality analysis, the cell population is selected for primary cell bank building and expansion culture.

[0093] ​​​​In some embodiments, the extraction and purification of the fusion protein comprises depth filtration, S / D incubation virus inactivation, affinity chromatography, intermediate depth filtration, cation exchange chromatography, virus removal filtration, ultrafiltration and diafiltration, bulk preparation, and sub-pooling.

[0094] In some embodiments of the fifth aspect, the method further comprises separation of monomer and multimer of the purified fusion protein (e.g., by size exclusion chromatography (SEC)). In some specific embodiments, the monomer has a SEC purity of about 99.3% at 280 nm with a retention time of about 7.29 min. In some specific embodiments, the multimer has a peak with no retention time of about 7.29 min, wherein the content at a retention time of about 5.78 min is about 68.2% at 280 nm and the content at a retention time of about 6.30 min is about 31.8%.

[0095] In some embodiments, the obtained fusion protein has a size exclusion chromatography (SEC) purity of greater than or equal to 45%, e.g., greater than or equal to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0096] In a sixth aspect, the present application provides a composition comprising the fusion protein described in the first aspect above, e.g., the composition is used for enriching / isolating natural killer (NK) cells in a peripheral blood mononuclear cell (PBMC) sample. 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 present in the composition at a concentration of 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, 16 mg / ml, 16.5 mg / ml, 17 mg / ml, 17.5 mg / ml, 18 mg / ml, 18.5 mg / ml, 19 mg / ml, 20 mg / ml, 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 can be present in the composition at a concentration greater than 50 mg / ml. The composition can further comprise at least one additional component, e.g., a buffer and / or an osmotic pressure regulator. The buffer can comprise, e.g., a weak acid or a salt thereof, a weak base or a salt thereof, and other buffers well known in the art or combinations thereof, e.g., borate, citric acid, citrate, phosphoric acid, phosphate, Tris, etc. The osmotic pressure regulator can comprise, e.g., a salt (e.g., sodium salt, potassium salt, magnesium salt, etc.), a sugar (e.g., glucose, sucrose, etc.), an amino acid (e.g., glycine, histidine, etc.), and other osmotic pressure regulators well known in the art or combinations thereof. For example, the composition comprises the fusion protein and a histidine-histidine hydrochloride buffer solution. In some embodiments, the composition further comprises sucrose to improve the stability of the fusion protein. In some specific embodiments, the composition comprises about 5 mg / ml of the fusion protein, about 10 mM histidine-histidine hydrochloride, about 9% sucrose. In some embodiments, the composition has a pH of about 5.5.

[0097] In a seventh aspect, the present application provides a method for enriching / isolating natural killer (NK) cells in a peripheral blood mononuclear cell (PBMC) sample, the method comprising:

[0098] adding the fusion protein described in the first aspect above to a peripheral blood sample with a sedimentation agent to sediment T cells and red blood cells in the peripheral blood sample; and

[0099] obtaining the upper layer of the peripheral blood sample comprising NK cells.

[0100] In some embodiments of the seventh aspect, the sedimentation agent can include hydroxyethyl starch, gelatin, dextran, polyvinylpyrrolidone, methylcellulose, and combinations, analogs, or derivatives thereof. In some particular embodiments, the sedimentation agent can be hydroxyethyl starch.

[0101] The sedimentation agent can be added at a final concentration of 0.5-4%, for example, at a final concentration of 0.5-1%, 1-2%, or 2-4%. For example, the sedimentation agent can be hydroxyethyl starch at a final concentration of about 2%.

[0102] In some embodiments of the seventh aspect, the fusion protein is added to the peripheral blood sample in monomeric form with the sedimentation agent, wherein the monomer is in a concentration of about 5 mg / mL and is added to the peripheral blood sample in a volume of 5 mL to 25 mL per 100 mL of peripheral blood, for example, about 5 mL, about 5.5 mL, about 6 mL, about 6.5 mL, about 7 mL, about 7.5 mL, about 8 mL, about 8.5 mL, about 9 mL, about 9.5 mL, about 10 mL, about 10.5 mL, about 11 mL, about 11.5 mL, about 12 mL, about 12.5 mL, about 13 mL, about 13.5 mL, about 14 mL, about 14.5 mL, about 15 mL, about 15.5 mL, about 16 mL, about 16.5 mL, about 17 mL, about 17.5 mL, about 18 mL, about 18.5 mL, about 19 mL, about 19.5 mL, about 20 mL, about 20.5 mL, about 21 mL, about 21.5 mL, about 22 mL, about 22.5 mL, about 23 mL, about 23.5 mL, about 24 mL, about 24.5 mL, or about 25 mL per 100 mL of peripheral blood, preferably about 5 mL, about 15 mL, or about 25 mL per 100 mL of peripheral blood.

[0103] In some embodiments of the seventh aspect, the fusion protein is added to peripheral blood in a mixture of monomer and multimer, wherein the mass ratio of the monomer to the multimer 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 multimer 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 multimer are added per 1.2 mL of peripheral blood.

[0104] In some embodiments of the seventh aspect, a multimer of the fusion protein is added to a peripheral blood sample, wherein the multimer can be in a form of about 5 mg / mL and is added to the peripheral blood sample in a volume of 5 mL to 25 mL per 100 mL of peripheral blood, for example about 5 mL, about 5.5 mL, about 6 mL, about 6.5 mL, about 7 mL, about 7.5 mL, about 8 mL, about 8.5 mL, about 9 mL, about 9.5 mL, about 10 mL, about 10.5 mL, about 11 mL, about 11.5 mL, about 12 mL, about 12.5 mL, about 13 mL, about 13.5 mL, about 14 mL, about 14.5 mL, about 15 mL, about 15.5 mL, about 16 mL, about 16.5 mL, about 17 mL, about 17.5 mL, about 18 mL, about 18.5 mL, about 19 mL, about 19.5 mL, about 20 mL, about 20.5 mL, about 21 mL, about 21.5 mL, about 22 mL, about 22.5 mL, about 23 mL, about 23.5 mL, about 24 mL, about 24.5 mL, or about 25 mL per 100 mL of peripheral blood, preferably about 5 mL, about 15 mL, or about 25 mL per 100 mL of peripheral blood.

[0105] In some embodiments of the present application, the present application achieves one or more beneficial technical effects including but not limited to the following:

[0106] 1. Since no magnetic beads are used, the method of the present application for enriching / isolating natural killer (NK) cells eliminates the step of removing magnetic beads and the control of magnetic bead residues;

[0107] 2. The NK%, double negative% effect obtained by treating a peripheral blood sample using the fusion protein of the present application is better than that of commercially available products, the T% is comparable, and the robustness is good in multiple donors; and / or

[0108] 3. The use of a mixture of monomers and multimers of the fusion protein of the present application can promote cell sedimentation, improve processing efficiency, and reduce the purity requirement of the fusion protein (e.g., PPC3S).

[0109] It is to be understood that the foregoing detailed description has been presented for the purposes of clarity and understanding, and is not intended to be limiting in any fashion. Various modifications and changes can be made to the described embodiments by persons of ordinary skill in the art.

[0110] Examples

[0111] The application will be further described with reference to the following examples. It should be understood that these examples are intended to be illustrative only and are not intended to limit the scope of the application.

[0112] Generally, the design of an exemplary fusion protein PPC3S of the present application is described in Example 1, and the construction of the expression vector and expression cell bank for PPC3S is described in Example 2.

[0113] The cell recovery, expansion and expression, and the preparation process of the fusion protein purification are described in Example 3, in which the PPC3S molecule is stored in a histidine-hydrochloric acid histidine buffer solution, with sucrose added to improve stability. After purification, the quality of the molecule is tested, including but not limited to: pH, binding activity, purity (SEC-HPLC), purity (non-reduced CE), bacterial endotoxin, microbial limit, sterility, etc.

[0114] The effect of PPC3S on the processing of PBMCs reaches the goal of molecular design, and the content of NK cells and T cells in the processed PBMCs meets the control requirements of PBMCs (<2.0%). The effect of combined use with hydroxyethyl starch and verification in different donor-derived peripheral blood are described in Example 4.

[0115] The effect of combined use of monomers and multimers of PPC3S, and the comparison of the effect of combined use with commercially available reagents and hydroxyethyl starch are described in Example 5.

[0116] Example 1 - Design of fusion protein

[0117] This example provides an exemplary fusion protein (hereinafter referred to as "PPC3S"), which is an artificially designed molecule that can cross-link red blood cells and T cells (CD3 + ) to form a rosette-like complex, and then remove most of the red blood cells and T cells through a cell sedimentation method, including the addition of hydroxyethyl starch, PPC3S multimers, to make the processed PBMCs more suitable for culture into NK cells.

[0118] PPC3S is a multivalent structure that targets CD235a and CD3, and is based on the framework of IgG, in which:

[0119] (1) The heavy chain constant region (CH1-hinge-CH2-CH3) part is designed as human IgG4 subtype, i.e. IGHG4 (UniProt accession number P01861-1, length 327 aa), as shown in SEQ ID No. 10. The affinity of human IgG4 to CD16a is much lower than that of human IgG1 to CD16a (≤1 / 10), so it can reduce the cross-linking of NK cells with T cells, red blood cells, thereby reducing the killing and loss of NK cells, and improving the yield of NK cells. At the same time, IgG4 has high affinity to protein A, and high-purity PPC3S can be obtained by affinity chromatography to meet the requirements of commercial production;

[0120] (2) To match the heavy chain constant region (CH1-hinge-CH2-CH3) of human IgG4, the light chain constant region of human IgG4, i.e. IGKC (UniProt accession number P01834, length 107 aa), is selected. IGHG4 and IGKC can be matched, as exemplified by antibodies such as Nivolumab, Lambrolizumab, Gemtuzumab, etc. In these antibodies, IGHG4 is matched with IGKC.

[0121] (3) For the variable region sequence of anti-CD235a, according to the sequence of 10F7MN single chain antibody (scFv) of full mouse origin in NCBI database (single chain antibody 10F7MN, partial [synthetic construct]-Protein-NCBI (nih.gov)), the 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 anti-CD235a human IgG4 monoclonal antibody 10F7MN;

[0122] (4) For the domain of anti-CD3, the strategy of BiTE (i.e. tandem scFv) is referred, i.e. at the C-terminal of 10F7MN of IgG4 structure, the scFv of anti-CD3 is fused, and the sequence of the scFv of anti-CD3 is divided into 4 parts, from N-terminal to C-terminal, in turn: linker 1, anti-CD3-VH, linker 2, anti-CD3-VL. Among them, linker 1 and linker 2 are repeatable sequences composed of glycine and serine, which can reduce the influence on the affinity of the target as much as possible while ensuring the conformation of the scFv. The sequences of linker 1 and linker 2 are shown in SEQ ID No. 15 and SEQ ID No. 16. The variable region sequences of anti-CD3 antibody, i.e. anti-CD3-VH (shown in SEQ ID No. 17) and anti-CD3-VL (shown in SEQ ID No. 18) are integrated into the scFv sequence, shown in SEQ ID No. 19.

[0123] (5) Finally, the scFv of anti-CD3 is integrated into the C-terminal of the heavy chain of human IgG4 monoclonal antibody 10F7MN, and the sequence of the obtained fusion heavy chain is shown in SEQ ID No. 20. The combination of SEQ ID No. 13 and SEQ ID No. 20 is the PPC3S molecule.

[0124] Therefore, the PPC3S molecule of the present embodiment is a dual-target (CD235a, CD3) tetravalent (2 Fv binding CD235a, 2 scFv binding CD3) IgG4 molecule containing constant region from human and variable region from mouse, and the structural schematic diagram is shown in Figure 1.

[0125] More specifically, the CDR sequence information in the CD235a binding domain and the CD3 binding domain of the PPC3S molecule of the present application is shown in the following table.

[0126] CDR sequence of the PPC3S molecule of the present application

[0127] Example 2 - Construction of expression vector and cell bank of PPC3S molecule

[0128] The DNA sequences of the light chain and the heavy chain of PPC3S were artificially synthesized (Genscript), and cloned into PHS10.0 expression vector (Haoyang Biotech). The PHS10.0 expression vector has a double CMV promoter and an open reading frame to ensure that the heavy chain and the light chain of PPC3S are highly expressed in a 1:1 ratio and assembled into an IgG structure. After the recombinant plasmid is transformed into E. coli, the recombinant plasmid with the light chain and the heavy chain of PPC3S can be obtained by ampicillin resistance gene (Amp), pressure screening and amplification. After amplification and purification, the recombinant plasmid is electroporated into mammalian expression cells, and the cells stably expressing PPC3S can be obtained by blasticidin resistance gene (Bla) and pressure screening. The recombinant expression vector is named HSP6093-LC-HC-10.0, and its structure is schematically depicted in FIG. 2.

[0129] The recombinant expression vector was transfected into host cells CHO-K1, and 48 hours after transfection, CD CHO medium containing blasticidin and zeocin was added and plated into 96-well plates, and the culture medium was changed twice a week until the cell confluence recovered to more than 50%, as a minipool. The minipool was transferred to a new 96-well plate, and after changing the medium, the expression was about 24 hours, and the HTRF detection was performed using the human IgG HTR kit to determine the expression amount and sort, and the minipool with the highest expression amount was selected for mixed inoculation and placement to obtain 10 cell populations. Batch fed culture was performed on the cell populations, and the expression sample quality was detected to confirm the preferred cell population. The batch fed culture scheme is shown in Table 1.

[0130] Table 1. PPC3S batch fed culture scheme

[0131] The expression amount results of each cell population are shown in Table 2.

[0132] Table 2. PPC3S batch fed culture expression amount of each cell population

[0133] Considering the expression level and cell growth of the cell population, the D14 supernatant of HSP6093-B7Z4-M001, HSP6093-B7Z4-M003 and HSP6093-B7Z4-M007 was collected, and after one-step purification by protein A, the protein quality analysis was performed, and the analysis results are shown in Table 3.

[0134] Table 3. Quality analysis results of PPC3S batch fed culture expression protein of preferred cell population

[0135] Based on the above quality analysis data, HSP6093-B7Z4-M003 and HSP6093-B7Z4-M007 were selected for the original cell bank library, and the cells were amplified and subcultured in CD CHO medium (containing 4 mM glutamine, 400 μg / ml zeocin and 7 μg / ml blasticidin), and the culture conditions were maintained at 36.5°C, 6.0% CO2, and 110 rpm. After culturing to the target cell number, the cell viability was detected, the cells were collected by centrifugation, resuspended with a cryopreservation solution containing 10% DMSO, and then programmed cooling and cryopreservation. The cells were sampled and detected after recovery, and the cell density and viability were detected. The detection results are shown in Table 4.

[0136] Table 4. PCB viable cell density and viability of PPC3S

[0137] Example 3 - Preparation of PPC3S molecules

[0138] The preparation process of PPC3S is divided into cell culture process and protein purification process to obtain high-purity, low-impurity, and exogenous factor controlled samples for the preparation of PBMC under sterile conditions. The cell culture process is shown in Table 5. After process confirmation, the cells of HSP6093-B7Z4-M003 are preferred for the preparation of the original solution of PPC3S. Under the process conditions, the detection data of cell viability (VIA), viable cell density (VCD), lactic acid (Lac), etc. during the cell culture stage are shown in Table 6, and the expression amount and quality data are shown in Table 7.

[0139] Table 5. Cell culture process of PPC3S

[0140] Table 6. Cell culture monitoring data of PPC3S

[0141] Table 7. Expression amount and quality data of PPC3S

[0142] Because the sample obtained by the cell culture process is obtained by one-step affinity purification, the sample contains multimers, and the culture medium contains CHO-related HCP, HCD, exogenous viruses, etc. which need to be removed by purification process. The protein purification process is shown in Figure 3. After harvesting the cell culture medium, the original solution of PPC3S is obtained by steps such as deep filtration, S / D virus inactivation, affinity chromatography, intermediate product deep filtration, cation exchange chromatography, virus removal filtration, ultrafiltration and diafiltration, original solution preparation and sub-packaging. The components of the original solution include: 5 mg / ml PPC3S protein, 10 mM histidine-histidine hydrochloride, 9% sucrose, pH 5.5.

[0143] The two-step process of S / D incubation virus inactivation and virus removal filtration was selected. The virus removal verification was carried out under the experimental conditions of scale-down and addition of virus, and the detection results are shown in Table 8. The results show that the protein purification process can effectively remove the exogenous virus, and the removal efficiency LRV is not less than 9.4 logs, which meets the requirements of exogenous factor control.

[0144] Table 8. Virus removal verification results of PPC3S

[0145] Therefore, the detection items and acceptable standards of the PPC3S stock solution include but are not limited to: (1) pH, which should be 5.5±0.2; (2) SEC-HPLC purity, the main peak should be not less than 90.0%, and the HMW should be not higher than 10.0%; (3) NR-CE purity, the main peak should be not less than 80.0%, and the LMW report results; (4) bacterial endotoxin, which should be not higher than 1.0 EU / mg; (5) sterility, which should be sterile growth. In the non-GMP stage, microbial limit instead of sterility test is used, the total number of aerobic bacteria should be not higher than 3 cfu / 30 ml, and the total number of mold and yeast should be not higher than 3 cfu / 30 ml. The PPC3S stock solution of the present application meets the standards for these detection items.

[0146] Example 4 - Isolation effect of PPC3S molecular monomer

[0147] PPC3S can cross-link red blood cells (RBCs) and T cells (CD3 + ) together to form a rosette-like complex. Hydroxyethyl starch can accelerate the sedimentation of red blood cells and T cells cross-linked therewith, and NK cells are mostly dispersed in the plasma layer. By using PPC3S in combination with hydroxyethyl starch, PPC3S is used to cross-link red blood cells and T cells together, and then hydroxyethyl starch is used for sedimentation to remove most of the red blood cells and T cells. The experimental steps are as follows:

[0148] A typical batch of PPC3S (SEC purity 98.8%, NR-CE purity 90.9%, which can be considered to be mainly monomer) was used, and an equal amount of peripheral blood was added to a centrifuge tube, and different concentrations of PPC3S (5 mL / 100 mL of peripheral blood, 15 mL / 100 mL of peripheral blood, 25 mL / 100 mL of peripheral blood) were added. Mix well, incubate for 40 min. Add 6% hydroxyethyl starch solution, the final concentration of hydroxyethyl starch solution after addition is 2%, and after 0.5 h of standing and sedimentation, the upper plasma layer is aspirated, resuspended to 45 mL, centrifuged (300g, 10 min), the supernatant is discarded, and the cell pellet is resuspended. The NK purity (CD56 - CD56 + ) and T cell ratio (CD3 + ) are detected by flow cytometry. The specific experimental data are shown in Table 9.

[0149] Table 9. T cell isolation effect of PPC3S monomer and hydroxyethyl starch by classical method

[0150] The results show that by using PPC3S monomer and hydroxyethyl starch, the proportion of T cells in the mononuclear cells obtained in the three groups is ≤0.5%, and the purity of NK cells is >70%.

[0151] According to the results of the above experiments, the amount of PPC3S monomer added is 5 mL / 100 mL of peripheral blood, and the peripheral blood of 4 different donors (numbered 1, 2, 3, and 4) is processed to verify the effect of the combination of PPC3S monomer and hydroxyethyl starch. Among them, the experimental group uses PPC3S monomer and then uses hydroxyethyl starch to precipitate the cell cross-linking complex, and the sample serial numbers are A1-A4; at the same time, the control group uses solvent instead of PPC3S, and the other operation steps are the same, and the sample serial numbers are B1-B4. The separation effect is shown in Table 10 (according to the percentage of flow cytometry results, retaining one decimal place), and the flow cytometry results are shown in Figures 6 (A1 / B1), 7 (A2 / B2), 8 (A3 / B3), and 9 (A4 / B4).

[0152] Table 10. Separation effect of peripheral blood cells from different donors treated with PPC3S monomer combined with hydroxyethyl starch

[0153] The above verification results prove that the use of PPC3S monomer combined with hydroxyethyl starch can remove most of the T cells in peripheral blood, greatly increase the percentage of NK cells in mononuclear cells, and compared with flow cytometry and magnetic bead screening, this method has low cost, short time consumption, and simple operation.

[0154] Example 5 - Separation effect of PPC3S monomer and polymer mixed use

[0155] PPC3S obtained by one-step affinity purification was separated by SEC-HPLC method to obtain monomer and polymer (HMW), and the sample SEC detection results are shown in Figures 4 and 5. Among them: the monomer is named PPC3S-U2, the SEC purity is 99.3% (280 nm), and the retention time is 7.29 min; the polymer is named PPC3S-U3, there is no peak with a retention time of 7.29 min, and the content of the peak with a retention time of 5.78 min is 68.2% (280 nm), and the content of the peak with a retention time of 6.30 min is 31.8% (280 nm), which is inferred to be different degrees of polymers of PPC3S.

[0156] 5.1 Screening of design space

[0157] A single donor peripheral blood cells were mixed and divided into several parts as experimental materials. The isolated PPC3S-U2 and PPC3S-U3 were added into the peripheral blood at different ratios. The T cells (CD3 + ) were isolated from the peripheral blood according to the experimental procedure of Example 4, and the purity of the NK cells (CD3 - CD56 + ) was detected. The experimental scheme and flow detection results are shown in Table 11, wherein the experimental groups are numbered as A-I, and the group E is repeated twice as the center point. The peripheral blood cells treated with the solvent are used as the control group, numbered as K0.

[0158] The experimental procedure is as follows:

[0159] The peripheral blood lymphocytes (the living cell density is about 1.2 x 10 8 cells / ml, and the number ratio of red blood cells to white blood cells is about 14:1) were divided into 10 15ml centrifuge tubes, and 1.2ml was added to each tube. Different volumes of PPC3S-U2, PPC3S-U3, and commercially available sample control 25ul were added according to the experimental design. After adding, the PPC3S samples adhered to the tube wall were washed down with 2ml PBS pH 7.4, and after incubation at room temperature for 20 minutes, each group was diluted to 10ml with PBS pH 7.4, and then 3.5ml lymph separation medium was added, and centrifuged at 1200g for 20 minutes. The white membrane layer cells of each group were collected, resuspended to 15ml with PBS pH 7.4, centrifuged at 1000g for 10 minutes to discard the supernatant, resuspended to 1ml with PBS pH 7.4, and sent for flow detection.

[0160] Table 11. Screening study scheme and separation effect of PPC3S monomer and polymer mixed use

[0161] PPC3S-U2 and PPC3S-U3 addition characteristic analysis: taking the addition amount of PPC3S-U2 and PPC3S-U3 as the process parameter, and taking T% and NK% as the key quality attributes, the experimental results of Table 11 were subjected to polynomial fitting, and the fitting results are shown in Figure 10, and the theoretical process optimum point is shown in Figure 11. The fitting coefficients of the T% model and the NK% model are 0.69 and 0.95, respectively, which can be used as the process design space, and the key data points are selected for process verification. When the addition amount of monomer PPC3S-U2 is 0.94mg / portion of peripheral blood, and the addition amount of polymer PPC3S-U3 is 1.09mg / portion of peripheral blood, the mixed use of PPC3S monomer and polymer can achieve the maximum effect, that is, the purity of NK cells (29.4%) and the content of T cells (0.18%) at this time.

[0162] 5.2 Confirmation of design space

[0163] The design space was confirmed by experiments, and the peripheral blood lymphocyte samples of 3 donors were used to confirm its durability. The parameter selection is shown in Figure 12, where the white part is the area of the quality requirement (T% <1.0%, NK% >30%) preferred by the design space. The parameter and experimental group list is shown in Table 12, the experimental result summary is shown in Table 13, the experimental operation steps are the same as described in this embodiment, and flow detection is performed (results not shown). In addition, the settling effect (fastest 4, slowest 1) of 20 minutes after treatment was sorted, as shown in Figure 13, where E is a commercially available settling reagent control group.

[0164] Table 12. Separation effect confirmation experiment research scheme of PPC3S molecule monomer and polymer mixed use

[0165] Table 13. Separation effect confirmation experiment result summary of PPC3S molecule monomer and polymer mixed use

[0166] 5.3 Summary analysis of confirmation experiment results

[0167] According to the confirmation experiment scheme design of Table 12, the separation effects of groups A, B, C and D are slightly different in peripheral blood lymphocytes from different donors, wherein the proportion of T% is significantly reduced compared with K0, i.e. the starting sample, from 57.1% to 65.0% to below 2.5%, and the optimal effect can reach 0.5%. In addition, each group can also reduce the relative proportion of double negative cells. In the K0, i.e. the starting sample, the double negative % is close to or higher than the NK%. After treatment by groups A, B, C and D, the double negative % is lower than the NK%, confirming that the experimental goal has been achieved. The proportion is usually evaluated based on the red-to-white ratio of peripheral blood lymphocytes from different donors, and the proportion of group B or C is more suitable, which can be used for small-scale confirmation before large-scale preparation.

[0168] In addition, based on the use principle of the molecule, the sedimentation and hemolysis of each group were compared. The sedimentation effect is basically consistent, C > B > D > A > E (commercially available settling reagent), that is, the higher the proportion of PPC3S-U3 multimers, the faster the sedimentation. This also means that during the preparation of PPC3S, it is beneficial to speed up the sedimentation and improve the experimental efficiency by appropriately retaining some multimers. Considering the combined use of hydroxyethyl starch, the monomer proportion should be as high as possible, and the multimer can be appropriately retained (such as preparing 10% multimer and 90% monomer PPC3S) to promote the sedimentation rate.

[0169] Therefore, the present application discloses a fusion protein (for example, PPC3S molecule) combining CD235a and CD3, and discloses a method using monomers thereof and using a mixture of multimers and monomers thereof, which can effectively remove T cells, increase the proportion of NK cells, and accelerate cell sedimentation.

[0170] Sequence description of the present application:

[0171] The use of any and all examples, or exemplary language (e.g., "such as") provided herein only intends to better illuminate the application and does not, and is not intended to, place any limit on the scope of the application unless otherwise specifically indicated. The language of the specification should not be interpreted as indicating any non-claimed element as essential to the practice of the application.

[0172] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. In addition, any theory, mechanism, proof, or finding stated herein is intended to further enhance the understanding of the application, and is not intended to limit the application to a certain theory, mechanism, proof, or finding. Although the present application has been shown and described in detail in the accompanying drawings and foregoing description, the present application should be considered as illustrative and not restrictive.

Claims

1. A fusion protein (e.g. in the form of a bispecific antibody) binding CD235a and CD3, comprising a CD235a binding domain and a CD3 binding domain, wherein: the CD235a binding domain comprises a first light chain variable region comprising a LCDR1 of the amino acid sequence RASSNVKYMY (SEQ ID No. 22), a LCDR2 of the amino acid sequence YTSNLAS (SEQ ID No. 23), and a LCDR3 of the amino acid sequence QQFTSSPYT (SEQ ID No. 24), and a first heavy chain variable region comprising a HCDR1 of the amino acid sequence SYFMH (SEQ ID No. 25), a HCDR2 of the amino acid sequence MIRPNGGTTDYNEKFKN (SEQ ID No. 26), and a HCDR3 of the amino acid sequence WEGSYYALDY (SEQ ID No. 27), and the CD3 binding domain comprises a second light chain variable region comprising a LCDR1 of the amino acid sequence RASSSVSYMN (SEQ ID No. 28), a LCDR2 of the amino acid sequence DTSKVAS (SEQ ID No. 29), and a LCDR3 of the amino acid sequence QQWSSNPLT (SEQ ID No. 30), and a second heavy chain variable region comprising a HCDR1 of the amino acid sequence RYTMH (SEQ ID No. 31), a HCDR2 of the amino acid sequence YINPSRGYTNYNQKFKD (SEQ ID No. 32), and a HCDR3 of the amino acid sequence YYDDHYCLDY (SEQ ID No. 33), wherein the amino acid sequences of the HCDRs and LCDRs are according to the definition of Kabat; preferably, the CD235a binding domain comprises a first light chain variable region of the amino acid sequence as set forth in SEQ ID No. 6 and a first heavy chain variable region of the amino acid sequence as set forth in SEQ ID No. 7, and / or the CD3 binding domain comprises a second light chain variable region of the amino acid sequence as set forth in SEQ ID No. 18 and a second heavy chain variable region of the amino acid sequence as set forth in SEQ ID No. 17; optionally, the fusion protein is present in a monomeric form or a multimeric form.

2. The fusion protein according to claim 1, wherein the fusion protein further comprises a heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is of the human IgG4 subtype, more preferably, the amino acid sequence of the heavy chain constant region is as set forth in SEQ ID No. 10 or SEQ ID No. 21; and / or the amino acid sequence of the light chain constant region is as set forth in SEQ ID No.

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

3. A polynucleotide encoding the fusion protein of any one of claims 1-2.

4. An expression vector comprising the polynucleotide of claim 3, preferably the expression vector is a recombinant plasmid.

5. A host cell comprising the polynucleotide of claim 3 or the expression vector of claim 4, preferably, the host cell is a mammalian cell, more preferably, a CHO-K1 cell.

6. A method of preparing the fusion protein of any one of claims 1-2, the method comprising: culturing the host cell of claim 5; and harvesting a cell culture broth comprising the cultured host cell, and extracting and purifying the fusion protein from the cell culture broth; Optionally, the method further comprises separation of monomer and multimer (e.g., by size exclusion chromatography (SEC)) of the fusion protein after extraction and purification, preferably, the monomer has a SEC purity of about 99.3% at 280 nm with a retention time of about 7.29 min, and the multimer has no peak at a retention time of about 7.29 min, wherein the content at a retention time of about 5.78 min at 280 nm is about 68.2%, and the content at a retention time of about 6.30 min is about 31.8%.

7. A composition comprising the fusion protein of any one of claims 1-2, e.g., the composition is for enriching / isolating natural killer (NK) cells in a peripheral blood mononuclear cell (PBMC) sample; Preferably, the composition comprises about 5 mg / ml of the fusion protein; or Preferably, the composition further comprises at least one additional component, e.g., a buffer and / or an osmotic pressure regulator; the buffer is, e.g., histidine-histidine hydrochloride, and the osmotic pressure regulator is, e.g., sucrose; For example, 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.

8. A method for enriching / isolating natural killer (NK) cells in a peripheral blood mononuclear cell (PBMC) sample, the method comprising: adding the fusion protein of any one of claims 1-2 to a peripheral blood sample with a sedimentation agent (e.g., hydroxyethyl starch, gelatin, dextran, polyvinylpyrrolidone, and / or methylcellulose) to sediment T cells and red blood cells in the peripheral blood sample; and obtaining a supernatant of the peripheral blood sample comprising NK cells.

9. The method of claim 8, wherein the sedimentation agent is hydroxyethyl starch; or optionally, the sedimentation agent is added at a final concentration of 0.5%-4%, e.g., at a final concentration of 0.5%-1%, 1%-2%, or 2%-4%; preferably, the sedimentation agent is hydroxyethyl starch at a final concentration of about 2%.

10. The method of any one of claims 8-9, wherein: the fusion protein is added to the peripheral blood sample in monomeric form with the sedimentation agent, wherein the monomer is in a concentration of about 5 mg / mL and is added to the peripheral blood sample in a volume of 5 mL to 25 mL per 100 mL of peripheral blood, more preferably in a volume of about 5 mL, about 15 mL, or about 25 mL per 100 mL of peripheral blood; or the fusion protein is added to the peripheral blood sample in multimeric form with the sedimentation agent, wherein the multimer is in a concentration of about 5 mg / mL and is added to the peripheral blood sample in a volume of 5 mL to 25 mL per 100 mL of peripheral blood, more preferably in a volume of about 5 mL, about 15 mL, or about 25 mL per 100 mL of peripheral blood. The fusion protein is added to the peripheral blood sample in a mixture of monomers and multimers in a mass ratio of 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, preferably 0.82-3.25 mg of the monomer and 0.84-3.35 mg of the multimer per 1.2 mL of peripheral blood, more preferably about 0.94 mg of the monomer and about 1.09 mg of the multimer. The fusion protein is added to the peripheral blood sample in a mixture of monomers and multimers in a mass ratio of 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, preferably 0.82-3.25 mg of the monomer and 0.84-3.35 mg of the multimer per 1.2 mL of peripheral blood, more preferably about 0.94 mg of the monomer and about 1.09 mg of the multimer.

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