Fusion protein binding CD235a and CD3 and preparation method and application thereof

Abstract

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

Description

Technical Field

[0001] The present application generally relates to the field of biotechnology, and in particular, to a fusion protein combining CD235a and CD3, a preparation method and uses thereof, and the like. Background Art

[0002] Natural killer (NK) cells are a type of off-the-shelf cell therapy product suitable for healthy allogeneic donors, including peripheral blood mononuclear cells (PBMC), umbilical cord blood (UCB), induced pluripotent stem cells (iPSCs) and NK cell lines (such as NK92). Among them, PBMC comes from the donor's single peripheral blood, and the NK cells derived from it have the characteristics of wide sources and good functional activity, so they become the preferred key raw materials. Combined with the mature PBMC-derived NK cell culture and expansion process, cheap and easy-to-use off-the-shelf NK cell therapy products can be obtained.

[0003] The following challenges are faced when processing donor blood into PBMC suitable for culturing and expanding NK cells: the proportion of NK cells in the material is not high, and its T cells (CD3 + ) account for about 50% to 84%, NK cells (CD3 - 、CD16 + and / or CD56 + ) accounts for about 7% to 40%. If the proportion of T cells in the obtained mononuclear cells is high, the proliferation activity of T cells in the culture process is higher than that of NK cells, the proportion of T cells will increase and will seriously affect the purity of subsequent NK cell culture, and T cells will cause safety risks in clinical use.

[0004] Therefore, there is a need in the art for improved methods for increasing the proportion of NK cells in biological raw materials, especially separation tools and methods for increasing the proportion of NK cells in PBMCs. Summary of the invention

[0005] In a first aspect, the present application provides a fusion protein (eg, in the form of a bispecific antibody) that binds to CD235a and CD3, wherein the fusion protein comprises 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, wherein the first light chain variable region comprises LCDR1 with an amino acid sequence of RASSNVKYMY (SEQ ID No. 22), LCDR2 with an amino acid sequence of YTSNLAS (SEQ ID No. 23), and LCDR3 with an amino acid sequence of QQFTSSPYT (SEQ ID No. 24), the first heavy chain variable region comprises HCDR1 with an amino acid sequence of SYFMH (SEQ ID No. 25), HCDR2 with an amino acid sequence of MIRPNGGTTDYNEKFKN (SEQ ID No. 26), and HCDR3 with an amino acid sequence of WEGSYYALDY (SEQ ID No. 27), and

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

[0008] The amino acid sequences of HCDR and LCDR are based on the Kabat definition.

[0009] In some embodiments of the first aspect, the fusion protein is capable of specifically binding to CD235a and CD3, for example, is 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 embodiments, the fusion protein exists in monomeric or multimeric 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 of 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 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 the light chain of the CD235a binding domain, and the first heavy chain variable region of the CD235a binding domain is associated with the heavy chain constant region to form the heavy chain of the CD235a binding domain. 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 of a second heavy chain variable region-a first linker-a second light chain variable region formed by a first linker. For example, the first linker is a GS-type flexible linker, and preferably, the amino acid sequence of the first linker is shown in SEQ ID No. 16. In some specific embodiments, the amino acid sequence of the CD3 binding domain is 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, and preferably the amino acid sequence of the second linker is 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 a mouse and a constant region from a human.

[0018] In a second aspect, the present application provides a polynucleotide encoding the fusion protein described in the first aspect above.

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

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

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

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

[0023] The cell culture fluid containing the cultured host cells is harvested, and the fusion protein is extracted and purified from the cell culture fluid.

[0024] In some embodiments of the fifth aspect, the method further comprises separating monomers and polymers of the extracted and purified fusion protein (e.g., by size exclusion chromatography (SEC) separation). In some specific embodiments, as shown by size exclusion chromatography, the SEC purity of the monomer of the fusion protein at 280 nm is about 99.3%, and the retention time is about 7.29 min. In some specific embodiments, as shown by size exclusion chromatography, 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 at 280 nm is about 68.2%, and the content at a retention time of about 6.30 min is about 31.8%.

[0025] 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 / separate natural killer (NK) cells in a peripheral blood mononuclear cell (PBMC) sample. In some specific 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 pressure regulator. The buffer is, for example, histidine-histidine hydrochloride. The osmotic pressure regulator is, for example, sucrose. In some specific 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, the present application provides a method for enriching / isolating natural killer (NK) cells in a peripheral blood mononuclear cell (PBMC) sample, the method comprising:

[0027] adding the fusion protein and sedimentation agent described in the first aspect above to a peripheral blood sample to sediment T cells and red blood cells in the peripheral blood sample; and

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

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

[0030] In some embodiments of the seventh aspect, the fusion protein can be added to the peripheral blood sample in monomer form with a sedimentation agent. In some specific embodiments, the monomer of the fusion protein is in a form with 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.

[0031] In some embodiments of the seventh aspect, the fusion protein is added to the peripheral blood sample with a mixed form of monomers and polymers and a sedimentation agent. In some specific embodiments, the mass ratio of the monomer and polymer of the fusion protein 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, 0.82-3.25mg of the monomer and 0.84-3.35mg of the polymer are added to every 1.2mL of peripheral blood. In some more specific embodiments, about 0.94mg of the monomer and about 1.09mg of the polymer are added to every 1.2mL of peripheral blood. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0033] Figure 2 The schematic diagram of the structure of the recombinant expression vector HSP6093-LC-HC-10.0 used for recombinantly expressing PPC3S of the present application is shown.

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

[0035] Figure 4 The SEC purity confirmation result of the monomer (PPC3S-U2) of the PPC3S of the present application after separation by SEC-HPLC is shown.

[0036] Figure 5 The SEC purity confirmation result of the multimer (PPC3S-U3) of the PPC3S of the present application after separation by SEC-HPLC is shown.

[0037] Figures 6 to 9 The flow cytometry results of the experiment using PPC3S monomer molecules and then hydroxyethyl starch to precipitate the cell cross-linked complex are shown respectively, wherein the sample numbers of the experimental group are A1 to A4, and the solvent is used instead of PPC3S in the control group, and the other operation steps are the same, and the sample numbers of the control group are B1 to B4.

[0038] Figure 10 to Figure 11 The results of the PPC3S-U2 and PPC3S-U3 addition characteristic analysis are shown. The addition amounts of PPC3S-U2 and PPC3S-U3 are used as process parameters, and T% and NK% are used as key quality attributes. Polynomial fitting is performed on the experimental results in Table 11. The fitting results are shown in Fig.10 , the theoretical process optimum is shown in Fig.11 .

[0039] Fig.12 Shown are the results of validation experiments for the process design space of the PPC3S molecule for treating PBMCs.

[0040] Fig.13 Shown are the results of sedimentation of peripheral blood lymphocyte samples from three donors using a combination of monomers and polymers of the PPC3S molecule. DETAILED DESCRIPTION

[0041] definition

[0042] Unless otherwise defined, all scientific and technical terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terms in the art, professionals can refer specifically to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0043] Although the numerical ranges and parameter approximations shown in the broad scope of the present application, the numerical values ​​shown in the specific examples are recorded as accurately as possible. However, any numerical value is bound to contain a certain error, which is caused by the standard deviation in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, the range of "1 to 10" recorded should be considered to include any and all sub-ranges between the minimum value 1 and the maximum value 10 (including the endpoints); that is, all sub-ranges starting with a minimum value of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum value of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0044] The terms "include" and "comprising" used herein should be interpreted as inclusive and open-ended, rather than exclusive. Specifically, when used in the specification and claims, the terms "include" and "comprising" and their variations mean including the specified features, steps or components. These terms should not be interpreted to exclude the presence of other features, steps or components.

[0045] The term "specific binding" as used herein is a term well known in the art, and methods for determining such specific binding of an antibody to an antigen are also well known in the art. For example, in some embodiments, "specific binding" refers to an antibody binding to an intended target but not significantly binding to other targets. An antibody binds to an intended target epitope with significantly increased affinity and / or for a longer duration than binding to other epitopes.

[0046] The term "fusion protein" as used herein refers to the purposeful connection of two or more genes encoding functional proteins to express the protein. The protein product obtained by artificially connecting the coding regions of two or more genes end to end and expressing the genes under the control of regulatory sequences is a fusion protein.

[0047] The term "linker" or "peptide linker" used herein refers to a short peptide used to connect two functional proteins in the context of this application, and the length can be from 3 amino acids to 76 amino acids. The peptide linker can provide certain flexibility for each functional protein in the fusion protein, so that it can play its own function.

[0048] The term "natural killer cells" used in this article, namely NK cells (natural killer cells), are a type of lymphocytes that are not MHC-restricted, do not require prior sensitization, do not require the participation of antibodies, and can directly kill tumor cells and virus-infected cells in a non-specific manner.

[0049] The term "mononuclear cell (MNC)" used herein refers to cells with a single nucleus in peripheral blood, cord blood, bone marrow, etc., including lymphocytes and monocytes.

[0050] PBMCs derived from allogeneic healthy donor peripheral blood refer to human peripheral blood mononuclear cells, which are collected from the peripheral blood of allogeneic healthy donors and are the raw materials of allogeneic donor-derived natural killer cells. In some embodiments, the present application screens the HLA and KIR of PBMCs. In some embodiments, the present application screens the variants (176V, 176F) of CD16a of PBMCs. In some embodiments, the present application removes most (≥99%) of the T cells while isolating PBMCs.

[0051] The term "monoclonal antibody" as used herein refers to an immunoglobulin with a clear amino acid sequence, expressed by a polyclonal or monoclonal host cell, such as a mammalian cell (such as a CHO cell, a 293 cell), and specifically targeting a target. In some embodiments, the host cell, such as a mammalian cell, is genetically engineered to highly express a specific target protein under screening pressure. In some embodiments, the specific targets of the immunoglobulin may be human CD235a and CD3. In some embodiments, the immunoglobulin is a human-mouse chimeric type, that is, it comprises an immunoglobulin portion from a human and an immunoglobulin portion from a mouse source, for example, a constant region from a human and a variable region from a mouse. In some embodiments, the Fc of the immunoglobulin is a human G4 subtype (IgG4).

[0052] The term "single-chain antibody (scFv)" as used herein refers to a single polypeptide chain consisting of a VH domain and a VL domain connected via a peptide linker. (scFv)2 comprises two VH domains connected by a peptide linker and two VL domains, wherein the two VL domains are combined with the two VH domains via a disulfide bridge.

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

[0054] The term "CD235a" as used herein refers to glycophorin A, which is a major, intrinsic membrane protein of erythrocytes and is expressed on the surface of mature erythrocytes. On average, there are 1×10 5 ~1×10 6 The N-terminus of this molecule is a glycosylated fragment, which is located outside the red blood cell membrane and is a mononuclear blood group receptor.

[0055] In a first aspect, the present application provides a fusion protein (eg, in the form of a bispecific antibody) that binds to CD235a and CD3, wherein the fusion protein comprises 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, wherein the first light chain variable region comprises LCDR1 with an amino acid sequence of RASSNVKYMY (SEQ ID No. 22), LCDR2 with an amino acid sequence of YTSNLAS (SEQ ID No. 23), and LCDR3 with an amino acid sequence of QQFTSSPYT (SEQ ID No. 24), the first heavy chain variable region comprises HCDR1 with an amino acid sequence of SYFMH (SEQ ID No. 25), HCDR2 with an amino acid sequence of MIRPNGGTTDYNEKFKN (SEQ ID No. 26), and HCDR3 with an amino acid sequence of WEGSYYALDY (SEQ ID No. 27), and

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

[0058] The amino acid sequences of HCDR and LCDR are based on the Kabat definition.

[0059] Figure 1 An exemplary construction scheme of the fusion protein of the present application is shown. Figure 1 In the embodiment, the fusion protein is in the form of a bispecific antibody, the bispecific antibody includes two light chains and two heavy chains, and the N-terminus is a CD235a binding domain (including a light chain variable region and a heavy chain variable region that bind to CD235a), and the C-terminus is a CD3a binding domain (including a light chain variable region and a heavy chain variable region that bind to CD3). Specifically, the light chain of this exemplary bispecific antibody includes a light chain constant region (human IgG4 subtype) and a light chain variable region of a CD235a binding domain (the light chain variable region includes three CDR regions of LCDR1, LCDR2 and LCDR3), and the heavy chain includes a heavy chain constant region (human IgG4 subtype), a heavy chain variable region of a CD235a binding domain (the heavy chain variable region includes three CDR regions of HCDR1, HCDR2 and HCDR3) and a CD3 binding domain in the form of scFv (including LCDR1, LCDR2 and LCDR3 and HCDR1, HCDR2 and HCDR3 regions).

[0060] It should be understood that Figure 1 The construction scheme of the bispecific antibody shown in is merely exemplary and non-restrictive. Those skilled in the art can reasonably know other construction schemes of bispecific antibodies. For example, the bispecific antibody of the present application can be obtained by the following methods (for example only, not limited thereto):

[0061] (i) based on an anti-CD235a full-length antibody, linking an antibody fragment that binds to CD3 (e.g., a single-chain antibody scFv that binds to CD3 and includes a heavy chain variable region and a light chain variable region) to the Fc fragment of the anti-CD235a antibody;

[0062] (ii) based on an anti-CD3 full-length antibody, linking an antibody fragment that binds to CD235a (e.g., a single-chain antibody scFv that binds to CD235a and comprises a heavy chain variable region and a light chain variable region) to the Fc fragment of the anti-CD3 antibody; or

[0063] (iii) replacing one antigen-binding arm of an anti-CD235a antibody (or anti-CD3 antibody) with a set of heavy chain variable regions and light chain variable regions of an anti-CD3 antibody (or anti-CD235a antibody), thereby enabling the two arms of the parent antibody to bind to CD235a and CD3, respectively.

[0064] 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. In some embodiments, 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.

[0065] In some embodiments, the fusion protein exists in monomeric or multimeric 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 optional embodiments, the heavy chain constant region may include a mutation in its hinge region, such as a mutation of serine (S) at the 10th amino acid position in the hinge region to proline (P), and the amino acid position is numbered according to the EU numbering system. For example, the amino acid sequence of the heavy chain constant region comprising a hinge region S10P mutation can be shown in SEQ ID No.21. In some specific embodiments, the amino acid sequence of the light chain constant region is shown in SEQ ID No.11.

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

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

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

[0071] The inventors have designed and prepared the fusion protein described above, and through the study of NK cell culture process, found that the fusion protein can improve the purity of NK cells in PBMC, and by combining with a suitable culture process, PBMC can be cultured into a high-purity NK cell product. In some specific embodiments, the inventors have found that an exemplary fusion protein of the present application (i.e., PPC3S), as an antibody based on human IgG4, can convert red blood cells (CD235a + ) and T cells (CD3 + ) are cross-linked together to form a rosette-like complex. Subsequently, sedimentation agents such as hydroxyethyl starch can be added to accelerate the sinking of red blood cells and cross-linked T cells, while NK cells are mostly diffused in the plasma layer. Then, PBMCs with red blood cells and T cells removed can be transferred to sterile containers by various means, packaged as key raw materials for NK cells, and frozen at deep low temperatures (≤-150°C).

[0072] The inventors also found that an advantage of the present application is that the fusion protein (e.g., PPC3S) of the present application is a molecule with a clear target gene, 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) have their own advantages in separation effect and separation rate, and both have practical value, and can replace the role of a part of the sedimentation agent (e.g., hydroxyethyl starch).

[0073] In particular, the PPC3S monoclonal antibody of the present application is a full-length IgG4 antibody expressed by mammalian cells and prepared through a purification process, targeting human CD235a and CD3, and comprising a variable region from mouse and a constant region from human.

[0074] CD235a, also known as glycophorin A (GYPA), is a major, intrinsic membrane protein of erythrocytes, expressed on the surface of mature erythrocytes, with an average of 1×10 5 ~1×10 6CD235a molecules (Merry et al. Biochem J. (1986) 233: 93-98; Loken et al. Blood (1987) 69: 255-263). In addition, CD235a is highly expressed in kidney, bladder, and urethral tissues, and is almost not expressed in lymphocytes (https: / / www.proteinatlas.org / ENSG00000170180-GYPA). CD235a is a component of the ankyrin 1 complex, which is related to the stability and shape of the erythrocyte membrane. Its N-terminus is a glycosylated fragment, which is outside the erythrocyte membrane and is a mononuclear blood group receptor. Its 20th to 91st amino acids are the extracellular domain, 92nd to 114 amino acids are the transmembrane region, and 115th to 150 are the intracellular domain. See SEQ ID No. 1 for details. In the CD235 family, there is a very high homology CD235b (glycophorin B, GYPB), and the extracellular N segment sequence is very consistent, see SEQ ID No. 2 for details, and its function is similar to CD235a. In apheresis peripheral blood, CD235a is expressed in red blood cells, but not in lymphocytes, and is a very selective target.

[0075] CD3 is a component of the T cell receptor-CD3 (TCR-CD3) complex on the surface of T lymphocytes and plays an important role in acquired immune response. When antigen presenting cells activate T cell receptors, TCR-mediated signals are transmitted to the cell through the δ, ε, γ, and ζ subunits of CD3, thereby activating the downstream signaling pathways of T cells. In cell identification, CD3 is a well-known surface marker of T cells in the art. + T cells, naive and memory CD4 + In conventional T cell positive or negative selection kits, CD3 antibody-coupled magnetic beads and supporting reagents are usually used for processing.

[0076] Based on the design principle of the product design, the inventors have selected CD235a and CD3 as targets to separate red blood cells (RBCs) from T cells (CD3 + ) are cross-linked together to form huge cell complexes and settle, thus achieving the purpose of separation.

[0077] There are some studies in the field on the drugability of antibodies derived from different sources of CD235a:

[0078] In patent US2022 / 0153859A1, the humanized antibody for CD235a is named 10F7-M10, the light chain variable region sequence is shown in SEQ ID No. 3, and the heavy chain variable region sequence is shown in SEQ ID No. 4. Its dissociation constant KD for CD235a is 140nM.

[0079] In patent US9879090B2, the VHH antibody of CD235a is named IH5, which is a single-domain antibody. The heavy chain variable region sequence is shown in SEQ ID No. 5. Its dissociation constant KD for CD235a is 33.7 nM.

[0080] In patent WO2017 / 015141A1, the mouse antibody of CD235a is named 10F7, and its light chain variable region sequence is detailed in SEQ ID No.6, and its heavy chain variable region sequence is detailed in SEQ ID No.7. In this patent, it is humanized into scFv, and through binding activity, functional studies, and stability, a combination of Glycophorin A binding activity is confirmed, and the sequence scFv-10F7-EPO is constructed, and its light chain variable region sequence is detailed in SEQ ID No.8, and its heavy chain variable region sequence is detailed in SEQ ID No.9.

[0081] In the literature (Catimel et al. J. Immunol. Methods (1993) 165 (2): 183-192), there are two antibodies for CD235a: one of which is called 1C3 / 86, which is measured to have 4.80×10 5 CD235a molecules, with a dissociation constant KD of 23×10 -8 M, which is 230nM; another one is called 10F7MN, which is 4.66×10 5 CD235a molecules, with a dissociation constant KD of 9.5×10 -8 M, that is 95nM.

[0082] However, none of these studies discloses or teaches the fusion protein of the present application.

[0083] In a second aspect, the present application provides a polynucleotide encoding the fusion protein described in the first aspect above.

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

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

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

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

[0088] The cell culture fluid containing the cultured host cells is harvested, and the fusion protein is extracted and purified from the cell culture fluid.

[0089] In some embodiments, the host cell is cultured as more than one cell population. In some specific embodiments, the more than one cell population is subjected to batch feeding culture. In some more specific embodiments, the cell population is selected for protein quality analysis based on the expression level and cell growth of each cell population, and based on the protein quality analysis results, the cell population is selected for original cell bank construction and expansion culture.

[0090] In some embodiments, extraction and purification of the fusion protein includes depth filtration, S / D incubation virus inactivation, affinity chromatography, intermediate depth filtration, cation exchange chromatography, virus removal filtration, ultrafiltration and diafiltration, stock solution preparation and packaging.

[0091] In some embodiments of the fifth aspect, the method further comprises separating the monomers and polymers of the extracted and purified fusion protein (e.g., by size exclusion chromatography (SEC) separation). In some specific embodiments, the monomer has a SEC purity of about 99.3% at 280 nm and 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 at 280 nm is about 68.2%, and the content at a retention time of about 6.30 min is about 31.8%.

[0092] In some embodiments, 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, the present application provides a composition comprising the fusion protein described in the first aspect above, for example, the composition is used to enrich / separate 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-50 mg / ml. In some embodiments, the fusion protein is present in the composition 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, In some embodiments, the fusion protein may be present in the composition at a concentration greater than 50 mg / ml. The composition may also include at least one additional component, such as a buffer and / or an osmotic pressure regulator. The buffer may include, for example, a weak acid or a salt thereof, a weak base or a salt thereof, and other buffers or combinations thereof well known in the art, such as borate, citric acid, citrate, phosphoric acid, phosphate, tris(hydroxymethyl)aminomethane (Tris), etc. Osmotic pressure regulators can, for example, include 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 osmotic pressure regulators or combinations thereof well known in the art. For example, the composition comprises a 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, and about 9% sucrose. In some embodiments, the composition has a pH of about 5.5.

[0094] 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:

[0095] adding the fusion protein and sedimentation agent described in the first aspect above to a peripheral blood sample to sediment T cells and red blood cells in the peripheral blood sample; and

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

[0097] In some embodiments of the seventh aspect, the sedimentation agent may include hydroxyethyl starch, gelatin, dextran, polyvinyl pyrrolidone, methyl cellulose, and combinations, analogs or derivatives thereof. In some specific embodiments, the sedimentation agent may be hydroxyethyl starch.

[0098] The sedimentation 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 sedimentation agent may 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 with a 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 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, about 22 mL, about 23 mL, about 24 mL, about 25 mL, about 26 mL, about 27 mL, about 28 mL, about 29 mL, about 30 mL, about 31 mL, about 32 mL, about 33 mL, about 34 mL, about 35 mL, about 36 mL, about 37 mL, about 38 mL, about 39 mL, about 40 mL, about 41 mL, about 42 mL, about 43 mL, about 44 mL, about 45 mL, about 46 mL L, about 13.5mL, about 14mL, about 14.5mL, about 15mL, about 15.5mL, about 16mL, about 16.5mL, about 17mL, about 17.5mL, about 18mL, about 18.5mL, about 19mL, about 19.5mL, about 20mL, about 20.5mL, about 21mL, about 21.5mL, about 22mL, about 22.5mL, about 23mL, about 23.5mL, about 24mL, about 24.5mL or about 25mL, preferably in a volume of about 5mL, about 15mL or about 25mL per 100mL of peripheral blood.

[0100] In some embodiments of the seventh aspect, the fusion protein is added to peripheral blood in the form of a mixture of monomers and polymers with a sedimentation 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 multimer of the fusion protein is added to a peripheral blood sample, wherein the multimer can be in the form of about 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, 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 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, about 22 mL, about 23 mL, about 24 mL, about 25 mL, about 26 mL, about 27 mL, about 28 mL, about 29 mL, about 30 mL, about 31 mL, about 32 mL, about 33 mL, about 34 mL, about 35 mL, about 36 mL, about 37 mL, about 38 mL, about 39 mL, about 40 mL, about 41 mL, about 42 mL, about 43 mL, about 44 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 is added, preferably in a volume of about 5 mL, about 15 mL or about 25 mL per 100 mL of peripheral blood.

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

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

[0104] 2. The NK% and double negative% obtained by treating peripheral blood samples with the fusion protein of the present application are better than those of commercially available products, and the T% is comparable to that of commercially available products, and the durability is good in multiple donors; and / or

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

[0106] It should be understood that the above detailed description is only for the purpose of enabling those skilled in the art to more clearly understand the content of the present application, and is not intended to be limiting in any aspect. Those skilled in the art can make various modifications and changes to the embodiments.

[0107] Example

[0108] The present application is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application.

[0109] In general, the design of an exemplary fusion protein PPC3S of the present 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 preparation process of cell recovery, amplification and expression, and purification of the fusion protein is described in Example 3, wherein the PPC3S molecule is stored in a histidine-histidine hydrochloride buffer solution, and sucrose is added to improve stability. The quality inspection items of the purified molecule include, but are not limited to: pH, binding activity, purity (SEC-HPLC), purity (non-reduced CE), bacterial endotoxin, microbial limit, sterility, etc.;

[0111] The treatment effect of PPC3S on PBMCs reached the goal of molecular design, and the content of NK cells and T cells in the treated PBMCs reached the control requirements of PBMCs (<2.0%). The effect of combined use with hydroxyethyl starch and the verification in peripheral blood from different donors are detailed in Example 4.

[0112] The effects of the combined use of PPC3S monomers and polymers were compared with the effects of the combined use of commercially available reagents and hydroxyethyl starch, as shown in Example 5.

[0113] Example 1 - Design of fusion protein

[0114] This example provides an exemplary fusion protein (hereinafter referred to as "PPC3S"), which is an artificially designed molecule that can bind red blood cells to T cells (CD3 + ) are cross-linked together to form a rosette-like complex, and then a cell sedimentation method is used, including the addition of hydroxyethyl starch and PPC3S polymers, to remove most of the red blood cells and T cells, making the treated PBMCs more suitable for culturing into NK cells.

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

[0116] (1) The heavy chain constant region (CH1-hinge-CH2-CH3) is designed as a human IgG4 subtype, i.e., IGHG4 (UniProt accession number is P01861-1, length is 327aa), see SEQ ID No. 10 for details. The affinity of human IgG4 to CD16a is much lower than that of human IgG1 to CD16a (≤1 / 10), so it can reduce the cross-linking of NK cells with T cells and red blood cells, thereby reducing the killing and loss of NK cells and increasing the yield of NK cells. At the same time, IgG4 has a high affinity with protein A, and high-purity PPC3S can be obtained by affinity chromatography to meet the requirements of commercial production;

[0117] (2) To match the heavy chain constant region of human IgG4 (CH1-hinge-CH2-CH3), the light chain constant region of human IgG4, i.e., IGKC (UniProt accession number P01834, length 107aa), was selected. IGHG4 and IGKC can match, as exemplified by antibodies such as Nivolumab, Lambrolizumab, and Gemtuzumab, in which IGHG4 matches IGKC.

[0118] (3) Regarding the variable region sequence of anti-CD235a, according to the 10F7MN sequence in the NCBI database (single chain antibody 10F7MN, partial [synthetic construct] -Protein-NCBI (nih.gov)), it is the sequence of a single-chain antibody (scFv) of full mouse origin, see SEQ ID No.12 for details, and its light chain and heavy chain variable region sequences are completely consistent with SEQ ID No.6 and SEQ ID No.7, respectively. Therefore, the inventors fused SEQ ID No.6 to the N-terminus of IGKC (SEQ ID No.11) to obtain SEQ ID No.13; and fused SEQ ID No.7 to the N-terminus of IGHG4 (SEQ ID No.10) to obtain SEQ ID No.14. SEQ ID No.13 and SEQ ID No.14 can be considered as the light chain and heavy chain of the human IgG4 monoclonal antibody 10F7MN against CD235a;

[0119] (4) For the anti-CD3 domain, the BiTE (i.e., tandem scFv) strategy was used as a reference, that is, the anti-CD3 scFv was fused to the C-terminus of the 10F7MN of the IgG4 structure. The anti-CD3 scFv sequence was divided into four parts, from the N-terminus to the C-terminus: linker 1, anti-CD3-VH, linker 2, and anti-CD3-VL. Among them, linker 1 and linker 2 are repeatable sequences composed of glycine and serine, which can minimize the impact on target affinity while ensuring the conformation of scFv. The sequences of linker 1 and linker 2 are shown in SEQ ID No. 15 and SEQ ID No. 16. The anti-CD3 antibody variable region sequences, i.e., anti-CD3-VH (see SEQ ID No. 17 for details) and anti-CD3-VL (see SEQ ID No. 18 for details) were integrated into the scFv sequence, see SEQ ID No. 19 for details.

[0120] (5) Finally, the anti-CD3 scFv was integrated into the C-terminus of the heavy chain of the human IgG4 monoclonal antibody 10F7MN, and the resulting fusion heavy chain sequence is shown in SEQ ID No. 20. The combination of SEQ ID No. 13 and SEQ ID No. 20 is the PPC3S molecule.

[0121] Therefore, the PPC3S molecule of this example is a dual-target (CD235a, CD3) tetravalent (2 Fvs bind CD235a, 2 scFvs bind CD3) IgG4 molecule containing a constant region from humans and a variable region from mice. The structural diagram is shown in Figure 1 .

[0122] More specifically, the CDR sequence information in the CD235a binding domain and CD3 binding domain of the PPC3S molecule of the present application is shown in the following table.

[0123] CDR sequences of the PPC3S molecule of this application

[0124]

[0125]

[0126] Example 2-Construction of expression vector and cell bank of PPC3S molecule

[0127] The DNA sequences of the light chain and heavy chain of PPC3S were artificially synthesized (GenScript) and cloned into the PHS10.0 expression vector (Haoyang Bio). The PHS10.0 expression vector has a dual CMV promoter and an open reading frame to ensure that the heavy chain and 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 Escherichia coli, the recombinant plasmid with the light chain and heavy chain of PPC3S can be obtained by pressure screening and amplification through the ampicillin resistance gene (Amp). After the amplified and purified recombinant plasmid is electroporated into mammalian expression cells, it can be pressure screened through the blasticidin resistance gene (Bla) to obtain cells that stably express PPC3S. The recombinant expression vector was named HSP6093-LC-HC-10.0, Figure 2 Its structure is schematically depicted.

[0128] The recombinant expression vector was transfected into the host cell CHO-K1. 48 hours after transfection, CD CHO medium containing blasticidin and Zeocin was added and plated into a 96-well plate. The culture medium was changed twice a week until the cell confluence recovered to more than 50%, as a minipool. The minipool was transferred to a new 96-well plate, expressed for about 24 hours after the medium was changed, and the expression level was detected and sorted by HTRF using a human IgG HTR kit. The minipool with the highest expression level was selected for mixed inoculation and separation to obtain 10 cell groups. The cell group was batch fed cultured, and the quality of the expression sample was tested to confirm the preferred cell group. The batch fed culture scheme is shown in Table 1.

[0129] Table 1. PPC3S fed-batch culture protocol

[0130]

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

[0132] Table 2. Expression levels of each cell population in PPC3S fed-batch culture

[0133]

[0134] Taking the expression level and cell growth of the cell population into consideration, the D14 supernatants of HSP6093-B7Z4-M001, HSP6093-B7Z4-M003 and HSP6093-B7Z4-M007 were collected, purified by protein A in one step, and then the protein quality was analyzed. The analysis results are shown in Table 3.

[0135] Table 3. Quality analysis results of proteins expressed by the optimal cell population for PPC3S fed-batch culture

[0136]

[0137] Based on the above quality analysis data, HSP6093-B7Z4-M003 and HSP6093-B7Z4-M007 were selected for the construction of the original cell library, and the cells were amplified and passaged in CD CHO medium (containing 4mM glutamine, 400μg / ml zeocin and 7μg / ml blasticidin), and the culture conditions were maintained at 36.5°C, 6.0% CO2, and 110rpm. After culturing to the target cell number, the cell viability was detected, the cells were collected by centrifugation, resuspended in a freezing solution containing 10% DMSO, and then programmed to cool and freeze. The cells were sampled and tested, and the cell density and viability were detected after recovery. The test results are shown in Table 4.

[0138] Table 4. PCB viable cell density and viability of PPC3S

[0139]

[0140] Example 3 - Preparation of PPC3S molecules

[0141] The preparation process of PPC3S is divided into a cell culture process and a protein purification process to obtain a high-purity, low-impurity, exogenous factor-controlled sample for the preparation of PBMC under sterile conditions. Its cell culture process is shown in Table 5. After process confirmation, HSP6093-B7Z4-M003 cells are preferably used for the preparation of PPC3S stock solution. Under this process condition, the cell viability (VIA), viable cell density (VCD), lactic acid (Lac) and other detection data in the cell culture stage are shown in Table 6, and the expression and quality data are shown in Table 7.

[0142] Table 5. Cell culture process of PPC3S

[0143]

[0144] Table 6. Cell culture monitoring data of PPC3S

[0145]

[0146] Table 7. Expression and quality data of PPC3S

[0147]

[0148] Because the sample obtained by the cell culture process is obtained by one-step affinity purification, the sample contains polymers, and the culture medium contains CHO-related HCP, HCD, exogenous viruses, etc., which need to be removed through a fine purification process. Figure 3As shown, after the cell culture fluid is harvested, it is subjected to the steps of deep filtration, S / D incubation virus inactivation, affinity chromatography, intermediate product deep filtration, cation exchange chromatography, virus removal filtration, ultrafiltration and diafiltration, stock solution preparation and subpackaging to obtain the stock solution of PPC3S. The components of the stock solution include: 5 mg / ml PPC3S protein, 10 mM histidine-histidine hydrochloride, 9% sucrose, pH 5.5.

[0149] Among them, the two-step process of S / D incubation virus inactivation and virus removal filtration was selected to perform virus removal verification under the experimental conditions of reduced scale and virus addition, and the test results are shown in Table 8. The results show that the protein purification process can effectively remove exogenous viruses, and the removal efficiency LRV is not less than 9.4logs, which meets the requirements for the control of exogenous factors.

[0150] Table 8. Virus clearance validation results of PPC3S

[0151]

[0152] Therefore, the detection items and acceptable standards for the PPC3S stock solution include but are not limited to: pH, which should be 5.5±0.2; (2) SEC-HPLC purity, the main peak should not be less than 90.0%, and the HMW should not be higher than 10.0%; (3) NR-CE purity, the main peak should not be less than 80.0%, and the LMW report result; (4) Bacterial endotoxin, which should not be higher than 1.0EU / mg; (5) Sterility, which should be sterile growth. In the non-GMP stage, microbial limits are used instead of sterility tests. The total aerobic bacteria count should not be higher than 3cfu / 30ml, and the total mold and yeast count should not be higher than 3cfu / 30ml. The PPC3S stock solution of this application meets the standards for these detection items.

[0153] Example 4-Separation effect of PPC3S molecular monomer

[0154] PPC3S can bind red blood cells (RBCs) to T cells (CD3 + ) are cross-linked together to form a rosette-like complex. Hydroxyethyl starch can accelerate the sinking of red blood cells and T cells cross-linked with them, and NK cells are mostly diffused 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 to settle, removing most of the red blood cells and T cells. The experimental steps are as follows:

[0155] Use a typical batch of PPC3S (SEC purity 98.8%, NR-CE purity 90.9%, which can be considered to be mainly monomeric), add an equal amount of peripheral blood to a centrifuge tube, and add different concentrations of PPC3S (5mL / 100mL peripheral blood, 15mL / 100mL peripheral blood, 25mL / 100mL peripheral blood). Mix well and incubate for 40 minutes. Add 6% hydroxyethyl starch solution during incubation. The final concentration of hydroxyethyl starch solution after addition is 2%. After standing and settling for 0.5h, aspirate the upper plasma layer, resuspend to 45mL, centrifuge (300g, 10min), discard the supernatant, resuspend the cell pellet, and detect NK purity (CD3 - CD56 + ) and T cell ratio (CD3 + ), the specific experimental data are shown in Table 9.

[0156] Table 9. Effect of PPC3S molecular monomer and hydroxyethyl starch on the separation of T cells by the classical method

[0157]

[0158] The results showed that by using PPC3S monomer molecules in combination with hydroxyethyl starch, the proportion of T cells in mononuclear cells obtained in the three groups was ≤0.5%, and the purity of NK cells was >70%.

[0159] According to the results of the above experiments, the amount of PPC3S monomer molecules added was 5mL / 100mL of peripheral blood, and the peripheral blood of 4 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. Among them, the experimental group used PPC3S monomer molecules and then used hydroxyethyl starch to precipitate the cell cross-linking complex, and the sample numbers were A1 to A4; at the same time, the solvent was used instead of PPC3S in the control group, and the other operation steps were the same, and the sample numbers were B1 to B4. The separation effect is summarized in Table 10 (according to the percentage of flow cytometry results, retaining 1 decimal place), and the flow cytometry detection results are detailed in Figure 6 (A1 / B1), Figure 7 (A2 / B2), Figure 8 (A3 / B3), Fig. 9 (A4 / B4).

[0160] Table 10. Separation effect of peripheral blood cells from different donors treated with PPC3S monomer molecule combined with hydroxyethyl starch

[0161]

[0162] The above verification results prove that the use of PPC3S monomer molecules combined with hydroxyethyl starch can remove most of the T cells in peripheral blood and significantly increase the percentage of NK cells in mononuclear cells. Compared with flow cytometry and magnetic bead screening, this method is low-cost, time-saving and easy to operate.

[0163] Example 5 - Separation effect of mixed use of PPC3S molecule monomer and polymer

[0164] The PPC3S obtained by one-step affinity purification was separated by SEC-HPLC to obtain monomers and polymers (HMW). The SEC test results of the samples are detailed in Figure 4 , Figure 5 Among them: the monomer was named PPC3S-U2, the SEC purity was 99.3% (280nm), and the retention time was 7.29min; the polymer was named PPC3S-U3, there was no peak with a retention time of 7.29min, the content of which was 68.2% (280nm) at a retention time of 5.78min, and the content of which was 31.8% (280nm) at a retention time of 6.30min. It was inferred that they were polymers of different degrees of PPC3S.

[0165] 5.1 Design space screening

[0166] A portion of peripheral blood cells from a single donor was mixed and then divided into multiple portions as experimental materials. The PPC3S-U2 and PPC3S-U3 separated above were added to the peripheral blood in different proportions. The T cells (CD3 + ) and detect NK cells (CD3 - CD56 + The experimental scheme and flow cytometry results are shown in Table 11, where the experimental groups are numbered A to I, with group E as the center point and repeated twice, and peripheral blood cells treated with solvent as the control group, numbered K0.

[0167] The experimental steps are as follows:

[0168] Peripheral blood lymphocytes (viable cell density about 1.2×10 8Cells / ml, red blood cells to white blood cells ratio of about 14:1) were placed in 10 15ml centrifuge tubes, and 1.2ml was added to each tube. Different volumes of PPC3S-U2, PPC3S-U3, and 25μl of commercially available sample control were added according to the experimental design. After addition, the PPC3S sample adhering to the tube wall was washed off with 2ml PBS pH 7.4. After incubation at room temperature for 20 minutes, each group was diluted to 10ml with PBS pH 7.4, and then 3.5ml of lymphocyte separation fluid was added and centrifuged at 1200g for 20 minutes. The buffy coat cells of each group were collected, resuspended to 15ml with PBS pH 7.4, centrifuged at 1000g for 10 minutes, and the supernatant was discarded, and then resuspended to 1ml with PBS pH 7.4 and sent for flow cytometry.

[0169] Table 11. Preliminary screening study plan and separation results of the mixed use of PPC3S monomers and polymers

[0170]

[0171]

[0172] Analysis of PPC3S-U2 and PPC3S-U3 addition characteristics: Taking the addition amount of PPC3S-U2 and PPC3S-U3 as process parameters, T% and NK% as key quality attributes, a polynomial fitting was performed on the experimental results in Table 11. The fitting results are detailed in Fig.10 , the theoretical process optimum is shown in Fig.11 The fitting coefficients of the T% model and the NK% model were 0.69 and 0.95, respectively, which can be used as process design space, and key data points are selected for process verification. Among them, when the addition amount of monomer PPC3S-U2 is 0.94 mg / part of peripheral blood and the addition amount of polymer PPC3S-U3 is 1.09 mg / part of peripheral blood, the mixed use of PPC3S molecular monomer and polymer can achieve the maximum effect, that is, at this time, the NK cell purity (29.4%) and T cell content (0.18%).

[0173] 5.2 Confirmation of design space

[0174] The above design space was confirmed by experiments, and the durability was confirmed using peripheral blood lymphocyte samples from three donors. The parameters were selected based on the following criteria: Fig.12 , where the white part is the area of ​​the preferred quality requirements (T% <1.0%, NK%>30%) of the design space, the parameters and experimental group list are shown in Table 12, the experimental results are summarized in Table 13, the experimental operation steps are the same as those described in this embodiment, and flow detection is performed (results are not shown). In addition, the sedimentation effect 20 minutes after treatment (fastest 4, slowest 1) is ranked, see Fig.13 , where E is the commercially available sedimentation reagent control group.

[0175] Table 12. Experimental study plan to confirm the separation effect of mixed use of PPC3S molecule monomer and polymer

[0176]

[0177] Table 13. Summary of experimental results for confirming the separation effect of mixed monomers and aggregates of PPC3S molecules

[0178]

[0179] 5.3 Summary and analysis of confirmation experiment results

[0180] According to the design of the confirmation experiment scheme in Table 12, the separation effects of groups A, B, C, and D were slightly different in peripheral blood lymphocytes from different donors. Compared with K0, the starting sample, the proportion of T% was significantly reduced from 57.1% to 65.0% to below 2.5%, and the optimal effect could reach 0.5%. In addition, each group can also reduce the relative proportion of double-negative cells. In K0, the starting sample, the double-negative% was close to or higher than NK%. After treatment with groups A, B, C, and D, the double-negative% was lower than NK%, confirming that the experimental goal has been achieved. The selection of the ratio is usually evaluated based on the red-to-white ratio of peripheral blood lymphocytes from different donors. Among them, the ratio of group B or group C has better applicability, and a small test can be carried out to confirm the scope of use before large-scale preparation.

[0181] In addition, based on the principle of molecular use, the sedimentation and hemolysis of each group were compared. The sedimentation effect was basically the same, C>B>D>A>E (commercially available sedimentation reagent), that is, when the proportion of PPC3S-U3 polymer added was higher, the sedimentation was faster. This also means that in the process of preparing PPC3S, appropriately retaining some polymers is conducive to accelerating sedimentation and improving experimental efficiency. Comprehensive separation effect and sedimentation speed record, although group A sedimented slowly, the separation effect can ensure T% <2.0%. Considering the combined use of hydroxyethyl starch, the monomer ratio should be as high as possible, and the polymer can be appropriately retained (such as the preparation process can obtain PPC3S with 10% polymer and 90% monomer) to promote the sedimentation rate.

[0182] Therefore, the present application discloses a fusion protein (such as PPC3S molecule) combining CD235a and CD3, and discloses a method of using its monomer and a mixture of its polymer and monomer, which can effectively remove T cells, increase the proportion of NK cells, and accelerate cell sedimentation.

[0183] Sequence description of this application:

[0184]

[0185]

[0186]

[0187]

[0188]

[0189] The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present application and does not limit the scope of the present application unless otherwise required. Language in the specification should not be construed as indicating that any non-claimed element is essential to the practice of the present application.

[0190] All publications and patent applications cited in this specification are incorporated herein by reference, as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In addition, any theory, mechanism, proof or discovery described herein is intended to further enhance the understanding of the application, and is not intended to limit the application to such theory, mechanism, proof or discovery in any way. Although the application has been shown and described in detail in the accompanying drawings and the preceding description, the application should be considered illustrative rather than restrictive.

Claims

1. A fusion protein (e.g., in the form of a bispecific antibody) that binds to CD235a and CD3, the 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, wherein the first light chain variable region comprises LCDR1 with an amino acid sequence of RASSNVKYMY (SEQ ID No. 22), LCDR2 with an amino acid sequence of YTSNLAS (SEQ ID No. 23), and LCDR3 with an amino acid sequence of QQFTSSPYT (SEQ ID No. 24), the first heavy chain variable region comprises HCDR1 with an amino acid sequence of SYFMH (SEQ ID No. 25), HCDR2 with an amino acid sequence of MIRPNGGTTDYNEKFKN (SEQ ID No. 26), and HCDR3 with an amino acid sequence of WEGSYYALDY (SEQ ID No. 27), and The CD3 binding domain comprises a second light chain variable region and a second heavy chain variable region, the second light chain variable region comprises a LCDR1 with an amino acid sequence of RASSSVSYMN (SEQ ID No.28), a LCDR2 with an amino acid sequence of DTSKVAS (SEQ ID No.29), and a LCDR3 with an amino acid sequence of QQWSSNPLT (SEQ ID No.30), the second heavy chain variable region comprises a HCDR1 with an amino acid sequence of RYTMH (SEQ ID No.31), a HCDR2 with an amino acid sequence of YINPSRGYTNYNQKFKD (SEQ ID No.32), and a HCDR3 with an amino acid sequence of YYDDHYCLDY (SEQ ID No.33), Among them, the amino acid sequences of HCDR and LCDR are based on the Kabat definition; Preferably, the CD235a binding domain comprises a first light chain variable region with an amino acid sequence as shown in SEQ ID No.6 and a first heavy chain variable region with an amino acid sequence as shown in SEQ ID No.7, and / or the CD3 binding domain comprises a second light chain variable region with an amino acid sequence as shown in SEQ ID No.18 and a second heavy chain variable region with an amino acid sequence as shown in SEQ ID No.17; Optionally, the fusion protein exists in monomeric or 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 human IgG4 subtype, more preferably, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID No.10 or SEQ ID No.21; and / or the amino acid sequence of the light chain constant region is as shown in SEQ ID No.11; Optionally, the first light chain variable region of the CD235a binding domain is associated with the light chain constant region to form the light chain of the CD235a binding domain, and the first heavy chain variable region of the CD235a binding domain is associated with the heavy chain constant region to form the heavy chain of the CD235a binding domain; preferably, the amino acid sequence of the light chain of the CD235a binding domain is as shown in SEQ ID No.13 and / or the amino acid sequence of the heavy chain of the CD235a binding domain is as shown in SEQ ID No.14; Optionally, the CD3 binding domain is in the form of a single-chain antibody (scFv), for example, a structure of a second heavy chain variable region-a first linker-a second light chain variable region formed by a first linker; for example, the first linker is a GS-type flexible linker, preferably, the amino acid sequence of the first linker is as shown in SEQ ID No.16; Optionally, the amino acid sequence of the CD3 binding domain is as shown in SEQ ID No.19; Optionally, the CD3 binding domain is associated with the heavy chain constant region of the CD235a binding domain to form a fusion heavy chain and the fusion heavy chain is combined with the light chain of the CD235a binding domain to form the fusion protein, preferably, the CD3 binding domain is associated with the CD235a binding domain via a second linker; for example, the second linker is a GS-type flexible linker, preferably the amino acid sequence of the second linker is shown in SEQ ID No.15; optionally, the amino acid sequence of the fusion heavy chain is shown in SEQ ID No.20; Optionally, the fusion protein is a tetravalent IgG4 molecule comprising a variable region from mouse and a constant region from human.

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

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

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

6. A method for preparing the fusion protein according to any one of claims 1 to 2, the method comprising: Cultivating the host cell according to claim 5; as well as harvesting a cell culture fluid containing cultured host cells, and extracting and purifying the fusion protein from the cell culture fluid; Optionally, the method further comprises separating monomers and polymers of the extracted and purified fusion protein (e.g., by size exclusion chromatography (SEC) separation), preferably, the monomer has a SEC purity of about 99.3% at 280 nm and a retention time of about 7.29 min, and the polymer has no peak with 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 to 2, for example, the composition is used to enrich / isolate 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, such as a buffer and / or an osmotic pressure regulator; the buffer is, for example, histidine-histidine hydrochloride, and the osmotic pressure regulator is, for example, 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 to 2 and a sedimentation agent (e.g., hydroxyethyl starch, gelatin, dextran, polyvinyl pyrrolidone and / or methylcellulose) to a peripheral blood sample to sediment T cells and red blood cells in the peripheral blood sample; and Obtain the upper layer of the peripheral blood sample containing NK cells.

9. The method according to claim 8, wherein the sedimentation agent is hydroxyethyl starch; or optionally, the sedimentation agent is added at a final concentration of 0.5%-4%, for example, 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 according to any one of claims 8-9, wherein: Adding the fusion protein in monomer form with the sedimentation agent to the peripheral blood sample, 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 the form of a mixture of monomers and polymers with the sedimentation 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, preferably 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, more preferably about 0.94 mg of the monomer and about 1.09 mg of the polymer.

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