A method for preparing universal CAR-T cells and applications thereof

Abstract

The present application relates to the technical field of CAR-T cell treatment, and particularly relates to a method for preparing universal CAR-T cells and application thereof. The VH domain and VL domain of the beta2M binding molecule provided in the present application contain CDR sequences, and can specifically bind to beta2M. The binding molecule is added to CAR-T cells, and the binding molecule can specifically bind to CAR-T cells without beta2M gene knockout, so as to separate CAR-T cells with beta2M gene knockout from CAR-T cells without beta2M gene knockout, and realize purification of universal CAR-T cells.

Description

[0001] Priority Claim

[0002] This application claims priority to Chinese Patent Application No. 202111253059.8, filed on October 27, 2021, entitled “A Method for Preparing Universal CAR-T Cells and Applications Thereof”, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to the field of CAR-T cell therapy, in particular to a method for preparing universal CAR-T cells and applications thereof. BACKGROUND

[0004] Since August 2017, the FDA has approved two CAR-T products targeting CD19 for listing (kyriamh of Novartis and yescarta of Kite). Therefore, the demand for CAR-T cells in the treatment of hematological tumors is increasing. However, current adoptive cell therapy is still based on autologous cell reinfusion therapy. Each patient has to prepare CAR-T cells individually, which is complicated and very expensive to produce. The listed price of kyriamh product of Novartis is $475,000 per case, and the price of yescarta of Kite is also $375,000. Most tumor patients have undergone repeated treatments before CAR-T cell therapy, especially radiotherapy and chemotherapy, which results in very few T lymphocytes obtained from their peripheral blood, and these T lymphocytes are very difficult to expand in vitro, making it very difficult to prepare autologous CAR-T cells. In addition, many infant patients and severe patients cannot obtain enough T lymphocytes from their peripheral blood before preparing CAR-T cells. Therefore, allogeneic CAR-T cells (i.e., universal CAR-T cells) can greatly reduce the production cost of CAR-T cells and provide treatment opportunities for patients who cannot prepare autologous CAR-T cells. Universal CAR-T cell therapy has natural advantages over autologous CAR-T cell therapy, so its research and development are attracting more and more attention.

[0005] With the continuous improvement of gene editing technology, the efficiency of knocking out T cell β2M gene has become higher and higher. However, gene editing operation has a great impact on the state and growth rate of T cells. Generally, the higher the efficiency of β2M gene knockout, the worse the state of T cells after gene editing and the slower the cell growth rate. Therefore, in order to balance the two, the efficiency of β2M gene knockout will be controlled to a certain extent during the preparation of universal CAR-T cells. Therefore, if the prepared universal CAR-T cells are to be used for clinical cell therapy, T cells without β2M gene knockout must be removed. Otherwise, it will cause graft-versus-host disease (GvHD) or host-versus-graft disease (HvGD) in clinical application.

[0006] In view of this, the present application is proposed. SUMMARY

[0007] The present application aims to provide a β2M binding molecule. The binding molecule can be used to remove T cells without knocking out β2M gene in universal CAR-T cells, achieve purification of universal CAR-T cells, and thus reduce or avoid occurrence of graft versus host disease (GvHD) or host versus graft disease (HvGD).

[0008] Another object of the present application is to provide the above-mentioned β2M binding molecule for use in β2M detection and purification of universal CAR-T cells, and for use in preparation of products for detecting β2M or purifying universal CAR-T cells. The products include formulations or kits that can be popularized and sold on the market.

[0009] Another object of the present application is to provide a method for purifying universal CAR-T cells using the above-mentioned formulations or kits. The method has good purification effect and is simple to operate, and is suitable for popularization.

[0010] Another object of the present application is to provide a method for preparing universal CAR-T cells using the above-mentioned β2M binding molecule. The method can directly obtain universal CAR-T cells that meet the clinical purity requirements, and avoids the cumbersome purification process before use.

[0011] To solve the above technical problems and achieve the above objects, the present application provides the following technical solutions.

[0012] In a first aspect, the present application provides a β2M binding molecule comprising a module a that specifically binds to β2M.

[0013] The module a comprises a VH domain, and the VH domain comprises a CDR-H1 having an amino acid sequence shown in SEQ ID No. 1, a CDR-H2 having an amino acid sequence shown in SEQ ID No. 2, and a CDR-H3 having an amino acid sequence shown in SEQ ID No. 3.

[0014] In an optional embodiment, the module a further comprises a VL domain, and the VL domain comprises a CDR-L1 having an amino acid sequence shown in SEQ ID No. 4, a CDR-L2 having an amino acid sequence of AAA, and a CDR-L3 having an amino acid sequence shown in SEQ ID No. 5.

[0015] In another aspect, the present application further provides a β2M binding molecule comprising a module a that specifically binds to β2M.

[0016] The module a comprises a VH domain and a VL domain, the VH domain comprising a CDR-H1, a CDR-H2 and a CDR-H3;

[0017] The VL domain comprises a CDR-L1, a CDR-L2 and a CDR-L3;

[0018] The amino acid sequence of the CDR-H1, the CDR-H2 and the CDR-H3 comprises the complementarity determining region sequence of SEQ ID No. 6;

[0019] The amino acid sequence of the CDR-L1, the CDR-L2 and the CDR-L3 comprises the complementarity determining region sequence of SEQ ID No. 7.

[0020] In an alternative embodiment, the amino acid sequence of the CDR-H1, the CDR-H2 and the CDR-H3 is defined by SEQ ID No. 6 according to the Kabat, Chothia or AbM numbering system;

[0021] The amino acid sequence of the CDR-L1, the CDR-L2 and the CDR-L3 is defined by SEQ ID No. 7 according to the Kabat, Chothia or AbM numbering system.

[0022] In an alternative embodiment, the VH domain of the module a has the amino acid sequence shown in SEQ ID No. 6.

[0023] In an alternative embodiment, the VL domain of the module a has the amino acid sequence shown in SEQ ID No. 7.

[0024] In an alternative embodiment, the β2M binding molecule is selected from a scFv molecule, a Fv molecule, a Fab molecule or a whole antibody molecule that specifically binds to a β2M antigen.

[0025] In another aspect, the present application provides the use of a β2M binding molecule according to any of the preceding embodiments for the manufacture of a β2M detection product, or for the in vitro detection of β2M not for the purpose of disease diagnosis or treatment.

[0026] In another aspect, the present application provides the use of a β2M binding molecule according to any of the preceding embodiments for the purification of universal CAR-T cells or for the manufacture of a product for the purification of universal CAR-T cells.

[0027] In another aspect, the present application provides a preparation for the detection of β2M or a preparation for the purification of universal CAR-T cells, said preparation comprising a β2M binding molecule according to any of the preceding embodiments, said β2M binding molecule preferably being coupled to biotin.

[0028] In an alternative embodiment, the preparation for purifying universal CAR-T cells further comprises a second binding molecule that specifically binds to the universal CAR-T cells.

[0029] In an alternative embodiment, the second binding molecule is preferably conjugated with biotin.

[0030] In an alternative embodiment, the second binding molecule is selected from other molecules that can specifically bind to the CAR-T cells positively expressing TCR.

[0031] The ratio of the amount of the second binding molecule to the amount of the β2M binding molecule can be routinely selected by a person skilled in the art according to actual needs.

[0032] In an alternative embodiment, the target protein specifically bound by the second binding molecule is TCR or CD3.

[0033] In an alternative embodiment, the second binding molecule comprises a module c that specifically binds to TCR or CD3; the module c comprises a VH domain having the amino acid sequence shown in SEQ ID No. 8, and a VL domain having the amino acid sequence shown in SEQ ID No. 9.

[0034] In another aspect, the present application provides a kit for purifying universal CAR-T cells, which comprises the preparation for purifying universal CAR-T cells according to any one of the preceding embodiments and optional consumables.

[0035] In another aspect, the present application provides a kit for detecting β2M, which comprises the preparation for detecting β2M according to any one of the preceding embodiments and optional consumables.

[0036] In another aspect, the present application provides a method for purifying universal CAR-T cells using the preparation for purifying universal CAR-T cells according to any one of the preceding embodiments or using the kit for purifying universal CAR-T cells according to any one of the preceding embodiments, which comprises: incubating the universal CAR-T cells to be purified in the preparation for purifying universal CAR-T cells, and then using anti-biotin conjugated magnetic beads for magnetic adsorption, taking the cell suspension, and obtaining the purified universal CAR-T cells.

[0037] In the present application, the term "anti-biotin conjugated magnetic beads" refers to magnetic beads conjugated with materials that can specifically bind to biotin, such as avidin conjugated magnetic beads.

[0038] In another aspect, the present application provides a method for preparing universal CAR-T cells, comprising the following steps:

[0039] (1) the β2M binding molecule and the optional anti-CD3 antibody described in the preceding embodiments are prepared;

[0040] (2) the β2M binding molecule and the anti-CD3 antibody are respectively conjugated with biotin;

[0041] (3) the β2M gene and the TCR gene of the CAR-T cells are knocked out;

[0042] (4) the β2M binding molecule and the anti-CD3 antibody conjugated with biotin are co-cultured with the CAR-T cells after the gene knockout;

[0043] (5) magnetic adsorption is performed using anti-biotin conjugated magnetic beads, and the cell suspension is taken to obtain the purified universal CAR-T cells.

[0044] In a ninth aspect, the present application provides a method for purifying universal CAR-T cells, which comprises the step of contacting the β2M binding molecule described above with the universal CAR-T cells to be purified.

[0045] The VH domain and the VL domain of the β2M binding molecule provided by the present application contain specific CDR sequences, which can specifically bind to β2M. When the binding molecule is added to the CAR-T cells with incomplete β2M gene knockout, the binding molecule can specifically bind to the CAR-T cells with unknocked-out β2M gene, thereby separating the CAR-T cells with knocked-out β2M gene from the CAR-T cells with unknocked-out β2M gene, and realizing the purification of universal CAR-T cells.

[0046] The method for purifying universal CAR-T cells provided by the present application using the preparation or kit containing the above-mentioned β2M binding molecule can significantly improve the purity of universal CAR-T cells after the universal CAR-T cells are purified by the β2M binding molecule provided by the present application. Especially after being used in combination with the second binding molecule with the target protein being CD3, the purity of the universal CAR-T cells can be as high as 99.96%, and the in-vivo and in-vitro tumor killing activity characterization proves that the purification method of universal CAR-T cells provided by the present application does not cause adverse effects on the universal CAR-T cells.

[0047] The present application also provides a preparation method of universal CAR-T cells, which uses the β2M binding molecule and the optional anti-CD3 antibody provided by the present application, directly co-cultures with the CAR-T cells after knocking out the β2M gene and the TCR gene without separating the CAR-T cells, and then separates the universal CAR-T cells, thereby omitting the independent purification process and reducing the labor intensity of the first-line workers. BRIEF DESCRIPTION OF DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the specific embodiments of the present application or the prior art, the drawings needed to be used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0049] Figure 1 The pCDHF plasmid map used in Experimental Example 2 of the present application;

[0050] Figure 2 The kinetic affinity analysis fitting curve of the anti-human β2M antibody β2M-2B1;

[0051] Figure 3 The pX330-spCAS9-HF1 plasmid map used in Embodiment 2 of the present application;

[0052] Figure 4 The TCR / β2M gene double knockout efficiency in Experimental Example 2 of the present application;

[0053] Figure 5 The CAR-T cell purity comparison chart before and after purification in Experimental Example 2 of the present application;

[0054] Figure 6 The activated effect comparison chart of CAR-T cells after purification in Experimental Example 3 of the present application;

[0055] Figure 7 The in vitro tumor killing effect chart of CAR-T cells after purification in Experimental Example 4 of the present application;

[0056] Figure 8 The in vitro tumor killing cytokine release comparison chart of CAR-T cells after purification in Experimental Example 4 of the present application;

[0057] Figure 9 The in vivo tumor killing effect chart of CAR-T cells after purification in Experimental Example 5 of the present application;

[0058] Figure 10 The survival curve chart of mice according to the survival of mice in Experimental Example 5 of the present application. DETAILED DESCRIPTION

[0059] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are some embodiments of the present application, not all embodiments. The components of the embodiments of the present application described and shown in the drawings here can be arranged and designed in various different configurations.

[0060] Therefore, the following detailed description of embodiments of the application provided in the accompanying drawings is not intended to limit the scope of the application claimed, but merely represents selected embodiments of the application. All other embodiments obtained based on the embodiments of the application by one of ordinary skill in the art without creative work are within the scope of the application.

[0061] It should be noted that similar reference numbers and letters represent similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0062] The term "amino acid" refers to a naturally occurring carboxyl alpha-amino acid. Naturally occurring amino acids include alanine (three letter code: Ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0063] The term "universal CAR-T cell" generally refers to a CAR-T cell made from T cells obtained from healthy volunteers, in which the relevant genes (mainly genes associated with GVHD and HvGD diseases, such as genes encoding TCR (such as alpha chain, beta chain), HLA (such as beta2 microglobulin (beta2M gene)), or CD52 molecules, etc.) are knocked out, and a CAR (chimeric antigen receptor) gene is introduced.

[0064] In order to remove CAR-T cells that have not knocked out the beta2M gene, the present application takes the beta2M protein as an antigen, and constructs a binding molecule specific to the beta2M protein, i.e., a beta2M binding molecule, based on antibody structure.

[0065] In a first aspect, in a specific embodiment, the present application provides a module (a1) that specifically binds to beta2M, the module (a1) comprising a VH domain, the VH domain comprising a CDR-H1 having an amino acid sequence as shown in SEQ ID No. 1, a CDR-H2 having an amino acid sequence as shown in SEQ ID No. 2, and a CDR-H3 having an amino acid sequence as shown in SEQ ID No. 3.

[0066] wherein SEQ ID No. 1-3 are defined by the light chain variable region sequence according to the IMGT numbering system.

[0067] In another embodiment, the present application provides a module (a2) that specifically binds to β2M, the module (a2) comprising a VL domain comprising a CDR-L1 having an amino acid sequence of SEQ ID No. 4, a CDR-L2 having an amino acid sequence of AAA, and a CDR-L3 having an amino acid sequence of SEQ ID No. 5.

[0068] wherein SEQ ID No. 4, AAA of CDR-L2, and SEQ ID No. 5 are defined by the light chain variable region sequence according to the IMGT numbering system.

[0069] In another embodiment, the present application provides a module (a3) that specifically binds to β2M, the module (a3) comprising a VH domain comprising a CDR-H1 having an amino acid sequence of SEQ ID No. 1, a CDR-H2 having an amino acid sequence of SEQ ID No. 2, and a CDR-H3 having an amino acid sequence of SEQ ID No. 3, and a VL domain comprising a CDR-L1 having an amino acid sequence of SEQ ID No. 4, a CDR-L2 having an amino acid sequence of AAA, and a CDR-L3 having an amino acid sequence of SEQ ID No. 5.

[0070] In another aspect, in another embodiment, the CDR-H1, the CDR-H2, and the CDR-H3 have amino acid sequences of the complementarity determining region sequences of SEQ ID No. 6;

[0071] the CDR-L1, the CDR-L2, and the CDR-L3 have amino acid sequences of the complementarity determining region sequences of SEQ ID No. 7.

[0072] In another embodiment, the CDR-H1, the CDR-H2, and the CDR-H3 can also be defined by SEQ ID No. 6 according to the Kabat, Chothia, or AbM numbering system.

[0073] In another embodiment, the CDR-L1, the CDR-L2, and the CDR-L3 can also have amino acid sequences defined by SEQ ID No. 7 according to the Kabat, Chothia, or AbM numbering system.

[0074] The above complementarity determining regions have the sequences defined according to the Kabat, Chothia, or AbM numbering system, as shown in the following table:

[0075]

[0076] IMGT is based on the numbering system of the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

[0077] Kabat is the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0078] Chothia is the immunoglobulin numbering system proposed by Chothia et al., which is based on the location of structural loops to identify the boundaries of CDR regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0079] AbM defines CDRs in a manner derived from the work of Martin (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272), which incorporates parts of both the Kabat and Chothia definitions.

[0080] In combination with the CDR-containing amino acid sequences of each of the modules of the first aspect, in a specific embodiment of the second aspect, the present application provides each of the following modules:

[0081] Module (a11) comprises a VH domain having the amino acid sequence shown as SEQ ID No. 6.

[0082] Module (a21) comprises a VL domain having the amino acid sequence shown in SEQ ID No. 7.

[0083] Module (a31) comprises a VH domain having the amino acid sequence shown in SEQ ID No. 6 and a VL domain having the amino acid sequence shown in SEQ ID No. 7.

[0084] In combination with each module provided in the second aspect, the third aspect of the present application provides a scFV molecule comprising a VH domain and a VL domain connected by a linker; the VH domain has the amino acid sequence shown in SEQ ID No. 6 and the VL domain has the amino acid sequence shown in SEQ ID No. 7.

[0085] The amino acid residue composition and length of the linker can be adjusted by routine means according to actual needs by those skilled in the art, including but not limited to composed of glycine (Gly) and serine (Ser) with a length of 15-25 amino acid residues.

[0086] As an exemplary linker, it includes (GGGGS)n, (GGGS)n, (GGS)n, or (GS)n, wherein n is selected from 1, 2, 3, 4, 5 or 6. The fourth aspect of the present application provides a Fv molecule comprising a VH domain and a VL domain connected by a short peptide; the VH domain has the amino acid sequence shown in SEQ ID No. 6 and the VL domain has the amino acid sequence shown in SEQ ID No. 7.

[0087] The short peptide can be adjusted by routine means according to actual needs by those skilled in the art, including but not limited to a short peptide chain composed of 3-9 amino acid residues.

[0088] The fifth aspect of the present application provides a Fab molecule comprising the VH domain, the VL domain, a light chain constant region (CL) and a heavy chain constant region (CH1); the VH domain has the amino acid sequence shown in SEQ ID No. 6 and the VL domain has the amino acid sequence shown in SEQ ID No. 7.

[0089] For CH1 and CL, one skilled in the art can select according to actual needs, for example, the CH1 is selected from any one or several of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE or IgM, and the CL is selected from a kappa chain or a lambda chain, and one skilled in the art can also adjust the sequences and modifications of the selected CH1 and CL according to actual needs by conventional means.

[0090] In a sixth aspect, the present application also provides a complete antibody molecule, which comprises two identical heavy chains and two identical light chains, the heavy chain comprises a VH domain and a heavy chain constant region, and the light chain comprises a VL domain and a light chain constant region; the VH domain has the amino acid sequence shown in SEQ ID No. 6, and the VL domain has the amino acid sequence shown in SEQ ID No. 7.

[0091] For the specific sequence composition of the heavy chain constant region and the light chain constant region, one skilled in the art can select according to actual needs, for example, the heavy chain constant region can be selected from any one or several of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE or IgM, and the light chain constant region can be selected from a kappa chain or a lambda chain.

[0092] In a specific embodiment, the heavy chain constant region and the light chain constant region of the complete antibody molecule are selected from murine IgG.

[0093] In addition, one skilled in the art can also adjust the sequences or modifications of the selected heavy chain constant region and light chain constant region according to actual needs by conventional means.

[0094] In a seventh aspect, the present application provides the use of each module, scFV molecule, Fv molecule, Fab molecule or complete antibody molecule described in the foregoing embodiments in the preparation of a β2M detection product, or in the in vitro detection of β2M for purposes other than disease diagnosis.

[0095] In an eighth aspect, the present application provides the use of each module, scFV molecule, Fv molecule, Fab molecule or complete antibody molecule described in the foregoing embodiments in the purification of universal CAR-T cells or in the preparation of a product for purifying universal CAR-T cells.

[0096] In a specific embodiment, the gene knocked out in the universal CAR-T cell at least comprises a β2M gene.

[0097] In a specific embodiment, the gene knocked out in the universal CAR-T cell further comprises a TCR gene. For example, it can be the alpha chain encoding gene or the beta chain encoding gene of a TCR molecule.

[0098] In a ninth aspect, in one specific embodiment, the present application provides a preparation for purifying universal CAR-T cells, which comprises the module, scFV molecule, Fv molecule, Fab molecule or whole antibody molecule of any of the preceding embodiments; and the module, scFV molecule, Fv molecule, Fab molecule or whole antibody molecule is coupled with biotin. It can be understood that the biotin-coupled magnetic beads are a common separation means in the art.

[0099] In a tenth aspect, in one specific embodiment, the present application provides a kit for purifying universal CAR-T cells, which comprises the preparation of the preceding embodiments and consumables. It can be understood that the consumables comprise the kit components in the art, such as well plates, reaction vessels, liquid taking devices, etc.

[0100] In an eleventh aspect, in one specific embodiment, when the gene knocked out in the universal CAR-T cell further comprises a TCR gene; the preparation or the kit further comprises a second binding molecule coupled with biotin. The second binding molecule can specifically bind to the CAR-T cell positively expressing TCR.

[0101] The target protein of the second binding molecule is TCR or CD3, which can be selected from CD3 antibodies or TCR antibodies, for example. The ratio of the use amount of the second binding molecule to the β2M binding molecule can be routinely selected by those skilled in the art according to actual needs.

[0102] In another specific embodiment, the target protein specifically bound by the second binding molecule is TCR.

[0103] In another specific embodiment, the target protein specifically bound by the second binding molecule is CD3, and the second binding molecule comprises a module c specifically binding to CD3.

[0104] The module c comprises a VH domain having the amino acid sequence shown in SEQ ID No. 8, and a VL domain having the amino acid sequence shown in SEQ ID No. 9.

[0105] In a twelfth aspect, in one specific embodiment, the present application provides a method for purifying universal CAR-T cells, which uses the preparation or the kit described above, incubates the universal CAR-T cells in the preparation, and then uses anti-biotin-coupled magnetic beads for magnetic adsorption, takes the cell suspension, and obtains the purified universal CAR-T cells.

[0106] In another specific embodiment, the present application provides a method for preparing universal CAR-T cells, which comprises the following steps:

[0107] (1) the β2M-binding molecule and optionally the anti-CD3 antibody described in the preceding embodiments are prepared;

[0108] (2) the β2M-binding molecule and the anti-CD3 antibody are each conjugated with biotin;

[0109] (3) the β2M gene and the TCR gene of the CAR-T cells are knocked out;

[0110] (4) the β2M-binding molecule and the anti-CD3 antibody conjugated with biotin are co-cultured with the CAR-T cells after the genes are knocked out;

[0111] (5) magnetic adsorption is performed using anti-biotin conjugated magnetic beads, and the cell suspension is taken to obtain the purified universal CAR-T cells.

[0112] In a thirteenth aspect, the present application provides, in one specific embodiment, a method for purifying universal CAR-T cells, which comprises: contacting the universal CAR-T cells to be purified with any of the modules, Fv molecules, Fab molecules, whole antibody molecules, or preparations described above, so as to separate and remove the CAR-T cells that have not been knocked out of the β2M gene therefrom.

[0113] In another specific embodiment, when the universal CAR-T cells needed are also knocked out of the TCR gene, the purification method can further comprise contacting the universal CAR-T cells to be purified with a second binding molecule, so as to separate and remove the CAR-T cells that have not been knocked out of the TCR gene (i.e., CAR-T cells positive for TCR expression) therefrom.

[0114] In another specific embodiment, the target protein of the second binding molecule can be TCR or CD3. On the CAR-T cells that have not been knocked out of the TCR gene, TCR and CD3 exist in the form of a complex, and the use of a binding molecule that binds to TCR or CD3 can achieve the removal of the CAR-T cells that have not been knocked out of the TCR gene.

[0115] In another specific embodiment, the second binding molecule can be used after, before, or simultaneously with any of the modules, Fv molecules, Fab molecules, whole antibody molecules, or preparations described above.

[0116] For example, any of the modules, Fv molecules, Fab molecules, whole antibody molecules, or preparations described above, and the second binding molecule are simultaneously contacted with the universal CAR-T cells to be purified;

[0117] For example, after any of the modules, Fv molecules, Fab molecules, whole antibody molecules, or preparations described above are contacted with the universal CAR-T cells to be purified, the second binding molecule is further contacted with the universal CAR-T cells to be purified;

[0118] For example, after contacting the universal CAR-T cells to be purified with the second binding molecule, any of the modules, Fv molecules, Fab molecules, whole antibody molecules or preparations described above are used to contact the universal CAR-T cells to be purified.

[0119] In another specific embodiment, the second binding molecule comprises a module c that specifically binds to CD3;

[0120] The module c comprises a VH domain having the amino acid sequence shown in SEQ ID No. 8, and a VL domain having the amino acid sequence shown in SEQ ID No. 9. Some embodiments of the present application are described in detail below with reference to the accompanying drawings. The embodiments described below and the features in the embodiments can be combined with each other without conflict.

[0121] The modules, scFV molecules, Fv molecules, Fab molecules and whole antibody molecules provided in the first aspect described above can be obtained by artificial synthesis, and the whole antibody molecules can also be obtained by constructing hybridoma cells and expressing and secreting.

[0122] Example 1

[0123] The present embodiment provides a preparation method of an anti-human β2M antibody, and the specific steps are as follows:

[0124] The recombinant β2M protein antigen (produced by Shenzhen Fipeng Biological Co., Ltd., product number BA-PAB-MU0001) was mixed with Freund's adjuvant (sigma, product number F5881) in equal amounts for emulsification. Healthy BALB / C mice (Guangdong Experimental Animal Center, 6 weeks old, female) were immunized by subcutaneous injection at multiple points on the back; 14 days later, the mice were immunized intraperitoneally; after four times of booster immunization, the tail blood of the mice was collected for titer detection, and the mouse spleen with the highest titer was selected for cell fusion with mouse myeloma cells SP2 / 0.

[0125] The spleen of the mouse was taken out under sterile condition on the third day after the last immunization, put in a dish, washed once with RPMI1640 (gibco, item number 12633012) basic culture solution, grinded on a nylon net in a small beaker to prepare a cell suspension. Centrifuged, the supernatant was discarded, and the cell was resuspended with RPMI1640 culture solution. The spleen cells were mixed with the prepared mouse myeloma cells SP20 at a ratio of 10:1, and then cell fusion was performed with 1ml of 50% PEG1500 (sigma, item number 81210), and the cell fusion was terminated after 1 minute by adding 15ml of RPMI1640 complete culture solution. Centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, and the cell was resuspended with 50ml of RPMI1640 screening culture solution, and then divided into 10 96-well plates, 100ul / well, and cultured at 37℃, 5% CO2. The culture was changed to HAT culture solution twice on the 6th day. The cell supernatant was taken on the second day, and the human β2M antigen was detected by ELISA method. The positive wells were subjected to monoclonalization by limiting dilution method; after 3 times of subcloning, a plurality of cell strains capable of stably secreting monoclonal antibodies were screened, and one of them was named β2M-2B1 cell strain.

[0126] The subcloned hybridoma cells of 1 to 5 million β2M-2B1 cell strains were used to extract total RNA using QIAGEN RNAeasy Mini kit, and then first strand cDNA was generated using Superscript III RT kit (Invitrogen), and then double-stranded cDNA of anti-human β2M IgG variable region was amplified by PCR reaction and expansion using conventional primers from mouse IgG heavy chain (IgG1, IgG2a, IgG2b) and light chain (κ or λ) constant region, and the PCR cycle conditions were 95℃ for 1 minute for 1 cycle; 95℃ for 1 minute, 63℃ for 1 minute and 72℃ for 1 minute for 25 cycles. The obtained PCR product was cloned into T vector (Invitrogen) and sequenced.

[0127] The sequencing results are as follows:

[0128] The anti-human β2M antibody β2M-2B1 belongs to mouse IgG1 subtype, and the light chain is κ chain;

[0129] The sequence of the heavy chain variable region is as follows:

[0130] EVQLQQSGPELVKPGASVRMSCKASGYTFSSYVLHWVKQKPGQGLEWIGYFNPYNDGTKYNEKFKGKATLTSDKSSSTAYMEFSSLTSEDSAVYYCARRGNTYDNFDYWGQGTTLTVSS (SEQ ID No. 6).

[0131] According to IMGT numbering system, it comprises heavy chain complementarity determining region 1 (CDR-H1): GYTFSSYV, i.e. SEQ ID No. 1, heavy chain complementarity determining region 2 (CDR-H2): FNPYNDGT, i.e. SEQ ID No. 2 and heavy chain complementarity determining region 3 (CDR-H3): ARRGNTYDNFDY, i.e. SEQ ID No. 3.

[0132] The sequence of the light chain variable region is as follows:

[0133] DVQMTQSPASQSASLGESVTITCLASQTIGTWLAWYQQKPGKSPQLLIYAAASLADGVPSRFSGSGSGTKFSFKISSLQAEDFVSYYCQQLYSSPLTFGGGTMLEIKR (SEQ ID No. 7).

[0134] According to IMGT numbering system, it comprises light chain complementarity determining region 1 (CDR-L1): QTIGTW, i.e. SEQ ID No. 4, light chain complementarity determining region 2 (CDR-L2): AAA and light chain complementarity determining region 3 (CDR-L3): QQLYSSPLT, i.e. SEQ ID No. 5.

[0135] Example 2

[0136] In this example, the anti-human β2M antibody β2M-2B1 obtained in Example 1 is biotinylated to prepare a β2M binding preparation, and the specific steps are as follows:

[0137] The anti-human β2M antibody β2M-2B1 is diluted to 1 mg / ml with 0.1 mol / L sodium bicarbonate (pH 8.0), and 1 mg of BNHS is dissolved in 1 ml of DMSO (dimethyl sulfoxide). 120 μl of BNHS solution (containing 120 μg of BNHS) is added to 1 ml of the antibody (i.e. 1 mg), and the biotin and the antibody are mixed at a mass ratio of 1:8.3. The mixture is stirred at room temperature (20-25°C) for 2 hours (the stirring time should not be too long, otherwise the antibody will be inactivated), and then the mixture is loaded into a dialysis bag and dialyzed against 0.01 mol / L PBS solution (pH 7.4) at 4°C overnight. The next day, the dialysis is continued for another 4-6 hours to remove free biotin. According to the final volume of the dialyzed mixture, an equal volume of buffer solution is added, and the mixture is stored at 4°C in the dark. The buffer solution is prepared at a ratio (by volume) of PBS+0.01% preservative+1% BSA.

[0138] Example 3

[0139] The embodiment provides a preparation method of the anti-human CD3 antibody, and the whole steps are the same as those in Embodiment 1, except that the immunogen is CD3. One cell strain obtained is selected and named as CD3-2C1 cell strain.

[0140] Sequencing shows that the CD3-2C1 antibody belongs to a mouse IgG1 subtype, and the light chain is a kappa chain; the VH domain has the amino acid sequence shown in SEQ ID No. 8, and the VL domain has the amino acid sequence shown in SEQ ID No. 9.

[0141] Embodiment 4

[0142] The embodiment biotin-couples the anti-human CD3 antibody CD3-2C1 obtained in Embodiment 3 to prepare a CD3 binding preparation, and the coupling method is the same as that in Embodiment 2.

[0143] Experimental Example 1

[0144] The anti-human β2M antibody β2M-2B1 prepared in Embodiment 1 is used to detect a solution of recombinant human β2M in vitro, and the detection method is as follows:

[0145] 5 μg / ml Ag (recombinant human β2M, His tag), mIgG (10 μg / ml, 2x) and the anti-human β2M antibody β2M-2B1 (10 μg / ml, 2x) are co-cultured, 2-fold gradient dilution is performed, then the binding of the β2M-2B1 and the β2M is monitored by using a HIS1K biosensor, and the affinity of the anti-human β2M antibody β2M-2B1 is determined by using an Octet HIS1K.

[0146] The experimental conditions are shown in Table 1:

[0147] Table 1: Experimental conditions for detection by using a HIS1K biosensor in Experimental Example 1

[0148] N Data Name Assay Time (sec) Flow Rate 1 Baseline 60 1000 2 Association 180 1000 3 Dissociation 300 1000 4 Baseline 1 90 1000 5 Loading 120 1000 6 Custom 5 1000

[0149] Buffer: 1x DPBS + 0.02% Tween-20.

[0150] The fitting curve of the kinetic affinity analysis of the anti-human β2M antibody β2M-2B1 after 2-fold gradient dilution is shown in Figure 2 The experimental results of the affinity of the anti-human β2M antibody β2M-2B1 to human β2M are shown in Table 2, and the results show that the anti-human β2M antibody β2M-2B1 has a very high affinity to human β2M (KD<1.0E-12 M), which proves that the anti-human β2M antibody β2M-2B1 provided in Embodiment 1 can be used for detecting β2M, or for preparing a product for detecting β2M.

[0151] Table 2 Anti-human β2M antibody β2M-2B1 affinity results for human β2M

[0152]

[0153] Experimental Example 2

[0154] In this experimental example, the biotin-conjugated β2M-2B1 preparation prepared in Example 2 and the biotin-conjugated CD3-2C1 preparation prepared in Example 4 were used to purify universal CAR-T cells with TCR and β2M genes knocked out, and the purification effect was investigated.

[0155] 1.1 TCR / β2M gene knockout

[0156] The lentivirus prepared from the target plasmid of Figure 1 was used to infect T cells to obtain CART-CD19 cells (CART cells targeting CD19). 1 x 10 6 CART-CD19 cells, 1 μg of pX330-spCAS9-HF1-TRAC TRAC sgRNA expression plasmid (knock out the α gene of T cell receptor (MHC class II molecule), the nucleic acid sequence expressing sgRNA is shown in SEQ ID NO. 10), 1 μg of pX330-spCAS9-HF1-β2M β2M sgRNA expression plasmid (knock out the β2 microglobulin gene of CAR-T cells (MHC class I molecule), the nucleic acid sequence expressing sgRNA is shown in SEQ ID NO. 11) were mixed uniformly in 200 μL of OPTI-MEM, 4 μL of PEI or 6 μL of lipo2000 reagent was added, and the mixture was mixed uniformly and incubated at room temperature for 15 min, then dropped into the hole, and the cells were replaced with fresh culture medium after 6-8 h. Figure 3

[0157] SEQ ID NO. 10: acaa aacugugcuagacaug;

[0158] SEQ ID NO. 11: cgcgagcacagcuaaggcca.

[0159] The TCR / β2M double-knockout efficiency is shown in Figure 4 The results show that the proportion of TCR / β2M gene KO cells analyzed by flow cytometry (FACS) is 71.35% after 48 h of medium replacement. In addition, the editing tool can also enter the cell in the form of sgRNA and RNP through electroporation and viral infection.

[0160] ​1.2 Purification of KO cells (UCAR-T cells)

[0161] Cells cultured for 4 days after electroporation were centrifuged and resuspended in a certain amount of DPBS to a density of 1×10⁻⁶. 8 Cells / ml were purified in one step using CD3-2C1-Biotin (an anti-human CD3 antibody conjugated with biotin, from Example 4) and β2M-2B1-Biotin (an anti-human β2M antibody conjugated with biotin, from Example 2). The purification method was as follows: 100 μl / ml CD3-2C1-Biotin and 100 μl / ml β2M-2B1-Biotin were added and incubated on ice in the dark for 20 min. After washing once with PBS, anti-Biotin Beads (purchased from Miltenyi Biotec, catalog number 130-090-485) were added and incubated on ice for 15 min. Then, the cells were placed in a magnet for 5 min, and the cell suspension containing UCAR-T cells was collected. 2 × 10⁻⁶ cells were taken. 5 Cell purity was determined by flow cytometry after incubation with APC-anti-human TCRa / β (Biolegend, B259839) and PE-anti-human β2M (Biolegend, B226121) antibodies for 15 min.

[0162] The image shows a comparison of CAR-T cell purity before and after purification. Figure 5 As shown, the results indicate that the TCR / β2M gene was knocked out and the cell purity was 99.96%.

[0163] It should be noted that the one-step purification method of CD3-2C1-Biotin and β2M-2B1-Biotin described above can also be replaced by two-step purification using CD3-2C1-Biotin and β2M-2B1-Biotin respectively, and the order of addition of the two purification antibodies has no significant impact on the purification results.

[0164] Experimental Example 3

[0165] The activation activity of the purified universal CAR-T cells from step 1.2 of Experiment Example 2 was detected, and the steps are as follows:

[0166] In this experiment, the effector cells were UCAR-T cells, CAR-T cells, and T cells (the latter two being controls). The cells were seeded in 24-well plates at a rate of 5.0 × 10⁶ cells per well. 5 Cells were collected in 500 μl volumes and treated with PHA-P at a concentration of 2.5 μg / ml. After 48 h, the cell surface activation molecules CD25 and CD69 were detected.

[0167] The comparison of the activation effect of purified CAR-T cells is shown in the figure below. Figure 6As shown, the results indicate that the universal CAR-T cannot be reactivated after two-step purification, while the control group was significantly activated.

[0168] Experiment Example 4

[0169] The in vitro tumor-killing activity of the purified universal CAR-T cells from step 1.2 of Experiment Example 2 was detected as follows:

[0170] This experiment used K562 cells and K562-CD19 cells (denoted as K19) as target cells. The purified effector cells UCAR-T used in this experiment were named UCART-19. CAR-T cells without gene knockout (named CART-19) and T cells were selected as controls. First, the target cells were stained with cytocalcein™ violet 550. Then, the effector cell density was adjusted to 5 × 10⁻⁶ cells / year. 6 Cells / ml, target cell density 5×10⁶ 5 Cells / ml. The three types of effector cells and target cells were added to 96-well plates at ratios of (0:1), (0.25:1), (1:1), (5:1), and (10:1), respectively, and co-cultured for 6 h and 24 h. Apoptosis was observed under a microscope during the above time periods. The mixed cells were centrifuged, and the supernatant was used to detect IL-2 and IFN-γ using the Human IL-2 Ready-SET-Go and Human IFN gamma ELISA Ready-SET-Go ELISA kits. The precipitate was resuspended in 100 μl of binding buffer, centrifuged at 300g for 5 min, and 1.2 μl of APC-Annexin V and 1.2 μl of PI dye were added. The cells were incubated in the dark for 15 min, and 100 μl of binding buffer was added to detect the apoptosis efficiency by flow cytometry.

[0171] The in vitro tumor-killing effect of purified CAR-T cells and the comparison of cytokine release are shown in the following figures. Figure 7 and Figure 8 As shown in the figure, the results indicate that the purified universal CAR-T cells have comparable in vitro tumor-killing effects to conventional CAR-T cells.

[0172] Experimental Example 5

[0173] The in vivo tumor-killing activity of the purified universal CAR-T cells from step 1.2 of Experiment Example 2 was detected as follows:

[0174] Twenty NPG mice were injected with 5.0 × 10⁶ raji-luc cells. 5, 5 days later, the mice were imaged by a small animal live fluorescence imaging instrument, and were divided into 5 groups according to the imaging results, and were injected with PBS, T-cell, CAR-T, UCART-19 1# cells and UCART-19 2# cells, respectively, with a number of 5.0x10 6 After 5 days, the mice were imaged by a small animal live fluorescence imaging instrument, and were divided into 5 groups according to the imaging results, and were injected with PBS, T-cell, CAR-T, UCART-19 1# cells and UCART-19 2# cells, respectively, with a number of 5.0x10 Figure 9 As shown in FIG. 6, the in vivo tumor killing effects of CAR-T, UCART-19 1# and UCART-19 2# were equivalent; and as shown in FIG. 7, the survival rates of the mice in the three groups were the same; it can be seen that the purified universal CAR-T cells and the conventional CAR-T cells have equivalent in vivo tumor killing effects. Figure 10 As shown in FIG. 6, the in vivo tumor killing effects of CAR-T, UCART-19 1# and UCART-19 2# were equivalent; and as shown in FIG. 7, the survival rates of the mice in the three groups were the same; it can be seen that the purified universal CAR-T cells and the conventional CAR-T cells have equivalent in vivo tumor killing effects.

[0175] Comparative Example 1

[0176] The anti-human β2M antibody β2M-2B1 in Example 2 was replaced with a commercially available anti-human β2M antibody (Biolegend, 395702), and PE was coupled to prepare a β2M binding preparation; in addition, the anti-human CD3 antibody CD3-2C1 in Example 4 was replaced with a commercially available anti-human CD3 antibody (Biolegend, 317302), and PE was coupled to prepare a CD3 binding preparation; the coupling method was the same as that in Example 2. The principle of the action of PE and biotin is the same.

[0177] The universal CAR-T cells with knocked-out TCR and β2M genes were purified using the above-prepared β2M binding preparation and CD3 binding preparation, and the purification effect was investigated.

[0178] The results show that the purification effect of the above β2M binding preparation and CD3 binding preparation on the universal CAR-T cells is only 72.73%, which indicates that the anti-human β2M antibody β2M-2B1 and the anti-human CD3 antibody CD3-2C1 of the present application have a significant purification effect on the universal CAR-T cells compared with the commercially available anti-human β2M antibody and the anti-human CD3 antibody.

[0179] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part or all of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A β2 microglobulin-binding molecule, characterized in that, It is module a, which specifically binds to β2 microglobulin; module a is an scFv molecule, an Fv molecule, a Fab molecule, or a complete antibody molecule; Module a includes a VH domain, which includes CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID No. 1, the amino acid sequence of CDR-H2 is shown in SEQ ID No. 2, and the amino acid sequence of CDR-H3 is shown in SEQ ID No.

3. SEQ ID Nos. 1-3 are defined by the heavy chain variable region sequences according to the IMGT numbering system. Module a further includes a VL domain, which includes CDR-L1, CDR-L2, and CDR-L3. The amino acid sequence of CDR-L1 is shown in SEQ ID No. 4, the amino acid sequence of CDR-L2 is AAA, and the amino acid sequence of CDR-L3 is shown in SEQ ID No.

5. SEQ ID No. 4, AAA of CDR-L2, and SEQ ID No. 5 are defined by the light chain variable region sequence according to the IMGT numbering system.

2. A β2 microglobulin-binding molecule, characterized in that, The β2 microglobulin-binding molecule is selected from scFv molecules, Fv molecules, Fab molecules, or intact antibody molecules that specifically bind to the β2 microglobulin antigen. The β2 microglobulin binding molecule includes a VH domain and a VL domain, wherein the VH domain includes CDR-H1, CDR-H2 and CDR-H3; The VL structural domain includes CDR-L1, CDR-L2 and CDR-L3; Wherein, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are the complementarity-determining region sequences in the heavy chain variable region sequence shown in SEQ ID No. 6; The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are the complementarity-determining region sequences in the light chain variable region sequence shown in SEQ ID No. 7; The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are defined by SEQ ID No. 6 according to the Kabat, Chothia, or AbM numbering system; The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are defined by SEQ ID No. 7 according to the Kabat, Chothia or AbM numbering system.

3. The β2-microglobulin binding molecule according to claim 1 or 2, characterized in that, The VH domain is the amino acid sequence shown in SEQ ID No.

6.

4. The β2-microglobulin binding molecule according to claim 1 or 2, characterized in that, The VL domain is the amino acid sequence shown in SEQ ID No.

7.

5. The use of the β2-microglobulin binding molecule according to any one of claims 1 to 4 in the preparation of β2-microglobulin detection products, or in the in vitro detection of β2-microglobulin for purposes other than disease diagnosis or treatment.

6. The use of the β2 microglobulin-binding molecule according to any one of claims 1 to 4 in purifying universal CAR-T cells for non-disease diagnosis or treatment purposes or in preparing products for purifying universal CAR-T cells.

7. A formulation for detecting β2-microglobulin or for purifying universal CAR-T cells, characterized in that, The formulation includes a first binding molecule, wherein the first binding molecule is the β2 microglobulin binding molecule according to any one of claims 1 to 4.

8. The formulation according to claim 7, characterized in that, The β2 microglobulin-binding molecule is coupled with biotin.

9. The formulation according to claim 8, characterized in that, When the gene knocked out in the universal CAR-T cells also includes the TCR gene; the formulation for purifying universal CAR-T cells also includes a second binding molecule that specifically binds to TCR-positive CAR-T cells; The target protein that specifically binds to the second binding molecule is CD3, and the second binding molecule is an anti-CD3 antibody; The amino acid sequence of the VH domain of the anti-CD3 antibody is SEQ ID No. 8, and the amino acid sequence of the VL domain of the anti-CD3 antibody is SEQ ID No.

9.

10. The formulation according to claim 9, characterized in that, The second binding molecule is coupled with biotin.

11. A kit for purifying universal CAR-T cells or for detecting β2-microglobulin, characterized in that, The kit comprises the formulation as described in any one of claims 7 to 10 and optional consumables.

12. A method for purifying universal CAR-T cells using the formulation according to any one of claims 7 to 10, or using the kit according to claim 11, characterized in that, After incubating the universal CAR-T cells to be purified in a preparation for purifying universal CAR-T cells, magnetic adsorption was performed using antibiotin-conjugated magnetic beads. The cell suspension was then collected to obtain purified universal CAR-T cells. This method is for purposes other than disease diagnosis or treatment.

13. A method for preparing universal CAR-T cells, said method for purposes other than disease diagnosis or treatment, characterized in that, Includes the following steps: (1) Prepare the β2 microglobulin binding molecule according to any one of claims 1 to 4, and prepare an anti-CD3 antibody; the amino acid sequence of the VH domain of the anti-CD3 antibody is SEQ ID No. 8, and the amino acid sequence of the VL domain is SEQ ID No. 9; (2) The β2 microglobulin-binding molecule and the anti-CD3 antibody are respectively conjugated to biotin; (3) Knockout of β2 microglobulin gene and TCR gene in CAR-T cells; (4) The biotin-conjugated β2 microglobulin-binding molecule and anti-CD3 antibody were co-cultured with gene-knockout CAR-T cells; (5) Use avidin-conjugated magnetic beads for magnetic adsorption, and take the cell suspension to obtain purified universal CAR-T cells.

14. A method for purifying universal CAR-T cells, the method being used for purposes other than disease diagnosis or treatment, characterized in that, It includes: The step of contacting the β2 microglobulin-binding molecule according to any one of claims 1 to 4 with the universal CAR-T cells to be purified.

15. The method according to claim 14, characterized in that, The steps include: contacting the β2 microglobulin-binding molecule and the second binding molecule with the universal CAR-T cells to be purified. Alternatively, after contacting the universal CAR-T cells to be purified with the second binding molecule, the β2 microglobulin binding molecule can be used to contact the universal CAR-T cells to be purified again. Alternatively, after contacting the β2 microglobulin binding molecule with the universal CAR-T cells to be purified, a second binding molecule may be used to contact the universal CAR-T cells to be purified. The second binding molecule specifically binds to TCR-positive CAR-T cells; The second binding molecule is an anti-CD3 antibody; The amino acid sequence of the VH domain of the anti-CD3 antibody is SEQ ID No. 8, and the amino acid sequence of the VL domain of the anti-CD3 antibody is SEQ ID No. 9.

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